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Supplementary Figure 5 from Tumor-Induced Pressure in the Bone Microenvironment Causes Osteocytes to Promote the Growth of Prostate Cancer Bone Metastases

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posted on 2023-03-30, 23:06 authored by Joseph L. Sottnik, Jinlu Dai, Honglai Zhang, Brittany Campbell, Evan T. Keller

Supplementary Figure 5: MMPs promote PCa migration and invasion. A) CM was isolated from MLO-Y4 and PCa cell lines, normalized by total protein concentration, and compared by zymography for MMP expression. Densitometry of zymograph assays is also presented. B) Densitometry of zymograph in Figure 4A of MMP activity. C) mRNA from MLO-Y4 cells exposed to pressure for 24 hours was analyzed by real time PCR for MMP2 and MMP9 expression and normalized to GAPDH. D) MLO-Y4 CM (0 mmHg) was incubated with batimastat to inhibit OCy-derived MMP. CM with batimastat or DMSO control where utilized to determine alterations in PCa migration and invasion. E) PCa cells were incubated in the indicated CM and recombinant MMP2 added at the indicated concentrations. MMP2 concentrations from 40 mmHg CM were recapitulated in 0 mmHg CM through addition of recombinant (exogenous) MMP2. Invasion of PCa towards CM with additional MMP2 was measured. PCR, densitometry, and MMP2 addition experiments were analyzed using One-way ANOVA with Bonferroni post-test. Student's two-tailed t-test was used to analyze MMP inhibition experiments. All bars represent significant relationships of p>0.05.

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ARTICLE ABSTRACT

Cross-talk between tumor cells and their microenvironment is critical for malignant progression. Cross-talk mediators, including soluble factors and direct cell contact, have been identified, but roles for the interaction of physical forces between tumor cells and the bone microenvironment have not been described. Here, we report preclinical evidence that tumor-generated pressure acts to modify the bone microenvironment to promote the growth of prostate cancer bone metastases. Tumors growing in mouse tibiae increased intraosseous pressure. Application of pressure to osteocytes, the main mechanotransducing cells in bone, induced prostate cancer growth and invasion. Mechanistic investigations revealed that this process was mediated in part by upregulation of CCL5 and matrix metalloproteinases in osteocytes. Our results defined the critical contribution of physical forces to tumor cell growth in the tumor microenvironment, and they identified osteocytes as a critical mediator in the bone metastatic niche. Cancer Res; 75(11); 2151–8. ©2015 AACR.