Figure 1.

GPC3 transgenic HM-1 cell generation and GPC3 overexpression induces M1 macrophage infiltration. Two colonies of cells stably expressing GPC3 (HM-1GPC3#1 and HM-1GPC3#2) were selected, and GPC3 expression was confirmed using western blotting with an anti-GPC3 antibody (A). Tumor-tissue sections collected 8 days after inoculation with cancer cells were stained with an anti-F4/80 antibody (light spot). Cell nuclei were counterstained with Hoechst 3358. Images were captured using a confocal microscope at ×200 magnification (B). FCM of infiltrating macrophages in control and HM-1GPC3#1 tumors 8 days after inoculation with 1×10⁶ cancer cells; cytograms show morphological parameters of cell size (FSC) and cell granularity (SSC) of dissociated cells (top panel in C). In dot plots on ungated samples, lines were drawn around different cell populations; lymphocytes (R3), monocytes and macrophages (R2), and granulocytes (R1). The percentage of cells in R2 regions is presented in the graph (bottom in C). Tumor-infiltrating macrophages were analyzed with anti-CD86 and F4/80 antibodies using FCM (top panel in D). The graph presented in the bottom section in D contains combined reproducible data (n=3). Results are mean value ± SD. (n=3).