Single-cell sequencing reveals the immune microenvironment landscape related to anti-PD-1 resistance in metastatic colorectal cancer with high microsatellite instability

Abstract

Background The objective response rate of microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC) patients with first-line anti-programmed cell death protein-1 (PD-1) monotherapy is only 40–45%. Single-cell RNA sequencing (scRNA-seq) enables unbiased analysis of the full variety of cells comprising the tumor microenvironment. Thus, we used scRNA-seq to assess differences among microenvironment components between therapy-resistant and therapy-sensitive groups in MSI-H/mismatch repair-deficient (dMMR) mCRC. Resistance-related cell types and genes identified by this analysis were subsequently verified in clinical samples and mouse models to further reveal the molecular mechanism of anti-PD-1 resistance in MSI-H or dMMR mCRC. Methods The response of primary and metastatic lesions to first-line anti-PD-1 monotherapy was evaluated by radiology. Cells from primary lesions of patients with MSI-H/dMMR mCRC were analyzed using scRNA-seq. To identify the marker genes in each cluster, distinct cell clusters were identified and subjected to subcluster analysis. Then, a protein‒protein interaction network was constructed to identify key genes. Immunohistochemistry and immunofluorescence were applied to verify key genes and cell marker molecules in clinical samples. Immunohistochemistry, quantitative real-time PCR, and western blotting were performed to examine the expression of IL-1β and MMP9. Moreover, quantitative analysis and sorting of myeloid-derived suppressor cells (MDSCs) and CD8+ T cells were performed using flow cytometry. Results Tumor responses in 23 patients with MSI-H/dMMR mCRC were evaluated by radiology. The objective response rate was 43.48%, and the disease control rate was 69.57%. ScRNA-seq analysis showed that, compared with the treatment-resistant group, the treatment-sensitive group accumulated more CD8+ T cells. Experiments with both clinical samples and mice indicated that infiltration of IL-1β-driven MDSCs and inactivation of CD8+ T cells contribute to anti-PD-1 resistance in MSI-H/dMMR CRC. Conclusions CD8+ T cells and IL-1β were identified as the cell type and gene, respectively, with the highest correlation with anti-PD-1 resistance. Infiltration of IL-1β-driven MDSCs was a significant factor in anti-PD-1 resistance in CRC. IL-1β antagonists are expected to be developed as a new treatment for anti-PD-1 inhibitor resistance.

Action of circulating and infiltrating B cells in the immune microenvironment of colorectal cancer by single-cell sequencing analysis

“…On the contrary, the role of plasma cells in tumor was greatly reduced, mainly plasma cells expressing IgA, and the evolution of GCB into plasma cells gradually decreased in tumor tissues, instead, it gradually developed into memory B cells[ 24 ]. In conclusion, B cells also play an important role in the tumor immune microenvironment of CRC, which is not a simple bystander cell, but an active participant in fundamentally coordinating the immune response[ 25 - 28 ].…”

Xiaoliu Pingyi Recipe Inhibits Lung Pre-Metastatic Niche Formation and Prevents Myeloid-Derived Suppressor Cells Recruitment

“…The 30 μg protein samples were subjected to SDS-PAGE and subsequently transferred to PVDF membranes (G6015, Servicebio) before blocking (GF1815, Genefist Life Science CO., LTD) and incubating with primary antibodies. 22 Primary antibodies included anti-S100A8 antibody (1:1000; A1688, Abclonal), anti-S100A9 antibody (1:1000; A9842, Abclonal), anti-matrix metalloproteinase 9 (MMP9) antibody (1:1000; ab76003, Abcam), anti-lysyl oxidase (LOX) antibody (1:1000; A11504, Abclonal), anti-signal transducer and activator of Transcription 3 (STAT3) antibody (1:2000; A1192, Abclonal), anti-p-STAT3 antibody (1:2000; AP0705, Abclonal), and anti-β-actin antibody (1:2000; AC006, Abclonal). The secondary antibody (1:5000; GB23303, Servicebio) was added and incubated at room temperature for 1 hour.…”

Myeloid-Derived Suppressor Cells: Therapeutic Target for Gastrointestinal Cancers

“…Finally, MDSCs also are associated with immune checkpoint blockade resistance in colorectal and pancreatic cancer. Infiltration of IL-1B-positive MDSCs identified from scRNA-seq is associated with resistance to checkpoint blockade in microsatellite instability high (MSI-H) metastatic colorectal cancer patient [ 50 ]. In the COMBAT trial, treatment of pancreatic tumors with a C-X-C motif chemokine receptor 4 (CXCR4) antagonist decreased infiltration of MDSCs and was synergistic with immune checkpoint blockade in pancreatic cancer [ 51 ].…”