Intravital Imaging to Monitor Therapeutic Response in Moving Hypoxic Regions Resistant to PI3K Pathway Targeting in Pancreatic Cancer.

Animal Experiments All animal experiments were conducted in compliance with Garvan Ethics Committee guidelines (13/17 and 16/13 protocols) and in accordance with the Australian code of practice for the care and use of animals for scientific purposes, with genotyping performed by Garvan Molecular Genetics (Sydney, NSW, Australia). Both male and female KP fl C and KPC GEM were monitored for swollen abdomen, cachexia, and reduced mobility until tumor was evident by palpation, and hypoxia markers were administered ( Hingorani et al., 2003 , Hingorani et al., 2005 , Morton et al., 2010 ). For xenografts experiments, 1 × 10 6 KPC primary PDAC cells in PBS were subcutaneously injected into the rear flank of 6- to 10-week-old female BALB/c-Fox1nuAusb mice. FACS Cells were fixed in 70% ethanol, prior to staining with propidium iodide (Sigma-Aldrich; P4170; 1 μg/mL) with RNaseA (Sigma-Aldrich; R6513; 500 μg/mL), and analysis on a FACSCanto II (BD Biosciences). Quantification was performed in FlowJo (Tree Star), and cell cycle phase was determined in ModFit (Verity Software House). Intravital FLIM/PLIM Imaging Xenografts of KPC cells expressing the Eevee-Akt-mT2 FRET biosensor were imaged at a final volume of 350 mm 3 , as per the tumor growth studies. Mice were anesthetized with xylazine (10 mg/kg) and Zoletil (50 mg/kg) and kept on a heated stage at 37°C. Anesthesia was maintained with isoflurane (3 L; O 2 , 1 L; vacuum, 1 L/min). Tumors were surgically exposed, as described previously ( Conway et al., 2017 , Vennin et al., 2017 ), prior to application of MitoImage NanO2 (10 μg; Ibidi; 74151). After 30 min, dual FLIM/PLIM imaging was performed on a Leica SP8 microscope with a 0.95 numerical aperture (NA), 25× water objective. Analysis of FLIM and PLIM decays was performed in FLIMfit ( Warren et al., 2013 ) and is presented as mean lifetime per cell.

Organotypic Assay

Organotypic matrices were generated as described previously ( Conway et al., 2014 , Morton et al., 2010 , Vennin et al., 2017 ). Quantification of the invasive and proliferative indices was performed on pan-cytokeratin (excludes fibroblasts) or Ki67-stained sections, respectively. IC 50 Curves IC 50 curves were performed in a 96-well plate format, with 1,000 cells/well. Cells were incubated for 24 hr after seeding before addition of inhibitors. Plates were assessed for relative cell density using the CellTiter 96 AQueous Cell Proliferation Assay (Promega; G1111) at 72 hr after addition of each inhibitor, with parallel plates incubated under standard normoxic or hypoxic (0.1% oxygen) conditions. IC 50 curves were then fit in Prism (GraphPad Software), normalizing to the vehicle (DMSO) control treatments from the respective normoxic or hypoxic (0.1% oxygen) conditions.

Statistical Analysis

Statistical tests were performed in Prism (GraphPad Software) with statistical significance given as ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 in all cases. A one-sample t test was performed on normalized data. For IC 50 curve comparisons, an extra sum-of-squares F test was performed between the best-fit parameters of each curve. Deming regression curves for the dual FLIM/PLIM analysis were plotted with the “mcr” package of R, using the analytical method to calculate the 95% confidence intervals. To assess whether the Deming regression slopes were significantly non-zero, a one-sample t test was performed. DNA cell cycle analysis was assessed for significance using a two-way ANOVA test with a Tukey correction for multiple comparisons. In all other cases, a Student two-tailed parametric t test was performed. Detailed protocols can be found in Supplemental Experimental Procedures .

Supplemental Information Document S1. Supplemental Experimental Procedures, Figures S1–S6, and Tables S1 and S2 Document S2. Article plus Supplemental Information