Papel da atividade neuronal sensorial e resposta quimioterápica à cisplatina no fenômeno de transição epitélio-mesenquimal (EMT) no câncer de pulmão de células não-pequenas (NSCLC))

Abstract

The phenomenon of epithelial-mesenchymal transition (EMT) is traditionally associated with cell migration during embryonic development or wound healing, however, today it is known that this mechanism plays a key role in the invasion and metastasis of solid tumors. Thus, using the state of the art, the main objective of this study was to evaluate the in-vitro participation of this event, in the non-small cell lung cancer (NSCLC) cell line, LLC1, in the monolayer (MN) and cancer stem cells enrichment model (CSC's) through the tumorspheres (TE) assay. Furthermore, we seek to elucidate how cisplatin treatment or sensory neuron depletion can modulate the EMT in lung cancer in-vivo. To achieve the objectives proposed here, we determined the IC50 for LLC1 cells treated with cisplatin. The transcriptional state of the EMT regulatory genes (Zeb1, Zeb2, Twist, Snail, and FoxA2) and the downstream targets of these transcription factors (E-cadherin, Tgfb1, Vimentin, Fibronectin, and N-cadherin), in addition to pluripotency genes (Sox2, Oct-4, and Nanog), were evaluated by qPCR. We evaluated the expression of the epithelial marker E-cadherin and the mesenchymal proteins fibronectin, vimentin, and N-cadherin by immunofluorescence. To characterize cell subpopulations with CSCs-like profiles (CD44+/CD133+/ALDH+/CD24+), and the proliferative state (Ki-67+) we performed immunophenotyping by flow cytometry. LLC1 cells were treated with the conditioned medium isolated from the in-vitro culture of sensory neurons extracted from the dorsal root ganglion of C57BL/6J animals, or placed in co-culture in the transwell system. Finally, LLC1 cells were inoculated subcutaneously (S.C) in C57Bl/6J animals that had their sensory neurons depleted through pharmacological treatment with resiniferotoxin (RTX) or that were treated only with the chemotherapy drug cisplatin. Thus, we characterized the microenvironment of tumors arising from the growth of LLC1 cells through immunophenotyping for lung CSCs and immune cells, in addition to evaluating the transcriptomic profile of the genes involved in the EMT phenomenon, pluripotency and programmed cell death by apoptosis. Among the results obtained, the following stand out: (I) positive regulation of the expression of regulatory transcripts of the EMT phenomenon and pluripotency genes after treatment with cisplatin in-vitro and presence of cells with a CSC profile even after treatment with cisplatin; (II) increase in IC50 in MN and TE treated with cisplatin diluted in conditioned medium; (III) presence of a hybrid phenotype in LLC1 cells in co-culture with sensory neurons, with expression of the epithelial E-cadherin and mesenchymal protein, vimentin, in tumor cells; (IV) absence of sensory fibers in the primary tumor of animals treated with cisplatin, similar to RTX depletion; (V) increase in volume, weight and area of

tumors in animals that had sensory neurons depleted with RTX; (VI) decrease of E-cadherin, N-cadherin, fibronectin, and vimentin proteins, reduction of CSCs-like profile and of immune system cells, in primary tumors of animals treated with cisplatin or whose neurons were depleted with RTX. Thus, our findings strongly support the idea that sensory nerve fibers establish a crosstalk with the tumor microenvironment and that cisplatin treatment can affect the presence of these fibers in the primary tumor. In this way, it is expected that the data generated may contribute to the understanding of how sensory nerve fibers can modulate the cellular complexity of the tumor, opening space for potential therapeutic approaches.