Abstract
BackgroundRecent developments in abscopal effect strongly support the use of radiotherapy for the treatment of metastatic disease. However, deeper understanding of the molecular mechanisms underlying the abscopal effect are required to best benefit a larger proportion of patients with metastasis. Several groups including ours, reported the involvement of wild-type (wt) p53 in radiation-induced abscopal effects, however very little is known on the role of wtp53 dependent molecular mechanisms.MethodsWe investigated through in vivo and in vitro approaches how wtp53 orchestrates radiation-induced abscopal effects. Wtp53 bearing (A549) and p53-null (H1299) NSCLC lines were xenotransplanted in nude mice, and cultured in 2D monolayers and 3D tumor spheroids. Extracellular vesicles (EVs) were isolated from medium cell culture by ultracentrifugation protocol followed by Nanoparticle Tracking Analysis. Gene expression was evaluated by RT-Real Time, digital qRT-PCR, and dot blot technique. Protein levels were determined by immunohistochemistry, confocal anlysis, western blot techniques, and immunoassay.ResultsWe demonstrated that single high-dose irradiation (20 Gy) induces significant tumor growth inhibition in contralateral non-irradiated (NIR) A549 xenograft tumors but not in NIR p53-null H1299 or p53-silenced A549 (A549sh/p53) xenografts. We further demonstrates that irradiation of A549 cells in vitro induces a senescence-associated secretory phenotype (SASP) producing extracellular vesicles (EVs) expressing CD63 and carrying DNA:RNA hybrids and LINE-1 retrotransposon. IR-A549 EVs also hamper the colony-forming capability of recipient NIR A549 cells, induce senescent phenotype, nuclear expression of DNA:RNA hybrids, and M1 macrophage polarization.ConclusionsIn our models, we demonstrate that high radiation dose in wtp53 tumors induce the onset of SASP and secretion of CD63+ EVs loaded with DNA:RNA hybrids and LINE-1 retrotransposons that convey senescence messages out of the irradiation field triggering abscopal effect in NIR tumors.
Highlights
Recent developments in abscopal effect strongly support the use of radiotherapy for the treatment of metastatic disease
In our models, we demonstrate that high radiation dose in wtp53 tumors induce the onset of senescenceassociated secretory phenotype (SASP) and secretion of CD63+ Extracellular vesicles (EVs) loaded with DNA:RNA hybrids and long interspersed element-1 (LINE-1) retrotransposons that convey senescence messages out of the irradiation field triggering abscopal effect in NIR tumors
Tumor-associated macrophages (TAM) and IL6 were detected in NIR A549 xenograft tumors To microscopically investigate the RT-induced abscopal effect” (AE), A549 xenografts were RT treated/untreated, as in Fig. 1a, and tumors collected at various time-points after irradiation for histological examination
Summary
Recent developments in abscopal effect strongly support the use of radiotherapy for the treatment of metastatic disease. A growing body of evidence sustains the immune system activation as the dominant player in radiation-induced AE It has been well-established that the release of a number of moieties endowed with immunostimulatory properties, released from irradiated lesions in the tumor microenvironment and systemic circulation, are capable of conveying death messages (apoptotic and / or necrotic signals) inducing the immunogenic cell death (ICD) [9, 10]. These molecules, known as damageassociated molecular patterns (DAMPs), promote and convert the IR site into an immunogenic hub through the innate and adaptive immune response [11]
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