Disruptive advances in exosome lyophilization: Unlocking new frontiers in precision oncology.

e14522 Background: Exosomes derived from pulsed dendritic cells (DEX) are pivotal in regulating the tumor microenvironment (TME), influencing immune responses and metabolic pathways that drive tumor progression. Lyophilization provides a breakthrough in preserving their structure, bioactivity, and therapeutic potential, eliminating the need for cold chain reliance and facilitating broader accessibility. This method is especially beneficial in low-resource settings, ensuring that DEX-based therapies can reach a wider range of aggressive cancers, including pediatric, adolescent, and elderly patients. Moreover, lyophilization offers scalability, making this approach globally applicable, ensuring it reaches underserved populations. The preservation of functional exosomes through lyophilization offers a novel treatment option for cancers that are challenging to treat with conventional therapies, enhancing patient outcomes across diverse clinical settings. Methods: DEX were generated from semi-immortalized dendritic cells through specific pulses designed to maximize secretome functionality. Samples were processed under fresh, cryopreserved (-80°C), and lyophilized conditions using advanced cryoprotectants. Key proteins (CD63, TSG101) were analyzed by Western Blot, and proteomic markers were evaluated using timsTOF Pro 2 (Bruker). A375 (melanoma) and AGS (gastric) tumor cell lines were treated with concentrations of 10, 50, and 100 µg/mL for 72 hours. Key cytokines (IL-10, TGF-β, IFN-γ) were assessed by Cytometric Bead Array (CBA), and apoptosis was evaluated via Annexin V/PI staining and caspase-3 activation. Results: Quantification and Functional Stability of DEX: o Fresh: 435 proteins, 2,125 peptides. o Cryopreserved: 308 proteins, 1,296 peptides. o Lyophilized: 456 proteins, 2,300 peptides. Modulation of TME: o Reduction in IL-10 (-40.8% ± 1.4, p < 0.01) and TGF-β (-38.5% ± 1.2, p < 0.01). o Increase in IFN-γ (+53.2% ± 2.0, p < 0.001). Tumor Apoptosis: o A375 increased by 38.1% ± 2.3 and AGS by 36.7% ± 1.9 (p < 0.01). Conclusions: The lyophilization of DEX represents a biotechnological advancement, preserving the stability and functionality of exosomes without the constraints of cold chain logistics. This scalable approach allows for global application, enabling DEX therapies to reach underserved populations. Its ability to modulate the TME and induce apoptosis in tumor cells makes it a tool in precision oncology, offering personalized treatment options for pediatric, adolescent, and elderly patients. The immunomodulatory effects of DEX further enhance its potential in targeted cancer therapies, addressing aggressive and resistant tumors. This method holds promise for revolutionizing cancer treatment globally, ensuring that cutting-edge therapies are accessible to all patients.