The core problem the researchers set out to solve is one that plagues much of modern pharmacology: getting enough drug to the diseased tissue without poisoning the rest of the body. Pirfenidone is one of only two antifibrotic agents approved for pulmonary fibrosis, yet its clinical benefit is limited by poor accumulation at the sites of active scarring and by systemic side effects that arise when the drug circulates throughout the body after oral or intravenous administration. Patients may tolerate the treatment poorly, and the lesions themselves receive only a fraction of the administered dose.

The team’s solution combines two ideas that have each attracted intense interest in recent years. The first is the exosome, a nanoscale vesicle naturally released by cells to shuttle proteins, lipids, and genetic material between them. Because exosomes are biological in origin, they are relatively invisible to the immune system and can cross biological barriers that defeat synthetic nanoparticles. The researchers harvested exosomes from 293T cells, a standard laboratory cell line, and verified their identity and quality using transmission electron microscopy to visualize their cup-shaped morphology, nanoparticle tracking analysis to measure their size distribution and concentration, Western blotting to confirm the presence of characteristic exosomal marker proteins, and high-performance liquid chromatography to quantify how much pirfenidone each vesicle had been loaded with.

The second idea is molecular targeting. Fibrotic lung tissue is distinguished by the activity of fibroblast activation protein, or FAP, a molecule displayed on the surface of the activated fibroblasts that drive scarring but largely absent from healthy tissue. Using copper-free click chemistry, a bioorthogonal reaction that joins molecules together efficiently under gentle physiological conditions, the team decorated the surface of the exosomes with anti-FAP antibodies. The result is a drug-loaded vesicle that behaves like a guided missile: once in the bloodstream or the airways, it seeks out cells bearing FAP and docks onto them, concentrating its therapeutic payload precisely where the fibrotic process is most active.

How the payload gets to the lung proved to be just as important as the vesicle carrying it. The researchers compared two routes of administration in a bleomycin-induced mouse model of pulmonary fibrosis, a widely used experimental system in which a single dose of the chemotherapy drug bleomycin triggers progressive lung scarring that mimics the human disease. One group of mice received the engineered exosomes by tail vein injection, the standard route for systemic drug delivery. Another group inhaled the same vesicles as an aerosol generated by a nebulizer, a device that converts liquid formulations into fine droplets that can be breathed deep into the lungs.

The difference was striking. In vivo fluorescence imaging, which tracks where labeled vesicles travel in a living animal, showed that nebulization dramatically enhanced both the accumulation of the engineered exosomes in the lungs and the length of time they remained there, compared with systemic injection. Confocal microscopy of lung tissue sections confirmed the same pattern at the cellular level. When the researchers assessed therapeutic outcomes, the nebulized formulation produced better lung histopathology, meaning less architectural distortion of the tissue, and reduced collagen deposition, the hallmark protein accumulation that stiffens fibrotic lungs. The inhaled route also produced stronger suppression of the TGF-β1/p-Smad3 signaling axis, a central molecular pathway through which the cytokine TGF-β1 drives fibroblasts to become collagen-secreting myofibroblasts. Because this pathway sits at the heart of the fibrotic cascade, its more complete inhibition is a meaningful mechanistic win.

But the study went beyond drug delivery and probed how the treatment reshapes the fibrotic microenvironment itself. Using single-cell RNA sequencing, a technique that profiles gene expression in individual cells within a tissue, the researchers found that nebulized delivery of the engineered exosomes attenuated the activation of fibroblasts and myofibroblasts, the cellular engines of scarring. At the same time, the treatment promoted polarization of immune cells toward a repair-associated state, shifting the balance of the lung’s cellular ecosystem from one that perpetuates injury toward one that promotes healing. Proteomic analysis of the tissue, combined with immunofluorescence staining to localize specific proteins, identified AXL, a receptor tyrosine kinase, as a candidate regulatory molecule associated with these beneficial effects.

To test whether AXL was genuinely involved rather than merely correlated, the team turned to cell-based functional experiments. In MLE12 cells, a mouse lung epithelial cell line, and in bone marrow-derived macrophages driven to polarize toward pro-fibrotic phenotypes, the researchers demonstrated that modulating AXL influenced the cellular behaviors that fuel fibrosis. Manipulating the receptor affected pro-fibrotic cellular functions, altered TGF-β1/p-Smad3 signaling, and changed the levels of oxidative stress, a form of cellular damage that both accompanies and promotes fibrotic remodeling. These findings position AXL-associated signaling as a potential mechanistic node in the fibrotic microenvironment and, intriguingly, as a therapeutic target in its own right, suggesting that the exosome platform may work partly by engaging endogenous repair pathways rather than simply delivering a passive drug cargo.

The implications of the study extend in several directions at once. Clinically, an inhaled, targeted formulation of pirfenidone could allow lower doses to achieve greater effect at the lesion while sparing patients the systemic exposure responsible for many side effects, a meaningful prospect for a disease whose treatments are often poorly tolerated. Technologically, the work demonstrates a practical pipeline for engineering exosomes with disease-specific ligands via click chemistry, loading them with small-molecule drugs, and administering them noninvasively through the airways. Scientifically, the identification of AXL as a regulator of the fibrotic microenvironment adds a new thread to the growing understanding of pulmonary fibrosis as a disease of cellular ecology rather than simple scar accumulation. The authors note that their findings establish nebulized anti-FAP-engineered exosomes as a promising targeted delivery platform and highlight microenvironment-focused intervention as a strategy worth pursuing. As with any preclinical mouse study, translation to human patients will require further validation, dose optimization, and safety testing, but the combination of a proven drug, a homing mechanism aimed at a validated disease marker, and a patient-friendly inhalation route gives this platform an unusually clear path toward clinical evaluation.

Subject of Research: Targeted nebulized exosome delivery of pirfenidone for pulmonary fibrosis therapy

Article Title: Mechanisms of nebulized anti-FAP-engineered exosomes encapsulating pirfenidone for targeted therapy of pulmonary fibrosis

Article References: Chen, H., Zhang, G., Kang, C., Liu, H., Tian, W., & Peng, P. (2026). Mechanisms of nebulized anti-FAP-engineered exosomes encapsulating pirfenidone for targeted therapy of pulmonary fibrosis. Journal of Translational Medicine. https://doi.org/10.1186/s12967-026-08894-5

Image Credits: AI Generated

DOI: 10.1186/s12967-026-08894-5

Keywords: pulmonary fibrosis, exosomes, pirfenidone, nebulization, drug delivery, fibroblast activation protein, TGF-beta signaling, AXL, single-cell RNA sequencing, myofibroblasts, targeted therapy, nanomedicine

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

VSIR, also called VISTA, is a type I transmembrane protein of the B7/CD28 family. Immunologists have long studied it for its immunosuppressive functions within the tumor microenvironment, where it attenuates effector T cell activation, fosters regulatory T cell expansion, and supports the suppressive activity of myeloid-derived suppressor cells and tumor-associated macrophages. But recent work has documented frequent upregulation of VSIR on tumor cells themselves across multiple cancer types, and in melanoma, tumor-cell-specific VSIR expression has been shown to promote tumor onset in preclinical models. In gastric cancer, elevated VSIR correlates with advanced stage, lymph node metastasis, and poor clinical outcomes. These observations raised an provocative question: could VSIR, beyond its checkpoint role, be driving tumor progression through mechanisms intrinsic to the cancer cell? The research team, led by Jinshan Liu, Guoquan Huang, and colleagues at institutions affiliated with Chongqing Medical University, set out to answer it by searching for the signaling pathways that VSIR might control.

Their attention soon turned to AXL, a receptor tyrosine kinase of the TAM family that has emerged as a central regulator of tumor progression, metastasis, and therapeutic resistance across many cancers. When bound by its ligand GAS6, AXL activates the PI3K/AKT, MAPK/ERK, and STAT3 pathways, enhancing cell survival, epithelial-mesenchymal transition, and immune evasion. In gastric cancer, AXL overexpression has been documented in both cell lines and patient specimens and is associated with aggressive phenotypes including peritoneal dissemination. Mining The Cancer Genome Atlas stomach adenocarcinoma cohort through the GEPIA2 platform, the researchers confirmed that AXL expression climbs steadily with tumor stage and that patients with high AXL levels suffer significantly worse overall survival, with a hazard ratio of 1.4. The clinical signal was unmistakable, but the regulatory circuitry that produces AXL overexpression in an inflammatory setting had not been mapped.

The team’s first mechanistic clue came from transcriptome sequencing of gastric cancer cells. When HGC-27 gastric cancer cells were engineered to overexpress VSIR and then stimulated with LPS to mimic bacterial inflammation, AXL mRNA levels rose dramatically. The effect was strikingly conditional: without LPS, VSIR overexpression alone failed to induce AXL, but once the inflammatory stimulus was applied, VSIR markedly enhanced AXL transcription. Conversely, silencing VSIR with two independent small interfering RNAs abolished the LPS-driven induction of AXL at both the mRNA and protein levels. The results established VSIR as both necessary and sufficient, within the inflammatory context, to promote AXL expression, forging a direct molecular link between innate immune signaling and AXL-driven tumor progression.

To identify the transcription factor mediating this effect, the researchers cross-referenced four independent prediction databases—hTFtarget, CHEA, FIMO_JASPAR, and ENCODE—and found that two candidates, STAT3 and EGR1, were consistently predicted to bind regulatory regions of the AXL gene. Chromatin profiling from the Cistrome Database revealed strong enrichment of STAT3-binding signals at an enhancer upstream of AXL, coinciding precisely with the active histone marks H3K27ac and H3K4me1, the biochemical signatures of functional enhancer elements. Chromatin immunoprecipitation followed by quantitative PCR in HGC-27 cells confirmed that STAT3 physically occupies this enhancer region. The evidence became causal when the team cloned the enhancer fragment into a luciferase reporter: co-transfection with a STAT3 expression plasmid dramatically boosted reporter activity, while site-directed mutation of the predicted STAT3-binding motif, changing the sequence CAT CTG GAA AG to ACC ACA ACC CA, abolished that activation. Knocking down STAT3 with siRNA reduced endogenous AXL mRNA and protein, and parallel experiments in mouse bone marrow-derived macrophages showed that LPS-induced AXL upregulation was completely eliminated when STAT3 was depleted. Together, these experiments established STAT3 as the essential transcriptional mediator linking LPS stimulation to AXL expression in both myeloid and cancer cells.

But how does VSIR, a membrane checkpoint protein, activate STAT3? The answer, the researchers found, lies in the IL-6 pathway. Under LPS stimulation, VSIR-overexpressing cells produced substantially more IL-6 mRNA, and western blotting revealed enhanced phosphorylation of STAT3—without any measurable change in the phosphorylation of p65 or JAK1, indicating a specific rather than generalized activation of inflammatory signaling. When the team silenced TLR4, the VSIR-driven boost in IL-6 production vanished, placing VSIR functionally downstream of the TLR4 receptor. The emerging picture is a coherent cascade: LPS engages TLR4, VSIR potentiates IL-6 release, IL-6 activates STAT3, and STAT3 directly switches on AXL transcription.

Remarkably, transcription proved to be only half the story. Co-immunoprecipitation experiments revealed that VSIR physically interacts with the AXL protein itself, and immunofluorescence microscopy confirmed the colocalization of the two molecules in both HGC-27 and MKN-45 gastric cancer cells. Cycloheximide chase assays, which track protein decay over time, demonstrated that VSIR overexpression significantly prolongs the half-life of AXL. The proteasome inhibitor MG132 erased the difference in AXL levels between VSIR-proficient and VSIR-deficient cells, pointing to the ubiquitin-proteasome pathway as the route of degradation, and direct ubiquitination assays showed that VSIR overexpression markedly attenuates the polyubiquitination of endogenous AXL. In other words, VSIR both writes more AXL mRNA and protects the resulting protein from destruction, a dual mechanism that ensures robust, sustained AXL signaling precisely when inflammatory conditions would otherwise favor tumor growth.

Clinical validation came from multiplex immunofluorescence staining of patient tissues, including samples from twelve gastric cancer patients treated at the First Affiliated Hospital of Chongqing Medical University. Tumor samples showed a significant positive correlation among VSIR, AXL, and phosphorylated STAT3, mirroring the in vitro findings. More striking still, when the team compared HER2-positive tumors from patients resistant to HER2-targeted therapy, treatment-naive HER2-positive tumors, HER2-negative tumors, and normal gastric mucosa, the fraction of cells simultaneously positive for VSIR, AXL, and HER2 was markedly highest in the resistant HER2-positive group. Because elevated VSIR and AXL were also present in treatment-naive HER2-positive tumors, the authors conclude that this co-expression pattern is a general feature of HER2-positive gastric cancer rather than a specific marker of acquired resistance—implying that these tumors depend on dual engines of growth: HER2-driven proliferation and VSIR-driven, AXL-mediated survival.

The therapeutic implications were tested directly in a subcutaneous xenograft model in nude mice, with animals randomized into five treatment groups. VSIR overexpression robustly accelerated tumor growth, and while the HER2 inhibitor tucatinib alone produced only modest suppression, the AXL inhibitor AXL-IN-13 sharply curtailed VSIR-driven tumor expansion, reducing tumor volume and weight to levels comparable to or below controls. The dual blockade of HER2 and AXL produced the most potent anti-tumor effect of all, nearly abolishing the pro-tumorigenic activity of VSIR, with Ki-67 immunohistochemistry confirming sharply reduced proliferation in the treated tumors. Rescue experiments in vitro reinforced the causal chain: when VSIR was knocked down in MKN-45 cells, migration and invasion declined sharply, but re-expressing AXL restored these metastatic capabilities. Taken together, the study recasts VSIR as a tumor-intrinsic oncogenic amplifier that operates independently of its immune checkpoint function, positions the VSIR-AXL axis as a druggable vulnerability in an inflammation-associated malignancy, and supplies a strong preclinical rationale for combining AXL inhibitors with HER2-directed therapy in patients whose tumors currently escape single-agent blockade.

Subject of Research: The role of VSIR in regulating AXL expression and promoting gastric cancer proliferation through inflammatory STAT3 signaling

Article Title: Mechanistic insights into VSIR-mediated AXL regulation and gastric cancer proliferation

Article References: Liu, J., Huang, G., Li, Y., Tan, Y., Qin, B., Hao, C., Peng, J., Zhu, H., Zhang, B., Cheng, Y., & Qian, K. (2026). Mechanistic insights into VSIR-mediated AXL regulation and gastric cancer proliferation. iScience, 29(10), Article 117580. https://doi.org/10.1016/j.isci.2026.117580

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117580

Keywords: gastric cancer, VSIR, VISTA, AXL, STAT3, IL-6, TLR4, Helicobacter pylori, HER2, immune checkpoint, protein stability, targeted therapy