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Defining how interactions between tumor subpopulations contribute to invasion is essential for understanding how tumors metastasize. Here, we find that the heterogeneous expression of the transcription factor ΔNp63 confers distinct proliferative and invasive epithelial-to-mesenchymal transition (EMT) states in subpopulations that establish a leader–follower relationship to collectively invade. A ΔNp63-high EMT program coupled the ability to proliferate with an IL1α- and miR-205–dependent suppression of cellular protrusions that are required to initiate collective invasion. An alternative ΔNp63-low EMT program conferred cells with the ability to initiate and lead collective invasion. However, this ΔNp63-low EMT state triggered a collateral loss of fitness. Importantly, rare growth-suppressed ΔNp63-low EMT cells influenced tumor progression by leading the invasion of proliferative ΔNp63-high EMT cells in heterogeneous primary tumors. Thus, heterogeneous activation of distinct EMT programs promotes a mode of collective invasion that overcomes cell intrinsic phenotypic deficiencies to induce the dissemination of proliferative tumor cells. Significance: These findings reveal how an interaction between cells in different EMT states confers properties that are not induced by either EMT program alone.

Most ductal breast carcinoma cells are weakly invasive in vitro and in vivo, suggesting that components of their microenvironment may facilitate a transition from in situ to invasive stages during progression. Here, we report that coculture of mammary fibroblasts specifically triggers invasive behavior in basal-type breast cancer cells through a ligand independent mechanism. When cultured alone in organotypic culture, both basal- and luminal-type breast cancer cells formed noninvasive spheroids with characteristics of ductal carcinoma in situ (DCIS). In contrast, when cocultured with mammary fibroblasts, basal-type spheroids exhibited invasive character whereas the luminal-type spheroids retained a benign and noninvasive duct-like architecture. Real-time imaging and functional studies revealed that the specificity of invasion was linked to a unique capacity of basal-type breast cancer cells to move within spheroids. Mammary fibroblasts induced invasion by triggering basal-type breast cancer cells to convert from a noninvasive program of mammary epithelial morphogenesis to an invasive program of sprouting endothelial angiogenesis. Contrary to the existing invasion models, soluble ligands produced by the fibroblasts were not sufficient to trigger invasion. Instead, basal-type invasion relied upon a Cdc42-dependent reorganization of collagen fibers in the extracellular matrix by fibroblasts. Inhibiting basal-type cell movement with clinically relevant drugs blocked invasion both in organotypic culture and in animals, suggesting a new treatment strategy for early-stage patients. Together our findings establish that fibroblast recruitment by basal-type breast cancer cells into early-stage tumors is sufficient to trigger their conversion from a benign, noninvasive DCIS-like stage to a malignant invasive stage. Furthermore, our findings suggest that different subtypes of breast cancer may require distinct types of contributions from the microenvironment to undergo malignant progression. Cancer Res; 71(21); 6857–66. ©2011 AACR.

Diverse genetic and epigenetic traits can promote variant behaviors in subpopulations of cells within primary tumors. In some cases, these heterogeneous subpopulations may compete against each other for clonal dominance to improve tumor fitness. By comparison, the extent to which tumor cell subpopulations cooperate during malignant progression is not well understood. Here, we find that a commensal relationship can exist between epigentically distinct subpopulations of tumor cells during collective invasion. Using quantitive phenotypic analysis and live-cell imaging, we discovered that a subpopulation of cells, termed trailblazer cells, has an enhanced ability to collectively invade. Trailblazer phenotype was heritable and trailblazer subpopulations were derived from 5 triple-negative breast cancer cell lines. The percentage of trailblazers cells within a population ranged from between 0.5% and 90% of the parental population. Gene expression profiling identified a 29-gene expression signature that distinguished trailblazer cells from non-trailblazer cells and identified patients with poor outcome, A cohort of genes trailblazer signature genes were necessary for the formation of actin-rich projections that facilitate path generation during collective invasion. In addition, the trailblazer gene DOCK10 was necessary for spontaneous metastasis to the lung. The path generating function of a small subpopulation of trailblazer cells was sufficient to induce the collective invasion of large population of naturally occurring non-trailblazer cells in organotypic culture. Trailblazer cells were also sufficient to induce the invasion of a mouse model of ductal carcinoma in situ. Taken together, our results suggest that a rare subpopulation of tumor cells with an enhanced ability to collectively invade can promote metastatic progression through cell autonomous and non-cell autonomous mechanisms. Thus, our results support a paradigm shift towards identifying both the cell autonomous and the non cell-autonomous regulatory mechanisms of cell biological processes within heterogeneous tumors to understand neoplastic progression and develop new intervention modalities. Citation Format: Amanda Prechtl, Jill Westcott, Tuyen Dang, Erin Maine, Gray Pearson. Non-cell autonomous regulation of collective invasion be an epigenetically distinct subpopulation of tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3161. doi:10.1158/1538-7445.AM2014-3161

The invasion of tumor cells out of ductal epithelium is a poorly understood process that dramatically increases the odds of patient death. We have defined a new non cell-autonomous control mechanism for neoplastic invasion that trigger tumors to switch from a benign to a malignant stage of growth. Using high resolution live-imaging and functional studies we show that an epigenetically distinct and autonomously invasive subpopulation of tumor cells induces the invasion of a large bulk population of autonomously noninvasive tumor cells by adopting migratory principles observed during sprouting angiogenesis. The molecular dissection of this cooperative interaction revealed a new “invasion signature” of 29 genes that is distinct from traditional epithelial-to-mesenchymal transition markers. Patients expressing this invasion signature had a worse clinical outcome, which suggests that this commensal relationship between tumor cell subpopulations contributes to tumor progression. Functional analysis showed that multiple components of the invasion signature were specifically required for the cooperative induction of invasion, but not cell motility, thus revealing new roles for these genes as non-cell autonomous regulators of invasion. These results are of broad interest because they are the first demonstration of a commensal relationship between tumor cell subpopulations during the induction of invasion. Further, these findings establish a mechanistic underpinning that obviates the notion that the exclusive role of heterogeneity in tumors is to act as a source for unique traits that can be selected for and expanded to improve the cell autonomous fitness of neoplastic cell populations. Thus, our results support a paradigm shift towards identifying both the cell autonomous and the non cell-autonomous regulatory mechanisms of cell biological processes within heterogeneous tumors to understand neoplastic progression and develop new intervention modalities. Citation Format: Gray Pearson, Amanda Prechtl, Jill Westcott, Tuyen Dang, Erin Maine. A commensal relationship between tumor cell subpopulations promotes collective invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4930. doi:10.1158/1538-7445.AM2013-4930 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Breast cancer patient mortality is a consequence of the metastatic dissemination of neoplastic cells into foreign tissues, not the primary tumor growth. Fibroblasts located in the breast microenvironment may trigger neoplastic cells to invade out of the mammary duct and into the stroma, an early step in breast cancer metastasis. Investigating how mammary fibroblasts promote breast cancer invasion may improve both treatment and the accuracy of diagnosis of breast cancer patients. To elucidate the mechanism of tumor cell invasion, we use organotypic cultures to generate three-dimensional interactions between breast cancer cells and the microenvironment and xenograft models for our in vivo studies. Live cell imaging, immunofluorescence and other biochemical methods were used to determine the interaction between breast cancer cells and mammary fibroblasts. We have found that mammary fibroblasts specifically induce the invasion of basal-type breast cancer cells. The propensity of the mammary fibroblasts to induce the invasion of the basal-type breast cancer cells correlated with the reorganization of the collagen in the microenvironment. Secreted factors from the mammary fibroblasts were insufficient to induce invasion of the neoplastic cells. Instead, we discovered that the Cdc42 dependent reorganization of stromal collagen by the mammary fibroblasts was necessary for the induction of basal-type breast cancer cell invasion. To determine how the basal-type breast cancer cells opportunistically invaded into paths created in the stromal collagen, we imaged an organotypic culture model in real-time. The live-imaging revealed that basal-type breast cancer cells within spheroids were motile and constantly exchanging cell-to-cell partners within the confines of the sphere. In contrast, the luminal-type breast cancer cells were not motile within the spheroids. When co-cultured with mammary fibroblasts, the basal-type breast cancer cells that invaded through paths created in the collagen were motile cells. The static luminal-type breast cancer cells were not able to leave the spheroids and invade into the stroma. Thus, the intrinsic ability of the basal-type breast cancer cells to move within multicellular spheroids promoted the invasion in response to the accumulation of nearby mammary fibroblasts. Cell motility is a complex process with many regulatory components including microRNA dependent control of gene expression. Expression profiling of patient and breast cancer cell lines has demonstrated that there is differential microRNA expression between the basal and luminal breast cancer subtypes. We are currently exploring how intrinsic subtype specific microRNA expression contributes to the differential motility observed between the basal and luminal-type breast cancer cells. In conclusion, our data suggest that mammary fibroblasts are sufficient to induce motile basal-type breast cancer cells to invade into the stroma and we are investigating how basal-type breast cancer microRNA expression promotes cell motility. Citation Format: Tuyen T. Dang, Gray W. Pearson. Breast cancer intrinsic subtype specific interactions with the microenvironment dictate the mechanism of tumor invasion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A38.