Distinguishing characteristics have been found in the left-sided and right-sided colorectal cancer (CRC), which have different embryonic origins, molecular and clinical features. These result in differences in the efficacy of targeted therapy and immunotherapy. Multi-omics characterization, predicated upon tumor laterality, may facilitate a more precise and personalized approach to the treatment of patients with CRC. Encompassing whole-exome, proteomics, and phosphoproteomics sequencing, we conducted a comprehensive investigation of tumor and matched normal adjacent tissues from a total of 80 pairs of patients with CRC. Results revealed that the pathogenesis of left-sided CRC was predominantly associated with chromosomal instability, while right-sided CRC was primarily linked to microsatellite instability. Regarding the tumor microenvironment, left-sided CRC exhibited predominant microvascular endothelial cell proliferation, while right-sided CRC displayed enhanced MHC Class II-associated antigen presentation mediated by M1 macrophages. Additionally, the proportion of deficient mismatch repair that developed into microsatellite instability-high was observed to be lower in the left-sided CRC compared to the right-sided, indicating divergent DNA damage repair systems between laterality subtypes that contribute to differential immunotherapy efficacy. This integrated proteogenomic study provides a comprehensive and nuanced understanding of the molecular heterogeneity between left- and right-sided CRC, offering opportunities for optimizing these patients' treatment outcomes through tailored therapeutic strategies.

The inhibitory effect of the AKT inhibitor Uprosertib on the proliferation and migration of CRC cell lines (HT29 and SW480).

In vivo assessment of ovarian metastasis rates and tumor burden in mice following AKT3 knockdown or overexpression.

The inhibitory effect of Uprosertib on CRC cell invasion and cytoskeletal organization.

Correlation analysis of AKT3 with key ECM/receptor genes in TCGA and validation of ITGB1 downregulation upon AKT3 knockdown.

Identification of CAF subsets and analysis of ligand-receptor interactions between CAFs and epithelial cells.

Pathway enrichment analysis and validation of AKT3, SNAIL, and CDH2 expression in CROM patient samples and organoids.

Objective:Although distance from the inferior tumor edge to the anal verge(DTAV)is a key predictor for sphincter-preserving surgery(SPS)in mid-low rectal cancer,its utility is limited in the"decision-gray zone"(DTAV,3-8 cm).Therefore,this study aimed to develop and validate a multiparametric magnetic resonance imaging-based nomogram for individualized preoperative prediction of SPS feasibility. Methods:This dual-center retrospective study included 335 patients with rectal adenocarcinoma(DTAV 3-8 cm).Patients were divided into training(n=263)and external validation(n=72)cohorts,and predictors were identified using multivariate logistic regression analysis.Model discrimination was assessed using area under the receiver operating curve(AUC)and calibration via the Hosmer-Lemeshow test.Subgroup analyses were performed across DTAV strata. Results:Four independent predictors were identified:larger DTAV[odds ratio(OR)=5.00,P<0.001)],larger pubococcygeal overlap distance(PCOD)(OR=1.08,P=0.001),transverse diameter of mesorectal fat(TMS)(OR=1.07,P=0.017),and subcutaneous adipose tissue thickness(SAT)(OR=0.94,P=0.016).The Sphincter Preservation Assessment in Rectal Cancer(SPARC)nomogram achieved an AUC of 0.928[95%confidence interval(95%CI):0.890-0.956]in the training cohort,outperforming DTAV alone(AUC=0.884,P=0.031)and maintaining an AUC of 0.916(95%CI:0.827-0.969)in external validation.Subgroup analysis showed notably improved predictions in the 5-8 cm DTAV subgroup.Decision curve analysis demonstrated a pronounced net clinical benefit across a wide range of threshold probabilities.Interobserver agreement was excellent(intraclass correlation coefficient,0.890-0.997). Conclusions:The SPARC nomogram reliably predicted SPS feasibility by integrating tumor location with pelvic anatomy and fat distribution.This provides valuable and evidence-based preoperative guidance,especially within the DTAV 3-8 cm gray zone.

scRNA-seq quality control metrics and cell type distribution in colorectal ovarian metastasis (CROM) samples.

3139 Background: The detection of minimal residual disease (MRD) in colorectal cancer (CRC) is crucial for predicting postoperative recurrence, particularly liver metastasis. While peripheral blood (PB) liquid biopsy is widely used, portal vein blood (PVB) may offer higher sensitivity due to direct drainage of the hepatic circulation. Methods: We prospectively enrolled 297 CRC patients undergoing curative surgery. Intraoperative portal vein blood (PVB) and matched preoperative peripheral blood (PB) samples were collected simultaneously. Cell-free DNA was sequenced using a targeted 689-gene panel (1.5 Mb). Variants were filtered to retain only those with increased allele frequency (AF) in PVB relative to PB or exclusive detection in PVB (AF ≥1%), followed by stringent germline and noise removal. Binary mutation matrices were constructed for both blood sources. Machine learning (random forest, SVM, logistic regression, gradient boosting) was used to identify predictive gene signatures separately for PVB and PB. Model performance was compared using five-fold cross-validation. Results: PVB-derived ctDNA detected a significantly higher number of tumor-specific mutations compared to PB (mean 3.2 vs. 1.4 mutations per patient, p<0.001). PVB contained significantly higher ctDNA concentration (median 8.4 ng/mL vs 2.1 ng/mL in PB, p<0.001). A 20-gene panel selected from PVB data demonstrated superior predictive accuracy for liver metastasis compared to PB-derived markers. When validated in the same cohort, the PVB panel achieved an AUC of 0.849 (95% CI: 0.802–0.891) versus 0.714 (95% CI: 0.653–0.771) for PB-based prediction (p=0.003). Sensitivity for predicting liver metastasis was 70.0% for PVB versus 48.5% for PB. The PVB panel identified 26.6% of mutation carriers as high-risk, with 100% specificity (no recurrence in mutation-negative patients). In contrast, the PB model failed to achieve comparable risk stratification, with lower positive predictive value and higher false-negative rates. Conclusions: Portal vein blood ctDNA analysis is significantly more sensitive and accurate than peripheral blood in predicting postoperative liver metastasis in CRC. PVB-based liquid biopsy provides superior risk stratification, which could better inform adjuvant therapy decisions—identifying high-risk patients for intensified surveillance or treatment, while reducing overtreatment in low-risk patients. These findings support the clinical integration of PVB sampling for MRD detection in CRC surgical practice.

The effect of AKT3 knockdown on the proliferation, migration, invasion, and cytoskeletal structure of CRC cell lines.

Colorectal ovarian metastasis (CROM), a distinct metastatic subtype of colorectal cancer, is associated with early disease onset and aggressive progression. CROM lacks specific treatment options, highlighting the need to elucidate the underlying biological mechanisms and potential therapeutic vulnerabilities. In this study, we performed integrated analyses of single-cell RNA sequencing (scRNA-seq) datasets from 155,163 cells across 35 patients from the in-house cohort and public datasets, with matched bulk transcriptomic profiling. The analysis identified AKT3+ epithelial-mesenchymal transition (EMT)-like cells at the invasive tumor-stroma interface as metastasis-initiating cells. Functional validation using in vivo xenograft models demonstrated that AKT3 deficiency reduced ovarian colonization, whereas AKT3 overexpression conferred a mesenchymal phenotype with invasive capacity. Furthermore, reciprocal cross-talk between AKT3+ mesenchymal-like cells and cancer-associated fibroblasts (CAF) played a key role in remodeling the tumor microenvironment. Multiplex immunofluorescence staining of primary tumor specimens revealed spatially coordinated AKT3+/SNAIL+/ITGB1+ tumor buds adjacent to αSMA+ CAFs at the invasive front. Critically, AKT3 inhibition or knockdown in patient-derived CROM organoids (CROM-PDO) significantly suppressed malignant phenotypes, recapitulating the AKT3 dependency. Collectively, these findings elucidate an AKT3-driven feedforward loop coupling EMT plasticity with CAF activation as a critical driver of CROM and propose CROM-PDOs as a robust platform for developing precision therapies targeting this aggressive colorectal cancer subtype. SIGNIFICANCE:Single-cell analyses reveal AKT3-expressing mesenchymal-like cells as drivers of colorectal ovarian metastases, which depends on the dynamic cross-talk between cancer cells and cancer-associated fibroblasts within the immunosuppressive ovarian niche.

BACKGROUND:Many patients with cancer benefit little from immune checkpoint blockade (ICB), a major obstacle to immunotherapy for decades. Finding alternative immune checkpoints that control CD8+ T-cell exhaustion is urgent if we are to improve the efficacy of immunotherapies, particularly in microsatellite stable (MSS) colorectal cancer (CRC) that is resistant to ICB. METHODS:Spatial proximity is essential for suppressive ligand-receptor signaling. Here, we mapped the spatial tumor microenvironment of patients with MSS CRC at single-cell resolution and analyzed the cells interacting with exhausted CD8+ T cells to identify immune checkpoint ligand-receptor pairs. To investigate the function of this previously unrecognized immune checkpoint, we performed validation studies spanning cellular experiments, mouse models, and clinical patient samples. RESULTS:We found that a subset of MSS CRC exhibits substantial CD8+ T-cell infiltration, but their function is suppressed. We identified CLEC2B (ligand)-KLRB1 (receptor) as a novel inhibiting ligand-receptor pair for CD8+ T cells. KLRB1 acts as an immune checkpoint receptor, increasing CD8+ T-cell exhaustion and facilitating immune escape in various human cancers. Binding of CLEC2B to KLRB1 initiates immunosuppressive signaling in CD8+ T cells. Clinically, CLEC2B-KLRB1 expression correlates positively with cancer progression and poor response to ICB, demonstrating that KLRB1+ CD8+ T cells are a key marker of the poorly responsive ICB subtype. Furthermore, blocking CLEC2B-KLRB1 signaling with antibodies enhances the antitumor function of CD8+ T cells, providing a potential immunotherapy target for ICB non-responders. CONCLUSIONS:Our study revealed CLEC2B-KLRB1 as a previously unrecognized immune checkpoint axis that drives T-cell exhaustion specifically in ICB poor responsive MSS CRC. Blockade of KLRB1 with a therapeutic antibody reinvigorated CD8+ T-cell antitumor immunity, positioning this axis as a promising target for enhancing immunotherapy efficiency in malignancies, including CRC and other ICB-resistant cancers.

The liver is a major metabolic organ, responsible for synthesizing and breaking down diverse metabolites. Recently, the liver's immunological functions have gradually been unveiled: combating pathogens and maintaining tissue homeostasis. Age-related functional alterations in these immune cells emerge as potential drivers of hepatic dysfunction and age-associated pathologies. However, systematic investigations into spatiotemporal immune cell dynamics during liver aging remain limited. To address this gap, we analyzed young and old mouse livers using single-cell/nuclei and spatial transcriptomics, revealing T cells as the immune cell population with the most pronounced transcriptomic alterations, marked by enrichment of exhausted CD8+ T cells in aged livers. Spatial mapping showed exhausted CD8+ T cells accumulating in portal vein (PV) zone, co-localizing with periportal hepatocytes (PP hepatocytes). Up-regulation of LPIN1 in PP hepatocyte promoted T cell exhaustion. CD8+ T cell exhaustion was tightly associated with disease progression. Therefore, our findings suggest that targeting LPIN1 may alleviate T cell exhaustion, offering potential therapeutic strategies for age-related liver diseases.

Validation that AKT3 overexpression enhances the invasion and migration capabilities of colorectal cancer cells.

Colorectal cancer (CRC) becomes highly lethal upon progression to advanced or metastatic stages. Treatment options are particularly limited for refractory metastatic CRC (mCRC) harboring KRAS mutations. In this study, we established a series of patient-derived organoids (PDOs) and patient-derived xenografts (PDXs) from mCRC patients to identify effective therapeutic compounds. We employed RNA sequencing to characterize the transcriptomic profiles of KRAS-mutant microsatellite stable (MSS) PDOs and analyzed single-cell RNA sequencing data to examine features of KRAS-mutant CRC epithelial cells. Transcriptomic analysis revealed that KRAS mutations induce elevated global transcription activity in both PDOs and epithelial cells. A large-scale drug screen of 786 Food and Drug Administration (FDA)-approved anticancer agents identified the acridine compound amsacrine hydrochloride as a potent inhibitor of PDOs and cell lines. We subsequently synthesized a series of acridine derivatives for further screening. Finally, LS-1-2 was discovered to overcome chemotherapy resistance and suppress liver metastasis in KRAS-mutated CRC. Mechanistically, LS-1-2 binds to non-muscle myosin heavy chain IIA (NMHC IIA), blocking its phosphorylation. This inhibition disrupts the PI3K/ERK/FOXO/PLK1 signaling pathway and attenuates KRAS-driven hypertranscription. In conclusion, the acridine derivative LS-1-2 emerges as a promising candidate from this preclinical investigation, providing a rationale for future clinical trials in KRAS-mutant CRC.

Abstract The liver is the primary target organ for hematogenous metastasis of colorectal cancer, and colorectal cancer liver metastasis is one of the key and challenging aspects in its treatment. In order to improve the diagnosis and comprehensive treatment of colorectal cancer liver metastasis, the guideline development group has summarized advanced experiences and the latest achievements from both domestic and international sources, and has once again revised and updated the Guideline for the diagnosis and comprehensive treatment of colorectal cancer liver metastases (2025 edition) to continuously provide guidance and reference for clinical practice in this field.

The treatment for locally advanced gallbladder cancer (GBC) remains controversial, particularly regarding the extent of surgery. This study aimed to evaluate the association between surgical extent and survival outcomes in patients with locally advanced GBC. Patients with locally advanced GBC were identified from the Surveillance, Epidemiology, and End Results (SEER) database. Overall survival (OS) and cancer-specific survival (CSS) were evaluated using Kaplan-Meier curves, Cox proportional hazards models, competing-risk models, and landmark analyses. Propensity score matching (PSM) was performed to minimize confounding, and subgroup analyses were conducted across key clinicopathological variables. Patients in the surgery group had better OS and CSS than those in the non-surgery group (median OS, 16.0 versus 9.0 months; median CSS, 17.0 versus 10.0 months; both P < 0.001), particularly when combined with multimodal therapy. Among surgically treated patients, 388 (19.8%) received radical resection (RR), while 1567 (80.2%) underwent simple resection (SR). RR was associated with improved OS and CSS compared with SR (median OS, 19.0 versus 15.0 months; median CSS, 21.0 versus 16.0 months; both P < 0.05). On multivariate analysis, RR remained independently associated with improved OS and CSS. The survival benefit of RR remained robust in 1- and 3-year landmark analyses. These results remained consistent after PSM and in competing-risk models. Subgroup analyses demonstrated consistent survival benefits from RR across various clinicopathological variables. These findings suggest that surgical intervention is associated with a survival advantage and that RR could be considered an important component of curative-intent treatment for selected patients with locally advanced GBC.

Characterization of EMT-like epithelial subsets, including CNV analysis and marker expression profiling.