Differential gene expression of tumor-infiltrating CD8⁺T cells in advanced versus early-stage colorectal cancer and identification of a gene signature of poor prognosis

Abstract

BackgroundCytotoxic CD8+ T cell-mediated response is the most important arm of adaptive immunity, which dictates the capacity of the host immune response in eradicating tumor cells. Due to tumor intrinsic and/or extrinsic factors, the density and function of CD8+ tumor-infiltrating lymphocytes (TILs) could be compromised, leading to poor prognosis and survival.MethodsUsing RNA-Seq, transcriptomes of sorted CD3+CD8+ TILs from treatment-naïve colorectal cancer (CRC) patients at advanced stages (III and IV) were compared with those from patients with early stages (I and II). A signature referred to as ‘poor prognosis CD8 gene signature (ppCD8sig)’ was identified and analyzed in The Cancer Genome Atlas CRC dataset. Scores for the ppCD8sig were calculated and classified as high, intermediate and low, and its prognostic significance was assessed using multivariate analysis and Cox proportional hazard model. Densities of CD3+ and CD8+ T cell infiltration in tumors from patients with high and low ppCD8sig scores were assessed by flow cytometry and immunostaining.ResultsGenes related to epigenetic regulation and response to hypoxia were upregulated in CD8+ TILs from patients with advanced stages, while genes related to T cell activation, cell proliferation and cell cycle were downregulated. Patients with high ppCD8sig score had poorer disease-specific survival (DSS) and shorter progression-free interval (PFI). The ppCD8sig was an independent prognostic indicator for DSS (HR 1.83, 95% CI 1.40 to 2.38, p<0.0001) and PFI (HR 1.42, 95% CI 1.04 to 1.93, p=0.026). Additionally, patients with high ppCD8sig score were more likely to have advanced stages (χ2 p<0.0001) and residual disease after primary therapy (χ2 p=0.046). Patients with high ppCD8sig score had reduced levels of CD3+ and CD8+ TILs and low Immunoscores (IS), compared to patients with low ppCD8sig score.ConclusionsOur data provided insights into the altered regulation of biological mechanisms and signaling pathways in CD8+ TILs during CRC progression, and revealed a gene signature as an independent prognostic indicator. Patients with high ppCD8sig score had lower levels of TILs and low IS. These data further confirm the prognostic value of the identified ppCD8sig and potentially highlight its clinical relevance.

Differential gene expression of tumor-infiltrating CD33+ myeloid cells in advanced- versus early-stage colorectal cancer

“…Relative gene expression was normalized to β-actin and calculated using 2 −ΔΔCT method. Additionally, the relative expression for each gene was calculated by normalizing the expression in CD33 + cells to data from sorted tumor-infiltrating CD8 + T cells from the same patients [ 16 ], after the initial normalization to β-actin . Primer sequences are presented in Table 2 .…”

“…The Z-scores were calculated from the TPM values as previously described [18]. Differential expression analyses were also performed to compare the transcriptomes of sorted tumor-infiltrating CD33 high myeloid cells with transcriptome of sorted tumor-infiltrating CD8 + T cells (used as controls) from the same patient cohort [16]. Volcano plots were generated using OriginPro 2020 software (OriginLab Corporation, Massachusetts, USA) with Log2 FC > 2 and P value cutoff < 0.05.…”

RNA-Seq Analysis of Colorectal Tumor-Infiltrating Myeloid-Derived Suppressor Cell Subsets Revealed Gene Signatures of Poor Prognosis

“…Tissue specimens were frozen in 1 ml of freezing medium (10% dimethyl sulphoxide (DMSO; Sigma-Aldrich, Missouri, USA), 50% fetal calf serum (FCS; HyClone, GE Healthcare Life Sciences, Utah, USA), and 40% RPMI-1640 medium (Life Technologies, New York, USA)), then stored in liquid nitrogen to be used in batches for subsequent analyses. Tissue specimens were thawed and processed, as previously described, followed by cell staining/ sorting (14,(19)(20)(21).…”

Transcriptome of Tumor-Infiltrating T Cells in Colorectal Cancer Patients Uncovered a Unique Gene Signature in CD4+ T Cells Associated with Poor Disease-Specific Survival

“…Molecular and cellular profiling of immune cell populations, specifically T cells, bear great significance in understanding CRC development and progression. We have recently performed transcriptomic profiling of sorted CD4 + [ 15 ] and CD8 + [ 16 ] tumor-infiltrating lymphocytes (TILs) from bulk tumors of CRC patients with varying disease stages. We reported downregulation of Th1-mediated immune response and cytotoxicity-mediated genes but upregulation of epigenetic silencing-related genes in CD4 + TILs from CRC patients with advanced stage disease [ 15 ].…”

“…We reported downregulation of Th1-mediated immune response and cytotoxicity-mediated genes but upregulation of epigenetic silencing-related genes in CD4 + TILs from CRC patients with advanced stage disease [ 15 ]. Moreover, epigenetic regulation-related genes and response to hypoxia were upregulated, while T cell/cell proliferation- and cell cycle-related genes were downregulated in CD8 + TILs in advanced stage CRC patients [ 16 ]. These findings highlighted the significance of CD4 + and CD8 + TILs in CRC patients during disease progression.…”