Features of the intratumoral T cell receptor repertoire associated with antigen exposure in cancer patients

The clinical success of immunotherapies demonstrates the importance of the immune system in tumour control, but the response rates remain low and many biological mechanisms underlying how these therapies work are still uncharacterised. In particular, the specificity of the anti-tumour immune response pre-existing in treatment-naive patients or induced by treatment remains poorly described. In this thesis, I explore how T cell receptor (TCR) sequencing data in multi-omics contexts can be utilised to identify features associated with antigen exposure in cancer patients. In treatment-naive non-small cell lung cancer (NSCLC) patients, multi-region TCR sequencing revealed a pattern of heterogeneity in the TCR repertoire resembling the heterogeneity observed in the mutational profile of these tumours and a range of clonotype frequency values associated with tumour specificity. A novel method was built in order to identify distinct TCR populations that spatially follow the pattern of the well-established clonal/subclonal mutational dichotomy. The impact of immune checkpoint blockade therapy on the TCR repertoire distribution was assessed in advanced renal cell carcinoma in the context of anti- PD1 treatment. TCRs with frequency distribution characteristics similar to what was observed in NSCLC were maintained upon treatment and associated with clinical response. In addition, RNA-sequencing analysis identified a gene expression profile consistent with specific activation of T cells through TCR signalling. Finally, the same methodology was applied to bone marrow samples harvested from B cell acute lymphoblastic leukaemia (B-ALL) patients. A statistical framework was developed in order to efficiently distinguish leukaemic re-arrangements from the non- leukaemic TCR repertoire of B-ALL patients. Subsequently, longitudinal analysis revealed TCR distributions that suggested the presence of cytotoxic T cells which was further characterised in matched single-cell RNA sequencing data

Liquid Biopsies to Occult Brain Metastasis

Brain metastasis (BrM) is a major problem associated with cancer-related mortality, and currently, no specific biomarkers are available in clinical settings for early detection. Liquid biopsy is widely accepted as a non-invasive method for diagnosing cancer and other diseases. We have reviewed the evidence that shows how the molecular alterations are involved in BrM, majorly from breast cancer (BC), lung cancer (LC), and melanoma, with an inception in how they can be employed for biomarker development. We discussed genetic and epigenetic changes that influence cancer cells to breach the blood-brain barrier (BBB) and help to establish metastatic lesions in the uniquely distinct brain microenvironment. Keeping abreast with the recent breakthroughs in the context of various biomolecules detections and identifications, the circulating tumor cells (CTC), cell-free nucleotides, non-coding RNAs, secretory proteins, and metabolites can be pursued in human body fluids such as blood, serum, cerebrospinal fluid (CSF), and urine to obtain potential candidates for biomarker development. The liquid biopsy-based biomarkers can overlay with current imaging techniques to amplify the signal viable for improving the early detection and treatments of occult BrM

Gut γδ T cells as guardians, disruptors and instigators of cancer

Colorectal cancer is the third most common cancer worldwide with nearly 2 million cases per year. Immune cells and inflammation are a critical component of colorectal cancer progression, and they are used as reliable prognostic indicators of patient outcome. With the growing appreciation for immunology in colorectal cancer, interest is growing on the role γδ T cells have to play, as they represent one of the most prominent immune cell populations in gut tissue. This group of cells consists of both resident populations—γδ intraepithelial lymphocytes (γδ IELs)—and transient populations that each has unique functions. The homeostatic role of these γδ T cell subsets is to maintain barrier integrity and prevent microorganisms from breaching the mucosal layer, which is accomplished through crosstalk with enterocytes and other immune cells. Recent years have seen a surge in discoveries regarding the regulation of γδ IELs in the intestine and the colon with particular new insights into the butyrophilin family. In this review, we discuss the development, specialities, and functions of γδ T cell subsets during cancer progression. We discuss how these cells may be used to predict patient outcome, as well as how to exploit their behavior for cancer immunotherapy

Role of N-glycosylation in oral cancer

Oral squamous cell carcinoma represents more than 90% head and neck cancers with high incidence rate and morbidity. To date, little is known about the molecular mechanisms responsible for OSCC initiation and progression to advanced disease. Thus, identifying key pathways involved in OSCC pathobiology is likely to lead to the identification of new druggable targets and future anti-cancer therapies. Work from our laboratory has linked the metabolic pathway of protein N-glycosylation with OSCC biology. Specifically, overexpression of the first N-glycosylation gene, DPAGT1, in human OSCC tumor specimens was shown to be associated with aberrant activation of canonical Wnt signaling and inhibition of mature E-cadherin junctions. The purpose of this study was to examine how increased N-glycosylation was associated with OSCC growth and metastatic properties in cellular and murine models. We show that high level of DPAGT1 expression correlates with increased cell surface modification of malignant OSCC HSC-3 cells with complex N-glycans. Further, HSC-3 cells are hypersenitive to the N-glycosylation inhibitor, tunicamycin, suggesting that aggressive properties of OSCC cells depend, in part, on the N-glycosylation pathway. Lastly, we show that orthotopic HSC-3 cell-derived tumor xenografts are inhibited by tunicamycin both in overall growth and metastases, indicating that targeting DPAGT1 and N-glycosylation may represent a new strategy for the treatment of OSCC in human patients

Quantitative approaches for profiling the T cell receptor repertoire in human tissues

The study of B and T cell receptor repertoires from high throughput sequencing is a recent development that allows for unprecedented resolution and quantification of the adaptive immune response. The immense diversity and long tailed distribution of these repertoires has up until now limited such studies to expanded clonal signatures or to analysis of imprecise signals with limited dynamic range collected by techniques such as radioactive and fluorescent labeling. This thesis presents a number of quantitative methods to characterize the repertoire and examine the questions of sequence diversity and inter-repertoire divergence of T cell repertoires. These approaches attempt to accurately parametrize the inherent distribution of T cell clones drawing from statistical tools derived from ecological literature and information theory. The methods presented are applied to T cell analyses of various tissue compartments of the human body, including peripheral blood mononucleocytes, thymic tissues, spleen, inguinal lymph nodes, lung lymph nodes and the brain. A number of applications are explored with strong implications for translational use in medicine. Novel insights are made into the mechanism of maintenance and compartmentalization of na{\"i}ve T cells from human donors of many different ages. Diversity and divergence of the tumor infiltrating sequence repertoire is measured in low grade gliomas and glioblastomas from cancer patients, and potential sequence based biomarkers are assessed for studying glioma phenotype progression. A careful investigation of the immune response to allogeneic stimulus reveals the effect of HLA on sequence sharing and diversity of the alloresponse, and quantifies for the first time using sequence data the fraction of T cells in a repertoire that are alloreactive. The use of repertoire sequencing and mathematical models within immunology is a new and emerging concept within the rapidly expanding field of systems immunology and will undoubtedly have a profound impact on the future of immunology research. It is hoped that the tools presented in this thesis will give insight into how to quantitatively explore the breadth and depth of the T cell receptor repertoire, and provide future directions for TCR repertoire analysis

The NK cells phenotype and function in resected non small cell lung cancer: differences between squamous and adenocarcinoma.

The tumor microenvironment has come to light as a key player in carcinogenesis and progression. Tumors affect many host cell types, in particular immune cells. The immune system appears to select particularly fit tumor cells in the process of immuno-­editing, while the tumor cells influence the polarization of immune cells towards phenotypes that favor tumor growth and vascularization. Here we investigated the phenotype of tumor infiltrating natural killer (NK) cells, focusing on angiogenesis associated cytokines and activities in patient-­derived material from non-­small cell lung cancer (NSCLC). Samples from the tumor and adjacent normal issues, as well as peripheral blood and lung samples as well as from non-­oncologic patients with bullous emphysema were collected and rapidly processed to obtain single cell suspensions. Flow cytometry (FC) analyses were performed to evaluate specific markers (CD3, CD56, CD16) to identify NK cell subsets. We observed that in the NSCLC samples, the CD56+CD16-­ NK phenotype, associated with cytokine production, predominated in the tumor samples while the CD56dimCD16+ cytotoxic phenotype dominated in the adjacent normal tissues and in lung tissue derived from non-­oncologic patients. This was independent of tumor histotype and smoking status. We examined the angiogenic potential of tumor infiltrating NK cells by intracellular staining for production of VEGF, PlGF, IL-­8 (CXCL8), IFNand other markers. The CD56+CD16-­ subset was clearly associated with production of angiogenic cytokines in all samples. However, patients with squamous carcinoma histotypes showed remarkably and significantly higher production of angiogenic factors in tumor infiltrating, adjacent tissue and especially in peripheral blood CD56+CD16-­ NK cells than patients with adenocarcinomas. Following surgical intervention, these levels were reduced in disease-free patients. Moreover, supernatants derived from the tumor infiltrating CD56+CD16-­ NK cells were able to induce endothelial cell chemotaxis and formation of capillary-­like structures in vitro; this was particularly evident for NK cells isolated from squamous cell carcinomas. Our data suggest that squamous NSCLC tumors have a significant systemic effect on NK cells, enhancing angiogenic cytokine production in a manner dependent on the presence of disease. NK cells appear to participate in tumor neovascularization and could represent a peripheral marker for disease progression, angiogenesis and response to therapies in some tumor subsets

The NK cells phenotype and function in resected non small cell lung cancer: differences between squamous and adenocarcinoma.

The tumor microenvironment has come to light as a key player in carcinogenesis and progression. Tumors affect many host cell types, in particular immune cells. The immune system appears to select particularly fit tumor cells in the process of immuno-\uadediting, while the tumor cells influence the polarization of immune cells towards phenotypes that favor tumor growth and vascularization. Here we investigated the phenotype of tumor infiltrating natural killer (NK) cells, focusing on angiogenesis associated cytokines and activities in patient-\uadderived material from non-\uadsmall cell lung cancer (NSCLC). Samples from the tumor and adjacent normal issues, as well as peripheral blood and lung samples as well as from non-\uadoncologic patients with bullous emphysema were collected and rapidly processed to obtain single cell suspensions. Flow cytometry (FC) analyses were performed to evaluate specific markers (CD3, CD56, CD16) to identify NK cell subsets. We observed that in the NSCLC samples, the CD56+CD16-\uad NK phenotype, associated with cytokine production, predominated in the tumor samples while the CD56dimCD16+ cytotoxic phenotype dominated in the adjacent normal tissues and in lung tissue derived from non-\uadoncologic patients. This was independent of tumor histotype and smoking status. We examined the angiogenic potential of tumor infiltrating NK cells by intracellular staining for production of VEGF, PlGF, IL-\uad8 (CXCL8), IFN\uf0e3\uf020and other markers. The CD56+CD16-\uad subset was clearly associated with production of angiogenic cytokines in all samples. However, patients with squamous carcinoma histotypes showed remarkably and significantly higher production of angiogenic factors in tumor infiltrating, adjacent tissue and especially in peripheral blood CD56+CD16-\uad NK cells than patients with adenocarcinomas. Following surgical intervention, these levels were reduced in disease-free patients. Moreover, supernatants derived from the tumor infiltrating CD56+CD16-\uad NK cells were able to induce endothelial cell chemotaxis and formation of capillary-\uadlike structures in vitro; this was particularly evident for NK cells isolated from squamous cell carcinomas. Our data suggest that squamous NSCLC tumors have a significant systemic effect on NK cells, enhancing angiogenic cytokine production in a manner dependent on the presence of disease. NK cells appear to participate in tumor neovascularization and could represent a peripheral marker for disease progression, angiogenesis and response to therapies in some tumor subsets

Phenotypic, functional, and metabolic heterogeneity of immune cells infiltrating non–small cell lung cancer.

Lung cancer is the leading cancer in the world, accounting for 1.2 million of new cases annually, being responsible for 17.8% of all cancer deaths. In particular, non–small cell lung cancer (NSCLC) is involved in approximately 85% of all lung cancers with a high lethality probably due to the asymptomatic evolution, leading patients to be diagnosed when the tumor has already spread to other organs. Despite the introduction of new therapies, which have improved the long-term survival of these patients, this disease is still not well cured and under controlled. Over the past two decades, single-cell technologies allowed to deeply profile both the phenotypic and metabolic aspects of the immune cells infiltrating the TME, thus fostering the identification of predictive biomarkers of prognosis and supporting the development of new therapeutic strategies. In this review, we discuss phenotypic and functional characteristics of the main subsets of tumorinfiltrating lymphocytes (TILs) and tumor-infiltrating myeloid cells (TIMs) that contribute to promote or suppress NSCLC development and progression. We also address two emerging aspects of TIL and TIM biology, i.e., their metabolism, which affects their effector functions, proliferation, and differentiation, and their capacity to interact with cancer stem cells

Cancer progression: a single cell perspective

Tumor tissues are constituted by a dynamic diversity of malignant and non-malignant cells, which shape a puzzling biological ecosystem affecting cancer biology and response to treatments. Over the course of the tumoral disease, cancer cells acquire genotypic and phenotypic changes, allowing them to improve cellular fitness and overcome environmental and treatment constraints. This progression is depicted by an evolutionary process in which single cells expand as a result of an interaction between single-cell changes and the lovelopments have made it possible to depict the development of cancer at the single-cell level, offering a novel method for understanding the biology of this complex disease. Here, we review those complex interactions from the perspective of single cells and introduce the concept of omics for single-cell studies. This review emphasizes the evolutionary dynamics that control cancer progression and the capacity of single cells to escape the local environment and colonize distant sites. We are assisting a rapid progression of studies carried out at the single-cell level, and we survey relevant single-cell technologies looking at multi-omics studies. These path for precision medicine in cancer

Sarcoma ecosystems : spatial characterization and prognostic significance

Sarcoma is a highly heterogeneous disease with complex biological activities and unique tumor microenvironments (TME) in distinct subtypes. The limited treatment options and inadequate responses to current therapies necessitate a deeper understanding of sarcoma biology and personalized treatment strategies. Our research comprehensively explores the sarcoma TME through advanced spatial analysis and investigates sarcoma's molecular and genetic profiles through transcriptome and genome sequencing. In paper I, we focused on undifferentiated pleomorphic sarcoma (UPS) using multiplex immunofluorescence (mIF) staining for in-depth spatial analysis of B cell populations and lymphocyte aggregates (LAs). LAs in UPS were found to be associated with longer overall survival (OS) and metastasis-free survival (MFS). Moreover, we unveiled distinct maturation profiles among B cell subsets, indicative of different phenotypes that contribute to functional ecosystems in TME. LA-positive tumors displayed a more well-differentiated B cell profile throughout the entire tumor section, not limited in LA regions. We introduced the B-index, an integrated measurement tool combining B cell abundance and maturity, which demonstrated predictive power for both MFS and OS. Using the TissUUmap tool, we identified B cell desert areas characterized by extremely low B cell infiltration. LA-positive tumors displayed smaller and more fragmented B cell desert areas. In paper II, we performed double immunohistochemistry to study CD11c-positive antigen-presenting cells (APCs) and CD8- positive cells in 177 soft tissue sarcoma (STS) patients. We found that CD11c-CD8 interactions in the TME were associated with improved MFS and OS. Transcriptomic analysis in The Cancer Genome Atlas (TCGA) sarcoma cohort supported the prognostic significance of combining CD11c with CD8, irrespective of FOXP3 levels and in the presence of CD274 (PD-L1). In paper III, we conducted transcriptome and targeted DNA sequencing in 91 synovial sarcomas, identifying three distinct Synovial Sarcoma Clusters (SSCs) mirroring histological subtypes. SSC-I was characterized by high cell proliferation and immune evasion with an unfavorable prognosis. SSC-II was dominated by vascularstromal components and correlated with better outcomes. SSC-III displayed biphasic differentiation, genomic complexity, and immune checkpoint-mediated immune suppression, leading to adverse outcomes, even after a good histological response to neoadjuvant treatment. In paper IV, we analyzed Ewing sarcoma (ES) transcriptome signatures in four previously published cohorts and identified 29 prognostic RNA-binding protein (RBP) genes, from which we constructed and validated an RBP-associated prognostic risk model (RPRM). The RPRM demonstrated stable predictive value for prognosis, with NSUN7 emerging as an independent and favorable prognostic marker. In summary, our research integrates spatial analysis of the sarcoma TME to identify unique immune features and prognostic markers. Moreover, we use transcriptomic and genomic analyses to categorize specific sarcoma types for more detailed survival stratification. This work provides a deeper insight into the sarcoma TME and suggests an improved grouping strategy, aiming to shape the development of personalized immunotherapy in the future