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Breast cancer (BCa) proliferates within a complex, three-dimensional microenvironment amid heterogeneous biochemical and biophysical cues. Understanding how mechanical forces within the tumor microenvironment (TME) regulate BCa phenotype is of great interest. We demonstrate that mechanical strain enhanced the proliferation and migration of both estrogen receptor (ER)+ and triple-negative (TNBC) human and mouse BCa cells. Furthermore, a critical role for exosomes derived from cells subjected to mechanical strain in these pro-tumorigenic effects was identified. Exosome production by TNBC cells increased upon exposure to oscillatory strain (OS), which correlated with elevated cell proliferation. Using a syngeneic, orthotopic mouse model of TNBC, we identified that preconditioning BCa cells with OS significantly increased tumor growth and myeloid-derived suppressor cells (MDSCs) and M2 macrophages at the TME. This pro-tumorigenic myeloid cell enrichment also correlated with a decrease in CD8+ T cells. An increase in PD-L1+ exosome release from BCa cells following OS supported additive T cell inhibitory functions in the TME. The role of exosomes in MDSC and M2 macrophage was confirmed in vivo by cytotracking fluorescent exosomes, derived from labeled 4T1.2 cells, preconditioned with oscillatory strain. Additionally, in vivo internalization and intratumoral localization of tumor-cell derived exosomes was observed within MDSCs, M2 macrophages, and CD45-negative cell populations following direct injection of fluorescently-labeled exosomes. Our data demonstrate that exposure to mechanical strain promotes invasive and pro-tumorigenic phenotypes in BCa cells, indicating that mechanical strains can impact the growth and proliferation of cancer cell, alters exosome production by BCa, and induces immunosuppression in the TME by dampening anti-tumor immunity.

Introduction: Breast cancer (BCa) occurs with a complex, three-dimensional microenvironment that involves heterogeneous biochemical and biophysical cues. Understanding how mechanical properties within the tumor microenvironment (TME) regulate breast cancer phenotype and immunosuppression is of great interest. Materials and Methods: BCa cells (MCF-7, MDA-MB-231 or 4T1.2) cultured to confluence on collagen coated FlexCell culture plates were subjected to 10% uniaxial cyclic/oscillatory strain at 0.3 Hz, or 10% constant strain, or no strain for 48 hours. They were isolated for analysis of proliferation (MTT assay and cell count by trypan blue), and migration (transwell and wound healing assay). Exosomes from conditioned media were isolated by differential centrifugation or using the Total Exosome Isolation kit. The purified exosomes were quantified by NanoSight and characterized by ImageStream. 5 × 105 4T1.2 cells or PKH67-labeled strained or control cells were injected into the mammary fat pad of BALB/c mice. Tumor volume was measured at the indicated time points after injection. Tumor-infiltrating immune cells and the internalization of exosomes were analyzed by flow cytometry on day 14 post implantation. In some experiments, on day 6 after tumor injection, 7.5 × 108 PHK67-labeled tumor cell-derived exosomes or PBS were injected into the tumor nodule. Tumor tissues were harvested for analysis of the internalization of exosomes by immune cells and tumor cells on days 2 and 8 after exosome injection. Results: We show that mechanical strain enhanced the proliferation and migration of BCa cells in vitro. Exosome concentrations produced by triple negative breast cancer (TNBC) cells were increased following exposure to oscillatory strain. Phenotyping exosomes by ImageStream showed that the percentages of CD81+PD-L1+ and CD63+PD-L1+ exosomes were increased after exposure to oscillatory strain. Using a syngeneic orthotopic mouse model of TNBC, we showed that preconditioning with mechanical strain increased tumor growth. The percentages of tumor-infiltrating monocytic myeloid-derived suppressor cells (M-MDSC) and recruited macrophages were increased while CD8+ T cells decreased in the TME of mice implanted with 4T1.2 cells preconditioned with oscillatory strain. Further, exosome internalizations by M-MDSC and recruited macrophages were elevated when tumor cells were preconditioned with oscillatory strain. Moreover, exosomes internalization by immune cells and tumor cells in TME were identified by PKH67 positive signals on days 2 and 8 after injection of PKH67-labeled exosomes into tumor nodules by flow cytometry analyses and confocal microscope imaging. Conclusions: Our data indicate that exposure to mechanical strain promotes invasive and pro-tumorigenic phenotypes in BCa, alters exosome production by BCa and induces immunosuppression in the TME. Citation Format: Yong Wang, Kayla F. Goliwas, Paige E. Severino, Kenneth Hough, Derek Van Vessem, Hong Wang, Sultan Tousif, Roy P. Koomullil, Andra R. Frost, Selvarangan Ponnazhagen, Joel L. Berry, Jessy S. Deshane. Mechanical strain induces phenotypic changes in breast cancer cells and promotes immunosuppression in the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1711.

Introduction: Cells within the breast tumor mass are met with a variety of biophysical or mechanical signals that is associated with elevated compression or solid stress within the tumor interior, tension at the tumor periphery, and altered interstitial fluid pressure. Understanding how the mechanical stress within the tumor microenvironment (TME) regulates cancer cell phenotype is of interest, yet the response of breast cancer (BCa) cells to these forces is largely unknown. Our study aims to identify the impact of mechanical stress on BCa cell phenotype by mimicking the tension at the tumor periphery. Materials and Methods: BCa cells (MCF-7 or 4T1.2) were cultured to confluence on collagen coated FlexCell culture plates. These plates were then subjected to 10% uniaxial cyclic/oscillatory strain at 0.3 Hz, or 10% constant strain, or no strain for 48 hours. Strained or control cells were isolated for analysis of proliferation (MTT assay), and migration (8.0 μm pore transwell). Exosomes from conditioned media were isolated via differential centrifugation and purified exosomes were characterized by ImageStream. 5x105 4T1.2 cells or PKH-labeled strained or control cells were injected into the mammary fat pad of BALB/c mice. Tumor volume was measured over 14 days. Tumor-infiltrating immune cells and the uptake of exosomes were analyzed by flow cytometry on day-14 post implantation. Results: We determined significant increases in proliferation and migration of 4T1.2 and MCF-7 cells in vitro following exposure to oscillatory forces. The populations of CD63+, CD63+CD24+ and CD63+PD-L1+ exosomes were increased when 4T1.2 cells were exposed to oscillatory strain compared to unstrained control cells. Further, we investigated how oscillatory forces affect tumor growth and tumor-immune cell interactions in vivo by using a syngeneic, orthotopic mouse model of BCa. Mice implanted with 4T1.2 cells that were pre-exposed to oscillatory forces showed a significant increase in primary tumor growth at 8 and 11 days post tumor challenge. The percentages of tumor-infiltrating monocytic myeloid-derived suppressor cells (M-MDSC) and recruited macrophages were increased in the TME of mice implanted with 4T1.2 cells that were pre-exposed to oscillatory forces, while the granulocytic MDSC subset was not significantly different between the two groups. A marginal decrease in the percentage of CD8+ T cells was noted in the TME of mice implanted with strained 4T1.2 when compared to controls, suggesting immune suppression in the TME. Furthermore, exosome uptakes by M-MDSC and recruited macrophages were increased in the TME of mice implanted with PKH-labeled 4T1.2 cells, exposed to oscillatory strain. Conclusion: Together, these data indicate that exposure to mechanical stress changes BCa cell phenotype to an invasive and protumorigenic phenotype that promotes immunosuppressive effects in the TME. Citation Format: Yong Wang, Paige E. Severino, Kayla Goliwas, Kenneth Hough, Derek Van Vessem, Hong Wang, Andra R. Frost, Selvarangan Ponnazhagen, Joel L. Berry, Jessy S. Deshane. Mechanical strain induces phenotypic changes in breast cancer cells and promotes immunosuppression in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1177.