Reinforced immunotherapy of M1 macrophage-derived exosomes with CEL on TNBC via regulating macrophage functions

Introduction: Triple-negative breast cancer (TNBC) demonstrates poor prognosis due to its heterogeneity-related biological barrier, immunosuppressive tumor microenvironment (TME) and escape of cancer cells on immune surveillance. Exosomes are membrane-encased vehicles with properties of inter- and intra-cellular communication and regulation for therapeutic use, even as drug vehicles. M1 macrophage-derived exosomes (M1-Exos) can communicate with adjacent M2 macrophages and reprogram them to M1 subtypes to reshape TME. Celastrol (CEL) is a highly promising natural antitumor drug and plays an important role in immunotherapy but with high toxicity and low water solubility. Objectives: This work aims to provide reinforced immunotherapy to reshape tumor immunosuppressive microenvironment of TNBC by employing M1-Exos as a nanoscale co-delivery vehicle for CEL, offering increased tumor targeting and antitumor efficacy while reducing systemic toxicity to achieve improved treatment outcomes. Methods: A versatile combinatorial delivery system, tLyP-1-M1-Exos/CEL, was engineered by modifying tLyP-1 peptide and loading CEL to M1-Exos, then the size distribution, morphological features were characterized by transmission electron microscope, nanoparticle tracking analysis, and biomarkers were characterized by western blot. The efficacy and mechanism were assessed using CCK-8, qPCR, immunofluorescence, in vivo fluorescence imaging system, flow cytometry, western blot, H&E staining and other methods at cellular level and in nude mice. Results: The synthesized tLyP-1-M1-Exos/CEL exhibited specific tumor-targeting ability, greater tumor-suppressing properties and lower toxicity. The expression of M1 markers was upregulated and the expression of M2 markers was downregulated in M2-phenotype macrophages co-incubated with tLyP-1-M1-Exos/CEL. tLyP-1-M1-Exos/CEL decreased the expression of MHC I by inhibiting IRF1-CIITA signal pathway, making tumor cells more susceptible to be phagocytosed by macrophages. Conclusion: Our study showed a reinforced immunotherapy that the synthesized tLyP-1-M1-Exos/CEL could not only reverse M2 macrophages into M1 to reshape immunosuppressive TME but also decrease the expression of MHC Ⅰ on tumor surface to enhance macrophage phagocytic autoimmunity.