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.

Immunofluorescence staining of tumor macrophages in CRC with CD68 (green), CD163 (red) and calprotectin (blue)
UMAP plot of tumour macrophages in CRC