Gut microbiota modulate T cell trafficking into human colorectal cancer.

OBJECTIVE: Tumour-infiltrating lymphocytes (TILs) favour survival in human colorectal cancer (CRC). Chemotactic factors underlying their recruitment remain undefined. We investigated chemokines attracting T cells into human CRCs, their cellular sources and microenvironmental triggers. DESIGN: Expression of genes encoding immune cell markers, chemokines and bacterial 16S ribosomal RNA (16SrRNA) was assessed by quantitative reverse transcription-PCR in fresh CRC samples and corresponding tumour-free tissues. Chemokine receptor expression on TILs was evaluated by flow cytometry on cell suspensions from digested tissues. Chemokine production by CRC cells was evaluated in vitro and in vivo, on generation of intraperitoneal or intracecal tumour xenografts in immune-deficient mice. T cell trafficking was assessed on adoptive transfer of human TILs into tumour-bearing mice. Gut flora composition was analysed by 16SrRNA sequencing. RESULTS: CRC infiltration by distinct T cell subsets was associated with defined chemokine gene signatures, including CCL5, CXCL9 and CXCL10 for cytotoxic T lymphocytes and T-helper (Th)1 cells; CCL17, CCL22 and CXCL12 for Th1 and regulatory T cells; CXCL13 for follicular Th cells; and CCL20 and CCL17 for interleukin (IL)-17-producing Th cells. These chemokines were expressed by tumour cells on exposure to gut bacteria in vitro and in vivo. Their expression was significantly higher in intracecal than in intraperitoneal xenografts and was dramatically reduced by antibiotic treatment of tumour-bearing mice. In clinical samples, abundance of defined bacteria correlated with high chemokine expression, enhanced T cell infiltration and improved survival. CONCLUSIONS: Gut microbiota stimulate chemokine production by CRC cells, thus favouring recruitment of beneficial T cells into tumour tissues.

Mesenchymal stromal cells induce epithelial-to-mesenchymal transition in human colorectal cancer cells through the expression of surface-bound TGF-ß.

Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor-associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow-derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial-to-mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT-related genes, downregulation of E-cadherin and acquisition of mesenchymal morphology. These effects required cell-to-cell contact and were mediated by surface-bound TGF-ß newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC-conditioned CRC cells were larger and characterized by higher vessel density, decreased E-cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC-conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT-related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells.

Identification of TPM2 and CNN1 as Novel Prognostic Markers in Functionally Characterized Human Colon Cancer-Associated Stromal Cells.

Stromal infiltration is associated with poor prognosis in human colon cancers. However, the high heterogeneity of human tumor-associated stromal cells (TASCs) hampers a clear identification of specific markers of prognostic relevance. To address these issues, we established short-term cultures of TASCs and matched healthy mucosa-associated stromal cells (MASCs) from human primary colon cancers and, upon characterization of their phenotypic and functional profiles in vitro and in vivo, we identified differentially expressed markers by proteomic analysis and evaluated their prognostic significance. TASCs were characterized by higher proliferation and differentiation potential, and enhanced expression of mesenchymal stem cell markers, as compared to MASCs. TASC triggered epithelial-mesenchymal transition (EMT) in tumor cells in vitro and promoted their metastatic spread in vivo, as assessed in an orthotopic mouse model. Proteomic analysis of matched TASCs and MASCs identified a panel of markers preferentially expressed in TASCs. The expression of genes encoding two of them, calponin 1 (CNN1) and tropomyosin beta chain isoform 2 (TPM2), was significantly associated with poor outcome in independent databases and outperformed the prognostic significance of currently proposed TASC markers. The newly identified markers may improve prognostication of primary colon cancers and identification of patients at risk.

Low Expression of Programmed Death 1 (PD-1), PD-1 Ligand 1 (PD-L1), and Low CD8+ T Lymphocyte Infiltration Identify a Subgroup of Patients With Gastric and Esophageal Adenocarcinoma With Severe Prognosis.

Prognosis of gastric and esophageal cancer is poor and treatment improvements are needed. Programmed cell death 1 receptor (PD-1) interaction with its ligand PD-L1 in tumor micro-environment promotes immune tolerance and blocking monoclonal antibodies have entered clinical practice. However, clinical significance of PD-1 and PD-L1 expression in gastric and esophageal adenocarcinomas, particularly in non-Asian patients, is still unclear. Three tissue microarrays including 190 clinically annotated esophageal (n = 31) and gastric (n = 159) adenocarcinomas and 58 paired mucosa specimens, were stained with PD-1, PD-L1, and CD8-specific reagents in indirect immunohistochemistry assays. PD-L1 expression was detectable in 23.2% of cancer specimens. High PD-1 expression was detectable in 37.3% of cases and high CD8+ infiltration in 76%. PD-L1 and high PD1 expression significantly correlated with each other (r s = 0.404, P < 0.0001) and both significantly correlated with CD8+ infiltration (r s = 0.435, P = 0.0003, and r s = 0.444; P = 0.0004, respectively). CD8+ lymphocyte infiltration correlated with improved survival in univariate (P = 0.009), but not multivariate analysis. Most interestingly, multivariate analysis and Kaplan-Meier curves indicate that combined low PD-1/PD-L1 expression and low CD8+ lymphocyte infiltration significantly correlate with poor prognosis. Our data document the clinical significance of a microenvironmental signature including PD-1/PD-L1 expression and CD8+ lymphocyte infiltration in gastric and esophageal adenocarcinomas and contribute to identify a patients' subset requiring more aggressive peri-operative treatments.

A replication-incompetent CD154/40L recombinant vaccinia virus induces direct and macrophage-mediated antitumor effects in vitro and in vivo.

CD40 triggering may result in antitumor effects of potentially high clinical relevance. To gain insights important for patient selection and to identify adequate targeting techniques, we investigated CD40 expression in human cancer tissues and generated a replication-incompetent recombinant vaccinia virus expressing CD40 ligand (rVV40L). Its effects were explored in vitro and in vivo upon direct CD40 targeting on malignant cells or macrophage activation. CD40 expression was analyzed by immunohistochemistry in tumor and stromal cells in a multi-tumor array including 836 specimens from 27 different tumor types. Established tumor cell lines were used to explore the capacity of rVV40L to induce malignant cell apoptosis and modulate functional profiles of polarized macrophages. CD40 expression was detectable in significantly higher numbers of stromal as compared to malignant cells in lung and breast cancers. CD40 ligation following rVV40L infection induced apoptosis in CD40(+) cancer cells, but only in the presence of intact specific signal transduction chain. Importantly, rVV40L infection promoted the induction of TNF-α-dependent antitumor activity of M1-like macrophages directed against CD40(-) targets. CD40-activated M1-like macrophages also displayed enhanced ability to CXCL10-dependently recruit CD8+ T cells and to efficiently present cancer cell intracellular antigens through cross-priming. Moreover, rVV-driven CD40L expression partially "re-educated" M2-like macrophages, as suggested by detectable CXCL10 and IL-12 production. Most importantly, we observed that intra-tumoral injection of rVV40L-infected human macrophages inhibits progression of human CD40(-) tumors in vivo. First evidences of anticancer activity of rVV40L strongly encourage further evaluations.

Liver biopsy derived induced pluripotent stem cells provide unlimited supply for the generation of hepatocyte-like cells.

BACKGROUND & AIMS: Hepatocyte-like cells (HLCs) differentiated from induced pluripotent stem cells (iPSCs) have emerged as a promising cell culture model to study metabolism, biotransformation, viral infections and inherited liver diseases. iPSCs provide an unlimited supply for the generation of HLCs, but incomplete HLC differentiation remains a major challenge. iPSC may carry-on a tissue of origin dependent expression memory influencing iPSC differentiation into different cell types. Whether liver derived iPSCs (Li-iPSCs) would allow the generation of more fully differentiated HLCs is not known. METHODS: In the current study, we used primary liver cells (PLCs) expanded from liver needle biopsies and reprogrammed them into Li-iPSCs using a non-integrative Sendai virus-based system. Li-iPSCs were differentiated into HLCs using established differentiation protocols. The HLC phenotype was characterized at the protein, functional and transcriptional level. RNA sequencing data were generated from the originating liver biopsies, the Li-iPSCs, fibroblast derived iPSCs, and differentiated HLCs, and used to characterize and compare their transcriptome profiles. RESULTS: Li-iPSCs indeed retain a liver specific transcriptional footprint. Li-iPSCs can be propagated to provide an unlimited supply of cells for differentiation into Li-HLCs. Similar to HLCs derived from fibroblasts, Li-HLCs could not be fully differentiated into hepatocytes. Relative to the originating liver, Li-HLCs showed lower expression of liver specific transcription factors and increased expression of genes involved in the differentiation of other tissues. CONCLUSIONS: PLCs and Li-iPSCs obtained from small pieces of human needle liver biopsies constitute a novel unlimited source for the production of HLCs. Despite the preservation of a liver specific gene expression footprint in Li-iPSCs, the generation of fully differentiated hepatocytes cannot be achieved with the current differentiation protocols.

Maintenance of Primary Human Colorectal Cancer Microenvironment Using a Perfusion Bioreactor-Based 3D Culture System.

Colorectal cancer (CRC) is a leading cause of cancer-related death. Conventional chemotherapeutic regimens have limited success rates, and a major challenge for the development of novel therapies is the lack of adequate in vitro models. Nonmalignant mesenchymal and immune cells of the tumor microenvironment (TME) are known to critically affect CRC progression and drug responsiveness. However, tumor drug sensitivity is still evaluated on systems, such as cell monolayers, spheroids, or tumor xenografts, which typically neglect the original TME. Here, it is investigated whether a bioreactor-based 3D culture system can preserve the main TME cellular components in primary CRC samples. Freshly excised CRC fragments are inserted between two collagen scaffolds in a "sandwich-like" format and cultured under static or perfused conditions up to 3 d. Perfused cultures maintain tumor tissue architecture and densities of proliferating tumor cells to significantly higher extents than static cultures. Stromal and immune cells are also preserved and fully viable, as indicated by their responsiveness to microenvironmental stimuli. Importantly, perfusion-based cultures prove suitable for testing the sensitivity of primary tumor cells to chemotherapies currently in use for CRC. Perfusion-based culture of primary CRC specimens recapitulates TME key features and may allow assessment of tumor drug response in a patient-specific context.

In Vitro Modeling of Tumor-Immune System Interaction.

Immunotherapy has emerged during the past two decades as an innovative and successful form of cancer treatment. However, frequently, mechanisms of actions are still unclear, predictive markers are insufficiently characterized, and preclinical assays for innovative treatments are poorly reliable. In this context, the analysis of tumor/immune system interaction plays key roles, but may be unreliably mirrored by in vivo experimental models and standard bidimensional culture systems. Tridimensional cultures of tumor cells have been developed to bridge the gap between in vitro and in vivo systems. Interestingly, defined aspects of the interaction of cells from adaptive and innate immune systems and tumor cells may also be mirrored by 3D cultures. Here we review in vitro models of cancer/immune cell interaction and we propose that updated technologies might help develop innovative treatments, identify biologicals of potential clinical relevance, and select patients eligible for immunotherapy treatments.

Hemidesmus indicus induces immunogenic death in human colorectal cancer cells.

The ability of anticancer treatments to promote the activation of tumor-reactive adaptive immune responses is emerging as a critical requirement underlying their clinical effectiveness. We investigated the ability of Hemidesmus indicus, a promising anticancer botanical drug, to stimulate immunogenic cell death in a human colorectal cancer cell line (DLD1). Here we show that Hemidesmus treatment induces tumor cell cytotoxicity characterized by surface expression of calreticulin, increased HSP70 expression and release of ATP and HMGB1. Remarkably, the exposure to released ICD-inducer factors from Hemidesmus-treated DLD1 cells caused a modest induction of CD14-derived dendritic cells maturation, as demonstrated by the increased expression of CD83. Moreover, at sub-toxic concentrations, H.i. treatment of monocytes and dendritic cells induced their mild activation, suggesting its additional direct immunostimulatory activity. These data indicate that Hemidesmus indicus induces immunogenic cell death in human tumor cells and suggest its potential relevance in innovative cancer immunotherapy protocols.

Ex-vivo assessment of drug response on breast cancer primary tissue with preserved microenvironments.

Interaction between cancerous, non-transformed cells, and non-cellular components within the tumor microenvironment plays a key role in response to treatment. However, short-term culture or xenotransplantation of cancer specimens in immunodeficient animals results in dramatic modifications of the tumor microenvironment, thus preventing reliable assessment of compounds or biologicals of potential therapeutic relevance. We used a perfusion-based bioreactor developed for tissue engineering purposes to successfully maintain the tumor microenvironment of freshly excised breast cancer tissue obtained from 27 breast cancer patients and used this platform to test the therapeutic effect of antiestrogens as well as checkpoint-inhibitors on the cancer cells. Viability and functions of tumor and immune cells could be maintained for over 2 weeks in perfused bioreactors. Next generation sequencing authenticated cultured tissue specimens as closely matching the original clinical samples. Anti-estrogen treatment of cultured estrogen receptor positive breast cancer tissue as well as administration of pertuzumab to a Her2 positive breast cancer both had an anti-proliferative effect. Treatment with anti-programmed-death-Ligand (PD-L)-1 and anti-cytotoxic T lymphocyte-associated protein (CTLA)-4 antibodies lead to immune activation, evidenced by increased lymphocyte proliferation, increased expression of IFNγ, and decreased expression of IL10, accompanied by a massive cancer cell death in ex vivo triple negative breast cancer specimens. In the era of personalized medicine, the ex vivo culture of breast cancer tissue represents a promising approach for the pre-clinical evaluation of conventional and immune-mediated treatments and provides a platform for testing of innovative treatments.

Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment.

Culture of cancerous cells in standard monolayer conditions poorly mirrors growth in three-dimensional architectures typically observed in a wide majority of cancers of different histological origin. Multicellular tumor spheroid (MCTS) culture models were developed to mimic these features. However, in vivo tumor growth is also characterized by the presence of ischemic and necrotic areas generated by oxygenation gradients and differential access to nutrients. Hypoxia and necrosis play key roles in tumor progression and resistance to treatment. To provide in vitro models recapitulating these events in highly controlled and standardized conditions, we have generated colorectal cancer (CRC) cell spheroids of different sizes and analyzed their gene expression profiles and sensitivity to treatment with 5FU, currently used in therapeutic protocols. Here we identify three MCTS stages, corresponding to defined spheroid sizes, characterized by normoxia, hypoxia, and hypoxia plus necrosis, respectively. Importantly, we show that MCTS including both hypoxic and necrotic areas most closely mimic gene expression profiles of in vivo-developing tumors and display the highest resistance to 5FU. Taken together, our data indicate that MCTS may mimic in vitro generation of ischemic and necrotic areas in highly standardized and controlled conditions, thereby qualifying as relevant models for drug screening purposes.

The hyaluronan-mediated motility receptor RHAMM promotes growth, invasiveness and dissemination of colorectal cancer.

In colorectal cancer (CRC), RHAMM is an independent adverse prognostic factor. The aim of the study was therefore to investigate on the role of RHAMM as a potential direct driver of cell proliferation and migration in CRC cell lines and to identify pathways dependent on RHAMM in human CRC. Proliferation, cell cycle alterations and invasive capacity were tested in two RHAMM- and control- knockdown CRC cell lines by flow cytometry and in vitro assays. Tumorigenicity and metastasis formation was assessed in immunodeficient mice. RNA-Seq and immunohistochemistry was performed on six RHAMM+/- primary CRC tumors. In vitro, silencing of RHAMM inhibited CRC cell migration and invasion by 50% (p<0.01). In vivo, RHAMM knockdown resulted in slower growth, lower tumor size (p<0.001) and inhibition of metastasis (p<0.001). Patients with RHAMM-high CRC had a worse prognosis (p=0.040) and upregulated pathways for cell cycle progression and adhesion turnover. RHAMM overexpression is correlated with increased migration and invasion of CRC cells, leads to larger, fast growing tumors, and its downregulation essentially abolishes metastasis in mouse models. RHAMM is therefore a promising therapeutic target in all CRC stages as its inhibition affects growth and dissemination of the primary CRC as well as the metastases.

The Interplay Between Neutrophils and CD8+ T Cells Improves Survival in Human Colorectal Cancer.

Purpose: Tumor infiltration by different T lymphocyte subsets is known to be associated with favorable prognosis in colorectal cancer. Still debated is the role of innate immune system. We investigated clinical relevance, phenotypes, and functional features of colorectal cancer-infiltrating CD66b+ neutrophils and their crosstalk with CD8+ T cells.Experimental Design: CD66b+ and CD8+ cell infiltration was analyzed by IHC on a tissue microarray including >650 evaluable colorectal cancer samples. Phenotypic profiles of tissue-infiltrating and peripheral blood CD66b+ cells were evaluated by flow cytometry. CD66b+/CD8+ cells crosstalk was investigated by in vitro experiments.Results: CD66b+ cell infiltration in colorectal cancer is significantly associated with increased survival. Interestingly, neutrophils frequently colocalize with CD8+ T cells in colorectal cancer. Functional studies indicate that although neutrophils are devoid of direct antitumor potential, coculture with peripheral blood or tumor-associated neutrophils (TAN) enhances CD8+ T-cell activation, proliferation, and cytokine release induced by suboptimal concentrations of anti-CD3 mAb. Moreover, under optimal activation conditions, CD8+ cell stimulation in the presence of CD66b+ cells results in increasing numbers of cells expressing CD45RO/CD62L "central memory" phenotype. Importantly, combined tumor infiltration by CD66b+ and CD8+ T lymphocytes is associated with significantly better prognosis, as compared with CD8+ T-cell infiltration alone.Conclusions: Neutrophils enhance the responsiveness of CD8+ T cells to T-cell receptor triggering. Accordingly, infiltration by neutrophils enhances the prognostic significance of colorectal cancer infiltration by CD8+ T cells, suggesting that they might effectively promote antitumor immunity. Clin Cancer Res; 23(14); 3847-58. ©2017 AACR.

Expression of the hyaluronan-mediated motility receptor RHAMM in tumor budding cells identifies aggressive colorectal cancers.

Expression of the hyaluronan-mediated motility receptor (RHAMM, CD168) predicts adverse clinicopathological features and decreased survival for colorectal cancer (CRC) patients. Using full tissue sections, we investigated the expression of RHAMM in tumor budding cells of 103 primary CRCs to characterize the biological processes driving single-cell invasion and early metastatic dissemination. RHAMM expression in tumor buds was analyzed with clinicopathological data, molecular features and survival. Tumor budding cells at the invasive front of CRC expressed RHAMM in 68% of cases. Detection of RHAMM-positive tumor budding cells was significantly associated with poor survival outcome (P = .0312), independent of TNM stage and adjuvant therapy in multivariate analysis (P = .0201). RHAMM-positive tumor buds were associated with frequent lymphatic invasion (P = .0007), higher tumor grade (P = .0296), and nodal metastasis (P = .0364). Importantly, the prognostic impact of RHAMM expression in tumor buds was maintained independently of the number of tumor buds found in an individual case (P = .0246). No impact of KRAS/BRAF mutation, mismatch repair deficiency and CpG island methylation was observed. RHAMM expression identifies an aggressive subpopulation of tumor budding cells and is an independent adverse prognostic factor for CRC patients. These data support ongoing efforts to develop RHAMM as a target for precision therapy.

Bioreactor-engineered cancer tissue-like structures mimic phenotypes, gene expression profiles and drug resistance patterns observed "in vivo".

Anticancer compound screening on 2D cell cultures poorly predicts "in vivo" performance, while conventional 3D culture systems are usually characterized by limited cell proliferation, failing to produce tissue-like-structures (TLS) suitable for drug testing. We addressed engineering of TLS by culturing cancer cells in porous scaffolds under perfusion flow. Colorectal cancer (CRC) HT-29 cells were cultured in 2D, on collagen sponges in static conditions or in perfused bioreactors, or injected subcutaneously in immunodeficient mice. Perfused 3D (p3D) cultures resulted in significantly higher (p < 0.0001) cell proliferation than static 3D (s3D) cultures and yielded more homogeneous TLS, with morphology and phenotypes similar to xenografts. Transcriptome analysis revealed a high correlation between xenografts and p3D cultures, particularly for gene clusters regulating apoptotic processes and response to hypoxia. Treatment with 5-Fluorouracil (5-FU), a frequently used but often clinically ineffective chemotherapy drug, induced apoptosis, down-regulation of anti-apoptotic genes (BCL-2, TRAF1, and c-FLIP) and decreased cell numbers in 2D, but only "nucleolar stress" in p3D and xenografts. Conversely, BCL-2 inhibitor ABT-199 induced cytotoxic effects in p3D but not in 2D cultures. Our findings advocate the importance of perfusion flow in 3D cultures of tumor cells to efficiently mimic functional features observed "in vivo" and to test anticancer compounds.

Absence of myeloperoxidase and CD8 positive cells in colorectal cancer infiltrates identifies patients with severe prognosis.

Colorectal cancer (CRC) infiltration by cells expressing myeloperoxidase (MPO) or CD8 positive T lymphocytes has been shown to be independently associated with favorable prognosis. We explored the relationship occurring between CD8+ and MPO+ cell CRC infiltration, its impact on clinical-pathological features and its prognostic significance in a tissue microarray (TMA) including 1,162 CRC. We observed that CRC showing high MPO+ cell infiltration are characterized by a prognosis as favorable as that of cancers with high CD8+ T cell infiltration. However, MPO+ and CD8+ CRC infiltrating cells did not synergize in determining a more favorable outcome, as compared with cancers showing MPOhigh/CD8low or MPOlow/CD8high infiltrates. Most importantly, we identified a subgroup of CRC with MPOlow/CD8low tumor infiltration characterized by a particularly severe prognosis. Intriguingly, although MPO+ and CD8+ cells did not co-localize in CRC infiltrates, an increased expression of TIA-1 and granzyme-B was detectable in T cells infiltrating CRC with high MPO+ cell density.

Efficacy, safety and complications of autologous fat grafting to healthy breast tissue: a systematic review.

BACKGROUND: Fat grafting for primary breast augmentation is growing in popularity due to its autologous properties and its side benefit of removing unwanted fat from other areas, although volume gain is unpredictable and patient safety remains unclear. OBJECTIVE: The aim of this study was to provide an evidence-based overview of autologous fat grafting to healthy breast tissue with focus on volume gain, safety and complications. DESIGN: A systematic review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. DATA SOURCES: The MEDLINE, Cochrane Library and EMBASE databases were searched for clinical studies on autologous fat grafting to healthy breast tissue within the last 30 years. DATA EXTRACTION: Clinical articles were evaluated for indication, pre- and postoperative work-up, surgical technique, volume gain (efficacy), complications, radiographic changes and oncological safety. The level of evidence was assessed according to the Oxford Centre for Evidence-based Medicine 2011. RESULTS: A total of 36 articles involving 1453 patients with a mean follow-up period of 16.3 months (1-156 months) were included. No randomised controlled studies were found. Six percent of the patients undergoing fat grafting to healthy breast tissue experienced major complications requiring a surgical intervention or hospitalisation. Two patients with breast cancer (0.1%) after fat grafting for cosmetic purposes were reported. Average breast volume gain ranged from 55% to 82% relative to the grafted fat volume. CONCLUSIONS: The prevalence of complications and re-operations in fat grafting to healthy breast tissue compared favourably to implant-based breast augmentation. Although no increased incidence of breast cancer was found, long-term breast cancer screening and the implementation of publicly accessible registries are critically important to proving the safety of fat grafting.

"In vitro" 3D models of tumor-immune system interaction.

Interaction between cancer cells and immune system critically affects development, progression and treatment of human malignancies. Experimental animal models and conventional "in vitro" studies have provided a wealth of information on this interaction, currently used to develop immune-mediated therapies. Studies utilizing three-dimensional culture technologies have emphasized that tumor architecture dramatically influences cancer cell-immune system interaction by steering cytokine production and regulating differentiation patterns of myeloid cells, and decreasing the sensitivity of tumor cells to lymphocyte effector functions. Hypoxia and increased production of lactic acid by tumor cells cultured in 3D architectures appear to be mechanistically involved. 3D culture systems could be further developed to (i) include additional cell partners potentially influencing cancer cell-immune system interaction, (ii) enable improved control of hypoxia, and (iii) allow the use of freshly derived clinical cancer specimens. Such advanced models will represent new tools for cancer immunobiology studies and for pre-clinical assessment of innovative treatments.

3'-UTR poly(T/U) tract deletions and altered expression of EWSR1 are a hallmark of mismatch repair-deficient cancers.

The genome-wide accumulation of DNA replication errors known as microsatellite instability (MSI) is the hallmark lesion of DNA mismatch repair (MMR)-deficient cancers. Although testing for MSI is widely used to guide clinical management, the contribution of MSI at distinct genic loci to the phenotype remains largely unexplored. Here, we report that a mononucleotide (T/U)16 tract located in the 3' untranslated region (3'-UTR) of the Ewing sarcoma breakpoint region 1 (EWSR1) gene is a novel MSI target locus that shows perfect sensitivity and specificity in detecting mismatch repair-deficient cancers in two independent populations. We further found a striking relocalization of the EWSR1 protein from nucleus to cytoplasm in MMR-deficient cancers and that the nonprotein-coding MSI target locus itself has a modulatory effect on EWSR1 gene expression through alternative 3' end processing of the EWSR1 gene. Our results point to a MSI target gene-specific effect in MMR-deficient cancers.