LAMP2A regulates cisplatin resistance in colorectal cancer through mediating autophagy.

BACKGROUND: Drug resistance is an important constraint on clinical outcomes in advanced cancers. LAMP2A is a limiting protein in molecular chaperone-mediated autophagy. This study was aimed to explore LAMP2A function in cisplatin (cis-diamminedichloroplatinum, DDP) resistance colorectal cancer (CRC) to seek new ideas for CRC clinical treatment. METHODS: In this study, LAMP2A expression was analyzed by molecular experimental techniques,such as qRT-PCR and western blot. Then, LAMP2A in cells was interfered by cell transfection experiments. Subsequently, the function of LAMP2A on proliferation, migration, invasion, DDP sensitivity, and autophagy of CRC/DDP cells were further investigated by a series of experiments, such as CCK-8, transwell, and western blot. RESULTS: We revealed that LAMP2A was clearly augmented in DDP-resistant CRC and was related to poor patient prognosis. Functionally, LAMP2A insertion remarkably CRC/DDP proliferation, migration, invasion ability and DDP resistance by strengthen autophagy. In contrast, LAMP2A knockdown limited the proliferation, migration, and invasion while heightened cellular sensitivity to DDP by restraining autophagy in CRC/DDP cells. Furthermore, LAMP2A silencing was able to curb tumor formation and enhance sensitivity to DDP in vivo. CONCLUSION: In summary, LAMP2A boosted malignant progression and DDP resistance in CRC/DDP cells through mediating autophagy. Clarifying LAMP2A function in DDP resistance is promising to seek cancer therapies biomarkers targeting LAMP2A activity.

Role played by MDSC in colitis-associated colorectal cancer and potential therapeutic strategies.

Colitis-associated colorectal cancer has been a hot topic in public health issues worldwide. Numerous studies have demonstrated the significance of myeloid-derived suppressor cells (MDSCs) in the progression of this ailment, but the specific mechanism of their role in the transformation of inflammation to cancer is unclear, and potential therapies targeting MDSC are also unclear. This paper outlines the possible involvement of MDSC to the development of colitis-associated colorectal cancer. It also explores the immune and other relevant roles played by MDSC, and collates relevant targeted therapies against MDSC. In addition, current targeted therapies for colorectal cancer are analyzed and summarized.

Differential expression of angiogenesis-related genes 'VEGF' and 'angiopoietin-1' in metastatic and EMAST-positive colorectal cancer patients.

Abnormal angiogenesis leads to tumor progression and metastasis in colorectal cancer (CRC). This study aimed to elucidate the association between angiogenesis-related genes, including VEGF-A, ANGPT-1, and ANGPT-2 with both metastatic and microsatellite alterations at selected tetranucleotide repeats (EMAST) subtypes of CRC. We conducted a thorough assessment of the ANGPT-1, ANGPT-2, and VEGF-A gene expression utilizing publicly available RNA sequencing and microarray datasets. Then, the experimental validation was performed in 122 CRC patients, considering their disease metastasis and EMAST+/- profile by using reverse transcription polymerase chain reaction (RT-PCR). Subsequently, a competing endogenous RNA (ceRNA) network associated with these angiogenesis-related genes was constructed and analyzed. The expression level of VEGF-A and ANGPT-2 genes were significantly higher in tumor tissues as compared with normal adjacent tissues (P-value < 0.001). Nevertheless, ANGPT-1 had a significantly lower expression in tumor samples than in normal colon tissue (P-value < 0.01). We identified a significantly increased VEGF-A (P-value = 0.002) and decreased ANGPT-1 (P-value = 0.04) expression in EMAST+ colorectal tumors. Regarding metastasis, a significantly increased VEGF-A and ANGPT-2 expression (P-value = 0.001) and decreased ANGPT-1 expression (P-value < 0.05) were established in metastatic CRC patients. Remarkably, co-expression analysis also showed a strong correlation between ANGPT-2 and VEGF-A gene expressions. The ceRNA network was constructed by ANGPT-1, ANGPT-2, VEGF-A, and experimentally validated miRNAs (hsa-miR-190a-3p, hsa-miR-374c-5p, hsa-miR-452-5p, and hsa-miR-889-3p), lncRNAs (AFAP1-AS1, KCNQ1OT1 and MALAT1), and TFs (Sp1, E2F1, and STAT3). Network analysis revealed that colorectal cancer is amongst the 82 significant pathways. We demonstrated a significant differential expression of VEGF-A and ANGPT-1 in colorectal cancer patients exhibiting the EMAST+ phenotype. This finding provides novel insights into the molecular pathogenesis of colorectal cancer, specifically in EMAST subtypes. Yet, the generalization of in silico findings to EMAST+ colorectal cancer warrants future experimental investigations. In the end, this study proposes that the EMAST biomarker could serve as an additional perspective on CMS4 biology which is well-defined by activated angiogenesis and worse overall survival.

Preoperative three-dimensional lung volumetry predicts respiratory complications in patients undergoing major liver resection for colorectal metastases.

Colorectal liver metastases (CRLM) are the predominant factor limiting survival in patients with colorectal cancer and liver resection with complete tumor removal is the best treatment option for these patients. This study examines the predictive ability of three-dimensional lung volumetry (3DLV) based on preoperative computerized tomography (CT), to predict postoperative pulmonary complications in patients undergoing major liver resection for CRLM. Patients undergoing major curative liver resection for CRLM between 2010 and 2021 with a preoperative CT scan of the thorax within 6 weeks of surgery, were included. Total lung volume (TLV) was calculated using volumetry software 3D-Slicer version 4.11.20210226 including Chest Imaging Platform extension ( http://www.slicer.org ). The area under the curve (AUC) of a receiver-operating characteristic analysis was used to define a cut-off value of TLV, for predicting the occurrence of postoperative respiratory complications. Differences between patients with TLV below and above the cut-off were examined with Chi-square or Fisher's exact test and Mann-Whitney U tests and logistic regression was used to determine independent risk factors for the development of respiratory complications. A total of 123 patients were included, of which 35 (29%) developed respiratory complications. A predictive ability of TLV regarding respiratory complications was shown (AUC 0.62, p = 0.036) and a cut-off value of 4500 cm3 was defined. Patients with TLV < 4500 cm3 were shown to suffer from significantly higher rates of respiratory complications (44% vs. 21%, p = 0.007) compared to the rest. Logistic regression analysis identified TLV < 4500 cm3 as an independent predictor for the occurrence of respiratory complications (odds ratio 3.777, 95% confidence intervals 1.488-9.588, p = 0.005). Preoperative 3DLV is a viable technique for prediction of postoperative pulmonary complications in patients undergoing major liver resection for CRLM. More studies in larger cohorts are necessary to further evaluate this technique.

Fusobacterium nucleatum infection modulates the transcriptome and epigenome of HCT116 colorectal cancer cells in an oxygen-dependent manner.

Fusobacterium nucleatum, a gram-negative oral bacterium, has been consistently validated as a strong contributor to the progression of several types of cancer, including colorectal (CRC) and pancreatic cancer. While previous in vitro studies have shown that intracellular F. nucleatum enhances malignant phenotypes such as cell migration, the dependence of this regulation on features of the tumor microenvironment (TME) such as oxygen levels are wholly uncharacterized. Here we examine the influence of hypoxia in facilitating F. nucleatum invasion and its effects on host responses focusing on changes in the global epigenome and transcriptome. Using a multiomic approach, we analyze epigenomic alterations of H3K27ac and global transcriptomic alterations sustained within a hypoxia and normoxia conditioned CRC cell line HCT116 at 24 h following initial infection with F. nucleatum. Our findings reveal that intracellular F. nucleatum activates signaling pathways and biological processes in host cells similar to those induced upon hypoxia conditioning in the absence of infection. Furthermore, we show that a hypoxic TME favors F. nucleatum invasion and persistence and therefore infection under hypoxia may amplify malignant transformation by exacerbating the effects induced by hypoxia alone. These results motivate future studies to investigate host-microbe interactions in tumor tissue relevant conditions that more accurately define parameters for targeted cancer therapies.

Cancer-derived exosomal lncRNA SNHG3 promotes the metastasis of colorectal cancer through hnRNPC-mediating RNA stability of ß-catenin.

Metastasis is the leading cause of death in colorectal cancer (CRC) patients. By mediating intercellular communication, exosomes exhibit considerable value in regulating tumor metastasis. Long non-coding RNAs (lncRNAs) are abundant in exosomes and participate in regulating tumor progression. However, it is poorly understood how the cancer-secreted exosomal lncRNAs affect CRC proliferation and metastasis. Here, by analyzing the public databases we identified a lncRNA SNHG3 and demonstrated that SNHG3 was delivered through CRC cells-derived exosomes to promote metastasis in CRC. Mechanistically, exosomal SNHG3 was internalized by CRC cells and afterward upregulated the expression of ß-catenin by facilitating the intranuclear transport of hnRNPC. Consequently, the RNA stability of ß-catenin was enhanced which led to the activation of EMT and metastasis of CRC cells. Our findings expand the oncogenic mechanisms of exosomal SNHG3 and identify it as a diagnostic marker for CRC.

Nuclear Softness Promotes the Metastatic Potential of Large-Nucleated Colorectal Cancer Cells via the ErbB4-Akt1-Lamin A/C Signaling Pathway.

Abnormal nuclear enlargement is a diagnostic and physical hallmark of malignant tumors. Large nuclei are positively associated with an increased risk of developing metastasis; however, a large nucleus is inevitably more resistant to cell migration due to its size. The present study demonstrated that the nuclear size of primary colorectal cancer (CRC) cells at an advanced stage was larger than cells at an early stage. In addition, the nuclei of CRC liver metastases were larger than those of the corresponding primary CRC tissues. CRC cells were sorted into large-nucleated cells (LNCs) and small-nucleated cells (SNCs). Purified LNCs exhibited greater constricted migratory and metastatic capacity than SNCs in vitro and in vivo. Mechanistically, ErbB4 was highly expressed in LNCs, which phosphorylated lamin A/C at serine 22 via the ErbB4-Akt1 signaling pathway. Furthermore, the level of phosphorylated lamin A/C was a negative determinant of nuclear stiffness. Taken together, CRC LNCs possessed greater constricted migratory and metastatic potential than SNCs due to ErbB4-Akt1-mediated lamin A/C phosphorylation and nuclear softening. These results may provide a potential treatment strategy for tumor metastasis by targeting nuclear stiffness in patients with cancer, particularly CRC.

Role of MEK1 and DIAPH3 expression in colorectal adenoma-carcinoma sequence.

BACKGROUND: It is well established that most colorectal carcinomas arise from conventional adenomas through the adenoma-carcinoma sequence (ACS) model. mitogen-activated protein kinases (MAPKs) pathway has been reported as a crucial player in tumorigenesis. The MAPK signaling pathway is activated by different extracellular signals involving the "mitogen-activated/extracellular signal-regulated kinase 1 (MEK1)", and this induces the expression of genes involved in proliferation and cellular transformation. Diaphanous-related formin-3 (DIAPH3) acts as a potential metastasis regulator through inhibiting the cellular transition to amoeboid behavior in different cancer types. OBJECTIVE: The aim of the study was to investigate the pattern of immunohistochemical expression of MEK1 and DIAPH3 in colorectal adenoma (CRA) and corresponding colorectal carcinoma (CRC) specimens. METHODS: The immunohistochemical expression of DIAPH3 and MEK1 was examined in 43 cases of CRC and their associated adenomas using tissue microarray technique. RESULTS: MEK1 was overexpressed in 23 CRC cases (53.5%) and in 20 CRA cases (46.5%). DIAPH3 was overexpressed in 11 CRA cases (about 29%) which were significantly lower than CRC (22 cases; 58%) (P = 0.011). Both MEK1 and DIAPH3 overexpression were significantly correlated in CRC (P = 0.009) and CRA cases (P = 0.002). Tumors with MEK1 overexpression had a significantly higher tumor grade (P = 0.050) and perineural invasion (P = 0.017). CONCLUSIONS: Both MEK1 and DIAPH3 are overexpressed across colorectal ACS with strong correlation between them. This co- expression suggests a possible synergistic effect of MEK1 and DIAPH-3 in colorectal ACS. Further large-scale studies are required to investigate the potential functional aspects of MEK1 and DIAPH3 in ACS and their involvement in tumor initiation and the metastatic process.

Protein expression of the amino acid transporter SLC7A5 in tumor tissue is prognostic in early-stage colorectal cancer.

Proteins overexpressed in early-stage cancers may serve as early diagnosis and prognosis markers as well as targets for cancer therapies. In this study, we examined the expression of an essential amino acid carrier SLC7A5 (LAT1, CD98, or 4F2 light chain) in cancer tissue from two well-annotated cohorts of 575 cases of early-stage and 106 cases of late-stage colorectal cancer patients. Immunohistochemistry showed SLC7A5 overexpression in 72.0% of early-stage and 56.6% of late-stage cases. SLC7A5 expression was not influenced by patient gender, age, location, or mismatch repair status, although it appeared to be slightly less prevalent in tumors of mucinous differentiation or with lymphovascular invasion. Statistical analyses revealed a positive correlation between SLC7A5 overexpression and both overall survival and disease-free survival in early-stage but not late-stage cancers. Co-expression analyses of the TCGA and CPTAC colorectal cancer cohorts identified a network of gene transcripts positively related to SLC7A5, with its heterodimer partner SLC3A2 having the highest co-expression score. Network analysis uncovered the SLC7A network to be significantly associated with ncRNA such as tRNA processing and the mitotic cell cycle. Since SLC7A5 is also a marker of activated lymphocytes such as NK, T, and B lymphocytes, SLC7A5 overexpression in early colorectal cancers might trigger a strong anti-tumor immune response which could results in better clinical outcome. Overall, our study provides clear evidence of differential SLC7A5 expression and its prognostic value for early-stage colorectal cancer, although the understanding of its functions in colorectal tumorigenesis and cancer immunity is currently rather limited and awaits further characterization.

Investigation of ENO2 as a promising novel marker for the progression of colorectal cancer with microsatellite instability-high.

BACKGROUND: Microsatellite instability-high (MSI-H) has emerged as a significant biological characteristic of colorectal cancer (CRC). Studies reported that MSI-H CRC generally had a better prognosis than microsatellite stable (MSS)/microsatellite instability-low (MSI-L) CRC, but some MSI-H CRC patients exhibited distinctive molecular characteristics and experienced a less favorable prognosis. In this study, our objective was to explore the metabolic transcript-related subtypes of MSI-H CRC and identify a biomarker for predicting survival outcomes. METHODS: Single-cell RNA sequencing (scRNA-seq) data of MSI-H CRC patients were obtained from the Gene Expression Omnibus (GEO) database. By utilizing the copy number variation (CNV) score, a malignant cell subpopulation was identified at the single-cell level. The metabolic landscape of various cell types was examined using metabolic pathway gene sets. Subsequently, functional experiments were conducted to investigate the biological significance of the hub gene in MSI-H CRC. Finally, the predictive potential of the hub gene was assessed using a nomogram. RESULTS: This study revealed a malignant tumor cell subpopulation from the single-cell RNA sequencing (scRNA-seq) data. MSI-H CRC was clustered into two subtypes based on the expression profiles of metabolism-related genes, and ENO2 was identified as a hub gene. Functional experiments with ENO2 knockdown and overexpression demonstrated its role in promoting CRC cell migration, invasion, glycolysis, and epithelial-mesenchymal transition (EMT) in vitro. High expression of ENO2 in MSI-H CRC patients was associated with worse clinical outcomes, including increased tumor invasion depth (p = 0.007) and greater likelihood of perineural invasion (p = 0.015). Furthermore, the nomogram and calibration curves based on ENO2 showed potential prognosis predictive performance. CONCLUSION: Our findings suggest that ENO2 serves as a novel prognostic biomarker and is associated with the progression of MSI-H CRC.

Cetuximab plus FOLFOXIRI versus cetuximab plus FOLFOX as conversion regimen in RAS/BRAF wild-type patients with initially unresectable colorectal liver metastases (TRICE trial): A randomized controlled trial.

BACKGROUND: It remains unclear whether intensification of the chemotherapy backbone in tandem with an anti-EGFR can confer superior clinical outcomes in a cohort of RAS/BRAF wild-type colorectal cancer (CRC) patients with initially unresectable colorectal liver metastases (CRLM). To that end, we sought to comparatively evaluate the efficacy and safety of cetuximab plus FOLFOXIRI (triplet arm) versus cetuximab plus FOLFOX (doublet arm) as a conversion regimen (i.e., unresectable to resectable) in CRC patients with unresectable CRLM. METHODS AND FINDINGS: This open-label, randomized clinical trial was conducted from April 2018 to December 2022 in 7 medical centers across China, enrolling 146 RAS/BRAF wild-type CRC patients with initially unresectable CRLM. A stratified blocked randomization method was utilized to assign patients (1:1) to either the cetuximab plus FOLFOXIRI (n = 72) or cetuximab plus FOLFOX (n = 74) treatment arms. Stratification factors were tumor location (left versus right) and resectability (technically unresectable versus ≥5 metastases). The primary outcome was the objective response rate (ORR). Secondary outcomes included the median depth of tumor response (DpR), early tumor shrinkage (ETS), R0 resection rate, progression-free survival (PFS), overall survival (not mature at the time of analysis), and safety profile. Radiological tumor evaluations were conducted by radiologists blinded to the group allocation. Primary efficacy analyses were conducted based on the intention-to-treat population, while safety analyses were performed on patients who received at least 1 line of chemotherapy. A total of 14 patients (9.6%) were lost to follow-up (9 in the doublet arm and 5 in the triplet arm). The ORR was comparable following adjustment for stratification factors, with 84.7% versus 79.7% in the triplet and doublet arms, respectively (odds ratio [OR] 0.70; 95% confidence intervals [CI] [0.30, 1.67], Chi-square p = 0.42). Moreover, the ETS rate showed no significant difference between the triplet and doublet arms (80.6% (58/72) versus 77.0% (57/74), OR 0.82, 95% CI [0.37, 1.83], Chi-square p = 0.63). Although median DpR was higher in the triplet therapy group (59.6%, interquartile range [IQR], [50.0, 69.7] versus 55.0%, IQR [42.8, 63.8], Mann-Whitney p = 0.039), the R0/R1 resection rate with or without radiofrequency ablation/stereotactic body radiation therapy was comparable with 54.2% (39/72) of patients in the triplet arm versus 52.7% (39/74) in the doublet arm. At a median follow-up of 26.2 months (IQR [12.8, 40.5]), the median PFS was 11.8 months in the triplet arm versus 13.4 months in the doublet arm (hazard ratio [HR] 0.74, 95% CI [0.50, 1.11], Log-rank p = 0.14). Grade ≥ 3 events were reported in 47.2% (35/74) of patients in the doublet arm and 55.9% (38/68) of patients in the triplet arm. The triplet arm was associated with a higher incidence of grade ≥ 3 neutropenia (44.1% versus 27.0%, p = 0.03) and diarrhea (5.9% versus 0%, p = 0.03). The primary limitations of the study encompass the inherent bias in subjective surgical decisions regarding resection feasibility, as well as the lack of a centralized assessment for ORR and resection. CONCLUSIONS: The combination of cetuximab with FOLFOXIRI did not significantly improve ORR compared to cetuximab plus FOLFOX. Despite achieving an enhanced DpR, this improvement did not translate into improved R0 resection rates or PFS. Moreover, the triplet arm was associated with an increase in treatment-related toxicity. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03493048.

Integrating artificial intelligence techniques for advancements in colorectal cancer management: navigating past and predicting future direction.

Artificial Intelligence (AI) in the last few years has emerged as a valuable tool in managing colorectal cancer, revolutionizing its management at different stages. In early detection and diagnosis, AI leverages its prowess in imaging analysis, scrutinizing CT scans, MRI, and colonoscopy views to identify polyps and tumors. This ability enables timely and accurate diagnoses, initiating treatment at earlier stages. AI has helped in personalized treatment planning because of its ability to integrate diverse patient data, including tumor characteristics, medical history, and genetic information. Integrating AI into clinical decision support systems guarantees evidence-based treatment strategy suggestions in multidisciplinary clinical settings, thus improving patient outcomes. This narrative review explores the multifaceted role of AI, spanning early detection of colorectal cancer, personalized treatment planning, polyp detection, lymph node evaluation, cancer staging, robotic colorectal surgery, and training of colorectal surgeons.

IL-2RG as a possible immunotherapeutic target in CRC predicting poor prognosis and regulated by miR-7-5p and miR-26b-5p.

Despite advances in treatment strategies, colorectal cancer (CRC) continues to cause significant morbidity and mortality, with mounting evidence a close link between immune system dysfunctions issued. Interleukin-2 receptor gamma (IL-2RG) plays a pivotal role as a common subunit receptor in the IL-2 family cytokines and activates the JAK-STAT pathway. This study delves into the role of Interleukin-2 receptor gamma (IL-2RG) within the tumor microenvironment and investigates potential microRNAs (miRNAs) that directly inhibit IL-2RG, aiming to discern their impact on CRC clinical outcomes. Bioinformatics analysis revealed a significant upregulation of IL-2RG mRNA in TCGA-COAD samples and showed strong correlations with the infiltration of various lymphocytes. Single-cell analysis corroborated these findings, highlighting IL-2RG expression in critical immune cell subsets. To explore miRNA involvement in IL-2RG dysregulation, mRNA was isolated from the tumor tissues and lymphocytes of 258 CRC patients and 30 healthy controls, and IL-2RG was cloned into the pcDNA3.1/CT-GFP-TOPO vector. Human embryonic kidney cell lines (HEK-293T) were transfected with this construct. Our research involved a comprehensive analysis of miRPathDB, miRWalk, and Targetscan databases to identify the miRNAs associated with the 3' UTR of human IL-2RG. The human microRNA (miRNA) molecules, hsa-miR-7-5p and hsa-miR-26b-5p, have been identified as potent suppressors of IL-2RG expression in CRC patients. Specifically, the downregulation of hsa-miR-7-5p and hsa-miR-26b-5p has been shown to result in the upregulation of IL-2RG mRNA expression in these patients. Prognostic evaluation of IL-2RG, hsa-miR-7-5p, and hsa-miR-26b-5p, using TCGA-COAD data and patient samples, established that higher IL-2RG expression and lower expression of both miRNAs were associated with poorer outcomes. Additionally, this study identified several long non-coding RNAs (LncRNAs), such as ZFAS1, SOX21-AS1, SNHG11, SNHG16, SNHG1, DLX6-AS1, GAS5, SNHG6, and MALAT1, which may act as competing endogenous RNA molecules for IL2RG by sequestering shared hsa-miR-7-5p and hsa-miR-26b-5p. In summary, this investigation underscores the potential utility of IL-2RG, hsa-miR-7-5p, and hsa-miR-26b-5p as serum and tissue biomarkers for predicting CRC patient prognosis while also offering promise as targets for immunotherapy in CRC management.

Cetuximab inhibits colorectal cancer development through inactivating the Wnt/ß-catenin pathway and modulating PLCB3 expression.

Colorectal cancer (CRC) often necessitates cetuximab (an EGFR-targeting monoclonal antibody) for treatment. Despite its clinical utility, the specific operative mechanism of cetuximab remains elusive. This research investigated the influence of PLCB3, a potential CRC oncogene, on cetuximab treatment. We extracted differentially expressed genes from the GSE140973, the overlapping genes combined with 151 Wnt/ß-Catenin signaling pathway-related genes were identified. Then, we conducted bioinformatics analysis to pinpoint the hub gene. Subsequently, we investigated the clinical expression characteristics of this hub gene, through cell experimental, scrutinized the impact of cetuximab and PLCB3 on CRC cellular progression. The study identified 26 overlapping genes. High expression of PLCB3, correlated with poorer prognosis. PLCB3 emerged as a significant oncogene associated with patient prognosis. In vitro tests revealed that cetuximab exerted a cytotoxic effect on CRC cells, with PLCB3 knockdown inhibiting CRC cell progression. Furthermore, cetuximab treatment led to a reduction in both ß-catenin and PLCB3 expression, while simultaneously augmenting E-cadherin expression. These findings revealed PLCB3 promoted cetuximab inhibition on Wnt/ß-catenin signaling. Finally, simultaneous application of cetuximab with a Wnt activator (IM12) and PLCB3 demonstrated inhibited CRC proliferation, migration, and invasion. The study emphasized the pivotal role of PLCB3 in CRC and its potential to enhance the efficacy of cetuximab treatment. Furthermore, cetuximab suppressed Wnt/ß-catenin pathway to modulate PLCB3 expression, thus inhibiting colorectal cancer progression. This study offered fresh perspectives on cetuximab mechanism in CRC.

Integrating spatial and single-cell transcriptomics reveals tumor heterogeneity and intercellular networks in colorectal cancer.

Single cell RNA sequencing (scRNA-seq), a powerful tool for studying the tumor microenvironment (TME), does not preserve/provide spatial information on tissue morphology and cellular interactions. To understand the crosstalk between diverse cellular components in proximity in the TME, we performed scRNA-seq coupled with spatial transcriptomic (ST) assay to profile 41,700 cells from three colorectal cancer (CRC) tumor-normal-blood pairs. Standalone scRNA-seq analyses revealed eight major cell populations, including B cells, T cells, Monocytes, NK cells, Epithelial cells, Fibroblasts, Mast cells, Endothelial cells. After the identification of malignant cells from epithelial cells, we observed seven subtypes of malignant cells that reflect heterogeneous status in tumor, including tumor_CAV1, tumor_ATF3_JUN | FOS, tumor_ZEB2, tumor_VIM, tumor_WSB1, tumor_LXN, and tumor_PGM1. By transferring the cellular annotations obtained by scRNA-seq to ST spots, we annotated four regions in a cryosection from CRC patients, including tumor, stroma, immune infiltration, and colon epithelium regions. Furthermore, we observed intensive intercellular interactions between stroma and tumor regions which were extremely proximal in the cryosection. In particular, one pair of ligands and receptors (C5AR1 and RPS19) was inferred to play key roles in the crosstalk of stroma and tumor regions. For the tumor region, a typical feature of TMSB4X-high expression was identified, which could be a potential marker of CRC. The stroma region was found to be characterized by VIM-high expression, suggesting it fostered a stromal niche in the TME. Collectively, single cell and spatial analysis in our study reveal the tumor heterogeneity and molecular interactions in CRC TME, which provides insights into the mechanisms underlying CRC progression and may contribute to the development of anticancer therapies targeting on non-tumor components, such as the extracellular matrix (ECM) in CRC. The typical genes we identified may facilitate to new molecular subtypes of CRC.

Identification of DNA methylation characteristics associated with metastasis and prognosis in colorectal cancer.

Colorectal cancer (CRC) is prone to metastasis and recurrence after surgery, which is one of the main causes for its poor treatment and prognosis. Therefore, it is essential to identify biomarkers associated with metastasis and recurrence in CRC. DNA methylation has a regulatory role in cancer metastasis, tumor immune microenvironment (TME), and prognosis and may be one of the most valuable biomarkers for predicting CRC metastasis and prognosis. We constructed a diagnostic model and nomogram that can effectively predict CRC metastasis based on the differential methylation CpG sites (DMCs) between metastatic and non-metastatic CRC patients. Then, we identified 17 DMCs associated with progression free survival (PFS) of CRC and constructed a prognostic model. The prognosis model based on 17 DMCs can predict the PFS of CRC with medium to high accuracy. The results of immunohistochemical analysis indicated that the protein expression levels of the genes involved in prognostic DMCs were different between normal and colorectal cancer tissues. According to the results of immune-related analysis, we found that the low-risk patients had better immunotherapy response. In addition, high risk scores were negatively correlated with high tumor mutation burden (TMB) levels, and patients with low TMB levels in the high-risk group had the worst PFS. Our work shows the clinical value of DNA methylation in predicting CRC metastasis and PFS, as well as their correlation with TME, immunotherapy, and TMB, which helps understand the changes of DNA methylation in CRC metastasis and improving the treatment and prognosis of CRC.

TGFß-Responsive Stromal Activation Occurs Early in Serrated Colorectal Carcinogenesis.

Activated TGFß signaling in the tumor microenvironment, which occurs independently of epithelial cancer cells, has emerged as a key driver of tumor progression in late-stage colorectal cancer (CRC). This study aimed to elucidate the contribution of TGFß-activated stroma to serrated carcinogenesis, representing approximately 25% of CRCs and often characterized by oncogenic BRAF mutations. We used a transcriptional signature developed based on TGFß-responsive, stroma-specific genes to infer TGFß-dependent stromal activation and conducted in silico analyses in 3 single-cell RNA-seq datasets from a total of 39 CRC samples and 12 bulk transcriptomic datasets consisting of 2014 CRC and 416 precursor samples, of which 33 were serrated lesions. Single-cell analyses validated that the signature was expressed specifically by stromal cells, effectively excluding transcriptional signals derived from epithelial cells. We found that the signature was upregulated during malignant transformation and cancer progression, and it was particularly enriched in CRCs with mutant BRAF compared to wild-type counterparts. Furthermore, across four independent precursor datasets, serrated lesions exhibited significantly higher levels of TGFß-responsive stromal activation compared to conventional adenomas. This large-scale analysis suggests that TGFß-dependent stromal activation occurs early in serrated carcinogenesis. Our study provides novel insights into the molecular mechanisms underlying CRC development via the serrated pathway.

Oat Beta-Glucan as a Metabolic Regulator in Early Stage of Colorectal Cancer-A Model Study on Azoxymethane-Treated Rats.

Factors that reduce the risk of developing colorectal cancer include biologically active substances. In our previous research, we demonstrated the anti-inflammatory, immunomodulatory, and antioxidant effects of oat beta-glucans in gastrointestinal disease models. The aim of this study was to investigate the effect of an 8-week consumption of a diet supplemented with low-molar-mass oat beta-glucan in two doses on the antioxidant potential, inflammatory parameters, and colonic metabolomic profile in azoxymethane(AOM)-induced early-stage colorectal cancer in the large intestine wall of rats. The results showed a statistically significant effect of AOM leading to the development of neoplastic changes in the colon. Consumption of beta-glucans induced changes in colonic antioxidant potential parameters, including an increase in total antioxidant status, a decrease in the superoxide dismutase (SOD) activity, and a reduction in thiobarbituric acid reactive substance (TBARS) concentration. In addition, beta-glucans decreased the levels of pro-inflammatory interleukins (IL-1α, IL-1ß, IL-12) and C-reactive protein (CRP) while increasing the concentration of IL-10. Metabolomic studies confirmed the efficacy of oat beta-glucans in the AOM-induced early-stage colon cancer model by increasing the levels of metabolites involved in metabolic pathways, such as amino acids, purine, biotin, and folate. In conclusion, these results suggest a wide range of mechanisms involved in altering colonic metabolism during the early stage of carcinogenesis and a strong influence of low-molar-mass oat beta-glucan, administered as dietary supplement, in modulating these mechanisms.

An inflammation-related subtype classification for analyzing tumor microenvironment and clinical prognosis in colorectal cancer.

Background: The inflammatory response plays an essential role in the tumor microenvironment (TME) of colorectal cancer (CRC) by modulating tumor growth, progression, and response to therapy through the recruitment of immune cells, production of cytokines, and activation of signaling pathways. However, the molecular subtypes and risk score prognostic model based on inflammatory response remain to be further explored. Methods: Inflammation-related genes were collected from the molecular signature database and molecular subtypes were identified using nonnegative matrix factorization based on the TCGA cohort. We compared the clinicopathological features, immune infiltration, somatic mutation profile, survival prognosis, and drug sensitivity between the subtypes. The risk score model was developed using LASSO and multivariate Cox regression in the TCGA cohort. The above results were independently validated in the GEO cohort. Moreover, we explored the biological functions of the hub gene, receptor interacting protein kinase 2 (RIPK2), leveraging proteomics data, in vivo, and in vitro experiments. Results: We identified two inflammation-related subtypes (inflammation-low and inflammation-high) and have excellent internal consistency and stability. Inflammation-high subtype showed higher immune cell infiltration and increased sensitivity to common chemotherapeutic drugs, while inflammation-low subtype may be more suitable for immunotherapy. Besides, the two subtypes differ significantly in pathway enrichment and biological functions. In addition, the 11-gene signature prognostic model constructed from inflammation-related genes showed strong prognostic assessment power and could serve as a novel prognostic marker to predict the survival of CRC patients. Finally, RIPK2 plays a crucial role in promoting malignant proliferation of CRC cell validated by experiment. Conclusions: This study provides new insights into the heterogeneity of CRC and provides novel opportunities for treatment development and clinical decision making.