# Integrative Transcriptomic and Single-Cell Protein Characterization of Colorectal Carcinoma Delineates Distinct Tumor Immune Microenvironments Associated with Overall Survival

## TL;DR

This study uses advanced techniques to analyze the immune environment in colorectal cancer tumors and finds that immune activity is linked to patient survival.

## Contribution

The study identifies two distinct immune microenvironment states in colorectal cancer associated with survival outcomes using integrative transcriptomic and single-cell analysis.

## Key findings

- Two CRC groups were identified: one with an immune-activated phenotype and another with an immunosuppressed microenvironment.
- The immunosuppressed group had lower overall survival probability (HR 1.66, p=0.007).
- Immune-activated tumors showed higher CD8+ T cells and tertiary lymphoid structures.

## Abstract

Colorectal carcinoma (CRC) is a heterogeneous group of tumors with varying therapeutic response and prognosis, and evidence suggests the tumor immune microenvironment (TIME) plays a pivotal role. Using advanced molecular and spatial biology technologies, we aimed to evaluate the TIME in patients with CRC to determine whether specific alterations in the immune composition correlated with prognosis.

We identified primary and metastatic tumor samples from 31 consented patients, which were profiled with whole-exome sequencing and bulk RNA-seq. Immune cell deconvolution followed by gene set enrichment analysis and unsupervised clustering was performed. A subset of tumors underwent in situ analysis of the TIME spatial composition at single-cell resolution through Imaging Mass Mass Cytometry.

Gene set enrichment analysis revealed two distinct groups of advanced CRC, one with an immune activated phenotype and the other with a suppressed immune microenvironment. The activated TIME phenotype contained increased Th1 cells, activated dendritic cells, tertiary lymphoid structures, and higher counts of CD8+ T cells whereas the inactive or suppressed TIME contained increased macrophages and a higher M2/M1 ratio. Our findings were further supported by RNA-seq data analysis from the TCGA CRC database, in which unsupervised clustering also identified two separate groups. The immunosuppressed CRC TIME had a lower overall survival probability (HR 1.66, p=0.007).

This study supports the pertinent role of the CRC immune microenvironment in tumor progression and patient prognosis. We characterized the immune cell composition to better understand the complexity and vital role that immune activity states of the TIME play in determining patient outcome.