ReCAST: Single-Cell-Informed Tumour Microenvironment Profiling for Translational Prognostic Assessment in Esophageal Cancer

Background/Objectives: Single-cell transcriptomics provides detailed maps of the tumour microenvironment, but translating these maps to large clinical cohorts requires methods that can recover biologically meaningful cell states from routinely available bulk transcriptomes and establish whether these states provide information beyond standard clinical factors. The objective of this study was to develop ReCAST, a donor-aware method that estimates tumour microenvironment cell states from bulk tumour transcriptomes, and to test whether these estimates improved survival prediction in esophageal cancer beyond routine clinical variables.

Methods

ReCAST constructed outcome-independent cell-state anchors from single-cell transcriptomes by first aggregating expression within donors and then across donors, followed by robust non-negative projection of bulk tumour profiles. The framework was evaluated using synthetic mixtures, 720 genuine-cell pseudo-bulk mixtures from 60 held-out donors, cross-study mixtures from an independent single-cell cohort, an independent adenocarcinoma single-cell cohort, and independently measured tumour compositions in 182 TCGA esophageal cancers. Biological validity was examined against Human Protein Atlas cell-type annotations. ReCAST-derived tumour microenvironment features were subsequently tested for incremental prognostic value beyond age, sex, histology, and stage using repeated cross-validation, and the clinical prediction models were evaluated by locked external validation in an independent cohort of 60 patients with squamous-cell carcinoma.