No abstract

The efficiency of delivering a therapeutic agent into a tumor is among the crucial factors determining the prospects for its clinical use. This problem is particularly acute in the case of targeted antitumor agents since many of them are high-molecular-weight compounds. In this work, the penetration of therapeutic agents of two distinct molecular weights into the spheroids of ovarian adenocarcinoma overexpressing human epidermal growth factor receptor 2 (HER2) was studied. It was shown that the low-molecular-weight chemotherapy drug, doxorubicin (~0.5 kDa), effectively penetrates through almost the entire depth of a 300 to 400 μm spheroid, while the penetration depth of the HER2-specific recombinant targeted toxin, DARPin-LoPE (~42 kDa), is only a few surface layers of cells and does not exceed 70 μm. The low penetration of the targeted toxin into spheroid was shown along with a significant decrease in its efficiency against the three-dimensional tumor spheroid as compared with the two-dimensional monolayer culture. The approaches to increasing the accumulation of agents in the tumor are presented and prospects of their use in order to improve the effectiveness of therapy are discussed.

Tumor resistance to therapy is associated with the 3D organization and peculiarities of the tumor microenvironment, of which intercellular adhesion is a key participant. In this work, the abundance of contact proteins was compared in SKOV-3 and SKOV-3.ip human ovarian adenocarcinoma cell lines, cultivated in monolayers, tumor spheroids and collagen hydrogels. Three-dimensional models were characterized by extremely low expression of basic molecules of adherens junctions E-cadherin and demonstrated a simultaneous decrease in desmosomal protein desmoglein-2, gap junction protein connexin-43 and tight junction proteins occludin and ZO-1. The reduction in the level of contact proteins was most pronounced in collagen hydrogel, accompanied by significantly increased resistance to treatment with doxorubicin and targeted anticancer toxin DARPin-LoPE. Thus, we suggest that 3D models of ovarian cancer, especially matrix-based models, tend to recapitulate tumor microenvironment and treatment responsiveness to a greater extent than monolayer culture, so they can be used as a highly relevant platform for drug efficiency evaluation.

Today’s research on the processes of carcinogenesis and the vital activity of tumor tissues implies more attention be paid to constituents of the tumor microenvironment and their interactions. These interactions between cells in the tumor microenvironment can be mediated via different types of protein junctions. Connexins are one of the major contributors to intercellular communication. They form the gap junctions responsible for the transfer of ions, metabolites, peptides, miRNA, etc., between neighboring tumor cells as well as between tumor and stromal cells. Connexin hemichannels mediate purinergic signaling and bidirectional molecular transport with the extracellular environment. Additionally, connexins have been reported to localize in tumor-derived exosomes and facilitate the release of their cargo. A large body of evidence implies that the role of connexins in cancer is multifaceted. The pro- or anti-tumorigenic properties of connexins are determined by their abundance, localization, and functionality as well as their channel assembly and non-channel functions. In this review, we have summarized the data on the contribution of connexins to the formation of the tumor microenvironment and to cancer initiation and progression.

The use of 3D in vitro tumor models has become a common trend in cancer biology studies as well as drug screening and preclinical testing of drug candidates. The transition from 2D to 3D matrix-based cell cultures requires modification of methods for assessing tumor growth. We propose the method for assessing the growth of tumor cells in a collagen hydrogel using macro-scale registration and quantification of the gel epi-fluorescence. The technique does not require gel destruction, can be used for real-time observation of fast (in seconds) cellular responses and demonstrates high agreement with cell counting approaches or measuring total DNA content. The potency of the method was proven in experiments aimed at testing cytotoxic activity of chemotherapeutic drug (cisplatin) and recombinant targeted toxin (DARPin-LoPE) against two different tumor cell lines genetically labelled with fluorescent proteins. Moreover, using fluorescent proteins with sensor properties allows registration of dynamic changes in cells’ metabolism, which was shown for the case of sensor of caspase 3 activity.