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Updated: May 26, 2026
Dissociation of Human and Mouse Tumor Tissue Samples for Single-cell RNA Sequencing
Published on: August 16, 2024
Integrative single-cell RNA-seq and transcriptomic analysis uncovers cellular heterogeneity and immunotherapy
Yirou Wang1, Yingying Zhang2, Ziyong Mao1
1BamRock Institute of Innovation, Suzhou, 215000, China.
Objective:
To investigate the molecular and cellular features associated with immunotherapy resistance in colorectal cancer (CRC) and identify potential biomarkers.
Methods:
The single-cell RNA sequencing data from 14 pre-treatmant CRC samples (11 complete respondence and 3 stable disease) were collected from GSE236581. Clustering, pseudotime trajectory analysis, and intercellular communication analysis were performed to explore the tumor microenvironment and malignant cell characteristics. Bulk transcriptome datasets TCGA-COAD and GSE39582 were collected to assist in screening and validating key genes involved in tumorigenesis and prognosis. Key findings were validated in independent cohorts and via immunohistochemistry and T cell cytotoxicity experiments.
Results:
Unsupervised clustering identified 21 cell clusters and 8,810 malignant epithelial cells. Differential gene expression analysis revealed 7,648 genes significantly differing between groups, with 568 upregulated in stable disease samples, enriched in ECM organization, ERK1/2 signaling, and metabolic reprogramming. Exploratory pseudotime analysis of cross-sectional pre-treatment samples revealed differences in inferred cellular state distributions in stable disease cells, accompanied by reduced leukocyte chemotaxis‑related signatures. Immune profiling highlighted enhanced CD8 + T-cell cytotoxicity in responders, while stable disease TME contained immunosuppressive macrophages. Intercellular communication analysis revealed stronger malignant cell-CD8 + T-cell interactions in stable disease samples. ASPHD1, associated with poor survival, was upregulated in CRC tissues and may serve as a candidate biomarker associated with immunotherapy response.
Conclusion:
Our findings highlighted the molecular and cellular features associated with immunotherapy resistance in CRC, involving malignant cell adaptations and TME remodeling. ASPHD1 was identified as a candidate gene linked to immunotherapy response in CRC. Given the exploratory nature of this work, these findings and the functional significance of ASPHD1 warrant further validation through larger cohorts and in-depth experimental studies.
Insights
This study reveals that colorectal cancer (CRC) cells adapt to immunotherapy by altering their tumor microenvironment (TME). ASPHD1 is identified as a potential biomarker for predicting immunotherapy response in CRC patients.
Area of Science:
- Oncology
- Immunology
- Genomics
Background:
- Colorectal cancer (CRC) poses a significant health challenge, with immunotherapy offering new treatment avenues.
- Understanding resistance mechanisms in CRC is crucial for improving patient outcomes.
- Identifying reliable biomarkers can guide treatment selection and predict response.
Purpose of the Study:
- To elucidate the molecular and cellular characteristics underlying immunotherapy resistance in colorectal cancer.
- To identify potential predictive biomarkers for immunotherapy response in CRC.
- To explore the interplay between malignant cells and the tumor microenvironment (TME) in treatment resistance.
Main Methods:
- Single-cell RNA sequencing (scRNA-seq) of pre-treatment CRC samples to analyze tumor heterogeneity and TME.
- Pseudotime trajectory and intercellular communication analyses to infer cellular states and interactions.
- Bulk transcriptome analysis of TCGA-COAD and GSE39582 datasets for gene validation.
- Immunohistochemistry and T cell cytotoxicity assays for experimental validation.
Main Results:
- Distinct cell clusters and malignant epithelial cell populations were identified in CRC.
- Gene expression differences in resistant tumors were linked to extracellular matrix (ECM) organization and metabolic reprogramming.
- Resistant tumors showed reduced leukocyte chemotaxis, while responders exhibited enhanced CD8+ T-cell cytotoxicity.
- Immunosuppressive macrophages and altered malignant cell-CD8+ T-cell interactions were observed in resistant TME.
- ASPHD1 was found to be upregulated in CRC and associated with poor survival, suggesting its role as a potential biomarker.
Conclusions:
- Immunotherapy resistance in CRC involves adaptive changes in malignant cells and TME remodeling.
- ASPHD1 emerges as a candidate biomarker associated with immunotherapy response in CRC.
- Further validation in larger cohorts and experimental studies is warranted to confirm these findings and the functional role of ASPHD1.
