Studies on immune tumour microenvironment in colorectal cancer (PI Diana Domanska and Frode Jahnsen)

The use of immunotherapy has revolutionized cancer treatment by modulating immune responses against tumours. The first generation of antibody-based immunotherapies – immune checkpoint blockade (ICB) – acts by blocking receptor and/or ligand interacting molecules, such as PD-1 and CTLA-4, molecules that are involved in dampening T cell activation. ICB was the first tissue-agnostic approval of a cancer therapy based on the presence of a single biomarker, microsatellite instability (MSI) or deficient mismatch repair (dMMR). However, although patient subgroups within various cancer types have experienced durable clinical responses with ICB treatment, most cancer patients do not respond to ICB and approximately half of the cancer patients eligible for ICB have primary resistance.
Retrospective studies of patients receiving ICB have shown that the immune tumour microenvironment (iTME) consists of distinct subclasses where some subclasses are associated responsiveness to ICB, whereas others are not. The classification of iTME is mainly based on medium-resolution data of bulk RNA sequencing and immunohistochemistry using techniques such as CYBERSORT, XCell )and Immunoscore. Due to the nature of the datasets being used, the information regarding cellular proportions, cellular heterogeneity and deeper spatial distribution are very limited. The iTME is very diverse and complex and a deeper analysis of this complexity will help us to better identify the patients that will benefit from ICB and guide the search for novel targets for immunotherapeutic intervention.
Colorectal cancer (CRC) is one of the most common types of cancer worldwide. CRC has very limited clinical benefit of ICB. In metastatic CRC, only a small proportion of patients are eligible for ICB treatment: ≤5% of patients with metastatic CRC have MSI/dMMR tumours, and only half of these respond. It is therefore an unmet clinical need to develop new immune-oncology strategies for CRC.
In this project, we will develop an integrated approach to reconstruct cancer tissue architecture by combining single cell technologies and spatial transcriptomics with high dimensional immunoimaging. Computationally integrating these data will allow us to generate a whole-transcriptome map of the cancer tissue at single cell resolution. In combination with clinical and tumour-genomic data from large patient cohorts of CRC we will i) develop a classification system of the iTME with unprecedented resolution, ii) use our high resolution data to predict new immunotherapeutic targets and establish “mini-tumours” to validate our predictions, iii) develop strategies to personalized treatment.

Specific aims
Aim 1: Integrating technologies to produce whole transcriptome single cell resolution of cancer tissue. We will perform single cell (sc) RNA sequencing (seq) and spatial transcriptomics (10X Genomics Visium chips) to generate cellular-resolution spatiotemporal data of the iTME of CRC.
Aim 2: High-resolution spatial analysis of cancer tissue from patients with different clinical outcome. We will apply Visium chips on archival material of cancer tissue from clinically well-characterized CRC patients, selected to represent short and long-term survivors. We will integrate these data with results from Aim 1 to propose a high-resolution classification system of iTME related to clinical outcome (discovery phase).
Aim 3: Develop marker panels of iTME to stratify cancer patients for personalized treatment.
Based on data from Aim 1&2 we will develop panels of antibodies and RNA probes to identify subclasses of iTME on tissue sections (test phase). The panels will be validated on tissue microarrays (TMA) of large patient cohorts designed to consider tumour heterogeneity (validation phase).
Aim 4: Establish mini-tumours for ex vivo functional proof-of-concept of new immune-oncology treatment strategies. We will establish 3D patient-derived organoids (mini-tumours) co-cultured with tumour infiltrating immune cells from patient tumour tissue for in vitro functional validation of novel immunotherapeutic strategies based on results from Aim 1&2. Drug testing of patient-derived organoids will be used to identify novel treatment strategies.

Studies of human gut plasma cells: implications for vaccination and treatment of chronic inflammation

Morbidity and mortality caused by infections in the gastrointe¬stinal tract is a major health problem worldwide. Gut infections are responsible for approxi¬mately 10 million deaths annually of children younger than 5 years of age. For the majority of these diseases, there are no available effective vaccines. The gut is the habitat for trillions of commensal microbes (microbiota) that are beneficial for the host. However, disruption to the normal balance between the gut microbiota and the host (termed dysbiosis) has been associated with inflammatory bowel disease, obesity, allergy and asthma, autoimmune diseases and neurological disorders. The microbiota has thus become a very attractive target for therapeutic interventions. Plasma cells (PCs) are one of the most prominent immune cell-types in the gastrointestinal tract. They produce secretory antibodies (mainly immunoglobulin A (IgA)) that are actively transported into the gut lumen where they protect against invading enteropathogens. Moreover, we and others have shown that secretory antibodies reinforce homeostasis in the gut by regulating the composition of the microbiota.
The prevailing scientific dogma states that PCs of the gut have short half-lives. However, we have provided conclusive evidence that intestinal PCs per¬sist for decades in humans. This finding fundamentally changes the concept of gut humoral immunity with implications both for vaccine development and for therapeutic strategies to target the microbiota. This project directly builds on this paradigm-shifting discovery and our main objectives are to: 1) Identify molecular mechanisms underlying the selection of long-lived intestinal PCs; 2) Identify and characterize members of the microbiota that are targeted by long-lived PCs.
We believe results from this project will have far-reaching clinical implications: 1) Understanding the molecular mechanisms that drive the selection of long-lived PCs is of great importance for the design of vaccines with long-lasting protective effects. 2) There is good reason to believe that commensals that induce persistent IgA responses are particularly important for gut homeostasis and that they therefore would be very attractive targets for therapeutic interventions in diseases associated with dysbiosis.

Dissecting differentiation and diversification of gut macrophages

Macrophages reside in virtually all organs and play a key role in host defense and tissue homeostasis. Tissue macrophages are particularly important in the gut where they defend the body against a wide variety of pathogens and toxic substances.
However, gut macrophages also contribute to pathology. Aberrant activation of macrophages plays an important role in disorders such as inflammatory bowel disease (IBD; ulcerative colitis and Crohn’s disease), necrotizing enterocolitis in infants and mobility dysfunctions. Importantly also, tumor-associated macrophages in colorectal cancer can promote cancer growth.
Although gut macrophages are instrumental in both host defense and pathology, knowledge about their origin, heterogeneity and functional properties is scarce. To increase our understanding of macrophages in the intestine we recently performed transcriptional, phenotypical and functional profiling of tissue macrophages in the human small intestine. We found that macrophages consist of several transcriptional states and by examining gut macrophages in a unique organ transplantation setting we found that blood monocytes emigrated into duodenal transplants and differentiated, through short-lived intermediates, into mature macrophages that survive for months.
To get a deeper understanding of macrophage diversity and functions we have applied single cell RNA sequencing to analyze macrophages isolated from the intestinal mucosa and the underlying smooth muscle layers (muscularis propria). We find that there is an unexpected macrophage heterogeneity within both compartments and between the compartments. Our findings raise several questions we will address in this project: What are the microenvironmental signals that imprint macrophage identity and diversity? Since macrophages are so diverse, is it possible to reprogram macrophages? And do they all originate from the same precursor cells?
We will apply an integrated approach combining high throughput single-cell technologies with unique clinical material and 3D organ cultures. Computational integration of the data will provide a spatiotemporal reconstruction of how monocyte-to-macrophage differentiation and diversification in the human gut is regulated and reveal functional properties.

Understanding the development of resident memory T cells in the human small intestine using integrative multiomic approaches (PI Raquel Bartolome Casado)

Tissue-resident memory T cells (TRM) are permanently lodged in barrier tissues like the gut, and protect the host against pathogens. However, the biological pathways that enable the long-term survival of TRM are poorly understood. By exploiting a unique human intestinal transplantation setting where we can directly distinguish persisting TRM from circulating T cells, we have recently shown that the human small intestine contains large populations of long-lived CD4+ and CD8+ T cells. We will now use this transplantation model to identify mechanisms and anatomical niches that promote and support the development and maintenance of TRM cells. To this end, we will partner with Sarah Teichmann at the Wellcome Sanger Institute in Cambridge, UK, who is a co-founder of the Human Cell Atlas Consortium and a world-leading expert in the field of single-cell genomics and its application to T-cell biology. Postdoc Raquel Bartolome-Casado will spend two years (from June-2021) in Dr. Teichmann’s lab to apply cutting-edge multiomic single-cell technologies, including parallel epigenetic, transcriptomic and T cell receptor analysis, to decipher the developmental pathways of human gut TRM cells. This project will provide unprecedented knowledge on the mechanisms of immune memory development in the human gut, which has major implications for long-lasting oral vaccination strategies and treatments of intestinal immune disorders involving persistent pathogenic T cells.

Identifying novel pathways to treat Hirschsprung-associated enterocolitis

Hirschsprung’s disease (HD) is a common cause of neonatal bowel obstruction. It is characterised by an absence of enteric neurons (aganglionosis) in the distal bowel. The aganglionosis involves the internal anal sphincter and extends orally, most often to the sigmoid colon. The aganglionic segment of the colon fails to relax, causing a functional, severe bowel obstruction. Current treatment is surgical resection of aganglionic bowel. Unfortunately, many children have problems after surgery, particularly in the first years of life. Postoperative problems include constipation, obstructive defecation, fecal incontinence and Hirschsprung-associated enterocolitis (HAEC).
HAEC is a dangerous clinical symptom complex characterized by abdominal distension, fever and diarrhoea. In addition, HAEC has been shown to be associated with reduced functional outcome, particularly fecal incontinence. HAEC occurs in 30–60% of HD patients and may occur both pre-and postoperatively. In some children, HAEC is the presenting symptom of HD, whereas others have one or more episodes after surgery. HAEC is most frequent the first year postoperatively, but some children continue to have relapsing HAEC episodes for many years. HAEC aetiology is poorly understood. Interestingly, this symptom complex does not occur in otherwise healthy children with severe chronic constipation or other bowel disorders. The aim of this project is to increase current understanding of HAEC aetiology with the goal to improve treatment for this condition, which causes severe morbidity and even may be life-threatening.

We hypothesize that the lack of enteric neurons affects the development and function of immune cells in the gut and that disturbances of mucosal immunity in patients with HD is an underlying cause for HAEC.
The aim of this project is to study the mucosal immune system in the gut of HD patients by computational integration of single cell RNA sequencing, Spatial Transcriptomics and high dimensional immune-imaging of colonic tissue from HD patients and age-matched controls.

Identifying biomarkers to predict graft versus host disease in hematopoietic stem cell transplanted patients

30-50% of allo-HSCT patients suffer from graft versus host disease (GVHD); a disease with very high mortality rate. Biomarkers that identify patients at risk for GVHD is important to treat these patients before or early in progression of disease. We have collected blood samples at multiple timepoints from more than 100 patients after transplantation. We are currently examining these samples by two complementary approaches: 1) T cells are assessed with CYTOF technology that are able to phenotype the cells simultaneously with 40 antibodies. 2) Plasma proteins are measured with a commercially available kit from Olink. This allows us to quantitate 1536 proteins in every sample. Comparing the phenotype of T cells and levels of proteins over time in patients that develop GVHD with those that do not develop GVHD, our goal is to identify clinical relevant biomarkers.