Abstract:In a multi-level ''deconstruction'' of omental metastases, we previously identified a prognostic matrisome gene expression signature in high-grade serous ovarian cancer (HGSOC) and twelve other malignancies. Here, our aim was to understand how six of these extracellular matrix (ECM) molecules, COL11A1, cartilage oligomeric matrix protein, FN1, versican, cathepsin B, and COL1A1, are upregulated in cancer. Using biopsies, we identified significant associations between TGFbR activity, Hedgehog (Hh) signaling, and… Show more
“…We observed a dense and thick collagen fiber network enriched within extra-tumoral spaces. These results were consistent with previous studies demonstrating collagen fiber localization beneath OC outgrowths originating from the fallopian tube 23 and stroma surrounding metastatic omental implants 44 . Collagen fiber formation occurs when collagen concentration increases 45 , indicating that extra-tumoral fibrils reflect the presence of highly elastic ECM.…”
Ovarian carcinoma (OC) form outgrowths that extend from the outer surface of an afflicted organ into the peritoneum. OC outgrowth formation is poorly understood because there is a limited availability of OC cell culture models to examine the behavior of cell assemblies that form outgrowths. Prompted by immuno-chemical evaluation of extracellular matrix (ECM) components, laminin gamma 1 and collagens, in human tissues representing untreated and chemotherapy-recovered OC, we developed laminin and collagen-rich ECM-reconstituted cell culture models amenable to studies of cell assemblies that can form outgrowths. We demonstrate that ECM promotes outgrowth formation in fallopian tube non-ciliated epithelial cells (FNE) expressing mutant p53R175H and various OC cell lines. Outgrowths were initiated by cell assemblies that had undergone outward translocation and, upon mechanical detachment, could intercalate into mesothelial cell monolayers. Electron microcopy, optical coherence tomography (OCT) and small-amplitude oscillatory shear experiments revealed that elevating ECM levels increased ECM fibrous network thickness and led to high shear elasticity of ECM environment. These physical characteristics were associated with suppression of outgrowths. Culture environment with low ECM content mimicked viscoelasticity and fibrous networks of ascites and supported cell proliferation, cell translocation and outgrowth formation. These results highlight the importance of ECM microenvironments in modulating OC growth and could provide additional explanation of why primary and recurrent ovarian tumors form outgrowths that protrude into the peritoneal cavity containing ascites as opposed to breaking through the basement membrane, invading collagen-dense tissues an intravasating into vasculature.
“…We observed a dense and thick collagen fiber network enriched within extra-tumoral spaces. These results were consistent with previous studies demonstrating collagen fiber localization beneath OC outgrowths originating from the fallopian tube 23 and stroma surrounding metastatic omental implants 44 . Collagen fiber formation occurs when collagen concentration increases 45 , indicating that extra-tumoral fibrils reflect the presence of highly elastic ECM.…”
Ovarian carcinoma (OC) form outgrowths that extend from the outer surface of an afflicted organ into the peritoneum. OC outgrowth formation is poorly understood because there is a limited availability of OC cell culture models to examine the behavior of cell assemblies that form outgrowths. Prompted by immuno-chemical evaluation of extracellular matrix (ECM) components, laminin gamma 1 and collagens, in human tissues representing untreated and chemotherapy-recovered OC, we developed laminin and collagen-rich ECM-reconstituted cell culture models amenable to studies of cell assemblies that can form outgrowths. We demonstrate that ECM promotes outgrowth formation in fallopian tube non-ciliated epithelial cells (FNE) expressing mutant p53R175H and various OC cell lines. Outgrowths were initiated by cell assemblies that had undergone outward translocation and, upon mechanical detachment, could intercalate into mesothelial cell monolayers. Electron microcopy, optical coherence tomography (OCT) and small-amplitude oscillatory shear experiments revealed that elevating ECM levels increased ECM fibrous network thickness and led to high shear elasticity of ECM environment. These physical characteristics were associated with suppression of outgrowths. Culture environment with low ECM content mimicked viscoelasticity and fibrous networks of ascites and supported cell proliferation, cell translocation and outgrowth formation. These results highlight the importance of ECM microenvironments in modulating OC growth and could provide additional explanation of why primary and recurrent ovarian tumors form outgrowths that protrude into the peritoneal cavity containing ascites as opposed to breaking through the basement membrane, invading collagen-dense tissues an intravasating into vasculature.
“…Figure 3 A shows a collagen gel containing fibroblasts and HGSOC cells as detailed in the tri-culture set up protocol 4a. Figure 3 B is an example of a complete tri-culture and relative H&E ( Figures 3 C, ( Delaine-Smith et al, 2021 )). Figure 4 represents an adipocyte gel (A) and a tetra-culture 24h with relative H&E (B-C) after the protocol completion.…”
Section: Expected Outcomesmentioning
confidence: 99%
“…Scale bar: 8 mm. (C) Tri-culture H&E, scale bar: 500 μm (image adapted from Delaine-Smith et al, 2021 ). Figure 4 Tetra-culture setup (A) Example of an adipocyte gel anchored to a 96-well plate before the addition of fibroblasts and mesothelial cells.…”
Section: Expected Outcomesmentioning
confidence: 99%
“…This protocol is optimized for building 3D models of high-grade serous ovarian cancer omental metastasis but can be adapted for modeling other cancers. For complete details on the use and execution of this profile, please refer to Delaine-Smith et al (2021) and Malacrida et al (2021) .…”
mentioning
confidence: 99%
“…For complete details on the use and execution of this profile, please refer to Delaine-Smith et al (2021) and Malacrida et al (2021) .…”
Summary
Three-dimensional (3D), multicellular
in
vitro
models provide a useful platform for studying human cancer biology, particularly through deconvolution of the tumor microenvironment, or where animal models do not recapitulate the human condition. Here, we detail a protocol for building human multicellular models made of patient-derived primary cells and malignant cell lines, which recapitulate features of the tumor microenvironment. This protocol is optimized for building 3D models of high-grade serous ovarian cancer omental metastasis but can be adapted for modeling other cancers.
For complete details on the use and execution of this profile, please refer to
Delaine-Smith et al. (2021)
and
Malacrida et al. (2021)
.
Ovarian carcinoma (OC) form outgrowths that extend from the outer surface of an afflicted organ into the peritoneum. OC outgrowth formation is poorly understood because there is a limited availability of OC cell culture models to examine the behavior of cell assemblies that form outgrowths. Prompted by immuno-chemical evaluation of extracellular matrix (ECM) components, laminin γ1 and collagens, in human tissues representing untreated and chemotherapyrecovered OC, we developed laminin and collagen-rich ECM-reconstituted cell culture models amenable to studies of cell assemblies that can form outgrowths. We demonstrate that ECM promotes outgrowth formation in fallopian tube non-ciliated epithelial cells (FNE) expressing mutant p53 R175H and various OC cell lines. Outgrowths were initiated by cell assemblies that had
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