Exploring immune gene expression in the tumor microenvironment of colorectal cancer

Biomarker potential and association with Fusobacterium spp.

Sammendrag

Colorectal cancer (CRC) remains a global health burden due to late-stage diagnosis and limitations of current screening methods. While colonoscopy remains the gold standard for colorectal cancer detection due to its high sensitivity, its invasive nature and associated patient burden limit widespread compliance. In contrast, fecal immunochemical testing (FIT) is noninvasive, but its limited sensitivity for early-stage lesions and advanced adenomas underscores the need for more accurate non-invasive biomarkers. The primary aim of this PhD project was to explore the potential of immune gene detection in feces as a non-invasive biomarker for colorectal cancer. Additionally, the project aimed to study the association between the immune gene expression and the colorectal cancer-associated bacterium Fusobacterium. In the first study, we profiled immune gene expression in CRC tumors. We analyzed the expression of 579 immune genes using nCounter technology, focusing on differential expression between tumor and adjacent non-tumorous tissue. A total of 202 differentially expressed genes (DEGs) were identified. Tumors consistently overexpressed pro-inflammatory mediators such as CXCL1, CXCL2, CXCL8, IL1B, IL6, and immune modulators like PTGS2 and SPP1. Reverse transcription qPCR (RT-qPCR) confirmed the findings. Furthermore, RTqPCR was performed on additional samples, including biopsies from polyp patients and healthy controls, as well as from multiple tumor sites within the same CRC patients. The results revealed elevated levels of several immune genes in adenomatous polyps, suggesting a role in early tumorigenesis. The second study explored associations between Fusobacterium and molecular alterations, immunological gene expression, and macrophage polarization in CRC tumors. Tumors with high Fusobacterium levels were linked to microsatellite instability (MSI). Analysis of differential immune gene expression, alongside correlation analyses, identified 25 genes associated with high levels of Fusobacterium, including IL6, CXCL8, SPP1, and IDO1. F. animalis was the most prevalent species found in tumor samples from this patient cohort. Although Fusobacterium adhesin protein 2 (fap2) was not transcriptionally elevated in tumor tissues, the total abundance of fap2 in each sample correlated with the expression of the same genes, suggesting that the load of active Fusobacterium cells is related to, and possibly influences, immune gene expression. The third study addressed the detection of immune gene RNA transcripts in stool. We evaluated RNA extraction and RT-PCR protocols, identifying the Norgen Stool RNA kit combined with SuperScript™ III One-Step RT-PCR as optimal. Using this method, we detected significantly higher levels of PTGS2, IL1B, and CXCL8 in fecal samples from CRC patients compared to polyp patients and controls. IL6 and SPP1 had a lower detection rate in fecal samples, but they were primarily restricted to the CRC group. These findings demonstrate that colorectal cancer-associated immune gene expression patterns can be detected in both tumor tissue and stool samples. Furthermore, they underscore the interplay between Fusobacterium colonization and host immune signaling. These insights lay the groundwork for the future development of non-invasive screening biomarkers based on immune-related transcripts and Fusobacterium-associated signatures.