Intraoperative tumor margin assessment during breast-conserving surgical resection is critically needed as positive margins are found in up to 34% of patients. Spectroscopic photoacoustic (sPA) molecular imaging combined with translatable antibody (Ab)-indocyanine green (ICG) contrast agent targeted to B7-H3, a molecular marker differentially expressed in breast cancer, may aid in margin assessment in murine breast cancers. ICG was conjugated to anti-B7-H3 antibodies through standard NHS chemistry. FVB/N Tg(MMTV/PyMT634Mul mice, a transgenic mouse model for breast cancer development, 5 weeks of age with ductal carcinoma in situ (DCIS) or small invasive carcinomas were given 33 μg of B7-H3-ICG 3-5 days before surgical resection. During excision of sequential sections of the lower mammary glands, fluorescence, multi-wavelength (680-900 nm, 10 nm increments) sPA, and B-mode ultrasound imaging were performed. Section specific molecular B7-H3 signal was compared to fluorescence imaging and histological (H&E) analysis. B-mode US and sPA imaging signal were able to relay accurate anatomical and molecular tissue information. sPA molecular imaging was able to detect B7-H3-ICG uptake in DCIS and early invasive carcinomas less than 1 mm in diameter. Furthermore, histological analysis of the excised tissues was able to show strong correlation between sPA imaging signal and disease state and location. While fluorescence imaging was able to detect ICG signal, there was insufficient resolution and specificity to identify and aid in resection of small cancerous foci. Adding molecular sPA imaging to help guide intraoperative margin assessment may increase the occurrence of negative margins which will decrease local disease recurrence.
Improved techniques for breast cancer screening are critically needed as current methods lack diagnostic accuracy. Using spectroscopic photoacoustic (sPA) molecular imaging with a priori knowledge of optical absorption spectra allows suppression of endogenous background signal, increasing the overall sensitivity and specificity of the modality to exogenous contrast agents. Here, sPA imaging was used to monitor antibody-indocyanine green (ICG) conjugates as they undergo optical absorption spectrum shifts after cellular endocytosis and degradation to allow differentiation between normal murine mammary glands from breast cancer by enhancing molecular imaging signal from target (B7-H3)-bound antibody-ICG. First, B7-H3 was shown to have highly specific (AUC of 0.93) expression on both vascular endothelium and tumor stroma in malignant lesions through quantitative immunohistochemical staining of B7-H3 on 279 human samples (normal (n=53), benign lesions (11 subtypes, n=182), breast cancers (4 subtypes, n=97)), making B7-H3 a promising target for sPA imaging. Second, absorption spectra of intracellular and degraded B7-H3-ICG and Isotype control (Iso-ICG) were characterized through in vitro and in vivo experiments. Finally, a transgenic murine breast cancer model (FVB/N-Tg(MMTVPyMT)634Mul) was imaged, and sPA imaging in found a 3.01 (IQR 2.63, 3.38, P<0.001) fold increase in molecular B7-H3-ICG signal in tumors (n=80) compared to control conditions (B7-H3-ICG in tumor negative animals (n=60), Iso-ICG (n=30), blocking B7-H3+B7-H3-ICG (n=20), and free ICG (n=20)) despite significant tumor accumulation of Iso-ICG, confirmed through ex vivo histology. Overall, leveraging anti-B7-H3 antibody-ICG contrast agents, which have dynamic optical absorption spectra representative of molecular interactions, allows for highly specific sPA imaging of murine breast cancer.
Delivery of contrast agents and their interaction with cells is emerging as an important tool in cancer imaging and
therapy. An alternative to traditional molecular targeting schemes that induce endocytotic uptake of contrast agents in
cells is presented here. Specifically, the application of high-intensity, focused ultrasound (HIFU) was used to enhance
uptake of gold nanorods in pancreatic cancer cells in vitro. A significant increase was observed in gold nanorod uptake
when cells were incubated with nanorods and treated with HIFU. Additionally, inclusion of liquid-filled, perfluorocarbon
(PFC) microdroplets in cell samples incubated with nanorods and treated with HIFU exhibited greater uptake of gold
over those samples exposed to HIFU without microdroplets. Furthermore, the level of acoustic pressure required to
increase nanoparticle uptake did not significantly decrease cell viability. Therefore, improved intracellular delivery of
nanoparticle contrast agents is possible using HIFU without compromising cell viability. Since nanoparticle delivery is
mechanically induced, this method can apply to a broad range of cancer imaging and therapy applications.
The complementary information provided by the ultrasound and photoacoustic imaging modalities has sparked their use
in combination in recent years. We introduce a dual contrast agent capable of providing image contrast enhancement for
both modalities simultaneously. The dual contrast agent is a small liquid perfluorocarbon droplet encased with bovine
serum albumin and loaded with plasmonic nanoparticles. The plasmonic metal nanoparticles themselves act as a
photoacoustic contrast agent. Furthermore, the perfluorocarbon droplet creates acoustic impedance mismatch between
itself and the surrounding tissue, allowing it to act as an ultrasound contrast agent. Experiments demonstrating the
performance of this dual contrast agent in ultrasound and photoacoustic imaging are presented. Differences in contrast
mechanisms of the dual agent are highlighted, and finally, the applications for the agent are discussed.
A dual contrast agent that combines perfluorocarbon droplets and metal nanoparticles has been developed for combined
ultrasound and photoacoustic imaging. Metal nanoparticles were incorporated in dodecafluoropentane (DDFP) droplets
encapsulated in a bovine serum albumin (BSA) shell. To embed aqueous colloidal suspensions of metal nanoparticles in
DDFP, a phase transfer of the plasmonic nanoparticles was completed in two different strategies, a single and a double
ligand exchange of capping materials. Emulsion techniques were used to encapsulate phase transferred metal
nanoparticles within the DDFP droplets. Spectrophotometry and cryogenic transmission electron microscopy were used
to characterize and to confirm successful fabrication of the dual contrast agent.
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