Two Proteins Found on Nearly All Liver Cancer Cells Point to a Dual-Target CAR-T Strategy

Hepatocellular carcinoma, the most common form of primary liver cancer, remains one of the most formidable targets in oncology. It ranks sixth worldwide in incidence and third in cancer-related deaths, and projections suggest that more than one million people will be affected annually by 2025, with incidence expected to keep rising over the next three decades. Against this backdrop, a team of researchers at Zhongshan Hospital, Fudan University, has delivered a finding that could reshape how cell-based immunotherapies for liver cancer are designed. Their study, published in Clinical Cancer Bulletin, provides the first direct demonstration that two cell-surface proteins, Glypican-3 and AXL, are co-expressed in the vast majority of hepatocellular carcinoma tumors, laying a quantitative foundation for a dual-target chimeric antigen receptor T cell, or CAR-T, approach.

The logic behind the study is deceptively simple but clinically consequential. CAR-T therapy works by engineering a patient’s own T cells to recognize a specific antigen on the surface of tumor cells. The strategy has produced remarkable results in blood cancers, but solid tumors have proven far more resistant, in part because tumor cells can simply stop displaying the antigen the therapy targets, a phenomenon known as target escape. One established countermeasure is to engineer CAR-T cells that recognize two different membrane molecules simultaneously, so that tumor cells remain visible to the immune system as long as they express either one of the two antigens. To justify such a design, researchers first need to know how often the two candidate antigens appear together in the same patients’ tumors, data that had been largely missing for GPC-3 and AXL in liver cancer.

The research team, led by Chenli Qiu, Jieyi Shi and Su Yan, who contributed equally, with senior authors Xiaoyan Zhang, Xiaowu Huang and Jianqing Xu, analyzed surgical specimens from 140 patients with hepatocellular carcinoma who underwent hepatectomy at Zhongshan Hospital in 2019. All patients had complete clinical information, and the samples were obtained following Institutional Review Board approval and written informed consent. The investigators used immunohistochemistry on 5-micrometer formalin-fixed, paraffin-embedded tissue sections, a standard pathology technique that uses antibodies to reveal where specific proteins sit within tissue. Sections were subjected to heat-induced antigen retrieval in Tris/EDTA buffer at 99 degrees Celsius, blocked with bovine serum albumin, and incubated overnight at 4 degrees Celsius with primary antibodies against AXL and GPC-3 before signal detection with HRP-labeled secondary antibodies and DAB chromogen.

Quantification followed a rigorous semiquantitative scheme. Only cytoplasmic or membranous staining counted as positive, and two pathologists independently scored each slide on a four-point scale, from 0 for no expression through weak, moderate and strong expression. The slides were digitized on a TissueFAXS platform, and categorical comparisons were analyzed with Pearson’s chi-square test, ordered data with the Goodman–Kruskal Gamma test, and survival outcomes with Kaplan–Meier curves and the log-rank test. This level of methodological detail matters because immunohistochemical scoring is inherently interpretive, and the discrepancy between different studies often traces back to the antibodies used and the thresholds applied, a point the authors themselves acknowledge.

The headline numbers are striking. GPC-3 was positive in 108 of 140 tumors, a rate of 77.1 percent, with strong expression accounting for the largest share, 42.1 percent of all cases. AXL was even more prevalent, positive in 120 of 140 tumors, or 85.7 percent, with strong expression in 48.6 percent. Critically, only 9 of the 140 samples, 6.4 percent, were negative for both proteins, meaning that 93.6 percent of hepatocellular carcinomas expressed at least one of the two antigens and 69.3 percent expressed both simultaneously. In adjacent non-tumor liver tissue, by contrast, GPC-3 was weakly positive in just one of 140 samples, and AXL staining, while present at low levels in some surrounding tissue, was consistently weaker than in the tumors themselves.

That contrast between tumor and normal tissue is what makes these two proteins attractive drug targets. GPC-3 is an oncofetal protein, a glycosylphosphatidylinositol-anchored membrane molecule that drives embryonic morphogenesis through the canonical Wnt/beta-catenin and Hedgehog pathways. It is essentially silent in the healthy adult liver but reactivated in hepatocellular carcinoma, where it appears in the cytoplasm and on the cell membrane in 70 to 100 percent of cases in previous analyses. Mechanistically, GPC-3 acts as a co-receptor that recruits Wnt, insulin-like growth factors and growth factors such as HGF, FGF-2 and TGF to their respective receptors, fueling proliferation, migration and survival. Overexpression also disrupts the Bax/Bcl-2 axis to increase resistance to apoptosis, promotes epithelial–mesenchymal transition through the ERK pathway, and modulates the CXCL12/CXCR4 axis to drive angiogenesis and metastasis.

AXL tells a complementary story. It is a receptor tyrosine kinase of the TAM family, spanning TYRO3, AXL and MERTK, and it is implicated in cancer cell proliferation, stemness, invasion, angiogenesis and therapeutic resistance. When its ligand GAS6 binds, AXL dimerizes and activates downstream PI3K/Akt and MAPK/ERK signaling, pathways central to tumor growth and spread. AXL is overexpressed across many malignancies, at roughly 47 percent in renal cancer, 69 percent in lung cancer, 57.6 percent in breast cancer and 76.7 percent in colon cancer, and prior studies had placed its expression in hepatocellular carcinoma at 22 to 43 percent. The higher rate observed in the new study may reflect differences in patient populations, detection antibodies and scoring criteria. Notably, AXL positivity was associated with microvascular invasion in this cohort, though neither protein correlated with overall survival over the 50-month follow-up, possibly because the negative sample groups were small and follow-up relatively short.

The therapeutic implications extend beyond CAR-T design. A phase I dose-escalation study of autologous CAR-T cells targeting GPC-3 in advanced hepatocellular carcinoma already showed a manageable safety profile, with an overall objective response rate of 50 percent that reached 57.1 percent in the highest dose cohort. On the AXL side, the selective inhibitor bemcentinib has received US FDA fast-track designation in combination with a PD-1/PD-L1 agent, and AXL-directed CAR-T cells have been reported in non-small cell lung cancer and triple-negative breast cancer. Intriguingly, the authors highlight reports that anti-PD-L1 antibodies can promote liver cancer cell proliferation by activating a PD-L1–AXL signal relay, which suggests that the high AXL expression documented in this study may partly explain the suboptimal performance of immune checkpoint inhibitors in some hepatocellular carcinoma patients, and that AXL-targeted therapy could sensitize tumors to checkpoint blockade.

Dual-targeting is not a panacea, however, and the authors are candid about the challenges. AXL is also present on macrophages and dendritic cells, raising the specter of on-target, off-tumor toxicity if engineered T cells attack healthy immune populations. Proposed engineering countermeasures include optimizing CAR structural design, incorporating logic gates that require combinations of antigens to trigger killing, and building suicide gene systems that can eliminate the infused cells if toxicity emerges. Antigen escape, limited persistence of CAR-T cells in the hostile solid tumor microenvironment, manufacturing cost and toxicity management remain the central obstacles that any GPC-3/AXL dual-target program would need to address in preclinical and clinical testing.

The study’s limitations are equally clear: it was a single-center, retrospective analysis with a relatively modest sample size and short follow-up, and it found no correlation between either protein and most clinicopathological features such as cirrhosis, macrovascular invasion, tumor size, BCLC stage, recurrence or mortality. Yet the core result stands on firm ground. Nearly 94 percent of hepatocellular carcinomas in this cohort displayed at least one of the two antigens, and more than two-thirds displayed both, making GPC-3 and AXL together a coverage profile that few single or paired targets in liver cancer can match. As immunotherapy increasingly becomes a pillar of hepatocellular carcinoma treatment alongside surgery, locoregional and systemic options, this co-expression map offers exactly the kind of evidence base needed to move dual-target CAR-T cells for liver cancer from concept toward the clinic.

Subject of Research: Co-expression of Glypican-3 and AXL in hepatocellular carcinoma and their potential as dual targets for CAR-T cell immunotherapy

Article Title: GPC-3 and AXL are essential factors and therapeutic targets for hepatocellular carcinoma

Article References: Qiu, C., Shi, J., Yan, S., Zhu, C., Zhang, S., Feng, Y., Zhang, X., Huang, X., & Xu, J. (2025). GPC-3 and AXL are essential factors and therapeutic targets for hepatocellular carcinoma. Clinical Cancer Bulletin, 4(1), Article 22. https://doi.org/10.1007/s44272-025-00051-1

Image Credits: AI Generated

DOI: 10.1007/s44272-025-00051-1

Keywords: hepatocellular carcinoma, Glypican-3, AXL, CAR-T cell therapy, immunotherapy, biomarkers, liver cancer, targeted therapy, immunohistochemistry, receptor tyrosine kinase, oncofetal protein, dual-target therapy