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Single-cell DNA sequencing reveals a late-dissemination model in metastatic colorectal cancer.

ABSTRACT: Metastasis is a complex biological process that has been difficult to delineate in human colorectal cancer (CRC) patients. A major obstacle in understanding metastatic lineages is the extensive intra-tumor heterogeneity at the primary and metastatic tumor sites. To address this problem, we developed a highly multiplexed single-cell DNA sequencing approach to trace the metastatic lineages of two CRC patients with matched liver metastases. Single-cell copy number or mutational profiling was performed, in addition to bulk exome and targeted deep-sequencing. In the first patient, we observed monoclonal seeding, in which a single clone evolved a large number of mutations prior to migrating to the liver to establish the metastatic tumor. In the second patient, we observed polyclonal seeding, in which two independent clones seeded the metastatic liver tumor after having diverged at different time points from the primary tumor lineage. The single-cell data also revealed an unexpected independent tumor lineage that did not metastasize, and early progenitor clones with the "first hit" mutation in APC that subsequently gave rise to both the primary and metastatic tumors. Collectively, these data reveal a late-dissemination model of metastasis in two CRC patients and provide an unprecedented view of metastasis at single-cell genomic resolution.

SUBMITTER: Leung ML

PROVIDER: S-EPMC5538546 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Single-cell DNA sequencing reveals a late-dissemination model in metastatic colorectal cancer.

Leung Marco L ML Davis Alexander A Gao Ruli R Casasent Anna A Wang Yong Y Sei Emi E Vilar Eduardo E Maru Dipen D Kopetz Scott S Navin Nicholas E NE

Genome research 20170525 8

Metastasis is a complex biological process that has been difficult to delineate in human colorectal cancer (CRC) patients. A major obstacle in understanding metastatic lineages is the extensive intra-tumor heterogeneity at the primary and metastatic tumor sites. To address this problem, we developed a highly multiplexed single-cell DNA sequencing approach to trace the metastatic lineages of two CRC patients with matched liver metastases. Single-cell copy number or mutational profiling was perfor ...[more]

PMID: 28546418

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Project description:BackgroundThe 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.MethodsThe 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.ResultsTumor 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.ConclusionsCD8+ 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.

Dataset Information

Single-cell DNA sequencing reveals a late-dissemination model in metastatic colorectal cancer.

Publications

Single-cell DNA sequencing reveals a late-dissemination model in metastatic colorectal cancer.

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