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1.
Int J Mol Sci ; 24(3)2023 Feb 01.
Article in English | MEDLINE | ID: mdl-36769077

ABSTRACT

Targeting the tumor vasculature through specific endothelial cell markers involved in different signaling pathways represents a promising tool for tumor radiosensitization. Two prominent targets are endoglin (CD105), a transforming growth factor ß co-receptor, and the melanoma cell adhesion molecule (CD1046), present also on many tumors. In our recent in vitro study, we constructed and evaluated a plasmid for simultaneous silencing of these two targets. In the current study, our aim was to explore the therapeutic potential of gene electrotransfer-mediated delivery of this new plasmid in vivo, and to elucidate the effects of combined therapy with tumor irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice in the syngeneic murine mammary adenocarcinoma tumor model TS/A. Histological analysis of tumors (vascularization, proliferation, hypoxia, necrosis, apoptosis and infiltration of immune cells) was performed to evaluate the therapeutic mechanisms. Additionally, potential activation of the immune response was evaluated by determining the induction of DNA sensor STING and selected pro-inflammatory cytokines using qRT-PCR. The results point to a significant radiosensitization and a good therapeutic potential of this gene therapy approach in an otherwise radioresistant and immunologically cold TS/A tumor model, making it a promising novel treatment modality for a wide range of tumors.


Subject(s)
Gene Transfer Techniques , Genetic Therapy , Animals , Mice , Genetic Therapy/methods , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/therapy , Neovascularization, Pathologic/pathology , Endoglin/genetics , Plasmids
2.
Bioelectrochemistry ; 142: 107932, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34474205

ABSTRACT

Due to a lack of data on predictors of electroporation-based treatment outcomes, we investigated the potential predictive role of contrast-enhanced harmonic ultrasound (CEUS) in mice B16F10 melanoma treated by gene electrotransfer (GET) to silence melanoma cell adhesion molecule (MCAM) and radiotherapy, which has not been evaluated yet. CEUS evaluation was verified by tumor histological analysis. Mice bearing subcutaneous tumors were treated with GET to silence MCAM, irradiation or the combination of GET to silence MCAM and irradiation (combined treatment). CEUS of the tumors used to evaluate tumor perfusion was performed before and up to 10 days after the beginning of the experiment, and the CEUS results were compared with tumor growth and the number of blood vessels analyzed in the histological tumor sections. CEUS revealed a decrease in tumor perfusion in the combined therapy groups compared with the control groups and correlated with tumor histological analyses, which showed a decreased vascular density. In this study a trend of inverse correlation was observed between tumor perfusion and treatment efficacy. The greater the perfusion of the tumor, the shorter the expected doubling time. Furthermore, decreased perfusion showed a trend to correlate with higher antitumor efficacy. Thus, CEUS could be used to predict tumoral vascular density and treatment effectiveness.


Subject(s)
Electroporation/methods , Melanoma, Experimental , Ultrasonography/methods , Animals , Cell Line, Tumor , Female , Melanoma, Experimental/radiotherapy , Melanoma, Experimental/ultrastructure , Mice , Mice, Inbred C57BL
3.
Int J Mol Sci ; 22(6)2021 Mar 17.
Article in English | MEDLINE | ID: mdl-33802812

ABSTRACT

Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.


Subject(s)
CD146 Antigen/genetics , Electroporation , Endoglin/genetics , Gene Silencing , Plasmids/genetics , Radiation, Ionizing , Transfection , Animals , Cell Death , Cell Line , Cytokines/metabolism , Endothelial Cells/radiation effects , Membrane Proteins , Mice , Neovascularization, Physiologic/radiation effects
4.
Radiol Oncol ; 54(2): 168-179, 2020 03 27.
Article in English | MEDLINE | ID: mdl-32229678

ABSTRACT

Background Management of locoregionally recurrent head and neck squamous cell carcinomas (HNSCC) is challenging due to potential radioresistance. Pulsed low-dose rate (PLDR) irradiation exploits phenomena of increased radiosensitivity, low-dose hyperradiosensitivity (LDHRS), and inverse dose-rate effect. The purpose of this study was to evaluate LDHRS and the effect of PLDR irradiation in isogenic HNSCC cells with different radiosensitivity. Materials and methods Cell survival after different irradiation regimens in isogenic parental FaDu and radioresistant FaDu-RR cells was determined by clonogenic assay; post irradiation cell cycle distribution was studied by flow cytometry; the expression of DNA damage signalling genes was assesed by reverse transcription-quantitative PCR. Results Radioresistant Fadu-RR cells displayed LDHRS and were more sensitive to PLDR irradiation than parental FaDu cells. In both cell lines, cell cycle was arrested in G2/M phase 5 hours after irradiation. It was restored 24 hours after irradiation in parental, but not in the radioresistant cells, which were arrested in G1-phase. DNA damage signalling genes were under-expressed in radioresistant compared to parental cells. Irradiation increased DNA damage signalling gene expression in radioresistant cells, while in parental cells only few genes were under-expressed. Conclusions We demonstrated LDHRS in isogenic radioresistant cells, but not in the parental cells. Survival of LDHRS-positive radioresistant cells after PLDR was significantly reduced. This reduction in cell survival is associated with variations in DNA damage signalling gene expression observed in response to PLDR most likely through different regulation of cell cycle checkpoints.


Subject(s)
Head and Neck Neoplasms/radiotherapy , Neoplasm Recurrence, Local/radiotherapy , Radiation Tolerance , Squamous Cell Carcinoma of Head and Neck/radiotherapy , Cell Cycle/radiation effects , Cell Line, Tumor , Cell Survival/radiation effects , DNA Damage/genetics , G1 Phase/radiation effects , G2 Phase/radiation effects , Gene Expression , Humans , Mitosis/radiation effects , Radiotherapy Dosage , Time Factors , Tumor Stem Cell Assay/methods
5.
Vaccines (Basel) ; 8(1)2020 Mar 19.
Article in English | MEDLINE | ID: mdl-32204304

ABSTRACT

In this study, radiotherapy was combined with the gene electrotransfer (GET) of plasmid encoding shRNA against melanoma cell adhesion molecule (pMCAM) with dual action, which was a vascular-targeted effect mediated by the silencing of MCAM and an immunological effect mediated by the presence of plasmid DNA in the cytosol-activating DNA sensors. The effects and underlying mechanisms of therapy were evaluated in more immunogenic B16F10 melanoma and less immunogenic TS/A carcinoma. The silencing of MCAM potentiated the effect of irradiation (IR) in both tumor models. Combined therapy resulted in 81% complete responses (CR) in melanoma and 27% CR in carcinoma. Moreover, after the secondary challenge of cured mice, 59% of mice were resistant to challenge with melanoma cells, and none were resistant to carcinoma. Combined therapy reduced the number of blood vessels; induced hypoxia, apoptosis, and necrosis; and reduced cell proliferation in both tumor models. In addition, the significant increase of infiltrating immune cells was observed in both tumor models but more so in melanoma, where the expression of IL-12 and TNF-α was determined as well. Our results indicate that the combined therapy exerts both antiangiogenic and immune responses that contribute to the antitumor effect. However, tumor immunological status is crucial for a sufficient immune system contribution to the overall antitumor effect.

6.
Radiat Oncol ; 14(1): 214, 2019 Nov 27.
Article in English | MEDLINE | ID: mdl-31775835

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.


Subject(s)
Carcinoma, Squamous Cell/radiotherapy , Head and Neck Neoplasms/radiotherapy , Pharyngeal Neoplasms/radiotherapy , Radiation Tolerance , Radiation, Ionizing , Apoptosis , Carcinoma, Squamous Cell/metabolism , Cell Cycle , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation , DNA Breaks, Double-Stranded , DNA Repair , DNA-Binding Proteins/metabolism , Gene Expression Profiling , Gene Expression Regulation , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms/metabolism , Histones/metabolism , Humans , Inhibitory Concentration 50 , Neoplasm Recurrence, Local , Pharyngeal Neoplasms/metabolism , Phenotype
7.
Oncol Rep ; 41(3): 1658-1668, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30628709

ABSTRACT

Electrochemotherapy is an established local ablative method used for the treatment of different tumor types, including tumors of the head and neck area. Clinical studies have demonstrated a lower response rate of tumors that recur in pre­irradiated area. The aim of the present study was to explore the response of experimentally induced radioresistant cells and tumors to electrochemotherapy with cisplatin or bleomycin. The radioresistant cells (FaDu­RR) were established by fractionated irradiation of parental human squamous cell carcinoma cell line, FaDu. We compared the 2 cell lines in response to chemotherapy and electrochemotherapy with cisplatin or bleomycin in vitro and in vivo. Using specific mass spectrometry­based analytical methods we determined the difference in the uptake of chemotherapeutics in tumors after electrochemotherapy. Additionally, we compared the capacity of the cells to repair DNA double­strand breaks (DSB) after exposure to the drugs used in electrochemotherapy with the γH2AX foci resolution determined by immunofluorescence microscopy. Our results indicate radio­ and cisplatin cross­resistance, confirmed with the lower response rate of radioresistant tumors after electrochemotherapy with cisplatin. On the other hand, the sensitivity to electrochemotherapy with bleomycin was similar in both cell lines and tumors. While the uptake of chemotherapeutics after electrochemotherapy was comparable in both tumor models, there was a difference between the cell lines in capacity to repair DNA DSB­the radioresistant cells had a lower level of DSB and faster DNA repair rate after exposure to both, cisplatin or bleomycin. Due to the higher complete response rate after electrochemotherapy with bleomycin than with cisplatin, we conclude that the results favor bleomycin­over cisplatin­based electrochemotherapy for treatment of radioresistant tumors and/or tumors that regrow after radiotherapy.


Subject(s)
Antineoplastic Agents/pharmacology , Electrochemotherapy/methods , Head and Neck Neoplasms/drug therapy , Neoplasm Recurrence, Local/prevention & control , Squamous Cell Carcinoma of Head and Neck/drug therapy , Animals , Antineoplastic Agents/therapeutic use , Bleomycin/pharmacology , Bleomycin/therapeutic use , Cisplatin/pharmacology , Cisplatin/therapeutic use , DNA Breaks, Double-Stranded/drug effects , DNA Breaks, Double-Stranded/radiation effects , Female , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/radiotherapy , Humans , Mice , Mice, SCID , Radiation Tolerance , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/radiotherapy , Treatment Outcome , Xenograft Model Antitumor Assays
8.
Mar Drugs ; 16(10)2018 Oct 03.
Article in English | MEDLINE | ID: mdl-30282908

ABSTRACT

The alkylpyridinium polymer APS8, a potent antagonist of α7 nicotinic acetylcholine receptors (nAChRs), selectively induces apoptosis in non-small cell lung cancer cells but not in normal lung fibroblasts. To explore the potential therapeutic value of APS8 for at least certain types of lung cancer, we determined its systemic and organ-specific toxicity in mice, evaluated its antitumor activity against adenocarcinoma xenograft models, and examined the in-vitro mechanisms of APS8 in terms of apoptosis, cytotoxicity, and viability. We also measured Ca2+ influx into cells, and evaluated the effects of APS8 on Ca2+ uptake while siRNA silencing of the gene for α7 nAChRs, CHRNA7. APS8 was not toxic to mice up to 5 mg/kg i.v., and no significant histological changes were observed in mice that survived APS8 treatment. Repetitive intratumoral injections of APS8 (4 mg/kg) significantly delayed growth of A549 cell tumors, and generally prevented regrowth of tumors, but were less effective in reducing growth of HT29 cell tumors. APS8 impaired the viability of A549 cells in a dose-dependent manner and induced apoptosis at micro molar concentrations. Nano molar APS8 caused minor cytotoxic effects, while cell lysis occurred at APS8 >3 µM. Furthermore, Ca2+ uptake was significantly reduced in APS8-treated A549 cells. Observed differences in response to APS8 can be attributed to the number of α7 nAChRs expressed in these cells, with those with more AChRs (i.e., A549 cells) being more sensitive to nAChR antagonists like APS8. We conclude that α7 nAChR antagonists like APS8 have potential to be used as therapeutics for tumors expressing large numbers of α7 nAChRs.


Subject(s)
Apoptosis/drug effects , Cell Proliferation/drug effects , Lung Neoplasms/drug therapy , Polymers/pharmacology , Pyridinium Compounds/pharmacology , alpha7 Nicotinic Acetylcholine Receptor/metabolism , A549 Cells , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/metabolism , Animals , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lung Neoplasms/metabolism , Mice , RNA, Small Interfering/metabolism
9.
Technol Cancer Res Treat ; 17: 1533033818797066, 2018 01 01.
Article in English | MEDLINE | ID: mdl-30176769

ABSTRACT

Hypoxia is a condition, common to most malignant tumors, where oxygen tension in the tissue is below the physiological level. Among consequences of tumor hypoxia is also altered cancer cell metabolism that contributes to cancer therapy resistance. Therefore, precise assessment of tumor hypoxia is important for monitoring the tumor treatment progression. In this study, we propose a simple model for prediction of hypoxic level in tumors based on multiparametric magnetic resonance imaging. The study was performed on B16F1 murine melanoma tumors ex vivo that were first magnetic resonance scanned and then analyzed for hypoxic level using hypoxia-inducable factor 1-alpha antibody staining. Each tumor was analyzed in identical sections and in identical regions of interest for pairs of hypoxic level and magnetic resonance values (apparent diffusion coefficient and T2). This was followed by correlation analysis between hypoxic level and respective magnetic resonance values. A moderate correlation was found between hypoxic level and apparent diffusion coefficient (ρ = 0.56, P < .00001) and lower between hypoxic level and T2 (ρ = 0.38, P < .00001). The data were analyzed further to obtain simple predictive models based on the multiple linear regression analysis of the measured hypoxic level (dependent variable) and apparent diffusion coefficient and T2 (independent variables). Among the hypoxic level models, the most efficient was the 3-parameter model given by relation ( HL = kADC ADC + kT2 T2 + b), where kADC = 26%/µm2/ms, kT2 = 0.8%/ms, and b = -32%. The model can be used for calculation of the predicted hypoxic level map based on magnetic resonance-measured apparent diffusion coefficient and T2 maps. Similar prediction models, based on tumor apparent diffusion coefficient and T2 maps, can be done also for other tumor types in vivo and can therefore help in assessment of tumor treatment as well as to better understand the role of hypoxia in cancer progression.


Subject(s)
Diffusion Magnetic Resonance Imaging/methods , Melanoma, Experimental/diagnostic imaging , Prognosis , Tumor Hypoxia/physiology , Animals , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Melanoma, Experimental/pathology , Melanoma, Experimental/physiopathology , Melanoma, Experimental/therapy , Mice
10.
Technol Cancer Res Treat ; 17: 1533033818784208, 2018 01 01.
Article in English | MEDLINE | ID: mdl-29969947

ABSTRACT

Vascular-targeted therapies exhibit radiosensitizing effects by remodeling tumor vasculature, thus facilitating the increased oxygenation of the remaining tumor tissue. To examine these phenomena, the effects of antiendoglin gene therapy alone and in combination with irradiation were monitored for 5 consecutive days on a murine mammary adenocarcinoma (TS/A) tumor model growing in a dorsal window chamber. The vascularization of the tumors was assessed by the determination of the tumor vascular area and by measurement of tumor perfusion by using laser Doppler flowmetry to provide insight into intratumoral gene electrotransfer effects. The changes in the vascular area after this specific therapy correlated with laser Doppler measurements, indicating that either of the methods can be used to demonstrate the induced changes in the vascularization and perfusion of tumors. Gene electrotransfer with an endothelial-specific promoter resulted in a vascular-targeted effect on tumor vasculature within the first 24 hours and did not restore within 5 days. The combination with the irradiation did not result in a more pronounced vascular effect, and irradiation alone only abrogated the formation of new vessels and prevented an increase in the tumor perfusion over time. The results indicate that tumors grown in a dorsal window chamber facilitate intravital measurements of the vascularization of tumors and blood perfusion, enabling the monitoring of the antiangiogenic or vascular disruptive effects of different therapies.


Subject(s)
Genetic Therapy/methods , Intravital Microscopy/methods , Mammary Neoplasms, Experimental/pathology , Neovascularization, Pathologic/diagnostic imaging , Adenocarcinoma/diagnostic imaging , Adenocarcinoma/pathology , Animals , Electroporation , Endoglin/antagonists & inhibitors , Female , Laser-Doppler Flowmetry/methods , Mammary Neoplasms, Experimental/diagnostic imaging , Mice , Neovascularization, Pathologic/pathology
11.
Radiol Oncol ; 51(1): 30-39, 2017 Mar 01.
Article in English | MEDLINE | ID: mdl-28265230

ABSTRACT

BACKGROUND: Tumor irradiation combined with adjuvant treatments, either vascular targeted or immunomodulatory, is under intense investigation. Gene electrotransfer of therapeutic genes is one of these approaches. The aim of this study was to determine, whether gene electrotransfer of plasmid encoding shRNA for silencing endoglin, with vascular targeted effectiveness, can radiosensitize melanoma B16F10 tumors. MATERIALS AND METHODS: The murine melanoma B16F10 tumors, growing on the back of C57Bl/6 mice, were treated by triple gene electrotransfer and irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice. Furthermore, histological analysis of tumors (necrosis, apoptosis, proliferation, vascularization, presence of hypoxia and infiltration of immune cells,) was used to evaluate the therapeutic mechanisms. RESULTS: Gene electrotransfer of plasmid silencing endoglin predominantly indicated vascular targeted effects of the therapy, since significant tumor growth delay and 44% of tumor free mice were obtained. In addition, irradiation had minor effects on radioresistant melanoma, with 11% of mice tumor free. The combined treatment resulted in excellent effectiveness with 88% of mice tumor free, with more than half resistant to secondary tumor challenge, which was observed also with the plasmid devoid of the therapeutic gene. Histological analysis of tumors in the combined treatment group, demonstrated similar mode of action of the gene electrotransfer of plasmid encoding shRNA for silencing endoglin and devoid of it, both through the induction of an immune response. CONCLUSIONS: The results of this study indicate that irradiation can in radioresistant melanoma tumors, by release of tumor associated antigens, serve as activator of the immune response, besides directly affecting tumor cells and vasculature. The primed antitumor immune response can be further boosted by gene electrotransfer of plasmid, regardless of presence of the therapeutic gene, which was confirmed by the high radiosensitization, resulting in prolonged tumor growth delay and 89% of tumor free mice that were up to 63% resistant to secondary challenge of tumor. In addition, gene electrotransfer of therapeutic plasmid for silencing endoglin has also a direct effect on tumor vasculature and tumors cells; however in combination with radiotherapy this effect was masked by pronounced immune response.

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