Tracing the stemness and malignant transition in a heritable colorectal cancer Lynch Syndrome by single-cell RNA-seq analysis
Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Background
Lynch Syndrome (LS) is an autosomal dominant disease characterized by germline heterozygous mutations in DNA mismatch repair (MMR) genes. High-risk LS patients may proceed to colorectal cancer (CRC). However, the drivers or biomarkers of LS benign colon tissue approaching malignant CRC are not completely understood. The similarity and difference between LS-related and nonLS-related CRC are also not well interrogated (LS-CRC vs. nonLS-CRC). This study aimed to understand the cellular changes during malignant transition in LS.
Methods
Single-cell RNA sequencing (scRNA-seq) was used to analyze paired biopsy samples from 3 patients with LS (cancer tissues vs. adjacent normal tissues, n=3). Single-nuclear RNA sequencing (snRNA-seq) was used to analyze a frozen biopsy sample from a patient with LS (n=1). scRNA-seq or snRNA-seq datasets from CRC were downloaded from the open source. Integrative computational analysis was performed to conclude the distinct pattern in the single-cell atlas. Immuno-histo-fluorescence staining (IHF) were also performed for three key markers.
Results
In the single-cell atlas, we observed an increase in primitive cancer stem-cell-like cells with high expression of a general cancer biomarker CarcinoEmbryonic Antigen-related Cell Adhesion Molecule 5 ( CEACAM5 ) in the epithelium of the LS. Infiltration of immune cells and DNA repair biological activity are dramatically increased in LS carcinoma. The burden in LS is fundamentally elevated compared to that in CRC or healthy donors when the mutations are in the coding-sequence-wide range. Furthermore, T cell and macrophage-related tumor immunity is readily mobilized in carcinomas compared to paraCArcinomas.
Conclusions
This study provides single-cell transcriptomic resource using affected tissues from patients with Lynch Syndrome and describes an integrative profile covering the alterations (cancer stem cell markers, mutation burden, and tumor immunity) during the malignant transition from healthy to Lynch Syndrome and to colorectal cancer at the single-cell level.
Highlights and Figure/Table Index (Take-Home Messages)
Colon tissues from HD (n=4), patients with LS (n=6) and with CRC (n=3) were collected for snRNA-seq or scRNA-seq analysis; Diagnosis of the 6 LS patients are clearly supported by our pedigree documentations (Fig. 1 and Supp. Fig. 1) and briefed in Table 1;
Among the 6 patients with LS, 1 for snRNA-seq analysis (a carcinoma tissue only), 3 for scRNA-seq analysis (3 paired para-carcinoma and carcinoma tissues) and 3 for experimental validations; (Fig. 1)
Following studies are focused on the comparisons between HD, LS and CRC; In addition, comparisons between paired carcinoma and para-carcinoma tissues from patients with LS were also performed; (Fig. 2-7)
When HD and LS are compared, a cancer stem cell marker CEACAM5 is readily detected by snRNA-seq analysis; (Fig. 5)
When carcinoma and para-carcinoma from LS patients are compared, increased immune cell infiltration of and enhanced DNA repair activity in the malignant tissues is readily detected by scRNA-seq analysis; in addition, three upregulated markers in LS carcinoma ( BACE2 , GRPC5A and OLFM4 ) were identified and validated by immunohistoinfloresnce; (Fig. 3 and Fig. 4)
SComatic algorithm-based mutation calling analysis suggests a comparable mutation burden between carcinoma and para-carcinoma from LS patients, but such mutation burdens are grossly greater than that from HD or that from patients with CRC; (Fig. 6)
