Model of Ovarian Adenocarcinoma Spheroids for Assessing Photodynamic Cytotoxicity.

The aim of the study was to compare the relevance of ovarian adenocarcinoma spheroids with that of a monolayer culture for assessing photodynamic effect of the tetrakis(4-benzyloxyphenyl)tetracyanoporphyrazine photosensitizer. MATERIALS AND METHODS: The work was performed on SKOV-3 human ovary adenocarcinoma cells grown in vitro in a monolayer culture and in the form of tumor spheroids obtained using culture plates with ultra-low attachment. We determined the photoinduced toxicity of porphyrazine on a monolayer culture using the MTT assay; the effect on the spheroids was tested by assessing the dynamics of their growth. Cellular uptake of porphyrazine was analyzed by confocal microscopy. RESULTS: Porphyrazine has a pronounced photodynamic effect on SKOV-3 cells. When exposed to light at a dose of 20 J/cm2, the IC50 value 24 h after exposure was 2.3 µM for SKOV-3 monolayer culture. For the spheroids, the effect manifested after a latency period: significant growth retardation of the treated spheroids appeared no sooner than 5 and 9 days after exposure. Notably, no decrease in the initial size of the treated spheroids was observed under any of the photodynamic regimes. The penetration depth of porphyrazine into spheroids was 50-100 µm during 24 h incubation. CONCLUSION: The limited penetration of the photosensitizer into the body of spheroids and its predominant accumulation in the surface layers can be one of the key factors behind the significant differences in the photodynamic response between the surface and deep layers of a spheroid. For cells located close to the spheroid surface, the photodynamic effect is comparable to that for a monolayer culture, while in deeper layers, the cells remain viable and support/maintain the growth of the spheroid even under intense photo-exposure. The fact that the in vitro distribution is similar to the inhomogeneous accumulation of photosensitizers in tumors in vivo allows us to consider spheroids more relevant than a monolayer culture for studying photodynamic anti-tumor effects.

Spheroids of HER2-Positive Breast Adenocarcinoma for Studying Anticancer Immunotoxins In Vitro.

Tumor response to therapeutic treatment is largely determined by its heterogeneity and the presence of intercellular junctions, hindering the penetration of large molecules deep into the three-dimensional structure of the tumor. In that context, 3D in vitro tumor models such as cancer cell spheroids are becoming increasingly popular. We obtained spheroids of human breast adenocarcinoma SKBR-3 overexpressing the HER2 cancer marker. The toxicity of HER2-targeted immunotoxin 4D5scFv-PE40 against spheroids was shown to be several orders of magnitude lower compared to a monolayer cell culture. The significant difference in the severity of the immunotoxin effect can be explained by the fact that it ineffectively penetrates the spheroid and predominantly influences the cells of the outer layers. The resulting tumor spheroid model can be used in development of drugs for targeted therapy as well as to study ways to improve the efficiency of anticancer agents by targeting cell-cell contacts.

Far-red fluorescent cell line for preclinical study of HER2-targeted agents.

We have created novel HER2-overexpressing human ovarian adenocarcinoma cell line stably transfected with far-red fluorescent protein TurboFP635. Growth characteristics, adhesion capacity and morphology of the cells do not differ from that of parental cell line. The obtained cell line (SKOV-kat) was confirmed to have high level of expression of the tumor marker HER2 comparable to that of initial cell line SKOV-3. The SKOV-kat cell line is designed for further establishment of experimental fluorescent models of HER2-overexpressing human ovarian cancer that make it possible to optimize and reduce the cost of preclinical study of agents for treatment of this disease.

[Development of a new photosensitizer on the basis of ytterbium porphyrazine complex].

The tetraphenyltetracyanoporphyrazine complex of ytterbium has been studied as a potential photosensitizer for fluorescence diagnostics and photodynamic therapy (PDT) of cancer. It has been shown that the new compound has an intensive absorption and fluorescence in the "tissue optical window". In particular, the absorption maximum of the complex is at the wavelength of 590 nm, and the fluorescence emission maximum is at 640 nm. A strong fluorescence enhancement with a 50-fold increase in the quantum yield has been revealed in blood serum. The experiments on human cancer cells line have demonstrated that the complex penetrates the cells in vitro and is located around the nuclei. The biodistribution and pharmacokinetics of the complex in animals have been investigated in vivo by a new method of transillumination fluorescence imaging using a peculiar setup. It has been found that the period of maximum uptake of the complex in mouse cervical carcinoma is from 3 to 6 h after i.v. injection, with the half-life in the tumor being 24 h. However, the selectivity of the complex in the tumor is not high enough. The time of clearance from the body is about 48 h. The area of the strongest fluorescence in the abdominal cavity in in vivo images is anatomically recognized as the intestine. This indicates that the new compounds undergo mainly the hepatic clearance mainly. The conventional methods ex vivo (confocal microscopy and point spectroscopic measurements) have detected the largest content of the complex in the intestine, liver, skin and tumor tissue. In general, the optical characteristics of the ytterbium porphyrazine complex as well as the features of its interaction with biological objects make it promising drug candidate for the photodynamic therapy and/or fluorescence diagnostics of cancer. However, a search for other novel formulations possessing a higher tumor selectivity remains an urgent problem.