Titolo della tesi: Multi-omics and immuno-informatics to untackle the Non-Small Cell Lung Cancer microenvironment and identify biomarkers of response to immunotherapy
Introduction
The actin regulatory protein hMENA undergoes tissue-specific splicing, with the epithelial hMENA11a highly expressed in tumor cells of early non-small cell lung cancer (NSCLC) patients with a favorable prognosis. Differently, the mesenchymal hMENAΔv6 is expressed in invasive cancer cells and in pro-tumoral cancer associated fibroblasts (CAFs).
Immune checkpoint blockade (ICB) has significantly prolonged survival of NSCLC patients, although only a minor percentage of patients have a durable response. The challenge is to understand the complex mechanisms of resistance. Despite NSCLC typically harbors many genomic alterations and a high mutational load, the effectiveness of ICB treatment mainly depends on the composition of the Tumor Immune Microenvironment (TIME) and the pathways activated through the constant dialogue among tumor, immune, and stromal cells. In the TIME, which comprises stromal and non-cellular components, the secretion of specific factors, an increase in immunosuppressive cells, and the creation of physical stromal barriers may contribute to an hostile and immunosuppressive microenvironment.
Therefore, it is imperative to gain a deeper understanding of the TIME landscape to unveil the intricate interactions among the various components. Recent advancements in multi-omics and single-cell technologies, as well as high-resolution spatial biology assays, have become paramount for addressing these crucial questions.
Aim
During my PhD as a Bioinformatician I actively worked and contributed to distinct research projects, all focused on the investigation of the NSCLC TIME. My primary objective was to integrate diverse omics technologies and delineate specific T cell subsets across various tissue districts. The overarching goal was to pinpoint potential biomarkers indicative of response to ICB treatment.
Results and methods
We profiled at single-cell level lymphocytes obtained from PBMC, tumor and adjacent non-tumoral tissues from NSCLC patients, focusing on the function of the co-stimulatory protein CD28 within the CD8+PD1+ cell population. Utilizing unsupervised clustering, we uncovered the presence of five distinct subsets of CD8+PD1+CD28+ lymphocytes and an additional five subsets of CD8+PD1+CD28-. Subsequent analyses and validations carried out on more extensive datasets uncovered gene signatures that exhibit associations with the survival outcomes and response to ICB therapy of NSCLC patients (published paper, Franzese, Palermo et al. JECCR, 2023);
Data from our lab’s in-vitro experiments and RNA-Seq analysis revealed the activation of a viral response, in absence of infection, exclusively in hMENA11a silenced cells. The translation of this data in a internal cohort of NSCLC patients and in publicly available transcriptomic data from two clinical trials with NSCLC patients who received anti-PD-L1 treatment, revealed that low expression of the actin cytoskeleton regulator hMENA11a transcript, coupled with high expression of IFN target genes and high macrophage score, were indicative of patients resistant to ICB therapy. This discovery emphasized the involvement of type I IFN in the promotion of EMT and in the peculiar pro-tumoral polarization of macrophages in the TME. This data highlighted the role of the actin cytoskeleton disturbance in activating immune suppressive pathways that result in the resistance to ICB therapy (published paper, Trono, Tocci et al. JITC, 2023);
I designed and analysed an extensive RNA-Seq experiment from two NSCLC cell lines wherein the hMENA isoforms were depleted. Additionally, I utilized transcript counts obtained from the Nanostring nCounter system and GeoMx DSP spatial transcriptomics data obtained from tumor tissue of NSCLC patients undergoing ICB treatment.We observed that hMENA isoform pattern, in conjunction with the expression levels of fibronectin and Lymphotoxin Beta Receptor (LTβR), demonstrated a broad capability to predict the response to ICB therapy, particularly in tumors where the presence of TLS correlates with an anti-tumour immune response i.e NSCLC, melanoma and triple-negative breast cancer (paper in revision).
Conclusions
The data gathered throughout the entire PhD program culminated in the publication of three research articles. This compelling evidence collectively aims to make a significant contribution by elucidating the heterogeneity within the Tumor-Immune Microenvironment (TIME). The findings reveal a novel role of the splicing of the actin cytoskeleton regulatory protein hMENA, demonstrating its ability not only to regulate actin cytoskeleton dynamics but also to influence crucial signal pathways governing the dialogue among tumor cells, Cancer-Associated Fibroblasts (CAF), and immune cells. Our data uncover pivotal factors associated with resistance to immunotherapy in Non-Small Cell Lung Cancer (NSCLC).