Anlotinib potentiates anti-PD1 immunotherapy via transferrin receptor-dependent CD8+ T-cell infiltration in hepatocellular carcinoma

Abstract

Background: The therapeutic potential of immune checkpoint blockade (ICB) extends across various cancers; however, its effectiveness in treating hepatocellular carcinoma (HCC) is frequently curtailed by both inherent and developed resistance.

Objective: This research explored the effectiveness of integrating anlotinib (a broad-spectrum tyrosine kinase inhibitor) with programmed death-1 (PD-1) blockade and offers mechanistic insights into more effective strategies for treating HCC.

Methods: Using patient-derived organotypic tissue spheroids and orthotopic HCC mouse models, we assessed the effectiveness of anlotinib combined with PD-1 blockade. The impact on the tumour immune microenvironment and underlying mechanisms were assessed using time-of-flight mass cytometry, RNA sequencing, and proteomics across cell lines, mouse models, and HCC patient samples.

Results: The combination of anlotinib with an anti-PD-1 antibody enhanced the immune response against HCC in preclinical models. Anlotinib remarkably suppressed the expression of transferrin receptor (TFRC) via the VEGFR2/AKT/HIF-1α signaling axis. CD8⁺ T-cell infiltration into the tumour microenvironment correlated with low expression of TFRC. Anlotinib additionally increased the levels of the chemokine CXCL14, crucial for attracting CD8⁺ T cells. CXCL14 emerged as a downstream effector of TFRC, exhibiting elevated expression following the silencing of TFRC. Importantly, low TFRC expression was also associated with a better prognosis, enhanced sensitivity to combination therapy, and a favourable response to anti-PD-1 therapy in patients with HCC.

Conclusions: Our findings highlight anlotinib's potential to augment the efficacy of anti-PD-1 immunotherapy in HCC by targeting TFRC and enhancing CXCL14-mediated CD8⁺ T-cell infiltration. This study contributes to developing novel therapeutic strategies for HCC, emphasizing the role of precision medicine in oncology.

Highlights: Synergistic effects of anlotinib and anti-PD-1 immunotherapy demonstrated in HCC preclinical models. Anlotinib inhibits TFRC expression via the VEGFR2/AKT/HIF-1α pathway. CXCL14 upregulation via TFRC suppression boosts CD8+ T-cell recruitment. TFRC emerges as a potential biomarker for evaluating prognosis and predicting response to anti-PD-1-based therapies in advanced HCC patients.

Keywords: hepatocellular carcinoma; immune checkpoint blockade; transferrin receptor; tumour microenvironment; tyrosine kinase inhibitor.

FIGURE 1

Anlotinib improves the anti‐tumour activity of PD‐1 blockade in vitro and vivo. (A) Schematic showing the preparation of HCC patient‐derived PDOTs (40–100 μm diameter). Viability assessment of PDOTs cultured in a microfluidic chip for 7 days using fluorescent staining (live = green, dead = red). Scale bars: 200 μm. (B) Immunofluorescent staining of CD45⁺ (green) and CD8⁺ (green) immune cells within PDOTs. Scale bars: 25 μm. (C) Heat map illustrating the viability of PDOTs cultured under different treatments: control (DMSO+IgG), anlotinib (10 μmol), anti‐PD‐1 (250 μg/mL), and combined anlotinib+anti‐PD‐1. (D) Representative image and quantitative analysis of the viability of PDOTs (n = 24) after different treatments as indicated by staining for acridine orange (AO, live cells shown in green) and propidium iodide (PI, dead cells shown in red). Scale bars: 100 μm; (E) Representative images of orthotopic Hep1‐6 tumours in C57BL/6 mice after various treatments (n = 6 per group), accompanied by graphs of tumour volumes (left) and weights (right) for the depicted tumours. (F, G) Representative H&E and Tunel images of the primary tumours from C57BL/6 mice. Scale bar: 100 μm. T, tumour; N, necrosis. Data are presented as mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

Anlotinib plays a crucial role in the regulation of tumour immunity. (A) Heatmap of selected immune markers for the 49 clusters in the CyTOF data. (B) tSNE plots of immune cell subpopulations. Box plots of differential immune cell subpopulations in each indicated group. (C) Representative images and bar plots illustrating the number of CD8⁺ and CD4⁺ T cells in Hepa1‐6 tumours from mice that received the indicated treatments, Scale bar: 100 μm. (D) Venn diagram of enriched cell subpopulations within each indicated group. (E) t‐SNE plot of the CD8⁺ T‐cell subpopulation distribution. Boxplot illustrating the fractions of cells in clusters 39 and 40 within each indicated group. (F) Schedule of tumour implantation and injection of antibodies for cell depletion in mice. Depletion of CD8⁺ or CD4⁺ cells during combination therapy with anlotinib and anti‐PD‐1. Bar plot showing the tumour size in each group (n = 6) following CD8⁺ and/or CD4⁺ T‐cell depletion. Data are presented as mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

FIGURE 3

Anlotinib triggers immune response signalling and induces the production of proinflammatory cytokines and chemokines. (A) Venn diagram revealing the common up/downregulated genes (adjusted p‐value < 0.05, fold‐change ≥ 2) detected using RNA‐seq in groups treated with anlotinib; 63 genes were shared among the groups (53 genes upregulated, 10 genes downregulated). (B) Results of GO and KEGG enrichment analyses based on these differentially expressed genes after treatment with anlotinib versus control. (C) GSEA analysis of the differentially expressed genes induced by anlotinib monotherapy. Four of the top ten most positively regulated “Hallmark signatures”. (D) A heatmap of the degree of immune cell infiltration in the anlotinib monotherapy group as indicated by mMCP‐counter algorithms. (E) Ternary plot displaying the distribution of Hepa1‐6 tumour samples with varying proportions of PDCD1, CD8, and CTLA‐4 expression, where each axis represents the percentage (0–100%) of the respective marker. Different coloured dots represent individual tumour samples, with red indicating the anlotinib group and blue representing the control group. (F) Representative images of multiplex immunofluorescence results for Hepa1‐6 tumour tissues stained for CD8 (green), PD‐1 (red), and nuclei (DAPI; blue) and quantification of PD‐1⁺CD8⁺ T cells in mice bearing Hepa1‐6 tumours from the indicated treatment groups. (G) Representative images of multiplex immunofluorescence results for Hepa1‐6 tumour tissues stained for CD8 (green), PD‐1 (red), and nuclei (DAPI; blue) and quantification of PD‐1⁺CD8⁺ T cells in mice bearing Hepa1‐6 tumours from the indicated treatment groups. Scale bar: 50 μm. Data are presented as mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

FIGURE 4

Anlotinib targets TFRC to regulate the immune response and inflammation via the VEGFR2/AKT/HIF‐1α Axis. (A) The PPI network of DEGs associated with anlotinib monotherapy vs. control group. (B) Venn diagram representing a number of overlapping differentially expressed target genes in anlotinib‐treated and combination (Anlotinib + anti‐PD‐1)‐treated tumours. Bar diagram of three common differentially expressed target genes between the anlotinib treatment and control groups. (C) qRT‐PCR and Western blot analyses of the TFRC expression levels in PLC/PRF/5 and HepG2 cells treated with increasing concentrations of anlotinib (0–20 μmol) for 24 h. (D) Western blot analysis of VEGFR2, p‐VEGFR2, FGFR1, p‐FGFR1, PDGFR‐β, and p‐PDGFR‐β expression in PLC/PRF/5 and HepG2 cell lines treated with anlotinib gradients for 24 h, with densitometry analysis from three representative experiments. (E) The expression and phosphorylation levels of VEGFR2, AKT, HIF‐1α, and TFRC in the PLC/PRF/5 and HepG2 cell lines after treatment with a concentration gradient anlotinib were detected by Western blot assays. (F) The expression and phosphorylation levels of VEGFR2, AKT, HIF‐1α, and TFRC in H22 and Hepa1‐6 tumours following the indicated treatments were detected using the Western blot assays. Data are presented as mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

FIGURE 5

CXCL14 facilitates the infiltration of CD8⁺ T cells via inhibition of TFRC expression by Anlotinib. (A) Correlation of the TFRC mRNA levels with 24 types of immune cells. (B) Representative images and tumour volumes of TFRC wild type or TFRC‐KO tumours from C57BL/C mice from the indicated treatment groups (n = 6 mice per group). (C–D) Schematic representation and quantitative analysis of CD8⁺ T‐cell migration towards tumour cells and the subsequent tumour cell viability assay. Activated CD8⁺ T cells were placed in the upper chamber of a migration setup, with HepG2 tumour cells treated with either vehicle, TFRC knockdown, or anlotinib in the lower chamber. After a 48 h co‐culture period, the migrated CD8⁺ T cells were quantified. Tumour cell viability was assessed by subjecting the HepG2 cells in the lower chamber to crystal violet staining. (E) Venn diagram representing a number of overlapping differentially expressed target genes in anlotinib‐treated and combination (Anlotinib + anti‐PD‐1)‐treated tumours. (F) Schematic representation and quantitative analysis of the effects of CXCL14 on CD8⁺ T‐cell migration. Activated CD8⁺ T cells were placed in the upper chamber, with the lower chamber containing varying concentrations of CXCL14 to assess its chemotactic effect on T cells. The bar graph indicates the quantity of CD8⁺ T cells present in the lower chamber medium after a 48‐h incubation. Bar diagram of three differentially expressed cytokines that were common between the anlotinib treatment and control groups. (G) Representative immunohistochemical staining of TFRC protein expression, CXCL14 secretion and CD8⁺ T cells in TFRC wild type or TFRC‐KO tumours from C57BL/C mice following exposure to the indicated treatments, Scale bar: 100 μm. Data are presented as mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

FIGURE 6

TFRC is associated with the overall survival and response of patients with HCC to anti‐PD‐1‐based immunotherapy. (A) IHC analysis of TFRC protein expression, CXCL14 secretion and CD8⁺ T‐cell infiltration in the primary HCC used to establish the PDOTs. Scale bar: 25 μm. (B) Correlations among the TFRC and CXCL14 protein expression and CD8⁺ T‐cell infiltration in HCC tissues. p‐value by Pearson's χ² analysis. (C) Bar graph showing the TKI (tumour‐killing index) of different samples from PDOTs following anti‐PD‐1 treatment. Box plot analysis of the TKI in groups with high and low TFRC expression. (D) Kaplan–Meier curves for the overall patient survival stratified according to the TFRC expression (high/low) in the TCGA database. (E) Dot plot depicting the TFRC mRNA expression levels from the GSE117358 dataset, classified as non‐responders and responders to anti‐PD‐1 therapy (**p < 0.01). (F) IHC analysis of the TFRC expression in primary HCC (left), scale bar: 100 μm. CT images of lesions before and after treatment with anlotinib plus pembrolizumab (right). Data are presented as mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

FIGURE 7

Schematic depiction of the mechanism underlying how anlotinib enhances the tumour response to anti‐PD‐1 immunotherapy.