Prostate Cancer-associated Fibroblasts Research Articles

GPR30 Promotes Prostate Stromal Cell Activation via Suppression of ERα Expression and Its Downstream Signaling Pathway.

Cancer-associated fibroblasts (CAFs) play a vital role in malignant transformation and progression of prostate cancer (PCa), and accumulating evidence suggests an enhancing effect of estrogens on PCa. The present study aimed to investigate the possible origin of prostate CAFs and the effects of estrogen receptors, G protein-coupled receptor 30 (GPR30) and estrogen receptor (ER)-α, on stromal cell activation. High expression of fibroblast activation protein (FAP), CD44, and nonmuscle myosin heavy chain B (SMemb) accompanied by low expression of smooth muscle differentiation markers was found in the stromal cells of PCa tissues and in cultured human prostate CAFs. Additionally, SMemb expression, which is coupled to cell phenotype switching and proliferation, was coexpressed with FAP, a marker of activated stromal cells, and with the stem cell marker CD44 in the stromal cells of PCa tissue. Prostate CAFs showed high GPR30 and low ERα expression. Moreover, GPR30 was coexpressed with FAP, CD44, and SMemb. Furthermore, the study demonstrated that the overexpression of GPR30 or the knockdown of ERα in prostate stromal cells induced the up-regulation of FAP, CD44, Smemb, and the down-regulation of smooth muscle markers. The conditioned medium from these cells promoted the proliferation and migration of LNCaP and PC3 PCa cells. GPR30 knockdown or ERα overexpression showed opposite effects. Finally, we present a novel mechanism whereby GPR30 limits ERα expression via inhibition of the cAMP/protein kinase A signaling pathway. These results suggest that stem-like cells within the stroma are a possible source of prostate CAFs and that the negative regulation of ERα expression by GPR30 is centrally involved in prostate stromal cell activation.

LIM domain only 2 over-expression in prostate stromal cells facilitates prostate cancer progression through paracrine of Interleukin-11.

Mechanisms of stromal-epithelial crosstalk are essential for Prostate cancer (PCa) tumorigenesis and progression. Peripheral zone of the prostate gland possesses a stronger inclination for PCa than transition zone. We previously found a variety of genes that differently expressed among different prostate stromal cells, including LIM domain only 2 (LMO2) which highly expressed in peripheral zone derived stromal cells (PZSCs) and PCa associated fibroblasts (CAFs) compared to transition zone derived stromal cells (TZSCs). Studies on its role in tumors have highlighted LMO2 as an oncogene. Herein, we aim to study the potential mechanisms of stromal LMO2 in promoting PCa progression. The in vitro cells co-culture and in vivo cells recombination revealed that LMO2 over-expressed prostate stromal cells could promote the proliferation and invasiveness of either prostate epithelial or cancer cells. Further protein array screening confirmed that stromal LMO2 stimulated the secretion of Interleukin-11 (IL-11), which could promote proliferation and invasiveness of PCa cells via IL-11 receptor α (IL11Rα) – STAT3 signaling. Moreover, stromal LMO2 over-expression could suppress miR-204-5p which was proven to be a negative regulator of IL-11 expression. Taken together, results of our study demonstrate that prostate stromal LMO2 is capable of stimulating IL-11 secretion and by which activates IL11Rα – STAT3 signaling in PCa cells and then facilitates PCa progression. These results may make stromal LMO2 responsible for zonal characteristic of PCa and as a target for PCa microenvironment-targeted therapy.

The Runt-related transcription factor 1 in prostate cancer-associated fibroblasts.

Although most carcinomas arise from a series of genetic alterations in epithelial cells, cancer is a disease that affects not just the transformed cells themselves, but the entire tissue. During tumor development, reciprocal signaling patterns between the cancer cells and the surrounding tissue are altered in a way that affects tissue architecture and cell fate (1). As a result of signals derived from the tumor and a response to tumor development that resembles the response to a wound (2, 3), epithelial tumors of a number of organs, including prostate, are often surrounded by an activated stroma that can constitute as much as half of the tissue mass. This activated stroma is characterized by changes in extracellular matrix proteins, increased angiogenesis, and a heightened inflammatory reaction (4). The cells within this activated stroma include endothelial cells and pericytes that form the tumor vasculature, infiltrated inflammatory cells, and the predominant population, the activated myofibroblasts (3, 5). Activated myofibroblasts resemble normal fibroblasts, cells that secrete extracellular matrix proteins and thereby create a 3D environment that provides tissue with strength and flexibility (4). However, in the tumor microenvironment, the fibroblasts undergo a myodifferentiation process in which they express smooth muscle actin, proliferate, and produce extracellular matrix proteins, including collagen (2). The cancer-associated fibroblasts of the activated stroma have been shown in mouse models to promote tumor growth and progression (6, 7). In patients, the presence of activated fibroblasts is associated with aggressive behavior (8), angiogenesis (7, 9), inflammation (9), and poor prognosis (10). Preventing the host stroma from contributing to cancer growth could represent an important therapeutic strategy. Realizing this goal requires a better understanding of the development of a reactive stroma. In PNAS, Kim et al. advance our understanding of the myofibroblasts that surround tumors and the molecular basis for their function (11).

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

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

297: The metabolic cooperation between prostate carcinoma cells and cancer associated fibroblasts: pyruvate kinase M2 at the crossroads

297: The metabolic cooperation between prostate carcinoma cells and cancer associated fibroblasts: pyruvate kinase M2 at the crossroads

Cofilin drives cell-invasive and metastatic responses to TGF-β in prostate cancer.

Cofilin (CFL) is an F-actin-severing protein required for the cytoskeleton reorganization and filopodia formation, which drives cell migration. CFL binding and severing of F-actin is controlled by Ser3 phosphorylation, but the contributions of this step to cell migration during invasion and metastasis of cancer cells are unclear. In this study, we addressed the question in prostate cancer cells, including the response to TGF-β, a critical regulator of migration. In cells expressing wild-type CFL, TGF-β treatment increased LIMK-2 activity and cofilin phosphorylation, decreasing filopodia formation. Conversely, constitutively active CFL (SerAla) promoted filipodia formation and cell migration mediated by TGF-β. Notably, in cocultures of prostate cancer epithelial cells and cancer-associated fibroblasts, active CFL promoted invasive migration in response to TGF-β in the microenvironment. Further, constitutively active CFL elevated the metastatic ability of prostate cancer cells in vivo. We found that levels of active CFL correlated with metastasis in a mouse model of prostate tumor and that in human prostate cancer, CFL expression was increased significantly in metastatic tumors. Our findings show that the actin-severing protein CFL coordinates responses to TGF-β that are needed for invasive cancer migration and metastasis.

The primary culture and characteristics of prostate cancer-associated fibroblasts

Objective To culture the prostate cancer-associated fibroblasts (CAF) and normal prostate fibroblasts (NPF),and compare their biological characteristics.Methods The CAF and NPF cells were isolated by percoll gradient centrifugation.The cell morphology was detected by phase contrast microscope; the Pan-CK (epithelium marker),Vimentin (fibroblast marker),and smooth muscle actin (SMA) (smooth muscle marker) was stained by immunofluorescence; the cell growth ability was detected by methyl thiazol tetrazolium (MTT) assay.The non-malignant prostatic BPH-1 cells were co-cultured with CAF or NPF cells,and the malignant transformation was detected by soft agar assay.Results The morphology of the CAF and NPF cells was similar.In the immunofluorescence assay,the Vimentin was expressed at high levels in both cells; the Pan-CK did not express in either cells; the SMA was expressed at a higher level in CAF cells.The CAF cells grew faster than the NPF cells (At 4 d,the A values were 0.98 ± 0.07 vs.0.74 ± 0.04,P ＜ 0.05 ; At 6 d,the A values were 2.06 ± 0.13 vs.1.59 ± 0.14,P ＜0.05),and could induce the malignant transformation of BPH-1 cells.Conclusion The CAFs cells are more active than NPF cells,and could promote the malignant transformation of prostate non-malignant cells. Key words: Prostate cancer; Cancer-associated fibroblasts; Primary culture

A reciprocal role of prostate cancer on stromal DNA damage

DNA damage found in prostate cancer-associated fibroblasts (CAF) promotes tumor progression. In the absence of somatic mutations in CAF, epigenetic changes dictate how stromal co-evolution is mediated in tumors. Seventy percent of prostate cancer patients lose expression of transforming growth factor-beta type II receptor (TGFBR2) in the stromal compartment (n = 77, p value = 0.0001), similar to the rate of glutathione S-transferase P1 (GSTP1) silencing. Xenografting of human prostate cancer epithelia, LNCaP, resulted in the epigenetic Tgfbr2 silencing of host mouse prostatic fibroblasts. Stromal Tgfbr2 promoter hypermethylation initiated by LNCaP cells was found to be dependent on IL-6 expression, based on neutralizing antibody studies. We further found that pharmacologic and transgenic knockout of TGF-β responsiveness in prostatic fibroblasts induced Gstp1 promoter methylation. It is known that TGF-β promotes DNA stability, however the mechanism is not well understood. Both prostatic human CAF and mouse transgenic knockout of Tgbr2 had elevated DNA methyltransferase I (DNMT1) activity and histone H3 lysine 9 trimethylation (H3K9me3) to suggest greater promoter methylation. Interestingly, the conditional knockout of Tgfbr2 in mouse prostatic fibroblasts, in modeling epigenetic silencing of Tgfbr2, had greater epigenetic gene silencing of multiple DNA damage repair and oxidative stress response genes, based on promoter methylation array analysis. Homologous gene silencing was validated by RT-PCR in mouse and human prostatic CAF. Not surprisingly, DNA damage repair gene silencing in the prostatic stromal cells corresponded with the presence of DNA damage. Restoring the expression of the epigenetically silenced genes in wild type fibroblasts with radiation-induced DNA damage reduced tumor progression. Tumor progression was inhibited even when epigenetic silencing was reversed in the Tgfbr2 knockout prostatic fibroblasts. Thus, fibroblastic epigenetic changes causative of DNA damage, initiated by association with cancer epithelia, is a dominant mediator of tumor progression over TGF-β responsiveness.

TIMP-1 Promotes Accumulation of Cancer Associated Fibroblasts and Cancer Progression

Treatment options for late stage prostate and colon cancer are limited and there is an urgent need to develop more effective and targeted novel therapies, which starts with identification and validation of novel therapeutic targets. Recent clinical studies have demonstrated that tissue inhibitor matrix metalloproteinase-1 (TIMP-1) levels are elevated in cancer patient plasma and elevated TIMP-1 levels are associated with worse clinical outcomes. However, it is unknown whether TIMP-1 serves merely as a biomarker of cancer progression or has a functional role in promoting cancer progression and can serve as a cancer therapeutic target, which is the main objective of this study. Here, we show that stroma of human prostate and colon cancer express higher levels of TIMP-1 compared to their normal counterparts and increased expression of TIMP-1 promotes in vivo growth of both cancer types. We demonstrate for the first time that increased TIMP-1 expression stimulates accumulation of cancer associated fibroblasts (CAFs) within prostate and colon cancer tissues and that TIMP-1 enhances prostate CAF proliferation and migration in vitro and promotes ERK1/2 kinase activation in these CAF cells. Our results establish the novel promotive effects of TIMP-1 on cancer progression and on accumulation of CAFs that in turn provides a pro-tumor microenvironment. Together, these results establish the potential of TIMP-1 as a novel target for cancer therapy and the mechanism underlying the pro-tumor activity of TIMP-1.

Androgen receptor in human prostate cancer-associated fibroblasts promotes prostate cancer epithelial cell growth and invasion

The androgens and androgen receptor (AR) play key roles in the prostate cancer (PCa) development and progression via epithelium-stroma cross talk. Prostate cancer-associated fibroblasts (CAFs) are dominant components in PCa stroma and are essential in the malignant progression by supporting tumorigenesis and metastasis. However, the AR roles in CAFs are still obscure. We isolated and immortalized the CAFs from human PCa tissues and found the CAFs are AR positive. We then knocked down their AR with siRNA and co-cultured the resultant CAFs with PCa cell line PC3. The MTT, invasion, and colony formation assays were performed to study the PC3 biological behavior. The results showed that the PCa epithelial growth, invasion, and colony formation abilities decreased when knocking down the CAFs AR. By using the real-time quantitative polymerase chain reaction, we found the IGF1, FGF7, FGF10, SDF1, HGF, and TGFb2 expression levels decreased in the AR knocked down CAFs. These results suggested that the AR in CAFs promoted PCa epithelial growth and invasion via regulating a series of growth factors. Targeting the AR in CAFs might be a potential therapeutic option for PCa in future.

Cancer-associated fibroblasts and M2-polarized macrophages synergize during prostate carcinoma progression

Inflammation is now acknowledged as an hallmark of cancer. Cancer-associated fibroblasts (CAFs) force a malignant cross talk with cancer cells, culminating in their epithelial-mesenchymal transition and achievement of stemness traits. Herein, we demonstrate that stromal tumor-associated cells cooperate to favor malignancy of prostate carcinoma (PCa). Indeed, prostate CAFs are active factors of monocyte recruitment toward tumor cells, mainly acting through stromal-derived growth factor-1 delivery and promote their trans-differentiation toward the M2 macrophage phenotype. The relationship between M2 macrophages and CAFs is reciprocal, as M2 macrophages are able to affect mesenchymal-mesenchymal transition of fibroblasts, leading to their enhanced reactivity. On the other side, PCa cells themselves participate in this cross talk through secretion of monocyte chemotactic protein-1, facilitating monocyte recruitment and again macrophage differentiation and M2 polarization. Finally, this complex interplay among cancer cells, CAFs and M2 macrophages, cooperates in increasing tumor cell motility, ultimately fostering cancer cells escaping from primary tumor and metastatic spread, as well as in activation of endothelial cells and their bone marrow-derived precursors to drive de novo angiogenesis. In keeping with our data obtained in vitro, the analysis of patients affected by prostate cancers at different clinical stages revealed a clear increase in the M2/M1 ratio in correlation with clinical values. These data, coupled with the role of CAFs in carcinoma malignancy to elicit expression of stem-like traits, should focus great interest for innovative strategies aimed at the co-targeting of inflammatory cells and fibroblasts to improve therapeutic efficacy.

EphA2-mediated mesenchymal–amoeboid transition induced by endothelial progenitor cells enhances metastatic spread due to cancer-associated fibroblasts

Tumor progression is deeply influenced by epigenetic changes induced by tumor stroma. Cancer-associated fibroblasts (CAFs) have been reported to promote epithelial-mesenchymal transition in cancer cells, thereby enhancing their aggressiveness and stem-like properties. As CAFs are able to recruit endothelial progenitor cells (EPCs) to tumor site, we aim to investigate their interplay for prostate carcinoma progression. Both prostate CAFs and cancer cells actively recruit EPCs, known to affect tumor progression through increased vasculogenesis. EPCs synergize with CAFs to further promote epigenetic plasticity of cancer cells, through a mesenchymal-to-amoeboid transition. Indeed, after fibroblasts have engaged epithelial-mesenchymal transition in cancer cells, a further shift towards amoeboid motility is promoted by EPCs through contact-mediated triggering of the bidirectional ephrinA1/EphA2 signaling. The activation of ephrinA1 reverse pathway enhances EPC-induced neo-vascularization, thus promoting tumor growth, while EphA2 forward signaling elicits mesenchymal-amoeboid transition in cancer cells, favoring their adhesion to endothelium, transendothelial migration, and lung metastatic colonization. We therefore underscore that the metastatic advantage given by tumor microenvironment embraces different motility strategies and propose EphA2-targeted tools as useful adjuvants in anti-metastatic treatments.

Abstract 1476: Prostate cancer-associated fibroblasts secrete Annexin A1 that facilitates both generation of cancer stem cell-like cells (CSCs) from prostatic malignant epithelial cells and self-renewal/differentiation of CSCs

Abstract Previously we described epithelial cell lines (Hormones & Cancer. 1: 44-54, 2010), primary cultures of cancer-associated fibroblasts (CAFs) (Cancer Res. 68: 198-205, 2008) and a primary cancer stem cell (CSC) subpopulation (Cancer Res. 70: 7294-303, 2010), all derived from the cPten-/-L mouse model of prostate adenocarcinoma (Cancer Res. 67: 7525-33, 2007). Most notably, we described evidence that CAFs secrete factors that enhance both the stemness and growth potentials of the CSCs (Cancer Res. 70: 7294-303, 2010). We have now identified the phospholipid binding Annnexin A1 as one of the pertinent secreted factors. When a malignant epithelial cell line (cE1) derived from castration-resistant prostate cancer (CRPC) from the model was fractionated into three different subpopulations based on the levels of cell surface expression of Sca-1(S) and CD49f (C) antigens: SChi, SCme and SC− (high, medium and low expression of S and C, respectively), we found that SCme cells were preferentially susceptible to epithelial-mesenchymal transition (EMT)-like transformation when treated with conditioned medium (CM) from either CAF cultures or Annexin A1 mimetic peptide (Ac1-25). Furthermore, following EMT induction, these trans-differentiated cells acquired stem cell-like properties similar to the SChi group based on expression of S and C and statistically significant up-regulation of Oct4 (p<0.05), Sox2 (p<0.001) and Nanog (p<0.0001). When transplanted within collagen grafts under the renal capsule of Nod.Scid mice, CM- and Ac1-25-EMT-ed SCme cells were able to form invasive lesions in vivo displaying cells with expression of both basal (p63) and luminal (cytokeratin 8, CK8)) cell-specific markers, as compared to untreated control grafts, which lacked glandular structures and had reduced p63 positive cells. Another effect of Annexin A1 was unraveled when CSCs (Lin− SChi subpopulation) from the primary tumors of the model were exposed to Acl-25. In vitro spheroid formation assays showed that in the presence of Acl-25 single spheroids generated from the CSCs could form multiple substructures potentially resulting from activation of additional stem-like progenitors, each capable of forming a prostasphere while remaining attached to the original spheroid structure. Immunofluorescent staining of cryosectioned spheroids revealed the presence of p63+ basal cells and an expanded population of p63/CK8 double positive transit-amplifying cells. Together these results describe two ways in which Annexin A1 may play a role in the development of a more malignant and stem cell-enriched tumor phenotype, or, to hypothetically take it a step further, possibly two ways in which Annexin A1, present in the tumor microenvironment, can lead to an increase or maintenance of a cancer stem cell(-like) population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1476. doi:1538-7445.AM2012-1476

Reduction of pro-tumorigenic activity of human prostate cancer-associated fibroblasts using Dlk1 or SCUBE1

SUMMARYHuman prostatic cancer-associated fibroblasts (CAFs) can elicit malignant changes in initiated but non-tumorigenic human prostate epithelium, demonstrating that they possess pro-tumorigenic properties. We set out to reduce the pro-tumorigenic activity of patient CAFs using the Dlk1 and SCUBE1 molecules that we had previously identified in prostate development. Our hypothesis was that mesenchymally expressed molecules might reduce CAF pro-tumorigenic activity, either directly or indirectly. We isolated primary prostatic CAFs and characterised their expression of CAF markers, expression of Notch2, Dlk1 and SCUBE1 transcripts, and confirmed their ability to stimulate BPH1 epithelial cell proliferation. Next, we expressed Dlk1 or SCUBE1 in CAFs and determined their effects upon tumorigenesis in vivo following recombination with BPH1 epithelia and xenografting in SCID mice. Tumour size was reduced by about 75% and BPH1 proliferation was reduced by about 50% after expression of Dlk1 or SCUBE1 in CAFs, and there was also a reduction in invasion of BPH1 epithelia into the host kidney. Inhibition of Notch signalling, using inhibitor XIX, led to a reduction in BPH1 cell proliferation in CAF-BPH1 co-cultures, whereas inhibition of Dlk1 in NIH3T3-conditioned media led to an increase in BPH1 growth. Our results suggest that pro-tumorigenic CAF activity can be reduced by the expression of developmental pathways.

Prostate cancer‐derived angiogenin stimulates the invasion of prostate fibroblasts

Prostate fibroblasts promote prostate cancer progression by secreting factors that enhance tumour growth and induce the migration and invasion of prostate cancer cells. Considering the role of fibroblasts in cancer progression, we hypothesized that prostate cancer cells recruit these cells to their vicinity, where they are most directly available to influence cancer cell behaviour. To test this hypothesis, we performed modified Boyden chamber assays assessing the migration and collagen I invasion of normal primary prostate fibroblasts (PrSCs) and prostate cancer-associated fibroblasts (PCAFs) in response to media conditioned by the metastatic prostate cancer cell lines PC-3, LNCaP and DU145. During 4-hr incubations, PrSCs and PCAFs migrated and invaded in response to the conditioned media. To identify candidate proteins in the conditioned media that produced these effects, we performed cytokine antibody arrays and detected angiogenin in all three media. Angiogenin-blocked PC-3-conditioned medium, obtained using an anti-angiogenin polyclonal antibody or angiogenin siRNA, significantly reduced PC-3-induced PrSC and PCAF collagen I invasion. Furthermore, angiogenin alone at 1, 2 and 5 ng/ml significantly stimulated PCAF collagen I invasion. These results suggest that PC-3-derived angiogenin stimulates the invasion of normal prostate fibroblasts and PCAFs and is sufficient for invasion of the latter. Because prostate fibroblasts play key roles in prostate cancer progression, targeting their invasion using an anti-angiogenin-based therapy may be a strategy for preventing or treating advanced prostate cancer.

Abstract LB-301: Potential role of prostate cancer-associated fibroblasts in inducing stem cell-like properties to the malignant epithelial cells of the prostate

Abstract Epithelial-mesenchymal transdifferentiation (EMT) is a well recognized mechanism for initiating metastasis of epithelial cancers. Shared attributes between EMT and cancer stem cells (CSCs) are being uncovered and extracellular signals from the microenvironment which regulate EMT are being investigated to define their role in a potential Cancer Cell-EMT-CSC lineage. Recently we have described epithelial cell lines (Hormones & Cancer. 1: 44–54, 2010), primary cultures of cancer-associated fibroblasts (CAFs) (Cancer Res. 68: 198–205, 2008) and CSC subpopulation (Cancer Res. 70: 7294–303, 2010), all derived from the cPten-/-L mouse model of prostate adenocarcinoma (Cancer Res. 67: 7525–33, 2007) that could be used to evaluate and characterize factors that can support or accelerate EMT induction to epithelial cancer cells, and further define putative factors that may facilitate de-differentiation to CSC phenotypes and biology. Here we report that a malignant epithelial cell line (cE1) derived from the androgen depletion-independent recurrent tumor of the model can be further fractionated into three different subpopulations based on the levels of cell surface expressions of SCA-1(S) and CD49f (C) antigens: SC− (low expression of S and C), SChi (high expression of S and C), and SCme (medium expression of S and C). Of these fractions, SCme population is relatively more sensitive to undergo EMT-like changes when exposed to TGF-β1 than the other fractions. SChi fraction displays increased ability to form spheroids in 3-D cultures akin to the CSCs isolated from the prostate tumors of the model. SC− cells appear to represent bulk of the cells with reduced propensity for EMT or spheroid formation. SCme cells were treated with conditioned medium (CM) from either CAF cultures or NPF (normal prostate fibroblast) cultures, and the morphological changes were monitored over a period of 14 days. It is remarkable that extensive EMT-like changes were induced by CM from CAFs and much less by that from NPFs. Furthermore, following EMT induction, these transformed cells appear to be more similar to the SChi group based on expression of S and C and statistically significant upregulation of Oct4 (p<0.05), Sox2 (p<0.001) and Nanog (p<0.0001). While the factors that are secreted by CAFs and responsible for the observed shift towards CSC phenotype remain to be defined, our preliminary results from mass spectrometric analyses indicate excess production of members of the Annexin family of proteins by CAFs. We believe the study system we described could be explored for this and other cues on the intersections of signaling networks regulating EMT of the cancer epithelial cells and then potentially to further de-differentiation to CSC-like phenotypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-301. doi:10.1158/1538-7445.AM2011-LB-301

Differential Inductive Signaling of CD90+ Prostate Cancer-Associated Fibroblasts Compared to Normal Tissue Stromal Mesenchyme Cells

Prostate carcinomas are surrounded by a layer of stromal fibroblastic cells that are characterized by increased expression of CD90. These CD90+ cancer-associated stromal fibroblastic cells differ in gene expression from their normal counterpart, CD49a+CD90lo stromal smooth muscle cells; and were postulated to represent a less differentiated cell type with altered inductive properties. CD90+ stromal cells were isolated from tumor tissue specimens and co-cultured with the pluripotent embryonal carcinoma cell line NCCIT in order to elucidate the impact of tumor-associated stroma on stem cells, and the ‘cancer stem cell.’ Transcriptome analysis identified a notable decreased induction of smooth muscle and prostate stromal genes such as PENK, BMP2 and ChGn compared to previously determined NCCIT response to normal prostate stromal cell induction. CD90+ stromal cell secreted factors induced an increased expression of CD90 and differential induction of genes involved in extracellular matrix remodeling and the RECK pathway in NCCIT. These results suggest that, compared to normal tissue stromal cells, signaling from cancer-associated stromal cells has a markedly different effect on stem cells as represented by NCCIT. Given that stromal cells are important in directing organ-specific differentiation, stromal cells in tumors appear to be defective in this function, which may contribute to abnormal differentiation found in diseases such as cancer.Electronic supplementary materialThe online version of this article (doi:10.1007/s12307-010-0061-4) contains supplementary material, which is available to authorized users.

CD90/THY1 is overexpressed in prostate cancer-associated fibroblasts and could serve as a cancer biomarker

A by-product in the processing of prostate tissue for cell sorting by collagenase digestion is the media supernatant that remains after the cells are harvested. These supernatants contain proteins made by the cells within the tissue. Quantitative proteomic analysis of N-glycosylated proteins detected an increased amount of CD90/THY1 in cancer supernatants compared with non-cancer supernatants. Immunohistochemistry showed that in all carcinomas, regardless of Gleason grade, a layer of CD90-positive stromal fibroblastic cells, ∼5 to 10 cells deep, was localized to tumor glands. In contrast, a no more than 1-cell wide girth of CD90-positive stromal cells was found around benign glands. The increased number of CD90-positive stromal cells in cancer correlated with overexpression of CD90 mRNA detected by gene expression analysis of stromal cells obtained by laser-capture microdissection. There is increasing evidence that cancer-associated stroma has a function in both tumor progression and carcinogenesis. Most experiments to identify cancer biomarkers have focused on the cancer cells. CD90, being a marker for prostate cancer-associated stroma, might be a potential biomarker for this cancer. A non-invasive test could be provided by a urine test. Proteomic analysis of urine from patients with prostate cancer identified CD90; conversely, CD90 was not detected in the urine of post-prostatectomy patients. Furthermore, this urinary CD90 protein was a variant CD90 protein not known to be expressed by such cells as lymphocytes that express CD90. These CD90 results were obtained from ∼90 cases consisting of proteomic analysis of tissue and urine, immunohistochemistry, western blot analysis of tissue media, flow cytometry of cells from digested tissue, and reverse transcriptase polymerase chain reaction analysis of isolated stromal cells.

Reciprocal Activation of Prostate Cancer Cells and Cancer-Associated Fibroblasts Stimulates Epithelial-Mesenchymal Transition and Cancer Stemness

Although cancer-associated fibroblasts (CAF) are key determinants in the malignant progression of cancer, their functional contribution to this process is still unclear. Analysis of the mutual interplay between prostate carcinoma cells and CAFs revealed a mandatory role of carcinoma-derived interleukin-6 in fibroblast activation. In turn, activated fibroblasts through secretion of metalloproteinases elicit in cancer cells a clear epithelial-mesenchymal transition (EMT), as well as enhancement of tumor growth and development of spontaneous metastases. CAF-induced EMT leads prostate carcinoma cells to enhance expression of stem cell markers, as well as the ability to form prostaspheres and to self-renew. Hence, the paracrine interplay between CAFs and cancer cells leads to an EMT-driven gain of cancer stem cell properties associated with aggressiveness and metastatic spread.

501 Analysis of the contractility of prostatic cancer-associated fibroblasts in a 3D collagen gel contraction assay

A 41-year-old man was accidentally exposed to carbon monoxide (CO) gas and found in a state of cardiopulmonary arrest while he took bath. After admission, he was resuscitated and underwent artificial ventilation in a comatose state and died about 19 h later. Computed tomography (CT) examination disclosed bilateral low density area in the basal ganglia and the thalamus, a well-known finding in the CO intoxication. Necropsy, histological examination, DNA ladder assay gave the first line of evidence for the presence of apoptosis as well as necrosis in the human case of CO intoxication. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) positive apoptotic cells were more predominant in the CA2 area than in CA1 area. There is general co-relation between the ratio of TUNEL-positive cells and the DNA laddering on the agarose gel. Basal ganglia and thalamus, which showed bilateral low density area in CT, were revealed to be severe edema. The two types of cell death occurred in the cortex, basal ganglia, hippocampus, thalamus, and cerebellum. Hypoxia caused by CO–hemoglobin formation alone cannot explain the phenomena.