Spatial and functional dissection of cancer-associated fibroblasts-mediated immune modulation in H. pylori-associated gastric cancer [LACE-seq]

We identify four transcriptionally and spatially distinct CAF subtypes with differential immune associations across histological and infection-defined subtypes of GC. Notably, in H. pylori-positive tumors, THBS1+ CAFs exhibit a strong spatial association with regulatory T cells and are linked to local immunosuppression via WNT-FZD interactions. In parallel, the RNA-binding protein ZFP36 post-transcriptionally represses FN1 expression, thereby attenuating FN1+ CAF-mediated cytotoxic T cell engagement. Overall design: LACE-seq was performed in the human gastric cancer cell line HGC-27 to map endogenous ZFP36–RNA interactions using four independent biological immunoprecipitation (IP-only) replicates processed in parallel (no IgG/Input controls). Cells were cultured under standard conditions (RPMI-1640 + 10% FBS, 37 °C, 5% CO2) and harvested at ~70–80% confluence, then subjected to laser-assisted UV crosslinking on ice to covalently fix RNA–protein complexes. Crosslinked lysates were prepared under RNase-controlled conditions and incubated with a ZFP36-specific antibody immobilized on protein A/G magnetic beads; after stringent high-salt and detergent washes to reduce nonspecific binding, complexes were eluted, digested with proteinase K to release crosslinked RNA fragments, and purified by silica-membrane columns. RNA fragments underwent 3'/5' adaptor ligation, reverse transcription, and limited-cycle PCR amplification with unique sample indices, followed by size selection (enriching ~50–150-nt inserts) and library QC (Bioanalyzer profile and qPCR quantification). Indexed libraries were pooled equimolarly and sequenced on an Illumina platform using paired-end reads, with replicate processing steps randomized where feasible to mitigate batch effects.