Chemotactic factors underlying tumor infiltration by immunocompetent cells in human colorectal cancer

Colorectal cancer (CRC) is a common digestive tract malignancy and a major cause of cancer mortality. Several studies have convincingly shown that CRC infiltration by immunocompetent cells and, in particular, cytotoxic CD8+ T cells (CTLs), IFN-γ-producing T-helper 1 cells (Th1), Foxp3+ regulatory T cells (Tregs), and CD16+ MPO+ neutrophils, is significantly associated with prolonged patient survival. However, the chemotactic factors driving these cell populations into the tumor site, their cellular sources and their microenvironmental triggers remain to be elucidated. During my PhD training I have investigated the chemokine/chemokine receptor network promoting CRC infiltration by immune cells associated to favorable prognosis. In particular, I addressed: 1. The expression of immune cell markers and their correlation with chemokine expression in primary CRC tissues; 2. The identification of chemokine receptors relevant for CRC infiltration by beneficial immune cells; 3. The chemokine sources in CRC; 4. The microenvironmental stimuli triggering chemokine production in CRC tissues; 5. The effects of chemokine production on immune cell recruitment into CRC. The expression of a panel of genes encoding 39 chemokines and 7 markers specific for defined immune cell populations was assessed by quantitative PCR array in 62 samples of freshly excised primary CRC and autologous healthy colonic tissue. Correlations between expression of chemokine genes and immune cell markers were then evaluated. Furthermore, chemokine receptor profiles were analysed by flow cytometry on cell suspensions obtained upon digestion of clinical specimens or on corresponding cell populations from autologous peripheral blood. Based on chemokine receptor expression on tumor infiltrating cells and correlations between expression of chemokines and immune cell markers, I could identify for each immune cell subset a putative “chemokine signature”: 1) CCL3, CCL5, CCL8 CXCL9, CXCL10 and CXCL12, associated with recruitment of cytotoxic CTLs; 2) CCL5, CCL22, CXCL9, and CXCL12 correlating with infiltration by Th1; 3) CCL22 and CXCL12 potentially attracting Tregs; 4) CXCL2 and CXCL5 promoting chemotaxis of CD16+ MPO+ neutrophils. I have further investigated potential chemokine sources and stimuli leading to chemokine release within CRC tissues. I found that CRC cells purified from primary tumor specimens express many of the genes encoding identified immune cell recruiting chemokines, including CCL3, CCL5, CXCL2, CXCL5, CXCL9 and CXCL10. In vitro experiments showed that chemokine production by CRC cells is triggered upon their exposure to microbial stimuli, such as Toll-like receptor agonists, or CRC-associated bacteria, including Fusobacterium nucleatum, Bacteroides Fragilis, Bacteroides vulgatus, and Escherichia Coli, thus suggesting that components of the gut flora may critically influence chemokine production in CRC tissues. This was indeed confirmed by “in vivo” experiments showing that chemokine gene expression in xenografts, generated upon injection of human CRC cells in immunodeficient NSG mice, appeared to be related to the presence of commensal bacteria. In particular, chemokine gene expression levels in intracecal xenografts, were found to be ≥10 fold higher as compared to those of subcutaneous xenografts, and they were significantly reduced upon antibiotic treatment of tumor bearing mice. Most importantly, a correlation between extent of immune cell infiltration and bacterial load was also observed in human CRC samples. Indeed, CRC samples characterized by high expression of chemokine and immune cell markers, displayed significantly higher bacterial loads, as assessed by analysis of bacterial 16S ribosomal RNA, as compared to samples showing low chemokine expression and immune cell infiltration. In addition, a significant correlation between bacterial load and expression of the Th1 marker IRF1, CCL3 and CCL5, was also detected. Our in vitro and in vivo results cumulatively suggest that bacteria-induced chemokine production by tumor cells may lead to tumor infiltration by beneficial immune cells. Consistent with this hypothesis, in preliminary “in vitro” experiments, I found that supernatants of bacteria-stimulated CRC cells promote chemotaxis of CTLs and Th1 cells to a higher extent than untreated tumor cells. Additional “in vivo” studies are clearly warranted. In particular, I plan to evaluate intratumoral recruitment of CRC-derived CTLs and Th1 cells upon adoptively transfer into intracecal xenografts-bearing mice. Bacterial species or strains mostly contributing to high chemokine expression and immune cell infiltration in human CRC samples also remain to be identified. Microbiome analysis of CRC samples characterized by high or low immune cell infiltration might be envisaged in future studies. The results of the present work together with the proposed additional studies will contribute to the understanding of the interplay occurring between gut flora and immune system in CRC, and may pave the way towards innovative treatments aimed at modifying the gut flora in order to promote CRC infiltration by beneficial immune cell subsets

Gut microbiota modulate T cell trafficking into human colorectal cancer.

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.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.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.Gut microbiota stimulate chemokine production by CRC cells, thus favouring recruitment of beneficial T cells into tumour tissues

Oviductal microvesicles and their effect on in vitro maturation of canine oocytes

The effect of conditioned medium (CM) or microvesicles (MVs), secreted by multicellular spheroids of oviductal cells, and the involvement of some microRNAs (miRNAs) were investigated in canine oocyte maturation. To generate CM, spheroids were cultured for 3 days. MVs were obtained by ultracentrifugation of CM at 100,000 g and measured for size and concentration by NanoSight instrument. Cumulus-oocyte complexes (COCs) were matured at 38.5 degrees C with 5% CO2 and 5% of O-2 in synthetic oviductal fluid (SOF) in biphasic systems: for 24 h, with 5.0 mu g/mL of LH and for other 48 h with 10% oestrous bitch serum. SOF was used as control (CTR) or supplemented with 10% CM or 25-50-75-100-150 x 10(6) MVs/mL labeled with PKH-26. Results show that multicellular aggregates secreted shedding vesicles. By fluorescence microscopy, the incorporation of labeled MVs was visible only at 72 h in oocyte cytoplasm. These MVs had a positive effect (P < 0.05) on maturation rate (MII) at the concentration of 75 and 100 x 10(6) MVs/mL compared to CM and CTR (20.34% and 21.82% vs 9.09% and 8.66% respectively). The concentration of 150 x 106 MVs/mL provided only 9.26% of MII. The expression of three specific miRNAs (miR-30b, miR-375 and miR-503) was studied. The lower rate of MII with the higher concentration of MVs is possibly due to the high level of miR-375. In conclusion, the oviductal MVs could be involved in cellular trafficking during oocyte maturation and their possible use in vitro could facilitate the exploitment of canine reproductive biotechnologies

Real-life prospective study on asthma control in Italy: Cross-sectional phase results

Objectives: To estimate the prevalence of partly controlled and uncontrolled asthmatic patients, to evaluate quality of life and healthcare resource consumption. Methods: Cross-sectional phase followed by a 12-month prospective phase. Asthma Control Test and the EQ-5D were used. Results: 2853 adult patients recruited in 56 Hospital Respiratory Units in Italy were evaluated: 64.4% had controlled asthma, 15.8% partly controlled asthma and 19.8% were uncontrolled. The mean (SD) EQ-5D score was 0.86 (0.17) in controlled, 0.75 (0.20) in partly controlled and 0.69 (0.23) in uncontrolled patients (p < 0.001 between groups). The number of patients requiring hospitalization or emergency room visits was lower in controlled (1.8% and 1.6%, respectively) than in partly controlled (5.1% and 11.5%) and uncontrolled (6.4% and 18.6%). A combination of an inhaled corticosteroid and a long-acting beta-2 agonist was the reported therapy by 56.0% of patients, with the rate of controlled asthma and improved quality of life being higher in patients on extrafine beclomethasone/formoterol compared to budesonide/formoterol (p < 0.05) and fluticasone/salmeterol (p < 0.05 for quality of life). Conclusions: Asthma control is achieved in a good proportion of Italian patients. Differences may be detected in a real-life setting in favor of extrafine beclomethasone/formoterol combination. © 2011 Elsevier Ltd. All rights reserved

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; 1-12. (c)2017 AACR

Prevalence and Risk Factors of Bullying and Sexual and Racial Harassment in Healthcare Workers: A Cross-Sectional Study in Italy

Background: This cross-sectional study aims to evaluate the prevalence and socio-demographic factors associated with workplace bullying, sexual harassment and racial harassment among Italian health workers. Methods: We recruited 3129 participants using an online Italian translation of the ‘Workplace Violence in the Health Sector Country Case Studies Research Instruments Survey’ (WVHS) questionnaire. Data were analyzed with univariate (chi-square) and multivariate (multiple logistic regression) analysis. Results: Univariate analysis shows that females are significantly more affected by bullying (16.4% vs. 12.3%) and sexual harassment (2.4% vs. 1.3%). On the other hand, males are significantly more affected by racial harassment (3.1% vs. 2.0%). Multivariate analysis shows higher odds of being affected by bullying (OR = 1.30; 95% CI (1.03, 1.64)) and sexual harassment (OR = 2.08; 95% CI (1.04, 4.00)) for females, and higher odds of undergoing racial harassment (OR = 1.55; 95% CI (0.95, 2.53)) for males. Conclusion: This analysis of work situations looks to identify those risk factors, existing or potential, that increase the probability of episodes of violence. A group of work or other subjects identified by direction will have to evaluate the vulnerability of workplaces and establish more effective preventive actions to be adopted

A novel radioguided surgery technique exploiting beta- decay

Radio-guided surgery (RGS) is a technique that helps the surgeon to perform a complete lesion resection. Currently, RGS uses γ emitting tracers, to mark the cancerous tissue from the healthy organs, and a γ radiation detection probe. To overcome the limitations due to the high penetration of γ radiation, a novel approach based on β-radiation has been developed (Sci Rep. 2014;4:4401), allowing to include cases with high uptake of nearby healthy organs, and to benefit of a low medical team exposure

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 &lt; 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

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 OX40(high)/CD8(high) infiltration were characterized by significantly prolonged overall survival, as compared to tumors with OX40(low)/CD8(high), OX40(high)/CD8(low) or OX40(low)/CD8(low) 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 OX40(high)/CD8(high) density infiltrates showed an overall survival similar to that of all stage I CRC included in the study. CONCLUSIONS: OX40(high)/CD8(high) density tumor infiltration represents an independent, favorable, prognostic marker in CRC with an overall survival similar to stage I cancers