Summary Antitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We re-engineered co-detection by indexing (CODEX) for paraffin-embedded tissue microarrays, enabling simultaneous profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers. We identified nine conserved, distinct cellular neighborhoods (CNs)—a collection of components characteristic of the CRC iTME. Enrichment of PD-1 + CD4 + T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferior outcomes. This study provides a framework for interrogating how complex biological processes, such as antitumoral immunity, occur through concerted actions of cells and spatial domains.

During preparation of our article, Shigemi Kinoshita was inadvertently omitted as an author. The author list and Author Contributions section of the online article have been updated. All co-authors have approved the changes. We apologize for the omission.

During preparation of our article, Shigemi Kinoshita was inadvertently omitted as an author. The author list and Author Contributions section of the online article have been updated. All co-authors have approved the changes. We apologize for the omission.

Antitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We optimized CO-Detection by indEXing (CODEX) for paraffin-embedded tissue microarrays, enabling profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers simultaneously. We identified nine conserved, distinct cellular neighborhoods (CNs)-a collection of components characteristic of the CRC iTME. Enrichment of PD-1 + CD4 + T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferioroutcomes. This study provides a framework for interrogating complex biological processes, such as antitumoral immunity, demonstrating an example of how tumors can disrupt immune functionality through interference in the concerted action of cells and spatial domains.

Background Previous assessments of peritumoral inflammatory infiltrate in colorectal cancer (CRC) have focused on the role of CD8 + T lymphocytes. We sought to compare the prognostic value of CD8 with downstream indicators of active immune cell function, specifically granzyme B (GZMB) and CD68 in the tumour microenvironment. Methods Immunohistochemical (IHC) staining was performed for CD8, GZMB, CD68 and CD163 on next-generation tissue microarrays (ngTMAs) in a primary cohort ( n = 107) and a TNM stage II validation cohort ( n = 151). Using digital image analysis, frequency of distinct immune cell types was calculated for tumour proximity (TP) zones with varying radii (10 μm-100 μm) around tumour cells. Results Associations notably of advanced TNM stage were observed for low density of CD8 ( p = 0.002), GZMB ( p < 0.001), CD68 ( p = 0.034) and CD163 ( p = 0.011) in the primary cohort. In the validation cohort only low GZMB ( p = 0.036) was associated with pT4 stage. Survival analysis showed strongest prognostic effects in the TP25μm zone at the tumour centre for CD8 , GZMB and CD68 (all p < 0.001) in the primary cohort and for CD8 ( p = 0.072), GZMB ( p = 0.035) and CD68 ( p = 0.004) in the validation cohort with inferior prognostic effects observed at the tumour invasive margin. In a multivariate survival analysis, joint analysis of GZMB and CD68 was similarly prognostic to CD8 in the primary cohort ( p = 0.007 vs. p = 0.002) and superior to CD8 in the validation cohort ( p = 0.005 vs. p = 0.142). Conclusion Combined high expression of GZMB and CD68 within 25 μm to tumour cells is an independent prognostic factor in CRC and of superior prognostic value to the well-established CD8 in TNM stage II cancers. Thus, assessment of antitumoral effect should consider the quality of immune activation in peritumoral inflammatory cells and their actual proximity to tumour cells. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-022-10048-x.