Proposed mechanism of the ketogenic diet-microbiota-MMA-immune axis in CRC. (Part 1) A ketogenic diet remodels gut microbiota homeostasis by depleting MMA-producing bacteria, thereby reducing the accumulation of the oncometabolite (MMA). (Part 2) At the molecular level, MMA acts as a ligand that binds to Rap1, activating the downstream MAPK/ERK signalling cascade. This signalling event drives the transcriptional reprogramming of TAMs towards the pro-tumorigenic M2 phenotype. (Part 3) Clinically, elevated serum MMA in CRC patients correlates with increased M2 macrophage infiltration in the tumour microenvironment and poor prognosis.

Background:Lateral lymph node metastasis (LLNM) and skip lymph node metastasis (SLNM) in papillary thyroid carcinoma (PTC) are generally unpredictable, especially in adolescents and young adults (AYAs). This study aims to investigate the risk factors associated with LLNM and skip SLNM in AYAs with PTC. Methods:This retrospective analysis was performed on the medical records of AYAs with PTC who underwent total or near-total thyroidectomy or lobectomy, along with central lymph node dissection (CLND) and lateral lymph node dissection (LLND) at The First Affiliated Hospital of Soochow University from January 2017 to December 2023. Variables identified as significant in the univariate analysis were further assessed using multivariate analysis. Continuous variables deemed significant were analyzed using receiver operating characteristic (ROC) curve analysis to establish optimal cut-off values. Logistic regression analysis was then employed to identify independent risk factors for LLNM and SLNM in AYAs with PTC. Results:Among the 545 AYAs with PTC, 480 individuals (88.1%) exhibited LLNM, with 54 of these 480 patients (11.3%) demonstrating SLNM. Univariate and multivariate analyses identified the diameter of the largest tumor [P<0.001, odds ratio (OR) =2.768, 95% confidence interval (CI): 1.544-4.962] and the number of central lymph node metastasis (CLNM) (P=0.001, OR =3.888, 95% CI: 1.701-8.888) as independent predictors for LLNM. Furthermore, the number of LLNM (P=0.007, OR =0.295, 95% CI: 0.122-0.712) was identified as an independent factor for SLNM. ROC curve analysis determined that the optimal cut-off values for the diameter of the largest tumor, the number of CLNM, and the CLNM/ CLND ratio as risk factors for LLNM in AYAs with PTC were 1.1 cm, 3, and 0.3, respectively. Similarly, the optimal cut-off values for the diameter of the largest tumor, the number of LLNM, and the LLNM/LLND ratio for predicting SLNM were 1.2 cm, 5, and 0.2, respectively. Conclusions:This study demonstrated that the diameter of the largest tumor greater than 1.1 cm and a number of CLNM equal to or greater than 3 were independent risk factors for LLNM in AYAs with PTC. Additionally, a number of LLNM equal to or less than 5 was identified as a risk factor for SLNM.

S1. Representative images of colon length and rectal bleeding at the endpoint of modelling in AOM/DSS model mice subjected to three different treatment methods.

S8. IHC staining of NRF2 in the normal BCAA diet group and the low BCAA diet group.

S6. Stable cells with circ_0126925 knockdown or overexpression.

e15580 Background: Microsatellite-stable (MSS) colorectal cancer (CRC) remains largely refractory to immune checkpoint blockade (ICB). While the gut microbiota regulates the tumor immune microenvironment, mechanisms linking antibiotic-induced dysbiosis to ICB efficacy remain unclear. We investigated whether vancomycin sensitizes MSS CRC to anti-PD-1 therapy and elucidated underlying mechanisms involving microbial metabolites and dendritic cell (DC) function. Methods: MSS CRC subcutaneous syngeneic models were established and treated with vancomycin, anti-PD-1, or their combination. Tumor growth was monitored longitudinally. Integrated multi-omics analysis (16S rRNA sequencing, metagenomics, and metabolomics) profiled gut microbiota and metabolic alterations. Molecular mechanisms were validated in bone marrow-derived DCs (BMDCs) and tumor-draining lymph nodes (TDLNs) using RNA sequencing, molecular docking, co-immunoprecipitation, and ubiquitination assays. Results: Compared with monotherapy, the combination of vancomycin and anti-PD-1 significantly suppressed tumor growth and prolonged survival. Microbiome analysis revealed that vancomycin dramatically altered gut microbiota composition, enriching beneficial bacterial taxa. Metabolomics identified marked downregulation of systemic and intratumoral asparagine levels in the combination group. Mechanistically, high asparagine levels impaired DC function specifically within TDLNs. Asparagine supplementation upregulated the E3 ubiquitin ligase FBXO21 in DCs. FBXO21 directly interacted with p100, promoting K48-linked ubiquitination and processing to p52, thereby aberrantly activating the non-canonical NF-κB pathway. This activation caused defective DC maturation and impaired antigen cross-presentation in TDLNs, hindering T cell priming. Conversely, vancomycin-induced asparagine depletion downregulated FBXO21, inhibited p100-to-p52 processing, and suppressed non-canonical NF-κB signaling, effectively restoring DC antigen-presenting capacity in TDLNs and promoting effector CD8+ T cell infiltration into tumors. Conclusions: Vancomycin enhances anti-PD-1 efficacy in MSS CRC by remodeling the gut microbiota and depleting asparagine. We identified a novel axis wherein asparagine downregulation inhibits FBXO21-mediated p100 processing and non-canonical NF-κB activation, restoring DC function in TDLNs. Targeting the microbiota-asparagine-FBXO21 axis offers a promising therapeutic strategy to overcome ICB resistance in MSS CRC.

Table S2. Primers used in RT-qPCR and vitro transcription.

Reliable evaluation methods serve an important role in improving the prognosis of patients with colorectal cancer (CRC), guiding the development of treatment plans and prolonging patient survival. In the present study, several preoperative inflammatory indicators and tumor markers were evaluated for their ability to predict CRC prognosis. A total of 224 eligible patients with CRC were enrolled and divided into the training (n=150) and validation (n=74) groups. The training group underwent both Least Absolute Shrinkage and Selection Operator (LASSO) regression and Cox regression analyses to discern pivotal prognostic factors, to formulate a nomogram for overall survival prediction. The results showed that LASSO regression, along with univariate and multivariate Cox regression analyses, identified neutrophil-lymphocyte ratio (NLR), carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) as effective risk factors for CRC. The concordance index of the nomogram was 0.716 in the training group and 0.700 in the validation group. The areas under the curve for predicting 3-year survival were 0.748 and 0.776 in the training and validation groups, respectively, and for 5-year survival were 0.749 and 0.731, respectively. In conclusion, NLR, CA19-9 and CEA may serve as effective additions to traditional clinical assessment methods and a nomogram incorporating these three preoperative indicators could be used efficiently to predict the prognosis of patients with CRC.

S4. HFD-modulated gut microbiota of mice and obese patients promote malignant progression of colorectal tumors in Abx pretreated AOM/DSS model mice.

S2. IF of the tight junction proteins ZO-1 and Occludin in AOM/DSS model mice subjected to three different treatment methods and IHC staining of the Ki-67 in the colon of mice subjected to two different treatment methods.

Persistent luminescence nanoparticles (PLNPs) have attracted significant attention in biosensing and bioimaging. However, the variety of PLNPs that can be directly synthesized via bottom-up approaches remains largely limited to ZnGa2O4:Cr, Zn2GeO4:Mn, and their analogs. Herein, we report new spinel-structured CaSc2O4:Tb (CSO) PLNPs synthesized via a straightforward one-step hydrothermal method. The morphology of CSO PLNPs can be precisely tuned from spindle-like to bipyramidal and rod-like structures by adjusting the pH values of the synthesis solution, which in turn directly modulates their persistent luminescence intensity and decay duration. The CSO PLNPs were further functionalized with an azo-bond-containing BHQ-1 quencher to construct CSO-BHQ nanoprobes for profiling bacterial extracellular metabolism by targeting azoreductase (AzoR) activity. As a key enzyme in extracellular bacterial metabolism, AzoR participates in critical processes including azo-antibiotic degradation and bacterial communication, and its secretion level serves as an indicator of metabolic activity. Detecting AzoR secretion across different bacterial growth phases shows that enzyme production peaked during the exponential growth phase, reflecting the most active stage of extracellular metabolism. Moreover, by assessing extracellular metabolic activity with the CSO-BHQ nanoprobes, we can also determine bacterial antibiotic susceptibility. For instance, S. aureus was found to be insensitive to ampicillin but highly susceptible to vancomycin, while E. coli showed the opposite sensitivity profile. This work not only introduces a new class of easily synthesized PLNPs but also highlights their promising utility in tracking bacterial metabolism and rapidly identifying antibiotic susceptibility.

Table S3. Information of antibodies.

Figure S7. Mass spectrometry analysis of the BCAT2, keratin 19 and ACOT7 peptides pulled down by antisense circ_0126925 probes.

Table S1. Clinical information of 96 patients

Table S5. Digoxin labeled probes, Cy3 labeled probes, Desthio Biotin-TEG labeled probes

S3. In AOM/DSS model mice, antibiotic treatment significantly attenuated the colon tumorigenesis.

S5. In vitro, circ_38945 promotes proliferation and migration.

Acyl-CoA oxidase 1 (ACOX1), a member of the acyl-coenzyme A oxidase family, is considered a crucial regulator whose dysregulation is implicated in the occurrence and progression of various cancers. This study aims to elucidate the impact of ACOX1 in CRC, shedding light on its potential as a therapeutic target. Through analysis of the GEO dataset, it was found that ACOX1 is significantly downregulated in colorectal cancer (CRC), and this lower expression level is associated with a worse prognosis. Additionally, in vitro as well as in vivo, ACOX1 overexpression dramatically reduced the proliferation and metastasis of CRC cells. Mass spectrometry revealed the crucial role of ACOX1 in fatty acid β-oxidation, as its overexpression led to a substantial increase in reactive oxygen species (ROS) derived from fatty acid β-oxidation. Further experiments demonstrated that ACOX1 overexpression, through modulation of fatty acid metabolism, increased ROS levels, reduced the phosphorylation activation of the key autophagy regulator mTOR, enhanced autophagy, and ultimately suppressed the growth and metastasis of CRC. In conclusions, ACOX1 expression is decreased in CRC. ACOX1 may regulate autophagy by reprogramming lipid metabolism to modulate the ROS/mTOR signaling pathway, consequently inhibiting the proliferation and migration of CRC.

Recent evidence indicates that a high-fat diet can promote tumor development, especially colorectal cancer, by influencing the microbiota. Regulatory circular RNA (circRNA) plays an important role in modulating host-microbe interactions; however, the specific mechanisms by which circRNAs influence cancer progression by regulating these interactions remain unclear. Here, we report that consumption of a high-fat diet modulates the microbiota by specifically upregulating the expression of the noncoding RNA hsa_circ_0126925 (herein, referred to as circ_0126925) in colorectal cancer. Acting as a scaffold, circ_0126925 hinders the recruitment of the E3 ubiquitin ligase tripartite motif-containing protein 21 (TRIM21) to branched-chain amino acid transaminase 2 (BCAT2), leading to reduced degradation of BCAT2. This reduction in targeted degradation of BCAT2 can protect tumors from limited branched-chain amino acid (BCAA) interference by improving the metabolism of BCAAs in colorectal cancer. Taken together, these data demonstrate that circ_0126925 plays a critical role in promoting the progression of colorectal cancer by maintaining BCAA metabolism and provide insight into the functions and crosstalk of circ_0126925 in host-microbe interactions in colorectal cancer. Implications: This study preliminarily confirms that circRNAs do indeed respond to microbiota/microbial metabolites, providing further evidence for the potential development of circRNAs as diagnostic tools and/or therapeutic agents to alleviate microbiome-related pathology in humans.