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.

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 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.

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.

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.

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.

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.

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.

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.

Imaging of radicals following injury or acute stress in peripheral nerves with activatable fluorescent probes.

Peripheral nerve injury evokes a complex cascade of chemical reactions including generation of molecular radicals. Conversely, the reactions within nerve induced by stress are difficult to directly detect or measure to establish causality. Monitoring these reactions in vivo would enable deeper understanding of the nature of the injury and healing processes. Here, we utilized near-infrared fluorescence molecular probes delivered via intra-neural injection technique to enable live, in vivo imaging of tissue response associated with nerve injury and stress. These initially quenched fluorescent probes featured specific sensitivity to hydroxyl radicals and become fluorescent upon encountering reactive oxygen species (ROS). Intraneurally delivered probes demonstrated rapid activation in injured rat sciatic nerve but minimal activation in normal, uninjured nerve. In addition, these probes reported activation within sciatic nerves of living rats after a stress caused by a pinprick stimulus to the abdomen. This imaging approach was more sensitive to detecting changes within nerves due to the induced stress than other techniques to evaluate cellular and molecular changes. Specifically, neither histological analysis of the sciatic nerves, nor the expression of pain and stress associated genes in dorsal root ganglia could provide statistically significant differences between the control and stressed groups. Overall, the results demonstrate a novel imaging approach to measure ROS in addition to the impact of ROS within nerve in live animals.

Enhancing in vivo tumor boundary delineation with structured illumination fluorescence molecular imaging and spatial gradient mapping.

Fluorescence imaging, in combination with tumor-avid near-infrared (NIR) fluorescent molecular probes, provides high specificity and sensitivity for cancer detection in preclinical animal models, and more recently, assistance during oncologic surgery. However, conventional camera-based fluorescence imaging techniques are heavily surface-weighted such that surface reflection from skin or other nontumor tissue and nonspecific fluorescence signals dominate, obscuring true cancer-specific signals and blurring tumor boundaries. To address this challenge, we applied structured illumination fluorescence molecular imaging (SIFMI) in live animals for automated subtraction of nonspecific surface signals to better delineate accumulation of an NIR fluorescent probe targeting α4ß1 integrin in mice bearing subcutaneous plasma cell xenografts. SIFMI demonstrated a fivefold improvement in tumor-to-background contrast when compared with other full-field fluorescence imaging methods and required significantly reduced scanning time compared with diffuse optical spectroscopy imaging. Furthermore, the spatial gradient mapping enhanced highlighting of tumor boundaries. Through the relatively simple hardware and software modifications described, SIFMI can be integrated with clinical fluorescence imaging systems, enhancing intraoperative tumor boundary delineation from the uninvolved tissue.

Fluorescence Imaging of Inflammation in Live Animals.

Inflammation is associated with many diseases, such as stroke, cancer, and atherosclerosis. Noninvasive in vivo monitoring of inflammation can provide deeper understanding of such diseases, which might help to develop better treatment. Inflammation normally causes neutrophils and macrophages to generate reactive oxygen species (ROS) as the destruction tool, which can be used as a biomarker for inflammation. Near-infrared (NIR) window is optimal for in vivo fluorescence imaging owing to the reduced autofluorescence and low attenuation of light in biological tissues. Among NIR fluorescent probes, activatable probes have the promise of achieving high imaging contrast. In this chapter, we describe the method for in vivo fluorescence imaging of inflammation using a ROS-activatable NIR probe.

Fluorescence Lifetime Imaging of Cancer In Vivo.

Optical imaging of fluorescent reporters in animal models of cancer has become a common tool in oncologic research. Fluorescent reporters including fluorescent proteins, organic dyes, and inorganic photonic materials are used in fluorescence spectroscopy, microscopy, and whole body preclinical imaging. Fluorescence lifetime imaging provides additional, quantitative information beyond that of conventional fluorescence intensity signals, enabling signal multiplexing, background separation, and biological sensing unique to fluorescent materials.

Anti-tax interacting protein-1 (TIP-1) monoclonal antibody targets human cancers.

Radiation-inducible neo-antigens are proteins expressed on cancer cell surface after exposure to ionizing radiation (IR). These neo-antigens provide opportunities to specifically target cancers while sparing normal tissues. Tax interacting protein-1 (TIP-1) is induced by irradiation and is translocated to the surface of cancer cells. We have developed a monoclonal antibody, 2C6F3, against TIP-1.Epitope mapping revealed that 2C6F3 binds to the QPVTAVVQRV epitope of the TIP-1 protein. 2C6F3 binds to the surface of lung cancer (A549, LLC) and glioma (D54, GL261) cell lines. 2C6F3 binds specifically to TIP-1 and ELISA analysis showed that unconjugated 2C6F3 efficiently blocked binding of radiolabeled 2C6F3 to purified TIP-1 protein. To study in vivo tumor binding, we injected near infrared (NIR) fluorochrome-conjugated 2C6F3 via tail vein in mice bearing subcutaneous LLC and GL261 heterotopic tumors. The NIR images indicated that 2C6F3 bound specifically to irradiated LLC and GL261 tumors, with little or no binding in un-irradiated tumors.We also determined the specificity of 2C6F3 to bind tumors in vivo using SPECT/CT imaging. 2C6F3 was conjugated with diethylene triamine penta acetic acid (DTPA) chelator and radiolabeled with 111Indium (111In). SPECT/CT imaging revealed that 111In-2C6F3 bound more to the irradiated LLC tumors compared to un-irradiated tumors. Furthermore, injection of DTPA-2C6F3 labeled with the therapeutic radioisotope, 90Y, (90Y-DTPA-2C6F3) significantly delayed LLC tumor growth. 2C6F3 mediated antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP) in vitro.In conclusion, the monoclonal antibody 2C6F3 binds specifically to TIP-1 on cancer and radio-immunoconjugated 2C6F3 improves tumor control.

Ex Vivo and In Vivo Evaluation of Overexpressed VLA-4 in Multiple Myeloma Using LLP2A Imaging Agents.

UNLABELLED: Very-late-antigen-4 (VLA-4, α4ß1 integrin, CD49d/CD29) is a transmembrane adhesion receptor that plays an important role in cancer and immune responses. Enhanced VLA-4 expression has been observed in multiple myeloma (MM) cells and surrounding stroma. VLA-4 conformational activation has been associated with MM pathogenesis. VLA-4 is a promising MM imaging and therapeutic biomarker. METHODS: Specificity of (64)Cu-LLP2A ((64)Cu-CB-TE1A1P-PEG4-LLP2A), a high-affinity VLA-4 peptidomimetic-based radiopharmaceutical, was evaluated in α4 knock-out mice and by competitive blocking in wild-type tumor-bearing mice. (64)Cu-LLP2A PET/CT (static and dynamic) imaging was conducted in C57BL6/KaLwRij mice bearing murine 5TGM1-GFP syngeneic tumors generated after intravenous injection via the tail. Blood samples were collected for serum protein electrophoresis. Bone marrow and splenic cells extracted from tumor-bearing and control mice (n= 3/group) were coincubated with the optical analog LLP2A-Cy5 and mouse B220, CD4, Gr1, and Mac1 antibodies and analyzed by fluorescence-activated cell sorting. Human radiation dose estimates for (64)Cu-LLP2A were extrapolated from mouse biodistribution data (6 time points, 0.78 MBq/animal, n= 4/group). Ten formalin-fixed paraffin-embedded bone marrow samples from deceased MM patients were stained with LLP2A-Cy5. RESULTS: (64)Cu-LLP2A and LLP2A-Cy5 demonstrated high specificity for VLA-4-positive mouse 5TGM1-GFP myeloma and nonmalignant inflammatory host cells such as T cells and myeloid/monocytic cells. Ex vivo flow cytometric analysis supported a direct effect of myeloma on increased VLA-4 expression in host hematopoietic microenvironmental elements. SUVs and the number of medullar lesions detected by (64)Cu-LLP2A PET corresponded with increased monoclonal (M) protein (g/dL) in tumor-bearing mice over time (3.29 ± 0.58 at week 0 and 9.97 ± 1.52 at week 3). Dynamic PET with (64)Cu-LLP2A and (18)F-FDG demonstrated comparable SUV in the prominent lesions in the femur. Human radiation dose estimates indicated urinary bladder wall as the dose-limiting organ (0.200 mGy/MBq), whereas the dose to the red marrow was 0.006 mGy/MBq. The effective dose was estimated to be 0.017 mSv/MBq. Seven of the ten human samples displayed a high proportion of cells intensely labeled with LLP2A-Cy5 probe. CONCLUSION: (64)Cu-LLP2A and LLP2A-Cy5 demonstrated binding specificity for VLA-4 in an immune-competent murine MM model. (64)Cu-LLP2A displayed favorable dosimetry for human studies and is a potential imaging candidate for overexpressed VLA-4.

Evaluation of Dynamic Optical Projection of Acquired Luminescence for Sentinel Lymph Node Biopsy in Large Animals.

Open surgery requiring cytoreduction still remains the primary treatment course for many cancers. The extent of resection is vital for the outcome of surgery, greatly affecting patients' follow-up treatment including need for revision surgery in the case of positive margins, choice of chemotherapy, and overall survival. Existing imaging modalities such as computed tomography, magnetic resonance imaging, and positron emission tomography are useful in the diagnostic stage and long-term monitoring but do not provide the level of temporal or spatial resolution needed for intraoperative surgical guidance. Surgeons must instead rely on visual evaluation and palpation in order to distinguish tumors from surrounding tissues. Fluorescence imaging provides high-resolution, real-time mapping with the use of a contrast agent and can greatly enhance intraoperative imaging. Here we demonstrate an intraoperative, real-time fluorescence imaging system for direct highlighting of target tissues for surgical guidance, optical projection of acquired luminescence (OPAL). Image alignment, accuracy, and resolution was determined in vitro prior to demonstration of feasibility for operating room use in large animal models of sentinel lymph node biopsy. Fluorescence identification of regional lymph nodes after intradermal injection of indocyanine green was performed in pigs with surgical guidance from the OPAL system. Acquired fluorescence images were processed and rapidly reprojected to highlight indocyanine green within the true surgical field. OPAL produced enhanced visualization for resection of lymph nodes at each anatomical location. Results show the optical projection of acquired luminescence system can successfully use fluorescence image capture and projection to provide aligned image data that is invisible to the human eye in the operating room setting.

Temperature-dependent shape-responsive fluorescent nanospheres for image-guided drug delivery.

Temperature-responsive nanoparticles used in conjunction with hyperthermia promise to provide synergistic effects for increasing drug efficacy. We propose a near-infared (NIR) fluorescent system based on a upper critical solution temperature (UCST) polymer, ISP2, integrated with a NIR fluorescent dye HITC for in vivo tracking. The system forms a nanoparticle that increases its volume as temperature increases, similar to the expansion of a Hoberman sphere. The nanospheres nearly doubled in size, from 80 nm to 140 nm, during a temperature increase from 40°C to 60°C.