Imaging CAR-NK cells targeted to HER2 ovarian cancer with human sodium-iodide symporter-based positron emission tomography.

Chimeric antigen receptor (CAR) cell therapies utilize CARs to redirect immune cells towards cancer cells expressing specific antigens like human epidermal growth factor receptor 2 (HER2). Despite their potential, CAR T cell therapies exhibit variable response rates and adverse effects in some patients. Non-invasive molecular imaging can aid in predicting patient outcomes by tracking infused cells post-administration. CAR-T cells are typically autologous, increasing manufacturing complexity and costs. An alternative approach involves developing CAR natural killer (CAR-NK) cells as an off-the-shelf allogeneic product. In this study, we engineered HER2-targeted CAR-NK cells co-expressing the positron emission tomography (PET) reporter gene human sodium-iodide symporter (NIS) and assessed their therapeutic efficacy and PET imaging capability in a HER2 ovarian cancer mouse model.NK-92 cells were genetically modified to express a HER2-targeted CAR, the bioluminescence imaging reporter Antares, and NIS. HER2-expressing ovarian cancer cells were engineered to express the bioluminescence reporter Firefly luciferase (Fluc). Co-culture experiments demonstrated significantly enhanced cytotoxicity of CAR-NK cells compared to naive NK cells. In vivo studies involving mice with Fluc-expressing tumors revealed that those treated with CAR-NK cells exhibited reduced tumor burden and prolonged survival compared to controls. Longitudinal bioluminescence imaging demonstrated stable signals from CAR-NK cells over time. PET imaging using the NIS-targeted tracer 18F-tetrafluoroborate ([18F]TFB) showed significantly higher PET signals in mice treated with NIS-expressing CAR-NK cells.Overall, our study showcases the therapeutic potential of HER2-targeted CAR-NK cells in an aggressive ovarian cancer model and underscores the feasibility of using human-derived PET reporter gene imaging to monitor these cells non-invasively in patients.

Theranostics - a sure cure for cancer after 100 years?

Cancer has remained a formidable challenge in medicine and has claimed an enormous number of lives worldwide. Theranostics, combining diagnostic methods with personalized therapeutic approaches, shows huge potential to advance the battle against cancer. This review aims to provide an overview of theranostics in oncology: exploring its history, current advances, challenges, and prospects. We present the fundamental evolution of theranostics from radiotherapeutics, cellular therapeutics, and nanotherapeutics, showcasing critical milestones in the last decade. From the early concept of targeted drug delivery to the emergence of personalized medicine, theranostics has benefited from advances in imaging technologies, molecular biology, and nanomedicine. Furthermore, we emphasize pertinent illustrations showcasing that revolutionary strategies in cancer management enhance diagnostic accuracy and provide targeted therapies customized for individual patients, thereby facilitating the implementation of personalized medicine. Finally, we describe future perspectives on current challenges, emerging topics, and advances in the field.

A synthetic notch (synNotch) system linking intratumoral immune-cancer cell communication to a synthetic blood biomarker assay.

Introduction: Cellular immunotherapy has greatly improved cancer treatment in recent years. For instance, chimeric antigen receptor (CAR) T cell therapy has been proven highly effective in treating hematological malignancies, and many CAR cell designs are being explored for solid tumors. However, many questions remain why responses differ across patients and some tumor types are resistant. Improved and relatively inexpensive ways to monitor these cells could provide some answers. Clinically, blood tests are regularly used to monitor these therapies, but blood signals often do not reflect the activity of immune cells within the tumor(s). Here, using the synthetic Notch (synNotch) receptor that tethers antigen binding to customized transgene expression, we linked intratumoral immune-cancer cell communication to a simple secreted reporter blood test. Specifically, we engineered immune cells with a CD19-targeted synNotch receptor and demonstrated that binding to CD19 on cancer cells in vivo resulted in the production of secreted embryonic alkaline phosphatase (SEAP) at levels that are readily detected in the blood. Methods and Results: Jurkat T cells were engineered via sequential lentiviral transduction of two components: an anti-CD19 synNotch receptor and a synNotch response element encoding SEAP. Co-culture of engineered cells with CD19+, but not CD19-, Nalm6 cells, resulted in significantly elevated SEAP in media. Nod-scid-gamma (NSG) mice were subcutaneously injected with either CD19+ or CD19- Nalm6 cells. Intratumoral injection of engineered T cells (1x107) resulted in significantly elevated blood SEAP activity in mice bearing CD19+ tumors (n = 7), but not CD19- tumors (n = 5). Discussion: Our synNotch reporter system allows for the monitoring of antigen-dependent intratumoral immune-cancer cell interactions through a simple and convenient blood test. Continued development of this system for different target antigens of interest should provide a broadly applicable platform for improved monitoring of many cell-based immunotherapies during their initial development and clinical translation, ultimately improving our understanding of design considerations and patient-specific responses.

Development of a Suite of Gadolinium-Free OATP1-Targeted Paramagnetic Probes for Liver MRI.

Five amphiphilic, anionic Mn(II) complexes were synthesized as contrast agents targeted to organic anion transporting polypeptide transporters (OATP) for liver magnetic resonance imaging (MRI). The Mn(II) complexes are synthesized in three steps, each from the commercially available trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) chelator, with T1-relaxivity of complexes ranging between 2.3 and 3.0 mM-1 s-1 in phosphate buffered saline at an applied field strength of 3.0 T. Pharmacokinetics were assessed in female BALB/c mice by acquiring T1-weighted images dynamically for 70 min after agent administration and determining contrast enhancement and washout in various organs. Uptake of Mn(II) complexes in human OATPs was investigated through in vitro assays using MDA-MB-231 cells engineered to express either OATP1B1 or OATP1B3 isoforms. Our study introduces a new class of Mn-based OATP-targeted contrast that can be broadly tuned via simple synthetic protocols.

A Protocol for Simultaneous In Vivo Imaging of Cardiac and Neuroinflammation in Dystrophin-Deficient MDX Mice Using [18F]FEPPA PET.

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by dystrophin loss-notably within muscles and the central neurons system. DMD presents as cognitive weakness, progressive skeletal and cardiac muscle degeneration until pre-mature death from cardiac or respiratory failure. Innovative therapies have improved life expectancy; however, this is accompanied by increased late-onset heart failure and emergent cognitive degeneration. Thus, better assessment of dystrophic heart and brain pathophysiology is needed. Chronic inflammation is strongly associated with skeletal and cardiac muscle degeneration; however, neuroinflammation's role is largely unknown in DMD despite being prevalent in other neurodegenerative diseases. Here, we present an inflammatory marker translocator protein (TSPO) positron emission tomography (PET) protocol for in vivo concomitant assessment of immune cell response in hearts and brains of a dystrophin-deficient mouse model [mdx:utrn(+/-)]. Preliminary analysis of whole-body PET imaging using the TSPO radiotracer, [18F]FEPPA in four mdx:utrn(+/-) and six wildtype mice are presented with ex vivo TSPO-immunofluorescence tissue staining. The mdx:utrn(+/-) mice showed significant elevations in heart and brain [18F]FEPPA activity, which correlated with increased ex vivo fluorescence intensity, highlighting the potential of TSPO-PET to simultaneously assess presence of cardiac and neuroinflammation in dystrophic heart and brain, as well as in several organs within a DMD model.

Visualizing cell-cell communication using synthetic notch activated MRI.

Cell-cell communication plays a fundamental role in multicellular organisms. Cell-based cancer immunotherapies rely on the ability of innate or engineered receptors on immune cells to engage specific antigens on cancer cells to induce tumor kill. To improve the development and translation of these therapies, imaging tools capable of noninvasively and spatiotemporally visualizing immune-cancer cell interactions would be highly valuable. Using the synthetic Notch (SynNotch) system, we engineered T cells that upon interaction with a chosen antigen (CD19) on neighboring cancer cells induce the expression of optical reporter genes and the human-derived, magnetic resonance imaging (MRI) reporter gene organic anion transporting polypeptide 1B3 (OATP1B3). Administration of engineered T cells induced the antigen-dependent expression of all our reporter genes in mice bearing CD19-positive tumors but not CD19-negative tumors. Notably, due to the high spatial resolution and tomographic nature of MRI, contrast-enhanced foci within CD19-positive tumors representing OATP1B3-expressing T cells were clearly visible and their distribution was readily mapped. We then extended this technology onto human natural killer-92 (NK-92) cells, observing similar CD19-dependent reporter activity in tumor-bearing mice. Furthermore, we show that when delivered intravenously, engineered NK-92 cells can be detected via bioluminescence imaging in a systemic cancer model. With continued work, this highly modular imaging strategy could aid in the monitoring of cell therapies in patients and, beyond this, augment our understanding of how different cell populations interact within the body during normal physiology or disease.

Spatiotemporally dynamic electric fields for brain cancer treatment: anin vitroinvestigation.

Objective. The treatment of glioblastoma (GBM) using low intensity electric fields (∼1 V cm-1) is being investigated using multiple implanted bioelectrodes, which was termed intratumoral modulation therapy (IMT). Previous IMT studies theoretically optimized treatment parameters to maximize coverage with rotating fields, which required experimental investigation. In this study, we employed computer simulations to generate spatiotemporally dynamic electric fields, designed and purpose-built an IMT device forin vitroexperiments, and evaluated the human GBM cellular responses to these fields.Approach. After measuring the electrical conductivity of thein vitroculturing medium, we designed experiments to evaluate the efficacy of various spatiotemporally dynamic fields: (a) different rotating field magnitudes, (b) rotating versus non-rotating fields, (c) 200 kHz versus 10 kHz stimulation, and (d) constructive versus destructive interference. A custom printed circuit board (PCB) was fabricated to enable four-electrode IMT in a 24-well plate. Patient derived GBM cells were treated and analyzed for viability using bioluminescence imaging.Main results. The optimal PCB design had electrodes placed 6.3 mm from the center. Spatiotemporally dynamic IMT fields at magnitudes of 1, 1.5, and 2 V cm-1reduced GBM cell viability to 58%, 37% and 2% of sham controls respectively. Rotating versus non-rotating, and 200 kHz versus 10 kHz fields showed no statistical difference. The rotating configuration yielded a significant reduction (p< 0.01) in cell viability (47 ± 4%) compared to the voltage matched (99 ± 2%) and power matched (66 ± 3%) destructive interference cases.Significance. We found the most important factors in GBM cell susceptibility to IMT are electric field strength and homogeneity. Spatiotemporally dynamic electric fields have been evaluated in this study, where improvements to electric field coverage with lower power consumption and minimal field cancellations has been demonstrated. The impact of this optimized paradigm on cell susceptibility justifies its future use in preclinical and clinical trial investigations.

Complementary early-phase magnetic particle imaging and late-phase positron emission tomography reporter imaging of mesenchymal stem cells in vivo.

Stem cell-based therapies have demonstrated significant potential in clinical applications for many debilitating diseases. The ability to non-invasively and dynamically track the location and viability of stem cells post administration could provide important information on individual patient response and/or side effects. Multi-modal cell tracking provides complementary information that can offset the limitations of a single imaging modality to yield a more comprehensive picture of cell fate. In this study, mesenchymal stem cells (MSCs) were engineered to express human sodium iodide symporter (NIS), a clinically relevant positron emission tomography (PET) reporter gene, as well as labeled with superparamagnetic iron oxide nanoparticles (SPIOs) to allow for detection with magnetic particle imaging (MPI). MSCs were additionally engineered with a preclinical bioluminescence imaging (BLI) reporter gene for comparison of BLI cell viability data to both MPI and PET data over time. MSCs were implanted into the hind limbs of immunocompromised mice and imaging with MPI, BLI and PET was performed over a 30-day period. MPI showed sensitive detection that steadily declined over the 30-day period, while BLI showed initial decreases followed by later rapid increases in signal. The PET signal of MSCs was significantly higher than the background at later timepoints. Early-phase imaging (day 0-9 post MSC injections) showed correlation between MPI and BLI data (R2 = 0.671), while PET and BLI showed strong correlation for late-phase (day 10-30 post MSC injections) imaging timepoints (R2 = 0.9817). We report the first use of combined MPI and PET for cell tracking and show the complementary benefits of MPI for sensitive detection of MSCs early after implantation and PET for longer-term measurements of cell viability.

Dual Magnetic Particle Imaging and Akaluc Bioluminescence Imaging for Tracking Cancer Cell Metastasis.

Magnetic particle imaging (MPI) provides hotspot tracking and direct quantification of superparamagnetic iron oxide nanoparticle (SPIO)-labelled cells. Bioluminescence imaging (BLI) with the luciferase reporter gene Akaluc can provide complementary information on cell viability. Thus, we explored combining these technologies to provide a more holistic view of cancer cell fate in mice. Akaluc-expressing 4T1Br5 cells were labelled with the SPIO Synomag-D and injected into the mammary fat pads (MFP) of four nude mice. BLI was performed on days 0, 6 and 13, and MPI was performed on days 1, 8 and 14. Ex vivo histology and fluorescence microscopy of MFP and a potential metastatic site was conducted. The BLI signal in the MFP increased significantly from day 0 to day 13 (p < 0.05), mirroring tumor growth. The MPI signal significantly decreased from day 1 to day 14 (p < 0.05) due to SPIO dilution in proliferating cells. Both modalities detected secondary metastases; however, they were visualized in different anatomical regions. Akaluc BLI complemented MPI cell tracking, allowing for longitudinal measures of cell viability and sensitive detection of distant metastases at different locations. We predict this multimodal imaging approach will help to evaluate novel therapeutics and give a better understanding of metastatic mechanisms.

A Genetically Encoded Magnetic Resonance Imaging Reporter Enables Sensitive Detection and Tracking of Spontaneous Metastases in Deep Tissues.

Metastasis is the leading cause of cancer-related death. However, it remains a poorly understood aspect of cancer biology, and most preclinical cancer studies do not examine metastasis, focusing solely on the primary tumor. One major factor contributing to this paradox is a gap in available tools for accurate spatiotemporal measurements of metastatic spread in vivo. Here, our objective was to develop an imaging reporter system that offers sensitive three-dimensional (3D) detection of cancer cells at high resolutions in live mice. An organic anion-transporting polypeptide 1b3 (oatp1b3) was used as an MRI reporter gene, and its sensitivity was systematically optimized for in vivo tracking of viable cancer cells in a spontaneous metastasis model. Metastases with oatp1b3-MRI could be observed at the single lymph node level and tracked over time as cancer cells spread to multiple lymph nodes and different organ systems in individual animals. While initial single lesions were successfully imaged in parallel via bioluminescence, later metastases were largely obscured by light scatter from the initial node. Importantly, MRI could detect micrometastases in lung tissue comprised on the order of 1,000 cancer cells. In summary, oatp1b3-MRI enables longitudinal tracking of cancer cells with combined high resolution and high sensitivity that provides 3D spatial information and the surrounding anatomical context. SIGNIFICANCE: An MRI reporter gene system optimized for tracking metastasis in deep tissues at high resolutions and able to detect spontaneous micrometastases in lungs of mice provides a useful tool for metastasis research.

Gadolinium-free Magnetic Resonance Imaging of the Liver via an Oatp1-Targeted Manganese(III) Porphyrin.

Controversy surrounding gadolinium-based contrast agents (GBCAs) has rendered their continued utility highly contentious, but the liver-specific GBCA Gd(III) ethoxybenzyl-diethylene triamine pentaacetic acid (Gd(III)-EOB-DTPA) remains in use because it provides unique diagnostic information that could not be obtained by any other means. To address the need for an alternate liver-specific MRI probe, we synthesized Mn(III) 20-(4-ethoxyphenyl) porphyrin-5,10,15-tricarboxylate (Mn(III)TriCP-PhOEt), which exhibited significantly higher r1 relaxivity than Gd(III)-EOB-DTPA in vitro, while also targeting hepatocyte-specific organic anion-transporting polypeptide 1 (Oatp1) channels as a marker of viability. In mice, Mn(III)TriCP-PhOEt resulted in significant and specific increases in liver signal intensity on T1-weighted images and significant decreases in liver T1 time relative to pre-contrast measurements. Our findings suggest that Mn(III)TriCP-PhOEt operates as a specific and sensitive MR probe for Oatp1-targeted imaging in vivo.

A Human-derived Dual MRI/PET Reporter Gene System with High Translational Potential for Cell Tracking.

PURPOSE: Reporter gene imaging has been extensively used to longitudinally report on whole-body distribution and viability of transplanted engineered cells. Multi-modal cell tracking can provide complementary information on cell fate. Typical multi-modal reporter gene systems often combine clinical and preclinical modalities. A multi-modal reporter gene system for magnetic resonance imaging (MRI) and positron emission tomography (PET), two clinical modalities, would be advantageous by combining the sensitivity of PET with the high-resolution morphology and non-ionizing nature of MRI. PROCEDURES: We developed and evaluated a dual MRI/PET reporter gene system composed of two human-derived reporter genes that utilize clinical reporter probes for engineered cell detection. As a proof-of-concept, breast cancer cells were engineered to co-express the human organic anion transporter polypeptide 1B3 (OATP1B3) that uptakes the clinical MRI contrast agent gadolinium ethoxybenzyl-diethylenetriaminepentaacetic acid (Gd-EOB-DTPA), and the human sodium iodide symporter (NIS) which uptakes the PET tracer, [18F] tetrafluoroborate ([18F] TFB). RESULTS: T1-weighted MRI results in mice exhibited significantly higher MRI signals in reporter-gene-engineered mammary fat pad tumors versus contralateral naïve tumors (p < 0.05). No differences in contrast enhancement were observed at 5 h after Gd-EOB-DTPA administration using either intravenous or intraperitoneal injection. We also found significantly higher standard uptake values (SUV) in engineered tumors in comparison to the naïve tumors in [18F]TFB PET images (p < 0.001). Intratumoral heterogeneity in signal enhancement was more conspicuous in relatively higher resolution MR images compared to PET images. CONCLUSIONS: Our study demonstrates the ability to noninvasively track cells engineered with our human-derived dual MRI/PET reporter system, enabling a more comprehensive evaluation of transplanted cells. Future work is focused on applying this tool to track therapeutic cells, which may one day enable the broader application of cell tracking within the healthcare system.

Self-immolative polyplexes for DNA delivery.

Nucleic acids have immense potential for the treatment and prevention of a wide range of diseases, but delivery vehicles are needed to assist with their entry into cells. Polycations can reversibly complex with nucleic acids via ionic interactions to form polyplexes and transport them into cells, but they are still hindered by the need to balance cytotoxicity and delivery effectiveness. In this work, we describe a new self-immolative polyglyoxylamide (PGAm) platform designed to address these challenges by complexing nucleic acids via multivalent interactions in the polymeric form and releasing them upon depolymerization. Nine PGAms were synthesized and characterized, with different end-caps and variable cationic pendent groups. The PGAms underwent depolymerization under mildly acidic conditions, with rates dependent on their pendent groups and end-caps. They complexed plasmid DNA, forming cationic nanoparticles, and released it upon depolymerization. Cytotoxicity assays of the PGAms and polyplexes in HEK 293T cells showed a decrease in toxicity following depolymerization, and all samples exhibited much lower toxicity than a commercial non-degradable linear polyethyleneimine (jetPEI) transfection agent. Transfection assays revealed that selected PGAms provided similar levels of reporter gene expression to jetPEI in vitro with a PGAm analogue of poly[2-(dimethylamino)ethyl methacrylate] having particularly interesting activity that was dependent on depolymerization, along with low cytotoxicity. Overall, these results indicate that end-to-end depolymerization of self-immolative polymers can provide a new and promising tool for nucleic acid delivery.

Visualizing CAR-T cell Immunotherapy Using 3 Tesla Fluorine-19 MRI.

PURPOSE: Chimeric antigen receptor (CAR) T cell cancer immunotherapies have shown remarkable results in patients with hematological malignancies and represent the first approved genetically modified cellular therapies. However, not all blood cancer patients respond favorably, serious side effects have been reported, and the treatment of solid tumors has been a challenge. An imaging tool for visualizing the variety of CAR-T cell products in use and being explored could provide important patient-specific data on CAR-T cell location to inform on potential success or failure of treatment as well as off-target toxicities. Fluorine-19 (19F) magnetic resonance imaging (MRI) allows for the noninvasive detection of 19F perfluorocarbon (PFC) labeled cells. Our objective was to visualize PFC-labeled (PFC +) CAR-T cells in a mouse model of leukemia using clinical field strength (3 Tesla) 19F MRI and compare the cytotoxicity of PFC + versus unlabeled CAR-T cells. PROCEDURES: NSG mice (n = 17) received subcutaneous injections of CD19 + human B cell leukemia cells (NALM6) expressing firefly luciferase in their left hind flank (1 × 106). Twenty-one days later, each mouse received an intratumoral injection of 10 × 106 PFC + CD19-targeted CAR-T cells (n = 6), unlabeled CD19-targeted CAR-T cells (n = 3), PFC + untransduced T cells (n = 5), or an equivalent volume of saline (n = 3). 19F MRI was performed on mice treated with PFC + CAR-T cells days 1, 3, and 7 post-treatment. Bioluminescence imaging (BLI) was performed on all mice days - 1, 5, 10, and 14 post-treatment to monitor tumor response. RESULTS: PFC + CAR-T cells were successfully detected in tumors using 19F MRI on days 1, 3, and 7 post-injection. In vivo BLI data revealed that mice treated with PFC + or PFC - CAR-T cells had significantly lower tumor burden by day 14 compared to untreated mice and mice treated with PFC + untransduced T cells (p < 0.05). Importantly, mice treated with PFC + CAR-T cells showed equivalent cytotoxicity compared to mice receiving PFC - CAR-T cells. CONCLUSIONS: Our studies demonstrate that clinical field strength 19F MRI can be used to visualize PFC + CAR-T cells for up to 7 days post-intratumoral injection. Importantly, PFC labeling did not significantly affect in vivo CAR-T cell cytotoxicity. These imaging tools may have broad applications for tracking emerging CAR-T cell therapies in preclinical models and may eventually be useful for the detection of CAR-T cells in patients where localized injection of CAR-T cells is being pursued.

Molecular imaging of cellular immunotherapies in experimental and therapeutic settings.

Cell-based cancer immunotherapies are becoming a routine part of the armamentarium against cancer. While remarkable successes have been seen, including durable remissions, not all patients will benefit from these therapies and many can suffer from life-threatening side effects. These differences in efficacy and safety across patients and across tumor types (e.g., blood vs. solid), are thought to be due to differences in how well the immune cells traffic to their target tissue (e.g., tumor, lymph nodes, etc.) whilst avoiding non-target tissues. Across patient variability can also stem from whether the cells interact with (i.e., communicate with) their intended target cells (e.g., cancer cells), as well as if they proliferate and survive long enough to yield potent and long-lasting therapeutic effects. However, many cell-based therapies are monitored by relatively simple blood tests that lack any spatial information and do not reflect how many immune cells have ended up at particular tissues. The ex vivo labeling and imaging of infused therapeutic immune cells can provide a more precise and dynamic understanding of whole-body immune cell biodistribution, expansion, viability, and activation status in individual patients. In recent years numerous cellular imaging technologies have been developed that may provide this much-needed information on immune cell fate. For this review, we summarize various ex vivo labeling and imaging approaches that allow for tracking of cellular immunotherapies for cancer. Our focus is on clinical imaging modalities and summarize the progression from experimental to therapeutic settings. The imaging information provided by these technologies can potentially be used for many purposes including improved real-time understanding of therapeutic efficacy and potential side effects in individual patients after cell infusion; the ability to more readily compare new therapeutic cell designs to current designs for various parameters such as improved trafficking to target tissues and avoidance of non-target tissues; and the long-term ability to identify patient populations that are likely to be positive responders and at low-risk of side effects.

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.

Sampling Thoracic Duct Lymph After Esophagectomy: A Pilot Study Investigating the "Gut-Lymph" Concept.

Background: Gut-lymph in animal models of acute disease is altered by intestinal ischemia and contributes to the development of systemic inflammation and organ dysfunction. Investigating gut-lymph in humans is hampered difficulty in accessing the thoracic duct (TD) for lymph sampling. The aims of this study were to develop and pilot a technique of intraoperative TD cannulation with delayed embolization to serially measure TD lymph pressure, flow, and composition (including markers of intestinal injury) during the early postoperative period and in response to enteral feeding and vasopressor treatment. Methods: A Seldinger technique was used for percutaneous TD cannulation during an Ivor Lewis esophagogastrectomy. Lymph flow rate and pressure were measured. TD lymph and plasma were sampled at 12 hourly intervals for up to 120 hours after surgery and before TD embolization. Biochemistry, lipids, cytokines, and markers of intestinal injury were measured before and after enteral feeding commenced at 36 hours. Results: Intraoperative TD cannulation was technically feasible in three of four patients. Delayed TD embolization was only successful in one of three patients, with two patients requiring a re-thoracotomy to treat chylothorax. Profound changes in TD composition, but not flow rate, occurred over time and in response to enteral feeding and vasopressors. TD lymph compared with plasma had significantly higher lipase (1.4-17 × ), interleukin-6 (8-108 × ), tumor necrosis factor-α (2.7-17 × ), d-lactate (0.3-23 × ), endotoxin (0.1-41 × ), and intestinal fatty acid binding protein (1.1-853 × ). Conclusions: Although TD cannulation and lymph sampling were successful, TD embolization failed in two of three patients. The composition of sampled TD lymph changed dramatically in response to enteral feeding, indicating intestinal ischemia that could be exacerbated by nonselective vasopressors. The higher concentration of proinflammatory cytokines and gut injury markers in TD lymph, compared with plasma, lends support to the gut-lymph concept.

Brightening up Biology: Advances in Luciferase Systems for in Vivo Imaging.

Bioluminescence imaging (BLI) using luciferase reporters is an indispensable method for the noninvasive visualization of cell populations and biochemical events in living animals. BLI is widely performed with preclinical rodent models to understand disease processes and evaluate potential cell- or gene-based therapies. However, in vivo BLI remains constrained by low photon production and tissue attenuation, limiting the sensitivity of reporting from small numbers of cells in deep locations and hindering its application to larger animal models. This Review highlights recent advances in the development of luciferase systems that improve the sensitivity of in vivo BLI and discusses the expanding array of biological applications.

A method for the efficient iron-labeling of patient-derived xenograft cells and cellular imaging validation.

There is momentum towards implementing patient-derived xenograft models (PDX) in cancer research to reflect the histopathology, tumor behavior, and metastatic properties observed in the original tumor. To study PDX cells preclinically, we used both bioluminescence imaging (BLI) to evaluate cell viability and magnetic particle imaging (MPI), an emerging imaging technology to allow for detection and quantification of iron nanoparticles. The goal of this study was to develop the first successful iron labeling method of breast cancer cells derived from patient brain metsastases and validate this method with imaging during tumor development. The overall workflow of this labeling method is as follows: adherent and non-adherent luciferase expressing human breast cancer PDX cells (F2-7) are dissociated and concurrently labeled after incubation with micron-sized iron oxide particles (MPIO; 25 µg Fe/ml), with labeling validated by cellular imaging with MPI and BLI. In this study, NOD/SCID/ILIIrg-/- (n = 5) mice Received injections of 1 × 106 iron-labeled F2-7 cells into the fourth mammary fat pad (MFP). BLI was performed longitudinally to day 49 and MPI was performed up to day 28. In vivo BLI revealed that signal increased over time with tumor development. MPI revealed decreasing signal in the tumors over time. Here, we demonstrate the first application of MPI to monitor the growth of a PDX MFP tumor and the first successful labeling of PDX cells with iron oxide particles. Imaging of PDX cells provides a powerful system to better develop personalized therapies targeting breast cancer brain metastasis.

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.