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.

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 ].…”

Genomic characterization and immunotherapy for microsatellite instability-high in cholangiocarcinoma

“…This deficiency, known as deficient DNA mismatch repair (dMMR), is characterized by microsatellite instability-high (MSI-H) in tumors and has a unique genomic status and tumor microenvironments [ 3 , 4 ]. Patients with MSI-H cancers, including gastrointestinal (GI) cancer, have experienced excellent effects from programmed cell death protein-1 (PD-1)-based immune checkpoint inhibitor (ICI) immunotherapy [ 5 – 7 ]. Based on this evidence, the Food and Drug Administration (FDA) approved programmed cell death protein-1 (PD-1) inhibitors monotherapy (and cytotoxic T lymphocyte-associated antigen 4 inhibitors) for patients with MSI-H/dMMR colorectal cancer and multiple other cancers, regardless of the location [ 3 , 8 – 11 ].…”