Pparg Activation-Mediated Lipid Metabolism Reprogramming Confers an Invasive Phenotype and Immune Evasion in Early-Onset Colorectal Cancer
Description
This dataset contains single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics data to systematically compare the tumor microenvironment (TME) between early-onset colorectal cancer (EOCRC) and late-onset CRC (LOCRC). scRNA-seq: Twelve CRC tissue samples were collected from AOM-DSS-induced C57BL/6 mice: young group (3-4 months, n=6) and old group (18-20 months, n=6), female:male=1:1. Single-cell suspensions were prepared by enzymatic digestion. Libraries were constructed using the SeekOne DD kit (High Precision Life Technology Co., LTD). Quality control was performed with Scanpy (v1.11.3) and omicverse (v1.7.5): cells with mitochondrial%<25%, ribosomal%<25%, hemoglobin%<5%, UMI>500, genes>200 were retained. Doublets were removed using DoubletFinder (v2.0.4). Batch correction used Harmony (v1.2.0) on 30 PCs. Clustering at resolution 0.2, with cell type annotation via SingleR (ImmGen/MouseRNAseq references) plus manual marker gene verification. Ten major cell types were identified: epithelial cells, T cells, B cells, macrophages/monocytes, fibroblasts, plasma cells, neutrophils, endothelial cells, smooth muscle cells, and enteric neurons. Malignant epithelial cells were distinguished via inferCNV and sub-clustered into 9 subpopulations (Mal1-Mal9). T cells were sub-classified into CD4+ (Naive, Tem, Tfh, Th17, Treg) and CD8+ (Cytotoxic, Tcm, Tem, Tex) subsets. Spatial transcriptomics: Spatial transcriptomic sequencing was performed on two CRC patient tissue samples (EOCRC, n=1; LOCRC, n=1, Table S6) using the High Precision platform (High Precision Life Technology Co., LTD).FFPE blocks (6mmx6mm) were sectioned at 5um. Paired probes (Human WT Probes V2P1/V2P2) were hybridized overnight at 50C, followed by ligation, CytAssist-mediated probe release, and second-strand extension. Libraries were sequenced on Genemind SURF5000 with paired-end sequencing. Data were analyzed at bin20 (~20um/spot): EOCRC yielded 66,344 spots and LOCRC yielded 48,018 spots, with 18,107 genes detected. Cell type deconvolution used NNLS with a reference from GSE132465 (51,055 cells, 8 cell types after removing Enteric_Neuron). Lipid metabolism scoring used 52 genes from 4 pathways (MSigDB/KEGG); immune scoring used an IFN-gamma gene set. Ligand-receptor spatial communication was analyzed with 29 curated LR pairs across 13 pathways. Key findings: EOCRC showed significantly lower T cell infiltration with immune functions enriched in lipid metabolism pathways. The Mal3 subpopulation, enriched in EOCRC, drives lipid metabolic reprogramming via the Pparg-Emp1 axis and interacts with CD8+ T cells through LGALS9-CD45, promoting immunosuppressive TME. Spatial data confirmed EOCRC tumors are epithelial-dominant with elevated lipid metabolism and reduced immune infiltration versus LOCRC (P<0.001).
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Steps to reproduce
This study employed the azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colorectal cancer (CRC) mouse model to investigate age-dependent differences in tumorigenesis between early-onset CRC (EOCRC) and late-onset CRC (LOCRC). The AOM-DSS model is a well-established inflammation-driven carcinogenesis model that recapitulates key features of human CRC, including tumor initiation through genetic damage and promotion through chronic inflammation. Young (3-4 months old, n=8) and old (18-20 months old, n=8) female and male C57BL/6 mice (female:male=1:1) were used. The age stratification was based on established references for modeling EOCRC versus LOCRC. All mice were maintained under a 12-hour light/dark cycle at 21+/-2C with relative humidity of 45+/-10% under specific pathogen-free conditions with ad libitum access to food and water. The AOM-DSS protocol was performed as follows: on Day 1, mice received a single intraperitoneal injection of AOM (10 mg/kg; Sigma-Aldrich, USA). After one week, mice underwent three cycles of DSS treatment (MP Biomedicals, UK) to simulate CRC development. Each cycle consisted of 1 week of 2% DSS in drinking water followed by 2 weeks of regular water for recovery. Disease progression was monitored weekly through body weight measurement and stool consistency assessment. After the final cycle, mice were euthanized by cervical dislocation under isoflurane anesthesia (150 mg/kg; 792632, Sigma-Aldrich, USA), and colons were harvested for downstream analyses including scRNA-seq (n=6 per group), histopathological examination (H&E staining), immunohistochemistry, and primary tumor cell isolation. For scRNA-seq, 6 mice per group (young: 3-4 months; old: 18-20 months) were used. Tumor tissues were enzymatically digested into single-cell suspensions for library construction. For primary tumor cell experiments, tumors were microscopically dissected, minced to 1-2mm3, and digested with collagenase IV (0.1%), hyaluronidase (0.05%), dispase (0.05%), and DNase I (20ug/mL) at 37C. Cells were cultured in DMEM/F12 with 10% FBS, EGF (20ng/mL), and bFGF (10ng/mL) on Matrigel-coated plates. All functional experiments used passages P2-P4 to preserve original tumor phenotypes. Cell purity was verified by EpCAM+ flow cytometry. All animal procedures were approved by the institutional animal ethics committee (approval number:LX4825041403 and conducted in accordance with ARRIVE guidelines and national regulations for laboratory animal welfare.
Institutions
- Sir Run Run Shaw HospitalZhejiang, Hangzhou