Activation of NF-κB/ALDH1A1 Signaling Promotes Non-Mutational Resistance to EGFR-TKIs in Non-Small Cell Lung Cancer  

Activation of NF-κB/ALDH1A1 Signaling Promotes Non-Mutational Resistance to EGFR-TKIs in Non-Small Cell Lung Cancer | 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 Activation of NF-κB/ALDH1A1 Signaling Promotes Non-Mutational Resistance to EGFR-TKIs in Non-Small Cell Lung Cancer Lingyu Zhang, Guibin Weng, Chunjiang Liu, Qiumei Li, Jieyu Li, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6132050/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study investigates the molecular mechanisms underlying acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC). First, we utilized NSCLC cells, lung cancer organoids (LCOs), xenograft tumor, and patient-derived orthotopic xenografts tumor models to establish TKI resistance. Their stem cell-like properties, drug sensitivity, tumor formation in vitro and in vivo and transcriptomic profiles were analyzed. The differentially expressed genes related to TKI resistance and associated pathways were identified. Functional experiments, including gain- and loss-of-function assays, dual-luciferase reporter analysis, chromatin immunoprecipitation, Western blot, and RT-qPCR, revealed the roles of NF- κ B/ALDH1A1 signaling in acquired resistance. Compared to the parental cells and LCOs, the TKI-resistant R-PC9 and LCO/R exhibited increased RELA phosphorylation, higher frequency of ALDH1 + cells, enhanced sphere formation. ALDH1A1 over-expression promoted sphere formation and enhanced resistance to TKIs while ALDH1A1 or RELA silencing had opposite effects. Furthermore, RELA activation enhanced ALDH1A1 activity, and pharmacological inhibition of either NF- κ B or ALDH1A1 activity enhanced the sensitivity to TKIs both in vitro and in vivo. These findings indicated that aberrant activation of the NF- κ B/ALDH1A1 signaling promoted non-mutational resistance to EGFR-TKIs and provided a therapeutic target for EGFR-resistant NSCLC. NSCLC EGFR-TKIs Resistance NF-κB ALDH1A1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Somatic mutations, including the L858R mutation in exon 21 and the deletion in exon 19, in the epidermal growth factor receptor (EGFR) activate the EGFR-related signaling, leading to the development of non-small cell lung cancer (NSCLC). Approximately 15% of Caucasian patients with NSCLC have such mutations and East-Asian NSCLC patients have a 50% prevalence of such mutations [ 1 , 2 ]. NSCLC patients with such mutations in the EGFR usually respond to EGFR-TKIs, like erlotinib (Erlo), afatinib (Afa), and osimertinib (Osi). Actually, treatment with EGFR-TKIs has significantly extended the progression-free survival (PFS) to 10–14 months, outperforming traditional chemotherapy [ 3 , 4 ]. Unfortunately, many NSCLC patients develop resistance to EGFR-TKIs following treatment with the TKI, which may stem from selective pressure for preexisted subclones of non-responding cancer cells or from evolutions of initially responsive cancer cells. This latter development of acquired resistance may be more prevalent than the emergence of pre-existing resistant subclones [ 5 , 6 ]. Previous studies have revealed that the molecular mechanisms underlying the development of acquired resistance to TKIs may be attributed to secondary EGFR mutations, changes in other receptor tyrosine kinases (RTKs) or downstream signaling molecules, alternative signaling activation, and phenotypic shifts [ 6 , 7 ]. However, approximately one-third of NSCLC cases with resistance to TKIs do not have such changes. Apparently, other molecular mechanisms may contribute to the development of resistance to TKIs and need to be investigated. Cancer stem cells (CSCs) are a small subset of cancer cells and have a high tumorigenicity by initiating tumor formation, tumor growth, epithelial-to-mesenchymal transition process, metastasis, and relapse as well as drug resistance [ 8 ]. Lung CSCs can be identified by the expression of surface markers, such as CD133, CD44, ALDH1A1, and ABCG2, and are regulated by the Notch, Wnt, and cell cycle signaling pathways, which are also critical for the development and maintenance of drug resistance [ 9 – 12 ]. Previous studies have developed anti-CSC therapeutics that target the surface markers of CSCs and associated pathways in different cancer types in animals and those anti-CSC therapies are being tested in clinical trials [ 13 – 15 ]. Thus, further understanding the role of CSC in EGFR-TKI resistance is critical for the development of new therapeutic strategies for the treatment of TKI-resistant NSCLC. Stem cells can self-renew and form 3D organoids in vitro culture in the presence of optimal supporting factors and the formed organoids can sustain near physiological tissues [ 16 ]. Similarly, CSCs can also form organoids and carry many genetic and histological features of the parental tumors [ 17 – 19 ]. It is notable that stem cells can differentiate into different types of cells so that an organoid from stem cells or organ progenitors can contain more than one organ-specific type of cells [ 20 ]. Conceivably, the organoid is a well-characterized in vitro model for scrutinizing the self-renewal, differentiation and drug resistance of CSCs. To explore new mechanisms underlying the acquisition of EGFR-TKI resistance in lung CSCs, we established Osi resistant lung cancer organoids (LCOs). Combining Smart-RNA sequencing with biological experiments, our research discovered that the NF-κB signaling and EGFR-TKI-related signaling were enriched in ALDH + stem-like cells, thereby mediating resistance to EGFR-TKIs in NSCLC cells. Furthermore, treatment withALDH1A1 and NF-κB inhibitors attenuated the resistance to EGFR-TKIs by suppressing the NF-κB/ALDH1A1 axis and the stemness of EGFR-TKI resistant NSCLC. 2. Materials and Methods 2.1 Human tissue samples Surgical lung cancer tissue samples were obtained from individual patients in the Department of Thoracic Surgery of Fujian Provincial Tumor Hospital. Their demographic and clinical characteristics, including providing tissues for the establishment of organoids are shown in Supplementary Table 1. Individual patients with NSCLC were diagnosed by radiological imaging and pathology. Their tumors were staged using the TNM system. Written informed consent was collected from individual patients. The experimental protocol was approved by the Ethics Committee of Fujian Provincial Tumor Hospital (Approval Number: K2023-118-01). 2.2 Establishment and culture of EGFR-TKI resistant lung cancer organoids The establishment of LCOs was performed as described previously 20 . The established LCOs were counted, embedded in 30 µL of Matrigel (356231, Corning) at 10,000 crypts/mL and cultured using the LCO kit (K2318-LA, BioGenous). To establish EGFR-TKI-resistant LCOs, the primarily cultured cancer cells were treated with different concentrations (1/4 IC50 of LCO1, LCO2 and LCO3) of osimertinib in a stepwise-increase manner. After culture for 4 months (about 12 generations), the cells were able to grow in media containing 8, 6, or 10 µM osimertinib and were considered as EGFR-TKI resistant LCOs (collectively referred to as LCOs/OR). 2.3 Cell line and culture Human NSCLC H1650 (del E746-A750), PC-9 (EGFR exon 19 deletion) and H1975 (L858R/T790M) cells with varying levels of sensitivity to EGFR-TKIs were obtained from Shanghai Cell Bank (Shanghai, China) and identified by STR. The cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin) at 37.0°C in a 5% CO2 incubator. 2.4 Establishment of EGFR-TKIs-resistant NCI-PC9 cells To establish an EGFR-TKIs resistant subline, PC9 cells were treated with 0.01 µM, 0.05 µM and 0.012 µM of erlotinib, afatinib, and osimertinib, respectively. These drug concentrations were almost 1/4 IC50 for PC9, and their concentrations increased gradually. After culture for 6 months (about 20 generations), the cells were able to grow in 6 µM (erlotinib) and 5 µM (afatinib, osimertinib) and considered as EGFR-TKI resistant PC9 cells (collectively referred to as R-PC9). The newly established erlotinib- (afatinib, osimertinib)-resistant PC9 cells were named PC9/ER (PC9/AR, PC9/OR) cells. Simultaneously, some parental PC9 cells were cultured in drug-free medium and served as the controls. 2.5 CCK8 assay The impact of reagents on the viability of PC9, PC9/ER, PC9/AR and PC9/OR cells was tested by CCK8 assay using the specific kit. Briefly, the different groups of cells were treated with the same reagent(s) for 72 h. Individual wells were added with the CCK8 solution and incubated at 37°C for 4h. The absorbance at 450 nm in individual wells was measured using a microplate reader (Thermo Multiskan GO, USA). The potential synergism, additivity and antagonism of these reagents were analyzed by the combination indexes (CI) using the CompuSyn synergism/antagonism analysis software (Version 1.0, ComboSyn, Paramus, NJ, USA) (30). 2.6 ATP assay The viability of the LCOs was determined using the CellTiter-Glo 3D Cell Viability Assay (Vazyme), following the manufacturer's protocol. In brief, the CellTiter-Glo reagent was mixed with the organoid culture medium in a 1:1 volume ratio. Luminescence was subsequently measured using a multi-plate reader. 2.7 Colony formation assay PC9, PC9/ER, PC9/AR and PC9/OR cells (800 cells/well) were cultured in 12-well plates for 24 h, and the cells were treated with indicated concentrations of compounds for 7 days. The cells were washed with PBS and fixed with 4% paraformaldehyde, followed by staining with crystal violet solution. Typical images were photographed and the survival colony rates were calculated, relative to the number of colonies in the control group. 2.8 Antibodies Information regarding all antibodies used in this study is shown in Supplemental Table 2. 2.9 Western blot assay The cells or LCOs were harvested and lyzed on ice for 20 min in NP-40 lysis buffer containing 1 × PMSF, phosphatase inhibitors, and protease inhibitors. After centrifuged, their protein concentrations were determined using a BCA method. The lysate samples (30 µg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% dry milk in TBST and incubated at 4°C with primary antibodies overnight. After being washed, the bound antibodies were reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies, and visualized with ECL (P0018S, Beyotime, Jiangsu, China) in a chemiluminescence imaging system (BIO-RAD, USA). 2.10 Whole-exome sequencing (WES) The LCOs and their parent tumor tissues were harvested and their DNA was extracted, followed by WES in Novogene (Beijing) using Illumina PE150. The clean data were mapped to the reference genome ((GRCh37/hg19/GRCh38) using the Burrows Wheeler Aligner (BWA) software (Li et al., 2018) and Samblaster (Faust et al., 2014) to generate BAM files. Somatic variants were identified, according to the reference and tumor or organoid sequencing data (MuTect) with default parameters (Cibulskis et al., 2013). The functional predictions and annotations were analyzed using ANNOVAR (Wang et al., 2010). To detect somatic copy number alternations (CNAs), the BAM files were analyzed to compare the organoids with tumor tissues using Control-FREEC (Boeva et al., 2012). Mutational signatures were analyzed using the BSgenome in the R package. 2.11 Smart-seq2 SMART-seq2 data were analyzed by Wancheng Biotechnology, Shanghai, China. Total RNA was extracted from parental LCO1-3 and LCO/OR1-3 in the transwell system using lysis solution (Wancheng Biotechnology), According to the manufacturer’s instructions. The prepared RNA was sequenced on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA). The levels of gene transcripts were quantified using StringTie software and the differentially expressed genes (DEGs) between groups were identified using the edgeR and DEseq2. The potential functions of DEGs were analyzed by KEGG and Reactome enrichment analysis (R-package: ComplexHeatmap). 2.12 LCO and cell viral transduction All recombinant lentiviral and control vectors were constructed by GenePharma (Shanghai, China). When transducing organoids, the LCOs were digested into single cells and then mixed with the viral solution for 4 hours. The cells were mixed with Matrigel, and cultured for 3 to 7 days in the presence of 2 µg/mL puromycin. In addition, different groups of cells were cultured in 6-well plates up to a confluence of 50% and transduced with virus for 48 to 96 hours, followed by treatment with 2 µg/mL puromycin. The established stable cell lines expressing the gene of interest were cultured for subsequent experiments. The shRNA target sequences used in this study are listed in the Supplemental Table 3. 2.13 Nuclear–cytoplasmic fractionation Cell fractionation was performed utilizing the Cytoplasmic and Nuclear Protein Extraction Reagents as per the manufacturer's guidelines (Thermo Fisher Scientific, cat# 78833). The resulting cellular fractions were centrifuged at 16,000 g for 20 minutes at 4°C. Subsequently, their supernatants were carefully collected for further analysis. 2.14 Aldefluor assay The activity of ALDH enzyme in different groups of cells was analyzed by the Aldefluor assay using an ALDE-FLUOER kit (01700; Stem Cell Technologies), following the manufacturer's protocol. In brief, individual groups of cells were suspended and cultured with the activated ALDE-FLUOER reagent at 37°C for 45 minutes. The diethylaminobenzaldehyde (DEAB) was used as a control. The frequency of ALDH1 + cells were analyzed by flow cytometry (BD Biosciences, NJ, USA). 2.15 Hematoxylin and eosin (H&E) and immunohistochemistry staining The LCOs were firstly isolated from Matrigel using Cell Recovery Solution (Corning). The PDOs and tissue samples were fixed with fresh 4% paraformaldehyde at 4°C for one day, and paraffin-embedded. The tissue sections (5 µm) were routine-stained with H&E and immunohistochemistry staining using primary anti-TTF-1 and anti-CK7. H&E and immunohistochemical images were acquired under an inverted microscope (Leica). 2.16 Immunofluorescence After treatment, the cells, LCOs and tissues were fixed, ruptured, and blocked sequentially. The samples were probed with primary anti-NF-κB, anti-p-NF-κB, anti-MDR1, anti-ALDH1A1, overnight at 4℃. After being washed with PBS, the samples were reacted with Alexa Fluor 482 /628/ conjugated secondary antibody (Termo Fisher Scientifc), followed by nuclear-stained with DAPI (1:1000, Beyotime). The fluorescent imaging was captured by a confocal Microscope. 2.17 Spheroid colony formation PC9 and R-PC9 cells (3000 cells/well) were cultured in a mixed matrix gel in 24-well plates. Following a 7-day incubation in DF-12 medium, the spheres (> 50 mm in diameter) in individual wells were quantified to measure the capacity of cells for their self-renewal. Images were taken, and the numbers of spheres were counted. 2.18 Luciferase reporter assay The different groups of cells were transfected with pNF-kB-Luc, pRL-TK (Genepharma, shanghai, China) using Lipofectamine 3.0 (HanBio, shanghai, China), following the protocol provided by the manufacturer. After culturing for 48 h., the luciferase activities in individual wells were measured using the Dual-Luciferase reporter assay kit (Promega). The firefly luciferase activity in each sample was normalized to Renilla luciferase activity. 2.19 Chromatin immunoprecipitation assay The potential target DNA of p65 was analyzed by chromatin immunoprecipitation (ChIP) assay using the EZ-ChIP kit (17–295; Millipore), according to the manufacturer’s protocol. Briefly, 1x107 cells were fixed in 1% formaldehyde, lyzed, and sonicated using an ultrasonic processor (Biorupter Pico, Biorupter Pico). The cellular samples were incubated with anti-p65 antibody (5 µl per 1 mg of total protein) and reacted with rabbit IgG-beads (2 µl per 1 mg of total protein), followed by centrifugation. After being washed, the bound DNA was extracted and used for PCR amplification with specific primers (Supplemental Table 4). 2.20 Caspase3 apoptosis assay After drug treatment, the cells were treated with a green fluorescent caspase-3 probe (0.5 µM, 40273ES60, YEASEN, Shanghai, China). The apoptotic cells were photoimaged under a laser confocal microscope (TCS SP8, Leica). 2.21 TUNELAssay The frequency of apoptotic cells in individual tissue sections was quantified by the TUNEL assay using the In Situ Cell Death Detection Kit, following the manufacturer’s protocol. Briefly, after incubation of tissue sections with the specific solutions at 37°C for 30 minutes, the sections were reacted with Alexa Fluor-labeled secondary antibodies (Invitrogen) and nuclear-counterstained with DAPI. The fluorescent signals were photoimaged under a laser scanning confocal microscope (TCS SP8, Leica). 2.22 RNA-seq and data analysis The transcriptomic profiles of parental PC9, PC9/ER, PC9/AR and PC9/OR cells were analyzed by RNA-seq. Briefly, total RNA was extracted from individual types of cells and submitted to Novogene Bioinformatics Technology (Beijing, China) for sequencing. The raw data were analyzed for their quality and gene expression quantification by Annoroad (Beijing). The DEGs were defined by log2 fold change ≥ 1 and p-value < 0.05 using DESeq R package. The DEGs were analyzed by KEGG pathway, Venn diagram and Heatmaps ( https://magic.novogene.com ). 2.23 RT-qPCR assay The relative levels of gene mRNA transcripts to the control GAPDH in the different groups of cells were quantified by RT-qPCR using specific primers (Supplemental Table 5) and the SYBR Green PCR Master Mix (Life Technologies). The PC9 and R-PC9 cells were treated with different concentrations of EGCG-NPs and EGFR-TKIs for 48 h. Their total RNA was extracted with TRIzol reagent (Invitrogen, USA) and reversely transcribed into cDNA using a PrimeScript RT Reagent Kit (TaKaRa #RR047A), followed by PCR. The data were analyzed the 2-ΔΔCt method. 2.24 Tumorigenicity study Animal studies were carried out in accordance with the guidelines for the care and use of human specimens and animals, and approved by the Institutional Review Board of Fujian Medical University (Approval Number: IACUC FJMU 2024 − 0177). Male athymic nude mice and NSG mice were obtained from Fuzhou Nordens Biotechnology and housed in a specific pathogen-free facility in campus. To establish xenograft tumors, individual male athymic nude mice at 5 weeks of age were implanted subcutaneously with 5 × 106 PC9/OR cells into their right flanks. The development of tumors was monitored frequently. When the tumors reached about 100 mm 3 , the mice were randomized and treated with vehicle (CTL, DMSO, Corn Oil), Osi (5 mg/kg/d, P.O by gavage), DSF (5 mg/kg/d, P.O), EGCG (8 mg/kg, i.p., every other day), Osi + DSF (P.O, daily for 16 days), or a combination of Osi + EGCG. For a PDOX model, the same age of individual male NSG mice were injected subcutaneously with 5x106 LCO1/OR cells in Matrigel (0. 1 mL 50%; 356231, BD Biosciences) into their right flank. For PDOX generation, tumors were passaged to additional athymic male nude mice when they reached about 1000 mm3. The dosing regimen was consistent with the aforementioned plan. The tumor volumes were measured and calculated using the equation [(width) 2 × (height)]/2. Tumor sizes and mouse body weights were measured every other day. After euthanizing the mice at the end of the study period, their tumors were dissected for Western blot to examine the expression levels of p-RELA and ALDH1A1. Additionally, the frequency of apoptotic cells in tumor tissues was analyzed by the TUNEL assay. 2.25 Statistical analysis Each experiment was repeated three times. Data are expressed as the mean ± standard deviations (SD). The difference between the control and experimental groups was analyzed by a Student t test using GraphPad Prism 8.0 software and the different levels of significance were defined as * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.001, ns., no significant, P ≥ 0.05. 3. Results 3.1 Aberrant activation of the NF-κB signaling is associated with the development of resistance to EGFR-TKIs in NSCLC To unravel the intricate mechanisms underlying the acquisition of EGFR-TKI resistance in lung CSCs, we developed three LCOs derived from surgical NSCLC specimens with the EGFR exon 19 deletion (Supplemental Table 1). These LCOs faithfully recapitulated the characteristics of primary tissues (Fig. 1 A, Supplemental Fig. 1A-D). Subsequently, we investigated the drug responses of these LCOs. Enhanced CT scans exhibited a significant reduction in tumor size, confirming the efficacy of the EGFR-TKI. Consistently, these LCOs were resistant to varying concentrations of TKIs (Supplemental Fig. 2, Fig. 1 B-C). Next, we generated three Osi-resistant LCO variants (LCO1-3) by gradually increasing Osi concentrations in the culture medium. The Osi-resistant LCOs displayed markedly higher resistance to Osi, with 7- to 12-fold greater IC50 Osi than their parental LCOs (Fig. 1 B-C). Notably, HE staining revealed no obviously histological change in LCOs before and after developing drug resistance (Supplemental Fig. 1E). Immunohistochemistry exhibited a substantial elevation in multidrug resistance 1 (MDR1) expression, a key marker of drug resistance [ 21 ] (Supplemental Fig. 3A). Conversely, the levels of KI67 expression, a proliferation marker, were reduced in drug-resistant LCOs (LCOs/OR), implying that drug resistance was associated with inhibition of cell proliferation (Supplemental Fig. 3B). To elucidate the molecular underpinnings of Osi resistance acquisition in LCOs/OR, we analyzed the transcriptomic profiles of both the parental LCOs (LCO1-3) and their Osi-resistant counterparts (LCO1-3/OR) by RNA-seq. Principal component analysis (PCA) of the RNA-seq data revealed a striking similarity in the gene expression profiles between each pair of parental LCOs and its corresponding LCOs/OR (Supplemental Fig. 1F). Gene enrichment analysis uncovered a significant increase in the expression of genes in multiple stemness-related signaling pathways in the LCO1-3/OR when compared to the parental LCOs, and the NF-κB pathway was remarkably enriched in the LCO1-3/OR (Fig. 1 D). Western blot and immunofluorescent analyses unveiled an obvious elevation in the relative levels of phosphorylated RELA to RELA expression in LCOs/OR, relative to that in their parental LCOs (Fig. 1 E-F and Supplemental Fig. 3C). We also established stable Erlo-resistant PC9/ER, Afa-resistant PC9/AR and Osi-resistant PC9/OR cell lines, collectively known as R-PC9 (Supplemental Fig. 4A) using the dose-escalation method [ 22 ] (Supplemental Fig. 4B-C). Whole exome sequencing (WES) revealed no major single-nucleotide variant pattern shifts between PC9/ER and PC9 cells, and yet variations were identified in PC9/AR and PC9/OR (Supplemental Fig. 5). Notably, aside from the intrinsic EGFR exon 19 deletion, there was no mutation in reported TKI-resistant gene sets (i.e., EGFR T790M, EGFR C797S, HER2 amp) in these cell lines. Similar to the LCOs, R-PC9 cells displayed a slower rate of proliferation, evidenced by less EDU labeling (Supplemental Fig. 4D). Surprisingly, there was no significant enrichment in the NF-κB pathway in the R-PC9 lines (Supplemental Fig. 6). Given the differences, we isolated and analyzed nuclear-cytoplasmic fractionations on R-PC9 cells, and the results indicated a significant increase in the relative ratios of nuclear phosphorylated RELA to RELA expression, but not cytoplasmic RELA phosphorylation, in R-PC9 cells, relative to that in their parental PC9 cells (Fig. 1 G). These data highlighted the advantages of the organoid model and indicated that aberrant activation of the NF-κB signaling was associated with the development of EGFR-TKI resistance in NSCLC. 3.2 Up-regulated ALDH1A1 activity in the EGFR-TKI-resistant NSCLC cells We next studied how EGFR-TKI activated the NF-κB signaling in NSCLC. Considering that tumor cells with EGFR-TKI resistance may manifest stem-cell-like properties [ 23 , 24 ], we analyzed whether PC9/AR, PC9/ER and PC9/OR cells exhibited a CSC phenotype. First, the EGFR-TKI resistant cells formed significantly more numbers of spheres than PC9 cells (Fig. 2 A). Second, significantly higher expression levels of CSC-related markers, particularly for ALDH1A1, were detected in these resistant cells (Fig. 2 B). The ALDH1A1 is one of the top-ranking isozymes in the aldehyde dehydrogenase (ALDH) family, one of the biomarkers for CSCs and activates the NF-κB signaling, contributing to the immunosuppressive environment in malignant tumors [ 25 ]. In addition, the Aldefluor assay was used to evaluate ALDH1 activity by flow cytometry. The results indicated a marked increase in the proportion of ALDH1 + cells within the resistant R-PC9 cell population relative to the PC9 cells (Fig. 2 C). Similarly, significantly increased ALDH1A1 mRNA transcripts were detected in the EGFR-TKI resistant LCOs/R, relative to that in their parental LCOs and the levels of ALDH1A1 mRNA transcripts were the most up-regulated among the tested stemness factors in these LCOs (Fig. 2 D). Further IHC staining also revealed that the levels of ALDH1A1 expression increased in the LCOs/OR compared with those in the parental LCOs (Fig. 2 E). Collectively, these findings indicated that ALDH1A1 expression was significantly up-regulated in the EGFR-TKI resistant NSCLC and suggest that Up-regulated ALDH1A1 may contribute to the development of EGFR-TKI resistance in NSCLC and enhance their stemness during the development of TKI resistance. 3.3 ALDH1A1 supports the development of resistance to TKIs in NSCLC. To investigate the function of ALDH1A1, we performed the gain and loss of function experiments in PC9 and R-PC9 cells, respectively. After induction of ALDH1A1 over-expression in PC9 cells or silencing in R-PC9 cells (Supplemental Fig. 7A), we found that ALDH1A1 over-expression increased its activity and the numbers of formed spheres in PC9 cells (Fig. 3 A, Supplemental Fig. 7B). Moreover, ALDH1A1 over-expression significantly reduced the effect of Erlo, Afa and Osi on inhibiting the proliferation of PC9 cells (Fig. 3 B). Conversely, ALDH1A1 silencing mitigated the ALDH1A1 activity (Supplemental Fig. 7C-E), decreased the numbers of formed spheres (Supplemental Fig. 7F-G) and enhanced the effects of TKIs on inhibiting the proliferation of R-PC9 cells (Fig. 3 C). Given that ALDH1A1 silencing enhanced the sensitivity to EGFR-TKIs in R-PC9 cells, we tested whether pharmacological inhibition of ALDH1A1 enzymatic activity could modulate drug resistance in PC9 cells. We found that treatment with disulfiram (DSF, a selective ALDH1A1 inhibitor) inhibited the proliferation of R-PC9 and PC9 cells in a dose-dependent manner and the sensitivity of drug-resistant R-PC9 cells to DSF was higher than that of PC9 cells (Supplemental Fig. 8A). Furthermore, treatment with different concentrations of DSF, in combination with different concentrations of each TKI, unveiled that high concentrations of TKIs and DSF had antagonistic effect on inhibiting the proliferation of R-PC9 cells while treatment with low concentrations of TKIs and DSF resulted in a synergistic effect on inhibiting the proliferation of R-PC9 cells (Fig. 3 D). Moreover, treatment with a low concentration of DSF synergistically enhanced the inhibition of TKIs on the proliferation and clonal formation of R-PC9 cells (Supplemental Fig. 8B-F). More importantly, treatment with both the TKI and DSF increased the relative levels of cleaved PARP and cleaved Caspase3 in R-PC9 cells (Fig. 3 E). Thus, pharmacological inhibition of ALDH1A1 enzymatic activity enhanced the cytotoxic effect of EGFR-TKIs on the viability of drug-resistant NSCLC cells by triggering their apoptosis. We further validated the regulatory function of ALDH1A1 in TKI resistance in LCOs. We also observed that ALDH1A1 silencing in LCOs/OR significantly enhanced their sensitivity to Osi (Fig. 3 F, Supplemental Fig. 9A-B). Similarly, LCOs/OR wer e more sensitive to DSF than their parental LCOs (Supplemental Fig. 9C). Remarkably, treatment with both low concentrations of Osi and DSF synergistically inhibited the proliferation of LCOs/OR (Fig. 3 G-H, Supplemental Fig. 9D-E). More interestingly, treatment with both Osi and DSF synergistically enhanced Caspase3 expression in LCOs (Fig. 3 I, Supplemental Fig. 9F-G). Collectively, these data indicated that ALDH1A1 positively supported the development of resistance to TKIs in NSCLC and inhibition of ALDH1A1 partially restored the sensitivity to TKIs in NSCLC. 3.4 RELA regulates the activity of ALDH1A1 in NSCLC RELA, also known as p65, is a transcription factor. Phosphorylated RELA can bind to the promoters of its regulated genes, regulating the expression of those genes, such as inflammatory cytokines [ 26 ]. To understand how the ALDH1A1 expression was up-regulated in drug-resistant NSCLC, we performed bioinformatics to identify the potential binding sites of transcription factors in the ALDH1A1 promoter. We performed ChIP assays and found that anti-p65 effectively precipitated the ALDH1A1 promoter region in NSCLC cells (Fig. 4 A-B). These findings underscored the likelihood of RELA to regulate ALDH1A1 expression in NSCLC. Immunofluorescence revealed that RELA expression was co-localized with ALDH1A1 expression in NSCLC from the patients, who were refractory to EGFR-TKI targeted therapy, but not those responding to EGFR-TKI therapy (Fig. 4 C, Supplemental Fig. 10A-B). These suggest that ALDH1A1 may be a target gene of RELA during the development of EGFR-TKI resistance in NSCLC. Notably, the percentages of ALDH1 + CSCs in clinical NSCLC specimens with EGFR L858 mutation or 19del mutation were correlated positively with the percentages of RELA + tumor cells (Supplemental Fig. 10C). Furthermore, RELA silencing decreased the frequency of ALDH1 + cells in PC9/OR (Fig. 4 D-E). Consistently, RELA silencing decreased the levels of ALDH1A1 gene expression, ALDH1 + cells, and sphere formation and partially restored the sensitivity to Osi in LCO1/OR (Fig. 4 F-H). Collectively, these findings indicated that RELA positively regulated ALDH1A1 activity, contributing to the development of EGFR-TKI resistance in NSCLC. 3.5 EGCG potentiates the sensitivity to EGFR-TKIs by inhibiting the NF-κB signaling and downstream ALDH1A1 activity in NSCLC. We next investigated whether pharmacologic inhibition of the NF-κB could modulate the resistance to EGFR-TKIs in NSCLC. Currently, the clinically available inhibitors of the NF-κB signaling only indirectly target either IKK or the proteasome, which leads to non-specific effects and limited therapeutic efficacy in patients [ 27 ]. Previous studies have shown that treatment with EGCG effectively inhibits the proliferation of lung cancer cells by down-regulating the NF-κB signaling[ 28 , 29 ]. Accordingly, we chose EGCG as an inhibitor of the NF-κB signaling. Luciferase reporter assays unveiled that treatment with EGCG inhibited the transcriptional activity of the NF-κB promoter and enhanced the inhibition of TKI on the transcriptional activity of the NF-κB promoter in R-PC9 cells (Fig. 5 A). A similar pattern of luciferase activities was observed in LCO1/OR (Fig. 5 B). EGCG treatment limited the RELA nuclear transportation and reduced the binding of RELA to the ALDH1A1 promoter in R-PC9 cells (Fig. 5 C-D). Furthermore, treatment with both EGCG and EGFR-TKIs effectively decreased the percentages of ALDH1 + cells in R-PC9 cells (Fig. 5 E). Compared with R-PC9 cells, other NSCLC H1975 and H1650 cell lines displayed lower levels of RELA expression and phosphorylation as well as low frequency ofALDH1 + cells (Supplemental Fig. 11A-C). Interestingly, the levels of phosphorylated RELA and the ALDH1A1 activity were positively correlated with the sensitivity to EGCG in these cell lines (Supplemental Fig. 11D-E). Apparently, low RELA phosphorylation and low ALDH1A1 activity led to a lower sensitivity to EGCG in H1650 and H1975 cells. Consistently, LCOs/OR exhibited greater sensitivity to EGCG compared to LCO/pare cells, which contained a lower proportion of ALDH1 + cells (Supplemental Fig. 11F, Fig. 2 E). Thus, EGCG treatment decreased RELA phosphorylation and subsequent ALDH1A1 activity in NSCLC cells. Next, we treated R-PC9 cell lines with varying concentrations of EGCG and/or EGFR-TKIs to determine the combination index. The results indicated that EGCG and EGFR-TKIs at high concentrations exhibited antagonistic effects while they at low concentrations induced synergistic effects in R-PC9 cells and LCO1/OR. Actually, treatment with low doses of EGCG and EGFR-TKIs synergistically enhanced their inhibition on the proliferation of R-PC9 cells (Supplemental Fig. 12A) and increased the levels of cleaved PARP and cleaved caspase3 in R-PC9 cells (Fig. 5 G). A similar pattern of synergistic antitumor effect of EGCG and Osi was observed in the LCOs/OR (Fig. 5 F-H, Supplemental Fig. 12B-E). Mechanistically, we found that treatment with EGCG failed to synergistically enhance the inhibition of Osi on the proliferation of RELA-silencing R-PC9 cells and LCOs (Fig. 5 I, Supplemental Fig. 13A-B). These indicated that the EGCG-enhanced inhibition of TKIs on the proliferation of NSCLC cells depended on high levels of RELA expression in the TKI-resistant cells. Similarly, treatment with EGCG also failed to significantly increase the inhibition of different doses of Osi on the proliferation of ALDH1A1-silencing LCO1/OR (Fig. 5 J). A similar pattern of the lack of synergistic effects of EGCG and TKIs was observed on the proliferation of ALDH1A1-silencing R-PC9 and LCO/OR (Supplemental Fig. 13C-D). Therefore, EGCG treatment potentiated the sensitivity to EGFR-TKIs, dependent on high levels of RELA expression and downstream ALDH1A1 activity in the EGFR-TKI resistant NSCLC cells. Conceivably, treatment with EGCG to inhibit the NF-κB signaling may inhibit and prevent the development of resistance to EGFR-TKIs in EGFR-mutant NSCLC. 3.6 Pharmacological inhibition of NF-κB or ALDH1A1 enhances response to EGFR TKI in vivo Given that high frequency of stem-like cells was presented in EGFR-TKI resistant R-PC9 cells, we tested the tumorigenicity of R-PC9 cells by implanting different numbers of R-PC9 cells into nude mice. The results indicated that implantation with 2x106 PC9/OR cells induced solid tumors in two out of five nude mice while injection with 8x106 parental PC9 cells only triggered solid tumor formation in one out of five mice, clearly implying high tumorigenicity of R-PC9 cells (Fig. 6 A). To determine the effect of DSF on the growth of R-PC9 tumors in vivo, we established a nude mouse model of xenograft PC9/OR. After establishment of solid tumors, the tumor-bearing mice were randomized and treated with vehicle, Osi and/or DSF and their tumor sizes were monitored (Fig. 6 B-C). Quantitative analysis indicated that treatment of either 5 mg/kg Osi or 2 mg/kg DSF for 16 days moderately inhibited the growth of implanted PC9/OR tumors and treatment with both Osi and DSF synergistically suppressed the growth of implanted PC9/OR tumors in mice (Fig. 6 B-C). However, these treatments did not change the body weights among these groups of mice (Fig. 6 D). Western blot analyses displayed that treatment of both Osi and DSF obviously decreased RELA phosphorylation and ALDH1A1 expression, but increased the numbers of apoptotic cells in the PC9/OR tumor tissues (Fig. 6 E, Supplemental Fig. 14A). We further tested the effect of Osi and/or DSF treatment on the growth of implanted patient-derived organoid-based xenograft (PDOX) tumors in NSG mice. First, we detected obviously up-regulated levels of RELA and ALDH1A1 expression and co-localization in the LCO1/OR-derived PDOX tumors, relative to those LCO1-derived tumors (Fig. 6 F). Consistently, treatment with either Osi or DSF moderately decreased the tumor sizes and treatment with both Osi and DSF further reduced the tumor sizes in this model (Fig. 6 G-H). However, treatment with Osi and/or DSF did not change the body weights in these groups of mice (Fig. 6 I). Treatment with both Osi and DSF obviously reduced the levels of RELA phosphorylation and ALDH1A1 expression in the LCO1/OR-derived tumors, accompanied by increased numbers of apoptotic cells (Fig. 6 J, Supplemental Fig. 14B). These data indicated that targeting ALDH1A1 by DSF treatment enhanced the sensitivity to Osi in the EGFR-TKI resistant NSCLC in mice. Given that EGCG effectively suppressed the growth of tumor cells in vitro, we tested whether treatment with EGCG could enhance the antitumor effect of Osi in the PC9/OR-based tumor in nude mice. Like in vitro results, treatment with either 5 mg/kg Osi or 10 mg/kg EGCG slightly reduced the tumor sizes and treatment with both significantly decreased the growth of implanted PC9/OR tumors, but failed to alter the body weights in mice, accompanied by increased numbers of apoptotic cells in the tumors (Fig. 6 K-M, Supplemental Fig. 14C). A similar pattern of anti-tumor effects was observed in the LCO1/OR-derived PODX tumors (Fig. 6 O-Q). Interestingly, treatment with both Osi and EGCG obviously reduced the levels of ALDH1A1 expression and RELA phosphorylation in both the PC9/OR and LCO1/OR derived tumors (Fig. 6 N, R), but significantly increased the numbers of apoptotic tumor cells in these tumors (Supplemental Fig. 14D). These in vivo data further supported the notion that activation of the NF-κB/ALDH1A1 signaling contributed to the development of EGFR-TKI resistance in NSCLC while treatment with DSF or EGCG to inhibit the ALDH1A1 or the NF-κB restored the sensitivity to EGFR-TKIs in NSCLC tumors. 4. Discussion The EGFR mutations and the genetic alternations in other genes account for about 45% of cases with acquired resistance to EGFR-TKIs and have promoted the development of next generation targeted therapeutics and combinational therapies for NSCLC [ 31 – 32 ]. Furthermore, another 15% of lung cancer cases are attributed to the histologic transformation (e.g., SCLC) and also be treated by combined chemotherapy [ 33 , 34 ]. However, how the remaining about 40% of lung cancer cases develop the EGFR-TKI resistance is still unknown and they have no effective therapy due to the lack of molecular targets. In our study, we employed NSCLC cell lines, organoids, in vivo xenograft tumor, and PDOX tumor models to explore the potential mechanisms underlying the development of resistance to EGFR-TKIs. We identified a unique mechanism, which explained that long-term TKI treatment induced aberrant activation of the NF-κB signaling to up-regulate ALDH1A1 activity, increasing the numbers of stem-like cells and acquiring non-mutational resistance to EGFR-TKIs in NSCLC. The NF-κB is a heterodimeric complex of proteins in the REL family, including p50, p52, RELA, c-REL, and RELB, and interacts with inhibitory IκB in the cytoplasm [ 35 ]. When activated, the NF-kB is separated from IkBs, and translocates into the nuclei to regulate the targeted gene expression by binding to the gene promoter [ 36 ]. Previous studies have shown that suppression of the NF-κB and FAS significantly enhances the antitumor effect of erlotinib in NSCLC [ 37 ] and targeting the NF-κB/miR-155/FOXO3a pathway reduces the EGFR mutation-independent resistance to gefitinib, and the stemness of NSCLC cells [ 38 ]. Furthermore, inhibition of the EGFR can trigger the EGFR-TRAF2-RIP1-IKK complex to activate the NF-κB-mediated transcriptional survival program. Additionally, the TET2/NF-κB axis can act as an alternative pathway to confer non-mutational acquired resistance to EGFR-TKIs [ 39 ]. Moreover, IL-6 through the JAK2/STAT3 pathway can promote the resistance to gefitinib in NSCLC by activating the NF-κB signaling [ 40 , 41 ]. In this study, we discovered a novel aspect that aberrant activation of the NF-κB/ALDH1A1 signaling contributed to the development of resistance to EGFR-TKIs in NSCLC while pharmacological inhibition of NF-kB or ALDH1A1 activity restored the sensitivity to EGFR-TKIs in NSCLC. Hence, our findings revealed new therapeutic targets of the NF-kB/ALDH1A1 axis for preventing and inhibiting the resistance to EGFR-TKIs in NSCLC. A recent study has implicated the role of lung CSCs in the development of TKI resistance, evidenced by the facts that Oct-4 can induce gefitinib resistance in EGFR-mutant NSCLC cells by modulating their CSC properties, and up-regulated Oct-4 expression is detected in TKI-resistant NSCLC tissues [ 42 ]. Additionally, afatinib-resistant NSCLC cells display CSC traits, such as increased colony formation, proliferation, ALDH1A1, and CD44 expression [ 43 ]. This suggests that TKI resistance may stem partly from CSCs. Consistently, we found that ALDH1A1 expression was up-regulated in a subset of TKI-resistant PC9 cells and increased frequency of ALDH1 + cells occurred in R-PC9 cells. These also implied that some ALDH1 + NSCLC cells had the ability to withstand EGFR-TKI drug stress. Similarly, the findings from our LCO model indicated that the CSC-related ALDH1A1 stabilized the resistance characteristics. The transcriptome analysis revealed that aberrant activation of the NF-κB pathway up-regulated ALDH1A1 expression, increasing the frequency of ALDH1 + stem-like cells to promote the resistance to EGFR-TKIs in NSCLC. Pharmacological inhibition of ALDH1A1 decreased the frequency of ALDH1 + stem-like cells and partially restored the sensitivity to EGFR-TKIs in NSCLC in vitro and in vivo. Therefore, targeting ALDH1A1 may be a valuable strategy for preventing and inhibiting TKI resistance in NSCLC. In our study, we identified that during the development of TKI resistance, the activated RELA promoted the expression of ALDH1A1 in the TKI-resistant NSCLC cells while ALDH1A1 silencing or inhibition partially restored the sensitivity to EGFR-TKIs so that the ALDH1A1 may be a crucial enzyme for the development of drug resistance in NSCLC. Strikingly, the levels of RELA phosphorylation were obviously up-regulated and co-localized with high levels of ALDH1A1 expression in NSCLC tumors from patients resistant to EGFR-TKI therapy. This co-localization suggests a functional interaction between these two proteins, which may significantly contribute to the development of drug resistance. Thus, targeting the NF-κB/ALDH1A1 axis may be a promising strategy to combat or delay the onset of drug resistance in NSCLC. Currently, there is no effective treatment for patients resistant to EGFR-TKIs, particularly for those with no new EGFR mutant-resistant NSCLC. Fortunately, previous studies have shown that several natural products can synergistically enhance the antitumor effect of popular chemotherapy drugs [ 44 , 45 ]. EGCG, a component in green tea, has been shown to inhibit the proliferation of various NSCLC cells by inhibiting the NF-κB pathway [ 29 , 46 , 47 ]. Our data indicated that EGCG monotherapy not only decreased the nuclear translocation of RELA and the frequency of ALDH1 + stem-like cells, but also synergistically enhanced antitumor effect of TKIs in TKI-resistant NSCLC although treatment with high concentrations of EGCG mitigated the sensitivity to EGFR-TKIs in NSCLC. These data underscored the significant role of the NF-κB activation in the development of resistance to EGFR-TKIs in NSCLC and suggest that the anti-TKI resistant effect of EGCG may be attributed to its inhibition on the NF-κB pathway in NSCLC. Interestingly, the R-PC9 cells had acquired the resistance to TKIs and exhibited higher sensitivity to EGCG than their parental PC9 cells, whereas the H1975 and H1650 cells, harboring the T790M mutation and the deletion of E746-A750 in the EGFR displayed less sensitivity to EGCG. This reduced sensitivity is likely due to the lower levels of RELA phosphorylation, and lower ALDH1A1 activity in these cell lines. The diminished activity of the NF-κB and ALDH1A1 may underlie the lower sensitivity to EGCG for its inhibition on the proliferation and survival of NSCLC cells. Therefore, the level of the NF-κB and ALDH1A1 activities may be a valuable biomarker for predicting the therapeutic responses to EGCG in NSCLC. Collectively, these findings suggest that the NF-κB/ALDH1A1 axis may serve as an alternative pathway conferring non-mutational, acquired resistance to EGFR-TKIs in NSCLC. Conceivably combination of inhibitors for the NF-κB and ALDH1A1 activity with EGFR-TKIs may be valuable for preventing and inhibiting EGFR-TKI resistance in NSCLC. Conclusion Our studies revealed that the NF-κB/ALDH1A1 axis promoted the development of non-mutational EGFR-TKI resistance in NSCLC and might be a pivotal target for the development of new therapeutics to treat EGFR-TKI resistant NSCLC. Mechanistically, aberrant activation of the NF-κB and subsequently induced ALDH1A1 expression enhanced the stemness of NSCLC CSCs. Evidently, pharmacologic inhibition of NF-κB or ALDH1A1 partially restored the sensitivity to EGFR-TKIs in NSCLC. Therefore, our findings not only provided new insights into the unequal mechanisms underlying the development of non-mutational EGFR-TKI resistance in NSCLC, but also uncovered new therapeutic targets for the intervention of EGFR-TKI resistant NSCLC, particularly for combination of inhibitors for the NF-κB and ALDH1A1 activity with TKIs to achieve long-term remission of patients with TKI-resistant NSCLC. Abbreviations NSCLC Non-Small Cell Lung Cancer EGFR Epidermal Growth Factor Receptor TKIs Tyrosine Kinase Inhibitors LCOs Lung Cancer Organoids PFS Progression-free survival RTKs Receptor tyrosine kinases CSCs Cancer Stem Cells MDR1 Multidrug Resistance Protein 1 RNA-seq RNA Sequencing WES Whole-Exome Sequencing PDOX Patient-Derived Orthotopic Xenograft DSF Disulfiram EGCG Epigallocatechin Gallate IHC Immunohistochemistry IF Immunofluorescence CI Combination Index Afa Afatinib Erlo Erlotinib Osi Osimertinib R-PC9 EGFR-TKI resistant PC9 Cells PC9/OR Osimertinib-resistant PC9 PC9/ER Erlotinib -resistant PC9 PC9/AR Afatinib -resistant PC9 SDS‒PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis PVDF Polyvinylidene fluoride HRP Horseradish peroxidase CNAs Copy number alterations ChIP Chromatin immunoprecipitation Declarations Funding This study was supported by the Natural Science Foundation of Fujian Province (Grant No. 2022J05075), Fujian Provincial Health Technology Project (Grant No. 2022GGA030), Scientific Research Foundation of Fujian Cancer Hospital (Grant No. 2023YN08), High-level Talent Development Program of Fujian Cancer Hospital (Grant No. 2022YNG03), Joint Funds for the innovation of science and Technology, Fuiian province(Grant No. 2021Y9198), the Startup Fund for Scientific Research, Fujian Medical University (Grant No. 2021QH1137), the Natural Science Foundation of Fujian Province (Grant No. 2022J01742). Author Contributions Statement Yunbin Ye, Lingyu Zhang and Dali Zheng conceived and designed the experiments. Lingyu zhang, Wanson Lin and Yang Wang performed bioinformatics analyses; Lingyu zhang, Chunjiang Liu, Qiumei Li, Jieyu Li and Chuanzhong performed the in vitro experiments. Lingyu zhang, Guibin Weng and Chunjiang Liu performed the in vivo experiments. Yunbin Ye, Wanson Lin, and Lingyu Zhang performed and guided the statistical analysis. Guibin Weng provided or collected the study materials or patient samples; Yunbin Ye, Dali Zheng, Lingyu zhang, Qiumei Li and Wanson Lin wrote and reviewed the manuscript. All the authors read and approved the final manuscript. Data availability All the data generated in this study were shown in the main text and supplementary material. Additional information is also available upon reasonable request to the corresponding authors. Conflict of interests The authors declare no conflict of interests. Ethics approval and consent to participate All experiments were approved by the Medical Ethics Committee of the Fujian Provincial Tumor Hospital (K2023-118-01). Experiments were conducted in accordance with relevant institutional guidelines and regulations. Consent for publication Not applicable. References D'Angelo SP, et al. Incidence of EGFR exon 19 deletions and L858R in tumor specimens from men and cigarette smokers with lung adenocarcinomas. J Clin Oncol. 2011;15:2066-70. Sharma SV, et al. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer. 2007;3:169-81. Morgillo F, et al. Mechanisms of resistance to EGFR-targeted drugs: lung cancer. ESMO Open. 2016;3:e000060. Yun CH, et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci U S A. 2008;6:2070-5. Camidge DR, et al. Acquired resistance to TKIs in solid tumours: learning from lung cancer. Nat Rev Clin Oncol. 2014;8:473-81. Lin JJ, Shaw AT. 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Clin Cancer Res. 2021;23:6500-13. Zhou Y, et al. Epigallocatechin gallate circumvents drug-induced resistance in non-small-cell lung cancer by modulating glucose metabolism and AMPK/AKT/MAPK axis. Phytother Res. 2023;12:5837-53. Milligan SA, et al. The green tea polyphenol EGCG potentiates the antiproliferative activity of c-Met and epidermal growth factor receptor inhibitors in non-small cell lung cancer cells. Clin Cancer Res. 2009;15:4885-94. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfiles.docx SupplementaryTable15.docx Cite Share Download PDF Status: Posted Version 1 posted 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-6132050","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":423325364,"identity":"6d08b66e-c6bc-46bd-82f0-9b65b507af27","order_by":0,"name":"Lingyu Zhang","email":"","orcid":"","institution":"Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lingyu","middleName":"","lastName":"Zhang","suffix":""},{"id":423325365,"identity":"57b2c3d5-f163-41ea-b4b6-1523a0d5e7b3","order_by":1,"name":"Guibin Weng","email":"","orcid":"","institution":"Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Guibin","middleName":"","lastName":"Weng","suffix":""},{"id":423325366,"identity":"bbe7acc8-6faa-4989-96a7-4a971063798e","order_by":2,"name":"Chunjiang Liu","email":"","orcid":"","institution":"Fuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Chunjiang","middleName":"","lastName":"Liu","suffix":""},{"id":423325367,"identity":"bc8edc92-11cb-46c5-a7ef-62aae701dc1b","order_by":3,"name":"Qiumei Li","email":"","orcid":"","institution":"Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qiumei","middleName":"","lastName":"Li","suffix":""},{"id":423325368,"identity":"31cf745a-48bb-4cb4-b531-d606e4f1fadc","order_by":4,"name":"Jieyu Li","email":"","orcid":"","institution":"Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jieyu","middleName":"","lastName":"Li","suffix":""},{"id":423325369,"identity":"7c3cab52-4604-4cfd-9d37-2d3631539932","order_by":5,"name":"Yang Wang","email":"","orcid":"","institution":"Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Wang","suffix":""},{"id":423325370,"identity":"a0d3bfe8-4962-48f3-a118-9f3574926f8c","order_by":6,"name":"Chuanzhong Huang","email":"","orcid":"","institution":"Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chuanzhong","middleName":"","lastName":"Huang","suffix":""},{"id":423325371,"identity":"dd46b727-142d-4148-94b2-f0043f3abb71","order_by":7,"name":"Wanson Lin","email":"","orcid":"","institution":"Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wanson","middleName":"","lastName":"Lin","suffix":""},{"id":423325372,"identity":"508a4974-8717-42da-9d34-21b6f4caface","order_by":8,"name":"Dali Zheng","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dali","middleName":"","lastName":"Zheng","suffix":""},{"id":423325373,"identity":"493a2340-4ba0-4103-adba-bd2a4b9d31ff","order_by":9,"name":"Yunbin Ye","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYFCCAwwMjA0SDPzMBxtAXMYGorVItiUSrQWqzOBYApyNH8g3njGTLtxhkWd8jLl1Mw+DjeyGA8zPHuC3AKhl5hmJYrNjjG23eRjSjDccYDM3wKeFmQGohbdNInHb/UaQlsOJGw7wsEng08IG07K5DWzLf8JaeGBaNrCBtRwgrEWC4VixNe8ZicQZQL/cnGOQbDzzMJsZXi3yMw5vvM27oy6xv4392Y03FXayfcebn+HVwiBxAjl4QGxmvOqBgL/9ASElo2AUjIJRMNIBAF5ZSX1KhgalAAAAAElFTkSuQmCC","orcid":"","institution":"Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yunbin","middleName":"","lastName":"Ye","suffix":""}],"badges":[],"createdAt":"2025-03-01 02:38:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6132050/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6132050/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78248082,"identity":"857958cf-a70a-4410-b3ab-bebf08c7912b","added_by":"auto","created_at":"2025-03-11 09:38:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5913265,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAberrant activation of NF-κB is present in the EGFR-TKI resistant NSCLC. A. \u003c/strong\u003eHE staining and immunohistochemistry of TTF-1 and CK7 displayed that the morphology of LCOs was highly consistent with surgicalspecimens from NSCLC patients. O, LCOs; T, patients’ primary specimens. Scale bar = 50 μm; \u003cstrong\u003eB. \u003c/strong\u003eThe morphologies of andresponses to different doses of Osi in LCO1-3. \u003cstrong\u003eC \u003c/strong\u003eThe dose-response curves of parental and Osi-resistant LCOs to Osi. TheLCOs were cultured with various concentrations of Osi for 72 h and the survival of LCOs was determined by ATP assays. All LCOs/OR were less sensitive to Osi than the parent LCOs. \u003cstrong\u003eD. \u003c/strong\u003eKEGG analysis of DEGs for their involved in the enrichedpathways in LCOs/OR. \u003cstrong\u003eE. \u003c/strong\u003eWestern blot analysis of the relative levels of RELA expression and phosphorylation in theindicated LCOs using anti\u003cstrong\u003e-\u003c/strong\u003eSer536 phosphorylation antibody. \u003cstrong\u003eF. \u003c/strong\u003eImmunofluorescent images of the levels of p-RELA in LCOs and LCOs/OR, magnification x 200. \u003cstrong\u003eG. \u003c/strong\u003eWestern blot analyses of the relative levels of nuclear and cytosolic RELA andp-RELA in PC9 and R-PC9 cells. Lamin B and β-actin were the controls for nuclear and cytosolic proteins, respectively.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6132050/v1/9c3ada1f6eb36a9790bb1fb8.jpg"},{"id":78246572,"identity":"1394f294-be88-4dd4-b410-e91b8ffb46a4","added_by":"auto","created_at":"2025-03-11 09:30:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4434718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUP-regulated AlDH1A1 expression is associated with increased frequency of ALDH1+ stem-like cells in EGFR-TKI resistant NSCLC\u003c/strong\u003e. \u003cstrong\u003eA. \u003c/strong\u003eSphere formation in PC9 ，PC9/ER, PC9/AR and PC9/OR cells and quantitative analysis. Scale bars, 100 μm. \u003cstrong\u003eB. \u003c/strong\u003eRT-qPCR analysis of the relative levels of CSC-related gene mRNA transcripts in PC9 and R-PC9cells. \u003cstrong\u003eC. \u003c/strong\u003eFlow cytometry analysis of the proportion of ALDH1+ cells in PC9, PC9/AR, PC9ER and PC9/OR cells. Diethylaminobenzaldehyde (DEAB) served as a control staining to determine the gate. \u003cstrong\u003eD. \u003c/strong\u003eRT-qPCR analysis of the relative levels of the indicated gene mRNA transcripts in LCOs and LCOs/OR. \u003cstrong\u003eE. \u003c/strong\u003eImmunohistochemical analysis of ALDH1A1expression in LCOs and LCOs/OR. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001; ns, no significant.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6132050/v1/e986f04e55749be88e337399.jpg"},{"id":78245039,"identity":"dd150289-20e6-46da-be9b-69e847f01b5b","added_by":"auto","created_at":"2025-03-11 09:22:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4502381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAltered ALDH1A1 expression modulates the sensitivity to EGFR-TKIs in R-PC9 and LCOs/OR. A. \u003c/strong\u003eALDH1A1 over-expression enhanced spheroid colony formation in PC9 cells\u003cstrong\u003e. B. \u003c/strong\u003eALDH1A1 over-expression decreased the\u003c/p\u003e\n\u003cp\u003esensitivity to TKIs in PC9 cells. \u003cstrong\u003eC. \u003c/strong\u003eALDH1A1 silencing enhanced the sensitivity to TKIs in resistant R-PC9 cells. \u003cstrong\u003eD. \u003c/strong\u003ePC9/AR, PC9/ER and PC9/OR cells were treated with different concentrations of EGFR-TKIs and/or DSF for 72h and theircell viability was assessed using CCK-8 assay; combination index (CI) values were analyzed using CompuSyn software for non-constant drug ratio. \u003cstrong\u003eE. \u003c/strong\u003eWestern blot analysis of caspase3 (C3), cleaved caspase 3 (CC3), cleaved PARP (C-PARP) and PARP levels in PC9/AR, PC9/ER and PC9/OR cells after treatment with indicated agents. \u003cstrong\u003eF. \u003c/strong\u003eALDH1A1 silencing enhanced the sensitivity to Osi in LCO1/OR. \u003cstrong\u003eG. \u003c/strong\u003eLCO1/OR cells were treated with different concentrations of Osi and/or DSF for 72h and cell viability was assessed using ATP assay; CI values were analyzed using CompuSyn software for non-constant drugratio. \u003cstrong\u003eH. \u003c/strong\u003eTreatment with DSF synergistically enhanced the antitumor effect of Osi in LCOs. \u003cstrong\u003eI. \u003c/strong\u003eTreatment with DSF synergistically increased the levels of caspase3 expression in LCOs/OR. CI \u0026lt;1 indicates synergism, CI \u0026gt; 1 indicates antagonism. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001; ns, no significant.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6132050/v1/abc3ce148d505d088ba200ea.jpg"},{"id":78245049,"identity":"abea6ad5-158f-4d56-8fd3-0cef202ad410","added_by":"auto","created_at":"2025-03-11 09:22:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5590521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivated RELA regulates the activity of ALDH1A1 in NSCLC. A. \u003c/strong\u003eBioinformatics revealed the existence of P65-binding elements in the promoters of ALDH1A1 gene. \u003cstrong\u003eB. \u003c/strong\u003eChIP unveiled higher levels of RELA binding to the promoter of ALDH1A1 gene using anti-p65 Ab or control IgG. \u003cstrong\u003eC. \u003c/strong\u003eImmunofluorescence exhibited the co-localization of ALDH1A1and RELA proteins in NSNCLC tissues (magnification x 40). Illustrating the sensitivity or resistance of patients to first-generation of EGFR-TKIs. The white box indicates enlarged areas. \u003cstrong\u003eD. \u003c/strong\u003eWestern blot analysis of the relative levels ofRELA expression in the indicated cells. \u003cstrong\u003eE. \u003c/strong\u003eFlow cytometry analysis of the proportion of ALDH1+ cells in R-PC9 and RELA-silencing R-PC9 cells. \u003cstrong\u003eF. \u003c/strong\u003eRT-qPCR analysis of the relative levels of ALDH1A1 and RELA mRNA transcripts inRELA-silencing LCO1-3/OR. \u003cstrong\u003eG. \u003c/strong\u003eFlow cytometry quantitative analysis of the percentages of ALDH1+ cells in LCO1/OR and RELA-silencing LCO1/OR and the representative images of LCO1/OR. Scale bar, 100 μm. \u003cstrong\u003eH. \u003c/strong\u003eRELA silencing enhanced thesensitivity to Osi in LCO1/OR 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; ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6132050/v1/431373438c8650dbfa8a4853.jpg"},{"id":78245047,"identity":"8c1e9f15-e63f-49e7-99a2-4a55c581500d","added_by":"auto","created_at":"2025-03-11 09:22:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5011618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEGCG treatment potentiates the sensitivity to EGFR-TKIs by inhibiting the NF-κB activity in NSCLC. A-B.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLuciferase reporter assays unveiled that treatment with EGCG decreased the NF-κB promoter activity and enhanced the inhibition of some TKIs on the NF-κB promoter activity in R-PC9 cells (A) and LCO1/OR(B). \u003cstrong\u003eC. \u003c/strong\u003eWestern blot analysis displayed that EGCG treatment mitigated the nuclear translocation of p-RELA in PC9/ER, PC9/AR and PC9/OR. \u003cstrong\u003eD. \u003c/strong\u003eChIP assays revealed that EGCG treatment decreased the binding of p-RELA to the ALDH1A1 promoter using anti-p65 Ab or control IgG. \u003cstrong\u003eE. \u003c/strong\u003eFlow cytometry analysis indicated that EGCG treatment dramatically decreased the frequency of ALDH1+ stem-like cells in R-PC9 cells. \u003cstrong\u003eF. \u003c/strong\u003eR-PC9 and LCO1/OR cells were treated with varying doses of Osi and/or DSF for 72 hours. The levels of cell viability were determined utilizing CCK8 or ATP assay. The CI value of less than 1 indicates synergistic interaction between the drugs. \u003cstrong\u003eG \u003c/strong\u003eWestern blot analysis of caspase3 (C3), cleaved caspase3 (CC3), cleaved PARP (C-PARP), and PARP levels in PC9/AR, PC9/ER and PC9/OR cells after treatment with different concentrations of agents. \u003cstrong\u003eH. \u003c/strong\u003eImmunofluorescence exhibited that EGCG treatment enhanced the expression of caspase3 in the Osi-treated LCO1/OR. \u003cstrong\u003eI-J. \u003c/strong\u003eEGCG treatment failed to enhance the sensitivity to Osi in the ALDH1A1-silencing and RELA-silencing LCO1/OR cells. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. ns, no significant.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6132050/v1/ca7e2decce298e831623be8d.jpg"},{"id":78245051,"identity":"5098fb0f-b072-4760-8074-e925b33cde5a","added_by":"auto","created_at":"2025-03-11 09:22:37","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5953165,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacological inhibition of NF-κB/ or ALDH1A1 enhances the response to EGFR-TKIs in EGFR-mutant NSCLC tumors in mice. A. \u003c/strong\u003ePC9/OR cells had higher tumorigenicity in nude mice. \u003cstrong\u003eB\u003c/strong\u003e. A image of all xenograft tumors in themice implanted with PC9/OR and treated with the indicated drugs. \u003cstrong\u003eC. \u003c/strong\u003eThe dynamic growth of different groups of xenografttumors, n = 5 per group. \u003cstrong\u003eD. \u003c/strong\u003eChange in mouse body weights. \u003cstrong\u003eE. \u003c/strong\u003eWestern blot analysis of the relative levels of RELA, p-RELAand ALDH1A1 proteins in xenograft tumors. \u003cstrong\u003eF. \u0026nbsp;\u003c/strong\u003eImmunofluorescent analysis of ALDH1A1 and RELA expression in the PDOX tumors derived from LCO1 and LCO1/OR (magnification x 40). R, resistant; S, sensitive. \u003cstrong\u003eG. \u003c/strong\u003eA image of all PDOX tumors. \u003cstrong\u003eH. \u003c/strong\u003eThe dynamic growth of different groups of PDOX tumors in mice \u003cstrong\u003e(\u003c/strong\u003en = 3 per group). \u003cstrong\u003eI. \u003c/strong\u003eChanges in the mousebody weights. \u003cstrong\u003eJ. \u003c/strong\u003eWestern blot analysis of the relative levels of RELA, p-RELA and ALDH1A1 proteins in PDOX tumors. \u003cstrong\u003eK. \u003c/strong\u003eA image of all xenograft tumors from the PC9/OR cells. \u003cstrong\u003eL\u003c/strong\u003e. The dynamic growth of different groups of xenograft tumors, n = 5 per group. \u003cstrong\u003eM. \u003c/strong\u003eChanges in mouse body weights. \u003cstrong\u003eN. \u003c/strong\u003eWestern blot analysis of the relative levels of RELA, p-RELA and ALDH1A1 proteins in xenograft tumors from PC9/OR cells. \u003cstrong\u003eO. \u003c/strong\u003eA image of all PDOX tumors. \u003cstrong\u003eP. \u003c/strong\u003eThe dynamic growth of different groups of PDOX tumors, n = 3 per group. \u003cstrong\u003eQ. \u003c/strong\u003eChanges in mouse body weights. \u003cstrong\u003eR \u003c/strong\u003eWestern blot analysis of the relative levels of RELA, p-RELA and ALDH1A1 proteins in PDOX tumors. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 ，**\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6132050/v1/8f2c3cb4f9557abc3c3cd5d2.jpg"},{"id":78251703,"identity":"1dacb37a-1fe4-4783-86ad-58f607ec08e2","added_by":"auto","created_at":"2025-03-11 10:02:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":32790519,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6132050/v1/c183c6e4-71e1-4fe1-8f40-39df81f6eb24.pdf"},{"id":78246577,"identity":"99066c36-b39d-4b43-bc9d-09ed1352c167","added_by":"auto","created_at":"2025-03-11 09:30:37","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2488760,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-6132050/v1/a04ea16fb519fafd5df19cf0.docx"},{"id":78245037,"identity":"c9974707-f9af-4af1-ab2a-f3b67a92c017","added_by":"auto","created_at":"2025-03-11 09:22:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25773,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable15.docx","url":"https://assets-eu.researchsquare.com/files/rs-6132050/v1/227c5e46516f99ef129cd405.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Activation of NF-κB/ALDH1A1 Signaling Promotes Non-Mutational Resistance to EGFR-TKIs in Non-Small Cell Lung Cancer ","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSomatic mutations, including the L858R mutation in exon 21 and the deletion in exon 19, in the epidermal growth factor receptor (EGFR) activate the EGFR-related signaling, leading to the development of non-small cell lung cancer (NSCLC). Approximately 15% of Caucasian patients with NSCLC have such mutations and East-Asian NSCLC patients have a 50% prevalence of such mutations [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. NSCLC patients with such mutations in the EGFR usually respond to EGFR-TKIs, like erlotinib (Erlo), afatinib (Afa), and osimertinib (Osi). Actually, treatment with EGFR-TKIs has significantly extended the progression-free survival (PFS) to 10\u0026ndash;14 months, outperforming traditional chemotherapy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnfortunately, many NSCLC patients develop resistance to EGFR-TKIs following treatment with the TKI, which may stem from selective pressure for preexisted subclones of non-responding cancer cells or from evolutions of initially responsive cancer cells. This latter development of acquired resistance may be more prevalent than the emergence of pre-existing resistant subclones [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Previous studies have revealed that the molecular mechanisms underlying the development of acquired resistance to TKIs may be attributed to secondary EGFR mutations, changes in other receptor tyrosine kinases (RTKs) or downstream signaling molecules, alternative signaling activation, and phenotypic shifts [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, approximately one-third of NSCLC cases with resistance to TKIs do not have such changes. Apparently, other molecular mechanisms may contribute to the development of resistance to TKIs and need to be investigated.\u003c/p\u003e \u003cp\u003eCancer stem cells (CSCs) are a small subset of cancer cells and have a high tumorigenicity by initiating tumor formation, tumor growth, epithelial-to-mesenchymal transition process, metastasis, and relapse as well as drug resistance [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Lung CSCs can be identified by the expression of surface markers, such as CD133, CD44, ALDH1A1, and ABCG2, and are regulated by the Notch, Wnt, and cell cycle signaling pathways, which are also critical for the development and maintenance of drug resistance [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Previous studies have developed anti-CSC therapeutics that target the surface markers of CSCs and associated pathways in different cancer types in animals and those anti-CSC therapies are being tested in clinical trials [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Thus, further understanding the role of CSC in EGFR-TKI resistance is critical for the development of new therapeutic strategies for the treatment of TKI-resistant NSCLC.\u003c/p\u003e \u003cp\u003eStem cells can self-renew and form 3D organoids in vitro culture in the presence of optimal supporting factors and the formed organoids can sustain near physiological tissues [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Similarly, CSCs can also form organoids and carry many genetic and histological features of the parental tumors\u003c/p\u003e \u003cp\u003e[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It is notable that stem cells can differentiate into different types of cells so that an organoid from stem cells or organ progenitors can contain more than one organ-specific type of cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Conceivably, the organoid is a well-characterized \u003cem\u003ein vitro\u003c/em\u003e model for scrutinizing the self-renewal, differentiation and drug resistance of CSCs.\u003c/p\u003e \u003cp\u003eTo explore new mechanisms underlying the acquisition of EGFR-TKI resistance in lung CSCs, we established Osi resistant lung cancer organoids (LCOs). Combining Smart-RNA sequencing with biological experiments, our research discovered that the NF-κB signaling and EGFR-TKI-related signaling were enriched in ALDH\u0026thinsp;+\u0026thinsp;stem-like cells, thereby mediating resistance to EGFR-TKIs in NSCLC cells. Furthermore, treatment withALDH1A1 and NF-κB inhibitors attenuated the resistance to EGFR-TKIs by suppressing the NF-κB/ALDH1A1 axis and the stemness of EGFR-TKI resistant NSCLC.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Human tissue samples\u003c/h2\u003e \u003cp\u003eSurgical lung cancer tissue samples were obtained from individual patients in the Department of Thoracic Surgery of Fujian Provincial Tumor Hospital. Their demographic and clinical characteristics, including providing tissues for the establishment of organoids are shown in Supplementary Table\u0026nbsp;1. Individual patients with NSCLC were diagnosed by radiological imaging and pathology. Their tumors were staged using the TNM system. Written informed consent was collected from individual patients. The experimental protocol was approved by the Ethics Committee of Fujian Provincial Tumor Hospital (Approval Number: K2023-118-01).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Establishment and culture of EGFR-TKI resistant lung cancer organoids\u003c/h2\u003e \u003cp\u003eThe establishment of LCOs was performed as described previously \u003cb\u003e20\u003c/b\u003e. The established LCOs were counted, embedded in 30 \u0026micro;L of Matrigel (356231, Corning) at 10,000 crypts/mL and cultured using the LCO kit (K2318-LA, BioGenous). To establish EGFR-TKI-resistant LCOs, the primarily cultured cancer cells were treated with different concentrations (1/4 IC50 of LCO1, LCO2 and LCO3) of osimertinib in a stepwise-increase manner. After culture for 4 months (about 12 generations), the cells were able to grow in media containing 8, 6, or 10 \u0026micro;M osimertinib and were considered as EGFR-TKI resistant LCOs (collectively referred to as LCOs/OR).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell line and culture\u003c/h2\u003e \u003cp\u003eHuman NSCLC H1650 (del E746-A750), PC-9 (EGFR exon 19 deletion) and H1975 (L858R/T790M) cells with varying levels of sensitivity to EGFR-TKIs were obtained from Shanghai Cell Bank (Shanghai,\u003c/p\u003e \u003cp\u003eChina) and identified by STR. The cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS and antibiotics (100 units/mL penicillin and 100 \u0026micro;g/mL streptomycin) at 37.0\u0026deg;C in a 5% CO2 incubator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Establishment of EGFR-TKIs-resistant NCI-PC9 cells\u003c/h2\u003e \u003cp\u003eTo establish an EGFR-TKIs resistant subline, PC9 cells were treated with 0.01 \u0026micro;M, 0.05 \u0026micro;M and 0.012 \u0026micro;M of erlotinib, afatinib, and osimertinib, respectively. These drug concentrations were almost 1/4 IC50 for PC9, and their concentrations increased gradually. After culture for 6 months (about 20 generations), the cells were able to grow in 6 \u0026micro;M (erlotinib) and 5 \u0026micro;M (afatinib, osimertinib) and considered as EGFR-TKI resistant PC9 cells (collectively referred to as R-PC9). The newly established erlotinib- (afatinib, osimertinib)-resistant PC9 cells were named PC9/ER (PC9/AR, PC9/OR) cells. Simultaneously, some parental PC9 cells were cultured in drug-free medium and served as the controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 CCK8 assay\u003c/h2\u003e \u003cp\u003eThe impact of reagents on the viability of PC9, PC9/ER, PC9/AR and PC9/OR cells was tested by CCK8 assay using the specific kit. Briefly, the different groups of cells were treated with the same reagent(s) for 72 h. Individual wells were added with the CCK8 solution and incubated at 37\u0026deg;C for 4h. The absorbance at 450 nm in individual wells was measured using a microplate reader (Thermo Multiskan GO, USA). The potential synergism, additivity and antagonism of these reagents were analyzed by the combination indexes (CI) using the CompuSyn synergism/antagonism analysis software (Version 1.0, ComboSyn, Paramus, NJ, USA) (30).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 ATP assay\u003c/h2\u003e \u003cp\u003eThe viability of the LCOs was determined using the CellTiter-Glo 3D Cell Viability Assay (Vazyme), following the manufacturer's protocol. In brief, the CellTiter-Glo reagent was mixed with the organoid culture medium in a 1:1 volume ratio. Luminescence was subsequently measured using a multi-plate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Colony formation assay\u003c/h2\u003e \u003cp\u003ePC9, PC9/ER, PC9/AR and PC9/OR cells (800 cells/well) were cultured in 12-well plates for 24 h, and the cells were treated with indicated concentrations of compounds for 7 days. The cells were washed with PBS and fixed with 4% paraformaldehyde, followed by staining with crystal violet solution. Typical images were photographed and the survival colony rates were calculated, relative to the number of colonies in the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Antibodies\u003c/h2\u003e \u003cp\u003eInformation regarding all antibodies used in this study is shown in Supplemental Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Western blot assay\u003c/h2\u003e \u003cp\u003eThe cells or LCOs were harvested and lyzed on ice for 20 min in NP-40 lysis buffer containing 1 \u0026times; PMSF, phosphatase inhibitors, and protease inhibitors. After centrifuged, their protein concentrations were determined using a BCA method. The lysate samples (30 \u0026micro;g/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% dry milk in TBST and incubated at 4\u0026deg;C with primary antibodies overnight. After being washed, the bound antibodies were reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies, and visualized with ECL (P0018S, Beyotime, Jiangsu, China) in a chemiluminescence imaging system (BIO-RAD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Whole-exome sequencing (WES)\u003c/h2\u003e \u003cp\u003eThe LCOs and their parent tumor tissues were harvested and their DNA was extracted, followed by WES in Novogene (Beijing) using Illumina PE150. The clean data were mapped to the reference genome ((GRCh37/hg19/GRCh38) using the Burrows Wheeler Aligner (BWA) software (Li et al., 2018) and Samblaster (Faust et al., 2014) to generate BAM files. Somatic variants were identified, according to the reference and tumor or organoid sequencing data (MuTect) with default parameters (Cibulskis et al., 2013). The functional predictions and annotations were analyzed using ANNOVAR (Wang et al., 2010). To detect somatic copy number alternations (CNAs), the BAM files were analyzed to compare the organoids with tumor tissues using Control-FREEC (Boeva et al., 2012). Mutational signatures were analyzed using the BSgenome in the R package.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Smart-seq2\u003c/h2\u003e \u003cp\u003eSMART-seq2 data were analyzed by Wancheng Biotechnology, Shanghai, China. Total RNA was extracted from parental LCO1-3 and LCO/OR1-3 in the transwell system using lysis solution (Wancheng Biotechnology), According to the manufacturer\u0026rsquo;s instructions. The prepared RNA was sequenced on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA). The levels of gene transcripts were quantified using StringTie software and the differentially expressed genes (DEGs) between groups were identified using the edgeR and DEseq2. The potential functions of DEGs were analyzed by KEGG and Reactome enrichment analysis (R-package: ComplexHeatmap).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 LCO and cell viral transduction\u003c/h2\u003e \u003cp\u003eAll recombinant lentiviral and control vectors were constructed by GenePharma (Shanghai, China). When\u003c/p\u003e \u003cp\u003etransducing organoids, the LCOs were digested into single cells and then mixed with the viral solution for 4 hours. The cells were mixed with Matrigel, and cultured for 3 to 7 days in the presence of 2 \u0026micro;g/mL puromycin. In addition, different groups of cells were cultured in 6-well plates up to a confluence of 50% and transduced with virus for 48 to 96 hours, followed by treatment with 2 \u0026micro;g/mL puromycin. The established stable cell lines expressing the gene of interest were cultured for subsequent experiments. The shRNA target sequences used in this study are listed in the Supplemental Table\u0026nbsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Nuclear\u0026ndash;cytoplasmic fractionation\u003c/h2\u003e \u003cp\u003eCell fractionation was performed utilizing the Cytoplasmic and Nuclear Protein Extraction Reagents as per the manufacturer's guidelines (Thermo Fisher Scientific, cat# 78833). The resulting cellular fractions were centrifuged at 16,000 g for 20 minutes at 4\u0026deg;C. Subsequently, their supernatants were carefully collected for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Aldefluor assay\u003c/h2\u003e \u003cp\u003eThe activity of ALDH enzyme in different groups of cells was analyzed by the Aldefluor assay using an ALDE-FLUOER kit (01700; Stem Cell Technologies), following the manufacturer's protocol. In brief, individual groups of cells were suspended and cultured with the activated ALDE-FLUOER reagent at 37\u0026deg;C for 45 minutes. The diethylaminobenzaldehyde (DEAB) was used as a control. The frequency of ALDH1\u0026thinsp;+\u0026thinsp;cells were analyzed by flow cytometry (BD Biosciences, NJ, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Hematoxylin and eosin (H\u0026amp;E) and immunohistochemistry staining\u003c/h2\u003e \u003cp\u003eThe LCOs were firstly isolated from Matrigel using Cell Recovery Solution (Corning). The PDOs and tissue samples were fixed with fresh 4% paraformaldehyde at 4\u0026deg;C for one day, and paraffin-embedded. The tissue sections (5 \u0026micro;m) were routine-stained with H\u0026amp;E and immunohistochemistry staining using primary anti-TTF-1 and anti-CK7. H\u0026amp;E and immunohistochemical images were acquired under an inverted microscope (Leica).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Immunofluorescence\u003c/h2\u003e \u003cp\u003eAfter treatment, the cells, LCOs and tissues were fixed, ruptured, and blocked sequentially. The samples were probed with primary anti-NF-κB, anti-p-NF-κB, anti-MDR1, anti-ALDH1A1, overnight at 4℃. After being washed with PBS, the samples were reacted with Alexa Fluor 482 /628/ conjugated secondary antibody (Termo Fisher Scientifc), followed by nuclear-stained with DAPI (1:1000, Beyotime). The fluorescent imaging was captured by a confocal Microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17 Spheroid colony formation\u003c/h2\u003e \u003cp\u003ePC9 and R-PC9 cells (3000 cells/well) were cultured in a mixed matrix gel in 24-well plates. Following a\u003c/p\u003e \u003cp\u003e7-day incubation in DF-12 medium, the spheres (\u0026gt;\u0026thinsp;50 mm in diameter) in individual wells were quantified to measure the capacity of cells for their self-renewal. Images were taken, and the numbers of spheres were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.18 Luciferase reporter assay\u003c/h2\u003e \u003cp\u003eThe different groups of cells were transfected with pNF-kB-Luc, pRL-TK (Genepharma, shanghai, China) using Lipofectamine 3.0 (HanBio, shanghai, China), following the protocol provided by the manufacturer. After culturing for 48 h., the luciferase activities in individual wells were measured using the Dual-Luciferase reporter assay kit (Promega). The firefly luciferase activity in each sample was normalized to Renilla luciferase activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.19 Chromatin immunoprecipitation assay\u003c/h2\u003e \u003cp\u003eThe potential target DNA of p65 was analyzed by chromatin immunoprecipitation (ChIP) assay using the EZ-ChIP kit (17\u0026ndash;295; Millipore), according to the manufacturer\u0026rsquo;s protocol. Briefly, 1x107 cells were fixed in 1% formaldehyde, lyzed, and sonicated using an ultrasonic processor (Biorupter Pico, Biorupter Pico). The cellular samples were incubated with anti-p65 antibody (5 \u0026micro;l per 1 mg of total protein) and reacted with rabbit IgG-beads (2 \u0026micro;l per 1 mg of total protein), followed by centrifugation. After being washed, the bound DNA was extracted and used for PCR amplification with specific primers (Supplemental Table\u0026nbsp;4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.20 Caspase3 apoptosis assay\u003c/h2\u003e \u003cp\u003eAfter drug treatment, the cells were treated with a green fluorescent caspase-3 probe (0.5 \u0026micro;M, 40273ES60, YEASEN, Shanghai, China). The apoptotic cells were photoimaged under a laser confocal microscope (TCS SP8, Leica).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e2.21 TUNELAssay\u003c/h2\u003e \u003cp\u003eThe frequency of apoptotic cells in individual tissue sections was quantified by the TUNEL assay using the In Situ Cell Death Detection Kit, following the manufacturer\u0026rsquo;s protocol. Briefly, after incubation of tissue sections with the specific solutions at 37\u0026deg;C for 30 minutes, the sections were reacted with Alexa Fluor-labeled secondary antibodies (Invitrogen) and nuclear-counterstained with DAPI. The fluorescent signals were photoimaged under a laser scanning confocal microscope (TCS SP8, Leica).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e2.22 RNA-seq and data analysis\u003c/h2\u003e \u003cp\u003eThe transcriptomic profiles of parental PC9, PC9/ER, PC9/AR and PC9/OR cells were analyzed by RNA-seq.\u0026nbsp;Briefly, total RNA was extracted from individual types of cells and submitted to Novogene Bioinformatics Technology (Beijing, China) for sequencing. The raw data were analyzed for their quality and gene expression quantification by Annoroad (Beijing). The DEGs were defined by log2 fold change\u0026thinsp;\u0026ge;\u0026thinsp;1\u003c/p\u003e \u003cp\u003eand p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 using DESeq R package. The DEGs were analyzed by KEGG pathway, Venn diagram and Heatmaps (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://magic.novogene.com\u003c/span\u003e\u003cspan address=\"https://magic.novogene.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e2.23 RT-qPCR assay\u003c/h2\u003e \u003cp\u003eThe relative levels of gene mRNA transcripts to the control GAPDH in the different groups of cells were quantified by RT-qPCR using specific primers (Supplemental Table\u0026nbsp;5) and the SYBR Green PCR Master Mix (Life Technologies). The PC9 and R-PC9 cells were treated with different concentrations of EGCG-NPs and EGFR-TKIs for 48 h. Their total RNA was extracted with TRIzol reagent (Invitrogen, USA) and reversely transcribed into cDNA using a PrimeScript RT Reagent Kit (TaKaRa #RR047A), followed by PCR. The data were analyzed the 2-ΔΔCt method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e2.24 Tumorigenicity study\u003c/h2\u003e \u003cp\u003e Animal studies were carried out in accordance with the guidelines for the care and use of human specimens and animals, and approved by the Institutional Review Board of Fujian Medical University (Approval Number: IACUC FJMU 2024\u0026thinsp;\u0026minus;\u0026thinsp;0177). Male athymic nude mice and NSG mice were obtained from Fuzhou Nordens Biotechnology and housed in a specific pathogen-free facility in campus.\u003c/p\u003e \u003cp\u003eTo establish xenograft tumors, individual male athymic nude mice at 5 weeks of age were implanted subcutaneously with 5 \u0026times; 106 PC9/OR cells into their right flanks. The development of tumors was monitored frequently. When the tumors reached about 100 mm\u003cb\u003e3\u003c/b\u003e, the mice were randomized and treated with vehicle (CTL, DMSO, Corn Oil), Osi (5 mg/kg/d, P.O by gavage), DSF (5 mg/kg/d, P.O), EGCG (8 mg/kg, i.p., every other day), Osi\u0026thinsp;+\u0026thinsp;DSF (P.O, daily for 16 days), or a combination of Osi\u0026thinsp;+\u0026thinsp;EGCG.\u003c/p\u003e \u003cp\u003eFor a PDOX model, the same age of individual male NSG mice were injected subcutaneously with 5x106 LCO1/OR cells in Matrigel (0. 1 mL 50%; 356231, BD Biosciences) into their right flank. For PDOX generation, tumors were passaged to additional athymic male nude mice when they reached about 1000 mm3. The dosing regimen was consistent with the aforementioned plan.\u003c/p\u003e \u003cp\u003eThe tumor volumes were measured and calculated using the equation [(width) 2 \u0026times; (height)]/2. Tumor\u003c/p\u003e \u003cp\u003esizes and mouse body weights were measured every other day. After euthanizing the mice at the end of the study period, their tumors were dissected for Western blot to examine the expression levels of p-RELA and ALDH1A1. Additionally, the frequency of apoptotic cells in tumor tissues was analyzed by the TUNEL assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e2.25 Statistical analysis\u003c/h2\u003e \u003cp\u003eEach experiment was repeated three times. Data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SD). The difference between the control and experimental groups was analyzed by a Student t test using GraphPad\u003c/p\u003e \u003cp\u003ePrism 8.0 software and the different levels of significance were defined as *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; *** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; **** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ns., no significant, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003e3.1 Aberrant activation of the NF-κB signaling is associated with the development of resistance to EGFR-TKIs in NSCLC\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo unravel the intricate mechanisms underlying the acquisition of EGFR-TKI resistance in lung CSCs, we developed three LCOs derived from surgical NSCLC specimens with the EGFR exon 19 deletion (Supplemental Table\u0026nbsp;1). These LCOs faithfully recapitulated the characteristics of primary tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplemental Fig.\u0026nbsp;1A-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, we investigated the drug responses of these LCOs. Enhanced CT scans exhibited a significant reduction in tumor size, confirming the efficacy of the EGFR-TKI. Consistently, these LCOs were resistant to varying concentrations of TKIs (Supplemental Fig.\u0026nbsp;2, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C). Next, we generated three Osi-resistant LCO variants (LCO1-3) by gradually increasing Osi concentrations in the culture medium. The Osi-resistant LCOs displayed markedly higher resistance to Osi, with 7- to 12-fold greater IC50 Osi than their parental LCOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C). Notably, HE staining revealed no obviously histological change in LCOs before and after developing drug resistance (Supplemental Fig.\u0026nbsp;1E). Immunohistochemistry exhibited a substantial elevation in multidrug resistance 1 (MDR1) expression, a key marker of drug resistance [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] (Supplemental Fig.\u0026nbsp;3A). Conversely, the levels of KI67 expression, a proliferation marker, were reduced in drug-resistant LCOs (LCOs/OR), implying that drug resistance was associated with inhibition of cell proliferation (Supplemental Fig.\u0026nbsp;3B).\u003c/p\u003e \u003cp\u003eTo elucidate the molecular underpinnings of Osi resistance acquisition in LCOs/OR, we analyzed the transcriptomic profiles of both the parental LCOs (LCO1-3) and their Osi-resistant counterparts (LCO1-3/OR) by RNA-seq.\u0026nbsp;Principal component analysis (PCA) of the RNA-seq data revealed a striking similarity in the gene expression profiles between each pair of parental LCOs and its corresponding LCOs/OR (Supplemental Fig.\u0026nbsp;1F). Gene enrichment analysis uncovered a significant increase in the expression of genes in multiple stemness-related signaling pathways in the LCO1-3/OR when compared to the parental LCOs, and the NF-κB pathway was remarkably enriched in the LCO1-3/OR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Western blot and immunofluorescent analyses unveiled an obvious elevation in the relative levels of phosphorylated RELA to RELA expression in LCOs/OR, relative to that in their parental LCOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F and Supplemental Fig.\u0026nbsp;3C).\u003c/p\u003e \u003cp\u003eWe also established stable Erlo-resistant PC9/ER, Afa-resistant PC9/AR and Osi-resistant PC9/OR cell lines, collectively known as R-PC9 (Supplemental Fig.\u0026nbsp;4A) using the dose-escalation method [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] (Supplemental Fig.\u0026nbsp;4B-C). Whole exome sequencing (WES) revealed no major single-nucleotide variant pattern shifts between PC9/ER and PC9 cells, and yet variations were identified in PC9/AR and PC9/OR (Supplemental Fig.\u0026nbsp;5). Notably, aside from the intrinsic EGFR exon 19 deletion, there was no mutation in reported TKI-resistant gene sets (i.e., EGFR T790M, EGFR C797S, HER2 amp) in these cell lines. Similar to the LCOs, R-PC9 cells displayed a slower rate of proliferation, evidenced by less EDU labeling (Supplemental Fig.\u0026nbsp;4D). Surprisingly, there was no significant enrichment in the NF-κB pathway in the R-PC9 lines (Supplemental Fig.\u0026nbsp;6). Given the differences, we isolated and analyzed nuclear-cytoplasmic fractionations on R-PC9 cells, and the results indicated a significant increase in the relative ratios of nuclear phosphorylated RELA to RELA expression, but not cytoplasmic RELA phosphorylation, in R-PC9 cells, relative to that in their parental PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). These data highlighted the advantages of the organoid model and indicated that aberrant activation of the NF-κB signaling was associated with the development of EGFR-TKI resistance in NSCLC.\u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;\u003c/b\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003eare here\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Up-regulated ALDH1A1 activity in the EGFR-TKI-resistant NSCLC cells\u003c/h2\u003e \u003cp\u003eWe next studied how EGFR-TKI activated the NF-κB signaling in NSCLC. Considering that tumor cells with EGFR-TKI resistance may manifest stem-cell-like properties [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], we analyzed whether PC9/AR, PC9/ER and PC9/OR cells exhibited a CSC phenotype. First, the EGFR-TKI resistant cells formed significantly more numbers of spheres than PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Second, significantly higher expression levels of CSC-related markers, particularly for ALDH1A1, were detected in these resistant cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The ALDH1A1 is one of the top-ranking isozymes in the aldehyde dehydrogenase (ALDH) family, one of the biomarkers for CSCs and activates the NF-κB signaling, contributing to the immunosuppressive environment in malignant tumors [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In addition, the Aldefluor assay was used to evaluate ALDH1 activity by flow cytometry. The results indicated a marked increase in the proportion of ALDH1\u0026thinsp;+\u0026thinsp;cells within the resistant R-PC9 cell population relative to the PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Similarly, significantly increased ALDH1A1 mRNA transcripts were detected in the EGFR-TKI resistant LCOs/R,\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003erelative to that in their parental LCOs and the levels of ALDH1A1 mRNA transcripts were the most up-regulated among the tested stemness factors in these LCOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Further IHC staining also revealed that the levels of ALDH1A1 expression increased in the LCOs/OR compared with those in the parental LCOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Collectively, these findings indicated that ALDH1A1 expression was significantly up-regulated in the EGFR-TKI resistant NSCLC and suggest that Up-regulated ALDH1A1 may contribute to the development of EGFR-TKI resistance in NSCLC and enhance their stemness during the development of TKI resistance.\u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;\u003c/b\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003eis here\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.3 ALDH1A1 supports the development of resistance to TKIs in NSCLC.\u003c/h2\u003e \u003cp\u003eTo investigate the function of ALDH1A1, we performed the gain and loss of function experiments in PC9 and R-PC9 cells, respectively. After induction of ALDH1A1 over-expression in PC9 cells or silencing in R-PC9 cells (Supplemental Fig.\u0026nbsp;7A), we found that ALDH1A1 over-expression increased its activity and the numbers of formed spheres in PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Supplemental Fig.\u0026nbsp;7B). Moreover, ALDH1A1 over-expression significantly reduced the effect of Erlo, Afa and Osi on inhibiting the proliferation of PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Conversely, ALDH1A1 silencing mitigated the ALDH1A1 activity (Supplemental Fig.\u0026nbsp;7C-E), decreased the numbers of formed spheres (Supplemental Fig.\u0026nbsp;7F-G) and enhanced the effects of TKIs on inhibiting the proliferation of R-PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven that ALDH1A1 silencing enhanced the sensitivity to EGFR-TKIs in R-PC9 cells, we tested whether pharmacological inhibition of ALDH1A1 enzymatic activity could modulate drug resistance in PC9 cells. We found that treatment with disulfiram (DSF, a selective ALDH1A1 inhibitor) inhibited the proliferation of R-PC9 and PC9 cells in a dose-dependent manner and the sensitivity of drug-resistant R-PC9 cells to DSF was higher than that of PC9 cells (Supplemental Fig.\u0026nbsp;8A). Furthermore, treatment with different concentrations of DSF, in combination with different concentrations of each TKI, unveiled that high concentrations of TKIs and DSF had antagonistic effect on inhibiting the proliferation of R-PC9 cells while treatment with low concentrations of TKIs and DSF resulted in a synergistic effect on inhibiting the proliferation of R-PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Moreover, treatment with a low concentration of DSF synergistically enhanced the inhibition of TKIs on the proliferation and clonal formation of R-PC9 cells (Supplemental Fig.\u0026nbsp;8B-F). More importantly, treatment with both the TKI and DSF increased the relative levels of cleaved PARP and cleaved Caspase3 in R-PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Thus, pharmacological\u003c/p\u003e \u003cp\u003einhibition of ALDH1A1 enzymatic activity enhanced the cytotoxic effect of EGFR-TKIs on the viability of drug-resistant NSCLC cells by triggering their apoptosis.\u003c/p\u003e \u003cp\u003eWe further validated the regulatory function of ALDH1A1 in TKI resistance in LCOs. We also observed that ALDH1A1 silencing in LCOs/OR significantly enhanced their sensitivity to Osi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, Supplemental Fig.\u0026nbsp;9A-B). Similarly, LCOs/OR wer\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ee\u003c/span\u003e more sensitive to DSF than their parental LCOs (Supplemental Fig.\u0026nbsp;9C). Remarkably, treatment with both low concentrations of Osi and DSF synergistically inhibited the proliferation of LCOs/OR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H, Supplemental Fig.\u0026nbsp;9D-E). More interestingly, treatment with both Osi and DSF synergistically enhanced Caspase3 expression in LCOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, Supplemental Fig.\u0026nbsp;9F-G). Collectively, these data indicated that ALDH1A1 positively supported the development of resistance to TKIs in NSCLC and inhibition of ALDH1A1 partially restored the sensitivity to TKIs in NSCLC.\u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;\u003c/b\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u003cb\u003eis here\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.4 RELA regulates the activity of ALDH1A1 in NSCLC\u003c/h2\u003e \u003cp\u003eRELA, also known as p65, is a transcription factor. Phosphorylated RELA can bind to the promoters of its regulated genes, regulating the expression of those genes, such as inflammatory cytokines [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To understand how the ALDH1A1 expression was up-regulated in drug-resistant NSCLC, we performed bioinformatics to identify the potential binding sites of transcription factors in the ALDH1A1 promoter. We performed ChIP assays and found that anti-p65 effectively precipitated the ALDH1A1 promoter region in NSCLC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). These findings underscored the likelihood of RELA to regulate ALDH1A1 expression in NSCLC. Immunofluorescence revealed that RELA expression was co-localized with ALDH1A1 expression in NSCLC from the patients, who were refractory to EGFR-TKI targeted therapy, but not those responding to EGFR-TKI therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Supplemental Fig.\u0026nbsp;10A-B). These suggest that ALDH1A1 may be a target gene of RELA during the development of EGFR-TKI resistance in NSCLC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotably, the percentages of ALDH1\u0026thinsp;+\u0026thinsp;CSCs in clinical NSCLC specimens with EGFR L858 mutation or 19del mutation were correlated positively with the percentages of RELA\u0026thinsp;+\u0026thinsp;tumor cells (Supplemental Fig.\u0026nbsp;10C). Furthermore, RELA silencing decreased the frequency of ALDH1\u0026thinsp;+\u0026thinsp;cells in PC9/OR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E). Consistently, RELA silencing decreased the levels of ALDH1A1 gene expression, ALDH1\u0026thinsp;+\u0026thinsp;cells, and sphere formation and partially restored the sensitivity to Osi in LCO1/OR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-H). Collectively,\u003c/p\u003e \u003cp\u003ethese findings indicated that RELA positively regulated ALDH1A1 activity, contributing to the development of EGFR-TKI resistance in NSCLC.\u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;\u003c/b\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cb\u003eis here\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5 EGCG potentiates the sensitivity to EGFR-TKIs by inhibiting the NF-κB signaling and downstream ALDH1A1 activity in NSCLC.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe next investigated whether pharmacologic inhibition of the NF-κB could modulate the resistance to EGFR-TKIs in NSCLC. Currently, the clinically available inhibitors of the NF-κB signaling only indirectly target either IKK or the proteasome, which leads to non-specific effects and limited therapeutic efficacy in patients [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Previous studies have shown that treatment with EGCG effectively inhibits the proliferation of lung cancer cells by down-regulating the NF-κB signaling[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Accordingly, we chose EGCG as an inhibitor of the NF-κB signaling. Luciferase reporter assays unveiled that treatment with EGCG inhibited the transcriptional activity of the NF-κB promoter and enhanced the inhibition of TKI on the transcriptional activity of the NF-κB promoter in R-PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). A similar pattern of luciferase activities was observed in LCO1/OR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). EGCG treatment limited the RELA nuclear transportation and reduced the binding of RELA to the ALDH1A1 promoter in R-PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). Furthermore, treatment with both EGCG and EGFR-TKIs effectively decreased the percentages of ALDH1\u0026thinsp;+\u0026thinsp;cells in R-PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared with R-PC9 cells, other NSCLC H1975 and H1650 cell lines displayed lower levels of RELA expression and phosphorylation as well as low frequency ofALDH1\u0026thinsp;+\u0026thinsp;cells (Supplemental Fig.\u0026nbsp;11A-C). Interestingly, the levels of phosphorylated RELA and the ALDH1A1 activity were positively correlated with the sensitivity to EGCG in these cell lines (Supplemental Fig.\u0026nbsp;11D-E). Apparently, low RELA phosphorylation and low ALDH1A1 activity led to a lower sensitivity to EGCG in H1650 and H1975 cells. Consistently, LCOs/OR exhibited greater sensitivity to EGCG compared to LCO/pare cells, which contained a lower proportion of ALDH1\u0026thinsp;+\u0026thinsp;cells (Supplemental Fig.\u0026nbsp;11F, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Thus, EGCG treatment decreased RELA phosphorylation and subsequent ALDH1A1 activity in NSCLC cells.\u003c/p\u003e \u003cp\u003eNext, we treated R-PC9 cell lines with varying concentrations of EGCG and/or EGFR-TKIs to determine the combination index. The results indicated that EGCG and EGFR-TKIs at high concentrations exhibited antagonistic effects while they at low concentrations induced synergistic effects in R-PC9 cells and LCO1/OR. Actually, treatment with low doses of EGCG and EGFR-TKIs synergistically enhanced\u003c/p\u003e \u003cp\u003etheir inhibition on the proliferation of R-PC9 cells (Supplemental Fig.\u0026nbsp;12A) and increased the levels of cleaved PARP and cleaved caspase3 in R-PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). A similar pattern of synergistic antitumor effect of EGCG and Osi was observed in the LCOs/OR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-H, Supplemental Fig.\u0026nbsp;12B-E).\u003c/p\u003e \u003cp\u003eMechanistically, we found that treatment with EGCG failed to synergistically enhance the inhibition of Osi on the proliferation of RELA-silencing R-PC9 cells and LCOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, Supplemental Fig.\u0026nbsp;13A-B). These indicated that the EGCG-enhanced inhibition of TKIs on the proliferation of NSCLC cells depended on high levels of RELA expression in the TKI-resistant cells. Similarly, treatment with EGCG also failed to significantly increase the inhibition of different doses of Osi on the proliferation of ALDH1A1-silencing LCO1/OR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). A similar pattern of the lack of synergistic effects of EGCG and TKIs was observed on the proliferation of ALDH1A1-silencing R-PC9 and LCO/OR (Supplemental Fig.\u0026nbsp;13C-D). Therefore, EGCG treatment potentiated the sensitivity to EGFR-TKIs, dependent on high levels of RELA expression and downstream ALDH1A1 activity in the EGFR-TKI resistant NSCLC cells. Conceivably, treatment with EGCG to inhibit the NF-κB signaling may inhibit and prevent the development of resistance to EGFR-TKIs in EGFR-mutant NSCLC.\u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;\u003c/b\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e \u003cb\u003eis here\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Pharmacological inhibition of NF-κB or ALDH1A1 enhances response to EGFR TKI \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eGiven that high frequency of stem-like cells was presented in EGFR-TKI resistant R-PC9 cells, we tested the tumorigenicity of R-PC9 cells by implanting different numbers of R-PC9 cells into nude mice. The results indicated that implantation with 2x106 PC9/OR cells induced solid tumors in two out of five nude mice while injection with 8x106 parental PC9 cells only triggered solid tumor formation in one out of five mice, clearly implying high tumorigenicity of R-PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To determine the effect of DSF on the growth of R-PC9 tumors in vivo, we established a nude mouse model of xenograft PC9/OR. After establishment of solid tumors, the tumor-bearing mice were randomized and treated with vehicle, Osi and/or DSF and their tumor sizes were monitored (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C). Quantitative analysis indicated that treatment of either 5 mg/kg Osi or 2 mg/kg DSF for 16 days moderately inhibited the growth of implanted PC9/OR tumors and treatment with both Osi and DSF synergistically suppressed the growth of implanted PC9/OR tumors in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C). However, these treatments did not change the body weights among these groups of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Western blot analyses displayed that treatment of both Osi and DSF obviously decreased RELA phosphorylation and ALDH1A1 expression, but increased the numbers of apoptotic cells in the PC9/OR tumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, Supplemental Fig.\u0026nbsp;14A).\u003c/p\u003e \u003cp\u003eWe further tested the effect of Osi and/or DSF treatment on the growth of implanted patient-derived organoid-based xenograft (PDOX) tumors in NSG mice. First, we detected obviously up-regulated levels of RELA and ALDH1A1 expression and co-localization in the LCO1/OR-derived PDOX tumors, relative to those LCO1-derived tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Consistently, treatment with either Osi or DSF moderately decreased the tumor sizes and treatment with both Osi and DSF further reduced the tumor sizes in this model (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-H). However, treatment with Osi and/or DSF did not change the body weights in these groups of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Treatment with both Osi and DSF obviously reduced the levels of RELA phosphorylation and ALDH1A1 expression in the LCO1/OR-derived tumors, accompanied by increased numbers of apoptotic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ, Supplemental Fig.\u0026nbsp;14B). These data indicated that targeting ALDH1A1 by DSF treatment enhanced the sensitivity to Osi in the EGFR-TKI resistant NSCLC in mice.\u003c/p\u003e \u003cp\u003eGiven that EGCG effectively suppressed the growth of tumor cells in vitro, we tested whether treatment with EGCG could enhance the antitumor effect of Osi in the PC9/OR-based tumor in nude mice. Like in vitro results, treatment with either 5 mg/kg Osi or 10 mg/kg EGCG slightly reduced the tumor sizes and treatment with both significantly decreased the growth of implanted PC9/OR tumors, but failed to alter the body weights in mice, accompanied by increased numbers of apoptotic cells in the tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK-M, Supplemental Fig.\u0026nbsp;14C). A similar pattern of anti-tumor effects was observed in the\u003c/p\u003e \u003cp\u003eLCO1/OR-derived PODX tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO-Q). Interestingly, treatment with both Osi and EGCG obviously reduced the levels of ALDH1A1 expression and RELA phosphorylation in both the PC9/OR and LCO1/OR derived tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN, R), but significantly increased the numbers of apoptotic tumor cells in these tumors (Supplemental Fig.\u0026nbsp;14D).\u003c/p\u003e \u003cp\u003eThese in vivo data further supported the notion that activation of the NF-κB/ALDH1A1 signaling contributed to the development of EGFR-TKI resistance in NSCLC while treatment with DSF or EGCG to inhibit the ALDH1A1 or the NF-κB restored the sensitivity to EGFR-TKIs in NSCLC tumors.\u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;\u003c/b\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e \u003cb\u003eis here\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe EGFR mutations and the genetic alternations in other genes account for about 45% of cases with acquired resistance to EGFR-TKIs and have promoted the development of next generation targeted therapeutics and combinational therapies for NSCLC [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e–\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Furthermore, another 15% of lung cancer cases are attributed to the histologic transformation (e.g., SCLC) and also be treated by combined\u003c/p\u003e \u003cp\u003echemotherapy [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, how the remaining about 40% of lung cancer cases develop the EGFR-TKI resistance is still unknown and they have no effective therapy due to the lack of molecular targets. In our study, we employed NSCLC cell lines, organoids, in vivo xenograft tumor, and PDOX tumor models to explore the potential mechanisms underlying the development of resistance to EGFR-TKIs. We identified a unique mechanism, which explained that long-term TKI treatment induced aberrant activation of the NF-κB signaling to up-regulate ALDH1A1 activity, increasing the numbers of stem-like cells and acquiring non-mutational resistance to EGFR-TKIs in NSCLC.\u003c/p\u003e \u003cp\u003eThe NF-κB is a heterodimeric complex of proteins in the REL family, including p50, p52, RELA, c-REL, and RELB, and interacts with inhibitory IκB in the cytoplasm [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. When activated, the NF-kB is separated from IkBs, and translocates into the nuclei to regulate the targeted gene expression by binding to the gene promoter [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Previous studies have shown that suppression of the NF-κB and FAS significantly enhances the antitumor effect of erlotinib in NSCLC [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and targeting the NF-κB/miR-155/FOXO3a pathway reduces the EGFR mutation-independent resistance to gefitinib, and the stemness of NSCLC cells [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Furthermore, inhibition of the EGFR can trigger the EGFR-TRAF2-RIP1-IKK complex to activate the NF-κB-mediated transcriptional survival program. Additionally, the TET2/NF-κB axis can act as an alternative pathway to confer non-mutational acquired resistance to EGFR-TKIs [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Moreover, IL-6 through the JAK2/STAT3 pathway can promote the resistance to gefitinib in NSCLC by activating the NF-κB signaling [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In this study, we discovered a novel aspect that aberrant activation of the NF-κB/ALDH1A1 signaling contributed to the development of resistance to EGFR-TKIs in NSCLC while pharmacological inhibition of NF-kB or ALDH1A1 activity restored the sensitivity to EGFR-TKIs in NSCLC. Hence, our findings revealed new therapeutic targets of the NF-kB/ALDH1A1 axis for preventing and inhibiting the resistance to EGFR-TKIs in NSCLC.\u003c/p\u003e \u003cp\u003eA recent study has implicated the role of lung CSCs in the development of TKI resistance, evidenced by the facts that Oct-4 can induce gefitinib resistance in EGFR-mutant NSCLC cells by modulating their CSC properties, and up-regulated Oct-4 expression is detected in TKI-resistant NSCLC tissues [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Additionally, afatinib-resistant NSCLC cells display CSC traits, such as increased colony formation, proliferation, ALDH1A1, and CD44 expression [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. This suggests that TKI resistance may stem partly from CSCs. Consistently, we found that ALDH1A1 expression was up-regulated in a subset of TKI-resistant PC9 cells and increased frequency of ALDH1 + cells occurred in R-PC9 cells. These also implied that some ALDH1 + NSCLC cells had the ability to withstand EGFR-TKI drug stress. Similarly, the findings from our LCO model indicated that the CSC-related ALDH1A1 stabilized the resistance\u003c/p\u003e \u003cp\u003echaracteristics. The transcriptome analysis revealed that aberrant activation of the NF-κB pathway up-regulated ALDH1A1 expression, increasing the frequency of ALDH1 + stem-like cells to promote the resistance to EGFR-TKIs in NSCLC. Pharmacological inhibition of ALDH1A1 decreased the frequency of ALDH1 + stem-like cells and partially restored the sensitivity to EGFR-TKIs in NSCLC in vitro and in vivo. Therefore, targeting ALDH1A1 may be a valuable strategy for preventing and inhibiting TKI resistance in NSCLC.\u003c/p\u003e \u003cp\u003eIn our study, we identified that during the development of TKI resistance, the activated RELA promoted the expression of ALDH1A1 in the TKI-resistant NSCLC cells while ALDH1A1 silencing or inhibition partially restored the sensitivity to EGFR-TKIs so that the ALDH1A1 may be a crucial enzyme for the development of drug resistance in NSCLC. Strikingly, the levels of RELA phosphorylation were obviously up-regulated and co-localized with high levels of ALDH1A1 expression in NSCLC tumors from patients resistant to EGFR-TKI therapy. This co-localization suggests a functional interaction between these two proteins, which may significantly contribute to the development of drug resistance. Thus, targeting the NF-κB/ALDH1A1 axis may be a promising strategy to combat or delay the onset of drug resistance in NSCLC.\u003c/p\u003e \u003cp\u003eCurrently, there is no effective treatment for patients resistant to EGFR-TKIs, particularly for those with no new EGFR mutant-resistant NSCLC. Fortunately, previous studies have shown that several natural products can synergistically enhance the antitumor effect of popular chemotherapy drugs [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. EGCG, a component in green tea, has been shown to inhibit the proliferation of various NSCLC cells by inhibiting the NF-κB pathway [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Our data indicated that EGCG monotherapy not only decreased the nuclear translocation of RELA and the frequency of ALDH1 + stem-like cells, but also synergistically enhanced antitumor effect of TKIs in TKI-resistant NSCLC although treatment with high concentrations of EGCG mitigated the sensitivity to EGFR-TKIs in NSCLC. These data underscored the significant role of the NF-κB activation in the development of resistance to EGFR-TKIs in NSCLC and suggest that the anti-TKI resistant effect of EGCG may be attributed to its inhibition on the NF-κB pathway in NSCLC.\u003c/p\u003e \u003cp\u003eInterestingly, the R-PC9 cells had acquired the resistance to TKIs and exhibited higher sensitivity to EGCG than their parental PC9 cells, whereas the H1975 and H1650 cells, harboring the T790M mutation and the deletion of E746-A750 in the EGFR displayed less sensitivity to EGCG. This reduced sensitivity is likely due to the lower levels of RELA phosphorylation, and lower ALDH1A1 activity in these cell lines. The diminished activity of the NF-κB and ALDH1A1 may underlie the lower sensitivity to EGCG for its inhibition on the proliferation and survival of NSCLC cells. Therefore, the level of the NF-κB and\u003c/p\u003e \u003cp\u003eALDH1A1 activities may be a valuable biomarker for predicting the therapeutic responses to EGCG in NSCLC.\u003c/p\u003e \u003cp\u003eCollectively, these findings suggest that the NF-κB/ALDH1A1 axis may serve as an alternative pathway conferring non-mutational, acquired resistance to EGFR-TKIs in NSCLC. Conceivably combination of inhibitors for the NF-κB and ALDH1A1 activity with EGFR-TKIs may be valuable for preventing and inhibiting EGFR-TKI resistance in NSCLC.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eOur studies revealed that the NF-κB/ALDH1A1 axis promoted the development of non-mutational EGFR-TKI resistance in NSCLC and might be a pivotal target for the development of new therapeutics to treat EGFR-TKI resistant NSCLC. Mechanistically, aberrant activation of the NF-κB and subsequently induced ALDH1A1 expression enhanced the stemness of NSCLC CSCs. Evidently, pharmacologic inhibition of NF-κB or ALDH1A1 partially restored the sensitivity to EGFR-TKIs in NSCLC. Therefore, our findings not only provided new insights into the unequal mechanisms underlying the development of non-mutational EGFR-TKI resistance in NSCLC, but also uncovered new therapeutic targets for the intervention of EGFR-TKI resistant NSCLC, particularly for combination of inhibitors for the NF-κB and ALDH1A1 activity with TKIs to achieve long-term remission of patients with TKI-resistant NSCLC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNSCLC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Non-Small Cell Lung Cancer\u003c/p\u003e\n\u003cp\u003eEGFR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Epidermal Growth Factor Receptor\u003c/p\u003e\n\u003cp\u003eTKIs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Tyrosine Kinase Inhibitors\u003c/p\u003e\n\u003cp\u003eLCOs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Lung Cancer Organoids\u003c/p\u003e\n\u003cp\u003ePFS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Progression-free survival\u003c/p\u003e\n\u003cp\u003eRTKs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Receptor tyrosine kinases\u003c/p\u003e\n\u003cp\u003eCSCs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cancer Stem Cells\u003c/p\u003e\n\u003cp\u003eMDR1 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Multidrug Resistance Protein 1\u003c/p\u003e\n\u003cp\u003eRNA-seq \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;RNA Sequencing\u003c/p\u003e\n\u003cp\u003eWES \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Whole-Exome Sequencing\u003c/p\u003e\n\u003cp\u003ePDOX \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Patient-Derived Orthotopic Xenograft\u003c/p\u003e\n\u003cp\u003eDSF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Disulfiram\u003c/p\u003e\n\u003cp\u003eEGCG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Epigallocatechin Gallate\u003c/p\u003e\n\u003cp\u003eIHC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Immunohistochemistry\u003c/p\u003e\n\u003cp\u003eIF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Immunofluorescence\u003c/p\u003e\n\u003cp\u003eCI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Combination Index\u003c/p\u003e\n\u003cp\u003eAfa \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Afatinib\u003c/p\u003e\n\u003cp\u003eErlo \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Erlotinib\u003c/p\u003e\n\u003cp\u003eOsi \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Osimertinib\u003c/p\u003e\n\u003cp\u003eR-PC9 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; EGFR-TKI resistant PC9 Cells\u003c/p\u003e\n\u003cp\u003ePC9/OR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Osimertinib-resistant \u0026nbsp;PC9 \u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePC9/ER \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Erlotinib -resistant \u0026nbsp;PC9\u003c/p\u003e\n\u003cp\u003ePC9/AR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Afatinib -resistant \u0026nbsp;PC9\u003c/p\u003e\n\u003cp\u003eSDS‒PAGE \u0026nbsp; \u0026nbsp;Sodium dodecyl sulfate-polyacrylamide gel electrophoresis\u003c/p\u003e\n\u003cp\u003ePVDF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Polyvinylidene fluoride\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHRP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Horseradish peroxidase\u003c/p\u003e\n\u003cp\u003eCNAs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Copy number alterations\u003c/p\u003e\n\u003cp\u003eChIP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Chromatin immunoprecipitation\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Natural Science Foundation of Fujian Province (Grant No. 2022J05075), Fujian Provincial Health Technology Project (Grant No. 2022GGA030), Scientific Research Foundation of Fujian Cancer Hospital (Grant No. 2023YN08), High-level Talent Development Program of Fujian Cancer Hospital \u0026nbsp; (Grant No. \u0026nbsp;2022YNG03), \u0026nbsp;Joint Funds \u0026nbsp;for the \u0026nbsp;innovation \u0026nbsp;of science \u0026nbsp;and \u0026nbsp;Technology, Fuiian province(Grant No. 2021Y9198), the \u0026nbsp;Startup \u0026nbsp;Fund \u0026nbsp;for \u0026nbsp;Scientific \u0026nbsp;Research, Fujian Medical University (Grant No. 2021QH1137), the Natural Science Foundation of Fujian Province (Grant No. 2022J01742).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYunbin Ye, Lingyu Zhang and Dali Zheng conceived and designed the experiments. Lingyu zhang, Wanson Lin and Yang Wang performed bioinformatics analyses; Lingyu zhang, Chunjiang Liu, Qiumei Li, Jieyu Li and Chuanzhong performed the in vitro experiments. Lingyu zhang, Guibin Weng and Chunjiang Liu performed the in vivo experiments. Yunbin Ye, Wanson Lin, and Lingyu Zhang performed and guided the statistical analysis. Guibin Weng provided or collected the study materials or patient samples; Yunbin Ye, Dali Zheng, Lingyu zhang, Qiumei Li and Wanson Lin wrote and reviewed the manuscript. All the authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data generated in this study were shown in the main text and supplementary material. Additional information is also available upon reasonable request to the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of\u003c/strong\u003e\u003cstrong\u003einterests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were approved by the Medical Ethics Committee of the Fujian Provincial Tumor Hospital (K2023-118-01). Experiments were conducted in accordance with relevant institutional guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eD\u0026apos;Angelo SP, et al. 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Clin Cancer Res. 2009;15:4885-94.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"NSCLC, EGFR-TKIs, Resistance, NF-κB, ALDH1A1","lastPublishedDoi":"10.21203/rs.3.rs-6132050/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6132050/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigates the molecular mechanisms underlying acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC). First, we utilized NSCLC cells, lung cancer organoids (LCOs), xenograft tumor, and patient-derived orthotopic xenografts tumor models to establish TKI resistance. Their stem cell-like properties, drug sensitivity, tumor formation in vitro and in vivo and transcriptomic profiles were analyzed. The differentially expressed genes related to TKI resistance and associated pathways were identified. Functional experiments, including gain- and loss-of-function assays, dual-luciferase reporter analysis, chromatin immunoprecipitation, Western blot, and RT-qPCR, revealed the roles of NF- κ B/ALDH1A1 signaling in acquired resistance. Compared to the parental cells and LCOs, the TKI-resistant R-PC9 and LCO/R exhibited increased RELA phosphorylation, higher frequency of ALDH1\u0026thinsp;+\u0026thinsp;cells, enhanced sphere formation. ALDH1A1 over-expression promoted sphere formation and enhanced resistance to TKIs while ALDH1A1 or RELA silencing had opposite effects. Furthermore, RELA activation enhanced ALDH1A1 activity, and pharmacological inhibition of either NF- κ B or ALDH1A1 activity enhanced the sensitivity to TKIs both in vitro and in vivo. These findings indicated that aberrant activation of the NF- κ B/ALDH1A1 signaling promoted non-mutational resistance to EGFR-TKIs and provided a therapeutic target for EGFR-resistant NSCLC.\u003c/p\u003e","manuscriptTitle":"Activation of NF-κB/ALDH1A1 Signaling Promotes Non-Mutational Resistance to EGFR-TKIs in Non-Small Cell Lung Cancer ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-11 09:22:29","doi":"10.21203/rs.3.rs-6132050/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0e4d4c63-7200-40b5-a053-07a087aa5cd0","owner":[],"postedDate":"March 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-19T05:00:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-11 09:22:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6132050","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6132050","identity":"rs-6132050","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}