IL-17A-mediated mitochondrial dysfunction induces pyroptosis in colorectal cancer cells and promotes CD8 + T-cell tumour infiltration.

BACKGROUND: Interleukin-17A (IL-17A), a proinflammatory cytokine primarily secreted by Th17 cells, γδT cells and natural killer T (NKT) cells, performs essential roles in the microenvironment of certain inflammation-related tumours by regulating cancer growth and tumour elimination proved in previous literature. In this study, the mechanism of IL-17A that induces mitochondrial dysfunction promoted pyroptosis has been explored in colorectal cancer cells. METHOD: The records of 78 patients diagnosed with CRC were reviewed via the public database to evaluate clinicopathological parameters and prognosis associations of IL-17A expression. The colorectal cancer cells were treated with IL-17A, and the morphological characteristics of those cells were indicated by scanning electron microscope and transmission electron microscope. After IL-17A treatment, mitochondrial dysfunction was tested by mitochondrial membrane potential (MMP) and reactive oxygen species (ROS). The expression of pyroptosis associated proteins including cleaved caspase-4, cleaved gasdermin-D (GSDMD), IL-1ß, receptor activator of nuclear NOD-like receptor family pyrin domain containing 3 (NLRP3), apoptosis-associated speck like protein containing a card (ASC), and factor-kappa B was measured through western blotting. RESULTS: Positive IL-17A protein expression was observed in CRC compared to the non-tumour tissue. IL-17A expression indicates a better differentiation, earlier stage, and better overall survival in CRC. IL-17A treatment could induce mitochondrial dysfunction and stimulate intracellular reactive oxygen species (ROS) production. Furthermore, IL-17A could promote pyroptosis of colorectal cancer cells and significantly increase the secretion of inflammatory factors. Nevertheless, the pyroptosis induced by IL-17A could be inhibited through the pre-treatment with Mito-TEMPO (a mitochondria-targeted superoxide dismutase mimetic with superoxide and alkyl radical scavenging properties) or Z-LEVD-FMK (caspase-4 inhibitor, fluoromethylketone). Additionally, after being treated with IL-17A, an increasing number of CD8 + T cells showed in mouse-derived allograft colon cancer models. CONCLUSION: IL-17A, as a cytokine mainly secreted by γδT cells in the colorectal tumour immune microenvironment, can regulate the tumour microenvironment in multiple ways. IL-17A could induce mitochondrial dysfunction and pyroptosis through the ROS/NLRP3/caspase-4/GSDMD pathway, and promote intracellular ROS accumulation. In addition, IL-17A can promote the secretion of inflammatory factors such as IL-1ß、IL-18 and immune antigens, and recruit CD8 + T cells to infiltrate tumours.

CCL7 and TGF-ß secreted by MSCs play opposite roles in regulating CRC metastasis in a KLF5/CXCL5-dependent manner.

CXCL5 is overexpressed in colorectal cancer (CRC) and promotes distant metastasis and angiogenesis of tumors; however, the underlying mechanism that mediates CXCL5 overexpression in CRC remains unclear. Here, we successfully extracted and identified primary mesenchymal stromal cells (MSCs) and verified the promoting effects of tumor-associated MSCs on CRC proliferation and metastasis in vivo and in vitro. We found that MSCs not only promoted the expression of CXCL5 by secreting CCL7 but also secreted TGF-ß to inhibit this process. After secretion, CCL7/CCR1 activated downstream CBP/P300 to acetylate KLF5 to promote CXCL5 transcription, while TGF-ß reversed the effect of KLF5 on transcription activation by regulating SMAD4. Taken together, our results indicate that MSCs in the tumor microenvironment promoted the progression and metastasis of CRC and regulated the expression of CXCL5 in CRC cells by secreting CCL7 and TGF-ß. KLF5 is the key site of these processes and plays a dual role in CXCL5 regulation. MSCs and their secreted factors may serve as potential therapeutic targets in the tumor environment.

High ligation versus low ligation of the inferior mesenteric artery in laparoscopic rectal cancer surgery: a retrospective study on surgical and long-term outcome.

BACKGROUND: In laparoscopic low anterior resection for rectal cancer surgery, there has been controversy to whether the inferior mesenteric artery (IMA) should be ligated at the origin of its aorta (high ligation (HL)) or below the branches of the left colonic artery (LCA) (low ligation (LL)). This study was intended to clarify oncological outcome and long-term prognosis of retrospective analysis. METHODS: Analyzed the cases who underwent laparoscopic low anterior resection (LAR) in Shanghai Ruijin Hospital from January 2015 to December 2016, 357patients scheduled into 2 groups according to the level of IMA ligation: HL (n = 247) versus LL (n = 110). RESULTS: The primary endpoint is long-term outcomes, and the secondary endpoint is the incidence rate of major postoperative complications. There were no significant differences in 5-year overall survival (P = 0.92) and 5-year disease-free survival (P = 0.41). There were no differences between the clinical baseline levels in each group. The incidence of low anterior resection syndrome (LARS) in the two groups was statistically significant (P = 0.037). No significant differences were observed in operative time (P = 0.092) and intraoperative blood loss (P = 0.118). In the HL group, 6 cases (2.4%) had additional colonic excision due to poor anastomotic blood supply; none of the colonic anastomosis in the low ligation group had ischemic manifestations, and length from the proximal margin (P = 0.076), length from the distal margin (P = 0.184), the total number of lymph nodes excised (P = 0.065), and anastomotic leakage incidence (P = 0.33). CONCLUSION: Low ligation of the IMA which reserved LCA with vascular root lymph node dissection in laparoscopic low anterior resection for rectal cancer surgery may help protect the blood supply of the anastomosis, and will not increase postoperative complications while enhance recovery, without compromising radical excision and long-term prognosis.

Intracorporeal versus extracorporeal anastomosis in laparoscopic right colectomy: a retrospective study.

BACKGROUND: The surgical procedure for laparoscopic right colectomy (LRC) is not standardized. Some published studies show the superiority of ileocolic anastomosis (IIA), but the evidence so far is insufficient. This study aimed to investigate the potential advantages in postoperative recovery and safety of IIA in LRC. METHODS: A total of 114 patients who underwent LRC with IIA (n = 58) or extracorporeal ileocolic anastomosis (EIA, n = 56) between January 2019 and September 2021 were enrolled. We collected certain factors as clinical features, intraoperative characteristics, oncological outcomes, postoperative recovery, and short-term outcomes. Our primary outcome was time to gastrointestinal (GI) function recovery. Secondary outcomes were postoperative complications within 30 days, postoperative pain, and length of hospital stay. RESULTS: Faster GI recovery and less postoperative pain were observed in patients with IIA compared to EIA [time to first flatus: (2.4 ± 0.7) vs (2.8 ± 1.0) days, p < 0.01; time to liquid intake: (3.5 ± 0.7) vs (4.0 ± 1.1) days, p = 0.01; postoperative visual analogue scale score: (3.9 ± 1.0) vs (4.3 ± 0.6), p = 0.02]. No significant differences were detected in oncological outcomes or postoperative complications. IIA, rather than EIA, tended to be performed in patients with higher body mass index [(23.93 ± 3.52) vs (22.36 ± 2.87) kg/m2, p = 0.01]. CONCLUSIONS: IIA is associated with faster GI function recovery and less postoperative pain and may be more favorable for obese patients.

Combination of FOXD1 and Plk2: A novel biomarker for predicting unfavourable prognosis of colorectal cancer.

Colorectal cancer (CRC) is a worldwide disease with worse survival. Our objective is to identify previously unrecognized prognostic factors to better evaluate disease progression. Seven GEO datasets were collected and analysed using R software, followed by KEGG enrichment analysis and TFs network construction. LASSO-COX analysis was performed to select the most useful prognostic features. COX model was used to analyse prognostic factors associated with OS. The survival curve was constructed using Kaplan-Meier analysis. A Nomogram model was also constructed to predict prognosis. A total of 3559 differentially expressed genes (DEGs) and 66 differentially expressed transcription factors were identified. FOXD1 was identified as the most differentially expressed factor of TFs covering the most downstream DEGs and independent risk prognostic factor. Next, FOXD1 expression was detected using immunohistochemical staining in 131 CRC patients' tissue and the association between FOXD1 expression and clinicopathologic features was analysed. High expression of FOXD1 was correlated with TNM stage and pathological differentiation. Multivariate COX regression analyses confirmed that FOXD1 high-expression, TNM stage and tumour differentiation were independent prognostic risk factor of OS and DFS. Patients with high expression of FOXD1 were more likely to have poor overall survival and disease-free survival. The combination of FOXD1 and Plk2 which we have previously reported allowed us to predict the survival of post-surgical CRC patients more accurately, adding to the former prognostic model based on the TNM Stage. The results showed that patients with high expression of both FOXD1 and Plk2 have the worst survival. A combination of FOXD1 and Plk2 can better evaluate patients' survival.

Platelet infiltration predicts survival in postsurgical colorectal cancer patients.

Platelets promote tumor growth and metastasis in several tumor types. Recent research has found platelets can extravasate and infiltrate into the tumor stroma and interact with the tumor microenvironment. The prognostic role of platelet infiltration in colorectal cancer (CRC) remains controversial. A pan-cancer survival analysis was performed to find the potential prognostic value of platelet infiltration in patients with cancer. A survival analysis and a nomogram prognostic model were established to further confirm the results with data from our center. The correlations between patient outcomes and tumor-infiltrating platelets (TIPs) were identified by immunohistochemical staining for CD42b. The prognostic accuracy and discriminative ability of the nomogram were determined by the concordance index (C-index) and a calibration curve. The pan-cancer survival analysis showed platelet infiltration can lead to a poor prognosis in patients with several types of cancers, including CRC. Platelet infiltration was associated with overall survival (OS) and disease-free survival (DFS) in both primary and validation cohorts. The C-index values of the nomogram for predicting OS and DFS were 0.774 and 0.769, respectively, which were higher than that of the TNM staging system alone. Our study found platelet infiltration has a potential prognostic value regarding postsurgical survival in CRC patients. The proposed nomogram resulted in a more accurate prognostic prediction for postsurgical CRC patients.

High versus low ligation of the inferior mesenteric artery during laparoscopic rectal cancer surgery: A prospective study of surgical and oncological outcomes.

BACKGROUND AND OBJECTIVES: There is controversy regarding whether the inferior mesenteric artery (IMA) should be ligated at its origin from the aorta (high ligation, HL) or below the branch of the left colic artery (low ligation, LL) during surgery for rectal cancer. METHODS: This prospective study randomized 95 patients with histologically proven rectal cancer (clinical stages I-III based on the 8th American Joint Committee on Cancer guidelines) to undergo HL (n = 47) or LL with lymph node dissection at the root of the IMA (n = 48). RESULTS: Only two intraoperative adverse events were observed (two HL patients experienced anastomotic ischemia and underwent extended bowel excision and splenic flexure mobilization). The LL group had a significantly shorter time to first flatus (p < .0001). No significant differences were observed in operative time (p = .14), intraoperative blood loss (p = .21), distance from the upper margin (p = .77), distance from the lower margin (p = .35), harvested lymph nodes (p = .33), or anastomotic leakage (p = .44), 2-year overall survival (p = .97), or 2-year disease-free survival (p = .42). CONCLUSION: During laparoscopic low anterior resection, a combination of LL at the IMA and vascular root lymph node dissection may help protect the blood supply of the anastomosis, reduce postoperative complications, and enhance recovery, without compromising radical excision.

Effect of different reinforcement methods on anastomotic leakage prevention after laparoscopic double anastomosis.

BACKGROUND AND OBJECTIVES: To investigate the effects of different suture reinforcement methods for anastomotic leakage and other postoperative complications after the use of a laparoscopic double stapling technique (DST). METHODS: We collected the data of 124 patients who underwent laparoscopic radical resection of colorectal cancer from July 2017 to September 2018 at our institution. Patients were divided into three groups according to the suture reinforcement methods: intermittent, continuous suture reinforcement, and non-reinforcement (n = 41, 41, and 42, respectively). One-way analysis of variance, χ2 , Fisher's exact, and nonparametric tests were used for statistical analysis. RESULTS: Among the 124 patients, there were no statistically significant differences in operation times, intraoperative blood loss, postoperative hospital stays and recovery of bowel movement. Nine patients were diagnosed with anastomotic leakage (AL). The incidences of serious AL in the intermittent and continuous suture reinforcement groups were lower than that in the control group, with lower reoperation rate, shorter average lengths of stay and lower treatment costs of two experimental groups. CONCLUSION: Intermittent and continuous sutures after laparoscopic DST is effective, safe, and feasible on anastomotic leakage prevention. These procedures could be popularized in rectal surgery on patients with high risk of AL.

CCR6 promotes tumor angiogenesis via the AKT/NF-κB/VEGF pathway in colorectal cancer.

Chemokines and chemokine receptors play an important role in tumorigenesis. Angiogenesis is a vital part of the occurrence, development and metastasis of cancer. CCR6 is an important factor during tumor progression; however, its function in tumor angiogenesis is not fully understood. In our study, we found that CCR6 was significantly overexpressed in colorectal cancer (CRC) tissues and predicted a poor prognosis in CRC patients. We then verified the function of CCR6 on tumor angiogenesis in vivo and in vitro. We observed that silencing CCR6 could decrease angiogenesis by inhibiting the proliferation and migration of human umbilical vein endothelial cells (HUVECs), whereas overexpression of CCR6 can promote angiogenesis. Additionally, we investigated the molecular mechanisms and demonstrated that activation of the AKT/NF-κB pathway maybe involved in CCR6-mediated tumor angiogenesis, which was able to promote the secretion of vascular endothelial growth factor A (VEGF-A). In conclusion, CCR6 facilitates tumor angiogenesis via the AKT/NF-κB/VEGF pathway in colorectal cancer. CCR6 inhibition may be a novel option for anti-vascular treatment in CRC.

Tumor-derived CXCL5 promotes human colorectal cancer metastasis through activation of the ERK/Elk-1/Snail and AKT/GSK3ß/ß-catenin pathways.

BACKGROUND: Metastasis is a major cause of death in human colorectal cancer patients. However, the contribution of chemokines in the tumor microenvironment to tumor metastasis is not fully understood. METHODS: Herein, we examinined several chemokines in colorectal cancer patients using chemokine ELISA array. Immunohistochemistry was used to detect expression of CXCL5 in colorectal cancer patients tissues. Human HCT116 and SW480 cell lines stably transfected with CXCL5, shCXCL5 and shCXCR2 lentivirus plasmids were used in our in vitro study. Immunoblot, immunofluorescence and transwell assay were used to examine the molecular biology and morphological changes in these cells. In addition, we used nude mice to detect the influence of CXCL5 on tumor metastasis in vivo. RESULTS: We found that CXCL5 was overexpressed in tumor tissues and associated with advanced tumor stage as well as poor prognosis in colorectal cancer patients. We also demonstrated that CXCL5 was primarily expressed in the tumor cell cytoplasm and cell membranes, which may indicate that the CXCL5 was predominantly produced by cancer epithelial cells instead of fibroblasts in the tumor mesenchyme. Additionally, overexpression of CXCL5 enhanced the migration and invasion of colorectal cancer cells by inducing the epithelial-mesenchymal transition (EMT) through activation of the ERK/Elk-1/Snail pathway and the AKT/GSK3ß/ß-catenin pathway in a CXCR2-dependent manner. The silencing of Snail and ß-catenin attenuated CXCL5/CXCR2-enhanced cell migration and invasion in vitro. The elevated expression of CXCL5 can also potentiate the metastasis of colorectal cancer cells to the liver in vivo in nude mice intrasplenic injection model. CONCLUSION: In conclusion, our findings support CXCL5 as a promoter of colorectal cancer metastasis and a predictor of poor clinical outcomes in colorectal cancer patients.

Cadherin-12 enhances proliferation in colorectal cancer cells and increases progression by promoting EMT.

Cadherin-12 (CDH12) is a subtype of N-cadherin family. In this study, we investigated the expression of CDH12 and the role of CDH12 in prognosis of colorectal cancer (CRC) patients. In addition, we observed the influence of CDH12 on proliferation and progression of CRC cell lines. By using immunohistochemical staining, we analyzed CRC samples and adjacent non-tumor tissues collected from 78 patients who underwent laparoscopic surgery in Shanghai Minimally Invasive Center, China. Statistical analyses were used to analyze relationship between CDH12 and tumor features. Kaplan-Meier method was used to analyze patients' survival. Proliferation ability of CRC cells was tested by CCK-8 assay, and transwell assays were performed to detect migration and invasion ability. Western blot assay was performed to investigate epithelial-mesenchymal transition (EMT) variants. We found that expression of CDH12 in tumor tissue was higher than in adjacent normal tissue. High expression of CDH12 was associated with tumor invasion depth and predicts poor prognosis of CRC patients. Ectopic/repressing expression of CDH12 increased/decreased the proliferation and migration ability of CRC cells. CDH12 is able to increase cancer cell migration and invasion via promoting EMT by targeting transcriptional factor Snail. These findings may conclude that CDH12 may act as a predictor in CRC patients' prognosis and an oncogene in CRC cell proliferation and migration. CDH12 may influence CRC cell progression through promoting EMT by targeting Snail. In addition, CDH12 is promoted by MCP1 through induction of MCPIP.

Antitumor efficacy of CC motif chemokine ligand 19 in colorectal cancer.

OBJECTIVES: To investigate the function of CC motif chemokine ligand 19 (CCL19) in colorectal cancer (CRC). METHODS: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot and immunohistochemistry were performed separately to detect the expression of CCL19 in colorectal carcinoma tissues. The expression of CCL19 and its receptor (CCR7) in CRC cell lines were screened by Western blot. SW620, SW1116 and LoVo cell lines were screened and processed with recombinant human CCL19 (rhCCL19) or si-CCL19 RNA. Cell proliferation assay and transwell assay were performed to evaluate the proliferation, migration and invasion of CRC cells, respectively. And the role of proangiogenesis was checked by endothelial tube formation assay. RESULTS: qRT-PCR, Western blot and immunohistochemistry revealed that both CCL19 mRNA and protein were obviously expressed in a lower degree in CRC tissues than normal tissues (P < 0.01). The CCL19 expression correlated with tumor size (P = 0.03) and invasion depth (P = 0.04) in a negative manner and CCL19-positive patients had longer lifespans (P < 0.05). SW620 and SW1116 cells were screened as CCL19/CCR7 high-expression cells, while LoVo was selected as CCL19/CCR7 low-expression cell among seven CRC cell lines by Western blot. The proliferation, migration, invasion and proangiogenesis of SW620 and SW1116 cells were distinctly suppressed after they were stimulated by rhCCL19 (P < 0.05), and the data presented dose-dependency. Oppositely, these abilities were significantly enhanced after CCL19 gene was silenced (P < 0.05). However, the effects of rhCCL19 and si-CCL19 RNA on LoVo were not significant (P > 0.05). CONCLUSION: Our research findings indicate that CCL19 may play a suppressive role in colorectal tumorigenesis.

Long-term outcome of laparoscopic-assisted right-hemicolectomy with D3 lymphadenectomy versus open surgery for colon carcinoma.

PURPOSE: To investigate the applicability, safety, short-term and long-term outcomes of laparoscopic surgery in the treatment of right-sided colon carcinomas with D3 lymphadenectomy. METHODS: Between June 2003 and September 2010, 324 patients with right-sided colon carcinoma underwent surgical treatment in the same hospital, 177 cases were treated by laparoscopic surgery (LRH group) and 147 cases by open surgery (ORH group). We performed a retrospective analysis of the differences between the two groups in terms of the clinical data. RESULTS: There were no significant differences between the two groups in the demographic data; however, the recovery time was significantly shorter in the LRH group, the number of overall lymph nodes harvested and principle lymph nodes harvested in the LRH group was significantly higher than in the ORH group, the incidence of postoperative complications was 12.99 % in the LRH group and 22.45 % in the ORH group (P < 0.05), and the recurrence rate in the LRH group was lower than that in the ORH group, although the difference was not significant (15.25 vs 19.73 %). The cumulative overall survival for all stages at 1, 3 and 5 years in the LRH group (97.18, 83.73 and 70.37 %) were not significantly different compared to those in the ORH group (94.56, 77.84 and 66.97 %). CONCLUSIONS: Laparoscopic-assisted right hemicolectomy with D3 lymphadenectomy for colon carcinomas is safe and effective, while it is also superior to open surgery regarding the short-term outcomes, and the long-term outcomes are similar to those of open surgery.

Cadherin-12 contributes to tumorigenicity in colorectal cancer by promoting migration, invasion, adhersion and angiogenesis.

BACKGROUND: Cadherin 12 (CDH12), which encodes a type II classical cadherin from the cadherin superfamily, may mediate calcium-dependent cell adhesion. It has been demonstrated that CDH12 could play an important role in the invasion and metastasis of salivary adenoid cystic carcinoma. We decided to investigate the relationship between CDH12 expression level and clinicopathologic variables in colorectal carcinoma (CRC) patients and to explore the functions of CDH12 in tumorigenesis in CRC. METHODS: The expression levels of CDH12 in colorectal carcinoma tissues were detected by immunohistochemistry. Real-time PCR and Western Blot were used to screen CDH12 high-expression cell lines. CCK-8 assay was used to detect the proliferation ability of CRC cells being transfected by shRNAs against CDH12. The wound assay and transwell assay were performed to test migration and invasion ability. The importance of CDH12 in cell-cell junctions was detected by cell adhesion assay and cell aggregation assay. Endothelial tube formation assay was used to test the influence of CDH12 on angiogenesis. RESULTS: Statistical analysis of clinical cases revealed that the positive rate of CDH12 was higher in the CRC tumor tissues compared with the adjacent non-tumor tissues. The expression levels of CDH12 in CRC patients are significantly correlated with invasion depth. Consistently, the ability of proliferation, migration and invasion were suppressed when CDH12 was decreased in CRC cells transfected with shRNAs. Cell adhesion assay and cell aggregation assay presented that tumor cells tend to disperse with the lack of CDH12. Endothelial tube formation assay showed that down-regulation of CDH12 could obviously inhibit the process of angiogenesis, implying that CDH12 may play an important role in tumor metastasis CONCLUSION: Our results showed that CDH12 promotes proliferation, migration, invasion, adhesion and angiogenesis, suggesting that CDH12 may be an oncogene in colorectal cancer. CDH12 is expected to become a new diagnostic and prognostic marker and a novel target of the treatment of colorectal cancer.

Long-term results of laparoscopy-assisted radical right hemicolectomy with D3 lymphadenectomy: clinical analysis with 177 cases.

PURPOSES: To study the feasibility, safety, and short-/long-term outcomes of laparoscopy-assisted right hemicolectomy with D3 lymphadenectomy for colon cancer. METHODS: The clinical data of 177 cases that underwent laparoscopy-assisted radical right hemicolectomy with D3 lymphadenectomy for colon cancer between Jun 2003 and Sep 2010 was collected; the safety of operation, status of recovery, complication, oncological outcomes, and results of short-/long-term follow-up were analyzed. RESULTS: No case died in this study; five cases (2.82 %) were converted to open surgery. Four cases (2.26 %) underwent hand-assisted laparoscopic right hemicolectomy. The average operation time was 133 ± 36 min, and the blood loss was 94 ± 34 ml. The average time for passage of flatus, liquid food eating, and hospitalization were 2.1 ± 0.7, 3.2 ± 0.5, and 10.4 ± 2.7 day, respectively. The total number of lymph nodes removed was 15.2 ± 10.1. Postoperative complications were observed in 23 of 177 patients (12.99 %). The median follow-up period was 54 months; port-site recurrence was observed in one patient; local recurrence was found in five cases (2.82 %); distant metastasis was found in 21 cases (11.86 %). The cumulative overall survival of all stages at 12, 36, 60, and 72 months was 97.18 %, 83.73 %, 70.37 %, and 68.99 %, respectively. The cancer-specific survival was 98.73 % (12 months), 87.81 % (36 months), and 80.17 % (60 months). CONCLUSIONS: Laparoscopy-assisted right hemicolectomy with D3 lymphadenectomy can be successfully performed for right colon cancer with the advantages of minimally invasive surgery. Moreover, the results implied appropriate short- and long-term outcomes.

Colorectal cancer tissue-originated spheroids reveal tumor intrinsic signaling pathways and mimic patient clinical chemotherapeutic response as a rapid and valid model.

Locally advanced colorectal cancer requires preoperative chemotherapy to reduce local recurrence and metastasis rates, but it remains difficult to predict the tumor will be sensitive to which treatments. The patient-derived organoids (PDOs) are considered an effective platform for predicting tumor drug responses in precision oncology. However, it has the limitation of being time-consuming in practical applications, especially in neoadjuvant treatment. Here we used cancer tissue-originated spheroids (CTOS) method to establish organoids from a heterogeneous population of colorectal cancer specimens, and evaluated the capacity of CTOS to predict clinical drug responses. By analyzing the relationship of the activities of drug-treated CTOS, drug targets and target-related pathways, tumor intrinsic effective-target-related pathways can be identified. These pathways were highly matched to the abnormal pathways indicated by whole-exome sequencing. Based on this, we used half effective concentration gradients to classify CTOS as sensitive or resistant to chemotherapy regimens within a week, for predicting neoadjuvant treatment outcomes for colorectal cancer patients. The drug sensitivity test results are highly matched to the clinical responses to treatment in individual patients. Thus, our data suggested that CTOS models can be effectively screened ex vivo to identify pathways sensitive to chemotherapies. These data also supported organoid research for personalized clinical medication guidance immediately after diagnosis in patients with advanced colorectal cancer.

Cytokine concentration in peripheral blood of patients with colorectal cancer.

Introduction: The role of tumour secretory cytokines and peripheral circulatory cytokines in tumour progression has received increasing attention; however, the role of tumour-related inflammatory cytokines in colorectal cancer (CRC) remains unclear. In this study, the concentrations of various cytokines in the peripheral blood of healthy controls and patients with CRC at different stages were compared. Methods: Peripheral blood samples from 4 healthy participants and 22 colorectal cancer patients were examined. Luminex beads were used to evaluate concentration levels of 40 inflammatory cytokines in peripheral blood samples. Results: In peripheral blood, compared with healthy controls and early stage (I + II) CRC patients, advanced CRC (III + IV) patients had increased concentrations of mononuclear/macrophage chemotactic-related proteins (CCL7, CCL8, CCL15, CCL2, and MIF), M2 polarization-related factors (IL-1ß, IL-4), neutrophil chemotactic and N2 polarization-related cytokines (CXCL2, CXCL5, CXCL6, IL-8), dendritic cells (DCs) chemotactic-related proteins (CCL19, CCL20, and CCL21), Natural killer (NK) cell related cytokines (CXCL9, CXCL10), Th2 cell-related cytokines (CCL1, CCL11, CCL26), CXCL12, IL-2, CCL25, and CCL27, and decreased IFN-γ and CX3CL1 concentrations. The differential upregulation of cytokines in peripheral blood was mainly concentrated in CRC patients with distant metastasis and was related to the size of the primary tumour; however, there was no significant correlation between cytokine levels in peripheral blood and the propensity and mechanism of lymph node metastasis. Discussion: Different types of immune cells may share the same chemokine receptors and can co-localise in response to the same chemokines and exert synergistic pro-tumour or anti-tumour functions in the tumour microenvironment. Chemokines and cytokines affect tumour metastasis and prognosis and may be potential targets for treatment.

FOXD1 promotes chemotherapy resistance by enhancing cell stemness in colorectal cancer through ß­catenin nuclear localization.

Forkhead box D1 (FOXD1) serves a critical role in colorectal cancer (CRC). FOXD1 expression is an independent prognostic factor in patients with CRC; however, the molecular mechanism and signaling pathway of FOXD1 that regulates cell stemness and chemoresistance has not been fully characterized. The aim of the present study was to further validate the effect of FOXD1 on the proliferation and migration of CRC cells, and to delve into the possible potential of FOXD1 in the clinical treatment of CRC. The effect of FOXD1 on cell proliferation was assessed using Cell Counting Kit 8 (CCK­8) and colony formation assays. The effect of FOXD1 on cell migration was assessed by wound­healing and Transwell assays. The effect of FOXD1 on cell stemness was assessed by spheroid formation in vitro and limiting dilution assays in vivo. The expression of stemness associated proteins, leucine rich repeat containing G protein­coupled receptor 5 (LGR5), OCT4, Sox2 and Nanog, and epithelial­mesenchymal transition associated proteins, E­cadherin, N­cadherin and vimentin, were detected by western blotting. Proteins interrelationships were assessed by a co­immunoprecipitation assay. Oxaliplatin resistance was assessed using CCK­8 and apoptosis assays in vitro, and using a tumor xenograft model in vivo. By constructing FOXD1 overexpression and knockdown stably transfected strains of colon cancer cells, it was revealed that the overexpression of FOXD1 increased CRC cell stemness and chemoresistance. By contrast, knockdown of FOXD1 produced the opposite effects. These phenomena were caused by the direct interaction between FOXD1 and ß­catenin, thus promoting its nuclear translocation and the activation of downstream target genes, such as LGR5 and Sox2. Notably, inhibition of this pathway with a specific ß­catenin inhibitor (XAV­939) could impair the effects induced by the overexpression of FOXD1. In summary, these results indicated that FOXD1 may promote cell stemness and the chemoresistance of CRC by binding directly to ß­catenin and enhancing ß­catenin nuclear localization; therefore, it may be considered a potential clinical target.

Leveraging a KRAS-based signature to predict the prognosis and drug sensitivity of colon cancer and identifying SPINK4 as a new biomarker.

KRAS is one of the leading mutations reported in colon cancer. However, there are few studies on the application of KRAS related signature in predicting prognosis and drug sensitivity of colon cancer patient. We identified KRAS related differentially expressed genes (DEGs) using The Cancer Genome Atlas (TCGA) database. A signature closely related to overall survival was recognized with Kaplan-Meier survival analysis and univariate cox regression analysis. Then we validated this signature with overall expression score (OE score) algorithm using both scRNA-seq and bulk RNA-seq data. Based on this signature, we performed LASSO cox regression to establish a prognostic model, and corresponding scores were calculated. Differences in genomic alteration, immune microenvironment, drug sensitivity between high- and low-KRD score groups were investigated. A KRAS related signature composed of 80 DEGs in colon cancer were recognized, among which 19 genes were selected to construct a prognostic model. This KRAS related signature was significantly correlated with worse prognosis. Furthermore, patients who scored lower in the prognostic model presented a higher likelihood of responding to chemotherapy, targeted therapy and immunotherapy. Furthermore, among the 19 selected genes in the model, SPINK4 was identified as an independent prognostic biomarker. Further validation in vitro indicated the knockdown of SPINK4 promoted the proliferation and migration of SW48 cells. In conclusion, a novel KRAS related signature was identified and validated based on clinical and genomic information from TCGA and GEO databases. The signature was proved to regulate genomic alteration, immune microenvironment and drug sensitivity in colon cancer, and thus might serve as a predictor for individual prognosis and treatment.

Negative regulation of STAT3 protein-mediated cellular respiration by SIRT1 protein.

In mammals, the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is regulated by the deacetylase SIRT1. However, whether the newly described nongenomic actions of STAT3 toward mitochondrial oxidative phosphorylation are dependent on SIRT1 is unclear. In this study, Sirt1 gene knock-out murine embryonic fibroblast (MEF) cells were used to delineate the role of SIRT1 in the regulation of STAT3 mitochondrial function. Here, we show that STAT3 mRNA and protein levels and the accumulation of serine-phosphorylated STAT3 in mitochondria were increased significantly in Sirt1-KO cells as compared with wild-type MEFs. Various mitochondrial bioenergetic parameters, such as the oxygen consumption rate in cell cultures, enzyme activities of the electron transport chain complexes in isolated mitochondria, and production of ATP and lactate, indicated that Sirt1-KO cells exhibited higher mitochondrial respiration as compared with wild-type MEFs. Two independent approaches, including ectopic expression of SIRT1 and siRNA-mediated knockdown of STAT3, led to reduction in intracellular ATP levels and increased lactate production in Sirt1-KO cells that were approaching those of wild-type controls. Comparison of profiles of phospho-antibody array data indicated that the deletion of SirT1 was accompanied by constitutive activation of the pro-inflammatory NF-κB pathway, which is key for STAT3 induction and increased cellular respiration in Sirt1-KO cells. Thus, SIRT1 appears to be a functional regulator of NF-κB-dependent STAT3 expression that induces mitochondrial biogenesis. These results have implications for understanding the interplay between STAT3 and SIRT1 in pro-inflammatory conditions.