None of the genes defining the myofibroblast-like phenotype mapped to regions of DNA copy number variation, arguing against a role for genomic rearrangements during the acquisition of the myofibroblastic phenotype

None of the genes defining the myofibroblast-like phenotype mapped to regions of DNA copy number variation, arguing against a role for genomic rearrangements during the acquisition of the myofibroblastic phenotype. Open in a separate window Figure 3 DNA copy variation(A) Segmented copy numbers for each cell line were inferred with the GLAD (gain and loss analysis of DNA) algorithm and normalized to a median of two copies. an invasive advantage to the metastatic tumor cells. We further show that some of the constituents of the mesenchymal signature, including the expression of the well characterized myofibroblastic marker was subcloned into the pQCXIN retroviral-based vector (Clontech, Palo Alto, CA). For knockdown, the pLKO.1 plasmid (clone TRCN0000053608) encoding short hairpin RNA (shRNAs) targeting was obtained Mouse monoclonal to PBEF1 from Open Biosystems (Open Biosystems, Huntsville, AL). Viral supernatants were generated by transfecting 293-FT cells with the shRNA constructs in combination with Regorafenib Hydrochloride the packaging vectors pVSVG and pDR2. Results A xenograft mouse model for RCC metastasis SN12C cells, when implanted in the renal sub-capsule of NOD/SCID mice, showed limited lung metastatic activity (3C10 nodules). Lung-derived nodules, when expanded in culture and reinoculated into the kidney capsule showed higher lung metastatic activity (80 nodules), designated LM1 (22). Another round of selection yielded a second generation of highly metastatic cells (400 nodules), designated LM2 (Fig. 1A). Interestingly, the LM cells also showed an increased metastatic spread to other organs ((22), our results). Of note, the growth rate of the highly metastatic cells at the primary site was comparable to that of the parental SN12C cells (Fig. 1B). Histologic review of lung metastasis showed that this tumor cells, in addition to colonizing the lung parenchyma, associated in thrombus-like structures inside lymphatic as well as blood vessels (Fig. 1C). Open in a separate window Physique 1 A xenograft mouse model for RCC metastasisNOD/SCID mice were injected with 106 tumor cells as indicated in the kidney subcapsule. (A Regorafenib Hydrochloride Primary tumor Regorafenib Hydrochloride (B) Lung metastasis (C) H&E staining. Black arrow indicates thrombi inside the blood vessels. Blue arrow indicates vascular thrombus invading the vessel wall. A mesenchymal expression signature correlates with RCC metastatic phenotype To evaluate genome wide changes in gene expression during metastatic conversion we subjected the three cell types, SN12C, LM1, and LM2, to gene expression profiling using the Affymetrix U133 plus 2.0 arrays. Class comparison between SN12 and LM2 cells identified 349 unique genes that showed differential expression; 191 were upregulated and 158 were downregulated in the most aggressive metastatic cells. Analysis of the gene expression patterns revealed a mesenchymal signature as the dominant feature Regorafenib Hydrochloride of the gene list that characterized the metastatic cells (Table 1) (Fig. S1). Epithelial-mesenchymal transition (EMT) has been proposed to be one of the initial actions during metastatic conversion that endows tumor cells with the ability to invade surrounding tissues (24). We, therefore, investigated if the observed mesenchymal phenotype of LM cells correlated with an increase in invasiveness. Surprisingly, the ability of LM cells to invade through both an artificial basement membrane (Boyden chamber assay) and an artifical endothelium (HUVECs) was not significantly altered when compared with parental SN12C cells (Fig. S2). This result suggested that this SN12C cells may already have undergone the initial stages of EMT. Consistent with this assumption, we found that epithelial markers such as E-cadherin were downregulated while mesenchymal markers such as N-cadherin and vimentin were already upregulated in the SN12C poorly metastatic cells (Fig. S2C). Emerging evidence suggests that epithelial cells are also an important source of myofibroblasts in fibrosis and cancer (25). Therefore, we reasoned that a significant function of tumor EMT would be to provide tumor cells with stromal myofibroblastic properties, which would bypass the Regorafenib Hydrochloride requirement of an activated stroma during metastatic colonization. Detailed analysis of mesenchymal genes upregulated in the LM cells revealed a high enrichment for expression of genes related to fibrosis and desmoplastic response (and because this molecule could serve as a functional link between fibrosis and metastasis during kidney cancer progression, as strong evidence suggests to be a crucial myofibroblast-expressed factor that regulates metastasis (28). To determine whether this gene plays a causal role in lung metastasis we stably decreased its expression in LM2 cells using shRNA and found that levels had a profound affect on metastatic activity (Fig. 2A). To investigate if was sufficient to induce metastatic spread, we engineered SN12C cells to overexpress at levels comparable to those seen in LM2 cells. Injection of SN12C-cells into the kidney sub-capsule showed a very modest lung colonization advantage in.