Correlation of tumor-associated macrophage density and proportion of M2 subtypes with the pathological stage of colorectal cancer

BACKGROUND Colorectal cancer(CRC)is a prevalent global malignancy with complex prognostic factors.Tumor-associated macrophages(TAMs)have shown paradoxical associations with CRC survival,particularly concerning the M2 subset.AIM We aimed to establish a simplified protocol for quantifying M2-like TAMs and explore their correlation with clinicopathological factors.METHODS A cross-sectional study included histopathological assessment of paraffinembedded tissue blocks obtained from 43 CRC patients.Using CD68 and CD163 immunohistochemistry,we quantified TAMs in tumor stroma and front,focusing on M2 proportion.Demographic,histopathological,and clinical parameters were collected.RESULTS TAM density was significantly higher at the tumor front,with the M2 proportion three times greater in both zones.The tumor front had a higher M2 proportion,which correlated significantly with advanced tumor stage(P=0.04),pathological nodal involvement(P=0.04),and lymphovascular invasion(LVI,P=0.01).However,no significant association was found between the M2 proportion in the tumor stroma and clinicopathological factors.CONCLUSION Our study introduces a simplified protocol for quantifying M2-like TAMs in CRC tissue samples.We demonstrated a significant correlation between an increased M2 proportion at the tumor front and advanced tumor stage,nodal involvement,and LVI.This suggests that M2-like TAMs might serve as potential indicators of disease progression in CRC,warranting further investigation and potential clinical application.

Calculus bovis inhibits M2 tumor-associated macrophage polarization via Wnt/β-catenin pathway modulation to suppress liver cancer

BACKGROUND Calculus bovis(CB),used in traditional Chinese medicine,exhibits anti-tumor effects in various cancer models.It also constitutes an integral component of a compound formulation known as Pien Tze Huang,which is indicated for the treatment of liver cancer.However,its impact on the liver cancer tumor microenvironment,particularly on tumor-associated macrophages(TAMs),is not well understood.AIM To elucidate the anti-liver cancer effect of CB by inhibiting M2-TAM polarization via Wnt/β-catenin pathway modulation.METHODS This study identified the active components of CB using UPLC-Q-TOF-MS,evaluated its anti-neoplastic effects in a nude mouse model,and elucidated the underlying mechanisms via network pharmacology,transcriptomics,and molecular docking.In vitro assays were used to investigate the effects of CB-containing serum on HepG2 cells and M2-TAMs,and Wnt pathway modulation was validated by real-time reverse transcriptase-polymerase chain reaction and Western blot analysis.RESULTS This study identified 22 active components in CB,11 of which were detected in the bloodstream.Preclinical investigations have demonstrated the ability of CB to effectively inhibit liver tumor growth.An integrated approach employing network pharmacology,transcriptomics,and molecular docking implicated the Wnt signaling pathway as a target of the antineoplastic activity of CB by suppressing M2-TAM polarization.In vitro and in vivo experiments further confirmed that CB significantly hinders M2-TAM polarization and suppresses Wnt/β-catenin pathway activation.The inhibitory effect of CB on M2-TAMs was reversed when treated with the Wnt agonist SKL2001,confirming its pathway specificity.CONCLUSION This study demonstrated that CB mediates inhibition of M2-TAM polarization through the Wnt/β-catenin pathway,contributing to the suppression of liver cancer growth.

Tumor-associated macrophages regulate gastric cancer cell invasion and metastasis through TGFβ2/NF-κB/Kindlin-2 axis

Objective: Recent studies have shown that tumor-associated macrophages(TAMs) play an important role in cancer invasion and metastasis. Our previous studies have reported that TAMs promote the invasion and metastasis of gastric cancer(GC) cells through the Kindlin-2 pathway. However, the mechanism needs to be clarified.Methods: THP-1 monocytes were induced by PMA/interleukin(IL)-4/IL-13 to establish an efficient TAM model in vitro and M2 macrophages were isolated via flow cytometry. A dual luciferase reporter system and chromatin immunoprecipitation(Ch IP) assay were used to investigate the mechanism of transforming growth factor β2(TGFβ2) regulating Kindlin-2 expression. Immunohistochemistry was used to study the relationships among TAM infiltration in human GC tissues, Kindlin-2 protein expression, clinicopathological parameters and prognosis in human GC tissues. A nude mouse oncogenesis model was used to verify the invasion and metastasis mechanisms in vivo.Results: We found that Kindlin-2 expression was upregulated at both m RNA and protein levels in GC cells cocultured with TAMs, associated with higher invasion rate. Kindlin-2 knockdown reduced the invasion rate of GC cells under coculture condition. TGFβ2 secreted by TAMs regulated the expression of Kindlin-2 through the transcription factor NF-кB. TAMs thus participated in the progression of GC through the TGFβ2/NF-κB/Kindlin-2 axis. Kindlin-2 expression and TAM infiltration were significantly positively correlated with TNM stage, and patients with high Kindlin-2 expression had significantly poorer overall survival than patients with low Kindlin-2 expression. Furthermore, Kindlin-2 promoted the invasion of GC cells in vivo.Conclusions: This study elucidates the mechanism of TAMs participating in GC cell invasion and metastasis through the TGFβ2/NF-κB/Kindlin-2 axis, providing a possibility for new treatment options and approaches.

2-Hexyl-4-Pentylenic Acid(HPTA) Stimulates the Radiotherapy-induced Abscopal Effect on Distal Tumor through Polarization of Tumor-associated Macrophages

Objective The aim of this study was to explore the effects of 2-hexyl-4-pentylenic acid(HPTA)in combination with radiotherapy(RT)on distant unirradiated breast tumors.Methods Using a rat model of chemical carcinogen(7,12-dimethylbenz[a]anthracene,DMBA)-induced breast cancer,tumor volume was monitored and treatment response was evaluated by performing HE staining,immunohistochemistry,immunofluorescence,q RT-PCR,and western blot analyses.Results The results demonstrated that HPTA in combination with RT significantly delayed the growth of distant,unirradiated breast tumors.The mechanism of action included tumor-associated macrophage(TAM)infiltration into distant tumor tissues,M1 polarization,and inhibition of tumor angiogenesis by IFN-γ.Conclusion The results suggest that the combination of HPTA with RT has an abscopal effect on distant tumors via M1-polarized TAMs,and HPTA may be considered as a new therapeutic for amplifying the efficacy of local RT for non-targeted breast tumors.

Usefulness of urinary trypsinogen-2 and trypsinogen activation peptide in acute pancreatitis:A multicenter study in Japan

BACKGROUND Rapid urinary trypsinogen-2 dipstick test and levels of urinary trypsinogen-2 and trypsinogen activation peptide(TAP) concentration have been reported as prognostic markers for the diagnosis of acute pancreatitis.AIM To reconfirm the validity of all these markers in the diagnosis of acute pancreatitis by undertaking a multi-center study in Japan.METHODS Patients with acute abdominal pain were recruited from 17 medical institutions in Japan from April 2009 to December 2012. Urinary and serum samples were collected twice, at enrollment and on the following day for measuring target markers. The diagnosis and severity assessment of acute pancreatitis were assessed based on prognostic factors and computed tomography(CT) Grade of the Japanese Ministry of Health, Labour, and Welfare criteria.RESULTS A total of 94 patients were enrolled during the study period. The trypsinogen-2 dipstick test was positive in 57 of 78 patients with acute pancreatitis(sensitivity,73.1%) and in 6 of 16 patients with abdominal pain but without any evidence of acute pancreatitis(specificity, 62.5%). The area under the curve(AUC) score of urinary trypsinogen-2 according to prognostic factors was 0.704, which was highest in all parameter. The AUC scores of urinary trypsinogen-2 and TAP according to CT Grade were 0.701 and 0.692, respectively, which shows higher than other pancreatic enzymes. The levels of urinary trypsinogen-2 and TAP were significantly higher in patients with extended extra-pancreatic inflammation as evaluated by CT Grade.CONCLUSION We reconfirmed urinary trypsinogen-2 dipstick test is useful as a marker for the diagnosis of acute pancreatitis. Urinary trypsinogen-2 and TAP may be considered as useful markers to determine extra-pancreatic inflammation in acute pancreatitis.

Urinary trypsinogen-2 for diagnosing acute pancreatitis:a meta-analysis

BACKGROUND: Currently, serum amylase and lipase are the most popular laboratory markers for early diagnosis of acute pancreatitis with reasonable sensitivity and specificity. Urinary trypsinogen-2 (UT-2) has been increasingly used but its clinical value for the diagnosis of acute pancreatitis and post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis has not yet been systematically assessed. DATA SOURCES: A comprehensive search was carried out using PubMed (MEDLINE), Embase, and Web of Science for clinical trials, which studied the usefulness of UT-2 as a diagnostic marker for acute pancreatitis. Sensitivity, specificity and the diagnostic odds ratios (DORs) with 95% confidence interval (CI) were calculated for each study and were compared with serum amylase and lipase. Summary receiver-operating curves were conducted and the area under the curve (AUC) was evaluated. RESULTS: A total of 18 studies were included. The pooled sensitivity and specificity of UT-2 for the diagnosis of acute pancreatitis were 80% and 92%, respectively (AUC=0.96, DOR=65.63, 95% CI: 31.65-139.09). The diagnostic value of UT-2 was comparable to serum amylase but was weaker than serum lipase. The pooled sensitivity and specificity for the diagnosis of post-ERCP pancreatitis were 86% and 94%, respectively (AUC=0.92, DOR=77.68, 95% CI: 24.99-241.48).CONCLUSIONS: UT-2 as a rapid test could be potentially used for the diagnosis of post-ERCP pancreatitis and to an extent, acute pancreatitis. Further studies are warranted to confirm these results.

Use of the urinary trypsinogen-2 dipstick test in early diagnosis of pancreatitis after endoscopic retrograde cholangiopancreatography (ERCP)

Background: Acute pancreatitis is one of the most serious complications of ERCP. Early diagnosis of post ERCP pancreatitis helps physicians to provide intensive care and possible medical treatment as early as possible. Trypsinogen-2 in urine is a good diagnostic and prognostic marker of acute pancreatitis. Objectives: To evaluate the diagnostic value of urinary trypsinogen-2 dipstick test for early diagnosis of post ERCP pancreatitis. Methods: A total of 37 patients with obstructive jaundice were tested with the urinary trypsinogen-2 dipstick test and serum levels of amylase and lipase before ERCP and 6 hours after ERCP. Results: Post ERCP pancreatitis was diagnosed in 6 (16%) of 37 patients. The sensitivity, specificity, positive predictive value and negative predictive value of urinary trypsinogen-2 dipstick test at 6 hours after ERCP were 100%, 97%, 86%, 100% respectively. At the cutoff level (130 U/L) for lipase, the positive predictive value and negative predictive value all were (100%), however, the positive predictive value and negative predictive value for amylase levels at cutoff (122 U/L) were 60%, 100% respectively. Serum lipase level was the best test for diagnosing post ERCP pancreatitis followed by the urinary trypsinogen-2 dipstick test. Conclusions: The urinary trypsinogen-2 dipstick test can be used as a rapid and easy test for early diagnosis of post ERCP pancreatitis with high sensitivity and specificity.

Unveiling the anticancer effect of traditional Chinese herbal medicine

In this issue of World Journal of Gastroenterology,Huang et al reported that Calculus bovis(CB),a traditional Chinese herbal medicine,impedes the growth of liver cancers in vivo.Through further in vitro studies,they showed that CB suppressed the M2 polarization of tumor-associated macrophages by suppressing the Wnt signaling pathway,which consequently inhibited the growth of liver cancer.Although the effects of traditional Chinese herbal medicine are often not scientifically proven,Huang et al successfully identified the molecular mechanism involved in the anticancer effect of CB,and it is anticipated that the molecular mechanisms involved in the effects of other traditional Chinese herbal medicines will be scientifically elucidated,as demonstrated in this article.

Pancreatic melatonin enhances anti-tumor immunity in pancreatic adenocarcinoma through regulating tumor-associated neutrophils infiltration and NETosis

Tumor microenvironment contributes to poor prognosis of pancreatic adenocarcinoma(PAAD)patients.Proper regulation could improve survival.Melatonin is an endogenous hormone that delivers multiple bioactivities.Here we showed that pancreatic melatonin level is associated with patients'survival.In PAAD mice models,melatonin supplementation suppressed tumor growth,while blockade of melatonin pathway exacerbated tumor progression.This anti-tumor effect was independent of cytotoxicity but associated with tumor-associated neutrophils(TANs),and TANs depletion reversed effects of melatonin.Melatonin induced TANs infiltration and activation,therefore induced cell apoptosis of PAAD cells.Cytokine arrays revealed that melatonin had minimal impact on neutrophils but induced secretion of Cxcl2 from tumor cells.Knockdown of Cxcl2 in tumor cells abolished neutrophil migration and activation.Melatonin-induced neutrophils presented an N1-like anti-tumor phenotype,with increased neutrophil extracellular traps(NETs)causing tumor cell apoptosis through cell-to-cell contact.Proteomics analysis revealed that this reactive oxygen species(ROS)-mediated inhibition was fueled by fatty acid oxidation(FAO)in neutrophils,while FAO inhibitor abolished the anti-tumor effect.Analysis of PAAD patient specimens revealed that CXCL2 expression was associated with neutrophil infiltration.CXCL2,or TANs,combined with NET marker,can better predict patients'prognosis.Collectively,we discovered an anti-tumor mechanism of melatonin through recruiting N1-neutrophils and beneficial NET formation.

Gold nanoparticle-directed autophagy intervention for antitumor immunotherapy via inhibiting tumor-associated macrophage M2 polarization

Tumor-associated macrophages(TAMs),one of the dominating constituents of tumor microenvironment,are important contributors to cancer progression and treatment resistance.Therefore,regulation of TAMs polarization from M2 phenotype towards M1 phenotype has emerged as a new strategy for tumor immunotherapy.Herein,we successfully initiated antitumor immunotherapy by inhibiting TAMs M2 polarization via autophagy intervention with polyethylene glycol-conjugated gold nanoparticles(PEG-Au NPs).PEG-Au NPs suppressed TAMs M2 polarization in both in vitro and in vivo models,elicited antitumor immunotherapy and inhibited subcutaneous tumor growth in mice.As demonstrated by the m RFP-GFP-LC3 assay and analyzing the autophagy-related proteins(LC3,beclin1 and P62),PEGAu NPs induced autophagic flux inhibition in TAMs,which is attributed to the PEG-Au NPs induced lysosome alkalization and membrane permeabilization.Besides,TAMs were prone to polarize towards M2phenotype following autophagy activation,whereas inhibition of autophagic flux could reduce the M2polarization of TAMs.Our results revealed a mechanism underlying PEG-Au NPs induced antitumor immunotherapy,where PEG-Au NPs reduce TAMs M2 polarization via induction of lysosome dysfunction and autophagic flux inhibition.This study elucidated the biological effects of nanomaterials on TAMs polarization and provided insight into harnessing the intrinsic immunomodulation capacity of nanomaterials for effective cancer treatment.

Identification of novel HLA-A 0201-restricted epitopes from anterior gradient-2 as a tumor-associated antigen against colorectal cancer

Anterior gradient-2 （AGR2） promotes tumor growth, cell migration and cellular transformation and its enhanced expression is almost completely restricted to malignant tissues, thus making AGR2 an interesting target for the development of immunotherapeutic strategies. We investigated whether the AGR2 molecule comprises human leukocyte antigen （HLA）-A 0201-binding epitopes recognized by human cytotoxic T lymphocytes （CTLs）, which could be targeted in dendritic cell （DC）-based cancer immunotherapy against colorectal cancer （CRC）. We reviewed the sequence of AGR2 for peptides that could potentially bind to HLA-A 0201 with the aid of a computer-based program. Five candidate peptides with different binding scores were synthesized and tested. These peptides were then assessed for their immunogenicity to elicit specific immune responses mediated by CTLs in vitro by means of enzyme-linked immunospot assays and CTL assays. AGR2 was highly expressed in several CRC cell lines, including DK01, DLD1, KM 12C, HCT-8 and HT-29. DCs pulsed with AGR2-P2 （aa 11-19; LLVALSYTL） or AGR2-P4 （aa 127-135; RIMFVDPSL） generated potent CTLs that could lyse T2 cells pulsed with AGR2-P2 or AGR2-P4 and HLA-A0201＋ AGR2-positive CRC cell lines in a strong dose-dependent and HLA-A 0201-restricted manner. In conclusion, these novel epitopes derived from AGR2 protein may be attractive candidates for DC-based immunotherapy for CRC.

Dicer-independent snRNA/snoRNA-derived nuclear RNA 3 regulates tumor-associated macrophage function by epigenetically repressing inducible nitric oxide synthase transcription

Background:Small RNAs(sRNAs)extensively mediate gene-specific chromatin regulation in lower organisms.As a dominant type of functional sRNAs in mature mammals,microRNAs mainly regulate gene expression at posttranscription level in the cytoplasm.Currently,whether there exists a type of nuclear-localized sRNAs mediating gene-specific epigenetic regulation in mature mammalian cells remains largely unclear.Here,we profiled sRNAs enriched in the nucleus and investigated their function in mediating genespecific epigenetic regulation in anti-tumor immunity.Methods:We established cytoplasmic and nuclear transcriptomes of sRNAs of dendritic cells(DCs)using high-throughput sequencing.Transcription abundances of sRNAs and mRNAs were analyzed by reverse transcriptionquantitative polymerase chain reaction(RT-qPCR)assay.The associations between sRNAs and Argonaute(AGO)proteins were detected by RNA immunoprecipitation analysis.Synthesized sRNAs and locked nucleic acid(LNA)-modified sRNA inhibitors were used to screen the function of sRNAs in innate immune cells.The effect of sRNA on the enrichment of either chromatin remodeler or histone modification at the gene promoter was analyzed by chromatin immunoprecipitation(ChIP)-qPCR assay.Chromatin accessibility qPCR assay was used to detect the accessibility of gene promoters.A B16 melanomabearing mouse model was established to determine the function of sRNAs in tumor-associated macrophages(TAMs)and their effect on tumor growth.Results:We identified a new class of nucleus-localized sRNAs,named snRNA/snoRNA-derived nuclear RNAs(sdnRNAs).Some sdnRNAs were Dicerindependent and had no association with Argonaute proteins.sdnRNA-3,the most abundant Dicer-independent sdnRNAs identified in our analysis,was selected as a representative to examine the biological function of sdnRNAs.sdnRNA-3 selectively inhibited the transcription of Nos2 in macrophages during innate immune response by repressing the chromatin accessibility at Nos2 gene promoter.sdnRNA-3 promoted the enrichments of repressive chromatinremodeling regulator Mi-2βand the repressive histone modification H3K27me3 at Nos2 gene promoter.In the B16 melanoma mouse model,we found higher expression of sdnRNA-3 in M2 TAMs than M1 TAMs and DCs.Transfer of sdnRNA-3-silenced macrophages inhibited tumor growth with increased expression of inducible nitric oxide synthase(iNOS)in TAMs.Conclusions:Our results demonstrated that the sdnRNA-3 repressed the transcription of Nos2 by repressing chromatin accessibility at the promoter,providing new insights into the regulation of macrophage function in tumor immunity.

Endoscopic retrograde cholangiopancreatography with rendezvous cannulation reduces pancreatic injury

AIM:To examine whether rendezvous endoscopic retrograde cholangiopancreatography(ERCP)is associated with less pancreatic damage,measured as leakage of proenzymes,than conventional ERCP.METHODS:Patients(n=122)with symptomatic gallstone disease,intact papilla and no ongoing inflammation,were prospectively enrolled in this case-controldesigned study.Eighty-one patients were subjected to laparoscopic cholecystectomy and if intraoperative cholangiography suggested common bile duct stones(CBDS),rendezvous ERCP was performed intraoperatively(n=40).Patients with a negative cholangiogram constituted the control group(n=41).Another 41 patients with CBDS,not subjected to surgery,underwent conventional ERCP.Pancreatic proenzymes,procarboxypeptidase B and trypsinogen-2 levels in plasma,were analysed at 0,4,8 and 24 h.The proenzymes were determined in-house with a double-antibody enzyme linked immunosorbent assay.Pancreatic amylase was measured by an enzymatic colourimetric modular analyser with the manufacturer’s reagents.All samples were blinded at analysis.RESULTS:Post ERCP pancreatitis(PEP)occurred in3/41(7%)of the patients cannulated with conventional ERCP and none in the rendezvous group.Increased serum levels indicating pancreatic leakage were significantly higher in the conventional ERCP group compared with the rendezvous ERCP group regarding pancreatic amylase levels in the 4-and 8-h samples(P=0.0015;P=0.03),procarboxypeptidase B in the4-and 8-h samples(P<0.0001;P<0.0001)and trypsinogen-2 in the 24-hour samples(P=0.03).No differences in these markers were observed in patients treated with rendezvous cannulation technique compared with patients that underwent cholecystectomy alone(control group).Post procedural concentrations of pancreatic amylase and procarboxypeptidase B were significantly correlated with pancreatic duct cannulation and opacification.CONCLUSION:Rendezvous ERCP reduces pancreatic enzyme leakage compared with conventional ERCP cannulation technique.Thus,laparo-endoscopic technique can be recommended with the ambition to minimise the risk for post ERCP pancreatitis.

Membrane Proteins as Potential Colon Cancer Biomarkers: Verification of 4 Candidates from a Secretome Dataset

Colorectal cancer (CRC) is an important health issue in Taiwan. There were over ten thousand newly diagnosed CRC patients each year. The outcome of late stage CRC still remains to be improved, and tumor markers are expected to improve CRC detection and management. From a colorectal cancer cell secretome database, we chose four proteins as candidates for clinical verification, including tumor-associated calcium signal transducer 2 (TROP2, TACSTD2), transmembrane 9 superfamily member 2 (TM9SF2), and tetraspanin-6 (TSPAN6), and tumor necrosis factor receptor superfamily member 16 (NGFR). Different groups of 30 CRC patients’ tissue samples collected from Chang Gung Memorial Hospital were analyzed by immunohistochemistry (IHC) for the four proteins, and the results were scored by pathologist. For all the four candidate proteins, marked differences of IHC score existed between tumor and adjacent non-tumor counterpart. However, there were only trends between higher protein expression levels and worse outcome. Three proteins (TROP2, TM9SF2 and NGFR) had trends between higher tissue expression and tumor stage or lymph node metastasis. Our study revealed that tissue expression of four proteins (TROP2, TM9SF2, TSPAN6, and NGFR) was markedly different between tumor and adjacent non-tumor counterparts. Overexpression of all these four proteins showed some trends with poorer survival.

Ultrasound-visualized nanocarriers with siRNA for targeted inhibition of M2-like TAM polarization to enhance photothermal therapy in NSCLC

Photothermal therapy(PTT)has received a lot of attention as a promising strategy for eliminating tumors quickly.However,the unavoidable inflammatory response during the treatment might result in a high concentration of M2-like tumor-associated macrophages(TAMs),increasing the risk of tumor recurrence and metastasis.To address this problem,gold-based nanocarriers(PGMP-small interfering RNA(siRNA)nanoparticles(NPs))containing STAT6siRNA,that inhibited M2-like TAM polarization,were designed and investigated for PTT and gene therapy of non-small cell lung cancer(NSCLC).In an NSCLC model,the nanocarriers demonstrated excellent siRNA delivery ability and a high gene transfection rate of up to 90%in macrophages,thus inhibiting the polarization of about 87%of M2-like TAMs and effectively suppressing the invasion and metastasis of NSCLC.Meanwhile,the unique gold nanosphere structure offered improved PTT and contrast-enhanced ultrasound imaging,thus contributing to the efficient elimination and real-time monitoring of the tumor tissues.These nanocarriers with combined gene and photothermal therapeutic capabilities improved the efficacy of single-modality treatment,and showed the potential to inhibit cancer cell recurrence and metastasis to ultimately cure NSCLC.