Supplementary Fig 5. Effects of CLK1 modulation on YAP1 downstream target gene expression.

Colorectal cancer is a highly heterogeneous malignancy characterized by complex interactions between tumor cells and the immune system. The tumor microenvironment (TME) plays a crucial role in colorectal cancer progression and response to therapy. However, the mechanisms regulating TME composition remain poorly understood because of the genetic and phenotypic diversity of tumor cells. In this study, we investigated the tumor-intrinsic factors contributing to TME formation and evaluated genotype-based combination strategies to enhance the efficacy of immunotherapy in colorectal cancer. Using RNA sequencing, single-cell analysis, and immunohistochemistry (IHC), we identified pro-oncogenic proteins associated with low immune activation. Functional studies using in vitro co-culture systems, subcutaneous colorectal tumor models, flow cytometry, and IHC revealed a role for CDC-like kinase 1 (CLK1) in tumor progression and immunosuppressive TME remodeling. Mechanistically, CLK1 activation led to hyperactivation of the Hippo signaling pathway, promoting nuclear translocation of Yes-associated protein (YAP) and subsequent transcriptional upregulation of the chemokine CXCL1. Elevated CLK1 expression correlated with increased infiltration of myeloid-derived suppressor cells (MDSC) and impaired antitumor immune responses. Knockdown (KD) of CLK1 significantly reduced MDSC recruitment and restored CD8+ T-cell activity. Moreover, combined CLK1 KD and anti-PD-1 therapy enhanced intratumoral CD8+ T-cell infiltration to a greater extent and elicited robust antitumor responses in murine colorectal cancer models. Collectively, our findings identify the CLK1-Hippo/YAP-CXCL1 signaling axis as a regulator of immune evasion and TME remodeling in colorectal cancer and highlight the potential of therapeutically targeting this axis to improve the efficacy of immune checkpoint blockade.

Cellular senescence, a fundamental hallmark of aging, plays a paradoxical, often pro-tumorigenic role in cancer. This malignancy is largely driven by the senescence-associated secretory phenotype (SASP), yet the mechanisms that govern the production of a pro-tumorigenic SASP remain poorly understood. This study uncovers an epitranscriptomic axis in colorectal cancer (CRC) where the TRMT6/TRMT61A tRNA N1-methyladenosine (m1A) methyltransferase complex is aberrantly elevated, driving a senescent state in malignant cells. Mechanistically, TRMT6/61A-dependent m1A deposition on specific tRNAs enhances the translational efficiency of their cognate codons. This codon-biased translational control selectively boosts the synthesis of ARG2. Accumulation of ARG2 subsequently activates mTOR and NF-κB signaling and thereby establishes a robust SASP, which actively reprograms the tumor microenvironment by promoting the growth and invasiveness of neighboring cancer cells, activating cancer-associated fibroblasts, and polarizing immunosuppressive M2 macrophages. Collectively, these findings define the TRMT6/61A-ARG2 pathway as a driver for pro-tumorigenic senescence in an m1A-dependent manner, revealing a new layer of translational control in aging-associated pathology and offering a compelling rationale for developing senomorphic therapies.

Supplementary Fig 2. CLK1 plays a crucial role in facilitating in vivo tumor growth.

BACKGROUND:Neoadjuvant immune checkpoint blockade has shown remarkable activity in mismatch repair-deficient (dMMR) or microsatellite instability-high (MSI-H), locally advanced colorectal cancer. Preclinical studies suggest that COX-2 inhibition might modulate the inflammatory tumour microenvironment and augment the effect of PD-1 blockade. We aimed to investigate whether the addition of the COX-2 inhibitor celecoxib to neoadjuvant toripalimab would increase the pathological complete response in this population. METHODS:PICC-2 was a multicentre, open-label, randomised, controlled, phase 2 trial conducted at three academic hospitals in China. Eligible patients were aged 18-75 years; had histologically confirmed dMMR or MSI-H colorectal cancer, of clinical stage T3-T4 or any clinical T stage with lymph node positivity (N+); had an Eastern Cooperative Oncology Group performance status score of 0 or 1; and had adequate haematological, hepatic, and renal function. Participants were randomly assigned (1:1) via an interactive web response system, stratified by tumour location and clinical T stage, to receive neoadjuvant toripalimab plus celecoxib or toripalimab monotherapy every 14 days for 12 cycles, followed by surgery. Toripalimab 3 mg/kg was administered intravenously on day 1 in both groups; patients in the toripalimab plus celecoxib group also received celecoxib 200 mg orally twice daily on days 1-14. The primary endpoint was the proportion of patients with pathological complete response, defined as no presence of residual viable tumour in the primary tumour and all sampled lymph nodes at surgery, assessed by central blinded independent pathological review in the intention-to-treat population. Safety was assessed in all patients who received at least one dose of study treatment. This study is registered with ClinicalTrials.gov, NCT03926338, and is ongoing. FINDINGS:Between May 5, 2022, and Jan 20, 2025, 110 patients were randomly assigned to toripalimab plus celecoxib (n=55) or toripalimab monotherapy (n=55). Overall, 65 (59%) patients were male, all patients were Chinese, 76 (69%) had cT4 tumours, and 105 (95%) had clinically node-positive disease. At the data cutoff date (Oct 10, 2025), median follow-up was 18·1 months (IQR 11·5-25·3). 49 (89%) of 55 patients in each group completed all 12 planned cycles of neoadjuvant therapy. Surgery was done in 53 (96%) of 55 patients in the toripalimab plus celecoxib group and 51 (93%) of 55 in the monotherapy group. Pathological complete response was observed in 49 of 55 patients (89% [95% CI 78-96]) in the toripalimab plus celecoxib group versus 38 of 55 (69% [55-81]) in the monotherapy group (between-group difference 19 percentage points [95% CI 4-34]; p=0·014). Grade 3 treatment-related adverse events occurred in three (5%) patients and four (7%) patients, respectively; grade 3 treatment-related adverse events were tumour perforation (two [4%]) and bowel obstruction (one [2%]) in the toripalimab plus celecoxib group, and rash (one [2%]), tumour perforation (one [2%]), increased aminotransferase (one [2%]), and hypothyroidism (one [2%]) in the monotherapy group. No grade 4 or 5 treatment-related adverse events occurred. INTERPRETATION:In patients with dMMR or MSI-H locally advanced colorectal cancer, neoadjuvant toripalimab plus celecoxib significantly increased the proportion of patients attaining pathological complete response compared with toripalimab monotherapy, with a similar safety profile. These findings support further investigation of this combination strategy in larger phase 3 trials. FUNDING:The National Natural Science Foundation of China, Guangdong Basic and Applied Basic Research, the National Key Clinical Discipline of China, the Program of Guangdong Provincial Clinical Research Center for Digestive Diseases, and the Chinese Society of Clinical Oncology-Junshi Biosciences Oncology Immunity Research. TRANSLATION:For the Chinese translation of the abstract see Supplementary Materials section.

BACKGROUND:The efficacy of neoadjuvant chemotherapy combined with dual immune checkpoint blockade in proficient mismatch repair/microsatellite-stable (pMMR/MSS) locally advanced colorectal cancer (LACRC) remains uncertain. METHODS:In this open-label, randomized phase 2 trial (ClinicalTrials.gov: NCT05571644), eligible patients were randomly assigned (1:1:1) to receive 6 cycles of neoadjuvant mFOLFOXIRI plus cadonilimab, mFOLFOXIRI, or mFOLFOX6, followed by surgery and adjuvant chemotherapy. The primary endpoint was pathological complete response (pCR) in the intention-to-treat population. Secondary endpoints included major pathological response (MPR), safety, locoregional recurrence, disease-free survival, and overall survival. FINDINGS:Between July 2023 and August 2024, 123 patients were randomly assigned (41 per group). Neoadjuvant mFOLFOXIRI plus cadonilimab significantly improved pCR compared with mFOLFOX6 (26.8% versus 9.8%; odds ratio [OR], 3.4; 95% confidence interval [CI], 1.04 to 13.24; p = 0.046), whereas mFOLFOXIRI alone did not (14.6% versus 9.8%; OR, 1.6; 95% CI, 0.4 to 6.7; p = 0.50). MPR rates were 68.3%, 46.3%, and 43.9%, respectively, and were significantly higher with mFOLFOXIRI plus cadonilimab than with mFOLFOX6 (p = 0.026). Downstaging to ypStage 0-I occurred in 66%, 45%, and 41% of patients in the three groups, respectively, and was significantly higher in the mFOLFOXIRI plus cadonilimab group (p = 0.027). Grade ≥3 adverse events were manageable, with liver enzyme elevation and neutropenia being the most common. CONCLUSIONS:Neoadjuvant mFOLFOXIRI plus cadonilimab significantly improved pCR compared with mFOLFOX6 in LACRC, with a manageable safety profile, supporting further investigation. FUNDING:This work was funded by the National Science Fund for Distinguished Young Scholars (82425045) and the National Natural Science Foundation of China (82272800).

Supplementary Fig 1. CLK1 is associated with a decreased response to immunotherapy.

Objectives: This study aimed to investigate the impact of TDs on the survival of patients with locally advanced rectal cancer (LARC). Additionally, we propose a novel staging method that combines TDs and lymph node metastases (LNMs) to enhance prognostic accuracy. Methods: Patients with LARC were retrospectively identified from the Surveillance, Epidemiology, and End Results (SEER) database and a Sun Yat-sen University (SYSU) cohort. Propensity score matching (PSM) was utilized to minimize selection bias when evaluating TDs. We quantitatively stratified TDs counts and integrated them with regional LNMs to formulate a novel tumor node metastasis (TNM) staging system. Furthermore, a prognostic nomogram incorporating TDs was constructed and validated to predict survival. Results: Overall, 19,991 patients were included in the SEER database, with 2667 (13.3%) TDs-positive and 17,324 (86.7%) TDs-negative tumors. After PSM, multivariate Cox analysis reveals that TDs are an independent adverse prognostic factor (HR = 1.521, 95% CI: 1.366–1.693, p < 0.001). Patients with high-risk group (TDs > 4) at any TNM stage exhibit OS comparable to or worse than that of stage IIIC disease. For patients staged as T4N2M0, the high-risk group (TDs > 4) demonstrates OS equivalent to stage IV disease. The nomogram achieved C-indices of 0.713 (training cohort, n = 8586) and 0.789 (external validation cohort, n = 304), with AUCs of 0.774 (3-year) and 0.710 (5-year). Conclusions: The presence of TDs is associated with poorer OS, and integrating TDs with LNMs improves the accuracy of TNM staging. The nomogram (C-index = 0.789) provides enhanced prognostic stratification and survival prediction.

ABSTRACT Novel synthetic methods offer significant potential to accelerate drug development, yet there remains a largely unexplored area for efficiently synthesizing metal‐based anticancer agents. Herein, we report a novel solvothermal domino reaction of pyridine‐2‐methylamine (and its 4‐OCH 3 ‐substituted derivative), benzaldehyde, and CuCl 2 ∙2H 2 O, which simultaneously achieves ligand synthesis and coordination assembly in one pot and facilely affords innovative dinuclear dicoordinate copper(I) complexes ( Cu1 and Cu2 ) with the in situ‐formed bulky steric‐hindering tetraarylethane ligands featuring the bis‐imidazo[1,5‐ a ]pyridine scaffold. The unique geometry of Cu1 and Cu2 confers physiological stability and vacant coordination sites for efficiently catalyzing Fenton‐like reactions. Further studies reveal that Cu2 effectively elevates intracellular copper ion levels to induce cuproptosis, concurrently disrupting cellular redox homeostasis to trigger ferroptosis. The concurrent cuproptosis‐ferroptosis activation finally elicits significantly enhanced immunogenic cell death (ICD), which facilitates the antitumor activity of Cu2 . Moreover, in combination with immune checkpoint inhibitor α PD‐1, Cu2 exhibits improved immunotherapy effects. This work introduces the first small‐molecule copper complex that achieves immunotherapy potentiation through cuproptosis‐ferroptosis‐ICD induction and provides a new pathway for accessing innovative metal‐based antitumor agents through such a rationally designed domino reaction.

Supplementary Fig 4. Spatial transcriptomic analysis of CLK1 expression and its correlation with immune markers.

The oncologic safety of laparoscopic-assisted surgery for synchronous colorectal cancer (SCRC), a distinct high-risk subgroup, remains under-investigated. This study aimed to compare the short-term and long-term outcomes of laparoscopic-assisted versus open surgery for SCRC. A retrospective analysis of a cohort of 178 consecutive patients undergoing curative-intent surgery for SCRC between June 2007 and April 2019 was performed. To balance baseline covariates for the primary analysis, overlap weighting (OW) was applied to the cohort (133 laparoscopic, 45 open). A 1:1 propensity score matching (PSM) analysis was conducted as a sensitivity analysis. The primary outcomes were 5-year overall survival (OS) and 5-year disease-free survival (DFS). In the primary OW analysis, the laparoscopic group showed significantly less estimated blood loss (median, 100 mL vs. 200 mL; P = 0.003) and shorter time to first liquid diet (median, 4 vs. 5 days; P = 0.005) than the open surgery group. The two groups did not differ significantly in terms of 5-year OS (75.1

Immune checkpoint inhibitors (ICIs) have reshaped the treatment in multiple tumors. However, a substantial proportion of patients exhibit limited responses. The lack of harmonized, large-scale pan-cancer ICI datasets coupled with accessible analysis tools hinders the systematic discovery of response biomarkers and resistance mechanisms. Therefore, we developed a comprehensive resource named ICIsAtlas, which encompassed curated transcriptomic and clinical data from 1,268 ICI-treated patients across eight tumor types, with an accompanying R package implementing a complete workflow for deconvolution and biomarker evaluation. Applying the ICIsAtlas framework, a systematic pan-cancer analysis was performed. We identified the universal signatures that were specific for ICI response, including cooperative interactions among favorable immune cells. In addition, we discovered a competitive cell community and SERPING1 + VEGFA+ Natural Killer (NK) cell-mediated immunosuppressive niche in non-responders, which were further validated with single-cell and multiplex immunohistochemistry data. The ICIsAtlas resource and R package represent a powerful, publicly available platform for hypothesis generation and biomarker discovery, which could be used to develop the next-generation biomarkers and therapeutic targets to improve tumor immunotherapy.

Circulating tumor DNA (ctDNA) shows promise for predicting recurrence in colorectal cancer liver metastases (CRLM) patients. We investigated the prognostic value of perioperative ctDNA, particularly 2 week postoperative minimal residual disease (MRD), in CRLM patients undergoing liver resection. We prospectively collected blood samples from 94 CRLM patients before (after neoadjuvant therapy, if any) and 2 weeks after liver metastasectomy. 63 patients had both assessments. ctDNA status and variant allele fraction (VAF) were determined using targeted sequencing. Disease-free survival (DFS) was analyzed using Kaplan–Meier curves and Cox regression. Baseline characteristics were compared based on ctDNA status. Preoperative ctDNA status was not associated with DFS (p = 0.77). However, postoperative 2 week MRD status was a strong predictor; MRD-positive patients (N = 31) had significantly worse DFS than MRD-negative patients (N = 32) (p < 0.001). In multivariate analysis, postoperative MRD positivity (HR = 5.52, 95

Supplementary Table S1. Primers and shRNA sequences used in the study.

Supplementary Table S2. Information on the antibodies used in the study

Supplementary Fig 3. CLK1 negatively regulates CD8+ T cell activity.

Importance Previous studies have demonstrated the advantages of short-term histopathological outcomes and complications associated with transanal total mesorectal excision (TME) compared with laparoscopic TME. However, the long-term oncological outcomes of transanal TME remain ambiguous. This study aims to compare 3-year disease-free survival of transanal TME with laparoscopic TME. Objective To evaluate 3-year disease-free survival between transanal TME and laparoscopic TME in patients with rectal cancer. Design, Setting, and Participants This randomized, open-label, noninferiority, phase 3 clinical trial was performed in 16 different centers in China. Between April 2016 and June 2021, a total of 1115 patients with clinical stage I to III mid-low rectal cancer were enrolled. The last date of participant follow-up was in June 2024. Interventions Participants were randomly assigned in a 1:1 ratio before their surgical procedure to undergo either transanal TME (n = 558) or laparoscopic TME (n = 557). Main Outcomes and Measures The primary end point was 3-year disease-free survival, with a noninferiority margin of -10% for the comparison between transanal TME and laparoscopic TME. Secondary outcomes included 3-year overall survival and 3-year local recurrence. Results In the primary analysis set, the median patient age was 60 years. A total of 692 male and 397 female patients were included in the analysis. Three-year disease-free survival was 82.1% (97.5% CI, 78.4%-85.8%) for the transanal TME group and 79.4% (97.5% CI, 75.6%-83.4%) for the laparoscopic TME group, with a difference of 2.7% (97.5% CI, -3.0% to 8.1%). The lower tail of a 2-tailed 97.5% CI for the group difference in 3-year disease-free survival was above the noninferiority margin of -10 percentage points. Furthermore, the 3-year local recurrence was 3.6% (95% CI, 2.0%-5.1%) for transanal TME and 4.4% (95% CI, 2.6%-6.1%) for laparoscopic TME. Three-year overall survival was 92.6% (95% CI, 90.4%-94.8%) for transanal TME and 90.7% (95% CI, 88.3%-93.2%) for laparoscopic TME. Conclusions and Relevance In patients with mid-low rectal cancer, 3-year disease-free survival for transanal TME was noninferior to that of laparoscopic TME.

Peritoneal metastasis (PM) after radical surgery is an important cause of treatment failure in colorectal cancer (CRC). Intraoperative intraperitoneal perfusion chemotherapy may be an effective method for preventing postoperative PM in patients with CRC. This study aimed to explore the safety and feasibility of intraoperatively preventive intraperitoneal perfusion chemotherapy using lobaplatin for CRC. Between 12 December 2017 and 17 October 2019, 720 eligible CRC patients with T4 or N + clinical TNM stage were recruited from 25 hospitals in China. Eligible patients were randomised in a 1:1 ratio to undergo resection of CRC only (control group) or resection of CRC with intraperitoneal perfusion chemotherapy with lobaplatin intraoperatively (lobaplatin group). The primary endpoint of this trial was the rate of PM after surgery, while secondary endpoints included safety, overall survival (OS) time, recurrence-free survival (RFS) time, peritoneal recurrence-free survival (PRFS) time, and the rate of liver metastasis. Of 716 patients included in the full analysis set (FAS), 352 were assigned to the lobaplatin group and 364 to the control group. In the FAS population, adding intraoperatively preventive intraperitoneal perfusion chemotherapy with lobaplatin decreased the primary end point rate of 3-year PM (3.56

BACKGROUND:Immunotherapy is beneficial for some colorectal cancer (CRC) patients, but immunosuppressive networks limit its effectiveness. Cancer-associatedfibroblasts (CAFs) are significant in immune escape and resistance toimmunotherapy, emphasizing the urgent need for new treatment strategies. METHODS:Flow cytometric, Western blotting, proteomics analysis, analysis of public database data, genetically modified cell line models, T cell coculture, crystal violetstaining, ELISA, metabonomic and clinical tumour samples were conducted to assess the role of EDEM3 in immune escape and itsmolecular mechanisms. We evaluated theeffects of FMD plus 2-DG on antitumour immunity using multipleximmunofluorescence, flow cytometry, cytokine profiling, TUNEL assays, xenografttumours, and in vivo studies. RESULTS:We show thatCAFs upregulate PD-L1 glycosylation and contribute to immune evasion byglycosyltransferase EDEM3. Additionally, EDEM3 plays a role in tumour immunityduring tumour progression. However, the EDEM3-mediated upregulation of PD-L1 expression underpins PD-1/PD-L1 blockade resistance in vivo. This finding contradictsthe previous trend that positive PD-L1 expression indicates a strong responseto PD-1/PD-L1 blockade. Mechanistically, high-EDEM3 expression facilitates M2-like This finding contradictsthe previous trend that positive PD-L1 expression indicates a strong responseto PD-1/PD-L1 blockade.Mechanistically, polarizationand chemotactic migration of macrophages, which are enriched in theperipheral region of tumours compared to thecore region, precluding access of CD8+ T cells to tumourfoci. Furthermore, we EDEM3 predominantly activates the recruited M2-like macrophagesvia a glucose metabolism-dependent mechanism. Manipulationof glucose utilization by a fasting-mimicking diet(FMD) plus 2-DG treatmentsynergistically with PD-1 antibody elicits potent antitumour activity byeffectively decreasing tumour glycosylated PD-L1 expression, augmenting the CD8+effector T cell infiltration and activation while concurrently reducing the infiltration.TheCAFs-EDEM3-M2-like macrophage axis plays a critical role in promotingimmunotherapy resistance. infiltration.TheCAFs-EDEM3-M2-like macrophage axis plays a critical role in promotingimmunotherapy resistance. CONCLUSIONS:Our study suggests that blocking EDEM3-induced M2-like macro phage trafficking by FMD plus 2-DG is a promising and effective strategy to overcomeresistance to checkpoint blockade therapy offeringhope for improved treatment outcomes. KEY POINTS:Cancer-associated fibroblasts (CAFs) can enhance PD-L1 glycosylation through the glycosyltransferase EDEM3, contributing to immune evasion during tumour progression. EDEM3 predominantly activates the recruit M2-like macrophages via a glucose metabolism-dependent mechanism. Blocking glucose utilization antagonizes recruiting and polarizing M2-like macrophages synergistically with PD-1 antibody to improve anticancer immunity.