Background According to current guideline, patients with resected specimens showing high-risk features are recommended additional surgery after local excision (LE) of T1 colorectal cancer, despite the low incidence of recurrence. However, surgical resection in patients with low rectal cancer (RC) is challenging and may compromise anal function, leading to a low quality of life. To reduce unnecessary surgical resection in these patients, we used artificial intelligence (AI) to develop and validate a prediction model for the risk of recurrence after LE. Materials and methods We constructed an artificial neural network (ANN) to predict recurrence using pathological images from endoscopically or transanal surgically resected T1 RC specimens. Data were retrospectively obtained from two hospitals between 2001 and 2015. The model was constructed using 496 images obtained from the Guangdong Provincial People's Hospital (GDPH), and then validated using independent external datasets (150 images from Sun Yat-sen Memorial Hospital [SYSMH]) to verify its generalizability. Results The ANN model yielded good discrimination, achieving areas under the receiver operating characteristic curves (AUC) of 0.979 in the training cohort (GDPH). The AUC for the validation cohort (SYSMH) was 0.978. More importantly, the AI-based prediction model avoided more than 34.9 % of unnecessary additional surgeries compared with the current US guideline in all enrolled patients. Conclusions We propose a novel ANN model for the risk of recurrence prediction in patients with T1 RC to provide physicians and patients guidance for decisions after LE. Furthermore, this may lead to a reduction in unnecessary invasive surgeries in patients with T1 RC.

Pancreatic cancer (PC) is among the most aggressive malignancies, with a five-year survival rate of <7% in China. The substantial stromal component and activation of epithelial-mesenchymal transition (EMT) in PC contribute to drug resistance and poor outcomes. Between January 2017 and December 2020, 62 pancreatic specimens were obtained via surgical resection from two hospitals. In alignment with the GEPIA database, the expression levels of a disintegrin and metalloproteinase 12 (ADAM12) and heparin-binding epidermal growth factor (HB-EGF) were significantly elevated in 43 PC tissues compared with 19 benign pancreatic masses. Furthermore, elevated expression of ADAM12 and HB-EGF was significantly associated with lymph node metastasis, advanced TNM stage, and reduced survival rates. Additionally, high ADAM12 expression was correlated with the upregulation of EGFR and EMT markers. Collectively, the present findings suggested that ADAM12 is involved in PC progression and may facilitate the shedding of HB-EGF, thereby inducing EMT through the EGFR pathway. These results suggest that targeting the ADAM12/HB-EGF/EGFR signaling pathway could represent a potential therapeutic strategy, warranting further in vivo and in vitro investigations to elucidate the underlying mechanisms.

Correction for 'Nanoparticles modified by triple single chain antibodies for MRI examination and targeted therapy in pancreatic cancer' by Jinmao Zou et al., Nanoscale, 2020, 12, 4473-4490, https://doi.org/10.1039/C9NR04976B.

BACKGROUND:Pancreatic cancer (PC) is characterized by abnormally fibrotic mesenchyme, which notably influences on the effectiveness of immunotherapy. Low-dose arsenic trioxide (ATO, 1.0 μM) can inhibit the activation of pancreatic stellate cells (PSCs) and affect fibrosis, which is a potential strategy for enhancing the sensitivity to immunotherapy. METHODS:Extracellular matrix (ECM) models were employed to assess the regulatory effects of ATO on ECM and peripheral blood mononuclear cells. Orthotopic C57BL/6J models were utilized to evaluate the influence of ATO on CD8+T cell infiltration and immunotherapy in PC. Additionally, nanomaterials loaded with ATO designed to specifically target PSCs (scAbFAP-α-HMSNs-PAA-ATO) were produced to enhance targeting effects of ATO. RESULTS:Low-dose ATO (1.0 μM) suppressed PSCs activation, exhibiting potential for synergistic immunotherapy. Under low-dose ATO intervention, ECM underwent remodeling, leading to increases in CD8+T cell infiltration, thereby enhancing anti-PD-L1 therapy effect. We further demonstrated that low-dose ATO remodeled ECM by regulating the expression of LOXL3 in PSCs. scAbFAP-α-HMSNs-PAA-ATO exhibited improved targeting capabilities, and enhanced capacity to inhibit fibrosis and sensitize immunotherapy. CONCLUSIONS:Our research reveals that low-dose ATO, by regulating LOXL3, remodels the ECM and enhances CD8+T cell infiltration, thus sensitizing the efficacy of immunotherapy, which provides a novel strategy for comprehensive treatment to PC.

Glycolytic metabolism is a hallmark of pancreatic ductal adenocarcinoma (PDAC), and tumor-associated stromal cells play important roles in tumor metabolism. We previously reported that tumor-associated macrophages (TAMs) facilitate PDAC progression. However, little is known about whether TAMs are involved in regulating glycolysis in PDAC. Here, we found a positive correlation between CD68+ TAM infiltration and FDG maximal standardized uptake (FDG SUVmax) on PET-CT images of PDAC. We discovered that the glycolytic gene set was prominently enriched in the high TAM infiltration group through Gene Set Enrichment Analysis using The Cancer Genome Atlas database. Mechanistically, TAMs secreted IL-8 to promote GLUT3 expression in PDAC cells, enhancing tumor glycolysis both in vitro and in vivo, whereas this effect could be blocked by the IL-8 receptor inhibitor reparixin. Furthermore, IL-8 promoted the translocation of phosphorylated STAT3 into the nucleus to activate the GLUT3 promoter. Overall, we demonstrated that TAMs boosted PDAC cell glycolysis through the IL-8/STAT3/GLUT3 signaling pathway. Our cumulative findings suggest that the abrogation of TAM-induced tumor glycolysis by reparixin might exhibit an antitumor impact and offer a potential therapeutic target for PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is characterised by immune evasion that contribute to poor prognosis. Cancer-associated fibroblasts (CAFs) play a pivotal role in orchestrating the PDAC tumour microenvironment. We investigated the role of CAF-derived extracellular vesicle (EV)-packaged long non-coding RNAs (lncRNAs) in immune evasion and explored gene therapy using engineered EVs loading small interfering RNAs (siRNAs) as a potential therapeutic strategy. Our findings highlight the significance of EV-packaged lncRNA RP11-161H23.5 from CAF in promoting PDAC immune evasion by downregulating HLA-A expression, a key component of antigen presentation. Mechanistically, RP11-161H23.5 forms a complex with CNOT4, a subunit of the mRNA deadenylase CCR4-NOT complex, enhancing the degradation of HLA-A mRNA by shortening its poly(A) tail. This immune evasion mechanism compromises the anti-tumour immune response. To combat this, we propose an innovative approach utilising engineered EVs as natural and biocompatible nanocarriers for siRNA-based gene therapy and this strategy holds promise for enhancing the effectiveness of immunotherapy in PDAC. Overall, our study sheds light on the critical role of CAF-derived EV-packaged lncRNA RP11-161H23.5/CNOT4/HLA-A axis in PDAC immune evasion and presents a novel avenue for therapeutic intervention.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies, highlighting the urgent need to elucidate the underlying oncogenic mechanisms. VIRMA is a classic isoform of methyltransferases that participates in epigenetic transcriptomic modification in eukaryotic mRNAs. However, the exact roles of VIRMA in PDAC remain unclear. Here, we identified that VIRMA is highly expressed in PDAC, and histone modifications of the promoter may partly account for this dysregulation. Moreover, VIRMA is closely related to glycolysis and poor prognosis in PDAC. We further determined that STRA6 is a direct downstream target of VIRMA in PDAC by RNA sequencing (RNA-seq) and m6A sequencing (m6A-seq). VIRMA is involved in gene expression regulation via 3’ UTR targeting of STRA6 mRNA. Furthermore, the m6A reader IGF2BP2 was shown to critically contribute to the stability of STRA6 mRNA. We describe the role of VIRMA in promoting signaling via the STRA6/STAT3 axis, which results in increased levels of HIF-1α, a key activator of glycolysis. In vivo and in vitro experiments reveal that the VIRMA-STRA6-STAT3-HIF-1α axis plays an instrumental role in glycolysis and tumor progression in PDAC. In conclusion, we demonstrate that VIRMA can increase glycolysis in PDAC by upregulating STRA6, a cell surface membrane protein that stimulates the STAT3 pathway, thereby activating HIF-1α and leading to pancreatic cancer malignancy. Overall, our data strongly suggest that the VIRMA-STRA6-STAT3-HIF-1α axis is a viable therapeutic target in PDAC.

Colon cancer, a frequently encountered malignancy, exhibits a comparatively poor survival prognosis. Perineural invasion (PNI), highly correlated with tumor progression and metastasis, is a substantial effective predictor of stage II-III colon cancer. Nonetheless, the lack of effective and facile predictive methodologies for detecting PNI prior operation in colon cancer remains a persistent challenge. Pre-operative computer tomography (CT) images and clinical data of patients diagnosed with stage II-III colon cancer between January 2015 and December 2023 were obtained from two sub-districts of Sun Yat-sen Memorial Hospital (SYSUMH). The LASSO/RF/PCA filters were used to screen radiomics features and LR/SVM models were utilized to construct radiomics model. A comprehensive model, shown as nomogram finally, combining with radiomics score and significant clinical features were developed and validated by area under the curve (AUC) and decision curve analysis (DCA). The total cohort, comprising 426 individuals, was randomly divided into a development cohort and a validation cohort as a 7:3 ratio. Radiomics scores were extracted from LASSO-SVM models with AUC of 0.898/0.726 in the development and validation cohorts, respectively. Significant clinical features (CA199, CA125, T-stage, and N-stage) were used to establish combining model with radiomics scores. The combined model exhibited superior reliability compared to single radiomics model in AUC value (0.792 vs. 0.726, p = 0.003) in validation cohorts. The radiomics-clinical model demonstrated an AUC of 0.918/0.792, a sensitivity of 0.907/0.813 and a specificity of 0.804/0.716 in the development and validation cohorts, respectively. The study developed and validated a predictive nomogram model combining radiomics scores and clinical features, and showed good performance in predicting PNI pre-operation in stage II-III colon cancer patients.

BACKGROUND:Perineural invasion (PNI) has a high incidence and poor prognosis in pancreatic ductal adenocarcinoma (PDAC). Our study aimed to identify the underlying molecular mechanism of PNI and propose effective intervention strategies.METHODS:To observe PNI in vitro and in vivo, a Matrigel/ dorsal root ganglia (DRG) model and a murine sciatic nerve invasion model were respectively used. Magnetic resonance (MR) imaging and positron emission tomography/computed tomography (PET-CT) imaging were also used to evaluate tumor growth. Publicly available datasets and PDAC tissues were used to verify how the nerve cells regulate PDAC cells' PNI.RESULTS:Our results showed that glutamate from nerve cells could cause calcium influx in PDAC cells via the N-methyl-d-aspartate receptor (NMDAR), subsequently activating the downstream Ca2+ dependent protein kinase CaMKII/ERK-MAPK pathway and promoting the mRNA transcription of gene METTL3. Next, METTL3 upregulates the expression of hexokinase 2 (HK2) through N6-methyladenosine (m6A) modification in mRNA, enhances the PDAC cells' glycolysis, and promotes PNI. Furthermore, the IONPs-PEG-scFvCD44v6-scAbNMDAR2B nanoparticles dual targeting CD44 variant isoform 6 (CD44v6) and t NMDAR subunit 2B (NMDAR2B) on PDAC cells were synthesized and verified showing a satisfactory blocking effect on PNI.CONCLUSIONS:Here, we firstly provided evidence that glutamate from the nerve cells could upregulate the expression of HK2 through mRNA m6A modification via NMDAR2B and downstream Ca2+ dependent CaMKII/ERK-MAPK pathway, enhance the glycolysis in PDAC cells, and ultimately promote PNI. In addition, the dual targeting nanoparticles we synthesized were verified to block PNI effectively in PDAC.

Nearly one fourth of patients with pancreatic ductal adenocarcinoma (PDAC) occur to liver metastasis after surgery, and liver metastasis is a risk factor for prognosis for those patients with surgery therapy. However, there is no effective way to predict liver metastasis post-operation. Clinical data and preoperative magnetic resonance imaging (MRI) of PDAC patients diagnosed between July 2010 and July 2020 were retrospectively collected from three hospital centers in China. The significant MRI radiomics features or clinicopathological characteristics were used to establish a model to predict liver metastasis in the development and validation cohort. A total of 204 PDAC patients from three hospital centers were divided randomly (7:3) into development and validation cohort. Due to poor predictive value of clinical features, MRI radiomics model had similar receiver operating characteristics curve (ROC) value to clinical-radiomics combing model in development cohort (0.878 vs. 0.880, p = 0.897) but better ROC in validation dataset (0.815 vs. 0.732, p = 0.022). Radiomics model got a sensitivity of 0.872/0.750 and a specificity of 0.760/0.822 to predict liver metastasis in development and validation cohort, respectively. Among 54 patients randomly selected with post-operation specimens, fibrosis markers (α-smooth muscle actin) staining was shown to promote radiomics model with ROC value from 0.772 to 0.923 (p = 0.049) to predict liver metastasis. This study developed and validated an MRI-based radiomics model and showed a good performance in predicting liver metastasis in resectable PDAC patients.

BACKGROUND:Intrahepatic cholangiocarcinoma (ICC) is a malignant disease characterized by onset occult, rapid progression, high relapse rate, and high mortality. However, data on how the tumor microenvironment (TME) regulates ICC metastasis at the transcriptomic level remains unclear. This study aimed to explore the mechanisms and interactions between hepatocytes and ICC cells.METHODS:We analyzed the interplay between ICC and liver microenvironment through cytokine antibody array analysis. Then we investigated the role of N6-methyladenosine (m6A) modification and the downstream target in vitro, in vivo experiments, and in clinical specimens.RESULTS:Our study demonstrated that cytokine CCL3, which is secreted by hepatocytes, promotes tumor metastasis by regulating m6A modification via vir-like m6A methyltransferase associated (VIRMA) in ICC cells. Moreover, immunohistochemical analyses showed that VIRMA correlated with poor outcomes in ICC patients. Finally, we confirmed both in vitro and in vivo that CCL3 could activate VIRMA and its critical downstream target SIRT1, which fuels tumor metastasis in ICC.CONCLUSIONS:In conclusion, our results enhanced our understanding of the interaction between hepatocytes and ICC cells, and revealed the molecular mechanism of the CCL3/VIRMA/SIRT1 pathway via m6A-mediated regulation in ICC metastasis. These studies highlight potential targets for the diagnosis, treatment, and prognosis of ICC.

BackgroundThis study aims to (1) identify preoperative testing-based characteristics associated with enhanced prognosis and survival for cholangiocarcinoma patients, and (2)create a distinctive nomogram to anticipate each patient's cancer-specific survival (CSS).MethodsRetrospective analysis was performed on 197 CCA patients who underwent radical surgery at Sun Yat-sen Memorial Hospital; they were divided into a 131-person "training cohort" and a 66-person "internal validation cohort." The prognostic nomogram was created following a preliminary Cox proportional hazard regression search for independent factors influencing the patients' CSS. Its applicable domain was examined via an external validation cohort, which included 235 patients from the Sun Yat-sen University Cancer Center.ResultsThe median follow-up period for the 131 patients in the training group was 49.3 months (range, 9.3 to 133.9 months). One-, three-, and five-year CSS rates were 68.7%, 24.5%, and 9.2%, respectively, with the median CSS length being 27.4 months (range: 1.4 to 125.2 months). PLT, CEA, AFP, tumor location, differentiation, lymph node metastasis, chemotherapy, and TNM stage were determined to be independent risk factors for CCA patients by univariate and multivariate Cox proportional hazard regression analysis. We were able to accurately predict postoperative CSS after incorporating all of these characteristics into a nomogram. The AJCC's 8th edition staging method's C-indices were statistically substantially (P < 0.001) lower than the nomogram's C-indices (0.84, 0.77, and 0.74 in the training, internal and external validation cohorts respectively).ConclusionsA realistic and useful model for clinical decision-making and the optimization of therapy is presented as a nomogram that includes serum markers and clinicopathologic features for predicting postoperative survival in cholangiocarcinoma.

Pancreatic adenocarcinoma (PAAD) is a digestive tumor with extremely high malignancy. Previous studies have reported that Glucose transporter 1 (GLUT1) contributes to the aggressive tumor progression in various cancer types and indicates an unfavorable prognosis. However, the function of GLUT1 in PAAD remains largely unclear. Through pan-cancer analysis of GLUT1 expression, GLUT1 expression was significantly higher in several cancer types including PAAD. Survival analysis based on the GLUT1 expression showed that GLUT1 could serve as a predictor of poor prognosis. We further predicted and screened the candidate non-coding RNAs (ncRNAs) upstream of the GLUT1 mRNA through correlation analysis, and found that the CASC19/miR-140-5p axis contributing to the regulation of GLUT1 expression. Our study suggested a link exists between GLUT1 expression and selected immunity-related indicators. Subsequent analysis revealed overexpression of GLUT1 in pancreatic cancer specimens and patients with highly expressed GLUT1 expression had worse prognosis. Based on the significantly different expression of GLUT1, the possibility that GLUT1 participated in tumor progression was identified. Using online public databases, genes co-expressed with GLUT1 were screened and enriched to metastasis-related pathways by enrichment analysis. Additionally, functional assays verified that GLUT1 could function in the metastatic process of PAAD cancer cells. Therefore, we proposed that GLUT1 might serve as a role in tumor immunity and tumor metastasis, and was expected to be a prognostic factor in PAAD.

目的 探讨铁死亡相关基因对胰腺癌患者预后预测的价值,并进一步探索其分子机制.方法 从公共数据库中下载胰腺癌患者的基因表达及相应的临床病理数据,采用R软件"limma"包筛选胰腺癌组织中差异表达基因,使用单因素COX回归分析筛选预后相关的铁死亡相关基因,利用LASSO COX回归分析构建预后模型.通过Kaplan-Meier生存分析检验该模型在预测胰腺癌预后中的意义,用ROC曲线评估模型预测胰腺癌预后的准确性.在ICGC验证队列中通过Kaplan-Meier生存分析、ROC曲线评估该模型在外部队列中的预测价值.依照风险值中位数,将TCGA训练队列分为两组,比较其基因表达差异、差异基因富集通路差异,评估免疫细胞浸润丰度.结果 通过单因素COX回归分析,37个铁死亡相关基因被鉴定为预后基因(P<0.05).利用LASSO COX回归分析,构建了一个基于15个铁死亡相关基因的预后预测模型.根据模型计算将队列中所有患者分为高风险组和低风险组,Kaplan-Meier生存分析表明高风险组的胰腺癌患者较低风险组生存时间短(HR=2.16,P<0.05).ROC曲线分析证明了预测模型在胰腺癌预后预测中的准确性:ROC曲线下面积值分别为0.74(1年)、0.82(3年)、0.88(5年).对高低风险组差异表达基因进行功能富集分析,发现两组胰腺癌患者免疫微环境之间存在差异,高风险组中幼稚B细胞、浆细胞、CD8+T细胞浸润程度较低风险组低,但M0和M1巨噬细胞的浸润程度高(P<0.05).此外,在PD-1和CTLA4在TCGA-PAAD队列低危组和高危组中的表达中,TCGA-PAAD队列低风险组患者中PD-1和CTLA4的表达水平较高风险组患者更高(P<0.05).结论 本研究构建了胰腺癌中铁死亡相关基因预后预测模型,具有预测胰腺癌患者预后的作用,并发现TCGA队列中高低风险组免疫微环境存在差异,可为免疫治疗提供参考.

Perineural invasion (PNI) occurs in most pancreatic ductal adenocarcinomas (PDACs). The relationship between cancer cells and peripheral nerves, however, is unknown. Therefore, we focused on the cooperation of PDAC cells and peripheral nerve astrocytes, Schwann cells (SCs), in PNI. The mutual tumor-supportive secretory cytokines between SCs (sNF96.2) and PDAC cells (PANC-1, BxPC-3) were screened by human cytokine arrays and verified. The prognostic value of selected cytokines and SC-associated markers was confirmed in PDAC patients. TIMP1 and CCL7 were found to form a paracrine feedback loop between PDAC cells and SCs. PDAC cell-derived TIMP1 promotes SCs proliferation and migration via CD63/PI3K/AKT signaling. CCL7 secreted from SCs enhances PDAC cell migration, invasion and expression of TIMP1 via CCR2/STAT3. PDAC cell-SC cooperation in PNI was blocked when TIMP1 knockdown in vitro and in vivo. Finally, TIMP1, CCL7 and SC-associated markers were correlated with PNI and prognosis in PDAC patients. In conclusion, SCs collaborate with PDAC cells through the TIMP1-CCL7 paracrine feedback loop to promote PNI. TIMP1 knockdown in PDAC cells suppresses PNI. Strategies to disrupt the TIMP1-CCL7 feedback loop might be developed to inhibit PNI in PDAC.

Pancreatic stellate cells (PSCs) constitute the fibrotic tumor microenvironment composed of the stroma matrix, which blocks the penetration of gemcitabine (GEM) in pancreatic adenocarcinoma (PDAC) and results in chemoresistance. We analyzed the expression of α-SMA, collagen type I, and fibronectin by immunohistochemistry of pancreatic cancer tissues and demonstrated that the abundant interstitial stroma is associated with dismal survival. Two desmoplastic pancreatic tumor models are treated with arsenic trioxide (ATO) and GEM to confirm the sensitizing effect of ATO on GEM. RNA-seq was performed to analyze the potential fibrotic genes regulated by ATO. Western blotting, CCK-8 methods, colony formation, and wound healing and transwell assays were utilized to verify that ATO attenuates the tumor-promoting ability of PSCs by inhibiting its activation and decreasing matrix secretion via the PI3K/AKT/AP4/galectin-1 pathway. Furthermore, we developed targeted ATO-loaded nanoparticles self-assembled by poly (D,L-lactide) and poly(ethylene glycol) (PEG-PDLLA) and modified by the single-chain antibody of FAP-α (scAbFAP-α) (scAb-ATO-NPs) to promote the delivery efficiency of ATO to PSCs and enhance anti-tumor effects with gemcitabine. Herein, we elucidate the mechanism of how ATO inhibits the activation of PSCs and enhances the therapeutic effect of GEM. We propose a novel cocktail therapy including scAb-ATO-NPs and GEM, indicating a new perspective in the treatment of PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is one of the highest mortalities malignant tumors, which is characterized by difficult diagnosis, rapid progression and high recurrence rate. Nevertheless, PDAC responds poorly to conventional therapies, which highlights the urgency to identify novel prognostic and therapeutic targets. LEMT2 was a newly discovered protein-encoding gene with little cancer research and an unclear mechanism. Thus, this study aimed to illustrate LETM2 as the crucial oncogene for tumor progression in PDAC. In this study, we analyzed the expression level and prognostic value of LETM2 in multiple cancers using pan-cancer analysis. The analyses based on the TCGA-GTEx dataset indicated that the LETM2 expression was obviously elevated in several cancers, and it was the most significantly related to the dismal prognosis of PDAC. Subsequently, we demonstrated the functional role and mechanism of LETM2 by clinical sample evaluation, and in in vitro and in vivo experiments. Immunohistochemical analyses showed that high expression of LETM2 was correlated with poor outcomes of PDAC. Moreover, we demonstrated that LETM2 knockdown significantly inhibited tumor proliferation and metastasis, and promoted cell apoptosis, while LETM2 overexpression exerted the opposite effects. Finally, the impairment caused by LETM2-knockdown could be recovered via excitation of the PI3k-Akt pathway in vitro and in vivo animal models, which suggested that LETM2 could activate the downstream PI3K-Akt pathway to participate in PDAC progression. In conclusion, the study enhanced our understanding of LETM2 as an oncogene hallmark of PDAC. LETM2 may facilitate tumor progression by activating the PI3K-Akt signaling pathway, which provides potential targets for the diagnosis, treatment, and prognosis of pancreatic cancer.

目的 构建人永生化胰腺星形细胞(PSCs)系并探讨其生物学功能.方法 采用outgrowth方法提取人原代胰腺星形细胞(hPSCs),慢病毒感染方法导入SV40LT基因和hTERT基因,嘌呤霉素筛选稳定高表达两个基因的细胞(im PSCs),qRT-PCR和Western bolt方法检测表达水平;CCK8法、核型分析、免疫荧光、体内成瘤实验检测im PSCs生长曲线,染色体数目,胶原基质蛋白表达和有无恶性表型转化;共培养方法、CCK8法、细胞划痕、迁移侵袭方法检测imPSCs促瘤作用.结果 hPSCs呈多角形和梭形,慢病毒感染的im PSCs,显微镜下有亮绿色荧光;im PSCs细胞高表达SV40LT基因和hTERT基因;im PSCs生长曲线呈典型的"S"型,染色体数目增多,体内不能成瘤,表达α-SMA、Collagen I、Fibronectin,具有PSCs的生物学功能和特点;其与胰腺癌细胞共培养后,明显促进肿瘤生长,与人原代PSCs功能相当.结论 采用慢病毒转染SV40LT基因和hTERT基因可成功构建人胰腺星形细胞永生化细胞系,为胰腺星形细胞本身及胰腺癌微环境相关研究提供实验材料和模型.

Objective Growing evidence has highlighted that the immune and stromal cells that infiltrate in pancreatic cancer microenvironment significantly influence tumor progression. However, reliable microenvironment-related prognostic gene signatures are yet to be established. The present study aimed to elucidate tumor microenvironment-related prognostic genes in pancreatic cancer. Methods We applied the ESTIMATE algorithm to categorize patients with pancreatic cancer from TCGA dataset into high and low immune/stromal score groups and determined their differentially expressed genes. Then, univariate and LASSO Cox regression was performed to identify overall survival-related differentially expressed genes (DEGs). And multivariate Cox regression analysis was used to screen independent prognostic genes and construct a risk score model. Finally, the performance of the risk score model was evaluated by Kaplan-Meier curve, time-dependent receiver operating characteristic and Harrell's concordance index. Results The overall survival analysis demonstrated that high immune/stromal score groups were closely associated with poor prognosis. The multivariate Cox regression analysis indicated that the signatures of four genes, including TRPC7, CXCL10, CUX2, and COL2A1, were independent prognostic factors. Subsequently, the risk prediction model constructed by those genes was superior to AJCC staging as evaluated by time-dependent receiver operating characteristic and Harrell's concordance index, and both KRAS and TP53 mutations were closely associated with high risk scores. In addition, CXCL10 was predominantly expressed by tumor associated macrophages and its receptor CXCR3 was highly expressed in T cells at the single-cell level. Conclusions This study comprehensively investigated the tumor microenvironment and verified immune/stromal-related biomarkers for pancreatic cancer.

The therapeutic effect of gemcitabine (GEM) in pancreatic ductal adenocarcinoma (PDAC) is limited due to low drug sensitivity and high drug resistance. Tissue inhibitor of matrix metalloprotease 1 (TIMP1) is reportedly associated with GEM resistance in PDAC. However, the effect of TIMP1 down-regulation in combination with GEM treatment is unknown. We analyzed the expression of TIMP1 in human PDAC tissue using western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry. TIMP1 was highly expressed in PDAC specimens. Kaplan-Meier survival analysis suggested that a higher level of TIMP1 was correlated with poorer overall survival in 103 PDAC patients. The mRNA and protein expression profiles of TIMP1 were explored in the HTERT-HPNE human pancreatic ductal epithelium cell line, five PDAC cell lines (MIA PaCa-2, PANC-1, BxPC-3, Capan2, and SW1990), and two GEM-resistant PDAC cell lines (MIA PaCa-2R and PANC-1R). Compared with HTERT-HPNE, TIMP1 was highly expressed in the PDAC cell lines. In addition, TIMP1 was upregulated in GEM-resistant PDAC cell lines compared with their parental cells. When TIMP1 was knocked-down using short hairpin RNA, GEM-induced cytotoxicity and apoptosis were increased, while colony formation was repressed in MIA PaCa-2, PANC-1, and their GEM-resistant cells. When Bax was activated by BAM7 or Bcl-2 was inhibited by venetoclax, CCK-8 assays demonstrated that GEM sensitivity was restored in GEM-resistant cells. When Bax was down-regulated by siRNA, CCK-8 assays verified that GEM sensitivity was decreased in PDAC cells. The observations that TIMP1 knockdown enhanced GEM sensitivity and reversed chemoresistance by inducing cells apoptosis indicated cooperative antitumor effects of shTIMP1 and GEM therapy on PDAC cells. The combination may be a potential strategy for PDAC therapy.