Abstract 275: Deciphering biphasic responses to Sonic Hedgehog pathway inhibition in breast cancer: Insights from a tumor-fibroblast spheroid model and PDXs

Abstract A significant subset (30-40%) of triple-negative breast cancer (TNBC) cases exhibit hyperactivation of the sonic hedgehog (SHH) pathway, correlating with poor clinical outcomes. However, the therapeutic efficacy of SHH inhibitors in TNBC has yielded inconsistent results, showcasing biphasic tumor responses going from complete remission in some patients to faster disease progression in others. Such tumor response discrepancies underscore the need for more relevant culture models to elucidate how stromal cells respond to SHH signals and impact tumor cell reprogramming. We have developed a tumor-fibroblast spheroid model of robust paracrine SHH signaling to examine alterations in aggressive tumor cell phenotypes resulting from altered SHH pathway activity levels to understand better how SHH therapy leads to biphasic effects in tumor tissues. Specifically, tumor stemness alterations were examined as a function of SHH signal strength altered by tumor-fibroblast proximity, and findings were confirmed in two PDX models of TNBC of divergent sensitivity to pharmacological SHH pathway inhibition. In vitro, results show that tumor stemness potency is enriched by tumor-fibroblast proximity and inversely correlates with SHH pathway activity levels. Tumor stemness potency was determined via a mammosphere forming assay and resistance to chemotherapy using docetaxel as treatment. CD44+ sorted tumor cells derived from adjacent fibroblast cultures showed higher mammosphere forming efficiency and docetaxel sensitivity than those from mixed cultures. These effects correlated with differences in SHH pathway activity strengths and were reverted by pharmacological inhibition of SHH confirming pathway activity involvement. In two PDX models, both exhibiting high SHH pathway activity and similar tumor vascularization, we observed variable response to pharmacological SHH inhibition. PDX2 displayed favorable responses with minimal alpha-smooth muscle actin-positive (SMA+) fibroblast infiltration, while PDX1 exhibited no benefit from SHH therapy, accompanied by a high level of SMA+ fibroblasts. Spatial transcriptomic analysis confirmed stemness enrichments in the PDX1 model treated with SHH inhibitor- a scenario our mixed co-culture model resembled. Stemness-enriched tumor regions were positive for the Ki67 proliferation marker and confined to areas of high SMA+ cell abundance. Plotting Ki67 vs SMA+ cell levels revealed a positive correlation, suggesting that SMA levels may predict tumor response to SHH pathway inhibition. In summary, our studies demonstrate that pharmacological SHH inhibition can induce altered tumor plasticity by favoring stemness phenotypes. Strategies targeting tumor stemness may mitigate biphasic effects in response to SHH inhibitors, offering insights for optimizing TNBC therapeutic approaches. Citation Format: Heizel M. Rosado-Galindo, Miosotis Acevedo-Esquillin, Wandaliz Torres-García, Ana M. Reyes-Ramos, Gabriela Ortiz-Soto, Michelle M. Martínez-Montemayor, Maribella Domenech. Deciphering biphasic responses to Sonic Hedgehog pathway inhibition in breast cancer: Insights from a tumor-fibroblast spheroid model and PDXs abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 275.