Clinical and pathological data were collected from 257 patients with stage I–III primary CRC who underwent surgical treatment at the Affiliated Hospital of Qingdao University (Qingdao, China) and Shandong Electric Power Central Hospital (Shandong China) from 2015 to 2019. Adjacent non-tumor tissues (distance from the tumor tissues: 2 cm) were collected as normal controls. Clinical and pathological data were collected as previously described by Zhang et al. (Xiangyan et al. 2023). Patients were followed up until July 2024, and the survival status of the patients was recorded. Additionally, tumor biopsy specimens were collected from 30 patients with locally advanced rectal cancer (LARC). All 30 patients underwent standardized neoadjuvant chemoradiotherapy prior to total mesorectal excision (TME), followed by standardized FOLFOX adjuvant chemotherapy. The neoadjuvant treatment consists in long course radiation therapy (a total dose of 50 Gy in 25 fractions), and combined with oral capecitabine (825 mg/m2 twice a day) on radiotherapy days only. Moreover, the pathological response to neoadjuvant treatment was assessed according to the American Joint Committee on Cancer and College of American Pathologists tumor regression grade (AJCC/CAP TRG) system (Stephen and Carolyn 2010). TRG 0–1 was considered as radio-sensitive, and TRG 2–3 was considered as radio-resistance (Bingjie et al. 2023). The protocol for this study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University (approval number: QYFYWZLL 29685) and Shandong Electric Power Central Hospital (approval number: SDDLZXYY 2025-0101). All patients and/or their guardians provided written informed consent.

RKO, HT29, SW480, and HCT116 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells (except for HCT116 cells) were maintained in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific). The HCT116 cells were maintained in 5'Macoy (Thermo Fisher Scientific) supplemented with 20% fetal bovine serum at 37 °C and 5% CO2. The ferroptosis inhibitor ferrostatin-1 and ferroptosis activator RSL3 were purchased from MedChemExpress (Princeton, NJ, USA).

The transfection vectors UBAP2L-overexpression (UBAP2L-OE), UBAP2L-knockdown (UBAP2L-KD), and UBAP2L-control (UBAP2L-Ctrl) were provided by Gene BioChem Corporation (Shanghai, China). RKO and SW480 cells were transfected with the vectors according to the instructions provided by the manufacturer. The infected cells were selected by puromycin (Solarbio, Beijing, China).

Cell lysates were prepared using radioimmunoprecipitation assay buffer (R0020; Solarbio) supplemented with a protease inhibitor cocktail (CWBIO) and phosphatase inhibitors (CWBIO) for 30 min. Protein concentration was determined using a bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). Total proteins lysates (30 μg) were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Blots were blocked with 5% fat-free milk for 2 h at room temperature. Thereafter, the membranes were incubated at 4 °C overnight with primary antibodies against UBAP2L (1:1000; clone: 40,199; CST, USA), GPX4 (1:5000; clone:T5659F; Abmart, Shanghai, China), and anti-actin rabbit polyclonal (1:5,000; clone:E-AB-2058; Elabscience, Wuhan, China).

Cells (1000 per well) were plated in 96-well plates. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay (Dojindo, Dalian, China) at indicated time points according to the instructions provided by the manufacturer. Subsequently, the cells were irradiated (dose: 4 Gy). Absorbance at 450 nm wavelength was measured after incubation with CCK-8 reagent (10 μL) and cell culture medium (90 μL) for 1.5 h at 37 °C. Light absorbance was measured using an automated microplate reader (Infinitf 200; Tecan, Port Melbourne, Australia).

Sections of paraffin-embedded CRC tissues and adjacent tissues were used to perform IHC staining (n = 257). After deparaffinization and rehydration by xylene and a series of graded ethanol washes, sections were treated with Tris-ethylenediaminetetraacetic acid (pH = 9.0) at 100 °C for 10 min. The sections were subsequently treated with 0.3% hydrogen peroxidase (20 min, room temperature), followed by incubation with primary antibody targeting UBAP2L (1:300; clone: CSB-PA622754LAO1HU; CUSABIO) and GPX4 (1:600; clone: T5659F; Abmart) for 1 h at 37 °C. Next, the sections were incubated with secondary antibody (ZSGB Biotech, Beijing, China), stained with diaminobenzidine tetrahydrochloride (ZSGB Biotech), and counterstained with hematoxylin.

A semi-quantitative method based on the staining intensity and the percentage of positive tumor cells was used for UBAP2L and GPX4 IHC scoring. Staining intensity in tumor cells was scored on a scale from 0 to 3 as follows: 0 (no staining), 1 (light yellow staining), 2 (brown yellow staining), and 3 (dark brown staining), respectively. The percentage of positive tumor cells was scored as follows: 0 (negative), 1 (1–25%), 2 (26–50%), 3 (51%–75%), and 4 (> 75%). Then the two scores are multiplied, a multiplied score < 4 or ≥ 5 denoted low and high expression, respectively (Xiangyan et al. 2023).

Forty female BALB/c nude mice (age: 4–6 weeks; body weight: 18–20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed at 24–26 °C on a 12-h light/dark cycle. Mice were fed a full-fat diet and autoclaved water. A maximum of six mice were placed in each cage. A total of 1 × 107 infected RKO cells (Control, UBAP2L-KD and UBAP2L-OE) were suspended in phosphate-buffered saline (100 μL) and injected into the shoulder of the mice. After injected, mice were assigned into six groups (n = 6 per group): (i) Control; (ii) UBAP2L-KD; (iii) UBAP2L-OE; (iv) Radiotherapy; (v) UBAP2L-KD combined radiotherapy; and vi) UBAP2L-OE combined radiotherapy. Radiotherapy was performed as follows: The mice were anesthetized with isoflurane and fixed on a square lead plate, with the tumor region exposed. The radiation parameters were set to: 100 cm source-to-skin distance, 0.2 Gy/min dose rate, and 6 Gy total dose at a time for 6 consecutive days. After radiotherapy, all mice’s tumor size was measured every 6 days, and the maximum tumor diameter was recorded. The observation was terminated when any tumor reached a maximum diameter close to 1.5 cm. The mice were euthanized with CO2 anesthesia (50% cage volume/min), and the tumors were excised, weighed, photographed, and fixed in formalin for 24 h for paraffin embedding. Hematoxylin-and-eosin staining and IHC were performed. Animal experiments were reviewed and approved by the Ethics Committee on Animal Experiments of The Affiliated Hospital of Qingdao University (approval number: AHQU-MAL20240910XC).

The statistical analysis was performed using SPSS version 19.0 software (IBM Corp., Armonk, NY, USA) and all experiments were repeated at least thrice. The difference in UBAP2L expression between CRC tissues and control tissues, as well as the correlation between clinical pathological features, were evaluated using the chi-squared test. Survival analysis was conducted using the Kaplan–Meier method. P-values < 0.05 indicate statistically significant differences.