Fig 1.
Schematic outline of the experimental design.
Sporadic and rhesus Lynch (heterozygous MLH1 nonsense mutation, c.1029, C>G) animals bred and housed at UTMDACC KCCMR were used to genomically characterize colorectal tumors using an in-house MSI marker panel, IHC of MMR proteins, epigenetic assessment, whole transcriptomics analysis, and CMS classification. These analyses established the framework for utilizing rhesus as a surrogate to study MMRd CRC.
Fig 2.
Clinical, pathological, and molecular characteristics of the Rhesus cohort.
(A) Animal ages at the time of diagnosis of CRC and subsequent euthanasia. The average age at death for the rhesus CRC cohort was 19.14 years. Red dots indicate the age of animals with MLH1 germline mutation; (B) Gender of rhesus cohort. The majority of animals in this cohort were female; (C) Lynch syndrome MLH1 germline mutation status. A total of eight (20%) carried a heterozygous MLH1 nonsense mutation (c.1029, C>G); (D) IHC assessment of rhesus CRC. The majority of rhesus tumor samples displayed loss of MLH1 and PMS2; (E) MSI testing of rhesus tumors. Newly designed MSI testing panel for rhesus CRC included six markers (RheBAT25, RheBAT26, RheBAT40, RheD10S197, RheD18S58, and RheTGFβRII) that were orthologs of commonly tested MSI loci in human tumors (BAT25, BAT26, BAT40, D10S197, D18S58, and TGF βRII). Overall, RheBAT25, RheBAT26, and RheD18S58 MSI markers were the most mutated MSI markers in rhesus CRC; (F) Summary of MSI status of rhesus tumors. Rhesus CRC were predom-inantly MSI-H (75%), and only six tumors (15%) were MSI-L, and four (10%) MSS.
Fig 3.
Methylation analysis of rhesus CRC.
(A) 3D PCA of DNA methylation in rhesus specimens characterizing the trends exhibited by differentially methylated region profiles of sporadic MSI-H (green pyramid), sporadic MSS and MSI-L (purple cube), Lynch syndrome (blue sphere), and normal tissue (red diamond) samples. Each shape represents a tissue sample type. Each group clusters separately; however, sporadic MSS and MSI-L CRC samples are closer to normal tissue samples; (B) Hierarchical clustering of DNA methylation profiles assesses by CpG methylation using Pearson’s correlation. Distance displays the relationship between rhesus tumors and matched normal tissue samples with parameters set as distance method: “correlation”, clustering method: “ward”; (C) Significant differentially methylated regions (DMRs) of rhesus tumors displaying MSI-H and MSI-L/MSS phenotypes at FDR of 5%. PK1B, B4GALT7, GPR35, MYT1L and CYB5D2 are among hyper-methylated genes, and GRB10, SOD1, TMSB10 and CD52 are hypo-methylated.
Fig 4.
Transcriptomic analysis of rhesus CRC.
(A) 3D principal component analysis (PCA) of rhesus CRC gene expression profiles show clear separation among sporadic MSI-H samples (green pyramids), sporadic MSS and MSI-L (purple spheres), Lynch syndrome (blue cubes), and normal tissue (red diamonds). Normal tissue samples clustered separately from tumor tissue samples; (B) Pearson’s correlation coefficient of mean expression levels across 101 significant genes from COAD-READ MSI-H tumor samples, COADREAD MSS tumor samples, COADREAD normal tissue samples, rhesus LS tumor samples, and rhesus normal tissue samples; (C) Significant differentially expressed genes (DEGs) between tumor and normal tissue samples. DEGs were found based on BH-adjusted P-value≤0.05 between rhesus colorectal normal and tumor. Pearson’s correlation was used to perform hierarchical clustering between rhesus tumor and normal tissue samples. Columns represent samples, and rows represent statistically significant differentially expressed genes. MSI bar displays MSI status of samples based on PCR-based MSI testing. Gray color represents normal, pink MSI-H, and magenta MSS and MSI-L tissue samples. MSI type bar displays MLH1 genotyping data with gray color representing normal, orange LS, purple sporadic MSI-L and MSS and red sporadic MSI-H tissue samples.
Fig 5.
Gene set enrichment analysis in rhesus CRC.
(A-C) Significant gene expression pathways relevant to CRC biology are highlighted in (A) MSI-H (sporadic and LS) and (B) in MSI-L/MSS (sporadic and LS) compared to normal tissue samples. (C) Highlighted pathways are up regulated in MSI-H (sporadic and LS) rhesus CRC compared to MSI-L/MSS (sporadic and LS) rhesus CRC. BH-adjusted P-value≤0.05 was set as threshold for analysis; (D) CMS classification of rhesus CRC. The outer ring of circos plot represents CMS subtypes present in rhesus CRC with 52% of samples (n = 10) classifying as CMS2. Middle ring represents MSI status of samples, and inner ring indicates clinical categories of samples.