Recent advances in cancer-associated fibroblast: Biomarkers, signaling pathways, and therapeutic opportunities.

ABSTRACT: Anti-cancer therapies usually focus on tumor cells, but non-tumor stromal components in the tumor microenvironment also play vital roles in tumor initiation and progression, which may be the prognostic factors and potential therapeutic targets. Cancer-associated fibroblasts (CAFs) are the essential component in the tumor environment, exhibiting high heterogeneity in their cell origin and phenotype with diverse functions that influence tumor angiogenesis, immune systems, and metabolism. Single-cell RNA sequencing and genetically engineered mouse models have increased our understanding of CAF diversity, and many subtypes have been defined. However, the precise functions of these subtypes need to be studied and validated. Studies of signaling pathways and epigenetic changes in CAFs facilitate understanding of the phenotypes of CAFs and the crosstalk between tumor cells and CAFs to provide potential therapeutic targets. Some clinical trials, including phase III trials targeting CAFs, have been performed recently. However, few of these trials have generated promising results, which indicates that the complexity of CAFs in the tumor microenvironment remains largely unknown, and in-depth investigations of CAFs should be performed. This review summarizes the research on CAFs, focusing on the heterogeneity of their phenotypes and functions, specific signaling pathways, and the therapeutic strategies involving CAFs. Additionally, we briefly discuss the current technologies commonly used in CAF studies and describe the challenges and future perspectives of CAF research.

Effectiveness of no drainage after elective day-case laparoscopic cholecystectomy, even with intraoperative gallbladder perforation: a randomized controlled trial.

BACKGROUND: Laparoscopic cholecystectomy (LC) has been carried out as day-case surgery. Current guidelines do not mention the role of drainage after LC. In particular, data stay blank with no prospective study on drainage management when gallbladder perforation (GP) accidentally occurs intraoperatively. METHODS: A randomized controlled trial was conducted to compare clinical outcomes of drainage and no drainage after elective day-case LC. Intraoperative GP was recorded. The primary and secondary outcomes were major and minor complications, respectively. RESULTS: Two hundred patients were randomized. No major complications occurred in either group. In secondary outcomes, nausea/vomiting, pain, hospital stay, and cost were similar in the drainage group and no drainage group; postoperative fever, WBC, and CRP levels were significantly lower in the no drainage group. GP occurred in 32 patients. Male patients with higher BMI and CRP and abdominal pain within 1 month were more likely to occur GP. Subgroup analysis of GP, primary outcomes, and most secondary outcomes had no difference. Postoperative WBC and CRP were higher in the drainage group. Postoperative fever occurred in 63 patients. Univariate analysis of fever showed that blood loss, drainage, postoperative WBC, CRP, and hospital stay were significant. Multivariable logistic regression analysis demonstrated that drainage was an independent risk factor for fever after LC (OR 3.418, 95% CI 1.392-8.390; p = 0.007). CONCLUSIONS: No drainage after elective day-case LC is safe and associated with fewer complications, even in intraoperative GP. The trial proves that drainage is an independent risk factor for postoperative fever. The use of a drain after LC may lead to an unsuccessful day-case procedure by causing fever, elevated CRP, and extended hospital stay (NCT03909360).

Is it justified to assess the resectability of pancreatic cancer combined with biological and conditional factors?

Background: This study aimed to investigate the biological and conditional resectability criteria for pancreatic ductal adenocarcinoma (PDAC), as proposed by the International Association of Pancreatology (IAP), as well as to identify the role of biological and conditional factors in assessing the resectability of PDAC. Methods: The clinical data of PDAC patients who underwent upfront open/laparoscopic pancreaticoduodenectomy (PD/LPD) or distal pancreatectomy (DP/LDP) at our hospital between January 2013 to June 2019 were retrospectively analyzed. Patients who were diagnosed with anatomically resectable PDAC, as defined by National Comprehensive Cancer Network (NCCN) guideline of PDAC guideline Version 1.2020, were enrolled. Based on IAP-criteria, these patients were divided into two groups, including IAP-resectable (IAP-R) and IAP Borderline Resectable (IAP-BR). Clinical characteristics and outcomes were compared between the two groups. In order to identify independent biological and conditional predictors of recurrence-free survival (RFS) and overall survival (OS) of enrolled patients, an analysis was performed through the use of a Cox proportional-hazard model. Results: Overall, 97 patients were included in this study. Among them, 38 patients were IAP-R and 59 patients were IAP-BR. Compared to the IAP-R group, the IAP-BR group had a higher early recurrence rate (62.7% vs. 42.1%; P=0.047), and the median RFS (9.2 vs. 18.3 months, P<0.01) and OS (19.1 vs. 30.6 months, P<0.05) were also significantly worse. Preoperative CA19-9 serum levels that exceeded 500 U/mL and/or an imaging diagnosis of regional lymph nodes metastasis were independently associated with OS and RFS of anatomically resectable PDAC. Conclusions: The prognosis of patients with PDAC that undergo resection can be predicted more accurately by assessing the resectability of pancreatic cancer combined with anatomical and biological factors according to IAP criteria. Whether conditional factors should be included in the resectability criteria needs to be validated by prospective and large cohorts.

Gene expression profile of human colorectal cancer identified NKTR as a biomarker for liver metastasis.

OBJECTIVE: Liver metastasis is one of the prognostic factors of colorectal cancer (CRC). The aim of this study is to identify biomarkers that facilitate easier detection of liver metastasis. METHODS: Significance Analysis of Microarrays (SAM) and Array Data Analyzer (ADA) were applied used for the analysis of differentially differently expressed mRNAs. mRNA expression was verified by quantitative real-timer reverse transcriptiontase polymerase chain reaction (qRT-PCR). Immunohistochemistry were was used to show natural killer-tumor recognition (NKTR) expression in CRC. NKTR-knockdown CRC cells were constructed obtained by using short hairpin RNA (shRNA). Followed by CCK-8 assay, plate colony formation test, and transwell assay were used to evaluate the influence of NKTR on cell proliferation, migration, and invasion in vitro. RESULTS: SAM yielded showed 256 up-regulated and 224 down-regulated differentially differently expressed genes. Seven genes were identified by using ADA, tools and four genes were verified by using qRT-PCR. Three genes (metastasis associated lung adenocarcinoma transcript 1 (MALAT1), nuclear factor I/B (NKTR), and nuclear factor I/B (NFIB)) showed a statistically significant considerabley difference between CRC with and liver metastasis and CRC without liver metastasis. Immunohistochemical analysis showed that NKTR expression was much lower in primary CRC with liver metastasis than that in primary CRC without liver metastasis. The NKTR protein plays a role in the lytic function of natural killer (NK) cells and it has been rarely studied in the CRC. The down-regulation of NKTR by shRNA interference in CRC cells increased cell proliferation, migration, and invasion in vitro.

Lactate promotes the growth of patient-derived organoids from hepatopancreatobiliary cancers via ENO1/HIF1α pathway and does not affect their drug sensitivities.

The long culture duration of patient-derived organoids (PDOs) have severely limited their clinical applications. The aim of this study was to determine the effect of lactate supplementation on the growth, genetic profiles and drug sensitivities of PDOs from hepatopancreatobiliary tumors. LM3, Huh7, Panc02, and RBE cell lines were cultured as organoids in the presence or absence of lactate, and total protein was extracted to measure the expression of α-enolase (ENO1), hypoxia-inducible factor-1α (HIF1α), AKT, and PI3 kinase (PI3K). Thirteen hepatopancreatobiliary tumor specimens were collected during surgical resection and cultured as PDOs with or without L-lactate. Hematoxylin and eosin (H&E) staining and immunohistochemical staining were performed on the original tissues and PDOs to compare their pathological structures, and their genetic profiles were analyzed by whole-exome sequencing (WES). The sensitivity of the PDOs to gemcitabine, 5-fluorouracil, cisplatin, paclitaxel, ivosidenib, infigratinib, and lenvatinib were evaluated in terms of cell viability. Peripheral blood mononuclear cells (PBMCs) were isolated and co-cultured with PDOs to test the sensitivity of PDOs to tislelizumab. The addition of 20 mM lactate significantly promoted the growth of LM3 and Huh 7 organoids by 217% and 36%, respectively, compared to the control group, and the inhibition of lactate transporter decreased their growth. The HIF1α/ENO1/AKT/PI3K pathway was also activated by lactate. The inhibition of enolase also partly decreased the growth of organoids treated with lactate. Furthermore, 20 mM lactate increased the viability of 9 PDOs from 135% to 317% without affecting their pathological features. The genetic similarity, in terms of single nucleotide variations, insertions, and deletions, between original tissues and lactate-treated PDOs ranged from 83.2% to 94.1%, and that between the untreated and lactate-treated PDOs was at least 93.2%. Furthermore, the addition of lactate did not significantly change the dose-response curves of the PDOs to chemotherapeutic drugs, targeted drugs, and immune checkpoint inhibitor, especially for the drugs to which the cells were sensitive. Thus, lactate can be added to the culture medium of PDOs to promote their growth without altering their genetic profiles and drug sensitivities.

Establishment and drug screening of patient-derived extrahepatic biliary tract carcinoma organoids.

BACKGROUND: Patient-derived organoids (PDO) have been proposed as a novel in vitro method of drug screening for different types of cancer. However, to date, extrahepatic biliary tract carcinoma (eBTC) PDOs have not yet been fully established. METHODS: We collected six samples of gallbladder carcinoma (GBC) and one sample of extrahepatic cholangiocarcinoma (eCCA) from seven patients to attempt to establish eBTC PDOs for drug screening. We successfully established five GBC and one eCCA PDOs. Histological staining was used to compare structural features between the original tissues and cancer PDOs. Whole exome sequencing (WES) was performed to analyze the genetic profiles of original tissues and cancer PDOs. Drug screening, including gemcitabine, 5-fluorouracil, cisplatin, paclitaxel, infigratinib, and ivosidenib, was measured and verified by clinical effects in certain cases. RESULTS: Different PDOs exhibited diverse growth rates during in vitro culture. Hematoxylin and eosin staining demonstrated that the structures of most cancer PDOs retained the original structures of adenocarcinoma. Immunohistological and periodic acid-schiff staining revealed that marker expression in cancer PDOs was similar to that of the original specimens. Genetic profiles from the four original specimens, as well as paired cancer PDOs, were analyzed using whole exome sequencing. Three of the four PDOs exhibited a high degree of similarity when compared to the original specimens, except for GBC2 PDO, which only had a concordance of 74% in the proportion of single nucleotide polymorphisms in the coding sequence. In general, gemcitabine was found to be the most efficient drug for eBTC treatment, as it showed moderate or significant inhibitory impact on cancer growth. Results from drug screening were confirmed to a certain extent by three clinical cases. CONCLUSIONS: Our study successfully established a series of eBTC PDOs, which contributed to the field of eBTC PDOs. Additional enhancements should be explored to improve the growth rate of PDOs and to preserve their immune microenvironment.

The Cystic Fibrosis Transmembrane Conductance Regulator 470 Met Allele Is Associated with an Increased Risk of Chronic Pancreatitis in Both Asian and Caucasian Populations: A Meta-Analysis.

Background: The Met470Val polymorphism (1540A>G [rs213950]) within the cystic fibrosis transmembrane conductance regulator (CFTR) protein has been reported to be associated with chronic pancreatitis (CP). The results remain inconclusive, and therefore, we performed this meta-analysis to clarify the association between M470V and CP risk. Methodology/Results: We conducted a meta-analysis of 7 case-control studies, including a total of 1121 CP patients and 2209 controls from Asian and Caucasian populations. We calculated the odds ratio (OR) and 95% confidence intervals (95% CI). Met470Val was found to be significantly associated with an increased risk of CP under all the genetic models (M vs. V, OR = 1.260, 95% CI: 1.134-1.399; MV vs. VV, OR = 1.292, 95% CI: 1.091-1.530; MM vs. VV, OR = 1.579, 95% CI: 1.274-1.956; MV/MV vs. VV, OR = 1.366, 95% CI: 1.165-1.603; MM vs. MV/VV, OR = 1.346, 95% CI: 1.114-1.621). Met470Val was also found to be significantly associated with an increased risk of idiopathic CP (ICP) in allele contrast, codominant, and recessive models (M vs. V, OR = 1.298, 95% CI: 1.020-1.653; MV vs. VV, OR = 1.297, 95% CI: 1.074-1.566; MM vs. VV, OR = 1.473, 95% CI: 1.165-1.862; MM vs. MV/VV, OR = 1.254, 95% CI: 1.023-1.538). Conclusions: The CFTR 470 M allele is significantly associated with an increased risk of CP in both Asian and Caucasian populations. The CFTR 470 M allele is also significantly associated with risk of ICP.

Effects of modified FOLFOX-6 chemotherapy on cellular immune function in patients with gastric cancer.

Tumor immunosuppression serves an important role in the occurrence and development of gastric cancer. However, the effect of chemotherapy on the immune function of patients remains unclear. The present study aimed to investigate changes in cellular immune function and regulatory T cells (Tregs) in patients with gastric cancer prior to and following chemotherapy. In the peripheral blood of patients with gastric cancer, the percentage of CD4+ T cells was substantially decreased compared with that of healthy controls (11.39±5.91 vs. 22.34±3.37%, respectively; P<0.05). High frequencies of CD8+ T cells and Tregs were also observed in the peripheral blood of patients. Although the number of T cells decreased following chemotherapy (the proportions of CD4+ and CD8+ cells were 8.99±7.31 and 16.00±4.51%, respectively), the ratio of CD4+/CD8+ T cells increased (0.31±0.17 vs. 0.56±0.22; P<0.05). Furthermore, the level of C-C motif chemokine ligand 20 (CCL20) was increased in patients prior to chemotherapy compared with healthy controls. As the sole receptor for CCL20, a high level of expression of C-C motif chemokine receptor 6 on circulating Tregs was also identified in the patients, which decreased following chemotherapy. These results suggest that chemotherapy may efficiently promote cellular immune function and inhibit immunosuppression in patients with gastric cancer.

Anatomic isolated caudate lobectomy: Is it possible to establish a standard surgical flow?

AIM: To establish the surgical flow for anatomic isolated caudate lobe resection. METHODS: The study was approved by the ethics committee of the Second Affiliated Hospital Zhejiang University School of Medicine (SAHZU). From April 2004 to July 2014, 20 patients were enrolled who underwent anatomic isolated caudate lobectomy at SAHZU. Clinical and postoperative pathological data were analyzed. RESULTS: Of the total 20 cases, 4 received isolated complete caudate lobectomy (20%) and 16 received isolated partial caudate lobectomy (80%). There were 4 cases with the left approach (4/20, 20%), 6 cases with the right approach (6/20, 30%), 7 cases with the bilateral combined approach (7/20, 35%), 3 cases with the anterior approach (3/20, 15%), and the hanging maneuver was also combined in 2 cases. The median tumor size was 5.5 cm (2-12 cm). The median intra-operative blood loss was 600 mL (200-5700 mL). The median intra-operative blood transfusion volume was 250 mL (0-2400 mL). The median operation time was 255 min (110-510 min). The median post-operative hospital stay was 14 d (7-30 d). The 1- and 3-year survival rates for malignant tumor were 88.9% and 49.4%, respectively. CONCLUSION: Caudate lobectomy was a challenging procedure. It was demonstrated that anatomic isolated caudate lobectomy can be done safely and effectively.

Cancer-Associated Fibroblasts in Pancreatic Cancer Are Reprogrammed by Tumor-Induced Alterations in Genomic DNA Methylation.

Stromal fibrosis is a prominent histologic characteristic of pancreatic ductal adenocarcinoma (PDAC), but how stromal fibroblasts are regulated in the tumor microenvironment (TME) to support tumor growth is largely unknown. Here we show that PDAC cells can induce DNA methylation in cancer-associated fibroblasts (CAF). Upon direct contact with PDAC cells, DNA methylation of SOCS1 and other genes is induced in mesenchymal stem cells or in CAF that lack SOCS1 methylation at baseline. Silencing or decitabine treatment to block the DNA methylation enzyme DNMT1 inhibited methylation of SOCS1. In contrast, SOCS1 gene methylation and downregulation in CAF activated STAT3 and induced insulin-like growth factor-1 expression to support PDAC cell growth. Moreover, CAF facilitated methylation-dependent growth of PDAC tumor xenografts in mice. The ability of patient-derived CAF with SOCS1 methylation to promote PDAC growth was more robust than CAF without SOCS1 methylation. Overall, our results reveal how PDAC cells can reprogram CAF to modify tumor-stromal interactions in the TME, which promote malignant growth and progression. Cancer Res; 76(18); 5395-404. ©2016 AACR.

Identification of serologic biomarkers for predicting microvascular invasion in hepatocellular carcinoma.

Microvascular invasion (MVI) of hepatocellular carcinoma (HCC) is a major risk factor for early recurrence and poor survival after curative surgical therapies. However, MVI can only be diagnosed by pathological examination following resection. The aim of this study is to identify serologic biomarkers for predicting MVI preoperatively to help facilitate treatment decisions. We used the sero-proteomic approach to identify antigens that induce corresponding antibody responses either specifically in the serum from MVI (+) patients or from MVI (-) patients. Six antigens were subsequently identified as HSP 70, HSP 90, alpha-enolase (Eno-1), Annexin A2, glutathione synthetase and beta-actin by mass spectrometry. The antibodies titers in sera corresponding to four of these six antigens were measured by ELISA and compared between 35 MVI (+) patients and 26 MVI (-) patients. The titers of anti-HSP 70 antibodies were significantly higher in MVI (-) patients than those in MVI (+) patients; and the titers of anti-Eno-1 antibodies were significantly lower in MVI (-) patients than those in MVI (+) patients. The results were subjected to multivariate analysis together with other clinicopathologic factors, suggesting that antibodies against HSP 70 and Eno-1 in sera are potential biomarkers for predicting MVI in HCC prior to surgical resection. These biomarkers should be further investigated as potential therapeutic targets.

Semaphorin 3D autocrine signaling mediates the metastatic role of annexin A2 in pancreatic cancer.

Most patients with pancreatic ductal adenocarcinoma (PDA) present with metastatic disease at the time of diagnosis or will recur with metastases after surgical treatment. Semaphorin-plexin signaling mediates the migration of neuronal axons during development and of blood vessels during angiogenesis. The expression of the gene encoding semaphorin 3D (Sema3D) is increased in PDA tumors, and the presence of antibodies against the pleiotropic protein annexin A2 (AnxA2) in the sera of some patients after surgical resection of PDA is associated with longer recurrence-free survival. By knocking out AnxA2 in a transgenic mouse model of PDA (KPC) that recapitulates the progression of human PDA from premalignancy to metastatic disease, we found that AnxA2 promoted metastases in vivo. The expression of AnxA2 promoted the secretion of Sema3D from PDA cells, which coimmunoprecipitated with the co-receptor plexin D1 (PlxnD1) on PDA cells. Mouse PDA cells in which SEMA3D was knocked down or ANXA2-null PDA cells exhibited decreased invasive and metastatic potential in culture and in mice. However, restoring Sema3D in AnxA2-null cells did not entirely rescue metastatic behavior in culture and in vivo, suggesting that AnxA2 mediates additional prometastatic mechanisms. Patients with primary PDA tumors that have abundant Sema3D have widely metastatic disease and decreased survival compared to patients with tumors that have relatively low Sema3D abundance. Thus, AnxA2 and Sema3D may be new therapeutic targets and prognostic markers of metastatic PDA.

Exome capture sequencing of adenoma reveals genetic alterations in multiple cellular pathways at the early stage of colorectal tumorigenesis.

Most of colorectal adenocarcinomas are believed to arise from adenomas, which are premalignant lesions. Sequencing the whole exome of the adenoma will help identifying molecular biomarkers that can predict the occurrence of adenocarcinoma more precisely and help understanding the molecular pathways underlying the initial stage of colorectal tumorigenesis. We performed the exome capture sequencing of the normal mucosa, adenoma and adenocarcinoma tissues from the same patient and sequenced the identified mutations in additional 73 adenomas and 288 adenocarcinomas. Somatic single nucleotide variations (SNVs) were identified in both the adenoma and adenocarcinoma by comparing with the normal control from the same patient. We identified 12 nonsynonymous somatic SNVs in the adenoma and 42 nonsynonymous somatic SNVs in the adenocarcinoma. Most of these mutations including OR6X1, SLC15A3, KRTHB4, RBFOX1, LAMA3, CDH20, BIRC6, NMBR, GLCCI1, EFR3A, and FTHL17 were newly reported in colorectal adenomas. Functional annotation of these mutated genes showed that multiple cellular pathways including Wnt, cell adhesion and ubiquitin mediated proteolysis pathways were altered genetically in the adenoma and that the genetic alterations in the same pathways persist in the adenocarcinoma. CDH20 and LAMA3 were mutated in the adenoma while NRXN3 and COL4A6 were mutated in the adenocarcinoma from the same patient, suggesting for the first time that genetic alterations in the cell adhesion pathway occur as early as in the adenoma. Thus, the comparison of genomic mutations between adenoma and adenocarcinoma provides us a new insight into the molecular events governing the early step of colorectal tumorigenesis.

Vaccine therapy for pancreatic cancer.

Pancreatic cancer is a lethal disease and currently available therapies have significant limitations. Pancreatic cancer is thus an ideal setting for the development of novel treatment modalities such as immunotherapy. However, relevant obstacles must be overcome for immunotherapeutic regimens against pancreatic cancer to be successful. Vaccine therapy relies on the administration of biological preparations that include an antigen that (at least ideally) is specifically expressed by malignant cells, boosting the natural ability of the immune system to react against neoplastic cells. There are a number of ways to deliver anticancer vaccines. Potent vaccines stimulate antigen presentation by dendritic cells, hence driving the expansion of antigen-specific effector and memory T cells. Unlike vaccines given as a prophylaxis against infectious diseases, anticancer vaccines require the concurrent administration of agents that interfere with the natural predisposition of tumors to drive immunosuppression. The safety and efficacy of vaccines against pancreatic cancer are nowadays being tested in early phase clinical trials.

Adenosquamous carcinoma of the uncinate process of the pancreas with synchronous gastrointestinal stromal tumor of the stomach: Case report and review of the literature.

Recently, the coexistence of gastrointestinal stromal tumors (GISTs) with other neoplasms has been studied with increasing frequency. Coexistence of pancreatic cancer with GISTs remains a rarity; however, here, we report a very rare case of adenosquamous carcinoma (ASC) of the uncinate process of the pancreas with synchronous GISTs of the stomach in a 62-year-old female. The patient presented with epigastric discomfort and vomiting. Radiographic imaging revealed two masses; one located at the body of the stomach and the other located at the uncinate process of the pancreas. Intraoperatively, a fine needle aspiration biopsy was conducted in the uncinate process of the pancreas, which revealed the malignancy of the masses. A pancreaticoduodenectomy and partial gastrectomy were then conducted, and subsequent pathological examinations identified an ASC of the pancreas and a GIST of the stomach. In our case, contrary to the majority of previous cases of synchronous GISTs and other malignancies, GIST was not an incidental finding. The initial suspicion on the GIST as the underlying cause of clinical symptoms led to the discovery of the ASC of the uncinate process of the pancreas.

MicroRNA-9 up-regulation is involved in colorectal cancer metastasis via promoting cell motility.

Human microRNA-9 (miR-9) has been reported to be involved in the metastasis of several malignancies including brain breast cancer. However, its role in the metastasis of colorectal cancer (CRC) remains to be revealed. Here, we evaluated miR-9 expression in metastatic CRC and investigated its effects on the motility and proliferation of RKO cells. The expressions of miR-9 in 15 primary CRC specimens without distant metastasis (NM group) and 10 primary CRC specimens (M group) with distant metastasis (M group) were determined by quantitative real-time PCR. The alternations in the motility and morphology of RKO cells before and after miR-9 transfection were analyzed by migration assay and F-actin staining. The relationship between miR-9 and α-catenin was identified by Western blotting. Cell growth was examined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide) assay. Significant difference of miR-9 expression was observed in M group compared to the NM group (P < 0.001). Ectopic expression of miR-9 enhanced the motility of RKO cells as well as changed their morphological appearance, while cell growth remained unchanged. The overexpression of miR-9 could also down-regulate α-catenin expression. These data suggest that miR-9 may potentially participate in the metastatic process of CRC though facilitating cell motility.