Microbiota-induced S100A11-RAGE axis underlies immune evasion in right-sided colon adenomas and is a therapeutic target to boost anti-PD1 efficacy.

BACKGROUND: Tumourigenesis in right-sided and left-sided colons demonstrated distinct features. OBJECTIVE: We aimed to characterise the differences between the left-sided and right-sided adenomas (ADs) representing the early stage of colonic tumourigenesis. DESIGN: Single-cell and spatial transcriptomic datasets were analysed to reveal alterations between right-sided and left-sided colon ADs. Cells, animal experiments and clinical specimens were used to verify the results. RESULTS: Single-cell analysis revealed that in right-sided ADs, there was a significant reduction of goblet cells, and these goblet cells were dysfunctional with attenuated mucin biosynthesis and defective antigen presentation. An impairment of the mucus barrier led to biofilm formation in crypts and subsequent bacteria invasion into right-sided ADs. The regions spatially surrounding the crypts with biofilm occupation underwent an inflammatory response by lipopolysaccharide (LPS) and an apoptosis process, as revealed by spatial transcriptomics. A distinct S100A11+ epithelial cell population in the right-sided ADs was identified, and its expression level was induced by bacterial LPS and peptidoglycan. S100A11 expression facilitated tumour growth in syngeneic immunocompetent mice with increased myeloid-derived suppressor cells (MDSC) but reduced cytotoxic CD8+ T cells. Targeting S100A11 with well-tolerated antagonists of its receptor for advanced glycation end product (RAGE) (Azeliragon) significantly impaired tumour growth and MDSC infiltration, thereby boosting the efficacy of anti-programmed cell death protein 1 therapy in colon cancer. CONCLUSION: Our findings unravelled that dysfunctional goblet cells and consequential bacterial translocation activated the S100A11-RAGE axis in right-sided colon ADs, which recruits MDSCs to promote immune evasion. Targeting this axis by Azeliragon improves the efficacy of immunotherapy in colon cancer.

A co-carrier for plasmid DNA and curcumin delivery to treat pancreatic cancer via dendritic poly(l-lysine) modified amylose.

Pancreatic cancer is one of the most lethal malignancies in the world and remains one of the leading causes of cancer related death. For its treatment, a lot of investigations have dealt not only with individual chemotherapy by using polymeric carriers to deliver anticancer drugs, but also with individual gene therapy by using polymeric carriers to deliver nucleic acids such as small interfering RNA (siRNA) and plasmid DNA. However, relatively few studies have been focused on the co-delivery of gene and anticancer drug by multifunctional polymeric carriers for its synergistic therapy. In this work, a DPLL-functionalized amylose (ADP) was prepared by the click reaction between azidized amylose and propargyl focal point poly(l-lysine) dendrons, and then used to co-deliver plasmid pIRES2-EGFP-TNFα and curcumin for pancreatic cancer treatment. Due to the internal hydrophobic cavity of amylose component, ADP could load efficiently curcumin with anticancer activity and showed a sustained release behavior. Moreover, the curcumin-loaded ADP could form colloidally stable nanocomplexes with plasmid DNA in aqueous system due to the existence of cationic poly(l-lysine) dendrons and exhibited high gene transfection efficiency. The in vitro and in vivo tests confirmed the effectiveness of using ADP to co-deliver plasmid pIRES2-EGFP-TNFα and curcumin for synergistic therapy of pancreatic cancer.

Indocyanine green for radical lymph node dissection in patients with sigmoid and rectal cancer: randomized clinical trial.

BACKGROUND: D3 lymph node dissection is recommended for patients with advanced sigmoid and rectal cancer in Japan. This trial aimed to investigate the feasibility of indocyanine green (ICG) as a tracer to increase the nodal harvest during D3 lymph node dissection in patients with sigmoid and rectal cancer. METHODS: This prospective randomized clinical trial was performed between May 2021 and April 2022. The inclusion criteria were patients with stage I-III sigmoid or rectal cancer eligible for laparoscopic resection. Patients were 1: 1 randomized to either the ICG group (endoscopic ICG injection at the tumour site and intraoperative imaging to guide dissection) or the control group (routine laparoscopic white-light imaging). All patients were treated with D3 dissection, and the primary outcome was the number of harvested lymph nodes at the D3 level. RESULTS: Out of 210 patients screened, a total of 66 patients were enrolled and randomized. Patients in the two groups presented similar ages and clinical stages (ICG group versus control group, median age of 58.0 versus 58.5 years; stage III 36.4 per cent versus 36.4 per cent, whereas the rate of rectal cancer was 27.3 per cent versus 48.5 per cent respectively). ICG imaging was helpful for completely dissecting D3 lymph nodes and could identify a median of more than 2 (range 1-6) D3 lymph nodes neglected by routine laparoscopic white-light imaging during surgery. The median number of D3 lymph nodes harvested in the ICG group was significantly higher than that in the control group (7.0 versus 5.0, P = 0.003); however, there was no significant difference in the median numbers of positive D1, D2, and D3 lymph nodes between the two groups. CONCLUSION: ICG is safe and feasible to guide D3 lymph node dissection and can increase the number of harvested D3 lymph nodes in patients with sigmoid and rectal cancer. Registration number: NCT04848311 (http://www.clinicaltrials.gov).

FAM98A promotes resistance to 5-fluorouracil in colorectal cancer by suppressing ferroptosis.

BACKGROUND: FAM98A is a microtubule-associated protein involved in cell proliferation and migration, and is frequently dysregulated in epithelial cancers. But its role in the development of colorectal cancer (CRC) cancer remains unknown. METHODS: Immunohistochemical analysis was performed to examine the expression of FAM98A in CRC samples. We also investigated the effects of abnormal expression on the biological behavior of colorectal cancer cells both in vitro and in vivo. Immunoblotting and immunoprecipitation were used to screen FAM98A-related signalling pathways and downstream factors. RESULTS: FAM98A was upregulated in CRC tissues and CRC cell lines. Overexpression of FAM98A promoted cell proliferation and recovered 5-FU suppressed CRC cell proliferation both in vitro and in vivo. In addition, the Enhanced expression of FAM98A inhibited ferroptosis in CRC cells by activating the translation of xCT in stress granules (SGs). Furthermore, we identified that metformin could reverse FAM98A-mediated 5-FU resistance in CRC cells. CONCLUSIONS: Our results for the first time indicate that FAM98A plays a critical role in promoting CRC progression, which provides a novel target for clinical drug resistance of colorectal cancer. And metformin may sensitize 5-FU in the treatment of colorectal cancer.

Application of a cationic amylose derivative loaded with single-walled carbon nanotubes for gene delivery therapy and photothermal therapy of colorectal cancer.

Single-walled carbon nanotubes (SWNTs) are cylindrical graphitic helix molecules that exhibit superb mechanical and physical properties. Many polymers, such as polyethylene glycol and glycated chitosan, have been used to modify SWNTs to enhance the stability and biocompatibility of delivery systems; thus, a novel modification for SWNTs with amylose derivatives containing poly(L-lysine) dendrons (ADP@SWNT) is developed. Infrared spectra analysis, 1 H NMR analysis, circular dichroism spectra analysis and thermogravimetric analysis are used to characterize and confirm complex formation. The aqueous dispersion stability, cytotoxicity, gene transfection efficiency and photothermal effect of the complex are studied in vitro and in vivo. Results suggest that the ADP@SWNT complex is successfully synthesized with good water dispersion stability and pDNA transfection capacity. ADP@SWNT/TNFα inhibits tumor growth and metastasis both in vivo and in vitro, and the anti-tumor effect is enhanced by NIR irradiation, suggesting its high potential for application in tumor therapy.

Real-time in vivo distal margin selection using confocal laser endomicroscopy in transanal total mesorectal excision for rectal cancer.

BACKGROUND: Transanal total mesorectal excision (TaTME) allows patients with ultralow rectal cancer to be treated with sphincter-saving surgery. However, accurate delineation of the distal resection margin (DRM), which is essential to achieve R0 resection for low rectal cancer in TaTME, is technically demanding. AIM: To assess the feasibility of optical biopsy using probe-based confocal laser endomicroscopy (pCLE) to select the DRM during TaTME for low rectal cancer. METHODS: A total of 43 consecutive patients who were diagnosed with low rectal cancer and scheduled for TaTME were prospectively enrolled from January 2019 to January 2021. pCLE was used to determine the distal edge of the tumor as well as the DRM during surgery. The final pathological report was used as the gold standard. The diagnostic accuracy of pCLE examination was calculated. RESULTS: A total of 86 pCLE videos of 43 patients were included in the analyses. The sensitivity, specificity and accuracy of real-time pCLE examination were 90.00% [95% confidence interval (CI): 76.34%-97.21%], 86.96% (95%CI: 73.74%-95.06%) and 88.37% (95%CI: 79.65%-94.28%), respectively. The accuracy of blinded pCLE reinterpretation was 86.05% (95%CI: 76.89%-92.58%). Furthermore, our results show satisfactory interobserver agreement (κ = 0.767, standard error = 0.069) for the detection of cancer tissue by pCLE. There were no positive DRMs (≤ 1 mm) in this study. The median DRM was 7 mm [interquartile range (IQR) = 5-10 mm]. The median Wexner score was 5 (IQR = 3-6) at 6 mo after stoma closure. CONCLUSION: Real-time in vivo pCLE examination is feasible and safe for selecting the DRM during TaTME for low rectal cancer (clinical trial registration number: NCT04016948).

Erratum: MicroRNA-647 targets SRF-MYH9 axis to suppress invasion and metastasis of gastric cancer: Erratum.

[This corrects the article DOI: 10.7150/thno.20512.].

MicroRNA-647 Targets SRF-MYH9 Axis to Suppress Invasion and Metastasis of Gastric Cancer.

MicroRNAs (miRNAs) play important roles in regulating tumour development and progression. Here we show that miR-647 is repressed in gastric cancer (GC), and associated with GC metastasis. Moreover, we identify that miR-647 can suppress GC cell migration and invasion in vitro. Mechanistically, we confirm miR-647 directly binds to the 3' untranslated regions of SRF mRNA, and SRF binds to the CArG box located at the MYH9 promoter. CCG-1423, an inhibitor of RhoA/SRF-mediated gene transcription, inhibits the expression of MYH9, especially in SRF downregulated cells. Overexpression of miR-647 inhibits MGC 80-3 cells' metastasis in orthotropic GC models, but increasing SRF expression in these cells reverses this change. Importantly, we found the synergistic inhibition effect of CCG-1423 and agomir-647, an engineered miRNA mimic, on cancer metastasis in orthotropic GC models. Our study demonstrates that miR-647 functions as a tumor metastasis suppressor in GC by targeting SRF/MYH9 axis.

Hepatitis B Virus X Protein (HBx) Is Responsible for Resistance to Targeted Therapies in Hepatocellular Carcinoma: Ex Vivo Culture Evidence.

PURPOSE: Molecular targeted therapy is an important approach for advanced hepatocellular carcinoma (HCC). Hepatitis B virus-related HCC (HBV-HCC) accounts for approximately 50% of all HCC cases. Bortezomib, a proteasome inhibitor (PI), is used extensively for the treatment of hematologic malignancies, but its application in HCC, particularly in HBV-HCC, has not been fully explored. EXPERIMENTAL DESIGN: The effects of bortezomib on HCC tissues were evaluated by TUNEL assays. The growth inhibitory activity was measured using cell viability assays, and apoptosis was measured using flow cytometry. The levels of HBx, P-Raf/Raf, and P-Erk/Erk expression were measured by Western blot analysis. The ability of the MEK inhibitor PD98059 to enhance the cell killing activity of bortezomib was evaluated using ex vivo and in vivo methods. RESULTS: The potency of bortezomib varied among HCC samples and cell lines, and HBV/HBx expression was associated with resistance to bortezomib. Bortezomib increased the levels of P-Raf and P-Erk in HBV/HBx-positive cells but not in HBV/HBx-negative HCC cells or in breast cancer or glioblastoma multiform cells. HBx was also upregulated after exposure to bortezomib, which was associated with the inhibition of proteasome activity. P-Erk upregulation mediated by bortezomib was effectively suppressed by the addition of the MEK inhibitor PD98059. Moreover, bortezomib and PD98059 synergistically inhibited HCC cell proliferation, as measured using both ex vivo and in vivo models. CONCLUSIONS: Our studies demonstrate for the first time that HBx causes resistance to bortezomib in HCC, and this resistance can be antagonized by a MEK signaling inhibitor, providing a novel therapeutic approach.

Cationic dendronization of amylose via click chemistry for complexation and transfection of plasmid DNA.

For the development of effective and safe gene carrier based on starch, the amylose from potato starch was azidized by reacting with 3-azidopropylamine in the presence of N, N'-carbonyldiimidazole and then conjugated with propargyl focal point poly(amidoamine) (PAMAM) dendrons by a Cu(I)-catalyzed azide-alkyne cycloaddition. Such a cationic dendronization was verified by Fourier transform infrared spectroscopy and proton nuclear magnetic resonance analyses. For the resultant amylose conjugates with various contents and generations of PAMAM dendron, their buffering capacity, binding ability with plasmid DNA and in vitro cytotoxicity were investigated. These amylose conjugates were found to exhibit good buffering capacity and biocompatibility. In particular, they could condense effectively plasmid DNA into the nanocomplexes, as confirmed by agarose gel electrophoresis, zeta potential, and particle size analyses as well as transmission electron microscopy observation. For their nanocomplexes with plasmid DNA, the in vitro transfection properties in human embryonic kidney 293T cells were studied by fluorescence microscopy and flow cytometry. It was found that the transfection efficiency could be optimized by the dendronization extent of amylose and the complexation extent of dendronized amylose with plasmid DNA.

The long non-coding RNA HOTTIP promotes progression and gemcitabine resistance by regulating HOXA13 in pancreatic cancer.

BACKGROUND: The human genome encodes many long non-coding RNAs (lncRNAs). However, their biological functions, molecular mechanisms, and the prognostic value associated with pancreatic ductal adenocarcinoma (PDAC) remain to be elucidated. Here, we identify a fundamental role for the lncRNA HOXA transcript at the distal tip (HOTTIP) in the progression and chemoresistance of PDAC. METHODS: High-throughput microarrays were performed to detect the expression profiles of lncRNAs and messenger RNAs in eight human PDAC tissues and four pancreatic tissues. Quantitative real-time PCR was used to determine the levels of HOTTIP and HOXA13 transcripts in PDAC cell lines and 90 PDAC samples from patients. HPDE6 cells (immortalized human pancreatic ductal epithelial cells) and corresponding adjacent non-neoplastic tissues were used as controls, respectively. The functions of HOTTIP and HOXA13 in cell proliferation, invasion, and epithelial-mesenchymal transition were evaluated by targeted knockdown in vitro. CCK-8 assays, colony formation assays, and xenografts in nude mice were used to investigate whether targeted silencing of HOTTIP could sensitize pancreatic cancer cells to gemcitabine. Immunohistochemistry was performed to investigate the relationship between HOXA13 expression and patient outcome. RESULTS: Microarray analyses revealed that HOTTIP was one of the most significantly upregulated lncRNAs in PDAC tissues compared with pancreatic tissues. Quantitative PCR further verified that HOTTIP levels were increased in PDAC cell lines and patient samples compared with controls. Functionally, HOTTIP silencing resulted in proliferation arrest by altering cell-cycle progression, and impaired cell invasion by inhibiting epithelial-mesenchymal transition in pancreatic cancer. Additionally, inhibition of HOTTIP potentiated the antitumor effects of gemcitabine in vitro and in vivo. Furthermore, knockdown of HOXA13 by RNA interference (siHOXA13) revealed that HOTTIP promoted PDAC cell proliferation, invasion, and chemoresistance, at least partly through regulating HOXA13. Immunohistochemistry results revealed that higher HOXA13 expression was correlated with lymph node metastasis, poor histological differentiation, and decreased overall survival in PDAC patients. CONCLUSIONS: As a crucial tumor promoter, HOTTIP promotes cell proliferation, invasion, and chemoresistance by modulating HOXA13. Therefore, the HOTTIP/HOXA13 axis is a potential therapeutic target and molecular biomarker for PDAC.

Nanocomplexation of thrombin with cationic amylose derivative for improved stability and hemostatic efficacy.

As a topical hemostatic agent, thrombin has wide application for many surgical treatments. However, native thrombin always suffers from its physical and chemical instabilities. In this work, a nanocomplexation strategy was developed for modifying the stability and hemostatic efficacy of thrombin, in which a water-soluble cationic amylose derivative containing poly(l-lysine) dendrons was prepared by a click reaction and then used to complex thrombin in an aqueous system. For resultant thrombin nanocomplexes, their morphology and particle size distribution were investigated. Their stabilities were studied in terms of activity retention percentages under different storage time, pH values, and illumination time. In addition, their ability to achieve in vitro fibrinogen and blood coagulation were evaluated. Via a rat hepatic hemorrhage model and a rat iliac artery hemorrhage model, these thrombin nanocomplexes were confirmed to have good tissue biocompatibility and in vivo hemostatic effectiveness.

Click modification of helical amylose by poly(l-lysine) dendrons for non-viral gene delivery.

Although amylose as a naturally-occurring helical polysaccharide has been widely used for biomedical applications, few studies have dealt with its chemical modification for non-viral gene delivery. In this work, the click modification of amylose by poly(l-lysine) dendrons was carried out and then characterized by Fourier transform infrared spectroscopy, wide-angle X-ray diffraction and elemental analyses. Such a modified polysaccharide exhibited excellent ability to condense plasmid pMSCV-GFP-PARK2 into compact and spherical nanoparticles. Moreover, it displayed much lower cytotoxicity when compared to branched polyethylenimine (bPEI, 25kDa), a commercially available gene vector. Similar to bPEI, it had a dose-dependent gene transfection activity in human embryonic kidney 293T cells, as observed by confocal laser scanning microscopy and flow cytometry. At each optimized N/P ratio, the percentage of transfected cells by this modified polysaccharide was found to be comparable to that by bPEI. Western blot and cell apoptosis analyses confirmed its effectiveness for the delivery of plasmid pMSCV-GFP-PARK2 to 293T cells.

Macrophage migration inhibitory factor is overexpressed in pancreatic cancer tissues and impairs insulin secretion function of ß-cell.

BACKGROUND: Understanding the pathogenic mechanism of pancreatic cancer associated diabetes (PCDM) might help yield biomarkers for the early diagnosis of pancreatic cancer (PC) from population with new-onset diabetes. In the current study, we sought to determine the role of macrophage migration inhibitory factor (MIF) in PCDM pathogenesis. METHODS: The protein and mRNA levels of MIF in paraffin-embedded human PC samples, chronic pancreatitis specimens, and normal pancreas were measured by immunohistochemistry and quantitative reverse-transcriptase polymerase chain reaction. We measured serum levels of MIF in PC patients and controls. The biologic impacts of MIF overexpression on insulin secretion function of mice islets and ß cells (HIT-T15) were investigated in vitro. RESULTS: MIF expression was significantly increased in pancreatic cancer tissues compared with chronic pancreatitis or normal pancreas specimens. The insulin secretion function of both islets and HIT-T15 cells was impaired by indirect co-cultured with PC cells or treated with conditioned media from them. Stable MIF knock-down significantly decreased the diabetogenic effect of PC cells, while MIF knock-in HPDE6 cells demonstrated a strong inhibitory effect on insulin secretion function of islets and HIT-T15 cells. MIF impaired ßcell function by depressing the Ca⁺ currents, decreasing L-type Ca⁺ channel α1 subunit protein expression level, and enhancing p-Src activity. Mean serum level of MIF was significant higher in new-onset diabetes associated PC patients in comparison with other groups. CONCLUSIONS: MIF is up-regulated in patients with pancreatic cancer and causes dysfunction of insulin secretion in ß-cells.

Effect of recombinant plasmid pEGFP-AFP-hTNF on liver cancer cells (HepG2 Cells) in vitro when delivered by PEG-PEI/Fe3O4 nanomagnetic fluid.

BACKGROUND/PURPOSE: Gene delivery into liver cancer cells has been a problem. This study aimed to understand the effect of using PEGI/Fe3O4 nanomagnetic fluid as a gene vector for liver cancer gene therapy. An AFP enhancer aids in the expression of tumor-specific foreign genes in AFP-producing cancer cells like HepG2 cells, and was utilized in the delivery method in this study. METHODS: We constructed recombinant plasmid PEGFP-AFP-hTNFα, which was transfected into AFP positive HepG2 cells and AFP negative Hela cells by PEG-PEI/Fe3O4 nanomagnetic fluid. Fluorescence microscopy was used to evaluate the transfection rate of the hTNFα gene in the HepG2 cells 12 hours after transfection. Reverse transcription polymerase chain reaction (RT-PCR) and western blot were used to detect expression of hTNFα gene in the HepG2 cells 48 hours after transfection. Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the inhibitory effect of hTNFα on the proliferation of HepG2 cells. Flow cytometry was used to analyze the apoptosis of HepG2 cells. RESULTS: Plasmid PEGFP-AFP-hTNFα delivered by PEG-PEI/Fe3O4 nanomagnetic fluid was successfully transfected into HepG2 cells and expressed in the HepG2 cells. The transfection efficacy of hTNFα gene delivered by PEG-PEI/Fe3O4 nanomagnetic fluid was superior to that of hTNFα gene delivered by lipofectamine in HepG2 cells. RT-PCR and western blot demonstrated that hTNFα gene was expressed in HepG2 cells that were transfected with complexes of nanomagnetic fluid/PEGFP-AFP-hTNFα. MTT and flow cytometry showed that the hTNFα gene markedly exerted a cell killing effect. CONCLUSION: PEG-PEI/Fe3O4 nanomagnetic fluid successfully transfected PEGFP-AFP-hTNFα into HepG2 cells and induced expression of hTNFα gene in the HepG2 cells, thus showing promise as a gene vector for liver cancer gene therapy. Furthermore, an AFP enhancer can specifically increase the expression of target genes in cells positive for AFP.