Valentina Mele

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

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‐β 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.

CD133+, CD166+CD44+, and CD24+CD44+ Phenotypes Fail to Reliably Identify Cell Populations with Cancer Stem Cell Functional Features in Established Human Colorectal Cancer Cell Lines

Increasing evidence that cancers originate from small populations of so‐called cancer stem cells (CSCs), capable of surviving conventional chemotherapies and regenerating the original tumor, urges the development of novel CSC‐targeted treatments. Screening of new anticancer compounds is conventionally conducted on established tumor cell lines, providing sufficient material for high‐throughput studies. Whether tumor cell lines might comprise CSC populations resembling those of primary tumors, however, remains highly debated. We have analyzed the expression of defined phenotypic profiles, including CD133+, CD166+CD44+, and CD24+CD44+, reported as CSC‐specific in human primary colorectal cancer (CRC), on a panel of 10 established CRC cell lines and evaluated their correlation with CSC properties. None of the putative CSC phenotypes consistently correlated with stem cell‐like features, including spheroid formation ability, clonogenicity, aldehyde dehydrogenase‐1 activity, and side population phenotype. Importantly, CRC cells expressing putative CSC markers did not exhibit increased survival when treated with chemotherapeutic drugs in vitro or display higher tumorigenicity in vivo. Thus, the expression of CD133 or the coexpression of CD166/CD44 or CD24/CD44 did not appear to reliably identify CSC populations in established CRC cell lines. Our findings question the suitability of cell lines for the screening of CSC‐specific therapies and underline the urgency of developing novel platforms for anticancer drug discovery.

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.

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.

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.

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.

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.

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

ABSTRACT 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.

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.

Abstract 615: Scaffold-based perfusion bioreactor system for in vitro maintenance of primary breast cancer tissue microenvironment suitable for personalized medicine

INTRODUCTION Two-dimensional (2D) in vitro culture systems and in vivo animal models are the primary tools used to test cancer cell responses to drugs but they are not suited for the development of immune-mediate therapies. Here we present an innovative method for in vitro culture primary breast cancer (BrCa) tissues in porous 3D scaffolds by using a perfusion-based bioreactor system (U-CUP). MATERIALS & METHODS Freshly excised breast cancer specimens were fragmented and cultured in a 3D “sandwich-like format” between collagen porous scaffolds under perfusion flow. DMEM/F12, supplemented with 10% autologous human serum, was used as a culture medium. Malignant and non-malignant cells survival, expansion into the scaffold and the ability to recapitulate features of the original BrCa specimens were histologically assessed. For estrogen receptor (ER) positive tissues we tested the response to hormonal therapy by adding the anti-ER drug Fulvestrant. Furthermore, the maintenance of immune-infiltrating cells allowed testing immune blockade therapy in vitro using anti PD-L1 on PD-L1 positive samples. Response to treatment was evaluated by histology and qRT-PCR for markers of immune-response. RESULTS By culturing BrCa using the U-CUP we were able to preserve viability and to promote the expansion of breast cancer cells from surgical specimens together with accompanying stromal and immune cells into the porous scaffold. Expanding cancer cells were viable after 21 days and recapitulating the initial histology with formation of glands. Administration of anti-ER treatment was associated with decreased expansion of cancer tissue into the scaffold after 21 days. After 7 days of anti PD-L1 antibody treatment we observed a reduced number of tumor cells due to the activation of infiltrating lymphocytes, as shown by increased expression of IFNg and decreased expression of IL10. CONCLUSIONS The scaffold-based perfusion bioreactor represents a successful organotypic tumor model allowing in vitro long-term culture of breast cancer specimens. Our findings shed the light on a promising system for selecting personalized treatment based on a patient9s tumor specific microenvironment. Citation Format: Manuele Giuseppe Muraro, Simone Muenst, Valentina Mele, Luca Quagliata, Alexandar Tzankov, Walter P. Weber, Giulio C. Spagnoli, Savas D. Soysal. Scaffold-based perfusion bioreactor system for in vitro maintenance of primary breast cancer tissue microenvironment suitable for personalized medicine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 615.