Artificial Microvesicles Isolated from Mesenchymal Stem Cells with IL2 Overexpression Activate CD8+ T-Killers to Kill Triple Negative Breast Cancer Cells

Abstract

Almost all human cells release extracellular vesicles (EVs) participating in intercellular communication. EVs are rounded structures surrounded by the cytoplasmic membrane, which embody cytoplasmic contents of the parent cells. Since the orientation of surface receptors persists during EV formation, it is EVs that are of interest as a therapeutic tool for cell-free therapy that has targeted delivery. The ideal cell type for the EV production are mesenchymal stem cells (MSCs) as they exhibit a homing behaviour to tumor niches. In this study, human MSCs were isolated from adipose tissue (hADSCs) and genetically modified to overexpress interleukin-2 (IL2) (hADSCs-IL2) or red fluorescent protein (hADSCs-RFP). Extracellular vesicle release from native and genetically modified hADSCs was induced by cytochalasin B treatment. Size and immunophenotype of isolated Cytochalasin B induced microvesicles (CIMVs) were analysed using flow cytometry. To evaluate immunomodulating properties of microvesicles from native (native CIMVs) or genetically modified (CIMVs-IL2) hADSCs, peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and co-cultured with CIMVs (50 μg/ml) for 72 hours. Activation of PBMC populations was determined by flow cytometry using staining with antibodies specific for surface markers of various human immune cell populations. To analyze the antitumor activity of PBMCs after interaction with CIMVs-IL2, activated PBMCs were transferred to triple negative breast cancer cells MDA-MB-231 which were pre-seeded on 16-well xCelligence plate (3 × 103 cells per well) or 6-well plate (5 × 104 cells per well) in 1:3 ratio (tumor cells:PBMCs). Proliferative activity of tumor cells was evaluated using an RTCA xCelligence instrument (ACEA Biosciences, USA) for 72 hours. Tumor cell viability was assessed 24 hours after culturing with PBMCs using the APC Annexin V Apoptosis Detection Kit with PI (#640932, Biolegend, USA) on FACS Aria III flow cytometer (BD, USA). Isolated CIMVs were mostly 50-200 nm in diameter and positive for CD44, CD90 and CD105 MSC surface markers, but CD29 and CD73 levels were significantly decreased. The number of activated T-killers (CD3+CD8+CD4-CD38+HLA-DR+) was significantly increased after incubation with CIMVs-IL2 (205.6 ± 4.5%) compare to native PBMCs (100.0 ± 9%) and PBMCs incubated with native CIMVs (116.7 ± 4.5%) (Fig. 1A). The number Th1 cells was decreased in the sample of PBMCs incubated with CIMVs-IL2 (92.3 ± 1.6%) compared with native PBMCs (100.0 ± 1%) (Fig. 1B), while the number of Th2 cells (109.0 ± 0.1%) was increased compared to native PBMCs (100.0 ± 1%) (Fig. 1C), number of Tregs was unchanged (Fig. 1D). After the addition of PBMCs, activated with CIMVs-IL2, the proliferation of MDA-MB-231 cells has been significantly decreases. Twenty four hours after the addition of CIMVs-IL2-activated PBMCs the proliferation of MDA-MB-231 was decreased 4-fold (to 25.0 ± 5.6%) compared to native MDA-MB-231 (100.0 ± 2.9%) and MDA-MB-231 incubated with native PBMCs (110.8 ± 8.7%) and PBMCs after native CIMVs (103.7 ± 2.4%) (Fig. 1E,G). This decrease in proliferation is likely due to the fact that part of the tumor cells underwent apoptosis and then died which is confirmed by the MDA-MB-231 cell viability after 24 hours of cultivation with PBMCs activated with CIMVs-IL2 (Fig. 1F). The number of healthy MDA-MB-231 cells after 24 hours of cultivation with PBMCs after CIMVs-IL2 was reduced by 35% (46.1 ± 3.7%) compared to native MDA-MB-231 (78.2 ± 2.1%) and MDA-MB-231 incubated with native PBMCs (70.1 ± 2.8%) and PBMCs after native CIMVs (62.1 ± 3.2%). Viable MDA-MB-231 cells in the sample of PBMCs after CIMVs-IL2 continue to proliferate, however their proliferative activity remains reduced (41.2 ± 4.7%) after 72 hours of cultivation compared to native MDA-MB-231 (100.0 ± 21.0%) and MDA-MB-231 incubated with native PBMCs (109.7 ± 2.5%) and PBMCs after native CIMVs (82.5 ± 20.5%) (Fig. 1H). The use of CIMVs-IL2 can be effective in the treatment of triple negative breast cancer, since CIMVs-IL2 are able to activate and stimulate the proliferation of T-killers, which in turn are able to induce apoptosis in breast cancer cells. However, further studies of CIMVs efficiency in animal tumor models are required. This study was supported by the Russian Science Foundation grant 18-74-10044 and the Russian Government Program of Competitive Growth of KFU. Figure Disclosures No relevant conflicts of interest to declare.