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A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis.

Reticker-Flynn NE, Malta DF, Winslow MM, Lamar JM, Xu MJ, Underhill GH, Hynes RO, Jacks TE, Bhatia SN - Nat Commun (2012)

Bottom Line: Furthermore, we uncovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3, galectin-8 or laminin.We show that these molecules correlate with human disease and that their interactions are mediated in part by α3β1 integrin.Thus, our platform allowed us to interrogate interactions between metastatic cells and their microenvironments, and identified extracellular matrix and integrin interactions that could serve as therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

ABSTRACT
Extracellular matrix interactions have essential roles in normal physiology and many pathological processes. Although the importance of extracellular matrix interactions in metastasis is well documented, systematic approaches to identify their roles in distinct stages of tumorigenesis have not been described. Here we report a novel-screening platform capable of measuring phenotypic responses to combinations of extracellular matrix molecules. Using a genetic mouse model of lung adenocarcinoma, we measure the extracellular matrix-dependent adhesion of tumour-derived cells. Hierarchical clustering of the adhesion profiles differentiates metastatic cell lines from primary tumour lines. Furthermore, we uncovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3, galectin-8 or laminin. We show that these molecules correlate with human disease and that their interactions are mediated in part by α3β1 integrin. Thus, our platform allowed us to interrogate interactions between metastatic cells and their microenvironments, and identified extracellular matrix and integrin interactions that could serve as therapeutic targets.

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Metastasis-associated ECM molecules are present in the sites of metastases but not primary tumorsImmunostaining of the metastasis-associated ECM molecules in the lungs, lymph nodes, and distant metastases of mice bearing endogenous lung adenocarcinomas (KrasLSL-G12D/+;p53flox/flox mice). Insets are magnified views of boxed areas showing ECM molecule fibrils. Number of tissues examined for each organ: Lungs: 10; lymph nodes: 5; livers/kidneys: 22. ‘T’: tumor. Dotted line depicts edge of tumor and normal kidney. Scale bars are 50µm.
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Figure 4: Metastasis-associated ECM molecules are present in the sites of metastases but not primary tumorsImmunostaining of the metastasis-associated ECM molecules in the lungs, lymph nodes, and distant metastases of mice bearing endogenous lung adenocarcinomas (KrasLSL-G12D/+;p53flox/flox mice). Insets are magnified views of boxed areas showing ECM molecule fibrils. Number of tissues examined for each organ: Lungs: 10; lymph nodes: 5; livers/kidneys: 22. ‘T’: tumor. Dotted line depicts edge of tumor and normal kidney. Scale bars are 50µm.

Mentions: Next we sought to correlate our in vitro adhesion profiles with ECM expression in vivo. To investigate whether the identified ECM molecules may be important in natural tumorigenesis, organs containing primary autochthonous tumors and their metastases were resected from KrasLSL-G12D/+;p53flox/flox mice and stained. Trichrome staining of lungs with extensive tumor burden revealed a significant presence of ECM deposition in the tumor-bearing lung (Supplementary Fig. S5a and ref 31). Previously, we found that primary tumors that have acquired the ability to metastasize (TMet tumors) upregulate the chomatin-associated protein Hmga229. Therefore, we used Hmga2 immunohistochemistry in addition to histological characteristics to identify areas of highly aggressive cancer cells (Supplementary Fig. S5a). As anticipated, primary lung tumors were positive for collagen I (black arrowheads), collagen VI (open black arrowheads), and osteopontin (red arrowheads), with the most intense staining overlapping with the high-grade tumor areas (Supplementary Fig. S5a and S5c). In particular, osteopontin staining strongly co-localized with Hmga2pos regions, suggesting that increased osteopontin production is associated with metastatic primary lung tumors. Furthermore, little to no laminin, galectin-3, or galectin-8 staining was detected in the primary tumors (Fig. 4). Interestingly, fibronectin staining in the tumor was strong, revealing a correlation between increasingly metastatic populations and the presence of fibronectin early in the metastatic cascade (Fig. 4).


A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis.

Reticker-Flynn NE, Malta DF, Winslow MM, Lamar JM, Xu MJ, Underhill GH, Hynes RO, Jacks TE, Bhatia SN - Nat Commun (2012)

Metastasis-associated ECM molecules are present in the sites of metastases but not primary tumorsImmunostaining of the metastasis-associated ECM molecules in the lungs, lymph nodes, and distant metastases of mice bearing endogenous lung adenocarcinomas (KrasLSL-G12D/+;p53flox/flox mice). Insets are magnified views of boxed areas showing ECM molecule fibrils. Number of tissues examined for each organ: Lungs: 10; lymph nodes: 5; livers/kidneys: 22. ‘T’: tumor. Dotted line depicts edge of tumor and normal kidney. Scale bars are 50µm.
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Related In: Results  -  Collection

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Figure 4: Metastasis-associated ECM molecules are present in the sites of metastases but not primary tumorsImmunostaining of the metastasis-associated ECM molecules in the lungs, lymph nodes, and distant metastases of mice bearing endogenous lung adenocarcinomas (KrasLSL-G12D/+;p53flox/flox mice). Insets are magnified views of boxed areas showing ECM molecule fibrils. Number of tissues examined for each organ: Lungs: 10; lymph nodes: 5; livers/kidneys: 22. ‘T’: tumor. Dotted line depicts edge of tumor and normal kidney. Scale bars are 50µm.
Mentions: Next we sought to correlate our in vitro adhesion profiles with ECM expression in vivo. To investigate whether the identified ECM molecules may be important in natural tumorigenesis, organs containing primary autochthonous tumors and their metastases were resected from KrasLSL-G12D/+;p53flox/flox mice and stained. Trichrome staining of lungs with extensive tumor burden revealed a significant presence of ECM deposition in the tumor-bearing lung (Supplementary Fig. S5a and ref 31). Previously, we found that primary tumors that have acquired the ability to metastasize (TMet tumors) upregulate the chomatin-associated protein Hmga229. Therefore, we used Hmga2 immunohistochemistry in addition to histological characteristics to identify areas of highly aggressive cancer cells (Supplementary Fig. S5a). As anticipated, primary lung tumors were positive for collagen I (black arrowheads), collagen VI (open black arrowheads), and osteopontin (red arrowheads), with the most intense staining overlapping with the high-grade tumor areas (Supplementary Fig. S5a and S5c). In particular, osteopontin staining strongly co-localized with Hmga2pos regions, suggesting that increased osteopontin production is associated with metastatic primary lung tumors. Furthermore, little to no laminin, galectin-3, or galectin-8 staining was detected in the primary tumors (Fig. 4). Interestingly, fibronectin staining in the tumor was strong, revealing a correlation between increasingly metastatic populations and the presence of fibronectin early in the metastatic cascade (Fig. 4).

Bottom Line: Furthermore, we uncovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3, galectin-8 or laminin.We show that these molecules correlate with human disease and that their interactions are mediated in part by α3β1 integrin.Thus, our platform allowed us to interrogate interactions between metastatic cells and their microenvironments, and identified extracellular matrix and integrin interactions that could serve as therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

ABSTRACT
Extracellular matrix interactions have essential roles in normal physiology and many pathological processes. Although the importance of extracellular matrix interactions in metastasis is well documented, systematic approaches to identify their roles in distinct stages of tumorigenesis have not been described. Here we report a novel-screening platform capable of measuring phenotypic responses to combinations of extracellular matrix molecules. Using a genetic mouse model of lung adenocarcinoma, we measure the extracellular matrix-dependent adhesion of tumour-derived cells. Hierarchical clustering of the adhesion profiles differentiates metastatic cell lines from primary tumour lines. Furthermore, we uncovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3, galectin-8 or laminin. We show that these molecules correlate with human disease and that their interactions are mediated in part by α3β1 integrin. Thus, our platform allowed us to interrogate interactions between metastatic cells and their microenvironments, and identified extracellular matrix and integrin interactions that could serve as therapeutic targets.

Show MeSH
Related in: MedlinePlus