Fig. 3

Colon cancer exosome-associated HSP90B1 promotes the polarization of M1 macrophage into M2 phenotype in vitro and in vivo. (A-C) 20 µg of CT-26 cell-derived exosomes (CT-26/exo) or MCEC cell-derived exosomes (MCEC/exo) were subjected to mass spectrometry analyses (A) or western blot analyses (B-C) (n = 3 independent experiments). (D-F) CT-26 cells stably expressing shGFP, shHSP90B1-1, or shHSP90B1-2 were subjected to qPCR (D) or western blot (E-F) analyses (n = 3 independent experiments). (G-H) Mouse M1 macrophages were treated with 20 µg of CT-26-shGFP/exo, CT-26-shHSP90B1-1/exo, or CT-26-shHSP90B1-2/exo for 48 h. Cells were subjected to FACS analyses (n = 3 independent experiments). (I) Mouse M1 macrophages were treated with 20 µg of CT-26-shGFP/exo, CT-26-shHSP90B1-1/exo, or CT-26-shHSP90B1-2/exo for 48 h followed by an examination of as indicated cytokines levels in the medium (n = 3 independent experiments). (J-K) Mouse M1 macrophages (1 × 10⁶) were treated with 20 µg of CT-26-shGFP/exo, CT-26-shHSP90B1-1/exo, or CT-26-shHSP90B1-2/exo for 48 h, followed by co-culture with CD8 + T cells (1 × 10⁶) derived from mouse spleen for 48 h. Cells were subjected to FACS analyses (n = 3 independent experiments). (L-Q) Five-week-old female BALB/c mice (n = 8 per group) were injected with 20 µg of CT-26-shGFP/exo, CT-26-shHSP90B1-1/exo, or CT-26-shHSP90B1-2/exo, or PBS via the tail vein every 3 days. After 3 weeks, the mice were sacrificed, and liver tissues were collected. Immunofluorescence staining was performed to detect the number of M1 macrophages (CD86+/CD11b+) (L-M), M2 macrophages (CD206+/CD11b+) (N-O), and CD8 + T cells (P-Q) in the liver tissues. Scale bar = 50 μm. Data were presented as mean ± SD (C, D, F, H, I, K) or SEM (M, O, Q). Statistical analyses were performed with one-way ANOVA with Tukey’s test (D, F, H, I, K, M, O, Q) and unpaired two-tailed Student’s t-test (C)