MC38 colorectal tumor cell lines from two different sources display substantial differences in transcriptome, mutanome and neoantigen expression

Abstract

Introduction: The cell line MC38 is a commonly used murine model for colorectal carcinoma. It has a high mutational burden, is sensitive to immune checkpoint immunotherapy and endogenous CD8+ T cell responses against neoantigens have been reported.

Methods: Here, we re-sequenced exomes and transcriptomes of MC38 cells from two different sources, namely Kerafast (originating from NCI/NIH, MC38-K) and the Leiden University Medical Center cell line collection (MC38-L), comparing the cell lines on the genomic and transcriptomic level and analyzing their recognition by CD8+ T cells with known neo-epitope specificity.

Results: The data reveals a distinct structural composition of MC38-K and MC38-L cell line genomes and different ploidies. Further, the MC38-L cell line harbored about 1.3-fold more single nucleotide variations and small insertions and deletions than the MC38-K cell line. In addition, the observed mutational signatures differed; only 35.3% of the non-synonymous variants and 5.4% of the fusion gene events were shared. Transcript expression values of both cell lines correlated strongly (p = 0.919), but we found different pathways enriched in the genes that were differentially upregulated in the MC38-L or MC38-K cells, respectively. Our data show that previously described neoantigens in the MC38 model such as Rpl18^mut and Adpgk^mut were absent in the MC38-K cell line resulting that such neoantigen-specific CD8+ T cells recognizing and killing MC38-L cells did not recognize or kill MC38-K cells.

Conclusion: This strongly indicates that at least two sub-cell lines of MC38 exist in the field and underlines the importance of meticulous tracking of investigated cell lines to obtain reproducible results, and for correct interpretation of the immunological data without artifacts. We present our analyses as a reference for researchers to select the appropriate sub-cell line for their own studies.

Keywords: MC38; colorectal carcinoma; expression profile; murine tumor model; mutation analysis; neoantigens.

Figure 1

MC38 cell lines MC38-K and MC38-L differ substantially on genomic level. (A) Circos plots showing the somatic alterations of both cell lines compared to wild type C57BL/6 mice. Outer circle: SNVs (grey) and small indels (red); second circle from the outside: CNVs, log scaled, with grey dashed lines marking copy numbers 1, 25 and 200 (MC38-K only); middle: fusion gene events. (B) Number of SNVs, indels and fusion gene events detected in MC38-K or MC38-L only or shared by both cell lines. (C) Variant allele frequencies (VAF) distributions of SNVs in exons in DNA and RNA of both cell lines. VAF values of -1 indicate no coverage in RNA-seq. (D) Mutational signatures observed in both cell lines. Significant differences (adjusted p-value < 0.05) are indicated with a line and the respective p-values are depicted. Significance was determined with t-test followed by multiple testing correction with Benjamini-Hochberg correction.

Figure 2

Differential expression analysis of MC38 cell lines indicates distinct transcriptomic profiles. (A) Vulcano plot of the differential expression analysis between MC38-K and MC38-L cells. The top 25 differentially expressed (DE) genes are labeled. (B) DE genes were subjected to pathway enrichment analysis. Significantly enriched KEGG pathways are shown (adjusted p-value < 0.05).

Figure 3

Comparison of MC38-L and MC38-Kcell lines on immunogenic level. (A) Antigen specificities of established CD8+ T cell lines were analyzed by Rpl18 and Adpgk specific tetramers. (B) Established CD8+ T cell lines were analyzed for recognition of MC38-L or MC38-K tumor cells by induced cytokine production after coculture with live tumor cells. IFNγ secretion by Adpgk-TCR (C) or Rpl18-TCR (D) transduced T cells upon co-culture with different tumor cells via ELISPOT assay. Data indicate mean ± SD of biological replicates (n=2). P values determined by One-way ANOVA Tukey´s multiple comparison test. (E) In vitro cytotoxic activity of Adpgk-TCR or Rpl18-TCR transduced T cells after 12h co-culture with different tumor cells. Data indicate mean ± SD of biological replicates (n=3). P-values were determined with respect to OTI-TCR control by One-way ANOVA Bonferroni´s multiple comparison test. **p < 0.01, ***p < 0.001, ****p < 0.0001.