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Tissue Engineering

Stem Cells in Tissue Engineering and Disease Modeling

Collagen Mineralization and Breast Cancer-Derived Factors Synergistically Regulate Mesenchymal Stem Cell Phenotype in Bone Metastatic Niches

Thursday, October 12, 2023
1:00 PM – 1:15 PM PDT
Location: 322 (Center, Level 3)
Introduction::

Bone metastases occur in over 80% of advanced breast cancer patients. They result in significant morbidity and ultimately high mortality, but the underlying mechanisms remain poorly understood. While overt bone metastasis manifests by osteolysis in the clinic, cancer cells initially colonize osteogenic niches in bone. Mesenchymal Stem Cells (MSCs) and their osteoblast derivatives make up the osteogenic niche and regulate bone mineralization homeostasis. Although decreased bone mineral density predicts increased risk of bone metastasis, how the bone extracellular matrix (ECM), which is primarily composed of mineralized collagen I fibrils, regulates MSC phenotype and how these changes in turn impact tumor cells remains poorly understood. To better understand these connections, model systems are needed that enable selective control over the degree of collagen mineralization for mechanistic studies. Here, I am using biofunctional materials systems to study the hypothesis that reduced bone matrix mineralization and breast cancer-derived factors synergize to promote tumor growth and metastasis by inhibiting MSC differentiation into osteoblasts, while supporting their differentiation into cancer-associated fibroblasts (CAFs) through altered mechanosignaling. Results from these studies will advance our understanding of the cues that regulate early stages of bone metastasis and determine potential targets for future clinical intervention.





Materials and Methods:: Our group has developed a bone matrix model consisting of physiologically mineralized collagen fibrils within polydimethylsiloxane (PDMS) microwells. Intrafibrillar mineralization was achieved by incubation in a calcium, phosphate and polyaspartic acid-containing solution using our established protocols. Human MSCs (RoosterBio) were seeded on mineralized or non-mineralized collagen controls. Using this model system, MSC differentiation into osteoblasts and CAFs was analyzed through colorimetric, immunofluorescence, and ELISA-based assays. Tumor conditioned media (TCM) was collected from cultures of metastatic MDA-MB231s (ATCC), concentrated, and reconstituted in fresh media. MSCs were treated with TCM or control media in the presence and absence of osteogenic induction factors. Osteogenic differentiation of MSCs was assessed by an alkaline phosphatase (ALP) enzymatic activity assay. MSC fibronectin deposition was measured via immunofluorescence staining, while MSC secretion of interleukin-6 (IL-6) was assessed via an enzyme-linked immunoassay (ELISA). In addition, matrix-dependent differences in tumor cell growth were assessed after 5-day culture of MDA-MB231s (ATCC) on mineralized or control collagen microwells in the presence or absence of MSC-deposited matrix. Substrates incorporating matrix deposited by MSCs were obtained by culturing MSCs for 7 days, followed by detergent-based decellularization. Finally, to examine altered mechanotransduction as a mechanism mediating MSC phenotypic changes, we performed traction force microscopy (TFM) and interfered with mechanotransduction through inhibition of key steps in the mechanosignaling cascade. Briefly, for TFM experiments MSCs were pre-cultured in mineralized or control collagen for 7 days and subsequently transferred to 10 kPa polyacrylamide (PA) gels with fluorescent beads embedded, imaged, and analyzed as previously described.




Results, Conclusions, and Discussions:: Our results suggest that MSCs interacting with mineralized versus non-mineralized collagen differentiate more readily into osteoblasts as indicated by increased alkaline phosphatase (ALP) activity. Conversely, MSCs cultured on non-mineralized collagen likely differentiate into CAFs, as suggested by their increased deposition and alignment of fibronectin, increased inflammatory cytokine secretion, and increased total traction forces. Interestingly, decellularized matrix deposited by MSCs on non-mineralized collagen versus mineralized collagen promoted tumor cell growth. Treatment of MSCs with TCM mediated similar effects which were less pronounced when MSCs were cultured on substrates with physiological mineral content. Importantly, mineral-dependent MSC differentiation into osteoblasts was independent of actomyosin contractility consistent with our previous findings that mineralization inhibits collagen remodeling and strain-stiffening that otherwise promote mechanosignaling (data not shown). Overall, reduced bone matrix mineralization likely promotes tumor growth by inhibiting MSC osteogenic differentiation while promoting a CAF-like phenotype. Whereas tumor-secreted factors can induce similar changes, physiological bone matrix mineralization partly compensates for these effects. Collectively, our results suggest that reduced bone mineral density, a known risk factor for bone metastasis, guides the formation of a favorable niche for tumor cell growth during early stages of metastasis.




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