ROLE OF EXTRACELLULAR VESICLES IN THE TUMOUR MICROENVIRONMENT AND MULTIDRUG RESISTANCE

The tumor microenvironment represents a complex network that contributes significantly to cancer progression, invasion, and metastasis, in which tumor cells communicate with each other but also with stromal and immune cells. One of the main mechanisms of therapeutic failure and consequent mortality of cancer patients is the development of resistance to anticancer drugs. Although there are several mechanisms through which tumor cells develop resistance, the over-expression of ABC family transporters, particularly P glycoprotein (P-gp), is one of the main causes of multi-drug resistance (MDR). Furthermore, it is known that tumor-derived extracellular vesicles, (EVs), are highly specialized structures that carry numerous surface markers and signaling molecules, and which, in addition to their important role in communication between cells, can mediate the transfer of drug-resistance to sensitive tumor cells, through the horizontal transfer of specific bioactive factors including the drug efflux pump, survival factors, apoptosis inhibitors and non-coding RNAs (Bucci-Muñoz et al. Life 2023, 13, 1633). The aim of our work was, therefore, to isolate the extracellular vesicles released by a model of sensitive cell line and its MDR variant, characterize them and verify that the EVs released from resistant cells are responsible for the transfer of P-gp to sensitive cells, significantly contributing to the spread of resistance in the tumor microenvironment. For this purpose, we used the human leukemia cell line HL60 and its MDR variant HL60R, obtained by exposure to gradually increasing concentrations of doxorubicin, as a model of acquired resistance in a liquid tumor. Our data show that HL-60R cells release about 70% more EVs, compared to the sensitive HL-60 cells; this was further confirmed by scanning electron microscopy (SEM) analysis, in agreement with literature data indicating that resistant cells release a greater quantity of large vesicles than sensitive cells (Lopes-Rodrigues V.et al. Biochimica et Biophysica Acta (BBA) - General Subjects, 2016). By Western Blotting analysis we observed the presence of P-gp only in the HL-60R cell extracts and in the respective vesicles; the protein is absent in sensitive HL-60 cells and its related vesicles. To verify the ability of HL-60R to carry out the horizontal transfer of P-gp through the release of EVs, immunofluorescence analysis was performed on sensitive HL-60 cells, treated for different steps of time with EVs released from HL-60R and stained with polyclonal P-gp antibody. Our data show P-gp expression only in the positive control HL-60R and in HL-60 treated with EVs released from resistant cells. In contrast, if HL-60 cells were incubated with EVs released from the same sensitive cells, they do not express P-gp on their membrane; this confirms the western blotting data that indicated the absence of P-gp in HL-60 cells and its related EVs. Since EVs released from HL-60R transfer P-gp, we are now carrying out some functional assays to verify whether, after horizontal transfer of the efflux pump into sensitive HL-60 cells, they acquire the resistant phenotype regarding sensitivity to doxorubicin. In parallel, we are conducting the same type of analysis on another model of acquired drug resistance: the MCF-7 breast cancer line and its MDR variant MCF-7R. Previous data had demonstrated several common characteristics between these two different tumor models, such as the constitutive activation of the nuclear factor- κappa (NF-κ) and the overexpression of P-gp and different IAPs. (Poma P. et al. Crit Rev Oncog. 2021) Since EVs released by tumor cells are implicated in various stages of tumor progression and drug resistance, we believe that the inhibition of their secretion, and therefore of the horizontal transfer of their contents, is a very promising strategy to counteract these phenomena.