Background: Electrochemotherapy is an effective local ablative method for treatment of different types of tumours, where intratumoral or intravenous chemotherapeutic drug administrantion (cisplatin or bleomycin) is combined with electroporation. Effectiveness of this method depends on technical performance and on tumor characteristics. It was suggested in clinical studies that the pre-treatment with radio- or chemotherapy significantly lowered the response rate of the electrochemotherapy treated tumours. The acquired intrinsic resistance of tumour cells to chemotherapeutics could be one of the possible reasons for the observed, as recurrent tumours emerge from those resistant tumor cells that survived previous radiation therapy due to certain adaptations, like higher level of DNA damage repair. It is less known, how those adaptations affect the chemoresistance of tumour cells, and consequently the resistance of those cells to electrochemotherapy.
Aim: In the dissertation, our aim was to explore mechanisms of intrinsic chemo- and radioresistance of reccurrent tumours after previous irradiation, which could affect the effectiveness of electrochemotherapy.
Hypothesis: Two hypotheses were explored. (1) Radioresistant tumour cells are less responsive to electrochemotherapy due to intrinsic resistance to cisplatin or bleomycin; and (2) The development of chemoresistance in radioresistant tumour cells is a result of difference in DNA repair mechanisms and altered cellular uptake of chemotherapeutics.
Materials and methods: In our experiments, we compared the two human cell lines of squamous cell carcinoma of the pharynx: the parental (FaDu) and the radioresistant subline (FaDu-RR) that was established by fractionated irradiation of the FaDu line. With the clonogenic test, we first determined the degree of sensitivity of both cell lines to irradiation, continuous exposure to cisplatin or bleomycin, and to electrochemotherapy. To perform in vivo experiments, we induced FaDu and FaDu-RR tumours by subcutaneous injection of the two cell lines on the right flanks of the immunocompromised (SCID) mice. We compared the tumour models in some histological characteristics and in response to irradiation, concomitant irradiation with cisplatin, chemotherapy and to electrochemotherapy. The effect of the therapies was monitored by measuring the tumour growth delay, compared to control groups, and with survival analysis. In the second part of the study, we compared the two cell lines in capacity to repair DNA double strand breaks (by immunocytochemical determination of γH2AX foci) and in DNA repair signaling pathway gene expression (by PCR). Additionally, using highly specific mass spectrometry analytical methods, we determined the cisplatin or bleomycin tumour uptake after chemo- and electrochemotherapy.
Results: With fractionated irradiation, we successfully established radioresistant subline FaDuRR that developed defense mechanisms leading to radio- and cisplatin cross-resistance. In vivo, we showed that FaDu-RR tumours were more resistant to concurrent radiochemotherapy with cisplatin, as well as to electrochemotherapy with cisplatin. Importantly, both cell lines and tumour models were equally sensitive to electrochemotherapy with bleomycin. In the second part of the study, where we investigated potential resistance mechanisms, we have demonstrated that in addition to the reduced intracellular accumulation of cisplatin compared to the parent cell line, the radioresistant cells also demonstrated a more efficient repair of DNA damage and have altered spectrum of DNA damage repair gene expression.
Conclusions: Fractionated irradiation in the newly established cell line induced some adaptation mechanisms such as more effective repair of DNA damage and cross-resistance to cisplatin. In in vivo conditions, FaDu-RR tumours were equally sensitive to bleomycin-based electrochemotherapy and more resistant to cisplatin-based electrochemotherapy compared to FaDu tumours. Given the fact that the difference between tumour models was small, based on these results we can exclude a more significant role of intrinsic radioresistance of tumour cells in the response of previously irradiated tumours to electrochemotherapy. However, regarding a certain degree of cross-resistance in the radioresistant cells to cisplatin, the bleomycin-based electrochemotherapy should be considered in the treatment of previously irradiated tumours.
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