High sensitivity: high-resolution SPECT-CT/MR molecular imaging of angiogenesis in the Vx2 model

Abstract

Objectives: The use of antiangiogenic therapy in conjunction with traditional chemotherapy is becoming increasingly in cancer management, but the optimal benefit of these targeted pharmaceuticals has been limited to a subset of the population treated. Improved imaging probes that permit sensitive detection and high-resolution characterization of tumor angiogenesis could improve patient risk-benefit stratification. The overarching objective of these experiments was to develop a dual modality alpha(nu)beta3-targeted nanoparticle molecular imaging agent that affords sensitive nuclear detection in conjunction with high-resolution MR characterization of tumor angiogenesis.

Materials and methods: In part 1, New Zealand white rabbits (n = 21) bearing 14d Vx2 tumor received either alpha(nu)beta3-targeted 99mTc nanoparticles at doses of 11, 22, or 44 MBq/kg, nontargeted 99mTc nanoparticles at 22 MBq/kg, or alpha(nu)beta3-targeted 99mTc nanoparticles (22 MBq/kg) competitively inhibited with unlabeled alpha(nu)beta3-nanoparticles. All animals were imaged dynamically over 2 hours with a planar camera using a pinhole collimator. In part 2, the effectiveness of alpha(nu)beta3-targeted 99mTc nanoparticles in the Vx2 rabbit model was demonstrated using clinical SPECT-CT imaging techniques. Next, MR functionality was incorporated into alpha(nu)beta3-targeted 99mTc nanoparticles by inclusion of lipophilic gadolinium chelates into the outer phospholipid layer, and the concept of high sensitivity - high-resolution detection and characterization of tumor angiogenesis was shown using sequential SPECT-CT and MR molecular imaging with 3D neovascular mapping.

Results: alpha(nu)beta3-Targeted 99mTc nanoparticles at 22 MBq/kg produced the highest tumor-to-muscle contrast ratio (8.56 +/- 0.13, TMR) versus the 11 MBq/kg (7.32 +/- 0.12) and 44 MBq/kg (6.55 +/- 0.07) doses, (P < 0.05). TMR of nontargeted particles at 22.2 MBq/kg (5.48 +/- 0.09) was less (P < 0.05) than the equivalent dosage of alpha(nu)beta3-targeted 99mTc nanoparticles. Competitively inhibition of 99mTc alpha(nu)beta3-integrin-targeted nanoparticles at 22.2 MBq/kg reduced (P < 0.05) TMR (5.31 +/- 0.06) to the nontargeted control contrast level. Multislice CT imaging could not distinguish the presence of Vx2 tumor implanted in the popliteal fossa from lymph nodes in the same fossa or in the contralateral leg. However, the use of 99mTc alpha(nu)beta3-nanoparticles with SPECT-CT produced a clear neovasculature signal from the tumor that was absent in the nonimplanted hind leg. Using alpha(nu)beta3-targeted 99mTc-gadolinium nanoparticles, the sensitive detection of the Vx2 tumor was extended to allow MR molecular imaging and 3D mapping of angiogenesis in the small tumor, revealing an asymmetrically distributed, patchy neovasculature along the periphery of the cancer.

Conclusion: Dual modality molecular imaging with alpha(nu)beta3-targeted 99mTc-gadolinium nanoparticles can afford highly sensitive and specific localization of tumor angiogenesis, which can be further characterized with high-resolution MR neovascular mapping, which may predict responsiveness to antiangiogenic therapy.

Figure 1

Bis-pyridyl-lysine-caproyl-phosphatidylethanolamine was custom synthesized and incorporated into the phospholipid surfactant at 3 mole%.

Figure 2

^99mTc images of the Vx2 tumor neovasculature from the popliteal fossa obtained with pinhole collimation 15 minutes after intravenous ear vein injection of 11 MBq/kg (A), 22 MBq/kg (B), or 44 MBq/kg (C) dosages.

Figure 3

All figures present tumor-to-muscle ratio of counts versus time post injection. A, 11 MBq/kg versus 22 MBq/kg dosages. B, 22 MBq/kg versus 44 MBq/kg dosages. C, Nontargeted 22 MBq/kg versus targeted 22 MBq/kg. D, Competitive inhibition of targeted 22 MBq/kg with nonlabeled, integrin-targeted nanoparticles.

Figure 4

A–C, Axial, sagittal, and coronal reconstructions from tomographic CT images of the rabbit hindquarters revealed the leg, bones, and a nodular mass within the popliteal fossa. Note the tissue within the popliteal fossa (yellow arrow heads) cannot be discriminated as tumor or lymph node because relatively prominent lymph nodes are always associated with this region. D–F, In combination with the attenuation corrected SPECT images, the presence of neovascular signal from ^99mTc α_vβ₃-targeted nanoparticle signal (blue arrow heads) associated with an ∼1-cm tissue mass located superior to the lymph node proper is readily appreciated and distinguished. Other regions of increased nuclear signal are associated with growing bone (green arrow heads) and testis (red arrow heads), which are all are appreciated bilaterally. The pelvic signal (brown circle) reflects the clearance of ^99mTc into the bladder. The combination of high-sensitivity molecular imaging in conjunction with high-resolution CT imaging readily facilitates the discrimination of pathologic from physiologic sources of signal.

Figure 5

A, SPECT-CT image of nascent Vx2 adenocarcinoma in rabbit popliteal fossa after 11 MBq/kg dosage, α_vβ₃-targeted ^99mTc-gadolinium nanoparticles given via ear vein superimposed on the MR image obtained at 2 hours. B, T1w, fat-suppressed, gradient echo image of Vx2 adenocarcinoma of rabbit shown in panel A 2 hours after administration of remaining MR dosage, revealing the asymmetric, peripheral neovascular signal enhancement previously reported for this model. A slight misalignment of the SPECT-CT and MR images occurred, which reflected error in registering the true centers of high-resolution gadolinium MR signal and the low-resolution ^99mTc signal from the peripheral fiducial markers. C, MR 3D neovascular map of Vx2 adenocarcinoma of rabbit in panel A and B, illustrating heterogeneity of neovasculature in tumor periphery.

Figure 6

A, High-power (40×) fluorescent image shows colocalization of rhodamine-labeled α_vβ₃-targeted nanoparticles (shown in B, 40×) with FITC-labeled lectin, a vascular endothelial marker (shown in C, 40×) obtained in tumor periphery, illustrating the vascular constraint of nanoparticles in the tumor vasculature. D, Although FITC-labeled vasculature was prominent within the central partially necrotic core of the tumor, integrin-targeted rhodamine nanoparticles were rarely observed (40×).