High spatial resolution imaging of cisplatin and Texas Red cisplatin in tumour spheroids using laser ablation isotope dilution inductively coupled plasma mass spectrometry and confocal fluorescence microscopy.

Oncology research uses different imaging techniques to provide information about the spatial distribution of the chemotherapy drugs used for the targeted tissues. Among them, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is increasingly being used to track the spatial distribution of metal-based chemotherapeutics in different tissue samples. In this investigation, instrumental parameters were optimized for the bioimaging of Pt in HT29 tumour spheroids treated with cisplatin (CDDP) or Texas Red cisplatin (TR-CDDP) using LA-ICP-MS. A high spatial resolution, using pixel dimensions of 2.0 µm × 2.5 µm, and a high sensitivity, with the limits of detection (LOD) better than 0.78 mg kg-1 Pt, was achieved. Matrix-matched gelatine standards and/or isotope dilution (ID) analyses were used to quantify the amount of Pt. Differences between the results of the Pt concentrations determined by the two quantification were less than 4%. The results of the LA analysis revealed that the Pt in the CDDP-treated tumour spheroids was localized primarily in the outer rim of the spheroids and to a lesser extent in the intermediary layer and the necrotic core. Due to the steric effects, significantly lower Pt concentrations were accumulated in the spheroids treated with TR-CDDP (2.2 times lower than in CDDP-treated spheroids, normalized to the spheroid volume), while the Pt was mostly distributed in the areas of the outer rim. Finally, imaging with confocal fluorescence microscopy, which is commonly used in oncology research, was compared with that by LA-ICP-MS. The results of the two complementary techniques demonstrated good agreement in terms of the spatial distribution of the TR-CDDP, while the intensity of the fluorescence matched well with the concentrations of Pt determined with LA-ICP-MS.

Potentiation of electrochemotherapy effectiveness by immunostimulation with IL-12 gene electrotransfer in mice is dependent on tumor immune status.

Electrochemotherapy (ECT) exhibits high therapeutic effectiveness in the clinic, achieving up to 80% local tumor control but without a systemic (abscopal) effect. Therefore, we designed a combination therapy consisting of ECT via intratumoral application of bleomycin, oxaliplatin or cisplatin with peritumoral gene electrotransfer of a plasmid encoding interleukin-12 (p. t. IL-12 GET). Our hypothesis was that p. t. IL-12 GET potentiates the effect of ECT on local and systemic levels and that the potentiation varies depending on tumor immune status. Therefore, the combination therapy was tested in three immunologically different murine tumor models. In poorly immunogenic B16F10 melanoma, IL-12 potentiated the antitumor effect of ECT with biologically equivalent low doses of cisplatin, oxaliplatin or bleomycin. The most pronounced potentiation was observed after ECT using cisplatin, resulting in a complete response rate of 38% and an abscopal effect. Compared to B16F10 melanoma, better responsiveness to ECT was observed in more immunogenic 4 T1 mammary carcinoma and CT26 colorectal carcinoma. In both models, p. t. IL-12 GET did not significantly improve the therapeutic outcome of ECT using any of the chemotherapeutic drugs. Collectively, the effectiveness of the combination therapy depends on tumor immune status. ECT was more effective in more immunogenic tumors, but GET exhibited greater contribution in less immunogenic tumors. Thus, the selection of the therapy, namely, either ECT alone or combination therapy with p. t. IL-12, should be predominantly based on tumor immune status.

Experimental and Numerical Study of Electroporation Induced by Long Monopolar and Short Bipolar Pulses on Realistic 3D Irregularly Shaped Cells.

In this article, the reversible electroporation induced by rectangular long unipolar and short bipolar voltage pulses on 3D cells is studied. The cell geometry was reconstructed from 3D images of real cells obtained using the confocal microscopy technique. A numerical model based on the Maxwell and the asymptotic Smoluchowski equations has been developed to calculate the induced transmembrane voltage and pore density on the plasma membrane of real cells exposed to the pulsed electric field. Moreover, in the case of the high-frequency pulses, the dielectric dispersion of plasma membranes has been taken into account using the second-order Debye-based relationship. Several numerical simulations were performed and we obtained suitable agreement between the numerical and experimental results.

Effect of calcium electroporation on tumour vasculature.

Calcium electroporation (CaEP) is a novel anti-tumour treatment that induces cell death by internalization of large quantities of calcium. The anti-tumour effectiveness of CaEP has been demonstrated in vitro, in vivo, and in preliminary clinical trials; however, its effects on the vasculature have not been previously investigated. Using a dorsal window chamber tumour model, we observed that CaEP affected to the same degree normal and tumour blood vessels in vivo, as it disrupted the vessels and caused tumour eradication by necrosis. In all cases, the effect was more pronounced in small vessels, similar to electrochemotherapy (ECT) with bleomycin. In vitro studies in four different cell lines (the B16F1 melanoma, HUVEC endothelial, FADU squamous cell carcinoma, and CHO cell lines) confirmed that CaEP causes necrosis associated with acute and severe ATP depletion, a picture different from bleomycin with electroporation. Furthermore, CaEP considerably inhibited cell migratory capabilities of endothelial cells and their potential to form capillary-like structures. The finding that CaEP has anti-vascular effects and inhibits cell migration capabilities may contribute to the explanation of the high efficacy observed in preclinical and clinical studies.

Comparable effectiveness and immunomodulatory actions of oxaliplatin and cisplatin in electrochemotherapy of murine melanoma.

Interest in platinum-based chemotherapeutics such as oxaliplatin (OXA) and cisplatin (CDDP) has been reinvigorated by their newly described impacts on tumor-specific immune responses. In addition to CDDP, OXA is frequently used to treat cancers. Based on the characteristics of OXA, which are similar to those of CDDP, and the presumably more pronounced immunomodulatory effect of OXA, OXA is a candidate for electrochemotherapy (ECT). We compared the effectiveness of intratumoral ECT with OXA to that of ECT with CDDP in murine B16F10 melanoma to determine the equieffective dose. Special attention was given to the elicitation of immunogenic cell death and local immune response. Based on the in vitro and in vivo results pertaining to effectiveness and drug uptake in cells and tumors, ECT with OXA is as effective as ECT with CDDP when the OXA dose is increased 1.6-fold. Exposure of melanoma cells to ECT induces immunogenic cell death when either OXA or CDDP is used, which correlates with a comparable increase in lymphocyte infiltration into tumors after ECT with either OXA or CDDP. Based on these results, OXA is a valid platinum-based drug for use with ECT, and the effectiveness of ECT with OXA is comparable to that of the well-established ECT with CDDP. Furthermore, both drugs display equal and specific immune responses following ECT.