Feasibility and safety of electrochemotherapy (ECT) in the pancreas: a pre-clinical investigation

Roberto Girelli, Simona Prejanò, Ivana Cataldo, Vincenzo Corbo, Lucia Martini, Aldo Scarpa, Bassi Claudio, Roberto Girelli, Simona Prejanò, Ivana Cataldo, Vincenzo Corbo, Lucia Martini, Aldo Scarpa, Bassi Claudio

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease generally refractory to standard chemotherapeutic agents; therefore improvements in anticancer therapies are mandatory. A major determinant of therapeutic resistance in PDAC is the poor drug delivery to neoplastic cells, mainly due to an extensive fibrotic reaction. Electroporation can be used in vivo to increase cancer cells' local uptake of chemotherapeutics (electrochemotherapy, ECT), thus leading to an enhanced tumour response rate. In the present study, we evaluated the in vivo effects of reversible electroporation in normal pancreas in a rabbit experimental model. We also tested the effect of electroporation on pancreatic cancer cell lines in order to evaluate their increased sensitivity to chemotherapeutic agents.

Materials and methods: The application in vivo of the European Standard Operating Procedure of Electrochemotherapy (ESOPE) pulse protocol (1000 V/cm, 8 pulses, 100 μs, 5 KHz) was tested on the pancreas of normal New Zealand White Rabbits and short and long-term toxicity were assessed. PANC1 and MiaPaCa2 cell lines were tested for in vitro electrochemotherapy experiments with and without electroporation. Levels of cell permeabilization were determined by flow cytometry, whereas cell viability and drug (cisplatin and bleomycin) sensitivity of pulsed cells were measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.

Results: In healthy rabbits, neither systemic nor local toxic effects due to the electroporation procedure were observed, demonstrating the safety of the optimized electric parameters in the treatment of the pancreas in vivo. In parallel, we established an optimized protocol for ECT in vitro that determined an enhanced anti-cancer effect of bleomycin and cisplatin with respect to treatment without electroporation.

Conclusions: Our data suggest that electroporation is a safe procedure in the treatment of PDAC because it does not affect normal pancreatic parenchyma, but has a potentiating effect on cytotoxicity of bleomycin in pancreatic tumour cell lines. Therefore, ECT could be considered as a valid alternative for the local control of non-resectable pancreatic cancer.

Keywords: bleomycin; cisplatin; electrochemotherapy; electroporation; pancreatic adenocarcinoma; preclinical study; safety.

Figures

FIGURE 1.
FIGURE 1.
Serum levels of the liver enzymes, AST, ALT (A) and of the pancreatic amylase (B). Data points represent mean ± (standard error) SE, n = 3–6.
FIGURE 2.
FIGURE 2.
Photomicrographs of rabbit pancreata at different times after electroporation. (A) 24 hours: necrosis in the treated areas surrounded by significant hyperaemia and oedema. Acinar degranulation and vacuolation with granulocytes and lymphocytes infiltration (inset). (B) 72 hours: necrosis is still evident while acinar to ductal metaplasia appears along with intraductal protein plugs (inset). (C) 15 days: fibrotic areas are evident; chronic inflammatory cells are present while pancreatic acini are normal (inset). (D) 30 days: calcium deposition is detectable in fibrotic areas; pancreatic parenchyma is normal (inset).
FIGURE 3.
FIGURE 3.
Cell lines permeabilization as determined by Lucifer yellow uptake and flow cytometry. Mean ± standard deviation (S.D.) of n = 3 independent experiments; circle symbol, Panc1; square symbol, MiaPaCa2
FIGURE 4.
FIGURE 4.
Dose-response curve of bleomycin treatment. Cell viability was assessed at 72 hours of drug exposure using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega) and the Countess Cell Counter (Invitrogen, Milan, Italy). Dashed line = no electroporation; solid line = electroporation; grey lines indicate 95 % confident interval [CI]
FIGURE 5.
FIGURE 5.
Dose-response curve of cisplatin treatment. Cell viability was assessed at 72 hours of drug exposure using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega) and the Countess Cell Counter (Invitrogen, Milan, Italy). Dashed line = no electroporation; solid line = electroporation; grey lines = indicate 95% confident interval [CI]

References

    1. Ferlay J, Parkin DM, Steliarova-Foucher E. Estimates of cancer incidence and mortality in Europe in 2008. Eur J Cancer. 2010;46:765–81.
    1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, et al. Global cancer statistics. CA Cancer J Clin. 2011;61:69–90.
    1. Krejs GJ. Pancreatic cancer: epidemiology and risk factors. Dig Dis. 2010;28:355–8.
    1. Neoptolemos IP, Stocken DD, Friess H, Bassi C, MD, Dunn JA, Hickey H, et al. A randomized trial of chemoradiotherapy and chemotherapy after resection of pancreatic cancer. NEJM. 2004;350:1200–10.
    1. Gastrointestinal Tumour Study Group Radiation therapy combined with adriamycin or 5-Fluorouracil for the treatment of locally unresectable pancreatic carcinoma. Cancer. 1985;56:2563–8.
    1. Klaassen DJ, MacIntyre JM, Catton GE, Engstrom PF, Moertel CG. Treatment of locally unresectable cancer of the stomach and pancreas: a randomized comparison of 5-fluorouracil alone with radiation plus concurrent and maintenance 5-fluorouracil--an Eastern Cooperative Oncology Group study. J Clin Oncol. 1985;3:373–8.
    1. Chauffert B, Mornex F, Bonnetain F, Rougier P, Mariette C, Bouché O, et al. Phase III trial comparing intensive induction chemoradiotherapy (60 Gy, infusional 5-FU and intermittent cisplatin) followed by maintenance gemcitabine with gemcitabine alone for locally advanced unresectable pancreatic cancer. Definitive results of the 2000-01 FFCD/SFRO study. Ann Oncol. 2008;19:1592–9.
    1. Loehrer PJ, Sr, Feng Y, Cardenes H, Wagner L, Brell JM, et al. Gemcitabine alone versus gemcitabine plus radiotherapy in patients with locally advanced pancreatic cancer: An eastern cooperative oncology group trial. J Clin Oncol. 2011;29:4105–12.
    1. Conroy T, Desseigne F, Ychou M, Bouché O, Guimbaud R, Bécouarn Y, et al. For the Groupe Tumeurs Digestives of Unicancer and the PRODIGE Intergroup FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med. 2011;364:1817–25.
    1. Gillen S, Schuster T, Meyer zum Büschenfelde C, Friess H, Kleeff J. Preoperative/neoadjuvant therapy in pancreatic cancer: A systematic review and meta-analysis of response and resection percentages. PLoS Med. 2010;7(4):e1000267. 2010.
    1. Huguet F, André T, Hammel P, Artru P, Balosso J, Selle F, et al. Impact of chemoradiotherapy after disease control with chemotherapy in locally advanced pancreatic adenocarcinoma in GERCOR Phase II and III studies. J Clin Oncol. 2007;25:226–331.
    1. Krishnan S, Rana V, Janjan NA, Varadhachary GR, Abbruzzese JL, Das P, et al. Induction chemotherapy selects patients with locally advanced, unresectable pancreatic cancer for optimal benefit from consolidative chemoradiation therapy. Cancer. 2007;110:47–55.
    1. Chu Katrina F, Dupuy Damian E. Thermal ablation of tumours: biological mechanisms and advances in therapy. Nature Rev. 2014;14:199–208.
    1. Giardino A, Girelli R, Lusenti A, Auriemma A, Bassi C, Cantore M, et al. Triple approach strategy for patients with locally advanced pancreatic carcinoma. HPB (Oxford) 2013;15:623–7.
    1. Girelli R, Gobbo S, Malleo G, Regi P, Salvia R, Frigerio I, et al. Results of 100 pancreatic radiofrequency ablations in the context of a multimodal strategy for stage III ductal adenocarcinoma. Langenbecks Arch Surg. 2013;398:63–9.
    1. Cantore M, Frigerio I, Giardino A, Girelli R, Mambrini A, Orlandi M, et al. Combined modality treatment for patients with locally advanced pancreatic adenocarcinoma. Brit J Surg. 2012;99:1083–8.
    1. Girelli R, Frigerio I, Salvia R, Barbi E, Tinazzi Martini P, et al. Feasibility and safety of radiofrequency ablation for locally advanced pancreatic cancer. Brit J Surg. 2010;97:220–5.
    1. Shi X, Liu S, Kleeff J, Friess H, Büchler MW. Acquired resistance of pancreatic cancer cells towards 5-Fluorouracil and gemcitabine is associated with altered expression of apoptosis-regulating genes. Oncology. 2002;62:354–62.
    1. Arumugam T, Ramachandran V, Fournier KF, Wang H, Marquis L, Abbruzzese JL, et al. Epithelial to mesenchymal transition contributes to drug resistance in pancreatic cancer. Cancer Res. 2009;69:5820–8.
    1. Tamburrino A, Piro G, Carbone C, Tortora G, Melisi D. Mechanisms of resistance to chemotherapeutic and anti-angiogenic drugs as novel targets for pancreatic cancer therapy. Front Pharmacol. 2013;4:56.
    1. Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M, et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell. 2007;1:313–23.
    1. Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, et al. Identification of pancreatic cancer stem cells. Cancer Res. 2007;67:1030–7.
    1. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139:871–90.
    1. Bria E, Milella M, Gelibter A, Cuppone F, Pino MS, Ruggeri EM, et al. Gemcitabine-based combinations for inoperable pancreatic cancer: have we made real progress? A meta-analysis of 20 phase 3 trials. Cancer. 2007;110:525–33.
    1. Olson P, Hanahan D. Cancer. Breaching the cancer fortress. Science. 2009;324:1400–1.
    1. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH. Gene transfer into mouse lyoma cells by electroporation in high electric field. EMBO J. 1982;1:841–5.
    1. Neumann E, Kakorin S, Toesing K. Fundamentals of electroporative delivery of drugs and genes. Bioelectrochem Bioenerg. 1999;48:3–16.
    1. Orlowski S, Mir LM. Cell electropermeabilization: a new tool for biochemical and pharmacological studies. Biochim Biophys Acta. 1993;1154:51–63.
    1. Gehl J. Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol Scand. 2003;177:437–47.
    1. Mir LM, Orlowski S, Belehradek M, Paoletti C. Electrochemotherapy: potentiation of antitumor effect of bleomycin by local electric pulses. Eur J Cancer. 1991;1:68–72.
    1. Belehradek M, Domenge C, Luboinski B, Orlowski S, Belehradek J, Jr, Mir LM. Electrochemotherapy, a new antitumor treatment - first clinical phase I–II trial. Cancer. 1993;72:3694–700.
    1. Marty M, Sersa G, Garbay JR, Gehl J, Collins CG, Snoj M, et al. Electrochemotherapy – an easy, highly effective and safe treatment of cutaneous and subcutaneous metastases. Results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study. Eur J Cancer. 2006;S4:3–13.
    1. Sersa G, Stabuc B, Cemazar M, Jancar B, Miklavcic D, Rudolf Z. Electrochemotherapy with CDDP: potentiation of local CDDP antitumor effectiveness by application of electric pulses in cancer patients. Eur J Cancer. 1998;34:1213–8.
    1. Sersa G, Stabuc B, Cemazar M, Miklavcic D, Rudolf Z. Electrochemotherapy with cisplatin: clinical experience in malignant melanoma patients. Clin Cancer Res. 2000;6:863–7.
    1. Matthiessen LW, Johannesen HH, Hendel HW, Moss T, Kamby C, Gehl J. Electrochemotherapy for large cutaneous recurrence of breast cancer: A phase II clinical trial. Acta Oncol. 2012;51:713–21.
    1. Mevio N, Bertino G, Occhini A, Scelsi D, Tagliabue M, Mura F, et al. Electrochemotherapy for the treatment of recurrent head and neck cancers: preliminary results. Tumori. 2012;98:308–13.
    1. Curatolo P, Quaglino P, Marenco F, Mancini M, Nardò T, Mortera C, et al. Electrochemotherapy in the treatment of kaposi sarcoma cutaneous lesions: A two-center prospective phase II trial. Ann Surg Oncol. 2012;1:192–8.
    1. Campana LG, Valpione S, Mocellin S, Sundararajan R, Granziera E, Sartore L, et al. Electrochemotherapy for disseminated superficial metastases from malignant melanoma. Br J Surg. 2012;99:821–30.
    1. Fini M, Salamanna F, Parrilli A, Martini L, Cadossi M, Maglio M, et al. Electrochemotherapy is effective in the treatment of rat bone metastases. Clin Exp Met. 2013;30:1033–45.
    1. Edhemovic I, Gadzijev EM, Brecelj E, Miklavcic D, Kos B, Zupanic A, et al. Electrochemotherapy: a new technological approach in treatment of metastases in the liver. Technol Cancer Res Treat. 2011;10:475–85.
    1. Edhemovic I, Brecelj E, Gasljevic G, Marolt Music M, Gorjup V, Mali B, et al. Intraoperative electrochemotherapy of colorectal liver metastases. J Surg Oncol. 2014;110:320–7.
    1. Heller R, Jaroszeski M, Perrot R, Messina J, Gilbert R. Effective treatment of B16 melanoma by direct delivery of bleomycin using electrochemotherapy. Melanoma Res. 1997;7:10–8.
    1. Sersa G, Cemazar M, Miklavcic D, Mir LM. Electrochemotherapy: variable anti-tumor effect on different tumor models. Bioelectrochem Bioenerg. 1994;35:23–7.
    1. Pendez S, Jaroszeski MJ, Gilbert R, Hyacinthe M, Dang V, Hickey J, et al. Direct delivery of chemotherapeutic agents for the treatment of hepatoma and sarcoma in rat models. Radiol Oncol. 1998;21:53–64.
    1. Sersa G, Novakovic S, Miklavcic D. Potentiation of bleomycin antitumor effectiveness by electrochemotherapy. Cancer Lett. 1993;69:81–4.
    1. Nanda GS, Sun FX, Hofmann GA, Hofmann RM, Dev SB. Electroporation enhances therapeutic efficacy of anticancer drugs: treatment of human pancreatic tumor in animal model. Anticancer Res. 1988;18:1361–6.
    1. Jaroszeski MJ, Gilbert RA, Heller R. In vivo antitumor effects of electro-chemotherapy in a hepatoma model. Bioch Biophis Acta. 1997;1334:15–8.
    1. Ramirez LH, Orlowski S, An D, Gindoula G, Dzodic R, Ardouin P, et al. Electrochemotherapy on liver tumours in rabbit. Br. J Cancer. 1998;77:2104–11.
    1. Jaroszeski MJ, Illingworth P, Pottinger C, Hyacinthe M, Miller R. Electrically mediated drug delivery for treating subcutaneous and orthotopic pancreatic adenocarcinoma in a hamster model. Anticancer Res. 1999;19:989–94.
    1. Liu X, Tian X, Wang F, Ma Y, Kornmann M, Yang Y. BRG1 promotes chemoresistance of pancreatic cancer cells through crosstalking with Akt signalling. Eur J Cancer. 2014;50:2251–62.
    1. Granata V, Fusco R, Piccirillo M, Palaia R, Lastoria A, Petrillo A, et al. Feasibility and safety of intraoperative electrochemotherapy in locally pancreatic tumor: A preliminary experience. Eur J Inflamm. 2014;12:467–77.
    1. Allegretti JP, Panje WR. Electroporation therapy for head and neck cancer including carotid artery involvement. Laryngoscope. 2001;111:52–6.
    1. Bloom DC, Goldfarb PM. The role of intratumour therapy with electroporation and bleomycin in the management of advanced squamous cell carcinoma of the head and neck. Eur J Surg Oncol. 2005;31:1029–35.
    1. Miklavcic D, Sersa G, Brecelj E, Gehl J, Soden D, Bianchi G, Ruggieri P, Rossi CR, Campana LG, Jarm T. Electrochemotherapy: technological advancements for efficient electroporation-based treatment of internal tumors. Med Biol Eng Comput. 2012;50:1213–25.
    1. Todorovic V, Sersa G, Flisar K, Cemazar M. Enhanced cytotoxicity of bleomycin and cisplatin after electroporation in murine colorectal carcinoma cells. Radiol Oncol. 2009;43:264–73.
    1. Cemazar M, Tamzali Y, Sersa G, Tozon N, Mir LM, Miklavcic D, et al. Electrochemotherapy in veterinary oncology. J Vet Inter Med. 2008;22:826–31.

Source: PubMed

Szponzorok és közreműködők

Egészségi állapot

Kábítószer-beavatkozások

3
Iratkozz fel