PRL-3 overexpression in epithelial cells is induced by surrounding stromal fibroblasts

We isolate and culture carcinoma-associated fibroblasts (CAFs) from primary tumour (CAFpt), CAFs from corresponding synchronous liver metastasis (CAFlm) as well as normal colonic fibroblasts (NCF) from the same patient. From these cultures, conditioned media (CM) was obtained. Culture of a wide panel of colorectal and pancreatic cell lines in CM from CAFlm resulted in overexpression of mRNA PRL-3 and higher overexpression in CAFs than in non-activated fibroblasts. Moreover PRL-3 mRNA expression correlates with expression of α-SMA and deposition of collagen fibrils in the stroma. We demonstrate that products secreted by CAFs trigger PRL-3 overexpression in cancer cells. Identification of these factors may contribute to new stroma-targeted therapies for desmoplastic tumours

The Blockade of Tumoral IL1β-Mediated Signaling in Normal Colonic Fibroblasts Sensitizes Tumor Cells to Chemotherapy and Prevents Inflammatory CAF Activation

Heterotypic interactions between newly transformed cells and normal surrounding cells define tumor's fate in incipient carcinomas. Once homeostasis has been lost, normal resident fibroblasts become carcinoma-associated fibroblasts, conferring protumorogenic properties on these normal cells. Here we describe the IL1 beta-mediated interplay between cancer cells and normal colonic myofibroblasts (NCFs), which bestows differential sensitivity to cytotoxic drugs on tumor cells. We used NCFs, their conditioned media (CM), and cocultures with tumor cells to characterize the IL1 beta-mediated crosstalk between both cell types. We silenced IL1 beta in tumor cells to demonstrate that such cells do not exert an influence on NCFs inflammatory phenotype. Our results shows that IL1 beta is overexpressed in cocultured tumor cells. IL1 beta enables paracrine signaling in myofibroblasts, converting them into inflammatory-CAFs (iCAF). IL1 beta-stimulated-NCF-CM induces migration and differential sensitivity to oxaliplatin in colorectal tumor cells. Such chemoprotective effect has not been evidenced for TGF beta 1-driven NCFs. IL1 beta induces the loss of a myofibroblastic phenotype in NCFs and acquisition of iCAF traits. In conclusion, IL1 beta-secreted by cancer cells modify surrounding normal fibroblasts to confer protumorogenic features on them, particularly tolerance to cytotoxic drugs. The use of IL1 beta-blocking agents might help to avoid the iCAF traits acquisition and consequently to counteract the protumorogenic actions these cells. Keyword

Hepatic Carcinoma—Associated Fibroblasts Promote an Adaptative Response in Colorectal Cancer Cells That Inhibit Proliferation and Apoptosis: Nonresistant Cells Die by Nonapoptotic Cell Death

AbstractCarcinoma-associated fibroblasts (CAFs) are important contributors of microenvironment in determining the tumor’s fate. This study aimed to compare the influence of liver microenvironment and primary tumor microenvironment on the behavior of colorectal carcinoma. Conditioned medium (CM) from normal colonic fibroblasts (NCFs), CAFs from primary tumor (CAF-PT) or liver metastasis (CAF-LM) were obtained. We performed functional assays to test the influence of each CM on colorectal cell lines. Microarray and gene set enrichment analysis (GSEA) were performed in DLD1 cells cultured in matched CM. In DLD1 cells, CAF-LM CM compared with CAF-PT CM and NCF led to a more aggressive phenotype, induced the features of an epithelial-to-mesenchymal transition more efficiently, and stimulated migration and invasion to a greater extent. Sustained stimulation with CAF-LM CM evoked a transient G2/M cell cycle arrest accompanied by a reduction of apoptosis, inhibition of proliferation, and decreased viability of SW1116, SW620, SW480, DLD1, HT-29, and Caco-2 cells and provoked nonapoptotic cell death in those cells carrying KRAS mutations. Cells resistant to CAF-LM CM completely changed their morphology in an extracellular signal-regulated protein kinase-dependent process and depicted an increased stemness capacity alongside the Wnt pathway stimulation. The transcriptomic profile of DLD1 cells treated with CAF-LM CM was associated with Wnt and mitogen-activated protein kinase pathways activation in GSEA. Therefore, the liver microenvironment induces more efficiently the aggressiveness of colorectal cancer cells than other matched microenvironments do but secondarily evokes cell death. Resistant cells displayed higher stemness capacity

Spatial Immunology in Liver Metastases from Colorectal Carcinoma according to the Histologic Growth Pattern

In the era of immunotherapy, the tumor microenvironment (TME) has attracted special interest. However, colorectal liver metastases (CRC-LM) present histological peculiarities that could affect the interaction of immune and tumor cells such as fibrotic encapsulation and dense intratumoral stroma. We explored the spatial distribution of lymphocytic infiltrates in CRC-LM in the context of the histologic growth patterns using multispectral digital pathology providing data on three different scenarios, tumor periphery, invasive margin, and central tumoral areas. Our results illustrate a similar poor cell density of CD8(+) cells between different metastases subtypes in intratumoral regions. However, in encapsulated metastases, cytotoxic cells reach the tumor cells while remaining retained in stromal areas in non-encapsulating metastases. Some aspects are still unresolved, such as understanding the reason why most lymphocytes are largely retained in the capsule. Colorectal cancer liver metastases (CRC-LM) present differential histologic growth patterns (HGP) that determine the interaction between immune and tumor cells. We explored the spatial distribution of lymphocytic infiltrates in CRC-LM in the context of the HGP using multispectral digital pathology. We did not find statistically significant differences of immune cell densities in the central regions of desmoplastic ((d)HGP) and non-desmoplastic ((nd)HGP) metastases. The spatial evaluation reported that (d)HGP-metastases displayed higher infiltration by CD8(+) and CD20(+) cells in peripheral regions as well as CD4(+) and CD45RO(+) cells in (nd)HGP-metastases. However, the reactive stroma regions at the invasive margin (IM) of (nd)HGP-metastases displayed higher density of CD4(+), CD20(+), and CD45RO(+) cells. The antitumor status of the TIL infiltrates measured as CD8/CD4 reported higher values in the IM of encapsulated metastases up to 400 mu m towards the tumor center (p < 0.05). Remarkably, the IM of (d)HGP-metastases was characterized by higher infiltration of CD8(+) cells in the epithelial compartment parameter assessed with the ratio CD8(epithelial)/CD8(stromal), suggesting anti-tumoral activity in the encapsulating lesions. Taking together, the amount of CD8(+) cells is comparable in the IM of both HGP metastases types. However, in (d)HGP-metastases some cytotoxic cells reach the tumor nests while remaining retained in the stromal areas in (nd)HGP-metastases

A 5-gene classifier from the carcinoma-associated fibroblast transcriptomic profile and clinical outcome in colorectal cancer

Based on 108 differentially expressed genes between carcinoma-associated fibroblasts (CAFs) and paired normal colonic fibroblasts we recently reported, a 5-gene classifier for relapse prediction in Stage II/III colorectal cancer (CRC ) was developed. Its predictive value was validated in datasets GSE17538, GSE33113 and GSE14095. An additional validation was performed in a metacohort (n=317) and 142 CRC patients by means of RT-PCR. The 5-gene classifier was significantly associated with increased relapse risk and death from CRC across all validation series of Stage II/III patients used. Multivariate Cox regression analyses confirmed the independent prognostic value of the stromal classifier (HR=2.67; P=0.002). Post-test probabilities provided evidence of the suitability of the 5-gene classifier in clinical practice, identifying a subgroup of Stage-II patients who were at high risk of relapse. Moreover, the a priory worst prognosis mesenchymal subtype of tumours can be stratified according to the physiological status of their carcinoma-associated fibroblasts. In conclusion the CAFs-derived 5-gene classifier provides more accurate information about outcome than conventional clinicopathological criteria and it could be useful to take clinical decisions, especially in Stage II. Additionally, the classifier put into relevance the CAF's intratumoral heterogeneity and might contribute to find relevant targets for depleting adequate CAFS subtypes

Carcinoma-associated fibroblasts affect sensitivity to oxaliplatin and 5FU in colorectal cancer cells

The importance of tumor microenvironment (TME) as a relevant contributor to cancer progression and its role in the development of de novo resistance to targeted therapies has become increasingly apparent. However, the mechanisms of microenvironment-mediated drug resistance for nonspecific conventional chemotherapeutic agents, such as platinum compounds or antimetabolites, are still unclear. Here we describe a mechanism induced by soluble factors released by carcinoma-associated fibroblasts (CAFs) that induce the translocation of AKT, Survivin and P38 to the nucleus of tumor cells. These changes are guided to ensure DNA repair and the correct entrance and exit from mitosis in the presence of chemotherapy. We used conditioned media (CM) from normal-colonic fibroblasts and paired CAFs to assess dose response curves of oxaliplatin and 5-fluorouracil, separately or combined, compared with standard culture medium. We also evaluated a colony-forming assay and cell death to demonstrate the protective role of CAF-CM. Immunofluorescence confirmed the translocation of AKT, P38 and Survivin to the nucleus induced by CAF-soluble factors. We also have shown that STAT3 or P38 inhibition provides a promising strategy for overcoming microenvironment-mediated resistance. Conversely, pharmacologic AKT inhibition induces an antagonistic effect that relieves a cMET and STAT3-mediated compensatory feedback that might explain the failure of AKT inhibitors in the clinic so far

PRL-3 is essentially overexpressed in primary colorectal tumours and associates with tumour aggressiveness

Phosphatase PRL-3 has been involved in different types of cancer, especially in metastases from colorectal carcinoma (CRC). In this study, we explored both isoforms of PRL-3 as a biomarker to predict the recurrence of stage IIIB-C CRC. Overexpression of PRL-3 was investigated in primary human colorectal tumours (n=20) and hepatic metastases (n=36) xenografted in nude mice, samples characterised by absence of human non-tumoral cells, showing a high degree of expression in metastases (P=0.001). In 27 cases of matched normal colonic mucosa/primary tumour/hepatic metastases, PRL-3 overexpression occurs in primary tumours vs normal mucosa (P=0.001) and in hepatic metastases vs primary tumours (P=0.045). Besides, our results in a series of 80 stage IIIB-C CRC primary tumours showed that high levels of PRL-3 were an independent predictor of metastasis (P<0.0001; OR: 9.791) in multivariate analysis of a binary logistic regression and that PRL-3 expression tightly correlates with parameters of bad outcome. Moreover, PRL-3 expression associated with poor outcome in univariate (P<0.0001) and multivariate Cox models (hazard ratio: 3.322, 95%, confidence interval: 1.405-7.852, P=0.006). In conclusion, PRL-3 is a good marker of aggressiveness of locally advanced CRS and a promising predictor of distant metastases. Nevertheless, for prognosis purposes, it is imperative to validate the cutoff value of PRL-3 expression in a larger and consecutive series and adjuvant setting

Orthoxenografts of testicular germ cell tumors demonstrate genomic changes associated with cisplatin resistance and identify PDMP as a resensitizing agent

[Purpose] To investigate the genetic basis of cisplatin resistance as efficacy of cisplatin-based chemotherapy in the treatment of distinct malignancies is often hampered by intrinsic or acquired drug resistance of tumor cells.[Experimental Design] We produced 14 orthoxenograft transplanting human nonseminomatous testicular germ cell tumors (TGCT) in mice, keeping the primary tumor features in terms of genotype, phenotype, and sensitivity to cisplatin. Chromosomal and genetic alterations were evaluated in matched cisplatin-sensitive and their counterpart orthoxenografts that developed resistance to cisplatin in nude mice.[Results] Comparative genomic hybridization analyses of four matched orthoxenografts identified recurrent chromosomal rearrangements across cisplatin-resistant tumors in three of them, showing gains at 9q32-q33.1 region. We found a clinical correlation between the presence of 9q32-q33.1 gains in cisplatin-refractory patients and poorer overall survival (OS) in metastatic germ cell tumors. We studied the expression profile of the 60 genes located at that genomic region. POLE3 and AKNA were the only two genes deregulated in resistant tumors harboring the 9q32-q33.1 gain. Moreover, other four genes (GCS, ZNF883, CTR1, and FLJ31713) were deregulated in all five resistant tumors independently of the 9q32-q33.1 amplification. RT-PCRs in tumors and functional analyses in Caenorhabditis elegans (C. elegans) indicate that the influence of 9q32-q33.1 genes in cisplatin resistance can be driven by either up- or downregulation. We focused on glucosylceramide synthase (GCS) to demonstrate that the GCS inhibitor DL-threo-PDMP resensitizes cisplatin-resistant germline-derived orthoxenografts to cisplatin[Conclusions] Orthoxenografts can be used preclinically not only to test the efficiency of drugs but also to identify prognosis markers and gene alterations acting as drivers of the acquired cisplatin resistance.Several authors are grateful recipients of predoctoral fellowships: J.M. Piulats from the AECC and F.J. García-Rodríguez from the Instituto de Salud Carlos III (ISCIII). This study was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2002-02265 and FIS: BFU2007-67123; PI10-0222, PI13-01339, and PI16/01898, to A. Villanueva; PI15-00895, to J. Ceron; SAF2013-46063R, to F. Vi nals; PI030264, to ~ X. García-del-Muro), Fundacio La Marat o TV3 (051430, to F. Vi nals and X. ~ García-del-Muro), Generalitat de Catalunya (2014SGR364, to A. Villanueva and F. Vinals; FIS09/0059, to A. Morales), cofunded by FEDER funds/ ~ European Regional Development Fund (ERDF) — a way to Build Europe. A. Villanueva received a BAE11/00073 grant. We thank the staff of the Animal Core Facility of IDIBELL for mouse care and maintenance.Peer reviewe

The Tumor Microenvironment in Liver Metastases from Colorectal Carcinoma in the Context of the Histologic Growth Patterns

Colorectal carcinoma (CRC) is the third most common cancer. Likewise, it is a disease that has a long survival if it is prematurely detected. However, more than 50% of patients will develop metastases, mainly in the liver (LM-CRC), throughout the evolution of their disease, which accounts for most CRC-related deaths. Treatment it is certainly a controversial issue, since it has not been shown to increase overall survival in the adjuvant setting, although it does improve disease free survival (DFS). Moreover, current chemotherapy combinations are administered based on data extrapolated from primary tumors (PT), not considering that LM-CRC present a very particular tumor microenvironment that can radically condition the effectiveness of treatments designed for a PT. The liver has a particular histology and microenvironment that can determine tumor growth and response to treatments: double blood supply, vascularization through fenestrated sinusoids and the presence of different mesenchymal cell types, among other particularities. Likewise, the liver presents a peculiar immune response against tumor cells, a fact that correlates with the poor response to immunotherapy. All these aspects will be addressed in this review, putting them in the context of the histological growth patterns of LM-CRC, a particular pathologic feature with both prognostic and predictive repercussions