Photoacoustic Tomography Detects Early Vessel Regression and Normalization During Ovarian Tumor Response to the Antiangiogenic Therapy Trebananib

Abstract

The primary aim of this study was to assess the potential of in vivo photoacoustic tomography for direct functional measurement of ovarian tumor response to antiangiogenic therapy.

Methods: In vivo studies were performed with institutional animal care and use committee approval. We used an orthotopic mouse model of ovarian cancer treated with trebananib (n = 9) or vehicle (n = 9). Tumor-bearing mice were randomized into trebananib or vehicle groups at day 10 and dosed on days 12, 15, and 18 after implantation. Photoacoustic tomography and blood draws were performed at day 10 and then 24 h after each drug dose. Tumors were excised for histopathology after the final studies on day 19. Data analysis to test for statistical significance was performed blinded.

Results: Blockade of angiopoietin signaling using trebananib resulted in reduced total hemoglobin-weighted photoacoustic signal (n = 9, P = 0.01) and increased oxyhemoglobin-weighted photoacoustic signal (n = 9, P < 0.01). The latter observation indicated normalization of the residual tumor vessels, which was also implied by low levels of angiopoietin 1 in serum biomarker profiling (0.76 ± 0.12 ng/mL). These noninvasive measures reflected a 30% reduction in microvessel density and increased vessel maturation in ex vivo sections.

Conclusion: Photoacoustic tomography is able to evaluate both vessel regression and normalization in response to trebananib. Noninvasive imaging data were supported by modulation of serum markers in vitro and ex vivo histopathology.

Keywords: angiogenesis; angiopoietin; ovarian cancer; photoacoustics.

FIGURE 1

Study design for in vivo imaging and in vitro serum diagnostics. (A) Timeline of therapy response monitoring. (B) Schematic illustrating study performed. (C) Imaging at 757, 797, and 837 nm delineates HbO₂ and Hb. Spectra from http://omlc.ogi.edu/spectra/index.html. BLI = bioluminescence imaging.

FIGURE 2

PAT images of THb-weighted signals. Three-dimensional oblique and orthogonal views at day 10 show vasculature in normal tissue and chaotic neovasculature of developing tumor. Tumor (T) is outlined using a 3-dimensional ROI. All images are on same logarithmic intensity scale.

FIGURE 3

PAT displays changes in tumor vasculature induced by trebananib treatment. Both images of full mouse torso (2 × 2 cm) and zoomed 2-dimensional maximum-intensity projection (MIP) focused on tumor ROI (T) are shown. Top of images point toward head of mouse. K = kidneys; S = spine; SP = spleen; SV = superficial vessels.

FIGURE 4

PAT detects reduced vessel density and increased HbO₂ in trebananib-treated mice. (A) THb photoacoustic (PA) signal in tumor ROI is significantly elevated in vehicle- compared with trebananib-treated mice; no significant difference is observed in contralateral ROI. (B) In trebananib-treated group, HbO₂-weighted signal increases significantly, whereas Hb-weighted signal decreases significantly, over same time course. *P < 0.05. **P < 0.01.

FIGURE 5

Vessel-derived mAng1 and mAng2 along with tumor cell SEAP protein secretion are modulated by tumor growth and trebananib therapy. (A) Serum concentration of mAng1 is significantly elevated by day 19 in vehicle-treated group but was maintained at baseline in trebananib-treated group. (B) mAng2 is elevated in both groups. (C) Detection of SEAP is significantly lower in trebananib-treated mice by day 19 after treatment, and plasma SEAP is correlated with THb. SEAP in plasma quoted as ng protein detected in 5 μL of isolated plasma. *P < 0.05.

FIGURE 6

Hematoxylin and eosin (H&E) staining. Tumor cell death was more than 2-fold higher in trebananib-treated group. Scale bar was 2.5 mm whole field (black bar) and 400 urn in zoomed sections (white bar) at location of white arrows in whole field. **P < 0.01. T = trebananib; V = vehicle.

FIGURE 7

Immunofluorescence staining showed reduced density of CD31-positive tumor vasculature (green) for trebananib-treated group (A) but increased desmin-positive pericyte (red) coverage of vessels (B). Blue stain is filamentous (F-) actin. Scale bar was 150 μm in whole field, 100 μm in zoomed sections. **P < 0.01. ****P < 0.0001.