Prostate cancers frequently have a indolent course even if left without active treatment . However, clinically relevant disease with significant morbidity and mortality also occurs in a significant number of patients . The mechanisms responsible for this aggressive behavior remain elusive, albeit it is well established that the supporting tumor microenvironment has a decisive role in controlling prostate cancer growth, invasion and metastasis .
Cancer-implicated mammary and colonic fat pads [11, 21] are physically close to epithelial cells, whereas in prostate there is initially a capsular-like structure separating the PP fat from tumor cells. Nevertheless, frequently prostate tumors infiltrate the PP fat pad by transposing or infiltrating the physical barriers, resulting in immediate proximity to adipose tissue. Once extension beyond the capsule occurs, the PP adipose tissue-secreted factors, extracellular matrix components or direct cell-cell contact may influence the phenotypic behavior of malignant cells. Recent studies observed that PP adipose tissue thickness was linked to prostate cancer severity , while its secretory profile associated with advanced disease . In the present study, we found that PP adipose tissue-derived conditioned media may potentiate prostate cancer aggressiveness through modulation of metalloproteinases activity, and by promoting cancer cell proliferation and migration.
In tumors, cancer cells are not the only source of MMPs. In our study, MMP9 activity was significantly elevated in the PP adipose tissue of overweight/obese men (BMI ≥ 25 Kg/m2), implying excess body fat and the PP fat depot in the modulation of extra-capsular cancer cells' microenvironment. Concordantly, other studies found MMP9 to be positively correlated with BMI . Further research is warranted to uncover the effects of MMPs in association with distinct obesity grades. In our sample only two subjects presented BMI > 30 Kg/m2, limitating such approach.
Matrix metalloproteinases are proteolytic enzymes that regulate many cell mechanisms with prominence in cancer biology . Their expression in prostate tumors is related with disease progression and metastasis , whereas MMP9 was shown to increase growth factors bioavailability and to elicit epithelial-to-mesenchymal transition in tumor cells [25, 26], therefore promoting an aggressive phenotype. A recent report indicated that oesophageal tumors from obese patients express more MMP9 and that co-culture of VIS adipose tissue explants with tumor cells up-regulated MMP2 and MMP9 . Remains undetermined the influence of PP adipose tissue in the expression of MMPs by prostate cancer cells, which might further contribute towards an aggressive phenotype. Noteworthy, cancer-derived factors stimulate other surrounding cells, including adipose tissue cells, to synthesize MMPs .
In an effort to understand if the effects of PP adipose tissue extend to other aggressiveness characteristics, we used adipose tissue-derived CM to perform cell proliferation assays in prostate cancer cell lines. We found that CM from in vitro culture of adipose tissue explants stimulated the proliferation of hormone-refractory prostate cancer cells. Conversely, this media inhibited growth in hormone-sensitive cells.
It is well-established that adipose tissue secretes a wide array of molecules . These adipokines, exclusively or partially secreted by adipocytes or stromal-vascular fraction cells, are likely to have a role in modulating the risk of cancer progression [1, 29, 30]. Few studies examined the effect of adipocytes in prostate cancer cells growth [12, 13]. While a proliferative effect was observed in hormone-refractory PC-3 cells, these findings didn't replicate in LNCaP cells . In fact, the mitogenic and anti-apoptoptic effects of several adipokines, alone and combined, in prostate cancer cell growth (e.g. leptin, IL-6, insulin-like growth factor 1, IGF-1), seems to be limited to hormone-refractory prostate cancer cells [12, 31–34]. Previous studies also report on the suppression of LNCaP cell growth as response to adipokines (e.g. TNF-α, decreased expression of vascular endothelial growth factor, VEGF), not observed in hormone-refractory cells [13, 35–37].
Contrary to explants, CM from SVF cultures induces cancer cell proliferation, independently of cell line, except for the SVF from PP adipose tissue in PC-3 cells. Cells that constitute the SVF fraction of adipose tissue, where macrophages have a modulatory role, are known to secrete several angiogenic and antiapoptotic factors [38–40], which ultimately can impact prostate cancer cells growth. The lack of proliferative effect observed for the SVF fraction from PP adipose tissue may partially be due to the reported low number of macrophages in PP fat depot , diminishing the proliferative stimulus in prostate cancer cells.
Progression to an invasive and metastatic phenotype is responsible by prostate cancer mortality and morbidity. The increased cellular motility is another parameter associated with increased metastatic potential [41, 42]. By employing time-lapsed imaging, we found that factors produced by whole adipose tissue cultures (explants) increased significantly the migration speed and the final relative distance to origin of both PC-3 and LNCaP cells compared with control. Only the SVF fraction-derived CM effect in the final relative distance to origin of PC-3 cells, was not increased compared with control.
The mechanisms involved in tumor cell movement are far from fully elucidated, although various biophysical processes are considered to be involved : in order for a cell to move it must be polarized or have a sense of directionality; polarity is accompanied by 1) lamellipodia protrusion at the leading edge, followed by 2) detachment of the cell's rear end and subsequent 3) transcellular contractility. These mechanisms are modulated by the activation of several signaling pathways, such as PI3K, ERK/MAPK and c-Src tyrosine kinase , which are known downstream signals of adipokines . In fact, many adipokines (e.g. IGF-1, osteopontin, leptin, adiponectin, VEGF, thrombospondin, interleukin-8 and IL-6) have been shown to modulate different steps of cell motile behavior [44–56]. The repetitive and coordinated cycling of these processes results in productive locomotion of the cell. Several key pathways and molecules involved in this process can be induced by factors secreted by adipose tissue, hence supporting the increased motility we found in stimulated prostate cancer cells. Nevertheless, besides the influence of extrinsic factors, migratory tumor cells also present autocrine growth factor signaling systems . We disclose any potential bias from inadvertent selection using manual cell tracking analysis, urging careful interpretation of motility findings. Further studies using migration assays to extend and confirm our results are warranted.
Adipose tissue is a heterogeneous organ that consists of multiple cell types: adipocyte fraction, which contains lipid-loaded adipocytes, and stromal-vascular fraction, which includes preadipocytes, endothelial cells, fibroblasts, stem cells, macrophages and other immune cells . The fractions of adipose tissue differ in that while explants reflect an organotypic cell culture system of whole adipose tissue, the major characteristic of stromal-vascular fraction culture is the depletion of adipocytes and absence of extracellular matrix. In order to investigate which fraction influenced tumor cells, we cultured paired explants and stromal-vascular fraction cells. To allow comparison between depots and adipose tissue fractions, the cell count was adjusted per gram of adipose tissue. Interestingly, our findings showed that media from explants and PP adipose tissue depot presented the higher gelatinolytic activity per gram of adipose tissue, compared with SVF cultures- and VIS adipose tissue-derived media. Although the amount of MMP9 has been described to be higher in stromal-vascular fraction of adipose tissue compared with adipocytes , the latter have greater plasticity to increase MMPs expression when interacting with other cells in adipose tissue [22, 59]. The increased activity of metalloproteinases in CM from adipose tissue explants in culture compared with SVF, likely reflect the additive effect or interaction between cells of the stromal-vascular fraction plus adipocytes. We found that MMP2 activity was increased in PP versus VIS adipose tissue supernatants. Although there is no evidence of MMP2 role in adipose tissue/cancer cells crosstalk, recent findings suggest MMP2 is up-regulated in tumor cells co-cultured with adipose tissue explants and that its expression and activation is modulated by several adipokines (e.g. Wdnm1-like and visfatin) [27, 60, 61]. Additionally, other MMPs, notably MMP11, have been shown to be correlated with breast cancer-induced adipocyte's activated state [11, 62]. If confirmed, our findings may reveal a novel specific proteinase expression and activity pattern in PP adipose tissue favorable to prostate cancer progression.
In this study, proliferation was increased with CM from PP and VIS explants versus SVF CM in PC-3 cells, whereas LNCaP cells only proliferated significantly more with VIS explants compared to VIS SVF. As the highest proliferation was seen following stimulation with CM from explants we speculate adipocytes may be the main effectors. Other studies also found a proliferative effect of adipocytes in prostate cancer cells [12, 13]. Adipocytes add significantly to the proliferative effect in hormone-refractory prostate cancer cells, even though the adipokines responsible by these results have yet to be determined. Alternatively, since explants culture preserve the paracrine signals by maintaining the existing crosstalk among the different cell types , we hypothesize that the higher proliferative stimulus conferred by explants CM likely reflects a co-stimulatory and/or additive effect of adipokines produced by adipocytes and by the stromal vascular fraction cells.
Explants-derived CM, whether from VIS or PP origin exerted consistently, also across cell lines, an increased effect in migration speed and final relative distance to origin, when compared with SVF fraction. It is possible that explants CM, which reveal the secretory profile of adipocytes plus stromal-vascular cells, produce more motile factors and exclusive secretion of others (e.g. leptin and adiponectin), thereby resulting in increased total distance/mean speed and final relative distance to origin of prostate cancer cells.
The anatomical origin of adipose tissue accounts for increased gelatinolytic activity and different proliferative and migratory stimulus. CM from PP results in higher log10-transformed PC-3 and LNCaP cell count per gram of adipose tissue, only when SVF CM was used. Furthermore, adipose tissue from PP origin exerted the stronger motile effect (of both analyzed parameters) in PC-3 cells compared to VIS depot, independently of the culture type. In LNCaP cells only the PP explants-derived CM didn't impact the mean speed more than CM from VIS explants. These findings suggest that VIS and PP fat pads may have distinct relative cellular composition or are differently programmed to secrete molecules involved in the regulation of cell proliferation and motility. We recently found increased amount of adipose stem cells (CD34+/CD45-/CD31-/CD146-) in PP compared with VIS adipose tissue (Ribeiro R, unpublished observations).
Tumor cell progression depends on itself as well as on the surrounding microenvironment, which is able to influence proliferation, migration and metastatic behavior of tumor cells by modulating the extracellular matrix and growth factor production . If the tissues where tumor cells exist provide the missing extrinsic signals, then cells will proliferate and acquire an invasive phenotype, which may lead to metastasis. Whole periprostatic fat, not only stromal vascular fraction cells, seems to warrant the necessary factors to induce a specific microenvironment for prostate cancer tumor cells, which ultimately may result, as we found, in tumor cell survival, increased motility and availability of extracellular proteases. During cell migration, pericellular proteolysis of extracellular matrix is important for cell protrusion.
The increased production of MMPs found in PP adipose tissue can fuel invasive and metastatic behavior of PP fat-infiltrating prostate cancer cells.