Figure 6.

Trastuzumab plus saracatinib combinatorial treatment overcomes trastuzumab resistance in vivo. (a) Top, representative immunofluorescence images of SRC knockdown in PTEN.shRNA xenografts using intratumoral injection of SRC.shRNA-containing virus. Scale bar, 100 µm. Bottom, volume of trastuzumab-resistant PTEN-deficient tumors with or without SRC knockdown upon treatment with IgG or Ttzm. Tumor volume at various times of treatment is presented as percentage of original tumor size at day zero of treatment. (b) Top, representative immunohistochemistry (IHC) images of in vivo inhibition of SRC-Y416 phosphorylation by saracatinib or AKT-S473 phosphorylation by triciribine in BT474.TtzmR xenograft tumors. Scale bar, 100 µm. Bottom, TtzmR xenograft tumor volume in response to different treatments. (c) Left, representative in vivo luciferase images of mice at day 0 and 21 days after indicated treatment. Left side of animal, BT474 control.shRNA tumors; right side, BT474 PTEN.shRNA tumors. Right, tumor volume in response to different treatments. (d) Left, representative IHC staining of AKT-S473 phosphorylation after different treatments (vehicle or trastuzumab plus saracatinib) in BT474.PTEN.shRNA xenograft tumors. Scale bar, 50 µm. Right, overall AKT-S473 phosphorylation IHC staining intensity between trastuzumab-alone group and combination-treatment group. Phospho-AKT (pAKT) staining was compared between each group by Fisher’s exact test (P = 0.049, two-sided). (e) Top, representative tumor sections with TUNEL staining. Scale bar, 50 µm. Bottom, in situ TUNEL staining of apoptotic cells in tumors treated as indicated. All error bars, s.e.m. All in vivo data were generated from a minimum of five replicates. (f) Model of SRC as a common node downstream of multiple resistance pathways and conquering trastuzumab resistance by targeting SRC.