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Cancer Technologies

Cancer Mechanobiology

Dynamic Interstitial Fluid Flow Alters Breast Cancer Cells and Osteocytes in Bone Metastasis

Saturday, October 26, 2024
2:30 PM – 2:45 PM EST
Location: Room 336

Introduction:  Breast cancer (BCa) metastasis to bone is a common, yet incurable, diagnosis. BCa cells in the skeleton dysregulate bone tissue remodeling via secretion of cytokines, which ultimately causes extensive bone loss and fragility. Increased mechanical loading (akin to exercise), a strong anabolic signal in bone, may be osteoprotective and anti-tumorigenic. However, the underlying mechanisms are currently unknown, in part because their response to mechanical signals in bone, which are dynamic and higher in magnitude, is relatively unknown. Thus, we are investigating how anabolic bone signals impact breast cancer function. Osteocytes are specialized mechanosensory bone cells that that regulates the bone remodeling balance. We have previously shown that breast cancer cell-secreted soluble factors impaired the mechanoresponse of early-stage osteocytes. Because mature osteocytes constitute 95% of all bone cells, in this study, we also tested whether mature osteocytes were impacted by breast cancer cells.

Materials and

Methods:  CELLS: Human triple negative [MDA-MB-231 and a bone-homing subclone], and human ER+ [ZR-75-1] BCa cells. Mature osteocyte cells from mouse mesenchymal progenitor cells (MPCs) [2]. BONE-MIMICKING SCAFFOLD: PLGA scaffolds that incorporate bone hydroxyapatite crystals were fabricated via gas foaming-salt leaching. Cells were seeded drop-wise onto scaffolds (1 million BCa cells, or 500K MPCs). MPCs differentiated into mature osteocytes via osteogenic media for 2 weeks prior to experimental treatment. MECHANICAL LOADING: Cell-seeded scaffolds underwent pulsatile forced perfusion using a custom perfusion bioreactor for 3 days (2 hr/d, 1 Hz) at inlet velocities of 1, 5, or 15 mL/min (+ static control), which engenders anabolic wall shear stresses within scaffold pores. CONDITIONED MEDIA: 24 hours after the final loading bout, conditioned media from MDA-MB-231 and ZR-75-1 BCa cells was screened for soluble cytokines via cytokine array (Proteome Profiler XL Oncology Array) as well as collected for culture with MPCs. BREAST CANCER RNA-SEQ: Following perfusion, bone-homing MDA-MB-231 total RNA was extracted, and cDNA libraries were generated for sequencing. mRNA was isolated via capture bead technology and transcribed into cDNA. Resulting libraries were assessed for quality control and sequenced at 20 million reads per sample. OSTEOCYTE FUNCTION: MPC-seeded scaffolds were treated with 50% BCa conditioned media and 50% osteogenic media, and underwent perfusion as described. Following perfusion, mRNA was collected for qPCR analysis of genes related to the osteocyte mechanoresponse [sclerostin (SOST), receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG)].

Results, Conclusions, and Discussions: Most cytokines were altered with flow, but their secretion levels unexpectedly did not show a dose-response, suggesting they are more sensitive to other microenvironmental factors than wall shear stress magnitude. Preliminary RNAseq analysis indicates that the impacts of perfusion on bone-homing MDA-MB-2331 BCa cells was mixed. Specifically, several pro-tumorigenic markers – receptor activator of nuclear factor-κB ligand (RANK), matrix metalloproteinase-9 (MMP-9), and receptor for advanced glycation end products (RAGE) - were down-regulated by flow, which may partially explain the anti-tumorigenic effects of increased mechanical loading. However, BCa expression of SOST and DKK-1, potent negative regulators of bone formation, were unchanged with fluid shear, which was unexpected given our previous data showing BCa-induced osteocyte impairment. When mature osteocytes were exposed to MDA-MB-231 (triple negative) conditioned media, their mechanoresponse was impaired as indicated by no loading-induced changes in SOST, RANKL, or OPG, results that are similar to what we reported in early-stage osteocytes before. However, the impacts from ZR-75-1 (ER+) conditioned media were mixed, in osteocytes retained their ability to decrease SOST in response to loading even though RANKL and OPG remained unchanged, but. In conclusion, breast cancer cells seem to impair the mechanoresponse of osteocytes, but this dependent upon BCa sub-type.

Acknowledgements (Optional):  NSF (#2047187), Bone-homing MDA-MB-231 cell subclone – Dr. Julie Rhoades (Vanderbuilt University)