Emerging Applications for Optically Enabled Intravital Microscopic Imaging in Radiobiology

Radiation therapy is an effective cancer treatment used in over 50% of cancer patients. Preclinical research in radiobiology plays a major role in influencing the translation of radiotherapy-based treatment strategies into clinical practice. Studies have demonstrated that various components of tumors and their microenvironments, including vasculature, immune and stem cells, and stromal cells, can influence the response of solid tumors to radiation. Optically enabled imaging techniques used in experimental animal models of cancer offer a unique and powerful way to quantitatively track spatiotemporal changes in these tumor components in vivo at macro-, meso-, and microscopic resolutions following radiotherapy. In this review, we discuss the role of both well-established and emerging intravital microscopy techniques for studying tumors and their microenvironment in vivo, in response to irradiation. The development and application of new animal models, small animal microirradiation technologies, and multimodal optically enabled intravital microscopy techniques are emphasized within the framework of preclinical radiobiology research. We also comment on the potential influence that these newer imaging techniques may have on the clinical translation of new preclinical radiobiology discoveries

Region-specific Differentiation Potential of Adult Rat Spinal Cord Neural Stem/Precursors and Their Plasticity in Response to In Vitro Manipulation

This study characterized the differentiation of neural stem/precursor cells (NSPCs) isolated from different levels of the spinal cord (cervical vs lumbar cord) and different regions along the neuraxis (brain vs cervical spinal cord) of adult male Wistar enhanced green fluorescent protein rats. The differentiation of cervical spinal cord NSPCs was further examined after variation of time in culture, addition of growth factors, and changes in cell matrix and serum concentration. Brain NSPCs did not differ from cervical cord NSPCs in the percentages of neurons, astrocytes, or oligodendrocytes but produced 26.9% less radial glia. Lumbar cord NSPCs produced 30.8% fewer radial glia and 6.9% more neurons compared with cervical cord NSPCs. Spinal cord NSPC differentiation was amenable to manipulation by growth factors and changes in in vitro conditions. This is the first study to directly compare the effect of growth factors, culturing time, serum concentration, and cell matrix on rat spinal cord NSPCs isolated, propagated, and differentiated under identical conditions. (J Histochem Cytochem 57:405–423, 2009

The Necrosis-Avid Small Molecule HQ4-DTPA as a Multimodal Imaging Agent for Monitoring Radiation Therapy-Induced Tumor Cell Death

Purpose: Most effective antitumor therapies induce tumor cell death. Non-invasive, rapid and accurate quantitative imaging of cell death is essential for monitoring early response to antitumor therapies. To facilitate this, we previously developed a biocompatible necrosis-avid near-infrared fluorescence (NIRF) imaging probe, HQ4, which was radiolabeled with "'Indium-chloride el In-C13) via the chelate diethylene triamine pentaacetic acid (DTPA), to enable clinical translation. The aim of the present study was to evaluate the application of HQ4-DTPA for monitoring tumor cell death induced by radiation therapy. Apart from its NIRF and radioactive properties, HQ4-DTPA was also tested as a photoacoustic imaging probe to evaluate its performance as a multimodal contrast agent for superficial and deep tissue imaging. Materials and methods: Radiation-induced tumor cell death was examined in a xenograft mouse model of human breast cancer (MCF-7). Tumors were irradiated with three fractions of 9 Gy each. HQ4-DTPA was injected intravenously after the last irradiation, NIRF and photoacoustic imaging of the tumors were performed at 12, 20, and 40 h after injection. Changes in probe accumulation in the tumors were measured in vivo, and ex vivo histological analysis of excised tumors was performed at experimental endpoints. In addition, biodistribution of radiolabeled [In]DTPA-HQ4 was assessed using hybrid single-photon emission computed tomography computed tomography (SPECT CT) at the same time points. Results: In vivo NIRF imaging demonstrated a significant difference in probe accumulation between control and irradiated tumors at all time points after injection. A similar trend was observed using in vivo photoacoustic imaging, which was validated by ex vivo tissue fluorescence and photoacoustic imaging. Serial quantitative radioactivity measurements of probe biodistribution further demonstrated increased probe accumulation in irradiated tumors. Conclusion: HQ4-DTPA has high specificity for dead cells in vivo, potentiating its use as a contrast agent for determining the relative level of tumor cell death following radiation therapy using NIRF, photoacoustic imaging and SPECT in vivo. Initial preclinical results are promising and indicate the need for further evaluation in larger cohorts. If successful, such studies may help develop a new multimodal method for non-invasive and dynamic deep tissue imaging of treatment-induced cell death to quantitatively assess therapeutic response in patients

The necrosis-avid small molecule HQ4-DTPA as a multimodal imaging agent for monitoring radiation therapy-induced tumor cell death

textabstractPurpose: Most effective antitumor therapies induce tumor cell death. Non-invasive, rapid and accurate quantitative imaging of cell death is essential for monitoring early response to antitumor therapies. To facilitate this, we previously developed a biocompatible necrosis-avid near-infrared fluorescence (NIRF) imaging probe, HQ4, which was radiolabeled with 111Indium-chloride (111In-Cl3) via the chelate diethylene triamine pentaacetic acid (DTPA), to enable clinical translation. The aim of the present study was to evaluate the application of HQ4-DTPA for monitoring tumor cell death induced by radiation therapy. Apart from its NIRF and radioactive properties, HQ4-DTPA was also tested as a photoacoustic imaging probe to evaluate its performance as a multimodal contrast agent for superficial and deep tissue imaging. Materials and methods: Radiation-induced tumor cell death was examined in a xenograft mouse model of human breast cancer (MCF-7). Tumors were irradiated with three fractions of 9 Gy each. HQ4-DTPA was injected intravenously after the last irradiation, NIRF and photoacoustic imaging of the tumors were performed at 12, 20, and 40 h after injection. Changes in probe accumulation in the tumors were measured in vivo, and ex vivo histological analysis of excised tumors was performed at experimental endpoints. In addition, biodistribution of radiolabeled [111In]DTPA-HQ4 was assessed using hybrid single-photon emission computed tomography-computed tomography (SPECT-CT) at the same time points. Results: In vivo NIRF imagin

CONSORT Flow diagram for Part 1 of clinical study.

<p>Flow diagram of the progress through the phases of enrollment, allocation, follow-up, and data analysis for Part 1 of the clinical study.</p

Quantitative longitudinal tracking of bacterial load in chronic wounds.

<p><i>A</i>. Sequential white light (<i>top row</i>) and AF images (<i>middle row</i>) of a non-healing diabetic foot ulcer in a 67 y old female performed over 5.5 months. AF images revealed bluish-green fluorescence within the wound bed and bright red bacteria around the wound periphery. A fluorescence intensity-based segmentation algorithm was used to quantify bacterial load changes over time (<i>bottom row</i>), with the bacteria false-colored and overlaid on original AF images of the middle row. <i>B</i>. Quantitative changes in bacterial load over time measured by relative bacterial fluorescence amount (total red AF area in cm²) indicate clinically-significant microbial load in the wound periphery, both of which are missed during conventional clinical examination. <i>Scale bar</i>: <i>A</i>. ~<i>1 cm</i>.</p

Photographs of the handheld prototype PRODIGI imaging device and its use for real-time autofluorescence imaging of bacterial load invisible by white light examination.

<p><i>A</i>. Front view of PRODIGI showing wound fluorescence image displayed in real-time on the LCD screen in high definition. <i>B</i>. Back view of PRODIGI showing white light and 405 nm LED arrays providing illumination of the wound, while the fluorescence mission filter is placed in front of the CCD sensor. Inset shows side profile of the device. <i>C-E</i>. Photograph of PRODIGI device used to examine a diabetic foot ulcer with room lights on, in a hard shell carrying case in a typical wound clinic setting, and placed on typical wound care cart, respectively. Room lights are turned off for fluorescence imaging. <i>F</i>. PRODIGI white light image of type II diabetic foot ulcer in a 52 y old male patient. <i>G</i>. Corresponding AF image taken in < 1 sec showing bright red fluorescence of pathogenic bacteria in the wound periphery (yellow arrow) and in ‘off site’ areas (white arrow) away from the primary wound (confirmed by swab microbiology as mainly heavy growth S. aureus). Bacterial fluorescence appears red against a background of green fluorescence from connective tissues of the healthy skin, which provides anatomical context for localizing the bacteria within and around the wound. The bacterial regions were not seen under white-light visualization. <i>H</i>. A magnified view of <i>G</i>. showing S. aureus growing within the fissures of the wound periphery. Bright fluorescent ‘hot spots’ (yellow arrow) illustrate heterogeneity in the distribution of bioburden in the wound periphery. Fluorescence imaging allowed targeted swabbing of bacterial areas not possible by white light visualization. The heavy growth S. aureus growing in the off-site area was invisible by traditional clinical examination. <i>Scale bars</i>: <i>A</i>. <i>2 cm</i>, <i>B</i>. <i>2 cm</i>, <i>C</i>. <i>1 cm</i>.</p

Autofluorescence detection of clinically-significant bacterial load in wound periphery and off-site areas.

<p><i>A</i>. White light image of a type II diabetic foot ulcer in a 78 y old female. <i>B</i>. Corresponding AF image showing heavy growth of S. aureus in the wound periphery missed by white light imaging. <i>C</i>. White light shows unremarkable areas between toes, while in <i>D</i>. the corresponding AF imaging detected bacterial biofilm, confirmed by microbiology. <i>E</i>. Schematic illustrates different wound locations where fluorescence imaging detected clinically significant bacterial load. <i>F</i>. Comparison of accuracy for correctly detecting clinically-significant bioburden between standard WL and AF imaging in wound bed, wound periphery and off-site areas. <i>Scale bars</i>: <i>A</i>,<i>B</i>. <i>1 cm; C</i>. <i>2 cm</i>, <i>D</i>. <i>1 cm</i>.</p

Wound swabs taken under autofluorescence imaging guidance, indicating detected polymicrobial species prevalence.

<p>The percentage of bacteria for each species are plotted for <i>A</i>. all swabs, <i>B</i>. swabs obtained from the wound bed, <i>C</i>. from the wound periphery and <i>D</i>. ‘off-site’ areas away from the primary wound.</p