Fig 1.
Validating intrathoracic injection technique.
(A) A standard calibration curve was made using known quantities of Ad5CMVCre to then quantify unknown samples of Ad5CMVCre. Six different known concentrations of Ad5CMVCre were spiked into lung tissue to make the standard calibration curve: 3.12x105, 6.24x105, 1.56x106, 3.12x106, 1.56x107, and 3.12x107 pfu. (B) Amount of Ad5CMVCre (in pfu) measured in 14 mice that had their left lung injected with 3.12x107 pfu Ad5CMVCre virus. Measurements were made using the TaqMan real-time PCR assay. The bar plot shows the mean with standard error and overlaying dot plot shows measurements of individual animals (n = 14). pfu = plaque-forming unit, a standard measure of the number of particles capable of forming plaques per unit volume. (C-D) To characterize Ad5CMVCre activity in the left lung of KrasG12D+/-; p53fl/fl; myristolated-p110αfl/fl-ROSA-gfp mice, gfp immunohistochemistry was used to detect Cre-recombination effect within the left lung, 4 days after Ad5CMVCre injection. (C) A representative micrograph of no gfp/Cre-recombination in the left lung of a mouse that had no Ad5CMVCre virus injected serves as a negative control. (D.1) A representative micrograph of activation of gfp/Cre-recombination in the left lung of a mouse that underwent Ad5CMVCre virus injection. To validate that the virus did not enter the bloodstream to turn on other primary cancers, (D.2) is representative of no gfp/Cre-recombination activation in the right lung and (D.3) is representative of no gfp/Cre-recombination activation in the heart.
Fig 2.
Representative micro CT of lung tumor progression.
(A) Representative single 2D coronal slice (largest length x width) of serial micro CT scans of one mouse exposed to cyclical intermittent hypoxia (upper panel) and one mouse exposed to room air (Sham) (lower panel) at 12, 19, 26, 33, and 40 days after Ad5CMVCre intrathoracic injection. (B) Using ITK snap, manual segmentation of the lung tumor, outlined in red, allows for precise 2D contouring of lung tumors in consecutive 100um coronal slices. C & D. ITK snap then automatically stitches all 2D segmented tumors and calculates a 3D tumor volume that is then used to compare CIH vs Sham tumor progression. A representative 3D tumor anterior (C) and posterior (D) visualization of a lung tumor from the same mouse in (B) is colored in red.
Fig 3.
Representative H&E stain of left lung tumor of mouse exposed to cyclical intermittent hypoxia (A) compared to Sham (B).
Primary tumor is outlined by red arrows [1.25X magnification].
Fig 4.
Tumor volume measured by micro CT scans.
Tumor volume and associated 95% confidence intervals estimated from linear mixed model analysis are shown. Data for individual mice are shown. We found a significantly faster increase in tumor volume in the CIH versus Sham groups (p = 0.0003) and significant differences between the two groups across all timepoints (overall p<0.0001). In pairwise comparisons at each individual timepoint, the CIH group had trending or significantly greater tumor volume at day 26 (p = 0.055), day 33 (p = 0.004) and day 40 (p<0.0001). Sample size is as follows: for CIH at Day 12 (n = 11), Day 19 (n = 11), Day 26 (n = 11), Day 33 (n = 11), Day 40 (n = 10); for Sham at Day 12 (n = 13), Day 19 (n = 13), Day 26 (n = 13), Day 33 (n = 13), Day 40 (n = 13).
Fig 5.
Variability in tumor volume as measured by micro CT scans.
The measured tumor volume within the CIH and Sham groups in each time point is demonstrated. We found significantly greater variability in tumor volumes for the CIH group compared to Sham at day 19 (p<0.0001), day 26 (p<0.0001), day 33 (p = 0.025) and day 40 (p = 0.004).
Fig 6.
Metastases visualized at harvest.
(A) Metastases to right lung (black arrows) is visualized at harvest in addition to primary tumor (white arrows) in the left lung (site of Ad5CMVCre virus injection). (B) Metastases to mediastinal lymph nodes (black chevron), rib (yellow chevron) and sternum (white chevron) is visualized at harvest.
Fig 7.
(A) H&E stain confirms primary tumor in left lung (red chevrons outline border of primary tumor) as well as metastases to right lung (black chevron-RL) and mediastinal lymph nodes (black chevron-MLN). Surfactant protein C (SPC) immunohistochemistry demonstrates metastases to the heart in a CIH mouse (B.1) and Sham mouse (B.3). We provide SPC immunohistochemistry to CIH lung (B.2) and Sham lung (B.4) for positive control. All micrographs taken at 40X, bar is 50 microns.