Molecular epidemiology study on genetically regulated gene expression in the colonic mucosa and its role in disease susceptibility

[spa] La expresión genética es un proceso celular clave, que además está relacionado con la susceptibilidad genética a enfermedades y rasgos complejos. La mayoría de genes se someten a splicing alternativo (AS). Las variantes genéticas que regulan la expresión genética y el AS se llaman ¿quantitative trait loci¿ (e/sQTLs). Técnicas estadísticas permiten predecir in silico la expresión genética en un tejido concreto a partir de datos genéticos. Esta aproximación se lleva a cabo en los estudios de asociación de transcriptoma completo (TWAS). Esta Tesis se compone de tres objetivos principales y presenta tres artículos. 1) Generar perfiles de expresión genética de la mucosa colónica de individuos sanos, así como sus diferencias a lo largo del colon y sus e/sQTLs asociados; 2) Desarrollar una aplicación web que permita explorar los datos de expresión genética en el colon; 3) Llevar a cabo un TWAS para proponer genes de susceptibilidad a enfermedad inflamatoria intestinal (EII). Como resumen de los resultados, 1) se generaron catálogos de e/sQTLs a partir de nuevos datos de expresión genética en colon de 445 individuos, y se encontraron más de 4,000 genes que varían sus niveles de expresión a lo largo del colon; 2) se desarrolló el "Colon Transcriptome Explorer", disponible públicamente en https://barcuvaseq.org/cotrex/; 3) se propusieron más de doscientos genes de susceptibilidad genética a EII. En conclusión, nuestros estudios proporcionan nuevos datos y evidencias sobre los genes involucrados en mecanismos de susceptibilidad a enfermedades relacionadas con el colon, y servirán de guía a otros investigadores para proponer nuevas hipótesis en este campo

Leveraging single-cell genomics to uncover clinical and preclinical responses to cancer immunotherapy

Immune checkpoint inhibitors (ICIs) provide durable clinical responses in about 20% of cancer patients, but have been largely ineffective for non-immunogenic cancers that lack intratumoral T cells. Most tumors have somatic mutations that encode for mutant proteins that are tumor-specific and not expressed on normal cells (termed neoantigens). Cancers, such as melanoma, with the highest mutational burdens are more likely to respond to single agent ICIs. However, most cancers, including pancreatic ductal adenocarcinoma (PDAC), have lower mutational loads, resulting in fewer T cells infiltrating the tumor. Studies have previously demonstrated that an allogeneic GM-CSF-based vaccine enhances T cell infiltration into human pancreatic cancer. Recent work with Panc02 cells, which express around 60 neoantigens similar to human PDAC, showed that PancVAX, a neoantigen-targeted vaccine, when paired with immune modulators cleared tumors in Panc02-bearing mice. This data suggests that cancer vaccines targeting tumor neoantigens induce neoepitope-specific T cells, which can be further activated by ICIs, leading to tumor rejection. Currently, the impact of ICIs and neoantigen-targeted vaccines on immune cell expression states and the underlying mechanism of therapeutic response remains poorly defined. Comprehensive characterization of responding immune cells, particularly T cells, will be critical in understanding mechanisms of response and providing a rationale for combinatorial therapies. In this work, we develop innovative computational methods and analysis pipelines to analyze the tumor-immune microenvironment at single-cell resolution. We establish an algorithm to quantify differential heterogeneity in single-cell RNA-seq data, demonstrate the use of non-negative matrix factorization and transfer learning algorithms to identify previously unknown and conserved ICI responses between species, and develop a novel algorithm to physicochemically compare single-cell T cell receptor sequences. We leverage these methods in various contexts to yield new insight into the biological mechanisms underlying positive immunotherapeutic responses in diverse tumor types, including PDAC

Biomarkers of the effect of gastrin on Barrett’s oesophagus

The incidence of adenocarcinoma of the oesophagus (ACO) is rapidly rising, with the UK white male population currently experiencing the highest incidence worldwide. ACO arises on a background of Barrett’s oesophagus (BO), a condition characterised by metastatic remodelling of the lower oesophagus in response to reflux of gastric contents. Previous work within our laboratory has shown that Barrett’s epithelium expresses the gastrin activated CCK2 receptor, that gastrin stimulates proliferation of BO cells, and that Barrett’s patients with the highest circulating gastrin concentrations are more likely to exhibit dysplasia. Patients diagnosed with BO are often prescribed proton pump inhibitors (PPIs) to reduce damage when acid reflux occurs. A common consequence of PPI usage is a significant increase in circulating gastrin concentration. The idea underlying the present study is that gastrin drives the progression of BO to ACO. If so, there is a need for biomarkers of the effect of gastrin on Barrett’s epithelium that might be useful in tracking the progression of BO to ACO and in monitoring the effectiveness of novel anti-gastrin therapies. Circulating gastrin concentration was analysed by radioimmunoassay and related to putative biomarker mRNA and miRNA expression quantified by qPCR in gastric and oesophageal biopsies from BO patients. Using gastric adenocarcinoma cell lines expressing the CCK2 receptor and treated with gastrin, putative protein biomarkers were analysed using western blot. Treatment with PPIs was associated with significant increases in circulating gastrin in control and Barrett’s patients. Abundance of CgA and HDC mRNA was also significantly elevated in gastric biopsies of both groups treated with PPIs. In gastric biopsies from patients with preneoplastic conditions CgA was significantly decreased in the highest risk group. Interestingly, BO biopsies exhibited a significantly lower abundance of CgA, MMP-1, MMP-7, COX-2 and SHH transcripts in patients with high circulating gastrin (>100 pM). In serum, miR-21, -221 and 222 abundances were significantly elevated when circulating gastrin was 30-100 pM but not >100 pM; tissue miR-221 and 222 was lower with gastrin >100 pM. The expression of putative biomarkers in vitro varied between cell lines, although TIMP-1 and MMP-1 exhibited consistent responses to gastrin. Unexpectedly, in stomach and BO biopsies, CCK2 receptor mRNA abundance was significantly depressed in patients with elevated circulating gastrin concentration. Whilst changes in CCK2 receptor expression relative to gastrin have been suggested previously, the regulatory mechanisms are not completely understood. This result was surprising and may account for the pattern of association of putative gastrin-regulated biomarkers and circulating gastrin. The result also has wider-reaching implications for the interpretation of previous studies and for the design of future studies of the effect of gastrin on the progression of preneoplastic conditions in the gastrointestinal tract