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Background The characterization of the highly heterogeneous tumor microenvironment (TME) is key to obtaining a comprehensive understanding of the biological pathways and molecular factors that influence tumor progression and metastasis. Secreted factors, such as chemokines and cytokines, play a crucial role in orchestrating immune responses against tumors. 1 Identifying the spatial expression patterns of such secreted molecules is pivotal for deciphering cell communication, immune cell recruitment, and activation mechanisms. Clinically promising cancer immunotherapies harnessing cytokines and chemokines are expanding,2 yet the spatial visualization of such secreted molecules poses a significant challenge.Here, we integrated multiplexed immunofluorescence and RNA in situ hybridization to simultaneously detect protein and RNA biomarkers to spatially map key secreted factors within the tumor and its microenvironment.Methods Fresh-frozen breast tumor sections were stained and imaged on the automated COMET™ platform combining RNAscope™ 3 HiPlex Pro and sequential immunofluorescence (seqIF™)4 assays to enable simultaneous detection of up to 12 RNA and 24 protein targets on the same tissue section at sub-cellular resolution. The HORIZON™ software was used to analyze the spatial distribution of various cell populations at a single-cell level.Results We conducted spatial profiling of fresh-frozen breast tumor, mapping tumor epithelial cells and neighboring immune cell populations, including helper, regulatory, and cytotoxic T cells, B cells, and antigen-presenting cells. Concomitant in situ detection of key cytokines, such as IFNG, IL-1B, TNFA, and TGFB1, enabled us to characterize T cell differentiation, activation and effector function within the TME. Furthermore, we explored the immune cell infiltration profile by utilizing RNA probes targeting chemokines like CXCL9 and CXCL10, crucial mediators of chemotaxis underlying the immune cell recruitment. We extended our characterization of the TME to map cancer-associated fibroblasts targeting transcripts of the secreted factors COL11A1 and COL1A1, critical players in the metastatic process.Conclusions This fully automated multiomics approach enables simultaneous visualization of multiple RNA and protein targets within the tumor and its microenvironment on fresh-frozen cancer samples. Spatial profiling of secretory molecule expressions within the TME is paramount to provide functional insights guiding the advancement in personalized cancer immunotherapies.References Propper DJ, Balkwill FR. Harnessing cytokines and chemokines for cancer therapy. Nat Rev Clin Oncol. 2022;19:237–253. https://doi.org/10.1038/s41571-021-00588-9 Yi M, Li T, Niu M, et al. Targeting cytokine and chemokine signaling pathways for cancer therapy. Sig Transduct Target Ther. 2024;9:176. https://doi.org/10.1038/s41392-024-01868-3 Wang F, Flanagan J, Su N, Wang LC, Bui S, Nielson A, Wu X, Vo HT, Ma XJ, Luo Y. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn. 2012 Jan;14(1):22–9. doi: 10.1016/j.jmoldx.2011.08.002.Rivest F, et al. Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics. Sci Rep. 2023;13(1):16994. doi: 10.1038/s41598-023-43435-w.