CCKBR+ cancer cells contribute to the intertumor heterogeneity of gastric cancer and confer sensitivity to FOXO inhibition

Background: The presence of heterogeneity and poor diagnosis in gastric adenocarcinoma poses a significant challenge to cancer treatment. To address the limited therapeutic options and enhance complex clinical management, we investigated the effects of targeting FOXO to inhibit CCKBR+ cancer cells in vitro and in vivo. Methods: We conducted single-cell sequencing (scRNA-seq) analysis on malignant epithelial cells from five gastric adenocarcinoma patients, revealing the notable heterogeneity within gastric adenocarcinoma (GA). We identified the oncological features of CCKBR+ tumors through immunohistochemistry and clinical statistical analysis. To validate the biological similarity of CCKBR+ tumors, we performed single-cell sequencing on datasets GSE183904 and TCGA. The mutational hallmarks of CCKBR+ tumors were detected using OncoDriveFML and GISTIC 2.0. To further investigate the potential target of CCKBR+ tumors, we utilized organoids and tumor xenograft mouse models, assessing the impact of FOXO inhibition. Finally, we employed CUT&Tag and flow cytometry to determine the genes regulated by FOXO in CCKBR+ tumors. Results: We identified a subset of CCKBR+ stem cell-like cancer cells associated with poorly differentiated features and a worse prognosis in gastric adenocarcinoma. This finding was validated through additional single-cell RNA sequencing (scRNA-seq), TCGA analysis, and histopathology, underscoring the heterogeneity of GA. The unique propensity for gene mutations, such as TP53, APC, SMAD4, and GLI3, was observed in CCKBR+ tumors. Mechanistically, our investigation revealed that FOXOs serve as key transcription factors in maintaining the homeostasis of CCKBR+ tumors. Inhibition of FOXO was found to suppress the growth of CCKBR+ organoids and xenograft tumors. Notably, FOXOs were identified as regulators of α2,3 sialylation levels in CCKBR+ tumor cells, shedding light on a crucial aspect of their homeostasis. Conclusions: CCKBR+ tumor cells significantly contribute to the intertumor heterogeneity of gastric cancer, leading to lower differentiation potential and a poorer prognosis. FOXO plays a crucial role in maintaining the homeostasis of CCKBR+ tumors by upregulating sialylation levels mediated by ST3GAL3/4/5 and ST6GALNAC6. Targeting FOXO effectively suppresses the progression of CCKBR+ tumors. This insight opens new avenues for developing targeted therapeutic strategies aimed at treating cancers associated with CCKBR, providing a novel perspective for precision medicine in this context. We conducted single-cell sequencing (scRNA-seq) analysis on malignant epithelial cells from five gastric adenocarcinoma patients. 10X Genomics Chromium Single-Cell 3’ kit (V3) was used to load 10x Chromium for capturing a maximum of 13000 single cell form single cell suspensions, and following cDNA amplification and library construction were performed according to the manufacturer’s instructions. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. Cell Ranger (vision 5.0.0) pipeline was used for sample demultiplexing, barcode processing and gene counting. For dimensional reduction, clustering and downstream analysis, Cell Ranger output was loaded to Seurat (version 3.1.1) after genome aligning. The STAR aligner was used for generating elementary data, including reads to transcriptome, normalized matrix of gene counts versus cells across samples.