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Abstract HPV infection renders oropharyngeal squamous cell carcinomas more radiosensitive, which results in a favorable prognosis for HPV-positive patients treated with radiation alone or with concurrent platinum-based chemotherapy. The degree of radiosensitivity in fractionated regimens has not yet been fully explored; therefore, in this study, the radiosensitivity of HPV-negative tumors (FaDu) was compared to that of HPV-positive tumors (2A3) subjected to concurrent cisplatin chemotherapy and fractionated versus isoeffective single-dose tumor irradiation in immunodeficient mice. HPV-positive tumors were approximately 5 times more radiosensitive than HPV-negative tumors, irrespective of the irradiation regimen. In both tumor models, concurrent cisplatin chemotherapy and the fractionated regimen induced significant tumor radiosensitization, with a 3- to 4-fold increase in the tumor growth delay compared to that of single-dose irradiation. Furthermore, the degree of radiosensitization induced by cisplatin chemotherapy concurrent with the fractionated irradiation regimen was much higher in HPV-positive tumors, where a synergistic antitumor effect was observed. Specifically, after combined therapy, a 26% higher survival rate was observed in mice with HPV-positive tumors than in mice with HPV-negative tumors. These data suggest that HPV-positive tumors are more radiosensitive to fractionated regimen than to single-dose irradiation with concurrent cisplatin chemotherapy acting synergistically to irradiation.

Abstract Background Treatment options for recurrent head and neck tumours in the previously irradiated area are limited, including re-irradiation due to radioresistance of the recurrent tumour and previous dose received by surrounding normal tissues. As an in vitro model to study radioresistance mechanisms, isogenic cells with different radiosensitivity can be used. However, they are not readily available. Therefore, our objective was to establish and characterize radioresistant isogenic human pharyngeal squamous carcinoma cells and to evaluate early radiation response in isogenic parental, radioresistant and radiosensitive cells. Methods Radioresistant cells were derived from parental FaDu cells by repeated exposure to ionizing radiation. Radiosensitivity of the established isogenic radioresistant FaDu-RR cells was evaluated by clonogenic assay and compared to isogenic parental FaDu and radiosensitive 2A3 cells. Additional phenotypic characterization of these isogenic cells with different radiosensitivity included evaluation of chemosensitivity, cell proliferation, cell cycle, radiation-induced apoptosis, resolution of DNA double-strand breaks, and DNA damage and repair signalling gene expression before and after irradiation. Results In the newly established radioresistant cells in response to 5 Gy irradiation, we observed no alteration in cell cycle regulation, but delayed induction and enhanced resolution of DNA double-strand breaks, lower induction of apoptosis, and pronounced over-expression of DNA damage signalling genes in comparison to parental cells. On the other hand, radiosensitive 2A3 cells were arrested in G2/M-phase in response to 5 Gy irradiation, had a prominent accumulation of and slower resolution of DNA double-strand breaks, and no change in DNA damage signalling genes expression. Conclusions We concluded that the emergence of the radioresistance in the established radioresistant isogenic cells can be at least partially attributed to the enhanced DNA double-strand break repair, altered expression of DNA damage signalling and repair genes. On the other hand, in radiosensitive isogenic cells the reduced ability to repair a high number of induced DNA double-strand breaks and no transcriptional response in DNA damage signalling genes indicate on a lack of adaptive response to irradiation. Altogether, our results confirmed that these isogenic cells with different radiosensitivity are an appropriate model to study the mechanisms of radioresistance.

Endoglin is a transforming growth factor-β (TGF- β) co-receptor that participates in the activation of a signaling pathway that mediates endothelial cell proliferation and migration in angiogenic tumor vasculature. Therefore, silencing of endoglin expression is an attractive approach for antiangiogenic therapy of tumors. The aim of our study was to evaluate the therapeutic potential of small interfering RNA (siRNA) molecules against endoglin in vitro and in vivo. Therapeutic potential in vitro was assessed in human and murine endothelial cells (HMEC-1, 2H11) by determining endoglin expression level, cell proliferation and tube formation. In vivo, the therapeutic potential of siRNA molecules was evaluated in TS/A mammary adenocarcinoma growing in BALB/c mice. Results of our study showed that siRNA molecules against endoglin have a good antiangiogenic therapeutic potential in vitro, as expression of endoglin mRNA and protein levels in mouse and human microvascular endothelial cells after lipofection were efficiently reduced, which resulted in the inhibition of endothelial cell proliferation and tube formation. In vivo, silencing of endoglin with triple electrotransfer of siRNA molecules into TS/A mammary adenocarcinoma also significantly reduced the mRNA levels, number of tumor blood vessels and the growth of tumors. The obtained results demonstrate that silencing of endoglin is a promising antiangiogenic therapy of tumors that could not be used as single treatment, but as an adjunct to the established cytotoxic treatment approaches.

A 15-year- old male castrated English setter was presented for evaluation of a rapidly growing oral mass. Patient’s history was otherwise unremarkable, except of moderate proteinuria of 5 years duration. Clinical examination findings were within normal limits, except of an ulcerated located at the left mandibular canine tooth, which was histologically confirmed as a high grade infiltrative fibrosarcoma with high mitotic index (61/10 HPF) and multifocal necrotic areas. The client declined full staging, so only hematological and biochemistry examinations of blood were performed, which were without any clinically significant alterations. Furthermore a fine needle aspiration biopsy of regional lymph nodes was performed, which revealed reactive lymphadenopathy without signs of metastases. After declining other more invasive therapeutic procedures, the clients elected treatment with combination of electrochemotherapy and IL-12 electrogene therapy. Four consecutive treatment sessions were performed, resulting not only in complete response of the primary tumor, but also in regression of untreated distant metastasis, which was diagnosed approximately one month after the initial examination. Furthermore, the percentage of circulating CD8+ cells was increased after each therapy session, indicating possible systemic induction of immune response by IL-12 gene therapy. This case shows that this type of therapy can represent an alternative type of both local and systemic treatment in selected tumor cases, where clients seek a less invasive nonsurgical treatment. Key words: dog; fibrosarcoma; electroporation; electrochemotherapy; electrogene therepy, interleukin-12 PALIATIVNO ZDRAVLJENJE NERESEKTABILNEGA FIBROSARKOMA SPODNJE ČELJUSTI PRI PSU S KOMBINACIJO ELEKTROKEMOTERAPIJE Z BLEOMICINOM IN GENSKEGA ELEKTROPRENOSA IL-12 Izvleček: Petnajstleni kastrirani angleški seter je bil na našo kliniko sprejet z namenom pregleda hitro rastoče novotvorbe v ustni votlini. Pes ni imel pomembnejših sočasnih bolezni, razen zmerne proteinurije zadnjih pet let. Fizikalni pregled ni pokazal nikakršnih odstopanj z izjemo ulcerirane novotvorbe na področju levega mandibularnega grabilca. Histološka diagnoza novotvorbe je bila infiltrativni fibrosarkom visoke stopnje z visokim mitotičnim indeksom (61/10 v polju visoke povečave) in multifokalnimi nekrotičnimi področji. Skrbnik psa je zavrnil popolno določitev stadija bolezni, zato smo izvedli le osnovne hematološke in biokemijske preisakve, ki niso pokazale pomembnejših odstopanj. Izvedli smo tudi tankoligelno biopsijo regionalnih bezgavk, ki je pokazala reaktivno limfadenopatijo brez znakov prisotnosti zasevkov. Po predstavitvi vseh možnosti zdravljenja se je lastnik odločil za zdravljenje s kombinacijo elektrokemoterapije in genskega elektroprenosa IL-12. Izvedli smo štiri zaporedne cikluse kombinirane terapije, s katero smo dosegli ne le popolni odgovor primarnega tumorja, pač pa tudi regresijo nezdravljenih oddaljenih podkožnih metastaz, ki so se pojavile približno mesec dni po začetku terapije. Poleg tega smo po vsaki terapiji ugotovili povečan delež cirkulirajočih CD8+ celic, kar lahko nakazuje na to, da je genska terapija z IL-12 sprožila sistemski imunski odziv. Predstavljeni primer kaže, da lahko kombinacija elektrokemoterapije in genskega elektroprenosa IL-12 predstavlja alternativno obliko tako lokalnega kot sistemskega zdravljenja določenih novotvorb, zlasti v primerih, ko skrbnik živali želi manj invazivne terapevtske postopke. Ključne besede: pes; fibrosarkom; elektroporacija; elektrokemoterapija, genski eletroprenos; interlevkin-12