Modeling Immune-ECM Crosstalk and Therapeutic Response in an Aged ER+/PR+ Breast Cancer Model

Introduction: : Breast cancer is one of the most prevalent cancers worldwide, with hormone receptor-positive (HR+) subtypes accounting for the majority of cases. While hormone-targeting therapies have improved patient outcomes, therapeutic resistance remains a barrier, particularly in older populations. While aging research has historically focused on intrinsic cellular and genetic alterations, emerging evidence highlights the role of the aged extracellular matrix (ECM) and immune system in shaping the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) trend toward an M2-like, immunosuppressive phenotype in aged tissues and are essential for immune evasion, ECM remodeling, and therapeutic resistance. However, the relationship between the aged ECM, macrophage behavior, and cancer progression remains understudied. Utilizing a previously validated platform, we examine how the aged ECM influences macrophage polarization and how these macrophages influence cancer cell behavior and response to targeted therapies. By integrating immune and ECM aging, we seek to uncover how macrophage-ECM-cancer crosstalk contributes to therapeutic resistance in aged patients. The findings aim to inform the development of age-relevant therapeutic strategies for aged patients with HR+ breast cancer.

Materials and

Methods: : Fourth mammary glands were harvested from aged (24–28 months) C57BL/6 mice and processed to generate decellularized extracellular matrices (dECM). Tissues were decellularized and delipidized using a previously established protocol, followed by enzymatic digestion, creating solubilized dECM (sdECM). The sdECM was mixed with human type I collagen and gelatin methacrylate (GelMA) to form age-mimetic hydrogels. Rheological testing and CD spectra were performed to assess hydrogel stiffness and secondary structure. iPSC-derived macrophages were resuspended within the aged hydrogel, and primary ER+/PR+/HER2- breast cancer organoids were generated and embedded within the aged hydrogels with and without macrophages. Tumor cell viability and growth were assessed between groups, and macrophage polarization analyzed. High-throughput drug screening is performed using an FDA-approved drug library (Selleck). Drug responses is analyzed in the presence or absence of macrophages to evaluate immune effects on therapeutic efficacy.

Results, Conclusions, and Discussions:: Characterization of age-mimetic hydrogels revealed rheological properties comparable to native mammary tissue (Fig. 1A). Circular dichroism secondary structure analysis confirmed that addition of sdECM preserved similar secondary structure (Fig. 1B), validating the hydrogel as a biologically relevant ECM age-mimetic environment. Initial studies with aged ER+/PR+ organoids showed that age-mimetic hydrogels supported cellular viability and enhanced invasion in the aged gel compared to the young (Fig. 1C), showcasing the hydrogels' relevancy for modeling age-specific tumor behavior.
Preliminary analysis of ECM-driven immune modulation was performed with iPSC-derived macrophages exposed to age-respective sdECM and/or aged ER+/PR+ conditioned media. Aged sdECM alone increased expression of CD206 (M2-macrophage marker), whereas young sdECM did not, and conditioned media alone also enriched CD206 expression (Fig. 1D, 1E). Synergistically combined, aged sdECM and conditioned media produced the highest CD206 levels. In contrast, young sdECM and conditioned media reduced CD206 levels when compared to the conditioned media alone.
CD80 (M1-macrophage marker) was also assessed, where it was found that aged sdECM and tumor conditioned media alone were able to increase its levels, with the highest expression level occurring when combined synergistically. Additionally, the young sdECM combined with conditioned media was once again able to reduce expression levels when compared to conditioned media alone. As both M1 and M2 macrophage markers were present in individual cells, it is indicated that the aged ECM and tumor secretome factors cooperatively and individually induce a TAM phenotype.
These preliminary findings validate our age-mimetic model for studying ECM-immune interactions in the aged population. Additionally, the model holds promise for uncovering age-specific therapeutic vulnerabilities and improving strategies tailored toward the aged population for hormone-receptor positive therapies.

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