Periprostatic adipocytes act as a driving force for prostate cancer progression in obesity

Obesity favours the occurrence of locally disseminated prostate cancer in the periprostatic adipose tissue (PPAT) surrounding the prostate gland. Here we show that adipocytes from PPAT support the directed migration of prostate cancer cells and that this event is strongly promoted by obesity. This process is dependent on the secretion of the chemokine CCL7 by adipocytes, which diffuses from PPAT to the peripheral zone of the prostate, stimulating the migration of CCR3 expressing tumour cells. In obesity, higher secretion of CCL7 by adipocytes facilitates extraprostatic extension. The observed increase in migration associated with obesity is totally abrogated when the CCR3/CCL7 axis is inhibited. In human prostate cancer tumours, expression of the CCR3 receptor is associated with the occurrence of aggressive disease with extended local dissemination and a higher risk of biochemical recurrence, highlighting the potential benefit of CCR3 antagonists in the treatment of prostate cancer.

Du-145 cells were pre-incubated or not with the SB225002 (CXCR1/2 inhibitor, 50nM), AMD3100 (CXCR4 inhibitor, 100nM), sc-202525 (CCR2 inhibitor, 25nM) or UCB35625 (CCR1/3 inhibitor, 200nM) for 30 minutes and migration towards Ad-CM (obtained from in vitro differentiated 3T3-F442A mature adipocytes) was performed for 12 h in the presence of inhibitors. (c) Similar experiments were performed in the presence of cells treated or not with control isotype, anti-CXCR1, anti-CXCR2, anti-CXCR4, anti-CCR1 or anti-CCR3 mAbs, all used at 10μg ml -1 . Data are expressed as the percentage of migrating cells relative to the migration of untreated cells (set to 100%) and are shown as mean±s.e.m (n=3). The statistical significance of differences between means of migrating cells (in %) in treated versus untreated (NT) cells was evaluated with Student's t-tests. Statistical analysis: * statistically significant by Student's t-test, p<0.05, *** p<0.001, NS for not significant. n stands for the number of replicated independent experiments.

sc-202525
Supplementary Figure 2. Pharmacological inhibitors used in migration assays are not toxic for prostate tumor cells. The effect of the chemokine receptors inhibitors on PC-3 cells viability was determined using MTT assays. Cell viability was determined with cells exposed during 24 h to increasing doses of the pharmacological inhibitors. Data are expressed as the number of treated viable cells relative to the viability of untreated cells (set to 1) and are shown as mean±s.e.m (n=3). The statistical significance of differences between means of viable cells (in %) in treated versus untreated (NT) cells was evaluated with Student's t-tests. Statistical analysis: NS for not significant. n stands for the number of replicated independent experiments.  Figure 3. CCR3 receptor is a key player in prostate cancer cell migration towards Ad-CM compared to CXCR2, CXCR4 and CCR2. PC-3 cells were pre-incubated or not with the SB225002 (CXCR1/2 inhibitor, 50nM), AMD3100 (CXCR4 inhibitor, 100nM), sc-202525 (CCR2 inhibitor, 25nM) or UCB35625 (CCR1/3 inhibitor, 200nM) for 30 minutes and migration towards Ad-CM (obtained from in vitro differentiated 3T3-F442A mature adipocytes) was performed for 12 h in the presence of inhibitors (used alone or in combination). Bar plots represent the percentage of migrating cells relative to the migration of untreated cells (set to 100%). Data are shown as mean±s.e.m (n=3). The statistical significance of differences between means of migrating cells (in %) in treated versus untreated (NT) cells was evaluated with Student's t-tests. Statistical analysis: Statistical analysis: * statistically significant by Student's t-test p<0.05, ** p<0.01, *** p<0.001, NS for not significant. n stands for the number of replicated independent experiments.   . Cells were pre-incubated with the inhibitors or mAbs for 30 minutes and the inhibitors or mAbs remained present during the migration experiments (12 h for prostate, breast, pancreas, colon cancer models and 24 h for melanoma cells). The line surrounding the histogram indicates the mAb that induces the highest inhibitory effect in cell migration. Bar plots represent the percentage of migrating cells relative to the migration of untreated cells (set to 100%). Data are shown as mean±s.e.m (n=3). The statistical significance of differences between means of migrating cells (in %) in treated versus untreated (NT) cells was evaluated with Student's t-tests. Statistical analysis: * statistically significant by Student's t-test p<0.05, ** p<0.01, *** p<0.001, NS for not significant. n stands for the number of replicated independent experiments. Pearson's correlation between leptin (marker of adipocytes hypertrophy) and CCL7 expression. The correlation between leptin and CCL7 expression suggests that CCL7 secretion is related to the hypertrophic state of adipocytes.
Supplementary Figure 9. The directed migration of prostate cancer cells towards the secretions of SVF does not involve the CCL7/CCR3 axis. In vitro migration of PC-3 cells treated or not with the CCR3 inhibitor (UCB35625, 200nM) towards medium without serum (negative control) or CM obtained from adipocytes or SVF cells isolated from the VAT of C57BL/6 lean or obese mice (six animals per group). Data are shown as mean±s.e.m (n=3). Statistical analysis: * statistically significant by Student's t-test p<0.05, ** p<0.01, *** p<0.001, NS for not significant. n stands for the number of replicated independent experiments. Note that the % of migrating cells was significantly higher when SVF-CM was used as a chemoattractant compared to 0% serum. The migration of prostate cancer cells towards SVF-CM was unaffected by obesity and CCR3 antagonist.  Figure 11. CXCR4/CXCL12 axis is poorly involved in the migration towards Ad-CM obtained from lean and obese mice. (a) CXCL12 secretion by primary adipocytes or SVF cells isolated from mu-VAT of C57BL/6 lean or obese mice (six animals per group). Data are shown as mean±s.e.m (n=3). (b) Adipocytes were isolated from the mu-vat of lean or obese C57bl6 mice (six animals per group). In vitro migration of PC-3 cells towards mu-VAT adipocyte-CM was performed in the presence or absence of blocking mAbs directed against CXCR4 or CXCL12, or control IgG (10μg ml -1 ). Data are shown as mean±s.e.m (n=3). The statistical significance of differences between means of migrating cells (in %) in treated versus untreated cells was evaluated with Student's t-tests. The differences between the percentages of migrating cells towards Ad-CM isolated from mu-VAT from obese versus lean mice are also shown. (c) Inhibition of the CCR3/CCL7, but not CXCR4/CXCL12 axis totally abrogates the enhanced chemotaxis observed in obesity. The histograms represent the ratio of migration towards conditioned medium of primary isolated adipocytes from obese to lean mice in each treated conditions. Statistical analysis: * statistically significant by Student's t-test p<0.05, *** p<0.001, NS for not significant. n stands for the number of replicated independent experiments. ). A: picture of the whole slice after staining with CCR3; (B, C): zoom of the intra-tumoral CCR3 staining in two representative zones; (D, E): zoom of the CCR3 staining at the invasive front in two representative zones. Scale bars, for A, 500 µm and for B, C, D, E, 100 μm. (b) Histograms show CCR3 expression in tumor cells at the invasive front compared to intra-tumoral areas for four lean (BMI<25) and four overweight/obese patients (BMI>25). Data are shown as mean±s.e.m (n=4 tumors per group). Tumor glands (showed with black arrows) were defined with two pathologists and the stained areas of tumors were digitalized and protein expression was quantified using ImageJ software plugins. Note that some blood vessels (blue stars) or nerve tissues are also strongly stained but quantification only considered tumor glands. Statistical analysis: * statistically significant by Student's t-test p<0.05, ** p<0.01, *** p<0.001.  Results obtained for adiponectin and resistin, two well-known adipokines, are also indicated. The number of experiments in which proteins have been identified (NE), the best score (score), the number of peptides (PN) and the average sequence coverage for each protein are indicated. The presence of chemokines and adipokines in the conditioned medium was confirmed by ELISA and multiplex analysis. Data obtained (mean of two independent experiments) are as following: CCL2 (6.3ng ml -1 for 1x10 5 cells), CCL7 (28.3ng ml -1 for 1x10 5 cells), CXCL1 (32.6ng ml -1 for 1x10 5 cells), CXCL12 (0.8ng ml -1 for 1x10 5 cells), Adiponectin (32ng ml -1 for 1x10 5 cells) and Resistin (1.7ng ml -1 for 1x10 5 cells).
Moreover seeing that CCL5 (absent of our proteomic analysis) has been identified in the secretion of mammary adipocytes we have determined CCL5 concentration in Ad-CM by ELISA. The results showed that the level of CCL5 was almost undetectable (mean concentration 0.01ng ml -1 for 1x10 5 cells).

Supplementary Table 2:
Immunohistochemical staining for CCR3, as well as hematoxylin counterstaining, was performed in normal epithelium (10 samples) and human prostate cancer tissues (91 tumors in duplicate). Two pathologists, who were blind to clinical data, independently scored CCR3 expression. The Gleason score characterizes the glandular architecture of the prostate based on a score that represents the level of cancer de-differentiation. The Gleason score is comprised of two numbers, each representing the most common Gleason patterns ranging from 1 to 5, where 1 represents a highly differentiated carcinoma and 5 represents an aggressive de-differentiated one. Therefore the highest Gleason score is 10 and the lowest 2. A related issue is the clinical usefulness of the percentage of high-grade carcinoma (Gleason patterns 4 and 5) in a tumor specimen. The correlation between CCR3 expression and Gleason score has been evaluated by Spearman rank correlation assuming a monotonic relation between considered variables.