Erianin Potentiates the Efficacy of Immune Checkpoint inhibitor in Cholangiocarcinoma by Inhibiting FAK-mediated cell growth and senolytic activity

Erianin Potentiates the Efficacy of Immune Checkpoint inhibitor in Cholangiocarcinoma by Inhibiting FAK-mediated cell growth and senolytic activity | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 30 March 2026 V1 Latest version Share on Erianin Potentiates the Efficacy of Immune Checkpoint inhibitor in Cholangiocarcinoma by Inhibiting FAK-mediated cell growth and senolytic activity Authors : Liyun Zheng , Wenjing yang , Minhua Wu , Xiaojie Zhang , Mengzhu Han , Mengyuan Wang , Qiaoyou Weng , … Show All … , Bufu Tang , Zhongwei Zhao , Minjiang Chen , Jianfei Tu , Jiansong Ji , and Shiji Fang [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.177487426.67212017/v1 127 views 88 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Objective : Cholangiocarcinoma (CCA) is a highly aggressive malignancy with limited therapeutic options and poor prognosis. Erianin has demonstrated broad-spectrum antitumor activity, yet its role in CCA and tumor immunity remains unclear. Methods : The cytotoxic and growth-inhibitory effects of erianin were evaluated in CCA cell lines and xenograft models. Apoptosis induction, mitochondrial dysfunction, and suppression of focal adhesion kinase (FAK) signaling were examined. The effects of erianin on cellular senescence, senescence-associated secretory phenotype (SASP) factor secretion, and senescent cell clearance were assessed. Pharmacological FAK inhibition was applied to validate the FAK-mediated cell growth and senolytic activity of erianin. The therapeutic efficacy of erianin alone or combined with anti-PD-L1 therapy was evaluated in senescent tumor-bearing mice. Results : Erianin exhibited significant cytotoxicity and induced mitochondrial apoptosis in CCA cells. Rather than inducing senescence, erianin eliminated senescent cells by promoting apoptosis and reduced SASP factor secretion. Erianin effectively inhibited FAK phosphorylation, and FAK blockade further enhanced its antiproliferative and senolytic activity. Moreover, erianin reprogrammed senescence-induced macrophage polarization from an M2- to an M1-like phenotype and increased antitumor immune cell infiltration within the tumor microenvironment. Importantly, erianin synergized with anti-PD-L1 therapy to achieve superior tumor control and prolonged survival in vivo . Conclusions : Erianin exerts potent antitumor effects in CCA by inhibiting FAK-mediated cell growth and senolytic activity, thereby enhancing antitumor immunity and immune checkpoint inhibitor efficacy. These findings identify erianin as a promising senolytic and immunotherapy adjuvant for CCA. Erianin Potentiates the Efficacy of Immune Checkpoint inhibitor in Cholangiocarcinoma by Inhibiting FAK-mediated cell growth and senolytic activity Liyun Zheng 1,2# , Wenjing yang 1# , Minhua Wu 1,2# , Xiaojie Zhang 1 , Mengzhu Han 1 , Mengyuan Wang 1 , Qiaoyou Weng 1 , Bufu Tang 3 , Zhongwei Zhao 1,2 , Minjiang Chen 1,2 , Jianfei Tu 1,2* , Jiansong Ji 1,2* , Shiji Fang 1,2* 1 Zhejiang Key Laboratory of Imaging and Interventional Medicine, the Fifth Affiliated Hospital of Wenzhou Medical University, Lishui 323000, China. 2 Cancer center, Lishui Central Hospital, the Fifth Affiliated Hospital of Wenzhou Medical University, Lishui 323000, China. 3 Department of Interventional Radiology, Zhongshan Hospital, Shanghai Institute of Medical Imaging, Shanghai Institution of Medical Imaging, Shanghai, National Clinical Research Center of Interventional Medicine, Fudan University, Shanghai 200032, China. Jianfei Tu, PhD, Zhejiang Key Laboratory of Imaging and Interventional Medicine, Fifth Affiliated Hospital of Wenzhou Medical University, No 289, KuoCang road, Lishui 323000, China. Tel.: +86 0578 2285011. E-mail: [email protected] Jiansong Ji, MD, PhD, Zhejiang Key Laboratory of Imaging and Interventional Medicine, Fifth Affiliated Hospital of Wenzhou Medical University, No 289, KuoCang road, Lishui 323000, China. Tel.: +86 0578 2285011. E-mail: [email protected] Shiji Fang, PhD, Zhejiang Key Laboratory of Imaging and Interventional Medicine, Fifth Affiliated Hospital of Wenzhou Medical University, No 289, KuoCang road, Lishui 323000, China. Tel.: +86 0578 2285011. E-mail: [email protected] # Liyun Zheng, Wenjing Yang and Minhua Wu equally contributed to this work. Abstract Objective : Cholangiocarcinoma (CCA) is a highly aggressive malignancy with limited therapeutic options and poor prognosis. Erianin has demonstrated broad-spectrum antitumor activity, yet its role in CCA and tumor immunity remains unclear. Methods : The cytotoxic and growth-inhibitory effects of erianin were evaluated in CCA cell lines and xenograft models. Apoptosis induction, mitochondrial dysfunction, and suppression of focal adhesion kinase (FAK) signaling were examined. The effects of erianin on cellular senescence, senescence-associated secretory phenotype (SASP) factor secretion, and senescent cell clearance were assessed. Pharmacological FAK inhibition was applied to validate the FAK-mediated cell growth and senolytic activity of erianin. The therapeutic efficacy of erianin alone or combined with anti-PD-L1 therapy was evaluated in senescent tumor-bearing mice. Results : Erianin exhibited significant cytotoxicity and induced mitochondrial apoptosis in CCA cells. Rather than inducing senescence, erianin eliminated senescent cells by promoting apoptosis and reduced SASP factor secretion. Erianin effectively inhibited FAK phosphorylation, and FAK blockade further enhanced its antiproliferative and senolytic activity. Moreover, erianin reprogrammed senescence-induced macrophage polarization from an M2- to an M1-like phenotype and increased antitumor immune cell infiltration within the tumor microenvironment. Importantly, erianin synergized with anti-PD-L1 therapy to achieve superior tumor control and prolonged survival in vivo . Conclusions : Erianin exerts potent antitumor effects in CCA by inhibiting FAK-mediated cell growth and senolytic activity, thereby enhancing antitumor immunity and immune checkpoint inhibitor efficacy. These findings identify erianin as a promising senolytic and immunotherapy adjuvant for CCA. Key words: Erianin; senescence; Focal adhesion kinase; immunotherapy; Cholangiocarcinoma. Introduction Cholangiocarcinoma (CCA) is a highly aggressive malignancy originating from the epithelial cells of the biliary tract and ranks as the second most common primary liver cancer 1 . The incidence of CCA has been steadily rising worldwide, and the prognosis remains poor. Surgical resection is the only curative treatment. However, the majority of patients present with unresectable at diagnosis. Although chemotherapy with gemcitabine and cisplatin (GEMCIS) has long been the standard of treatment, it provides only modest survival benefits. The recent success of the TOPAZ-1 2 and KEYNOTE-966 3 phase III trials established immune checkpoint inhibitors (ICIs) such as durvalumab and pembrolizumab, in combination with GEMCIS, as new first-line regimens for CCA. Nevertheless, the objective response rate remains low, and only a subset of patients achieves durable clinical benefit 4 . Accumulating evidence indicates that the tumor microenvironment (TME) of CCA is characterized by dense desmoplasia, extensive fibroblast activation, and infiltration of immunosuppressive immune cells, including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells, and regulatory T cells (Tregs) 4-5 . These components collectively form a physical and immunological barrier that hinders the infiltration and function of cytotoxic T lymphocytes (CTLs), thereby limiting the efficacy of immunotherapy 4-5 . Among various oncogenic pathways implicated in this process, focal adhesion kinase (FAK) signaling plays a pivotal role in promoting tumor growth, metastasis, and immune exclusion 6 . FAK, a non-receptor tyrosine kinase encoded by PTK2, mediates intracellular signaling downstream of integrins and growth factor receptors to regulate cell adhesion, cytoskeletal dynamics, proliferation, and survival 7 . Aberrant activation of FAK has been observed in multiple solid tumors, including intrahepatic CCA, where it correlates with advanced stage and poor prognosis 6 . Inhibition of FAK signaling disrupts tumor-stroma interactions, remodels the extracellular matrix, enhances CD8 + T-cell infiltration, and sensitizes tumors to ICIsin several preclinical models 8-10 . These findings position FAK as an attractive therapeutic target and a rational node for combination immunotherapy in CCA. Another key contributor to resistance of immunotherapy is unfavorable senescence 11 . Senescence is a state of stable cell cycle arrest triggered by various stressors such as DNA damage, oxidative stress, or oncogene activation. Although transient senescence acts as a tumor-suppressive mechanism, the long-term persistence of senescent cells leads to the secretion of a complex network of pro-inflammatory cytokines, chemokines, and proteases collectively termed the senescence-associated secretory phenotype (SASP) 12 . The SASP profoundly reshapes the TME by promoting angiogenesis, matrix remodeling, and the recruitment of immunosuppressive cells such as M2-like macrophages and Tregs, thereby fostering tumor progression and immune evasion 13 . In this context, senescent tumor not only contribute to therapeutic resistance but also establish an immunosuppressive niche 14 . Therefore, pharmacological agents capable of eliminating senescent cells or attenuating SASP signaling have emerged as promising candidates to restore immune surveillance and enhance immunotherapy responsiveness. Erianin is a natural bibenzyl compound extracted from the traditional Chinese medicinal herb Dendrobium chrysotoxum 15-16 . It has attracted increasing attention for its broad-spectrum anticancer properties. Previous studies have reported that erianin inhibits cell proliferation and induces apoptosis, cell cycle arrest, or ferroptosis in various cancers 17-24 . Mechanistically, erianin has been shown to modulate multiple oncogenic pathways such as ERK, PI3K/Akt, and STAT3, leading to the suppression of tumor growth and metastasis 16, 25 . Notably, previous reports have suggested that erianin can impair tumor cell adhesion and migration, hinting at a potential interaction with the FAK signaling axis 22, 26 . However, the precise role of erianin in CCA and its impact on the tumor microenvironment, particularly in relation to FAK-mediated signaling and senescence-associated immunosuppression, remain largely unexplored. Given the crucial role of FAK signaling in CCA progression and immune evasion, and the dual oncogenic and immunosuppressive functions of cellular senescence, we hypothesized that erianin may suppress CCA progression by inhibiting FAK-mediated pathways, eliminating senescent cells, and reprogramming the tumor immune microenvironment, thereby potentiating the efficacy of immune checkpoint blockade. To test this hypothesis, we systematically evaluated the cytotoxicity, apoptosis of CCA cells following erianin treatment, explored the involvement of the FAK signaling cascade, and assessed its effects on cellular senescence and SASP modulation. Furthermore, using both xenograft and senescent tumor models, we investigated the antitumor efficacy of erianin alone and in combination with anti-PD-L1 therapy in vivo . Collectively, this study uncovers a previously unrecognized function of erianin as a senolytic agent that not only inhibits tumor cell proliferation but also clears senescent cells and reshapes the immune landscape. Our findings highlight a novel combinational strategy of erianin plus ICIsfor the treatment of CCA, providing mechanistic insights and a translational basis for developing natural compound-based immune-sensitizing therapies. Material and methods Cell Lines and Reagents Human CCA cell lines TFK-1 (RRID: CVCL_2214) and RBE (RRID: CVCL_4896) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium (Gibco, Cat#11875-093, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Cat#10099-141, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Cat#15140-122, USA) at 37 °C in a humidified incubator containing 5% CO 2 . The murine cell line SB-1 cells were maintained in DMEM (high glucose) (Gibco, Cat#11965-092, USA) under the same conditions. All cell lines were authenticated by STR profiling and used within 15 passages to ensure phenotypic stability. erianin (≥98% purity) (MedChemExpress, Cat#HY-N0186, USA) was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Cat#D2650, USA) to prepare a 10 mM stock solution stored at −20 °C, and working solutions were freshly diluted in complete medium before each experiment. The FAK inhibitor IN10018 (MedChemExpress, Cat#HY-10018, USA), the pan-caspase inhibitor z-VAD-fmk (Selleck Chemicals, Cat#S7023, USA), and the mitochondrial membrane potential inhibitor SFA (MedChemExpress, Cat#HY-12345, USA) were used in subsequent experiments. Doxorubicin (Dox) was obtained from Sigma-Aldrich (Cat#D1515, USA). The anti-PD-L1 antibody was purchased from BioXcell (Cat#BE0101, USA). All reagents were of analytical or cell culture grade, and stock solutions were stored according to the manufacturers’ instructions. The Cell Counting Kit-8 (CCK-8) (Cat#C0038), EdU Cell Proliferation Kit (Cat#C0071L), Calcein/PI Cell Viability/Cytotoxicity Assay Kit(Cat# C2015S), Annexin V-FITC/PI Apoptosis Detection Kit (Cat#C1062L), JC-1 Mitochondrial Membrane Potential Assay Kit (Cat#C2006), SA-β-Gal Staining Kit (Cat#C0602), and RIPA Lysis Buffer (Cat#P0013B) were obtained from Beyotime (China). ELISA kits for IL-6 (Cat#DY206), IL-8 (Cat#DY208), and CCL2 (Cat#DY279) were purchased from R&D Systems (USA). The BCA Protein Assay Kit (Cat# PC0020), Rystal Violet (Cat#C8470) was obtained from Solarbio (Shanghai, China). Primary antibodies were obtained from Cell Signaling Technology (CST, USA), including Bax (Cat#5023), Bcl-2 (Cat#4223), Cleaved Caspase-3 (Cat#9661), p-FAK (Y397, Cat#8556), FAK (Cat#3285), p-Src (Y416, Cat#2101), Src (Cat#2109), p-Paxillin (Y118, Cat#2541), Paxillin (Cat#12065), p53 (Cat#2527), p21 (Cat#2947), and p16 (Cat#80772). For flow cytometry analyses, antibodies against CD45 (Cat#103106), CD3 (Cat#100236), CD4 (Cat#100406), CD8 (Cat#344702), NK1.1 (Cat#108702), F4/80 (Cat#123108), CD86 (Cat#305412), CD206 (Cat#321206), and Foxp3 (Cat#320102) were purchased from BioLegend (USA). For immunofluorescence analysis, antibodies against F4/80 (Cat#28463-1-AP), Ki67 (Cat#27309-1-AP), p21 (Cat#10355-1-AP), CD206 (Cat#60143-1-Ig), and CD86 (Cat#13395-1-AP) were purchased from Proteintech (USA). Fluorescent secondary antibodies, including Alexa Fluor 488, Alexa Fluor 594, and Alexa Fluor 647conjugated antibodies, were also obtained from Proteintech (USA). Cell Viability and Cytotoxicity Assay Cell viability was assessed using the Cell Counting Kit-8 according to the manufacturer’s protocol. TFK-1 and RBE cells were seeded in 96-well plates at 5 × 10 3 cells/well and treated with different concentrations of erianin. Absorbance at 450 nm was measured using a microplate reader. The IC 50 values were calculated using GraphPad Prism 8.0. Morphological changes were observed under an inverted microscope. Colony Formation and EdU Incorporation Assay For colony formation, cells were plated in 6-well plates and treated with erianin for 10-14 days, followed by fixation in 4% paraformaldehyde and staining with 0.5% crystal violet. Colonies were counted under a microscope. Cell proliferation was analyzed using an EdU Cell Proliferation Kit. After 24 h of erianin exposure, cells were incubated with 50 μM EdU for 3 h, fixed, and counterstained with DAPI. Fluorescence images were captured using a microscope. Apoptosis and Mitochondrial Membrane Potential (MMP) Analysis Apoptosis was quantified using the Annexin V-FITC/PI Apoptosis Detection Kit following the manufacturer’s protocol. The mitochondrial membrane potential (MMP) was assessed with the JC-1 Assay Kit. Flow cytometry was employed to determine the proportion of Annexin V-positive apoptotic cells and to analyze the JC-1 monomer fluorescence shift, which indicates mitochondrial depolarization. Western blotting analysis Total cellular proteins were extracted using RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using the BCA Protein Assay Kit. Equal amounts of protein (20 μg) were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% nonfat milk, membranes were incubated overnight at 4 °C with primary antibodies against Bax, Bcl-2, cleaved caspase-3, p-FAK, total FAK, p-Src, Src, p-Paxillin, Paxillin, E-cadherin, N-cadherin, MMP2, MMP3, p53, p21, p16, cleaved PARP1, and GAPDH. HRP-conjugated secondary antibodies were used, and signals were visualized with enhanced chemiluminescence. Bioinformatic and Target Prediction Analysis Potential molecular targets of erianin were predicted using the SwissTargetPrediction database (http://www.swisstargetprediction.ch/). The identified targets were further subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using the Sangerbox online platform (http://sangerbox.com/). Pathways related to cellular senescence and focal adhesion were prioritized for subsequent experimental validation. Senescence Detection and SASP Analysis Senescence-associated β-galactosidase (SA-β-Gal) activity was detected using a Senescence β-Gal Staining Ki following the manufacturer’s instructions. For senescence induction, cells were treated with doxorubicin (0.5 μM, 72 h). SASP cytokine levels (IL-6, IL-8, CCL2) in the culture supernatant were measured by ELISA. Co-Culture of Senescent Cells and Macrophages To evaluate macrophage polarization, senescent or erianin-treated CCA cells were co-cultured with THP-1-derived macrophages using a Transwell system. After 48 h, macrophages were analyzed by flow cytometry for CD86 (M1 marker) and CD206 (M2 marker) expression. mRNA levels (Arg-1, IL-10, TGFβ, iNOS, TNFα, IL-112) were determined by RT-PCR, and macrophage phenotype was further confirmed by immunofluorescence. For mRNA quantification, total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions, and reverse transcription was performed using the PrimeScript RT Reagent Kit. The relative mRNA expression levels of Arg-1, IL-10, TGF-β, iNOS, TNF-α, and IL-12 were normalized to GAPDH using the 2 ⁻ΔΔCt method. Animal Experiments All animal procedures were approved by the Institutional Animal Care and Use Committee of the fifth affiliated hospital of Wenzhou medical University. For subcutaneous xenografts, 5 × 10 6 TFK-1 cells were injected into the right flank of 6-week-old BALB/c nude mice. Mice were randomized into three groups and treated intraperitoneally with vehicle, erianin (50 or 100 mg/kg), three times. For senescent tumor models, senescence was induced by intraperitoneal doxorubicin (100 mg/kg). Non-senescent tumor bearing mice were set as control. To evaluate the immune response and therapeutic efficacy, senescent SB-1 tumor-bearing C57BL/6 mice were established using the same senescence induction protocol as described above. Mice were then randomly assigned into four groups and treated with vehicle, erianin, anti-PD-L1 antibody (2 mg/kg; BioXCell, USA), or their combination. Non-senescent tumor bearing mice were set as control. Tumor volume and survival were monitored throughout the experiment. the formula for Tumor volume is V = (length × width 2 )/2. Tumors were harvested for Ki67, TUNEL, and immunohistochemical staining. Histological, Immunohistochemical, and TUNEL Staining Paraffin-embedded tissue sections (4 μm) were deparaffinized and subjected to antigen retrieval. After blocking, sections were incubated with primary antibodies against Ki67, p21, F4/80，CD86, and CD206 at 4 °C overnight. After washing, appropriate Alexa Fluor-conjugated secondary antibodies were applied. Nuclei were counterstained with DAPI and visualized. For immunohistochemistry (IHC), sections were incubated with HRP-labeled secondary antibodies, developed with a DAB substrate, counterstained with hematoxylin, dehydrated, and mounted for microscopic examination. For TUNEL staining, paraffin-embedded tumor sections were deparaffinized, rehydrated, and incubated with proteinase K for 20 minutes at room temperature. After PBS washes, sections were treated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT) and fluorescein-labeled dUTP at 37 °C for 1 hour in the dark. Nuclei were counterstained with DAPI, and apoptotic cells were visualized and imaged using a fluorescence microscope. Statistical Analysis Data were presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 8.0. Differences between two groups were assessed by Student’s t-test, while multiple comparisons were analyzed by one-way ANOVA followed by Tukey’s post hoc test. A p -value < 0.05 was considered statistically significant. Cytotoxicity of erianin in CCA cells Two human CCA cell lines (TFK-1 and RBE) were selected to tested the cytotoxicity of erianin. The IC 50 of erianin in TFK-1 and RBE cells were 0.72±0.19 μg/mL and 0.69±0.16 μg/mL (Figure1 A) . Upon treatment with eriannin at concentrations of 0.25, 0.5, and 1 μg/mL, both cell lines exhibited progressive morphological changes, including cell rounding, shrinkage, and detachment in clusters. Higher concentrations resulted in extensive cell detachment, loss of adherence, and marked reduction in cell density, leaving only sparse, floating cell debris in the culture medium (Figure1 B) . Live/dead staining showed that erianin induced cell death in TFK-1 cells at 0.25, 0.5, and 1.0 μg/mL with death rates of 18.5 ± 2.1%, 41.1 ± 1.7%, and 79.4 ± 2.3%, and in RBE cells with death rates of 20.7 ± 1.9%, 53.2 ± 2.7%, and 84.1 ± 3.1% (Figure1 C, D) . These observations indicate that erianin exerts cytotoxic effects on CCA cells, consistent with the CCK-8 assay results. To test the proliferative inhibition of erianin, EdU staining and clonogenic assays were performed. EdU staining showed that erianin reduced the proportion of EdU-positive cells in both TFK-1 and RBE cells, with percentages decreasing from 47.17±5.59% and 50.89 ± 3.53% at 0 μg/mL to 8.28 ±0.76% and 8.87 ± 1.27% at 1.0 μg/mL, respectively. (Figure1 E, F) . Consistently, clonogenic assays confirmed that erianin strongly suppressed colony formation in TFK-1 and RBE cells (Figure S1) . These observations suggest that erianin suppresses cell proliferation. Figure 1. erianin exerts cytotoxicity and antiproliferation in CCA cells. (A) Cell viability of TFK-1 and RBE cells treated with increasing concentrations of erianin, as determined by CCK-8 assay. (B) Morphological changes of TFK-1 and RBE cells after treatment with 0, 0.25, 0.5, and 1.0 μg/mL erianin. (C) Live/dead staining showing erianin-induced cell death in TFK-1 and RBE cells. Live cells are labeled in green, and dead cells are labeled in red. (D) Quantification of dead cell percentages in TFK-1 and RBE cells treated with 0, 0.25, 0.5, and 1.0 μg/mL erianin. (E) Quantification of EdU-positive cells with erianin treatment. (F) EdU incorporation assay showing inhibition of DNA synthesis by erianin in TFK-1 and RBE cells. EdU-positive nuclei are shown in red, and Hoechst-stained nuclei are shown in blue. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with control. Erianin induced apoptosis and mitochondrial damages in CCA cells Flow cytometric analysis showed that erianin increased the proportion of Annexin V-positive cells. The percentage of Annexin V-positive cells increased from 6.10 ± 1.51% to 44.32 ± 2.17% in TFK-1 and from 7.10 ± 1.63% to 46.97 ± 7.09% in RBE cells at 0, 0.25, 0.5, and 1.0 μg/mL, respectively (Figure2 A, B) . Western blot analysis showed that erianin treatment increased the expression of pro-apoptotic proteins Bax and cleaved caspase-3 while decreasing the expression of the anti-apoptotic protein Bcl-2 (Figure2 C) . Furthermore, erianin treatment at 0.25, 0.5, and 1.0 μg/mL significantly decreased mitochondrial membrane potential (MMP) in TFK-1 and RBE cells, as evidenced by an increase in JC-1 monomers (Figure2 D, E) . These results indicate that erianin-induced apoptosis may be mediated by mitochondrial damage, a hallmark of the canonical intrinsic apoptotic pathway 27 . Importantly, pretreatment with the pan-caspase inhibitor zVAD-fmk or the MMP inhibitor SFA partially alleviated erianin-induced cytotoxicity and increased cell viability compared with erianin alone (Figure2 F) . These findings indicate that erianin-induced proliferation inhibition is partially dependent on apoptosis. Figure 2. erianin induces apoptosis and mitochondrial damage in CCA cells. (A) Flow cytometric analysis of Annexin V-FITC/PI staining showing dose-dependent induction of apoptosis in TFK-1 and RBE cells after treatment with 0, 0.25, 0.5, and 1.0 μg/mL erianin for 24 h. (B) Quantification of Annexin V-positive cells. Data represent mean ± SD (n = 3). (C) Western blot analysis showing increased expression of pro-apoptotic proteins Bax and cleaved caspase-3, and decreased expression of the anti-apoptotic protein Bcl-2 in TFK-1 and RBE cells treated with erianin. (D) Flow cytometric analysis of mitochondrial membrane potential (MMP) using JC-1 staining. The proportion of JC-1 monomers increased with higher concentrations of erianin, indicating mitochondrial depolarization. (E) Quantification of JC-1 monomer/aggregate ratios in cells treated with erianin. (F) Pretreatment with the pan-caspase inhibitor z-VAD-fmk (20 μM) or mitochondrial membrane potential inhibitor SFA (10 μM) partially alleviated erianin-induced cytotoxicity, as determined by CCK-8 assay. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control. Erianin inhibited the growth of CCA in vivo. To evaluate the anti-tumor efficacy of erianin in vivo , a subcutaneous TFK-1 xenograft model was established in nude mice. The mice were randomly assigned to three groups and treated with 0, 50, or 100 mg/kg of erianin, and tumor volumes were monitored over time. As shown in Figure 3A, B tumors from erianin-treated mice exhibited significantly smaller volumes compared with controls at day 21 post-treatment. Compared with the control group, tumor volumes were reduced approximately 2.14 and 4.59 times in mice treated with 50 and 100 mg/kg erianin, respectively (Figure3 C) . Similarly, the tumor weights were markedly decreased in a dose-dependent manner, consistent with the observed reduction in tumor volume (Figure3 D) . Importantly, the body weight of mice remained stable throughout the experiment (Figure3 E) , indicating that erianin treatment was well tolerated. IHC analysis showed that the proportion of Ki67-positive cells within tumors decreased to 34.10 ± 1.64% and 14.28 ± 6.81% in mice treated with 50 mg/kg and 100 mg/kg erianin, respectively, which was markedly lower than in the control group (Figure3 F, G) . Consistently, the percentage of TUNEL-positive cells increased to 18.28 ± 2.58% and 33.47 ± 4.69% in the 50 mg/kg and 100 mg/kg groups (Figure3 H, I) , indicating that erianin effectively suppressed proliferation and induced apoptosis in vivo . Figure 3. erianin inhibits tumor growth and induces apoptosis in vivo . (A) Subcutaneous TFK-1 xenograft-bearing nude mice treated with 0 mg/kg, 50 mg/kg, or 100 mg/kg of erianin for 21 days. (B) Tumors from each treatment group at the endpoint. (C) Tumor growth curves of each group. (D) Tumor weights of each group. (E) Body weights of mice during the treatment period, showing no significant toxicity. (F) Representative immunohistochemical staining for Ki67 in tumor tissues. (G) Quantification of Ki67-positive cells. (H) TUNEL staining of tumor tissues showing increased apoptotic cells in erianin-treated groups. Nuclei were counterstained with DAPI (blue), and TUNEL-positive cells appear green. (I) Quantification of TUNEL-positive cells. *P < 0.05, **P < 0.01, ***P < 0.001 versus control. Erianin eliminated the senescent cells in CCA cells Next, to investigate the effects of erianin, the potential molecular targets predicted by SwissTargetPrediction (http://www.swisstargetprediction.ch/) were further analyzed. A total of 105 potential targets were identified. Among them, kinases, cytochrome P450s, enzymes, and oxidoreductases each represented 13.3% of the total. Family A G protein-coupled receptors, primary active transporters, structural proteins, nuclear receptors, and secreted proteins each accounted for 6.7%, with the remaining 6.7% belonging to unclassified proteins (Figure S2 A) . KEGG pathway analysis indicated that the identified targets were mainly enriched in several key pathways, including pathways in cancer, focal adhesion, cellular senescence, Rap1 signaling pathway, cAMP signaling pathway, and chemokine signaling pathway, among others (Figure4 A) . Gene Ontology (GO) enrichment analysis revealed that the identified targets were primarily involved in biological processes related to regulation of cell death, phosphate-containing compound metabolic process, protein phosphorylation, regulation of intracellular signal transduction. In the cellular component category, these targets were mainly associated with the cytosol, plasma membrane part, cytoplasmic vesicle, synapse part, plasma membrane region. For molecular function, they were enriched in catalytic activity, small molecule binding, drug binding, nucleotide binding, nucleoside phosphate binding (Figure S2 B-D) . These findings suggest that the identified targets are closely associated with cell survival, signal transduction, and tumor-related biological processes. Cellular senescence is a process of cell cycle arrest triggered by various forms of cellular stress, including DNA damage, telomere shortening, oxidative stress, and oncogenic signaling. Therapeutic agents capable of inducing or clearing senescent cells have shown promising anti-tumor potential across multiple cancer types, owing to the paradoxical roles of senescence in both tumor suppression and promotion 11, 14 . Thus, we sought to investigate the effect of erianin on cellular senescence in CCA cells. To this end, TFK-1 and RBE cells were treated with a low dose of erianin for 5 days. β-Gal staining showed no significant increase in the number of β-Gal-positive cells (Figure S3 A) , indicating that erianin did not induce cellular senescence under these conditions. Moreover, erianin-treated cells exhibited a rounded morphology and did not display the enlarged and flattened shape typical of senescent cells (Figure S3 A) . Western blot analysis indicated that erianin failed to increase the expression of the senescence-related proteins p53, p21, and p16. Conversely, erianin treatment led to a sharp decrease in the levels of p53, p21, and p16 (Figure S3 B) . These findings suggest that erianin may not promote cellular senescence, but rather eliminate or suppress senescent cells. As expected, in the Dox induced senescent model, erianin treatment sharply decrease the percentage of the β-Gal-positive cells (Figure4 B, C) . Consistently, immunofluorescence (IF) analysis indicated that the number of Ki67-positive cells decreased in senescent cells and was further reduced in erianin-treated senescent cells (Figure4 D, E) . Western blot analysis confirmed that the expression levels of p53, p21, and p16 were significantly decreased following erianin treatment, further confirming that erianin effectively attenuated cellular senescence (Figure4 F) . In addition, erianin treatment markedly reduced the expression of SASP factors, such as IL-6, IL-8, and CCL2 (Figure4 G) , suggesting that erianin alleviated the pro-inflammatory microenvironment associated with cellular senescence. It has been reported that senescent cells are highly resistant to apoptosis 28-29 . To determine whether erianin could overcome this resistance, we next investigated whether erianin treatment induces apoptosis in senescent cells. Our results demonstrated that the proportion of Annexin V-positive cells in erianin-treated senescent cells increased to 16.84 ± 1.61% in TFK-1 and 20.75 ± 1.71% in RBE cells (Figure S4 A, B) . Consistently, Western blot analysis revealed that the expression levels of cleaved PARP1 and cleaved caspase-3 were significantly elevated in erianin-treated senescent cells (Figure S4 C) , indicating the activation of apoptosis. To validate the senescence-eliminating capacity of erianin in vivo , a senescent tumor model was established by intraperitoneal (i.p.) administration of Dox (10 mg/kg) every two days for three cycles 30 . Mice bearing senescent or non-senescent tumors were randomly assigned to three groups: control (non-senescent), Sen, and Sen + erianin (Figure4 H) . Compared with the control group, the tumor volume in the Sen group showed a slight decrease, whereas a sharp reduction was observed in the Sen + erianin group. The average tumor volume in the Sen + erianin group was nearly 2.40 times smaller than that in the Sen group (Figure4 I) . The tumor weight also exhibited a similar trend, showing a slight decrease in the Sen group and a pronounced reduction in the Sen + erianin group (Figure4 J) . The tumor weight was reduced by more than twofold in the Sen + erianin group compared with the Sen group. Notably, mice in the Sen + erianin group exhibited the longest overall survival (Figure4 K) , suggesting that erianin treatment significantly prolonged survival in the senescent tumor model. Consistently, immunohistochemical analysis revealed that the expression of Ki67 (Figure4 L, N) and p21 (Figure4 M, O) was markedly decreased in the Sen + erianin group relative to the Sen group. Collectively, these findings indicate that erianin acts as a senescence eliminator, effectively reducing senescent cell burden and improving tumor outcomes. Figure 4. Erianin eliminates senescent cells and suppresses senescence-associated tumor growth. (A) KEGG pathway enrichment analysis of potential erianin targets predicted by SwissTargetPrediction. (B) SA-β-Gal staining of senescent cells treated with erianin. Non-senescent cells were set as control. (C) Quantification of β-Gal staining in senescent cells treated with or without erianin treatment. (D) Immunofluorescence staining of non-senescent cells and senescent cells. (E) Ki67 staining of non-senescent cells and senescent cells. Nuclei were counterstained with DAPI. (F) Western blot analysis of p53, p21, and p16 expression in erianin treated senescent cells. (G) ELISA assays of IL-6, IL-8, and CCL2. (H) Schematic illustration of the Dox-induced senescent tumor model. (I, J) Tumor growth curves and final tumor weights. (K) Kaplan-Meier survival analysis indicating prolonged survival in erianin-treated mice. (L, M) Quantification of Ki67-positive and p21-positive cells in tumor tissues from different groups. (N, O) Representative immunohistochemical (Ki67) and immunofluorescence (p21) staining of the proliferation and senescence markers in the erianin-treated senescent tumor. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control or senescent group. FAK signaling mediates the antiproliferative and senolytic effects of erianin Focal adhesion is a critical signaling pathway that regulates the malignant biological behaviors of tumor cells, including proliferation, migration, and survival 7 . FAK is aberrantly upregulated in CCA and associated with tumor initiation, progression, and therapeutic resistance 6 . In addition, dysregulation of the FAK signaling pathway has been implicated in cellular senescence 31-32 . Thus, we hypothesize that erianin induces proliferation and migration inhibition and eliminates senescent cells, at least in part, by modulating the FAK signaling pathway. Indeed, erianin treatment resulted in a marked reduction in the phosphorylation of FAK, Src, and paxillin, while their total protein expression remained unchanged (Figure5 A) . IF staining confirmed that the fluorescence intensity of p-FAK was markedly decreased in erianin-treated cells (Figure5 B) . In addition, F-actin staining revealed a disrupted cytoskeletal organization and reduced stress fiber formation in erianin-treated cells, further suggesting suppression of focal adhesion signaling (Figure5 B) . Importantly, treatment with IN10018, a selective FAK inhibitor, partially reversed the erianin-induced inhibition of cell proliferation (Figure5 C, D) , as indicated by the elevated number of EdU-positive cells in the IN10018 + erianin group compared with the erianin group. Furthermore, Western blot analysis confirmed that the phosphorylation levels of FAK and Src were upregulated in senescent cells compared with non-senescent cells. In contrast, treatment with erianin or IN10018 significantly reduced the elevated phosphorylation of FAK and Src in non-senescent cells (Figure5 E) . β-Gal staining confirmed that treatment with either erianin or IN10018 significantly decreased the proportion of β-Gal-positive cells. Notably, their combination further decreased the percentage of β-Gal-positive cells compared with either treatment alone (Figure5 F, G) . Consistently, the release of IL-6, IL-8, and CCL2 from senescent cells was attenuated by treatment with erianin or IN10018, and this effect was further enhanced by their combination (Figure5 H) . Collectively, these findings indicate that FAK signaling mediates the inhibitory effects of erianin on proliferation and contributes to its senescence-eliminating activity. Figure 5. FAK signaling mediates the antiproliferative and senolytic effects of erianin. (A) Western blot analysis showing that erianin treatment decreased phosphorylation of FAK, Src, and paxillin in CCA cells. (B) Immunofluorescence staining of p-FAK and F-actin in erianin-treated cells. Nuclei were counterstained with DAPI (blue). (C) EdU staining showing that pharmacological inhibition of FAK with IN10018 enhanced erianin-induced antiproliferative effects, as reflected by a further decrease in EdU-positive cells. (D) Quantification of EdU-positive cells in each group. (E) Western blot analysis of the phosphorylation of FAK and Src in senescent cells. Treatment with erianin or IN10018 markedly reduced the phosphorylation of FAK and Src. C: control, E: Erianin, FAKi: IN10018. (F, G) SA-β-Gal staining and quantification of senescent cells with indicated treatments. (H) ELISA assays of IL-6, IL-8, and CCL2. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus control or as indicated. Erianin reprograms senescence-induced M2-like polarization While senescent cells lose their proliferative capacity, they remain metabolically active and continuously secrete various mediators, such as SASP factors, which can markedly remodel TME and modulate immune responses 11, 14 . Macrophages are the predominant immune cells within TME, and are usually polarized into an M2-like phenotype that promotes tumor progression and immune suppression 33 . SASP factors from senescent cells have been demonstrated to promote macrophage recruitment into TME and drive their polarization toward an M2-like phenotype 34 . Therefore, we next examined whether erianin could reprograms senescence-induced macrophages polarization within the TME. Immunofluorescence staining revealed increased F4/80 fluorescence intensities in the senescent TME, indicating enhanced macrophage infiltration (Figure6 A, B) . While CD86 showed a slight elevation, CD206 expression increased sharply, and the magnitude of change was substantially greater for CD206, indicating a predominant M2-like polarization (Figure6 A, B) . Importantly, erianin treatment led to a decrease in CD206 expression but an increase in CD86 expression (Figure6 C, D) , demonstrating a shift toward M1-like macrophage polarization within TME. To further validate these findings, an in vitro co-culture system was established (Figure6 E) . After 48 hours of co-culture, flow cytometric analysis revealed that erianin-treated senescent TFK-1 and RBE cells increased the proportion of CD86⁺ THP-1 macrophages to 43.08 ± 3.22% and 46.33 ± 2.10%, respectively, which were 2.12-fold and 2.05-fold higher than those observed in macrophages co-cultured with senescent cells alone (Figure6 F, H) . In contrast, the percentage of CD206⁺ macrophages decreased markedly, from 72.86 ± 6.66% to 34.45 ± 3.74% and from 69.68 ± 4.98% to 33.03 ± 2.13% in the TFK-1 or RBE co-culture system (Figure6 G, I) , indicating that erianin reversed the M2-dominant phenotype. Immunofluorescence analysis further confirmed elevated CD86 and reduced CD206 fluorescence in THP-1 macrophages co-cultured with erianin-treated senescent cells (Figure6 J) . The release levels of M1 markers, including IL1β, IL6, and TNFα, were markedly upregulated in THP-1 macrophages co-cultured with erianin-treated senescent TFK-1 and RBE cells (Figure6 K, L) . In contrast, M2-related genes, such as Arg1, IL10, and CD163, were significantly downregulated following erianin treatment (Figure6 K, L) . These results indicate that erianin reshapes the SASP profile of senescent cells, thereby promoting M1-like macrophage polarization in the senescent TME. Interestingly, we also found that similar with erianin, IN10018 treatment significantly reduced the percentage of CD206⁺ cells, and this effect was further strengthened when combined with IN10018. In contrast, the percentage of CD86⁺ macrophages increased in THP-1 cells co-cultured with IN10018-treated senescent cells, and further rose upon combined treatment, indicating a synergistic promotion of M1-like polarization (Figure 5S) . These results demonstrate that the FAK signaling pathway mediates the effect of erianin in reversing senescence-induced M2-like polarization. Figure 6. Erianin reprograms senescence-induced macrophage polarization. (A, B) Immunofluorescence staining of F4/80, CD86, and CD206 in non-senescent and senescent tumor tissues. (C, D) Quantification of F4/80 + CD86 + (M1-like) and F4/80 + CD206 + (M2-like) macrophages in tumors. (E) Schematic illustration of the co-culture system established between Dox-induced senescent tumor cells and THP-1 macrophages. (F, H) Flow cytometric analysis and quantification of CD86 + M1-like THP-1 macrophages in the co-culture system under the indicated treatments. (G, I) Flow cytometric analysis and quantification of CD206 + M2-like THP-1 macrophages in the co-culture system. (J) Immunofluorescence staining of CD86 and CD206 in THP-1 macrophages showing erianin-induced M1 polarization. (K, L) RT-PCR analysis of M1-associated (iNOS, TNF-α, IL-12) and M2-associated (Arg-1, IL-10, TGF-β) marker gene expression in THP-1 cells following the indicated treatments. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus control or as indicated. Erianin enhanced the anti-tumor efficacy of anti-PD-L1 within senescent TME. Having demonstrated that erianin effectively eliminates senescent cells and remodels their associated secretory phenotype to modulate macrophage function, we hypothesized that it might also impact other immune cell populations and thereby influence the efficacy of immunotherapy. To this end, SB-1 tumor-bearing C57BL/6 mice were employed (Figure7 A) . After successful tumor establishment, mice were randomly assigned into the following groups: control, Sen, Sen+erianin, Sen+PD-L1, and Sen+erianin+aPD-L1. As anticipated, the Sen+erianin+aPD-L1 group exhibited the best tumor control compared with the other groups (Figure7 B) . Compared with the control group, the Sen group exhibited slight decrease tumor growth, consistent with the tumor-control effects of senescence. In the senescent tumor model, erianin treatment markedly suppressed tumor progression, whereas aPD-L1 monotherapy produced a moderate inhibitory effect. Strikingly, the combination of erianin and aPD-L1 resulted in the strongest antitumor efficacy, as evidenced by the smallest tumor volume and weight among all groups. Quantitative analysis showed that tumor volume in the Sen+erianin+aPD-L1 group was reduced by more than 85% compared with the Sen group at the endpoint (Figure7 C) . Consistently, tumor weight exhibited a similar trend to tumor volume. Compared with the Sen group, tumor weight was reduced by 2.23-, 1.42-, and 5.83-fold in the Sen+erianin, Sen+aPD-L1, and Sen+erianin+aPD-L1 groups, respectively. Notably, the combination of erianin and aPD-L1 achieved a markedly stronger tumor inhibitory effect than either treatment alone in the senescent tumor model (Figure7 D) . Furthermore, survival analysis demonstrated that mice in the Sen+erianin+aPD-L1 group exhibited the longest median overall survival compared with all other groups (Figure7 E) . Collectively, these findings demonstrate that erianin not only suppresses senescence-associated tumor progression but also synergizes with ICIs to enhance antitumor efficacy in vivo . Histological analysis revealed a progressive reduction in Ki67-positive cells across treatment groups (Figure7 F). The percentage of Ki67-positive cells was decreased in the senescent tumor model and further reduced in the Sen+erianin and Sen+erianin+aPD-L1 groups. Quantitative analysis confirmed that the Sen+erianin+aPD-L1 group exhibited the lowest proportion of Ki67-positive cells among all groups (Figure7 G) , indicating a pronounced suppression of tumor cell proliferation. Consistently, TUNEL staining demonstrated a marked increase in apoptotic cells following treatment (Figure7 H) . The proportion of TUNEL-positive cells was markedly higher in the Sen+erianin and Sen+erianin+aPD-L1 groups compared with the Sen group, with the highest level observed in the Sen + erianin + aPD-L1 group, suggesting enhanced apoptosis induction. Moreover, immunofluorescence analysis showed that p21 expression was strongly upregulated in the Sen group, consistent with senescence activation. In contrast, erianin treatment markedly reduced p21 fluorescence intensity, and this decrease was further enhanced when combined with aPD-L1 in the senescent tumor model (Figure7 I) . Collectively, these findings indicate that erianin, particularly in combination with aPD-L1, suppresses tumor proliferation, promotes apoptosis, and attenuates senescence in vivo. To further investigate the infiltration of anti-tumor related immune cells within the TME, flow cytometric analysis was performed using tumor tissues. Compared with the control group, the Sen group exhibited higher levels of CD86 positive cells, and further increases were observed in the Sen+erianin and Sen+aPD-L1 groups. The highest infiltration of CD86 positive cells was detected in the Sen+erianin+aPD-L1 group (Figure7 J, S6) . Conversely, the Sen group displayed the higher proportion of CD206⁺ cells than the control group, indicating abundant M2-like macrophages with immunosuppressive properties in senescent tumor model. Treatment with erianin or aPD-L1 alone significantly reduced CD206 expression, while the combination of erianin and aPD-L1 led to the lowest percentage of CD206⁺ cells among all groups (Figure7 K, S6) . Furthermore, compared with the control group, the Sen group exhibited a slight rise, while further increases were observed in the Sen+erianin and Sen+aPD-L1 groups (Figure7 L, S6) . Notably, the Sen+erianin+aPD-L1 group showed the highest proportion of CD8⁺ cells, significantly higher than all other groups. compared with the Sen group, the percentage of the Treg cells decreases were observed in the Sen + erianin and Sen+aPD-L1 groups. Strikingly, the Sen+erianin+aPD-L1 group displayed the lowest proportion of Treg cells, which was significantly reduced compared with all other groups (Figure7 M, S6) . The control group displayed the low NK1.1⁺ cell level, while the Sen group showed a slight increase. Further elevation was observed in the Sen+erianin and Sen+aPD-L1 groups. Notably, the Sen+erianin+aPD-L1 group exhibited the highest proportion of NK1.1⁺ cells, significantly higher than all other groups (Figure S7) . Importantly, RT-PCR analysis (Figure7 N) revealed that aPD-L1 or erianin, as well as their combination, modulated the expression levels of immune-related genes. Compared with the control, the Sen group showed increased expression of M2-like phenotype markers Arg1 and TGF-β, but a decrease in the expression of iNOS and IL-12, which are associated with M1-like macrophages. In addition, the expression of anti-tumor effector genes, including TNF-α, IFN-γ, and GZMB, was markedly reduced, indicating an immunosuppressive tumor microenvironment induced by senescence. Treatment with erianin or aPD-L1 reversed these changes to varying degrees, restoring iNOS, IL-12, TNF-α, IFN-γ, and GZMB expression while suppressing Arg1 and TGF-β levels. Notably, the combined treatment of erianin and aPD-L1 achieved the most pronounced immunomodulatory effect. These findings indicate that erianin and aPD-L1 act synergistically to reprogram the TME from an immunosuppressive to an immunostimulatory state in the senescent tumor model. Figure 7. erianin synergizes with anti-PD-L1 therapy to enhance antitumor efficacy in senescent tumor-bearing mice. (A) Schematic illustration of the treatment schedule in Dox-induced senescent SB-1 tumor-bearing mice. Mice received erianin (50 mg/kg, intraperitoneally, i.p.) three times per week and/or anti–PD-L1 antibody (2 mg/kg, intravenously, i.v.) once per week. (B) Representative images of tumors excised from each treatment group at the study endpoint. (C) Tumor growth curves showing that erianin or anti-PD-L1 monotherapy moderately suppressed tumor growth, whereas combination therapy achieved superior tumor control. (D) Final tumor weights of each group. (E) Kaplan-Meier survival curves indicating prolonged survival in mice receiving the combination of erianin and anti-PD-L1 antibody compared with monotherapy. (F) Representative immunohistochemical and immunofluorescence staining of Ki67, TUNEL, and p21 in tumor sections. (G-I) Quantification of Ki67-positive cells, TUNEL-positive apoptotic cells, and p21 expression levels in tumor tissues. (J, K) Quantification of tumor-infiltrating macrophages, including F4/80 + CD86 + (M1-like) and F4/80 + CD206 + (M2-like) populations, in each treatment group. (L) Quantification of macrophage-associated cytokines (IL-6, TNF-α, IFN-γ, Arg-1, TGF-β, and IL-12) in tumor tissues. (M, N) Quantification of tumor-infiltrating CD8 + T cells and regulatory T cells (Tregs) showing enhanced cytotoxic T-cell infiltration and reduced immunosuppressive Tregs following combination treatment. *P < 0.05, **P < 0.01, ***P < 0.001 versus control or as indicated. Discussion Despite the recent introduction of ICIs into clinical practice, most patients obtained limited benefit due to the profoundly immunosuppressive TME and tumor-intrinsic resistance mechanisms 4-5 . The present study provides evidence that erianin, a natural bibenzyl compound isolated from Dendrobium chrysotoxum 15 , exerts potent antitumor activity in CCA through inhibition of FAK-mediated proliferation, elimination of senescent cells, and reprogramming of the TME to enhance the efficacy of ICIs. Our findings integrate and extend previous knowledge of both erianin’s biological functions and the pathogenic role of FAK signaling and cellular senescence in CCA. Erianin effectively induce various cell death pathways such as apoptosis, cell cycle arrest, and ferroptosis 35 . For instance, erianin triggered ferroptotic cell death characterized by accelerated GPX4 ubiquitination and degradation in colorectal cancer 36 . In osteosarcoma, erianin promotes G2/M-phase arrest and induces apoptosis and autophagy through ROS/JNK pathway activation 18 . Consistent with previous studies 37-38 , our study in CCA revealed that erianin primarily induces mitochondrial-dependent apoptosis. Importantly, its cytotoxicity was partially rescued by the pan-caspase inhibitor zVAD-fmk and the mitochondrial protector SFA, indicating that apoptosis predominates in CCA. Online target prediction suggested that erianin might be involved in the regulation of cellular senescence. Our study reveals that erianin does not induce senescence in CCA cells at low doses but rather eliminates pre-existing senescent cells. Erianin markedly reduced SA-β-gal-positive cells, downregulated p53, p21, and p16, and diminished SASP factor expression in senescent model, both in vitro and i n vivo . These features are characteristic of senolytic rather than senogenic activity. It well known that senolytic drugs, like navitoclax (BCL-2 inhibitor) 39-40 or dasatinib /quercetin (D+Q) 41 have been shown to potentiate cancer therapy by reshaping the TME. Therefore, erianin may serve as a potential adjuvant therapy to complement senescence-inducing treatment strategies by clearing accumulated senescent cells. Overactivation of the FAK-Src axis has been implicated in multiple malignancies, including CCA 6, 8 . FAK activation correlates with aggressive histological subtypes and poor prognosis in intrahepatic CCA. Pharmacological inhibition of FAK using defactinib or IN10018 markedly suppresses CCA cell growth and metastasis and enhances the efficacy of CDK4/6 inhibitors and immune therapy in preclinical models 6 . Our results align with and extend these findings. Notably, pharmacologic blockade of FAK using IN10018 recapitulated these phenotypes and synergized with erianin to further suppress proliferation. Moreover, inhibition of FAK with IN10018 phenocopied this senolytic effect, and combination treatment enhanced clearance of senescent cells and reduced SASP cytokines. Thus, we propose that erianin acts as a senolytic, adding a new dimension to the pharmacological landscape of senescence modulation. Compared with classical senolytics that target antiapoptotic BCL-2 family proteins 42 , erianin operates through disruption of adhesion and cytoskeletal signaling, leading to loss of pro-survival anchorage dependence in senescent cells. This mechanism is conceptually aligned with emerging evidence that senescent cells reorganize focal adhesion and cytoskeletal activity 43-44 . Our findings therefore not only reveal a novel senolytic mechanism but also establish FAK as a previously underappreciated vulnerability in senescent tumor cells. Accumulation of persistently senescent cells contributes to tumor progression and immune escape by reshaping the TME 11, 14 . Senolytic agents have been recognized as modulators of the tumor immune landscape, capable of reversing senescence-driven immunosuppression and reactivating antitumor immune responses 45 . Consequently, the combination of senolytics with ICIs represents a promising therapeutic strategy with favorable efficacy and safety profiles 14 . Notably, FAK inhibition has been shown to promote macrophage repolarization by disrupting focal adhesion driven cytokine networks 46 . Preclinical data in pancreatic and breast cancers have demonstrated that FAK inhibition enhances ICIs efficacy 9, 47-48 . Together, these findings support the hypothesis that erianin may enhance the antitumor efficacy of immunotherapy in CCA. Our findings corroborate and extend this concept to CCA. In senescent tumor-bearing mice, erianin treatment markedly potentiated the antitumor effects of anti-PD-L1 therapy. Mechanistically, this synergy likely arises from two convergent effects: (1) inhibition of tumor-intrinsic FAK signaling reduces PD-L1 mediated immune escape and enhances T-cell infiltration 49-50 , and (2) elimination of senescent cells alleviates M2 macrophage polarization, thereby restoring immune surveillance 51 . Together, these processes reprogram the TME from an immunosuppressive to an immunostimulatory state, providing a mechanistic rationale for combining FAK-targeting senolytics with ICIs in CCA. Compared with strategies designed to enhance the efficacy of ICIs, erianin offers several advantages. It is a naturally derived, low-toxicity compound, exhibits multimodal antitumor mechanisms, and can simultaneously target tumor cells, senescent cells, and immune components. Moreover, the FAK mediated senescence axis targeted by erianin represents a unique therapeutic niche that complements existing immunotherapy paradigms. These features collectively highlight erianin as a multifunctional immune-sensitizing agent with translational potential. Despite the promising findings, this study has several limitations. First, while we identified FAK as a key mediator of erianin’s activity, whether erianin directly binds to FAK or acts through upstream regulators such as integrins or lipid rafts remains to be elucidated. Second, erianin-induced cell death may not be limited to apoptosis. Crosstalk with ferroptosis or necroptosis pathways in CCA warrants further investigation. Third, future studies employing single-cell RNA sequencing or spatial transcriptomics will be essential to map the comprehensive immune landscape and identify additional responsive cell types. Conclusion In summary, our findings identify erianin as a multifunctional antitumor agent that exerts its effects in CCA through an integrated mechanism involving FAK inhibition, apoptosis induction, senescence elimination, and immune microenvironment reprogramming. By bridging tumor-intrinsic signaling suppression and immune modulation, erianin achieves a dual benefit of direct tumor control and enhanced responsiveness to immune checkpoint therapy. Author Contributions Liyun Zheng, Wenjing Yang, and Minhua Wu contributed equally to this work and were responsible for the methodology, investigation, and writing of the original draft preparation; Xiaojie Zhang, Mengzhu Han, Mengyuan Wang, Qiaoyou Weng, and Bufu Tang performed the validation, formal analysis, data curation, and visualization; Zhongwei Zhao, Minjiang Chen, and Jianfei Tu contributed to the resources, project administration, and manuscript review and editing; Jiansong Ji and Shiji Fang were responsible for conceptualization, supervision, funding acquisition, and the final review and editing of the manuscript. All authors have read and approved the final version of the manuscript. Ethics declaration This study received approval from the Institutional Review Board of the Fifth Affiliated Hospital of Wenzhou Medical University and was carried out in compliance with the ethical principles stated in the Declaration of Helsinki. All animal procedures were conducted in compliance with the Animal Care and Use Committee of the Fifth Affiliated Hospital of Wenzhou Medical University. Funding This work was supported by Lishui Science and Technology Bureau (2023LHT02); Zhejiang Traditional Chinese Medicine Administration (GZY-KJS-ZJ-2025-065,); Natural Science Foundation of Zhejiang Province (LLSSZ25H180001, LLSSZ25H280002); Chinese Medicine Research Program of Zhejiang Province (2025KY1926, 2024KY559, 2023KY424); Health Commission of Zhejiang Provincial (2023ZR059). Acknowledgments We acknowledge BioRender for its assistance in creating schematic diagrams. Data availability The data are available from the corresponding author on reasonable request. Declaration of Competing Interest The authors declare no competing interests. Reference 1. Brindley, P. J.; Bachini, M.; Ilyas, S. I.; Khan, S. A.; Loukas, A.; Sirica, A. E.; Teh, B. T.; Wongkham, S.; Gores, G. J., Cholangiocarcinoma. Nature reviews. 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Authors Affiliations Liyun Zheng Lishui Central Hospital View all articles by this author Wenjing yang Lishui Central Hospital View all articles by this author Minhua Wu Lishui Central Hospital View all articles by this author Xiaojie Zhang Lishui Central Hospital View all articles by this author Mengzhu Han Lishui Central Hospital View all articles by this author Mengyuan Wang Lishui Central Hospital View all articles by this author Qiaoyou Weng Lishui Central Hospital View all articles by this author Bufu Tang Shanghai Institute of Medical Imaging View all articles by this author Zhongwei Zhao Lishui Central Hospital View all articles by this author Minjiang Chen Lishui Central Hospital View all articles by this author Jianfei Tu Lishui Central Hospital View all articles by this author Jiansong Ji Lishui Central Hospital View all articles by this author Shiji Fang [email protected] Lishui Central Hospital View all articles by this author Metrics & Citations Metrics Article Usage 127 views 88 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Liyun Zheng, Wenjing yang, Minhua Wu, et al. Erianin Potentiates the Efficacy of Immune Checkpoint inhibitor in Cholangiocarcinoma by Inhibiting FAK-mediated cell growth and senolytic activity. Authorea . 30 March 2026. DOI: https://doi.org/10.22541/au.177487426.67212017/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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