Single-Cell RNA Sequencing: A Deep Dive into The Cellular Landscape and Interactions in Hepatocellular Carcinoma

Kejia Miao; Xudong Liu

doi:10.54097/hset.v66i.12010

Authors

Kejia Miao
Xudong Liu

DOI:

https://doi.org/10.54097/hset.v66i.12010

Keywords:

hepatocellular carcinoma (HCC), single-cell RNA sequencing (scRNA-seq), tumor microenvironment, intratumor heterogeneity, cellular composition.

Abstract

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, with its progression highly influenced by the cellular interplay within the tumor microenvironment that is underexplored. Aiming to bridge this gap, our study utilizes single-cell RNA sequencing (scRNA-seq) to examine the cellular heterogeneity of HCC and investigate the roles of distinct cell populations. scRNA-seq was performed on eight DEN mice HCC samples, followed by bioinformatic analysis with Seurat package. Nine distinct cell populations were identified, with three unique macrophage populations suggestive of their role as tumor-associated macrophages (TAMs). The detected endothelial cells and pericytes hint at ongoing neoangiogenesis, with implications that endothelial cells might function as tumor-associated endothelial cells (TECs) and pericytes as carcinoma-associated fibroblasts (CAFs). Our findings provide insights into the potential roles of various cell populations in the HCC tumor microenvironment, which paves the way for developing novel therapies. These postulations, while offering a deeper understanding of HCC's cellular landscape, necessitate experimental validation for confirmation.

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References

Bouwens, L., Baekeland, M., de Zanger, R., & Wisse, E. (1986). Quantitation, tissue distribution and proliferation kinetics of kupffer cells in normal rat liver. Hepatology, 6(4), 718–722. https://doi.org/10.1002/hep.1840060430

Cancer-associated fibroblasts: Overview, progress, challenges, and directions | Cancer Gene Therapy. (n.d.). Retrieved July 28, 2023, from https://www.nature.com/articles/s41417-021-00318-4

Capece, D., Fischietti, M., Verzella, D., Gaggiano, A., Cicciarelli, G., Tessitore, A., Zazzeroni, F., & Alesse, E. (2012). The Inflammatory Microenvironment in Hepatocellular Carcinoma: A Pivotal Role for Tumor-Associated Macrophages. BioMed Research International, 2013, e187204. https://doi.org/10.1155/2013/187204

CD72 protein expression summary—The Human Protein Atlas. (n.d.). Retrieved August 6, 2023, from https://www.proteinatlas.org/ENSG00000137101-CD72

Cell Preparation for Single Cell Protocols—Official 10x Genomics Support. (n.d.). 10x Genomics. Retrieved July 15, 2023, from https://www.10xgenomics.com/support/single-cell-gene-expression/documentation/steps/sample-prep/single-cell-protocols-cell-preparation-guide

Choose gene expression markers | PanglaoDB. (n.d.). Retrieved July 16, 2023, from https://panglaodb.se/markers.html?cell_type=%27choose%27

Current Strategies for the Treatment of Hepatocellular Carcinoma by Mo | IJN. (n.d.). Retrieved July 25, 2023, from https://www.dovepress.com/current-strategies-for-the-treatment-of-hepatocellular-carcinoma-by-mo-peer-reviewed-fulltext-article-IJN

De Sanctis, F., Ugel, S., Facciponte, J., & Facciabene, A. (2018). The dark side of tumor-associated endothelial cells. Seminars in Immunology, 35, 35–47. https://doi.org/10.1016/j.smim.2018.02.002

Ding, T., Xu, J., Wang, F., Shi, M., Zhang, Y., Li, S.-P., & Zheng, L. (2009). High tumor-infiltrating macrophage density predicts poor prognosis in patients with primary hepatocellular carcinoma after resection. Human Pathology, 40(3), 381–389. https://doi.org/10.1016/j.humpath.2008.08.011

Dll4-Notch signaling in regulation of tumor angiogenesis | SpringerLink. (n.d.). Retrieved July 27, 2023, from https://link.springer.com/article/10.1007/s00432-013-1534-x

Dow, M., Pyke, R. M., Tsui, B. Y., Alexandrov, L. B., Nakagawa, H., Taniguchi, K., Seki, E., Harismendy, O., Shalapour, S., Karin, M., Carter, H., & Font-Burgada, J. (2018). Integrative genomic analysis of mouse and human hepatocellular carcinoma. Proceedings of the National Academy of Sciences, 115(42), E9879–E9888. https://doi.org/10.1073/pnas.1811029115

Frisbie, L., Buckanovich, R. J., & Coffman, L. (2022). Carcinoma-Associated Mesenchymal Stem/Stromal Cells: Architects of the Pro-tumorigenic Tumor Microenvironment. Stem Cells, 40(8), 705–715. https://doi.org/10.1093/stmcls/sxac036

Genes & Expression—Site Guide—NCBI. (n.d.). Retrieved July 16, 2023, from https://www.ncbi.nlm.nih.gov/guide/genes-expression/

Goitsuka, R., Mamada, H., Kitamura, D., Cooper, M. D., & Chen, C. H. (2001). Genomic Structure and Transcriptional Regulation of the Early B Cell Gene chB11. The Journal of Immunology, 167(3), 1454–1460. https://doi.org/10.4049/jimmunol.167.3.1454

Guillot, A., & Tacke, F. (2019). Liver Macrophages: Old Dogmas and New Insights. Hepatology Communications, 3(6), 730–743. https://doi.org/10.1002/hep4.1356

Habenicht, L. K. L., Wang, Z., Zhang, X., Li, Y., Mogler, C., Huspenina, J. S., Schmid, R. M., Weber, C., Mohanta, S. K., Ma, Z., & Yin, C. (2022). The C1q-ApoE complex: A new hallmark pathology of viral hepatitis and nonalcoholic fatty liver disease. Frontiers in Immunology, 13. https://www.frontiersin.org/articles/10.3389/fimmu.2022.970938

Hanahan, D., & Weinberg, R. A. (2011). Hallmarks of cancer: The next generation. Cell, 144(5), 646–674. https://doi.org/10.1016/j.cell.2011.02.013

Hepatic macrophages in homeostasis and liver diseases: From pathogenesis to novel therapeutic strategies | Cellular & Molecular Immunology. (n.d.). Retrieved July 21, 2023, from https://www.nature.com/articles/cmi2015104

Hu, T., Wu, Z., Bush, S. J., Freem, L., Vervelde, L., Summers, K. M., Hume, D. A., Balic, A., & Kaiser, P. (2019). Characterization of Subpopulations of Chicken Mononuclear Phagocytes That Express TIM4 and CSF1R. The Journal of Immunology, 202(4), 1186–1199. https://doi.org/10.4049/jimmunol.1800504

Hu, Z.-W., Chen, L., Ma, R.-Q., Wei, F.-Q., Wen, Y.-H., Zeng, X.-L., Sun, W., & Wen, W.-P. (2021). Comprehensive analysis of ferritin subunits expression and positive correlations with tumor-associated macrophages and T regulatory cells infiltration in most solid tumors. Aging (Albany NY), 13(8), 11491–11506. https://doi.org/10.18632/aging.202841

IJMS | Free Full-Text | An Eye on Kupffer Cells: Development, Phenotype and the Macrophage Niche. (n.d.). Retrieved July 21, 2023, from https://www.mdpi.com/1422-0067/23/17/9868

Kolios, G., Valatas, V., & Kouroumalis, E. (2006). Role of Kupffer cells in the pathogenesis of liver disease. World Journal of Gastroenterology : WJG, 12(46), 7413–7420. https://doi.org/10.3748/wjg.v12.i46.7413

Liu, C., Li, Y., Yu, J., Feng, L., Hou, S., Liu, Y., Guo, M., Xie, Y., Meng, J., Zhang, H., Xiao, B., & Ma, C. (2013). Targeting the Shift from M1 to M2 Macrophages in Experimental Autoimmune Encephalomyelitis Mice Treated with Fasudil. PLOS ONE, 8(2), e54841. https://doi.org/10.1371/journal.pone.0054841

Malaguarnera, L., Rosa, M. D., Zambito, A. M., dell’Ombra, N., Nicoletti, F., & Malaguarnera, M. (2006). Chitotriosidase gene expression in Kupffer cells from patients with non-alcoholic fatty liver disease. Gut, 55(9), 1313–1320. https://doi.org/10.1136/gut.2005.075697

MCA | Mouse Cell Atlas. (n.d.). Retrieved July 16, 2023, from https://bis.zju.edu.cn/MCA/

Mossenta, M., Busato, D., Baboci, L., Di Cintio, F., Toffoli, G., & Dal Bo, M. (2019). New Insight into Therapies Targeting Angiogenesis in Hepatocellular Carcinoma. Cancers, 11(8), 1086. https://doi.org/10.3390/cancers11081086

Nguyen-Lefebvre, A. T., & Horuzsko, A. (2015). Kupffer Cell Metabolism and Function. Journal of Enzymology and Metabolism, 1(1), 101.

Novikova, M. V., Khromova, N. V., & Kopnin, P. B. (2017). Components of the hepatocellular carcinoma microenvironment and their role in tumor progression. Biochemistry (Moscow), 82(8), 861–873. https://doi.org/10.1134/S0006297917080016

Oxytocin system alleviates intestinal inflammation by regulating macrophages polarization in experimental colitis | Clinical Science | Portland Press. (n.d.). Retrieved July 22, 2023, from https://portlandpress.com/clinsci/article-abstract/133/18/1977/220436/Oxytocin-system-alleviates-intestinal-inflammation

Pan, Y., Cao, W., Mu, Y., & Zhu, Q. (2022). Microfluidics Facilitates the Development of Single-Cell RNA Sequencing. Biosensors, 12(7), Article 7. https://doi.org/10.3390/bios12070450

Pernot, S., Evrard, S., & Khatib, A.-M. (2022). The Give-and-Take Interaction Between the Tumor Microenvironment and Immune Cells Regulating Tumor Progression and Repression. Frontiers in Immunology, 13. https://www.frontiersin.org/articles/10.3389/fimmu.2022.850856

Ramón Y Cajal, S., Sesé, M., Capdevila, C., Aasen, T., De Mattos-Arruda, L., Diaz-Cano, S. J., Hernández-Losa, J., & Castellví, J. (2020). Clinical implications of intratumor heterogeneity: Challenges and opportunities. Journal of Molecular Medicine (Berlin, Germany), 98(2), 161–177. https://doi.org/10.1007/s00109-020-01874-2

Shiga, K., Hara, M., Nagasaki, T., Sato, T., Takahashi, H., & Takeyama, H. (2015). Cancer-Associated Fibroblasts: Their Characteristics and Their Roles in Tumor Growth. Cancers, 7(4), Article 4. https://doi.org/10.3390/cancers7040902

Tahmasebi Birgani, M., & Carloni, V. (2017). Tumor Microenvironment, a Paradigm in Hepatocellular Carcinoma Progression and Therapy. International Journal of Molecular Sciences, 18(2), Article 2. https://doi.org/10.3390/ijms18020405

Tang, X. (2013). Tumor-associated macrophages as potential diagnostic and prognostic biomarkers in breast cancer. Cancer Letters, 332(1), 3–10. https://doi.org/10.1016/j.canlet.2013.01.024

Targeting tumor‑associated macrophages in the tumor microenvironment (Review). (n.d.). Retrieved July 23, 2023, from https://www.spandidos-publications.com/10.3892/ol.2020.12097

TGF-β promotes pericyte-myofibroblast transition in subretinal fibrosis through the Smad2/3 and Akt/mTOR pathways | Experimental & Molecular Medicine. (n.d.). Retrieved July 28, 2023, from https://www.nature.com/articles/s12276-022-00778-0

van der Heide, D., Weiskirchen, R., & Bansal, R. (2019). Therapeutic Targeting of Hepatic Macrophages for the Treatment of Liver Diseases. Frontiers in Immunology, 10. https://www.frontiersin.org/articles/10.3389/fimmu.2019.02852

Wen, N., Cai, Y., Li, F., Ye, H., Tang, W., Song, P., & Cheng, N. (2022). The clinical management of hepatocellular carcinoma worldwide: A concise review and comparison of current guidelines: 2022 update. BioScience Trends, 16(1), 20–30. https://doi.org/10.5582/bst.2022.01061

Whiteside, T. L. (2008). The tumor microenvironment and its role in promoting tumor growth. Oncogene, 27(45), Article 45. https://doi.org/10.1038/onc.2008.271

Wu, F., Yang, J., Liu, J., Wang, Y., Mu, J., Zeng, Q., Deng, S., & Zhou, H. (2021). Signaling pathways in cancer-associated fibroblasts and targeted therapy for cancer. Signal Transduction and Targeted Therapy, 6(1), Article 1. https://doi.org/10.1038/s41392-021-00641-0

Wu, S.-D., Ma, Y.-S., Fang, Y., Liu, L.-L., Fu, D., & Shen, X.-Z. (2012). Role of the microenvironment in hepatocellular carcinoma development and progression. Cancer Treatment Reviews, 38(3), 218–225. https://doi.org/10.1016/j.ctrv.2011.06.010

Yang, J. D., Nakamura, I., & Roberts, L. R. (2011). The tumor microenvironment in hepatocellular carcinoma: Current status and therapeutic targets. Seminars in Cancer Biology, 21(1), 35–43. https://doi.org/10.1016/j.semcancer.2010.10.007

Yin, Z., Dong, C., Jiang, K., Xu, Z., Li, R., Guo, K., Shao, S., & Wang, L. (2019). Heterogeneity of cancer-associated fibroblasts and roles in the progression, prognosis, and therapy of hepatocellular carcinoma. Journal of Hematology & Oncology, 12(1), 101. https://doi.org/10.1186/s13045-019-0782-x

Yoshikawa, M., Mukai, Y., Okada, Y., Yoshioka, Y., Tsunoda, S., Tsutsumi, Y., Okada, N., Aird, W. C., Doi, T., & Nakagawa, S. (2008). Ligand-independent assembly of purified soluble magic roundabout (Robo4), a tumor-specific endothelial marker. Protein Expression and Purification, 61(1), 78–82. https://doi.org/10.1016/j.pep.2008.05.006

Zhou, J., Zhang, A., & Fan, L. (2020). HSPA12B Secreted by Tumor-Associated Endothelial Cells Might Induce M2 Polarization of Macrophages via Activating PI3K/Akt/mTOR Signaling. OncoTargets and Therapy, 13, 9103–9111. https://doi.org/10.2147/OTT.S254985