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. 2017 Jan 3;8(1):118-132.
doi: 10.18632/oncotarget.9497.

Zoledronic acid impairs stromal reactivity by inhibiting M2-macrophages polarization and prostate cancer-associated fibroblasts

Giuseppina Comito  1 , Coral Pons Segura  1 , Maria Letizia Taddei  1 , Michele Lanciotti  2 , Sergio Serni  2 , Andrea Morandi  1 , Paola Chiarugi  1   3 , Elisa Giannoni  1
Affiliations

Zoledronic acid impairs stromal reactivity by inhibiting M2-macrophages polarization and prostate cancer-associated fibroblasts

Giuseppina Comito et al. Oncotarget. .

Abstract

Zoledronic acid (ZA) is a biphosphonate used for osteoporosis treatment and also proved to be effective to reduce the pain induced by bone metastases when used as adjuvant therapy in solid cancers. However, it has been recently proposed that ZA could have direct anti-tumour effects, although the molecular mechanism is unknown. We herein unravel a novel anti-tumour activity of ZA in prostate cancer (PCa), by targeting the pro-tumorigenic properties of both stromal and immune cells. Particularly, we demonstrate that ZA impairs PCa-induced M2-macrophages polarization, reducing their pro-invasive effect on tumour cells and their pro-angiogenic features. Crucially, ZA administration reverts cancer associated fibroblasts (CAFs) activation by targeting the mevalonate pathway and RhoA geranyl-geranylation, thereby impairing smooth muscle actin-α fibers organization, a prerequisite of fibroblast activation. Moreover, ZA prevents the M2 macrophages-mediated activation of normal fibroblast, highlighting the broad efficacy of this drug on tumour microenvironment. These results are confirmed in a metastatic xenograft PCa mouse model in which ZA-induced stromal normalization impairs cancer-stromal cells crosstalk, resulting in a significant reduction of primary tumour growth and metastases. Overall these findings reinforce the efficacy of ZA as a potential therapeutic approach to reduce cancer aggressiveness, by abrogating the supportive role of tumour microenvironment.

Keywords: cancer-associated fibroblasts; macrophages; mevalonate pathway; prostate cancer; zoledronic acid.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. ZA suppresses monocyte differentiation toward M2 macrophages
A, B. Human monocytes isolated from normal donor buffy coat were cultured for 7 days with M-CSF (50ng/ml). Differentiation toward the M1 phenotype was induced by treatment with LPS (100 ng/ml) and IFNγ (100 ng/ml) for 24h. M2 macrophages were polarized by stimulating with IL-4 (20 ng/ml) for 24h. Alternatively, M2-like macrophages were obtained by treating isolated monocytes with CM from PC3 for 7 days. During differentiation macrophages were treated with different concentrations of ZA and the levels of IL-12 or IL-10 were measured by ELISA test. 1-way ANOVA, Dunnett's corrected, ***p< 0,001 vs untreated. C. Cells treated as in A were lysed and the expression of M-CSFR, COX-2, CD206, NOS2 and actin was evaluated by immunoblots.
Figure 2
Figure 2. M2 and M2-like macrophages-dependent increase of PC3 cells invasiveness is impaired by ZA treatment
A. Monocytes were differentiated for 7 days with M-CSF and then polarized into M2 macrophages by stimulating with IL-4 for 24h. Alternatively, monocytes were stimulated with CM from PC3 for 7 days to obtain M2-like macrophages. ZA was administrated during differentiation at different concentrations and then macrophages were serum-starved for 48h to obtain the corresponding CM. PC3 cells were incubated for 24h with CM from the above differentiated macrophages (treated or not with ZA), or serum starved as a control, and then allowed to invade toward medium containing 10% serum as chemoattractant for additional 24 h. (St. Med. Starvation Medium) B. Invading cells were counted and the mean of six randomly chosen fields was plotted in the bar graph. 1-way ANOVA, Dunnett's corrected, ***p< 0,001 vs untreated.
Figure 3
Figure 3. Treatment with ZA inhibits the M2/M2-like macrophages-induced pro-angiogenic effect
M2 and M2-like macrophages were obtained by treating monocytes for 7 days with M-CSF (then treated with IL-4 for additional 24h) or with CM from PC3, respectively. M2 and M2-like macrophages were treated with different concentrations of ZA during differentiation and then were serum-starved for 48h to obtain the corresponding CM. HUVEC A. and EPC B. cells were treated with CM from the above differentiated macrophages (treated or not with ZA) and in vitro angiogenesis was evaluated by capillary morphogenesis assay. The number of joints was quantified and plotted in the bar graph. 1-way ANOVA, Dunnett's corrected, ***p< 0,001 vs untreated.
Figure 4
Figure 4. ZA administration reverts CAF activation and impairs their pro-invasive effects on cancer cells
A. Subconfluent HPFs and CAFs were serum starved and treated for 5 days with different concentration of ZA. α-SMA was analyzed by immunoblot as a marker for fibroblast activation. Actin was used as loading control. B. HPFs and CAFs were treated in serum-free medium with ZA 200nM for 5 days and after were placed in DMEM supplemented with collagen. Serum-starved HPFs and CAFs were used as control cells. Contraction of the collagen discs is expressed as relative collagen area from ST group. 1-way ANOVA, Bonferroni's corrected***p< 0,001 vs CAF. C. PC3 cells were incubated for 24 h with CM from HPFs and CAFs treated as in A and then allowed to invade for additional 24 h toward medium containing 10% serum as chemoattractant. Invading cells were counted and the mean of six randomly chosen fields was plotted in the bar graph. 1-way ANOVA, Dunnett's corrected, ***p< 0,001 vs CM CAF.
Figure 5
Figure 5. ZA inhibits RhoA prenylation/activation, thereby abrogating α-SMA organization and CAF activation
A. CAFs were treated in serum-free medium with ZA 200nM for 5 days alone or in combination with geranylgeranyl pyrophosphate 20 μM (ZA+GGPP) or Rho Activator (ZA+RhoAct). Serum-starved HPFs and CAFs were used as control cells. ActiveGTP-bound RhoA was quantified by Rhotekin pull-down assay and normalized by total RhoA immunoblot upon densitometric analysis. 1-way ANOVA, Bonferroni's corrected, *p< 0,05 vs HPF, **p< 0,01 vs CAF, ***p< 0,001 vs CAF+ ZA. B-C. Fibroblasts were treated as in A. α-SMA was quantified by both immunoblot (B) and confocal microscopy. Bar, 50 μm (C).
Figure 6
Figure 6. ZA counteracts macrophage-dependent fibroblasts activation
A. M2 and M2-like macrophages were obtained by treating monocytes for 7 days with M-CSF (then treated with IL-4 for additional 24h) or with CM from PC3, respectively. M2 and M2-like macrophages were treated with different concentrations of ZA during differentiation and then were serum-starved for 48h to obtain the corresponding CM. Subconfluent HPFs were treated for 24 h with CM from the above differentiated macrophages (treated or not with ZA) or with 10 ng/ml TGF-β1 as a positive control, and serum starved for additional 24 h. Fibroblasts were lysed and immunoblots for α-SMA and actin were performed. B. M2 and M2-like macrophages were obtained as in A and were treated with ZA 200nM during differentiation and then were serum-starved for 48 h to obtain the corresponding CM. Subconfluent HPFs were treated for 24 h with CM from the above differentiated macrophages (treated or not with ZA) or with 10 ng/ml TGF-β1 as a positive control, α-SMA was quantified by confocal microscopy. Bar, 50 μm. C. PC3 cells were incubated for 24 h with CM from HPFs treated as in A (CM M2 AFs or CM M2-like AFs; AFs: Activated fibroblasts) and then allowed to invade for additional 24 h toward medium containing 10% serum as chemoattractant. Invading cells were counted and the mean of six randomly chosen fields was plotted in the bar graph. Representative photographs for each sample are shown under the corresponding bar. 1-way ANOVA, Dunnett's corrected, ***p< 0,001 vs untreated.
Figure 7
Figure 7. ZA in vivo administration prevents tumor growth and lung metastatic dissemination
A. A mixture of 1 × 106 PC3 cells and 0.5×106 CAFs were subcutaneously injected in both the lateral flanks of SCID bg/bg mice. Mice were treated once a week with intraperitoneal injection of PBS (control mice) or 100 μg/kg of ZA, for 6 weeks (n=5 per group), starting the treatment 24 h after cell injection. The volume of the primary tumour for control or ZA-treated mice was reported at different time points. 2-way ANOVA, Bonferroni's corrected ***p<0,001 vs untreated B. Mice were sacrificed after 6 weeks. Lungs were inspected and the number of micrometastases were counted and plotted. Representative paraffin-embedded lung tissue sections, stained with hematoxylin/eosin, are shown. Student t-test, **p<0,0036 vs PC3+CAF.
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