The lysosomal cysteine protease Cathepsin L promotes stemness and multidrug resistance of non-small cell lung cancer by targeting HGF activator

The lysosomal cysteine protease Cathepsin L promotes stemness and multidrug resistance of non-small cell lung cancer by targeting HGF activator | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The lysosomal cysteine protease Cathepsin L promotes stemness and multidrug resistance of non-small cell lung cancer by targeting HGF activator Hui Shi, Jianyu Xu, Juan Wu, Sha Hu, Xi Chen, Qianfang Hu, Qian Liu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7459610/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Nov, 2025 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted 7 You are reading this latest preprint version Abstract Multidrug resistance (MDR) in non-small cell lung cancer (NSCLC) is a major cause of chemotherapy failure, with lung cancer stem cells (CSCs) playing a central role in the development of MDR. Although protease family member Cathepsin L (CTSL) is known to be associated with tumor progression, its function in lung CSCs and MDR remains unclear. The chemotherapeutic sensitivities of spheroid from NSCLC cell lines were evaluated using the CCK8 assay. Western blot and qPCR analyses were performed to assess the expression levels of CTSL, stem cell markers (CD133 and CD44), stemness-maintaining molecules (OCT4 and SOX2), drug resistance proteins (MDR1 and ABCG2). In vivo experiments were conducted to validate the chemosensitizing effects of CTSL inhibitor, while ELISA was used to measure the secretion levels of HGF activator (HGFAC) and HGF. Our results demonstrated that spheroid cells exhibited pronounced MDR. High expression of CTSL correlated with poor prognosis in NSCLC patients and enhanced MDR in lung CSCs. Interfering with CTSL increased the sensitivity of lung CSCs to multiple chemotherapy drugs while reducing cell stemness and survival. Moreover, the combination of CTSL inhibitor and docetaxel effectively suppressed tumor growth in vivo. Additionally, RNA-seq analysis revealed that HGFAC expression is positively correlated with CTSL levels. Finally, CCK-8 and colony formation assays demonstrated that CTSL mediates chemoresistance by regulating HGFAC/HGF/Met expression in lung CSCs. Taken together, CTSL plays a pivotal role in NSCLC multidrug resistance. The CTSL-HGFAC axis represents a promising therapeutic target for reversing MDR in NSCLC. Non-small cell lung cancer Multidrug resistance Stemness Cathepsin L HGF activator Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Lung cancer is the leading cause of cancer-related deaths worldwide[ 1 ]. Non-small cell lung cancer (NSCLC) accounts for over 80% of all lung cancer cases, and nearly three-quarters of patients are diagnosed at an advanced stage [ 2 ]. At this stage, chemotherapy yields a 5-year survival rate of only 15%, resulting in a poor prognosis[ 3 ]. Common chemotherapy regimens for NSCLC typically include paclitaxel, docetaxel, gemcitabine, vinorelbine, or pemetrexed in combination with platinum-based drugs such as carboplatin or cisplatin[ 4 , 5 ]. However, most advanced NSCLC cases develop multidrug resistance (MDR) to these treatments. Therefore, investigating the molecular mechanisms underlying chemoresistance in NSCLC and identifying potential therapeutic targets is of utmost importance. Recent studies have increasingly demonstrated that lung cancer stem cells (CSCs)-a subpopulation characterized by self-renewal and differentiation capabilities-play a pivotal role in reducing the efficacy of chemotherapy. These cells can survive treatment, repopulate the tumor, and contribute to disease relapse and poor patient prognosis. Lung CSCs exhibit enhanced chemoresistance through mechanisms such as elevated drug efflux pump expression, activation of DNA repair pathways, and increased detoxification enzyme activity, collectively contributing to MDR in NSCLC [ 6 ]. Recent investigations have suggested that cysteine cathepsins are crucial regulators of CSC functions [ 7 ]. In particular, studies have shown that cathepsins, including Cathepsin S [ 8 ], Cathepsin C [ 9 ], Cathepsin K [ 10 ] and Cathepsin L (CTSL) [ 11 ], play important roles in tumor progression in non-small cell lung cancer. Notably, CTSL is upregulated in malignant gliomas and correlates with the aggressive progression of human glioblastoma. Our previous work revealed that glioma stem cells express high levels of CTSL and, along with the CD133 marker, exhibit remarkable radioresistance [ 12 ]. As a lysosomal endopeptidase, CTSL is involved in the turnover of intracellular and secreted proteins related to growth regulation [ 7 , 13 ]. Elevated CTSL levels have been observed in multiple cancers and are associated with poor survival outcomes [ 14 ]. Beyond its established roles in carcinogenesis and tumor growth, CTSL has recently been implicated in drug resistance in several cancers [ 1 , 15 – 17 ]. However, its role and mechanisms in mediating stemness and MDR in NSCLC remain to be fully elucidated. In the present study, we utilized NSCLC cell lines A549 and H1299 to generate spheroid and investigate the role of CTSL in stemness and multidrug resistance. Our investigation revealed a significant association between elevated CTSL expression and adverse prognostic outcomes in NSCLC patients, as well as an augmented MDR phenotype in NSCLC cells. Furthermore, the application of CTSL-targeting siRNA or specific inhibitors effectively sensitized NSCLC spheroid to a variety of chemotherapeutic agents, concurrently diminishing their stem cell characteristics and viability. These findings suggest that CTSL is a promising therapeutic target for overcoming MDR in NSCLC patients. Materials and Methods Database Analysis Transcriptomic datasets related to paclitaxel resistance in lung cancer (GSE77209) [ 18 ] and non-small cell lung CSCs (GSE50627) [ 19 ] were retrieved from the NCBI database ( https://www.ncbi.nlm.nih.gov/ ). Differentially expressed genes (DEGs) were identified using thresholds of |log₂Fold Change| >1.5 and adjusted P < 0.05. Drug resistance- and stem cell-associated genes were curated from the GeneCards platform ( https://www.genecards.org/ ). Survival disparities (overall survival, progression-free survival, disease-specific survival, and disease-free interval) between high- and low-expression groups were analyzed via the Gene Set Cancer Analysis (GSCA) portal ( https://guolab.wchscu.cn/GSCA/ ). Tumor stage-specific gene expression profiles were generated using GEPIA2.0 ( http://gepia.cancer-pku.cn/ ). Kaplan-Meier survival curves were constructed via the KM-Plot platform ( https://kmplot.com/analysis/ ) to evaluate PFS differences between expression cohorts, with hazard ratios (HRs) calculated using Cox proportional hazards models. Cell lines and culture The human non-small cell lung cancer lines, A549 and NCI-H1299 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 media (Sangon Biotech) and supplemented with 10% fetal bovine serum (HyClone), 1% Penicillin-Streptomycin. Cell culture was conducted at 37°C in a humidified 5% CO 2 incubator. Tumorsphere formation assay Cells were cultured as tumorspheres in RPMI-1640 containing recombinant human fibroblast growth factor (bFGF, 20 ng/mL; Invitrogen), recombinant human epidermal growth factor (EGF, 20 ng/mL; Invitrogen), and 1% N 2 supplement (Gibco Life Technologies). The cells were plated at a density of 100 cells per well in ultra-low attachment 96-well plates, and 7 days later, plates were examined for tumorsphere formation using an inverted microscope. Tumorspheres with diameter > 100 µm were counted. CCK-8 assay Cell Counting Kit-8 (CCK-8) assay was used to measure the viability and proliferation of cells. Adherent and spheroids of A549 and H1299 cells were inoculated into 96-well culture plates with the appropriate number of 3Í10 3 /well, then cultured overnight and treated the next day. The different concentrations of chemotherapies (PTX, HY-B0015; DTX, HY-B0011; PEM, HY-10820; GEM, HY-17026; DDP, HY-17394; CBP, HY-17393, MedChemExpress) were added to the cells, followed by incubation for another 48 h. Next, 10 µL CCK-8 solution was added to each well and incubated for 4 h at 37°C. The absorbance was measured at 450 nm. Western blot analysis Detailed procedure was as described in a previous study [ 20 ]. Primary antibody against CD44 (ab51037), CD133 (ab216323), OCT4 (ab181557), MDR1 (ab170904), and ABCG2 (ab207732) were purchased from Abcam. Primary antibody against SOX2 (#23064), Mcl-1 (#94296), Bcl-2 (#4223), HGF (#52445), Met (#8198), Phospho-Met (#3077), and GAPDH (#97166) were purchased from Cell Signaling Technology. CTSL (C4618) primary antibody was purchased from Sigma-Aldrich. Quantitative real‑time PCR (qRT-PCR) Detailed procedure for these steps has been previously reported [ 21 ]. LightCycler® 480 SYBR Green I Master Mix (Roche) was used. The delta-delta Ct method was used to calculate relative expression levels between cell lines according to standard procedures. The primer sequences employed for the PCR analysis were listed in Table 1 . All primers were synthesized by Sangon Biotech (Shanghai, China). Table 1 Primers for POU5F1, SOX2, ABCB1, ABCG2 and CTSL. Gene Sequence POU5F1 forward 5′-AGCACTTCTGTCATGCTGGA-3′ reverse 5′-TCAAGAGATTTATCGAGCACCTTCT-3′ SOX2 forward 5′-AAGGATAAGTACACGCTGCCC-3′ reverse 5′-GTTCATGTGCGCGTAACTGT-3′ ABCB1 forward 5′-TACTCACTTCAGGAAGCAACCA-3′ reverse 5′-CCAATCAGCCTCACCACAGAT-3′ ABCG2 forward 5′-CTGTTTTGTGTTTATGATGGTCTGT-3′ reverse 5′-ATGCTGCAAAGCCGTAAATCC-3′ CTSL forward 5′-AAACTGGGAGGCTTATCTCACT-3′ reverse 5′-GCATAATCCATTAGGCCACCAT-3′ siRNA transfection CTSL siRNA and negative control siRNA were purchased from Sangon Biotech (Shanghai, China). For transfection, siRNA was mixed with Lipofectamine® 3000 (Invitrogen, Carlsbad, CA, USA) and then transfected into spheroids of A549 or H1299 cells. After 6 h, the supernatant was replaced with fresh tumorsphere conditional medium and cultured for another 24 h. Three siRNA sequences used for transfection were listed in Table 2 . Table 2 Sequences for CTSL siRNA and CTSL shRNA. Name Sequence siNC sense 5′-UUCUCCGAACGUGUCACGUTT-3′ antisense 5′-ACGUGACACGUUCGGAGAATT-3′ CTSL -homo-450 sense 5′-GCGAUGCACAACAGAUUAUTT-3′ antisense 5′-AUAAUCUGUUGUGCAUCGCTT-3′ CTSL -homo-994 sense 5′-CCAAGUAUUCUGUUGCUAATT-3′ antisense 5′-UUAGCAACAGAAUACUUGGTT-3′ CTSL -homo-1112 sense 5′-CCUUCCUGUUCUAUAAAGATT-3′ antisense 5′-UCUUUAUAGAACAGGAAGGTT-3′ shNC 5′-CCTAAGGTTAAGTCGCCCTCG-3′ CTSL -shRNA1 5′-GCGATGCACAACAGATTAT-3′ CTSL -shRNA2 5′-TGCCTCAGCTACTCTAACATT-3′ CTSL -shRNA3 5′-CCAAGTATTCTGTTGCTAA-3′ CTSL overexpressing and knockdown cell line establishment The lentivirus carrying GFP-tagged CTSL was constructed by GeneChem (Shanghai, China). H1299 cells were seeded in 6-well plate and then infected with the lentivirus according to protocols as recommended by the manufacturer. The sequences of shRNA listed in Table 2 were synthesized and constructed into the transfer vector, the packaging plasmids pMD2.G (Addgene, Cambridge, UK) and pSPAX2 (Addgene) using Lipofectamine® 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. In order to obtain a stable CTSL overexpressing and knockdown cell line, the lentivirus infected cells were selected by incubation with complete medium of 2 µg/mL puromycin (MedChemExpress). Following the establishment of puromycin resistance, individual clones from each transfection group were isolated and assessed for CTSL expression using Western blot analysis. RNA sequencing analysis H1299-oe Control and H1299-oe CTSL cells (n = 3 each group) were collected. Total RNA was extracted using TRIzol reagent (Life Technologies, Thermo Scientific, CA, USA) follow manufacturer's instructions. Then RNA was quality checked and the raw data were analyzed by Iproteome Biotechnology (Shanghai, China). The DEGs were defined as CTSL related genes with the criteria of absolute value of |log₂Fold Change| >1.5 and P < 0.05. ELISA Human HGFAC and human HGF ELISA analysis were used to quantify secretory HGFAC and HGF in the conditioned media of different cells according to the manufacturer’s instructions (R&D Systems, MN, USA). Cells were seeded in six-well plates at a density of 5Í 10 5 cells per well. HGFAC and HGF were quantified in the conditioned medium 48 h after the cells were seeded. Colony Formation assay H1299-oeCTSL were seeded in 6-well plates at a density of 3Í 10 2 cells per well. The next day, the cells were pretreated with Capmatinib (a Met phosphorylation inhibitor, 1 nM for 12 h, HY-13404, MedChemExpress), then exposed to PTX (200 nM), DTX (6 nM), and GEM (3 µM). After incubation for one week, colonies were washed twice with PBS, fixed with methanol, and stained with 0.5% crystal violet (Sigma Aldrich). The colonies containing more than 50 cells were counted as surviving clones. Animal experiments All mice experiments were conducted in accordance with the humane treatment of animals under institutional guidelines approved by the Ethical Committee of Children’s Hospital of Soochow University. The mice were housed in individually ventilated cages in the Animal Laboratory of the Children’s Hospital of Soochow University. Six-week-old BALB/c Nude (SM-014) mice (Shanghai Model Organisms, Shanghai, China) were used in the study. Subcutaneous tumor transplantation was conducted using the H1299-Sp cells. Cells (1Í 10 6 ) were resuspended in 100 µL PBS and implanted into the right flank of nude mice under sterile conditions. After the formation of palpable tumors (tumor volume reached 100 mm 3 ), mice were randomized into four groups (5 mice per group): Control group (saline, i.p.), Z-FY-CHO (a specific CTSL inhibitor, HY-128140, MedChemExpress) group (5 mg/kg, i.p.), DTX (HY-B0011, MedChemExpress) group (5mg/kg, i.p.), Z-FY-CHO (5mg/kg, i.p.) plus DTX (5 mg/kg, i.p.) group. Mice were injected with vehicle or with drugs three times weekly. The size of the tumor of each mouse were measured as described previously[ 20 ]. Mice were sacrificed on day 15, and tumor weight were recorded. Statistical analysis All measurement data were presented as the mean ± S.D. at least three independent experiments were conducted. Intergroup comparisons were performed using two-tailed Student’s t -test for continuous variables and χ ² test or Fisher’s exact test for categorical variables, as appropriate. Differences were considered statistically significant at P values of < 0.05. All analyses were performed employing GraphPad Prism 10. Results Spheroid Cultivation Confers Stemness and MDR in NSCLC Cells To obtain tumorspheres from NSCLC cell lines in vitro, A549 and NCI-H1299 cells were first cultured as adherent monolayers (designated A549-Ad and H1299-Ad). Then cells were transferred to stem cell medium containing bFGF and EGF. After 3 days in culture, macroscopic and spherical or oval tumorspheres were formed (designated A549-Sp and H1299-Sp, Fig. 1 A). Western blot analysis revealed significantly elevated protein levels of stemness markers CD44 and CD133 in spheroid cultures compared with those grown in adherence (Fig. 1 B). Consistently, spheroid-derived cells demonstrated elevation of self-renewal and stemness capability markers OCT4 (POU5F1) and SOX2, with both mRNA and protein expression levels significantly exceeding those in adherent populations (Fig. 1 C-D). Drug sensitivity analysis revealed enhanced chemotherapy resistance in spheroids, evidenced by increased IC50 values for six frontline chemotherapeutic agents, including paclitaxel (PTX), docetaxel (DTX), pemetrexed (PEM), gemcitabine (GEM), carboplatin (CBP), and cisplatin (DDP). (Fig. 1 E; Supplementary Figure S1 ). The resistance index (RI) of the spheroids of NSCLC cells with different chemotherapies were listed in Table 3 . Interestingly, among these chemotherapeutic agents, these spheroid stem cells showed the highest resistance index to DTX. Furthermore, spheroid-enriched populations exhibited upregulated mRNA and protein levels of ABC transporters MDR1 (ABCB1) and ABCG2 (Fig. 1 F-G). Collectively, our results demonstrate that tumor sphere cultivation drives the acquisition of stem-like characteristics coupled with MDR in NSCLC models, providing a powerful platform for investigating chemoresistance mechanisms. Table 3 The IC50 values of the adherent and spheroids of A549 and H1299 cells with different chemotherapies, and the resistance index (RI). Group A549-Ad A549-Sp RI H1299-Ad H1299-Sp RI IC50 (µM) Mean ± SD IC50 (µM) Mean ± SD PTX 0.06 ± 0.01 0.14 ± 0.04 2.27 0.31 ± 0.05 1.33 ± 0.39 4.27 DTX 11.02×10 − 3 ± 2.73×10 − 3 0.92 ± 0.35 83.25 6.21×10 − 3 ± 1.25×10 − 3 0.65 ± 0.25 104.88 PEM 7.01 ± 1.92 20.31 ± 5.85 2.90 9.63 ± 4.12 128.10 ± 43.15 13.30 GEM 2.52 ± 0.49 15.98 ± 4.24 6.35 3.80 ± 1.22 179.10 ± 84.50 47.19 CBP 48.17 ± 7.25 87.88 ± 14.10 1.82 32.79 ± 3.14 236.30 ± 59.03 7.21 DDP 9.02 ± 0.85 14.68 ± 2.00 1.63 9.54 ± 1.17 377.10 ± 111.78 39.54 RI, Resistance index, RI = Sp (IC50) / Ad (IC50); SD, standard deviation; PTX, Paclitaxel; DTX, Docetaxel; PEM, Pemetrexed; GEM, Gemcitabine; CBP, Carboplatin; DDP, Cisplatin Elevated CTSL levels are positively associated with chemotherapy resistance and tumor progression in NSCLC patients To investigate molecular mechanisms underlying stemness acquisition and drug resistance in NSCLC, we analyzed paclitaxel-resistant and stemness-associated transcriptomic datasets (GSE77209, GSE50627) from the GEO repository. Differential gene expression analysis (threshold: |log₂Fold Change| >1.5, adjusted P < 0.05) identified candidate genes, which were cross-referenced with drug resistance- and stem cell-related genes from GeneCards to yield 13 overlapping candidates (Fig. 2 A). Subsequently, we used the GSCA database to evaluate the disease-free interval (DFI), disease-specific survival (DSS), overall survival (OS) and progression-free survival (PFS) between the cohort with high and low expression of these genes (Fig. 2 B). Notably, CTSL was identified as a robust prognostic biomarker significantly associated with adverse outcomes in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), the two major NSCLC histological subtypes. Furthermore, CTSL expression exhibited stage-dependent escalation during tumor progression (Fig. 2 C) and demonstrated a strong inverse correlation with PFS in NSCLC cohorts (Fig. 2 D). Experimental validation using WB and qPCR confirmed marked upregulation of CTSL at both mRNA and protein levels in A549-Sp and H1299-Sp compared to adherent groups (Fig. 2 E-F). Collectively, these findings establish CTSL as a dual-functional regulator orchestrating stemness maintenance and chemoresistance, thereby promoting NSCLC progression and adverse clinical prognosis. Suppression of CTSL reduces MDR in NSCLC spheroid cells To validate the functional role of CTSL in MDR, we silenced CTSL in A549-Sp and H1299-Sp cells using three independent siRNAs (Fig. 3 A). Following validation of silencing efficiency, siCTSL450 (demonstrating superior target suppression) was selected for mechanistic studies (Fig. 3 B). Dose-response assays revealed that CTSL depletion significantly potentiated chemosensitivity in spheroid cells, with marked reductions in IC50 values for six conventional chemotherapeutics: PTX, DTX, PEM, GEM, CBP and DDP (Fig. 3 C; Supplementary Figure S2 ). The sensitivity index (SI) of the spheroids of NSCLC cells with different chemotherapies were listed in Table 4 . The results indicated that knockdown of CTSL in NSCLC spheroid cells significantly increased their sensitivity to taxane-based chemotherapeutic agents, with the most pronounced effect observed for DTX, where drug resistance was reduced by approximately 70%. Consistent with these functional changes, we observed coordinated downregulation of ABC transporter expression - evidenced by decreased MDR1 and ABCG2 protein levels (Fig. 3 D) and reduced ABCB1/ABCG2 transcript abundance (Fig. 3 E). To verify these findings in vivo , we employed Z-FY-CHO (a selective CTSL inhibitor) in xenograft models. While monotherapy with either Z-FY-CHO or DTX moderately inhibited tumor growth in nude mice, combinatorial treatment elicited potent synergistic effects (Fig. 3 F-G). This therapeutic enhancement paralleled our in vitro- observations, further supporting CTSL's critical role in maintaining chemoresistance. Table 4 The IC50 values of the si NC and si CTSL 450 of A549-Sp and H1299-Sp cells with different chemotherapies, and the Sensitivity Index (SI). Group A549-Sp SI H1299-Sp SI si NC si CTSL 450 si NC si CTSL 450 IC50 (µM) Mean ± SD IC50 (µM) Mean ± SD PTX 0.12 ± 0.04 0.07 ± 0.02 0.63 1.33 ± 0.51 0.33 ± 0.08 0.25 DTX 0.90 ± 0.38 0.32 ± 0.14 0.35 0.68 ± 0.31 0.19 ± 0.07 0.28 PEM 18.08 ± 3.71 13.95 ± 3.14 0.77 134.90 ± 38.68 119.00 ± 40.68 0.88 GEM 14.10 ± 3.28 7.57 ± 1.67 0.54 172.70 ± 58.50 77.71 ± 20.59 0.45 CBP 82.23 ± 8.72 61.46 ± 8.41 0.75 243.40 ± 38.35 238.30 ± 46.83 0.98 DDP 14.33 ± 1.69 11.15 ± 0.82 0.78 367.70 ± 91.64 326.20 ± 88.19 0.90 SI, Sensitivity index, SI = si CTSL 450 (IC50) / si NC (IC50); SD, standard deviation; PTX, Paclitaxel; DTX, Docetaxel; PEM, Pemetrexed; GEM, Gemcitabine; CBP, Carboplatin; DDP, Cisplatin CTSL enhances stemness and survival in NSCLC spheroid cells Having established CTSL's role in LCSCs chemoresistance, we next sought to characterize its functional role in maintaining stemness and driving malignant progression. Pharmacological inhibition of CTSL with the specific inhibitor Z-FY-CHO markedly attenuated tumorsphere formation capacity (Fig. 4 A), a phenotype recapitulated by shRNA-mediated CTSL knockdown (Supplementary Figure S3). Notably, CTSL suppression induced parallel downregulation of both protein (Fig. 4 B) and transcript (Fig. 4 C) levels for core pluripotency factors OCT4 (POU5F1) and SOX2, indicating its critical role in maintaining NSCLC cell stemness. Intriguingly, CTSL depletion also reduced protein levels of pro-survival factors Mcl-1 and Bcl-2 (Fig. 4 D). Conversely, CTSL overexpression upregulated these pro-survival proteins (Fig. 4 E) and enhanced tumorsphere formation (Fig. 4 F). These findings collectively demonstrate that CTSL sustains LCSC populations through coordinated molecular strategy: preserving stemness and survival. Identification of HGFAC mediated HGF/Met axis as a target of CTSL in NSCLC cells Previously, we have systematically described the multiple carcinogenic functions of CTSL in NSCLC spheroid cells, including stem cell maintenance, promotion of cell survival, and induction of MDR. Subsequently, to examine the molecular mechanisms responsible for these phenomena, we performed transcriptomic profiling of CTSL-overexpressing H1299 cells, identifying 75 DEGs, which were cross-referenced with drug resistance- and stem cell-associated gene sets to yield 10 overlapping candidates (Fig. 5 A; Supplementary Figure S4A). Kaplan-Meier survival analysis revealed hepatocyte growth factor activator (HGFAC) as the most prognostically significant gene among these candidates (Fig. 5 B) (hazard ratio [HR] = 1.59; Supplementary Figure S4B-C), with its expression strongly correlating with CTSL levels. Consistently, GDSC database analysis further linked high HGFAC expression to DTX resistance (Fig. 5 C). Mechanistically, CTSL overexpression in H1299 cells increased both HGFAC secretion and hepatocyte growth factor (HGF) secretion (Fig. 5 D) and enhanced Met receptor phosphorylation (p-Met) (Fig. 5 E). Conversely, CTSL knockdown in A549-Sp and H1299-Sp cells reduced HGFAC and HGF production (Fig. 5 F) and suppressed Met signaling (Fig. 5 G). Functional rescue experiments demonstrated that exogenous HGF supplementation partially restored DTX sensitivity in CTSL-depleted H1299-Sp cells (Fig. 5 H). Colony formation assays confirmed that pharmacological inhibition of p-Met with capmatinib (Supplementary Figure S4D) reversed CTSL-driven chemoresistance to PTX, DTX, and GEM (Fig. 5 I). Taken together, these findings define a CTSL-HGFAC-HGF-Met signaling axis that maintains LCSC stemness and chemoresistance, thereby establishing a mechanistic rationale for targeting this pathway in NSCLC therapeutic strategies. Discussion Lung cancer, primarily NSCLC, is the most prevalent malignancy with a poor prognosis. [ 22 ]. While chemotherapy is a mainstay treatment, its effectiveness is frequently limited by MDR, in which lung CSCs play a critical role. This research identified CTSL as a critical functional contributor to MDR in NSCLC. Crucially, CTSL is associated with adverse clinical outcomes in NSCLC and functionally contributes to MDR in lung CSCs. CTSL expression directly increases CSC resistance to multiple chemotherapy drugs and augments their stemness properties, including viability and survival. Furthermore, combining a CTSL inhibitor with docetaxel effectively overcomes MDR and suppresses tumor growth in vivo . This study establishes CTSL as a pivotal functional driver of MDR in NSCLC and identifies its inhibition as a potent strategy for reversing chemotherapy resistance. CTSL is a universally expressed lysosomal peptidase involved in the terminal degradation of intracellular and endocytic proteins [ 23 ]. Upregulation of CTSL is common in various human cancers [ 24 – 26 ] and is widely associated with metastatic invasiveness and poor prognosis. Our previous reports have shown that the TGF-β/Smad signaling pathway regulates CTSL-mediated paclitaxel resistance in lung cancer, while Egr-1 and CREB are implicated in CTSL-mediated cisplatin resistance [ 15 ]. Furthermore, the epithelial-to-mesenchymal transition (EMT) phenotype induced by CTSL upregulation has been linked to the development of cisplatin or paclitaxel resistance in A549 cells [ 27 , 28 ]. Thus, targeting CTSL and understanding its mechanisms in NSCLC could improve diagnostic and therapeutic strategies. However, the specific ways in which CTSL affects stemness and drug resistance in NSCLC have not been fully elucidated. This study showed CTSL is markedly upregulated in high-grade NSCLC tissues and stem-like cell lines, correlating with worse patient PFS. Crucially, we demonstrate that pharmacologically or genetically inhibiting CTSL potently reduces tumor spheroid formation, MDR, and tumorigenicity in vitro and in vivo, while CTSL overexpression enhances these traits. This positions CTSL inhibition as a viable strategy for clinical intervention in chemotherapy-resistant NSCLC. MDR in NSCLC is intrinsically linked to the stemness properties (self-renewal, differentiation, tumorigenicity) of lung CSCs, maintained by complex signaling networks. Multiple oncogenes and signaling pathways contribute to maintaining CSC stemness and tumorigenicity. For example, HK2 inhibits the ubiquitination and degradation of CD133 by enhancing USP11 binding to CD133, thereby promoting CSC properties and tumor growth [ 29 ]. Similarly, TIPRL regulates stemness and survival in lung CSCs via activation of the CaMKK2-CaMK4-CREB feedback loop [ 30 ]. These findings offer promising new therapeutic targets for lung cancer. Our research establishes a direct role for CTSL in regulating NSCLC stemness. Inhibition of CTSL significantly diminished stemness-associated phenotypes and downregulated stemness-maintenance genes, whereas CTSL overexpression amplified these features. This provides direct evidence that CTSL is a key molecular driver sustaining the CSC population responsible for MDR. HGFAC is a protease that activates HGF through proteolytic cleavage[ 31 ]. The HGF/MET axis is a well-established regulator of tumor growth, survival, metastasis, and CSC functions. HGF binding to MET triggers receptor dimerization, autophosphorylation (e.g., Tyr1234/1235, Tyr1349/1356), and activation of downstream pathways (MAPK, PI3K/AKT, STAT3), promoting cell survival and chemoresistance[ 32 , 33 ]. Consequently, the HGFAC/HGF/MET axis plays a pivotal role in regulating tumor stem cell functions—such as self-renewal, proliferation, and differentiation—contributing to tumor initiation, progression, and chemoresistance [ 34 ]. A key finding of this study is that CTSL acts as a direct upstream regulator of HGFAC expression. This CTSL/HGFAC/HGF/MET axis constitutes the core mechanism linking CTSL to NSCLC stemness maintenance and MDR development, establishing the novel role of CTSL in controlling HGF/MET signaling in this context. Despite these insights, our study has some limitations. We did not fully elucidate the specific mechanism by which CTSL regulates HGFAC, and further experiments are needed to clarify how CTSL interacts with HGFAC to affect stemness and drug resistance. Nevertheless, our findings highlight the CTSL-HGFAC axis as a potential therapeutic target for overcoming chemoresistance in NSCLC. Thus,our future studies will specifically investigate the molecular mechanism by which CTSL regulates HGFAC expression, examining whether CTSL acts at the transcriptional level or post-transcriptionally. Furthermore, we will determine how CTSL and HGFAC interact with each other. These experiments are essential to fully understand how this axis governs stemness maintenance and drug resistance, and will inform the development of targeted inhibitors or combination strategies to disrupt the CTSL-HGFAC pathway for overcoming NSCLC chemoresistance. Conclusions In summary, our findings indicate that CTSL plays a vital role in enhancing CSC phenotypes and chemoresistance in lung cancer, suggesting that targeting CTSL may represent a promising strategy for improving chemotherapy outcomes in NSCLC patients. Abbreviations CBP carboplatin CCK-8 Cell Counting Kit-8 CSCs cancer stem cells CTSL Cathepsin L DDP cisplatin DEGs differentially expressed genes DFI disease-free interval DOX doxorubicin DSS disease-specific survival DTX docetaxel EMT epithelial-to-mesenchymal transition GEM gemcitabine GSCA Gene Set Cancer Analysis HGFAC HGF activator LUAD lung adenocarcinoma LUSC lung squamous cell carcinoma MDR multidrug resistance NSCLC non-small cell lung cancer OS overall survival PEM pemetrexed PFS progression-free survival PTX paclitaxel SI sensitivity index RI resistance index Declarations Data availability The data are available from the corresponding author on reasonable request. Acknowledgements The authors are grateful for the support from the Biorender for Graphical Abstract（ Created in https://BioRender.com）. Funding This work was supported by the grants from the National Natural Science Foundation of China (Grant No. 82172840 and 82272871), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (Grant No. 21KJB320017), Gusu Health Talents Project of Suzhou Municipal Health Commission (Grant No. GSWS2022062), Chinese Pharmaceutical Association Hospital Pharmacy Department (Grant No. CPA-Z05-ZC-2024002), and Bethune Charitable Foundation (Grant No. Z04J2023E095). The funders had no role in study design, data collection, analysis or interpretation of the data, preparation of the manuscript or decision to publish the results. Author information Authors and Affiliations Department of Pharmacy, Children’s Hospital of Soochow University, Suzhou 215025, China Wenjuan Wang &Hui Shi&Sha Hu& Xi Chen College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China Wenjuan Wang &Hui Shi&Sha Hu& Xi Chen Department of Pharmacy, Medical Science University and Suzhou Technology China Innovation Center, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School of Nanjing Medical University, Suzhou 215001, China. Xinyuan Ding& Jianyu Xu& Juan Wu& Qianfang Hu&Qian Liu Contributions Hui Shi: Writing original draft, methodology, investigation, and visualization. Jianyu Xu: Writing original draft, methodology, and investigation. Juan wu: Data curation, methodology, and funding acquisition. Sha Hu, Xi Chen, Qianfang Hu and Qian Liu: Methodology, investigation, validation, and data curation. Xinyuan Ding: Review and editing of manuscript critically for important intellectual content, and funding acquisition. Wenjuan Wang: Conception, design, funding acquisition, review and editing of manuscript. All authors read, revised and approved the final manuscript. Corresponding author Correspondence to Wenjuan Wang or Xinyuan Ding Conflict of interest The authors declare that there are no known competing financial interests or personal relationships that could have appeared to influence this work. References Abdelaziz RF, Hussein AM, Kotob MH, Weiss C, Chelminski K, Stojanovic T, Studenik CR, Aufy M (2023) Enhancement of Radiation Sensitivity by Cathepsin L Suppression in Colon Carcinoma Cells. Int J Mol Sci 24:17042. 10.3390/ijms242317106 Menon T, Gopal S, Rastogi Verma S (2023) Targeted therapies in non-small cell lung cancer and the potential role of AI interventions in cancer treatment. 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Supplementary Files SupplementaryFigures.pdf Figure S1.Drug sensitivity assays demonstrated increased IC50 values in spheroids of A549 and H1299 cells for six chemotherapeutics: paclitaxel (PTX), docetaxel (DTX), pemetrexed (PEM), gemcitabine (GEM), carboplatin (CBP), and cisplatin (DDP). Figure S2.Drug sensitivity assays demonstrated decreased IC50 values in spheroids after knockdown of CTSL for paclitaxel (PTX), docetaxel (DTX), pemetrexed (PEM), gemcitabine (GEM), carboplatin (CBP), and cisplatin (DDP). Figure S3.(A) Western Blot analysis of shRNA-mediated knockdown of CTSL in H1299-Sp cells. (B) Number of spheres was decreased after knockdown of CTSL in H1299-Sp cells. * P <0.05, ** P <0.01 Figure S4. (A) RNA-seq-derived differentially expressed genes (DEGs) in CTSL-overexpressing H1299 cells. (B-C) Kaplan-Meier survival curves demonstrating the prognostic significance of 10 candidates in NSCLC. (D) Western Blot analysis of p-Met expression in oeCTSL groups after treated with Capmatinib. ** P <0.01 GraphicalAbstract.pdf Cite Share Download PDF Status: Published Journal Publication published 03 Nov, 2025 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted Editorial decision: Revision requested 04 Sep, 2025 Reviews received at journal 30 Aug, 2025 Reviewers agreed at journal 30 Aug, 2025 Reviewers invited by journal 28 Aug, 2025 Editor assigned by journal 28 Aug, 2025 Submission checks completed at journal 26 Aug, 2025 First submitted to journal 26 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7459610","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509184142,"identity":"a8d08ad9-da18-4e7f-a632-ca23d1e31419","order_by":0,"name":"Hui Shi","email":"","orcid":"","institution":"Children’s Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Shi","suffix":""},{"id":509184143,"identity":"641110b0-1e59-4f5c-847f-31324405181a","order_by":1,"name":"Jianyu Xu","email":"","orcid":"","institution":"Medical Science University and Suzhou Technology China Innovation 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06:53:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7459610/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7459610/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11010-025-05423-8","type":"published","date":"2025-11-03T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90801842,"identity":"1fd773c6-48aa-4eb0-b26b-23d7352ef625","added_by":"auto","created_at":"2025-09-08 10:20:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":227339,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInduction of multidrug resistance through tumorsphere formation in NSCLC. \u003c/strong\u003e(A) Representative pictures of tumorsphere formation assay from A549 and NCI-H1299 cells (designated A549-Sp and H1299-Sp, respectively). (B) Western blot analysis of stemness markers CD44 and CD133 in spheroid-enriched (A549-Sp, H1299-Sp) versus adherent (A549-Ad, H1299-Ad) populations. (C) Western blot validation of OCT4 and SOX2 protein in spheroid cultures. Band intensities quantified relative to GAPDH. (D) qRT-PCR analysis of POU5F1 and SOX2 mRNA levels in spheroid versus adherent cells. (E) RI values of paclitaxel (PTX), docetaxel (DTX), pemetrexed (PEM), gemcitabine (GEM), carboplatin (CBP), and cisplatin (DDP) in spheroid versus adherent cells (CCK-8 assays). (F) mRNA levels of ABC transporters ABCB1 and ABCG2 in spheroid-enriched populations. (G) Western blot validation of MDR1 and ABCG2 protein in spheroid cultures. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, *** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7459610/v1/7422c2d25ea997fa1b936200.png"},{"id":90800968,"identity":"67737fc2-e522-4bb4-b69b-b205d8a73bd1","added_by":"auto","created_at":"2025-09-08 10:12:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":176273,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElevated CTSL levels are positively associated with chemotherapy resistance and tumor progression in NSCLC.\u003c/strong\u003e (A) Candidate Gene Identification. Transcriptomic datasets from GEO datasets were analyzed for differential gene expression. These genes were then cross-referenced with drug resistance- and stem cell-related genes from GeneCards, resulting in 13 overlapping candidates. (B) Survival Analysis of 13 Candidate Genes. Kaplan-Meier survival plots illustrate the association between gene expression levels and NSCLC patient survival outcomes. (C) The relationship between the expression level of CTSL and the progression of tumor stage in NSCLC patients. (D) The relationship between CTSL expression and the survival rate of NSCLC patients. (E) qPCR analyses confirmed CTSL mRNA levels in A549-Sp and H1299-Sp cells. (F) Western blot analyses confirmed CTSL protein levels in A549-Sp and H1299-Sp cells. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, *** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7459610/v1/dca9fff4e8292863cfae74f3.png"},{"id":90803031,"identity":"ae19cf3a-8628-4046-b927-c78855b6dd3f","added_by":"auto","created_at":"2025-09-08 10:28:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":254938,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuppression of CTSL reduces MDR in NSCLC spheroid cells. \u003c/strong\u003e(A) Western Blot analysis of siRNA-mediated knockdown of CTSL in A549-Sp and H1299-Sp cells. (B) qPCR analysis of knockdown efficacy of si\u003cem\u003eCTSL\u003c/em\u003e450 in A549-Sp and H1299-Sp cells. (C) SI values for paclitaxel (PTX), docetaxel (DTX), pemetrexed (PEM), gemcitabine (GEM), carboplatin (CBP), and cisplatin (DDP) in CTSL-depleted spheroids. (D) Western blot analysis of ABC transporter protein levels (MDR1 and ABCG2) post-CTSL knockdown. (E) qPCR validation of ABCB1 and ABCG2 mRNA downregulation in CTSL-silenced cells. (F) In vivo efficacy of CTSL inhibitor Z-FY-CHO, DTX or their combination in xenograft-bearing mouse. (G) Terminal tumor weights at study endpoint. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, *** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7459610/v1/1b6a9605f10eda39f7ed2485.png"},{"id":90801845,"identity":"65b2c798-cee5-4ee9-9775-9fa5023d7af7","added_by":"auto","created_at":"2025-09-08 10:20:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":204610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCTSL enhances stemness and survival in NSCLC spheroid cells.\u003c/strong\u003e (A) The formation of tumor spheres in NSCLC spheroid cells after CTSL was inhibited by Z-FY-CHO. (B-C) The protein and mRNA levels of pluripotency regulators OCT4 (POU5F1) and SOX2 after CTSL knockdown. (D) Western blot analysis of the protein levels of survival-promoting factors MCL1 and BCL2 in CTSL-downregulated NSCLC spheroid cells. (E) Western blot analysis of the protein levels of survival-promoting factors MCL1 and BCL2 in CTSL-overexpression NSCLC spheroid cells. (F)Tumorsphere formation capacity in CTSL-overexpressing lung cancer cells. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, *** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7459610/v1/776158125bd80552a089772f.png"},{"id":90800972,"identity":"288aec82-59ea-4715-864d-4c7a105c1c74","added_by":"auto","created_at":"2025-09-08 10:12:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":322298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of HGFAC mediated HGF/Met axis as a target of CTSL in NSCLC cells. \u003c/strong\u003e(A) Venn diagram showing overlap between RNA-seq-derived differentially expressed genes (DEGs, top 75) in CTSL-overexpressing H1299 cells and curated drug resistance-/stem cell-associated genes. (B) Kaplan-Meier survival curves demonstrating the prognostic significance of HGFAC expression in NSCLC. (C) GDSC drug sensitivity analysis linking high HGFAC expression to DTX resistance. (D) ELISA quantification of secreted HGFAC and HGF in H1299 cells overexpressing CTSL. (E) Western blot analysis of Met receptor phosphorylation (p-Met) in CTSL-overexpressing H1299 cells. (F) ELISA quantitative detection was used to compare the secretion of HGFAC and HGF after CTSL knockdown in A549-sp and H1299-sp. (G) Western blot analysis of the level of p-Met protein in H1299-Sp cells after CTSL knockdown. (H) CCK8 analysis of the rescue of DTX sensitivity in CTSL-depleted H1299-Sp cells by exogenous HGF supplementation. (I) Colony formation assays were conducted to analyze whether Met inhibition reversed CTSL-driven resistance to PTX, DTX and GEM. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7459610/v1/d595dca50d551706bfb04019.png"},{"id":95564208,"identity":"1830515e-36d7-49a2-96c7-8039802285de","added_by":"auto","created_at":"2025-11-10 16:08:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2417438,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7459610/v1/66fda153-26de-4d19-9dfb-feab4c3860ed.pdf"},{"id":90803032,"identity":"6e4a24a8-abec-4d2c-a2b8-85c07511ed7f","added_by":"auto","created_at":"2025-09-08 10:28:41","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2933722,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1.\u003c/strong\u003eDrug sensitivity assays demonstrated increased IC50 values in spheroids of A549 and H1299 cells for six chemotherapeutics: paclitaxel (PTX), docetaxel (DTX), pemetrexed (PEM), gemcitabine (GEM), carboplatin (CBP), and cisplatin (DDP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S2.\u003c/strong\u003eDrug sensitivity assays demonstrated decreased IC50 values in spheroids after knockdown of CTSL for paclitaxel (PTX), docetaxel (DTX), pemetrexed (PEM), gemcitabine (GEM), carboplatin (CBP), and cisplatin (DDP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S3.\u003c/strong\u003e(A) Western Blot analysis of shRNA-mediated knockdown of CTSL in H1299-Sp cells. (B) Number of spheres was decreased after knockdown of CTSL in H1299-Sp cells. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.01\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S4. \u003c/strong\u003e(A) RNA-seq-derived differentially expressed genes (DEGs) in CTSL-overexpressing H1299 cells. (B-C) Kaplan-Meier survival curves demonstrating the prognostic significance of 10 candidates in NSCLC. (D) Western Blot analysis of p-Met expression in oeCTSL groups after treated with Capmatinib. ** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.01\u003c/p\u003e","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7459610/v1/bc814d302d5caecbfa7a4846.pdf"},{"id":90800974,"identity":"5b379926-db28-44ee-814a-e0844830c5b1","added_by":"auto","created_at":"2025-09-08 10:12:41","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":699631,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7459610/v1/e4234f383c350be17544ecd7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The lysosomal cysteine protease Cathepsin L promotes stemness and multidrug resistance of non-small cell lung cancer by targeting HGF activator","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer is the leading cause of cancer-related deaths worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Non-small cell lung cancer (NSCLC) accounts for over 80% of all lung cancer cases, and nearly three-quarters of patients are diagnosed at an advanced stage [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. At this stage, chemotherapy yields a 5-year survival rate of only 15%, resulting in a poor prognosis[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Common chemotherapy regimens for NSCLC typically include paclitaxel, docetaxel, gemcitabine, vinorelbine, or pemetrexed in combination with platinum-based drugs such as carboplatin or cisplatin[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, most advanced NSCLC cases develop multidrug resistance (MDR) to these treatments. Therefore, investigating the molecular mechanisms underlying chemoresistance in NSCLC and identifying potential therapeutic targets is of utmost importance.\u003c/p\u003e\u003cp\u003eRecent studies have increasingly demonstrated that lung cancer stem cells (CSCs)-a subpopulation characterized by self-renewal and differentiation capabilities-play a pivotal role in reducing the efficacy of chemotherapy. These cells can survive treatment, repopulate the tumor, and contribute to disease relapse and poor patient prognosis. Lung CSCs exhibit enhanced chemoresistance through mechanisms such as elevated drug efflux pump expression, activation of DNA repair pathways, and increased detoxification enzyme activity, collectively contributing to MDR in NSCLC [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecent investigations have suggested that cysteine cathepsins are crucial regulators of CSC functions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In particular, studies have shown that cathepsins, including Cathepsin S [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], Cathepsin C [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], Cathepsin K [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and Cathepsin L (CTSL) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], play important roles in tumor progression in non-small cell lung cancer. Notably, CTSL is upregulated in malignant gliomas and correlates with the aggressive progression of human glioblastoma. Our previous work revealed that glioma stem cells express high levels of CTSL and, along with the CD133 marker, exhibit remarkable radioresistance [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. As a lysosomal endopeptidase, CTSL is involved in the turnover of intracellular and secreted proteins related to growth regulation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Elevated CTSL levels have been observed in multiple cancers and are associated with poor survival outcomes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Beyond its established roles in carcinogenesis and tumor growth, CTSL has recently been implicated in drug resistance in several cancers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, its role and mechanisms in mediating stemness and MDR in NSCLC remain to be fully elucidated.\u003c/p\u003e\u003cp\u003eIn the present study, we utilized NSCLC cell lines A549 and H1299 to generate spheroid and investigate the role of CTSL in stemness and multidrug resistance. Our investigation revealed a significant association between elevated CTSL expression and adverse prognostic outcomes in NSCLC patients, as well as an augmented MDR phenotype in NSCLC cells. Furthermore, the application of CTSL-targeting siRNA or specific inhibitors effectively sensitized NSCLC spheroid to a variety of chemotherapeutic agents, concurrently diminishing their stem cell characteristics and viability. These findings suggest that CTSL is a promising therapeutic target for overcoming MDR in NSCLC patients.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDatabase Analysis\u003c/h2\u003e\u003cp\u003eTranscriptomic datasets related to paclitaxel resistance in lung cancer (GSE77209) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and non-small cell lung CSCs (GSE50627) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] were retrieved from the NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Differentially expressed genes (DEGs) were identified using thresholds of |log₂Fold Change| \u0026gt;1.5 and adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Drug resistance- and stem cell-associated genes were curated from the GeneCards platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Survival disparities (overall survival, progression-free survival, disease-specific survival, and disease-free interval) between high- and low-expression groups were analyzed via the Gene Set Cancer Analysis (GSCA) portal (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://guolab.wchscu.cn/GSCA/\u003c/span\u003e\u003cspan address=\"https://guolab.wchscu.cn/GSCA/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Tumor stage-specific gene expression profiles were generated using GEPIA2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Kaplan-Meier survival curves were constructed via the KM-Plot platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://kmplot.com/analysis/\u003c/span\u003e\u003cspan address=\"https://kmplot.com/analysis/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to evaluate PFS differences between expression cohorts, with hazard ratios (HRs) calculated using Cox proportional hazards models.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell lines and culture\u003c/h3\u003e\n\u003cp\u003eThe human non-small cell lung cancer lines, A549 and NCI-H1299 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 media (Sangon Biotech) and supplemented with 10% fetal bovine serum (HyClone), 1% Penicillin-Streptomycin. Cell culture was conducted at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\n\u003ch3\u003eTumorsphere formation assay\u003c/h3\u003e\n\u003cp\u003eCells were cultured as tumorspheres in RPMI-1640 containing recombinant human fibroblast growth factor (bFGF, 20 ng/mL; Invitrogen), recombinant human epidermal growth factor (EGF, 20 ng/mL; Invitrogen), and 1% N\u003csub\u003e2\u003c/sub\u003e supplement (Gibco Life Technologies). The cells were plated at a density of 100 cells per well in ultra-low attachment 96-well plates, and 7 days later, plates were examined for tumorsphere formation using an inverted microscope. Tumorspheres with diameter\u0026thinsp;\u0026gt;\u0026thinsp;100 \u0026micro;m were counted.\u003c/p\u003e\n\u003ch3\u003eCCK-8 assay\u003c/h3\u003e\n\u003cp\u003eCell Counting Kit-8 (CCK-8) assay was used to measure the viability and proliferation of cells. Adherent and spheroids of A549 and H1299 cells were inoculated into 96-well culture plates with the appropriate number of 3\u0026Iacute;10\u003csup\u003e3\u003c/sup\u003e/well, then cultured overnight and treated the next day. The different concentrations of chemotherapies (PTX, HY-B0015; DTX, HY-B0011; PEM, HY-10820; GEM, HY-17026; DDP, HY-17394; CBP, HY-17393, MedChemExpress) were added to the cells, followed by incubation for another 48 h. Next, 10 \u0026micro;L CCK-8 solution was added to each well and incubated for 4 h at 37\u0026deg;C. The absorbance was measured at 450 nm.\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eDetailed procedure was as described in a previous study [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Primary antibody against CD44 (ab51037), CD133 (ab216323), OCT4 (ab181557), MDR1 (ab170904), and ABCG2 (ab207732) were purchased from Abcam. Primary antibody against SOX2 (#23064), Mcl-1 (#94296), Bcl-2 (#4223), HGF (#52445), Met (#8198), Phospho-Met (#3077), and GAPDH (#97166) were purchased from Cell Signaling Technology. CTSL (C4618) primary antibody was purchased from Sigma-Aldrich.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative real‑time PCR (qRT-PCR)\u003c/h2\u003e\u003cp\u003eDetailed procedure for these steps has been previously reported [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. LightCycler\u0026reg; 480 SYBR Green I Master Mix (Roche) was used. The delta-delta Ct method was used to calculate relative expression levels between cell lines according to standard procedures. The primer sequences employed for the PCR analysis were listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All primers were synthesized by Sangon Biotech (Shanghai, China).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimers for POU5F1, SOX2, ABCB1, ABCG2 and CTSL.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eSequence\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePOU5F1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eforward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-AGCACTTCTGTCATGCTGGA-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ereverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-TCAAGAGATTTATCGAGCACCTTCT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSOX2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eforward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-AAGGATAAGTACACGCTGCCC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ereverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-GTTCATGTGCGCGTAACTGT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eABCB1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eforward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-TACTCACTTCAGGAAGCAACCA-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ereverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CCAATCAGCCTCACCACAGAT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eABCG2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eforward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CTGTTTTGTGTTTATGATGGTCTGT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ereverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-ATGCTGCAAAGCCGTAAATCC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCTSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eforward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-AAACTGGGAGGCTTATCTCACT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ereverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-GCATAATCCATTAGGCCACCAT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003esiRNA transfection\u003c/h3\u003e\n\u003cp\u003eCTSL siRNA and negative control siRNA were purchased from Sangon Biotech (Shanghai, China). For transfection, siRNA was mixed with Lipofectamine\u0026reg; 3000 (Invitrogen, Carlsbad, CA, USA) and then transfected into spheroids of A549 or H1299 cells. After 6 h, the supernatant was replaced with fresh tumorsphere conditional medium and cultured for another 24 h. Three siRNA sequences used for transfection were listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSequences for CTSL siRNA and CTSL shRNA.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eSequence\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003esiNC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003esense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-UUCUCCGAACGUGUCACGUTT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eantisense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-ACGUGACACGUUCGGAGAATT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eCTSL\u003c/em\u003e-homo-450\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003esense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-GCGAUGCACAACAGAUUAUTT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eantisense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-AUAAUCUGUUGUGCAUCGCTT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eCTSL\u003c/em\u003e-homo-994\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003esense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CCAAGUAUUCUGUUGCUAATT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eantisense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-UUAGCAACAGAAUACUUGGTT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eCTSL\u003c/em\u003e-homo-1112\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003esense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CCUUCCUGUUCUAUAAAGATT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eantisense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-UCUUUAUAGAACAGGAAGGTT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eshNC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CCTAAGGTTAAGTCGCCCTCG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCTSL\u003c/em\u003e-shRNA1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-GCGATGCACAACAGATTAT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCTSL\u003c/em\u003e-shRNA2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-TGCCTCAGCTACTCTAACATT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCTSL\u003c/em\u003e-shRNA3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CCAAGTATTCTGTTGCTAA-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eCTSL overexpressing and knockdown cell line establishment\u003c/h3\u003e\n\u003cp\u003eThe lentivirus carrying GFP-tagged CTSL was constructed by GeneChem (Shanghai, China). H1299 cells were seeded in 6-well plate and then infected with the lentivirus according to protocols as recommended by the manufacturer. The sequences of shRNA listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e were synthesized and constructed into the transfer vector, the packaging plasmids pMD2.G (Addgene, Cambridge, UK) and pSPAX2 (Addgene) using Lipofectamine\u0026reg; 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer\u0026rsquo;s instructions. In order to obtain a stable CTSL overexpressing and knockdown cell line, the lentivirus infected cells were selected by incubation with complete medium of 2 \u0026micro;g/mL puromycin (MedChemExpress). Following the establishment of puromycin resistance, individual clones from each transfection group were isolated and assessed for CTSL expression using Western blot analysis.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eRNA sequencing analysis\u003c/h2\u003e\u003cp\u003eH1299-oe\u003cem\u003eControl\u003c/em\u003e and H1299-oe\u003cem\u003eCTSL\u003c/em\u003e cells (n\u0026thinsp;=\u0026thinsp;3 each group) were collected. Total RNA was extracted using TRIzol reagent (Life Technologies, Thermo Scientific, CA, USA) follow manufacturer's instructions. Then RNA was quality checked and the raw data were analyzed by Iproteome Biotechnology (Shanghai, China). The DEGs were defined as CTSL related genes with the criteria of absolute value of |log₂Fold Change| \u0026gt;1.5 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eELISA\u003c/h2\u003e\u003cp\u003eHuman HGFAC and human HGF ELISA analysis were used to quantify secretory HGFAC and HGF in the conditioned media of different cells according to the manufacturer\u0026rsquo;s instructions (R\u0026amp;D Systems, MN, USA). Cells were seeded in six-well plates at a density of 5\u0026Iacute; 10\u003csup\u003e5\u003c/sup\u003e cells per well. HGFAC and HGF were quantified in the conditioned medium 48 h after the cells were seeded.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eColony Formation assay\u003c/h2\u003e\u003cp\u003eH1299-oeCTSL were seeded in 6-well plates at a density of 3\u0026Iacute; 10\u003csup\u003e2\u003c/sup\u003e cells per well. The next day, the cells were pretreated with Capmatinib (a Met phosphorylation inhibitor, 1 nM for 12 h, HY-13404, MedChemExpress), then exposed to PTX (200 nM), DTX (6 nM), and GEM (3 \u0026micro;M). After incubation for one week, colonies were washed twice with PBS, fixed with methanol, and stained with 0.5% crystal violet (Sigma Aldrich). The colonies containing more than 50 cells were counted as surviving clones.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eAnimal experiments\u003c/h2\u003e\u003cp\u003eAll mice experiments were conducted in accordance with the humane treatment of animals under institutional guidelines approved by the Ethical Committee of Children\u0026rsquo;s Hospital of Soochow University. The mice were housed in individually ventilated cages in the Animal Laboratory of the Children\u0026rsquo;s Hospital of Soochow University. Six-week-old BALB/c Nude (SM-014) mice (Shanghai Model Organisms, Shanghai, China) were used in the study. Subcutaneous tumor transplantation was conducted using the H1299-Sp cells. Cells (1\u0026Iacute; 10\u003csup\u003e6\u003c/sup\u003e) were resuspended in 100 \u0026micro;L PBS and implanted into the right flank of nude mice under sterile conditions. After the formation of palpable tumors (tumor volume reached 100 mm\u003csup\u003e3\u003c/sup\u003e), mice were randomized into four groups (5 mice per group): Control group (saline, i.p.), Z-FY-CHO (a specific CTSL inhibitor, HY-128140, MedChemExpress) group (5 mg/kg, i.p.), DTX (HY-B0011, MedChemExpress) group (5mg/kg, i.p.), Z-FY-CHO (5mg/kg, i.p.) plus DTX (5 mg/kg, i.p.) group. Mice were injected with vehicle or with drugs three times weekly. The size of the tumor of each mouse were measured as described previously[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Mice were sacrificed on day 15, and tumor weight were recorded.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll measurement data were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. at least three independent experiments were conducted. Intergroup comparisons were performed using two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test for continuous variables and \u003cem\u003eχ\u003c/em\u003e\u0026sup2; test or Fisher\u0026rsquo;s exact test for categorical variables, as appropriate. Differences were considered statistically significant at \u003cem\u003eP\u003c/em\u003e values of \u0026lt;\u0026thinsp;0.05. All analyses were performed employing GraphPad Prism 10.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eSpheroid Cultivation Confers Stemness and MDR in NSCLC Cells\u003c/h2\u003e\u003cp\u003eTo obtain tumorspheres from NSCLC cell lines in vitro, A549 and NCI-H1299 cells were first cultured as adherent monolayers (designated A549-Ad and H1299-Ad). Then cells were transferred to stem cell medium containing bFGF and EGF. After 3 days in culture, macroscopic and spherical or oval tumorspheres were formed (designated A549-Sp and H1299-Sp, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Western blot analysis revealed significantly elevated protein levels of stemness markers CD44 and CD133 in spheroid cultures compared with those grown in adherence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Consistently, spheroid-derived cells demonstrated elevation of self-renewal and stemness capability markers OCT4 (POU5F1) and SOX2, with both mRNA and protein expression levels significantly exceeding those in adherent populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). Drug sensitivity analysis revealed enhanced chemotherapy resistance in spheroids, evidenced by increased IC50 values for six frontline chemotherapeutic agents, including paclitaxel (PTX), docetaxel (DTX), pemetrexed (PEM), gemcitabine (GEM), carboplatin (CBP), and cisplatin (DDP). (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE; Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The resistance index (RI) of the spheroids of NSCLC cells with different chemotherapies were listed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Interestingly, among these chemotherapeutic agents, these spheroid stem cells showed the highest resistance index to DTX. Furthermore, spheroid-enriched populations exhibited upregulated mRNA and protein levels of ABC transporters MDR1 (ABCB1) and ABCG2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-G). Collectively, our results demonstrate that tumor sphere cultivation drives the acquisition of stem-like characteristics coupled with MDR in NSCLC models, providing a powerful platform for investigating chemoresistance mechanisms.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe IC50 values of the adherent and spheroids of A549 and H1299 cells with different chemotherapies, and the resistance index (RI).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA549-Ad\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eA549-Sp\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eRI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eH1299-Ad\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eH1299-Sp\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eRI\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eIC50 (\u0026micro;M) Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eIC50 (\u0026micro;M) Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePTX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e4.27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDTX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e11.02\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026plusmn; 2.73\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e83.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e6.21\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026plusmn; 1.25\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e104.88\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePEM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e7.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e20.31\u0026thinsp;\u0026plusmn;\u0026thinsp;5.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e9.63\u0026thinsp;\u0026plusmn;\u0026thinsp;4.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e128.10\u0026thinsp;\u0026plusmn;\u0026thinsp;43.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e13.30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGEM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e15.98\u0026thinsp;\u0026plusmn;\u0026thinsp;4.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e3.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e179.10\u0026thinsp;\u0026plusmn;\u0026thinsp;84.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e47.19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCBP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e48.17\u0026thinsp;\u0026plusmn;\u0026thinsp;7.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e87.88\u0026thinsp;\u0026plusmn;\u0026thinsp;14.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e32.79\u0026thinsp;\u0026plusmn;\u0026thinsp;3.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e236.30\u0026thinsp;\u0026plusmn;\u0026thinsp;59.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e7.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDDP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e14.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.54 \u0026plusmn;\u0026thinsp;1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e377.10\u0026thinsp;\u0026plusmn;\u0026thinsp;111.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e39.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eRI, Resistance index, RI\u0026thinsp;=\u0026thinsp;Sp (IC50) / Ad (IC50); SD, standard deviation; PTX, Paclitaxel; DTX, Docetaxel; PEM, Pemetrexed; GEM, Gemcitabine; CBP, Carboplatin; DDP, Cisplatin\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eElevated CTSL levels are positively associated with chemotherapy resistance and tumor progression in NSCLC patients\u003c/h2\u003e\u003cp\u003eTo investigate molecular mechanisms underlying stemness acquisition and drug resistance in NSCLC, we analyzed paclitaxel-resistant and stemness-associated transcriptomic datasets (GSE77209, GSE50627) from the GEO repository. Differential gene expression analysis (threshold: |log₂Fold Change| \u0026gt;1.5, adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) identified candidate genes, which were cross-referenced with drug resistance- and stem cell-related genes from GeneCards to yield 13 overlapping candidates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Subsequently, we used the GSCA database to evaluate the disease-free interval (DFI), disease-specific survival (DSS), overall survival (OS) and progression-free survival (PFS) between the cohort with high and low expression of these genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Notably, CTSL was identified as a robust prognostic biomarker significantly associated with adverse outcomes in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), the two major NSCLC histological subtypes. Furthermore, CTSL expression exhibited stage-dependent escalation during tumor progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and demonstrated a strong inverse correlation with PFS in NSCLC cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Experimental validation using WB and qPCR confirmed marked upregulation of CTSL at both mRNA and protein levels in A549-Sp and H1299-Sp compared to adherent groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F). Collectively, these findings establish CTSL as a dual-functional regulator orchestrating stemness maintenance and chemoresistance, thereby promoting NSCLC progression and adverse clinical prognosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eSuppression of CTSL reduces MDR in NSCLC spheroid cells\u003c/h2\u003e\u003cp\u003eTo validate the functional role of CTSL in MDR, we silenced CTSL in A549-Sp and H1299-Sp cells using three independent siRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Following validation of silencing efficiency, siCTSL450 (demonstrating superior target suppression) was selected for mechanistic studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Dose-response assays revealed that CTSL depletion significantly potentiated chemosensitivity in spheroid cells, with marked reductions in IC50 values for six conventional chemotherapeutics: PTX, DTX, PEM, GEM, CBP and DDP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The sensitivity index (SI) of the spheroids of NSCLC cells with different chemotherapies were listed in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The results indicated that knockdown of CTSL in NSCLC spheroid cells significantly increased their sensitivity to taxane-based chemotherapeutic agents, with the most pronounced effect observed for DTX, where drug resistance was reduced by approximately 70%. Consistent with these functional changes, we observed coordinated downregulation of ABC transporter expression - evidenced by decreased MDR1 and ABCG2 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and reduced ABCB1/ABCG2 transcript abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). To verify these findings \u003cem\u003ein vivo\u003c/em\u003e, we employed Z-FY-CHO (a selective CTSL inhibitor) in xenograft models. While monotherapy with either Z-FY-CHO or DTX moderately inhibited tumor growth in nude mice, combinatorial treatment elicited potent synergistic effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G). This therapeutic enhancement paralleled our \u003cem\u003ein vitro-\u003c/em\u003eobservations, further supporting CTSL's critical role in maintaining chemoresistance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe IC50 values of the si\u003cem\u003eNC\u003c/em\u003e and si\u003cem\u003eCTSL\u003c/em\u003e\u003csub\u003e450\u003c/sub\u003e of A549-Sp and H1299-Sp cells with different chemotherapies, and the Sensitivity Index (SI).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eA549-Sp\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eSI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eH1299-Sp\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eSI\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003esi\u003cem\u003eNC\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003esi\u003cem\u003eCTSL\u003c/em\u003e\u003csub\u003e450\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003esi\u003cem\u003eNC\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003esi\u003cem\u003eCTSL\u003c/em\u003e\u003csub\u003e450\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eIC50 (\u0026micro;M) Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eIC50 (\u0026micro;M) Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePTX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDTX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePEM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e18.08\u0026thinsp;\u0026plusmn;\u0026thinsp;3.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13.95\u0026thinsp;\u0026plusmn;\u0026thinsp;3.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e134.90\u0026thinsp;\u0026plusmn;\u0026thinsp;38.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e119.00\u0026thinsp;\u0026plusmn;\u0026thinsp;40.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.88\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGEM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e14.10\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e172.70\u0026thinsp;\u0026plusmn;\u0026thinsp;58.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e77.71\u0026thinsp;\u0026plusmn;\u0026thinsp;20.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCBP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e82.23\u0026thinsp;\u0026plusmn;\u0026thinsp;8.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e61.46\u0026thinsp;\u0026plusmn;\u0026thinsp;8.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e243.40\u0026thinsp;\u0026plusmn;\u0026thinsp;38.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e238.30\u0026thinsp;\u0026plusmn;\u0026thinsp;46.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.98\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDDP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e14.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e367.70\u0026thinsp;\u0026plusmn;\u0026thinsp;91.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e326.20\u0026thinsp;\u0026plusmn;\u0026thinsp;88.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eSI, Sensitivity index, SI\u0026thinsp;=\u0026thinsp;si\u003cem\u003eCTSL\u003c/em\u003e\u003csub\u003e450\u003c/sub\u003e (IC50) / si\u003cem\u003eNC\u003c/em\u003e (IC50); SD, standard deviation; PTX, Paclitaxel; DTX, Docetaxel; PEM, Pemetrexed; GEM, Gemcitabine; CBP, Carboplatin; DDP, Cisplatin\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eCTSL enhances stemness and survival in NSCLC spheroid cells\u003c/h2\u003e\u003cp\u003eHaving established CTSL's role in LCSCs chemoresistance, we next sought to characterize its functional role in maintaining stemness and driving malignant progression. Pharmacological inhibition of CTSL with the specific inhibitor Z-FY-CHO markedly attenuated tumorsphere formation capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), a phenotype recapitulated by shRNA-mediated CTSL knockdown (Supplementary Figure S3). Notably, CTSL suppression induced parallel downregulation of both protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) and transcript (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) levels for core pluripotency factors OCT4 (POU5F1) and SOX2, indicating its critical role in maintaining NSCLC cell stemness. Intriguingly, CTSL depletion also reduced protein levels of pro-survival factors Mcl-1 and Bcl-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Conversely, CTSL overexpression upregulated these pro-survival proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and enhanced tumorsphere formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These findings collectively demonstrate that CTSL sustains LCSC populations through coordinated molecular strategy: preserving stemness and survival.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of HGFAC mediated HGF/Met axis as a target of CTSL in NSCLC cells\u003c/h2\u003e\u003cp\u003ePreviously, we have systematically described the multiple carcinogenic functions of CTSL in NSCLC spheroid cells, including stem cell maintenance, promotion of cell survival, and induction of MDR. Subsequently, to examine the molecular mechanisms responsible for these phenomena, we performed transcriptomic profiling of CTSL-overexpressing H1299 cells, identifying 75 DEGs, which were cross-referenced with drug resistance- and stem cell-associated gene sets to yield 10 overlapping candidates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; Supplementary Figure S4A). Kaplan-Meier survival analysis revealed hepatocyte growth factor activator (HGFAC) as the most prognostically significant gene among these candidates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) (hazard ratio [HR]\u0026thinsp;=\u0026thinsp;1.59; Supplementary Figure S4B-C), with its expression strongly correlating with CTSL levels. Consistently, GDSC database analysis further linked high HGFAC expression to DTX resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Mechanistically, CTSL overexpression in H1299 cells increased both HGFAC secretion and hepatocyte growth factor (HGF) secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and enhanced Met receptor phosphorylation (p-Met) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Conversely, CTSL knockdown in A549-Sp and H1299-Sp cells reduced HGFAC and HGF production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) and suppressed Met signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Functional rescue experiments demonstrated that exogenous HGF supplementation partially restored DTX sensitivity in CTSL-depleted H1299-Sp cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Colony formation assays confirmed that pharmacological inhibition of p-Met with capmatinib (Supplementary Figure S4D) reversed CTSL-driven chemoresistance to PTX, DTX, and GEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Taken together, these findings define a CTSL-HGFAC-HGF-Met signaling axis that maintains LCSC stemness and chemoresistance, thereby establishing a mechanistic rationale for targeting this pathway in NSCLC therapeutic strategies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eLung cancer, primarily NSCLC, is the most prevalent malignancy with a poor prognosis. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. While chemotherapy is a mainstay treatment, its effectiveness is frequently limited by MDR, in which lung CSCs play a critical role. This research identified CTSL as a critical functional contributor to MDR in NSCLC. Crucially, CTSL is associated with adverse clinical outcomes in NSCLC and functionally contributes to MDR in lung CSCs. CTSL expression directly increases CSC resistance to multiple chemotherapy drugs and augments their stemness properties, including viability and survival. Furthermore, combining a CTSL inhibitor with docetaxel effectively overcomes MDR and suppresses tumor growth \u003cem\u003ein vivo\u003c/em\u003e. This study establishes CTSL as a pivotal functional driver of MDR in NSCLC and identifies its inhibition as a potent strategy for reversing chemotherapy resistance.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eCTSL is a universally expressed lysosomal peptidase involved in the terminal degradation of intracellular and endocytic proteins [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Upregulation of CTSL is common in various human cancers [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and is widely associated with metastatic invasiveness and poor prognosis. Our previous reports have shown that the TGF-β/Smad signaling pathway regulates CTSL-mediated paclitaxel resistance in lung cancer, while Egr-1 and CREB are implicated in CTSL-mediated cisplatin resistance [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, the epithelial-to-mesenchymal transition (EMT) phenotype induced by CTSL upregulation has been linked to the development of cisplatin or paclitaxel resistance in A549 cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Thus, targeting CTSL and understanding its mechanisms in NSCLC could improve diagnostic and therapeutic strategies. However, the specific ways in which CTSL affects stemness and drug resistance in NSCLC have not been fully elucidated. This study showed CTSL is markedly upregulated in high-grade NSCLC tissues and stem-like cell lines, correlating with worse patient PFS. Crucially, we demonstrate that pharmacologically or genetically inhibiting CTSL potently reduces tumor spheroid formation, MDR, and tumorigenicity in vitro and in vivo, while CTSL overexpression enhances these traits. This positions CTSL inhibition as a viable strategy for clinical intervention in chemotherapy-resistant NSCLC.\u003c/p\u003e\u003cp\u003eMDR in NSCLC is intrinsically linked to the stemness properties (self-renewal, differentiation, tumorigenicity) of lung CSCs, maintained by complex signaling networks. Multiple oncogenes and signaling pathways contribute to maintaining CSC stemness and tumorigenicity. For example, HK2 inhibits the ubiquitination and degradation of CD133 by enhancing USP11 binding to CD133, thereby promoting CSC properties and tumor growth [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, TIPRL regulates stemness and survival in lung CSCs via activation of the CaMKK2-CaMK4-CREB feedback loop [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These findings offer promising new therapeutic targets for lung cancer. Our research establishes a direct role for CTSL in regulating NSCLC stemness. Inhibition of CTSL significantly diminished stemness-associated phenotypes and downregulated stemness-maintenance genes, whereas CTSL overexpression amplified these features. This provides direct evidence that CTSL is a key molecular driver sustaining the CSC population responsible for MDR.\u003c/p\u003e\u003cp\u003eHGFAC is a protease that activates HGF through proteolytic cleavage[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The HGF/MET axis is a well-established regulator of tumor growth, survival, metastasis, and CSC functions. HGF binding to MET triggers receptor dimerization, autophosphorylation (e.g., Tyr1234/1235, Tyr1349/1356), and activation of downstream pathways (MAPK, PI3K/AKT, STAT3), promoting cell survival and chemoresistance[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Consequently, the HGFAC/HGF/MET axis plays a pivotal role in regulating tumor stem cell functions\u0026mdash;such as self-renewal, proliferation, and differentiation\u0026mdash;contributing to tumor initiation, progression, and chemoresistance [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. A key finding of this study is that CTSL acts as a direct upstream regulator of HGFAC expression. This CTSL/HGFAC/HGF/MET axis constitutes the core mechanism linking CTSL to NSCLC stemness maintenance and MDR development, establishing the novel role of CTSL in controlling HGF/MET signaling in this context.\u003c/p\u003e\u003cp\u003eDespite these insights, our study has some limitations. We did not fully elucidate the specific mechanism by which CTSL regulates HGFAC, and further experiments are needed to clarify how CTSL interacts with HGFAC to affect stemness and drug resistance. Nevertheless, our findings highlight the CTSL-HGFAC axis as a potential therapeutic target for overcoming chemoresistance in NSCLC. Thus,our future studies will specifically investigate the molecular mechanism by which CTSL regulates HGFAC expression, examining whether CTSL acts at the transcriptional level or post-transcriptionally. Furthermore, we will determine how CTSL and HGFAC interact with each other. These experiments are essential to fully understand how this axis governs stemness maintenance and drug resistance, and will inform the development of targeted inhibitors or combination strategies to disrupt the CTSL-HGFAC pathway for overcoming NSCLC chemoresistance.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, our findings indicate that CTSL plays a vital role in enhancing CSC phenotypes and chemoresistance in lung cancer, suggesting that targeting CTSL may represent a promising strategy for improving chemotherapy outcomes in NSCLC patients.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCBP carboplatin\u003c/p\u003e\n\u003cp\u003eCCK-8 Cell Counting Kit-8\u003c/p\u003e\n\u003cp\u003eCSCs cancer stem cells\u003c/p\u003e\n\u003cp\u003eCTSL Cathepsin L\u003c/p\u003e\n\u003cp\u003eDDP cisplatin\u003c/p\u003e\n\u003cp\u003eDEGs differentially expressed genes\u003c/p\u003e\n\u003cp\u003eDFI disease-free interval\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDOX doxorubicin\u003c/p\u003e\n\u003cp\u003eDSS disease-specific survival\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDTX docetaxel\u003c/p\u003e\n\u003cp\u003eEMT epithelial-to-mesenchymal transition\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGEM gemcitabine\u003c/p\u003e\n\u003cp\u003eGSCA Gene Set Cancer Analysis\u003c/p\u003e\n\u003cp\u003eHGFAC HGF activator\u003c/p\u003e\n\u003cp\u003eLUAD lung adenocarcinoma\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLUSC lung squamous cell carcinoma\u003c/p\u003e\n\u003cp\u003eMDR multidrug resistance\u003c/p\u003e\n\u003cp\u003eNSCLC non-small cell lung cancer\u003c/p\u003e\n\u003cp\u003eOS overall survival\u003c/p\u003e\n\u003cp\u003ePEM pemetrexed\u003c/p\u003e\n\u003cp\u003ePFS progression-free survival\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePTX paclitaxel\u003c/p\u003e\n\u003cp\u003eSI sensitivity index\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRI resistance index\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe data are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful for the support from the Biorender for Graphical Abstract（ Created in \u0026nbsp;https://BioRender.com）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the grants from the National Natural Science Foundation of China (Grant No. 82172840 and 82272871), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (Grant No. 21KJB320017), Gusu Health Talents Project of Suzhou Municipal Health Commission (Grant No. GSWS2022062), Chinese Pharmaceutical Association Hospital Pharmacy Department (Grant No. CPA-Z05-ZC-2024002), and Bethune Charitable Foundation (Grant No. Z04J2023E095). The funders had no role in study design, data collection, analysis or interpretation of the data, preparation of the manuscript or decision to publish the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Pharmacy, Children\u0026rsquo;s Hospital of Soochow University, Suzhou 215025, China\u003c/p\u003e\n\u003cp\u003eWenjuan Wang \u0026amp;Hui Shi\u0026amp;Sha Hu\u0026amp; Xi Chen\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollege of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China\u003c/p\u003e\n\u003cp\u003eWenjuan Wang \u0026amp;Hui Shi\u0026amp;Sha Hu\u0026amp; Xi Chen\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDepartment of Pharmacy, Medical Science University and Suzhou Technology China Innovation Center, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School of Nanjing Medical University, Suzhou 215001, China.\u003c/p\u003e\n\u003cp\u003eXinyuan Ding\u0026amp; Jianyu Xu\u0026amp; Juan Wu\u0026amp; Qianfang Hu\u0026amp;Qian Liu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHui Shi: Writing original draft, methodology, investigation, and visualization. Jianyu Xu: Writing original draft, methodology, and investigation. Juan wu: Data curation, methodology, and funding acquisition. Sha Hu, Xi Chen, Qianfang Hu and Qian Liu: Methodology, investigation, validation, and data curation. Xinyuan Ding: Review and editing of manuscript critically for important intellectual content, and funding acquisition. Wenjuan Wang: Conception, design, funding acquisition, review and editing of manuscript. All authors read, revised and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Wenjuan Wang or Xinyuan Ding\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no known competing financial interests or personal relationships that could have appeared to influence this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdelaziz RF, Hussein AM, Kotob MH, Weiss C, Chelminski K, Stojanovic T, Studenik CR, Aufy M (2023) Enhancement of Radiation Sensitivity by Cathepsin L Suppression in Colon Carcinoma Cells. 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Lancet Oncol 18:e66. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s1470-2045(16)30680-5\u003c/span\u003e\u003cspan address=\"10.1016/s1470-2045(16)30680-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Non-small cell lung cancer, Multidrug resistance, Stemness, Cathepsin L, HGF activator","lastPublishedDoi":"10.21203/rs.3.rs-7459610/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7459610/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMultidrug resistance (MDR) in non-small cell lung cancer (NSCLC) is a major cause of chemotherapy failure, with lung cancer stem cells (CSCs) playing a central role in the development of MDR. Although protease family member Cathepsin L (CTSL) is known to be associated with tumor progression, its function in lung CSCs and MDR remains unclear. The chemotherapeutic sensitivities of spheroid from NSCLC cell lines were evaluated using the CCK8 assay. Western blot and qPCR analyses were performed to assess the expression levels of CTSL, stem cell markers (CD133 and CD44), stemness-maintaining molecules (OCT4 and SOX2), drug resistance proteins (MDR1 and ABCG2). In vivo experiments were conducted to validate the chemosensitizing effects of CTSL inhibitor, while ELISA was used to measure the secretion levels of HGF activator (HGFAC) and HGF. Our results demonstrated that spheroid cells exhibited pronounced MDR. High expression of CTSL correlated with poor prognosis in NSCLC patients and enhanced MDR in lung CSCs. Interfering with CTSL increased the sensitivity of lung CSCs to multiple chemotherapy drugs while reducing cell stemness and survival. Moreover, the combination of CTSL inhibitor and docetaxel effectively suppressed tumor growth in vivo. Additionally, RNA-seq analysis revealed that HGFAC expression is positively correlated with CTSL levels. Finally, CCK-8 and colony formation assays demonstrated that CTSL mediates chemoresistance by regulating HGFAC/HGF/Met expression in lung CSCs. Taken together, CTSL plays a pivotal role in NSCLC multidrug resistance. The CTSL-HGFAC axis represents a promising therapeutic target for reversing MDR in NSCLC.\u003c/p\u003e","manuscriptTitle":"The lysosomal cysteine protease Cathepsin L promotes stemness and multidrug resistance of non-small cell lung cancer by targeting HGF activator","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-08 10:12:37","doi":"10.21203/rs.3.rs-7459610/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-04T06:18:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-30T07:18:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216001808651637417694622850719804450285","date":"2025-08-30T07:05:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-28T06:25:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-28T06:22:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-26T15:40:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular and Cellular Biochemistry","date":"2025-08-26T06:49:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a6540813-8b9f-4e3f-b176-12739a8e2b08","owner":[],"postedDate":"September 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T16:05:32+00:00","versionOfRecord":{"articleIdentity":"rs-7459610","link":"https://doi.org/10.1007/s11010-025-05423-8","journal":{"identity":"molecular-and-cellular-biochemistry","isVorOnly":false,"title":"Molecular and Cellular Biochemistry"},"publishedOn":"2025-11-03 15:57:17","publishedOnDateReadable":"November 3rd, 2025"},"versionCreatedAt":"2025-09-08 10:12:37","video":"","vorDoi":"10.1007/s11010-025-05423-8","vorDoiUrl":"https://doi.org/10.1007/s11010-025-05423-8","workflowStages":[]},"version":"v1","identity":"rs-7459610","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7459610","identity":"rs-7459610","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}