Integrative Multiomics Approach Unveils Systemic Dysfunction in Colorectal Cancer (CRC)

A recent study, published in Clinical and Translational Medicine, provides new insights into the colorectal cancer (CRC) immune macroenvironment, identifying key clinical biomarkers that could help stratify CRC patients for immune checkpoint inhibitor (ICI) therapies.

CRC and the Tumor Macroenvironment

With an estimated 1,926,136 cases in 2022 alone, CRC remains one of the most common types of cancer (1). In the recent decade, ICI-based neoadjuvant therapy has demonstrated a promising therapeutic influence (2). 

The tumor microenvironment (TME)—a bionetwork of immune cells, stromal cells, and extracellular components that organize the surrounding architecture and determine tumor progression and therapeutic response—is central to identifying important ICI responders in oncological treatments (3). However, anti-tumor immunity is not limited to the TME.

A much broader tumor environment exists, termed the tumor macroenvironment, encompassing the primary tumor and the surrounding organs, draining lymph node, bone marrow, spleen, and blood, which also responds to tumorigenesis. During tumor progression in mouse models, the tumor macroenvironment has displayed a shift in the composition and function of immune cells, implying an alteration of the immune system (4).

Atopic lymphoid tissues within or surrounding tumors, termed tertiary lymphoid structures (TLS), reveal a new connection between tumors and the immune system. In the context of CRC, this aids our understanding of immune profile and tumor subtypes, contributing to diagnosis, selection of the appropriate treatment, and prognosis prediction (5).

Immune Profiling Methods for the CRC Macroenvironment

The researchers in the current study investigated variation with the CRC immune macroenvironment, identifying fundamental influencing factors that relate to the immune macroenvironment in response to ICI treatment in CRC patients.

The researchers took a multi-disciplinary approach, combining multiomic techniques, including cytometry by time-of-flight (CyTOF), single-cell RNA-sequencing (scRNA-seq), spatial transcriptomics (ST-seq), with multiplex immunohistochemistry (mIHC) for immunoprofiling.

Using a new anatomical dissection method the researchers dissected the bowel into the following four layers: epithelial layer, lamina propria, submucosa and muscularis propria. TissueGnostics’ TissueFAXS microscopy system was utilized to image the mIHC stained tumor and bowel sections, capturing whole cell slide images, while the quantities and distribution of marker-positive cells were analyzed using contextual image analysis software StrataQuest.

cyTOF, scRNA sequencing, ST-seq and immunofluorescent staining enabled the characterization and validation of immune cell heterogeneity, elucidating variation in the CRC immune macroenvironment, and connecting vital influencing factors in the immune macroenvironment to patient response to ICI treatment. 

Adapted from Immune profiling of the macroenvironment in colorectal cancer unveils systemic dysfunction and plasticity of immune cells by Ke et al, 2025. Figure 7. Immune profiling of the macroenvironment in colorectal cancer (CRC) patients with immune checkpoint inhibitor (ICI) treatment. 

New Insights into the CRC Macroenvironment

Immune profiling of non-cancerous human bowel samples revealed each bowel layer to have distinct immune cell niches. CD8+ Tem cells, CD8+ γδ T cells, CD8− γδ T cells, double negative T cells (DNT) and innate immune cells (Lin− CD7+) were enriched within the epithelial layer, while the lamina propria was enriched with mast cell, intermediate monocytes, CD16− NK cells, CD19+ or CD19− plasma cells and natural killer T (NKT) cells.

The major immune cell types in the submucosa included mostly CD4+ T cell subsets, such as CD4+ naïve T cell, Tem, Tcm, Treg, along with naïve B cells, memory B cells, and group 3 innate lymphoid cells (ILC3). The muscularis propria, however, was dominated by myeloid cell subsets.

Immune cell types documented in layers of tumor-adjacent bowel samples, on the other hand, were significantly different, showing a tumor-specific phenotype. Generally, when CD39+ CD4+ Tregs were increased in the marginal intestinal make-up, T cells were found to be terminally activated and exhausted. This could exacerbate tumor progression by furthering the formation of an immunosuppressive environment.

Overall, the CRC macroenvironment was found to increase T cell immunosuppressive marker expression and disturb immune cell composition.

Promising Biomarkers for the CRC Macroenvironment: SPP1–CD44, TLS and CD69

In-situ investigation of the SPP1–CD44 interaction revealed that only macrophage-enriched regions expressed SPP1 within the TME. CD44, on the other hand, was highly expressed in nearly all regions.

The SPP1-CD44 interaction identified in this study was found to have a key immunosuppressive effect directed by SPP1+ macrophages, and this rewired interaction may be a potential ICI therapy target.

TLS, however, were found to boost anti-tumor immunity in the TME, and a higher TLS signature was linked to better overall survival in CRC patients. TLS presence altered the functional capacity of T cells within the TME, and enrichment of the stromal cells was found to potentially inhibit the formation of TLS. TLS was found to be associated with the presence of CD8+ Tem cells in the PBMC, which could serve as a predictive TLS biomarker within the CRC macroenvironment. Further analysis revealed the expression of CD69 and PD-1 in CD8+ Tem from PBMC to be promising prognostic markers for treated-naïve CRC patients.

Cell abundance analysis revealed the enrichment of CD8+ T subsets, Lin− CD7+ cells and DNT cells in consensus molecular subtype 1 (CMS1) tumors, along with an increase in CD8+ T subsets and myeloid cell subsets in CMS3 tumors. Although CMS2 tumors did not show a discrete immune profile, CMS4 tumors had greater infiltration of B cell subsets and naïve T cells.

Examination of PMBC immune cells in relation to CMS group revealed that a reduction in the expression of CD69 considerably distorted the functional capacity of cells in PMBC. Thus, CD69 was considered a promising blood biomarker for the prediction of intrinsic CMS (iCMS) for CRC patients, offering a novel method for selecting iCMS3 patients for ICI treatment.

The Impact of ICI Treatment on the CRC Macroenvironment

CRC patients who received ICI treatment showed increased IFN-γ response pathway activation in the stromal region in tumor or bowel wall samples. Tumors with ICI treatment displayed high expression of the ISG15 gene, and ISG15+ cells increased in both the tumor and normal bowel wall, but ISG15+ CD68+ macrophages increased only in the normal bowel wall, as was shown using quantitative image analysis in StrataQuest.

ICI treatment, therefore, caused a systematic IFN-γ response through the formation of TLS and enhanced the expansion of CD8+ T cells, while ISG15 was characterized as a proinflammatory marker associated with poor response to immunotherapy in CRC patients.

Conclusion

The findings of this study offer fundamental insights into the CRC macroenvironment, revealing distinct immunotypes. TLS presence is positively associated with CMS cell enrichment, ICI efficacy, and overall patient survival rate. By strategically combining the power of multiomic technologies, including TissueFAXS slide scanning platform, important biomarkers were identified, and valuable understandings were provided for implementing the immune macroenvironment in clinical decision-making for CRC patients.

Resources

1. Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 74(1): 12-49.

2. Cercek A, Lumish M, Sinopoli J, et al. 2022. PD-1 blockade in mismatch repair-deficient, locally advanced rectal cancer. N Engl J Med. 386(25): 2363-2376.

3. Zeng D, Wu J, Luo H, et al. 2021. Tumor microenvironment evaluation promotes precise checkpoint immunotherapy of advanced gastric cancer. J Immunotherapy Cancer. 9(8): e002467.

4. Allen BM, Hiam KJ, Burnett CE, et al. 2020. Systemic dysfunction and plasticity of the immune macroenvironment in cancer models. Nat Med. 26(7): 1125-1134.

5. Ogino S, Nosho K, Irahara N, et al. 2009. Lymphocytic reaction to colorectal cancer is associated with longer survival, independent of lymph node count, microsatellite instability, and CpG island methylator phenotype. Clin Cancer Res. 15(20): 6412-6420.

6. Ke,H, Li, P, Li Z, et al. 2025. Immune profiling of the macroenvironment in colorectal cancer unveils systemic dysfunction and plasticity of immune cells." Clinical and Translational Med. 15(2): e70175.

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