Single-cell and spatial transcriptome analysis reveals the cellular heterogeneity of liver metastatic colorectal cancer.

In this study, we comprehensively charted the cellular landscape of colorectal cancer (CRC) and well-matched liver metastatic CRC using single-cell and spatial transcriptome RNA sequencing. We generated 41,892 CD45- nonimmune cells and 196,473 CD45+ immune cells from 27 samples of six CRC patients, and found that CD8_CXCL13 and CD4_CXCL13 subsets increased significantly in liver metastatic samples that exhibited high proliferation ability and tumor-activating characterization, contributing to better prognosis of patients. Distinct fibroblast profiles were observed in primary and liver metastatic tumors. F3+ fibroblasts enriched in primary tumors contributed to worse overall survival by expressing protumor factors. However, MCAM+ fibroblasts enriched in liver metastatic tumors might promote generation of CD8_CXCL13 cells through Notch signaling. In summary, we extensively analyzed the transcriptional differences of cell atlas between primary and liver metastatic tumors of CRC by single-cell and spatial transcriptome RNA sequencing, providing different dimensions of the development of liver metastasis in CRC.

Precision medicine-guided co-delivery of ASPN siRNA and oxaliplatin by nanoparticles to overcome chemoresistance of colorectal cancer.

The development of chemoresistance is a major hurdle for the treatment of colorectal cancer (CRC), which contributes remarkably to the poor clinical prognosis. Nanodrug delivery systems show great potential in overcoming chemoresistance, but limited by the lack of identification of chemoresistance targets from cancer patients. In the present study, we enrolled chemotherapy-resistant or sensitive CRC patients and used the next-generation RNA sequencing to reveal that Asporin (ASPN) is highly expressed in tumor tissues from oxaliplatin (OXA)-resistant patients and closely correlated with a poor prognosis of CRC. Downregulation of ASPN reversed OXA resistance and promoted cell apoptosis both in vitro and in vivo. To overcome ASPN-mediated OXA resistance, we constructed a nanoparticle-based co-delivery system (denoted as PPO-siASPN) for simultaneous delivery of OXA and siRNA targeting ASPN (siASPN). PPO-siASPN not only facilitated the intracellular delivery of OXA through the enhanced cellular uptake, but effectively suppressed ASPN expression for synergistic antitumor activity in vitro and in vivo. In the more clinically relevant patient-derived xenograft (PDX) mouse model, systemic administration of PPO-siASPN achieved a remarkable therapeutic effect. This study uncovered the critical role of ASPN in causing OXA resistance in CRC patients and suggests a promising nanoformulation that may be more effective than current standard-of-care medications.

Notch signaling mutations increase intra-tumor chemokine expression and predict response to immunotherapy in colorectal cancer.

BACKGROUND: The Notch signaling mutation is associated with enhanced anti-tumor immune response in colorectal cancer (CRC). In this study, we aim to investigate the underlying mechanism and the predictive potential of Notch signaling mutation for responding to immunotherapy in CRC. METHODS: We analyzed the immune response associated genes in CRC with Notch signaling mutation concomitant with or without microsatellite instability (MSI) using TCGA dataset and investigated the mutation profiles of the Notch signaling pathway using cBioPortal. The Notch signaling scores and immune cell infiltration scores in different groups were calculated. We applied the Kaplan-Meier method for survival analysis in CRC patients who underwent immunotherapy, and the log-rank test to determine the statistically significant differences in survival. Notch1-knock-down cell line was constructed to detect the pathway and gene variations. RESULTS: We found that Notch signaling pathway mutation was associated with activated immune response, especially in those with MSI. Such association is useful for predicting a prolonged overall survival of CRC patients who underwent immune checkpoint inhibitor treatment. The mutation resulted in the functional loss of Notch signaling and may modulate the tumor immune microenvironment by increasing the expression of chemokines that are important for recruiting immune cells. CONCLUSIONS: The Notch signaling mutation can modulate the chemotaxis of immune cells by upregulating the chemokine levels of the tumor immune microenvironment, and CRC patients with Notch signaling pathway mutation have better overall survival after immune checkpoint inhibitor treatment.

Overexpression of Ubiquitin-Specific Protease15 (USP15) Promotes Tumor Growth and Inhibits Apoptosis and Correlated With Poor Disease-Free Survival in Hepatocellular Carcinoma.

USP15 is a member of ubiquitin-specific proteases (USPs, the largest subfamily of deubiquitinases) and functions as a stabilize factor of target proteins in reversible ubiquitiantion progression. Dysregulated expression of USP15 has been observed in various cancers. However the expression profile and regulatory mechanism of USP15 in hepatocellular carcinoma (HCC) remains largely elusive. To exam the USP15 expression changes in the progression of HCC, we performed IHC analysis to test USP15 expression in a series of cancer-prone diseases including 2 normal liver tissues, 6 liver cirrhosis, 16 primary liver lesions and 15 metastases of hepatocellular carcinoma. The expression of USP15 was upregulated in various liver diseases in compared with normal tissue significantly (p < 0.05). Although no significant different of USP15 expression were discovered between cirrhotic tissue and primary tissue, its expression in HCC metastatic tissue was upregulated. Subsequently, we test the USP15 expression profile in a cohort of 66 HCC patients. USP15 expression was positively correlated with the recurrence of HCC significantly (p = 0.004). HCC patients with high USP15 expression had shorter disease free survival time in compare with those with low USP15 expression (56.9% VS 26.7%, P = 0.012). Subsequently, Cox multivariate analyses of clinical factors associated with disease free survival were performed and USP15 expression (p = 0.008) together with tumor size (p = 0.034) were proved to be independent predict factors in HCC. Then, we silenced USP15 expression in HCC cells and the results showed that downregulated USP15 expression resulting proliferation inhibition and apoptosis induction. In conclusion, our results suppose USP15 to be a potential target in HCC.

BARX2 expression is downregulated by CpG island hypermethylation and is associated with suppressed cell proliferation and invasion of gastric cancer cells.

BarH­like homeobox 2 (BARX2), a homeobox gene, is associated with several types of cancers. The present study aimed to determine whether DNA methylation downregulates BARX2 expression and whether BARX2 is associated with suppression of gastric carcinogenesis. BARX2 protein expression in normal and cancerous gastric tissues and various gastric cancer (GC) cell lines was detected using immunohistochemical and western blot assays. BARX2 mRNA levels were detected using both reverse transcription­polymerase chain reaction (RT­PCR) and quantitative PCR (qPCR). Promoter hypermethylation in GC cells was detected using methylation­specific PCR or bisulfite DNA sequencing PCR. Effects of BARX2 expression on GC cell proliferation, clonal formation, and migration were evaluated after lentivirus­BARX2 transfection. The effect of stable BARX2 transfection on tumor formation was assessed in a nude xenograft mouse model. BARX2 was strongly expressed in the normal gastric mucosa, but weakly or not expressed in GC tissues and most GC cell lines. BARX2 expression was negatively correlated with DNMT (a marker for DNA methylation) expression in the gastric tissues. The BARX2 promoter fragment was hypermethylated in the GC cell lines. Overexpression of BARX2 significantly inhibited GC cell proliferation, clonal formation, and migration. Stable BARX2 transfection inhibited tumor formation in xenograft mice, which was correlated with decreased expression of E­cadherin, proliferation markers, and matrix metalloproteinases. In conclusion, BARX2 expression is aberrantly reduced in GC, which is associated with increased DNA methylation of its promoter. BARX2 inhibits GC cell proliferation, migration, and tumor formation, suggesting that BARX2 acts as a tumor suppressor in gastric carcinogenesis.

Is Preoperative Fibrinogen Associated with the Survival Prognosis of Gastric Cancer Patients? A Multi-centered, Propensity Score-Matched Retrospective Study.

BACKGROUND: The prognostic significance of preoperative plasma fibrinogen in patients with operable gastric cancer remains under debate. This study aimed to elucidate the prognostic value of fibrinogen in gastric cancer patients underwent gastrectomy. METHODS: A total of 4351 patients with gastric cancer collected from three comprehensive medical centers were retrospectively evaluated. Patients were categorized by minimum P value using X-tile, while the baseline confounders for fibrinogen was balanced through propensity score matching (PSM). The relationships between fibrinogen and other clinicopathologic features were evaluated, and nomogram was constructed to assess its prognostic improvement compared with TNM staging system. RESULTS: Fibrinogen was significantly correlated with macroscopic type, tumor differentiation, tumor size, and T and N stage. The factors, fibrinogen and T stage as well as N stage, were identified to be independent prognostic factors after PSM. Nomogram based on fibrinogen demonstrated a smaller Akaike information criterion (AIC) and a larger concordance index (C-index) than TNM staging system, illustrating that fibrinogen might be able to improve the prognostic accuracy. CONCLUSIONS: Preoperative plasma fibrinogen levels in gastric cancer patients were significantly correlated with tumor progression, which could be regarded as a reliable marker for survival prognostic prediction.

Long non-coding RNA PVT1 as a novel potential biomarker for predicting the prognosis of colorectal cancer.

BACKGROUND: Long non-coding RNA (lncRNA) plays a very important role in the occurrence and development of various tumors, and is a potential biomarker for cancer diagnosis and prognosis. The purpose of this study was to investigate the relationship between the expression of lncRNA plasmacytoma variant translocation 1 (PVT1) and the prognostic significance in patients with colorectal cancer. METHODS: The expression of PVT1 was measured by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in cancerous and adjacent tissues of 210 colorectal cancer patients. The disease-free survival and overall survival of colorectal cancer patients were evaluated by Kaplan-Meier analysis, and univariate and multivariate analysis were performed by Cox proportional-hazards model. RESULTS: Our results revealed that PVT1 expression in cancer tissues of colorectal cancer was significantly higher than that of adjacent tissues (P<0.001). High PVT1 expression was increased by 51.4% (108/210), which was significantly correlated with the tumor differentiation, the depth of invasion, the stage of tumor, node, metastasis (TNM), and lymphatic metastasis. The Kaplan-Meier analysis showed that high PVT1 expression resulted in a shorter disease-free survival (Log-rank test P<0.001) and overall survival (Log-rank test P<0.001) compared with the low PVT1 expression group in colorectal cancer patients, whether at TNM I/II stage or at TNM III/IV stage. A multivariate Cox regression analysis demonstrated that high PVT1 expression was an independent predictor of poor prognosis in colorectal cancer patients. CONCLUSIONS: Our results suggest that high PVT1 expression might be a potential biomarker for assessing tumor recurrence and prognosis in colorectal cancer patients.

Gastric Cancer Screening by Combined Determination of Serum Helicobacter pylori Antibody and Pepsinogen Concentrations: ABC Method for Gastric Cancer Screening.

OBJECTIVE: Gastroscopy combined with gastric mucosa biopsies is currently regarded as a gold standard for diagnosis of gastric cancer. However, its application is restricted in clinical practice due to its invasive property. A new noninvasive population screening process combining the assay of anti-Helicobacter pylori antibody and serum pepsinogen (PG) (ABC method) is adopted to recognize the high-risk patients for further endoscopy examination, avoiding the unnecessary gastroscopy for most population and saving the cost consumption for mass screening annually. Nevertheless, controversies exist for the grouping of ABC method and the intervals of gastroscopy surveillance for each group. In this review, we summarized these popular concerned topics for providing useful references to the healthcare practitioner in clinical practice. DATA SOURCES: The PubMed databases were systematically searched from the inception dates to November 22, 2017, using the keywords "Helicobacter pylori," "Pepsinogens," and "Stomach Neoplasms." STUDY SELECTION: Original articles and reviews on the topics were selected. RESULTS: Anti-H. pylori antibody and serum PG concentration showed significant changes under the different status of H. pylori infection and the progression of atrophic gastritis, which can be used for risk stratification of gastric cancer in clinic. In addition, anti-H. pylori antibody titer can be used for further risk stratification of gastric cancer contributing to determine better endoscopy surveillance interval. CONCLUSIONS: The early detection and diagnosis of gastric cancer benefit from the risk stratification, but the cutoff values for H. pylori antibody and serum PG concentration require further modification.

Knockdown of long non­coding RNA PVT1 reverses multidrug resistance in colorectal cancer cells.

Multidrug resistance (MDR) is one of the primary causes of chemotherapy failure in colorectal cancer (CRC), and extensive biological studies into MDR are required. The non­coding RNA plasmacytoma variant translocation 1 (PVT1) has been demonstrated to be associated with low survival rates in patients with CRC. However, whether PVT1 serves a critical function in the MDR of CRC remains to be determined. To determine the association between PVT1 expression and 5­fluorouracil (5­FU) resistance in CRC, the expression levels of PVT1 mRNA in 5­FU­resistant CRC tissues and cell lines (HCT­8/5­FU and HCT­116/5­FU) were assessed by a reverse transcription­quantitative polymerase chain reaction (RT­qPCR). Cytotoxicity was evaluated using a Cell Counting Kit­8 assay and apoptosis rates were assessed via flow cytometry. In the present study, PVT1 mRNA was highly expressed in 5­FU­resistant CRC tissues and cell lines. HCT­8/5­FU and HCT­116/5­FU cells transfected with small interfering RNA PVT1 and treated with 5­FU exhibited higher apoptotic rates and lower survival rates. By contrast, overexpression of PVT1 in HCT­8 and HCT­116 cells transfected with lentiviral vector­PVT1­green fluorescent protein and treated with 5­FU exhibited lower apoptosis rates and higher survival rates. RT­qPCR and western blotting demonstrated that the overexpression of PVT1 increased the mRNA and protein expression levels of multidrug resistance­associated protein 1, P­glycoprotein, serine/threonine­protein kinase mTOR and apoptosis regulator Bcl2. The present study indicates that PVT1 overexpression may promote MDR in CRC cells, and suggested that inhibition of PVT1 expression may be an effective therapeutic strategy for reversing MDR in CRC.

Diagnosis and Treatment of 26 Cases of Abdominal Cocoon.

BACKGROUND AND AIMS: Abdominal cocoon (AC) is a rare abdominal disease with nonspecific clinical features, and it is difficult to be diagnosed before operation and hard to be treated in clinical practice. The aim of this study is to investigate the diagnosis and treatment of AC. METHODS: The clinical manifestations, findings during surgery, treatments, and follow-up results of 26 cases of AC were retrospectively studied from January 2001 to January 2015. RESULTS: All of 26 cases were diagnosed as AC definitely by laparotomy or laparoscopic surgery. Their clinical findings were various, with 7 intestines obstructed with bezoars and 4 intestines perforated by spiny material. Based on the existence of the second enterocoelia, all cases were categorized into 2 types: type I is absent of second enterocoelia (18 cases, 69.23%), while type II shows second enterocoelia (8 cases, 30.77%). Twenty cases (12 were type I and 8 were type II) underwent membrane excision and careful enterodialysis to release the small intestine entirely or partially, while the other 6 cases (all were type I) did not. In addition, all patients were treated with medical treatment and healthy diet and lifestyle. Finally, most of the patients recovered smoothly. CONCLUSIONS: AC can be categorized into two types; surgery is recommended for type II and part of type I with severe complications, but sometimes conservative therapy might be appropriate for type I. Laparoscopic surgery plays an important role in the diagnosis and treatment of AC. Furthermore, favorite health education, healthy diet and lifestyle are of significance in patients' recovery.

[Mechanism of colon cancer cell apoptosis induced by telocinobufagin: role of oxidative stress and apoptosis pathway].

OBJECTIVE: To investigate the effects of telocinobufagin on viability and apoptosis of colorectal cancer (CRC) cells and explore the mechanism of telocinobufagin-induced apoptosis. METHODS: MTT assay was performed to detect the viability of CRC cells exposed to telocinobufagin. Nuclear staining with Hoechst 33342 and flow cytometry were used to analyze the cell death of CRC cells. Expressions of proteins related with cell apoptosis and oxidative stress were determined with Western blotting. RESULTS: Telocinobufagin decreased the viability of CRC cells in a time- and dose-dependent manner. The presence of karyopycnosis and apoptotic bodies together with the results of flow cytometry suggested that telocinobufagin induced cell apoptosis to cause cell death. Western blotting showed that telocinobufagin exposure of the cells resulted in upregulated p53 and Bax protein expressions and promoted cleavage of caspase 9 and PARP. Telocinobufagin induced phosphorylation of Bad and PARP cleavage, and suppressed phosphorylation of IKBα and TAK1 and expression of survivin in the cells. CONCLUSION: Telocinobufagin can decrease the viability of CRC cells by inducing cell apoptosis, which involves p53-mediated Bax activation and inhibition of the IAP pathway.

Prognostic significance of B7-H3 expression in patients with colorectal cancer: A meta-analysis.

OBJECTIVE: The co-stimulatory molecule B7-H3 plays an important role in prognosis of several malignancies. However, its prognostic value in clinic in patient with colorectal cancer (CRC) is still controversial. This meta-analysis evaluated the relationship between B7-H3 expression and the outcomes of CRC patients. METHODS: PubMed, Google Scholar, Embase, CNKI and Wanfang database were searched for the studies on the relationship between the expression of B7-H3 and prognosis of CRC patients. Pooled odds ratios (ORs) analysis with 95% confidence interval (95% CIs) for lymph node metastasis, 24-month overall survival and 72-month overall survival were performed mainly using Review Manager 5.0. RESULTS: Six articles including 1,202 total CRC cases were included for the meta-analysis. Pooled analysis with fixed-effects model showed that B7-H3 expression had no relationship with lymphatic metastasis in CRC patients (Fixed-effects, OR= 1.18; 95 % CI:0.87-1.61, P=0.28). However, B7-H3 expression was associated with 24-month overall survival (Fixed-effects, OR=0.48, 95% CI:0.32-0.74, P<0.001) and 72-month overall survival (Fixed-effects, OR = 0.61, 95% CI: 0.43-0.85, P< 0.01) in CRC patients. CONCLUSION: The co-stimulatory molecule B7-H3 expression is negatively associated with lymph node metastasis in CRC. However, B7-H3 detection might be a feasible and effective means to predict the prognosis in CRC patients.

Comparative proteomics analysis of colorectal cancer.

BACKGROUND AND OBJECTIVE: Protein expression in colon and rectal cancer (CRC) and paired normal tissues was examined by two-dimensional gel electrophoresis (2-DE) to identify differentially expressed proteins. MATERIALS AND METHODS: Five fresh colorectal cancer and paired adjacent normal tissues were obtained and differentially expressed protein spots were determined using PDQuest software, with identification on the basis of MALDI- TOF mass spectra. RESULTS: Compared with normal colorectal mucosa, protein abnormal expression of 65 spots varying more than 1.5 times were found in 2-DE gels from colorectal cancer samples (P<0.05); forty-two proteins were up-regulated and 23 were down-regulated; twelve protein spots were identified using mass spectrometry, of which 8 were up-regulated, including HSPB1 and Annexin A4, while 4 were down-regulated, the results being consistent with Western blot findings. CONCLUSIONS: Two-dimensional electrophoresis reference maps for CRC tissues and adjacent normal mucosa (NMC) were established and 12 differentially expressed proteins were identified. Up-regulated HSPB1 and Annexin A4 may play many important roles in the pathogenesis of colorectal cancer.

Screening peptides binding specifically to colorectal cancer cells from a phage random peptide library.

The aim of this study was to screen for polypeptides binding specifically to LoVo human colorectal cancer cells using a phage-displayed peptide library as a targeting vector for colorectal cancer therapy. Human normal colorectal mucous epithelial cells were applied as absorber cells for subtraction biopanning with a c7c phage display peptide library. Positive phage clones were identified by enzyme-linked immunosorbent assay and immunofluorescence detection; amino acid sequences were deduced by DNA sequencing. After 3 rounds of screening, 5 of 20 phage clones screened positive, showing specific binding to LoVo cells and a conserved RPM motif. Specific peptides against colorectal cancer cells could be obtained from a phage display peptide library and may be used as potential vectors for targeting therapy for colorectal cancer.

Apigenin suppresses the growth of colorectal cancer xenografts via phosphorylation and up-regulated FADD expression.

Apigenin is a flavonoid belonging to the flavone structural class. It has been implicated as a chemopreventive agent against prostate and breast cancers. However, to the best of our knowledge, no published data are available regarding apigenin in colorectal cancer (CRC). The effects and mechanisms of apigenin on CRC may vary significantly. This study aimed to analyze the effects of apigenin on the growth of CRC xenografts in nude mice derived from SW480, as well as to investigate the underlying mechanisms. Whole-body fluorescence imaging is an inexpensive optical system used to visualize gene expression in small mammals using reporter genes, such as eGFP as a reporter. In our study, the expression of eGFP may reflect the size of the tumor. A terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay showed that apigenin promoted the apoptosis of CRC cells. Furthermore, the expression of five genes related to the proliferation and apoptosis of CRC, i.e., cyclin D1, BAG-1, Bcl-2, yrdC and Fas-associated protein with death domain (FADD), was detected by real-time quantitative RT-PCR. Among these genes, the up-regulated expression of FADD was noted in CRC xenograft tumors treated with apigenin. Immunohistochemistry and Western blotting confirmed the results at the protein level. Furthermore, Western blot analysis showed that apigenin induced the phosphorylation of FADD. Our findings suggest that apigenin enhances the expression of FADD and induces its phosphorylation, which may cause apoptosis of CRC cells and inhibition of tumor growth.

[Preoperative evaluation of the 64-slice spiral CT three dimensional angiography for vascular invasion in gastric cancer].

OBJECTIVE: To evaluate the preoperative diagnosis value of 64-slice spiral CT three dimensional angiography (3D CTA) for the vascular invasion in gastric cancer. METHODS: CT images of 40 patients diagnosed as gastric cancer by endoscope,who proceeded to surgical exploration from August 2006 to December 2007,were collected. These images were rebuilt by 3D CTA to judge vascular invasion by gastric cancer in comparison with the surgical finding as standard reference. RESULTS: Successful 3D CTA reconstructions were performed for all these 40 patient images. Out of 40 cases, 14 cases presented vascular invasion in the 3D CTA, and 12 of 14 cases were proved to have vascular invasion in the surgery. For assessing vascular invasion with CTA, the sensitivity was 98.1% and the specificity was 96.4% respectively (Chi Square chi(2)=0.0099,P>0.05). There was no significant differences regarding vascular invasion in gastric cancer between preoperative 3D CTA assessment and surgical finding. CONCLUSION: Sixty-four-slice spiral CT 3D angiography is effective in assessing vascular invasion in gastric cancer and is also valuable in clinical application.

[Screening peptides binding specifically to large intestinal cancer LoVo cells from phage random peptide library].

OBJECTIVE: To screen the polypeptides specifically binding to human large intestinal cancer LoVo cells from a phage-displayed peptide library for potential use as targeting vectors for large intestinal cancer therapy. METHODS: With the LoVo cells as the target cells and human normal large intestinal mucosal epithelial cells as the absorber cells for subtraction biopanning from a c7c phage-display peptide library, the positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence detection. The amino acid sequences of the identified peptides were deduced by DNA sequencing. RESULTS: After 3 rounds of screening, 5 positive phage clones showing specific binding to LoVo cells and containing conserved motif RPMP were obtained from the 20 randomly selected clones. CONCLUSION: Specific peptide against large intestinal cancer cells can be obtained from a phage-display peptide library for use as potential vectors for targeting therapy of large intestinal cancer.

[Changes in nutritional status and immune function in patients with colorectal cancer after operation].

OBJECTIVE: To evaluate the changes in the nutritional status and immune function in patients with colorectal cancer after operation. METHODS: The indexes of the nutritional status and immune function were measured in 40 patients with colorectal cancer before and after operation and compared with the control group. RESULTS: The levels of IgM, IgG, prealbumin and transferrin were lowered after operation in these colorectal cancer patients (P<0.05), but no significant difference was noted after operation in the control group. CONCLUSION: The nutritional status and immune function in colorectal cancer patients obviously deteriorate after operation.