Vascular regional analysis unveils differential responses to anti-angiogenic therapy in pancreatic xenografts through macroscopic photoacoustic imaging

Abstract

Background: Amongst the various imaging techniques that provide surrogate tumor radiographic indications to aid in planning, monitoring, and predicting outcomes of therapy, ultrasound-guided photoacoustic imaging (US-PAI) is a promising non-ionizing modality based on endogenous blood (hemoglobin) and blood oxygen saturation (StO₂) contrast. Adaptation of US-PAI to the clinical realm requires macroscopic system configurations for adequate depth visualization. Methods: Here we present a vascular regional analysis (VRA) methodology of obtaining areas of low and high vessel density regions within the tumor (LVD and HVD respectively) by frequency domain filtering of macroscopic PA images. In this work, we evaluated the various vascular and oxygenation profiles of different murine xenografts of pancreatic cancer (AsPC-1, MIA PaCa-2, and BxPC-3) that have varying levels of angiogenic potentials and investigated the effects of receptor tyrosine kinase inhibitor (sunitinib) on the tumor microvessel density and StO₂. Results: The administration of sunitinib resulted in transient deoxygenation and reduction in vessel density within 72 h in two (AsPC-1 and MIA PaCa-2) of the three tumor types. Utilizing VRA, the regional change in StO₂ (∆StO₂) revealed the preferential targeting of sunitinib in LVD regions in only the AsPC-1 tumors. We also identified the presence of vascular normalization (validated through immunohistochemistry) in the sunitinib treated AsPC-1 tumors at day 8 post-treatment where a significant increases in HVD ∆StO₂ (~20%) were seen following the 72-hour time point, indicative of improved vessel flow and functionality. Treated AsPC-1 vasculature displayed increased maturity and functionality compared to non-treated tumors on day 8, while these same metrics showed no conclusive evidence of vascular normalization in MIA PaCa-2 or BxPC-3 tumors. Conclusion: Overall, VRA as a tool to monitor treatment response allowed us to identify time points of vascular remodeling, highlighting its ability to provide insights into the tumor microenvironment for sunitinib treatment and other anti-angiogenic therapies.

Keywords: angiogenesis; endogenous contrast; pancreatic cancer; photoacoustics; sunitinib; vascular density; vascular normalization.

Figure 1

Study timeline describing the imaging, treatment, and histological examination of pancreatic tumor xenografts. D(0) represents Day 0, which is the day of the first treatment or vehicle administration. Cohort #1 was euthanized on D(3), cohort #2 was euthanized on D(8), and cohort #3 was monitored longitudinally up to D(20).

Figure 2

Image processing workflow displaying method for segmenting regions of high vascular density (HVD) and low vascular density (LVD)

Figure 3

A) 2D cross sectional US image in a representative MIA PaCa-2 tumor. B) H&E stain of the same tumor cross section shown in A,C. C) 2D cross sectional image of HbT overlayed on US in a representative MIA PaCa-2 tumor with white arrows pointing towards areas of HVD and yellow arrows pointing towards areas of LVD. D) IF stain of the same tumor cross section shown in A,C with blue representing DAPI and red representing CD31. White arrows pointing towards areas of HVD and yellow arrows pointing towards areas of LVD according to the HbT image. E) Plot of normalized average CD31 intensity in a 1 mm x 1 mm ROI versus the fraction of pixels in the ROI labelled as HVD. All scale bars shown represent 2 mm.

Figure 4

A-C) Plot of tumor volume for sunitinib (red) and no treatment - NT (black) groups for AsPC-1 (A), MIA PaCa-2 (B), and BxPC-3 (C) tumors with treatment starting at Day 0 (blue arrow). D-E) Violin plot of growth rate (D) and volume change from pre-treatment (D(-1)) to post-treatment or experiment end point (D(18-20)) (E) for sunitinib treated (red) and control tumors (black) in AsPC-1, MIA PaCa-2, and BxPC-3 tumors. All error bars shown represent SEM. p-values: * < 0.05, ** < 0.01, *** < 0.001, # or **** < 0.0001

Figure 5

A-B) Representative AsPC-1 tumors stained for Ki-67 (orange) overlaid on DAPI (blue) for the control (NT) (A) and sunitinib treated (B) group. C) Bar graph comparing Ki-67⁺ / DAPI⁺ cell ratio between the control (black) and sunitinib (red) groups. D-E) Representative MIA PaCa-2 tumors stained for Ki-67 (orange) overlaid on DAPI (blue) for the control (D) and sunitinib treated (E) group. F) Bar graph comparing Ki-67⁺ / DAPI⁺ cell ratio between the control (black) and sunitinib (red) groups for MIA PaCa-2. All scale bars = 125 μm, all error bars shown represent SEM. p-values: * < 0.05, ** < 0.01, *** < 0.001

Figure 6

A-B) Plot of StO₂ in sunitinib (80 mg/kg) treated (red line) and untreated control (no treatment, black line) AsPC-1 tumors in regions of high vascular density (HVD) regions (A) and (LVD) regions (B). C-D) Violin plot comparing StO₂ on D(1) (C) and ∆StO₂ between D(1) and D(-1) (D) for Sunitinib at 80 mg/kg (red) and No Treatment (black) AsPC-1 tumors in areas HVD and LVD. E-F) Violin plot comparing StO₂ on D(3) (E) and ∆StO₂ between D(3) and D(-1) (F) for Sunitinib at 80 mg/kg (red) and No Treatment (black) AsPC-1 tumors in areas HVD and LVD. G-H) Violin plot comparing StO₂ on D(8) (G) and ∆StO₂ between D(8) and D(-1) (H) for Sunitinib at 80 mg/kg (red) and No Treatment (black) AsPC-1 tumors in areas HVD and LVD. I) Regional 2D cross sectional images (top) and 3D rendered images (bottom) of StO₂ in sunitinib (80 mg/kg) and no treatment AsPC-1 tumors displaying high HVD and LVD areas. All error bars shown represent SEM. Scale bars = 2 mm. p-values: * < 0.05, ** < 0.01, *** < 0.001, # or **** < 0.0001

Figure 7

A-B) Plot of StO₂ in sunitinib (80 mg/kg) treated (red line) and untreated control (no treatment, black line) MIA PaCa-2 tumors in regions of high vascular density (HVD) regions (A) and (LVD) regions (B). C-D) Violin plot comparing StO₂ on D(1) (C) and ∆StO₂ between D(1) and D(-1) (D) for Sunitinib at 80 mg/kg (red) and No Treatment (black) MIA PaCa-2 tumors in areas HVD and LVD. E-F) Violin plot comparing StO₂ on D(3) (E) and ∆StO₂ between D(3) and D(-1) (F) for Sunitinib at 80 mg/kg (red) and No Treatment (black) MIA PaCa-2 tumors in areas HVD and LVD. G-H) Violin plot comparing StO₂ on D(8) (G) and ∆StO₂ between D(8) and D(-1) (H) for Sunitinib at 80 mg/kg (red) and No Treatment (black) MIA PaCa-2 tumors in areas HVD and LVD. I) Regional 2D cross sectional images (top) and 3D rendered images (bottom) of StO₂ in sunitinib (80 mg/kg) and no treatment MIA PaCa-2 tumors displaying high HVD and LVD areas. All error bars shown represent SEM. Scale bars = 2 mm. p-values: * < 0.05, ** < 0.01, *** < 0.001, # or **** < 0.0001

Figure 8

A-B) Representative AsPC-1 tumors stained for CD31 (red) and αSMA (green) overlaid on DAPI (blue) for the control (A) and sunitinib (B) treated group. C) Bar graph comparing αSMA ⁺ / CD31⁺ cell ratio between the control (black) and sunitinib (red) groups. D-E) Representative MIA PaCa-2 tumors stained with CD31 (red) and αSMA (green) overlaid on DAPI (blue) for the control (D) and sunitinib (E) treated group. F) Bar graph (Mean +/- SEM) comparing αSMA ⁺ / CD31⁺ cell ratio between the control (black) and sunitinib (red) groups for MIA PaCa-2. G-H) Scatter plot of αSMA⁺ / CD31⁺ versus the HVD ∆StO₂ between D(8) and D(-1) (G) HVD fraction on D(8) (H) for each histological sample with points corresponding to MIA PaCa-2 tumors outlined in green, and AsPC-1 tumors outlined in blue. I-J) Representative AsPC-1 tumors stained for CD31 (red) and TL (green) overlaid on DAPI (blue) for the control (I) and sunitinib (J) treated group. K) Bar graph comparing TL ⁺ / CD31⁺ cell ratio between the control (black) and sunitinib (red) groups. L-M) Representative MIA PaCa-2 tumors stained with CD31 (red) and TL (green) overlaid on DAPI (blue) for the control (L) and sunitinib (M) treated group. N) Bar graph comparing TL ⁺ / CD31⁺ cell ratio between the control (black) and sunitinib (red) groups for MIA PaCa-2. O-P) Scatter plot of ΤL⁺ / CD31⁺ versus the HVD ∆StO₂ between D(8) and D(-1) (O) HVD fraction on D(8) (P) for each histological sample with points corresponding to MIA PaCa-2 tumors outlined in green, and AsPC-1 tumors outlined in blue. It is to be noted that all four stains DAPI, TL, αSMA and CD31 are performed on the same section. For display purposes we showed DAPI, TL, CD 31 and DAPI, αSMA and CD31 images separately. All scale bars = 125 μm, All error bars shown represent SEM. p-values: * < 0.05, ** < 0.01, *** < 0.001

Figure 9

A-C) Bar graphs (Mean +/- SEM) comparing the pre-treatment LVD fraction (A), HbT (B), and StO₂ (C) in all AsPC-1 (blue) and MIA PaCa-2 (green) tumors and BxPC-3 (purple). D-E) scatter plots of LVD ΔHbT on D(3) versus pre-treatment LVD fraction (D), and HbT (E). F) scatter plot of LVD ΔStO₂ from D(-1) to D(3) in sunitinib-treated tumors. ). G-H) scatter plots of HVD ΔHbT on D(8) versus pre-treatment LVD fraction (D), and HbT (E). I) scatter plot of HVD ΔStO₂ from D(-1) to D(8) in sunitinib-treated AsPC-1 (blue), MIA PaCa-2 (green), and BxPC-3 (purple) tumors. p-values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001