cells

Review
Utilisation of Chick Embryo Chorioallantoic Membrane as a
Model Platform for Imaging-Navigated Biomedical Research
Lei Chen 1 , Shuncong Wang 1 , Yuanbo Feng 1 , Jinyong Zhang 2,3 , Yuqing Du 2 , Jiang Zhang 4 ,
Chantal Van Ongeval 1 , Yicheng Ni 1, * and Yue Li 2, *

1 KU Leuven, Biomedical Group, Campus Gasthuisberg, 3000 Leuven, Belgium; lei.chen@kuleuven.be (L.C.);
shuncong.wang@kuleuven.be (S.W.); yuanbo.feng@kuleuven.be (Y.F.);
chantal.vanongeval@uzleuven.be (C.V.O.)
2 Shanghai Key Laboratory of Molecular Imaging, Shanghai University of Medicine and Health Sciences,
Shanghai 201318, China; 192672234@st.usst.edu.cn (J.Z.); luojy@sumhs.edu.cn (Y.D.)
3 School of Medical Instrument and Food Engineering, University of Shanghai for Science & Technology,
Shanghai 200093, China
4 Faculty of Agricultural Biotechnology and Ecotechnology, Shanghai Vocational College of Agriculture and
Forestry, Shanghai 201600, China; zhangj@shafc.edu.cn
* Correspondence: yicheng.ni@kuleuven.be (Y.N.); liy_16@sumhs.edu.cn (Y.L.)

Abstract: The fertilised chick egg and particularly its chorioallantoic membrane (CAM) have drawn
continuing interest in biomedicine and bioengineering fields, especially for research on vascular
study, cancer, drug screening and development, cell factors, stem cells, etc. This literature review sys-
temically introduces the CAM’s structural evolution, functions, vascular features and the circulation
system, and cell regulatory factors. It also presents the major and updated applications of the CAM



in assays for pharmacokinetics and biodistribution, drug efficacy and toxicology testing/screening


in preclinical pharmacological research. The time course of CAM applications for different assays
Citation: Chen, L.; Wang, S.; Feng, Y.; and their advantages and limitations are summarised. Among these applications, two aspects are
Zhang, J.; Du, Y.; Zhang, J.; Van emphasised: (1) potential utility of the CAM for preclinical studies on vascular-disrupting agents
Ongeval, C.; Ni, Y.; Li, Y. Utilisation (VDAs), promising for anti-cancer vascular-targeted therapy, and (2) modern imaging technologies,
of Chick Embryo Chorioallantoic
including modalities and their applications for real-time visualisation, monitoring and evaluation of
Membrane as a Model Platform for
the changes in CAM vasculature as well as the interactions occurring after introducing the tested
Imaging-Navigated Biomedical
medical, pharmaceutical and biological agents into the system. The aim of this article is to help
Research. Cells 2021, 10, 463.
https://doi.org/10.3390/cells1002
those working in the biomedical field to familiarise themselves with the chick embryo CAM as an
0463 alternative platform and to utilise it to design and optimise experimental settings for their specific
research topics.
Received: 25 January 2021
Accepted: 19 February 2021 Keywords: chick embryo; chorioallantoic membrane (CAM); preclinical pharmacological research;
Published: 22 February 2021 vascular disrupting agents (VDAs); imaging techniques

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in
published maps and institutional affil- 1. Introduction
iations.
To produce new medications which are safe and efficacious, and can pass all regulatory
requirements, pharmaceutical development must proceed through several stages: (1)
drug candidate discovery, (2) molecule characterisation, (3) formulation for delivery, (4)
pharmacokinetics and biodistribution, (5) efficacy and toxicology testing, (6) investigational
Copyright: © 2021 by the authors. new drug (IND) application, (7) bioanalytical testing and (8) phase I–IV clinical trials.
Licensee MDPI, Basel, Switzerland. The intermediate in vivo preclinical studies of the above stages 4 and 5 are traditionally
This article is an open access article conducted, by the laboratories of academia or industry, on experimental animals without
distributed under the terms and
and with human disease simulation. However, animal welfare and protection are raising
conditions of the Creative Commons
more and more public concerns. The currently advocated guiding policy is to replace,
Attribution (CC BY) license (https://
reduce, and refine (3 Rs) animal use in research. Therefore, with plausible ethical, scientific,
creativecommons.org/licenses/by/
legal and economic reasons, it is necessary to develop scientific methods or platforms to
4.0/).

Cells 2021, 10, 463. https://doi.org/10.3390/cells10020463 https://www.mdpi.com/journal/cells

Cells 2021, 10, 463 2 of 40

reduce the need for animals or eventually to replace them entirely in scientific research and
the pharmaceutical industry. Thus, fertilised eggs or chick embryos have become a good
choice.
The egg embryo is considered a good pharmaceutical testing platform with the fol-
lowing advantageous features: (1) the egg embryo is a complete creature, with necessary
organs within an isolated environment; (2) the size of the egg embryo is small and easy to
handle; (3) the egg embryo contains rich nutrients and vigorous angiogenesis capacities;
(4) eggs do not possess a complete immune system at certain stages of chick development
and are much less expensive than immune-compromised animals; and (5) eggs are less
restricted with animal welfare concerns because they are not considered animals yet.
Embryologically, during the development of fertilised eggs, an extremely rich vascular
network is generated between the double layers of the chorioallantoic membrane (CAM).
This vascular network fuses closely underneath the eggshell and connects to the embryonic
circulation via the allantoic stalk. The CAM system has been widely used in in vivo assays
for studying angiogenesis [1] and for human tumour growth and therapies [2]. Indeed,
angiogenesis and anti-angiogenesis processes related to tissues, cells or soluble factors are
tested by the CAM [2]. Many substances have been reported to boost or inhibit angiogenesis
in the CAM, for instance growth factors, anti-cancer agents, pro-angiogenic molecules,
natural and synthetic molecules, antibodies, organic-metallic compounds, antibiotics,
etc. [3].
Meanwhile, the classical property of the CAM with a native vasculature already
allows testing the pharmacological effects of certain compounds in biomedical research.
Small-molecule vascular disrupting agents (VDAs) constitute a new therapy for cancer and
can selectively affect the tumour vasculature via some pathways to inhibit blood flow and
cause extensive necrosis within the tumour [3–5]. Therefore, VDAs are ideal candidates for
demonstration of the efficiency and feasibility of the CAM platform in preclinical efficacy
and toxicology testing of pharmaceuticals. To the best of our knowledge, only few studies
have been conducted in this area.
The crucial step in evaluation is to observe and monitor vascular alterations, including
angiogenesis, vascular disrupting processes following administration of tested medicines,
etc. Researchers have developed different techniques to measure and quantify these
properties in the CAM of the chick embryo. The techniques can be classified into destructive
(ex ovo) and non-destructive (in ovo) and qualitative, semi-quantitative and quantitative
ones [6,7].
Here, we systematically review existing CAM assays for different applications and
the available techniques for measuring and monitoring the vasculature in the CAM. Some
new imaging techniques are emphasised, which permit us to visualise the vascular struc-
ture at the microscopic level. Moreover, new techniques permit us to continuously and
dynamically visualise and record the changes in the vasculature and carry out quantified
analysis and assessment. Modern techniques and methods can save time, materials, and
labour, with reduced experimental deviations.
This review outlines the potential of the CAM platform to evaluate the pharmacologi-
cal effects of VDAs, from the most advanced imaging instrument for vasculature change
measurements.

2. CAM Assay
2.1. Development of Fertilised Chick Eggs in Incubation
The chick embryo normally experiences 21 days of development before hatching,
which corresponds to multiple stages (Figure 1). Usually, the first day of incubation is
defined as embryonic day one (ED 1) [8].
 Cells 2021, 10, 463 3 of 40
ls 2021, 10, x FOR PEER REVIEW 3 of 41

Figure 1. Schematic
Figure drawings
1. Schematic of chronological
drawings chick embryo
of chronological development.
chick embryo development.

In fact, embryonic development

In fact, embryonic starts in thestarts
development chickinegg the before ED before
chick egg 1. The EDprocess
1. Theof process of
blastodermblastoderm
generation generation
was described waslongdescribed
ago [9].long agomating,
After [9]. Afterthemating,
female the
ovumfemale
meetsovum meets
with the malewithsperm
the male
cell sperm
to formcell to form acell.
a fertilised fertilised cell. Cellinitiate
Cell divisions divisions initiate
around 5 haround
after 5 h after
insemination,insemination,
and then aand thenof
cluster a cluster
cells isofgenerated
cells is generated on the surface
on the surface of the
of the yolk yolk (Figure 2A).
(Figure
This cluster of cells is called the blastoderm, which grows
2A). This cluster of cells is called the blastoderm, which grows into the embryo (Figure into the embryo (Figure 2B,C).
2B,C). Later,Later, the blastoderm
the blastoderm continues
continues to divide
to divide intointo
threethree
germgerm layers:
layers: ectoderm,
ectoderm, mesoderm and
meso-
endoderm.
derm and endoderm.
Then, the blastoderm
Then, the blastoderm evolves into evolves into the
the embryo as embryo
follows:as follows:
The mesodermThe mesoderm
separates separates
into two layers, upper (somatic mesoderm) and lower
into two layers, upper (somatic mesoderm) and lower (splanchnic mesoderm). Spaces be- (splanchnic mesoderm). Spaces
between the two layers are surrounded and form the
tween the two layers are surrounded and form the coelom. The ectoderm and the somatic coelom. The ectoderm and the so-
matic mesoderm compose the somatopleure; the endoderm
mesoderm compose the somatopleure; the endoderm and the splanchnic mesoderm com- and the splanchnic mesoderm
compose the splanchnopleure.
pose the splanchnopleure. The coelom merges The coelom merges
the embryo the with
body embryothebody
middle with the of
parts middle parts
of the somatopleure
the somatopleure and splanchnopleure.
and splanchnopleure.
The extra-embryonic parts growparts
The extra-embryonic grow intomembranes,
into different different membranes,
including theincluding the yolk sac,
yolk sac,
the amnion, the chorion and the allantois. The chorion
the amnion, the chorion and the allantois. The chorion and the amnion are derived from and the amnion are derived from
the extra-embryonic somatopleure, and the yolk sac
the extra-embryonic somatopleure, and the yolk sac derives from the extra-embryonic derives from the extra-embryonic
splanchnopleure.
splanchnopleure. The embryoThe bodyembryo body getsfrom
gets separated separated from the extra-embryonic
the extra-embryonic tissues, only tissues, only
with the umbilicus connection. The endodermal and ectodermal tissue become epithelial epithelial
with the umbilicus connection. The endodermal and ectodermal tissue become
cells of the cells of the membranes,
membranes, and the mesodermal
and the mesodermal tissue createstissue creates
the blood fromtheand
blood from
to this ep-and to this
epithelium.
ithelium.
The yolk sac is the membrane enclosing the yolk, and digested yolk can pass into the
The yolk sac is the membrane enclosing the yolk, and digested yolk can pass into the
embryonic blood system through it [10].
embryonic blood system through it [10].
The amnion is a sac surrounding the embryo. It can secrete liquid to cushion the
The amnion is a sac surrounding the embryo. It can secrete liquid to cushion the em-
embryo and keep it from dehydration [10].
bryo and keep it from dehydration [10].
Chorioallantois is the membrane generated at the final stage and is formed by the
Chorioallantois is the membrane generated at the final stage and is formed by the
fusion of the chorion and the allantois. The allantois becomes a balloon-like shape outside
fusion of the chorion and the allantois. The allantois becomes a balloon-like shape outside
the embryo body by ED 4 and begins to fuse with the inside of the chorion by EDs 6–7,
the embryo body by ED 4 and begins to fuse with the inside of the chorion by EDs 6–7,
forming the chorioallantoic membrane (CAM).
forming the chorioallantoic membrane (CAM).
Cells 2021, 10, 463 4 of 40
Cells 2021, 10, x FOR PEER REVIEW 4 of 41

Figure 2. (A) A blastoderm floating on the surface of yolk on ED 2, (B,C) magnified views of the
Figure 2. (A) A blastoderm floating on the surface of yolk on ED 2, (B,C) magnified views of the
blastoderm in development on ED 2 and (D) illustration of membranes and blood circulation sys-
blastoderm in development on ED 2 and (D) illustration of membranes and blood circulation system
tem of an embryonic chick egg in the middle stage.
of an embryonic chick egg in the middle stage.
Three extra-embryonic membranes are there to support and nourish the embryo dur-
Three extra-embryonic membranes are there to support and nourish the embryo
ing growth:
during growth: thethe
yolk sac,sac,
yolk thethe
amnion
amnion and thethe
and CAMCAM [11].
[11].
These membranes feature internal variable structures in in
These membranes feature internal variable structures eggs
eggs during
during embryo
embryo devel-
develop-
opment.
ment. TheThesize,size, morphology
morphology and position
and position of theofthree
the three membranes
membranes keep changing.
keep changing. All
All these
these changes are aimed to adapt the embryo development
changes are aimed to adapt the embryo development and physiological functions. and physiological functions.
Theextra-embryonic
The extra-embryoniccirculation
circulationhas hasbeen
beendistinguished
distinguishedfrom fromthetheintra-embryonic
intra-embryonic
circulation [9]. The vitelline circulation and the allantoic circulation are
circulation [9]. The vitelline circulation and the allantoic circulation are extra-embryonic extra-embryonic
circulations. Major
circulations. Major blood
bloodvessels
vesselsconnect
connectdifferent
differentparts
partsof the embryonic
of the embryonic circulation (Fig-
circulation
ure 2D).
(Figure 2D).
The circulating blood
The circulating blood volume
volume of of the
the embryo
embryo fromfromED ED44until
untilED
ED1818hashasbeen
beendeter-
de-
mined (Table 1) [12]. The blood volume does not show an entire
termined (Table 1) [12]. The blood volume does not show an entire perfect curve of perfect curve of exponen-
tial growth.growth.
exponential The blood Thevolume reaches reaches
blood volume a peak value
a peakbetween ED 16 and
value between ED ED16 and18 and
ED 18de-
and decreases somewhat towards the end of hatching. This volume reduction is related the
creases somewhat towards the end of hatching. This volume reduction is related to to
degeneration
the degeneration of the extra-embryonic
of the extra-embryonic circulation
circulationsystem.
system.
Maina [13] comprehensively explained how the embryo absorbs vital nutrients from
Table
the 1. Circulating
albumen and theblood
yolkvolume
to build (ml)
newin chick embryo
tissues on different
and sustain embryonic
existing organsdays (EDs).
during incuba-
tion before EDinternal pipping
4 (the
5 chick
6 pecks7 the CAM
8 and
9 the
10 shell breaks).
12 14 The 16calcium
18
Blood Volume (mL) 0.04 0.10 0.17 0.26 0.37 0.51 0.68 1.15 2.15 3.13 2.58
Cells 2021, 10, x FOR PEER REVIEW 5 of 41

Cells 2021, 10, 463 Maina [13] comprehensively explained how the embryo absorbs vital nutrients 5from
of 40
the albumen and the yolk to build new tissues and sustain existing organs during incuba-
tion before internal pipping (the chick pecks the CAM and the shell breaks). The calcium
deposited in the eggshell provides the necessary source for bone formation in the chick
deposited in the eggshell provides the necessary source for bone formation in the chick
embryo.
embryo.
2.2. CAM Development and Physiological Function
Table 1. Circulating blood volume (mL) in chick embryo on different embryonic days (EDs).
The CAM is a double-layer membrane fused by the chorion and the allantois and has
an extremely
ED rich vascular 4 network.5 The
6 vascular
7 8network
9 in10the CAM
12 is14connected
16 with
18
the embryonic circulation through the allantoic stalk (Figure 3A). The CAM is responsible
Blood Volume (mL) 0.04 0.10 0.17 0.26 0.37 0.51 0.68 1.15 2.15 3.13 2.58
for embryonic respiration, protecting and nourishing the embryo during most of the chick
embryonic development.
2.2. CAM Development
The formation of and
the Physiological Function
allantois is shown in Figure 3B. The allantois of the embryo
The CAM is a double-layer membrane
emerges at about 3.5 embryonic days (ED 3.5). The fused by ventral
the chorion
walland theendodermal
of the allantois andhind
has
an extremely rich vascular network. The vascular network in the CAM is connected
gut of the embryo evaginates. This evagination pushes out a part of the embryo body into with
the embryonic circulation
extra-embryonic coelom.through the allantoic
Its proximal stalk(allantoic
portion (Figure 3A). Thelies
stalk) CAM is responsible
parallel and just
for embryonic
next respiration,
to the bottom protecting
of the yolk andwhen
sac, and nourishing the embryo
the distal portion during
extendsmost away of from
the chick
the
embryonic
embryo, development.
it grows bigger. It is named the allantoic vesicle [14].

Figure 3.chick
Figure 3. (A) A photo of a fertilised (A) Aegg
photo of a4fertilised
at day chick eggand
post-incubation at day
(B) a4 schematic
post-incubation anddisplaying
drawing (B) a schematic draw-
how the
ing displaying how the allantois is formed.
allantois is formed.

The
The allantoic
formation vesicle
of theenlarges
allantoisvery rapidlyinduring
is shown FigureEDs3B.4–10
The [14]. As shown
allantois of theinembryo
Figure
3A, a chicken
emerges embryo
at about at ED 4 forms
3.5 embryonic the(ED
days vascularised
3.5). The allantoic membrane.
ventral wall The background
of the endodermal hind
is
gutthe
ofyolk sac membrane
the embryo evaginates.(YSM),Thisalso highly vascularised
evagination pushes out[15].
a part of the embryo body into
By the end of ED
the extra-embryonic 7, mostItsofproximal
coelom. the chorion is in (allantoic
portion contact with thelies
stalk) shell membrane
parallel [16].
and just next
The process to form the CAM has been further described previously
to the bottom of the yolk sac, and when the distal portion extends away from the embryo, [9]. Through the
continuous
it grows bigger.enlargement
It is named of the
theallantoic
allantoicvesicle,
vesicle at about 100 h of incubation, the allantois
[14].
startsThe
to fuse with vesicle
allantoic the chorion, andvery
enlarges consequently,
rapidly duringthe CAM is formed.
EDs 4–10 [14]. AsThe CAMinconsists
shown Figure
of
3A,three layersembryo
a chicken (chorion, at stroma,
ED 4 formsallantoic membrane)allantoic
the vascularised and lies close to the eggshell
membrane. (Figure.
The background
4).
is the yolk sac membrane (YSM), also highly vascularised [15].
The
By the CAMendspreads
of ED 7,over mostthe yolkchorion
of the sac surface and covers
is in contact withitthe
completely between[16].
shell membrane ED 6
and EDThe7process to form
[16]. This the CAMwith
is consistent has been further described
Romanoff’s description previously
[9]: around[9]. EDs
Through
7–8, the
continuous
CAM extends enlargement
throughout ofthe
theblunt
allantoic
halfvesicle, at about
of the egg 100 h ofthe
and reaches incubation, the allantois
middle line. He con-
starts tothat
cluded fuseeventually,
with the chorion,
the CAM andcovers
consequently,
the entire theegg
CAM is formed.
around The CAM consists of
EDs 10–11.
threeAslayers (chorion,
illustrated stroma,4,allantoic
in Figure membrane) extends
the chorioallantois and lies to
close to the the
embrace eggshell (Figure
contents 4).
of the
whole The eggCAMat EDspreads over thethe
12, attaching yolk sac surface
entire surface and covers
of the innerit shell
completely
membranebetween
[17].ED
The 6
and ED 7 [16]. This is consistent with Romanoff’s description [9]:
CAM surface is 6 cm at ED 6, undergoing rapid extension, and becomes 65 cm at ED 14
2 around EDs 7–8,
2 the CAM
extends throughout the blunt half of the egg and reaches the middle line. He concluded
[18].
that eventually,
Although the theCAM
CAMbecomes
covers the entire egglarge,
increasingly around theEDs
two10–11.
extra-embryonic circulations
remain separate, but both are connected to the intra-embryonic circulation.
Coming to the end, apoptosis progresses in the CAM at ED 18, and apoptotic cells
are found in the mesenchyme [8]. Finally, during internal piping, chick lung ventilation
 initiates CAM degeneration [19]. The outer and inner shell membranes interface each
other with the eggshell, the chorion epithelium and the subchorionic sinuses (Figure 5),
which brings capillary blood close to the air via the air cell [13].
Cells 2021, 10, 463
The CAM is a highly vascularised and transparent membrane [11]. Arteries, 6veins of 40
and the capillary plexus exist in the CAM [2,20]. The chorioallantoic capillary volume and
surface size increase during CAM development [18].

Figure 4. The development ofFigure 4. The development

the chorioallantoic of the(CAM)
membrane chorioallantoic
(red lines)membrane
at different(CAM)
stages: (red lines)
(A) ED at different
5 initial stages:
coverage,
(A) ED 5 initial coverage, (B) ED 8 half coverage, (C) ED 12 and (D) ED 18 full coverage of the
(B) ED 8 half coverage, (C) ED 12 and (D) ED 18 full coverage of the chick embryo.
chick embryo.
As illustrated in Figure 4, the chorioallantois extends to embrace the contents of the
whole The
eggCAM
at EDperforms
12, attachingthe gas-exchange
the entire surface function
of thethrough themembrane
inner shell aerial vascular interface,
[17]. The CAM
when the chick 2 embryo starts developing before ED 6 [9].
surface is 6 cm at ED 6, undergoing rapid extension, and becomes 65 cm at ED 14 [18]. 2
Since the CAM
Although the CAM is subjacent
becomesto the inner shell
increasingly large,membrane at ED 5, this highly
the two extra-embryonic vascu-
circulations
larised membrane serves as the respiratory system of the
remain separate, but both are connected to the intra-embryonic circulation.embryo and is solely responsible
for gas exchange
Coming until
to the end,EDapoptosis
19 [20]. Here is the mechanism
progresses in the CAM of at
gasED
exchange: the respiratory
18, and apoptotic cells
exchange
are found in the mesenchyme [8]. Finally, during internal piping, chick lung the
of oxygen and carbon dioxide is a passive diffusion process between embryo
ventilation
and the environment,
initiates CAM degeneration the eggshell
[19]. Theand the CAM
outer confer
and inner a resistance
shell membranes function,
interface and theother
each vas-
culature
with the in the CAM
eggshell, theischorion
correlated with the and
epithelium O2 uptake ability of thesinuses
the subchorionic embryo in this 5),
(Figure diffusion
which
process. The general
brings capillary bloodtrend
closeoftothis
the function
air via theisair
a gradual
cell [13].increase from ED 6 to EDs 14–15,
and then a plateau
The CAM is a phase
highly[19,21,22].
vascularisedThe and
demand for O2 increases
transparent membrane throughout embryo
[11]. Arteries, de-
veins
velopment, with increased production of metabolic end products [23].
and the capillary plexus exist in the CAM [2,20]. The chorioallantoic capillary volume and
Meanwhile,
surface size increasethe during
allantois works
CAM as a deposit[18].
development for waste products excreted by embryo
kidneys,
The mostly urea at the
CAM performs theearly stage and function
gas-exchange chiefly uric acid atthe
through a later
aerialstage [9,14].
vascular interface,
whenThethe CAM has other
chick embryo physiological
starts developingfunctions:
before EDtransportation
6 [9]. of electrolytes (sodium
and chloride)
Since the from
CAMthe allantois and
is subjacent to thecalcium movement
inner shell membranefrom at
shell
EDto 5, bone for minerali-
this highly vascu-
larised membrane
sation. serves as the
Calcium movement canrespiratory
be certified system of thecalcium-transporting
by typical embryo and is solely responsible
cells in the
for gas exchange
chorionic cavity by until
EDED 12, 19
and [20].
the Here is the mechanism
movement rate can reach of gas
100 exchange:
nmol of calciumthe respiratory
per hour
exchange
per 1 cm 2 of oxygen and carbon
the CAM surface [8,14,24]. dioxide is a passive diffusion process between the embryo
and the environment, the eggshell and the CAM confer a resistance function, and the
vasculature in the CAM is correlated with the O2 uptake ability of the embryo in this
diffusion process. The general trend of this function is a gradual increase from ED 6 to
EDs 14–15, and then a plateau phase [19,21,22]. The demand for O2 increases throughout
embryo development, with increased production of metabolic end products [23].
 A full understanding of the vascular structure of the CAM is essential to investigate
the mechanisms of the vascular response in the CAM assay platform.
The vascular system is the first system built up in the chick embryo in order to facil-
itate respiration, because oxygen molecules only can diffuse about 100–200 μm in embry-
onic tissues [25].
Cells 2021, 10, 463 7 of 40
As shown in Figure 5, the CAM holds a rich vascular network within its mesodermal
layer, and paired allantoic (umbilical) arteries and veins supply blood to this system [18].

Figure 5. Images
Imagesillustrating
illustratingthe
thematuration
maturationofofthe CAM
the CAM vasculature.
vasculature.(A)(A)
Vascular remodelling,
Vascular remodelling,
growth and anastomosis can be seen at ED 7; (B) hierarchic vascular structures and fully differen-
growth and anastomosis can be seen at ED 7; (B) hierarchic vascular structures and fully differentiated
tiated vessels are noted on ED 10; (C) vessel tree reconstruction and flow direction for major
vessels are noted on ED 10; (C) vessel tree reconstruction and flow direction for major arterial arte-
and
rial and venous vessels (arrows); and (D) scanning electron microscope view of blood vessels
venous vessels (arrows); and (D) scanning electron microscope view of blood vessels on the CAM. on
the CAM.
Meanwhile, the allantois works as a deposit for waste products excreted by embryo
The mostly
kidneys, different stages
urea of the
at the earlyvascular development
stage and chiefly uricprocess
acid at in the CAM
a later stagepresent
[9,14]. specific
characteristics [26]. On ED 4, all vessels remain undifferentiated capillaries,
The CAM has other physiological functions: transportation of electrolytes (sodium whose walls
and
only have a single endothelial layer without a basal lamina. By ED 8,
chloride) from the allantois and calcium movement from shell to bone for mineralisation.primary vessels grow
and differentiate
Calcium movement intocan
an be
artery–venous systemcalcium-transporting
certified by typical and thus create a capillary network.
cells in These
the chorionic
small, by
cavity thin-walled
ED 12, and capillaries
the movement with arate
10–15
canμm lumenal
reach 100 nmol diameter migrate
of calcium and present
per hour per 1 cmat2
thethe
of superficial
CAM surface layer[8,14,24].
of the CAM, which is just beneath the chorionic epithelium (Figure
6).
2.3. Microcirculation
Meanwhile, as the and CAM
Morphology in the CAM
is expanding, endothelium mitosis undergoes a rapid phase
in theAcapillary network. Concurrently,
full understanding of the vascular thestructure
capillaryofendothelium in the CAM
the CAM is essential undergoes
to investigate
a sequence
the mechanismsof structural changesresponse
of the vascular [16]. A capillary
in the CAMnetwork
assaydevelops
platform.via respective angio-
genicThe
processes,
vascularincluding
system is sprouting, elongation,
the first system built upfusion
in the to theembryo
chick capillaryinplexus
order toand intus-
facilitate
susceptive growth
respiration, because[27]. The total
oxygen lengthonly
molecules of thecanCAM vessels
diffuse aboutis closely
100–200correlated with the
µm in embryonic
entire area
tissues [25].of the CAM [27].
As shown
Other in Figure
vessels located5,inthe
the CAM holds a rich
mesodermal vascular
layer network
are bigger within
generally, its mesodermal
with a 10–115 μm
layer, and and
diameter, paired
theallantoic
endothelium(umbilical) arteries
of the blood and veins
vessels supply blood
is surrounded by a to thisof
layer system [18].
mesenchy-
The different
mal cells stages of
and completely the vascular
wrapped by a development process 6).
basal lamina (Figure in the CAM present specific
characteristics [26]. On ED 4, all vessels remain undifferentiated capillaries, whose walls
only have a single endothelial layer without a basal lamina. By ED 8, primary vessels grow
and differentiate into an artery–venous system and thus create a capillary network. These
small, thin-walled capillaries with a 10–15 µm lumenal diameter migrate and present at
the superficial layer of the CAM, which is just beneath the chorionic epithelium (Figure 6).
 and sprouting angiogenesis [28]. After short-term sprouting, intussusceptive growth re-
mains dominant and increases density and complexity [27]. Intussusceptive growth is the
main process from EDs 7–11, with lower endothelial cell proliferation [29], and the prolif-
eration comes down after ED 11 [30]. Within this network, parts of the vessels become
Cells 2021, 10, 463 larger and extend laterally to form arteriolar and venular trees [27] (Figure 5C). EDs8 of
10–40
12 is an important period for vascular development in the CAM. During EDs 10–12, the
capillaries become adjacent to the chorionic epithelium (Figure 6).

Figure
Figure 6. Simplified schematic 6. Simplified
diagram schematic
(A) showing diagramcomponents
the structural (A) showing
of the
the structural
CAM, which components of the
amplifies the CAM,
embedded
which amplifies the embedded microscopic view of the haematoxylin-eosin-stained slice of the
microscopic view of the haematoxylin-eosin-stained slice of the CAM (B) where the ectoderm includes the chorion epithelium
CAM (B) where the ectoderm includes the chorion epithelium and sub-chorionic capillary sinus
and sub-chorionic capillary sinus layers of the CAM, and the endoderm corresponds to the allantoic epithelium of the CAM.
layers of the CAM, and the endoderm corresponds to the allantoic epithelium of the CAM. *Larger
*Larger vessels in the mesoderm (stroma).
vessels in the mesoderm (stroma).
Meanwhile, as the CAM is expanding, endothelium mitosis undergoes a rapid phase
Now, arteries and veins are distinguished in the mesodermal vessels. For example, a
in the capillary2network. Concurrently, the capillary endothelium in the CAM undergoes
piece of 1.7 cm CAM contains two major arteries and one major vein. Diameters of the
a sequence of structural changes [16]. A capillary network develops via respective an-
two main arterioles are 261 μm and 172 μm, and the major venule diameter is 390 μm [31].
giogenic processes, including sprouting, elongation, fusion to the capillary plexus and
Arteries have walls containing one or two layers of mesenchymal cells with more connec-
intussusceptive growth [27]. The total length of the CAM vessels is closely correlated with
tive tissue area
the entire surrounding
of the CAM the [27].
endothelium, tending to develop a fibroblast-type adventitia.
VeinsOther
have vessels
walls surrounded
located in the bymesodermal
connective tissues
layer areand incomplete
bigger mesenchymal
generally, with a 10–115cells,
µm
which develop into smooth muscle cells [26]. CAM vessels
diameter, and the endothelium of the blood vessels is surrounded by a layer grow rapidly up to
ofED 11, and
mesenchy-
then the endothelial
mal cells cell mitosis
and completely wrapped dramatically reduces(Figure
by a basal lamina and stays6). at a minimal growth rate
[26]. The
The capillary
density and fractal
plexus dimension
is formed of the vascular
and develops quickly network
from EDsincrease steadily from
4–5 by vasculogenesis
EDs 6 to 14 and stop at ED 15, for both complexity and branching
and sprouting angiogenesis [28]. After short-term sprouting, intussusceptive growth patterns [32,33].
The dominant
remains successiveanddevelopment of the CAM
increases density has been studied
and complexity [31]. At ED 14, growth
[27]. Intussusceptive the capil-
is
lary
the main process from EDs 7–11, with lower endothelial cell proliferation [29],the
plexus invades and is located at the surface of the ectoderm underneath andshell
the
membrane.
proliferationThe larger
comes down vessels
after in
EDthe11 mesoderm
[30]. Withincanthisfloat freelyparts
network, and move with the
of the vessels spon-
become
taneous
larger and movement of the embryo
extend laterally [16], whereas
to form arteriolar the capillary
and venular trees plexus is embedded
[27] (Figure 5C). EDs in the
10–12
most
is an superficial
important layer
period of for
the vascular
CAM [15], as illustratedininthe
development Figure
CAM.6. During EDs 10–12, the
The CAM
capillaries microcirculation
become adjacent to the is chorionic
supplied by two primary
epithelium arteries
(Figure 6). and drained by a sin-
gle vein.
Now, arteries and veins are distinguished in the mesodermalpre-
These primary vessels connect several vessels from the and post-capillaries
vessels. For example, a
of next 2
generations [18]. The left and right allantoic arteries
piece of 1.7 cm CAM contains two major arteries and one major vein. Diametersand vein remain apart during
of the
two main arterioles are 261 µm and 172 µm, and the major venule diameter is 390 µm [31].
Arteries have walls containing one or two layers of mesenchymal cells with more connective
tissue surrounding the endothelium, tending to develop a fibroblast-type adventitia. Veins
have walls surrounded by connective tissues and incomplete mesenchymal cells, which
develop into smooth muscle cells [26]. CAM vessels grow rapidly up to ED 11, and then
the endothelial cell mitosis dramatically reduces and stays at a minimal growth rate [26].
The density and fractal dimension of the vascular network increase steadily from EDs 6 to
14 and stop at ED 15, for both complexity and branching patterns [32,33].
Cells 2021, 10, 463 9 of 40

The successive development of the CAM has been studied [31]. At ED 14, the capillary
plexus invades and is located at the surface of the ectoderm underneath the shell membrane.
The larger vessels in the mesoderm can float freely and move with the spontaneous
movement of the embryo [16], whereas the capillary plexus is embedded in the most
superficial layer of the CAM [15], as illustrated in Figure 6.
The CAM microcirculation is supplied by two primary arteries and drained by a single
vein. These primary vessels connect several vessels from the pre- and post-capillaries of
next generations [18]. The left and right allantoic arteries and vein remain apart during
the fast growth of the CAM [31]. The interspace of major vessels allows inter-digitating
arteriolar and venular trees to grow and let relatively few vessels cross.
In the assay platform to test the response of blood vessels, these large and primary
vessels are the best target to be measured and evaluated in quantification by modern
imaging techniques (medical and bioengineering).
Now that the expansion of the CAM vasculature network is well established, branch-
ing patterns and microcirculation of pre- and post-capillaries of the CAM will be studied.
The morphology and mechanism of such microcirculation are described in detail. The CAM
consists of a superficial 2D network of very dense capillaries, named the capillary plexus,
shown by scanning electron microscopy in Figure 7 as a mesh morphology [34]. The cap-
illary mesh wraps a surrounding 3D space. The spatial configuration consists of two
horizontal planes, which are connected by pre-capillary bridging vessels. These bridg-
ing vessels rise in an oblique-to-vertical direction towards the superficial capillary plane.
Cells 2021, 10, x FOR PEER REVIEW 10 of 41
The medium and large arterial and venous vessels, which supply and drain the superficial
layer, are located within this 3D space [16].

Figure 7. Resin corrosion cast (Mercox

Figure cast)
7. Resin of the developing
corrosion cast (MercoxCAM
cast) vasculature at ED CAM
of the developing 12. (A,B) A three-dimensional
vasculature at ED 12. (A,B) A
three-dimensional structure containing a capillary plexus and a layer of supplying
structure containing a capillary plexus and a layer of supplying and collecting vessels is recognisable. (C,D) Numerous and collecting
vessels is recognisable. (C,D) Numerous pillars of different sizes caused by intussusceptive
pillars of different sizes caused by intussusceptive angiogenesis processes. Original magnification: (A) 3100, (B,C) 3200 and angio-
genesis processes.
(D) 3400. Reproduced with permission from [11]. Original magnification: (A) 3100, (B,C) 3200 and (D) 3400. Reproduced with
permission from [11].
The pre-capillary arterioles connect directly with the capillary network. However,
2.4.
the Growth and Regulation
post-capillary venulesFactors of Angiogenesis
connect in CAM network via the venous sinus
with the superficial
The angiogenesis process is controlled by the balance of multiple growth factors
which have proliferative and inhibitory regulatory activity [33]. A variety of growth fac-
tors have been revealed [11,14,36–39], which induce and promote CAM angiogenesis and
undertake precise spatial and temporal regulations to form a mature vascular network
(Table 2).
Cells 2021, 10, 463 10 of 40

system. The small sinus is formed by the confluence of capillaries at the beginning of
the post-capillary venules. The blood from the arterial tree streams into the superficial
capillary mesh and then drains into the venous system. It is possible to distinguish the
arterial and the venous blood in the circulatory system of the CAM by a smooth muscle
layer surrounding the endothelium of the arterioles, but not that of the venules. Thus
their morphology is different [16]. Another important conclusion is that blood vessels in
the CAM have no terminal vessels, tips or sprouts and are always exhibited as a closed
cycle [34].
More recently, morphology studies about microcirculation in the CAM have been
deepened to discover how arterial and venous vessel trees integrate (connect) and commu-
nicate with the capillary bed [31,35]. These are described on a smaller scale by scanning
electron microscopy [31]. The interdigitating pattern derives from arterial and venous
vessel trees, and long arterial pathways link with short venous pathways through the capil-
lary mesh, and vice versa. A distinct feature is the presence of new connections between
arteriole and venule trees, which feed and drain the underlying plexus not only at their
termini but also along proximal segments of the vessel tree.

2.4. Growth and Regulation Factors of Angiogenesis in CAM

The angiogenesis process is controlled by the balance of multiple growth factors which
have proliferative and inhibitory regulatory activity [33]. A variety of growth factors have
been revealed [11,14,36–39], which induce and promote CAM angiogenesis and undertake
precise spatial and temporal regulations to form a mature vascular network (Table 2).

Table 2. Representative growth factors for angiogenesis in the CAM.

Growth
Description Functions Ref.
Factors
To exert vascular permeability
Vascular endothelial growth
VEGF and endothelial cell [37]
factor
recruitment
FGF Fibroblast growth factor To elicit fibrocyte proliferation [11,39,40]
PDGF Platelet-derived growth factor To stimulate vascular stability [38]
To act on endothelial sprouting,
ANG Angiopoietin vessel wall remodelling and [37]
mural cell recruitment
As a cytokine to stimulate
HGF Hepatocyte growth factor proliferation and [37]
morphogenesis of epithelia
To induce expression of VEGF
HIF Hypoxia-inducible factor [14,37]
and its receptors
A proteolytic fragment of
To act as an endogenous
Endostain collagen XVIII (a component of [37]
anti-angiogenic molecule
the basement membrane)

Vascular endothelial growth factor (VEGF) plays a leading role among these factors
and is crucial for the angiogenic expansion at the early stage of CAM development. An
endogenous VEGF-A presents two peaks at EDs 8–9 and 11–12 [40].
VEGF is the prime factor to attract migrating endothelial cells and stabilise vessels
by bounding substrates during the formation of vascular tubes [36]. Some researchers
believe that VEGF causes vascular permeability, recruits endothelial cells and inhibits
vessel stabilisation. The fibroblast growth factor (FGF) family is the most potent cytokine
to stimulate mitogenesis of multiple cell types, such as endothelial cells, osteoblasts, bone
marrow stromal cells, mesenchymal stem cells and immune cells. It directly causes fibroge-
nesis [37]. At present, there is a general consensus that VEGF and FGF are main factors for
the vascular growth regulation in the chick CAM.
Cells 2021, 10, 463 11 of 40

FGF-1 and FGF-2 are major prototypic members of the FGF family. Both of them
initiate activation immediately after binding to their cell surface receptor, which is named
fibroblast growth factor receptor-1 (FGFR1), one of the receptor tyrosine kinases. FGF-1
is considered as a standard angiogenesis stimulator [39]. Endogenous FGF-2 may affect
the proliferation, movement, redistribution and invasion of endothelial cells [11]. FGF-2
becomes detectable in the CAM since ED 6, and maximal concentrations occur between ED
10 and ED 14. Meanwhile, FGF located in the CAM can regulate angiogenesis [38].
However, VEGF and FGF are not sufficient to finish angiogenesis, because they
function not only as promoters of endothelial cell proliferation but also as inhibitors of
vessel maturation. Through suppressing receptors on smooth muscle cells, VEGF inhibits
pericyte coverage of vascular sprouts and renders existing vessels unstable, whereas
platelet-derived growth factor (PDGF) creates vascular stability at the maturation stage
(late stage) of angiogenesis [37].
PDGF drives pericytes and smooth muscle cells to recruit, which form a layer of cells
around new capillaries, embed the endothelial lining and facilitate binding strongly to
the extracellular matrix. These are necessary conditions to stabilise new blood vessels.
Meanwhile, PDGF is functional to inhibit the recruitment of endothelial cells.
These facts show that a single factor is not sufficient to create a stable mature vascula-
ture. However, the coordination and balance of multiple factors can induce a successful
angiogenic response and mature vessel network in the CAM.
Not many concrete studies have been carried out on angiopoietins (ANGs). ANGs
play an important role in endothelial sprouting, mural cell recruiting and vessel wall
remodelling [36]. Angiopoietin-1 (ANG-1), a 498-amino-acid glycoprotein, is a ligand
of the endothelium-specific receptor Tie2 [41]. It recruits periendothelial cells, at the
late stage of vascular maturation, in the presence of VEGF. Unlike VEGF, ANG-1 has no
mitogenic effect in vivo [42]. However, another report indicated that ANG-2 can induce
rapid initiation of blood vessels [37].
Hepatocyte growth factor (HGF), a specific growth factor for the liver, shows the
highest expression at the beginning of chick embryo development. Meanwhile, HGF is a
cytokine which stimulates the proliferation and morphogenesis of epithelia [36]. HGF can
also directly act on endothelial cells, including stimulation of proliferation, cell mobility,
protease production and organisation of capillary-like tubes [43].
Hypoxia-inducible factor 1-alpha and 2-alpha (HIF-1α and HIF-2α) are activated by a
hypoxic environment. HIF may switch on the expression of angiogenic genes, for example
VEGF, which attract branching vessels to stretch towards hypoxic tissues [36,44]. A high
expression level of HIF-1α correlates with a peak in the angiogenic process in the CAM [14].
Among anti-angiogenic molecules, endostatin, a proteolytic fragment of collagen
XVIII (a component of the basement membrane), inhibits endothelial cell survival and mi-
gration. During angiogenesis in the CAM, endostatin progressively elevates its expression;
meanwhile, most pro-angiogenic factors show a steadily downgrading expression [36].
In addition to these endocrine and paracrine molecules and growth factors mentioned
above, the extracellular matrix of the CAM may modify its composition and express
fibronectin, collagen type IV, laminin and specific glycosaminoglycans. These substances
can assist the angiogenesis occurring in the matrix [14].
Recently, scientists have started to reveal cellular functional mechanisms and path-
ways of growth factors in CAM angiogenesis, for example EGFR, Ties receptor and
AKT/PKB signalling and Ras-MEK-MAPK, AKT, P38 and PKC pathways [11,37].
Besides molecular regulation on endothelial cells, effects of some molecules on per-
icytes and the basement membrane in the CAM have also been studied. Netrin-4 is a
laminin-disrupting molecule. Through disturbing the laminin network, it disrupts the
full basement membranes surrounding pericytes, which are resultantly detached from the
endothelium, leading to the collapse of the capillary network. Thus, netrin-4 treatment se-
riously impacts CAM angiogenesis and reduces the vascular area by altering the basement
membrane and pericytes [45].
Cells 2021, 10, 463 12 of 40

3. CAM Assays in Preclinical Biomedical and Pharmacological Research

For over 100 years, scientists have utilised the CAM in medical studies. Rous and
Murphy grafted chicken sarcoma cells onto the CAM and observed tumour growth [46].
However, it was more than half a century later when attention was drawn to vascular
questions about the CAM, which is recognised as an excellent assay system for research on
vascular responses [18,26,34,47]. The CAM is one of the ordinarily used in vivo models to
study blood vessel development and to test and evaluate pro-angiogenic or anti-angiogenic
properties of a number of substances. By utilising blood vessel features of the CAM,
innovative applications have been increasingly developed, e.g., in drug screening [48] and
vascular targeting therapies [49].
More recently, the CAM as a concrete experimental platform has attracted great inter-
ests from domains in bioengineering, vascular diseases, tissue transplantation, tumour and
metastasis, cancer therapies, biomaterials engineering, drug development, genomics, etc.
Although in ovo assays represent the main trend on a wider scale, innovative ex ovo (shell-
less) CAM assays have been successfully utilised to study the angiogenic response and the
initial tissue response to biomaterials engineering [50,51]. Such ex ovo modification models
can keep survival rates over 80% and provide several advantages, e.g., to better visualise
implanted biomaterials and growing embryos, to transplant combinatory biomaterials on
the bigger area of one CAM simultaneously and to observe the vascularisation process
during the whole time course of the test [51].
The CAM appears to be an excellent preclinical model for pharmacological assays due
to convenient experimental manipulation, common tissue composition and economical
accessibility. Meanwhile, it has been used as an intermediate step before advancing
to in vivo preclinical evaluation in mammals [24]. Studies on drug pharmacokinetics
and biodistribution, drug activity and toxicity are the most crucial stages in preclinical
pharmacological verification, as summarised here. We also outline the best time course,
advantages and limitations of CAM applications for different assays.

3.1. Drug Pharmacokinetics and Biodistribution

3.1.1. Drug Delivery System
The CAM is regarded as an excellent platform for testing multiple drug delivery
systems (DDSs), which are designed to distribute medical substances to specific sites of
disease or wounds [24]. One of the major functions for a DDS is to control the releasing
rate of a drug. As noted, pharmacokinetics and biodistribution in vivo are important
aspects that reflect the efficacy of a DDS. A DDS can be topically applied on the CAM or
injected into the amnion [14]. Topically applied drugs can enter the blood circulation after
absorption by the CAM. However, recently, direct drug injection into the CAM blood vessel
is being preferred [52,53], which is conformable to clinical usage.

3.1.2. Conventional Analysis Approach

There are different approaches to assess pharmacokinetics and biodistribution with
a CAM platform. In the traditional approach, following drug administration, blood is
sampled, selected organs are extracted and then quantitative analysis of the drug can be
carried out [24]. After sampling, the drug plasma level is measured by high-performance
liquid chromatography (HPLC), and pharmacokinetic parameters are determined by a
common algorism. After dissection and tissue sampling, the biodistribution of a drug can
be evaluated by HPLC quantification of its tissue concentration in different organs [54,55].

3.1.3. Advanced Approaches

The more advanced approach is either to directly observe intravascular fluorescence
after injection of fluorescent materials or to utilise biosensors [24].
Regarding the fluorescent approach, fluorescent drugs can be traced by using a fluo-
rescence microscope to measure drug penetration and distribution for pharmacokinetics
biodistribution studies. The techniques for fluorescence measurement have been de-
Cells 2021, 10, 463 13 of 40

scribed previously [24,48]. After intravascular injection of a photosensitiser into the CAM,
time-dependent fluorescence angiography is performed under a fluorescence microscope.
The extent of fluorescence inside and outside blood vessels can be analysed by these
fluorescent photographs and recorded in a semi-quantitative way. Then, by calculating
the photographic contrast, the extent of the photosensitiser diffused through the CAM
vasculature is determined and the profiles of photosensitiser pharmacokinetics can be
outlined.
Improved methodologies have been adopted to visualise vascular changes induced
by tumours and to study tumour invasion. A metastatic human melanoma cell line (C8161)
and a prostate cancer cell line (PC3) were labelled with green fluorescent protein and
implanted onto the CAM. At different time points of tumour cell growth, lens culinaris ag-
glutinin as a fluorescently tagged lectin was injected intravenously to label endothelial cells.
Through fluorescent endothelial cells, microvessels were imaged and measured by confocal
microscopy to evaluate angiogenesis. Furthermore, tumour cells grown on the CAM sur-
face and invaded into the CAM endoderm could also be imaged and evaluated by confocal
microscopy. Confocal z-stack imaging is a useful tool to analyse tumour invasion [56]. In
another research, lens culinaris agglutinin labelled with rhodamine was injected into the
blood circulation of the CAM to visualise and evaluate the microvasculature by confocal
microscopy [57].
On the other hand, with biosensors, the concentration of a drug can be determined
through conversion of a biological response into an electrical signal [58]. For instance, an
acetaminophen sensor was topically applied and integrated into the CAM of an embryo
for 7 days. Afterwards, blood levels of acetaminophen were determined with the biosensor,
which reflected the changes in acetaminophen levels [59]. A biosensor was also used to
measure the glucose concentration through non-invasive methods in a CAM model [60].

3.1.4. Vessel Permeability

Vessel permeability provides an important feature to study the pharmacokinetics of
drug diffusion through the CAM vasculature, while the change in vascular leakage in the
CAM can be monitored at different development phases, for example during endothelial
proliferation, cytodifferentiation and senescence of the chick embryo.
Vessel permeability can be measured in real time by transiting relatively small molecules
into the interstitial space, which include fluorescent dextrans of about 100 kDa, macro-
molecular drug carriers (70 kDa) and antibodies (150 kDa), whereas larger dextrans of
2000 kDa are enclosed in the vascular space. These dextrans of different sizes provide
real-time monitoring capacity to study vascular leakage in the CAM. Spatial and temporal
differences in vessel permeability may also be captured by an intravital imaging approach
with high resolution [15].

3.2. Biocompatibility
The CAM has been utilised as a model assay to study and evaluate allogenic and xeno-
geneic transplantations of different tissues and organs. As the earliest explorer, Murphy
transplanted multiple chicken tissues onto the CAM, including the spleen, liver, kidney,
bone marrow, etc., most of which survived after homologous grafting [61].
The CAM was also successfully transplanted with more allogenic tissues, including
the liver [62], mesonephros [63], adrenal gland and cerebellum [64]. In addition, the CAM
has been extensively used for xenogeneic transplantations of human bone [65], kidney [66],
endometrium [67], skin [68], ovaries [69], etc. An uncompleted immune system of the chick
embryo before ED 15 is the main reason for the successful results in these procedures.
In the CAM, these transplanted tissues can survive and grow by vascular anastomoses
between the transplanted tissue and the original CAM and most commonly by neoangio-
genesis from the CAM into implanted tissues [70]. The graft and the CAM are connected
by relatively large arteries or veins; thereby, the CAM can provide grafts with nutrients
and growth factors [63].
Cells 2021, 10, 463 14 of 40

Biocompatibility is essential for tissue engineering. In recent years, the CAM has been
used in innovative stem cell research. Extracellular matrices or synthetic polymers are
made with desired tissue-like structures and transplanted onto the CAM without rejection,
wherein cell growth, differentiation and angiogenesis can occur.
Using CAM or chick embryo to study biocompatible materials and stem cell inte-
gration has become a new future trend [15]. The angiogenic response has been tested
in implanted extracellular matrices extracted from the skin, brain, oesophagus, larynx,
trachea, aorta, diaphragm and cartilage, etc. These biomaterials acquire effective vascular
supplies with enough oxygen and other nutrients to sustain their biocompatibility and
availability [14].
More recently, fabricated synthetic polymers have been evaluated for angiogenesis.
Biodegradable poly(lactic acid)/poly(lactic-co-glycolic acid) (PLA/PLGA) scaffolds em-
bedded in the CAM were used to sequentially deliver VEGF, FGF-2 and PDGF with distinct
kinetics in order to observe angiogenic differentiation [37]. Other biomaterials such as
fibrin matrices [71], collagen matrices [72], etc., have also been used for similar purposes.
In view of the drawbacks with VEGF, such as time-consuming or complex steps
and leaky or haemorrhagic vessels, new substances were explored to substitute VEGF, of
which 2-deoxy-D-ribose (2dDR) and 17β-Estradiol (E2) were selected as two leads. An ex
ovo CAM model was used to evaluate the angiogenic activity of the two agents, which
were gradually released from tissue engineering scaffolds. Both 2dDR and E2 induced
angiogenesis during 7 days and were approximately 80% as effective as VEGF. Based
on dose-dependent angiogenic responses, the most effective doses of both agents were
determined [57].
When non-biological materials are introduced, inflammation and ulceration could
be responding problems. The trend of tissue engineering materials is to achieve biocom-
patibility without inflammation and to control the kinetic release of growth factors [71].
Tissue transplantation studies have also built a good basis for tumour transplantation and
relevant therapies.

3.3. The Efficacy of Drugs

The activity or toxicity of a drug can be evaluated in the CAM and grafted tumours
on the CAM [14]. To evaluate the efficacy and action mechanism of drugs in a CAM
model, pro- and anti- angiogenic substances have been chosen as a pioneer area to study,
including a variety of substances such as natural molecules, growth factors, anti-angiogenic
molecules, antibodies, synthetic small molecules, anti-cancer agents, antibiotics, etc. [14].

3.3.1. Pro-Angiogenic Agents

Pro-angiogenic agents can stimulate new blood vessels to grow from existing vessels,
resulting in a concurrent increase in the blood vasculature. More than 140 kinds of pro-
angiogenic stimulators have been tested by CAM assays [73]. To test their efficacy, pro-
angiogenic agents are usually applied directly on the surface of the CAM to monitor
the angiogenic response. These pro-angiogenic agents can be utilised as components of
microparticles or nanoparticles, drug conjugates or tissue scaffolds [15].
There are many techniques to apply agents onto the CAM (Table 3).

Table 3. Methods to apply pro-angiogenic agents on the CAM.

Model in Technical Materials Ref.

Filter disks [74]
Culture coverslide glasses [75]
Inert synthetic polymers: Elvax 40 (ethylene-vinyl acetate copolymer) [76]
Hydron (a poly-2-hydroxyethylmethacrylate polymer) [76]
Methylcellulose disks [77]
Cells 2021, 10, 463 15 of 40

Table 3. Cont.

Model in Technical Materials Ref.

Alginate pellet [78]
Gelatin sponges [79]
Cross-linked collagen hydrolysate [80]
Cross-linked collagen matrices [81]
Cross-linked and heparinised collagen matrices [81]
Fibrin matrices [71]
Poly(D,L-lactic acid) (PLA) [82]
Poly(ethylene glycol) (PEG) [83]
Modified Matrigel mixtures [84]
Paraffin and plastic embedding [85]

Rather than placing agents on the surface of the CAM, some methods directly injecte
cell suspensions or fluid substances into the allantoic vesicle so that their activity over the
vascular system is uniform [86].
Growth factors of different formulations such as VEGF121, VEGF165 and FGF en-
trapped in fibrin matrices have been screened on a CAM platform for their efficacy in
angiogenesis [71,87]. It is found that a single delivery of each of these factors resulted
in angiogenic activity, but the new vasculature was leaky and haemorrhagic in the fibrin
matrices. However, combined delivery of these factors or immobilising either VEGF 121 or
VEGF 165 can form non-leaky vessels with a normal morphology without haemorrhages
but with barrier function. These vessels look more mature than those produced as a re-
sponse to any single growth factor. In addition, VEGF alone can initiate the formation of
structurally intact vessels, provided it is released slowly in a low sustained dose [87].

3.3.2. Anti-Angiogenic Agents

As an in vivo model, the CAM has also been utilised to study the efficacy of anti-
angiogenic modulating agents. The efficiency of anti-angiogenic drugs or formulations was
evaluated by determining the extent of vascular occurrence in the CAM [88], the reduction
of blood vessels [24] and the presence of an avascular or a hypovascular zone at the drug
application site [89].
Around 360 anti-angiogenic agents have been tested in CAM assays [73]. They are
classified into two groups (Table 4): (a) direct inhibitors, which affect the survival or
functions of endothelial cells, and (b) indirect inhibitors, which block the activity of pro-
angiogenic molecules [89]. These are semi-synthetic or synthetic substances, biological
antagonists or endogenous factors which inhibit the immature neovasculature or the
angiogenic cascade [89].

Table 4. Direct and indirect angiogenesis inhibitors.

Direct Indirect
Targeting EGF-receptor tyrosine kinase
Angiostatin, bevacizumab (Avastin) Sunitinib
Arresten, canstatin, combrestatin Targeting the VEGF receptor
Endostatin, thrombospondin Targeting the PDGF receptor
Tumstatin, methoxyestradiol, vitaxin Targeting Erb-B2 Receptor Tyrosine Kinase 2 (ERBB-2)
Targeting the interferon alpha receptor *
* Interferon alpha can be considered both a direct angiogenesis inhibitor because it inhibits endothelial migration
and an indirect angiogenesis inhibitor because it inhibits synthesis of FGF by tumour cells [57,89].

Avastin (bevacizumab) is a typical example of anti-angiogenic agents in clinical appli-

cation. It is an anti-VEGF-neutralising monoclonal antibody and an anti-angiogenic agent
which is first utilised in clinical anti-cancer therapy [52,89].
Avastin has been extensively studied for its efficacy by using CAM assays [52,90]. It
was applied onto the CAM at ED 7, and fluorescence angiography was used to observe
Cells 2021, 10, 463 16 of 40

vascular changes through ED 9. Comparing images at EDs 7 and 9 showed that Avastin
strongly inhibited angiogenesis in the CAM [52] and caused an increment of over 32%
of the mean avascular surface as compared to the control group. Avastin also caused a
significant reduction in the capillary density at the tested CAM site [52].
The yolk sac membrane (YSM) was also used to substitute the CAM to test Avastin.
After Avastin treatment, the number of quaternary blood vessels in both the YSM and
the CAM significantly decreased, which validated pilot screening for anti-angiogenic
agents [88].
Sunitinib is an inhibitor for multiple receptor tyrosine kinases and can work excellently
to inhibit angiogenesis. It can be used as a negative control for angiogenic responses
compared with tested agents and a positive control (VEGF) in the CAM [57].
Another good example is paclitaxel, an anti-cancer drug with anti-angiogenic property.
The efficacy of several formulations of paclitaxel was analysed on a CAM model [24].
Paclitaxel was incorporated into microspheres made from either poly(ε-caprolactone)
(PCL) or Poly lactic acid-ethylene-vinyl acetate (PLA-EVA) copolymer. By using PLA-EVA
microspheres, the paclitaxel dose was about eight times smaller than the dose used for
PCL microspheres in order to induce the same level of vascular occlusion. These results
demonstrated that the CAM model can compare the performance of drugs with different
carriers. Some formulations were tested to adjust the release rate of paclitaxel in a CAM
assay where methoxypoly ethyleneglycol (MePEG), PCL, lipospheres and gelatine were
applied to validate its pharmacokinetics [24].
Other innovative anti-angiogenic agents have also been explored on the CAM for
photodynamic therapy (PDT) and radiotherapy. PDT is to combine photosensitisers (anti-
angiogenic compounds) with various activating light sources to suppress vascular growth,
whereas radiotherapy increases the effectiveness of anti-angiogenic treatment when X-rays
are combined with radio-sensitisers [91,92].

3.3.3. Wound Healing

The US Food and Drug Administration (FDA) used the CAM assay to preclinically
evaluate drugs to be approved for treating burn wounds and chronic cutaneous ulcers [93].
The CAM was used to study wound healing [94]. The CAM could constantly reproduce all
stages in human wound healing, including inflammation, re-epithelisation, angiogenesis,
fibronectin deposition and scar formation. During wound healing on the CAM, chorionic
epithelium hyperplasia and macrophage inflammatory infiltration can be observed with
tripled micro-vessels and fibroblasts in the wound area than in the adjacent control area.
A CAM assay may also test the mechanism of action of specific substances during
wound healing. Taking FGF-2 as an example, blocking antibodies were used to inhibit FGF-
2 after CAM wounding, which inhibited the development of micro-vessels and fibroblast
density. Thus, wound-healing delay was observed. Conversely, when FGF-2 was added
into the wounds, the repair was accelerated by 24 h compared to control wounds [95].
Another substance, activated protein C (APC), which is a serine protease anti-coagulate
and a potent anti-inflammatory mediator, was tested for wound healing [96]. Via a com-
plex mechanism, APC may help cutaneous wound healing by angiogenesis stimulation,
re-epithelialisation promotion and inflammation inhibition. This CAM assay suggests that
APC can be an interesting therapeutic to accelerate chronic wound healing.
In silico models have become innovative tools to mimic and understand cell behaviour
before real animal assays start. Concerning wound healing, an in silico numerical model
accurately simulated the sprouting angiogenesis process, which was regulated by the
chemoattractant effect of VEGF [97]. It was the first time to simulate the capillary network
presented in realistic CAM images. The basic methodology was to integrate the radial
point interpolation method (RPIM) with the characteristics of growth factor initiation,
endothelial cell movement and branching pattern. Through a comparison and validation
between the in silico model and the in vivo CAM, the parameters (total branching number,
total vessel length and average branching angle) were closely simulated. The capillary
Cells 2021, 10, 463 17 of 40

volume fraction was also compared. The in silico model made sprouting angiogenesis
more predictable and the combination with the in vivo assay more valuable [97].

3.3.4. Tumour Growth and Metastasis

A. Tumour angiogenesis and growth
The CAM is an excellent host to study cancer biology and tumour graft growth, be-
cause the immune system has not become competent and rejection has not been established
until ED 18 [73].
In 1913, Murphy first explored tumour-induced angiogenesis with the CAM [98].
Afterwards, others attempted implanting tumour cell lines or tissues from mice, chickens
and humans on the surface of the CAM in their studies [73,99–101]. Thereby, characteristics
of tumours could be observed, such as tissue development, angiogenesis, invasion, extrava-
sation and metastasis [15,101]. This makes it feasible to compare and evaluate tumours’
growth and histological features as well as their viability after being re-transplanted in the
original host and the effects on chick embryos [100].
The CAM tumour assay is much faster than a tumour assay in mammalian models,
which often take 3–6 weeks [14]. After tumour cell inoculation onto the CAM, the grafts
remain avascular for a few days, after which they are ready for rapid growth once pene-
trated by new blood vessels. Normally, it takes 2–5 days for tumour xenografts to become
visible with their blood supply originating from the CAM.
A detailed process of tumour tissue transplantation has been described previously [102].
Tumours grafted on the CAM do not surpass a mean diameter of 0.93 ± 0.29 mm during
the prevascular phase (approximate 72 h). After entering the vascularisation phase, it takes
24 h for tumours to start growing quickly and reach a mean diameter of 8.0 ± 2.5 mm by 7
days. Interestingly, when 1–4-mm-size tumour grafts are implanted on the CAM at ED 9,
they undergo a prevascular phase for 72 h and shrink rapidly due to necrosis and autolysis.
However, when vascularisation starts, rapid growth resumes.
Through a morphological study, the blood vessels in grafted tumours and CAM host
blood vessels were distinguished [70]. The pre-existing tumour native blood vessels in
the graft disintegrate in 24 h after implantation, and vascularisation reoccurs when CAM-
derived vessels penetrate into the graft. This mechanism is completely different from the
one in the grafts of chick tissue or adult tissue.
B. Tumour metastasis
The CAM can practically host the growth of inoculated xenogeneic tumour cells and
tissues, which efficiently helps to simulate and analyse metastases of human tumours [99].
Cancer metastasis is initiated by a change in cell–cell adhesive interactions [73]. During
this process, tumour cells first dissociate from the primary lesion, undergo local invasion
and migrate into the interstitial matrix. For haematogenous metastasis, tumour cells enter
the host circulation through intravasation, get stagnated in the microvasculature and leave
the blood circulation by extravasation. Then, the tumour cells may start local invasion
again to form secondary metastatic foci. Finally, these small foci may initiate an angiogenic
response for sustaining tumour growth.
Undergoing such metastasis, tumour cells can colonise in the CAM and in the internal
organs of the embryo, such as the brain, lungs and liver [103]. These metastatic cancer
cells simulate stem cells because of their ability to self-regenerate and to derive diverse
next generations [73]. Such a small group of cancer cells is named cancer stem cells
with stem-like properties. Organotropic breast cancer cell lines were studied for such
active properties [104]. The evaluation was undertaken by a limiting dilution assay in the
CAM, following in silico bioinformatics analysis, to validate the stem-cell-like prevalence
of different breast cancer cell lines as an updated approach to determine the stem cell
properties of tumour cells.
When studying metastasis either by a spontaneous model or by an experimental
model, as described below, the chick embryo only takes 7–8 days, considerably shorter
than 4–10 weeks with most typical mice models [73].
Cells 2021, 10, 463 18 of 40

Spontaneous metastasis model

Spontaneous metastasis is the process by which grafted tissue or inoculated tumour
cells intravasate into the vasculature of the CAM, which shows the metastatic potential of
a specific cell line or primary tumour fragment responsible for tumour progression [15,73].
Histological analysis of the CAM may reveal that tumour cells invade the chori-
onic epithelium from the surface and the blood vessels through the intermediate mes-
enchyme [73,103,105,106].
Tumour cells invade the vascularised mesenchyme underneath the chorionic epithe-
lium. By attaching to arterioles, cancer cells migrate to the dense bed of blood vessels to
realise intravasation. In vivo microscopy can monitor morphological changes in cancer
cells present in the CAM microcirculation, which mostly survive without significant cell
damage. On the CAM, tumour cells can be found in locations away from the inoculation
site and in internal organs within a few days after inoculation [11,14].
Experimental metastasis model
Experimental metastasis refers to injecting cancer cells into the blood circulation to
form metastatic tumours in distant organs. Experimental metastasis surpasses dissem-
ination steps of spontaneous metastasis [107]. Experimental metastasis studies on the
CAM may provide comprehensive information about critical metastatic steps, including (i)
cancer cell survival in the blood circulation and transportation to target organs, (ii) cancer
cell immobilisation in the microcirculation, (iii) cancer cell migration through the vessel
wall into the interstitial space (extravasation) and (iv) cancer cell proliferation in target
organs [73].
By comparing spontaneous and experimental metastases, different functions of non-
metastatic and metastatic cells can be determined at the steps of the dissemination cas-
cade [99]. The CAM is an attractive model to visualise the behaviour of grafted tumour
cells with microscopy in the settings of both spontaneous and experimental metastases.
Another mechanism of cell dissemination is angiotropism, in which tumour cells
preferentially localise in the existent blood vasculature, rather than randomly migrating
within the CAM mesoderm [85].
C. Drug assays
Anti-cancer drug therapies can be evaluated when tumours are grafted on the CAM [14].
The CAM has been used as a test platform for anti-angiogenic drugs to study the inhibition
of angiogenesis of tumour growth [11], chemosensitivity towards tumour invasion and
metastasis [101,108], inhibitory effects of chemotherapeutic drugs on metastatic foci [109]
and experimental radiation oncology research [110]. Common CAM-based drug assays are
listed in Table 5.

Table 5. Drug assays for tumour grafts or metastasis on a CAM platform.

Drugs Type of Tumour Ref.

Hydrophobic derivatives of boswellic acid PC-3 human prostate cancer [111]
Monoclonal antibodies (anti-GD2) or
Asn-Gly-Arg (NGR) peptides incorporated in Neuroblastoma [112]
liposomes
Photosensitiser Ce6 Bladder cancer [113]
Bis(methoxyethyl)-di-n-propylporphycene RR 1022, epithelial cells from
[114]
(BEPPn) Rous sarcoma virus (RSV)
Human malignant ovarian
Photosensitising drug: methylene blue (MB) [92]
adenocarcinoma
Sema3C Glioblastoma (U87 MG cells) [115]
Streptokinase and gemcitabine Lewis lung carcinoma [103]
Neutralising antibody against protein A Ovarian cancer [116]
Doxorubicin in nanoparticles Ovarian cancer [53]

Since the CAM is an isolated subject without external excretion, the metabolic half-life
of many molecules, such as small peptides, tends to be much longer in the chick embryo
Cells 2021, 10, 463 19 of 40

compared to other animal models. This makes it feasible to experimentally evaluate

potential anti-metastatic compounds available only in small quantities [14,73].

3.4. Screening of Drug Toxicity

3.4.1. Irritation
The CAM assay has been accepted for testing the drug irritation potential on human
skin. Traditional animal sensitisation approaches can be altered by the promising chimeric
skin–CAM system. The grafted skin remains viable as long as the survival of the chick
embryo with a constant blood supply [68].
The CAM is accepted to substitute the Draize test, which tests the potential irritation
by chemicals on rabbit eyes [14]. The Draize eye irritancy test placing substances into the
eyes of rabbits can now be replaced by the CAM to determine the irritation potential of
liquid, flake and powdered cosmetics [117].
In a comprehensive safety report, 32 chemicals and 187 cosmetic products were
evaluated by using CAM irritation tests. A good concordance was observed between
CAM and in vivo tests for all substances. In addition, the CAM can be a screening tool for
cleansing foam, hair-dye, hair-curling and shampoo formulations [118].
In addition, the CAM is an alternative platform for irritation testing of ocular tissues
such as the cornea and conjunctiva, because they share similar responsive inflammatory
reactions to irritant substances. By using the CAM, naltrexone, as a therapeutic agent for
diabetic keratopathy, has been tested for its irritant effects on blood vessels [119]. A new
molecule LQFM048, as a photoprotective agent, was also tested on the CAM with a purpose
to investigate its eye irritation potential. Measured parameters include haemorrhage,
coagulation and vascular lysis, resulting in no irritation on the CAM test [120].

3.4.2. Toxicity
The adverse effects on various organs, damage to the vasculature and even embryo
death can be evaluated as indexes for the toxicities of tested drugs or carriers on the
CAM [14]. Here are a few examples.
The influence and toxicity of cigarette smoke and nicotine were investigated in an
ex ovo chick embryo culture [121]. Shell-less chick embryo cultures were used to assess
the effects of acute glucose toxicity [122]. The toxicity of certain herbal medicines such
as Angelica sinensis injection (ASI) and Astragalus membranaceus injection (AMI) was
explored on a CAM platform [123]. Control, ASI, AMI and different ratios of ASI and
AMI were applied topically on the CAM via the drug carrier. After treatment, the number
of living chick embryos was counted. Results showed that ASI and AMI did not inhibit
the survival of chick embryos, and it was concluded that ASI and AMI could be further
investigated for clinical applications in angiogenesis modulation.
As an advanced anti-cancer therapy, the combination of a drug and a carrier can
be assessed. Biodegradable periodic mesoporous organosilica (PMO) nanoparticles were
developed, and doxorubicin (anti-cancer drug) was loaded inside. Human ovarian cancer
cells were planted on the CAM to induce rapid tumour formation. The efficacy and toxicity
of the drug were evaluated after nanoparticles were injected intravenously into the embryo.
The tumour underwent elimination, and no significant damage to various organs (heart,
liver, spleen, kidney, lung and intestine) occurred in the embryo. Toxic effects of the
drug-loaded nanoparticles were obviously missing. In contrast, when doxorubicin without
nanoparticles was injected directly, widespread organ damage was observed, even when a
low concentration was administered [53]. These results sugges that the CAM is a valuable
tumour platform to screen the toxicity and side effects of novel drugs and therapies.

3.4.3. Anti-Vascular Selectivity

The vascular system is vital to supply nutrients to normal tissues and particularly
tumours, so vascular-targeting therapy is a main branch in anti-cancer therapies. The toxic-
Cells 2021, 10, 463 20 of 40

ity and side effects of vascular-targeting drugs and substances must be first screened at
preclinical research levels.
Vascular-targeting strategies
Vascular-targeting therapies (VTTs) include two different approaches: the anti-angiogenic
approach and the vascular-disrupting approach [116]. Accordingly, there are two distinct
categories of agents: anti-angiogenic agents (AAs) and vascular-disrupting agents (VDAs).
AAs suppress the growth of the new tumour vasculature through the inhibition of vascular
endothelial growth factor (VEGF) and other pro-angiogenic molecules [3,124], whereas
VDAs act on endothelial cells and pericytes of the existent tumoral vasculature and conse-
quently vessel occlusion, shutdown of the circulation and pervasive necrosis are induced
inside the tumour [3,125].
Vascular-disrupting agents
VDAs are a type of novel vascular-targeting agents used to treat solid cancers. VDAs
can selectively act on the tumour vasculature by several pathways by which blood flow
inhibition and extensive secondary necrosis occur within tumours, while having little
effect on non-malignant tissues (which remain relatively intact). Tumour blood vessels are
abnormal in structure and function, which are fundamentally different from the vascular
networks of normal tissues, and thus selectively targeted by VDAs [3,125,126].
Classification and mechanisms of VDAs
Currently, two types of VDAs exist in preclinical and clinical R&D, small molecules
and ligand-directed VDAs. Small-molecule VDAs are in the advanced stage of clinical
trials, which can be further divided into two categories: tubulin-binding agents and
flavonoids [125] with distinct mechanisms of action.
A. Tubulin-binding agents
Tubulin-binding agents act directly at the endothelial cytoskeleton, bind to the colchicine-
binding site of the β-subunit of endothelial tubulin, initiate microtubule depolymerisation
and actin/tubulin disorganisation, disrupt endothelial conjunctions and cause cytoskele-
ton deformation and dysfunction. As a result (Figure 8), endothelial cells round up and
bleb on the surface, leading to increased vascular resistance, blood flow stasis, blood cell
stacking, leaky vasculature and intra-tumoral vessel occlusion, followed by coagulation
cascade, thrombus formation, cell–cell disconnection and exposure of the already abnormal
basement membrane. Ultimately, massive ischaemic and haemorrhagic tumour necrosis
occurs [3,126,127].
It must be emphasised that (1) such VDA-induced cytoskeletal disruption occurs
selectively in immature endothelial cells with a lack of contact with smooth muscle and
pericytes, instead of vessels in normal tissues, and (2) such VDA-induced tumour necrosis
is always incomplete, leaving viable cancer cells behind for tumour recurrence [3], which
presents a bottleneck problem with VDAs and calls for a smart solution [49].
Representative tubulin-binding agents are stilbenes of the combretastatin family and
heterocyclic compounds, including combretastatin A-4 phosphate (CA4P), a combretastatin
A-1 derivative OXi4503 and a serine-linked amino derivative AVE8062 [127], as listed in
Table 6.
 icytes, instead of vessels in normal tissues, and (2) such VDA-induced tumour necrosis is
always incomplete, leaving viable cancer cells behind for tumour recurrence [3], which
presents a bottleneck problem with VDAs and calls for a smart solution [49].
Representative tubulin-binding agents are stilbenes of the combretastatin family and
Cells 2021, 10, 463 heterocyclic compounds, including combretastatin A-4 phosphate (CA4P), a 21com- of 40
bretastatin A-1 derivative OXi4503 and a serine-linked amino derivative AVE8062 [127],
as listed in Table 6.

Figure 8. Schematic mechanisms of tumour vascular-targeting tubulin-binding agents.

Figure 8. Schematic mechanisms of tumour vascular-targeting tubulin-binding agents.

Table 6. Vascular-disrupting agents (VDAs) and developmental status.

VDAs Name Company Stage of Clinical Development Ref.

Tubulin Binding
CA4P (fosbretabulin) Mateon Therapeutics Phase 2/3 [3,49]
C118P Sanhome Pharmaceutical Phase 2 [128]
Combretastatin A4
University Bayreuth New formulation under research [129]
gold derivative
Deoxypodophyllotoxin (DPT) China Pharmaceutical University New formulation under research [130]
NPI-2358 (plinabulin) BeyondSpring Phase 3 [3]
BNC105P Bionomics Phase 2 [3]
EPC2407 (crolibulin) Immune Pharmaceuticals Phase 1/2 [127]
OXi4503 (CA1P) Mateon Therapeutics Phase 1/2 [3]
Chong Kun Dang
CKD-516 Phase 1 [3,131]
Pharmaceutical
MN-029 Medicinova Phase 1 [3]
ENMD-1198 EntreMed Phase 1 [132]
Shanghai Institute of Materia Medica,
C9 New formulation under research [133]
Chinese Academy of Sciences
Phenyl-3-(2-chloroethyl) urea (CEU) IMOTEP Inc. New formulation under research [134]
National Health Research Institutes,
BPR0C261 New formulation under research [135]
Taiwan
National Health Research Institutes,
BPR0L075 New formulation under research [136]
Taiwan
IMC-038525 ImClone Systems New formulation under research [137]
ABT-751 Abbott Phase 2 [3]
AVE8062 (ombrabulin) Sanofi-Aventis Development terminated [3]
CYT997 (lexibulin) Gilead Development terminated [3]
Dolastatin-10 Marine Biotech Phase 2 [3]
Cells 2021, 10, 463 22 of 40

Table 6. Cont.

VDAs Name Company Stage of Clinical Development Ref.

Tubulin Binding
MPC-6827 (verubulin, Azixa) Myrexis Phase 2 [3]
TZT-1027 (soblidotin) Daiichi-Sankyo Phase 2 [3]
ZD6126 (ANG453) AstraZeneca Phase 2 [3]
Flavonoids
ASA404 or DMXAA Antisoma Phase 3 [3]

B. Flavonoids
ASA404 or DMXAA (an analogue of flavone acetic acid) is an example of this class (Ta-
ble 6). Flavonoids have a mechanism which is tubulin independent but induces both direct
and indirect anti-vascular action. The direct action leads to rapid apoptosis of endothelial
cells, probably due to the induction of tumour necrosis factor-α and the recruitment of
activated neutrophils, whereas the indirect action stimulates the production of cytokines,
for example tumour necrosis factor-α and other cytokines and chemokines, resulting in
ischaemic tumour necrosis [3,126].
VDAs under research and clinical development
Several small-molecule VDAs have been assessed for cancer treatment. However, the
development many VDAs was terminated because of their unsatisfactory efficacy or their
toxicity and side effects (Table 6).
Combretastatin A4-phosphate (CA4P) is a most extensively studied VDA in clinical
trials for various tumour types, alone or in combination therapy. Thus, CA4P is chosen to
demonstrate the features, efficacy and safety of VDA treatment.
Many trails on a single use of CA4P in human patients have been performed. CA4P
causes an acute and serious shutdown of blood vessels and nearly overall stopping of
blood flow in the tumour and ultimately leads to selective tumour necrosis. However, such
impacts are not present in normal tissues. Meanwhile, CA4P is safe and well tolerated and
lacks haematologic toxicity, which is verified by studies under different dosing schedules
(weekly, 3-weekly, and daily for 5 days every 3 weeks). A maximum-tolerated dose
(MTD) of 50–60mg/m2 was set with consistent anti-vascular effects [125]. Through several
preclinical studies on dose–time correlations of CA4P treatment in tumours and normal
tissues, an estimated dose of 10 mg/kg of CA4P was set as the MTD for rats, equivalent to
that for humans [138].
It must be noted that CA4P, list other VDAs, can cause considerable tumour necrosis,
but tumour regrowth is attributed to peripheral sparing cancer cells (the viable rim) which
are supplied with nutrients and oxygen by the surrounding normal vasculature. Rapid re-
growth of tumours can ensue when a single VDA is administered [125,138]. It is a common
drawback of VDAs, which needs to be tackled [49]. These facts indicate a direction: VDA
combination therapies, for example CA4P plus chemotherapy, CA4P plus AAs, CA4P plus
AAs and chemotherapy, CA4P plus radiology, etc.
Combined with chemotherapy, CA4P showed promising results in clinical trials [3].
A safety and efficacy evaluation was carried out for CA4P with paclitaxel/carboplatin in
anaplastic thyroid carcinoma. The results showed that one-year survival for chemother-
apy/CA4P versus chemotherapy alone was 26% versus 9%, respectively. In addition, no
significant cardiovascular side effects arose and the therapy was well tolerated. Thus,
CA4P with carboplatin/paclitaxel can be a prospective therapy for anaplastic thyroid
carcinoma [139].
Patients with platinum-resistant ovarian cancer underwent a phase II trial with CA4P
in combination with carboplatin and paclitaxel. CA4P with paclitaxel and carboplatin
caused a higher response rate in patients than sole chemotherapy without CA4P and was
well tolerated [140].
Cells 2021, 10, 463 23 of 40

The VDA–AA combination could be an effective treatment without chemotherapy.

Human clear cell renal carcinoma tumours were tested. With the VDA–AA combination,
there was a significant tumour response than with single-agent treatments. CA4P plus
bevacizumab (Avastin) realised a tumour growth delay of 13 days [141].
The safety of CA4P in combination with carboplatin, paclitaxel and bevacizumab
(an AA) was studied in patients with advanced nonsquamous, non-small-cell lung cancer
(NSCLC). Overall and progression-free survival rates were comparable in groups, and
CA4P plus carboplatin, paclitaxel and bevacizumab appears a tolerable therapy with
acceptable toxicity [142].
There is an emerging interest in combining VDA therapy with AAs and chemotherapy
together, especially for patients with poor prognosis. Treatment for platinum-resistant
ovarian cancer was proposed [143]. Mateon Therapeutics Ltd. also submitted clinical trials
to the FDA (clinicalTrials.gov identifier NCT02641639). However, all these clinical trials
so far have failed to be approved by the FDA for commercialisation due to insufficient
therapeutic efficacy.
More recently, by combining a VDA with a radioactive necrosis-avid compound, a
dual-targeting broad-spectrum anti-cancer therapy, OncoCiDia, was introduced, which
is supposed to solve the bottleneck problem of incomplete tumour destruction with all
VDAs [5].
However, there is a pending critical uncertainty from scientific and clinical viewpoints:
tumour specificity [125]. Do VDAs selectively act on the tumour-related endothelium, or is
there a more general effect on normal blood vessels with a risk of subsequent ischaemic
complications and other safety risks? This uncertainty could be tackled by utilising a CAM
research platform.
VDAs assays on the CAM
So far, only a few researchers have explored the CAM model to evaluate vascular
responses of VDAs (Table 7).

Table 7. Studies of VDAs on the CAM.

VDAs Test Methods Ref.

Combretastatin A4-phosphate (CA4P) Topical paper [144]
CA4 gold derivative Topical thin silicon foil [129]
Deoxypodophyllotoxin (DPT) Topical filter paper disks [130]
C9 Topical [133]
Verubulin (MPC-6827) Topical silicon foil ring [145]
Phenyl-3-(2 chloroethyl) urea (CEU) Intravenous injection [134]

These studies have just been reported with preliminary evaluation of the vascular
responses towards VDAs. Testing methods are simple with VDAs applied topically on the
surface of the CAM to observe visual changes in vessels and their branches and to count the
number of blood vessel branches by photography [130] and even manual counting [144].
There have not been any more precise quantitative analyses of the vascular response to
VDAs, with a lack of analyses on efficacy and safety.

3.5. Time Course of CAM Applications for Different Assays

To achieve the best results of preclinical assays, a specific time course (ED period) of
the chick embryo is important to be defined for different CAM applications.

3.5.1. Drug Tests

Commonly, a drug can be applied on the surface of the CAM or injected into the
amnion and the allantois at EDs 7 and 8 [24] in order to examine the drug’s efficacy and
toxicity. However, drug topical application appears more flexible in terms of ED periods.
In one study, drugs for primary pancreatic cancer cells were tested. Tumours grown from
Cells 2021, 10, 463 24 of 40

pancreatic cancer cells were treated with gemcitabine or crizotinib or their combination,
topically applied daily from ED 10 until ED 18 on the CAM [146].
Other researchers prefer ED 9 as the optimal time to inject drugs intravenously into
the CAM to test drug formulations, since major blood vessels are mature at this time [147].
We believe this period can be expanded until ED 10.

3.5.2. Vascular Assays

The period of ED 11 until ED 13 has been opted for assays targeting the existent
vasculature [147,148]. This was also concluded and recommended from our own practical
experiences. After ED 10, major vessels mature and become stronger, so it is more con-
venient for vascular interventions and imaging monitoring. However, beyond ED 13, it
becomes difficult to illuminate through the entire egg to find the exact position of vessels.
For neovascular assays, the chosen time window is from ED 5 to ED 10, although it
was found that angiogenesis decreases as early as ED 5 by hyperglycaemia treatment [149].
Renal tumour fragments and endostatin were placed on the CAM on ED 7, and their
interactions were examined on the vascular network [150], while another study applied
gelatin sponges soaked with a blood vessel stimulator or inhibitor on the developing CAM
at ED 8 [89].
The quantity of intussusceptive pillars in the CAM peaked between ED 8 and 10 [40,151].
On ED 8, the capillary plexus appeared much denser, and numerous intercapillary tissue
islands started to be detected. Therefore, EDs 8–10 are more appropriate for neo-microvascular
assays.

3.5.3. Tumour Implantation

When reviewing the CAM model for tumour biology, a strong angiogenic response
occurs when tumour tissues are implanted on ED 8 to ED 10 onto the CAM, but there is
no such response on ED 11 to ED 12 [152]. This can be explained by the high mitotic rate
of CAM endothelia until ED 10, followed by a decrease later. Osteosarcoma cells were
implanted onto the CAM at ED 9 [153], and urological cancers cells were implanted onto
the CAM on ED 10 [154]. Although HuH7 liver cancer cells were successfully implanted on
the CAM since ED 7 [155], we still recommend EDs 8–10 as a good time course for tumour
implantation.

3.5.4. Cell Growth Factors and Antibody Inhibitors

Similar to the vigorous period for CAM angiogenesis during EDs 7–12, this time
window is also optimal for the investigation of the effects of cell growth factors and
antibody-mediated inhibitors [37]. Vascular endothelial growth factor (VEGF), fibroblast
growth factor (FGF) and platelet-derived growth factor (PDGF) are among classic cell
growth factors.
Recently, stem cells and their derivatives were tested on the CAM for new exploration
of tissue engineering. Although ED 5 and ED 11 have been tested, the CAM at EDs 8–12 is
considered to yield the most significant impacts of stem cells on angiogenesis [156], which
is identical to the conclusion of Bai et al. [37]. An antibody, anti-FGF2 (fibroblast growth
factor inhibitor), was tested on the CAM at ED 8 to treat myeloma plasma cells and cancer
metastasis [152]. A neutralising antibody (Protein A) was validated for its effects on OV-90
ovarian cancer cells implanted onto the CAM at ED 11 [116].

3.6. Advantages and Drawbacks

The CAM as a novel test platform possesses both advantageous features and certain
limitations, as summarised in Table 8.
Cells 2021, 10, 463 25 of 40

Table 8. Advantages and limitations of the CAM as a screening platform.

CAM Features Research Methodology

Small size and easy to handle Reproducibility and reliability
Contains rich nutrients and intensive
Great accessibility
angiogenesis capacity
Rapid vascular growth Rapid screening platform
Necessary organs isolated within the
Broad imaging modalities available
Advantages CAM, in vivo
High embryo survival rate No mobility of animal
Complete circulatory system
Cost-effectiveness
(intravascular delivery of drugs)
Naturally immune deficient, different
Topical and intravascular
tissues and species transplantations
administration of drugs
without immune responses
No requirement for animal protocol
approval, less animal welfare burden
Chicken origin of the assay limiting Very thin and fragile, careful
availability of reagents manipulation required
Complex choice of protocols
CAM under rapid change
available
Developed vascular network difficult to
Limitations Difficult real-time monitoring
be distinguished from new capillaries
Sensitive to environmental factors Short observation time after
(temperature, oxygen tension) treatment
Differences in drug metabolism with Post-grafting non-specific
mammals inflammatory reactions after ED 15
False angiogenesis due to dust and other Oral drug administration not
irritants feasible

4. Methods to Image and Evaluate the Changes in the Vasculature of the CAM
Advanced imaging techniques have enabled us to inspect and quantify the number,
spacing and growth of blood vessels on the CAM, analyse their structural and functional
normalities or abnormalities, measure blood flow and vascularity and facilitate the assess-
ment of vascular-targeting drugs in laboratory or preclinical assays [157].
The CAM is one of the most broadly used assays for vascular-related research [1].
Therefore, various imaging and evaluating techniques have been developed for deter-
mining the angiogenesis and vascular changes induced by different factors or substances
applied on the CAM.
The most conventional methods are destructive: the vasculature of the excised CAM
is observed through a microscope or camera; another approach is morphometric analysis
of the blood vessel network based on stereology [158]. Under such conditions, the CAM is
not intact, not in vivo, and the chick embryo is not vital.
Non-destructive techniques have been developed to provide versatile images of the
CAM vasculature. These can be divided into X-ray, magnetic resonance, gamma-rays and
acoustic and optical techniques, supplemented most recently by artificial intelligence (AI).
Those modalities can, respectively, measure structural changes in the vascular system, the
number and diameter of blood vessels and certain functional parameters, for example
blood flow, dynamics and blood oxygen levels [44,157].
Innovative methods for imaging the vasculature can visualise blood vessels in tumours
grafted on the CAM in order to assess the efficacy of vascular-targeting drugs for treatment
of cancer or other chronic diseases.

4.1. Light Microscopic Methods

Microscopies are traditional and powerful methods to visualise vascularisation and
vascular changes. Furthermore, new contrast or imaging agents selectively label certain
Cells 2021, 10, 463 26 of 40

vessels, which enable one to visualise selectively specific vasculature at the microscopic
level.
By microscopic methods, the simplest evaluation is to observe the presence or absence
of blood vessels and their changes with parameters such as the number or density of blood
vessels; vessel dimensions, including length, diameter and branch points; and the total area
of the CAM [44,73,157]. Vascular changes can be determined by using stereological princi-
ples and morphometric analysis. Qualitative, semi-quantitative and quantitative skills are
available, among which automated image analysis and statistical post-processing methods
are valuable tools incorporating appropriate computer software [44,157,159]. A ×10 mag-
nification stereomicroscope was utilised to analyse the vascular convergence towards the
graft, distribution, branching and density of CAM vessels at different time points. A ×250
magnification microscope was used to quantitatively evaluate microvessel density, which is
expressed as the percentage of intersectional points occupied by microvessels [159].
Different tumour cells were inoculated into the CAM. Macrovessel images next to
the graft were taken by a handy digital camera at ED s10, 12 and 14, and the images were
processed to quantify angiogenesis. In parallel, microvessels were evaluated by confocal
microscopy, which focused on the capillary plexus adjacent to the surface of the CAM [56].
A fluorescent microscope was also used to visualise expansion of the metastasis in
the CAM. The human epidermoid carcinoma cell line was injected intravenously, and a
fluorescent stereomicroscope imaged a section of the CAM at different intervals over days.
Contrasting the dark-green background from the eggshell, the tumour cells were visible as
bright-green spots and the vessels were visible as black lines [160].
One set of intravital microscopy was designed to image blood vessels which grew
or regressed in the CAM and to inspect neovascularisation in tumour grafts in the ex
ovo chick embryo [161]. It could catch real-time images for over 72 h and quantify the
angiogenesis progress, without affecting the host and tumour systems. This designed
unit was integrated with an upright epifluorescence or spinning disk confocal microscope.
Fluorescent nanoparticles or dyes were used to enable visualisation of blood vessels.
Such intravital microscopy can collect 3D spatial and temporal data. Rapid successive
fluorescence images can be acquired in a single plane to visualise the dynamics of blood
flow. The detailed structure at specific time points can be analysed by a 3D image stack
at high resolution. Vasculature changes over time can be figured out by acquiring 3D
stacks. Meanwhile, intravital microscopic imaging could display several unusual features
of tumour vessels in preclinical applications [157].
The microscopic methods provide the highest resolution; confocal microscopy has
about 100 nm resolution, and scanning electron microscopy (SEM) yields a few nanometres,
which is only restricted for ex vivo studies [157,161].

4.2. X-ray Methods

With the aid of a contrast medium, computed tomography (CT) is conventionally
used to document vascular characteristics such as blood flow, blood volume, mean transit
time of flow and microvascular permeability. Normally, a greyscale image is obtained by
means of contrast agent injection and the X-ray attenuation coefficients of the tissues. By
computer processing of a series of 2D sections and tomographic reconstruction, one 3D
image of the inside of the subject is generated [162,163]. Moreover, dual-energy CT, also
named spectral CT, is a novel technique which uses two independent X-ray photon energy
spectra. Materials with different attenuation properties can be interrogated at different
energies [164]. It could be applied for studies on a CAM platform.
In one study, 3D micro-CT was used to explore three topics on embryonic viability
and morphogenesis: (1) impact of the micro-CT X-ray radiation dose, (2) impact of the CT
contrast agent type and microinjected dose and 3) CT contrast media biodistribution and
persistence within the embryo. It was demonstrated that micro-CT is feasible for analysing
3D volumetric quantitative imaging of live embryo morphogenesis and that the efficacy
and safety of contrast-enhanced micro-CT appears acceptable for live embryos [165].
Cells 2021, 10, 463 27 of 40

Digital substraction angiography (DSA) is another X-ray-based imaging acquisition

technique to visualise the vascular system of the CAM in the developing chick embryo [166].
DSA is a dynamic technique which can effectively demonstrate blood flow patterns at
various phases of circulations in the CAM and chick embryo: from the CAM vein to the
heart and then to arteries of the embryo and CAM membrane arteries, etc. It is able to
determine vessels with a diameter of 100 µm and study morphological changes in the
vasculature in the embryo system. A modern DSA system utilises low-noise video and
digital electronic equipment with high-speed image processors to assess vascular effects
of various therapies. DSA is also used to evaluate the effect of ionising radiation on the
angiogenesis process in the CAM. Computerised analysis of angiographic images showed
that the anti-angiogenic effect of irradiation during various phases of CAM development is
insignificant during the studied time window [167]. Furthermore, the anti-angiogenetic
effect of ionising radiation on the angiogenesis of a transplanted tumour on the CAM
was evaluated and quantified by using DSA [168]. Irradiated areas of micro- and macro-
vessels (smaller or greater than 200 µm) presented a damageable effect reflecting statistical
reduction in both length and total vessel area.

4.3. Magnetic Resonance Methods

Regarding magnetic resonance imaging (MRI), the object is placed in a strong electro-
magnet, where the hydrogen atoms align parallel to the magnetic field. A short powerful
radio signal is released passing through the object, which is perpendicular to the electro-
magnetic field. The hydrogen atoms which have the same frequency as the radio signal
get excited and start to resonate with the exciting wave. When the radio signal is switched
off, the hydrogen atoms return to their original energy state after a certain period of time.
Meanwhile, the absorbed excitation energy is then released in the modality as radio signals,
which are detected to acquire an MRI picture at this level or slice. The releasing time
depends on the characteristics of the tissue and the number of atoms and is measured and
configured into 2D and 3D images [163]. The spatial resolution of clinical MRI is 100 to 500
µm. Micro-MRI has better spatial resolution, up to 10 µm, in preclinical modalities, with
poorer temporal resolution [157]. MRI can measure the blood volume and blood vessel
permeability through contrast-agent-enhanced dynamic imaging [163].
Combining specific and sensitive probes, high-resolution MRI is regarded as a promis-
ing technique for molecular imaging [169]. Three-dimensional magnetic resonance mi-
croscopy (MRM) has been successfully developed to image perfusion-fixed hearts of chick
embryos. The major heart chamber and even blood vessels of smaller diameters can be
visualised after the perfusion of the embryonic cardiovascular system. The technique
enables volumetric analysis of chick heart morphology at different stages.
MRI was utilised to study tissue bioengineering by monitoring bone implants on the
CAM. The bone formation process was assessed in polymeric scaffolds in vitro, which
had been implanted onto the CAM. Furthermore, to improve the in vivo assessment, a
novel MRI contrast agent was developed to facilitate detection of mineral deposits within
engineered bone with improved specificity [170].
Another innovative application of MRI is to monitor and quantify the perfusion
capacity of 3D biomaterials in a model of a scaffold placed on the CAM in vivo and non-
invasively [171]. The perfusion capacity was assessed by comparing the longitudinal
relaxation rate before and after injecting a paramagnetic MRI contrast agent. The relaxation
rate changes have a strong positive correlation with vessel density, which was histologically
proven, whereas vessel density correlates with perfusion capacities. The highest relaxation
rate changes were found at the interface where the scaffold was attached to the CAM, and
the surface of the scaffold showed the lowest relaxation rate changes. In addition, a large
difference of perfusion capacities can be observed among different biomaterials, suggesting
that MRI is sensitive to reveal such differences [152]. Therefore, by means of MRI in vivo,
the CAM can be a good platform to test a large variety of bioengineered materials.
Cells 2021, 10, 463 28 of 40

Unlike X-ray- and gamma-ray-based imaging modalities, which emit ionising radia-
tion harmful to organisms, including animals and humans, MRI is virtually harmless to
Cells 2021, 10, x FOR PEER REVIEW life and therefore can be applied to monitor the growth of chick embryos longitudinally
29 of or
41
on any ED (Figure 9).

Figure 9. Using a clinical 3.0T Siemens magnetic resonance imaging (MRI) scanner, a chick embryo
Figure 9. Using a clinical 3.0T Siemens magnetic resonance imaging (MRI) scanner, a chick embryo on ED 12 can be imaged
on ED 12 can be imaged entirely at both axial and coronal slices of 3 mm thickness with both T1
entirely at both axial and coronal slices of 3 mm thickness with both T1 and T2 weighted sequences, reflecting different
and T2 weighted sequences, reflecting different physiochemical properties of the tissue compo-
physiochemical properties of the tissue components.
nents.

4.4.
4.4. Gamma-Ray
Gamma-Ray Methods
Methods
Positron emission tomography
Positron emission tomography (PET)
(PET) is aisnewa new approach
approach utilising
utilising positron positron
and gammaand
gamma rays. A positron-emitting
rays. A positron-emitting radiotracer
radiotracer is intravenously
is intravenously administered,
administered, and its dis-
and its distribution
tribution is monitored
is monitored during a during
suitablea uptake
suitableperiod.
uptakeWhileperiod. While decaying,
decaying, the radionuclide
the radionuclide emits a
emits a positron, which interacts with an electron in the environment,
positron, which interacts with an electron in the environment, inducing the emission inducing the emis-of
sion of two gamma rays in opposite directions. These emissions
two gamma rays in opposite directions. These emissions can be detected by two detectors can be detected by two
detectors
on opposite on sides
opposite
of thesides
objectof the object [163].
[163].
For
For aanormal
normaltissue
tissuestudy,
study,PETPET can
canmeasure
measure thetheblood volume
blood volumeby the
by signal but can-
the signal but
not actually
cannot visualise
actually visualisevascular
vascular structures
structures [157].
[157].ForForcancer
cancerstudies,
studies, tumour
tumour functional
characteristics
characteristics such
such asas blood
blood flow or volume and glucose metabolism can be accurately
quantified by the application of radiotracers, e.g., 15 H2 O, 11CO
15H2O, 11
COand and1818
FDGFDG [163].
[163].
Tumour glucose metabolism and protein synthesis were successfully imaged by PET
in a U87 glioblastoma model on the CAM. The injection injection of of 18
18FDG into blood vessels of the

CAM enabled dynamic imaging imaging of glucose

glucose metabolism,
metabolism, and and thethe uptake
uptakeof of 18
18FDG over time

in individual tumours was verified [172]. The accuracy of tumour volume measurements
can be improved by by CTCT imaging.
imaging. Therefore,
Therefore,aacombination
combinationofofPET PETand andX-ray
X-rayCT CTbecomes
becomes a
standard approach to ensure and elevate the accuracy of imaging,
a standard approach to ensure and elevate the accuracy of imaging, which yields func- which yields functional
data with
tional dataanatomic information.
with anatomic In addition,
information. the novel
In addition, theapplication of PET/CT
novel application was tested
of PET/CT was
on theon
tested CAM to screen
the CAM novel
to screen PETPET
novel tracers [172].
tracers [172].AA CAM
CAMtumourtumourmodel
modelis is particularly
valuable for
valuable forrapid
rapidandandhigh-throughput
high-throughputscreening screening of of novel
novel radiotracers,
radiotracers, as well
as well as op-
as for for
optimisation
timisation of PET/CT
of PET/CT imaging
imaging protocols
protocols [153].
[153].

4.5. Acoustic
4.5. Acoustic Methods
Methods
Ultrasonography refers
Ultrasonography refers an
an ordinary
ordinary and
and widely
widely available imaging modality
available imaging modality in
in both
both
clinical and preclinical research. It is suitable to visualise tumour growth and vascularisa-
clinical and preclinical research. It is suitable to visualise tumour growth and vascularisa-
tion in the CAM assay. By using a commercial ultrasonographic scanner, tumour growth
tion in the CAM assay. By using a commercial ultrasonographic scanner, tumour growth
and angiogenesis were successfully monitored, and results of ultrasound images were
and angiogenesis were successfully monitored, and results of ultrasound images were
significantly correlated with those of histological analysis from the excised tumour [155].
significantly correlated with those of histological analysis from the excised tumour [155].
Photoacoustics is based on the generation of sound waves by laser radiation. A short
laser pulse heats absorbers inside the tissue, with the temperature raised by generated
energy. Due to up-and-down changes in the temperature, the absorber expands and sub-
sequently contracts, inducing a pressure wave or an acoustic wave. These time-trace
waves disperse through the tissue and can be seized by the detector at the tissue surface.
Cells 2021, 10, 463 29 of 40

Photoacoustics is based on the generation of sound waves by laser radiation. A short

laser pulse heats absorbers inside the tissue, with the temperature raised by generated
energy. Due to up-and-down changes in the temperature, the absorber expands and
subsequently contracts, inducing a pressure wave or an acoustic wave. These time-trace
waves disperse through the tissue and can be seized by the detector at the tissue surface.
The dispersion time determines the photoacoustic position [173]. The strength and the
profile of the captured signals carry the overall spatial information about the distribution
of laser absorption in the tissue [174].
Blood (haemoglobin), a typical absorber of ultrasound, can position and monitor
blood flow and concentration in vessels, tissues and tumours. A photoacoustic system
was used to visualise single blood vessels in the CAM at ED 9, which revealed that the
majority of vessels were sized from 200 to 500 µm in diameter, with other smaller vessels
ranging from 125 to 200 µm. Meanwhile, through 3D rendering of the reconstructed
dataset, the blood vessels’ structures of 3.5 mm depth became clearly visible [173]. In most
cases, photoacoustic signals were acquired at flat and reflective surfaces. However, in real
biological applications, the signals always interfere with the roughness of sample surfaces.
Considering this fact, a fibre-based noncontact photoacoustic tomography system was
designed to overcome the disadvantage. With a fibre-optic heterodyne interferometer as
a signal detector, the system measured the surface displacement of a sample. In fact, the
system could successfully measure the photoacoustic signals from the surface without any
physical contacting with the sample [175].
A similar principle called photothermal radiometry, which uses thermal waves to
image discrete blood vessels of the CAM in vivo, was applied. However, the authors used
an infrared (IR) sensor to remotely detect and record 2D IR images during excitation by
periodically modulated laser radiation [176].

4.6. Optical Techniques and Methods

Clinical settings of CT, MRI, PET and ultrasonography cannot reach enough resolution
to visualise microcirculation in the CAM. Besides, the contrast agent (or radiotracer)
injection may cause unwanted effects [177].
However, optical techniques may be advantageous by enabling high-resolution in vivo
blood flow imaging of CAM vessels. Blood has a very typical reactive spectrum of light,
and important parameters, including blood flow, volume, flow velocity, blood perfusion
and the structure of the vessel network in the CAM, can be obtained by optical imaging
techniques.
A few optical methods have been developed to provide information about the blood
movement, quantified blood flow and perfusion in CAM assays. These innovative applica-
tions are mainly derived from three directions: (1) the optical coherence tomography (OCT)
principle, (2) the laser Doppler principle and (3) the laser speckle principle.

4.6.1. Optical Coherence Tomography (OCT) Principle

The OCT technique is for non-invasive cross-sectional imaging in biomedical systems.
Low-coherence interferometry is utilised to produce 2D images of optical scattering from
microstructures of internal tissues. OCT has spatial resolutions longitudinally and laterally
in a few micrometres. OCT is analogous to pulse-echo in ultrasonic imaging [178], except
that it uses light to replace sound [179]. Cross-sectional images from different depths are
extracted by coherence gating, whereby 3D imaging is built from 2D data [180].
However, OCT is not eligible for blood flow imaging due to the interference of the
internal tissue background. Hence, optical Doppler tomography (ODT), which integrates
the Doppler principle, i.e., Doppler flowmetry, into OCT, was developed to measure the
blood flow velocity in biomedical systems. In addition, both blood microcirculation and
tissue structures surrounding the vessel can be imaged in vivo non-invasively due to the
high resolution of ODT [181]. The blood vessel wall, chorion membrane and yolk sac
membrane of the CAM and embryo are depicted in tomographic structural images. In
Cells 2021, 10, 463 30 of 40

ODT velocity images, blood moving at different velocities appears distinctive in contrast to
dark static regions in the CAM. The blood flow velocity is maximum at the vessel centre,
decreasing gradually towards the vessel wall, with a velocity resolution of 100 µm/s.
The ODT probes into tissues to a depth of about 1–2 mm.
A Doppler variance optical coherence tomography (DVOCT) method was developed
to apply to tumour vasculature imaging. A glioma F98 tumour spheroid was tested on
the CAM, and 3D mapping of the tumour was carried out. Both the microstructure and
the microvasculature of the tumour were visualised clearly. A dense vascular network
of capillaries up to 10 µm in diameter was visualised from the top view of the DVOCT
image with a spatial resolution of 3.5 µm and an average penetration depth of 1.5 mm. As
a real-time imaging system, DVOCT has the potential to assess tumour vasculature as a
non-invasive technique in clinical and preclinical settings [177].

4.6.2. Laser Doppler Principle

Doppler optical theory was first demonstrated to measure the red blood cell (RBC)
concentration and blood flow velocity four decades ago. Since then, laser Doppler instru-
ments have been developed [182]. With this approach, one part of the light is scattered by
static tissue and remains at its incoming frequency, whereas the other part of the light is
scattered by moving RBCs and reflected to a detector, but its frequency shifts due to the
Doppler effect. The mixture (interference) of these different frequencies at the detector
leads to a beat frequency equal to the Doppler shift between the two light parts. Because
the scattering light in tissue is virtually random, such photons have different Doppler shifts.
Thus a Doppler frequency spectrum is generated and detected by the detector, which
is converted into the power spectrum of the current fluctuations. The power spectrum
reflects information about the blood perfusion. Through signal processing, an RBC flux is
expressed by a combination between RBC concentration and velocity. Doppler technology
has huge publication records due to its maturity and wide applications [183].
Laser Doppler perfusion imaging (LDPI) with an area scanning beam represents a
recent advance in laser Doppler technology [184]. A CAM-LDPI platform was established
to assess drug absorption [185]. Vasoactive drugs were chosen, including caffeine, propra-
nolol, glucagon, theophylline, glyceryl trinitrate (GTN), etc., and common solvents. They
were applied on the CAM surface, and the blood perfusion was quantitatively measured
by LDPI in response to different concentrations of solvents and drugs. Perfusion is a
parameter related to the speed of multiple numbers of RBCs [186], and the CAM is useful
for the assessment of drug absorption by using LDPI to measure blood perfusion [184].
New generations of commercial LDPI work with rasters on a line of pixels in parallel at a
single time, thereby substantially reducing the acquisition time of single images.
A laser Doppler anemometer (LDA) and signal analysis method were developed to
measure the absolute blood flow velocity in animal and human near-surface arterioles and
venules [187]. The vessels of the CAM at EDs 9–13 with a diameter of 111–229 µm lying
at a depth of 70–200 µm were measured. The study found out how the LDA works, and
what considerably influences its measuring capability are periodic and aperiodic blood
flow pulsations [187].
Another technology, a complementary metal-oxide semiconductor (CMOS) acquires
data from all points through a strong powered expanded laser beam at the same time.
Temporal resolution is improved significantly: just 2–10 s are needed for acquisition and
display of a 256 × 256–pixel image, instead of 4 min by common point scanning LDPI [183].
Although LDPI is a standard method for optical measurement of blood perfusion and
has been improved significantly, it is still limited by low resolution and long measurement
times. Therefore, a new analysis technique, laser speckle perfusion imaging (LSPI), was
developed, which can create prompt, high-resolution perfusion images [184].
Cells 2021, 10, 463 31 of 40

4.6.3. Laser Speckle Principle

The speckle imaging principle was initially exploited for imaging retinal blood flow
in the early 1980s [188]. Digital laser speckle imaging is a novel technology, and the first
commercial product, full-field laser perfusion imager (FLPI), was introduced in 2007 by
Moor Instruments.
Illuminating tissue with coherent light induces a random interference pattern, named
speckle. The speckle is formed by constructive and destructive interferences, which are
caused by variable lengths of travelling light reverting to the detector due to different
pathways traversing in the object and irregular surfaces [189].
Any motion of the particles in tissue leads to speckle pattern changes. If there is flow
in the region of interest (ROI), the speckle pattern undergoes decorrelation (or blur) and
the intensity in ROI decreases. Thus, the level of decorrelation depends on the speed and
volume of the RBCs in the tissue area. Under this logic, low intensity is relevant to a high
amount of blood flow (because the light is scattered by moving RBCs), and conversely,
high intensity expresses low blood flow. The speckle size is determined by the aperture
size of the imager alone. Relative to the correlation time of intensity fluctuations, the
detector integration time is sufficiently short. For imaging blood perfusion in the skin,
the integration time is set to between 5 and 20 ms in a typical device [183]. By analysing
intensity fluctuations using computer software and algorithms, real-time graphs and other
temporal and spatial information about blood perfusion in the tissue can be obtained.
Because the speckle pattern reflects the dynamics of the scatters, speckle imaging is very
beneficial for studying the fluid within turbid tissues in biomedical applications [189].
LSPI can response to changes in both blood flow velocity (0–800 L/min, 1% concen-
tration) and RBC concentration (0.1%–5%, 200 L/min) and provide a quicker response
time and higher spatial resolution, which is more advantageous to LDI for certain uses.
Therefore, LSPI and its commercial instrument were validated for measuring tissue blood
perfusion [184]. However, they are still rather new and not yet mature [183]. People
are still striving to explore new applications of LSPI in preclinical tests, for example in
mice [190–192], rabbits [193,194] and even human skin [195].
Concerning the LSPI application on the CAM, only a few efforts have been docu-
mented. In 2005, when a commercial LSPI instrument had not been released, a team used
a customised speckle imaging device and software to monitor changes in the vascular
structure and blood flow after photodynamic therapy (PDT) in the CAM at EDs 10–13.
The laser was used to stimulate PDT and also to function as the coherent light source for
LSPI [91]. At EDs 9–11, tumour cells from a lung metastasis human salivary adenoid cystic
carcinoma cell line were inoculated on the CAM. At ED 11 or ED 12, a photosensitiser
of pyropheophorbide acid (Pyro-Acid) solution was applied topically on the vessel-rich
position surrounding the tumour graft on the CAM. The treatment lasted 30 min before
laser irradiation for 15 min. The blood velocity changes at the treated area were recorded
by speckle imaging at 0, 3, 6, 9, 12 and 15 min after PDT. The results showed that the blood
velocity in vessels slowed down (small vessels decreased to approximately 10% and big
vessels decreased to about 25% compared to the beginning) due to vascular damage such
as vascular constriction, blood stasis and coagulation. The results suggested that speckle
imaging is effective to assess the PDT efficacy in peripheral vessel damage of the tumour
through monitoring changes in the vascular structure and blood flow.
However, this trial had some deficiencies: (1) the measurement was rough, so the
extent of vessel changes and blood flow perfusion needs a precise standard; (2) only a basic
equation was used in the calculation, but optical properties of scatters (scatter size and
multiple scattering) were not considered; (3) peripheral vessels of the tumour on the CAM
were measured, but intratumoral vasculature was not considered; (4) all experiments were
in vitro (ex ovo); and (5) only a customised device was used. Due to the lack of a standard
instrument, speckle imaging is still difficult to be popularised in preclinical assays and
clinical applications.
Cells 2021, 10, 463 32 of 40

More recently, the impact of the optical properties of scatters was noted and ex-
perimentally investigated, which is relevant to the quantification of the inverse relation
between decorrelation time (τc) and blood flow velocity (V), i.e., 1/τc = αV. A sidestream
dark-field–laser speckle contrast imaging (SDF-LSCI) device was designed to record the
specific vessels of the CAM at ED 9, grown ex ovo. V (by SDF) and τc (by LSCI) of the
same vessels were independently estimated. Thus, a practical speckle model, considering
multiple scattering (the influence on α), was summarised, and a blood flow velocity map
was generated. The vascularised CAM can be an intermediate calibration model [196].
Non-invasive imaging methods are still not optimal to evaluate blood flow, blood
volume and vascular change and cannot detect functionally structural changes in deep
tissues [157]. However, due to its uniqueness, the CAM could be a good platform to foster
more precise methods and tools to assess changes in normal tissue vasculature and tumour
blood vessels before and after treatment with different therapies, and thus to conduct
pharmacological research.

4.7. Artificial Intelligence (AI) Techniques and Algorithms

Chick embryos are also used for virus culture during the vaccine preparation process
for common industrial production. The quality of embryos crucially impacts the quality of
the vaccine. Therefore, any dead, broken, contaminated and weak embryos unsuitable for
vaccine preparation, and for other CAM studies as well, need to be eliminated. The mor-
phology and features of blood vessels in the CAM during certain ED periods provide a
feasible tool for the detection of unsuitable embryos.
Machine inspection is a popular approach to determining the viability of embryos
by extracting imaging information about the major blood vessels of the embryo. Several
machine vision methods are in use, which perform image enhancement, segmentation and
classification. They mainly focus on the major artery and vein. The limitations of image
acquisition conditions, image quality and complexity of specific embryos largely influence
the quality of classification [197].
Therefore, improved algorithms have been successfully applied to analyse and recon-
struct images. The results demonstrate that the improved algorithms have a much higher
anti-noise capacity and can greatly improve the quality of image reconstruction [174].
Further innovation has transited to systemic AI methods, which consolidate advanced
image reconstruction and analysis. In recent years, as typical deep learning methods,
convolutional neural networks (CNNs) have shown promising performance in image
classification. A CNN refers to a multi-layer, self-supervised learning perceptron. It is
composed of a data layer, a multiple alternating convolution layer, a pooling layer, a fully
connected layer and an output layer [198]. Several CNNs have been proposed for embryo
activity or viability detection based on many existing models. In 2017, Geng et al. proposed
a deep-learning-based classification method for embryo viability detection using a unique
CNN structure. It greatly improved the detection accuracy up to 99.5% [197].
AI methods can sufficiently achieve viability detection, but they are not effective for
weak embryo detection. In 2020, based on previous CNN models, Chen et al. proposed a
weak embryo detection method based on the multiscale feature fusion convolutional neural
Weak Embryo Detection Network (WEDNet) [199]. The proposed residual multiscale fire
block (RMFB) to extract multiscale features from egg embryo images and feature fusion
performance could effectively use the overall colour information about the image and blood
vessel information at different scales. The detection accuracy of WEDNet, constructing
through the cascade of RMFB modules, could reach 99.35% [199].
Through AI techniques and algorithms, a completely non-invasive method to ex-
tract and analyse blood vessel information about chick embryos has been introduced.
Vaccine production as well as CAM studies can be improved by good embryo detection,
classification and selection.

Author Contributions: Conceptualisation, L.C., Y.N. and Y.L.; literature search, all; writing—original
draft preparation, L.C., Y.N., Y.L. and S.W.; review and Editing, all; supervision, C.V.O., Y.N. and Y.L.;
Cells 2021, 10, 463 33 of 40

administration, C.V.O., Y.N. and Y.L.; funding acquisition, Y.L. and Y.N. All authors have read and
agreed to the published version of the manuscript.
Funding: This study was partially funded by the FWO-NSFC project VS05621N, the KU Leu-
ven IOF−HB/12/018 OncoCiDia project, the National Natural Science Foundation of China (nos.
81603142, 81830052), the construction project of the Shanghai Key Laboratory of Molecular Imaging
(18DZ2260400), the Shanghai Municipal Education Commission (Class II Plateau Disciplinary Con-
struction Program of Medical Technology of SUMHS, 2018–2020) and the CZC Pharmaceutical and
Technology Co., Ltd. Nanjing.
Conflicts of Interest: All authors declare no conflict of interest.

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