In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies ^1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays ⁴. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor ^4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells ^7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.     

TEP/TDM au fluorure (18F) de sodium pour la détection des métastases osseuses du cancer de la prostate. Description de l’étude Fluprostic de comparaison de la TEP/TDM au fluorure (18F) de sodium à l’IRM corps entier dans cette indication

Fluorodeoxyglucose (¹⁸F) or FDG, the radioactive glucose analogue which is the reference radiopharmaceutical in oncologic PET, is not well suited for the detection of prostate cancer metastases the glucose metabolism of which is usually only slightly enhanced. Fluoride (¹⁸F) accumulates into the cortical bone, rapidly and intensely in reaction to a bony metastasis. In 2008, it has been granted a marketing authorisation in France, including imaging bone metastasis of prostate cancer. We report original clinical cases to illustrate its diagnostic performance. Whole-body MRI is developing and can also detect bone metastases. Recently diffusion-weighted MRI (DWI) has been proposed to increase the detection rate of metastases of the axial skeleton, which are largely predominant in prostate cancer. Using either hybrid PET/CT or MRI requires mobilising equipments, which are less available and more expensive than the gamma-cameras for classical bone scintigraphy, in the aim to achieve superior diagnostic performance. A clinical study protocol (STIC) has just been accepted for public funding. It aims to assess the impact on patient management of the discovery of the first macroscopic bony metastasis and the efficacy of diagnostic strategies including those innovations, individually and in association. In case of prostate cancer with a high risk of metastasis, but without any proven bone metastasis and no typical pattern on bone scintigraphy, fluoride (¹⁸F) PET/CT will be performed as well as whole-body MRI. Histopathology and/or data of a 6-month follow-up will be the standard of truth to evaluate the adequacy of impact on patient management and the benefit / cost ratio of those examinations. With this prospective national study, we hope to demonstrate in the real world a clinical role for this radiopharmaceutical, which was proposed several decades ago, but benefits from a renewed interest thanks to the development of PET/CT imaging.     

Angiostatin inhibits bone metastasis formation in nude mice through a direct anti-osteoclastic activity

Bone is a very common metastatic site for breast cancer. In bone metastasis, there is a vicious circle wherein bone-residing metastatic cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone promote tumor growth. The contribution of tumor angiogenesis in the growth of bone metastases is, however, unknown. By using an experimental model of bone metastasis caused by MDA-MB-231/B02 breast cancer cells that quite closely mimics the conditions likely to occur in naturally arising metastatic human breast cancers, we demonstrate here that when MDA-MB-231/B02 cells were engineered to produce at the bone metastatic site an angiogenesis inhibitor, angiostatin, there was a marked inhibition in the extent of skeletal lesions. Inhibition of skeletal lesions came with a pronounced reduction in tumor burden in bone. However, although angiostatin produced by MDA-MB-231/B02 cells was effective at inhibiting in vitro endothelial cell proliferation and in vivo angiogenesis in a Matrigel implant model, we have shown that it inhibited cancer-induced bone destruction through a direct inhibition of osteoclast activity and generation. Overall, these results indicate that, besides its well known anti-angiogenic activity, angiostatin must also be considered as a very effective inhibitor of bone resorption, broadening its potential clinical use in cancer therapy.     

Imaging Axl expression in pancreatic and prostate cancer xenografts

The receptor tyrosine kinase Axl is overexpressed in and leads to patient morbidity and mortality in a variety of cancers. Axl–Gas6 interactions are critical for tumor growth, angiogenesis and metastasis. The goal of this study was to investigate the feasibility of imaging graded levels of Axl expression in tumors using a radiolabeled antibody. We radiolabeled anti-human Axl (Axl mAb) and control IgG1 antibodies with ¹²⁵I with high specific radioactivity and radiochemical purity, resulting in an immunoreactive fraction suitable for in vivo studies. Radiolabeled antibodies were investigated in severe combined immunodeficient mice harboring subcutaneous CFPAC (Axl^high) and Panc1 (Axl^low) pancreatic cancer xenografts by ex vivo biodistribution and imaging. Based on these results, the specificity of [¹²⁵I]Axl mAb was also validated in mice harboring orthotopic Panc1 or CFPAC tumors and in mice harboring subcutaneous 22Rv1 (Axl^low) or DU145 (Axl^high) prostate tumors by ex vivo biodistribution and imaging studies at 72 h post-injection of the antibody. Both imaging and biodistribution studies demonstrated specific and persistent accumulation of [¹²⁵I]Axl mAb in Axl^high (CFPAC and DU145) expression tumors compared to the Axl^low (Panc1 and 22Rv1) expression tumors. Axl expression in these tumors was further confirmed by immunohistochemical studies. No difference in the uptake of radioactivity was observed between the control [¹²⁵I]IgG1 antibody in the Axl^high and Axl^low expression tumors. These data demonstrate the feasibility of imaging Axl expression in pancreatic and prostate tumor xenografts.     

TEP-TDM au FNa et IRM avec séquences de diffusion dans la détection des métastases osseuses : étude comparative prospective par lésion

IntroductionThe development of technologies aimed to detect bone metastases in nuclear medicine and radiology prompts us to compare their performance in their most effective form: positron emission tomography (PET) with NaF combined with computed tomography (CT) and magnetic resonance imaging (MRI) using anatomical and diffusion weighted sequences (T1-STIR, and DWI MRI), as well as to study several factors involved in the visualization of these lesions (anatomo-functional correlation, nature, size and localization).Materials and methodsThirteen patients underwent NaF PET-CT and T1-STIR-DWI MRI in a prospective study. One hundred and sixty-four lesions were found. For each, a malignancy score of 1 to 5 was assigned. Expert consensus and follow-up data for each patient, available in their medical records, determined the final diagnosis as gold standard.ResultsThe sensitivities, specificities, precision and AUC of the lesion-based analysis were respectively 78.3%, 93.8%, 89.3% and 0.85 for NaF PET-CT and 60.9%, 97.4%, 86.7% and 0.81 for T1-STIR-DWI MRI. Results were independent on the nature, size or location of the metastases. A significant change in AUC when CT was combined with PET, and when DWI was combined with T1-STIR was found for one of the two observers in both fields.ConclusionThe performance of PET-CT at NaF and T1-STIR-DWI MRI appeared equivalent. The combination of functional and morphological images is beneficial in both fields (nuclear and magnetic resonance) but its contribution varies depending on the observer.     

Integrated Multimodal Imaging of Dynamic Bone-Tumor Alterations Associated with Metastatic Prostate Cancer

Bone metastasis occurs for men with advanced prostate cancer which promotes osseous growth and destruction driven by alterations in osteoblast and osteoclast homeostasis. Patients can experience pain, spontaneous fractures and morbidity eroding overall quality of life. The complex and dynamic cellular interactions within the bone microenvironment limit current treatment options thus prostate to bone metastases remains incurable. This study uses voxel-based analysis of diffusion-weighted MRI and CT scans to simultaneously evaluate temporal changes in normal bone homeostasis along with prostate bone metatastsis to deliver an improved understanding of the spatiotemporal local microenvironment. Dynamic tumor-stromal interactions were assessed during treatment in mouse models along with a pilot prospective clinical trial with metastatic hormone sensitive and castration resistant prostate cancer patients with bone metastases. Longitudinal changes in tumor and bone imaging metrics during delivery of therapy were quantified. Studies revealed that voxel-based parametric response maps (PRM) of DW-MRI and CT scans could be used to quantify and spatially visualize dynamic changes during prostate tumor growth and in response to treatment thereby distinguishing patients with stable disease from those with progressive disease (p<0.05). These studies suggest that PRM imaging biomarkers are useful for detection of the impact of prostate tumor-stromal responses to therapies thus demonstrating the potential of multi-modal PRM image-based biomarkers as a novel means for assessing dynamic alterations associated with metastatic prostate cancer. These results establish an integrated and clinically translatable approach which can be readily implemented for improving the clinical management of patients with metastatic bone disease.

Trial Registration

ClinicalTrials.gov NCT02064283     

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis ¹. In some cancers (e.g. glioma ²), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality ³. It has been estimated that invasion and metastasis are responsible for 90% of cancer deaths ⁴. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment ⁵.A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling ⁶. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes ⁷. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer ⁸.We present a refined method ⁹ for examining the contributions of candidate proteins to collective invasion ¹⁰. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.     

Comparison of 99mTc-3PRGD2 Integrin Receptor Imaging with 99mTc-MDP Bone Scan in Diagnosis of Bone Metastasis in Patients with Lung Cancer: A Multicenter Study

Purpose

^99mTc-3PRGD2, a promising tracer targeting integrin receptor, may serve as a novel tumor-specific agent for single photon emission computed tomography (SPECT). A multi-center study was prospectively designed to evaluate the diagnostic accuracy of ^99mTc-3PRGD2 imaging for bone metastasis in patients with lung cancer in comparison with the conventional ^99mTc-MDP bone scan.

Methods

The patients underwent whole-body scan and chest tomography successively at both 1 h and 4 h after intravenous injection of 11.1 MBq/Kg ^99mTc-3PRGD2. ^99mTc-MDP whole-body bone scan was routinely performed within 1 week for comparison. Three experienced nuclear medicine physicians blindly read the ^99mTc-3PRGD2 and ^99mTc-MDP images. The final diagnosis was established based on the comprehensive assessment of all available data.

Results

A total of 44 patients (29 male, 59±10 years old) with suspected lung cancer were recruited from 4 centers. Eighty-nine bone lesions in 18 patients were diagnosed as metastases and 23 bone lesions in 9 patients were benign. In a lesion-based analysis, ^99mTc-3PRGD2 imaging demonstrated a sensitivity, specificity, and accuracy of 92.1%, 91.3%, and 92.0%, respectively. The corresponding diagnostic values for ^99mTc-MDP bone scan were 87.6%, 60.9%, and 82.1%, respectively in the same patients. ^99mTc-MDP bone scan had better contrast in most lesions, whereas the ^99mTc-3PRGD2 imaging seemed to be more effective to exclude pseudo-positive lesions and detect bone metastases without osteogenesis.

Conclusion

^99mTc-3PRGD2 is a novel tumor-specific agent based on SPECT technology with a promising value in diagnosis of bone metastasis in patients with lung cancer.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01737112","term_id":"NCT01737112"}}NCT01737112     

Efficiency of Non-Contrast-Enhanced Liver Imaging Sequences Added to Initial Rectal MRI in Rectal Cancer Patients

Purpose

The purpose of this study was to estimate the value of addition of liver imaging to initial rectal magnetic resonance imaging (MRI) for detection of liver metastasis and evaluate imaging predictors of a high risk of liver metastasis on rectal MRI.

Methods

We enrolled 144 patients who from October 2010 to May 2013 underwent rectal MRI with T2-weighted imaging (T2WI) and diffusion-weighted imaging (DWI) (b values = 50, 500, and 900 s/mm²) of the liver and abdominopelvic computed tomography (APCT) for the initial staging of rectal cancer. Two reviewers scored the possibility of liver metastasis on different sets of liver images (T2WI, DWI, and combined T2WI and DWI) and APCT and reached a conclusion by consensus for different analytic results. Imaging features from rectal MRI were also analyzed. The diagnostic performances of CT and an additional liver scan to detect liver metastasis were compared. Multivariate logistic regression to determine independent predictors of liver metastasis among rectal MRI features and tumor markers was performed. This retrospective study was approved by the Institutional Review Board, and the requirement for informed consent was waived.

Results

All sets of liver images were more effective than APCT for detecting liver metastasis, and DWI was the most effective. Perivascular stranding and anal sphincter invasion were statistically significant for liver metastasis (p = 0.0077 and p = 0.0471), while extramural vascular invasion based on MRI (mrEMVI) was marginally significant (p = 0.0534).

Conclusion

The addition of non-contrast-enhanced liver imaging, particularly DWI, to initial rectal MRI in rectal cancer patients could facilitate detection of liver metastasis without APCT. Perivascular stranding, anal sphincter invasion, and mrEMVI detected on rectal MRI were important imaging predictors of liver metastasis.     

In Vivo Quantification of Inflammation in Experimental Autoimmune Encephalomyelitis Rats Using Fluorine-19 Magnetic Resonance Imaging Reveals Immune Cell Recruitment outside the Nervous System

Progress in identifying new therapies for multiple sclerosis (MS) can be accelerated by using imaging biomarkers of disease progression or abatement in model systems. In this study, we evaluate the ability to noninvasively image and quantitate disease pathology using emerging “hot-spot” ¹⁹F MRI methods in an experimental autoimmune encephalomyelitis (EAE) rat, a model of MS. Rats with clinical symptoms of EAE were compared to control rats without EAE, as well as to EAE rats that received daily prophylactic treatments with cyclophosphamide. Perfluorocarbon (PFC) nanoemulsion was injected intravenously, which labels predominately monocytes and macrophages in situ. Analysis of the spin-density weighted ¹⁹F MRI data enabled quantification of the apparent macrophage burden in the central nervous system and other tissues. The in vivo MRI results were confirmed by extremely high-resolution ¹⁹F/¹H magnetic resonance microscopy in excised tissue samples and histopathologic analyses. Additionally, ¹⁹F nuclear magnetic resonance spectroscopy of intact tissue samples was used to assay the PFC biodistribution in EAE and control rats. In vivo hot-spot ¹⁹F signals were detected predominantly in the EAE spinal cord, consistent with the presence of inflammatory infiltrates. Surprising, prominent ¹⁹F hot-spots were observed in bone-marrow cavities adjacent to spinal cord lesions; these were not observed in control animals. Quantitative evaluation of cohorts receiving cyclophosphamide treatment displayed significant reduction in ¹⁹F signal within the spinal cord and bone marrow of EAE rats. Overall, ¹⁹F MRI can be used to quantitatively monitored EAE disease burden, discover unexpected sites of inflammatory activity, and may serve as a sensitive biomarker for the discovery and preclinical assessment of novel MS therapeutic interventions.     

Longitudinal Imaging Studies of Tumor Microenvironment in Mice Treated with the mTOR Inhibitor Rapamycin

Rapamycin is an allosteric inhibitor of mammalian target of rapamycin, and inhibits tumor growth and angiogenesis. Recent studies suggested a possibility that rapamycin renormalizes aberrant tumor vasculature and improves tumor oxygenation. The longitudinal effects of rapamycin on angiogenesis and tumor oxygenation were evaluated in murine squamous cell carcinoma (SCCVII) by electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) to identify an optimal time after rapamycin treatment for enhanced tumor radioresponse. Rapamycin treatment was initiated on SCCVII solid tumors 8 days after implantation (500–750 mm³) and measurements of tumor pO₂ and blood volume were conducted from day 8 to 14 by EPRI/MRI. Microvessel density was evaluated over the same time period by immunohistochemical analysis. Tumor blood volume as measured by MRI significantly decreased 2 days after rapamycin treatment. Tumor pO₂ levels modestly but significantly increased 2 days after rapamycin treatment; whereas, it decreased in non-treated control tumors. Furthermore, the fraction of hypoxic area (pixels with pO₂<10 mm Hg) in the tumor region decreased 2 days after rapamycin treatments. Immunohistochemical analysis of tumor microvessel density and pericyte coverage revealed that microvessel density decreased 2 days after rapamycin treatment, but pericyte coverage did not change, similar to what was seen with anti-angiogenic agents such as sunitinib which cause vascular renormalization. Collectively, EPRI/MRI co-imaging can provide non-invasive evidence of rapamycin-induced vascular renormalization and resultant transient increase in tumor oxygenation. Improved oxygenation by rapamycin treatment provides a temporal window for anti-cancer therapies to realize enhanced response to radiotherapy.     

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 µm in diameter on T₂-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.     

Multimodal Imaging of Orthotopic Mouse Model of Endometrial Carcinoma

Background

Orthotopic endometrial cancer models provide a unique tool for studies of tumour growth and metastatic spread. Novel preclinical imaging methods also have the potential to quantify functional tumour characteristics in vivo, with potential relevance for monitoring response to therapy.

Methods

After orthotopic injection with luc-expressing endometrial cancer cells, eleven mice developed disease detected by weekly bioluminescence imaging (BLI). In parallel the same mice underwent positron emission tomography–computed tomography (PET-CT) and magnetic resonance imaging (MRI) employing ¹⁸F-fluorodeoxyglocose (¹⁸F-FDG) or ¹⁸F- fluorothymidine (¹⁸F-FLT) and contrast reagent, respectively. The mice were sacrificed when moribund, and post-mortem examination included macroscopic and microscopic examination for validation of growth of primary uterine tumours and metastases. PET-CT was also performed on a patient derived model (PDX) generated from a patient with grade 3 endometrioid endometrial cancer.

Results

Increased BLI signal during tumour growth was accompanied by increasing metabolic tumour volume (MTV) and increasing MTV x mean standard uptake value of the tumour (SUV_mean) in ¹⁸F-FDG and ¹⁸F-FLT PET-CT, and MRI conspicuously depicted the uterine tumour. At necropsy 82% (9/11) of the mice developed metastases detected by the applied imaging methods. ¹⁸F-FDG PET proved to be a good imaging method for detection of patient derived tumour tissue.

Conclusions

We demonstrate that all imaging modalities enable monitoring of tumour growth and metastatic spread in an orthotopic mouse model of endometrial carcinoma. Both PET tracers, ¹⁸F-FDG and ¹⁸F-FLT, appear to be equally feasible for detecting tumour development and represent, together with MRI, promising imaging tools for monitoring of patient-derived xenograft (PDX) cancer models.     

Primed Infusion with Delayed Equilibrium of Gd.DTPA for Enhanced Imaging of Small Pulmonary Metastases

Objectives

To use primed infusions of the magnetic resonance imaging (MRI) contrast agent Gd.DTPA (Magnevist), to achieve an equilibrium between blood and tissue (eqMRI). This may increase tumor Gd concentrations as a novel cancer imaging methodology for the enhancement of small tumor nodules within the low signal-to-noise background of the lung.

Methods

A primed infusion with a delay before equilibrium (eqMRI) of the Gd(III) chelator Gd.DTPA, via the intraperitoneal route, was used to evaluate gadolinium tumor enhancement as a function of a bolus injection, which is applied routinely in the clinic, compared to gadolinium maintained at equilibrium. A double gated (respiration and cardiac) spin-echo sequence at 9.4T was used to evaluate whole lungs pre contrast and then at 15 (representative of bolus enhancement), 25 and 35 minutes (representative of eqMRI). This was carried out in two lung metastasis models representative of high and low tumor cell seeding. Lungs containing discrete tumor nodes where inflation fixed and taken for haematoxylin and eosin staining as well as CD34 staining for correlation to MRI.

Results

We demonstrate that sustained Gd enhancement, afforded by Gd equilibrium, increases the detection of pulmonary metastases compared to bolus enhancement and those tumors which enhance at equilibrium are sub-millimetre in size (<0.7 mm²) with a similar morphology to early bronchoalveolar cell carcinomas.

Conclusion

As Gd-chelates are routinely used in the clinic for detecting tumors by MRI, this methodology is readily transferable to the clinic and advances MRI as a methodology for the detection of small pulmonary tumors.     

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death ¹. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection ^2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature ⁴.Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin ^5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology ^7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin ⁹, allowed breakthroughs in the field.In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma ^10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.     

Functional imaging of angiogenesis in an orthotopic model of pancreatic cancer

Pancreatic cancer is a major unsolved health problem. The estimated overall 5-year survival rate of only 1-4% is due to aggressiveness of the disease and the lack of effective systemic therapies. Most pancreatic cancer-related deaths are due to the development of metastases, which represents the culmination of a complex interaction between the host organism and neoplastic cells within the primary tumor. Therefore, the study of tumor-host interaction in the context of the whole organism is necessary to evaluate the pathogenesis of tumor growth and metastasis so that effective therapies can be developed. Recent advances in functional imaging combined with animal models that faithfully recreate the biology of human tumors have elevated our ability to examine these complex interactions. In this review, we will use the example of orthotopic mouse models of pancreatic cancer as a tool to survey the challenges and possibilities of functional imaging of angiogenesis, a critical determinant of metastasis.     

Superparamagnetic iron oxide-enhanced MRI-guided stereotactic ablative radiation therapy for liver metastasis

BackgroundMRI-guided radiation therapy can image a target and irradiate it at the same time. Superparamagnetic iron oxide (SPIO) is a liver-specific contrast agent that can selectively visualize liver tumors, even if plain MRI does not depict them. The purpose of this study was to present a proof of concept of SPIO-enhanced MRI-guided radiation therapy for liver tumor.Case presentationMRI-guided stereotactic ablative radiation therapy (SABR) was planned for a patient with impaired renal function who developed liver metastases after nephroureterectomy for ureteral cancer. Because liver metastasis was not visualized on plain MRI, SPIO-enhanced MRI was performed at 0.35 T using true fast imaging with steady-state free precession (true FISP) pulse sequence and SABR was performed. Liver metastasis was clearly visualized by SPIO-enhanced MRI, and MRI-guided SABR was performed without adverse events.ConclusionEven if liver metastasis is not visualized by plain MRI, liver metastasis can be clearly depicted by administering SPIO, and MRI-guided radiation therapy can be performed.     

Multimodality Imaging of Tumor and Bone Response in a Mouse Model of Bony Metastasis

Cancer drug development generally performs in vivo evaluation of treatment effects that have traditionally relied on detection of morphologic changes. The emergence of new targeted therapies, which may not result in gross morphologic changes, has spurred investigation into more specific imaging methods to quantify response, such as targeted fluorescent probes and bioluminescent cells. The present study investigated tissue response to docetaxel or zoledronic acid (ZA) in a mouse model of bony metastasis. Intratibial implantations of breast cancer cells (MDA-MB-231) were monitored throughout this study using several modalities: molecular resonance imaging (MRI) tumor volume and apparent diffusion coefficient (ADC), micro-computed tomography (µCT) bone volume, bioluminescence imaging (BLI) reporting cancer cell apoptosis, and fluorescence using Osteosense 800 and CatK 680-FAST. Docetaxel treatment resulted in tumor cell kill reflected by ADC and BLI increases and tumor volume reduction, with delayed bone recovery seen in µCT prefaced by increased osteoblastic activity (Osteosense 800). In contrast, the ZA treatment group produced similar values in MRI, BLI, and Osteosense 800 fluorescence imaging readouts when compared to controls. However, µCT bone volume increased significantly by the first week post-treatment and the CatK 680-FAST signal was slightly diminished by 4 weeks following ZA treatment. Multimodality imaging provides a more comprehensive tool for new drug evaluation and efficacy screening through identification of morphology as well as function and apoptotic signaling.     

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level^1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) ⁶. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) ^{7, 8, 9,10,11}. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor^7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect ^{7, 10,11}. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.