Preclinical longitudinal imaging of tumor microvascular radiobiological response with functional optical coherence tomography

Abstract

Radiation therapy (RT) is widely used for cancer treatment, alone or in combination with other therapies. Recent RT advances have revived interest in delivering higher dose in fewer fractions, which may invoke both cellular and microvascular damage mechanisms. Microvasculature may thus be a potentially sensitive functional biomarker of RT early response, especially for such emerging RT treatments. However it is difficult to measure directly and non-invasively, and its time course, dose dependencies, and overall importance in tumor control are unclear. We use functional optical coherence tomography for quantitative longitudinal in vivo imaging in preclinical models of human tumor xenografts subjected to 10, 20 and 30 Gy doses, furnishing a detailed assessment of vascular remodeling following RT. Immediate (minutes to tens of minutes) and early (days to weeks) RT responses of microvascular supply, as well as tumor volume and fluorescence intensity, were quantified and demonstrated robust and complex temporal dose-dependent behaviors. The findings were compared to theoretical models proposed in the literature.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

(a) NRG mouse with grown BX-PC3 tumor (blue arrow).The back was shaved prior to DSWC installation. (b) Flipped skin on the opposite side of the back showing the grown tumor during DSWC installation procedure. (c) Mouse with DSWC installed. (d) Same mouse as shown in (c) 9 weeks post-DSWC installation and 7.5 weeks post-RT (single-dose 20 Gy). (e) NRG mouse placed in the small-animal irradiator. 225kVp X-rays were incident from the bottom; white-light images showing (f) tumor location within a DSWC covered with EBT-2 Gafchromic film. (g) EBT-2 Gafchromic film color change after 10 Gy single-dose RT. Note that DSWC glass coverslip retaining ring was removed prior to RT to avoid possible dosimetric complications. Scale bar is 3 mm.

Figure 2

(a) Experimental time course. At day -28 tumor cells were injected into the dorsal skin. DSWC was implanted after the tumor volume reached 3–5 mm in diameter. Tumors were irradiated ~10 days after DSWC installation (day 0 labeled with “R”). Right after irradiation tumor vasculature was monitored within 90 minutes (minutes time scale). For five to eight weeks following irradiation, tumor changes were monitored repeatedly with caliper measurements (tumor volume), svOCT imaging (vasculature) and epi-fluorescence microscope imaging (cancer cell fluorescence). Tumor resection for histological staining was performed at selected post-RT stages in several animals to support and validate the in-vivo observations. White light images of the (b) back and (c) front of the tumor within window chamber. (d) svOCT microvasculature map of the area labeled with black dotted rectangle in (c). (e) Tumor cells Ds-Red fluorescence image. Scale bars are 500 μm.

Figure 3

Schematic diagram of the swept-source OCT system setup with quadrature Mach-Zehnder fiber-based interferometer and optical amplification: SOA - semiconductor optical amplifier, PC - polarization controller, A - fiber attenuator, DB - dual balanced photo-detector, DAQ - data acquisition card, C - collimator, MZ Interferometer - Mach-Zehnder interferometer, L - lens, M - mirror, SG - scanning galvo, CR - circulator.

Figure 4

Real-time imaging of tumor microvasculature via svOCT. (a) White light image of window chamber with tumor tissue under the glass coverslip. (b) OCT volumetric image of (a) taken over a 6 × 6 mm2 field of view. (c) 8 sequential same-location B-scans for further inter-frame comparison required for svOCT analysis. (d) The svOCT algorithm for inter-frame comparison of pixel texture. (e)SVzx vascular cross-section obtained with svOCT algorithm. (f) Vascular en-face two-dimensional projection. (g) Depth-encoded vascular en-face two-dimensional projection. (h) Depth-encoded vascular volume of tissue volume shown in (b).

Figure 5

BX-PC3 pancreatic tumor vasculature development within 16 days after subcutaneous injection of cancer cells into dorsal skin. (a) 3 days; (b) 7 days; (c) 10 days; (d) 16 days after injection. (e) Healthy tissue vasculature for comparison. Scale bars are 0.5 mm.

Figure 6

Immediate (minutes - 10s of minutes) tumor microvascular response within 1.5 hours post-RT 10 Gy single-dose. (a) Before irradiation; (b) 30 min after; (c) 45 min after; (d) 60 min after; (e) 90 min after irradiation. Scale bars are 1 mm. The numbers below each panel represent VVD, relative to the pre-radiation value.

Figure 7

Longitudinal OCT imaging of radiation response of the tumor vasculature to single dose of 20 Gy. svOCT images were taken before and multiple times following irradiation. “Day −0” image was taken 1 hour pre-RT and “Day +0” represents 1.5 hours post-RT. Maximum suppression of tumor vasculature is ~ at day 8, followed by vascular re-growth from the vessels outside the tumor. Scale bars = 1 mm.

Figure 8

Tumor development dynamics as reflected via changes in vascular volume density (VVD), tumor volume and fluorescence intensity after 10, 20 and 30 Gy single-dose local irradiation. (a) Tumor microvasculature response. (b) Tumor volume response. (c) Tumor fluorescence response. Quantified values of VVD, volume and fluorescence pre- and post-RT changes of each tumor were normalized to values of these metrics obtained 1 hour before irradiation of the same tumor. Non-irradiated tumors (0 Gy data) continued to grow and were sacrificed at earlier time points for humane reasons (tumors grew too big and data is not shown for the entire course of 7.5 weeks post-RT). (d) Summary graph for VVD, volume and fluorescence response to 20 Gy single-dose irradiation. In (a)–(d), symbols are experimental points, and lines are a guide for the eye. n = 7–15 per group, error bars = mean ± standard deviation. (e) A recently proposed literature model of tumor growth and corresponding changes in microvasculature following 20 Gy single-dose irradiation (after ref.).

Figure 9

Representative images of H&E (top row) and TUNEL (bottom row) staining for tumors irradiated with (a) 0 Gy; (b) 10 Gy; (c) 20 Gy; (d) 30 Gy. Tumors were resected 2 weeks following irradiation. TUNEL positivity for the whole tumor section was 0.3%, 15.8%, 47.6%, and 97.7% for 0, 10, 20, and 30 Gy, respectively. Scale bar = 200 μm.