Bone adaptation to mechanical loading happens predominantly via modeling and remodeling, but the ... more Bone adaptation to mechanical loading happens predominantly via modeling and remodeling, but the latter is poorly understood. Haversian remodeling (cortical bone replacement resulting in secondary osteons) is thought to occur in regions of low strain as part of bone maintenance or high strain in response to microdamage. However, analyses of remodeling in primates have revealed an unappreciated association with the number of daily load cycles. We tested this relationship by raising 30 male domestic rabbits (Oryctolagus cuniculus) on disparate diets from weaning to adulthood (48 weeks), facilitating a naturalistic perspective on mandibular bone adaptation. A control group consumed only rabbit pellets and an ‘overuse’ group ate hay in addition to pellets. To process hay, which is tougher and stiffer, rabbits increase chewing investment and duration without increasing bite force (i.e. corpus mean peak strain is similar for the two foods). Corpus remodeling in overuse rabbits was ∼1.5 ti...
Oral squamous cell carcinoma (OSCC) represents 3% of all cancer deaths in the U.S. and is ranked ... more Oral squamous cell carcinoma (OSCC) represents 3% of all cancer deaths in the U.S. and is ranked one of the top 10 cancers worldwide. The 5-year survival rate has remained at a low 50% for the past several decades, necessitating discovery of novel biomarkers of aggressive disease and therapeutic targets. As overexpression of urinary type plasminogen activator and receptor (uPA/R) in OSCC is associated with malignant progression and poor outcome, cell lines were generated with either overexpression (SCC25-uPAR+) or silencing (SCC25-uPAR-KD) of uPAR. As SCC25-uPAR+ tumors behaved more aggressively both in vitro and in vivo, comparative cDNA microarray analysis was used to identify additional genes that may be associated with aggressive tumors. Four members of the human tissue kallikrein family (KLK 5, 7, 8, and 10) were identified and real-time RT-PCR (qPCR) was used to verify and quantify gene expression. qPCR analysis revealed 2.8-, 5.3-, 4.0-, and 3.5-fold increases in gene expression for KLK5, 7, 8, and 10, respectively, in SCC25-uPAR+ versus SCC25-uPAR-KD. Immunohistochemical analysis demonstrated strong reactivity for KLKs 5, 7, 8 and 10 in both orthotopic murine tumors and human OSCC tissues. Control experiments show lack of reactivity against KLK3 (prostate specific antigen). These results demonstrate that kallikreins 5, 7, 8, and 10 are abundantly expressed in human OSCC and may be implicated in malignant progression.
Oral squamous cell carcinoma (OSCC) has 50% 5-year survival rate, highlighting our limited unders... more Oral squamous cell carcinoma (OSCC) has 50% 5-year survival rate, highlighting our limited understanding of the molecular events that contribute to disease progression. Microarray analyses of primary oral tumors have identified urinary-type plasminogen activator (uPA) and its receptor (uPAR) as key genes associated with human OSCC progression. The uPAR functions as both a proteinase receptor and an integrin ligand, modifying proteolysis, migration, integrin signaling, and cellular transcription. In the current study, uPAR expression levels were modified in OSCC cells followed by analysis of tumor growth in an in vivo orthotopic xenograft model and by transcriptional profiling. Overexpression of uPAR resulted in more infiltrative and less differentiated tumors, with ill-defined borders, cytologic atypia, and enhanced vascularity. Analysis of serial sections of both murine experimental tumors and microarrayed human OSCC showed a statistically significant association between uPAR and α3 integrin colocalization in areas exhibiting extracellular signal-regulated kinase phosphorylation, suggesting that uPAR/α3 integrin interaction potentiates extracellular signal-regulated kinase signaling in vivo. This is supported by cDNA microarray analysis, which showed differential expression of 148 genes (113 upregulated and 35 downregulated). Validation of gene expression changes in human OSCC using immunohistochemistry and quantitative real-time PCR showed increased growth factors, proteinases/inhibitors, and matrix components in uPAR-overexpressing tumors. Together, these results support a model wherein increased uPAR expression promotes α3β1 integrin association, resulting in increased mitogen-activated protein kinase signaling and transcriptional activation, leading to the formation of more aggressive tongue tumors. This combined approach has efficacy to identify additional biomarkers and/or prognostic indicators associated with aggressive human OSCC. Mol Cancer Res; 8(2); 145–58
Ovarian cancer (OvCa) is frequently accompanied by accumulation of intraperitoneal ascites fluid ... more Ovarian cancer (OvCa) is frequently accompanied by accumulation of intraperitoneal ascites fluid early in disease progression. This fluid is rich in soluble and cellular components including tumor cells and multicellular aggregates (MCAs) of 150-300 um diameter shed from the primary tumor. In addition to chemical cues, ascites fluid buildup can also alter the force environment in the peritoneal cavity, thereby impacting the primary tumor, disseminating cells and MCAs, and host peritoneal tissues. Whereas the intraperitoneal pressure (IPP) of the normal peritoneal cavity is subatmospheric (-5 mmHg), the IPP measured in ovarian cancer patients with tense ascites is reported to be 24 mmHg. The potential effect of ascites-induced changes in peritoneal mechanobiology on tumor cells and host structures has not been investigated due to a lack of appropriate model systems. As a first approximation, we have begun preliminary investigations into the response of tumor and host structures to compressive and strain (stretching) forces. Our initial experiments used MCAs sealed in nonadherent cell culture bags placed into a temperature-controlled stainless-steel pressure vessel and subjected to a compressive force of 22-24 mmHg using an Instron system. While this approach is feasible for short-term experiments, longer-term compression experiments require a system with gas exchange to maintain cell viability. Thus, we fabricated a mold designed to fit within a Flexcell-400C Compression system Biopress+ Bioflex 6-well plate. This mold was used to produce porous hydrogels containing defined void areas so as to encapsulate MCAs within the hydrogel carrier and thereby ensure a more uniform encounter with the Flexcell compression plate. Our initial experiments investigated the effects of MCA compression on gene expression associated with epithelial-to-mesenchymal transition (EMT). Data indicate that short-term static compression (6h) downregulates CDH2 (N-cadherin, Ncad) with cell line-dependent inhibition of EMT regulators including SNAI1, SNAI2, and TWIST. In contrast, long-term compression (24h) upregulated expression of mesenchymal genes including CDH2, MMP14, Wnt5a, ROR1, and ROR2. To examine the impact of strain on receptivity of host peritoneal tissues to metastatic implantation, we used control or strained ex vivo explants of murine peritoneal tissue immobilized on silastic resin. Strained peritoneal tissue exhibited a 3-fold increase in stiffness as determined by atomic force microscopy. Concomitantly, adhesion of ovarian cancer cells to strained peritoneum increased by 4.5-fold. Together these data provide support for a more detailed investigation of the complex role of peritoneal mechanobiology as an important microenvironmental regulator of ovarian cancer metastatic success. Citation Format: Yuliya Klymenko, Rebecca Wates, Yueying LIu, Rachel Lombard, Holly Weiss-Bilka, Leigh Campbell, Diane Wagner, Matthew J. Ravosa, M. Sharon Stack. Modeling ascites-induced changes in peritoneal mechanobiology and ovarian cancer metastatic success. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A38.
Over the past. 30 years a series of morphological and experimental analyses have attempted to add... more Over the past. 30 years a series of morphological and experimental analyses have attempted to address questions about the functional and evolutionary significance of mandibular symphyseal fusion or complete ossification of the joint between the two dentaries (Scapino, 1965, 1981; Hylander, 1975a, 1977, 1979a,b, 1984, 1985; Beecher, 1977, 1979, 1983; Hirschfeld et al., 1977; Dessein, 1985; Hylander et al., 1987; Greaves, 1988, 1993; Ravosa, 1991; M. J. Ravosa, unpublished data; Ravosa and Hylander, 1993; Hylander and Johnson, 1994; Ravosa and Simons, 1994). This work has increased our understanding of the functional morphology of the mammalian masticatory apparatus, in part by highlighting the interaction of jaw mechanics, diet and allometry on symphyseal form. Many of these studies have also influenced adaptive explanations for the evolution of anthropoid craniodental morphology and impact directly on hypotheses regarding phylogenetic affinities among certain Eocene and Oligocene primates (Hiiemae and Kay, 1972, 1973; Gingerich, 1977, 1979; Beecher, 1977, 1979; Cachel, 1979a,b; Hylander, 1979a,b; Szalay and Delson, 1979; Rosenberger, 1981, 1986; Rosenberger et al., 1985; Rasmussen, 1986, 1990; Greaves, 1988, 1993; Simons, 1989, 1990, 1992; Ravosa, 1991; M. J. Ravosa, unpublished data; Rasmussen and Simons, 1992; Ravosa and Hylander, 1993; Ravosa and Simons, 1994).
Supplementary Data from Urinary-Type Plasminogen Activator Receptor/α3β1 Integrin Signaling, Alte... more Supplementary Data from Urinary-Type Plasminogen Activator Receptor/α3β1 Integrin Signaling, Altered Gene Expression, and Oral Tumor Progression
Skeletal functional morphology in primates underlies many fossil interpretations. Understanding t... more Skeletal functional morphology in primates underlies many fossil interpretations. Understanding the functional correlates of arboreal grasping is central to identifying locomotor signatures in extinct primates. We tested 3 predictions linking substrate orientation and digital grasping pressures: (1) below-branch pressures are greater than above-branch and vertical-branch pressures; (2) there is no difference in pressure exerted across digits within autopods at any substrate orientation, and (3) there is no difference in pressure exerted between homologous digits across autopods at any substrate orientation. Adult males and females from 3 strepsirrhine species crossed an artificial arboreal substrate oriented for above-, below- and vertical-branch locomotion. We compared digital pressures within and across behaviors via ANOVA and Tukey's Honest Significant Difference test. Results show limited support for all predictions: below-branch pressures exceeded vertical-branch pressures and above-branch pressures for some digits and species (prediction 1), lateral digits often exerted greater pressures than medial digits (prediction 2), and pedal digits occasionally exerted greater pressures than manual digits during above-branch and vertical orientations but less often for below-branch locomotion (prediction 3). We observed functional variability across autopods, substrate and species that could underlie morphological variation within and across primates. Future work should consider the complexity of arboreality when inferring locomotor modes in fossils.
Bone adaptation to mechanical loading happens predominantly via modeling and remodeling, but the ... more Bone adaptation to mechanical loading happens predominantly via modeling and remodeling, but the latter is poorly understood. Haversian remodeling (cortical bone replacement resulting in secondary osteons) is thought to occur in regions of low strain as part of bone maintenance or high strain in response to microdamage. However, analyses of remodeling in primates have revealed an unappreciated association with the number of daily load cycles. We tested this relationship by raising 30 male domestic rabbits (Oryctolagus cuniculus) on disparate diets from weaning to adulthood (48 weeks), facilitating a naturalistic perspective on mandibular bone adaptation. A control group consumed only rabbit pellets and an ‘overuse’ group ate hay in addition to pellets. To process hay, which is tougher and stiffer, rabbits increase chewing investment and duration without increasing bite force (i.e. corpus mean peak strain is similar for the two foods). Corpus remodeling in overuse rabbits was ∼1.5 ti...
Oral squamous cell carcinoma (OSCC) represents 3% of all cancer deaths in the U.S. and is ranked ... more Oral squamous cell carcinoma (OSCC) represents 3% of all cancer deaths in the U.S. and is ranked one of the top 10 cancers worldwide. The 5-year survival rate has remained at a low 50% for the past several decades, necessitating discovery of novel biomarkers of aggressive disease and therapeutic targets. As overexpression of urinary type plasminogen activator and receptor (uPA/R) in OSCC is associated with malignant progression and poor outcome, cell lines were generated with either overexpression (SCC25-uPAR+) or silencing (SCC25-uPAR-KD) of uPAR. As SCC25-uPAR+ tumors behaved more aggressively both in vitro and in vivo, comparative cDNA microarray analysis was used to identify additional genes that may be associated with aggressive tumors. Four members of the human tissue kallikrein family (KLK 5, 7, 8, and 10) were identified and real-time RT-PCR (qPCR) was used to verify and quantify gene expression. qPCR analysis revealed 2.8-, 5.3-, 4.0-, and 3.5-fold increases in gene expression for KLK5, 7, 8, and 10, respectively, in SCC25-uPAR+ versus SCC25-uPAR-KD. Immunohistochemical analysis demonstrated strong reactivity for KLKs 5, 7, 8 and 10 in both orthotopic murine tumors and human OSCC tissues. Control experiments show lack of reactivity against KLK3 (prostate specific antigen). These results demonstrate that kallikreins 5, 7, 8, and 10 are abundantly expressed in human OSCC and may be implicated in malignant progression.
Oral squamous cell carcinoma (OSCC) has 50% 5-year survival rate, highlighting our limited unders... more Oral squamous cell carcinoma (OSCC) has 50% 5-year survival rate, highlighting our limited understanding of the molecular events that contribute to disease progression. Microarray analyses of primary oral tumors have identified urinary-type plasminogen activator (uPA) and its receptor (uPAR) as key genes associated with human OSCC progression. The uPAR functions as both a proteinase receptor and an integrin ligand, modifying proteolysis, migration, integrin signaling, and cellular transcription. In the current study, uPAR expression levels were modified in OSCC cells followed by analysis of tumor growth in an in vivo orthotopic xenograft model and by transcriptional profiling. Overexpression of uPAR resulted in more infiltrative and less differentiated tumors, with ill-defined borders, cytologic atypia, and enhanced vascularity. Analysis of serial sections of both murine experimental tumors and microarrayed human OSCC showed a statistically significant association between uPAR and α3 integrin colocalization in areas exhibiting extracellular signal-regulated kinase phosphorylation, suggesting that uPAR/α3 integrin interaction potentiates extracellular signal-regulated kinase signaling in vivo. This is supported by cDNA microarray analysis, which showed differential expression of 148 genes (113 upregulated and 35 downregulated). Validation of gene expression changes in human OSCC using immunohistochemistry and quantitative real-time PCR showed increased growth factors, proteinases/inhibitors, and matrix components in uPAR-overexpressing tumors. Together, these results support a model wherein increased uPAR expression promotes α3β1 integrin association, resulting in increased mitogen-activated protein kinase signaling and transcriptional activation, leading to the formation of more aggressive tongue tumors. This combined approach has efficacy to identify additional biomarkers and/or prognostic indicators associated with aggressive human OSCC. Mol Cancer Res; 8(2); 145–58
Ovarian cancer (OvCa) is frequently accompanied by accumulation of intraperitoneal ascites fluid ... more Ovarian cancer (OvCa) is frequently accompanied by accumulation of intraperitoneal ascites fluid early in disease progression. This fluid is rich in soluble and cellular components including tumor cells and multicellular aggregates (MCAs) of 150-300 um diameter shed from the primary tumor. In addition to chemical cues, ascites fluid buildup can also alter the force environment in the peritoneal cavity, thereby impacting the primary tumor, disseminating cells and MCAs, and host peritoneal tissues. Whereas the intraperitoneal pressure (IPP) of the normal peritoneal cavity is subatmospheric (-5 mmHg), the IPP measured in ovarian cancer patients with tense ascites is reported to be 24 mmHg. The potential effect of ascites-induced changes in peritoneal mechanobiology on tumor cells and host structures has not been investigated due to a lack of appropriate model systems. As a first approximation, we have begun preliminary investigations into the response of tumor and host structures to compressive and strain (stretching) forces. Our initial experiments used MCAs sealed in nonadherent cell culture bags placed into a temperature-controlled stainless-steel pressure vessel and subjected to a compressive force of 22-24 mmHg using an Instron system. While this approach is feasible for short-term experiments, longer-term compression experiments require a system with gas exchange to maintain cell viability. Thus, we fabricated a mold designed to fit within a Flexcell-400C Compression system Biopress+ Bioflex 6-well plate. This mold was used to produce porous hydrogels containing defined void areas so as to encapsulate MCAs within the hydrogel carrier and thereby ensure a more uniform encounter with the Flexcell compression plate. Our initial experiments investigated the effects of MCA compression on gene expression associated with epithelial-to-mesenchymal transition (EMT). Data indicate that short-term static compression (6h) downregulates CDH2 (N-cadherin, Ncad) with cell line-dependent inhibition of EMT regulators including SNAI1, SNAI2, and TWIST. In contrast, long-term compression (24h) upregulated expression of mesenchymal genes including CDH2, MMP14, Wnt5a, ROR1, and ROR2. To examine the impact of strain on receptivity of host peritoneal tissues to metastatic implantation, we used control or strained ex vivo explants of murine peritoneal tissue immobilized on silastic resin. Strained peritoneal tissue exhibited a 3-fold increase in stiffness as determined by atomic force microscopy. Concomitantly, adhesion of ovarian cancer cells to strained peritoneum increased by 4.5-fold. Together these data provide support for a more detailed investigation of the complex role of peritoneal mechanobiology as an important microenvironmental regulator of ovarian cancer metastatic success. Citation Format: Yuliya Klymenko, Rebecca Wates, Yueying LIu, Rachel Lombard, Holly Weiss-Bilka, Leigh Campbell, Diane Wagner, Matthew J. Ravosa, M. Sharon Stack. Modeling ascites-induced changes in peritoneal mechanobiology and ovarian cancer metastatic success. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A38.
Over the past. 30 years a series of morphological and experimental analyses have attempted to add... more Over the past. 30 years a series of morphological and experimental analyses have attempted to address questions about the functional and evolutionary significance of mandibular symphyseal fusion or complete ossification of the joint between the two dentaries (Scapino, 1965, 1981; Hylander, 1975a, 1977, 1979a,b, 1984, 1985; Beecher, 1977, 1979, 1983; Hirschfeld et al., 1977; Dessein, 1985; Hylander et al., 1987; Greaves, 1988, 1993; Ravosa, 1991; M. J. Ravosa, unpublished data; Ravosa and Hylander, 1993; Hylander and Johnson, 1994; Ravosa and Simons, 1994). This work has increased our understanding of the functional morphology of the mammalian masticatory apparatus, in part by highlighting the interaction of jaw mechanics, diet and allometry on symphyseal form. Many of these studies have also influenced adaptive explanations for the evolution of anthropoid craniodental morphology and impact directly on hypotheses regarding phylogenetic affinities among certain Eocene and Oligocene primates (Hiiemae and Kay, 1972, 1973; Gingerich, 1977, 1979; Beecher, 1977, 1979; Cachel, 1979a,b; Hylander, 1979a,b; Szalay and Delson, 1979; Rosenberger, 1981, 1986; Rosenberger et al., 1985; Rasmussen, 1986, 1990; Greaves, 1988, 1993; Simons, 1989, 1990, 1992; Ravosa, 1991; M. J. Ravosa, unpublished data; Rasmussen and Simons, 1992; Ravosa and Hylander, 1993; Ravosa and Simons, 1994).
Supplementary Data from Urinary-Type Plasminogen Activator Receptor/α3β1 Integrin Signaling, Alte... more Supplementary Data from Urinary-Type Plasminogen Activator Receptor/α3β1 Integrin Signaling, Altered Gene Expression, and Oral Tumor Progression
Skeletal functional morphology in primates underlies many fossil interpretations. Understanding t... more Skeletal functional morphology in primates underlies many fossil interpretations. Understanding the functional correlates of arboreal grasping is central to identifying locomotor signatures in extinct primates. We tested 3 predictions linking substrate orientation and digital grasping pressures: (1) below-branch pressures are greater than above-branch and vertical-branch pressures; (2) there is no difference in pressure exerted across digits within autopods at any substrate orientation, and (3) there is no difference in pressure exerted between homologous digits across autopods at any substrate orientation. Adult males and females from 3 strepsirrhine species crossed an artificial arboreal substrate oriented for above-, below- and vertical-branch locomotion. We compared digital pressures within and across behaviors via ANOVA and Tukey's Honest Significant Difference test. Results show limited support for all predictions: below-branch pressures exceeded vertical-branch pressures and above-branch pressures for some digits and species (prediction 1), lateral digits often exerted greater pressures than medial digits (prediction 2), and pedal digits occasionally exerted greater pressures than manual digits during above-branch and vertical orientations but less often for below-branch locomotion (prediction 3). We observed functional variability across autopods, substrate and species that could underlie morphological variation within and across primates. Future work should consider the complexity of arboreality when inferring locomotor modes in fossils.
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