A Vulnerability of a Subset of Colon Cancers with Potential Clinical Utility.

BRAF(V600E) mutant colon cancers (CCs) have a characteristic gene expression signature that is also found in some tumors lacking this mutation. Collectively, they are referred to as "BRAF-like" tumors and represent some 20% of CCs. We used a shRNA-based genetic screen focused on genes upregulated in BRAF(V600E) CCs to identify vulnerabilities of this tumor subtype that might be exploited therapeutically. Here, we identify RANBP2 (also known as NUP358) as essential for survival of BRAF-like, but not for non-BRAF-like, CC cells. Suppression of RANBP2 results in mitotic defects only in BRAF-like CC cells, leading to cell death. Mechanistically, RANBP2 silencing reduces microtubule outgrowth from the kinetochores, thereby inducing spindle perturbations, providing an explanation for the observed mitotic defects. We find that BRAF-like CCs display far greater sensitivity to the microtubule poison vinorelbine both in vitro and in vivo, suggesting that vinorelbine is a potential tailored treatment for BRAF-like CCs.

Can medical imaging identify the histopathological growth patterns of liver metastases?

The histopathological growth patterns (HGPs) of liver metastases of colorectal cancer and of several other tumor types predict outcome of patients in multiple studies. The HGPs of liver metastases have a prognostic but also a predictive value with one of the growth patterns, the replacement growth pattern, related to resistance to systemic treatment. Given that the HGP can only be assessed in a reliable manner when a surgical resection of the metastasis has been performed, this biomarker cannot be exploited to the full. For example, HGPs can at this moment, not be used to decide whether patients with liver metastatic breast or colorectal cancer will benefit or not from locoregional treatment, such as surgery or radiotherapy, and from peri-operative systemic treatment. In this review we highlight studies that suggest that the HGPs of liver metastases can be identified by medical imaging. Although still to be confirmed by a prospective multicenter approach, some studies indeed achieve a high accuracy in predicting the HGPs by applying radiomic algorithms on CT- or MR-images of liver metastases. This is an important step towards a treatment planning of patients with liver metastatic cancer that takes into account the biology and the progression kinetics of the metastases.

Histopathological growth patterns of liver metastasis: updated consensus guidelines for pattern scoring, perspectives and recent mechanistic insights.

The first consensus guidelines for scoring the histopathological growth patterns (HGPs) of liver metastases were established in 2017. Since then, numerous studies have applied these guidelines, have further substantiated the potential clinical value of the HGPs in patients with liver metastases from various tumour types and are starting to shed light on the biology of the distinct HGPs. In the present guidelines, we give an overview of these studies, discuss novel strategies for predicting the HGPs of liver metastases, such as deep-learning algorithms for whole-slide histopathology images and medical imaging, and highlight liver metastasis animal models that exhibit features of the different HGPs. Based on a pooled analysis of large cohorts of patients with liver-metastatic colorectal cancer, we propose a new cut-off to categorise patients according to the HGPs. An up-to-date standard method for HGP assessment within liver metastases is also presented with the aim of incorporating HGPs into the decision-making processes surrounding the treatment of patients with liver-metastatic cancer. Finally, we propose hypotheses on the cellular and molecular mechanisms that drive the biology of the different HGPs, opening some exciting preclinical and clinical research perspectives.

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.

The Blockade of Tumoral IL1ß-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 IL1ß-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 IL1ß-mediated crosstalk between both cell types. We silenced IL1ß in tumor cells to demonstrate that such cells do not exert an influence on NCFs inflammatory phenotype. Our results shows that IL1ß is overexpressed in cocultured tumor cells. IL1ß enables paracrine signaling in myofibroblasts, converting them into inflammatory-CAFs (iCAF). IL1ß-stimulated-NCF-CM induces migration and differential sensitivity to oxaliplatin in colorectal tumor cells. Such chemoprotective effect has not been evidenced for TGFß1-driven NCFs. IL1ß induces the loss of a myofibroblastic phenotype in NCFs and acquisition of iCAF traits. In conclusion, IL1ß-secreted by cancer cells modify surrounding normal fibroblasts to confer protumorogenic features on them, particularly tolerance to cytotoxic drugs. The use of IL1ß-blocking agents might help to avoid the iCAF traits acquisition and consequently to counteract the protumorogenic actions these cells.

Aberrant gene expression in mucosa adjacent to tumor reveals a molecular crosstalk in colon cancer.

BACKGROUND: A colorectal tumor is not an isolated entity growing in a restricted location of the body. The patient's gut environment constitutes the framework where the tumor evolves and this relationship promotes and includes a complex and tight correlation of the tumor with inflammation, blood vessels formation, nutrition, and gut microbiome composition. The tumor influence in the environment could both promote an anti-tumor or a pro-tumor response. METHODS: A set of 98 paired adjacent mucosa and tumor tissues from colorectal cancer (CRC) patients and 50 colon mucosa from healthy donors (246 samples in total) were included in this work. RNA extracted from each sample was hybridized in Affymetrix chips Human Genome U219. Functional relationships between genes were inferred by means of systems biology using both transcriptional regulation networks (ARACNe algorithm) and protein-protein interaction networks (BIANA software). RESULTS: Here we report a transcriptomic analysis revealing a number of genes activated in adjacent mucosa from CRC patients, not activated in mucosa from healthy donors. A functional analysis of these genes suggested that this active reaction of the adjacent mucosa was related to the presence of the tumor. Transcriptional and protein-interaction networks were used to further elucidate this response of normal gut in front of the tumor, revealing a crosstalk between proteins secreted by the tumor and receptors activated in the adjacent colon tissue; and vice versa. Remarkably, Slit family of proteins activated ROBO receptors in tumor whereas tumor-secreted proteins transduced a cellular signal finally activating AP-1 in adjacent tissue. CONCLUSIONS: The systems-level approach provides new insights into the micro-ecology of colorectal tumorogenesis. Disrupting this intricate molecular network of cell-cell communication and pro-inflammatory microenvironment could be a therapeutic target in CRC patients.

Stromal interaction molecule 2 (STIM2) is frequently overexpressed in colorectal tumors and confers a tumor cell growth suppressor phenotype.

Allelic imbalances at chromosome 4p have been largely documented in many different tumor types. In colorectal cancer, loss of heterozygosity (LOH) at 4p15 has been associated with tumor aggressiveness and poor patient outcome, however no target genes in the region have been identified to date. Since stromal interaction molecule 2 (STIM2) is located at 4p15.2 and has been proposed as a candidate gene for this region in glioblastoma multiforme, we aimed at investigating the role of STIM2 in colorectal cancer. We studied STIM2 transcript expression levels in a collection of xenografted primary colorectal tumors (n = 20) and a well-annotated tumor series of colorectal cancer (n = 140). We observed an overexpression of STIM2 in 63.5% of the cases that was associated with a less invasive phenotype. In vitro and in vivo functional studies with colon cancer cell lines revealed that overexpression of STIM2 reduced cell proliferation and tumor growth, respectively. Our work presents several lines of evidence indicating that STIM2 overexpression is a frequent trait in colorectal cancer that results in cell growth suppression, certifying that even in the absence of somatic genetic or epigenetic alterations, recurrent regions of LOH should still be considered a hallmark for the presence of relevant genes for tumorigenesis.

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

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 (dHGP) and non-desmoplastic (ndHGP) metastases. The spatial evaluation reported that dHGP-metastases displayed higher infiltration by CD8+ and CD20+ cells in peripheral regions as well as CD4+ and CD45RO+ cells in ndHGP-metastases. However, the reactive stroma regions at the invasive margin (IM) of ndHGP-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 µm towards the tumor center (p < 0.05). Remarkably, the IM of dHGP-metastases was characterized by higher infiltration of CD8+ cells in the epithelial compartment parameter assessed with the ratio CD8epithelial/CD8stromal, 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 dHGP-metastases some cytotoxic cells reach the tumor nests while remaining retained in the stromal areas in ndHGP-metastases.

The prognostic impact of the tumour stroma fraction: A machine learning-based analysis in 16 human solid tumour types.

BACKGROUND: The development of a reactive tumour stroma is a hallmark of tumour progression and pronounced tumour stroma is generally considered to be associated with clinical aggressiveness. The variability between tumour types regarding stroma fraction, and its prognosis associations, have not been systematically analysed. METHODS: Using an objective machine-learning method we quantified the tumour stroma in 16 solid cancer types from 2732 patients, representing retrospective tissue collections of surgically resected primary tumours. Image analysis performed tissue segmentation into stromal and epithelial compartment based on pan-cytokeratin staining and autofluorescence patterns. FINDINGS: The stroma fraction was highly variable within and across the tumour types, with kidney cancer showing the lowest and pancreato-biliary type periampullary cancer showing the highest stroma proportion (median 19% and 73% respectively). Adjusted Cox regression models revealed both positive (pancreato-biliary type periampullary cancer and oestrogen negative breast cancer, HR(95%CI)=0.56(0.34-0.92) and HR(95%CI)=0.41(0.17-0.98) respectively) and negative (intestinal type periampullary cancer, HR(95%CI)=3.59(1.49-8.62)) associations of the tumour stroma fraction with survival. INTERPRETATION: Our study provides an objective quantification of the tumour stroma fraction across major types of solid cancer. Findings strongly argue against the commonly promoted view of a general associations between high stroma abundance and poor prognosis. The results also suggest that full exploitation of the prognostic potential of tumour stroma requires analyses that go beyond determination of stroma abundance. FUNDING: The Swedish Cancer Society, The Lions Cancer Foundation Uppsala, The Swedish Government Grant for Clinical Research, The Mrs Berta Kamprad Foundation, Sweden, Sellanders foundation, P.O.Zetterling Foundation, and The Sjöberg Foundation, Sweden.

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 alpha-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.

Noncanonical TGFß Pathway Relieves the Blockade of IL1ß/TGFß-Mediated Crosstalk between Tumor and Stroma: TGFBR1 and TAK1 Inhibition in Colorectal Cancer.

PURPOSE: The aim of the study is blocking the recruitment of a protective stroma by altering the crosstalk between normal stromal cells and tumor cells for stripping tumors of the protection conferred by the microenvironment. EXPERIMENTAL DESIGN: A transcriptomic analysis of cocultured normal colonic fibroblasts and colorectal tumor cells was performed. We focused on the study of molecules that mediate the communication between both compartments and that entail fibroblasts' activation and the alteration of the sensitivity to chemotherapy. We identified targets for the blocking of the tumor-stroma interaction. Finally, we tested, in vivo, the blockade of the tumor-stroma interaction in orthotopic models derived from patients and in models of acquired resistance to oxaliplatin. RESULTS: IL1ß/TGFß1 are the triggers for fibroblasts' recruitment and conversion into carcinoma-associated fibroblasts (CAF) in colorectal cancer. CAFs then secrete proinflammatory factors that alter sensitivity in tumor cells, activating JAK/STAT and PI3KCA/AKT pathways. Blocking such crosstalk with a neutralizing IL1ß antibody and a TGFBR1 inhibitor is relieved by the TAK1-mediated activation of the noncanonical TGFß pathway, which induces a change in the cytokine/chemokine repertoire that maintains a sustained activation of AKT in tumor cells. TAK1 plus TGFBR1 inhibition blocks IL1ß/TGFß1-mediated fibroblast activation, decreasing the secretion of proinflammatory cytokines. In turn, tumor cells became more sensitive to chemotherapy. In vivo, the combination of a TAK1 inhibitor plus TGFBR1 inhibitor reduced the metastatic capacity of tumor cells and the recruitment of fibroblasts. CONCLUSIONS: Our findings provide a translational rationale for the inhibition of TAK1 and TGFBR1 to remove the chemoprotection conferred by CAFs.

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. Clin Cancer Res; 24(15); 3755-66. ©2018 AACR.

Hemostatic status in long-term surviving xenografts.

BACKGROUND: Liver xenotransplantation presents, apart from immunologic problems, metabolic incompatibilities between species. The liver plays a key role in blood coagulation. The aim of this study is to describe the hemostatic status of long-term surviving xenografts in a hamster-to-rat liver xenotransplantation model. METHODS: Orthotopic liver transplantation with Tacrolimus and MMF was carried out with Golden Syrian hamsters, Brown Norway, or Dark Agouti rats as donors and Lewis rats as recipients. Prothrombine time (PT), activated partial thromboplastin time (APTT), antithrombin, protein-C, free protein-S, TAT-complexes, and factors V and VIII were assessed using standard methods. RESULTS: Protein-C was absent in rats, but values in xenotransplanted animals increased progressively toward those recorded in hamsters. Xenotransplanted animals also acquired PT, APTT, free protein-S, and antithrombin levels similar to those of donors and we observed a substantial activation of coagulation especially 7 days post-transplantation. Despite TAT high levels, we did not find thrombotic alterations in the histologic analysis of grafts. CONCLUSIONS: These results reflect a destabilization of the thrombotic-hemostatic balance, not associated with consumption coagulopathy, which gradually disappears. This deregulation is a general imbalance resulting from the replacement of all the components of hepatic synthesis. After 100 days of xenotransplantation, the absence of symptoms of thrombosis or hemorrhage suggests that the change of hemostatic status takes place under conditions of relative equilibrium.

Mutanome and expression of immune response genes in microsatellite stable colon cancer.

The aim of this study was to analyze the impact of the mutanome in the prognosis of microsatellite stable stage II CRC tumors. The exome of 42 stage II, microsatellite stable, colon tumors (21 of them relapse) and their paired mucosa were sequenced and analyzed. Although some pathways accumulated more mutations in patients exhibiting good or poor prognosis, no single somatic mutation was associated with prognosis. Exome sequencing data is also valuable to infer tumor neoantigens able to elicit a host immune response. Hence, putative neoantigens were identified by combining information about missense mutations in each tumor and HLAs genotypes of the patients. Under the hypothesis that neoantigens should be correctly presented in order to activate the immune response, expression levels of genes involved in the antigen presentation machinery were also assessed. In addition, CD8A level (as a marker of T-cell infiltration) was measured. We found that tumors with better prognosis showed a tendency to generate a higher number of immunogenic epitopes, and up-regulated genes involved in the antigen processing machinery. Moreover, tumors with higher T-cell infiltration also showed better prognosis. Stratifying by consensus molecular subtype, CMS4 tumors showed the highest association of expression levels of genes involved in the antigen presentation machinery with prognosis. Thus, we hypothesize that a subset of stage II microsatellite stable CRC tumors are able to generate an immune response in the host via MHC class I antigen presentation, directly related with a better prognosis.

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.

Acute xenograft rejection, late xenograft rejection and long term survival xenografts in the hamster-to-rat heart transplantation model: histological characterisation under low-dose of FK506.

BACKGROUND: Long-term survival studies have been conducted in hamster-to-rat cardiac models with a range of immunosuppressive treatments, but the histological pattern of Late Xenograft Rejection (LXR) has not been outlined. This study offers a detailed description of the histological changes in cardiac xenografts under three different immunological responses. MATERIALS AND METHODS: Heterotopic hamster-to-Lewis rat cardiac transplant. Recipients were administered an antiproliferative drug (MMF, 25 mg/kg, or CyP, 10 mg/kg, from day -7 to +7 or from day 0 to +7, according to group) and FK506 (0.2 mg/kg; from day 0 to +30 or continuously). Unmodified recipients were used as controls. Conventional histology and indirect immunofluorescence of IgM, IgG and C3 deposits were performed. RESULTS: In our study, xenografted rats that did not receive treatment developed a pattern of Acute Xenograft Rejection (AXR), with substantial tissue breakdown. Pretreated and treated animals until day 30 post-transplant developed LXR that may present two different histological patterns: one with vascular damage and predominant interstitial haemorrhage, and the other with extensive myocardial fibrosis. Long-term surviving rats (LTS) showed a morphology that was almost normal, with mild fibrosis and vascular endothelium preserved. CONCLUSIONS: AXR, LXR and LTS in the hamster-to-rat heart transplantation model present a common humoral mechanism although their histopathological patterns are different depending on the length of immunosuppressive treatment but not on the type of antiproliferative drug administered. Pretreatment exerts an effect on fibrosis formation.

Modulation of the pathology of late xenograft rejection by PAF-antagonist UR-12670 in the hamster-to-rat liver xenotransplant model.

PAF antagonists have been used in xenotransplantation to alleviate the pathogenesis of hyperacute rejection. This study evaluated the ability of the PAF antagonist UR-12670 to improve graft function in late xenograft rejection (LXR) in an orthotopic liver xenotransplantation model, and the involvement of PAF (platelet activating factor) in this type of rejection. The recipients of a hamster xenograft received standard immunosuppression (tacrolimus 0.2 mg/kg/30 days, MMF 25 mg/kg/8 days). Study groups: group A, without UR-12670, group B, UR-12670 (20 mg/kg/8 d) and group C, continuous administration of UR-12670 (20 mg/kg/d). Serum levels of xenoantibodies were evaluated by flow cytometry and tissue deposits by immunofluorescence. Immunoblot and indirect immunofluorescence assessed specificity of xenoantibodies. Conventional histology was performed. Continuous administration of UR-12670 improved the histological pattern of liver xenografts, especially necrosis, loss of hepatocytes, hemorrhage, sinusoidal congestion and lymphocyte infiltration. There was not a shift in specificity of xenoantibodies at different times posttransplantation, as demonstrated by immunoblotting and indirect immunofluorescence. UR-12670 administration had a beneficial effect on graft function and considerably improved the histopathological pattern, but it failed to induce tolerance after withdrawal of immunosuppression. UR-12670 had an immunomodulatory effect on cellular response but not on antibody production. There was not a change in the specificity of xenoantibodies produced at LXR compared with pretransplant antibodies.

A monotonic and prognostic genomic signature from fibroblasts for colorectal cancer initiation, progression, and metastasis.

UNLABELLED: The differential gene expression patterns between normal colonic fibroblasts (NCF), carcinoma-associated fibroblasts from primary tumors (CAF-PT), and CAFs from hepatic metastasis (CAF-LM) are hypothesized to be useful for predicting relapse in primary tumors. A transcriptomic profile of NCF (n = 9), CAF-PT (n = 14), and CAF-LM (n = 11) was derived. Prediction Analysis of Microarrays (PAM) was used to obtain molecular details for each fibroblast class, and differentially expressed transcripts were used to classify patients according to recurrence status. A number of transcripts (n = 277) were common to all three types of fibroblasts and whose expression level was sequentially deregulated according to the transition: NCF→CAF-PT→CAF-LM. Importantly, the gene signature was able to accurately classify patients with primary tumors according to their prognosis. This capacity was exploited to obtain a refined 19-gene classifier that predicted recurrence with high accuracy in two independent datasets of patients with colorectal cancer and correlates with fibroblast migratory potential. The prognostic power of this genomic signature is strong evidence of the link between the tumor-stroma microenvironment and cancer progression. Furthermore, the 19-gene classifier was able to identify low-risk patients very accurately, which is of particular importance for stage II patients, who would benefit from the omission of chemotherapy, especially T4N0 patients, who are clinically classified as being at high risk. IMPLICATIONS: A defined stromal gene expression signature predicts relapse in patients with colorectal cancer.

Differences between CAFs and their paired NCF from adjacent colonic mucosa reveal functional heterogeneity of CAFs, providing prognostic information.

Little is known about the difference in gene expression between carcinoma-associated fibroblasts (CAFs) and paired normal colonic fibroblasts (NCFs) in colorectal cancer. Paired CAFs and NCFs were isolated from eight primary human colorectal carcinoma specimens. In culture conditions, soluble factors secreted by CAFs in the conditioned media increased clonogenicity and migration of epithelial cancer cells lines to a greater extent than did NCF. In vivo, CAFs were more competent as tumour growth enhancers than paired NCFs when co-inoculated with colorectal cell lines. Gene expression analysis of microarrays of CAF and paired NCF populations enabled us to identify 108 deregulated genes (38 upregulated and 70 downregulated genes). Most of those genes are fibroblast-specific. This has been validated in silico in dataset GSE39396 and by qPCR in selected genes. GSEA analysis revealed a differential transcriptomic profile of CAFs, mainly involving the Wnt signallingsignalling pathway, focal adhesion and cell cycle. Both deregulated genes and biological processes involved depicted a considerable degree of overlap with deregulated genes reported in breast, lung, oesophagus and prostate CAFs. These observations suggest that similar transcriptomic programs may be active in the transition from normal fibroblast in adjacent tissues to CAFs, independently of their anatomic demarcation. Additionally NCF already depicted an activated pattern associated with inflammation. The deregulated genes signature score seemed to correlate with CAF tumour promoter abilities in vitro, suggesting a high degree of heterogeneity between CAFs, and it has also prognostic value in two independent datasets. Further characterization of the roles these biomarkers play in cancer will reveal how CAFs provide cancer cells with a suitable microenvironment and may help in the development of new therapeutic targets for cancer treatment.

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.