Corrigendum: Establishment of tumor treating fields combined with mild hyperthermia as novel supporting therapy for pancreatic cancer.

[This corrects the article DOI: 10.3389/fonc.2021.738801.].

[Corrigendum] Dexamethasone-induced inhibition of miR-132 via methylation promotes TGF-ß-driven progression of pancreatic cancer.

Subsequently to the publication of the above article, an interested reader drew to the authors' attention that two pairs of the culture plate images in Fig. 4A-C on p. 60 appeared to be the same, although the images were shown in different orientations; moreover, the 'NC/0 and DEX+miR132' and 'DEX and miR132' pairings of images in the scratch-wound assay experiments shown in Fig. 4B also appeared to be overlapping, such that these were apparently derived from the same original source where the results of differently performed experiments were intended to have been portrayed. After re­examining their original data, the authors have realized that some of the data in Fig. 4A and B were inadvertently assembled incorrectly. The revised version of Fig. 4, showing all the correct data for the culture plate images in Fig. 4A-C (specifically, the images fifth along on the right for Fig. 4B and C have been revised) and the correct images for 'NC/0' and 'DEX/0' in Fig. 4D is shown on on the next page. The authors are grateful to the Editor of International Journal of Oncology for allowing them this opportunity to publish a Corrigendum, and all the authors agree with its publication. Furthermore, the authors apologize to the readership for any inconvenience caused. [International Journal of Oncology 54: 53­64, 2019; DOI: 10.3892/ijo.2018.4616].

Corrigendum: Sulforaphane promotes dendritic cell stimulatory capacity through modulation of regulatory molecules, JAK/STAT3- and microRNA-signaling.

[This corrects the article DOI: 10.3389/fimmu.2020.589818.].

Tumor and stroma COL8A1 secretion induces autocrine and paracrine progression signaling in pancreatic ductal adenocarcinoma.

Several collagen subtypes are involved in pancreatic ductal adenocarcinoma (PDAC) desmoplasia, which constrains therapeutic efficacy. We evaluated collagen type VIII alpha 1 chain (COL8A1), whose function in PDAC is currently unknown. We identified COL8A1 expression in 7 examined PDAC cell lines by microarray analysis, western blotting, and RT‒qPCR. Higher COL8A1 expression occurred in 2 gemcitabine-resistant PDAC cell lines; pancreas tissue (n=15) from LSL-KrasG12D/+; p48-Cre mice with advanced PDAC predisposition; and PDAC parenchyma and stroma of a patient tissue microarray (n=82). Bioinformatic analysis confirmed higher COL8A1 expression in PDAC patient tissue available from TCGA (n=183), GTEx (n=167), and GEO (n=261) databases. siRNA or lentiviral sh-mediated COL8A1 inhibition in PDAC cells reduced migration, invasion and gemcitabine resistance and resulted in lower cytidine deaminase and thymidine kinase 2 expression and was rescued by COL8A1-secreting cancer-associated fibroblasts (CAFs). The activation of COL8A1 expression involved cJun/AP-1, as demonstrated by CHIP assay and siRNA inhibition. Downstream of COL8A1, activation of ITGB1 and DDR1 receptors and PI3K/AKT and NF-κB signaling occurred, as detected by expression, adhesion and EMSA binding studies. Orthotopic transplantation of PDAC cells with downregulated COL8A1 expression resulted in reduced tumor xenograft growth and lower gemcitabine resistance but was prevented by cotransplantation of COL8A1-secreting CAFs. Most importantly, COL8A1 expression in PDAC patient tissues from our clinic (n=84) correlated with clinicopathological data, and we confirmed these findings by the use of patient data (n=177) from the TCGA database. These findings highlight COL8A1 expression in tumor and stromal cells as a new biomarker for PDAC progression.

Corrigendum: Establishment of Tumor Treating Fields Combined With Mild Hyperthermia as Novel Supporting Therapy for Pancreatic Cancer.

[This corrects the article DOI: 10.3389/fonc.2021.738801.].

Establishment of Tumor Treating Fields Combined With Mild Hyperthermia as Novel Supporting Therapy for Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with poor prognosis and limited therapeutic options. Alternating electrical fields with low intensity called "Tumor Treating Fields" (TTFields) are a new, non-invasive approach with almost no side effects and phase 3 trials are ongoing in advanced PDAC. We evaluated TTFields in combination with mild hyperthermia. Three established human PDAC cell lines and an immortalized pancreatic duct cell line were treated with TTFields and hyperthermia at 38.5°C, followed by microscopy, assays for MTT, migration, colony and sphere formation, RT-qPCR, FACS, Western blot, microarray and bioinformatics, and in silico analysis using the online databases GSEA, KEGG, Cytoscape-String, and Kaplan-Meier Plotter. Whereas TTFields and hyperthermia alone had weak effects, their combination strongly inhibited the viability of malignant, but not those of nonmalignant cells. Progression features and the cell cycle were impaired, and autophagy was induced. The identified target genes were key players in autophagy, the cell cycle and DNA repair. The expression profiles of part of these target genes were significantly involved in the survival of PDAC patients. In conclusion, the combination of TTFields with mild hyperthermia results in greater efficacy without increased toxicity and could be easily clinically approved as supporting therapy.

Collagen Organization Does Not Influence T-Cell Distribution in Stroma of Human Pancreatic Cancer.

The dominant intrastromal T-cell infiltration in pancreatic cancer is mainly caused by the contact guidance through the excessive desmoplastic reaction and could represent one of the obstacles to an effective immune response in this tumor type. This study analyzed the collagen organization in normal and malignant pancreatic tissues as well as its influence on T-cell distribution in pancreatic cancer. Human pancreatic tissue was analyzed using immunofluorescence staining and multiphoton and SHG microscopy supported by multistep image processing. The influence of collagen alignment on activated T-cells was studied using 3D matrices and time-lapse microscopy. It was found that the stroma of malignant and normal pancreatic tissues was characterized by complex individual organization. T-cells were heterogeneously distributed in pancreatic cancer and there was no relationship between T-cell distribution and collagen organization. There was a difference in the angular orientation of collagen alignment in the peritumoral and tumor-cell-distant stroma regions in the pancreatic ductal adenocarcinoma tissue, but there was no correlation in the T-cell densities between these regions. The grade of collagen alignment did not influence the directionality of T-cell migration in the 3D collagen matrix. It can be concluded that differences in collagen organization do not change the spatial orientation of T-cell migration or influence stromal T-cell distribution in human pancreatic cancer. The results of the present study do not support the rationale of remodeling of stroma collagen organization for improvement of T-cell-tumor cell contact in pancreatic ductal adenocarcinoma.

Therapy of pancreatic cancer with alternating electric fields: Limitations of the method.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with a poor prognosis. More effective treatment options are urgently needed. The use of physical and weak alternating electric fields (TTFields) can inhibit cell division of PDAC carcinoma and is currently being investigated in clinical trials. Here, we analyzed this new physical treatment under non-ideal conditions such as may occur during patient treatment. Three established human PDAC cell lines BxPC-3, gemcitabine-resistant BxPC-3 (BxGem), AsPC-1, and a non-malignant primary pancreatic cell line CRL-4023 were treated with TTFields in vitro. MTT assays, electrical impedance measurement, cell staining with Annexin V/7AAD followed by FACS analysis, digital image analysis and immunohistochemistry were performed. Treatment with TTFields smaller than 0.7 V/cm and field lines in the direction of mitotic spindle orientation significantly inhibited proliferation of all PDAC cells at 150 kHz, but significantly increased viability of AsPC-1 cells at all frequencies between 100 kHz and 300 kHz and that of BxPC-3 and BxGem cells at 250 kHz. Apoptosis or necrosis were not induced. Non-malignant CRL-4023 cells were not affected at 150 kHz. TTFields damaged PDAC cell lines but even favored their viability at very weak field strength and unfavorable frequency or inadequate field direction.

Sulforaphane Promotes Dendritic Cell Stimulatory Capacity Through Modulation of Regulatory Molecules, JAK/STAT3- and MicroRNA-Signaling.

Introduction: The broccoli isothiocyanate sulforaphane was shown to inhibit inflammation and tumor progression, also in pancreatic cancer, while its effect on tumor immunity is poorly understood. We investigated the immunoregulatory effect of sulforaphane on human dendritic cells alone and in presence of pancreatic tumor antigens, as well as underlying molecular mechanisms. Methods: Sulforaphane-treated human dendritic cells were matured in vitro with a cytokine cocktail, and the expression of regulatory molecules was examined by flow cytometry. The subsequent T-cell response was analyzed by T-cell proliferation assay and CD25 expression. To confirm the findings, dendritic cells pulsed with pancreatic cancer-derived tumor antigens were used. To identify the involved pathway- and microRNA-signaling in sulforaphane-treated dendritic cells, inhibitors of various signaling pathways, western blot analysis, microRNA array, and bioinformatic analysis were applied. Results: Sulforaphane modulated the expression of the costimulatory CD80, CD83 and the suppressive B7-H1 molecules on dendritic cells and thereby promoted activation of T cells. The effect was verified in presence of pancreatic tumor antigens. Phosphorylation of STAT3 in dendritic cells was diminished by sulforaphane, and the inhibition of JAK/STAT3 led to downregulation of B7-H1 expression. Among the identified top 100 significant microRNA candidates, the inhibition of miR-155-5p, important for the expression of costimulatory molecules, and the induction of miR-194-5p, targeting the B7-H1 gene, were induced by sulforaphane. Conclusion: Our findings demonstrate that sulforaphane promotes T-cell activation by dendritic cells through the modulation of regulatory molecules, JAK/STAT3- and microRNA-signaling in healthy conditions and in context of pancreatic cancer-derived antigens. They explore the immunoregulatory properties of sulforaphane and justify further research on nutritional strategies in the co-treatment of cancer.

Fluorescence-guided fiber-optic micronavigation using microscopic identification of vascular boundary of liver segment and tumors.

Background: The exact identification of tumor boundaries and related liver segments is especially important for liver tumor surgery. This study aimed to evaluate a new approach for vascular boundary assessment and surgical navigation based on fiber-optic microscopy and microvascular fluorescence labeling. Methods: Antibody clones with fast binding ability were identified and selected using immunofluorescence. We evaluated the endothelial capture efficacy for an anti-mouse CD31 antibody labeled with different fluorophores and different degrees of labeling ex vivo. Segment boundary identification and navigation potential using endothelial capture were explored by two different fiber-optic microscopy systems. Finally, microvasculature labeling and fiber-optic microscopy were used to identify and treat microscopic liver tumors in vivo. Results: The following monoclonal antibodies were selected: anti-mouse CD31 (clone 390), anti-mouse CD54 (YN1/1.7.4), anti-human CD31 (WM59), and anti-human CD54 (HA58). These clones showed fast binding to endothelial cells and had long half-lives. The fluorophore choice and the degree of antibody labeling did not significantly affect capture efficacy in an isolated liver perfusion model. The microvascular system was clearly identified with wide-field fiber-optic microscopy after labeling the endothelium with low doses of specific antibodies, and the specifically labeled liver segment could be microscopically dissected. High antibody doses were required for confocal laser endomicroscopy. After microscopically identifying the vascular margin in vivo, tumor thermoablation strongly reduced tumor size or totally eliminated tumors. Conclusions: We demonstrated that vascular boundaries of liver tumors and locally perfused liver segments were accurately identified and surgical micronavigation was facilitated with fiber-optic microscopy and selected endothelium-specific antibodies.

Novel Broccoli Sulforaphane-Based Analogues Inhibit the Progression of Pancreatic Cancer without Side Effects.

The naturally occurring isothiocyanate sulforaphane, found in Brassicaceae vegetables, is promising in cancer treatment, e.g., by the normalization of enhanced levels of NF-κB-signaling in tumor stem cells. We chemically synthesized seven sulforaphane analogues by substitution of the sulfinyl group (S(O)) to either sulfimidoyl (S(NR)) or sulfonimidoyl (S (O) (NR)) groups, and characterized them in the cell lines of pancreatic cancer and several other tumor entities, including the NCI-60 cell panel. MTT and colony forming assays, flow cytometry, immunohistochemistry, microRNA arrays, bioinformatics, tumor xenotransplantation, and Kaplan Meier survival curves were performed. Compared to sulforaphane, the analogue SF102 was most efficient in inhibition of viability, colony formation, tumor growth, and the induction of apoptosis, followed by SF134. Side effects were not observed, as concluded from the body weight and liver histology of chick embryos and survival of C. elegans nematodes. Among 6659 differentially regulated microRNAs, miR29b-1-5p, and miR-27b-5p were downregulated by sulforaphane compared to controls, but upregulated by SF102 and SF134 compared to sulforaphane, suggesting differential signaling. Each substance was involved in the regulation of several NF-κB-related target genes. In conclusion, sulforaphane analogues are promising for the development of highly active new drugs in cancer treatment.

Novel plant microRNAs from broccoletti sprouts do not show cross-kingdom regulation of pancreatic cancer.

Food-derived plant microRNAs are suggested to control human genes by "cross-kingdom" regulation. We examined microRNAs in sprouts from Brassica rapa sylvestris, known as broccoletti, which are widely used as sulforaphane supplements, and assessed their influence on pancreatic cancer. RNA was isolated from 4-day-old sprouts, followed by deep sequencing and bioinformatic analysis. We identified 2 new and 745 known plant microRNA sequences in the miRbase database and predicted 15,494 human target genes and 76,747 putative 3'-UTR binding sites in these target genes. The most promising candidates were the already known microRNA sequence bra-miR156g-5p and the new sequence Myseq-330, both with predicted human target genes related to apoptosis. The overexpression of the respective oligonucleotides by lipofection did not alter the viability, apoptosis, clonogenicity, migration or associated protein expression patterns in pancreatic cancer cells. These data demonstrate that broccoletti sprouts contain microRNA sequences with putative binding sites in human genes, but the sequences evaluated here did not affect cancer growth. Our database of broccoletti-derived microRNA sequences provides a valuable tool for future analysis.

Broccoli sprout supplementation in patients with advanced pancreatic cancer is difficult despite positive effects-results from the POUDER pilot study.

Pancreatic ductal adenocarcinoma is a highly aggressive malignancy with short survival and limited therapeutic options. Broccoli sulforaphane is a promising new treatment due to the results of recent epidemiological, experimental and patient studies. Upon approval from the ethics committee and registration at ClinicalTrials.gov, 40 patients with palliative chemotherapy were placed into a placebo and treatment group in an unblinded fashion. Fifteen capsules with pulverized broccoli sprouts containing 90 mg/508 µmol sulforaphane and 180 mg/411 µmol glucoraphanin or methylcellulose were administered daily for up to 1 year. Twenty-nine patients were included in the treatment group and 11 patients were in the placebo group; these patients were followed for up to 1 year. The patient characteristics, overall survival and feasibility were assessed. Compared to those of the placebo group, the mean death rate was lower in the treatment group during the first 6 months after intake (day 30: 0%/18%, day 90: 0%/25%, and day 180: 25%/43%), and Kaplan-Meier analysis revealed a higher survival rate. There was a high drop-out rate (72% in the treatment group and 55% in the placebo group) after 1 year. We concluded from the Karnofsky index that the broccoli sprouts did not impact patient's self-care and overall abilities severely. The intake of 15 capsules daily was difficult for some patients, and the broccoli sprouts sometimes increased digestive problems, nausea and emesis. We did not obtain statistically significant results (p = 0.291 for the endpoint at day 180), but the knowledge about the feasibility is the basis for the development of new sulforaphane drugs.

Inhibition of miR30a-3p by sulforaphane enhances gap junction intercellular communication in pancreatic cancer.

The therapy resistance of pancreatic cancer is associated with the loss of gap junction intercellular communication and connexin 43 expression. The broccoli isothiocyanate sulforaphane restored these features and therapy sensitivity. We investigated whether microRNA signaling is involved. Established cell lines and a patient tissue array (n = 96) were evaluated by miRNA and gene array, bioinformatics, expression studies, in situ hybridization and immunohistochemistry. Sulforaphane inhibited the expression of our top candidate miR30a-3p. Upon transfection of miR30a-3p inhibitors, the gemcitabine bystander effect, Cx43 expression and intercellular communication increased, whereas miR30a-3p mimics inhibited the luciferase activity of a Cx43 3'-UTR construct. miR30a-3p-overexpressing tumor xenografts had a decreased tumor volume and increased gemcitabine sensitivity. In patient tissues, a higher expression of miR30a-3p and a lower expression of Cx43 correlated with malignancy. These findings provide new knowledge and suggest sulforaphane as cotreatment for pancreatic cancer.

Endothelial capture using antibodies and nanoparticles in human tissues: Antigen identification and liver segment imaging.

BACKGROUND: The unique phenomenon of endothelial antibody capture (endocapt) leads to site-specific accumulation of antibodies on the endothelium after its locoregional injection. The potential of this phenomenon has already been demonstrated in animal models. In the present study, the translational potential of several human endothelium-specific antibodies for their use in the endocapt-based approach was analysed. METHODS: The binding of different endothelium-surface specific monoclonal antibody clones was analysed in human tissue and in endothelial cells using image-based immunofluorescence and the determination of half-maximal effective concentration (EC50). The potential of endocapt-based locoregional application of antibodies or antibody-coated liposomes was analysed ex vivo using isolated mouse liver perfusion and in vivo using superselective injection in tumour models. RESULTS: Eight out of ten antibody clones were assigned to the group of "fast binding antibodies". Different antibody clones showed various binding kinetics to the same endothelial marker whereas the binding kinetics of single antibody clones was independent from the tissue type. Anti-CD49e, anti-CD31, anti-CD34 and anti-CD102 antibodies showed the lowest EC50 of antibody binding concentration and constant results in EC50 determination of antibody binding to cells and human tissue. Experimental studies using anti-mouse CD49e antibody and coated immunoliposomes confirmed their effective capture by endothelial cells in vitro and in vivo by which fluorescent liver segment labelling was achieved. CONCLUSIONS: Our findings identify the high potential of several human antibody clones, especially anti-CD49e, -CD31, -CD34 and -CD102, for endocapt technology. We also propose important translational implications of these antibodies for image-guided liver surgery and for use of nanoparticles/immunoliposomes. Toxicological studies are indispensable for further translational development of new antibodies for endocapt. STATEMENT OF SIGNIFICANCE: The phenomenon of endothelial antibody capture (endocapt) leads to site-specific accumulation of antibodies on the endothelium after its locoregional injection. This phenomenon broadly prevents systemic circulation of the antibody or antibody-drug conjugates. In the present study, our findings identify several human antibody clones promising for endothelial capture technology. This study provided the experimental demonstration of liver segment labelling ex vivo using isolated mouse liver perfusion and in vivo using superselective injection in tumor models. In addition, this study proposed the important translational implications of selected antibodies for image-guided liver surgery and for use of nanoparticles/immunoliposomes.

Sulforaphane Induces miR135b-5p and Its Target Gene, RASAL2, thereby Inhibiting the Progression of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal tumors, with poor therapeutic options in the advanced state. The broccoli-derived anti-inflammatory agent sulforaphane was shown to inhibit the progression of pancreatic cancer and other tumor entities. We examined the involvement of pancreatic cancer cell lines were evaluated by microRNA and gene expression arrays, bioinformatics, in silico analysis, qRT-PCR, western blotting, immunohistochemistry, in situ hybridization, self-renewal and differentiation assays, and in vivo xenograft studies. We selected the top nine differentially expressed microRNAs, and miR135b-5p was chosen as the most important candidate for the sulforaphane-induced upregulation of the tumor suppressor gene RASAL2. The expression of miR135b-5p and RASAL2 was almost absent in malignant pancreatic tissues and cell lines, but not in their normal counterparts. Lipofection of miR135b-5p enhanced RASAL2 expression and inhibited ERK1/2 signaling, viability, self-renewal capacity, and tumor growth. These results indicate that miR135b-5p acts as a tumor suppressor via the induction of RASAL2 in PDA.

MicroRNA-365a-3p inhibits c-Rel-mediated NF-κB signaling and the progression of pancreatic cancer.

NF-κB contributes to the aggressiveness of pancreatic ductal adenocarcinoma (PDA), which is counteracted by the bioactive agent sulforaphane. We investigated sulforaphane-induced microRNA signaling and its influence on progression features. Using established cell lines, microRNA and gene arrays, we predicted miR-365a as the top candidate for the sulforaphane-induced inhibition of the NF-κB subunit c-Rel. The lipofection of miR-365a-3p mimics inhibited the luciferase activity of a c-Rel 3'-UTR construct, as well as c-Rel expression, NF-κB activity, and tumor viability, migration, and clonogenicity, whereas apoptosis was induced. In vivo, miR-365a-3p reduced the volume of tumor xenografts and the expression of progression markers. In a tissue array, the expression of miR-365a-3p was absent in almost all 91 malignant tissues but not in 5 normal tissues, thus confirming the previous results. Our observations suggest that sulforaphane-induced miR-365a-3p expression inhibits NF-κB activity by downregulating c-Rel, which prevents the progression of PDA.

Dexamethasone-induced inhibition of miR-132 via methylation promotes TGF-ß-driven progression of pancreatic cancer.

Glucocorticoids (GCs) such as dexamethasone (DEX) are administered as cancer co­treatment for palliative purposes due to their pro­apoptotic effects in lymphoid cancer and limited side effects associated with cancer growth and chemotherapy. However, there is emerging evidence that GCs induce therapy resistance in most epithelial tumors. Our recent data reveal that DEX promotes the progression of pancreatic ductal adenocarcinoma (PDA). In the present study, we examined 1 primary and 2 established PDA cell lines, and 35 PDA tissues from patients who had received (n=14) or not received (n=21) GCs prior to surgery. Through microRNA microarray analysis, in silico, and RT­qPCR analyses, we identified 268 microRNAs differentially expressed between DEX­treated and untreated cells. With a focus on cancer progression, we selected miR­132 and its target gene, transforming growth factor-ß2 (TGF­ß2), as top candidates. miR­132 mimics directly bound to the 3' untranslated region (3'UTR) of a TGF­ß2 luciferase construct and enhanced expression, as shown by increased luciferase activity. By contrast, DEX inhibited miR­132 expression via promoter methylation. miR­132 mimics also reduced DEX­induced clonogenicity, migration and expression of vimentin and E­cadherin in vitro and in tumor xenografts. In patients, GC intake prior to surgery enhanced global hypermethylation and expression of TGF­ß2 in tissues; expression of miR­132 was detected but could not be quantified. Our results demonstrate that DEX­mediated inhibition of miR­132 is a key mediator in the progression of pancreatic cancer, and the findings provide a foundation for miRNA­based therapies.

Phenomenon of Endothelial Antibody Capture: Principles and Potential for Locoregional Targeting of Hepatic Tumors.

The systemic drug circulation represents a source of adverse effects during tumor targeting. We studied the binding efficacy of endothelium-specific antibodies after a very short contact with an antigen target, along with assessing the intravascular capture and targeting potential of these antibodies after locoregional injection. Fast-binding anti-CD 146 (clone ME-9F1) and anti-CD31 (clone 390) antibodies were selected based on histological analysis of their binding activity. The efficacy of antibody capture by hepatic endothelium under different conditions was analyzed using an isolated liver perfusion model. The local enrichment of R-phycoerythrin and 125 I-conjugated antibody was studied in vivo in two hepatic tumor models using biodistribution, scintigraphic imaging, and fluorescence microscopy. Upon injection into the tumor-feeding artery, the antibody was immediately captured in the microvasculature during the first passage. At doses not exceeding the saturation level of endothelial epitopes, the capture efficacy was almost 90%. We showed that the efficacy of endothelial capture is controlled by factors such as antibody affinity, number of binding sites on the endothelium, and microvascular flow rate. The targeting potential of endothelial capture was experimentally proven in vivo using scintigraphic imaging and biodistribution analysis after locoregional intra-arterial injection of 125 I-labeled antibodies in hepatic tumor models. Conclusion: The unique phenomenon of endothelial capture can broadly prevent systemic circulation of the antibody or antibody-drug conjugates applied by intravascular injection and may have specific relevance for targeting of hepatic tumors.

Simvastatin inhibits sonic hedgehog signaling and stemness features of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) has poor therapeutic options. Recent patient studies indicate that cholesterol-lowering statins have anti-tumor capacities. We examined several established and primary PDA and normal cell lines as well as PDA patient tissues (n = 68). We found that simvastatin inhibited viability, stemness, tumor growth and metastasis and that it enhanced the efficacy of gemcitabine. These changes were associated with modulation of Shh-related gene expression. Overexpression of Shh prevented the anti-cancer effect of simvastatin, and inhibition of Shh mimicked the simvastatin effect. In PDA tissues, expression levels of Shh, downstream mediators of Shh and progression markers, namely, cMet, CxCR4 and Vimentin, were lower when patients were prescribed statin medication prior to surgery. These results suggested that statins are cost effective and well-tolerated drugs for prevention and co-treatment of PDA.