Next-generation humanized patient-derived xenograft mouse model for pre-clinical antibody studies in neuroblastoma.

Faithful tumor mouse models are fundamental research tools to advance the field of immuno-oncology (IO). This is particularly relevant in diseases with low incidence, as in the case of pediatric malignancies, that rely on pre-clinical therapeutic development. However, conventional syngeneic and genetically engineered mouse models fail to recapitulate the tumor heterogeneity and microenvironmental complexity of human pathology that are essential determinants of cancer-directed immunity. Here, we characterize a novel mouse model that supports human natural killer (NK) cell development and engraftment of neuroblastoma orthotopic patient-derived xenograft (O-PDX) for pre-clinical antibody and cytokine testing. Using cytotoxicity assays, single-cell RNA-sequencing, and multi-color flow cytometry, we demonstrate that NK cells that develop in the humanized mice are fully licensed to execute NK cell cytotoxicity, permit human tumor engraftment, but can be therapeutically redirected to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Although these cells share phenotypic and molecular features with healthy controls, we noted that they lacked an NK cell subset, termed activated NK cells, that is characterized by differentially expressed genes that are induced by cytokine activation. Because this subset of genes is also downregulated in patients with neuroblastoma compared to healthy controls, we hypothesize that this finding could be due to tumor-mediated suppressive effects. Thus, despite its technical complexity, this humanized patient-derived xenograft mouse model could serve as a faithful system for future testing of IO applications and studies of underlying immunologic processes.

Optical See-Through Cancer Vision Goggles Enable Direct Patient Visualization and Real-Time Fluorescence-Guided Oncologic Surgery.

BACKGROUND: The inability to visualize the patient and surgical site directly, limits the use of current near infrared fluorescence-guided surgery systems for real-time sentinel lymph node biopsy and tumor margin assessment. METHODS: We evaluated an optical see-through goggle augmented imaging and navigation system (GAINS) for near-infrared, fluorescence-guided surgery. Tumor-bearing mice injected with a near infrared cancer-targeting agent underwent fluorescence-guided, tumor resection. Female Yorkshire pigs received hind leg intradermal indocyanine green injection and underwent fluorescence-guided, popliteal lymph node resection. Four breast cancer patients received 99mTc-sulfur colloid and indocyanine green retroareolarly before undergoing sentinel lymph node biopsy using radioactive tracking and fluorescence imaging. Three other breast cancer patients received indocyanine green retroareolarly before undergoing standard-of-care partial mastectomy, followed by fluorescence imaging of resected tumor and tumor cavity for margin assessment. RESULTS: Using near-infrared fluorescence from the dyes, the optical see-through GAINS accurately identified all mouse tumors, pig lymphatics, and four pig popliteal lymph nodes with high signal-to-background ratio. In 4 human breast cancer patients, 11 sentinel lymph nodes were identified with a detection sensitivity of 86.67 ± 0.27% for radioactive tracking and 100% for GAINS. Tumor margin status was accurately predicted by GAINS in all three patients, including clear margins in patients 1 and 2 and positive margins in patient 3 as confirmed by paraffin-embedded section histopathology. CONCLUSIONS: The optical see-through GAINS prototype enhances near infrared fluorescence-guided surgery for sentinel lymph node biopsy and tumor margin assessment in breast cancer patients without disrupting the surgical workflow in the operating room.

Protonation and Trapping of a Small pH-Sensitive Near-Infrared Fluorescent Molecule in the Acidic Tumor Environment Delineate Diverse Tumors in Vivo.

Enhanced glycolysis and poor perfusion in most solid malignant tumors create an acidic extracellular environment, which enhances tumor growth, invasion, and metastasis. Complex molecular systems have been explored for imaging and treating these tumors. Here, we report the development of a small molecule, LS662, that emits near-infrared (NIR) fluorescence upon protonation by the extracellular acidic pH environment of diverse solid tumors. Protonation of LS662 induces selective internalization into tumor cells and retention in the tumor microenvironment. Noninvasive NIR imaging demonstrates selective retention of the pH sensor in diverse tumors, and two-photon microscopy of ex vivo tumors reveals significant retention of LS662 in tumor cells and the acid tumor microenvironment. Passive and active internalization processes combine to enhance NIR fluorescence in tumors over time. The low background fluorescence allows tumors to be detected with high sensitivity, as well as dead or dying cells to be delineated from healthy cells. In addition to demonstrating the feasibility of using small molecule pH sensors to image multiple aggressive solid tumor types via a protonation-induced internalization and retention pathway, the study reveals the potential of using LS662 to monitor treatment response and tumor-targeted drug delivery.

Monitoring of biodistribution and persistence of conditionally replicative adenovirus in a murine model of ovarian cancer using capsid-incorporated mCherry and expression of human somatostatin receptor subtype 2 gene.

A significant limiting factor to the human clinical application of conditionally replicative adenovirus (CRAd)-based virotherapy is the inability to noninvasively monitor these agents and their potential persistence. To address this issue, we proposed a novel imaging approach that combines transient expression of the human somatostatin receptor (SSTR) subtype 2 reporter gene with genetic labeling of the viral capsid with mCherry fluorescent protein. To test this dual modality system, we constructed the Ad5/3Δ24pIXcherry/SSTR CRAd and validated its capacity to generate fluorescent and nuclear signals in vitro and following intratumoral injection. Analysis of 64Cu-CB-TE2A-Y3-TATE biodistribution in mice revealed reduced uptake in tumors injected with the imaging CRAd relative to the replication-incompetent, Ad-expressing SSTR2 but significantly greater uptake compared to the negative CRAd control. Optical imaging demonstrated relative correlation of fluorescent signal with virus replication as determined by viral genome quantification in tumors. Positron emission tomography/computed tomography studies demonstrated that we can visualize radioactive uptake in tumors injected with imaging CRAd and the trend for greater uptake by standardized uptake value analysis compared to control CRAd. In the aggregate, the plasticity of our dual imaging approach should provide the technical basis for monitoring CRAd biodistribution and persistence in preclinical studies while offering potential utility for a range of clinical applications.

An integrated single-cell RNA-seq map of human neuroblastoma tumors and preclinical models uncovers divergent mesenchymal-like gene expression programs.

Neuroblastoma is a common pediatric cancer, where preclinical studies suggest that a mesenchymal-like gene expression program contributes to chemotherapy resistance. However, clinical outcomes remain poor, implying we need a better understanding of the relationship between patient tumor heterogeneity and preclinical models. Here, we generated single-cell RNA-seq maps of neuroblastoma cell lines, patient-derived xenograft models (PDX), and a genetically engineered mouse model (GEMM). We developed an unsupervised machine learning approach ('automatic consensus nonnegative matrix factorization' (acNMF)) to compare the gene expression programs found in preclinical models to a large cohort of patient tumors. We confirmed a weakly expressed, mesenchymal-like program in otherwise adrenergic cancer cells in some pre-treated high-risk patient tumors, but this appears distinct from the presumptive drug-resistance mesenchymal programs evident in cell lines. Surprisingly however, this weak-mesenchymal-like program was maintained in PDX and could be chemotherapy-induced in our GEMM after only 24 hours, suggesting an uncharacterized therapy-escape mechanism. Collectively, our findings improve the understanding of how neuroblastoma patient tumor heterogeneity is reflected in preclinical models, provides a comprehensive integrated resource, and a generalizable set of computational methodologies for the joint analysis of clinical and pre-clinical single-cell RNA-seq datasets.

An integrated single-cell RNA-seq map of human neuroblastoma tumors and preclinical models uncovers divergent mesenchymal-like gene expression programs.

BACKGROUND: Neuroblastoma is a common pediatric cancer, where preclinical studies suggest that a mesenchymal-like gene expression program contributes to chemotherapy resistance. However, clinical outcomes remain poor, implying we need a better understanding of the relationship between patient tumor heterogeneity and preclinical models. RESULTS: Here, we generate single-cell RNA-seq maps of neuroblastoma cell lines, patient-derived xenograft models (PDX), and a genetically engineered mouse model (GEMM). We develop an unsupervised machine learning approach ("automatic consensus nonnegative matrix factorization" (acNMF)) to compare the gene expression programs found in preclinical models to a large cohort of patient tumors. We confirm a weakly expressed, mesenchymal-like program in otherwise adrenergic cancer cells in some pre-treated high-risk patient tumors, but this appears distinct from the presumptive drug-resistance mesenchymal programs evident in cell lines. Surprisingly, however, this weak-mesenchymal-like program is maintained in PDX and could be chemotherapy-induced in our GEMM after only 24 h, suggesting an uncharacterized therapy-escape mechanism. CONCLUSIONS: Collectively, our findings improve the understanding of how neuroblastoma patient tumor heterogeneity is reflected in preclinical models, provides a comprehensive integrated resource, and a generalizable set of computational methodologies for the joint analysis of clinical and pre-clinical single-cell RNA-seq datasets.

Intraoperative detection of liver tumors aided by a fluorescence goggle system and multimodal imaging.

Real-time image guidance in the operating room is needed to improve instantaneous surgical decisions. Toward this goal, we utilized a new fluorescence goggle system and a near-infrared fluorescent dye approved for human use, indocyanine green, to demonstrate the feasibility of detecting liver tumors intraoperatively. The fluorescence goggle provided successful imaging of multifocal breast cancer metastases in mouse liver. Diffused tumor deposits as small as 0.8 mm in diameter were detected, which were not obvious without the fluorescence goggle. A combination of surface-weighted fluorescence imaging and deep tissue-sensitive ultrasound imaging allowed comprehensive image guidance with the fluorescence goggle system for tumor resection in a rabbit VX2 liver metastasis model. This multimodal detection and guided surgical intervention strategy using ultrasonic imaging and real-time intraoperative fluorescence guidance is a promising and innovative technology platform for improving surgical outcome of human patients with primary or metastatic liver cancer.

Detection of MMP-2 and MMP-9 activity in vivo with a triple-helical peptide optical probe.

We report a novel activatable NIR fluorescent probe for in vivo detection of cancer-related matrix metalloproteinase (MMP) activity. The probe is based on a triple-helical peptide substrate (THP) with high specificity for MMP-2 and MMP-9 relative to other members of the MMP family. MMP-2 and MMP-9 (also known as gelatinases) are specifically associated with cancer cell invasion and cancer-related angiogenesis. At the center of each 5 kDa peptide strand is a gelatinase sensitive sequence flanked by 2 Lys residues conjugated with NIR fluorescent dyes. Upon self-assembly of the triple-helical structure, the 3 peptide chains intertwine, bringing the fluorophores into close proximity and reducing fluorescence via quenching. Upon enzymatic cleavage of the triple-helical peptide, 6 labeled peptide chains are released, resulting in an amplified fluorescent signal. The fluorescence yield of the probe increases 3.8-fold upon activation. Kinetic analysis showed a rate of LS276-THP hydrolysis by MMP-2 (k(cat)/K(M) = 30,000 s(-1) M(-1)) similar to that of MMP-2 catalysis of an analogous fluorogenic THP. Administration of LS276-THP to mice bearing a human fibrosarcoma xenografted tumor resulted in a tumor fluorescence signal more than 5-fold greater than that of muscle. This signal enhancement was reduced by treatment with the MMP inhibitor Ilomostat, indicating that the observed tumor fluorescence was indeed enzyme mediated. These results are the first to demonstrate that triple-helical peptides are suitable for highly specific in vivo detection of tumor-related MMP-2 and MMP-9 activity.

An End-To-End Pipeline for Fully Automatic Morphological Quantification of Mouse Brain Structures From MRI Imagery.

Segmentation of mouse brain magnetic resonance images (MRI) based on anatomical and/or functional features is an important step towards morphogenetic brain structure characterization of murine models in neurobiological studies. State-of-the-art image segmentation methods register image volumes to standard presegmented templates or well-characterized highly detailed image atlases. Performance of these methods depends critically on the quality of skull-stripping, which is the digital removal of tissue signal exterior to the brain. This is, however, tedious to do manually and challenging to automate. Registration-based segmentation, in addition, performs poorly on small structures, low resolution images, weak signals, or faint boundaries, intrinsic to in vivo MRI scans. To address these issues, we developed an automated end-to-end pipeline called DeepBrainIPP (deep learning-based brain image processing pipeline) for 1) isolating brain volumes by stripping skull and tissue from T2w MRI images using an improved deep learning-based skull-stripping and data augmentation strategy, which enables segmentation of large brain regions by atlas or template registration, and 2) address segmentation of small brain structures, such as the paraflocculus, a small lobule of the cerebellum, for which DeepBrainIPP performs direct segmentation with a dedicated model, producing results superior to the skull-stripping/atlas-registration paradigm. We demonstrate our approach on data from both in vivo and ex vivo samples, using an in-house dataset of 172 images, expanded to 4,040 samples through data augmentation. Our skull stripping model produced an average Dice score of 0.96 and residual volume of 2.18%. This facilitated automatic registration of the skull-stripped brain to an atlas yielding an average cross-correlation of 0.98. For small brain structures, direct segmentation yielded an average Dice score of 0.89 and 5.32% residual volume error, well below the tolerance threshold for phenotype detection. Full pipeline execution is provided to non-expert users via a Web-based interface, which exposes analysis parameters, and is powered by a service that manages job submission, monitors job status and provides job history. Usability, reliability, and user experience of DeepBrainIPP was measured using the Customer Satisfaction Score (CSAT) and a modified PYTHEIA Scale, with a rating of excellent. DeepBrainIPP code, documentation and network weights are freely available to the research community.

Ablation of VLA4 in multiple myeloma cells redirects tumor spread and prolongs survival.

Multiple myeloma (MM) is a cancer of bone marrow (BM) plasma cells, which is increasingly treatable but still incurable. In 90% of MM patients, severe osteolysis results from pathological interactions between MM cells and the bone microenvironment. Delineating specific molecules and pathways for their role in cancer supportive interactions in the BM is vital for developing new therapies. Very Late Antigen 4 (VLA4, integrin α4ß1) is a key player in cell-cell adhesion and signaling between MM and BM cells. We evaluated a VLA4 selective near infrared fluorescent probe, LLP2A-Cy5, for in vitro and in vivo optical imaging of VLA4. Furthermore, two VLA4-null murine 5TGM1 MM cell (KO) clones were generated by CRISPR/Cas9 knockout of the Itga4 (α4) subunit, which induced significant alterations in the transcriptome. In contrast to the VLA4+ 5TGM1 parental cells, C57Bl/KaLwRij immunocompetent syngeneic mice inoculated with the VLA4-null clones showed prolonged survival, reduced medullary disease, and increased extramedullary disease burden. The KO tumor foci showed significantly reduced uptake of LLP2A-Cy5, confirming in vivo specificity of this imaging agent. This work provides new insights into the pathogenic role of VLA4 in MM, and evaluates an optical tool to measure its expression in preclinical models.

Targeting the spliceosome through RBM39 degradation results in exceptional responses in high-risk neuroblastoma models.

Aberrant alternative pre-mRNA splicing plays a critical role in MYC-driven cancers and therefore may represent a therapeutic vulnerability. Here, we show that neuroblastoma, a MYC-driven cancer characterized by splicing dysregulation and spliceosomal dependency, requires the splicing factor RBM39 for survival. Indisulam, a "molecular glue" that selectively recruits RBM39 to the CRL4-DCAF15 E3 ubiquitin ligase for proteasomal degradation, is highly efficacious against neuroblastoma, leading to significant responses in multiple high-risk disease models, without overt toxicity. Genetic depletion or indisulam-mediated degradation of RBM39 induces significant genome-wide splicing anomalies and cell death. Mechanistically, the dependency on RBM39 and high-level expression of DCAF15 determine the exquisite sensitivity of neuroblastoma to indisulam. Our data indicate that targeting the dysregulated spliceosome by precisely inhibiting RBM39, a vulnerability in neuroblastoma, is a valid therapeutic strategy.

Optical imaging of mammary and prostate tumors in living animals using a synthetic near infrared zinc(II)-dipicolylamine probe for anionic cell surfaces.

In vivo optical imaging shows that a fluorescent imaging probe, comprised of a near-infrared fluorophore attached to an affinity group containing two zinc(II)-dipicolylamine (Zn-DPA) units, targets prostate and mammary tumors in two different xenograft animal models. The tumor selectivity is absent with control fluorophores whose structures do not have appended Zn-DPA targeting ligands. Ex vivo biodistribution and histological analyses indicate that the probe is targeting the necrotic regions of the tumors, which is consistent with in vitro microscopy showing selective targeting of the anionic membrane surfaces of dead and dying cells.

Selective imaging of solid tumours via the calcium-dependent high-affinity binding of a cyclic octapeptide to phosphorylated Annexin A2.

The heterogeneity and continuous genetic adaptation of tumours complicate their detection and treatment via the targeting of genetic mutations. However, hallmarks of cancer such as aberrant protein phosphorylation and calcium-mediated cell signalling provide broadly conserved molecular targets. Here, we show that, for a range of solid tumours, a cyclic octapeptide labelled with a near-infrared dye selectively binds to phosphorylated Annexin A2 (pANXA2), with high affinity at high levels of calcium. Because of cancer-cell-induced pANXA2 expression in tumour-associated stromal cells, the octapeptide preferentially binds to the invasive edges of tumours and then traffics within macrophages to the tumour's necrotic core. As proof-of-concept applications, we used the octapeptide to detect tumour xenografts and metastatic lesions, and to perform fluorescence-guided surgical tumour resection, in mice. Our findings suggest that high levels of pANXA2 in association with elevated calcium are present in the microenvironment of most solid cancers. The octapeptide might be broadly useful for selective tumour imaging and for delivering drugs to the edges and to the core of solid tumours.

Long fluorescence lifetime molecular probes based on near infrared pyrrolopyrrole cyanine fluorophores for in vivo imaging.

Fluorescence lifetime (FLT) properties of organic molecules provide a new reporting strategy for molecular imaging in the near infrared (NIR) spectral region. Unfortunately, most of the NIR fluorescent dyes have short FLT typically clustered below 1.5 ns. In this study, we demonstrate that a new class of NIR fluorescent dyes, pyrrolopyrrole cyanine dyes, have exceptionally long FLTs ranging from 3 to 4 ns, both in vitro (dimethyl sulfoxide and albumin/water solutions) and in vivo (mice). These results provide a new window for imaging molecular processes, rejecting backscattered light and autofluorescence, and multiplexing imaging information with conventional NIR fluorescent dyes that absorb and emit light at similar wavelengths.

Dynamic noninvasive monitoring of renal function in vivo by fluorescence lifetime imaging.

Kidneys normally filter the blood of excess salts and metabolic products, such as urea, while retaining plasma proteins. In diseases such as multiple myeloma and diabetes mellitus, the renal function is compromised and protein escapes into the urine. In this study, we present the use of fluorescence lifetime imaging (FLI) to image excess serum protein in urine (proteinuria). The near-infrared fluorescent dye LS-288 has distinct lifetimes when bound to protein versus free in solution, providing contrast between the protein-rich viscera and the mostly protein-free bladder. FLI with LS-288 in mice revealed that fluorescence lifetime (FLT) differences in the bladder relative to surrounding tissues was due to the fractional contributions of the bound and unbound dye molecules. The FLT of LS-288 decreased in the case of proteinuria while fluorescence intensity was unchanged. The results show that FLI can be useful for the dynamic imaging of protein-losing nephropathy due to diabetes mellitus and other renal diseases and suggest the potential use of the FLI to distinguish tumors from fluid-filled cysts in the body.

Interleukin-15 Enhances Anti-GD2 Antibody-Mediated Cytotoxicity in an Orthotopic PDX Model of Neuroblastoma.

PURPOSE: Immunotherapy with IL2, GM-CSF, and an anti-disialoganglioside (GD2) antibody significantly increases event-free survival in children with high-risk neuroblastoma. However, therapy failure in one third of these patients and IL2-related toxicities pose a major challenge. We compared the immunoadjuvant effects of IL15 with those of IL2 for enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) in neuroblastoma. EXPERIMENTAL DESIGN: We tested ADCC against neuroblastoma patient-derived xenografts (PDX) in vitro and in vivo and examined the functional and migratory properties of NK cells activated with IL2 and IL15. RESULTS: In cell culture, IL15-activated NK cells induced higher ADCC against two GD+ neuroblastoma PDXs than did IL2-activated NK cells (P < 0.001). This effect was dose-dependent (P < 0.001) and was maintained across several effector-to-tumor ratios. As compared with IL2, IL15 also improved chemotaxis of NK cells, leading to higher numbers of tumorsphere-infiltrating NK cells in vitro (P = 0.002). In an orthotopic PDX model, animals receiving chemoimmunotherapy with an anti-GD2 antibody, GM-CSF, and a soluble IL15/IL15Rα complex had greater tumor regression than did those receiving chemotherapy alone (P = 0.012) or combined with anti-GD2 antibody and GM-CSF with (P = 0.016) or without IL2 (P = 0.035). This was most likely due to lower numbers of immature tumor-infiltrating NK cells (DX5+CD27+) after IL15/IL15Rα administration (P = 0.029) and transcriptional upregulation of Gzmd. CONCLUSIONS: The substitution of IL15 for IL2 leads to significant tumor regression in vitro and in vivo and supports clinical testing of IL15 for immunotherapy in pediatric neuroblastoma.

Predicting in vivo fluorescence lifetime behavior of near-infrared fluorescent contrast agents using in vitro measurements.

Fluorescence lifetime (FLT) information is complementary to intensity measurement and can be used to improve signal-to-background contrast and provide environment sensing capability. In this study, we evaluate the FLTs of eight near-infrared fluorescent molecular probes in vitro in various solvent mediums and in vivo to establish the correlation between the in vitro and in vivo results. Compared with other mediums, two exponential fittings of the fluorescence decays of dyes dissolved in aqueous albumin solutions accurately predict the range of FLTs observed in vivo. We further demonstrate that the diffusion of a near-infrared (NIR) reporter from a dye-loaded gel can be detected by FLT change in mice as a model of controlled drug release. The mean FLT of the NIR probe increases as the dye diffuses from the highly polar gel interior to the more lipophilic tissue environment. The two-point analysis demonstrates an efficient in vitro method for screening new NIR fluorescent reporters for use as FLT probes in vivo, thereby minimizing the use of animals for FLT screening studies.

Bio-inspired imager improves sensitivity in near-infrared fluorescence image-guided surgery.

Image-guided surgery can enhance cancer treatment by decreasing, and ideally eliminating, positive tumor margins and iatrogenic damage to healthy tissue. Current state-of-the-art near-infrared fluorescence imaging systems are bulky and costly, lack sensitivity under surgical illumination, and lack co-registration accuracy between multimodal images. As a result, an overwhelming majority of physicians still rely on their unaided eyes and palpation as the primary sensing modalities for distinguishing cancerous from healthy tissue. Here we introduce an innovative design, comprising an artificial multispectral sensor inspired by the Morpho butterfly's compound eye, which can significantly improve image-guided surgery. By monolithically integrating spectral tapetal filters with photodetectors, we have realized a single-chip multispectral imager with 1000 × higher sensitivity and 7 × better spatial co-registration accuracy compared to clinical imaging systems in current use. Preclinical and clinical data demonstrate that this technology seamlessly integrates into the surgical workflow while providing surgeons with real-time information on the location of cancerous tissue and sentinel lymph nodes. Due to its low manufacturing cost, our bio-inspired sensor will provide resource-limited hospitals with much-needed technology to enable more accurate value-based health care.

Diagnosis of LVAD Thrombus using a High-Avidity Fibrin-Specific 99mTc Probe.

Treatment of advanced heart failure with implantable LVADs is increasing, driven by profound unmet patient need despite potential serious complications: bleeding, infection, and thrombus. The experimental objective was to develop a sensitive imaging approach to assess early thrombus accumulation in LVADs under operational high flow and high shear rates. Methods: A monomeric bifunctional ligand with a fibrin-specific peptide, a short spacer, and 99mTc chelating amino acid sequence (F1A) was developed and compared to its tetrameric PEG analogue (F4A). Results:99mTc attenuation by LVAD titanium (1 mm) was 23%. 99mTc-F1A affinity to fibrin was Kd ~10 µM, whereas, the bound 99mTc-F4A probe was not displaced by F1A (120,000:1). Human plasma interfered with 99mTc-F1A binding to fibrin clot (p<0.05) in vitro, whereas, 99mTc-F4A targeting was unaffected. The pharmacokinetic half-life of 99mTc-F4A was 28% faster (124±41 min) than 99mTc-F1A (176±26 min) with both being bioeliminated through the urinary system with negligible liver or spleen biodistribution. In mice with carotid thrombus, 99mTc-F4A binding to the injured carotid was much greater (16.3±3.3 %ID/g, p=0.01) than that measured with an irrelevant negative control, 99mTc-I4A (3.4±1.6 %ID/g). In an LVAD mock flow-loop (1:1, PBS:human plasma:heparin) operating at maximal flow rate, 99mTc-F4A bound well to phantom clots in 2 min (p<0.05), whereas 99mTc-F1A had negligible targeting. Excised LVADs from patients undergoing pump exchange or heart transplant were rewired, studied in the mock flow loop, and found to have spatially variable fibrin accumulations in the inlet and outlet cannulas and bearings. Conclusions:99mTc-F4A is a high-avidity prototype probe for characterizing thrombus in LVADs that is anticipated to help optimize anticoagulation, reduce thromboembolic events, and minimize pump exchange.

Evaluating Acetate Metabolism for Imaging and Targeting in Multiple Myeloma.

PURPOSE: We hypothesized that in multiple myeloma cells (MMC), high membrane biosynthesis will induce acetate uptake in vitro and in vivo Here, we studied acetate metabolism and targeting in MMC in vitro and tested the efficacy of 11C-acetate-positron emission tomography (PET) to detect and quantitatively image myeloma treatment response in vivo EXPERIMENTAL DESIGN: Acetate fate tracking using 13C-edited-1H NMR (nuclear magnetic resonance) was performed to study in vitro acetate uptake and metabolism in MMC. Effects of pharmacological modulation of acetate transport or acetate incorporation into lipids on MMC cell survival and viability were assessed. Preclinical mouse MM models of subcutaneous and bone tumors were evaluated using 11C-acetate-PET/CT imaging and tissue biodistribution. RESULTS: In vitro, NMR showed significant uptake of acetate by MMC and acetate incorporation into intracellular metabolites and membrane lipids. Inhibition of lipid synthesis and acetate transport was toxic to MMC, while sparing resident bone cells or normal B cells. In vivo, 11C-acetate uptake by PET imaging was significantly enhanced in subcutaneous and bone MMC tumors compared with unaffected bone or muscle tissue. Likewise, 11C-acetate uptake was significantly reduced in MM tumors after treatment. CONCLUSIONS: Uptake of acetate from the extracellular environment was enhanced in MMC and was critical to cellular viability. 11C-Acetate-PET detected the presence of myeloma cells in vivo, including uptake in intramedullary bone disease. 11C-Acetate-PET also detected response to therapy in vivo Our data suggested that acetate metabolism and incorporation into lipids was crucial to MM cell biology and that 11C-acetate-PET is a promising imaging modality for MM. Clin Cancer Res; 23(2); 416-29. ©2016 AACR.