Fig 1.
Schematic overview of the orthotopic murine model of PDAC with weekly BLI and DEXA scans.
Mice were injected with 1.0x105 KCKO-Luc cells and underwent weekly bioluminescent imaging (BLI) and dual energy X-ray absorptiometry (DEXA) analyses. Mice were monitored for failure to thrive criteria and received a final DEXA measurement before sacrifice on week 8 or week 10 post tumor inoculation. Tumors are excised and weighed postmortem. n = 10 NTC mice and n = 20 PDAC mice in total. Created with BioRender.com (2023).
Fig 2.
Limitations of bioluminescent imaging as a longitudinal biomarker of tumor mass and growth over time.
Longitudinal images of a representative PDAC mouse #14 from week 1 to week 8 post tumor inoculation using bioluminescent imaging (BLI). A standardized abdominal ROI (red circle) was used to quantify BLI values as total flux in radiance (photons/second). Note the robust BLI signal at week 1, the unexplained absence of BLI signal at week 4, and BLI signal at week 8 that is smaller than the BLI signal at week 1. Representative mouse is selected from all PDAC mice used in the study (n = 20) that received BLI scans.
Fig 3.
Longitudinal quantification of growing PDAC tumor mass via DEXA and validation with ex vivo tumor weight.
Longitudinal DEXA images of a representative non-tumor control (NTC) mouse (A) and PDAC mouse #5 (B) from baseline to week 8 post tumor inoculation are shown. An initial abdominal scout view ROI was used to identify PDAC tumors via manual segmentation from vertebrae T13-L6 and extension to the distal end of the femur (green highlighted region). An exclusion ROI (blue region) was utilized to remove interference from the skull and ear tag. Arrows indicate the presence of the tumor burden visible via DEXA starting at 3-weeks. The weekly percent change in scout ROI abdominal lean mass compared to baseline values from week 1–8 display the significant presence of a tumor burden at week 2 and on (p<0.05) (C). Sample size values represent the living PDAC mice at each weekly DEXA scan. Six PDAC mice met failure to thrive criteria, and their tumors were harvested for gross weight analysis. A representative image of the excised tumor from the median PDAC mouse weighing 1.6165 grams and 3–4 cm2 is shown (D), with all the ex vivo tumor weights from the PDAC mice that were not lost to attrition (E). n = 10 NTC and n = 10 PDAC mice were used for scout ROI analysis.
Fig 4.
Superiority of DEXA over BLI as an in vivo biomarker of tumor mass and longitudinal measure of tumor growth.
Ten additional mice received orthotopic KCKO-Luc cells and the growth of their PDAC was assessed via BLI and DEXA until their failure to thrive or sacrifice at 10 weeks post-implantation. DEXA quantification for this study was derived from manual ROI segmentation which reports the focused PDAC ROI in grams (PDAC mouse #13) (A). The asterisk denotes the primary tumor and arrows indicate local spread. Longitudinal DEXA quantification from each mouse is presented starting when tumor burden is visible (B) as well as the mean +/- SD and slope for all PDAC mice (n = 20) from weeks 3–10 (C). Weekly BLI signal is presented for each mouse in this study (n = 10) (D) as well as the mean +/- SD for all tumor-bearing mice (n = 20) (E) from weeks 1–10 with the slope. To assess the correlation between PDAC mass and biomarker outcome, linear regression analyses were performed comparing ex vivo tumor weight vs. terminal BLI (F) and terminal DEXA measures (G), and the graphed data are presented with the Pearson coefficient and p-value (n = 15 that were not lost to attrition). Two of the authors completed independent focused PDAC segmentation on end-of-life DEXA scans, and the interobserver reliability of the quantified tumor mass was determined via intraclass correlation coefficient (ICC r2 = 0.9736; p<0.0001) (H). n = 20 week 1–5, n = 16 week 6, n = 11 week 7, n = 7 week 8, n = 6 week 9, n = 5 week 10. Note: The discontinued readings for individual mice for panels B and D are due to meeting “failure to thrive criteria” and subsequent euthanasia, and the last DEXA and BLI measure is reported.