The ability of tumors to survive therapy is mediated not only by cell-intrinsic but also cell-extrinsic, microenvironmental mechanisms. Across many cancers, including triple-negative breast cancer (TNBC), a high stroma/tumor ratio correlates with poor survival. In many contexts, this correlation can be explained by the direct reduction of therapy sensitivity by stroma-produced paracrine factors through activating pro-survival signaling and stemness. We sought to explore whether this direct effect contributes to the link between stroma and poor responses to chemotherapies in TNBC. Our in vitro studies with panels of TNBC cell line models and stromal isolates failed to detect a direct modulation of chemoresistance. However, we found that fibroblasts often enhance baseline tumor cell proliferation. Consistent with this in vitro observation, we found evidence of stroma enhanced TNBC cell proliferation in vivo, in xenograft models and patient samples. Based on these observations, we hypothesized an indirect link between stroma and chemoresistance, where stroma-augmented proliferation potentiates the recovery of residual tumors between chemotherapy cycles. To test this hypothesis, we developed a spatial agent-based model of tumor response to repeated dosing of chemotherapy. The model was quantitatively parameterized from histological analyses and experimental studies. We found that even a slight enhancement of tumor cell proliferation within stroma-proximal niches can strongly enhance the ability of tumors to survive multiple cycles of chemotherapy under biologically and clinically feasible parameters. In summary, our study uncovered a novel, indirect mechanism of chemoresistance. Further, our study highlights the limitations of short-term cytotoxicity assays in understanding chemotherapy responses and supports the integration of experimental and in silico modeling.
Background: Stroma plays a crucial role in shaping tumor growth dynamics, invasion, and metastasis. High stroma to tumor ratio in the clinic is associated with poor outcomes and shorter remission periods for lung cancer patients. Recent studies suggest that stroma shelters tumor cells during drug administration, but the precise mechanisms to confer this protection remain unclear. We hypothesize that stroma secretes paracrine factors that enhance tumor cells' proliferation rate in proximity, hence outcompeting the death rate due to therapies. In this context, the residual disease is an inherent stage in tumors' eco-evolutionary dynamics under treatment. Therefore, understanding the nature of stroma-tumor cell interactions is crucial for designing effective therapies. Methods: The spatial relationship between the stroma and proliferating tumor cells was interrogated via spatial analysis of 2D samples from mouse xenograft models and subsequent spatial agent-based modeling (ABM). Results: First, we built a pipeline to quantify the spatial extent and amplitude of stroma shielding the presence or absence of treatment. The spatial distributions of tumor cells, their proliferating rates, and the spatial extent of stroma protective effects are used to parametrize the ABM and simulate the spatio-temporal dynamics of tumor growth with and without treatment. Our preliminary results show moderately higher clustering of proliferating and stroma cells than stroma and non-proliferating cells or random distribution in the absence of treatment. In contrast, during therapy, stroma-proliferating tumor cell clustering is considerably higher than stroma-non proliferating tumor cells and stroma-random distribution. Moreover, stroma's protective effect during treatment seems limited to cells either in direct contact with stromal cells or in their immediate proximity. Conclusions: We expect our results to lead to novel therapeutic interventions that aim to shift eco-evolutionary dynamics rather than maximize short-term tumor cell killing efficiency. Citation Format: Tatiana Miti, Anna Miller, Daria Myroshnychenko, Bina Desai, Andriy Marusyk, David Basanta. Integrating spatial analysis and agent-based modeling for studies of stroma sheltering effects in NSCLC [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A032.
Despite inducing strong and durable remissions, inhibitors of mutant ALK (ALKi) are not curative for advanced ALK+ lung cancers, as residual tumors eventually develop resistance and relapse. Apart from tumor cells’ intrinsic ability to escape from therapy, there is a growing body of evidence suggesting the contribution of extrinsic factors, produced by cancer-associated fibroblasts (CAFs). Despite the multitude of studies that demonstrated the ability of multiple CAF-produced factors to reduce the sensitivity of tumor cells to ALKi, the contribution of these effects toward responses in vivo remains unresolved. To study the impact of stroma on the ability of tumor cells to survive and develop resistance to ALKi, we derived multiple isolates of fibroblasts from clinical samples. Consistent with previous reports, we found that co-culture with CAFs or CAF-conditioned media protects tumor cells against ALKi. We observed that the degree of protection varies between different CAF isolates. This variability in the extent of protection could be attributed to variability in the levels of secreted hepatocyte growth factor (HGF) a known paracrine mediator of environmental resistance. Moreover, exogenous HGF phenocopied the effect of CAFs while blocking HGF-cMET signaling with neutralizing antibodies or pharmacologically abrogated the protective effect. To test the relevance of these findings in vivo, we took advantage of the inability of murine HGF to activate human cMET by comparing the response of ALK+ xenograft tumors to front-line ALKi alectinib between NSG mice and NSG-derivative strain with humanized HGF. In contrast to the in vitro data, HGF status had only a minimal impact on the remission-relapse dynamics of xenograft tumors. This lack of differences reflected a strong HGF-independent sheltering effect of the stromal niche. Our histological analyses of samples at different points of remission-relapse response revealed that while alectinib potently suppressed tumor cell proliferation, proximity to stroma reduced this cytostatic effect, without impacting cell proliferation in the absence of therapy. To gain insights into the mechanistic underpinning of this HGF-independent effect, we used spatial transcriptomics, comparing stroma-proximal and stroma-distant tumor regions. These analyses, as well as functional validation studies, indicate that stroma-sheltering effects are mediated by multiple mechanisms, acting in an additive fashion. In summary, our studies indicate that therapy resistance of tumor cells reflects the combined action of both intrinsic and microenvironmental factors. Our findings indicate that focusing on a single resistance mechanism at a time is unlikely to induce strong, durable responses. Instead, tackling the issue of therapy resistance necessitates the consideration of multiple resistance mechanisms as well as the moving target nature of tumors under therapy. Citation Format: Bina Desai, Tatiana Miti, Daria Miroshnychenko, Viktoriya Marusyk, Chandler Gatenbee, Menkara Henry, Uwe Rix, Alexander Anderson, Eric Haura, David Basanta, Andriy Marusyk. Stromal facilitated multifactorial resistance to tumor cells against targeted therapies in ALK+ NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 552.
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