Eleonora Cremonesi

Clinical impact of programmed cell death ligand 1 expression in colorectal cancer.

BACKGROUND Programmed cell death 1 (PD-1) receptor triggering by PD ligand 1 (PD-L1) inhibits T cell activation. PD-L1 expression was detected in different malignancies and associated with poor prognosis. Therapeutic antibodies inhibiting PD-1/PD-L1 interaction have been developed. MATERIALS AND METHODS A tissue microarray (n=1491) including healthy colon mucosa and clinically annotated colorectal cancer (CRC) specimens was stained with two PD-L1 specific antibody preparations. Surgically excised CRC specimens were enzymatically digested and analysed for cluster of differentiation 8 (CD8) and PD-1 expression. RESULTS Strong PD-L1 expression was observed in 37% of mismatch repair (MMR)-proficient and in 29% of MMR-deficient CRC. In MMR-proficient CRC strong PD-L1 expression correlated with infiltration by CD8(+) lymphocytes (P = 0.0001) which did not express PD-1. In univariate analysis, strong PD-L1 expression in MMR-proficient CRC was significantly associated with early T stage, absence of lymph node metastases, lower tumour grade, absence of vascular invasion and significantly improved survival in training (P = 0.0001) and validation (P = 0.03) sets. A similar trend (P = 0.052) was also detectable in multivariate analysis including age, sex, T stage, N stage, tumour grade, vascular invasion, invasive margin and MMR status. Interestingly, programmed death receptor ligand 1 (PDL-1) and interferon (IFN)-γ gene expression, as detected by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in fresh frozen CRC specimens (n = 42) were found to be significantly associated (r = 0.33, P = 0.03). CONCLUSION PD-L1 expression is paradoxically associated with improved survival in MMR-proficient CRC.

Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer

Background The immune contexture predicts prognosis in human colorectal cancer (CRC). Whereas tumour-infiltrating CD8+ T cells and myeloid CD16+ myeloperoxidase (MPO)+ cells are associated with favourable clinical outcome, interleukin (IL)-17-producing cells have been reported to correlate with severe prognosis. However, their phenotypes and functions continue to be debated. Objective To investigate clinical relevance, phenotypes and functional features of CRC-infiltrating, IL-17-producing cells. Methods IL-17 staining was performed by immunohistochemistry on a tissue microarray including 1148 CRCs. Phenotypes of IL-17-producing cells were evaluated by flow cytometry on cell suspensions obtained by enzymatic digestion of clinical specimens. Functions of CRC-isolated, IL-17-producing cells were assessed by in vitro and in vivo experiments. Results IL-17+ infiltrates were not themselves predictive of an unfavourable clinical outcome, but correlated with infiltration by CD8+ T cells and CD16+ MPO+ neutrophils. Ex vivo analysis showed that tumour-infiltrating IL-17+ cells mostly consist of CD4+ T helper 17 (Th17) cells with multifaceted properties. Indeed, owing to IL-17 secretion, CRC-derived Th17 triggered the release of protumorigenic factors by tumour and tumour-associated stroma. However, on the other hand, they favoured recruitment of beneficial neutrophils through IL-8 secretion and, most importantly, they drove highly cytotoxic CCR5+CCR6+CD8+ T cells into tumour tissue, through CCL5 and CCL20 release. Consistent with these findings, the presence of intraepithelial, but not of stromal Th17 cells, positively correlated with improved survival. Conclusions Our study shows the dual role played by tumour-infiltrating Th17 in CRC, thus advising caution when developing new IL-17/Th17 targeted treatments.

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.

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.

OX40 expression enhances the prognostic significance of CD8 positive lymphocyte infiltration in colorectal cancer

Background OX40 is a TNF receptor family member expressed by activated T cells. Its triggering by OX40 ligand promotes lymphocyte survival and memory generation. Anti-OX40 agonistic monoclonal antibodies (mAb) are currently being tested in cancer immunotherapy. We explored the prognostic significance of tumor infiltration by OX40+ cells in a large colorectal cancer (CRC) collective. Methods OX40 gene expression was analyzed in 50 freshly excised CRC and corresponding healthy mucosa by qRT-PCR. A tissue microarray including 657 clinically annotated CRC specimens was stained with anti-OX40, -CD8 and -FOXP3 mAbs by standard immunohistochemistry. The CRC cohort was randomly split into training and validation sets. Correlations between CRC infiltration by OX40+ cells alone, or in combination with CD8+ or FOXP3+ cells, and clinical-pathological data and overall survival were comparatively evaluated. Results OX40 gene expression in CRC significantly correlated with FOXP3 and CD8 gene expression. High CRC infiltration by OX40+ cells was significantly associated with favorable prognosis in training and validation sets in univariate, but not multivariate, Cox regression analysis. CRC with OX40high/CD8high infiltration were characterized by significantly prolonged overall survival, as compared to tumors with OX40low/CD8high, OX40high/CD8low or OX40low/CD8low infiltration in both uni- and multivariate analysis. In contrast, prognostic significance of OX40+ and FOXP3+ cell infiltration was not enhanced by a combined evaluation. Irrespective of TNM stage, CRC with OX40high/CD8high density infiltrates showed an overall survival similar to that of all stage I CRC included in the study. Conclusions OX40high/CD8high density tumor infiltration represents an independent, favorable, prognostic marker in CRC with an overall survival similar to stage I cancers.

Abstract 1275: Chemotactic factors underlying tumor infiltration by immunocompetent cells in colorectal cancer

Colorectal cancer (CRC) is a leading cause of cancer related death. Several studies have convincingly showed that CRC infiltration by immunocompetent cells and, in particular, cytotoxic CD8+ T cells, IFN-γ-producing cells, Foxp3+ regulatory T cells and CD16+ MPO+ neutrophils, is significantly associated with prolonged patient survival. However, the factors driving these cell populations into the tumor site remain to be elucidated. We are investigating the chemokine/chemokine receptor network promoting CRC infiltration by immune cells associated to favorable prognosis. We have analysed the expression of a panel of genes encoding for 39 chemokines and for 7 markers identifying specific immune cell types in 58 freshly excised specimens of CRC and corresponding control colonic tissues. In parallel, chemokine receptor profiles on clinically relevant immunocompetent cell populations have been characterized by flow cytometry. Our results suggest that tumor infiltration by immunocompetent cells associated to favorable prognosis is linked to the expression of three main chemokine patterns: 1) CCL3, CCL5, CXCL9, and CXCL10 capable of recruiting cytotoxic CD8+ and IFN-γ producing T cells; 2) CCL22 and CXCL12 attracting Foxp3+ regulatory T cells; 3) CXCL2, CXCL5 and CXCL8 inducing migration of CD16+ MPO+ neutrophils. We have started to investigate the chemokine sources. CRC cells purified from tumor specimens were found to express chemokines potentially recruiting T and myeloid cells, including CCL3, CCL5, CXCL9, CXCL10, CXCL12, and CXCL2, CXCL5, CXCL8. The stimuli driving chemokine expression by tumor cells remain however to be clarified and are currently under investigation. Citation Format: Eleonora Cremonesi, Francesca Amicarella, Elisabetta Padovan, Manuele G. Muraro, Valentina Mele, Paul Zajac, Christian Hirt, Raoul Droeser, Daniel Oertli, Markus Zuber, Michel Adamina, Raffaele Rosso, Jesus G. Garzon, Lubor Borsig, Giulio C. Spagnoli, Giandomenica Iezzi. Chemotactic factors underlying tumor infiltration by immunocompetent cells in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1275. doi:10.1158/1538-7445.AM2015-1275

Abstract A123: Modulation of immune cell trafficking into human colorectal cancer by gut microbiota

Colorectal cancer (CRC) is a leading cause of cancer-related death. CRC infiltration by immune cells, including cytotoxic CD8+ T cells (CTLs), IFN-gamma-producing T-helper 1 cells (Th1), Foxp3+ regulatory T cells (Tregs) and CD16+ MPO+ neutrophils, is associated with favorable prognosis. However, chemokines driving these cell populations into the tumor site, their cellular sources and their microenvironmental triggers remain to be elucidated. We investigated the chemokine/chemokine receptor network promoting CRC infiltration by immune cells associated to favorable prognosis. Analysis of freshly excised specimens of CRC and adjacent healthy colonic tissues revealed that tumor infiltration by beneficial immune cells is associated with the expression of four main chemokine patterns: 1) CCL3, CCL5, CCL8, CXCL9, CXCL10, and CXCL12 for CTLs; 2) CCL5, CCL22, CXCL9 and CXCL12 correlating with CRC infiltration by Th1; 3) CCL22 and CXCL12 attracting Tregs; 4) CXCL2 and CXCL5, promoting chemotaxis of CD16+ MPO+ myeloid cells. Most of identified chemokines were found to be expressed, although at different levels, in primary CRC cells purified from tumor specimens and from xenografts generated upon injection of CRC cells in immunodeficient mice. Notably, chemokine expression levels in orthotopic xenografts, developed upon intracecal injection of tumor cells, were significantly higher as compared to those of subcutaneous tumors. Antibiotic treatment of tumor bearing mice drastically reduced chemokine expression in orthotopic xenografts, thus suggesting a role for commensal bacteria in chemokines induction in tumor cells. Importantly, human CRC samples characterized by high expression of chemokine and immune cell markers, displayed significantly higher bacterial loads, as compared to samples showing low chemokine expression and immune cell infiltration. Furthermore, bacterial loads significantly correlated with expression of CCL3 and CCL5, and Th1 infiltration. These data suggest that stimuli derived from gut flora components may trigger chemokine production in CRC tissues thus leading to tumor infiltration by beneficial immune cells. Note: This abstract was not presented at the conference. Citation Format: Eleonora Cremonesi, Francesca Amicarella, Jesus Francisco Glaus Garzon, Valeria Governa, Manuele Giuseppe Muraro, Valentina Mele, Elisabetta Padovan, Daniel Oertli, Paul Zajac, Giulio Cesare Spagnoli, Lubor Borsig, Giandomenica Iezzi. Modulation of immune cell trafficking into human colorectal cancer by gut microbiota [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A123.

Abstract 2024: Resistance of colorectal cancer cells to 5-FU treatment in three dimensional cell culture models under perfusion involves BCL-2

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Assessment of the effectiveness of chemotherapeutic compounds in bidimensional cell cultures poorly mimics resistance development and the tumor-microenvironment complexity in vivo. In this study we addressed the suitability of a perfused bioreactor to sustain colorectal cancer cell growth and to test established treatment regimens in an in-vitro setting as compared to normal cell culture or xenografts of the same cell line. By culturing the HT29 colorectal cancer cell line in 3D on collagen scaffolds tissue-like structures characterized by a heterogeneous pattern of proliferating and apoptotic cells closely resembling xenografts from the same cell line as generated in immunodeficient mice could be successfully established. CDX2, a colorectal cancer tumor-marker , was expressed in 3D static and perfused cultures, but not in 2D cultures. Upon perfusion, homogeneous seeding on scaffolds could be obtained and significantly higher numbers of tumor cells were recovered, as compared to static cultures (13.7 fold increase, p<0.0001). After 48 hours treatment with clinically relevant concentrations of 5-FU no effect on numbers of cells cultured in 3D under perfusionand as xenografts was observed, as compared to a 55.8% inhibition of 2D cultures. The fraction of Ki67 negative cells (G0 cell phase), was increased after a 48 hours treatment in the 3D perfused and xenografts, but it reached again levels similar to untreated cells after 96 hours. In contrast,cells surviving n 2D cell cultures remained largely positive for Ki67. Importantly, in perfused cultures we could only observe a marginal effect on the expression of BCL-2, TRAF1, FLIP apoptosis resistance genes, as compared to a significant down regulation of their expression in 2D cell cultures and 3D static conditions. In the same line, a combination of ABT-199, a new clinically approved BCL-2 inhibitor, and 5-FU induced additional cytostatic and cytotoxic effects in 3D perfused but not in 2D cell cultures (83.3% inhibition vs. 39.8% p=0.003). Notably, we found that BCL-2 expression in tissues from neoadjuvant treated patients with resistance to treatment was higher than in stage and grade matched colorectal cancers from untreated patients or patients responding to treatment. Cultures in perfused bioreactors offer the possibility of generating tumour tissue-like structures over a short time period. These structures are characterized by sensitivity to chemotherapeutic treatments similar to that of xenografted tumors and significantly different from standard 2D cellular assays.In particular, our data suggest that inhibition of BCL-2 in combination with 5-FU treatment in colorectal cancer could be endowed with therapeutic potential. Citation Format: Christian Hirt, Adam Papadimitropoulos, Evangelos Panopoulos, Valentina Mele, Manuele Muraro, Eleonora Cremonesi, Raoul Droeser, Chantal Mengus, Michael Heberer, Daniel Oertli, Giandomenica Iezzi, Paul Zajac, Serenella Eppenberger-Castori, Luigi Tornillo, Luigi Terracciano, Ivan Martin, Giulio Spagnoli. Resistance of colorectal cancer cells to 5-FU treatment in three dimensional cell culture models under perfusion involves BCL-2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2024. doi:10.1158/1538-7445.AM2014-2024