Mice bearing pancreatic tumors were treated systemically with gemcitabine. * P< .05, Mann-Whitney U. Solid lines = mean; dashed lines = mean without responders. (A) Percent change in tumor volume in transplantation models (see Supplementary Online Material) treated with saline (blue) or 100mg/kg gemcitabine, Q3Dx4 (red). (B) Gemcitabine treatment did not induce tumor cell apoptosis in the transplantation models, as measured by immunohistochemistry (IHC) for cleaved caspase 3 (CC3). (C) Percent change in volume of tumors in KPC mice treated with saline (blue), 50mg/kg (green) or 100mg/kg of gemcitabine, Q3Dx4 (red). Two of seventeen KPC tumors responded transiently to gemcitabine, as assessed by ultrasonography (yellow). (D). Increased apoptosis was evident only in the KPC tumors that transiently responded to the drug (yellow).

Direct immunofluorescent detection of plant lectin (red) and doxorubicin (green) infused into transplanted (A) and KPC (B) tumors, along with H&E stained adjacent sections (inset). Scale bar, 200μm. Doxorubicin was effectively delivered to transplanted tumors (N=5), but poorly delivery to KPC tumors (N=4), relative to surrounding tissue. Perfusion of microbubbles (green) into transplanted (C) and KPC (D) tumors visualized by contrast ultrasonography. Transplanted tumors were well perfused (N=6) compared to KPC tumors (N=8). Tumors outlined in yellow. Scale bars, 1mm. DCE-MRI demonstrated increased perfusion and extravasation of Gd-DTPA (high delivery = white/yellow) in transplanted tumors (E, N=6) compared to KPC tumors (F, N=6). Tumors outlined in blue. Scale bars, 2mm.

CD31 IHC from transplanted (A), KPC (B) and human (C) pancreatic tumors (scale bar, 20μm). Arrows denote blood vessels. Neoplastic cells from transplanted tumors made direct contact with blood vessels while those in KPC and human tumors were more distantly spaced due to prominent stroma. (D) Mean Vessel Density (MVD) was measured in KPC tumors (KPC), syngeneic autografts (Syn), orthotopic xenografts (Ortho), adjacent surrounding tissues in KPC tumors (Adj), human pancreatic tumors (PDA) and normal pancreas from mice and humans (Norm). KPC and human pancreatic tumors were poorly vascularized compared to transplanted tumors and normal tissues (* P<.004, Mann-Whitney U). (E) The distance separating blood vessels and neoplastic cells was significantly higher in KPC tumors and human PDA than in transplanted tumors. (F) Computer-aided image analysis of MVD found human pancreatic tumors (N=18) to be poorly vascularized compared to normal pancreas (N=5) and chronic pancreatitis (N=5) samples (* P<.0015, ** P<.0001, Mann-Whitney U). Peripheral (P) and central (C) regions of tissues were distinguished.

KPC mice were treated for 8–12 days with: no treatment (NT), vehicle (V), gemcitabine (G), IPI-926 (I), or IPI-926 + gemcitabine (IG). (A) MVD was elevated following IPI-926 or IPI-926/Gem treatment (P<.05, Mann-Whitney U). (B) Doxorubicin fluorescence was elevated following IPI-926 alone or IPI-926/Gem treatment (*P<.02, Mann-Whitney U). (C) Following treatment with the indicated regimens, all mice were administered a single dose of gemcitabine and the concentration of fluorine-bearing metabolites was determined in extracted samples by ¹⁹F NMR. Gemcitabine metabolite concentration was elevated in IPI-926/gem treated tumors (P=.04, Mann-Whitney U). (D) IHC for cleaved caspase 3 revealed increased apoptosis in IPI-926/gem treated tumors (P=.008, Mann-Whitney U). (E) IPI-926/gem treatment significantly extended survival in KPC mice (P=.001 Log-Rank Test, Hazard Ratio = 0.157 ± 0.458 95%CI 6.36). (F) Fewer liver metastases were observed in IPI-926/gem KPC mice (*P=.015, Fisher’s Exact).