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 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.

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.

Tissue engineering has emerged as an indispensable tool for the reconstruction of organ-specific environments. Organ-derived extracellular matrices (ECM) and, especially, decellularized tissues (DCL) are recognized as the most successful biomaterials in regenerative medicine, as DCL preserves the most essential organ-specific ECM properties such as composition alongside biomechanics characterized by stiffness and porosity. Expansion of the DCL technology to cancer biology research, drug development, and nanomedicine is pending refinement of the existing DCL protocols whose reproducibility remains sub-optimal varying from organ to organ. We introduce a facile decellularization protocol universally applicable to murine organs, including liver, lungs, spleen, kidneys, and ovaries, with demonstrated robustness, reproducibility, high purification from cell debris, and architecture preservation, as confirmed by the histological and SEM analysis. The biomechanical properties of as-produced DCL organs expressed in terms of the local and total stiffness were measured using our facile methodology and were found well preserved in comparison with the intact organs. To demonstrate the utility of the developed DCL model to cancer research, we engineered three-dimensional tissue constructs by recellularization representative decellularized organs and collagenous hydrogel with human breast cancer cells of pronounced mesenchymal (MDA-MB-231) or epithelial (SKBR-3) phenotypes. The biomechanical properties of the DCL organs were found pivotal to determining the cancer cell fate and progression. Our histological and scanning electron microscopy (SEM) study revealed that the larger the ECM mean pore size and the smaller the total stiffness (as in lung and ovary), the more proliferative and invasive the mesenchymal cells became. At the same time, the low local stiffness ECMs (ranged 2.8–3.6 kPa) did support the epithelial-like SKBR-3 cells’ viability (as in lung and spleen), while stiff ECMs did not. The total and local stiffness of the collagenous hydrogel was measured too low to sustain the proliferative potential of both cell lines. The observed cell proliferation patterns were easily interpretable in terms of the ECM biomechanical properties, such as binding sites, embedment facilities, and migration space. As such, our three-dimensional tissue engineering model is scalable and adaptable for pharmacological testing and cancer biology research of metastatic and primary tumors, including early metastatic colonization in native organ-specific ECM.