The increased expression of vascular endothelial growth factor (VEGF) and its receptors is associated with angiogenesis in a growing tumor, presenting potential targets for tumor-selective imaging by way of targeted tracers. Though fluorescent tracers are used for targeted in vivo imaging, the lack of photostability and biocompatibility of many current fluorophores hinder their use in several applications involving long-term, continuous imaging. To address these problems, fluorescent nanodiamonds (FNDs), which exhibit infinite photostability and excellent biocompatibility, were explored as fluorophores in tracers for targeting VEGF receptors in growing tumors. To explore FND utility for imaging tumor VEGF receptors, we used click-chemistry to conjugate multiple copies of an engineered single-chain version of VEGF site-specifically derivatized with trans-cyclooctene (scVEGF-TCO) to 140 nm FND. The resulting targeting conjugates, FND-scVEGF, were then tested for functional activity of the scVEGF moieties through biochemical and tissue culture experiments and for selective tumor uptake in Balb/c mice with induced 4T1 carcinoma. We found that FND-scVEGF conjugates retain high affinity to VEGF receptors in cell culture experiments and observed preferential accumulation of FND-scVEGF in tumors relative to untargeted FND. Microspectroscopy provided unambiguous determination of FND within tissue by way of the unique spectral shape of nitrogen-vacancy induced fluorescence. These results validate and invite the use of targeted FND for diagnostic imaging and encourage further optimization of FND for fluorescence brightness.

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Significance: Decreasing the oxygen consumption rate (OCR) of tumor cells is a powerful method for ameliorating tumor hypoxia. However, quantifying the change in OCR is challenging in complex experimental systems. Aim: We present a method for quantifying the OCR of two tumor cell lines using oxygensensitive dual-emissive boron nanoparticles (BNPs). We hypothesize that our BNP results are equivalent to the standard Seahorse assay. Approach: We quantified the spectral emissions of the BNP and accounted for external oxygen diffusion to quantify OCR over 24 h. The BNP-computed OCR of two breast cancer cell lines, E0771 and 4T07, were compared with their respective Seahorse assays. Both cell lines were also irradiated to quantify radiation-induced changes in the OCR. Results: Using a Bland-Altman analysis, our BNPs OCR was equivalent to the standard Seahorse assay. Moreover, in an additional experiment in which we irradiated the cells at their 50% survival fraction, the BNPs were sensitive enough to quantify 24% reduction in OCR after irradiation. Conclusions: Our results conclude that the BNPs are a viable alternative to the Seahorse assay for quantifying the OCR in cells. The Bland-Altman analysis showed that these two methods result in equivalent OCR measurements. Future studies will extend the OCR measurements to complex systems including 3D cultures and in vivo models, in which OCR measurements cannot currently be made.

Real-time monitoring of physiological changes of tumor tissue during radiation therapy (RT) could improve therapeutic efficacy and predict therapeutic outcomes. Cherenkov radiation is a normal byproduct of radiation deposited in tissue. Previous studies in rat tumors have confirmed a correlation between Cherenkov emission spectra and optical measurements of blood-oxygen saturation based on the tissue absorption coefficients. The purpose of this study is to determine if it is feasible to image Cherenkov emissions during radiation therapy in larger human-sized tumors of pet dogs with cancer. We also wished to validate the prior work in rats, to determine if Cherenkov emissions have the potential to act an indicator of blood-oxygen saturation or water-content changes in the tumor tissue-both of which have been correlated with patient prognosis. Methods A DoseOptics camera, built to image the low-intensity emission of Cherenkov radiation, was used to measure Cherenkov intensities in a cohort of cancer-bearing pet dogs during clinical irradiation. Tumor type and location varied, as did the radiation fractionation scheme and beam arrangement, each planned according to institutional standard-of-care. Unmodulated radiation was delivered using multiple 6 MV X-ray beams from a clinical linear accelerator. Each dog was treated with a minimum of 16 Gy total, in �3 fractions. Each fraction was split into at least three subfractions per gantry angle. During each subfraction, Cherenkov emissions were imaged.

Background: Lymphovascular invasion (LVI), a critical feature of advanced cancers, is a major route of metastatic dissemination. Of the clinically-distinct types of breast cancer, the most lethal variant is inflammatory breast cancer (IBC) where LVI is a histopathological hallmark. Majority of IBC patients lack a distinct, solid tumor and instead present with diffuse tumor cell clusters cells, also termed as tumor emboli, in the breast and dermal lymphatics, which contributes to the clinical breast-skin symptoms and postulated to provide an efficient path to metastatic spread. The steps in lymphatic dissemination are challenging to study in vitro, and in vivo models are limited. Our goal was to develop new in vivo models that would enable direct visualization and quantitation of local tumor cell growth and tumor-lymphatic vessel interactions for improved understanding of the unique IBC tumor biology and for drug discovery. Methods: mCherry/GFP-expressing non-IBC (4T1, E0771) and IBC cells derived from patient untreated primary tumors (SUM149/basal and SUM190/Her2+) were implanted into the fascia in the center of a window chamber in the skin-fold or mammary fat pad. In order to allow direct study of tumor cell–lymphatic vessel interaction, we generated transgenic nude mice with fluorescent lymphatics [tdTomato fluorophore under control of a Prox1 promoter, encodes a transcription factor (prospero-related homeobox 1) necessary for the formation and maintenance of lymphatic vessels]. Multichannel microscopy was employed to serially quantify tumor growth, tumor spread and lymph-tumor interactions. Using this model, we also optimized implantation and live imaging of pre-formed IBC-cell derived tumor emboli generated by simulating the lymphatic sheer stress in culture. Results: Compared to non-IBC cells, which formed solid tumors that increased in size over a 14 day period, IBC models exhibited a diffuse and disseminating phenotype, within 24h, rather than a centralized tumor mass, similar to the disease presentation in patients. Using a threshold analysis in ImageJ, we quantified tumor area (as a metric of tumor growth), the tumor motility, and the linear density of lymph and blood vessels. Briefly, we binarized each fluorescent channel image such that the tumor or lymph signal was positive; then, an algorithm drew regions of interest (ROIs) around each defined tumor-cell cluster. The ROIs were used to calculate the parameters describing tumor growth and motility (distance of the tumor from the center of mass (CM)), and the area moment (the movement of the clusters relative to the CM) at each timepoint. The linear vascular density was calculated from the distance of segments drawn through the center of large vessels for each mouse, which provided quantitative data describing tumor cell or IBC emboli and lymphovascular interactions, suggesting that there is a specific microenvironment necessary for the unique phenotype and dissemination pattern exhibited by IBC tumor cells. Conclusions: Despite the poor prognosis of IBC, the clinical implications of how and why tumor emboli form and how they survive to migrate into the lymphatics is not defined. These in vivo models provide in-depth imaging and quantitative measurements of locoregional invasion, tumor cell-lymphatic or endothelial vessel interactions of implanted tumor cells or tumor emboli. This model has the potential to be extended to study other cancer cell types exhibiting LVI as well as a screening metric for IBC therapies. Supported by DOD-Breakthrough-W81XWH-17-1-029, IBC Research Foundation, DCI pilot funds, IBC Network Foundation, Duke Surgery Gardner Award. Citation Format: Ashlyn Rickard, Pranalee Patel, Scott J Sauer, Mark W Dewhirst, Gregory M Palmer, Gayathri R Devi. Multichannel serial imaging of transgenic, preclinical murine models provides the first quantitative analysis of the unusual growth kinetics and lymph-vascular invasion of patient-derived inflammatory breast cancer cells and tumor emboli [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-03.