(T1130-04-27) Anticancer Effects of Proteins and miRNAs Picked out by Proteomic Analysis of NK Cell-Derived EVs in Combination with Carboplatin in Resistant Lung Cancer: Role of PDL1/PD-1, SOD2, PARP, NF-kB and SET

Purpose: Lung cancer is still the deadliest cancer globally and accounts for 19 percent of all cancer deaths. Though there are various treatment options available (Chemotherapy, TKIs and Mabs), resistance develops after a few years of therapy. For this reason, immune therapy is being currently used in patients who meet the criteria. Extracellular vesicles (EVs) derived from immune cells (e.g., NK cells, dendritic cells) may be able to treat malignancies that are resistant to other treatments (e.g., chemotherapy) relying on the hypothesis that EVs carry almost the same cargo of the parent cells. The objective of this study is to combine immunotherapy with chemotherapy for enhanced anticancer activity as one is not better than the other. In this study, we evaluated natural killer cell-derived EVs (NK-EVs) as therapeutic agents besides being the delivery system for carboplatin and microRNAs that regulate the expression of cytotoxic proteins present in them.

Methods: NK92 cells were grown in flasks and seeded in PBS-vertical wheel 0.5 L bioreactor at the density of 20,000 cells/mL. IL-15, a cytokine was added in a separate bioreactor to see if it could enhance the anticancer activity. Further, samples were taken from the bioreactor to assess the concentration levels of lactate and glucose using a biochemical analyzer. Media was collected after two weeks to isolate NK-EVs by ultracentrifugation. After isolation, NK-EVs were characterized by Nanoparticle tracking analysis (NTA), Atomic Force Microscopy (AFM) and Western blotting for size, zeta potential, concentration in particles/mL, surface roughness and biomarkers. Proteomic analysis of the EVs followed by gene ontology studies was done to identify the proteins present in them and understand what miRNAs regulate them. Later, H1975 cells resistant to Osimertinib (EGFR-L858R mutated) were seeded in 96-well plates for 3D cytotoxicity studies using a magnetic nanoshuttle system (Greiner) followed by dual acridine orange/ethidium bromide fluorescent staining. The plates were treated with various concentrations of NK-EVs, Carboplatin and their combination for 48 hours. For in-vivo studies, H1975 cells (4 × 10⁶ cells) were subcutaneously injected into BALB/c athymic nude mice. The treatment was initiated to four groups (Untreated, NK-EVs alone, Carboplatin, and NK-EVs + Carboplatin combination). The tumor samples at the end of the study were taken for western blotting and proteomic analysis.

Results: NTA and AFM confirmed that EVs had a size of 80.5 ± 3.4 nm and Zeta potential of -21.29 ± 0.66 mV. Western blotting of EVs confirmed the presence of cytotoxic proteins perforin and granzyme along with general exosomal markers like tetraspanins, flotillin, TSG101, alix and negative for calnexin, used as a cell contamination marker. NK-EVs that were isolated from the bioreactor containing IL-15 showed higher expression of perforin and granzyme. The concentrations of glucose and lactate were measured in postculture medium and lactate/glucose ratio of 1.2 suggested aerobic metabolism with high metabolic activity for up to 4 days. Invitro cytotoxicity studies demonstrated that NK-EVs alone and in combination with carboplatin (IC50) effected cell death (P < 0.01) 1.8 ± 0.1-fold and 3.2 ± 0.15-fold respectively as compared to control suggesting the cytotoxic potential of NK-EVs. NanoShuttle magnetic 3D cell culture cytotoxicity studies supported the 2D data with 1.5 ± 0.1-fold and 3 ± 0.2-fold cell death respectively and was also supported by Acridine Orange/Ethidium Bromide dual staining (p < 0.001). The tumor volumes for treatment groups (NK-EVs, carboplatin and NK-EVs-carboplatin) were 84.3 ± 3.7 %, 65 ± 2.2 %, 30 ± 1.4 % (P < 0.001) for H1975 tumors compared to control group at the end of study indicating anti-cancer activity of NK-EVs. In combination with carboplatin, NK-EVs showed synergism in in-vitro studies and additive to synergistic effect in in-vivo studies suggesting the use of combination therapy. Western blotting of the tumor samples showed downregulation of PD-1 and PD-L1 along with other inflammatory markers such as SOD2, PARP, NF-kB, SET (P < 0.001). Importantly, elevated expression of PD-1, SOD2 and NF-kB has been reported to suppress activation of NK cell cytotoxicity, granzymes A and B secretion, and perforin expression and has been reported to induce inhibition of the innate anti-tumor activity of NK cells which has been reversed by delivery of the miRNA cargo in NK-EVs. MicroRNAs regulating cytotoxic proteins like perforin, granzyme and plastin were also identified using proteomics and the miRNAs: hsa-miR-1-3P/149-5P/320a/186-5p/339-5p, known for their tumor suppression were picked out for further studies. These miRNAs are directly involved in T cell or NK cell mediated cytotoxicity and are being currently investigated.

Conclusion: In order to successfully treat lung cancer and overcome resistance, combination of immunotherapy is required with existing approaches. In lung cancer, PD-L1 amplification along with other inflammation markers represent a major and undruggable target. NK cell derived EVs have anticancer potential and our results demonstrate the feasibility of NK-EVs as cancer immunotherapeutic agents. Further studies such as flow cytometry, immunofluorescence staining and RT-PCR etc. are currently undergoing to understand the molecular mechanisms of NK-EVs.