Abstract 2673: Tumor heterogeneity and stemness can be shaped by mechano-structural parameters in osteosarcoma

The aim of the study was to identify factors in the physical tumor microenvironment that might participate in Osteosarcoma Cancer Stem-like Cells (OS-CSC) development, which might underlie chemoresistance and recurrence of OS. It was hypothesized that within an OS tumor, spatial localized niches possessing distinct microenvironmental structural and mechanical properties could favor the genesis and survival of different OS cell populations, and certain niches might be more conducive for OS-CSCs formation and maintenance. Mineralized and porous collagen scaffolds mimicking heterogenous bone niches were fabricated by changing crosslinking and pore alignment and their distinct structural and mechanical properties were determined using electron microscopy, Atomic Force Microscopy (AFM) and compression tests. The 3D scaffolds were cultured with highly metastatic human OS cell lines and were assessed for cytoskeletal changes, cell stiffness, epigenetic pathways, chemoresistance and chemoresistance-related pathways, and the differential expression of stemness genes. A consistent trend of OS-CSC phenotype was observed in OS cells grown within softer bone niches (∼10 kPa) with aligned pores as compared to niches with non-aligned pores and increased stiffness (∼30 kPa). The cell population growing in softer, aligned scaffolds had significantly higher expression of stemness genes (OCT4, NANOG, SOX2, ALDH1A1) and drug efflux gene ABCB1 along with increased chemoresistance to doxorubicin and cisplatin, and gave rise to larger colonies in soft agar. Epigenetic modifications associated with cisplatin resistance in OS including decreased methyltransferase EZH2 and concurrent upregulation of LIF/Notch and PRKCA pathways were observed in softer, aligned niche cultures. Fluorescent imaging of scaffold cultures and AFM measurements showed rounder, softer cells in soft, aligned niches indicative of a CSC phenotype, and stretched, elongated, stiffer cells in the non-aligned, stiffer niche with higher YAP nuclear localization, indicative of a differentiated phenotype. RNA Sequencing showed differential expression of 87 genes between scaffold types, with enrichment in ECM-receptor interaction and PPAR signaling pathways; particularly interesting was the enhancement of stem cell reprogramming mediator RAD50 in aligned, soft cultures. In summary, our investigation demonstrates that both the mechanical and structural properties of bone niches influence the formation of distinct OS cell populations. A softer niche with an aligned structure is more conducive for maintaining OS-CSCs, and these findings could be translated to designing therapeutic strategies that modify the microarchitecture to potentially reduce CSC-favoring niches. Citation Format: Zunaira Shoaib, Aleczandria S. Tiffany, Brendan Harley, Joseph Irudayaraj, Timothy M. Fan. Tumor heterogeneity and stemness can be shaped by mechano-structural parameters in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2673.