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Despite the advent of immunotherapy, metastatic melanoma represents an aggressive tumor type with a poor survival outcome. The successful application of immunotherapy requires in-depth understanding of the biological basis and immunosuppressive mechanisms within the tumor microenvironment. In this study, we conducted spatially explicit analyses of the stromal-immune interface across 400 melanoma hematoxylin and eosin (H&E) specimens from The Cancer Genome Atlas. A computational pathology pipeline (CRImage) was used to classify cells in the H&E specimen into stromal, immune, or cancer cells. The estimated proportions of these cell types were validated by independent measures of tumor purity, pathologists' estimate of lymphocyte density, imputed immune cell subtypes, and pathway analyses. Spatial interactions between these cell types were computed using a graph-based algorithm (topological tumor graphs, TTG). This approach identified two stromal features, namely stromal clustering and stromal barrier, which represented the melanoma stromal microenvironment. Tumors with increased stromal clustering and barrier were associated with reduced intratumoral lymphocyte distribution and poor overall survival independent of existing prognostic factors. To explore the genomic basis of these TTG-derived stromal phenotypes, we used a deep learning approach integrating genomic (copy number) and transcriptomic data, thereby inferring a compressed representation of copy number-driven alterations in gene expression. This integrative analysis revealed that tumors with high stromal clustering and barrier had reduced expression of pathways involved in naïve CD4 signaling, MAPK, and PI3K signaling. Taken together, our findings support the immunosuppressive role of stromal cells and T-cell exclusion within the vicinity of melanoma cells. SIGNIFICANCE: Computational histology-based stromal phenotypes within the tumor microenvironment are significantly associated with prognosis and immune exclusion in melanoma., (©2019 American Association for Cancer Research.)

Drug resistance is the single most important driver of cancer treatment failure for modern targeted therapies, and the dialog between tumor and stroma has been shown to modulate the response to molecularly targeted therapies through proliferative and survival signaling. In this work, we investigate interactions between a growing tumor and its surrounding stroma and their role in facilitating the emergence of drug resistance. We used mathematical modeling as a theoretical framework to bridge between experimental models and scales, with the aim of separating intrinsic and extrinsic components of resistance in BRAF -mutated melanoma; the model describes tumor-stroma dynamics both with and without treatment. Integration of experimental data into our model revealed significant variation in either the intensity of stromal promotion or intrinsic tissue carrying capacity across animal replicates. Cancer Res; 77(19); 5409-18. ©2017 AACR ., (©2017 American Association for Cancer Research.)

There is an urgent need to identify new therapeutic opportunities for metastatic melanoma. Fragment-based screening has led to the discovery of orally available, ATP-competitive AKT kinase inhibitors, AT13148 and CCT129254. These compounds also inhibit the Rho-kinases ROCK 1 and ROCK 2 and we show they potently inhibit ROCK activity in melanoma cells in culture and in vivo. Treatment of melanoma cells with CCT129254 or AT13148 dramatically reduces cell invasion, impairing both "amoeboid-like" and mesenchymal-like modes of invasion in culture. Intravital imaging shows that CCT129254 or AT13148 treatment reduces the motility of melanoma cells in vivo. CCT129254 inhibits melanoma metastasis when administered 2 days after orthotopic intradermal injection of the cells, or when treatment starts after metastases have arisen. Mechanistically, our data suggest that inhibition of ROCK reduces the ability of melanoma cells to efficiently colonize the lungs. These results suggest that these novel inhibitors of ROCK may be beneficial in the treatment of metastasis., (©2015 American Association for Cancer Research.)

TGF-β can act as a tumor suppressor at early stages of cancer progression and as a tumor promoter at later stages. The E3 ubiquitin ligase Arkadia (RNF111) is a critical component of the TGF-β signaling pathway, being required for a subset of responses, those mediated by Smad3-Smad4 complexes. It acts by mediating ligand-induced degradation of Ski and SnoN (SKIL), which are 2 potent transcriptional repressors. Here, we investigate the role of Arkadia in cancer using model systems to address both potential tumor-suppressive and tumor-promoting roles. Stable reexpression of Arkadia in lung carcinoma NCI-H460 cells, which we show contain a hemizygous nonsense mutation in the Arkadia/RNF111 gene, efficiently restored TGF-β-induced Smad3-dependent transcription, and substantially decreased the ability of these cells to grow in soft agar in vitro. However, it had no effect on tumor growth in vivo in mouse models. Moreover, loss of Arkadia in cancer cell lines and human tumors is rare, arguing against a prominent tumor-suppressive role. In contrast, we have uncovered a potent tumor-promoting function for Arkadia. Using 3 different cancer cell lines whose tumorigenic properties are driven by TGF-β signaling, we show that loss of Arkadia function, either by overexpression of dominant negative Arkadia or by siRNA-induced knockdown, substantially inhibited lung colonization in tail vein injection experiments in immunodeficient mice. Our findings indicate that Arkadia is not critical for regulating tumor growth per se, but is required for the early stages of cancer cell colonization at the sites of metastasis.

Transforming growth factor beta (TGFbeta) has seemingly contradictory roles in tumor progression: it can promote metastatic invasion but also act as a tumor suppressor. Recently, two studies have used intravital imaging to unravel the role of TGFbeta at different stages of the metastatic process. TGFbeta promotes single cell motility, which enables invasion into blood vessels. However the activation of TGFbeta signaling is a transient event and is not maintained at distant sites. The downregulation of TGFbeta signaling at secondary sites then permits growth of secondary tumors. In the absence of TGFbeta, cells are restricted to collective movement and lymphatic spread. Here, we discuss these findings and their potential implications., ((c)2010 AACR.)

How melanoma acquire a metastatic phenotype is a key issue. One possible mechanism is that metastasis is driven by microenvironment-induced switching between noninvasive and invasive states. However, whether switching is a reversible or hierarchical process is not known and is difficult to assess by comparison of primary and metastatic tumors. We address this issue in a model of melanoma metastasis using a novel intravital imaging method for melanosomes combined with a reporter construct in which the Brn-2 promoter drives green fluorescent protein (GFP) expression. A subpopulation of cells containing little or no pigment and high levels of Brn2::GFP expression are motile in the primary tumor and enter the vasculature. Significantly, the less differentiated state of motile and intravasated cells is not maintained at secondary sites, implying switching between states as melanoma cells metastasize. We show that melanoma cells can switch in both directions between high- and low-pigment states. However, switching from Brn2::GFP high to low was greatly favored over the reverse direction. Microarray analysis of high- and low-pigment populations revealed that transforming growth factor (TGF)beta2 was up-regulated in the poorly pigmented cells. Furthermore, TGFbeta signaling induced hypopigmentation and increased cell motility. Thus, a subset of less differentiated cells exits the primary tumor but subsequently give rise to metastases that include a range of more differentiated and pigment-producing cells. These data show reversible phenotype switching during melanoma metastasis.

Although overexpression of the epidermal growth factor receptor (EGFR; ErbB1) has been correlated with poor prognosis in breast and other cancers, clinical trials of ErbB1 inhibitors have shown limited efficacy in inhibiting tumor proliferation. To evaluate other possible roles of ErbB1 in tumor malignancy besides proliferation, we have developed a series of tools for analysis of intravasation. Overexpression of ErbB1 in MTLn3 mammary adenocarcinoma cells results in increased intravasation and lung metastasis from tumors formed by injection of cells in the mammary fat pad. However, increased ErbB1 expression has no effect on primary tumor growth and lung seeding efficiency of cells injected i.v. Chemotactic responses to low concentrations of EGF in vitro and cell motility in vivo in the primary tumor measured using intravital imaging are significantly increased by ErbB1 overexpression. The increased cell motility is restricted to ErbB1-overexpressing cells in tumors containing mixtures of cells expressing different ErbB1 levels, arguing for a cell-autonomous effect of increased ErbB1 expression rather than alteration of the tumor microenvironment. In summary, we propose that ErbB1 overexpression makes more significant contributions to intravasation than growth in some tumors and present a novel model for studying ErbB1 contributions to tumor metastasis via chemotaxis and intravasation.

Previous studies have shown that macrophages and tumor cells are comigratory in mammary tumors and that these cell types are mutually dependent for invasion. Here we show that macrophages and tumor cells are necessary and sufficient for comigration and invasion into collagen I and that this process involves a paracrine loop. Macrophages express epidermal growth factor (EGF), which promotes the formation of elongated protrusions and cell invasion by carcinoma cells. Colony stimulating factor 1 (CSF-1) produced by carcinoma cells promotes the expression of EGF by macrophages. In addition, EGF promotes the expression of CSF-1 by carcinoma cells thereby generating a positive feedback loop. Disruption of this loop by blockade of either EGF receptor or CSF-1 receptor signaling is sufficient to inhibit both macrophage and tumor cell migration and invasion.

Progression of tumors to invasive and metastatic forms requires that tumor cells undergo dramatic morphologic changes, a process regulated by Rho GTPases. Elevated expression of RhoA and RhoC, as well as the Rho effector proteins ROCK I and ROCK II, are commonly observed in human cancers and are often associated with more invasive and metastatic phenotypes. To examine how ROCK contributes to the progression of solid tumors, we established a conditionally activated form of ROCK II by fusing the kinase domain to the estrogen receptor hormone-binding domain (ROCK:ER). ROCK:ER-expressing colon carcinoma cells grown as tumors in immunocompromised nude mice organized into discrete clusters surrounding blood vessels. However, ROCK:ER activation resulted in the aggressive dissemination of tumor cells into the surrounding stroma, indicating that increased ROCK signaling is sufficient to promote invasion from solid tumors. In addition, tumors in which ROCK:ER was activated were more highly vascularized, indicating that ROCK contributes to tumor angiogenesis. ROCK:ER activation resulted in changes to epithelial morphology and organization that facilitated motility in vitro, likely by inducing the redistribution of proteins such as ezrin, as well as adherens junction and extracellular matrix-binding proteins. These results suggest that ROCK inhibitors would be useful antimetastatic and antiangiogenic chemotherapeutic agents in tumors associated with elevated RhoA, RhoC, ROCK I, or ROCK II expression.

We subjected cells collected using an in vivo invasion assay to cDNA microarray analysis to identify the gene expression profile of invasive carcinoma cells in primary mammary tumors. Expression of genes involved in cell division, survival, and cell motility were most dramatically changed in invasive cells indicating a population that is neither dividing nor apoptotic but intensely motile. In particular, the genes coding for the minimum motility machine that regulates beta-actin polymerization at the leading edge and, therefore, the motility and chemotaxis of carcinoma cells, were dramatically up-regulated. However, ZBP1, which restricts the localization of beta-actin, the substrate for the minimum motility machine, was down-regulated. This pattern of expression implicated ZBP1 as a suppressor of invasion. Reexpression of ZBP1 in metastatic cells with otherwise low levels of ZBP1 reestablished normal patterns of beta-actin mRNA targeting and suppressed chemotaxis and invasion in primary tumors. ZBP1 reexpression also inhibited metastasis from tumors. These experiments support the involvement in metastasis of the pathways identified in invasive cells, which are regulated by ZBP1.