Mechanism of gambogic acid repressing invasion and metastasis of colorectal cancer by regulating macrophage polarization via tumor cell‐derived extracellular vesicle‐shuttled miR‐21

Abstract Colorectal cancer (CRC) is a major cause of mortality and morbidity. Gambogic acid (GA) is a promising antitumor drug for treating CRC. We aimed to elucidate its mechanism in CRC invasion/metastasis via tumor cell‐derived extracellular vesicle (EV)‐carried miR‐21. Nude mice peritoneal carcinomatosis (PC) model was subjected to GA treatment liver collection, followed by observation/counting of metastatic liver tissues/liver metastatic nodules by hematoxylin and eosin staining. miR‐21 expression in metastatic liver tissues/CD68 + CD86, CD68 + CD206 cell percentages and M2 macrophage marker CD206 level in tumor tissues/interleukin (IL)‐12 and IL‐10 levels were determined by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR)/flow cytometry/enzyme‐linked immunosorbent assay. HT‐29 cells were treated with GA/miR‐21 mimics/negative control for 48 h. miR‐21 expression/cell proliferation/migration/invasion/apoptosis were assessed by RT‐qPCR/cell counting kit‐8/scratch assay/transwell assay/flow cytometry. EVs were extracted from HT‐29 cells and identified by transmission electron microscope/nanoparticle tracking analysis/Western blot. IL‐4/IL‐13‐induced macrophages/PC nude mice were treated with GA and EVs, with the internalization of EVs by macrophages assessed through the uptake test. After intraperitoneal injection of GA, PC nude mice exhibited decreased tumor cell density/irregular cell number/liver metastatic nodule number/miR‐21 expression, and CRC cells manifested reduced CD68 + CD206 cells/IL‐10/miR‐21/proliferation/migration/invasion and increased CD68 + CD86 cells/IL‐12/apoptosis, while these trends were opposite after miR‐21 overexpression, implying that GA curbed CRC/cell invasion/metastasis and macrophage polarization by diminishing miR‐21 levels. miR‐21 was encapsulated in HT‐29 cell‐derived EVs. M2 polarization elevated CD206 cells/IL‐10, which were decreased by simultaneous GA treatment. EVs could be uptaken by macrophages. CRC cell‐EV‐miR‐21 annulled the suppression effects of GA on macrophage M2 polarization. GA suppressed macrophage M2 polarization by lessening tumor cell derived‐EV‐shuttled miR‐21, thereby weakening CRC invasion/metastasis.