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Colorectal cancer biomarker and application thereof in diagnosis, prevention, treatment and prognosis

A biomarker and colorectal cancer technology, applied in the field of unsupervised cluster analysis of biomarkers, can solve the problem of unclear subgroup map of ILCs and achieve the effect of survival rate prediction

Pending Publication Date: 2022-01-07
SHANGHAI INST OF IMMUNOLOGY +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Atlases of ILC subpopulations in steady state and neoplastic situations remain poorly defined in terms of composition, diversity and functional status of these cells in the human gut

Method used

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  • Colorectal cancer biomarker and application thereof in diagnosis, prevention, treatment and prognosis
  • Colorectal cancer biomarker and application thereof in diagnosis, prevention, treatment and prognosis
  • Colorectal cancer biomarker and application thereof in diagnosis, prevention, treatment and prognosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0147] Example 1 A healthy gut contains ILC1s, IL3Cs and ILCs / NK, but no ILC2s.

[0148] 1. Materials and methods

[0149] (1.1) Collection of clinical samples

[0150] Healthy blood samples for scRNAseq were obtained from individuals undergoing routine colonoscopies, usually in good health and without other relevant medical history, such as inflammatory bowel disease (IBD) or CRC.

[0151] (1.2) Separation of human lymphocytes

[0152] Prepare fresh intestinal tissue immediately after surgery. Adipose tissue and visible blood vessels are removed. Samples were weighed and washed in PBS, then cut into small pieces. Normal tissues were incubated in 10 mL of freshly prepared endothelial lymphocyte solution (PBS containing 5 mM EDTA, 15 mM HEPES, 10% FBS, 1 mM DTT) for 1 hour at 37°C with shaking at 200 rpm. CRC tissue was washed with shaking in 10 mL of freshly prepared PBS containing 65 mM DTT for 15 min at 37 °C. After the incubation, the tissue pieces were washed twice w...

Embodiment 2

[0160] Example 2 The present invention carried out single-cell transcriptome sequencing (scRNAseq) on 58,000 ILCs in blood samples of CRC patients, healthy blood, normal mucosa and CRC tissue samples ( Figure 6 D).

[0161] 1. Materials and methods

[0162] (1.1) Single-cell RNA sequencing

[0163] Purified ILCs were resuspended in PBS containing 0.04% BSA and kept on ice. For cell counting, cell density was adjusted to the concentration suggested by the 10X Genomics v3 kit and used for library construction. The Illumina platform (NovaSeq 6000) of Crystal Energy was used for library sequencing, and the sequencing depth was about 90,000 reads per cell.

[0164] (1.2) Raw sequence alignment, quality control and normalization

[0165] Using FastQC software v0.11.9

[0166] (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) for quality control of raw sequencing reads. Sequencing data in bcl files were converted to FASTQ format using bcl2fastq2 conversion software ...

Embodiment 3

[0170] Example 3 Hierarchical Clustering

[0171] 1. Materials and methods

[0172] (1.1) Unsupervised Hierarchical Clustering

[0173] Calculate gene expression values ​​for individual cells in each cell population. Only genes selected previously as variable features are used. The present invention uses the Heatmap.plus package to draw an unsupervised cluster map. Euclidean distances were calculated for genes in all cell populations. For normal mucosa, only cell populations clustered 0-4 were used for analysis.

[0174] (1.2) Dimensionality reduction and clustering

[0175] The top 2000 genes were screened using Seurat's "FindVariableGenes" function (Stuart et al., 2019) and used for principal component analysis (PCA). For ILCs in normal mucosa, the present invention retains the first 40 principal components. For normal blood, CRC blood and tumor tissue, the present invention retains the top 20 PCs. Clusters are identified using the "FindClusters" function, which is b...

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Abstract

The invention discloses a colorectal cancer biomarker and application thereof in diagnosis, prevention, treatment and prognosis. The invention also discloses different single cell characteristics of blood and intestinal ILCs subpopulations in the case of healthy conditions and colorectal cancer. The healthy intestinal tract is composed of ILC1s, ILC3s and ILC3s / NK, but does not have ILC2s. Subpopulations of other tumor-specific ILC1s and ILC2s are determined in a CRC patient. The invention also discloses selective expression of the SLAMF1 on tumor specific ILCs, and a higher level of SLAMF1 + ILCs is observed in the blood of a CRC patient. The survival rate of rectal cancer patients in the high SLAMF1 group is obviously higher than that of rectal cancer patients in the low SLAMF1 group, which indicates that the SLAMF1 is an anti-tumor biomarker in CRC. The invention also discloses the use of unsupervised hierarchical clustering methods to study helper ILCs heterogeneity during homeostasis and CRC in blood, normal mucous membrane, and gut tumors. By means of the biomarker, diagnosis, prevention, treatment, prognosis evaluation, survival rate prediction and the like can be effectively carried out on CRC.

Description

technical field [0001] The invention belongs to the field of medical technology, and relates to colorectal cancer biomarkers and their application, an unsupervised clustering analysis method of biomarkers, and reveals the relationship between tumor-specific innate lymphocytes and colorectal cancer progression and immunity through single-cell transcriptional maps . Background technique [0002] T cell-based immunotherapy has been used clinically to treat malignant tumors with great success, but only in a small proportion of patients (Baumeister et al., 2016; Chen and Mellman, 2017; Okazaki et al., 2013; Okazaki and Honjo , 2007; Schumacher and Schreiber, 2015; Sharma and Allison, 2015). Usually, when T cell immunotherapy is carried out, it is necessary to carry out treatment targeting other immune components at the same time, so as to increase the cure rate of patients receiving immunotherapy. Innate lymphocytes (ILCs) are tissue-intrinsic and antigen-independent lymphocyte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574C12Q1/6886A61K45/00A61P35/00
CPCG01N33/57484G01N33/57488G01N33/57419G01N33/57446C12Q1/6886A61K45/00A61P35/00C12Q2600/118C12Q2600/158
Inventor 苏冰齐晶晶沈蕾叶幼琼艾德琳·克里尼耶伯川德·艾斯卡利耶艾米莉·纳尔尼·曼奇内利埃里克·维维埃
Owner SHANGHAI INST OF IMMUNOLOGY
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