Magnetic microspheres can be used for magnetic particle imaging of cancer cells arrested in the mouse brain.

PURPOSE: Magnetic particle imaging (MPI) is a new imaging modality that sensitively and specifically detects superparamagnetic iron oxide nanoparticles (SPIOs). MRI cell tracking with SPIOs has very high sensitivity, but low specificity and quantification is difficult. MPI could overcome these limitations. There are no reports of micron-sized iron oxide particles (MPIO) for cell tracking by MPI. Therefore, the goal was to evaluate if MPIO can be used for in vivo detection and quantification of cancer cells distributed in the mouse brain by MPI. METHODS: In the first experiment mice were injected with either 2.5 × 105 or 5.0 × 105 MPIO-labeled cancer cells and MPI was performed ex vivo. In a second experiment, mice received either 2.5 × 105 or 5.0 × 104 MPIO-labeled cells and MPI was performed in vivo. In a third experiment, mice were injected with 5.0 × 104 cells, labeled with either MPIO or ferucarbotran, and MPI was performed in vivo. RESULTS: MPIO-labeled cells were visible in all MPI images of the mouse brain. The MPI signal and iron content measurements were greater for brains of mice that were injected with higher numbers of MPIO-labeled cells. Ferucarbotran-labeled cells were not detected in the brain by MPI. CONCLUSION: This is the first example of the use of MPIO for cell tracking with MPI. With an intracardiac cell injection, ~15% of cells will arrest in the brain vasculature. For our lowest cell injection of 5.0 × 104 cells, this was ~10 000 cells, distributed throughout the brain.

Cranial irradiation increases tumor growth in experimental breast cancer brain metastasis.

Whole-brain radiotherapy is the standard of care for patients with breast cancer with multiple brain metastases and, although this treatment has been essential in the management of existing brain tumors, there are many known negative consequences associated with the irradiation of normal brain tissue. In our study, we used in vivo magnetic resonance imaging analysis to investigate the influence of radiotherapy-induced damage of healthy brain on the arrest and growth of metastatic breast cancer cells in a mouse model of breast cancer brain metastasis. We observed that irradiated, but otherwise healthy, neural tissue had an increased propensity to support metastatic growth compared with never-irradiated controls. The elucidation of the impact of irradiation on normal neural tissue could have implications in clinical patient management, particularly in patients with residual systemic disease or with residual radio-resistant brain cancer.

Application of dual 19 F and iron cellular MRI agents to track the infiltration of immune cells to the site of a rejected stem cell transplant.

PURPOSE: Cellular MRI) was used to detect implanted human mesenchymal stem cells (hMSCs) and the resulting macrophage infiltration that occurs in response to xenotransplantation. METHODS: Human mesenchymal stem cells were prelabeled with a fluorine-19 (19 F) agent prior to implantation, allowing for their visualization and quantification over time. Following implantation of 1 × 10619 F-labeled hMSCs into the mouse hind limb, longitudinal imaging was performed to monitor the stem cell graft. Macrophages were labeled in situ by the intravenous administration of an ultrasmall superparamagentic iron oxide (USPIO), allowing for tracking of the inflammatory response. RESULTS: Quantification of 19 F MRI on day 0 agreed with the implanted number of cells, and 19 F signal decreased over time. By day 14, only 22% ± 11% of the original 19 F signal remained. In a second group, USPIO were administered intravenously after implantation of 19 F-labeled hMSCs. When imaged on day 2, a significant decrease in 19 F signal was observed compared to the first group alongside a large signal void region in the corresponding proton images. Immunohistochemistry confirmed the presence of iron-labeled macrophages in the stem cell tract. CONCLUSION: A dual-labeling technique was used to noninvasively track two distinct cell populations simultaneously. This information could be used to provide additional insight into the cause of graft failure. Magn Reson Med 78:713-720, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

MRI and histopathologic study of a novel cholesterol-fed rabbit model of xanthogranuloma.

PURPOSE: To develop a rabbit model of xanthogranuloma based on supplementation of dietary cholesterol. The aim of this study was to analyze the xanthogranulomatous lesions using magnetic resonance imaging (MRI) and histological examination. MATERIALS AND METHODS: Rabbits were fed a low-level cholesterol (CH) diet (n = 10) or normal chow (n = 5) for 24 months. In vivo brain imaging was performed on a 3T MR system using fast imaging employing steady state acquisition, susceptibility-weighted imaging, spoiled gradient recalled, T1 -weighted inversion recovery imaging and T1 relaxometry, PD-weighted and T2 -weighted spin-echo imaging and T2 relaxometry, iterative decomposition of water and fat with echo asymmetry and least-squares estimation, ultrashort TE MRI (UTE-MRI), and T2* relaxometry. MR images were evaluated using a Likert scale for lesion presence and quantitative analysis of lesion size, ventricular volume, and T1 , T2 , and T2* values of lesions was performed. After imaging, brain specimens were examined using histological methods. RESULTS: In vivo MRI revealed that 6 of 10 CH-fed rabbits developed lesions in the choroid plexus. Region-of-interest analysis showed that for CH-fed rabbits the mean lesion volume was 8.5 ± 2.6 mm(3) and the volume of the lateral ventricle was significantly increased compared to controls (P < 0.01). The lesions showed significantly shorter mean T2 values (35 ± 12 msec, P < 0.001), longer mean T1 values (1581 ± 146 msec, P < 0.05), and shorter T2* values (22 ± 13 msec, P < 0.001) compared to adjacent brain structures. The ultrashort T2* components were visible using UTE-MRI. Histopathologic evaluation of lesions demonstrated features of human xanthogranuloma. CONCLUSION: Rabbits fed a low-level CH diet develop sizable intraventricular masses that have similar histopathological features as human xanthogranuloma. Multiparametric MRI techniques were able to provide information about the complex composition of these lesions. J. Magn. Reson. Imaging 2016;44:673-682.

Molecular Imaging Reveals a High Degree of Cross-Seeding of Spontaneous Metastases in a Novel Mouse Model of Synchronous Bilateral Breast Cancer.

PURPOSE: Synchronous bilateral breast cancer (SBBC) patients present with cancer in both breasts at the time of diagnosis or within a short time interval. They show higher rates of metastasis and lower overall survival compared to women with unilateral breast cancer. Here we established the first preclinical SBBC model and used molecular imaging to visualize the patterns of metastasis from each primary tumor. PROCEDURES: We engineered human breast cancer cells to express either Akaluc or Antares2 for bioluminescence imaging (BLI) and tdTomato or zsGreen for ex vivo fluorescence microscopy. Both cell populations were implanted into contralateral mammary fat pads of mice (n=10), and dual-BLI was performed weekly for up to day 29 (n=3), 38 (n=4), or 42 (n=3). Primary tumors and lungs were fixed, and ex vivo fluorescence microscopy was used to analyze the cellular makeup of micrometastases. RESULTS: Signal from both Antares2 and Akaluc was first detected in the lungs on day 28 and was present in 9 of 10 mice at endpoint. Ex vivo fluorescence microscopy of the lungs revealed that for mice sacrificed on day 38, a significant percentage of micrometastases were composed of cancer cells from both primary tumors (mean 37%; range 27 to 45%), while two mice sacrificed on day 42 showed percentages of 51% and 70%. CONCLUSIONS: A high degree of metastatic cross-seeding of cancer cells derived from bilateral tumors may contribute to faster metastatic growth and intratumoral heterogeneity. We posit that our work will help understand treatment resistance and optimal planning of SBBC treatment.

Modular cell-assembled adipose matrix-derived bead foams as a mesenchymal stromal cell delivery platform for soft tissue regeneration.

With the goal of establishing a new clinically-relevant bioscaffold format to enable the delivery of high densities of human adipose-derived stromal cells (ASCs) for applications in soft tissue regeneration, a novel "cell-assembly" method was developed to generate robust 3-D scaffolds comprised of fused networks of decellularized adipose tissue (DAT)-derived beads. In vitro studies confirmed that the assembly process was mediated by remodelling of the extracellular matrix by the seeded ASCs, which were well distributed throughout the scaffolds and remained highly viable after 8 days in culture. The ASC density, sulphated glycosaminoglycan content and scaffold stability were enhanced under culture conditions that included growth factor preconditioning. In vivo testing was performed to compare ASCs delivered within the cell-assembled DAT bead foams to an equivalent number of ASCs delivered on a previously-established pre-assembled DAT bead foam platform in a subcutaneous implant model in athymic nude mice. Scaffolds were fabricated with human ASCs engineered to stably co-express firefly luciferase and tdTomato to enable long-term cell tracking. Longitudinal bioluminescence imaging showed a significantly stronger signal associated with viable human ASCs at timepoints up to 7 days in the cell-assembled scaffolds, although both implant groups were found to retain similar densities of human ASCs at 28 days. Notably, the infiltration of CD31+ murine endothelial cells was enhanced in the cell-assembled implants at 28 days. Moreover, microcomputed tomography angiography revealed that there was a marked reduction in vascular permeability in the cell-assembled group, indicating that the developing vascular network was more stable in the new scaffold format. Overall, the novel cell-assembled DAT bead foams represent a promising platform to harness the pro-regenerative paracrine functionality of human ASCs and warrant further investigation as a clinically-translational approach for volume augmentation.

Safe harbor-targeted CRISPR-Cas9 homology-independent targeted integration for multimodality reporter gene-based cell tracking.

Imaging reporter genes provides longitudinal information on the biodistribution, growth, and survival of engineered cells in vivo. A translational bottleneck to using reporter genes is the necessity to engineer cells with randomly integrating vectors. Here, we built homology-independent targeted integration (HITI) CRISPR-Cas9 minicircle donors for precise safe harbor-targeted knock-in of fluorescence, bioluminescence, and MRI (Oatp1a1) reporter genes. Our results showed greater knock-in efficiency using HITI vectors compared to homology-directed repair vectors. HITI clones demonstrated functional fluorescence and bioluminescence reporter activity as well as significant Oatp1a1-mediated uptake of the clinically approved MRI agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid. Contrast-enhanced MRI improved the conspicuity of both subcutaneous and metastatic Oatp1a1-expressing tumors before they became palpable or even readily visible on precontrast images. Our work demonstrates the first CRISPR-Cas9 HITI system for knock-in of large DNA donor constructs at a safe harbor locus, enabling multimodal longitudinal in vivo imaging of cells.

Enzyme-sensitive magnetic resonance imaging targeting myeloperoxidase identifies active inflammation in experimental rabbit atherosclerotic plaques.

BACKGROUND: Inflammation undermines the stability of atherosclerotic plaques, rendering them susceptible to acute rupture, the cataclysmic event that underlies clinical expression of this disease. Myeloperoxidase is a central inflammatory enzyme secreted by activated macrophages and is involved in multiple stages of plaque destabilization and patient outcome. We report here that a unique functional in vivo magnetic resonance agent can visualize myeloperoxidase activity in atherosclerotic plaques in a rabbit model. METHODS AND RESULTS: We performed magnetic resonance imaging of the thoracic aorta of New Zealand White rabbits fed a cholesterol (n=14) or normal (n=4) diet up to 2 hours after injection of the myeloperoxidase sensor bis-5HT-DTPA(Gd) [MPO(Gd)], the conventional agent DTPA(Gd), or an MPO(Gd) analog, bis-tyr-DTPA(Gd), as controls. Delayed MPO(Gd) images (2 hours after injection) showed focal areas of increased contrast (>2-fold) in diseased wall but not in normal wall (P=0.84) compared with both DTPA(Gd) (n=11; P<0.001) and bis-tyr-DTPA(Gd) (n=3; P<0.05). Biochemical assays confirmed that diseased wall possessed 3-fold elevated myeloperoxidase activity compared with normal wall (P<0.01). Areas detected by MPO(Gd) imaging colocalized and correlated with myeloperoxidase-rich areas infiltrated by macrophages on histopathological evaluations (r=0.91, P<0.0001). Although macrophages were the main source of myeloperoxidase, not all macrophages secreted myeloperoxidase, which suggests that distinct subpopulations contribute differently to atherogenesis and supports our functional approach. CONCLUSIONS: The present study represents a unique approach in the detection of inflammation in atherosclerotic plaques by examining macrophage function and the activity of an effector enzyme to noninvasively provide both anatomic and functional information in vivo.

Early identification of aortic valve sclerosis using iron oxide enhanced MRI.

PURPOSE: To test the ability of MION-47 enhanced MRI to identify tissue macrophage infiltration in a rabbit model of aortic valve sclerosis (AVS). MATERIALS AND METHODS: The aortic valves of control and cholesterol-fed New Zealand White rabbits were imaged in vivo pre- and 48 h post-intravenous administration of MION-47 using a 1.5 Tesla (T) MR clinical scanner and a CINE fSPGR sequence. MION-47 aortic valve cusps were imaged ex vivo on a 3.0T whole-body MR system with a custom gradient insert coil and a three-dimensional (3D) FIESTA sequence and compared with aortic valve cusps from control and cholesterol-fed contrast-free rabbits. Histopathological analysis was performed to determine the site of iron oxide uptake. RESULTS: MION-47 enhanced the visibility of both control and cholesterol-fed rabbit valves in in vivo images. Ex vivo image analysis confirmed the presence of significant signal voids in contrast-administered aortic valves. Signal voids were not observed in contrast-free valve cusps. In MION-47 administered rabbits, histopathological analysis revealed iron staining not only in fibrosal macrophages of cholesterol-fed valves but also in myofibroblasts from control and cholesterol-fed valves. CONCLUSION: Although iron oxide labeling of macrophage infiltration in AVS has the potential to detect the disease process early, a macrophage-specific iron compound rather than passive targeting may be required.

Comparing the fate of brain metastatic breast cancer cells in different immune compromised mice with cellular magnetic resonance imaging.

Metastasis is the leading cause of mortality in breast cancer patients, with brain metastases becoming increasingly prevalent. Studying this disease is challenging due to the limited experimental models and methods available. Here, we used iron-based cellular MRI to track the fate of a mammary carcinoma cell line (MDA-MB-231-BR) in vivo to characterize the growth of brain metastases in the nude and severely immune-compromised NOD/SCID/ILIIrg-/- (NSG) mouse. Nude and NSG mice received injections of iron-labeled MDA-MB-231-BR cells. Images were acquired with a 3T MR system and assessed for signal voids and metastases. The percentage of signal voids and the number and volume of metastases were quantified. Ex vivo imaging of the liver, histology, and immunofluorescence labeling was performed. Brain metastases grew more rapidly in NSG mice. At day 21 post cell injection, the average number of brain tumors in NSG mice was approximately four times greater than in nude mice. The persistence of iron-labeled cells, visualized as signal voids by MRI, was also examined. The percentage of voids decreased significantly over time for both nude and NSG mice. Body images revealed that the NSG mice also had metastases in the liver, lungs, and lymph nodes while tumors were only detected in the brains of nude mice. This work demonstrates the advantages of using the highly immune-compromised NSG mouse to study breast cancer metastasis, treatments aimed at inhibiting metastasis and outgrowth of breast cancer metastases in multiple organs, and the role that imaging can play toward credentialing these models that cannot be done with other in vitro or histopathologic methods alone.

Trimodal Cell Tracking In Vivo: Combining Iron- and Fluorine-Based Magnetic Resonance Imaging with Magnetic Particle Imaging to Monitor the Delivery of Mesenchymal Stem Cells and the Ensuing Inflammation.

The therapeutic potential of mesenchymal stem cells (MSCs) is limited, as many cells undergo apoptosis following administration. In addition, the attraction of immune cells (predominately macrophages) to the site of implantation can lead to MSC rejection. We implemented a trimodal imaging technique to monitor the fate of transplanted MSCs and infiltrating macrophages in vivo. MSCs were labeled with an iron oxide nanoparticle (ferumoxytol) and then implanted within the hind limb muscle of 10 C57BI/6 mice. Controls received unlabeled MSCs (n = 5). A perfluorocarbon agent was administered intravenously for uptake by phagocytic macrophages in situ; 1 and 12 days later, the ferumoxytol-labeled MSCs were detected by proton (1H) magnetic resonance imaging (MRI) and magnetic particle imaging (MPI). Perfluorocarbon-labeled macrophages were detected by fluorine-19 (19F) MRI. 1H/19F MRI was acquired on a clinical scanner (3 T) using a dual-tuned surface coil and balanced steady-state free precession (bSSFP) sequence. The measured volume of signal loss and MPI signal declined over 12 days, which is consistent with the death and clearance of iron-labeled MSCs. 19F signal persisted over 12 days, suggesting the continuous infiltration of perfluorocarbon-labeled macrophages. Because MPI and 19F MRI signals are directly quantitative, we calculated estimates of the number of MSCs and macrophages present over time. The presence of MSCs and macrophages was validated with histology following the last imaging session. This is the first study to combine the use of iron- and fluorine-based MRI with MPI cell tracking.

Cellular Magnetic Resonance Imaging for Tracking Metastatic Cancer Cells in the Brain.

Cellular magnetic resonance imaging (MRI) enables visualization of cells in vivo. This is accomplished by labeling cells with superparamagnetic iron oxide nanoparticles. Here, we describe the steps for labeling human cancer cells with iron for tracking them after injection into nude mice. We also provide details for validation of cell labeling, ultrasound guided intra-cardiac injection, and MRI.

Cellular MRI Reveals Altered Brain Arrest of Genetically Engineered Metastatic Breast Cancer Cells.

Purpose: The combined use of anatomical magnetic resonance imaging (MRI), cellular MRI, and bioluminescence imaging (BLI) allows for sensitive and improved monitoring of brain metastasis in preclinical cancer models. By using these complementary technologies, we can acquire measurements of viable single cell arrest in the brain after systemic administration, the clearance and/or retention of these cells thereafter, the growth into overt tumours, and quantification of tumour volume and relative cancer cell viability over time. While BLI is very useful in measuring cell viability, some considerations have been reported using cells engineered with luciferase such as increased tumour volume variation, changes in pattern of metastatic disease, and inhibition of in vivo tumour growth. Procedures: Here, we apply cellular and anatomical MRI to evaluate in vivo growth differences between iron oxide labeled naïve (4T1BR5) and luciferase-expressing (4T1BR5-FLuc-GFP) murine brain-seeking breast cancer cells. Balb/C mice received an intracardiac injection of 20,000 cells and were imaged with MRI on days 0 and 14. Mice that received 4T1BR5-FLuc-GFP cells were also imaged with BLI on days 0 and 14. Results: The number of signal voids in the brain (representing iron-labeled cancer cells) on day 0 was significantly higher in mice receiving 4T1BR5 cells compared to mice receiving 4T1BR5-FLuc-GFP cells (p < 0.0001). Mice that received 4T1BR5 cells also had significantly higher total brain tumour burden and number of brain metastases than mice that received 4T1BR5-FLuc-GFP cells (p < 0.0001). Conclusions: By employing highly sensitive cellular MRI tools, we demonstrate that engineered cells did not form tumours as well as their naïve counterparts, which appear to primarily be due to a reduction in cell arrest. These results indicate that engineering cancer cells with reporter genes may alter their tropism towards particular organs and highlight another important consideration for research groups that use reporter gene imaging to track metastatic cancer cell fate in vivo.

Longitudinal Visualization of Viable Cancer Cell Intratumoral Distribution in Mouse Models Using Oatp1a1-Enhanced Magnetic Resonance Imaging.

OBJECTIVES: Multimodality reporter gene imaging provides valuable, noninvasive information on the fate of engineered cell populations. To complement magnetic resonance imaging (MRI) measures of tumor volume and 2-dimensional reporter-based optical measures of cell viability, reporter-based MRI may offer 3-dimensional information on the distribution of viable cancer cells in deep tissues. MATERIALS AND METHODS: Here, we engineered human and murine triple-negative breast cancer cells with lentivirus encoding tdTomato and firefly luciferase for fluorescence imaging and bioluminescence imaging (BLI). A subset of these cells was additionally engineered with lentivirus encoding organic anion transporting polypeptide 1a1 (Oatp1a1) for MRI. Oatp1a1 operates by transporting gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) into cells, and it concomitantly improves BLI substrate uptake. After orthotopic implantation of engineered cells expressing or not expressing Oatp1a1, longitudinal fluorescence imaging, BLI, and 3-Tesla MRI were performed. RESULTS: Oatp1a1-expressing tumors displayed significantly increased BLI signals relative to control tumors at all time points (P < 0.05). On MRI, post-Gd-EOB-DTPA T1-weighted images of Oatp1a1-expressing tumors exhibited significantly increased contrast-to-noise ratios compared with control tumors and precontrast images (P < 0.05). At endpoint, tumors expressing Oatp1a1 displayed intratumoral MR signal heterogeneity not present at earlier time points. Pixel-based analysis of matched in vivo MR and ex vivo fluorescence microscopy images revealed a strong, positive correlation between MR intensity and tdTomato intensity for Oatp1a1-expressing tumors (P < 0.05), but not control tumors. CONCLUSIONS: These results characterize Oatp1a1 as a sensitive, quantitative, positive contrast MRI reporter gene for 3-dimensional assessment of viable cancer cell intratumoral distribution and concomitant BLI enhancement. This multimodality reporter gene system can provide new insights into the influence of viable cancer cell intratumoral distribution on tumor progression and metastasis, as well as improved assessments of anticancer therapies.

Iron nanoparticle-labeled murine mesenchymal stromal cells in an osteoarthritic model persists and suggests anti-inflammatory mechanism of action.

Osteoarthritis (OA) is characterized by cartilage degradation and chronic joint inflammation. Mesenchymal stem cells (MSCs) have shown promising results in OA, but their mechanism of action is not fully understood. We hypothesize that MSCs polarize macrophages, which are strongly associated with joint inflammation to more homeostatic sub-types. We tracked ferumoxytol (Feraheme™, iron oxide nanoparticle)-labeled murine MSCs (Fe-MSCs) in murine OA joints, and quantified changes to joint inflammation and fibrosis. 10-week-old C57BL/6 male mice (n = 5/group) were induced to undergo osteoarthritis by destabilization of medical meniscus (DMM) or sham surgery. 3 weeks post-surgery, mice were injected intra-articularly with either fluorescent dye-(DiR) labeled or DiR-Fe-MSC or saline to yield 4 groups (n = 5 per group for each timepoint [1, 2 and 4weeks]). 4 weeks after injection, mice were imaged by MRI, and scored for i) OARSI (Osteoarthritis Research Society International) to determine cartilage damage; ii) immunohistochemical changes in iNOS, CD206, F4/80 and Prussian Blue/Sca-1 to detect pro-inflammatory, homeostatic and total macrophages and ferumoxytol -labeled MSCs respectively, and iii) Masson's Trichrome to detect changes in fibrosis. Ferumoxytol-labeled MSCs persisted at greater levels in DMM vs. SHAM-knee joints. We observed no difference in OARSI scores between MSC and vehicle groups. Sca-1 and Prussian Blue co-staining confirmed the ferumoxytol label resides in MSCs, although some ferumoxytol label was detected in proximity to MSCs in macrophages, likely due to phagocytosis of apoptotic MSCs, increasing functionality of these macrophages through MSC efferocytosis. MRI hypertintensity scores related to fluid edema decreased in MSC-treated vs. control animals. For the first time, we show that MSC-treated mice had increased ratios of %CD206+: %F4/80+ (homeostatic macrophages) (p<0.05), and decreased ratios of %iNOS+: %F4/80+ macrophages (p<0.01), supporting our hypothesis that MSCs may modulate synovial inflammation.

Targeting FER Kinase Inhibits Melanoma Growth and Metastasis.

Melanoma is one of the most aggressive types of tumors and exhibits high metastatic potential. Fes-related (FER) kinase is a non-receptor tyrosine kinase that has been implicated in growth and metastasis of various epithelial tumors. In this study, we have examined the role that FER kinase plays in melanoma at the molecular level. FER-depleted melanoma cells exhibit impaired Wnt/ß-catenin pathway activity, as well as multiple proteomic changes, which include decreased abundance of L1-cell adhesion molecule (L1-CAM). Consistent with the pro-metastatic functions of these pathways, we demonstrate that depletion of FER kinase decreases melanoma growth and formation of distant metastases in a xenograft model. These findings indicate that FER is an important positive regulator of melanoma metastasis and a potential target for innovative therapies.

Invadopodia are chemosensing protrusions that guide cancer cell extravasation to promote brain tropism in metastasis.

Invadopodia are cell protrusions that mediate cancer cell extravasation but the microenvironmental cues and signaling factors that induce invadopodia formation during extravasation remain unclear. Using intravital imaging and loss of function experiments, we determined invadopodia contain receptors involved in chemotaxis, namely GABA receptor and EGFR. These chemotaxis capabilities are mediated in part by PAK1 which controls invadopodia responsiveness to ligands such as GABA and EGF via assembly, stability, and turnover of invadopodia in vivo. PAK1 knockdown rendered cells unresponsive to chemotactic stimuli present in the stroma, resulting in dramatically lower rates of cancer cell extravasation and metastatic colony formation compared to stimulated cancer cells. In an experimental mouse model of brain metastasis, inhibition of PAK1 significantly reduced overall tumor burden and reduced the average size of brain metastases. In summary, invadopodia contain chemotaxis receptors that can respond to microenvironmental cues to guide cancer cell extravasation, and when PAK1 is depleted, brain tropism of metastatic breast cancer cells is significantly reduced, blocking secondary colony growth at sites otherwise permissive for metastatic outgrowth.

Evaluating Nonintegrating Lentiviruses as Safe Vectors for Noninvasive Reporter-Based Molecular Imaging of Multipotent Mesenchymal Stem Cells.

Reporter gene-based molecular imaging can provide invaluable information on the fate of cellular therapies postimplantation. Integrating lentiviral vectors (ILVs) are commonly used for stably engineering cells; however, their potential for insertional mutagenesis poses a significant safety concern and barrier to widespread clinical adoption. In cells that slowly divide or are postmitotic in vivo, such as mesenchymal stem cells (MSCs), nonintegrating lentiviral vectors (NILVs) may be a safer alternative option because NILVs remain episomal and can provide prolonged expression profiles. Here, NILVs coexpressing fluorescence and bioluminescence imaging (BLI) reporters were engineered and used to longitudinally image the viability of human MSCs in vitro and in vivo in mice. In vitro, ILV-transduced cancer cells and MSCs maintained steady reporter gene expression over time, whereas NILV-transduced cells progressively lost signal. NILV reporter loss was accelerated in highly proliferative cancer cells compared with less proliferative MSCs. In vivo, ILV- and NILV-transduced MSCs were each detectable with BLI postintramuscular implantation, with significantly higher ILV-based signal compared with NILV-based signal. BLI signal was observed to similarly diminish over time for both cell populations, which was attributed to cell death. Despite the reduced signal intensity with NILVs and the minimal number of cells injected (40,000), live NILV-transduced MSCs were reliably visualized for up to 2 weeks. Safety is a concern for future clinical reporter gene applications. We present NILVs as a safe means of imaging reporter gene expression for slowly dividing or nondividing cells and showed effective tracking of the viability of a small number of live transplanted MSCs over time with optical imaging. Future work will evaluate improvements to episomal NILV reporter expression, explore sensitive clinically relevant reporters, and apply this approach to clinically relevant MSC applications in preclinical models. NILVs may have broad clinical applications for expression of imaging reporters and other gene products in MSC-based therapies.

Multimodality cellular and molecular imaging of concomitant tumour enhancement in a syngeneic mouse model of breast cancer metastasis.

The mechanisms that influence metastatic growth rates are poorly understood. One mechanism of interest known as concomitant tumour resistance (CTR) can be defined as the inhibition of metastasis by existing tumour mass. Conversely, the presence of a primary tumour has also been shown to increase metastatic outgrowth, termed concomitant tumour enhancement (CTE). The majority of studies evaluating CTR/CTE in preclinical models have relied on endpoint histological evaluation of tumour burden. The goal of this research was to use conventional magnetic resonance imaging (MRI), cellular MRI, and bioluminescence imaging to study the impact of a primary tumour on the development of brain metastases in a syngeneic mouse model. Here, we report that the presence of a 4T1 primary tumour significantly enhances total brain tumour burden in Balb/C mice. Using in vivo BLI/MRI we could determine this was not related to differences in initial arrest or clearance of viable cells in the brain, which suggests that the presence of a primary tumour can increase the proliferative growth of brain metastases in this model. The continued application of our longitudinal cellular and molecular imaging tools will yield a better understanding of the mechanism(s) by which this physiological inhibition (CTR) and/or enhancement (CTE) occurs.

Safe Harbor Targeted CRISPR-Cas9 Tools for Molecular-Genetic Imaging of Cells in Living Subjects.

Noninvasive molecular-genetic imaging of cells expressing imaging reporter genes is an invaluable approach for longitudinal monitoring of the biodistribution and viability of cancer cells and cell-based therapies in preclinical models and patients. However, labeling cells with reporter genes often relies on using gene transfer methods that randomly integrate the reporter genes into the genome, which may cause unwanted and serious detrimental effects. To overcome this, we have developed CRISPR-Cas9 tools to edit cells at the adeno-associated virus site 1 (AAVS1) safe harbour with a large donor construct (∼6.3 kilobases) encoding an antibiotic resistance gene and reporter genes for bioluminescence (BLI) and fluorescence imaging. HEK293T cells were transfected with a dual plasmid system encoding the Cas9 endonuclease and an AAVS1-targeted guide RNA in one plasmid, and a donor plasmid encoding a puromycin resistance gene, tdTomato and firefly luciferase flanked by AAVS1 homology arms. Puromycin-resistant clonal cells were isolated and AAVS1 integration was confirmed via PCR and sequencing of the PCR product. In vitro BLI signal correlated well to cell number (R2 = 0.9988; p < 0.05) and was stable over multiple passages. Engineered cells (2.5 × 106) were injected into the left hind flank of nude mice and in vivo BLI was performed on days 0, 7, 14, 21, and 28. BLI signal trended down from day 0 to day 7, but significantly increased by day 28 due to cell growth (p < 0.05). This describes the first CRISPR-Cas9 system for AAVS1 integration of large gene constructs for molecular-genetic imaging of cells in vivo. With further development, including improving editing efficiency, use of clinically relevant reporters, and evaluation in other cell populations that can be readily expanded in culture (e.g., immortalized cells or T cells), this CRISPR-Cas9 reporter gene system could be broadly applied to a number of in vivo cell tracking studies.