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. 2024 Feb;13(3):e6736.
doi: 10.1002/cam4.6736. Epub 2024 Jan 10.

Heterogeneity-induced NGF-NGFR communication inefficiency promotes mitotic spindle disorganization in exhausted T cells through PREX1 suppression to impair the anti-tumor immunotherapy with PD-1 mAb in hepatocellular carcinoma

Xin Wang  1 , Tongwang Yang  1   2   3 , Shangheng Shi  1 , Chuanshen Xu  1 , Feng Wang  1 , Deshu Dai  1 , Ge Guan  1 , Yong Zhang  1 , Shuxian Wang  1 , Jianhong Wang  1 , Bingliang Zhang  1 , Peng Liu  1 , Xiaoshuai Bai  1 , Yan Jin  1 , Xinqiang Li  1 , Cunle Zhu  1 , Dexi Chen  1   4 , Qingguo Xu  1   2 , Yuan Guo  1
Affiliations

Heterogeneity-induced NGF-NGFR communication inefficiency promotes mitotic spindle disorganization in exhausted T cells through PREX1 suppression to impair the anti-tumor immunotherapy with PD-1 mAb in hepatocellular carcinoma

Xin Wang et al. Cancer Med. 2024 Feb.

Abstract

Background: The mechanism of decreased T cells infiltrating tumor tissues in hepatocellular carcinoma is poorly understood.

Methods: Cells were separated from the single-cell RNA-sequence dataset of hepatocellular carcinoma patients (GSE149614) for cell-cell communication. Flow cytometry, EDU staining, H3-Ser28 staining, confocal immunofluorescence staining, western blotting and naked microsubcutaneous tumors were performed for the mechanism of NGF-NGFR promoting proliferation.

Results: The present study has revealed that during the process of T-cell infiltration from adjacent tissues to tumor tissues, an inefficiency in NGF-NGFR communication occurs in the tumor tissues. Importantly, NGF secreted by tumor cells interacts with NGFR present on the membranes of the infiltrated T cells, thereby promoting the proliferation through the activation of mitotic spindle signals. Mechanistically, the mediation of mitotic spindle signal activation promoting proliferation is executed by HDAC1-mediated inhibition of unclear trans-localization of PREX1. Furthermore, PD-1 mAb acts synergistically with the NGF-NGFR communication to suppress tumor progression in both mouse models and HCC patients. Additionally, NGF-NGFR communication was positively correlates with the PD-1/PDL-1 expression. However, expressions of NGF and NGFR are low in tumor tissues, which is responsible for the invasive clinicopathological features and the disappointing prognosis in HCC patients.

Conclusion: Inefficiency in NGF-NGFR communication impairs PD-1 mAb immunotherapy and could thus be utilized as a novel therapeutic target in the treatment of HCC patients in clinical practice.

Keywords: hepatocellular carcinoma; immunotherapy; nerve growth factor; nerve growth factor receptor; pd-1.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

FIGURE 1
FIGURE 1
The cell–cell communication between tumor cells and T cells. (A) The UMAP reveals clusters of cells in HCC patients. (B) The UMAP reveals the tumor cells and T cells in HCC patients. (C) The NGF signaling pathway network between the tumor cells and T cells. Clusters in the NGF signaling pathways are depicted in color. (D) Ligands and receptors contribute to the NGF signaling pathway between the tumor cells and T cells. (E) The circle plot depicts the NGF signaling pathway between the tumor cells and T cells. Clusters in the NGF signaling pathways are depicted in color and line. (F) NGF (green) and NGFR (red) co‐colocalization in the HCC patients. DAPI was used for staining the genomic DNA (blue). Scale bar, 25 μm.
FIGURE 2
FIGURE 2
Diffusion pseudo‐time analysis of T cells in HCC patients. (A) The UMAP depicts clusters of T cells in the tumor tissues and adjacent tissues. (B) The UMAP depicts T cells in tumor and adjacent tissues (Ba), tumor tissues (Bb), and adjacent tissues (Bc) of HCC patients. (C) T cells were pseudo‐time differentiated with one main distinct branch point adjacent to the tumor tissues. Ca cell differentiation map for pseudo‐time, Cb cell differentiation map for tumor and adjacent tissues, and Cc cell differentiation map for cluster. (D) Marker genes (top 10) of the sub‐population that were reprogrammed in this pseudo‐time process. (E) The UMAP for the expression of the T‐cell exhaustion markers LAYN (Ea), CTLA4 (Eb), and HAVCR2 (EC) in HCC patients.
FIGURE 3
FIGURE 3
NGF‐NGFR communication inefficiency suppressed the organization of the mitotic spindle. (Aa, b) α‐tubulin (green) and Lamin B (red) staining for observing the organization of the mitotic spindle and the nuclear envelope in Jurkat T cells. Scale bar, 10 μm. (B) Immunoblot assay for examining the monomeric α‐tubulin and polymeric α‐tubulin in Jurkat T cells. (C) Flow cytometry assay for revealing the cell cycle distribution of Jurkat T cells. (Da, b) H3‐Ser28 (red) staining for determining the mitotic index for the Jurkat T cells. Scale bar, 60 μm. (Dc) Ratio of H3‐Ser28 positive cells. (Ea, b) Edu (green) staining for determining the cell proliferation of Jurkat T cells. Scale bar, 60 μm. (Ec) Ratio of Edu‐positive cells. Genomic DNA was stained in blue using the DAPI solution. n = 12, *p < 0.05, **p < 0.01.
FIGURE 4
FIGURE 4
The molecular mechanism underlying the suppression of mitotic spindle formation is due to NGF‐NGFR communication inefficiency. (A) Co‐expression of PREX1 with NGF (Aa) and NGFR (Ab) in the tumors from TCGA. (B) Co‐expression of PREX1 with NGF (Ba) and NGFR (Bb) in the tumors from LIHC. (C) Co‐expression of PREX1 with NGF (Ca) and NGFR (Cb) in the tumors from GTEx. (D) Co‐expression of PREX1 with NGF (Da) and NGFR (Db) in the tumors from the liver. (E) Violin plot depicting the differences in the expression of PREX1 between NGF/NGFR low‐expression and high‐expression groups of tumor tissues. (F) The immunoblot assay for deciphering the effect of NGF‐NGFR communication inefficiency on PREX1 expression. (G) The immunoblot assay for determining the effect of NGF‐NGFR communication inefficiency on the nuclear translocation of HDAC1. Ga for the nuclear fraction and Gb for the cytoplasm fraction. (H) Immunofluorescence staining for determining the effect of NGF‐NGFR communication inefficiency on the nuclear translocation of HDAC1. Scale bar, 25 μm. Genomic DNA was stained with the DAPI solution.
FIGURE 5
FIGURE 5
The role of PREX1 expression in NGF‐NGFR communication inefficiency‐suppressed mitotic spindle organization. (A) An immunoblot assay was performed to determine the effect of PREX1‐siRNA on PREX1 expression. As for the total fraction, Ab for the polymeric fraction, and Ac for the monomeric fraction. (B) Immunoblot assay for determining the impact of PREX1‐siRNA on the polymerization of α‐tubulin. (C) α‐tubulin (green) and Lamin B (red) staining results reveal the effect of PREX1‐siRNA on the organization of the mitotic spindle and the nuclear envelope. Scale bar, 10 μm. (D) An immunoblot assay was performed to determine the impact of PREX1 overexpression on NGF‐NGFR communication inefficiency‐suppressed polymerization of α‐tubulin. (E) α‐tubulin (green) and Lamin B (red) staining results reveal the effect of PREX1 overexpression on NGF‐NGFR communication inefficiency‐suppressed organization of the mitotic spindle. Scale bar, 10 μm. Genomic DNA was stained using the DAPI solution.
FIGURE 6
FIGURE 6
Landscape of immune infiltration in the NGF and NGFR high‐expression and low‐expression LIHC patients. (A) Bar plot illustrating immune infiltration in the NGF and NGFR high‐expression and low‐expression LIHC patients (B) Heatmap illustrating immune infiltration in the NGF and NGFR high‐expression and low‐expression LIHC patients. The proportions of (C) central memory CD4+ T cells, (D) activated CD8+ T cells, (E) effector memory CD8+ T cells, (F) natural killer T cells, (G) natural killer cells, (H) and CD56dim natural killer cells, infiltrated in the NGF and NGFR high‐expression and low‐expression LIHC patients.
FIGURE 7
FIGURE 7
Synergistic role of NGF‐NGFR communication and PD‐1 mAb. (A, B) Immunodeficient mice, rebuilt with NGFR low‐expression CD3+ T cells and exposed to PD‐1 mAb therapy, were injected with NGFR‐shRNA‐Lv‐infected Huh7 cells. (A) The outline of the therapeutic plan for the immunodeficient mice. (B) Tumor growth curves are measured once a week. (C) CT imaging depicting the tumor diameter (red line). (D) NGF and NGFR expressions were recorded using the immunohistochemistry staining of the tumor tissues of HCC patients. Scale bar, 30 μm. (E) Tumor diameter in HCC patients before and after the PD‐1 mAb therapy. (F) Spearman's rank correlation for determining the diameter change and the NGF and NGFR expressions.
FIGURE 8
FIGURE 8
NGF‐NGFR communication in HCC patients. (Aa) NGF and (Ab)NGFR expressions in the patient data from TCGA. (Ba) NGF and (Bb) NGFR expressions in HCC patient data from GEO. (C) The relative mRNA expressions of NGF (Ca) and NGFR (Cb) in the data of 98 HCC patients were expressed as −ΔΔCT values. (D) The relative protein contents of NGF and NGFR in paired tumors and adjacent tissues of 42 HCC patients were determined using western blotting.
FIGURE 9
FIGURE 9
The molecular mechanism underlying the NGF‐NGFR communication inefficiency suppressed mitotic spindle signaling pathway activation in T‐cell proliferation.
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References

    1. Jiang ZR, Yang LH, Jin LZ, et al. Identification of novel cuproptosis‐related lncRNA signatures to predict the prognosis and immune microenvironment of breast cancer patients. Front Oncol. 2022;12:988680. doi:10.3389/fonc.2022.988680 - DOI - PMC - PubMed
    1. Lopez de Rodas M, Schalper KA. Tumour antigen‐induced T cell exhaustion ‐ the archenemy of immune‐hot malignancies. Nat Rev Clin Oncol. 2021;18(12):749‐750. doi:10.1038/s41571-021-00562-5 - DOI - PMC - PubMed
    1. Blank CU, Haining WN, Held W, et al. Defining ‘T cell exhaustion’. Nat Rev Immunol. 2019;19(11):665‐674. doi:10.1038/s41577-019-0221-9 - DOI - PMC - PubMed
    1. Wherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol. 2015;15(8):486‐499. doi:10.1038/nri3862 - DOI - PMC - PubMed
    1. Gao Y, Xu Q, Li X, et al. Heterogeneity induced GZMA‐F2R communication inefficient impairs antitumor immunotherapy of PD‐1 mAb through JAK2/STAT1 signal suppression in hepatocellular carcinoma. Cell Death Dis. 2012;13(3):213. doi:10.1038/s41419-022-04654-7 - DOI - PMC - PubMed

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