Tumor epithelial cells within a tumor coexist with a complex microenvironment in which a variety of interactions between its various components determine the behavior of the primary tumors. Cancer- associated fibroblasts (CAF) and M2 macrophages, characterized by high expression of different markers, including a-SMA, FSP1 and FAP, or CD163 and DCSIGN, respectively, are involved in the malignancy of different tumors. In the present study, expression of the above markers in CAF and M2 macrophages was analyzed using RT-PCR and immunohistochemistry in the normal mucosa and tumor tissue from a cohort of 289 colorectal cancer patients. Expression of CAF and M2 markers is associated with the clinical outcome of colorectal cancer patients. Moreover, the combination of CAF and M2 markers identifies three groups of patients with clear differences in the progression of the disease. This combined variable could be a decisive factor in the survival of advanced-stage patients. Taken together, these analyses demonstrate the prognostic involvement of interrelationships between DCSIGN, CD163, a-SMA, FSP1 and FAP markers in the survival of colon cancer patients.This work has been supported by Fundación Científica AECC, SAF2010-20750, S2010 ⁄BMD-2344, RTICC-RD06 ⁄ 0020 ⁄ 0020, PI12 ⁄ 02037 and Fundación Banco Santander. A.G.H. laboratory was supported by RD06 ⁄ 0020 ⁄ 0040. C.P. is the recipient of a Miguel Servet Contract from the Instituto de Salud Carlos III (CP09 ⁄ 00294) and V.G. is the recipient of a Fundación Científica AECC fellowshi

A significant proportion of extracellular nucleic acids in plasma circulate highly protected in tumor-specific exosomes, but it is unclear how the release of exosomes is modulated in carcinogenesis. We quantified by cytometry exosomes in plasma of 91 colorectal cancer patients to evaluate their potential as a tumor indicator and their repercussions on diagnosis and prognosis. We examined the involvement of TSAP6, a TP53-regulated gene involved in the regulation of vesicular secretion, in levels of circulating exosomes in plasma of colorectal patients and in HCT116 TP53-(wild-type and null) human colorectal cancer cell lines. The fraction of exosomes in cancer patients was statistically higher than in healthy controls (mean rank ¼ 53.93 vs. 24.35). High levels of exosomes in plasma of patients correlated with high levels of carcino-embryonic antigen (P ¼ 0.029) and with poorly differentiated tumors (P ¼ 0.039) and tended to have shorter overall survival than patients with low levels (P ¼ 0.056). Release of exosomes did not correlate with TSAP6 expression; and regulation of TSAP6 by TP53 was not shown either in tumor samples or in HCT116 cell lines. Although it was not suggested that the TP53/TSAP6 pathway regulates the release of exosomes into the plasma of colorectal cancer patients, the level of circulating exosomes may be used as a tumor indicator, because it correlates with poor prognosis parameters and shorter survival.

The identification of tumour biomarkers that detect the presence of disease using noninvasive diagnostic procedures is a key part of cancer research. We determined in plasma the vesicle-related microRNA (miRNA) expression profile of nonsmall cell lung cancer (NSCLC) and evaluate whether plasma miRNAs can be both discriminating (between patients and healthy controls) and prognostic markers.365 human miRNAs were analysed by Taqman1 low-density arrays (Applied Biosystems, Foster City, CA, USA) in the plasma from 28 NSCLC patients and 20 controls. Five selected miRNAs (let7f, miR-20b, miR-30e-3p, miR-223 and miR-301) were validated independently by real-time PCR in plasma from 78 NSCLC and 48 controls and correlated with pathologic parameters and survival.Levels of let-7f, miR-20b and miR-30e-3p were decreased in plasma vesicles of NSCLC patients. Moreover, levels of let-7f and miR-30e-3p distinguished between two groups of patients for stage of disease and therefore possibility of surgery. Plasma levels of miR-30e-3p and let-7f were associated with short disease-free survival and overall survival, respectively. NSCLC patients and healthy controls differ in vesicle-related miRNAs in plasma. Levels of let-7f and miR-30e-3p in NSCLC patients are associated with poor outcome. Thus, plasma vesiclerelated miRNAs obtained by noninvasive methods could serve as circulating tumour biomarkers of discriminating and prognostic value.

Crosstalk between tumor and stromal cells in the tumor microenvironment alter its properties in ways that facilitate the invasive behavior of tumor cells. Here, we demonstrate that cancer-associated fibroblasts (CAF) increase the stiffness of the extracellular matrix (ECM) and promote anisotropic fiber orientation, two mechanical signals generated through a Snail1/RhoA/aSMAdependent mechanism that sustains oriented tumor cell migration and invasiveness. Snail1-depleted CAF failed to acquire myofibroblastic traits in response to TGFb, including RhoA activation, aSMA-positive stress fibers, increased fibronectin fibrillogenesis, and production of a stiff ECM with oriented fibers. Snail1 expression in human tumor-derived CAF was associated with an ability to organize the ECM. In coculture, a relatively smaller number of Snail1-expressing CAF were capable of imposing an anisotropic ECM architecture, compared with nonactivated fibroblasts. Pathologically, human breast cancers with Snail1 þ CAF tended to exhibit desmoplastic areas with anisotropic fibers, lymph node involvement, and poorer outcomes. Snail1 involvement in driving an ordered ECM was further confirmed in woundhealing experiments in mice, with Snail1 depletion preventing the anisotropic organization of granulation tissue and delaying wound healing. Overall, our results showed that inhibiting Snail1 function in CAF could prevent tumor-driven ECM reorganization and cancer invasion. Cancer Res; 75(2); 284-95. Ó2014 AACR.

Purpose: Cancer-associated fibroblasts (CAF) actively participate in reciprocal communication with tumor cells and with other cell types in the microenvironment, contributing to a tumor-permissive neighborhood and promoting tumor progression. The aim of this study is the characterization of how CAFs from primary human colon tumors promote migration of colon cancer cells. Experimental design: Primary CAF cultures from 15 primary human colon tumors were established. Their enrichment in CAFs was evaluated by the expression of various epithelial and myofibroblast specific markers. Coculture assays of primary CAFs with different colon tumor cells were performed to evaluate promigratory CAF-derived effects on cancer cells. Gene expression profiles were developed to further investigate CAF characteristics. Results: Coculture assays showed significant differences in fibroblast-derived paracrine promigratory effects on cancer cells. Moreover, the association between CAFs' promigratory effects on cancer cells and classic fibroblast activation or stemness markers was observed. CAF gene expression profiles were analyzed by microarray to identify deregulated genes in different promigratory CAFs. The gene expression signature, derived from the most protumorogenic CAFs, was identified. Interestingly, this “CAF signature” showed a remarkable prognostic value for the clinical outcome of patients with colon cancer. Moreover, this prognostic value was validated in an independent series of 142 patients with colon cancer, by quantitative real-time PCR (qRT-PCR), with a set of four genes included in the “CAF signature.” Conclusions: In summary, these studies show for the first time the heterogeneity of primary CAFs' effect on colon cancer cell migration. A CAF gene expression signature able to classify patients with colon cancer into high- and low-risk groups was identified. Clin Cancer Res; 19(21); 5914–26. ©2013 AACR.