Tumor-immune partitioning and clustering algorithm for identifying tumor-immune cell spatial interaction signatures within the tumor microenvironment
Authors: Lau Mai Chan , Borowsky Jennifer , Väyrynen Juha P. , Haruki Koichiro , Zhao Melissa , Dias Costa Andressa , Gu Simeng , da Silva Annacarolina , Ugai Tomotaka , Arima Kota , Nguyen Minh N. , Takashima Yasutoshi , Yeong Joe , Tai David , Hamada Tsuyoshi , Lennerz Jochen K. , Fuchs Charles S. , Wu Catherine J. , Meyerhardt Jeffrey A. , Ogino Shuji , Nowak Jonathan A. 
Published: February 2025

Background
Growing evidence supports the importance of characterizing the organizational patterns of various cellular constituents in the tumor microenvironment in precision oncology. Most existing data on immune cell infiltrates in tumors, which are based on immune cell counts or nearest neighbor-type analyses, have failed to fully capture the cellular organization and heterogeneity.

Methods
We introduce a computational algorithm, termed Tumor-Immune Partitioning and Clustering (TIPC), that jointly measures immune cell partitioning between tumor epithelial and stromal areas and immune cell clustering versus dispersion. As proof-of-principle, we applied TIPC to a prospective cohort incident tumor biobank containing 931 colorectal carcinoma cases. TIPC identified tumor subtypes with unique spatial patterns between tumor cells and T lymphocytes linked to certain molecular pathologic and prognostic features. T lymphocyte identification and phenotyping were achieved using multiplexed (multispectral) immunofluorescence. In a separate hepatocellular carcinoma cohort, we replaced the stromal component with specific immune cell types—CXCR3+CD68+ or CD8+—to profile their spatial relationships with CXCL9+CD68+ cells.

Results
Six unsupervised TIPC subtypes based on T lymphocyte distribution patterns were identified, comprising two cold and four hot subtypes. Three of the four hot subtypes were associated with significantly longer colorectal cancer (CRC)-specific survival compared to a reference cold subtype. Our analysis showed that variations in T-cell densities among the TIPC subtypes did not strictly correlate with prognostic benefits, underscoring the prognostic significance of immune cell spatial patterns. Additionally, TIPC revealed two spatially distinct and cell density-specific subtypes among microsatellite instability-high colorectal cancers, indicating its potential to upgrade tumor subtyping. TIPC was also applied to additional immune cell types, eosinophils and neutrophils, identified using morphology and supervised machine learning; here two tumor subtypes with similarly low densities, namely ‘cold, tumor-rich’ and ‘cold, stroma-rich’, exhibited differential prognostic associations. Lastly, we validated our methods and results using The Cancer Genome Atlas colon and rectal adenocarcinoma data (n = 570). Moreover, applying TIPC to hepatocellular carcinoma cases (n = 27) highlighted critical cell interactions like CXCL9-CXCR3 and CXCL9-CD8.

Conclusions
Unsupervised discoveries of microgeometric tissue organizational patterns and novel tumor subtypes using the TIPC algorithm can deepen our understanding of the tumor immune microenvironment and likely inform precision cancer immunotherapy.