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Distinct routes to metastasis: plasticity-dependent and plasticity-independent pathways

View Article: PubMed Central - PubMed

ABSTRACT

The cascade that culminates in macrometastases is thought to be mediated by phenotypic plasticity, including epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT and MET). While there is substantial support for the role of EMT in driving cancer cell invasion and dissemination, much less is known about the importance of MET in the later steps of metastatic colonization. We created novel reporters, which integrate transcriptional and post-transcriptional regulation, to test whether MET is required for metastasis in multiple in vivo cancer models. In a model of carcinosarcoma, metastasis occurred via an MET-dependent pathway; however, in two prostate carcinoma models, metastatic colonization was MET-independent. Indeed, the MET-independent pathway was supported by reanalysis of pre-clinical and clinical data. These results provide evidence for both MET-dependent and MET-independent metastatic pathways.

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CS-99 metastasize via MET-dependent pathwaysA. Rint+/Gint− CS-99 cells were mixed 1:1 with CS-99 clones harboring E-cadDipIIIcI2/Gint and injected subcutaneously. CS-99 Rint+/Gint− and E-cadDipIIIcI2/Gint+ cells formed primary tumors with 100% penetrance. B. Flow cytometric analysis of the proportion of DsRed+ and GFP+ cells within primary tumors indicates that all primary tumors contain both DsRed+ control cells and E-cadDipIIIcI2/Gint+ cells. C. Representative images and D. quantification of CS-99 Rint+/Gint− control cells and E-cadDipIIIcI2/Gint+ suicide reporter cells grown in soft agar. E. Rint+/Gint− CS-99 cells were mixed 1:1 with CS-99 clones harboring E-cadDipIIIcI2/Gint and injected via the tail vein. With the exception of a single GFP+ colony, all metastases were DsRed+ and lacked EGFP expression. F. Quantification of CS-99 EGFP+ and DsRed+ lung metastases. Each uniquely-colored symbol represents the number of metastases from a single animal. The median number of Rint+/Gint− and E-cadDipIIIcI2/Gint+ metastases is indicated by a solid black bar.
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Figure 6: CS-99 metastasize via MET-dependent pathwaysA. Rint+/Gint− CS-99 cells were mixed 1:1 with CS-99 clones harboring E-cadDipIIIcI2/Gint and injected subcutaneously. CS-99 Rint+/Gint− and E-cadDipIIIcI2/Gint+ cells formed primary tumors with 100% penetrance. B. Flow cytometric analysis of the proportion of DsRed+ and GFP+ cells within primary tumors indicates that all primary tumors contain both DsRed+ control cells and E-cadDipIIIcI2/Gint+ cells. C. Representative images and D. quantification of CS-99 Rint+/Gint− control cells and E-cadDipIIIcI2/Gint+ suicide reporter cells grown in soft agar. E. Rint+/Gint− CS-99 cells were mixed 1:1 with CS-99 clones harboring E-cadDipIIIcI2/Gint and injected via the tail vein. With the exception of a single GFP+ colony, all metastases were DsRed+ and lacked EGFP expression. F. Quantification of CS-99 EGFP+ and DsRed+ lung metastases. Each uniquely-colored symbol represents the number of metastases from a single animal. The median number of Rint+/Gint− and E-cadDipIIIcI2/Gint+ metastases is indicated by a solid black bar.

Mentions: Using these clonal lines, we asked whether harboring the E-cadDipIIIc2 would affect CS-99 growth rates. In vitro WST-1 cell proliferation assays indicated that the E-cadDipIIIcI2/Gint clones and Rint+/Gint− cells grew at equal rates (S8A). To test whether or not MET was required for growth in vivo, we co-injected a 1:1 ratio of Rint+/Gint− control cells and E-cadDipIIIcI2/Gint+ cells s.c. in the flanks of nude mice. Palpable tumors formed within five to seven days. Analysis of the proportions of control cells and cells with the suicide reporter revealed that 100% of primary tumors contained substantial proportions of both cell types (Figure 6A). We observed only a very slight decrease in the proportion of E-cadDipIIIcI2/Gint cells relative to control Rint+/Gint− cells (Figure 6B). Indeed, cells harboring the suicide reporter cells formed colonies in soft agar only slightly less efficiently than control cells (Figure 6C and D). We concluded from this that MET was not required for growth and primary tumor formation in vitro or in vivo.


Distinct routes to metastasis: plasticity-dependent and plasticity-independent pathways
CS-99 metastasize via MET-dependent pathwaysA. Rint+/Gint− CS-99 cells were mixed 1:1 with CS-99 clones harboring E-cadDipIIIcI2/Gint and injected subcutaneously. CS-99 Rint+/Gint− and E-cadDipIIIcI2/Gint+ cells formed primary tumors with 100% penetrance. B. Flow cytometric analysis of the proportion of DsRed+ and GFP+ cells within primary tumors indicates that all primary tumors contain both DsRed+ control cells and E-cadDipIIIcI2/Gint+ cells. C. Representative images and D. quantification of CS-99 Rint+/Gint− control cells and E-cadDipIIIcI2/Gint+ suicide reporter cells grown in soft agar. E. Rint+/Gint− CS-99 cells were mixed 1:1 with CS-99 clones harboring E-cadDipIIIcI2/Gint and injected via the tail vein. With the exception of a single GFP+ colony, all metastases were DsRed+ and lacked EGFP expression. F. Quantification of CS-99 EGFP+ and DsRed+ lung metastases. Each uniquely-colored symbol represents the number of metastases from a single animal. The median number of Rint+/Gint− and E-cadDipIIIcI2/Gint+ metastases is indicated by a solid black bar.
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Figure 6: CS-99 metastasize via MET-dependent pathwaysA. Rint+/Gint− CS-99 cells were mixed 1:1 with CS-99 clones harboring E-cadDipIIIcI2/Gint and injected subcutaneously. CS-99 Rint+/Gint− and E-cadDipIIIcI2/Gint+ cells formed primary tumors with 100% penetrance. B. Flow cytometric analysis of the proportion of DsRed+ and GFP+ cells within primary tumors indicates that all primary tumors contain both DsRed+ control cells and E-cadDipIIIcI2/Gint+ cells. C. Representative images and D. quantification of CS-99 Rint+/Gint− control cells and E-cadDipIIIcI2/Gint+ suicide reporter cells grown in soft agar. E. Rint+/Gint− CS-99 cells were mixed 1:1 with CS-99 clones harboring E-cadDipIIIcI2/Gint and injected via the tail vein. With the exception of a single GFP+ colony, all metastases were DsRed+ and lacked EGFP expression. F. Quantification of CS-99 EGFP+ and DsRed+ lung metastases. Each uniquely-colored symbol represents the number of metastases from a single animal. The median number of Rint+/Gint− and E-cadDipIIIcI2/Gint+ metastases is indicated by a solid black bar.
Mentions: Using these clonal lines, we asked whether harboring the E-cadDipIIIc2 would affect CS-99 growth rates. In vitro WST-1 cell proliferation assays indicated that the E-cadDipIIIcI2/Gint clones and Rint+/Gint− cells grew at equal rates (S8A). To test whether or not MET was required for growth in vivo, we co-injected a 1:1 ratio of Rint+/Gint− control cells and E-cadDipIIIcI2/Gint+ cells s.c. in the flanks of nude mice. Palpable tumors formed within five to seven days. Analysis of the proportions of control cells and cells with the suicide reporter revealed that 100% of primary tumors contained substantial proportions of both cell types (Figure 6A). We observed only a very slight decrease in the proportion of E-cadDipIIIcI2/Gint cells relative to control Rint+/Gint− cells (Figure 6B). Indeed, cells harboring the suicide reporter cells formed colonies in soft agar only slightly less efficiently than control cells (Figure 6C and D). We concluded from this that MET was not required for growth and primary tumor formation in vitro or in vivo.

View Article: PubMed Central - PubMed

ABSTRACT

The cascade that culminates in macrometastases is thought to be mediated by phenotypic plasticity, including epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT and MET). While there is substantial support for the role of EMT in driving cancer cell invasion and dissemination, much less is known about the importance of MET in the later steps of metastatic colonization. We created novel reporters, which integrate transcriptional and post-transcriptional regulation, to test whether MET is required for metastasis in multiple in vivo cancer models. In a model of carcinosarcoma, metastasis occurred via an MET-dependent pathway; however, in two prostate carcinoma models, metastatic colonization was MET-independent. Indeed, the MET-independent pathway was supported by reanalysis of pre-clinical and clinical data. These results provide evidence for both MET-dependent and MET-independent metastatic pathways.

No MeSH data available.


Related in: MedlinePlus