I. Freitas
University of Pavia, Biology and Biotechnology, Faculty Member
Hypoxic tumor cells resist most therapies and cause tumor regrowth when their environment improves. Identifying the adaptation strategies to hypoxia would help develop better tailored cancer therapies. Ehrlich carcinomas implanted on mice... more
Hypoxic tumor cells resist most therapies and cause tumor regrowth when their environment improves. Identifying the adaptation strategies to hypoxia would help develop better tailored cancer therapies. Ehrlich carcinomas implanted on mice were analyzed histochemically for the following enzyme activities: lactate, succinate and glucose-6-phosphate dehydrogenases, dihydrofolate reductase, purine nucleoside phosphorylase, xanthine oxidoreductase, and acid phosphatase. With the exception of xanthine oxidoreductase, which was not active in tumor cells, and of succinate dehydrogenase the activity of which was not significatively altered, all other activities were much higher in perinecrotic cells with respect to cells close to blood vessels. These data suggest the integration of metabolic paths allowing purine and lipid biosyntheses. Degradation products from the necrosis are presumed to be employed as surrogates of blood-borne nutritive substances by cells distant from the vascularization.
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The therapeutical application of heat, in any manner obtained (radiofrequency, microwave, ultrasound hyperthermia) , causes progressive physiopathologic modifications to the tumoral mass.These consist in structural damages sufficiently... more
The therapeutical application of heat, in any manner obtained (radiofrequency, microwave, ultrasound hyperthermia) , causes progressive physiopathologic modifications to the tumoral mass.These consist in structural damages sufficiently known(18). The heating of a neoplastic mass usually brings about impairments of enzymatic cellular processes, with an increase of oxygen consumption, interesting anoxic or partially anoxic cells.These enzymatic impairments imbalance the normal homeostatic equilibrium, leading to cell. death(55). The thermal washout mechanisms, which protect the normal cells, for certain temperature limits, are missing for the neoplastic cells placed in different environment.The nutritive supply to the tumor microenvironment largely depends on an inefficient and inelastic vascular system (8). The temperature increase, beyond an optimal shoulder, 42.5°C, creates higher oxygen consumption, increase of microsomal activity and of anaerobic glycolysis, leading to two different processes: (A) increased production of lactic acid with consequently pH decrease, (B) a drastic reduction of ATP formation. The reduced availability of energy, due to high temperatures, impairs the enzymatic repair system of tumor DNA molecules.
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Solid tumors are "organoids" consisting of highly heterogeneous populations of malignant, stromal and inflammatory cells and dynamic extracellular matrix. In particular, distinct cellular microenvironments are observed. The... more
Solid tumors are "organoids" consisting of highly heterogeneous populations of malignant, stromal and inflammatory cells and dynamic extracellular matrix. In particular, distinct cellular microenvironments are observed. The survival strategies of malignant cells might therefore be highly differentiated, causing the high genotypic and phenotypic instability characteristic of malignant cells in vivo. A constant interplay between the tumor compartments and the host immune and hemostatic systems determines the behavior of the tumor. A description of typical microenvironments and of cellular and matrix interactions is provided. Based on these, it is here postulated that: (a) any cancer treatment, by influencing differently the various tumor compartments, will alter previously established equilibria; (b) the behavior (growth, invasiveness, metastatic potential, resistance to further treatment) of a malignancy after treatment might be altered with respect to what is assumed in te...
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The liver of tumor-bearing hosts manifests fetal phenotypes. We investigated the expression of differentiation markers on the liver in MMTV-neu (ErbB-2) transgenic mice, in the period from incipient neoangiogenesis to lung... more
The liver of tumor-bearing hosts manifests fetal phenotypes. We investigated the expression of differentiation markers on the liver in MMTV-neu (ErbB-2) transgenic mice, in the period from incipient neoangiogenesis to lung metastatization. We report AFP expression by hepatocytes in all lobular zones, CD34 cell arrest and subsequent hemopoiesis in periportal and mid-zone areas, oval-like cells (CD34+, CK19+, AFP+) and ductular reaction in portal tracts, portal CK19+ and GGT+ hepatoblast-like cells, and midzonal large dysplastic hepatocytes. We hypothesize that CD34 cells are recruited by the tumor from the marrow for angiogenic purposes and that their differentiation in the liver is influenced by altered liver microenvironment(s). AFP may act as a growth factor and biological response modifier for these cells and for the tumor. Dysplasia might be enhanced by metabolic stress. We conclude that the liver differentiation potential is lobular-zone-dependent and that the risk for eventual...
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Studies assessing the effects of partial-hepatic ischemia/reperfusion (I/R) injury focused on the damage to the ischemic-lobe, whereas few data are available on non-ischemic lobe. This study investigated whether acute liver I/R does... more
Studies assessing the effects of partial-hepatic ischemia/reperfusion (I/R) injury focused on the damage to the ischemic-lobe, whereas few data are available on non-ischemic lobe. This study investigated whether acute liver I/R does affect non-ischemic lobe function via modulation of extracellular matrix remodeling. Male Sprague-Dawley rats underwent left lateral- and median-lobe ischemia for 30 min and reperfusion for 60 min or sham operation. After reperfusion, blood samples and hepatic biopsies from both the ischemic (left-lobe, LL) and the non-ischemic lobe (right-lobe, RL) were collected. Serum hepatic enzymes and TNF-alpha, tissue matrix metalloproteinases (MMP-2, MMP-9), liver morphology, malondialdehyde (MDA), and myeloperoxidase (MPO) were evaluated. Liver I/R injury was confirmed by altered increased hepatic enzymes and TNF-alpha. I/R induced an altered morphology and an increase in MMP-2 and MMP-9 activity not only in left-ischemic lobe (LL) but also in the right-non-isch...
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Fluorescence microscopy (microfluorimetry) is an established technique that provides useful information about the biomolecules in a cellular environment[1,2]. The technique consists in exciting the samples (cells or part of them) with a... more
Fluorescence microscopy (microfluorimetry) is an established technique that provides useful information about the biomolecules in a cellular environment[1,2]. The technique consists in exciting the samples (cells or part of them) with a light source of suitable wavelength, and in detecting and processing the fluorescence emission.
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The tumor interstitial fluid (TIF) is a fluid phase present in the extracellular space of all tumors whose importance in oncology is seldom recognized. In order to stimulate other researchers to give it the due importance, a review of the... more
The tumor interstitial fluid (TIF) is a fluid phase present in the extracellular space of all tumors whose importance in oncology is seldom recognized. In order to stimulate other researchers to give it the due importance, a review of the available data (including our own) is provided. An hypothesis is presented for the genesis, fate and role of the TIF in the processes of invasion, growth and metastatization. Open questions regarding the TIF's role in tumor response to therapy are raised.
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The effects of Celsior solution were compared with those of the University of Wisconsin solution (UW) after 18 or 48 hours of cold storage in a perfused rat liver model. Lactate dehydrogenase (LDH), hyaluronic acid (HA) uptake,... more
The effects of Celsior solution were compared with those of the University of Wisconsin solution (UW) after 18 or 48 hours of cold storage in a perfused rat liver model. Lactate dehydrogenase (LDH), hyaluronic acid (HA) uptake, thiobarbituric acid-reactive substances (TBARS), tissue reduced (GSH) and oxidized glutathione (GSSG) and ATP were evaluated. Histochemical in situ evaluation ofLDH and reactive oxygen species (ROS) were also performed. No significant difference in LDH release, HA uptake, TBARS, ATP levels and GSH/GSSG ratio were observed between UW and Celsior solution when the livers were preserved for 18 hours. By contrast, when preservation was performed for 48 hours, LDH release, TBARS and ROS formation were higher and the ATP levels, GSH/GSSG ratio and HA uptake were lower in the liver preserved by Celsior as compared with UW. Celsior solution was as effective as UW in liver preservation up to 18 hours but the superiority of UW over Celsior solution was obtained when li...
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ABSTRACT In this paper we discuss the technique of laser fluorescence microscopy. First, we present the latest version of a laser microfluorometer, characterized by high spatial and temporal resolution, and by a high degree of versatility... more
ABSTRACT In this paper we discuss the technique of laser fluorescence microscopy. First, we present the latest version of a laser microfluorometer, characterized by high spatial and temporal resolution, and by a high degree of versatility and automation due to microprocessor control. We also present our latest results in the application of this technique to the study of the interaction properties of the photosensitizing antitumor drug Hematoporphyrin Derivative (HpD) at cellular level.
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Research Interests: Laser and Misure Ottiche
This work reports on studies of spectral properties and fluorescence decay times of HpD. The dependence of these parameters on physicochemical and biological conditions has been studied in solution, cells, and a culture medium. Particular... more
This work reports on studies of spectral properties and fluorescence decay times of HpD. The dependence of these parameters on physicochemical and biological conditions has been studied in solution, cells, and a culture medium. Particular attention was devoted to the presence of a HpD emission band in the 570-590-nm range. This band does not naturally occur in fresh HpD solution and seems more likely in tumor than in normal HpD-treated cells.
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The histochemical patterns of lactate dehydrogenase, LDH, are here proposed as indicators of the local levels of oxygenation of malignant tissue. This parameter has outstanding importance in determining the tumour aggressiveness and... more
The histochemical patterns of lactate dehydrogenase, LDH, are here proposed as indicators of the local levels of oxygenation of malignant tissue. This parameter has outstanding importance in determining the tumour aggressiveness and response to treatment. The tetrazolium salt reaction previously proposed for the mapping of hypoxia has been improved by the use of polyvinyl alcohol as a tissue stabilizer. The intracellular coloured products of this reaction appear in two distinct forms, diffuse and granular, which we previously postulated to be indicative of LDH isoenzymes soluble and bound, respectively. Solubility is promoted by H-LDH subunits preferentially synthesized under good oxygenation; binding to membranes is favoured by the presence of M-LDH subunits preferentially active under poor oxygeneration. A reversible shift between the two forms apparently regulates the cells' metabolic adaptation to different stress situations. We assume that the anoxic shock protein LDHk exis...
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Abstract Immune response to cancer is a dynamic process in which uncontrolled growth of cancer cells is countered by various protective mechanisms. However, the progression of the disease can be interpreted as failure of the antitumor... more
Abstract Immune response to cancer is a dynamic process in which uncontrolled growth of cancer cells is countered by various protective mechanisms. However, the progression of the disease can be interpreted as failure of the antitumor immune response. Recent studies indicate ...
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Autofluorescence spectroscopy is a promising and powerful approach for an in vivo, real time characterization of liver functional properties. In this work, preliminary results on the dependence of liver autofluorescence parameters on the... more
Autofluorescence spectroscopy is a promising and powerful approach for an in vivo, real time characterization of liver functional properties. In this work, preliminary results on the dependence of liver autofluorescence parameters on the nutritional status are reported, with particular attention to vitamin A and lipid accumulation in liver tissue. Normally fed and 24 h starving rats were used as animal models. Histochemical and autofluorescence analysis showed that lipids and vitamin A colocalize in the liver parenchyma. Fasting condition results in a parallel increase in both lipids and vitamin A. Autofluorescence imaging and microspectrofluorometric analysis carried out on unfixed, unstained tissue sections under 366 nm excitation, evidenced differences in both spectral shape and response to continuous irradiation between liver biopsies from fed and starving rats. As to photobleaching, in particular, fitting analysis evidenced a reduction of about 85% of the signal attributable solely to vitamin A during the first 10 s of irradiation. The tissue whole emission measured in fed and starving rat livers exhibited reductions of about 35% and 52%, respectively, that are closely related to vitamin A contents. The findings open interesting perspectives for the set up of an in situ, real time diagnostic procedure for the assessment of liver lipid accumulation, exploiting the photophysical properties of vitamin A.
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Research Interests: Chemistry, Aging, Histochemistry, Medicine, Free Radical, and 15 moreHumans, Reactive Oxygen Species, Nitric oxide, Animals, Astrocyte, Glial Cell, Experimental, Skeletal Muscle, Nitric Oxide Synthase, Polyvinyl alcohol, Endothelial cell, Singlet Oxygen, Catalytic Activity, Differential Interference Contrast, and Medical and Health Sciences
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Purpose. Warm hepatic ischemia-reperfusion (I/R) injury can lead to multiorgan dysfunction. The aim of the present study was to investigate whether acute liver I/R does affect the function and/or structure of remote organs such as lung,... more
Purpose. Warm hepatic ischemia-reperfusion (I/R) injury can lead to multiorgan dysfunction. The aim of the present study was to investigate whether acute liver I/R does affect the function and/or structure of remote organs such as lung, kidney, and heart via modulation of extracellular matrix remodelling.Methods. Male Sprague-Dawley rats were subjected to 30 min partial hepatic ischemia by clamping the hepatic artery and the portal vein. After a 60 min reperfusion, liver, lung, kidney, and heart biopsies and blood samples were collected. Serum hepatic enzymes, creatinine, urea, Troponin I and TNF-alpha, and tissue matrix metalloproteinases (MMP-2, MMP-9), myeloperoxidase (MPO), malondialdehyde (MDA), and morphology were monitored.Results. Serum levels of hepatic enzymes and TNF-alpha were concomitantly increased during hepatic I/R. An increase in hepatic MMP-2 and MMP-9 activities was substantiated by tissue morphology alterations. Notably, acute hepatic I/R affect the lung inasmuch...
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In the liver of adult mice bearing an Ehrlich carcinoma on the leg, progressively hypoxic and displaying reactive hepatitis but not metastatic dissemination, extramedullary hemopoiesis was detected. Electron microscopy revealed mainly... more
In the liver of adult mice bearing an Ehrlich carcinoma on the leg, progressively hypoxic and displaying reactive hepatitis but not metastatic dissemination, extramedullary hemopoiesis was detected. Electron microscopy revealed mainly erythropoietic islands and scattered megakaryocytes in maturation stages up to the platelet-releasing phase. Erythropoietic cells expressed an embryonic-type of hemoglobin, which is more adequate to oxygenate hypoxic environments than the adult type. They were positive for the peroxidase reaction due to the presence of hemoglobin and could furthermore be visualized by the blue-excited red autofluorescence of protoporphyrin IX. Extramedullary hemopoiesis, one of the various examples of reactivation of fetal features in the liver associated with carcinogenesis, is supposed to be compensatory for the loss of blood cells induced by the tumor. Reviewing this process has the purpose of raising the question whether the fetal features are better adapted than a...
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Several studies provide evidence that hypoxic cells present in animal and human solid tumors, may be critical for the successful treatment of cancer. In particular hypoxic cells are resistant to ionizing radiation, photodynamic treatment... more
Several studies provide evidence that hypoxic cells present in animal and human solid tumors, may be critical for the successful treatment of cancer. In particular hypoxic cells are resistant to ionizing radiation, photodynamic treatment and the large majority of chemotherapeutic drugs. Hypoxia is generally due to the inadequacy of vascular beds supporting the tumor and to an abnormal microcirculation. Three parameters, tumor interstitial fluid, hemorheological factors and lipoperoxidation, are considered and tentatively associated as playing a role in hypoxic cell treatment. Omega three fatty acids modify these factors and are discussed for their possible ability to enhance tumor cells susceptibility to radiotherapy.
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Visualization of lactate dehydrogenase (LDH) activity with Neotetrazolium as final electron acceptor under anaerobic conditions and an incubation medium containing polyvinyl alcohol showed that under normal physiological conditions a... more
Visualization of lactate dehydrogenase (LDH) activity with Neotetrazolium as final electron acceptor under anaerobic conditions and an incubation medium containing polyvinyl alcohol showed that under normal physiological conditions a zonal distribution of LDH activity is present in the liver lobule of male rats. Periportal hepatocytes contain more LDH activity than pericentral hepatocytes. This difference is due to the role of LDH both in gluconeogenesis (periportal cells) and glycolysis (pericentral cells). In livers containing metastases from colon carcinoma, areas of the parenchyma which are not affected by tumour growth maintain such zonation in the lobule, whereas areas close to metastatic foci show increased activity which is distributed uniformly over the lobule. This change may be explained by a Cori's cycle-like relationship between malignant cells and the surrounding hepatocytes due to glucose consumption and lactate production by the tumour cells. Within the metastati...
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Tumor stroma induction has been shown closely to resemble wound repair process, both involving the replacement of a fibrin gel by vascularized connective tissue. In such a process the initial phase of hyperpermeability of blood vessels... more
Tumor stroma induction has been shown closely to resemble wound repair process, both involving the replacement of a fibrin gel by vascularized connective tissue. In such a process the initial phase of hyperpermeability of blood vessels leads to diffuse oedema. It is here reported that cell loosening and a remarkably high mitotic burst were observed in Ehrlich carcinoma in regions in contact with the exudate, particularly at the perinecrotic (hypoxic) region. This suggests both an enhanced cell detachment from the tumour parenchyma and an improvement of the microenvironment, the exudate thus appearing as beneficial to the malignant cells contributing to the reoxygenation of formerly hypoxic regions. The temporary and well-localized concentration of mitoses in inner tumour areas has perhaps been disregarded by the pathologists engaged in mitosis counting for tumour grading. Peripheric and intraparenchymal concentrations of mast cells, lipid pools and platelets were seen in apparently ...
Research Interests: Fluorescence Microscopy, Biology, Mast Cells, Medicine, Mitosis, and 8 moreMice, Female, Animals, Anticancer, Edema, Necrosis, Oxygen Consumption, and Vacuoles
Nature and subcellular localization of 1H-NMR-detectable mobile lipid domains (ML) were investigated by NMR, Nile red fluorescence and electron microscopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3ras) transfected with a... more
Nature and subcellular localization of 1H-NMR-detectable mobile lipid domains (ML) were investigated by NMR, Nile red fluorescence and electron microscopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3ras) transfected with a high number of oncogene copies. Substantial ML levels (ratio of (CH2)n/CH3 peak areas R=1. 56+/-0.33) were associated in untransformed fibroblasts with both (a) intramembrane amorphous lipid vesicles, about 60 nm in diameter, distinct from caveolae; and (b) cytoplasmic, osmiophilic lipid bodies surrounded by own membrane, endowed of intramembrane particles. 2D NMR maps demonstrated that ML comprised both mono- and polyunsaturated fatty chains. Lower ML signals were detected in 3T3ras (R=0.76+/-0.37), under various conditions of cell growth. Very few (if any) lipid bodies and vesicles were detected in the cytoplasmic or membrane compartments of 3T3ras cells with R<0.4, while only intramembrane lipid vesicles were associated with moderate R values. ...
The photodynamic therapy of tumors is based on a photosensitization reaction that produces oxygen-derived cytotoxic species. The availability of oxygen is therefore a necessary condition to obtain the desired effect. However, most tumors... more
The photodynamic therapy of tumors is based on a photosensitization reaction that produces oxygen-derived cytotoxic species. The availability of oxygen is therefore a necessary condition to obtain the desired effect. However, most tumors develop regions that have outgrown their vascular supply, and therefore present severe hypoxia. In many hypoxic, yet viable areas, oxygen partial pressures almost two orders of magnitude lower that in normal tissues have been measured by other authors. It is here suggested that hypoxic cells are resistant to the therapy and hence are a source of postirradiation recurrence of the tumors. Methods are reviewed and discussed that can be used to: (a) improve the tumor oxygenation status prior to, or during irradiation; (b) destroy hypoxic cells; and, (c) allow the reoxygenation of the tumor by using fractionated irradiation protocols which increase tumor photosensitivity. Hyperthermia, a therapy to which hypoxic cells are particularly sensitive, is discu...
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compagni E carried out the experimental work; Zaouali MA,
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ABSTRACT Stroma formation in Ehrlich carcinoma, studied with histochemical and TEM techniques, is similar to wound healing. In this tumour mast cells, macrophages, adipocytes, platelets and fibrin seem to co-operate locally with malignant... more
ABSTRACT Stroma formation in Ehrlich carcinoma, studied with histochemical and TEM techniques, is similar to wound healing. In this tumour mast cells, macrophages, adipocytes, platelets and fibrin seem to co-operate locally with malignant cells in regulating stroma formation. The gaps opened in the tumor parenchyma by plasma outpouring from local blood vessels seem to offer easy routes for endothelial cell migration towards ill-nourished areas, and may explain the irregular aspect of tumor microvascularity.
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Graft steatosis is a risk factor for poor initial function after liver transplantation. Biliary complications are frequent even after normal liver transplantation. A subnormothermic machine perfusion (MP20) preservation procedure was... more
Graft steatosis is a risk factor for poor initial function after liver transplantation. Biliary complications are frequent even after normal liver transplantation. A subnormothermic machine perfusion (MP20) preservation procedure was developed by our group with high potential for reducing injury to hepatocytes and sinusoidal cells of lean and fatty livers respect to conventional cold storage (CS). We report the response of the biliary tree to CS or MP20, in lean and obese Zucker rat liver. Dipeptidylpeptidase-IV (DPP-IV), crucial for the inactivation of incretins and neuropeptides, was used as a marker. Liver morphology and canalicular network of lean livers were similar after CS/reperfusion or MP20/reperfusion. CS preservation of fatty livers induced serious damage to the parenchyma and to the canalicular activity/expression of DPP-IV whereas with MP20 the morphology and canalicular network were similar to those of untreated lean liver. CS and MP20 had similar effects on DPP-IV act...
The oxygenation, the growth rate and the metastatic potential of a solid tumor depend on its vascularization and, in particular, on angiogenesis; a therapeutic approach affecting angiogenesis has been suggested as an alternative to... more
The oxygenation, the growth rate and the metastatic potential of a solid tumor depend on its vascularization and, in particular, on angiogenesis; a therapeutic approach affecting angiogenesis has been suggested as an alternative to conventional ones. Especially the study of the metabolism in the cells of the vessel wall should be a useful prerequisite for this approach. In this connection, an enzyme histochemical study was performed to characterize the blood vessels in a solid tumor (Ehrlich carcinoma). The following enzymes were considered: (a) alkaline phosphatase, involved in the transcellular phosphate transport and in the response to inflammatory and growth promoting factors; (b) dihydrofolate reductase, involved in the metabolism of tetrahydrofolate (for the synthesis of nucleic acids and the metabolism of serine and glycine); (c) purine nucleoside phosphorylase, involved in the degradation of purines and, in particular, of extracellular ATP and ADP; (d) xanthine oxidoreductas...
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Using ultrastructural analysis, we studied the effects of hyperthermic treatment of one case of human liver metastasis from colon carcinoma. The results indicate that the main hyperthermic response involves the neoplastic and the... more
Using ultrastructural analysis, we studied the effects of hyperthermic treatment of one case of human liver metastasis from colon carcinoma. The results indicate that the main hyperthermic response involves the neoplastic and the histiocytic cell population. The drastic decrease in metastatic cells was accompanied by the appearance of cell fragments and apoptotic bodies. Consequently, the histiocytic component (Kupffer cells) showed increased frequency, indicating an activated state. The data are consistent with a direct action of the heat on tumor cells with subsequent activation of Kupffer cells.
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Several studies provide evidence that hypoxic cells present in animal and human solid tumors, may be critical for the successful treatment of cancer. In particular hypoxic cells are resistant to ionizing radiation, photodynamic treatment... more
Several studies provide evidence that hypoxic cells present in animal and human solid tumors, may be critical for the successful treatment of cancer. In particular hypoxic cells are resistant to ionizing radiation, photodynamic treatment and the large majority of chemotherapeutic drugs. Hypoxia is generally due to the inadequacy of vascular beds supporting the tumor and to an abnormal microcirculation. Three parameters, tumor interstitial fluid, hemorheological factors and lipoperoxidation, are considered and tentatively associated as playing a role in hypoxic cell treatment. Omega three fatty acids modify these factors and are discussed for their possible ability to enhance tumor cells susceptibility to radiotherapy.
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The earlier finding that the chromatin organization of Purkinje neurons is not the same in the whole population is confirmed in this study. This was found to be independent of the type of Feulgen reaction hydrolysis kinetics, analytical... more
The earlier finding that the chromatin organization of Purkinje neurons is not the same in the whole population is confirmed in this study. This was found to be independent of the type of Feulgen reaction hydrolysis kinetics, analytical method (microdensitometry or microfluorometry) and tissue preparation (sections, isolated cells, isolated nuclei). This heterogeneity, possibly linked to different functional stages, leads to Feulgen-DNA contents which, at optimal hydrolysis times, range from 2c to 4c values. The results obtained are discussed in relation to those in the literature.
Research Interests: Kinetics, Fluorescence Microscopy, DNA, Animals, Male, and 5 moreChromatin, Rats, Purkinje Cells, Densitometry, and Hydrolysis
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Hypoxia is thought to be a major cause of failure in cancer treatment. In this paper, we report methods transposable to clinical practice, for identifying hypoxic tumour cells. They consist of histochemical tests for revealing lactate... more
Hypoxia is thought to be a major cause of failure in cancer treatment. In this paper, we report methods transposable to clinical practice, for identifying hypoxic tumour cells. They consist of histochemical tests for revealing lactate dehydrogenase activity, endogenous lactate and accumulation of neutral fat. An ascites tumour (Yoshida hepatoma) and a solid tumour (Ehrlich carcinoma) were used as the experimental models. A gel film technique was used for visualizing lactate dehydrogenase and "nothing dehydrogenase" (or endogenous lactate). The fluorescent dyes Nile Red and Acridine Orange were used to demonstrate lipid accumulation and to visualize the tumour morphology, respectively. Tumour cells at the edge of areas of necrosis and at a distance of about 130 microns from a blood vessel were presumed to be hypoxic and showed the following features: 1) a dark blue granular pattern of lactate dehydrogenase (LDH) activity, ascribed to intense activity of the LDH5 and/or LDHk...