Irina I. Suvorova

BACKGROUND: Cultivation of dorsal root ganglia (DRGs) is a widely used approach for modeling physiological and pathological conditions of the peripheral nervous system in vitro. Existing scientific data indicates that postnatal DRGs contain a pool of glial stem cells capable of differentiating into neurons after injury. Therefore, isolated DRGs containing a stem cell pool may retain neurogenic potential ex vivo. However, no studies in the available scientific data have investigated the preservation of the regenerative potential of DRGs ex vivo. AIM: This study aimed to evaluate the neurogenic potential of isolated DRGs and establish a three-dimensional organotypic culture from these explants. METHODS: DRGs were isolated from the cervical, thoracic, and lumbar regions of the spine and cultured short-term and long-term as three-dimensional explants in Geltrex gel. The morphology, ultrastructure, and gene expression profile of the cultured DRGs were analyzed using light and electron microscopy, immunofluorescent staining, and quantitative reverse transcription polymerase chain reaction. RESULTS: DRGs cultured in Geltrex for 14 days consistently generated a three-dimensional periganglionic network composed of radially branching elongated cells. Immunofluorescent staining with Tuj1 antibodies (neuron-specific class III β-tubulin) demonstrated that the observed cells were neuronal. Immunostaining with O4 antibodies confirmed that Tuj1-positive cells were not migrating Schwann cells. Quantitative reverse transcription polymerase chain reaction analysis of total RNA extracted from the DRG explant cultures revealed a significant increase in mRNA levels of key neurogenesis-related genes, such as Neurog1, Neurog2, and Sox2, indicating activation of a neurogenic program in the cultured explants. Light and electron microscopy showed that adult sensory neurons degenerated within the DRG explants and did not contribute to the periganglionic network. Using available confocal capabilities, we evaluated three-dimensional parameters of the periganglionic network from two-dimensional microscopic images to demonstrate the topography of Tuj1-expressing neuronal cells in a DRG explant culture. CONCLUSION: We characterized a three-dimensional culture of DRGs. Isolated explants demonstrate regenerative capacity under in vitro conditions. They may represent a novel organotypic model with neurogenic potential. The obtained model may be relevant for studying regenerative processes in DRGs, expanding research opportunities beyond traditional stem cell–derived models.

Recent findings have indicated that pharmacological inhibition of the mTOR kinase can become a widely used experimental approach to generate dormant cancer cells in vitro. However, the suppression of mTOR, which is responsible for global translation, can significantly rewire basic cellular functions influencing the expression of housekeeping genes. To prevent incorrect selection of a reference gene in dormant tumor cells, we analyzed the expression stability of the widely used housekeeping genes GAPDH, ACTB, TUBA1A, RPS23, RPS18, RPL13A, PGK1, EIF2B1, TBP, CYC1, B2M, and YWHAZ in the T98G, A549, and PA-1 cancer cell lines treated with the dual mTOR inhibitor AZD8055. It has been revealed that the expression of the ACTB gene, encoding the cytoskeleton, and the RPS23, RPS18, and RPL13A genes, encoding ribosomal proteins, undergoes dramatic changes, and these genes are categorically inappropriate for RT-qPCR normalization in cancer cells treated with dual mTOR inhibitors. B2M and YWHAZ were determined to be the best reference genes in A549 cells, and the TUBA1A and GAPDH genes were the best reference genes in T98G cells. The optimal reference genes among the 12 candidate reference genes were not revealed in the PA-1 cell line. Validation of the stability of the 12 investigated genes demonstrated that the incorrect selection of a reference gene resulted in a significant distortion of the gene expression profile in dormant cancer cells.

The current focus in oncology research is the translational control of cancer cells as a major mechanism of cellular plasticity. Recent evidence has prompted a reevaluation of the role of the mTOR pathway in cancer development leading to new conclusions. The mechanistic mTOR inhibition is well known to be a tool for generating quiescent stem cells and cancer cells. In response to mTOR suppression, quiescent cancer cells dynamically change their proteome, triggering alternative non-canonical translation mechanisms. The shift to selective translation may have clinical relevance, since quiescent tumor cells can acquire new phenotypical features. This review provides new insights into the patterns of mTOR functioning in quiescent cancer cells, enhancing our current understanding of the biology of latent metastasis.

Numerous studies have already established that autophagy plays a central role in the survival of all cells, including malignant ones. Autophagy is a central cog in the general mechanism that provides the intracellular proteostasis determining cellular physiological and phenotypic characteristics. The accumulated data show that autophagy largely contributes to cancer cell stemness. Thus, autophagy modulation is considered one of the promising pharmacological targets in therapy aimed at cancer stem cell elimination. However, autophagy is a multi-stage intracellular process that involves numerous protein participants. In addition, the process can be activated simultaneously by various signaling modules. Therefore, it is no small feat to select an effective pharmacological drug against autophagy. Whats more, the search for potential chemotherapeutic agents that could eliminate cancer stem cells through pharmacological inhibition of autophagy is still under way. In the present work, we selected a panel of autophagy inhibitors (Autophinib, SBI-0206965, Siramesine, MRT68921, and IITZ-01), some of whom have been recently identified as effective autophagy inhibitors in cancer cells. Using A549 cancer cells, which express the core stem factors Oct4 and Sox2, we evaluated the effect of these drugs on the survival and preservation of the original properties of cancer stem cells. Among the agents selected, only Autophinib demonstrated a significant toxic effect on cancer stem cells. The obtained results demonstrate that autophagy inhibition by Autophinib downregulates the expression of the Sox2 protein in A549 cells, and that this downregulation correlates with a pronounced induction of apoptosis. Moreover, Autophinib-treated A549 cells are unable to form spheroids, which indicates a reduction in stemness. Thus, among the drugs studied, only Autophinib can be considered a potential agent against cancer stem cells.

The approach to the study of autophagy has been undergoing considerable change lately: from investigations of the protein components of autophagic machinery to its regulation at different molecular levels. Autophagy is being examinated not only as a separated degradative process per se in cells but as an executor mechanism of certain signaling pathways that converge on it, being activated under specific conditions. Additionally, autophagy is beginning to be observed as a key integral part of cellular reprogramming, the transition from one phenotypic state to another associated with rapid degradation of the previous proteostasis. Macrophages and microglia demonstrate a diversity of phenotypes reflecting their effective capability to phenotypic plasticity. Therefore, understanding the role of autophagy in macrophage and microglia functions needs to be addressed. In this review, we focus on autophagy as a fundamental intracellular process underlying macrophages and microglia polarization.

Currently, the success of targeted anticancer therapies largely depends on the correct understanding of the dormant state of cancer cells, since it is increasingly regarded to fuel tumor recurrence. The concept of cancer cell dormancy is often considered as an adaptive response of cancer cells to stress, and, therefore, is limited. It is possible that the cancer dormant state is not a privilege of cancer cells but the same reproductive survival strategy as diapause used by embryonic stem cells (ESCs). Recent advances reveal that high autophagy and mTOR pathway reduction are key mechanisms contributing to dormancy and diapause. ESCs, sharing their main features with cancer stem cells, have a delicate balance between the mTOR pathway and autophagy activity permissive for diapause induction. In this review, we discuss the functioning of the mTOR signaling and autophagy in ESCs in detail that allows us to deepen our understanding of the biology of cancer cell dormancy.

Embryonic stem cells (ESCs), and their artificial counterparts, induced pluripotent stem cells (iPSCs) give rise to all differentiated cell types in adult organism.Therefore, pluripotent cells are an inexhaustible cell source for regenerative medicine. However, the successful clinical application of ESCs and iPSCs is associated with the risk of teratoma formation after transplantation of their differentiated products. Oncogenic potential is believed to be associated with the preservation of pluripotent cells resistant to differentiation. For unknown reason under mitogenic stimuli these defective cells did not activate the mechanisms of exit from pluripotency and remained undifferentiated. During embryogenesis, there are special mechanisms for eliminating the abnormal cells unsuitable for embryo development, which are massively triggered before gastrulation, the initial stage of cell differentiation into germ layers. It is known that, prior to implantation, autophagy plays a critical role in embryo formation and can be considered as one of the main cellular strategies aimed at large-scale restructuring of intracellular material after fertilization. It can be proposed that unless massive intracellular reorganization of embryonic cells occur effective, such cells will have defective proteostasis, affecting their differentiation potential. Therefore, the high level of apoptosis observed before gastrulation in embryogenesis is associated with the elimination of mutant cells that are not suitable for differentiation. Damaged cells are marked with the activated p53 protein indicating the p53-dependent elimination mechanisms. And, apparently, the mechanism of the p53 activation is associated with damaged cellular proteostasis, regulated by autophagy. Thus, the p53-dependent autophagy can play a key role in determining the fate of pluripotent cells: induction of cell death and/or resistance to differentiation. We have shown that the p53 protein is tightly integrated with autophagy and, under defective proteostasis, p53 effectively induces autophagy-mediated cell death in pluripotent cells.