Microwave-Induced Tumor-Derived Microparticles Treat Lung Adenocarcinoma by Inducing Immunogenic Cell Death via HMGB1 Shuttling

Tumor-derived microparticles (TMPs), a subtype of extracellular vesicles, hold great promise in tumor immunotherapies and vaccines, and have demonstrated significant potential as drug delivery systems for clinical tumor treatment. However, concerns remain regarding the potential risks associated with nucleic acid incorporation, alongside the limited antitumor efficacy of ultraviolet irradiation (UV-TMPs) alone. Here, we introduce a microwave (MW)-assisted method for preparing TMPs, termed MW-TMPs. Brief exposure to short-wavelength MW radiation triggered various forms of programmed cell death, including apoptosis, necroptosis, and pyroptosis, promoting the release of TMPs. These resulting MW-TMPs contained fewer nucleic acids and exhibited superior antitumor activity in vivo compared to UV-TMPs. Mechanistically, MW-TMPs induced immunogenic cell death (ICD) by shuttling HMGB1 and enabled dual targeting of tumor cells by natural killer (NK) and T cells, while reprogramming of suppressive tumor immune microenvironments in LLC lung adenocarcinoma (LUAD) mouse models. When the intervention to LLC mouse models was completed, cell suspensions were prepared by tissue homogenate of fresh subcutaneous tumors. The single-cell suspension was added to the single-cell sorting honeycomb plate and excessive magnetic beads with barcodes were put into the plate. During the phase of cell lysis, the RNA was labeled as bounding to the barcode. Subsequently, reverse transcription of RNA and cDNA synthesis were carried out to obtain cDNA library for subsequent sequencing. Paired-end sequencing was conducted in the Nova seq6000 sequencing platform to get the transcriptome results. BD Rhapsody Analysis pipeline was applied to identify the UMI sequence and the cell tag sequence alignment to the reference genome (GRCm39). Overall design: LLC cells (5×10^5 cells suspended in 100 µl DMEM solution) were subcutaneously inoculated into the unilateral posterior flanks of C57BL/6 mice (6 weeks). After approximately one week of observation, they were randomized into several groups (n=6) and treated with MW-TMPs, UV-TMPs or PBS every two days totally for five times. The tumor volume and body weight were recorded every other day until the end of the intervention. When the intervention was completed, fresh subcutaneous tumors were stored in MACS tissue storage solution (#130100008) and cell suspensions were prepared by tissue homogenate. The single-cell suspension was added to the single-cell sorting honeycomb plate and excessive magnetic beads with barcodes were put into the plate. During the phase of cell lysis, the RNA was labeled as bounding to the barcode. Subsequently, reverse transcription of RNA and cDNA synthesis were carried out to obtain cDNA library for subsequent sequencing. Paired-end sequencing was conducted in the Nova seq6000 sequencing platform to get the transcriptome results. BD Rhapsody Analysis pipeline was applied to identify the UMI sequence and the cell tag sequence alignment to the reference genome (GRCm39). After removing low-quality cells, the normalization methods “LogNormalize” and “ScaleData” were used to standardize and scale each gene and then linear dimensional reduction was performed according to the PCA score. Uniform Manifold Approximation and Projection (UMAP) techniques were chosen to visualize the cell clusters (the visualized PCA dimension = 38).