Development of a novel preclinical pancreatic cancer research model: bioluminescence image-guided focal irradiation and tumor monitoring of orthotopic xenografts

Abstract

Purpose: We report on a novel preclinical pancreatic cancer research model that uses bioluminescence imaging (BLI)-guided irradiation of orthotopic xenograft tumors, sparing of surrounding normal tissues, and quantitative, noninvasive longitudinal assessment of treatment response.

Materials and methods: Luciferase-expressing MiaPaCa-2 pancreatic carcinoma cells were orthotopically injected in nude mice. BLI was compared to pathologic tumor volume, and photon emission was assessed over time. BLI was correlated to positron emission tomography (PET)/computed tomography (CT) to estimate tumor dimensions. BLI and cone-beam CT (CBCT) were used to compare tumor centroid location and estimate setup error. BLI and CBCT fusion was performed to guide irradiation of tumors using the small animal radiation research platform (SARRP). DNA damage was assessed by γ-H2Ax staining. BLI was used to longitudinally monitor treatment response.

Results: Bioluminescence predicted tumor volume (R = 0.8984) and increased linearly as a function of time up to a 10-fold increase in tumor burden. BLI correlated with PET/CT and necropsy specimen in size (P < .05). Two-dimensional BLI centroid accuracy was 3.5 mm relative to CBCT. BLI-guided irradiated pancreatic tumors stained positively for γ-H2Ax, whereas surrounding normal tissues were spared. Longitudinal assessment of irradiated tumors with BLI revealed significant tumor growth delay of 20 days relative to controls.

Conclusions: We have successfully applied the SARRP to a bioluminescent, orthotopic preclinical pancreas cancer model to noninvasively: 1) allow the identification of tumor burden before therapy, 2) facilitate image-guided focal radiation therapy, and 3) allow normalization of tumor burden and longitudinal assessment of treatment response.

Figure 1

BLI accurately predicts tumor volume (A). Mean photon counts (p/s/cm²/Sr) of MiaPaCa-2-ELN heterotopic tumors of varying sizes correlated well with measured tumor volume as noted by linear regression analysis (R = 0.8984; A). Normalized luminescence of pancreatic tumors was determined as a function of time and shows a relatively linear increase in signal up to 10-fold after which plateaued growth was noted (B).

Figure 2

Radiation treatment planning and delivery to orthotopic pancreatic tumors. (A) Coronal CBCT image of anesthetized mouse obtained on the SARRP platform showing bony anatomy without clear ability to identify pancreatic tumor. (B) Offline bioluminescent coronal image of anesthetized mouse seen in A. Checkerboard fusion of images from A and B aligned to match external animal contour and identifiable internal anatomy showing excellent registration (C). (D) Double-exposure radiographs obtained in the treatment position on the SARRP identifying the radiation portal and confirming the area to be irradiated with 5 Gy in a single fraction (D).

Figure 3

Radiation dose-response curve of heterotopic tumors. MiaPaCa-2-ELN flank tumors irradiated with 5, 10, or 15 Gy in 5-Gy daily fractions and assessed longitudinally show equal levels of significant inhibition with cumulative doses of 10 and 15 Gy and an intermediate level of growth inhibition with 5 Gy relative to untreated controls.

Figure 4

Staining with 4′,6-diamidino-2-phenylindole dihydrochloride and γ-H2Ax of abdominal organ dissected after focused abdominal radiation per Figure 3 shows intense colocalized staining of pancreatic tumor, as well as transverse colon and greater curvature of stomach, which were partly within the radiation field (A; original magnification, x40). Other abdominal organs, including liver, lesser curvature of the stomach, and spleen, show minimal to no γ-H2Ax staining (A). Quantification of γ-H2Ax foci in A reported as the average sum of foci divided by the average number of nuclei in areas containing a minimum of 40 cells showing significantly higher quantities of staining in the pancreatic tumor relative to the liver, greater and lesser curvature of the stomach, spleen, and unirradiated control (B; P < .05).

Figure 5

Longitudinal monitoring of bioluminescent orthotopic pancreatic tumors treated with or without 5 Gy of BLI-guided radiation shows treated tumors with a significantly lower tumor burden confirmed by pathologic (C) and mean photon emission mean (F) relative to respective controls. This is graphically depicted in the same animals in A and B and in D and E. When followed over time, irradiated tumors (■) showed a significant growth inhibition of 24 days compared to untreated tumors (●) using a 10x increase in tumor burden as an end point (G).