Subcutaneous (s.c.) tumor models are widely used in pre-clinical cancer metastasis research. Despite this, the dynamics and natural progression of circulating tumor cells (CTCs) and CTC clusters (CTCCs) in peripheral blood are poorly understood in these models. In this work, we used a new technique called ‘diffuse in vivo flow cytometry’ (DiFC) to study CTC and CTCC dissemination in an s.c. Lewis lung carcinoma (LLC) model in mice. Tumors were grown in the rear flank and we performed DiFC up to 31 days after inoculation. At the study endpoint, lungs were excised and bioluminescence imaging (BLI) was performed to determine the extent of lung metastases. We also used fluorescence macro-cryotome imaging to visualize infiltration and growth of the primary tumor. DiFC revealed significant heterogeneity in CTC and CTCC numbers amongst all mice studied, despite using clonally identical LLC cells and tumor placement. Maximum DiFC count rates corresponded to 0.1 to 14 CTCs per mL of peripheral blood. In general, CTC numbers did not necessarily increase monotonically over time and were poorly correlated with tumor volume. However, there was a good correlation between CTC and CTCC numbers in peripheral blood and lung metastases. We attribute the differences in CTC numbers primarily due to growth patterns of the primary tumor. This study is one of the few reports of CTC shedding dynamics in sub-cutaneous metastasis models and underscores the value of in vivo methods for continuous, non-invasive CTC monitoring.

The potential to image subsurface fluorescent contrast agents at high spatial resolution has facilitated growing interest in short-wave infrared (SWIR) imaging for biomedical applications. The early but growing literature showing improvements in resolution in small animal models suggests this is indeed the case, yet to date, images from larger animal models that more closely recapitulate humans have not been reported. We report the first imaging of SWIR fluorescence in a large animal model. Specifically, we imaged the vascular kinetics of an indocyanine green (ICG) bolus injection during open craniotomy of a mini-pig using a custom SWIR imaging instrument and a clinical-grade surgical microscope that images ICG in the near-infrared-I (NIR-I) window. Fluorescence images in the SWIR were observed to have higher spatial and contrast resolutions throughout the dynamic sequence, particularly in the smallest vessels. Additionally, vessels beneath a surface pool of blood were readily visualized in the SWIR images yet were obscured in the NIR-I channel. These first-in-large-animal observations represent an important translational step and suggest that SWIR imaging may provide higher spatial and contrast resolution images that are robust to the influence of blood. © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. Fig. 4 (a-d) Dynamic VTR values plotted as a function of time with corresponding vessel and tissue ROIs outlined above each respective vessel-to-tissue contrast plot.

The observed behavior of shortwave infrared (SWIR) light in tissue, characterized by relatively low scatter and subdiffuse photon transport, has generated considerable interest for the potential of SWIR imaging to produce high-resolution, subsurface images of fluorescence activity in vivo. These properties have important implications for fluorescence-guided surgery and preclinical biomedical research. Until recently, translational efforts have been impeded by the conventional understanding that fluorescence molecular imaging in the SWIR regime requires custom molecular probes that do not yet have proven safety profiles in humans. However, recent studies have shown that two readily available near-infrared (NIR-I) fluorophores produce measurable SWIR fluorescence, implying that other conventional fluorophores produce detectable fluorescence in the SWIR window. Using SWIR spectroscopy and wide-field SWIR imaging with tissue-simulating phantoms, we characterize and compare the SWIR emission properties of eight commercially available red/NIR-I fluorophores commonly used in preclinical and clinical research, in addition to a SWIR-specific fluorophore. All fluorophores produce measurable fluorescence emission in the SWIR, including shorter wavelength dyes such as Alexa Fluor 633 and methylene blue. This study is the first to report SWIR fluorescence from six of the eight conventional fluorophores and establishes an important comparative reference for developing and evaluating SWIR imaging strategies for biomedical applications. © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Whole-animal fluorescence cryo-imaging is an established technique that enables visualization of the biodistribution of labeled drugs, contrast agents, functional reporters and cells in detail. However, many tissues produce endogenous autofluorescence, which can confound interpretation of the cryo-imaging volumes. We describe a multi-channel, hyperspectral cryo-imaging system that acquires densely-sampled spectra at each pixel in the 3-dimensional stack. This information enables the use of spectral unmixing to isolate the fluorophore-of-interest from autofluorescence and/or other fluorescent reporters. In phantoms and a glioma xenograft model, we show that the approach improves detection limits, increases tumor contrast, and can dramatically alter image interpretation.

Purpose: This study demonstrates remote imaging for in vivo detection of radiation-induced tumor microstructural changes by tracking the diffusive spread of injected intratumor UV excited tattoo ink using Cherenkov-excited luminescence imaging (CELI). Methods: Micro-liter quantities of luminescent tattoo ink with UV absorption and visible emission were injected at a depth of 2mm into mouse tumors prior to receiving a high dose treatment of radiation. X-rays from a clinical linear accelerator were used to excite phosphorescent compounds within the tattoo ink through Cherenkov emission. The in vivo phosphorescence was detected using a time-gated intensified CMOS camera immediately after injection, and then again at varying time points after the ink had broken down with the apoptotic tumor cells. ex vivo tumors were imaged post-mortem using hyperspectral cryo-fluorescence imaging to quantify necrosis and compared to Cherenkov-excited light imaging of diffusive ink spread measured in vivo. Results: Imaging of untreated control mice showed that ink distributions remained constant after four days with less than 3% diffusive spread measured using full width at 20% max. For all mice, in vivo CELI measurements matched within 12% of the values estimated by the high-resolution ex vivo sliced luminescence imaging of the tumors. The tattoo ink spread in treated mice was found to correlate well with the nonperfusion necrotic core volume (R 2 =0.92) but not well with total tumor volume changes (R 2 =0.34). Conclusion: In vivo and ex vivo findings indicate that the diffusive spread of the injected tattoo ink can be related to radiation-induced necrosis, independent of total tumor volume change. Tracking the diffusive spread of the ink allows for distinguishing between an increase in tumor size due to new cellular growth and an increase in tumor size due to edema. Furthermore, the imaging resolution of CELI allows for in vivo tracking of subtle microenvironmental changes which occur earlier than tumor shrinkage and this offers the potential for novel, minimally invasive radiotherapy response assay without interrupting a singular clinical workflow.