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The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is frequently associated to high treatment resistance. Gemcitabine (GEM) alone or in combination is the most used chemotherapy for unresecable PDACs. Here we studied whether modulated electro-hyperthermia (mEHT), a non-invasive complementary treatment, can support the effect of GEM on PDAC cells
Pancreatic cancer is one of the most fatal malignant tumors worldwide. Pancreatic ductal adenocarcinomas (PDAC), representing more than 90% of all pancreatic cancers are characterized by high aggressiveness and mortality rates, and have limited response to available anticancer treatments [
Gemcitabine is a nucleoside analog (2′,2′-difluoro-2′-deoxycytidine) and is preferred for patients who are not suitable for aggressive chemotherapy [
Hyperthermia has gained increasing popularity in cancer treatment to complement traditional and even targeted therapies. Combining with radio- or chemotherapy it can be effective against several cancer types [
Modulated electro-hyperthermia (mEHT) is a loco-regional hyperthermia which uses radiofrequency to generate selective heat in malignant tissues as a result of the metabolic shift towards glycolysis in cancer cells even under oxidative conditions, known as the Warburg effect [
In this study, GEM sensitive (Capan1) and GEM resistant (Panc1) PDAC cell lines were tested using GEM either alone or in combination with standard mEHT to see whether hyperthermia can improve treatment efficacy and if so which molecular pathways are involved in this effect. We also took an effort to develop GEM-resistant cell lines to see if mEHT treatment could still be efficient on tumor clones selected out by the earlier administration of the drug.
Pancreatic adenocarcinoma cell lines, Capan1 (CLS Cell Lines Service GmbH, Eppelheim, Germany) and Panc1 (ATCC, Teddington, Middlesex, United Kingdom) were grown in Iscove’s Modified Dulbecco’s Medium and Dulbecco’s Modified Eagle Medium (IMDM LM-I1092 and DMEM LM-D1111, Biosera, Boussens, France), respectively; enriched with 10% heat inactivated fetal bovine serum (FBS FB-1001H, Biosera, Boussens, France) and 80 mg Genta-Gobens (Gentamicin, Laboratorios Normon, Madrid, Spain). In case of Capan1, the media were also supplemented with 1% 2 mM L-glutamine (XC-T1715 Biosera, Boussens, France). Cells were kept in a humidified incubator at 37°C with an atmosphere containing 5% CO2.
For treatments, we harvested subconfluent cell cultures using 0.25% trypsin and 0.22 mg/ml ethylenediaminetetraacetic acid (Trypsin-EDTA, XC-T1717/100, Biosera, Boussens, France). After centrifugation (300 g, 5 min), the total number of treated cells were determined.
GEM-resistant cell lines were established using cell selection method [
For mEHT treatment, Lab-EHY 200 device (Oncotherm Kft, Budaors, Hungary) was used with all accessories customized for cell suspension treatment. The treatment bag containing 1.5 ml of 106 cells/ml suspension was submerged into the treatment cuvette filled with distilled water. One temperature sensor was inserted directly inside the treatment bag and another one into the surrounding water. The treatments lasted for 65 min including 5–10 min preheating period at 10 W average input power, and 55–60 min maintaining period using 2.4 W average input power. The amplitude modulated electric field generated 42 ± 0.3°C inside the treatment bag.
For gemcitabine (Fresenius Kabi Oncology Plc., Hampshire, United States) treatment, a 1,000 µM stock solution was made in 0.1 M neutral phosphate-buffered saline (PBS) buffer. This stock solution was diluted in cell culture media to achieve the preferred concentration and added to the cells, or added right after the 60 min mEHT treatment when combined. Samples were collected and tested at 0, 24, 48 or 72 h from the start of any treatment. In the 60 min mEHT monotherapy group tumor cells were grown in normal culture media, until sampling. In the GEM treatment groups cells were grown in GEM containing media either from the beginning, or after 60 min mEHT, when the two treatments were combined (mEHT + GEM), for the rest of time.
To analyze cell morphology, one piece of 18 × 18 mm sterilized histology glass coverslips was placed into each well of a 6-well plate. We seeded 2 × 105 cells/2 ml of Capan1 or 105 cell/2 ml of Panc1 into wells supplemented for 24 and 48 h, with 0.5 nM GEM in Capan1 cultures and 12 nM or 100 nM GEM for Panc1. Hematoxylin-eosin staining was performed. Cells were washed twice with PBS then fixed in 4% formaldehyde solution at room temperature. After washing the coverslips with distilled water, cells were stained with hematoxylin (2 min) and eosin (2 min). Finally, running tap water was used for bluing the samples.
For immunocytochemistry, after formalin fixation, we performed a 30 min peroxide block with 3% hydrogen peroxide diluted in methanol. Cell membranes were permeabilized with TBST buffer for 30 min, made up of 0.01 mol/L Tris-buffered saline pH 7.4 (TBS) containing 0.5% Tween-20 (Fisher Scientific United Kingdom Ltd., Loughborough, United Kingdom). Nonspecific protein blocking was made in 3% bovine serum albumin (Probumin, BSA, 82-100-6, Merk, Darmstadt, Germany) for 30 min. The primary antibodies were diluted in 1% BSA, the secondary antibodies were diluted in TBST. Primary antibodies such as rabbit monoclonal cleaved Caspase-3 (1:300, clone: 5A1E, #9664, Cell Signaling, Danvers, MA, United States), rabbit monoclonal Cytochrome C (1:100, clone: 136F3, #4280S, Cell Signaling), rabbit monoclonal E-cadherin (1:100, clone: EP700Y, #246R-14, Cell Marque, Rocklin, CA, United States) and mouse monoclonal N-Cadherin (1:100, clone: 32/N-Cadherin from BD Bioscience, NJ, United States) were incubated for 2 h at room temperature. After rigorous washing, polymer-peroxidase labeled mouse or rabbit IgG (Histols MR-T, Histopathology Ltd., Pecs, Hungary) was used for 1 h at room temperature, in case of N-Cadherin this step was preceded by a 30-min incubation with signal enhancer (Histols MR-T, Histopathology Ltd.). The chromogen reaction was revealed using a DAB chromogen/hydrogen peroxide kit (DAB Quanto, TA-060QHDX, Thermo-Fisher, Cheshire, United Kingdom). Cell nuclei were counterstained with hematoxylin. All stained coverslip cultures were dehydrated, mounted onto glass slides, and digitalized (Pannoramic scanner, 3DHISTECH, Budapest, Hungary). The cleaved Caspase-3 slides were analyzed using the QuantCenter image analysis software package (3DHISTECH).
Control and mEHT treated cells were seeded in a 96-well plate, at a concentration of 104 cells/well in 200 μl cell culture media. Culture media were supplemented with gemcitabine at different concentrations between 0 and 100 µM and incubated for 24, 48, and 72 h.
Resazurin is a cell-permeable redox-sensitive dye, which is reduced to resorufin by aerobic respiratory enzymes within cells. In contrast to resazurin, resorufin fluoresces when exposed to green light, thereby it is widely used to measure the viability of cells. Resazurin sodium salt (R7017 Sigma Aldrich, St. Louis, MO, United States) was diluted in PBS to make a stock solution of 0.3 mg/ml concentration which was further diluted in 1:10 when added directly into cell culture media. The measurement was done using Fluoroskan FL Microplate Fluorometer (5200110 Thermo-Fisher, Cheshire, United Kingdom) at excitation and emission wavelength of 570/590 after 2 h incubation of resazurin.
The preparation of cells for flow cytometry was executed as we described before with minor changes [
Flow cytometry was performed on a CytoFLEX Flow Cytometer using CytExpert software (Beckman Coulter, Indianapolis, IN, United States).
Clonogenic assay was performed as we described before [
Western blot analysis was performed as we described before [
WES capillary Western blot device (ProteinSimple, San Jose, CA, United States) was used with the 12–230 kDa Jess/Wes separation module kit (SM-W004) according to the manufacturer’s instructions. The kit included a 25-capillary cartridge (12–230 kDa), pre-filled microplates with running buffer, wash buffer, 10× sample buffer, and an EZ standard pack with a 12–230 biotinylated ladder, a fluorescent 5× master mix, and a dithiothreitol (DTT) containing tube. The lyophilized DTT and biotinylated ladder were suspended in the right amount of distilled water. The fluorescent 5× master mix was suspended in 20 µl previously prepared DTT solution and 20 µl 10× Sample Buffer. The samples were diluted to a concentration of 1 µg/µl in 100× diluted “10× Sample Buffer,” five parts of sample solution and one part of fluorescent 5× master mix were mixed together, then the mixture was heated for 5 min at 95°C. The following primary antibodies were used: rabbit polyclonal BAX (1:30, #HPA027878, Sigma-Aldrich, St. Louis, MO, United States), mouse monoclonal p21waf1 (1:35, clone:70/Cip1/WAF1, #610234, BD Bioscience NJ 07417, United States), rabbit monoclonal Hsp27 (1:70, clone: D6W5V, #96357, Cell Signaling, Danvers, MA, United States), rabbit polyclonal Hsp70 (1:70, #4872, Cell Signaling, Danvers, MA, United States), rabbit monoclonal E-Cadherin (1:70, clone:EP700Y, #RM-2100, Thermo-Fisher, Cheshire, United Kingdom) and mouse monoclonal N-Cadherin (1:70, clone: 32/N-Cadherin, BD Bioscience NJ 07417, United States). We used peroxidase conjugated anti-rabbit and anti-mouse detection module reagents (ProteinSimple DM-001 and DM-002), and chemiluminescent substrate. The plates with all components were centrifuged at 2,500 rpm for 5 min, then the cartridges were inserted into the instrument which was used with the default settings of the software: stacking and separation at 395 V for 30 min; blocking for 5 min, primary and secondary antibodies both for 30 min; luminol/peroxide chemiluminescence detection for 15 min (exposure times were between 1 and 512 s). The electropherograms were checked then the automatic peak detection was manually corrected if it was required.
The experiments were carried out in minimum triplicate for each analysis. For statistical analysis we used nonparametric Kruskal-Wallis test, Dunn’s multiple comparison post-hoc test or nonparametric multiple T test integrated test calculated by GraphPad Prism software package (San Diego, CA, United States). Statistical significance was considered at
Each column in the graphs shows the mean and SD of the results gained from minimum three independent experiments including one or more parallel samples per group.
Both Capan1 and Panc1 PDAC cell lines are known to accumulate several tumor driver mutations resulting in a basic gemcitabine resistance, in particular for Panc1 [
Therefore, we tested gemcitabine cytotoxicity in the original Capan1 and Panc1 cell lines. We used gemcitabine in concentrations between 0–10,000 nM and cytotoxicity was measured by resazurin assay after 24, 48, and 72 h treatment (
Cell viability after GEM treatment of Capan1 and Panc1 cell lines using resazurin assay
To analyze the potential complementary effect of mEHT on GEM treatment, cell cultures were treated with mEHT for 60 min then we added GEM for 48 h. This standard mEHT treatment was applied in all relevant experiments in this study. Drug concentrations were selected based on the cytotoxicity curves: 0.5 nM for Capan1 and 12 nM or 100 nM GEM for Panc1 cells (
Panc1 cultures formed large sheets of adherent tumor cells as opposed to small cells groups of Capan1 cells. Treatments caused the detachment of damaged cells resulting in a decreased number of surviving adherent cell populations. Nevertheless, the morphological signs of cell death were visible from 1 day after treatments, particularly in the more adhesive Panc1 cultures (data not shown). H&E staining of the remaining adherent cells 48 h after treatment revealed obvious signs of apoptosis including elevated numbers of pyknotic tumor cells with chromatin condensation, high basophilia, and membrane blebbing (
Hematoxylin-eosin staining 48 h post treatment. The formalin-fixed cells showed signs of apoptosis like karyopyknosis and karyorrhexis aside other morphological changes: hyperchromic nuclei, elongated cell shape, spatial tapering morphology, and disruption of interactions between the cells. Scale bar = 50 µm.
To examine the mechanism of cell death, we prepared double-stained tumor cells after 24 and 48 h treatments using Annexin V and propidium iodide (PI). In flow cytometry measurements, those cells were considered as living cells which did not express any fluorescence signal, and apoptotic cells were determined that showed Annexin V with or without PI positivity (
Flow cytometry of Capan1 and Panc1 cell lines labeled with PI and Annexin V at 24 and 48 h after treatment. Representative flow cytometry analysis of Capan1 cells 48 h after treatment. The red boxes represent apoptotic cell fractions
24 h after treatment, mEHT alone and in combination with GEM the apoptotic subpopulation was significantly elevated compared to the control in both cell lines (
To further clarify the pathways of apoptosis after treatments, Cytochrome C, BAX, and Caspase-8 protein levels were measured using Wes Simple analysis. The proportion of cleaved Caspase-3 positive cell fraction was determined by immunocytochemistry.
In Capan1 cell cultures 48 h after treatment, the Kruskal-Wallis test showed a statistically significant difference in the number of nuclear cleaved Caspase-3 positive tumor cells (
Immunocytochemistry of Cytochrome C and cleaved Caspase-3. The granular Cytochrome C became diffuse cytoplasmic in both cell lines after treatment as a sign of mitochondrial release
Western blot analysis of extrinsic and intrinsic apoptosis pathways showed no statistical difference either in Caspase-8 or in Cytochrome C levels between the treated and the control groups (data not shown). In Panc1 cells, BAX levels were significantly elevated 24 h after mEHT treatment (
Though SubG1 phase apoptotic fraction did not show a major change in Capan1 cells 24 h after treatment or in Panc1 cells 48 h after treatments (
Cell cycle analysis for Capan1 and Panc1 cell lines 24 and 48 h after mEHT treatment. Capan1 cells did not show significant response after any treatment at 24 h
These results were in line with the significantly reduced G1, S, and G2/M phase cell fractions in Capan1 cells 48 h after both the mEHT and the mEHT + 0.5 nM GEM treatments, and with significantly fewer S phase in Panc1 cells in 12 nM GEM and mEHT + 12 nM GEM-treated groups 48 h after treatment (
None of the treatments changed the level of the cell cycle promoters CDK4 and Cyclin A measured by Wes Simple analysis (data not shown). However, the significant elevation of the cyclin-dependent kinase inhibitor p21waf1 protein levels was observed in Capan1 cells, 48 h after GEM and the combined mEHT + 0.5 nM GEM treatments compared to the controls (
Western blot analysis of p21waf1 protein in Capan1 and Panc1 cells. The level of p21waf1 increased significantly after mEHT + 0.5 nM GEM treatment in Capan1 cells at 48 h after treatment compared to the control, as seen on the representative WES protein loading
To analyze the treatment-associated cell stress, we looked for changes in heat shock proteins including Hsp27, Hsp70, and Hsp90. The combined mEHT + 0.5 nM GEM treatment of Capan1 cells elevated both in Hsp27 and Hsp70 levels after 48 h (
Western blot analysis of heat shock proteins. In case of Capan1 cells, the level of Hsp27 and Hsp70 proteins increased significantly 48 h after mEHT + 0.5 nM GEM treatment (
Reduced E-Cadherin and elevated N-Cadherin calcium-dependent cell adhesion molecule levels may be linked to increased migration, epithelial mesenchymal transition (EMT), and metastatic potential. Cell-cell adhesion between Panc1 cells is strong therefore some damaged apoptotic cells grab one of their neighbors and form long processes between them (
Western blot and immunocytochemistry analysis of cell membrane adhesion molecules. E-Cadherin level in Capan1 cells increased significantly 48 h after mEHT + 0.5 nM GEM treatment compared to the control (
Therapy resistance could be assessed by the survival of tumor progenitor/stem cells, therefore, we analyzed the efficiency of treatments on tumor progenitors using colony formation assay (
Representative images of colony forming assays of Capan1 and Panc1 cells stained with crystal violet, at the day 14
Despite the recent improvements in oncological chemo-, radio- or immunotherapies, PDACs still represent one of the most fatal malignant tumors [
Based on pilot studies, we have chosen LD20 gemcitabine concentration, which proved to be 200-times higher for Panc1 than for Capan1, which is in line with the higher GEM resistance of Panc1 cell line [
All effective treatment modules resulted in cell loss and morphological signs of apoptosis as we observed earlier [
Our results are also consistent with those results which revealed elevated apoptosis and reduced G2/M phase tumor cell fractions after mEHT + GEM treatment in SW1990 PDAC cell line, which also carries multiple cancer–related driver mutations [
Hsp27 has been described as a predictive and prognostic factor in PDAC cases [
Metastatic potential is one of the main reason for the poor prognosis of PDAC [
In conclusion, mEHT could induce cell viability loss, apoptotic cell death, and cell cycle block on the Capan1 PDAC cell line. When mEHT was followed by GEM, cell death induction was also observed in GEM-resistant Panc1 cells. The major pathway of apoptotic cell death was caspase-dependent which was confirmed by the increase in Caspase-8 positive cells and BAX levels the mitochondrial release of Cytochrome C and activation of Caspase-3. These were accompanied by the upregulation of p21waf1 protein and the decrease of S and G2/M phase cell fractions. In both cell lines, Hsp27 levels were induced after combination treatments, along with the reduction of the resistance-related tumor progenitor cell colonies and increased expression of the cell adhesion molecule E-Cadherin in Capan1 cells. Therefore, our results suggest a role of mEHT in sensitizing PDAC cells to an efficient GEM treatment.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Conceptualization: GF and TK; methodology: GF, GP, and AG; resources: GF and TK; writing—original draft preparation: GF; writing—review and editing: EK, TD, and TK; supervision: EK and TK; project administration: GF and TK; funding acquisition: GF and TK. All authors have read and agreed to the published version of the manuscript.
Prepared with the professional support of the Doctoral Student Scholarship Program of the Co-Operative Doctoral Program of the Ministry of Innovation and Technology financed from the National Research, Development and Innovation Fund.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.