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Revealing the role of necroptosis microenvironment: FCGBP + tumor-associated macrophages drive primary liver cancer differentiation towards cHCC-CCA or iCCA

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Abstract

Previous research has demonstrated that the conversion of hepatocellular carcinoma (HCC) to intrahepatic cholangiocarcinoma (iCCA) can be stimulated by manipulating the tumor microenvironment linked with necroptosis. However, the specific cells regulating the necroptosis microenvironment have not yet been identified. Additionally, further inquiry into the mechanism of how the tumor microenvironment regulates necroptosis and its impact on primary liver cancer(PLC) progression may be beneficial for precision therapy. We recruited a single-cell RNA sequencing dataset (scRNA-seq) with 34 samples from 4 HCC patients and 3 iCCA patients, and a Spatial Transcriptomic (ST) dataset including one each of HCC, iCCA, and combined hepatocellular-cholangiocarcinoma (cHCC-CCA). Quality control, dimensionality reduction and clustering were based on Seurat software (v4.2.2) process and batch effects were removed by harmony (v0.1.1) software. The pseudotime analysis (also known as cell trajectory) in the single cell dataset was performed by monocle2 software (v2.24.0). Calculation of necroptosis fraction was performed by AUCell (v1.16.0) software. Switch gene analysis was performed by geneSwitches(v0.1.0) software. Dimensionality reduction, clustering, and spatial image in ST dataset were performed by Seurat (v4.0.2). Tumor cell identification, tumor subtype characterization, and cell type deconvolution in spot were performed by SpaCET (v1.0.0) software. Immunofluorescence and immunohistochemistry experiments were used to prove our conclusions. Analysis of intercellular communication was performed using CellChat software (v1.4.0). ScRNA-seq analysis of HCC and iCCA revealed that necroptosis predominantly occurred in the myeloid cell subset, particularly in FCGBP + SPP1 + tumor-associated macrophages (TAMs), which had the highest likelihood of undergoing necroptosis. The existence of macrophages undergoing necroptosis cell death was further confirmed by immunofluorescence. Regions of HCC with poor differentiation, cHCC-CCA with more cholangiocarcinoma features, and the tumor region of iCCA shared spatial colocalization with FCGBP + macrophages, as confirmed by spatial transcriptomics, immunohistochemistry and immunofluorescence. Pseudotime analysis showed that premalignant cells could progress into two directions, one towards HCC and the other towards iCCA and cHCC-CCA. Immunofluorescence and immunohistochemistry experiments demonstrated that the number of macrophages undergoing necroptosis in cHCC-CCA was higher than in iCCA and HCC, the number of macrophages undergoing necroptosis in cHCC-CCA with cholangiocarcinoma features was more than in cHCC-CCA with hepatocellular carcinoma features. Further investigation showed that myeloid cells with the highest necroptosis score were derived from the HCC_4 case, which had a severe inflammatory background on pathological histology and was likely to progress towards iCCA and cHCC-CCA. Switchgene analysis indicated that S100A6 may play a significant role in the progression of premalignant cells towards iCCA and cHCC-CCA. Immunohistochemistry confirmed the expression of S100A6 in PLC, the more severe inflammatory background of the tumor area, the more cholangiocellular carcinoma features of the tumor area, S100A6 expression was higher. The emergence of necroptosis microenvironment was found to be significantly associated with FCGBP + SPP1 + TAMs in PLC. In the presence of necroptosis microenvironment, premalignant cells appeared to transform into iCCA or cHCC-CCA. In contrast, without a necroptosis microenvironment, premalignant cells tended to develop into HCC, exhibiting amplified stemness-related genes (SRGs) and heightened malignancy.

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Data availability

The ScRNA-seq datasets generated during the current study are available in the GEO database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE189903). The ST datasets generated during the current study are available in the Genome Sequence Archive database (https://ngdc.cncb.ac.cn/gsa-human/browse/HRA000437).

Abbreviations

scRNA-seq:

Single-cell RNA sequencing

ST:

Spatial transcriptome

PLC:

Primary liver cancer

HCC:

Hepatocellular carcinoma

iCCA/ICC:

Intrahepatic cholangiocarcinoma

CNV:

Copy number variation

TCGA:

The cancer genome atlas

TAM:

Tumor-associated macrophage

CAF:

Cancer associated fibroblasts

TIB:

Tumor immune barrier

LAM:

Lipid associated macrophages

TAME:

Tumor-adipose microenvironment

SRG:

Stemness-related gene

GEO:

Gene expression omnibus

HE:

Hematoxylin–eosin staining

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Acknowledgements

We thank all the team members for their hard work and efforts.

Funding

This study is supported by Shenzhen Science and Technology Program (Grant No. JSGG20201102162802008) and the Key Program for Clinical Research at Peking University Shenzhen Hospital (LCYJZD2021007).

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  1. Chun Wang and Jiakang Chen contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology, Peking University Shenzhen Hospital, Shenzhen, Guangdong Province, China

    Chun Wang, Cuimin Chen, Wenting Hu, Lili Tao & Jiakang Chen

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  1. Chun Wang
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Contributions

CW designed and directed the study and wrote the manuscript. JC designed the study, performed the bioinformatics analysis and data generation, and wrote the original manuscript. WH was responsible for immunofluorescence experiment. LT and CC participated in writing review, editing, and funding acquisition. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Jiakang Chen.

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The authors declare that they have no competing interests.

Ethics approval and consent to participate

The samples used to perform the Immunofluorescence and immunohistochemistry were obtained from patients who gave informed consent and signed an informed consent form. The study was approved by the Ethical Review Committee of Peking University Shenzhen Hospital(PKUSZH)) (Approval Number: 2023115), and all experiments were performed inaccordance with the Declaration of Helsinki ethical guidelines.

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Wang, C., Chen, C., Hu, W. et al. Revealing the role of necroptosis microenvironment: FCGBP + tumor-associated macrophages drive primary liver cancer differentiation towards cHCC-CCA or iCCA. Apoptosis 29, 460–481 (2024). https://doi.org/10.1007/s10495-023-01908-3

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