Phenotypic characterization of human prostatic stromal cells in primary cultures derived from human tissue samples

Emerging evidence has shown that the tumor microenvironment plays a crucial role in prostate cancer (PCa) development and progression. However, the mechanism(s) through which stromal cells regulate epithelial cells and the differences among prostatic stromal cells of different histological/pathological origin in PCa progression remain unclear. Therefore, it is necessary to characterize the stromal cell populations present in benign prostatic hyperplasia (BPH) and PCa. To this end, we used cultures from stromal cells obtained from BPH-derived (15 cases) and PCa-derived (30 cases) primary cultures. In culture, stromal cells are a mixture of fibroblasts, myofibroblasts (MFs) and muscle cells. Fibroblasts are characterized for the expression of vimentin, MFs for the co-expression of α-smooth muscle actin (α-SMA) and vimentin, whereas muscle cells for the expression of α-SMA and desmin. Fibroblasts were present in large amounts in the BPH-compared to the PCa-derived cultures, whereas MFs were more representative of PCa-as opposed to BPH-derived cultures. Some α-SMA-positive cells retained the expression of basal cytokeratin K14. This population was defined as myoepithelial cells and was associated with senescent cultures. The percentage of MFs was higher in high-grade compared to moderate-and low-grade PCa-derived cultures, whereas the number of myoepithelial cells was lower in high-grade compared to moderate-and low-grade PCa-derived cultures. In addition, we analyzed the expression of p75NTR, as well as the expression of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitors of MMPs (TIMPs). p75NTR expression was elevated in the stromal cultures derived from PCa compared to those derived from BPH and in cultures derived from cases with Gleason scores.7 compared to those derived from cases with Gleason scores <7, as well as in cultures with a high concentration of MFs compared to those with a high concentration of fibroblasts. MMP-2 was secreted by all primary cultures, whereas MMP-9 secretion was observed only in some PCa-derived stromal cells, when the percentage of MFs was significantly higher compared to BPH-derived cultures. TIMP1, TIMP2 and TIMP3 were secreted in elevated amounts in the BPH-compared to the PCa-derived stromal cultures, suggesting the differential regulation of extracellular matrix (ECM) degradation. When we used 22rv1 and PC3 PCa xenograft models for the isolation and characterization of murine cancer-associated fibroblasts (CAFs) we noted that the angiogenic wave was concurrent with the appearance of a reactive stroma phenotype, as determined by staining for α-SMA, vimentin, tenascin, calponin, desmin and Masson's trichrome. In conclusion, MF stromal cells from PCa participate in the progression and metastasis of PCa, modualting inflammation, angiogenesis and epithelial cancer cell proliferation

Estradiol, Progesterone, and Transforming Growth Factor α Regulate Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) Expression in Mouse Endometrial Cells

Insulin-like growth factor 1 (IGF1) Is Involved in the proliferation of mouse and rat endometrial cells in a paracrine or autocrine manner. Insulin-like growth factor binding protein-3 (IGFBP3) modulates actions of IGFs directly or indirectly. The present study aimed to determine whether IGFBP3 is Involved In the regulation of proliferation of mouse endometrial cells. Mouse endometrial epithelial cells and stromal cells were isolated, and cultured In a serum free medium. IGF1 stimulated DNA synthesis by endometrial epithelial and stromal cells, and IGFBP3 Inhibited IGF1-induced DNA synthesis. Estradiol-17 beta (E2) decreased the Igfbp3 mRNA level in endometrial stromal cells, whereas It Increased the Igf1 mRNA level. Transforming growth factor alpha (TGF alpha) significantly decreased IGFBP3 expression at both the mRNA and secreted protein levels in endometrial stromal cells. Progesterone (134) did not affect the E2-induced down-regulation of Igfbp3 mRNA expression in endometrial stromal cells, although P4 alone increased Igfbp3 mRNA levels. The present findings suggest that in mouse endometrial stromal cells E2 enhances IGF1 action through enhancement of IGF1 synthesis and reduction of IGFBP3 synthesis, and that TGF alpha affects IGF1 actions through modulation of IGFBP3 levels

Tumor-stroma interactions influence the response to PI3K targeted agents in preclinical models of colorectal cancer (CRC)

Introduction: One of the main obstacle to the successful development of therapeutic strategies remains the identification of biomarker underlying drug resistance. Recently, investigators have become more aware the role of the tumor microenvironment (TME) in cancer and the potential therapeutic opportunities that derive from suppressing potential resistance mechanisms arising microenvironmental interactions. The aim of this study was to set-up multicellular culture models to uncover the molecular mechanisms by which stromal/endothelial cells modulate response to signaling inhibitors and to identify potential therapeutic targets in PTEN-loss contexts. Methods and Materials: Isogenic CRC cell lines (X-MAN™ HCT116 and HCT116 PTEN-/-) were treated with MAPKi and PI3K/mTORi alone or in combination, in the presence or absence of stromal fibroblasts or fibroblast/endothelial cell conditioned medium (CM). Cytofluorimetric analysis and Crystal Violet assay were used to analyse functional response to targeted agents; pathways activation and cytokine/chemokine profile were analysed using Western blot and ELISA assay respectively. Results and Discussion: In co-culture CRC models, the response to MAPK and PI3K inhibitors is the result of interaction between tumor cells and their surrounding stroma. The response to PI3K/mTORi is mainly influenced by microenvironmental interactions: direct cell-to-cell tumor/stroma contact renders stromal cells resistant to PI3K/mTORi, while the presence of stromal cell-derived soluble factors sensitizes PTEN-competent CRC cells to PI3K/mTORi-mediated growth inhibition. This effect was confirmed using CM from different types of stromal cells (fibroblast/endothelial) that similarly affected the response of CRC cell lines to signalling inhibitors; this is probably due to similar profile of cytokine/chemokine production in stromal cell and is subjected to a “saturation” effect. The presence of stromal CM upregulates MAPK activation regardless of PTEN status, whereas mTOR pathway upregulation is observed mainly in PTEN-competent CRC cellsin PTEN-competent cells soluble factors released by stromal elements paradoxically impair PTEN function, leading to downstream mTORC1 complex formation and pathway activation. This paradoxical mTORC1 activation upon exposure to stroma-derived soluble factors results in functional hypersensitivity of PTEN-competent CRC cells to the growth inhibitory effects of double PI3K/mTOR inhibitors. . Conclusions: The presence of stromal cells (fibroblasts/endothelium) profoundly influences CRC response to PI3K/mTOR-targeting agents. Understanding the mechanisms underlying microenvironmental interactions (tumor, stroma, soluble factors) may be of fundamental importance to overcome therapeutic resistance and develop more effective therapies for patients affected by cancer

Identification of stromal cells in spleen which support myelopoiesis

Stromal cells in spleen organize tissue into red pulp, white pulp and marginal zone, and also interact with hematopoietic cells to regulate immune responses. This study has used phenotypic information of a previously described spleen stromal cell line called 5G3, which supports restricted hematopoiesis in vitro, to identify an equivalent stromal cell subset in vivo and to test its capacity to support hematopoiesis. Using stromal cell fractionation, phenotypic analysis, as well as cell growth and hematopoietic support assays, the Sca-1+gp38+Thy1.2+CD29+CD51+ fraction of spleen stroma has been identified as an equivalent stromal subset resembling the 5G3 cell counterpart. While heterogeneity may still exist within that subset, it has been shown to have superior hematopoietic support capacity compared with the 5G3 cell line, and all other spleen stromal cell fractions tested.This work was supported by project grants to HO from the Australian Research Council (#DP130101703) and the National Health and Medical Research Council of Australia (#585443). HL was supported by an Australian National University Postgraduate Scholarship

SerpinB2 regulates stromal remodelling and local invasion in pancreatic cancer

Pancreatic cancer has a devastating prognosis, with an overall 5-year survival rate of ~8%, restricted treatment options and characteristic molecular heterogeneity. SerpinB2 expression, particularly in the stromal compartment, is associated with reduced metastasis and prolonged survival in pancreatic ductal adenocarcinoma (PDAC) and our genomic analysis revealed that SERPINB2 is frequently deleted in PDAC. We show that SerpinB2 is required by stromal cells for normal collagen remodelling in vitro, regulating fibroblast interaction and engagement with collagen in the contracting matrix. In a pancreatic cancer allograft model, co-injection of PDAC cancer cells and SerpinB2(-/-) mouse embryonic fibroblasts (MEFs) resulted in increased tumour growth, aberrant remodelling of the extracellular matrix (ECM) and increased local invasion from the primary tumour. These tumours also displayed elevated proteolytic activity of the primary biochemical target of SerpinB2-urokinase plasminogen activator (uPA). In a large cohort of patients with resected PDAC, we show that increasing uPA mRNA expression was significantly associated with poorer survival following pancreatectomy. This study establishes a novel role for SerpinB2 in the stromal compartment in PDAC invasion through regulation of stromal remodelling and highlights the SerpinB2/uPA axis for further investigation as a potential therapeutic target in pancreatic cancer

Investigating prostate cancer tumour-stroma interactions - clinical and biological insights from an evolutionary game

BACKGROUND: Tumours are made up of a mixed population of different types of cells that include normal structures as well as ones associated with the malignancy, and there are multiple interactions between the malignant cells and the local microenvironment. These intercellular interactions, modulated by the microenvironment, effect tumour progression and represent a largely under appreciated therapeutic target. We use observations of primary tumor biology from prostate cancer to extrapolate a mathematical model: specifically; it has been observed that in prostate cancer three disparate cellular outcomes predominate: (i) the tumour remains well differentiated and clinically indolent - in this case the local stromal cells may act to restrain the growth of the cancer; (ii) early in its genesis the tumour acquires a highly malignant phenotype, growing rapidly and displacing the original stromal population (often referred to as small cell prostate cancer) - these less common aggressive tumours are relatively independent of the local microenvironment; and, (iii) the tumour co-opts the local stroma - taking on a classic stromagenic phenotype where interactions with the local microenvironment are critical to the cancer growth. METHODS: We present an evolutionary game theoretical construct that models the influence of tumour-stroma interactions in driving these outcomes. We consider three characteristic and distinct cellular populations: stromal cells, tumour cells that are self-reliant in terms of microenvironmental factors and tumour cells that depend on the environment for resources but can also co-opt stroma. 
RESULTS: Using evolutionary game theory we explore a number of different scenarios that elucidate the impact of tumour-stromal interactions on the dynamics of prostate cancer growth and progression and how different treatments in the metastatic setting can affect different types of tumors.
CONCLUSIONS: The tumour microenvironment plays a crucial role selecting the traits of the tumour cells that will determine prostate cancer progression. Equally important, treatments like hormone therapy affect the selection of these cancer phenotypes making it very important to understand how they impact prostate cancer’s somatic evolution

A novel and alternative in vitro method using microwave to study the epithelial-stromal interactions

The goal of the present work was to obtain a simple and reproducible experimental model that would maintain the characteristics of the extracellular physiological environment of breast epithelial cells, both in factors as well as stromal structure, on which we could grow and evaluate changes of normal and tumor breast cells. 3T3-L1 pre-adipocytes (breast stromal cell model) were cultured and irradiated in a microwave oven at different times and potencies. In order to lose their proliferation ability, cells had to be irradiated twice at 650 Watts with a two-minute pulse each. The characteristics of the treated stromal support were analyzed for cell morphology, presence of DNA and proteins. We then evaluated on this support the effect on proliferation and migration of both normal and tumor - murine and human - breast epithelial cells. Both cell types increased their proliferation, while only tumor cells increased migration, thus improving their metastatic capacity. We believe this is a new and simple experimental method of studying epithelial-stromal cell interaction.Fil: Sacca, Paula Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pistone Creydt, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Tesone, Amelia Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Calvo, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Tumor-associated Endo180 requires stromal-derived LOX to promote metastatic prostate cancer cell migration on human ECM surfaces

The diverse composition and structure of extracellular matrix (ECM) interfaces encountered by tumor cells at secondary tissue sites can influence metastatic progression. Extensive in vitro and in vivo data has confirmed that metastasizing tumor cells can adopt different migratory modes in response to their microenvironment. Here we present a model that uses human stromal cell-derived matrices to demonstrate that plasticity in tumor cell movement is controlled by the tumor-associated collagen receptor Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) and the crosslinking of collagen fibers by stromal-derived lysyl oxidase (LOX). Human osteoblast-derived and fibroblast-derived ECM supported a rounded ‘amoeboid-like’ mode of cell migration and enhanced Endo180 expression in three prostate cancer cell lines (PC3, VCaP, DU145). Genetic silencing of Endo180 reverted PC3 cells from their rounded mode of migration towards a bipolar ‘mesenchymal-like’ mode of migration and blocked their translocation on human fibroblast-derived and osteoblast-derived matrices. The concomitant decrease in PC3 cell migration and increase in Endo180 expression induced by stromal LOX inhibition indicates that the Endo180-dependent rounded mode of prostate cancer cell migration requires ECM crosslinking. In conclusion, this study introduces a realistic in vitro model for the study of metastatic prostate cancer cell plasticity and pinpoints the cooperation between tumor-associated Endo180 and the stiff microenvironment imposed by stromal-derived LOX as a potential target for limiting metastatic progression in prostate cancer

Control of human endometrial stromal cell motility by PDGF-BB, HB-EGF and trophoblast-secreted factors

Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of overlapping pathways in trophoblast-endometrial communication. In conclusion, trophoblast signals attract endometrial stromal cells, while PDGF-BB and HB-EGF, although not identified as trophoblast-derived, are local growth factors that may serve to fine-tune directed and non-directed migration at the implantation site

Mesenchymal Differentiation and Organ Distribution of Established Human Stromal Cell Lines in NOD/SCID Mice

Two human stromal cell lines were established previously from bone marrow-derived primary long-term cultures by immortalization using the SV40 large T antigen and cellular cloning. After irradiation, the fibroblast-like cell lines L87/4 and L88/5 support hematopoietic differentiation of allogeneic cord blood cells in vitro. The stromal cells do not express CD34 and CD50, but some adhesion molecules and integrins, such as CD44, CD54 and CD58. Their expression profiles on RNA and protein levels are suggestive of their osteogenic potency. The quality and quantity of osteocalcin and osteopontin protein expression depended on the culture conditions. Expression of the osteogenic markers increased over time in culture, especially in cells growing in clusters. The stromal cells also expressed collagens I and V, but did not show any expression of collagens II and III. The potentially osteoblastic stromal cells were transplanted into NOD/SCID recipient mice by intravenous injection and were found in various mesenchymal organs up to 10 weeks after transplantation. Osteocalcin-positive human stromal cells could be detected in the bone marrow, thymus, liver, brain and gut of the recipient animals. In summary, there is evidence that human bone-marrow-derived stromal cells have to be considered mesenchymal progenitors, persistently expressing osteogenic markers in vitro and in vivo. Copyright (C) 2001 S, Karger AG, Basel