Tumor-induced pressure in the bone microenvironment causes osteocytes to promote the growth of prostate cancer bone metastases

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.

Figure 1. Prostate cancer growth in bone leads to elevated intramedullary pressure

A) SCID mice (n = 4 per group) were challenged with 1×10⁶ tumor cells 24 hours prior to implantation of a wireless pressure monitoring device as depicted in the schematic. B) Mice challenged with DU145 showed a significant (p<0.05) increase in intramedullary pressure, with a peak average pressure of 37.84 mmHg 16 days after tumor challenge, compared to sham injected mice. C) Radiographs depict weakening of the cortical bone (arrow) beginning 2 weeks post tumor challenge, with loss of cortical bone 3 weeks post-challenge. Initiation of cortical thinning is associated with the drop in ImP observed. D) Mice challenged with ACE1 showed similar changes in pressure as observed in mice challenged with DU145. E) Radiographs from mouse challenged with ACE-1 show cortical bone decay similar to DU145 at 2 weeks. Together, these data provide show that PCa growth in bone leads to significant increases in intramedullary pressure. Two-way repeated measures ANOVA was utilized for statistical comparisons.

Figure 2. Application of hydrostatic pressure to osteocytes promotes PCa aggressiveness

A) Hydrostatic pressure was applied in vitro to MLO-Y4 osteocytes and DU145, LNCaP, and PC3 PCa cell lines. After 24 hours, cells were counted using trypan blue staining and live cell count depicted. B) CM was isolated from MLO-Y4 OCy under pressure at 0, 20, and 40 mmHg for 24 hours. Control media (RPMI with 0.1% FBS) was used as a negative control. OCy CM was then applied to PCa cell lines for 24 hours to determine changes in viability as measured by resazurin. CM was used as a chemoattractant in Boyden chamber assays to determine alterations in migration and invasion. One way ANOVA with Bonferroni post-test used for all analyses; bars represent significant differences of p<0.05.

Figure 3. Identification of CCL5/RANTES as an OCy secreted mediator of enhanced PCa invasion

A) CM prepared from MLO-Y4 cells pressurized at 0 and 40 mmHg for 24 hours was compared using a cytokine screening antibody array and subjected to densitometry. Results are shown as change in optical density (OD) of the cells subjected to 40 mmHg relative to the 0 mmHg. B) Real time PCR of CCL5 from pressurized MLO-Y4 cells was performed and normalized to GAPDH. C) CCL5 secreted from OCy, normalized by protein concentration, increases as the level of hydrostatic pressure increases as measured by ELISA. D) Recombinant murine CCL5 (rCCL5; 10 μg/ml) was used as a chemoattractant for PCa migration and invasion. Neutralization of rCCL5 was accomplished by incubating with anti-CCL5 neutralizing antibody (1 μg/ml) but not with isotype control antibody (1 μg/ml). rCCL5 neutralization inhibited migration across all cell lines. However, invasion of LNCaP and PC3 was not altered by the neutralization of rCCL5. E) CM derived from MLO-Y4 cells pressurized at 0, 20, and 40 mmHg was incubated with CCL5 neutralizing antibody before being used as a chemoattractant in migration and invasion assays. Neutralization of CM derived CCL5 led to significant inhibition of PCa migration and invasion despite increasing pressure. Secretion of CCL5 by MLO-Y4 is induced by tumor generated pressure leading to subsequent increases in tumor cell invasiveness. One way ANOVA with Bonferroni post-test (C/D) or two-way ANOVA (E) was performed; bars represent significant differences where p<0.05.

Figure 4. Inhibition of OCy derived MMPs mitigates pro-tumorigenic responses

A) MLO-Y4 was subjected to 0, 20, or 40 mmHg pressure for 24 hours, CM prepared and normalized by protein concentration as measured by BCA assay prior to analysis by zymography. BPH-1 was used as a positive control for MMP expression and plain cell culture media used as a negative control. Bands were subjected to densitometry and reported as percent active MMP2 and MMP9. CM was normalized by protein concentration, as measured by BCA, and MMP2 analyzed by ELISA. B) MLO-Y4 CM was incubated with batimastat, a broad spectrum MMP inhibitor, to inhibit OCy derived MMP. 40 mmHg CM with batimastat or DMSO control where then utilized to determine alterations in PCa migration and invasion. C) A proposed model system for tumor induced pressure promoting PCa aggressiveness. PCa proliferation promotes pressure in bone leading to induction of OCy production of CCL5, which can induce MMPs, and MMPs themselves. MMPs degrade the bone matrix releasing a variety of growth factors while the combination of CCL5 and MMPs promote further invasion into bone. One way ANOVA with Bonferroni post-test used for ELISA analysis; bars represent significant differences of p<0.05. Student’s two-tailed t-test was used to analyze MMP inhibition experiments. All bars represent significant differences where p<0.05.