ID: 2169

• Title:
  Effective colon carcinoma gene therapy with combination of IL-2 and IL-12
  
  Authors:
  

  The maximum word count is too low to fit all of the authors. The affiliations are listed in abstract box.

  
  
  Abstract:
  Komel,Tilen 1,2, Bosnjak,Masa 1, Kamensek,Urska 1, Znidar,Katarina 1, Kranjc Brezar,Simona 1, Dolinar,Klemen 3, Pirkmajer,Sergej 3, Sersa,Gregor 1,4 and Cemazar,Maja 1,5
  
  1 Institute of Oncology Ljubljana, Department of Experimental Oncology, Zaloska 2, SI-1000 Ljubljana, Slovenia
  
  2 University of Ljubljana, Faculty of Medicine, Vrazov trg 2, SI-1000 Ljubljana, Slovenia
  
  3 University of Ljubljana, Faculty of Medicine, Institute of Pathophysiology, Zaloska 4, SI-1000 Ljubljana, Slovenia
  
  4 University of Ljubljana, Faculty of Health Sciences, Zdravstvena pot 5, SI-1000 Ljubljana, Slovenia
  
  5 University of Primorska, Faculty of Health Sciences, Polje 42, SI-6310 Izola, Slovenia
  
  
  
  
  
  Gene therapy is emerging as an important approach to cancer treatment. One example is the introduction of genes that encode molecules such as interleukin 2 (IL-2) and interleukin 12 (IL-12) which in turn causes infiltration or activation of tumour-specific immune cells in tumour microenvironment. This therapy alone or in combination with other standard or immunotherapies could effectively eradicate the tumour cells. Electroporation was chosen for gene transfer because it is a safe and effective method to introduce genetic material (gene electrotransfer) into the cell. The aim of our study was to determine the effects of gene electrotransfer of plasmids encoding IL-2 (pIL-2) and IL-12 (pIL-12) in the murine colon carcinoma cell line CT26 in vitro and in vivo. Two electroporation protocols (EP) were tested: EP1 (600 V/cm, 5 ms, 1 Hz, 8 pulses) and EP2 (1300 V/cm, 100 µs, 1 Hz, 8 pulses). Cell viability was measured after the treatment using the PrestoBlueTM assay and the expression profile of IL-2 and IL-12 in the cells using qRT-PCR and ELISA. In addition, in vivo gene transfer of pIL-2 and pIL-12 (approval #U34401-3/2022/11) was also performed. Tumour growth after therapy was measured with a digital Vernier caliper and 3 days after therapy tumour samples were taken for immunohistochemistry and Magpix® analysis to determine infiltration by immune cells and the concentrations of the various cytokines, respectively. In vitro, the percentage of viable cells after gene electrotransfer of plasmids was significantly higher with the EP2 pulse protocol than with the EP1 pulse protocol. In contrast, the mRNA and protein expression of IL-2 and IL-12 was significantly higher in the cells after gene electrotransfer with EP1. Based on the in vitro results, EP1 was selected for the in vivo studies. Tumour growth delay was most evident in groups treated with gene electrotransfer of pIL-12 alone or in combination with pIL-2, where complete responses (absence of palpable tumour) were also observed. In these two groups elevated infiltration of various immune cells (macrophages, dendritic, CD4 and CD8 cells) was observed in comparison to electroporated and non-electroporated controls. Increased levels of several analytes such as Eotaxin, GM-CSF, IL-15, IL-13, IL-17, IL-6, IP-10, MIP-1a, MIP-1b and MIP-2 were observed in all groups treated with pulses. On the contrary, the levels of IL-12, IFNγ, G-CSF and TNFα were increased only in groups treated with gene electrotransfer of pIL-12 alone or in combination with pIL-2. Based on our results on mouse CT26 colon carcinoma, we can conclude that gene electrotransfer of plasmids encoding IL-2 and IL-12 into tumour cells is effective for both local and systemic treatment of tumours. Our combined approach induced immune cell activation, which in turn eradicated tumour cells.
  
  Keywords:
  gene electrotransfer, IL-2, IL-12, CT26 tumour model
  
  Refs:
  
  
  Topic 1:
  6. Cancer treatment and tumor ablation
  
  Topic 2:
  12. Biomedical applications