Plasminogen activator inhibitor-1 (PAI-1) gene transfection inhibits the liver metastasis of pancreatic cancer by preventing angiogenesis

We investigated the effect of TGF-beta1 on liver metastasis of pancreatic cancer using surgical specimens of pancreatic cancer and human pancreatic cancer cell lines Capan-2 and SW1990. Immunostaining of TGF-beta1 showed that TGF-beta1 positivity was significantly related to venous invasion and tumor staging, and also relatively associated with liver metastasis. Cellular invasion and protease production of MMP-2 and u-PA, and in vivo liver metastasis were significantly enhanced after treatment of cells with TGF-beta1. These findings suggest that TGF-beta1 might play an important role in enhancing liver metastasis of pancreatic cancer.

It is suggested that this liver metastasis model formed by a highly metastatic variant (KLM-1) is valuable for the study of the liver metastatic processes of human pancreatic cancer. Liver metastasis in the early postoperative period is one of the causes for the poor prognosis of patients with resected pancreatic cancer. Therefore, it is necessary to establish an experimental model to study the mechanisms of liver metastasis in pancreatic cancer. Human pancreatic cancer cell lines (PK-1, PK-9, and KLM-1) were injected into the portal vein of nude mice with or without pretreatment with antiasialo GM1, and colonies of liver metastases were counted for comparison of metastatic ability of these cell lines. Biological and histopathological characteristics of the highly liver metastatic cell line (KLM-1) were compared with its parent cell line (PK-1). PK-1 cells and PK-9 cells rarely formed liver metastasis foci, but pretreatment with antiasialo GM1 promoted liver metastasis. KLM-1 cells formed liver metastases at the rate of 70% even without antiasialo GM1 pretreatment. KLM-1 cells had such biological characteristics as short doubling time, short lag phase, and resistance to NK cytotoxicity. After intraportal injection of 125I-labeled KLM-1 cells, radioactivity as well as micrometastases were detected in the liver at 72 h.

Possible role of the 4G/5G polymorphism of the plasminogen activator inhibitor 1 gene in the development of asthma

TGF-βs are multifunctional polypeptides that regulate cell growth and differentiation, extracellular matrix deposition, cellular adhesion properties, angiogenesis and immune functions. In this study, we investigated the effect of TGF-β1 on liver metastasis and its mechanism by using human pancreatic cancer cell lines Panc-1, Capan-2, and SW1990. Capan-2 and SW1990 cells demonstrated enhanced liver metastatic potential by in vivo splenic injection with TGF-β1. Consequently, we examined the role of TGF-β1 on in vitro angiogenesis and received cytotoxicity by peripheral blood mononuclear leukocytes (PBMLs). While TGF-β1 slightly decreased cell proliferation, it also upregulated VEGF production in all cancer cells examined. The binding of PBMLs to cancer cells and cancer cell cytotoxicity during co-culture with PBMLs were remarkably decreased by treatment with TGF-β1. Panc-1 cells revealed no liver metastasis despite their high immunogenetic and angiogenetic abilities, which was attributed to a lack of expression of the cell surface carbohydrates that induce attachment to endothelial cells. We concluded that the presence of TGF-β1 in the microenvironment of tumour site might play an important role in enhancing liver metastasis of pancreatic cancer by modulating the capacity of angiogenesis and immunogenicity. © 2001 Cancer Research Campaign http://www.bjcancer.com

Several recent studies have reported that selectins are produced during ischemia-reperfusion injury, and that selectin ligands play an important role in cell binding to the endothelium and in liver metastasis. Portal clamping during pancreaticoduodenectomy with vessel resection for pancreatic head cancer causes hepatic ischemia-reperfusion injury, which might promote liver metastasis. We investigated the liver colonization of pancreatic cancer cells under hepatic ischemia-reperfusion and examined the involvement of E-selectin and its ligands. A human pancreatic cancer cell line (Capan-1) was injected into the spleen of mice after hepatic ischemia-reperfusion (I/R group). In addition, to investigate the effect of an anti-E-selectin antibody on liver colonization in the IR group, mice received an intraperitoneal injection of the anti-E-selectin antibody following hepatic ischemia-reperfusion and tumor inoculation (IR+Ab group). Four weeks later, mice were sacrificed and the number of tumor nodules on the liver was compared to mice without hepatic ischemia-reperfusion (control group). The incidence of liver metastasis in the I/R group was significantly higher (16 of 20, 80%) than that in the control group (6 of 20, 30%) (P<0.01). Moreover, mice in the I/R group had significantly more tumor nodules compared to those in the control group (median, 9.9 vs. 2.7 nodules) (P<0.01). In the I/R+Ab group, only 2 of 5 (40%) mice developed liver metastases. RT-PCR and southern blotting of the liver extracts showed that the expression of IL-1 and E-selectin mRNA after hepatic ischemia-reperfusion was significantly higher than the basal levels. Hepatic ischemia-reperfusion increases liver metastases and E-selectin expression in pancreatic cancer. These results suggest that E-selectin produced due to hepatic ischemia-reperfusion is involved in liver metastasis.

The Tumor Burden and Immune Dynamic Changes in Pancreatic Cancer Liver Metastasis

We developed a reliable new model system for assaying liver metastasis using NOD/SCID/gamma(c)(null) (NOG) mice. Seven human pancreatic cancer cell lines were examined for their ability to form diverse metastatic foci in the livers of NOD/SCID and NOG mice. Capan-2 and PL45 showed no metastasis when seeded at up to 10(5) cells in both strains, and no BxPC-3 metastasis was observed in NOD/SCID mice. The NOD/SCID mouse model detected liver metastasis only in the AsPC-1 cell line when inoculated with >10(3) cells. In contrast, when inoculated with only 10(2) MIA PaCa-2, AsPC-1 and PANC-1 cells, liver metastasis was evident in 71.4% (5/7), 57.1% (4/7) and 37.5% (3/8) of the NOG mice, respectively. Capan-1 and BxPC-3 cells metastasized when seeded at 10(3) cells in 50% (5/10) and in 12.5% (1/8) of the mice, respectively. Using the NOG mouse model system, we established a highly metastatic cell line, liver metastasized-BxPC-3 (LM-BxPC-3), from liver metastatic foci formed by the relatively poorly metastatic parental BxPC-3 cell line. The gene expression profiles of parental and LM-BxPC-3 cells were compared, and we identified forty-five genes that were either upregulated or downregulated >4-fold in the LM-BxPC-3 cell line. We validated 9 candidate protein-coding sequences, and examined the correlation between their expression pattern and the in vivo liver metastatic potential of all 7 pancreatic cancer cell lines. Only S100A4 expression correlated with the ability to form liver metastases, as evaluated in our quantitative model of metastasis in NOG mice. These results suggested that S100A4 is a key regulator of liver metastasis in pancreatic cancer, and demonstrated the feasibility of using the quantitative metastasis model to search for and develop new anti-cancer therapies and novel drugs against this and other key molecules.

To elucidate the mechanisms of metastasis, we established two sublines HPC-1H5 with a highly liver metastatic cell line and HPC-1P5a with a highly peritoneal disseminating cell line, which were sequentially selected from the parental pancreatic cancer cell line HPC-1. Using these three cell lines, we investigated several biological properties and mRNA levels of differentially-expressed genes involved in cancer metastasis by cDNA macroarray. Microscopic findings for the three cell lines were the same. The tumorigenicity, in vitro growth ability, motile activity, adhesive activity and the production of IL-8 of metastatic sublines were higher than those of parental HPC-1 cells. Particularly, HPC-1H5 cells showed clearly higher levels of IL-8 expression and tumors of HPC-1H5 cells grew faster and bigger than those of HPC-1P5a cells. In cDNA macroarray analysis of HPC-1H5 cells, 22 genes were up-regulated and 44 genes were down-regulated compared with parental HPC-1 cells. In HPC-1P5a cells, 9 genes were up-regulated and 28 genes were down-regulated compared with parental HPC-1 cells. This study provides a demonstration of global gene expression analysis of pancreatic cancer cells with liver metastasis and peritoneal dissemination. Furthermore, our results provide a new insight into the study of liver metastasis and peritoneal dissemination of human pancreatic cancer.

Background A more accurate prediction of liver metastasis (LM) in pancreatic cancer (PC) would help improve clinical therapeutic effects and follow-up strategies for the management of this disease. This study was to assess various prediction models to evaluate the risk of LM based on machine learning algorithms. Methods We retrospectively reviewed clinicopathological characteristics of PC patients from the Surveillance, Epidemiology, and End Results database from 2010 to 2018. The logistic regression, extreme gradient boosting, support vector, random forest (RF), and deep neural network machine algorithms were used to establish models to predict the risk of LM in PC patients. Specificity, sensitivity, and receiver operating characteristic (ROC) curves were used to determine the discriminatory capacity of the prediction models. Results A total of 47,919 PC patients were identified; 15,909 (33.2%) of which developed LM. After iterative filtering, a total of nine features were included to establish the risk model for LM based on machine learning. The RF showed the most promising results in the prediction of complications among the models (ROC 0.871 for training and 0.832 for test sets). In risk stratification analysis, the LM rate and 5-year cancer-specific survival (CSS) in the high-risk group were worse than those in the intermediate- and low-risk groups. Surgery, radiotherapy, and chemotherapy were found to significantly improve the CSS in the high- and intermediate-risk groups. Conclusion In this study, the RF model constructed could accurately predict the risk of LM in PC patients, which has the potential to provide clinicians with more personalized clinical decision-making recommendations.

Pancreatic cancer remains an extremely malignant digestive tract tumor, posing a significant global public health burden. Patients with pancreatic cancer, once metastasis occurs, lose all hope of cure, and prognosis is extremely poor. It is important to investigate liver metastasis of Pancreatic cancer in depth, not just because it is the most common form of metastasis in pancreatic cancer, but also because it is crucial for treatment planning and prognosis assessment. This study aims to delve into the mechanisms of pancreatic cancer liver metastasis, with the goal of providing crucial scientific groundwork for the development of future treatment methods and drugs. We explored the mechanisms of pancreatic cancer liver metastasis using single-cell sequencing data (GSE155698 and GSE154778) and bulk data (GSE71729, GSE19279, TCGA-PAAD). Initially, Seurat package was employed for single-cell data processing to obtain expression matrices for primary pancreatic cancer lesions and liver metastatic lesions. Subsequently, high-dimensional weighted gene co-expression network analysis (hdWGCNA) was used to identify genes associated with liver metastasis. Machine learning algorithms and COX regression models were employed to further screen genes related to patient prognosis. Informed by both biological understanding and the outcomes of algorithms, we meticulously identified the ultimate set of liver metastasis-related gene (LRG). In the study of LRG genes, various databases were utilized to validate their association with pancreatic cancer liver metastasis. In order to analyze the effects of these agents on tumor microenvironment, we conducted an in-depth analysis, including changes in signaling pathways (GSVA), cell differentiation (pseudo-temporal analysis), cell communication networks (cell communication analysis), and downstream transcription factors (transcription factor activity prediction). Additionally, drug sensitivity analysis and metabolic analysis were performed to reveal the effects of LRG on gemcitabine resistance and metabolic pathways. Finally, functional experiments were conducted by silencing the expression of LRG in PANC-1 and Bx-PC-3 cells to validate its influence to proliferation and invasiveness on PANC-1 and Bx-PC-3 cells. Through a series of algorithmic filters, we identified PAK2 as a key gene promoting pancreatic cancer liver metastasis. GSVA analysis elucidated the activation of the TGF-beta signaling pathway by PAK2 to promote the occurrence of liver metastasis. Pseudo-temporal analysis revealed a significant correlation between PAK2 expression and the lower differentiation status of pancreatic cancer cells. Cell communication analysis revealed that overexpression of PAK2 promotes communication between cancer cells and the tumor microenvironment. Transcription factor activity prediction displayed the transcription factor network regulated by PAK2. Drug sensitivity analysis and metabolic analysis revealed the impact of PAK2 on gemcitabine resistance and metabolic pathways. CCK8 experiments showed that silencing PAK2 led to a decrease in the proliferative capacity of pancreatic cancer cells and scratch experiments demonstrated that low expression of PAK2 decreased invasion capability in pancreatic cancer cells. Flow cytometry reveals that PAK2 significantly inhibited apoptosis in pancreatic cancer cell lines. Molecules related to the TGF-beta pathway decreased with the inhibition of PAK2, and there were corresponding significant changes in molecules associated with EMT. PAK2 facilitated the angiogenic potential of cancer cells and promotes the epithelial-mesenchymal transition process by activating the TGF-beta signaling pathway. Simultaneously, it decreased the differentiation level of cancer cells, consequently enhancing their malignancy. Additionally, PAK2 fostered communication between cancer cells and the tumor microenvironment, augments cancer cell chemoresistance, and modulates energy metabolism pathways. In summary, PAK2 emerged as a pivotal gene orchestrating pancreatic cancer liver metastasis. Intervening in the expression of PAK2 may offer a promising therapeutic strategy for preventing liver metastasis of pancreatic cancer and improving its prognosis.

Pancreatic stellate cells are involved in fibrosis of pancreatic cancer. An understanding of pancreatic cancer-cell interactions with stellate cells is critical to our ability to develop effective anti-tumor therapeutics for pancreatic cancer. We report here imaging of the interaction of pancreatic cancer cells and pancreatic stellate cells in liver metastasis. Human pancreatic cancer cell lines (XPA1 and MiaPaCa-2) were engineered to express green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm. Pancreatic stellate cells, engineered to express RFP, were co-injected with the cancer cells into the spleen of transgenic cyan fluorescent protein (CFP) nude mice. Three hours after splenic injection dual-color pancreatic cancer cells and pancreatic stellate cells were found distributed in the host liver. Seven days after cancer cell-stellate cell co-injection, most pancreatic cancer cells and stellate cells were dead. However, by 28 days after injection, liver metastases were observed in the host CFP nude mice. With the high-resolution intravital imaging afforded by the Olympus FV1000 confocal microscope, the interaction of the dual-colored pancreatic cancer cells and the RFP-expressing pancreatic stellate cells could be clearly imaged in the liver metastasis, suggesting that stellate cells participate in metastasis formation. Our hypothesis is that pancreatic stellate cells form a niche for liver metastasis of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1695. doi:1538-7445.AM2012-1695

Effects of the Antioxidative Vitamins A, C and E on Liver Metastasis and Intrametastatic Lipid Peroxidation in BOP-Induced Pancreatic Cancer in Syrian Hamsters

BACKGROUND: Pancreatic cancer is associated with extremely high mortality rates due to rapid progression and a high incidence of metastases. Liver and lung metastases in the early postoperative period are one of the causes for the poor prognosis of patients with resected pancreatic cancer. Information on the pathobiology of metastatic pancreatic cancer and elucidation of the underlying molecular mechanisms are urgently needed in order to develop novel and effective therapeutic approaches to treatment. Injection of human pancreatic cancer cells in the spleens of nude mice is one of the major liver metastasis models. However, this model yields few metastatic tumors in the livers and none in the lungs. NOG mice have severe immunodeficiency, including defects of T, B and NK cell functions, and dysfunction of dendritic cells. Human pancreatic cancer cells are reported to easily proliferate and metastasize to other organs in NOG mice. In this study, we established and characterized human pancreatic cancer cell lines from liver and lung metastases in NOG mice. METHODS: PANC-1 cells (1×105 cells) were injected into the spleens of NOG mice. The mice were euthanized 8 weeks after injection, and the livers and lungs were removed. Metastatic tumors of the liver and lung were cut into small pieces and dispersed into single cells in a medium containing antibiotics. Biological and morphological characteristics, and gene expression patterns of mesenchymal markers of the liver and lung metastatic cells, were compared with parental cells. RESULTS: These metastatic cells were confirmed as being of human-cell origin using PCR with primer pair of human mitochondrial DNA. There were no morphological changes between liver and lung metastatic cells and parental cells, but the metastatic cells proliferated more slowly than parental cells as determined by MTT assays and cell counts. By contrast, the metastatic cells exhibited greater migration than the parental cells, as determined in Boyden chamber assays and by time lapse analysis. Attachment assays revealed that adhesion of metastatic cells to the type I and IV collagen, laminin and fibronectin was stronger than that of parental cells. Nestin, a cancer stem cell marker, was highly expressed in the liver and lung metastatic cells. CONCLUSION: These findings suggest that human pancreatic cancer cell lines derived from liver and lung metastases in NOG mice have different characteristics from parental cells. These new cell lines may contribute to resolving the mechanisms of metastasis of pancreatic cancer and the roles of cancer stem cells in metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5240. doi:10.1158/1538-7445.AM2011-5240

The liver is the predominant metastatic site for diverse cancers, including pancreatic and colorectal cancers (CRC), etc. The high incidence of hepatic metastasis of pancreatic cancer is an important reason for its refractory and high mortality. Therefore, it is important to understand how metastatic pancreatic cancer affects the hepatic tumor immune microenvironment (TME) in patients. Here, we characterized the TME of liver metastases unique to pancreatic cancer by comparing them with CRC liver metastases. We integrated two single-cell RNA-seq (scRNA-seq) datasets including tumor samples of pancreatic cancer liver metastasis (P-LM), colorectal cancer liver metastasis (C-LM), primary pancreatic cancer (PP), primary colorectal cancer (PC), as well as samples of peripheral blood mono-nuclear cells (PBMC), adjacent normal pancreatic tissues (NPT), to better characterize the heterogeneities of the microenvironment of two kinds of liver metastases. We next performed comparative analysis on cellular compositions between P-LM and C-LM, found that Mph_SPP1, a subset of macrophages associated with angiogenesis and tumor invasion, was more enriched in the P-LM group, indicating this kind of macrophages provide a TME niche more vulnerable for pancreatic cancers. Analysis of the developmental trajectory implied that Mph_SPP1 may progressively be furnished with increased expression of genes regulating endothelium. Cell–cell communications analysis revealed that Mph_SPP1 potentially interacts with endothelial cells in P-LM via FN1/SPP1-ITGAV/ITGB1, implying this macrophage subset may construct an immunosuppressive TME for pancreatic cancer by regulating endothelial cells. We also found that Mph_SPP1 has a prognostic value in pancreatic adenocarcinoma that is not present in colon adenocarcinoma or rectum adenocarcinoma. This study provides a new perspective for understanding the characteristics of the hepatic TME in patients with liver metastatic cancer. And it provides a subset of macrophages specifically associated with the liver metastasis of pancreatic cancer, and its detection and intervention have potential value for preventing the metastasis of pancreatic cancer to the liver.

Aim: Patients were analyzed who underwent treatment of liver metastases from pancreatic cancer. Methods: Selection criteria were the possibility of R0 resection of the primary and/or the liver metastases, no other sites of metastases, and the presentation of liver metastases. A comparison of treatment by surgery versus chemotherapy regarding overall survival and disease-free interval was performed. Results: Between 1996 and 2008, a total number of 23 patients were retrospectively identified from a prospective database of 193 cases of pancreatic cancer. In 14 cases, liver metastases were found simultaneously, and in 9 cases metachronously, fulfilling the abovementioned selection criteria. Of these, 13 patients underwent surgery and 10 were treated by gemcitabine. There were no differences in survival in patients with synchronous liver metastases of pancreatic cancer treated by resection of the primary combined with partial hepatectomy versus treatment by gemcitabine (8 vs. 11 months). In patients with metachronous liver metastases, the median survival was increased after liver resection compared to patients who were treated with gemcitabine (31 vs. 11 months). Conclusions: Simultaneous resection of pancreatic cancer and liver metastases cannot be recommended. Resection of metachronous liver metastases of pancreatic cancer seems to improve survival in highly selected patients.