Fig. 6.

Anti-HSP90α antibody potently inhibits EndoMT-promoted and macrophage-associated tumor growth. a Abundant HSP90α secretion from the mouse macrophages after EndoMT CM stimulation. RAW264.7 cells were pre-incubated with 1% FBS-containing medium for 16 h. The medium was then replaced with Ctrl, Endo CM, or EndoMT CM for another 24 h. The treated macrophages were further incubated with 5 ml of fresh 1% FBS-containing medium for 24 h. The media were finally collected and subjected to immunoblot analysis. b, c Inhibitory effect of anti-HSP90α antibody on the tumor growth of Panc 02 cells plus EndoMT-derived cells. C57BL/6 mice were subcutaneously injected with Panc 02 cells alone or together with endothelial cells (designated as “Endo”) or EndoMT-derived cells (designated as “EndoMT”) (n = 8 per group). The mice inoculated with Panc 02 cells plus EndoMT-derived cells were further intravenously injected with control IgG or anti-HSP90α antibody (5 μg per g of body weight for each dosage) on day 4 post-inoculation. The injections were performed every 3 days for a period of 24 days. The sizes of developing tumors were superficially measured using a Vernier caliper since day 3 post-inoculation with the formula of ½ × length × width² (b). ^@P < 0.001 when “Panc 02 + EndoMT + HSP90α Ab” group was compared with “Panc 02 + EndoMT + IgG” group. Mice were sacrificed on day 30 post-inoculation and tumors were removed for weighing (c). ^@P < 0.001 when compared with “Panc 02” group. ^#P < 0.01 when compared with “Panc 02 + EndoMT + IgG” group. d Inhibitory effect of anti-HSP90α antibody on the elevation of serum HSP90α levels in the mice inoculated with Panc 02 cells plus EndoMT-derived cells as described above. Serum samples were collected from the mice on day 0 before cell-graft inoculation and on day 29 post-inoculation for HSP90α measurement by ELISA. ^αP < 0.001 when compared with “Panc 02” group. ^βP < 0.001 when compared with “Panc 02 + EndoMT + IgG” group. e, f Reduction of M2-type macrophage levels by anti-HSP90α antibody in the tumors derived from Panc 02 cells plus EndoMT-derived cells. Tumor tissue sections of the mice treated as described above were immunohistochemically staining with anti-CD163 (e) or anti-CD204 (f) antibody. ^@P < 0.001 when compared with “Panc 02” group. ^#P < 0.001 when compared with “Panc 02 + EndoMT + IgG” group