LRP8 Promotes Colorectal Cancer Progression by Suppressing Ferroptosis through the SLC3A2/GPX4 Signalling Axis LRP8/SLC3A2/GPX4 promoting CRC

LRP8 Promotes Colorectal Cancer Progression by Suppressing Ferroptosis through the SLC3A2/GPX4 Signalling Axis LRP8/SLC3A2/GPX4 promoting CRC | 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 Article LRP8 Promotes Colorectal Cancer Progression by Suppressing Ferroptosis through the SLC3A2/GPX4 Signalling Axis LRP8/SLC3A2/GPX4 promoting CRC Chengzhang Zhu, Zhengpeng Qian, Shijie Yang, Yongfeng Wang, Xiongfei Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6861954/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 Background: Colorectal cancer (CRC) persists as one of the most lethal malignancies worldwide, with therapeutic resistance representing a significant obstacle in clinical management. Ferroptosis, a form of programmed cell death triggered by iron accumulation and lipid peroxidation, has recently emerged as a promising target for cancer therapy. Although low-density lipoprotein receptor-related protein 8 (LRP8) has been implicated in oncogenic processes across cancer types, its involvement in CRC progression and ferroptosis regulation has not been fully elucidated. Methods: This study utilized an integrative multi-omics approach, incorporating transcriptomic profiling across the colorectal carcinogenesis spectrum (normal mucosa, adenoma, carcinoma; n=5 each) and proteomic analysis via 4D-DIA mass spectrometry. LRP8 expression patterns were examined in 40 paired CRC and adjacent normal tissues and a tissue microarray comprising 94 cases. Functional investigations were conducted in CRC cell lines following LRP8 knockdown or overexpression. Xenograft models were employed for in vivo validation. Mechanistic insights were gained through co-immunoprecipitation, redox assays, and transmission electron microscopy. Results: Transcriptomic data revealed a stepwise increase in LRP8 expression during CRC development. Clinical analyses demonstrated that elevated LRP8 levels correlated significantly with advanced tumour stage, lymphatic metastasis, and poorer patient prognosis. Functional assays indicated that LRP8 enhances oncogenic behaviors by interacting with SLC3A2. Reintroducing SLC3A2 in LRP8-depleted cells restored glutathione peroxidase 4 (GPX4) expression and mitigated oxidative stress, thereby rescuing ferroptosis resistance. In vivo, silencing LRP8 inhibited tumour growth and induced ferroptosis-associated alterations, including disrupted iron homeostasis and increased lipid peroxidation. Conclusion: LRP8 facilitates CRC progression by antagonizing ferroptosis via modulation of the SLC3A2/GPX4 signalling axis. These findings highlight LRP8 as a previously unrecognized regulator of ferroptotic vulnerability and a potential therapeutic target in CRC. Health sciences/Gastroenterology Health sciences/Medical research Health sciences/Oncology LRP8 colorectal cancer ferroptosis SLC3A2 GPX4 iron metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Globally, colorectal carcinoma (CRC) maintains its position as the third most frequently diagnosed malignancy and the second deadliest cancer type[1] Statistical models anticipate substantial growth in disease burden, estimating 3.2 million new cases and 1.6 million annual deaths by 2040—equivalent to a 63% incidence jump and 73.4% mortality surge [2]. Although current therapeutic strategies, including surgical resection, neoadjuvant chemoradiotherapy, and precision molecular interventions, have achieved incremental improvements in clinical outcomes, persistent issues such as locoregional recurrence, distant metastasis, and resistance to treatment continue to limit long-term survival[3, 4]. These challenges highlight the pressing need to identify novel molecular drivers of CRC and develop more effective, targeted treatment approaches. Ferroptosis, an iron-dependent programmed cell death mechanism driven by lipid peroxidation and antioxidant system failure [5-7], has become a potential cancer treatment strategy [8]. This process critically depends on the glutathione-GPX4 pathway, the primary cellular defence against oxidative membrane damage [9]. In CRC, GPX4 is frequently overexpressed in tumour tissues relative to adjacent normal mucosa, underscoring its potential role in tumour survival [10]. Experimental models have shown that induction of ferroptosis, whether through iron modulation, enhancement of reactive oxygen species (ROS), or inhibition of GPX4, effectively suppresses CRC progression [11]. Conversely, resistance to ferroptosis has been implicated in tumour relapse and treatment failure, positioning ferroptosis as a targetable vulnerability in CRC [12]. Using a multi-omics approach, we identified low-density lipoprotein receptor-related protein 8 (LRP8, also known as ApoER2) as a novel contributor to CRC pathogenesis and ferroptosis regulation. LRP8 has been shown to influence ferroptosis sensitivity by preserving the cellular pool of selenocysteine tRNAs required for the efficient translation of GPX4 [13] and by preventing ribosomal stalling at the UGA codon that encodes its essential selenocysteine residue [14]. LRP8 modulates lipid metabolism via the Wnt/β-catenin–stearoyl-CoA desaturase 1 (SCD1) signalling axis, further shaping the cellular redox landscape [15]. Beyond its role in ferroptosis, LRP8 has been implicated in promoting epithelial–mesenchymal plasticity, metastatic dissemination, and resistance to therapy across multiple malignancies, including gastric [16], ovarian [17], and breast cancers [18]. In CRC specifically, the miR-378a-5p/LRP8 regulatory axis has been associated with modulation of radiation response, suggesting broader oncogenic functions [19]. Despite these findings, the involvement of LRP8 in CRC ferroptosis regulation remains incompletely characterized. This study aims to elucidate the role of LRP8 in modulating ferroptotic sensitivity in CRC and to identify new therapeutic strategies that exploit this regulatory pathway. Materials and Methods Patient Samples and Tissue Microarray (TMA) Fresh CRC tissues and matched adjacent non-tumorous samples were collected from 40 patients undergoing curative resection at Gansu Provincial People's Hospital (Lanzhou, China) between April and August 2024. None of the patients received preoperative chemotherapy or radiotherapy. All patients provided written informed consent.This study received approval from the Ethics Committee of Gansu Provincial Hospital(Approval document No.2024-807) and was conducted in accordance with the ethical guidelines delineated in the Helsinki Declaration. Additionally, a commercially available tissue microarray (TMA) slide (Chip ID: HColA180Su21), comprising 94 pairs of CRC and adjacent normal tissues, was purchased from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China). All samples were processed according to standard procedures for downstream analyses, including qPCR and IHC. Clinicopathological features-including age, sex, tumour size, vascular/perineural invasion, lymph node status, TNM classification, PD-1/PD-L1 expression, and KRAS, NRAS, and BRAF mutation status were retrieved from medical records. Immunohistochemistry (IHC) Following optimized protocols, IHC staining was conducted on TMA and xenograft tumour sections using primary antibodies against LRP8(1:300, PA5-109269,Invitrogen),GPX4(1:50,30388-1-AP,Proteintech),SLC3A2(1:50,15193-1-AP,Proteintech) and Ki67(1:300,NBP2-22112,Novus).Two independent pathologists, blinded to clinical information, evaluated the staining results. Ten random high-power fields (×400) were assessed per slide, with 100 tumour cells counted per field. Staining was scored based on the percentage of positive cells and staining intensity. Proportion scores were assigned as follows: 0 (none), 1 (<25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). Intensity scores ranged from 0 (negative) to 3 (strong). Final IHC scores were calculated by multiplying the proportion and intensity scores. Cell Culture and Lentiviral Transduction CRC cell lines (SW480, SW620, HT-29, HCT-116, LOVO) and normal human colonic epithelial cells (NCM-460) were sourced from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). CRC cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C with 5% CO₂. NCM-460 cells were maintained in specialized medium as per the supplier's instructions. Short hairpin RNAs (shRNAs; sh1, sh2, sh3) targeting LRP8 or SLC3A2, a scrambled control (NC), and an overexpression vector (OE-LRP8) were cloned into lentiviral vectors (GeneChem, Shanghai, China) and transduced into CRC cells. Quantitative Real-Time PCR (qRT-PCR) Total RNA extraction was performed with the M5 HiPer Universal RNA Mini Kit (Polymed, China), and cDNA synthesis was used with PrimeScript™ RT reagent Kit (Takara, Japan). Quantitative PCR analysis was conducted with TB Green Premix Ex Taq™ (Takara, RR820A), and relative gene expression was calculated via the 2 ΔΔCt method. Western Blotting Cell lysates were prepared using ice-cold lysis buffer (20 mM Tris-HCl pH 7.5, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA, 50 mM NaF, 1 mM DTT, 10% glycerol) supplemented with protease inhibitors. Following protein quantification (BCA assay, Thermo Fisher), equal protein amounts were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% non-fat milk, membranes were incubated with primary antibodies against LRP8(1:500, PA5-109269,Invitrogen),GPX4(1:1000,30388-1-AP,Proteintech),SLC3A2(1:5000,15193-1-AP,Proteintech) and GAPDH(1:10000,ab181602,Abcam), then with HRP-conjugated secondary antibodies. Protein signals were visualized using enhanced chemiluminescence (Thermo Fisher). Colony Formation Assay Cells (1,000/well) were seeded in 6-well plates and incubated for 10–14 days. Colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Colonies with >50 cells were manually counted in five random fields. Flow Cytometry Analysis Cells were stained with Annexin V-FITC/PI (BD Biosciences, USA) and analyzed by flow cytometry (BD FACSCanto™ II). For cell cycle analysis, ethanol-fixed cells were stained with PI and RNase A, and DNA content was quantified by flow cytometry. EdU Incorporation Assay Cell proliferation was assessed using the EdU Apollo567 kit (RiboBio, China). Cells were treated with 50 μM EdU, then fixed, permeabilized, and stained. Nuclear counterstaining was performed using Hoechst 33342. Fluorescence images were captured using an Olympus microscope (Olympus). Migration and Invasion Assays Migration and invasion were assessed in Transwell chambers (8 μm pores; Corning, USA). For migration, serum-free cell suspensions were seeded in the upper chamber, with 10% FBS medium in the lower well. The chamber membrane was pre-coated with Matrigel (BD Biosciences, USA) for invasion. After 48 h, non-migrated cells were removed, and migrated/invaded cells were fixed, stained, and counted under a microscope. RNA Sequencing and Bioinformatics Novogene Co., Ltd. (Beijing, China) conducted RNA-seq using total RNA from 15 samples (normal mucosa, adenoma, and carcinoma; n = 5 each). After RNA quality control, sequencing was performed on the Illumina platform. Differentially expressed genes (DEGs) were identified, and functional annotations were performed using GO and KEGG analyses. Proteomics Analysis Proteomic profiling was performed using 4D-DIA mass spectrometry (Bruker timsTOF Pro). SW480 cells transfected with siLRP8 or siNC were analyzed by LC-MS/MS. Raw data were processed with Spectronaut (Biognosys, Sweden), and differential protein expression was analyzed with functional enrichment of affected pathways (GO, KEGG)[29,30]. Animal Experiments Fifteen male BALB/c nude mice (4 weeks old, n=5/group) were subcutaneously injected with 1×10⁶SW480 cells (wild-type, NC, or LRP8 knockdown). Tumour dimensions and body weight were measured every 2 days from day 10.After a 21-day alimentation period,All animals were euthanized by barbiturate overdose (intravenous injection, 150 mg/kg pentobarbital sodium),death was confirmed by loss of heartbeat, breathing and pupil response, and the tumor tissues were extirpated and weighed.Tumor tissues were immobilized in 4% paraformaldehyde for subsequent experimentation. All animal experimental procedures were sanctioned by the Bestcell Model Biological Center(IACUC Issue No.2025-04-18A). Transmission Electron Microscopy (TEM) Cells were fixed in glutaraldehyde, dehydrated, embedded in resin, and sectioned into ultrathin slices. After staining with uranyl acetate and lead citrate, mitochondrial ultrastructure was visualized using TEM (Thermo Fisher Scientific, USA). Biochemical Assays BCA assays were used for protein quantification. Glutathione (total, reduced, oxidised), total iron, ferrous iron (Fe²⁺), cysteine, and ROS levels were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute and Elabscience), following manufacturer protocols and analysed via spectrophotometry. Mitochondrial Membrane Potential (MMP) JC-1 staining (Cat. C2003S) assessed mitochondrial membrane potential. Cells were incubated with JC-1 dye, washed, and analyzed by flow cytometry(Thermo Fisher Scientific, USA). The red-to-green fluorescence ratio reflected MMP status. Statistical Analysis Statistical analyses were performed using GraphPad Prism 9.0 and SPSS 26.0. Data were expressed as mean ± SD. The students' t-test was used for two-group comparisons, and ANOVA was applied for multiple-group analyses. Associations between LRP8 expression and clinical variables were assessed using the χ² test. A p-value < 0.05 was considered statistically significant. Results Transcriptomic and Bioinformatic Analyses Identify LRP8 as a Key Gene Associated with CRC Progression To investigate dynamic gene expression changes during colorectal tumorigenesis, we conducted transcriptome sequencing on tissue samples representing three distinct stages: normal mucosa (N, n = 5), adenomas (T, n = 5), and carcinomas (C, n = 5) (Fig. 1A). A total of 3,685 DEGs were identified. Volcano plots illustrated substantial transcriptional shifts in both A vs. T and C vs. N comparisons (Fig. 1B), with the number of DEGs increasing progressively along the typical adenoma–carcinoma sequence (Fig. 1C). Unsupervised hierarchical clustering revealed clear segregation among the three sample types (Fig. 1D), and all sequencing datasets passed standard quality control assessments, confirming the robustness of the transcriptomic analysis (Fig. 1E). Venn diagrams demonstrated both shared and unique DEGs among the pairwise comparisons (Fig. 1F).Functional enrichment based on GO highlighted that the DEGs were predominantly involved in biological processes such as extracellular matrix organization, epithelial cell migration, and regulation of the Wnt signalling pathway (Fig. 1G–H). KEGG pathway analysis further revealed significant enrichment in pathways implicated in tumour biology, including IL-17 signalling, the p53 pathway, and focal adhesion (Fig. 1I).Complementary annotations via Disease Ontology, DisGeNET, and Reactome databases consistently associated the identified DEGs with colorectal cancer-related pathways and mechanisms (Fig. 1J–L). Notably, LRP8 expression exhibited a stepwise upregulation from normal mucosa to adenoma and carcinoma tissues, suggesting its potential involvement in the early and progressive stages of colorectal carcinogenesis. This observation positioned LRP8 as a promising candidate gene for subsequent functional validation. LRP8 Is Upregulated in Colorectal Cancer and Correlates with Poor Prognosis Subsequently, we analyzed LRP8 mRNA levels in both colon adenocarcinoma (COAD) (Fig. 2A) and rectal adenocarcinoma (READ) (Fig. 2 B) from TCGA. The results were significantly elevated compared with their corresponding adjacent normal tissues. Moreover, high LRP8 expression was associated with reduced overall survival in CRC patients, as demonstrated by Kaplan–Meier survival analysis (Fig. 2C). To validate these findings, we examined LRP8 expression in 40 paired CRC tumours and adjacent standard tissue samples. Immunofluorescence staining revealed that LRP8 protein was primarily localized to the cytoplasm and plasma membrane of cancer cells (Fig. 2D). RT-PCR confirmed a significant increase in LRP8 mRNA expression in tumor tissues compared to normal tissues (P = 0.006; Fig. 2E). Consistently, immunohistochemical (IHC) analysis of the same cohort demonstrated a marked overexpression of LRP8 in tumor specimens (Fig. 2F). This observation was further supported by semi-quantitative scoring of a tissue microarray containing 86 CRC cases, which revealed significantly higher LRP8 expression in tumor tissues (P < 0.0001; Fig. 2G). To explore the clinical relevance of LRP8, we examined its association with key clinicopathological features. Elevated LRP8 levels were significantly correlated with advanced TNM stage, presence of lymph node metastasis, increased infiltration of CD8⁺ T cells, PD-1/PD-L1 positivity, and KRAS mutations (Table 1). No significant associations were observed with patient age, sex, tumour size, or NRAS/BRAF mutation status. Importantly, Kaplan–Meier analysis of the tissue microarray cohort indicated that high LRP8 expression was an unfavourable prognostic indicator, predicting reduced 5-year overall survival (P < 0.05; Fig. 2H). LRP8 Promotes malignant behaviors of CRC Cells To elucidate the oncogenic role of LRP8 in colorectal carcinogenesis, we systematically characterized its expression profile and functional significance across multiple CRC cell lines. Comparative analysis revealed significant upregulation of LRP8 at both transcriptional and translational levels in all examined CRC cell lines (SW480, SW620, HT-29, HCT116, and LOVO) relative to normal colonic epithelial cells (NCM460) (Fig. 3A). Through comprehensive genetic perturbation studies employing three independent shRNA constructs(Fig. 3B,C), we demonstrated that LRP8 depletion in SW480 and SW620 cells resulted in profound suppression of malignant phenotypes, including impaired proliferative capacity (Fig. 3J), reduced clonogenic potential, attenuated migratory and invasive properties (Fig. 3H-I), as well as increased apoptotic susceptibility and G1 phase cell cycle arrest (Fig. 3K-L). Conversely, ectopic LRP8 expression in HCT116 cells significantly enhanced these oncogenic behaviors, as evidenced by accelerated cell cycle progression and diminished apoptosis (Fig. 3D,J-L). LRP8 Sustains GPX4 Expression to Inhibit Ferroptosis in CRC To explore the downstream effects of LRP8 in CRC, we performed proteomic profiling using 4D-DIA sequencing in SW480 cells following LRP8 knockdown by siRNA. The resulting volcano plot demonstrated a broad spectrum of differentially expressed proteins (DEPs), among which GPX4 and SLC3A2, key regulators of oxidative stress and ferroptosis, were significantly downregulated (Fig. 4D). GO and KEGG pathway enrichment analyses indicated that LRP8 silencing predominantly affected pathways related to oxidative stress response, ferroptosis, and metabolic regulation (Fig. 4E). Subcellular localization predictions revealed that LRP8 is mainly distributed in the nucleus (32.7%) and cytoplasm (30.29%), suggesting its potential involvement in organelle-specific regulatory mechanisms associated with ferroptotic cell death (Fig. 4F).Further GO enrichment analysis emphasised strong associations with ferroptosis-related biological processes, including organelle organisation, intracellular transport, and protein-binding functions (Fig. 4G). KEGG analysis showed that cell cycle progression and metabolic pathways were notably disrupted in LRP8-deficient cells, indicating broader impacts on cellular homeostasis (Fig. 4H). PPI network analysis identified clusters of DEPs with dense interconnections, particularly centered on ferroptosis-related proteins (Fig. 4I). A focused PPI subnetwork built around GPX4 highlighted its interactions with core antioxidant and metabolic regulators (Fig. 4K), reinforcing its potential role of LRP8 in ferroptosis. Subsequent qRT-PCR validation confirmed that LRP8 knockdown decreased GPX4, SLC3A2, YTHDF2, and DNMT1 expression, while SLC31A1 were upregulated (Fig. 4L). These results were consistent with the proteomic data and further supported the regulatory relationship between LRP8 and ferroptosis-associated genes. These findings demonstrate that LRP8 influences ferroptosis in CRC cells by controlling the expression of GPX4 and SLC3A2. To determine whether LRP8 suppression triggers ferroptosis in CRC cells, we generated stable LRP8-knockdown and LRP8-overexpressing cell lines (Fig. 5A–B). TEM of LRP8-silenced SW480 and SW620 cells revealed classic features of ferroptosis, including reduced mitochondrial volume, condensed membrane density, loss or disappearance of cristae, and outer membrane rupture. In contrast, LRP8-overexpressing cells exhibited intact mitochondrial structures without these abnormalities (Fig. 5C). JC-1 staining was used to assess mitochondrial membrane potential. LRP8 knockdown led to a pronounced reduction in JC-1 aggregates, indicating mitochondrial depolarisation and dysfunction. Conversely, LRP8 overexpression preserved membrane potential, as evidenced by stronger JC-1 aggregation (Fig. 5D). Silencing LRP8 significantly increased intracellular ferrous iron (Fe²⁺) levels (Fig. 5E), total iron content (Fig. 5G), and glutathione (GSH) levels (Fig. 5H), suggesting an accumulation of labile iron and heightened redox activity. Interestingly, LRP8 overexpression in HCT116 cells also elevated Fe²⁺, total iron, and GSH levels, indicating that LRP8 regulates iron metabolism and redox balance across multiple CRC cell types. To confirm that these changes were ferroptosis-specific, ferrostatin-1 (Fer-1), a selective ferroptosis inhibitor, was applied to LRP8-knockdown cells. Fer-1 treatment reversed mitochondrial damage, restoring organelle morphology (Fig. 5I) and normalizing JC-1 fluorescence patterns (Fig. 5J), indicating recovery of membrane potential. Fer-1 significantly reduced Fe²⁺ accumulation (Fig. 5K–L) in the LRP8-silenced cells, supporting the iron dependence of the observed phenotype. Treatment also restored GSH levels (Fig. 5M) and reduced oxidized glutathione (GSSG) levels (Fig. 5N), further alleviating oxidative stress. Together, these findings demonstrate that LRP8 influences ferroptosis sensitivity in CRC cells by regulating intracellular iron levels and redox status, and that ferroptotic responses induced by LRP8 loss are reversible through ferroptosis inhibition. LRP8 binds SLC3A2 to Promote Cysteine Uptake and Ferroptosis Resistance To investigate the molecular mechanisms by which LRP8 influences ferroptosis, we conducted mass spectrometry-based immunoprecipitation (MS-IP) in LRP8-overexpressing HCT116 cells to identify its interacting protein partners (Fig. 6A–B). Among the candidate interactors, SLC3A2 was identified as a potential binding partner (Fig. 6C). Functional enrichment analysis of the LRP8-associated protein complexes revealed significant enrichment in pathways related to protein trafficking, oxidative stress response, and endoplasmic reticulum (ER) stress (Fig. 6D–F), suggesting a broader role for LRP8 in maintaining cellular stress adaptation and metabolic homeostasis.Western blot analysis confirmed that LRP8 knockdown in SW480 and SW620 cells led to a marked reduction in SLC3A2 expression (Fig. 6G), supporting a regulatory relationship between these two proteins. In addition, protein–protein interaction (PPI) network analysis further reinforced the existence of a direct or functionally relevant interaction between LRP8 and SLC3A2(Fig. 6H).Subsequently,ELISA analysis confirmed that knockdown of SLC3A2 in SW480 and SW620 cells led to a significant decrease in Cyc content and a significant increase in ROS levels(Fig. 6I–J). These findings indicate that LRP8 may regulate cysteine uptake and redox homeostasis in CRC cells by interacting with SLC3A2, thereby contributing to ferroptosis susceptibility through modulation of system Xc⁻ activity. Building on the identified interaction between LRP8 and SLC3A2, we explored whether LRP8 promotes CRC progression by inhibiting SLC3A2-mediated ferroptosis. Efficient knockdown of SLC3A2 in HCT116 cells was validated at both the mRNA and protein levels (Fig. 7A). Co-transfection of LRP8-overexpressing cells with SLC3A2-targeting shRNA significantly reversed the upregulation of GPX4 and SLC3A2, implicating SLC3A2 as a downstream effector of LRP8's anti-ferroptotic activity (Fig.7B-C). Biochemical assays revealed that simultaneous LRP8 overexpression and SLC3A2 silencing resulted in significant reductions in intracellular GSH and Cys levels (Fig. 7D–E), accompanied by increased reactive oxygen species (ROS) accumulation (Fig. 7F), indicating a breakdown in redox homeostasis. Functionally, LRP8 overexpression enhanced proliferation, as evidenced by increased EdU and Ki67 positivity. However, this proliferative effect was markedly attenuated by SLC3A2 knockdown, indicating that SLC3A2 is necessary for LRP8-driven cell proliferation. Similarly, LRP8 overexpression promoted cell migration and invasion, as shown by transwell assays, whereas co-silencing SLC3A2 significantly suppressed these metastatic properties. Colony formation assays further demonstrated that LRP8-driven clonogenic growth was reduced upon SLC3A2 depletion. Additionally, cell cycle analysis showed that knockdown of SLC3A2 in LRP8-overexpressing cells caused a significant decrease in S and G2/M phase populations, suggesting impaired cell cycle progression. These findings indicate that SLC3A2 is a key mediator of LRP8’s oncogenic effects in CRC, facilitating ferroptosis resistance. LRP8 Silencing Triggers Ferroptosis in CRC Xenograft Models To explore the in vivo function of LRP8 in colorectal cancer (CRC), we established a subcutaneous xenograft model using SW480 cells with stable LRP8 knockdown. Tumours from the sh-LRP8 group exhibited significantly reduced volumes and growth rates compared to those in the negative control group, indicating that LRP8 is essential for efficient tumour progression (Fig. 8A–C). Immunohistochemistry confirmed successful knockdown, showing markedly decreased LRP8 expression in tumour sections from the sh-LRP8 cohort (Fig. 8D). TUNEL staining revealed a substantial increase in apoptotic cells within the sh-LRP8 tumors, suggesting that LRP8 contributes to tumor cell survival (Fig. 8E). Western blot analysis further validated these findings, demonstrating reduced levels of LRP8 and GPX4, a key regulator of ferroptosis, in tumor lysates from the LRP8-silenced group (Fig. 8F). Biochemical assays provided additional insight into the redox imbalance induced by LRP8 depletion. Tumours lacking LRP8 exhibited decreased concentrations of ferrous iron (Fe²⁺; Fig. 8J), total iron (t-Fe; Fig. 8K), GSH(Fig. 8L), and Cys (Fig. 8H). In contrast, oxidized glutathione (GSSG) levels were significantly elevated (Fig. 8M), accompanied by a marked increase in reactive oxygen species (ROS) accumulation (Fig. 8I). These metabolic shifts are indicative of ferroptotic cell death. Together, these in vivo results demonstrate that LRP8 promotes tumour growth in CRC by suppressing ferroptosis. Discussion Our integrated multi-omics and functional analyses establish LRP8 as a critical oncogenic driver in colorectal cancer. We firstly demonstrate that LRP8 overexpression, clinically correlated with advanced disease and poor prognosis. It overexpression promotes malignant transformation by enhancing proliferative, migratory, and anti-apoptotic capacities while maintaining redox homeostasis. The mechanistic elucidation reveals that LRP8 physically interacts with SLC3A2 to preserve system Xc⁻ integrity, thereby sustaining intracellular cysteine/GSH pools and GPX4-mediated lipid peroxide clearance. The finding is underscored by in vivo evidence showing LRP8 ablation induces tumor-suppressive ferroptosis, characterized by iron dyshomeostasis, mitochondrial degeneration, and lethal lipid peroxidation. This work defines a novel LRP8-SLC3A2-GPX4 regulatory axis with therapeutic potential for CRC treatment. While accumulating evidence highlights LRP8 as a pivotal oncoprotein in various malignancies [16, 17, 20, 21], its role in CRC progression remains incompletely characterised. Previous studies have demonstrated that LRP8 facilitates tumorigenesis in ovarian cancer by antagonising p53 signalling [21], promotes metastasis in lung cancer via Spon1 -mediated pathways [22], and silencing LRP8 promotes a luminal-epithelial differentiation state in TNBC cells, consequently enhancing their chemosensitivity [23]. In the present study, we observed a progressive increase in LRP8 expression throughout the colorectal adenoma-carcinoma sequence, with a marked upregulation in carcinoma tissues compared to normal mucosa and adenoma. Clinically, elevated LRP8 levels were significantly associated with advanced TNM stage, lymph node metastasis, and poor patient survival, underscoring its potential as a prognostic biomarker in CRC. In vitro experiments further revealed that LRP8 knockdown notably inhibited CRC cell proliferation, colony formation, migration, and invasion, while its overexpression amplified these malignant characteristics. These in vitro findings were corroborated in vivo, where LRP8 depletion significantly reduced tumour growth in xenograft models. In this study, SLC3A2 was identified as a critical downstream effector of LRP8, mediating ferroptosis resistance in CRC. As a chaperone protein, SLC3A2 plays a key role in the membrane localisation and stabilisation of light chains of amino acid transporters, such as SLC7A11 and SLC1A5, facilitating cystine uptake and maintaining intracellular redox balance [24]. The upregulation of SLC3A2 has been associated with ferroptosis suppression and enhanced glutathione synthesis in various tumour types, including laryngeal [25], breast [26], and treatment-resistant prostate cancers [27]. Proteomic analysis using immunoprecipitation followed by mass spectrometry (IP-MS) revealed a direct interaction between LRP8 and SLC3A2, which was further confirmed through co-immunoprecipitation assays. This interaction suggests that LRP8 may exert part of its anti-ferroptotic effects by modulation of SLC3A2. Rescue experiments further supported this hypothesis, showing that reintroducing SLC3A2 into LRP8-silenced cells alleviated ferroptosis-associated phenotypes, such as excessive ROS production, glutathione depletion, and mitochondrial damage. These findings establish SLC3A2 as a functional mediator of the LRP8-regulated ferroptosis pathway in CRC. Beyond its metabolic role, SLC3A2 also shapes the tumour immune microenvironment by regulating immune cell activation and antigen presentation [28]. This suggests that the LRP8–SLC3A2 axis may govern ferroptosis resistance and contribute to immune evasion in CRC. Targeting this axis could therefore represent a promising therapeutic strategy, disrupting tumour redox homeostasis while enhancing anti-tumour immune responses. In summary, our study systematically elucidates the oncogenic role of LRP8 in colorectal cancer through comprehensive multi-omics analyses and functional validation. We demonstrate that LRP8 exhibits progressive upregulation during CRC development, correlating with advanced disease stage and poor prognosis. Mechanistically, LRP8 promotes tumour progression by interacting with SLC3A2 to maintain redox homeostasis and suppress ferroptosis, while simultaneously enhancing CRC cells' proliferative, migratory, and invasive capacities. Identifying the LRP8-SLC3A2-GPX4 axis provides novel insights into the molecular regulation of ferroptosis resistance in CRC, revealing a previously unrecognized vulnerability that could be exploited therapeutically. Declarations Funding The work is supported by National Natural Science Foundation of China (No. 82360498) ,Gansu Joint Scientific Research Fund Major Project (No.23JRRA1537), the 2025 Central-Guided Local Science and Technology Development Found(No. 25ZYJA003), Gansu Provincial Health Industry Science and Technology Innovation Major Project(No.GSWSZD2024-01) , Gansu Province Key Talent Project(No.2025RCXM067) , and department of Health Commission of Gansu Province(No.GSWSKY2024-12). Ethics approval and consent to participate All patients provided written informed consent.This study received approval from the Ethics Committee of Gansu Provincial Hospital(Approval document No.2024-807) and was conducted in accordance with the ethical guidelines delineated in the Helsinki Declaration. All animal experimental procedures were sanctioned by the Bestcell Model Biological Center(IACUC Issue No.2025-04-18A). Conflict of interests None. Acknowlegement None. Availability of data and materials RNA sequencing:The datasets generated and analysed during the current study are available in the National Library of Medicine(https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1276701) repository. Project ID:PRJNA1276701 Proteomics data generated:The datasets generated and analysed during the current study are available in the Integrated Proteome Resources(https://www.iprox.cn/page/project.html?id=IPX0012425000) repository. Project ID:IPX0012425000 The designated point of contact for data requests:Dr Chengzhang Zhu;The First Clinical Medical College,Lanzhou Universit,Lanzhou, Gansu Province, 730000, China.;Email: [email protected] . Patient consent for publication Not applicable. Authors' contributions Chengzhang Zhu, Zhengpeng Qian are the co-first authors. Chengzhang Zhu: Conceptualization, Methodology, Investigation, Formal Analysis, Writing – Original Draft, Writing – Review & Editing. Zhengpeng Qian: Methodology, Validation, Investigation, Data Curation, Writing – Original Draft. Shijie Yang: Investigation, Resources, Visualization, Data Curation. Yongfeng Wang: Investigation, Software, Formal Analysis, Visualization. Xiongfei Yang: Investigation, Resources, Data Curation, Visualization. Binbin Du and Hui Cai: Supervision, Funding Acquisition, Project Administration, Writing – Review & Editing. References Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229-63. Epub 2024/04/04. doi: 10.3322/caac.21834. PubMed PMID: 38572751. Marmol I, Sanchez-de-Diego C, Pradilla Dieste A, Cerrada E, Rodriguez Yoldi MJ. Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer. Int J Mol Sci. 2017;18(1). Epub 2017/01/21. doi: 10.3390/ijms18010197. PubMed PMID: 28106826; PubMed Central PMCID: PMCPMC5297828. Scanu AM, De Miglio MR. Therapeutic Landscapes in Colorectal Carcinoma. Medicina (Kaunas). 2023;59(5). Epub 2023/05/27. doi: 10.3390/medicina59050821. PubMed PMID: 37241053; PubMed Central PMCID: PMCPMC10222447. Smith HG, Nilsson PJ, Shogan BD, Harji D, Gambacorta MA, Romano A, et al. Neoadjuvant treatment of colorectal cancer: comprehensive review. BJS Open. 2024;8(3). Epub 2024/05/15. doi: 10.1093/bjsopen/zrae038. PubMed PMID: 38747103; PubMed Central PMCID: PMCPMC11094476. Li J, Cao F, Yin HL, Huang ZJ, Lin ZT, Mao N, et al. Ferroptosis: past, present and future. Cell Death Dis. 2020;11(2):88. Epub 2020/02/06. doi: 10.1038/s41419-020-2298-2. PubMed PMID: 32015325; PubMed Central PMCID: PMCPMC6997353. Liang D, Minikes AM, Jiang X. Ferroptosis at the intersection of lipid metabolism and cellular signaling. Mol Cell. 2022;82(12):2215-27. Epub 2022/04/08. doi: 10.1016/j.molcel.2022.03.022. PubMed PMID: 35390277; PubMed Central PMCID: PMCPMC9233073. Tang D, Chen X, Kang R, Kroemer G. Ferroptosis: molecular mechanisms and health implications. Cell Res. 2021;31(2):107-25. Epub 2020/12/04. doi: 10.1038/s41422-020-00441-1. PubMed PMID: 33268902; PubMed Central PMCID: PMCPMC8026611. Jiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 2021;22(4):266-82. Epub 2021/01/27. doi: 10.1038/s41580-020-00324-8. PubMed PMID: 33495651; PubMed Central PMCID: PMCPMC8142022. Xue Q, Yan D, Chen X, Li X, Kang R, Klionsky DJ, et al. Copper-dependent autophagic degradation of GPX4 drives ferroptosis. Autophagy. 2023;19(7):1982-96. Epub 2023/01/10. doi: 10.1080/15548627.2023.2165323. PubMed PMID: 36622894; PubMed Central PMCID: PMCPMC10283421. Huang B, Wang H, Liu S, Hao M, Luo D, Zhou Y, et al. Palmitoylation-dependent regulation of GPX4 suppresses ferroptosis. Nat Commun. 2025;16(1):867. Epub 2025/01/21. doi: 10.1038/s41467-025-56344-5. PubMed PMID: 39833225; PubMed Central PMCID: PMCPMC11746948. Yan H, Talty R, Johnson CH. Targeting ferroptosis to treat colorectal cancer. Trends Cell Biol. 2023;33(3):185-8. Epub 2022/12/07. doi: 10.1016/j.tcb.2022.11.003. PubMed PMID: 36473802. Zeng K, Li W, Wang Y, Zhang Z, Zhang L, Zhang W, et al. Inhibition of CDK1 Overcomes Oxaliplatin Resistance by Regulating ACSL4-mediated Ferroptosis in Colorectal Cancer. Adv Sci (Weinh). 2023;10(25):e2301088. Epub 2023/07/10. doi: 10.1002/advs.202301088. PubMed PMID: 37428466; PubMed Central PMCID: PMCPMC10477855. Zhao Y, Shi G, Huang X, Zhang Z, Liao K, Xiong H, et al. LRP8 inhibits bladder cancer cell ferroptosis by activating the Wnt/beta-catenin-SCD1 positive feedback loop. Hum Mol Genet. 2025;34(10):843-51. Epub 2025/02/24. doi: 10.1093/hmg/ddaf024. PubMed PMID: 39989004. Li Z, Zhang J, Zheng H, Li C, Xiong J, Wang W, et al. Modulating lncRNA SNHG15/CDK6/miR-627 circuit by palbociclib, overcomes temozolomide resistance and reduces M2-polarization of glioma associated microglia in glioblastoma multiforme. J Exp Clin Cancer Res. 2019;38(1):380. Epub 2019/08/30. doi: 10.1186/s13046-019-1371-0. PubMed PMID: 31462285; PubMed Central PMCID: PMCPMC6714301. Wu GF, Luo ZG, Gao K, Ren Y, Shen C, Ying XR. LRP8 Regulates Lipid Metabolism to Stimulate Malignant Progression and Cisplatin Resistance in Bladder Cancer. Kaohsiung J Med Sci. 2025:e70042. Epub 2025/05/15. doi: 10.1002/kjm2.70042. PubMed PMID: 40372166. Liu B, Bukhari I, Li F, Ren F, Xia X, Hu B, et al. Enhanced LRP8 expression induced by Helicobacter pylori drives gastric cancer progression by facilitating beta-Catenin nuclear translocation. J Adv Res. 2025;69:299-312. Epub 2024/04/13. doi: 10.1016/j.jare.2024.04.002. PubMed PMID: 38609049; PubMed Central PMCID: PMCPMC11954824. Li C, Yang Y, Wang H, Song Y, Huang H. miR-362-3p suppresses ovarian cancer by inhibiting LRP8. Transl Oncol. 2022;15(1):101284. Epub 2021/11/29. doi: 10.1016/j.tranon.2021.101284. PubMed PMID: 34839107; PubMed Central PMCID: PMCPMC8636862. Li L, Qu WH, Ma HP, Wang LL, Zhang YB, Ma Y. LRP8, modulated by miR-1262, promotes tumour progression and forecasts the prognosis of patients in breast cancer. Arch Physiol Biochem. 2022;128(3):657-65. Epub 2020/01/30. doi: 10.1080/13813455.2020.1716019. PubMed PMID: 31994910. Hu G, Che P, Deng L, Liu L, Liao J, Liu Q. MiR-378a-5p exerts a radiosensitizing effect on CRC through LRP8/beta-catenin axis. Cancer Biol Ther. 2024;25(1):2308165. Epub 2024/02/23. doi: 10.1080/15384047.2024.2308165. PubMed PMID: 38389136; PubMed Central PMCID: PMCPMC10896128. Alborzinia H, Chen Z, Yildiz U, Freitas FP, Vogel FCE, Varga JP, et al. LRP8-mediated selenocysteine uptake is a targetable vulnerability in MYCN-amplified neuroblastoma. EMBO Mol Med. 2023;15(8):e18014. Epub 2023/07/12. doi: 10.15252/emmm.202318014. PubMed PMID: 37435859; PubMed Central PMCID: PMCPMC10405063. Xu Y, Zhou Y, Yi X, Nie X. LRP8 promotes tumorigenesis in ovarian cancer through inhibiting p53 signaling. Cell Biol Int. 2024;48(5):626-37. Epub 2024/01/24. doi: 10.1002/cbin.12133. PubMed PMID: 38263609. Qiu H, Shen X, Chen B, Chen T, Feng G, Chen S, et al. miR-30b-5p inhibits cancer progression and enhances cisplatin sensitivity in lung cancer through targeting LRP8. Apoptosis. 2021;26(5-6):261-76. Epub 2021/03/30. doi: 10.1007/s10495-021-01665-1. PubMed PMID: 33779882. Lin CC, Lo MC, Moody R, Jiang H, Harouaka R, Stevers N, et al. Targeting LRP8 inhibits breast cancer stem cells in triple-negative breast cancer. Cancer Lett. 2018;438:165-73. Epub 2018/09/19. doi: 10.1016/j.canlet.2018.09.022. PubMed PMID: 30227220; PubMed Central PMCID: PMCPMC6945120. Chen Z, Zhang J, Gao S, Jiang Y, Qu M, Gu J, et al. Suppression of Skp2 contributes to sepsis-induced acute lung injury by enhancing ferroptosis through the ubiquitination of SLC3A2. Cell Mol Life Sci. 2024;81(1):325. Epub 2024/07/31. doi: 10.1007/s00018-024-05348-3. PubMed PMID: 39079969; PubMed Central PMCID: PMCPMC11335248. Wu F, Xiong G, Chen Z, Lei C, Liu Q, Bai Y. SLC3A2 inhibits ferroptosis in laryngeal carcinoma via mTOR pathway. Hereditas. 2022;159(1):6. Epub 2022/01/22. doi: 10.1186/s41065-022-00225-0. PubMed PMID: 35057861; PubMed Central PMCID: PMCPMC8772086. Xiong H, Zhai Y, Meng Y, Wu Z, Qiu A, Cai Y, et al. Acidosis activates breast cancer ferroptosis through ZFAND5/SLC3A2 signaling axis and elicits M1 macrophage polarization. Cancer Lett. 2024;587:216732. Epub 2024/02/16. doi: 10.1016/j.canlet.2024.216732. PubMed PMID: 38360142. Ghoochani A, Hsu EC, Aslan M, Rice MA, Nguyen HM, Brooks JD, et al. Ferroptosis Inducers Are a Novel Therapeutic Approach for Advanced Prostate Cancer. Cancer Res. 2021;81(6):1583-94. Epub 2021/01/24. doi: 10.1158/0008-5472.CAN-20-3477. PubMed PMID: 33483372; PubMed Central PMCID: PMCPMC7969452. Nachef M, Ali AK, Almutairi SM, Lee SH. Targeting SLC1A5 and SLC3A2/SLC7A5 as a Potential Strategy to Strengthen Anti-Tumour Immunity in the Tumour Microenvironment. Front Immunol. 2021;12:624324. Epub 2021/05/07. doi: 10.3389/fimmu.2021.624324. PubMed PMID: 33953707; PubMed Central PMCID: PMCPMC8089370. Kanehisa M, Furumichi M, Sato Y, Matsuura Y, Ishiguro-Watanabe M. KEGG: biological systems database as a model of the real world. Nucleic Acids Res. 2025 Jan 6;53(D1):D672-D677. doi: 10.1093/nar/gkae909. PMID: 39417505; PMCID: PMC11701520. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000 Jan 1;28(1):27-30. doi: 10.1093/nar/28.1.27. PMID: 10592173; PMCID: PMC102409. Tables Table 1 The correlation between the expression level of LRP8 and clinicopathological characteristics as well as prognosis in CRC patients Characteristics No.of cases LRP8 expression c 2 P -value Low High Gender Male 46 14 32 1.139 0.286 Female 48 10 38 Age (years) ≤60 35 11 24 1.020 0.313 >60 59 13 46 Tumor size(cm) ≤5 45 9 36 1.389 0.238 >5 49 15 34 Mucinous cancer Yes 35 10 25 0.271 0.388 No 59 14 45 Remote relocation Yes 5 1 4 0.085 0.621 No 89 23 66 Right-sided Yes 43 14 29 2.058 0.116 No 51 10 41 Vascular invasion Yes 31 11 20 2.409 0.121 No 63 13 50 Nerve invasion Yes 17 6 11 1.040 0.308 No 77 18 59 T staging T1-T2 9 4 5 1.872 0.171 T3-T4 85 20 65 Lymph node status N0 59 21 38 8.437 0.004 N1-N2 35 3 32 Characteristics No.of cases LRP8 expression c 2 P -value Low High p TNM staging I/II 57 20 37 6.955 0.008 III/IV 37 4 33 CD8 positive rate <1% 29 3 26 5.088 0.024 ≧1% 65 21 44 PD1 positive rate <1% 30 12 18 4.851 0.028 ≧1% 64 12 52 PDL1 positive rate <1% 44 16 28 5.104 0.024 ≧1% 50 8 42 KRAS mutation Yes 32 4 28 4.334 0.037 No 62 20 42 NRAS mutation Yes 4 0 4 1.432 0.231 No 90 24 66 BRAF mutation Yes 2 1 1 0.643 0.442 No 92 23 69 Additional Declarations No competing interests reported. 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Hospital","correspondingAuthor":true,"prefix":"","firstName":"Hui","middleName":"","lastName":"Cai","suffix":""}],"badges":[],"createdAt":"2025-06-10 10:23:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6861954/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6861954/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87553424,"identity":"7487ddb1-a3c1-4958-aba0-0158fb9d418d","added_by":"auto","created_at":"2025-07-25 06:37:44","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":199907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic profiling reveals dynamic gene expression changes during colorectal tumorigenesis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Colorectal tissue samples were obtained from normal mucosa (N, \u003cem\u003en\u003c/em\u003e = 5), adenoma (T, \u003cem\u003en\u003c/em\u003e = 5), and carcinoma (C, \u003cem\u003en\u003c/em\u003e = 5) for transcriptomic analysis.\u003c/p\u003e\n\u003cp\u003e(B) Volcano plots depicting differentially expressed genes (DEGs) between T vs. N (left) and C vs. N (right) comparisons.\u003c/p\u003e\n\u003cp\u003e(C) Bar chart summarising the number of upregulated and downregulated DEGs in each comparison group.\u003c/p\u003e\n\u003cp\u003e(D) A hierarchical clustering heatmap shows global expression patterns of DEGs across the three groups (T, A, and C).\u003c/p\u003e\n\u003cp\u003e(E) Principal component analysis (PCA) illustrating distinct clustering of samples based on transcriptomic profiles.\u003c/p\u003e\n\u003cp\u003e(F) Venn diagram indicating the overlap of DEGs across A vs. T and C vs. N comparisons.\u003c/p\u003e\n\u003cp\u003e(G) Gene Ontology (GO) enrichment analysis of overlapping DEGs, highlighting key biological processes and cellular components involved in tumorigenesis.\u003c/p\u003e\n\u003cp\u003e(H) Directed acyclic graph (DAG) visualizing hierarchical relationships among enriched GO terms.\u003c/p\u003e\n\u003cp\u003e(I) KEGG pathway enrichment analysis identifies tumour-related signalling pathways that are significantly enriched among DEGs.\u003c/p\u003e\n\u003cp\u003e(J–L) Functional enrichment of DEGs using Disease Ontology (J), DisGeNET (K), and Reactome (L) databases, demonstrating strong associations with colorectal cancer-related disease terms and pathways.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6861954/v1/f0cf6ea65a0a844be69e0a6a.jpeg"},{"id":87553423,"identity":"bf850d32-cf8f-4026-a665-77879e46ac3f","added_by":"auto","created_at":"2025-07-25 06:37:44","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":246449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLRP8 is upregulated in colorectal cancer and associated with poor clinical outcomes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Analysis of LRP8 mRNA expression in colon adenocarcinoma (COAD) tissues compared to adjacent normal tissues using data from The Cancer Genome Atlas (TCGA).\u003c/p\u003e\n\u003cp\u003e(B) LRP8 mRNA expression in rectal adenocarcinoma (READ) versus matched normal tissues based on the TCGA dataset.\u003c/p\u003e\n\u003cp\u003e(C) Kaplan–Meier survival curves of colorectal cancer (CRC) patients stratified by LRP8 expression levels in the TCGA cohort.\u003c/p\u003e\n\u003cp\u003e(D) Representative immunofluorescence staining images showing increased LRP8 expression (red) in CRC tissues compared to adjacent normal mucosa. Nuclei are counterstained with DAPI (blue).\u003c/p\u003e\n\u003cp\u003e(E) RT-PCR analysis of LRP8 mRNA levels in 40 pairs of CRC and matched adjacent normal tissues.\u003c/p\u003e\n\u003cp\u003e(F) Representative immunohistochemistry (IHC) images displaying LRP8 expression in CRC and corresponding normal tissues.\u003c/p\u003e\n\u003cp\u003e(G) Quantitative analysis of IHC staining scores for LRP8 in 86 CRC samples obtained from a commercial tissue microarray.\u003c/p\u003e\n\u003cp\u003e(H) Kaplan–Meier survival analysis based on IHC data indicates that elevated LRP8 protein expression correlates with reduced overall survival in CRC patients.\u003c/p\u003e\n\u003cp\u003e*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6861954/v1/46e9b3aa4a4203ff0c28ffb4.jpeg"},{"id":87552614,"identity":"a7da3885-d296-4a4b-8a64-461e24609ae3","added_by":"auto","created_at":"2025-07-25 06:29:45","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":341749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLRP8 regulates CRC cell proliferation, migration, invasion, apoptosis, and cell cycle.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) \u003cstrong\u003eLRP8\u003c/strong\u003e expression levels were analyzed by qRT-PCR and western blot in NCM460 and CRC cell lines (SW480, SW620, HT-29, HCT116, LOVO).\u003c/p\u003e\n\u003cp\u003e(B–C) Validation of \u003cstrong\u003eLRP8\u003c/strong\u003e knockdown in SW480 (B) and SW620 (C) cells by qRT-PCR and western blot.\u003c/p\u003e\n\u003cp\u003e(D) Confirmation of successful \u003cstrong\u003eLRP8\u003c/strong\u003eoverexpression in HCT116 cells following lentiviral transduction.\u003c/p\u003e\n\u003cp\u003e(E–F) MOI titration to achieve stable \u003cstrong\u003eLRP8\u003c/strong\u003eknockdown in SW480 (E) and SW620 (F) cells.\u003c/p\u003e\n\u003cp\u003e(G) Wound healing assay to evaluate the impact of \u003cstrong\u003eLRP8\u003c/strong\u003eon cell migration.\u003c/p\u003e\n\u003cp\u003e(H) EdU incorporation assay assessing DNA synthesis in CRC cells upon \u003cstrong\u003eLRP8\u003c/strong\u003e modulation.\u003c/p\u003e\n\u003cp\u003e(I) Transwell assays to assess cell migration and invasion in response to \u003cstrong\u003eLRP8\u003c/strong\u003e modulation.\u003c/p\u003e\n\u003cp\u003e(J) Colony formation assay showing the effect of \u003cstrong\u003eLRP8\u003c/strong\u003eon CRC cell proliferation.\u003c/p\u003e\n\u003cp\u003e(K) Flow cytometric analysis of apoptosis in cells with \u003cstrong\u003eLRP8\u003c/strong\u003e silencing or overexpression.\u003c/p\u003e\n\u003cp\u003e(L) Cell cycle analysis via flow cytometry, showing G1-phase arrest in \u003cstrong\u003eLRP8\u003c/strong\u003e-knockdown cells.\u003c/p\u003e\n\u003cp\u003e*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6861954/v1/78c780bb7fb98dace1c32305.jpeg"},{"id":87552610,"identity":"ea34cbb4-fc8b-4260-9412-29688a01ddc8","added_by":"auto","created_at":"2025-07-25 06:29:44","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":225741,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLRP8 modulates ferroptosis-related proteins in CRC cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) SDS-PAGE and Coomassie Brilliant Blue staining were performed to evaluate protein integrity in CRC cell lines.\u003c/p\u003e\n\u003cp\u003e(B) Bar graph depicting the distribution of differentially expressed proteins (DEPs) across various comparison groups.\u003c/p\u003e\n\u003cp\u003e(C) Hierarchical clustering of DEPs, revealing distinct expression patterns in CRC cells.\u003c/p\u003e\n\u003cp\u003e(D) Volcano plot showing DEPs between \u003cstrong\u003eLRP8\u003c/strong\u003esiRNA and control groups, highlighting key proteins such as \u003cstrong\u003eGPX4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e(E) Bar chart of Clusters of Orthologous Groups (COG) analysis for the DEPs.\u003c/p\u003e\n\u003cp\u003e(F) Pie chart illustrating the subcellular localization of DEPs.\u003c/p\u003e\n\u003cp\u003e(G) Gene Ontology (GO) enrichment analysis of DEPs, identifying key biological processes.\u003c/p\u003e\n\u003cp\u003e(H) Bubble plot displaying Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEPs.\u003c/p\u003e\n\u003cp\u003e(I) Protein-protein interaction (PPI) network for DEPs, identifying key protein interactions.\u003c/p\u003e\n\u003cp\u003e(J) KEGG pathway map highlighting ferroptosis-related pathways, focusing on \u003cstrong\u003eGPX4\u003c/strong\u003e-associated signalling.\u003c/p\u003e\n\u003cp\u003e(K) Protein interaction network within ferroptosis-related pathways involving \u003cstrong\u003eGPX4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e(L) RT-qPCR validation of \u003cstrong\u003eGPX4\u003c/strong\u003e and \u003cstrong\u003eSLC3A2\u003c/strong\u003eexpression in \u003cstrong\u003eLRP8\u003c/strong\u003e knockdown CRC cells.\u003c/p\u003e\n\u003cp\u003e*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6861954/v1/84e6080c1f879c5e02b57677.jpeg"},{"id":87552607,"identity":"b9f7e089-4ea3-4b80-98be-25a0211d918c","added_by":"auto","created_at":"2025-07-25 06:29:44","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":392428,"visible":true,"origin":"","legend":"\u003cp\u003eLRP8 suppression triggers ferroptosis in colorectal cancer cells.\u003c/p\u003e\n\u003cp\u003e(A–B) qRT-PCR and western blot assessing GPX4 expression following LRP8 knockdown in three CRC cell lines.\u003c/p\u003e\n\u003cp\u003e(C) Transmission electron microscopy (TEM) revealing characteristic mitochondrial abnormalities associated with ferroptosis following \u003cstrong\u003eLRP8\u003c/strong\u003e silencing.\u003c/p\u003e\n\u003cp\u003e(D) JC-1 staining and flow cytometry assessing mitochondrial membrane potential in control and \u003cstrong\u003eLRP8\u003c/strong\u003e-deficient cells.\u003c/p\u003e\n\u003cp\u003e(E–F) Quantifying intracellular ferrous iron (Fe²⁺) and total iron (t-Fe) in \u003cstrong\u003eLRP8\u003c/strong\u003e-modulated cells.\u003c/p\u003e\n\u003cp\u003e(G–H) Measurement of total glutathione (GSH), reduced GSH, and oxidized GSH (GSSG) levels.\u003c/p\u003e\n\u003cp\u003e(I–J) TEM imaging and JC-1 staining of cells co-treated with the ferroptosis inhibitor Ferrostatin-1 (Fer-1), showing mitochondrial rescue.\u003c/p\u003e\n\u003cp\u003e(K–L) Iron content (Fe²⁺ and t-Fe) in cells after Fer-1 administration.\u003c/p\u003e\n\u003cp\u003e(M–N) GSH and GSSG levels in Fer–1–treated cells, confirming ferroptosis attenuation.\u003c/p\u003e\n\u003ch3\u003e*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/h3\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6861954/v1/d45412cfe1c1ef3d0259596e.jpeg"},{"id":87552612,"identity":"14200331-d321-4787-8d98-f2fd12d41def","added_by":"auto","created_at":"2025-07-25 06:29:44","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":148372,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLRP8 interacts with SLC3A2 to modulate stress responses and ferroptosis in colorectal cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot confirming the expression of Flag-tagged \u003cstrong\u003eLRP8\u003c/strong\u003e in HCT116 cells.\u003c/p\u003e\n\u003cp\u003e(B) Co-immunoprecipitation (Co-IP) assay demonstrating the physical interaction between \u003cstrong\u003eLRP8\u003c/strong\u003e and \u003cstrong\u003eSLC3A2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e(C) Volcano plot of mass spectrometry–identified proteins showing differentially expressed interactors.\u003c/p\u003e\n\u003cp\u003e(D) Gene Ontology (GO) enrichment analysis of \u003cstrong\u003eLRP8\u003c/strong\u003e-interacting proteins, emphasizing roles in protein localization, DNA damage response, and cellular stress adaptation.\u003c/p\u003e\n\u003cp\u003e(E) KEGG pathway analysis reveals enrichment in phagosome formation and endoplasmic reticulum (ER) protein processing.\u003c/p\u003e\n\u003cp\u003e(F) Subcellular compartment analysis indicating the localization patterns of \u003cstrong\u003eLRP8\u003c/strong\u003e-associated proteins.\u003c/p\u003e\n\u003cp\u003e(G) Western blot assessing \u003cstrong\u003eSLC3A2\u003c/strong\u003e expression following \u003cstrong\u003eLRP8\u003c/strong\u003eknockdown in SW480 and SW620 cells.\u003c/p\u003e\n\u003cp\u003e(H)protein–protein interaction (PPI) network analysis.\u003c/p\u003e\n\u003cp\u003e(I) ELISA analysis of Cyc content when knockdown of SLC3A2 in SW480 and SW620 cells.\u003c/p\u003e\n\u003cp\u003e(J) ELISA analysis of ROS levels when knockdown of SLC3A2 in SW480 and SW620 cells.\u003c/p\u003e\n\u003cp\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6861954/v1/ce416bd323b18d39bd8e79db.jpeg"},{"id":87553425,"identity":"40fd9e9c-4624-4918-bfde-3fb37d2da43f","added_by":"auto","created_at":"2025-07-25 06:37:44","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":130882,"visible":true,"origin":"","legend":"\u003cp\u003eLRP8 inhibits ferroptosis and enhances CRC cell proliferation and metastasis via the SLC3A2/GPX4 axis.\u003c/p\u003e\n\u003cp\u003eHCT116 cells were co-transfected with \u003cstrong\u003eLRP8 overexpression (OE-LRP8)\u003c/strong\u003e and \u003cstrong\u003eSLC3A2 shRNA (sh-SLC3A2)\u003c/strong\u003e constructs. Forty-eight hours post-transfection:\u003c/p\u003e\n\u003cp\u003e(A) Western blot confirming knockdown efficiency of \u003cstrong\u003eSLC3A2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e(B) Western blot showing \u003cstrong\u003eGPX4\u003c/strong\u003e protein expression following co-transfection.\u003c/p\u003e\n\u003cp\u003e(C) qPCR analysis validating reduced \u003cstrong\u003eSLC3A2\u003c/strong\u003e mRNA levels.(D–F) Biochemical assays measuring intracellular levels of total glutathione (GSH) (D), reactive oxygen species (ROS) (E) and cysteine(F) after dual modulation of \u003cstrong\u003eLRP8\u003c/strong\u003e and \u003cstrong\u003eSLC3A2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e(G) EdU incorporation assay evaluating proliferative capacity under \u003cstrong\u003eSLC3A2\u003c/strong\u003e knockdown with \u003cstrong\u003eLRP8\u003c/strong\u003e rescue.\u003c/p\u003e\n\u003cp\u003e(H) Transwell assays assessing migration and invasion following \u003cstrong\u003eSLC3A2\u003c/strong\u003e silencing.\u003c/p\u003e\n\u003cp\u003e(I) Colony formation assay demonstrating changes in clonogenic potential with \u003cstrong\u003eSLC3A2\u003c/strong\u003e knockdown.\u003c/p\u003e\n\u003cp\u003e(J) Flow cytometric analysis of apoptosis in cells with \u003cstrong\u003eSLC3A2\u003c/strong\u003e knockdown and \u003cstrong\u003eLRP8\u003c/strong\u003e overexpression.\u003c/p\u003e\n\u003cp\u003e(K) Cell cycle analysis by flow cytometry shows cell population distribution after \u003cstrong\u003eSLC3A2\u003c/strong\u003e silencing.\u003c/p\u003e\n\u003cp\u003e*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6861954/v1/50b0e05ef9a5c8fbd13a4f96.jpeg"},{"id":87553427,"identity":"70f34a08-ce5e-494a-b81b-a7c07a86ea40","added_by":"auto","created_at":"2025-07-25 06:37:45","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":258507,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLRP8 knockdown impairs tumour growth and promotes ferroptosis in a colorectal cancer xenograft model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of subcutaneous tumours derived from control and \u003cstrong\u003eLRP8-knockdown SW480\u003c/strong\u003ecells in nude mice (n = 5).\u003c/p\u003e\n\u003cp\u003e(B–C) Tumour volume measurements and growth curves were recorded over time.\u003c/p\u003e\n\u003cp\u003e(D) Immunohistochemical (IHC) staining of \u003cstrong\u003eLRP8\u003c/strong\u003e, \u003cstrong\u003eGPX4\u003c/strong\u003e, \u003cstrong\u003eSLC3A2\u003c/strong\u003e, and \u003cstrong\u003eKi-67\u003c/strong\u003e in xenograft tumour sections.\u003c/p\u003e\n\u003cp\u003e(E) TUNEL staining indicating apoptotic cell death in tumour tissues.\u003c/p\u003e\n\u003cp\u003e(F) Western blot analysis of \u003cstrong\u003eLRP8\u003c/strong\u003e, \u003cstrong\u003eGPX4\u003c/strong\u003e, and \u003cstrong\u003eSLC3A2\u003c/strong\u003eprotein levels in excised tumours.\u003c/p\u003e\n\u003cp\u003e(G) Tumour weight measurements in tumour tissues.\u003c/p\u003e\n\u003cp\u003e(H) Assessment of cysteine (Cys) content in tumour samples.\u003c/p\u003e\n\u003cp\u003e(I) Detection of reactive oxygen species (ROS) levels in tumour tissues.\u003c/p\u003e\n\u003cp\u003e(J–K) Quantifying ferrous iron (Fe²⁺) and total iron (t-Fe) content in tumour tissues.\u003c/p\u003e\n\u003cp\u003e(L–M) Measurement of reduced glutathione (GSH) and oxidized glutathione (GSSG) levels.\u003c/p\u003e\n\u003cp\u003e*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6861954/v1/b2ee5262318dae5fb13b62fa.jpeg"},{"id":89276426,"identity":"397a34fd-b05d-44f6-97af-8500122de1c3","added_by":"auto","created_at":"2025-08-18 09:32:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3575642,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6861954/v1/23bc5bca-e5c6-4648-89e5-3b2e374454f4.pdf"},{"id":87553426,"identity":"42787cb5-c71f-4653-8b71-043ed8d94124","added_by":"auto","created_at":"2025-07-25 06:37:44","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1006132,"visible":true,"origin":"","legend":"","description":"","filename":"Theuncroppedgelimagessupplementaryfile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6861954/v1/a6a1ee61cc89c1602764af0f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"LRP8 Promotes Colorectal Cancer Progression by Suppressing Ferroptosis through the SLC3A2/GPX4 Signalling Axis LRP8/SLC3A2/GPX4 promoting CRC","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlobally, colorectal carcinoma (CRC) maintains its position as the third most frequently diagnosed malignancy and the second deadliest cancer type[1]\u0026nbsp;Statistical models anticipate substantial growth in disease burden, estimating 3.2 million new cases and 1.6 million annual deaths by 2040\u0026mdash;equivalent to a 63% incidence jump and 73.4% mortality surge\u0026nbsp;[2]. Although current therapeutic strategies, including surgical resection, neoadjuvant chemoradiotherapy, and precision molecular interventions, have achieved incremental improvements in clinical outcomes, persistent issues such as locoregional recurrence, distant metastasis, and resistance to treatment continue to limit long-term survival[3, 4]. These challenges highlight the pressing need to identify novel molecular drivers of CRC and develop more effective, targeted treatment approaches.\u003c/p\u003e\n\u003cp\u003eFerroptosis, an iron-dependent programmed cell death mechanism driven by lipid peroxidation and antioxidant system failure\u0026nbsp;[5-7], has become a potential cancer treatment strategy\u0026nbsp;[8].\u0026nbsp;This process critically depends on the glutathione-GPX4 pathway, the primary cellular defence against oxidative membrane damage\u0026nbsp;[9]. In CRC, GPX4 is frequently overexpressed in tumour tissues relative to adjacent normal mucosa, underscoring its potential role in tumour survival\u0026nbsp;[10]. Experimental models have shown that induction of ferroptosis, whether through iron modulation, enhancement of reactive oxygen species (ROS), or inhibition of GPX4, effectively suppresses CRC progression\u0026nbsp;[11]. Conversely, resistance to ferroptosis has been implicated in tumour relapse and treatment failure, positioning ferroptosis as a targetable vulnerability in CRC\u0026nbsp;[12].\u003c/p\u003e\n\u003cp\u003eUsing a multi-omics approach, we identified low-density lipoprotein receptor-related protein 8 (LRP8, also known as ApoER2) as a novel contributor to CRC pathogenesis and ferroptosis regulation. LRP8 has been shown to influence ferroptosis sensitivity by preserving the cellular pool of selenocysteine tRNAs required for the efficient translation of GPX4 [13] and by preventing ribosomal stalling at the UGA codon that encodes its essential selenocysteine residue [14]. LRP8 modulates lipid metabolism via the Wnt/\u0026beta;-catenin\u0026ndash;stearoyl-CoA desaturase 1 (SCD1) signalling axis, further shaping the cellular redox landscape [15]. Beyond its role in ferroptosis, LRP8 has been implicated in promoting epithelial\u0026ndash;mesenchymal plasticity, metastatic dissemination, and resistance to therapy across multiple malignancies, including gastric [16], ovarian [17], and breast cancers [18]. In CRC specifically, the miR-378a-5p/LRP8 regulatory axis has been associated with modulation of radiation response, suggesting broader oncogenic functions [19]. Despite these findings, the involvement of LRP8 in CRC ferroptosis regulation remains incompletely characterized. This study aims to elucidate the role of LRP8 in modulating ferroptotic sensitivity in CRC and to identify new therapeutic strategies that exploit this regulatory pathway.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePatient Samples and Tissue Microarray (TMA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh CRC tissues and matched adjacent non-tumorous samples were collected from 40 patients undergoing curative resection at Gansu Provincial People\u0026apos;s Hospital (Lanzhou, China) between April and August 2024. None of the patients received preoperative chemotherapy or radiotherapy. All patients provided written informed consent.This study received approval from the Ethics Committee of Gansu Provincial Hospital(Approval document No.2024-807) and was conducted in accordance with the ethical guidelines delineated in the Helsinki Declaration.\u003c/p\u003e\n\u003cp\u003eAdditionally, a commercially available tissue microarray (TMA) slide (Chip ID: HColA180Su21), comprising 94 pairs of CRC and adjacent normal tissues, was purchased from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China). All samples were processed according to standard procedures for downstream analyses, including qPCR and IHC.\u003c/p\u003e\n\u003cp\u003eClinicopathological features-including age, sex, tumour size, vascular/perineural invasion, lymph node status, TNM classification, PD-1/PD-L1 expression, and KRAS, NRAS, and BRAF mutation status were retrieved from medical records.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing optimized protocols, IHC staining was conducted on TMA and xenograft tumour sections using primary antibodies against LRP8(1:300, PA5-109269,Invitrogen),GPX4(1:50,30388-1-AP,Proteintech),SLC3A2(1:50,15193-1-AP,Proteintech) and Ki67(1:300,NBP2-22112,Novus).Two independent pathologists, blinded to clinical information, evaluated the staining results. Ten random high-power fields (\u0026times;400) were assessed per slide, with 100 tumour cells counted per field.\u003c/p\u003e\n\u003cp\u003eStaining was scored based on the percentage of positive cells and staining intensity. Proportion scores were assigned as follows: 0 (none), 1 (\u0026lt;25%), 2 (26\u0026ndash;50%), 3 (51\u0026ndash;75%), and 4 (76\u0026ndash;100%). Intensity scores ranged from 0 (negative) to 3 (strong). Final IHC scores were calculated by multiplying the proportion and intensity scores.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Culture and Lentiviral Transduction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCRC cell lines (SW480, SW620, HT-29, HCT-116, LOVO) and normal human colonic epithelial cells (NCM-460) were sourced from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). CRC cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37\u0026deg;C with 5% CO₂. NCM-460 cells were maintained in specialized medium as per the supplier\u0026apos;s instructions.\u003c/p\u003e\n\u003cp\u003eShort hairpin RNAs (shRNAs; sh1, sh2, sh3) targeting LRP8 or SLC3A2, a scrambled control (NC), and an overexpression vector (OE-LRP8) were cloned into lentiviral vectors (GeneChem, Shanghai, China) and transduced into CRC cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative Real-Time PCR (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA extraction was performed with the M5 HiPer Universal RNA Mini Kit (Polymed, China), and cDNA synthesis was used with PrimeScript\u0026trade; RT reagent Kit (Takara, Japan). Quantitative PCR analysis was conducted with TB Green Premix Ex Taq\u0026trade; (Takara, RR820A), and relative gene expression was calculated via the 2\u003csup\u003e\u0026Delta;\u0026Delta;Ct\u0026nbsp;\u003c/sup\u003emethod.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell lysates were prepared using ice-cold lysis buffer (20 mM Tris-HCl pH 7.5, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA, 50 mM NaF, 1 mM DTT, 10% glycerol) supplemented with protease inhibitors. Following protein quantification (BCA assay, Thermo Fisher), equal protein amounts were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% non-fat milk, membranes were incubated with primary antibodies against LRP8(1:500, PA5-109269,Invitrogen),GPX4(1:1000,30388-1-AP,Proteintech),SLC3A2(1:5000,15193-1-AP,Proteintech) and GAPDH(1:10000,ab181602,Abcam), then with HRP-conjugated secondary antibodies. Protein signals were visualized using enhanced chemiluminescence (Thermo Fisher).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony Formation Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells (1,000/well) were seeded in 6-well plates and incubated for 10\u0026ndash;14 days. Colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Colonies with \u0026gt;50 cells were manually counted in five random fields.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow Cytometry Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were stained with Annexin V-FITC/PI (BD Biosciences, USA) and analyzed by flow cytometry (BD FACSCanto\u0026trade; II). For cell cycle analysis, ethanol-fixed cells were stained with PI and RNase A, and DNA content was quantified by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEdU Incorporation Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell proliferation was assessed using the EdU Apollo567 kit (RiboBio, China). Cells were treated with 50 \u0026mu;M EdU, then fixed, permeabilized, and stained. Nuclear counterstaining was performed using Hoechst 33342. Fluorescence images were captured using an Olympus microscope (Olympus).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMigration and Invasion Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMigration and invasion were assessed in Transwell chambers (8 \u0026mu;m pores; Corning, USA). For migration, serum-free cell suspensions were seeded in the upper chamber, with 10% FBS medium in the lower well. The chamber membrane was pre-coated with Matrigel (BD Biosciences, USA) for invasion. After 48 h, non-migrated cells were removed, and migrated/invaded cells were fixed, stained, and counted under a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Sequencing and Bioinformatics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNovogene Co., Ltd. (Beijing, China) conducted RNA-seq using total RNA from 15 samples (normal mucosa, adenoma, and carcinoma; n = 5 each). After RNA quality control, sequencing was performed on the Illumina platform. Differentially expressed genes (DEGs) were identified, and functional annotations were performed using GO and KEGG analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteomics Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteomic profiling was performed using 4D-DIA mass spectrometry (Bruker timsTOF Pro). SW480 cells transfected with siLRP8 or siNC were analyzed by LC-MS/MS. Raw data were processed with Spectronaut (Biognosys, Sweden), and differential protein expression was analyzed with functional enrichment of affected pathways (GO, KEGG)[29,30].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFifteen male BALB/c nude mice (4 weeks old, n=5/group) were subcutaneously injected with 1\u0026times;10⁶SW480 cells (wild-type, NC, or LRP8 knockdown). Tumour dimensions and body weight were measured every 2 days from day 10.After a 21-day alimentation period,All animals were euthanized by barbiturate overdose (intravenous injection, 150 mg/kg pentobarbital sodium),death was confirmed by loss of heartbeat, breathing and pupil response, and the tumor tissues were extirpated and weighed.Tumor tissues were immobilized in 4% paraformaldehyde for subsequent experimentation. All animal experimental procedures were sanctioned by the Bestcell Model Biological Center(IACUC Issue No.2025-04-18A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission Electron Microscopy (TEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were fixed in glutaraldehyde, dehydrated, embedded in resin, and sectioned into ultrathin slices. After staining with uranyl acetate and lead citrate, mitochondrial ultrastructure was visualized using TEM (Thermo Fisher Scientific, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBCA assays were used for protein quantification. Glutathione (total, reduced, oxidised), total iron, ferrous iron (Fe\u0026sup2;⁺), cysteine, and ROS levels were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute and Elabscience), following manufacturer protocols and analysed via spectrophotometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondrial Membrane Potential (MMP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJC-1 staining (Cat. C2003S) assessed mitochondrial membrane potential. Cells were incubated with JC-1 dye, washed, and analyzed by flow cytometry(Thermo Fisher Scientific, USA). The red-to-green fluorescence ratio reflected MMP status.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism 9.0 and SPSS 26.0. Data were expressed as mean \u0026plusmn; SD. The students\u0026apos; t-test was used for two-group comparisons, and ANOVA was applied for multiple-group analyses. Associations between LRP8 expression and clinical variables were assessed using the \u0026chi;\u0026sup2; test. A p-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTranscriptomic and Bioinformatic Analyses Identify LRP8 as a Key Gene Associated with CRC Progression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate dynamic gene expression changes during colorectal tumorigenesis, we conducted transcriptome sequencing on tissue samples representing three distinct stages: normal mucosa (N, n = 5), adenomas (T, n = 5), and carcinomas (C, n = 5) (Fig. 1A). A total of 3,685 DEGs were identified. Volcano plots illustrated substantial transcriptional shifts in both A vs. T and C vs. N comparisons (Fig. 1B), with the number of DEGs increasing progressively along the typical adenoma\u0026ndash;carcinoma sequence (Fig. 1C). Unsupervised hierarchical clustering revealed clear segregation among the three sample types (Fig. 1D), and all sequencing datasets passed standard quality control assessments, confirming the robustness of the transcriptomic analysis (Fig. 1E). Venn diagrams demonstrated both shared and unique DEGs among the pairwise comparisons (Fig. 1F).Functional enrichment based on GO highlighted that the DEGs were predominantly involved in biological processes such as extracellular matrix organization, epithelial cell migration, and regulation of the Wnt signalling pathway (Fig. 1G\u0026ndash;H). KEGG pathway analysis further revealed significant enrichment in pathways implicated in tumour biology, including IL-17 signalling, the p53 pathway, and focal adhesion (Fig. 1I).Complementary annotations via Disease Ontology, DisGeNET, and Reactome databases consistently associated the identified DEGs with colorectal cancer-related pathways and mechanisms (Fig. 1J\u0026ndash;L). Notably, LRP8 expression exhibited a stepwise upregulation from normal mucosa to adenoma and carcinoma tissues, suggesting its potential involvement in the early and progressive stages of colorectal carcinogenesis. This observation positioned LRP8 as a promising candidate gene for subsequent functional validation.\u003c/p\u003e\n\u003cp\u003eLRP8 Is Upregulated in Colorectal Cancer and Correlates with Poor Prognosis\u003c/p\u003e\n\u003cp\u003eSubsequently, we analyzed LRP8 mRNA levels in\u0026nbsp;both colon adenocarcinoma (COAD) (Fig. 2A) and rectal adenocarcinoma (READ) (Fig. 2 B) from TCGA. The results were significantly elevated compared with their corresponding adjacent normal tissues. Moreover, high LRP8 expression was associated with reduced overall survival in CRC patients, as demonstrated by Kaplan\u0026ndash;Meier survival analysis (Fig. 2C).\u003c/p\u003e\n\u003cp\u003eTo validate these findings, we examined LRP8 expression in 40 paired CRC tumours and adjacent standard tissue samples. Immunofluorescence staining revealed that LRP8 protein was primarily localized to the cytoplasm and plasma membrane of cancer cells (Fig. 2D). RT-PCR confirmed a significant increase in LRP8 mRNA expression in tumor tissues compared to normal tissues (P = 0.006; Fig. 2E). Consistently, immunohistochemical (IHC) analysis of the same cohort demonstrated a marked overexpression of LRP8 in tumor specimens (Fig. 2F). This observation was further supported by semi-quantitative scoring of a tissue microarray containing 86 CRC cases, which revealed significantly higher LRP8 expression in tumor tissues (P \u0026lt; 0.0001; Fig. 2G). To explore the clinical relevance of LRP8, we examined its association with key clinicopathological features. Elevated LRP8 levels were significantly correlated with advanced TNM stage, presence of lymph node metastasis, increased infiltration of CD8⁺ T cells, PD-1/PD-L1 positivity, and KRAS mutations (Table 1). No significant associations were observed with patient age, sex, tumour size, or NRAS/BRAF mutation status. Importantly, Kaplan\u0026ndash;Meier analysis of the tissue microarray cohort indicated that high LRP8 expression was an unfavourable prognostic indicator, predicting reduced 5-year overall survival (P \u0026lt; 0.05; Fig. 2H).\u003c/p\u003e\n\u003cp\u003eLRP8 Promotes malignant behaviors of CRC Cells\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo elucidate the oncogenic role of LRP8 in colorectal carcinogenesis, we systematically characterized its expression profile and functional significance across multiple CRC cell lines. Comparative analysis revealed significant upregulation of LRP8 at both transcriptional and translational levels in all examined CRC cell lines (SW480, SW620, HT-29, HCT116, and LOVO) relative to normal colonic epithelial cells (NCM460) (Fig. 3A). Through comprehensive genetic perturbation studies employing three independent shRNA constructs(Fig. 3B,C), we demonstrated that LRP8 depletion in SW480 and SW620 cells resulted in profound suppression of malignant phenotypes, including impaired proliferative capacity (Fig. 3J), reduced clonogenic potential, attenuated migratory and invasive properties (Fig. 3H-I), as well as increased apoptotic susceptibility and G1 phase cell cycle arrest (Fig. 3K-L). Conversely, ectopic LRP8 expression in HCT116 cells significantly enhanced these oncogenic behaviors, as evidenced by accelerated cell cycle progression and diminished apoptosis (Fig. 3D,J-L).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLRP8 Sustains GPX4 Expression to Inhibit Ferroptosis in CRC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the downstream effects of LRP8 in CRC, we performed proteomic profiling using 4D-DIA sequencing in SW480 cells following LRP8 knockdown by siRNA. The resulting volcano plot demonstrated a broad spectrum of differentially expressed proteins (DEPs), among which GPX4 and SLC3A2, key regulators of oxidative stress and ferroptosis, were significantly downregulated (Fig. 4D).\u003c/p\u003e\n\u003cp\u003eGO and KEGG pathway enrichment analyses indicated that LRP8 silencing predominantly affected pathways related to oxidative stress response, ferroptosis, and metabolic regulation (Fig. 4E). Subcellular localization predictions revealed that LRP8 is mainly distributed in the nucleus (32.7%) and cytoplasm (30.29%), suggesting its potential involvement in organelle-specific regulatory mechanisms associated with ferroptotic cell death (Fig. 4F).Further GO enrichment analysis emphasised strong associations with ferroptosis-related biological processes, including organelle organisation, intracellular transport, and protein-binding functions (Fig. 4G). KEGG analysis showed that cell cycle progression and metabolic pathways were notably disrupted in LRP8-deficient cells, indicating broader impacts on cellular homeostasis (Fig. 4H). PPI network analysis identified clusters of DEPs with dense interconnections, particularly centered on ferroptosis-related proteins (Fig. 4I). A focused PPI subnetwork built around GPX4 highlighted its interactions with core antioxidant and metabolic regulators (Fig. 4K), reinforcing its potential role of LRP8 in ferroptosis. Subsequent qRT-PCR validation confirmed that LRP8 knockdown decreased GPX4, SLC3A2, YTHDF2, and DNMT1 expression, while SLC31A1 were upregulated (Fig. 4L). These results were consistent with the proteomic data and further supported the regulatory relationship between LRP8 and ferroptosis-associated genes. These findings demonstrate that LRP8 influences ferroptosis in CRC cells by controlling the expression of GPX4 and SLC3A2.\u003c/p\u003e\n\u003cp\u003eTo determine whether LRP8 suppression triggers ferroptosis in CRC cells, we generated stable LRP8-knockdown and LRP8-overexpressing cell lines (Fig. 5A\u0026ndash;B). TEM of LRP8-silenced SW480 and SW620 cells revealed classic features of ferroptosis, including reduced mitochondrial volume, condensed membrane density, loss or disappearance of cristae, and outer membrane rupture. In contrast, LRP8-overexpressing cells exhibited intact mitochondrial structures without these abnormalities (Fig. 5C). JC-1 staining was used to assess mitochondrial membrane potential. LRP8 knockdown led to a pronounced reduction in JC-1 aggregates, indicating mitochondrial depolarisation and dysfunction. Conversely, LRP8 overexpression preserved membrane potential, as evidenced by stronger JC-1 aggregation (Fig. 5D). Silencing LRP8 significantly increased intracellular ferrous iron (Fe\u0026sup2;⁺) levels (Fig. 5E), total iron content (Fig. 5G), and glutathione (GSH) levels (Fig. 5H), suggesting an accumulation of labile iron and heightened redox activity. Interestingly, LRP8 overexpression in HCT116 cells also elevated Fe\u0026sup2;⁺, total iron, and GSH levels, indicating that LRP8 regulates iron metabolism and redox balance across multiple CRC cell types. To confirm that these changes were ferroptosis-specific, ferrostatin-1 (Fer-1), a selective ferroptosis inhibitor, was applied to LRP8-knockdown cells. Fer-1 treatment reversed mitochondrial damage, restoring organelle morphology (Fig. 5I) and normalizing JC-1 fluorescence patterns (Fig. 5J), indicating recovery of membrane potential. Fer-1 significantly reduced Fe\u0026sup2;⁺ accumulation (Fig. 5K\u0026ndash;L) in the LRP8-silenced cells, supporting the iron dependence of the observed phenotype. Treatment also restored GSH levels (Fig. 5M) and reduced oxidized glutathione (GSSG) levels (Fig. 5N), further alleviating oxidative stress. Together, these findings demonstrate that LRP8 influences ferroptosis sensitivity in CRC cells by regulating intracellular iron levels and redox status, and that ferroptotic responses induced by LRP8 loss are reversible through ferroptosis inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLRP8 binds SLC3A2 to Promote Cysteine Uptake and Ferroptosis Resistance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the molecular mechanisms by which LRP8 influences ferroptosis, we conducted mass spectrometry-based immunoprecipitation (MS-IP) in LRP8-overexpressing HCT116 cells to identify its interacting protein partners (Fig. 6A\u0026ndash;B). Among the candidate interactors, SLC3A2 was identified as a potential binding partner (Fig. 6C). Functional enrichment analysis of the LRP8-associated protein complexes revealed significant enrichment in pathways related to protein trafficking, oxidative stress response, and endoplasmic reticulum (ER) stress (Fig. 6D\u0026ndash;F), suggesting a broader role for LRP8 in maintaining cellular stress adaptation and metabolic homeostasis.Western blot analysis confirmed that LRP8 knockdown in SW480 and SW620 cells led to a marked reduction in SLC3A2 expression (Fig. 6G), supporting a regulatory relationship between these two proteins. In addition, protein\u0026ndash;protein interaction (PPI) network analysis further reinforced the existence of a direct or functionally relevant interaction between LRP8 and SLC3A2(Fig. 6H).Subsequently,ELISA analysis confirmed that knockdown of SLC3A2 in SW480 and SW620 cells led to a significant decrease in Cyc content and a significant increase in ROS levels(Fig. 6I\u0026ndash;J). These findings indicate that LRP8 may regulate cysteine uptake and redox homeostasis in CRC cells by interacting with SLC3A2, thereby contributing to ferroptosis susceptibility through modulation of system Xc⁻ activity.\u003c/p\u003e\n\u003cp\u003eBuilding on the identified interaction between LRP8 and SLC3A2, we explored whether LRP8 promotes CRC progression by inhibiting SLC3A2-mediated ferroptosis. Efficient knockdown of SLC3A2 in HCT116 cells was validated at both the mRNA and protein levels (Fig. 7A). Co-transfection of LRP8-overexpressing cells with SLC3A2-targeting shRNA significantly reversed the upregulation of GPX4 and SLC3A2, implicating SLC3A2 as a downstream effector of LRP8\u0026apos;s anti-ferroptotic activity (Fig.7B-C). Biochemical assays revealed that simultaneous LRP8 overexpression and SLC3A2 silencing resulted in significant reductions in intracellular GSH and Cys levels (Fig. 7D\u0026ndash;E), accompanied by increased reactive oxygen species (ROS) accumulation (Fig. 7F), indicating a breakdown in redox homeostasis. Functionally, LRP8 overexpression enhanced proliferation, as evidenced by increased EdU and Ki67 positivity. However, this proliferative effect was markedly attenuated by SLC3A2 knockdown, indicating that SLC3A2 is necessary for LRP8-driven cell proliferation. Similarly, LRP8 overexpression promoted cell migration and invasion, as shown by transwell assays, whereas co-silencing SLC3A2 significantly suppressed these metastatic properties. Colony formation assays further demonstrated that LRP8-driven clonogenic growth was reduced upon SLC3A2 depletion. Additionally, cell cycle analysis showed that knockdown of SLC3A2 in LRP8-overexpressing cells caused a significant decrease in S and G2/M phase populations, suggesting impaired cell cycle progression. These findings indicate that SLC3A2 is a key mediator of LRP8\u0026rsquo;s oncogenic effects in CRC, facilitating ferroptosis resistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLRP8 Silencing Triggers Ferroptosis in CRC Xenograft Models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the in vivo function of LRP8 in colorectal cancer (CRC), we established a subcutaneous xenograft model using SW480 cells with stable LRP8 knockdown. Tumours from the sh-LRP8 group exhibited significantly reduced volumes and growth rates compared to those in the negative control group, indicating that LRP8 is essential for efficient tumour progression (Fig. 8A\u0026ndash;C). Immunohistochemistry confirmed successful knockdown, showing markedly decreased LRP8 expression in tumour sections from the sh-LRP8 cohort (Fig. 8D). TUNEL staining revealed a substantial increase in apoptotic cells within the sh-LRP8 tumors, suggesting that LRP8 contributes to tumor cell survival (Fig. 8E). Western blot analysis further validated these findings, demonstrating reduced levels of LRP8 and GPX4, a key regulator of ferroptosis, in tumor lysates from the LRP8-silenced group (Fig. 8F). Biochemical assays provided additional insight into the redox imbalance induced by LRP8 depletion. Tumours lacking LRP8 exhibited decreased concentrations of ferrous iron (Fe\u0026sup2;⁺; Fig. 8J), total iron (t-Fe; Fig. 8K), GSH(Fig. 8L), and Cys (Fig. 8H). In contrast, oxidized glutathione (GSSG) levels were significantly elevated (Fig. 8M), accompanied by a marked increase in reactive oxygen species (ROS) accumulation (Fig. 8I). These metabolic shifts are indicative of ferroptotic cell death. Together, these in vivo results demonstrate that LRP8 promotes tumour growth in CRC by suppressing ferroptosis. \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur integrated multi-omics and functional analyses establish LRP8 as a critical oncogenic driver in colorectal cancer. We firstly demonstrate that LRP8 overexpression, clinically correlated with advanced disease and poor prognosis. It overexpression promotes malignant transformation by enhancing proliferative, migratory, and anti-apoptotic capacities while maintaining redox homeostasis. The mechanistic elucidation reveals that LRP8 physically interacts with SLC3A2 to preserve system Xc⁻ integrity, thereby sustaining intracellular cysteine/GSH pools and GPX4-mediated lipid peroxide clearance. The finding is underscored by in vivo evidence showing LRP8 ablation induces tumor-suppressive ferroptosis, characterized by iron dyshomeostasis, mitochondrial degeneration, and lethal lipid peroxidation. This work defines a novel LRP8-SLC3A2-GPX4 regulatory axis with therapeutic potential for CRC treatment.\u003c/p\u003e\n\u003cp\u003eWhile accumulating evidence highlights \u003cstrong\u003eLRP8\u003c/strong\u003e as a pivotal oncoprotein in various malignancies [16, 17, 20, 21], its role in CRC progression remains incompletely characterised. Previous studies have demonstrated that \u003cstrong\u003eLRP8\u003c/strong\u003e facilitates tumorigenesis in ovarian cancer by antagonising p53 signalling [21], promotes metastasis in lung cancer via \u003cstrong\u003eSpon1\u003c/strong\u003e-mediated pathways [22], and silencing LRP8 promotes a luminal-epithelial differentiation state in TNBC cells, consequently enhancing their chemosensitivity [23]. In the present study, we observed a progressive increase in \u003cstrong\u003eLRP8\u003c/strong\u003e expression throughout the colorectal adenoma-carcinoma sequence, with a marked upregulation in carcinoma tissues compared to normal mucosa and adenoma. Clinically, elevated \u003cstrong\u003eLRP8\u003c/strong\u003e levels were significantly associated with advanced TNM stage, lymph node metastasis, and poor patient survival, underscoring its potential as a prognostic biomarker in CRC. In vitro experiments further revealed that \u003cstrong\u003eLRP8\u003c/strong\u003e knockdown notably inhibited CRC cell proliferation, colony formation, migration, and invasion, while its overexpression amplified these malignant characteristics. These in vitro findings were corroborated in vivo, where \u003cstrong\u003eLRP8\u003c/strong\u003e depletion significantly reduced tumour growth in xenograft models.\u003c/p\u003e\n\u003cp\u003eIn this study, SLC3A2 was identified as a critical downstream effector of LRP8, mediating ferroptosis resistance in CRC. As a chaperone protein, SLC3A2 plays a key role in the membrane localisation and stabilisation of light chains of amino acid transporters, such as SLC7A11 and SLC1A5, facilitating cystine uptake and maintaining intracellular redox balance [24]. The upregulation of SLC3A2 has been associated with ferroptosis suppression and enhanced glutathione synthesis in various tumour types, including laryngeal [25], breast [26], and treatment-resistant prostate cancers [27]. Proteomic analysis using immunoprecipitation followed by mass spectrometry (IP-MS) revealed a direct interaction between LRP8 and SLC3A2, which was further confirmed through co-immunoprecipitation assays. This interaction suggests that LRP8 may exert part of its anti-ferroptotic effects by modulation of SLC3A2. Rescue experiments further supported this hypothesis, showing that reintroducing SLC3A2 into LRP8-silenced cells alleviated ferroptosis-associated phenotypes, such as excessive ROS production, glutathione depletion, and mitochondrial damage. These findings establish SLC3A2 as a functional mediator of the LRP8-regulated ferroptosis pathway in CRC. Beyond its metabolic role, SLC3A2 also shapes the tumour immune microenvironment by regulating immune cell activation and antigen presentation [28]. This suggests that the LRP8\u0026ndash;SLC3A2 axis may govern ferroptosis resistance and contribute to immune evasion in CRC. Targeting this axis could therefore represent a promising therapeutic strategy, disrupting tumour redox homeostasis while enhancing anti-tumour immune responses.\u003c/p\u003e\n\u003cp\u003eIn summary, our study systematically elucidates the oncogenic role of LRP8 in colorectal cancer through comprehensive multi-omics analyses and functional validation. We demonstrate that LRP8 exhibits progressive upregulation during CRC development, correlating with advanced disease stage and poor prognosis. Mechanistically, LRP8 promotes tumour progression by interacting with SLC3A2 to maintain redox homeostasis and suppress ferroptosis, while simultaneously enhancing CRC cells\u0026apos; proliferative, migratory, and invasive capacities. Identifying the LRP8-SLC3A2-GPX4 axis provides novel insights into the molecular regulation of ferroptosis resistance in CRC, revealing a previously unrecognized vulnerability that could be exploited therapeutically.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work is supported by National Natural Science Foundation of China (No. 82360498) ,Gansu Joint Scientific Research Fund Major Project (No.23JRRA1537), the 2025 Central-Guided Local Science and Technology Development Found(No. 25ZYJA003), Gansu Provincial Health Industry Science and Technology Innovation Major Project(No.GSWSZD2024-01) , Gansu Province Key Talent Project(No.2025RCXM067) , and department of Health Commission of Gansu Province(No.GSWSKY2024-12).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients provided written informed consent.This study received approval from the Ethics Committee of Gansu Provincial Hospital(Approval document No.2024-807) and was conducted in accordance with the ethical guidelines delineated in the Helsinki Declaration.\u003c/p\u003e\n\u003cp\u003eAll animal experimental procedures were sanctioned by the Bestcell Model Biological Center(IACUC Issue No.2025-04-18A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowlegement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA sequencing:The datasets generated and analysed during the current study are available in the National Library of Medicine(https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1276701) repository.\u003c/p\u003e\n\u003cp\u003eProject ID:PRJNA1276701\u003c/p\u003e\n\u003cp\u003eProteomics data generated:The datasets generated and analysed during the current study are available in the Integrated Proteome Resources(https://www.iprox.cn/page/project.html?id=IPX0012425000) repository.\u003c/p\u003e\n\u003cp\u003eProject ID:IPX0012425000\u003c/p\u003e\n\u003cp\u003eThe designated point of contact for data requests:Dr Chengzhang Zhu;The First Clinical Medical College,Lanzhou Universit,Lanzhou, Gansu Province, 730000, China.;Email:[email protected].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChengzhang Zhu, Zhengpeng Qian are the co-first authors.\u003c/p\u003e\n\u003cp\u003eChengzhang Zhu: Conceptualization, Methodology, Investigation, Formal Analysis, Writing \u0026ndash; Original Draft, Writing \u0026ndash; Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003eZhengpeng Qian: Methodology, Validation, Investigation, Data Curation, Writing \u0026ndash; Original Draft.\u003c/p\u003e\n\u003cp\u003eShijie Yang: Investigation, Resources, Visualization, Data Curation.\u003c/p\u003e\n\u003cp\u003eYongfeng Wang: Investigation, Software, Formal Analysis, Visualization.\u003c/p\u003e\n\u003cp\u003eXiongfei Yang: Investigation, Resources, Data Curation, Visualization.\u003c/p\u003e\n\u003cp\u003eBinbin Du and Hui Cai: Supervision, Funding Acquisition, Project Administration, Writing \u0026ndash; Review \u0026amp; Editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229-63. Epub 2024/04/04. doi: 10.3322/caac.21834. PubMed PMID: 38572751.\u003c/li\u003e\n\u003cli\u003eMarmol I, Sanchez-de-Diego C, Pradilla Dieste A, Cerrada E, Rodriguez Yoldi MJ. Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer. Int J Mol Sci. 2017;18(1). Epub 2017/01/21. doi: 10.3390/ijms18010197. PubMed PMID: 28106826; PubMed Central PMCID: PMCPMC5297828.\u003c/li\u003e\n\u003cli\u003eScanu AM, De Miglio MR. Therapeutic Landscapes in Colorectal Carcinoma. Medicina (Kaunas). 2023;59(5). Epub 2023/05/27. doi: 10.3390/medicina59050821. PubMed PMID: 37241053; PubMed Central PMCID: PMCPMC10222447.\u003c/li\u003e\n\u003cli\u003eSmith HG, Nilsson PJ, Shogan BD, Harji D, Gambacorta MA, Romano A, et al. Neoadjuvant treatment of colorectal cancer: comprehensive review. BJS Open. 2024;8(3). Epub 2024/05/15. doi: 10.1093/bjsopen/zrae038. PubMed PMID: 38747103; PubMed Central PMCID: PMCPMC11094476.\u003c/li\u003e\n\u003cli\u003eLi J, Cao F, Yin HL, Huang ZJ, Lin ZT, Mao N, et al. Ferroptosis: past, present and future. Cell Death Dis. 2020;11(2):88. Epub 2020/02/06. doi: 10.1038/s41419-020-2298-2. PubMed PMID: 32015325; PubMed Central PMCID: PMCPMC6997353.\u003c/li\u003e\n\u003cli\u003eLiang D, Minikes AM, Jiang X. Ferroptosis at the intersection of lipid metabolism and cellular signaling. Mol Cell. 2022;82(12):2215-27. Epub 2022/04/08. doi: 10.1016/j.molcel.2022.03.022. PubMed PMID: 35390277; PubMed Central PMCID: PMCPMC9233073.\u003c/li\u003e\n\u003cli\u003eTang D, Chen X, Kang R, Kroemer G. Ferroptosis: molecular mechanisms and health implications. Cell Res. 2021;31(2):107-25. Epub 2020/12/04. doi: 10.1038/s41422-020-00441-1. PubMed PMID: 33268902; PubMed Central PMCID: PMCPMC8026611.\u003c/li\u003e\n\u003cli\u003eJiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 2021;22(4):266-82. Epub 2021/01/27. doi: 10.1038/s41580-020-00324-8. PubMed PMID: 33495651; PubMed Central PMCID: PMCPMC8142022.\u003c/li\u003e\n\u003cli\u003eXue Q, Yan D, Chen X, Li X, Kang R, Klionsky DJ, et al. Copper-dependent autophagic degradation of GPX4 drives ferroptosis. Autophagy. 2023;19(7):1982-96. Epub 2023/01/10. doi: 10.1080/15548627.2023.2165323. PubMed PMID: 36622894; PubMed Central PMCID: PMCPMC10283421.\u003c/li\u003e\n\u003cli\u003eHuang B, Wang H, Liu S, Hao M, Luo D, Zhou Y, et al. Palmitoylation-dependent regulation of GPX4 suppresses ferroptosis. Nat Commun. 2025;16(1):867. Epub 2025/01/21. doi: 10.1038/s41467-025-56344-5. PubMed PMID: 39833225; PubMed Central PMCID: PMCPMC11746948.\u003c/li\u003e\n\u003cli\u003eYan H, Talty R, Johnson CH. Targeting ferroptosis to treat colorectal cancer. Trends Cell Biol. 2023;33(3):185-8. Epub 2022/12/07. doi: 10.1016/j.tcb.2022.11.003. PubMed PMID: 36473802.\u003c/li\u003e\n\u003cli\u003eZeng K, Li W, Wang Y, Zhang Z, Zhang L, Zhang W, et al. Inhibition of CDK1 Overcomes Oxaliplatin Resistance by Regulating ACSL4-mediated Ferroptosis in Colorectal Cancer. Adv Sci (Weinh). 2023;10(25):e2301088. Epub 2023/07/10. doi: 10.1002/advs.202301088. PubMed PMID: 37428466; PubMed Central PMCID: PMCPMC10477855.\u003c/li\u003e\n\u003cli\u003eZhao Y, Shi G, Huang X, Zhang Z, Liao K, Xiong H, et al. LRP8 inhibits bladder cancer cell ferroptosis by activating the Wnt/beta-catenin-SCD1 positive feedback loop. Hum Mol Genet. 2025;34(10):843-51. Epub 2025/02/24. doi: 10.1093/hmg/ddaf024. PubMed PMID: 39989004.\u003c/li\u003e\n\u003cli\u003eLi Z, Zhang J, Zheng H, Li C, Xiong J, Wang W, et al. Modulating lncRNA SNHG15/CDK6/miR-627 circuit by palbociclib, overcomes temozolomide resistance and reduces M2-polarization of glioma associated microglia in glioblastoma multiforme. J Exp Clin Cancer Res. 2019;38(1):380. Epub 2019/08/30. doi: 10.1186/s13046-019-1371-0. PubMed PMID: 31462285; PubMed Central PMCID: PMCPMC6714301.\u003c/li\u003e\n\u003cli\u003eWu GF, Luo ZG, Gao K, Ren Y, Shen C, Ying XR. LRP8 Regulates Lipid Metabolism to Stimulate Malignant Progression and Cisplatin Resistance in Bladder Cancer. Kaohsiung J Med Sci. 2025:e70042. Epub 2025/05/15. doi: 10.1002/kjm2.70042. PubMed PMID: 40372166.\u003c/li\u003e\n\u003cli\u003eLiu B, Bukhari I, Li F, Ren F, Xia X, Hu B, et al. Enhanced LRP8 expression induced by Helicobacter pylori drives gastric cancer progression by facilitating beta-Catenin nuclear translocation. J Adv Res. 2025;69:299-312. Epub 2024/04/13. doi: 10.1016/j.jare.2024.04.002. PubMed PMID: 38609049; PubMed Central PMCID: PMCPMC11954824.\u003c/li\u003e\n\u003cli\u003eLi C, Yang Y, Wang H, Song Y, Huang H. miR-362-3p suppresses ovarian cancer by inhibiting LRP8. Transl Oncol. 2022;15(1):101284. Epub 2021/11/29. doi: 10.1016/j.tranon.2021.101284. PubMed PMID: 34839107; PubMed Central PMCID: PMCPMC8636862.\u003c/li\u003e\n\u003cli\u003eLi L, Qu WH, Ma HP, Wang LL, Zhang YB, Ma Y. LRP8, modulated by miR-1262, promotes tumour progression and forecasts the prognosis of patients in breast cancer. Arch Physiol Biochem. 2022;128(3):657-65. Epub 2020/01/30. doi: 10.1080/13813455.2020.1716019. PubMed PMID: 31994910.\u003c/li\u003e\n\u003cli\u003eHu G, Che P, Deng L, Liu L, Liao J, Liu Q. MiR-378a-5p exerts a radiosensitizing effect on CRC through LRP8/beta-catenin axis. Cancer Biol Ther. 2024;25(1):2308165. Epub 2024/02/23. doi: 10.1080/15384047.2024.2308165. PubMed PMID: 38389136; PubMed Central PMCID: PMCPMC10896128.\u003c/li\u003e\n\u003cli\u003eAlborzinia H, Chen Z, Yildiz U, Freitas FP, Vogel FCE, Varga JP, et al. LRP8-mediated selenocysteine uptake is a targetable vulnerability in MYCN-amplified neuroblastoma. EMBO Mol Med. 2023;15(8):e18014. Epub 2023/07/12. doi: 10.15252/emmm.202318014. PubMed PMID: 37435859; PubMed Central PMCID: PMCPMC10405063.\u003c/li\u003e\n\u003cli\u003eXu Y, Zhou Y, Yi X, Nie X. LRP8 promotes tumorigenesis in ovarian cancer through inhibiting p53 signaling. Cell Biol Int. 2024;48(5):626-37. Epub 2024/01/24. doi: 10.1002/cbin.12133. PubMed PMID: 38263609.\u003c/li\u003e\n\u003cli\u003eQiu H, Shen X, Chen B, Chen T, Feng G, Chen S, et al. miR-30b-5p inhibits cancer progression and enhances cisplatin sensitivity in lung cancer through targeting LRP8. Apoptosis. 2021;26(5-6):261-76. Epub 2021/03/30. doi: 10.1007/s10495-021-01665-1. PubMed PMID: 33779882.\u003c/li\u003e\n\u003cli\u003eLin CC, Lo MC, Moody R, Jiang H, Harouaka R, Stevers N, et al. Targeting LRP8 inhibits breast cancer stem cells in triple-negative breast cancer. Cancer Lett. 2018;438:165-73. Epub 2018/09/19. doi: 10.1016/j.canlet.2018.09.022. PubMed PMID: 30227220; PubMed Central PMCID: PMCPMC6945120.\u003c/li\u003e\n\u003cli\u003eChen Z, Zhang J, Gao S, Jiang Y, Qu M, Gu J, et al. Suppression of Skp2 contributes to sepsis-induced acute lung injury by enhancing ferroptosis through the ubiquitination of SLC3A2. Cell Mol Life Sci. 2024;81(1):325. Epub 2024/07/31. doi: 10.1007/s00018-024-05348-3. PubMed PMID: 39079969; PubMed Central PMCID: PMCPMC11335248.\u003c/li\u003e\n\u003cli\u003eWu F, Xiong G, Chen Z, Lei C, Liu Q, Bai Y. SLC3A2 inhibits ferroptosis in laryngeal carcinoma via mTOR pathway. Hereditas. 2022;159(1):6. Epub 2022/01/22. doi: 10.1186/s41065-022-00225-0. PubMed PMID: 35057861; PubMed Central PMCID: PMCPMC8772086.\u003c/li\u003e\n\u003cli\u003eXiong H, Zhai Y, Meng Y, Wu Z, Qiu A, Cai Y, et al. Acidosis activates breast cancer ferroptosis through ZFAND5/SLC3A2 signaling axis and elicits M1 macrophage polarization. Cancer Lett. 2024;587:216732. Epub 2024/02/16. doi: 10.1016/j.canlet.2024.216732. PubMed PMID: 38360142.\u003c/li\u003e\n\u003cli\u003eGhoochani A, Hsu EC, Aslan M, Rice MA, Nguyen HM, Brooks JD, et al. Ferroptosis Inducers Are a Novel Therapeutic Approach for Advanced Prostate Cancer. Cancer Res. 2021;81(6):1583-94. Epub 2021/01/24. doi: 10.1158/0008-5472.CAN-20-3477. PubMed PMID: 33483372; PubMed Central PMCID: PMCPMC7969452.\u003c/li\u003e\n\u003cli\u003eNachef M, Ali AK, Almutairi SM, Lee SH. Targeting SLC1A5 and SLC3A2/SLC7A5 as a Potential Strategy to Strengthen Anti-Tumour Immunity in the Tumour Microenvironment. Front Immunol. 2021;12:624324. Epub 2021/05/07. doi: 10.3389/fimmu.2021.624324. PubMed PMID: 33953707; PubMed Central PMCID: PMCPMC8089370.\u003c/li\u003e\n\u003cli\u003eKanehisa M, Furumichi M, Sato Y, Matsuura Y, Ishiguro-Watanabe M. KEGG: biological systems database as a model of the real world. Nucleic Acids Res. 2025 Jan 6;53(D1):D672-D677. doi: 10.1093/nar/gkae909. PMID: 39417505; PMCID: PMC11701520.\u003c/li\u003e\n\u003cli\u003eKanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000 Jan 1;28(1):27-30. doi: 10.1093/nar/28.1.27. PMID: 10592173; PMCID: PMC102409.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1 The correlation between the expression level of LRP8 and clinicopathological characteristics as well as prognosis in CRC patients\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"568\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eNo.of cases\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLRP8\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eexpression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLow\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGender\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.286\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u0026le;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.313\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u0026gt;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor size(cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u0026le;5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.389\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.238\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u0026gt;5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMucinous cancer\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.271\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.388\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRemote relocation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.085\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.621\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRight-sided\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2.058\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.116\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVascular invasion\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2.409\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.121\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNerve invasion\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.308\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT staging\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eT1-T2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.872\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.171\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eT3-T4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLymph node status\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e8.437\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eN1-N2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"568\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eNo.of cases\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLRP8\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eexpression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLow\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003cstrong\u003eTNM staging\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eI/II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e6.955\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eIII/IV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD8 positive rate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u0026lt;1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e5.088\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.024\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e≧1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD1 positive rate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u0026lt;1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4.851\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.028\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e≧1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePDL1 positive rate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u0026lt;1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e5.104\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.024\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e≧1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKRAS mutation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4.334\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.037\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNRAS mutation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.432\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.231\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBRAF mutation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.643\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e0.442\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\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":"LRP8, colorectal cancer, ferroptosis, SLC3A2, GPX4, iron metabolism","lastPublishedDoi":"10.21203/rs.3.rs-6861954/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6861954/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eColorectal cancer (CRC) persists as one of the most lethal malignancies worldwide, with therapeutic resistance representing a significant obstacle in clinical management. Ferroptosis, a form of programmed cell death triggered by iron accumulation and lipid peroxidation, has recently emerged as a promising target for cancer therapy. Although low-density lipoprotein receptor-related protein 8 (LRP8) has been implicated in oncogenic processes across cancer types, its involvement in CRC progression and ferroptosis regulation has not been fully elucidated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThis study utilized an integrative multi-omics approach, incorporating transcriptomic profiling across the colorectal carcinogenesis spectrum (normal mucosa, adenoma, carcinoma; n=5 each) and proteomic analysis via 4D-DIA mass spectrometry. LRP8 expression patterns were examined in 40 paired CRC and adjacent normal tissues and a tissue microarray comprising 94 cases. Functional investigations were conducted in CRC cell lines following LRP8 knockdown or overexpression. Xenograft models were employed for in vivo validation. Mechanistic insights were gained through co-immunoprecipitation, redox assays, and transmission electron microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eTranscriptomic data revealed a stepwise increase in LRP8 expression during CRC development. Clinical analyses demonstrated that elevated LRP8 levels correlated significantly with advanced tumour stage, lymphatic metastasis, and poorer patient prognosis. Functional assays indicated that LRP8 enhances oncogenic behaviors by interacting with SLC3A2. Reintroducing SLC3A2 in LRP8-depleted cells restored glutathione peroxidase 4 (GPX4) expression and mitigated oxidative stress, thereby rescuing ferroptosis resistance. In vivo, silencing LRP8 inhibited tumour growth and induced ferroptosis-associated alterations, including disrupted iron homeostasis and increased lipid peroxidation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eLRP8 facilitates CRC progression by antagonizing ferroptosis via modulation of the SLC3A2/GPX4 signalling axis. These findings highlight LRP8 as a previously unrecognized regulator of ferroptotic vulnerability and a potential therapeutic target in CRC.\u003c/p\u003e","manuscriptTitle":"LRP8 Promotes Colorectal Cancer Progression by Suppressing Ferroptosis through the SLC3A2/GPX4 Signalling Axis LRP8/SLC3A2/GPX4 promoting CRC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-25 06:29:40","doi":"10.21203/rs.3.rs-6861954/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":"9034eb85-a34e-47f1-855b-5d66013d8bf4","owner":[],"postedDate":"July 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52004255,"name":"Health sciences/Gastroenterology"},{"id":52004256,"name":"Health sciences/Medical research"},{"id":52004257,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-09-20T09:53:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-25 06:29:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6861954","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6861954","identity":"rs-6861954","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}