Gambardella, Adriana Rosa (2023) IL-33 stimulates the anticancer activities of eosinophils through exosome-mediated reprogramming of tumor cells. [Tesi di dottorato]

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Abstract

Eosinophils (Eo) are a rare blood-circulating and tissue-infiltrating innate immune cell population representing 1–3% of total granulocytic leukocyte under physiological condition. Eo are important components of the tumor microenvironment (TME) where they play diverse roles depending on the type of cancer and on the environmental stimuli. IL-33 is an epithelial “alarmin” that constitutes a potent activator for eosinophils. Exosomes (Exo) are nanosized extracellular vesicles (EVs) secreted by all cell types that play central roles in intercellular communication in health and disease. Exo transfer their molecular bioactive cargo (nucleic acids, proteins, lipids and metabolites), that reflects the nature of donor cell and its physiological state, to recipient cells inducing downstream signaling cascades. Eosinophils are able producers of Exo through which they promote and autoregulate their functions. Our previous studies demonstrated that mouse eosinophils activated with IL-33 acquire anticancer properties through the formation of eosinophil-tumor cell synapses that result in degranulation and tumor cell killing. In the present study, we demonstrate that in mouse (melanoma, lung adenocarcinoma, fibrosarcoma) and in human (melanoma) tumor cells, the direct contact with IL-33 activated eosinophils (Eo33), respectively mouse and human, (but not with IL-5 stimulated eosinophils, namely Eo5) was responsible for changes in tumor cell expression of genes involved in tumor progression. Tumor cells cultured with Eo33 exhibited increased levels in the expression of cyclin dependent kinase inhibitors (CDKI), such as CDKN1A, CDKN2A, CDKN1B, CDKN2B, suggesting a possible propensity to cell cycle blockade. Analysis of the main molecules (E-Cadherin and N-Cadherin) that regulate the epithelial to mesenchymal transition (EMT) denoted a reversion of this process in mouse and human tumor cells co-cultured with Eo33, indicating that tumor cells might be less prone to tumor dissemination. Co-culture with Eo33 induced significantly higher expression of E-Cadherin (Cdh1) both at transcriptional and surface protein expression, with respect to Eo5. Concomitantly, Eo33 produced in target tumor cells significantly more marked down-modulation of N-Cadherin (Cdh2) compared to Eo5. The observation that the upregulation of E-Cadherin expression on mouse tumor cell surface occurred also in absence of direct contact with Eo33, led us to evaluate the possible involvement of an exosome-mediated pathway in the anticancer functions of IL-33 activated eosinophils. Moreover, we observed that stimulation with IL-33 also increased the secretion of exosomes by both mouse and human eosinophils, as demonstrated by flow cytometry-based enumeration of metabolically labeled C16+ Exo. This finding suggest that activation stimuli promote exosome secretion by eosinophils resulting in enhanced potential to shape target cells or tissues. We found that mouse and human IL-33 activated eosinophil-derived exosomes carry out antitumoral activities by influencing the transcriptional machinery of target tumor cells. We demonstrate that Eo33 drive the expression of CDKI genes in mouse and human tumor cells through the release of exosomes. This transcriptional reprogramming corresponded to cell cycle block in G0/G1 phase and an effective decrease in the proliferative rate of cancer cells, as suggested by flow cytometry experiments and MTS assay. Of note, the anti-proliferative effects of Exo33 did not seem to involve tumor apoptosis, as suggested by Annexin V+ staining of tumor cells cultured with GW exosome inhibitor-treated Eo33. Tumor 3D cultures provided some details of the impact of Eo33 -derived Exo on the ability of tumor cells to interact with each other to form single 3D spheroids. In mouse and human setting, the observation that Exo33 or the Eo33-derived secretome altered the formation and 3D architecture of the tumor spheroid by acting on tumor cell aggregation capability has inspired us to perform gene analysis of the expression of the main molecules that regulate cell adhesion pathways. We found increased expression of membrane integrins in mouse and human tumor cells exposed to Exo33 or to Eo33-derived secretome. Exo from either mouse and human Eo33 significantly increased Cdh1 expression while down-modulated Cdh2 in target tumor cells, with respect to Exo5. Our observations on EMT markers modulation as well as scratch assay experiments indicate that Exo33 may shape tumor cells towards a less aggressive phenotype. Our RNA-Seq analysis performed on mouse IL-5 and IL-33 stimulated eosinophils and on their respective released exosomes are in line with the functional experiments showing that tumor cells exposed to Exo33, but not to Exo5, modulated the expression of genes involved in tumor suppression, resulting in reduced tumor cell migratory ability and cell cycle arrest. LAGO and G-profiler essential GO (gene ontology) terms discriminated a set of biological processes leading to pro-tumoral activities in which mRNAs preferentially expressed in Exo5 seem to be involved, such as cell cycle enhancement, blood vessel morphogenesis and p53 binding processes. Conversely, mRNAs preferentially expressed in Exo33 appear to be involved in processes leading to a possible general decrease of tumor cell activities such as cell junction organization, cell-cell junction assembly (tight junction, adherents junction and desmosomes) that link cells to each other in tissues, regulate tissue homeostasis in critical cell processes that include tissue barrier functions. This observation may lead with the suggested Exo33 activity in reshaping tumor cell phenotype toward a less metastatic and invasive features, by inhibiting the EMT process. In addition, through GO analysis, we found that mRNAs preferentially expressed in Exo33 were reported to be associated with “negative regulation of signaling” that may lead to a possible decrease of tumor cell activities, resulting in anti-cancer outcomes. The intrinsic properties of Exo in regulating intracellular pathways have advanced their potential utility in the therapeutic control of many diseases and particularly in cancer.

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