Heterogeneity of Dendritic Cells in Colon and Non-small Cell Lung Cancer

BACKGROUND The activation of adaptive immune responses depends on dendritic cells (DCs) and macrophages, myeloid cells of the innate immune system specialized in antigen presentation and in the activation of T cells. This peculiarity of myeloid cells is fundamental not only during diseases infectious but also in the context of cancer, as phagocytes pick up antigens associated with cancer cells and present them to T cells within the tumor microenvironment or in tumor-draining lymph nodes to obtain antitumor responses. In particular, a positive correlation was observed between the overall tumor content of DC and the survival of cancer patients with different tumors, as well as a better reactivity to therapies based on the use of immunocheckpoint inhibitors (ICB). The presence of different dendritic cell subtypes has recently been revealed, as well as a specific adaptation of each subtype to the tumor environment. This is a critical point since the functional heterogeneity of DCs, macrophages and T cells in the tumor microenvironment is probably one of the factors responsible for the success or failure of anticancer immunotherapies. ICB-based therapies have revolutionized the treatment of patients with cancers, such as melanoma and lung cancer, but are currently only beneficial to a minority of patients. Improving understanding of the tumor immune microenvironment is the key to predicting clinical responses to existing therapies and possibly the development of new immunotherapies. RATIONALE Myeloid cells, such as dendritic cells and macrophages infiltrate many different types of tumors and can exert an antitumor function by activating T and NK cells, or they can carry out a pro-tumor activity by producing anti-inflammatory cytokines and inhibitory molecules. The presence within the tumor of several conventional DC subtypes has been associated with a better prognosis while a pro-tumor function has been proposed for unconventional DCs such as CD14 + CD1c + DC. The presence of macrophages has been associated with a protumoral action. The investigators hypothesize that the functional heterogeneity of myeloid cells and T cells within the tumor microenvironment is one of the factors responsible for the success or failure of antitumor immune responses. High resolution definition of myeloid cell subtypes and tumor associated T cells, their phenotype, distribution within the tumor, as well as their functional characteristics will help to understand the complexity of the tumor microenvironment. STUDY DESIGN Upon collection, each sample will be identified and labeled with a unique ID. The pathologist will sample the material and select the tissue that can be used for the experiment after having taken all that is needed for diagnostic purposes. The research sample will be placed in test tubes and kept on ice. It will then be sent to University of Milano-Bicocca laboratory. The tumor will be cut into pieces and single cell suspensions will be prepared with the human tumor dissociation kit and the gentleMACS ™ dissociator (Miltenyi Biotech) according to the standard protocol. Cell suspensions will then be isolated by density gradient centrifugation. The investigators will analyze approximately 60 patients for immunofluorescence studies and 4 patients for single cell transcriptomic analyzes (single cell RNA-seq). Single cell analysis performed by 4 patients has already shown to be sufficient to identify different subtypes of immune cells. Immune cells will be treated to obtain a cell suspension enriched in myeloid cells. This will allow for accurate subtypes analysis of DC, macrophages and CD4 + T cells and to identify even very rare populations that could be lost in unselected samples due to dilution. The pre-sorting strategy will be based on the expression of CD45 and MHC class II and on the absence of expression of CD3, CD19, CD56, Ly6G to exclude T, B, NK and neutrophils. Residual material will not be stored. It is assumed that patients will be enrolled and analyzed over a 5-year period. There are 5 diifferent tasks: Task1: Preparation and sequencing of the single cell RNA-seq library; Task 2: Single cell RNA-seq bioinformatics analysis; Task 3: Flow cytometric analysis of the DC subtypes present in the tumor microenvironment; Task 4: Spatial distributions of DC subtypes in the tumor microenvironment Task 5: Evaluation of the association between subtypes of myeloid cells present in the tumor microenvironment and survival; The study will end with the bioinformatics analysis of the data and the validation of them through flow cytometric analyzes