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We present two new synthetic strategies to rigid multivalent scaffolds of the general structure 1 based on adamantane. Both routes start from arylated adamantane derivatives and give the target compounds 12 and 18 in 5 and 7 steps, respectively. These scaffolds have been designed for the assembly of multivalent binders for cell surface epitopes. The adamantane nucleus exposes three carboxylic acid groups in a well-defined tripodal geometry for conjugation of targeting ligands. In addition, an amino group at the fourth bridgehead position provides a flexible linker for attachment of effector molecules such as contrast agents, radiotracers, or cytotoxins without interfering with the cell binding process.

Near-infrared (NIR) fluorescence has the potential to provide surgeons with real-time intraoperative image-guidance. Increasing the signal-to-background ratio of fluorescent agents involves delivering a controllable excitation fluence rate of proper wavelength and/or using complementary imaging techniques such as FLIM. In this study we describe a low-cost linear driver circuit capable of driving Light Emitting Diodes (LEDs) from DC to 35 MHz, at high power, and which permit fluorescence CW and lifetime measurements. The electronic circuit Gerber files described in this article and the list of components are available online at www.frangionilab.org.

Achieving an adequate signal to background ratio is of paramount importance for targeted diagnostics. We will present recent results in near-infrared time and spatial frequency methods applied to large field-of-view image-guided surgery.

We have developed a versatile nanoparticle construct using a compact cysteine coating on a CdSe(ZnCdS) core(shell) nanocrystal (QD-Cys) that is biologically compatible, exceptionally compact, highly fluorescent, and easily functionalized. The small hydrodynamic diameter of QD-Cys ( approximately 6 nm) allows for renal clearance of these nanoparticles in rat models. Moreover, the ability to directly conjugate to QD-Cys opens up the possibility of functionalized nanocrystals for in vivo targeted imaging, in which small targeting molecules can be appended to QD-Cys, and unbound QDs can be rapidly cleared to achieve high signal/noise ratios and to reduce background toxicity.

Invisible near-infrared light is safe and it penetrates relatively deeply through tissue and blood without altering the surgical field. Our hypothesis was that near-infrared fluorescence imaging would enable visualization of the ureteral anatomy and flow intraoperatively and in real time.CW800-CA (LI-COR, Lincoln, Nebraska), the carboxylic acid form of near-infrared fluorophore IRDye 800CW, was injected intravenously, and its renal clearance kinetics and imaging performance were quantified in 350 gm rats and 35 kg pigs. High performance liquid chromatography and electrospray time-of-flight mass spectrometry were used to characterize CW800-CA metabolism in urine. The clinically available near-infrared fluorophore indocyanine green was also used via retrograde injection into the ureter. Using the 2 near-infrared fluorophores the ureters were imaged under the conditions of steady state, intraluminal foreign bodies and injury.In rat models the highest signal-to-background ratio for visualization occurred after intravenous injection of 7.5 microg/kg CW800-CA with values of 4.0 or greater and 2.3 or greater at 10 and 30 minutes, respectively. In pig models 7.5 microg/kg CW800-CA clearly visualized the normal ureter and intraluminal foreign bodies as small as 2.5 mm in diameter. Retrograde injection of 10 microM indocyanine green also permitted the detection of normal ureter and pinpointed urine leakage caused by injury. Electrospray time-of-flight mass spectrometry, and absorbance and fluorescence spectral analysis confirmed that the fluorescent material in urine was chemically identical to CW800-CA.A convenient intravenous injection of CW800-CA or direct injection of indocyanine green permits high sensitivity visualization of the ureters under steady state and abnormal conditions using invisible light.

Background. Homogenous distribution of cardioplegia delivered to the myocardium has been identified as an important predictor of post-cardiopulmonary bypass ventricular recovery and function. Presently, a method to determine adequate distribution of cardioplegia in patients during cardiac surgery does not exist. The goal of this study was to evaluate the feasibility of quantifying cardioplegia delivery using a novel, noninvasive optical method. Such a system would permit instantaneous imaging of jeopardized myocardium and allow immediate, intraoperative corrective measures. Methods. We have previously developed a portable, intraoperative near-infrared (NIR) fluorescence imaging system for use in large animal cardiac surgery that simultaneously displays color video and NIR fluorescent images of the surgical field. By introducing exogenous, NIR fluorophores, specific cardiac functions can be visualized in real-time. Results. In a porcine cardiopulmonary bypass model, we demonstrate that the FDA-approved intravascular fluorophore indocyanine green (ICG) permits real-time assessment of cardioplegia delivery. ICG was injected into an aortic root and/or transatrial coronary sinus catheter during delivery of crystalloid cardioplegia solution. Segmental distribution was immediately noted at the time of injection. In a subset of animals, simulated coronary occlusions resulted in imaging defects consistent with poor cardioplegia delivery and jeopardized myocardium. Videodensitometric analysis was performed on-line to quantify distribution to the right ventricle and left ventricle. Conclusions. We report the development of a novel, noninvasive, intraoperative technique that can easily and safely provide a visual assessment of cardioplegia delivery (antegrade and/or retrograde) and that offers the potential to quantify the relative segmental distribution during cardiac surgical procedures.

Small-molecule ligands specific for prostate-specific membrane antigen (PSMA) have the potential to improve prostate cancer imaging. However, highly charged ligands are difficult to label with 99mTc and to purify. In this study, we present an adamantane-trimerized small molecule that has nanomolar binding to PSMA and also has 12 negative charges. Methods: To convert this molecule into a clinically viable SPECT diagnostic, we have developed a simple, cartridge-based, solid-phase prelabeling strategy that, within 25 min, converts readily available and inexpensive 99mTc-pertechnetate into a chemically pure complex, with a reactive N-hydroxysuccinimide (NHS) ester, in neat organic solvent. This stable intermediate can label any amine-containing small molecule or peptide with 99mTc in 1 step, with high specific activity and without the need for high-performance liquid chromatography (HPLC). Results: Solid-phase conversion of 99mTc-pertechnetate to 99mTc-MAS3-NHS (MAS3 is S-acetylmercaptoacetyltriserine) could be completed in 25 min, with >99% radiochemical purity and with no coligands present. This intermediate was then conjugated to adamantane-trimerized GPI (2[(3-amino-3-carboxypropyl)(hydroxy)(phosphinyl)-methyl]pentane-1,5-dioic acid) in 1 step with >95% yield and no need for HPLC purification. The final molecule bound specifically to living human tumor cells expressing PSMA on their surface. Quantitative comparison was made among GPI monomer, GPI trimer, and their 99mTc-derivatives. Conclusion: Our study describes a simple cartridge-based conversion of 99mTc-pertechnetate to a useful, preloaded NHS ester intermediate that takes only 25 min to prepare and results in >99% radiochemical purity. Using this chemistry, we produced a high-specific-activity, 99mTc-labeled, PSMA-targeted small molecule and demonstrate γ-ray radioscintigraphic imaging of living human prostate cancer cells.

We have developed a low-cost, safe, and easy to use NIR fluorescence intraoperative imaging system that permits the surgeon to see, in real time, surgical anatomy and invisible NIR fluorescence simultaneously.

With conventional methodology, sentinel lymph node (SLN) mapping of invasive urinary bladder cancer is technically challenging. This study was performed to determine the utility of invisible, near-infrared fluorescent (NIRF) light for patient-specific SLN mapping, in real time under complete image guidance.Lymphatic tracers, injection volume, NIRF excitation fluence rate, light collection of emitted fluorescence, and degree of bladder distension were systematically optimized in normal dogs and pigs. SLN mapping was then performed in pet dogs with naturally occurring invasive transitional cell carcinoma (InvTCC) of the urinary bladder, which closely mimics the human disease.NIRF albumin (hydrodynamic diameter [HD], 7.4 nm) and NIRF quantum dots (15-20 nm HD) injected into the bladder wall resulted in identification of draining lymph nodes (LNs) in under 3 min. In both species, considerable variability in the lymphatic drainage was observed among individuals. Optimal SLN mapping was achieved with the use of superficial, serosal injection of NIRF tracer, with the bladder distended to an intraluminal pressure of 20-40 cm H(2)O. In dogs with InvTCC, NIRF tracers identified SLNs that were confirmed histologically to harbor metastases.The use of invisible NIRF light permits real-time, patient-specific identification of SLNs that drain bladder cancer. Intraluminal bladder pressure is a key parameter that needs to be controlled for optimal results.

Understanding lymph drainage patterns of the peritoneum could assist in staging and treatment of gastrointestinal and ovarian malignancies. Sentinel lymph nodes (SLNs) have been identified for solid organs and the pleural space. Our purpose was to determine whether the peritoneal space has a predictable lymph node drainage pattern.Rats received intraperitoneal injections of near-infrared (NIR) fluorescent tracers: namely, quantum dots (designed for retention in SLNs) or human serum albumin conjugated with IRDye800 (HSA800; designed for lymphatic flow beyond the SLN). A custom imaging system detected NIR fluorescence at 10 and 20 minutes and 1, 4, and 24 hours after injection. To determine the contribution of viscera to peritoneal lymphatic flow, additional cohorts received bowel resection before NIR tracer injection. Associations with appropriate controls were assessed with the chi(2) test.Quantum dots drained to the celiac, superior mesenteric, and periportal lymph node groups. HSA800 drained to these same groups at early time points but continued flowing to the mediastinal lymph nodes via the thoracic duct. After bowel resection, both tracers were found in the thoracic, not abdominal, lymph node groups. Additionally, HSA800 was no longer found in the thoracic duct but in the anterior chest wall and diaphragmatic lymphatics.The peritoneal space drains to the celiac, superior mesenteric, and periportal lymph node groups first. Lymph continues via the thoracic duct to the mediastinal lymph nodes. Bowel lymphatics are a key determinant of peritoneal lymph flow, because bowel resection shifts lymph flow directly to the intrathoracic lymph nodes via chest wall lymphatics.

Sentinel lymph node (SLN) mapping is an important cancer surgery during which the first lymph node draining the site of a tumor is identified, resected, and analyzed for the presence or absence of malignant cells. Fluorescent semiconductor nanocrystals (quantum dots) of the appropriate size, charge, and emission wavelength permit this surgery to be performed rapidly, with high sensitivity, and under complete image guidance. We describe the materials and methods necessary for the production and characterization of type II near-infrared (NIR) fluorescent quantum dots, which have been optimized for SLN mapping. They contain a CdTe core, CdSe shell, and highly anionic, oligomeric phosphine organic coating. We also describe how to utilize such quantum dots in animal model systems of SLN mapping.

Mammography is currently the gold standard for the early detection of breast cancer.[1,2] However, mammography suffers from relatively low sensitivity and specificity,[3] and mammographic screening is limited in certain patient populations[4] and breast densities.[5] These limitations have spurred interest in alternate modalities to detect breast cancer. An important diagnostic feature of mammography is the presence of microcalcifications,[1] which result from deposition of calcium salts in breast tissue. Usually these salts are in the form of hydroxyapatite (HA),[6] which is also the first calcium salt deposited by osteoblasts during normal bone growth. In this communication, we describe a simplified, reproducible synthetic scheme for 3-amino tetramethyl 1-hydroxypropylidenebisphosphonate (methylester-protected pamidronate) and its use in preparing novel near-infrared (NIR) fluorescent optical contrast agents specific for HA. Such contrast agents show remarkable specificity for HA over other calcium salts. Our synthetic scheme permits preparative production in high-yield, and thus enables pre-clinical studies of contrast agent performance in large animal model systems approaching the size of humans. We also demonstrate that the agent Pam800 provides real-time, intraoperative NIR fluorescence imaging of HA in soft tissue and bone. Bisphosphonates are widely used for the treatment of bone metastases, and evidence suggests that these compounds provide benefit to breast cancer patients with metastases to bone.[7] Bisphosphonates are analogues of endogenous pyrophosphates in which the hydrolyzable oxygen atom that separates the two phosphate groups is replaced with a more stable carbon atom. The P-C-P structure is responsible for giving bisphosphonates their high affinity for bone, which can be further enhanced by addition of a hydroxyl group at the central carbon atom.[8] In vivo, bisphosphonates bind strongly to HA on the bone surface and are preferentially delivered to sites of increased bone formation or resorption. They are potent inhibitors of osteoclast-mediated bone resorption[9] and are effective in lowering serum calcium concentrations in patients with hypercalcemia of malignancy.[10,11] Treatment with bisphosphonates has also been shown to reduce significantly skeletal morbidity, and to improve quality of life in breast cancer patients with bone metastases.[11] We have previously reported NIR fluorescence imaging of HA in small animal model systems using our first generation molecule Pam78.[12-14] These studies were severely limited by the low yield of contrast agent synthesis, which precluded development of more relevant large animal models. The aim of this study was to develop a simplified synthetic scheme for 1-hydroxy-1,1-bisphosphonate derivatives conjugated to NIR fluorophores, and to validate contrast agent performance in vivo. Due to the extreme insolubility of pamidronate in organic solvents, and the base lability of heptamethine indocyanines, previously described synthetic schemes were carried out in aqueous media. As such, yields were low (≈ 18-21%) and purification was difficult.[13] This prompted us to develop methods for the synthesis of protected pamidronate, and its subsequent conjugation to heptamethine indocyanine fluorophores, under non-aqueous conditions. From the literature,[15] we found that by Michaelis-Arbuzov reaction of acyl halide with trialkyl phosphite and dialkyl phosphite, one can synthesize phosphate-protected bisphosphonates, a precursor for NIR fluorophore conjugation. However, previously published methods utilize strongly basic conditions and elevated temperature, and the reaction is complicated by rearrangement. This rearrangement generates isomeric compounds containing two chemically different phosphorus-carbon bonds, including a tetraalkyl phosphono-phosphate.[16,17] To obtain Pam800 (7), we first devised a synthesis of methylester-protected pamidronate (4a), based on the one-pot synthesis method of Tromelin et al.[18] In our method, β-alanine was converted to its acyl halide form, followed by one-pot treatment with trimethylphosphite and dimethyl phosphite at 0°C to room temperature (RT) over 30 min. Methylester-protected pamidronate (4a) was obtained without the use of any base or heating within an hour, in two chemical steps, with an overall yield of 91% (Scheme 1). While performing reactions at elevated temperature or in the presence of strong base, isomerization was a major obstacle, which could be avoided by performing a one-pot reaction of acyl halide and eliminating the isolation steps for the unstable intermediate α-ketophosphonate (3). Unwanted isomerized product (4b) was then less than 5% of total during analytical scale synthesis (Figure 1, Supporting Information, SI). Figure 1 Absorption and fluorescence spectra of Pam800 (7) at a concentration of 2 μM in PBS buffer. Peak absorption and fluorescence emission wavelengths were 778 nm and 799 nm, respectively. Scheme 1 Reagents and conditions The conjugation of NIR fluorophore IRDye®800CW NHS ester (5) to methylester-protected pamidronate (4a) was performed in DMSO in the presence of N-methyl morpholine. The reaction was performed in the dark at RT, and formation of Pam800 methyl ester (6a) was monitored by HPLC. Nucleophilic attack by the primary amine of methylester-protected pamidronate (4a) resulted in displacement of the NHS group and formation of a stable amide linkage between IRDye®800CW and protected pamidronate (Scheme 2). Note that approximately 8% of the undesired isomeric compound (6b) was observed (Figure ​(Figure2,2, SI). Figure 2 The specificity of Pam800 for HA and other calcium salts. A, Crystals of HA, calcium phosphate (CP; Ca3O8P2), calcium oxalate (CO; Ca(COO)2), calcium carbonate (CC; CaCO3), and calcium pyrophosphate (CPP; Ca2O7P2) were stained with 100 nM Pam800, washed, ... Scheme 2 Reagents and conditions Dealkylation of bisphosphonic ester functions was carried out by using bromotrimethylsilane in DMF followed by methanolysis. The final compound Pam800 (7) was purified by preparative HPLC and analyzed by LCMS and NMR. The typical overall yield of Pam800 (7) during preparative synthesis was 71%. Prior to in vivo experiments, Pam800 (7) was fully characterized for its spectral properties (Figure 1) and calcium salt specificity (Figure 2). Peak absorption (778 nm) and emission (799 nm) of Pam800 are located within the “NIR window,”[19] an area of the electromagnetic spectrum that maximizes photon penetration and recovery in living tissue. In PBS, Pam800′s extinction coefficient at 778 nm was 174,000 M-1cm-1 and its quantum yield was 6.4%. In 100% fetal bovine serum, its quantum yield was 6.0%. To determine the specificity of Pam800 for HA over other calcium salts, we incubated HA and the phosphate, oxalate, carbonate, and pyrophosphate salts of calcium with 100 nM Pam800 for 30 min at RT with constant motion, then washed 4 times with a 100-fold excess of PBS. As shown in Figure 2B, Pam800 has a greater than 8-fold specificity for HA over other calcium salts found in the body, and permits NIR fluorescence detection of HA with high sensitivity. We finally characterized Pam800 for its in vivo performance during image-guided surgery. Surgery was performed on 30 kg Yorkshire pigs whose organs are roughly the same size as human organs. Pam800 was administered intravenously to anesthetized pigs at a dose of 0.06 μmol/kg (1.8 mg total). After 2-4 hours of clearance, image-guided surgery was performed using a custom intraoperative NIR fluorescence imaging system.[20] As shown in Figure 3A, Pam800 provided high sensitivity detection of normal bones, and could guide surgery both prior to, and after, the skin incision, since NIR penetrates relatively deeply into living tissue. To replicate breast cancer microcalcification as would be encountered during breast surgery, 5 mg each of HA and calcium oxalate crystals were injected subcutaneously. Importantly, Pam800 could also detect soft tissue-embedded HA crystals with high sensitivity and specificity (Figure 3B). In particular, Pam800 correctly identified HA, which is common in malignant breast disease, but not calcium oxalate, which is typically deposited in benign lesions and is rarely seen in malignancies.[21,22] Figure 3 Real-Time Intraoperative NIR Fluorescence Imaging in 30 kg Yorkshire pigs. (A) After intravenous injection and 2-4 hours of clearance, Pam800 permits high-sensitivity, real-time image guidance of normal bones during surgery. Shown are bones of the forearm ... In conclusion, we have described the simple and reliable production of a methylester-protected pamidronate derivative that can be used in organic solvent to produce novel optical contrast agents, such as Pam800 (7). Pam800 can be produced in preparative quantities, at high yield, and provides real-time image-guidance to surgeons who require visualization of bone and/or tissue calcification. Finally we have developed a straightforward, reproducible large animal model system that could be used to validate optical contrast agents specific for breast cancer microcalcifications

The production of disease-targeted agents requires the covalent conjugation of a targeting molecule with a contrast agent or therapeutic, followed by purification of the product to homogeneity. Typical targeting molecules, such as small molecules and peptides, often have high charge-to-mass ratios and/or hydrophobicity. Contrast agents and therapeutics themselves are also diverse, and include lanthanide chelates for MRI, 99mTc chelates for SPECT, 90Y chelates for radiotherapy, 18F derivatives for PET, and heptamethine indocyanines for near-infrared fluorescent optical imaging. We have constructed a general-purpose HPLC/mass spectrometry platform capable of purifying virtually any targeted agent for any modality. The analytical sub-system is composed of a single dual-head pump that directs mobile phase to either a hot cell for the purification of radioactive agents or to an ES-TOF MS for the purification of nonradioactive agents. Nonradioactive agents are also monitored during purification by ELSD, absorbance and fluorescence. The preparative sub-system is composed of columns and procedures that permit rapid scaling from the analytical system. To demonstrate the platform's utility, we describe the preparation of five small molecule derivatives specific for prostate-specific membrane antigen (PSMA): a gadolinium derivative for MRI, indium, rhenium and technetium derivatives for SPECT, and an yttrium derivative for radiotherapy. All five compounds are derived from a highly anionic targeting ligand engineered to have a single nucleophile for N-hydroxysuccinimide-based conjugation. We also describe optimized column/mobile phase combinations and mass spectrometry settings for each class of agent, and discuss strategies for purifying molecules with extreme charge and/or hydrophobicity. Taken together, our study should expedite the development of disease-targeted, multimodality diagnostic and therapeutic agents. Copyright © 2006 John Wiley & Sons, Ltd.

Invisible near-infrared (NIR) fluorescent light permits high sensitivity, real-time image-guidance during oncologic surgery without changing the look of the surgical field. In this study, we complete pre-clinical development of the technology for sentinel lymph node (SLN) mapping using a large animal model of spontaneous melanoma.Sinclair swine with spontaneous melanoma metastatic to regional lymph nodes were used because of their similarity to human melanoma. Organic lymphatic tracers tested included FDA-approved indocyanine green adsorbed non-covalently to human serum albumin (HSA), and NIR fluorophore CW800 conjugated covalently to HSA (HSA800). The inorganic/organic hybrid tracer tested was type II NIR quantum dots with an anionic coating. Primary tumors received four peri-tumoral injections of each tracer, with a fluorophore dose of 100 pmol to 1 nmol per injection. SLN mapping and image-guided resection were performed in real-time.Each of the 3 lymphatic tracers was injected into n = 4 separate primary melanomas in a total of 6 animals. All 12 injections resulted in identification of the SLN(s) and their associated lymphatic channels within 1 minute in 100% of cases, despite highly pigmented skin and black fur. Hydrodynamic diameter had a profound impact on tracer behavior in vivo.This study completes the pre-clinical development of NIR fluorescence-guided SLN mapping and provides insight into imaging system optimization and tracer choice for future human clinical trials. The technology is likely to eliminate the need for radioactive and colored tracers, permits real-time image guidance throughout the procedure, and assists the pathologist in tissue analysis.

Challenges abound when academic institutions try to bring new in vivo diagnostic agents to the clinic.

Gelatin hydrogel microspheres (GHMs) have been reported as novel non-viral vectors for gene or protein delivery (GHM therapy). However, the components of an effective catheter-based delivery strategy for GHM therapy are unknown. We evaluated the effectiveness of three catheter-based strategies for cardiac GHM therapy: (1) antegrade injection (AI) via coronary arteries; (2) retrograde injection (RI) via coronary veins; and (3) direct myocardial injection (DI) via the coronary sinus. AI distributed microspheres homogeneously throughout the target area with 73+/-11% retention. RI scattered microspheres non-homogenously with 22+/-8% retention. DI distributed microspheres in the needle-advanced area with 47+/-14% retention. However, despite high efficiency, AI did not show biological effects of inducing angiogenesis from basic fibroblast growth factor bound to GHMs. Furthermore, focal micro-infarctions, owing to micro-embolism of aggregated GHMs into small coronary arterioles, were detected in the AI group. Conversely, only RI and DI groups displayed increased coronary flow reserve. DI groups also demonstrated increased capillary density. These results suggest that RI and DI are effective for cardiac GHM therapy, while AI appears inappropriate owing to the risk of focal infarctions.

Approximately 12,000 people are diagnosed with invasive transitional cell carcinoma of the urinary bladder (InvTCC) each year in the United States. Surgical removal of the bladder (cystectomy) and regional lymph node dissection are considered frontline therapy. Cystectomy causes extensive acute morbidity, and 50% of patients with InvTCC have occult metastases at the time of diagnosis. Better staging procedures for InvTCC are greatly needed. This study was performed to evaluate an intra-operative near infrared fluorescence imaging (NIRF) system (Frangioni laboratory) for identifying sentinel lymph nodes draining InvTCC. NIRF imaging was used to map lymph node drainage from specific quadrants of the urinary bladder in normal dogs and pigs, and to map lymph node drainage from naturally-occurring InvTCC in pet dogs where the disease closely mimics the human condition. Briefly, during surgery NIR fluorophores (human serum albumen-fluorophore complex, or quantum dots) were injected directly into the bladder wall, and fluorescence observed in lymphatics and regional nodes. Conditions studied to optimize the procedure including: type of fluorophore, depth of injection, volume of fluorophore injected, and degree of bladder distention at the time of injection. Optimal imaging occurred with very superficial injection of the fluorophore in the serosal surface of the moderately distended bladder. Considerable variability was noted from dog to dog in the pattern of lymph node drainage. NIR fluorescence was noted in lymph nodes with metastases in dogs with InvTCC. In conclusion, intra-operative NIRF imaging is a promising approach to improve sentinel lymph node mapping in invasive urinary bladder cancer.

Background: In recent years, animal studies and clinical trials have highlighted the regenerative potential of cardiac cellular therapy in the setting of both myocardial infarction and heart failure. Understanding the fate of stem/progenitor cells (SPCs) transplanted in the injured myocardium remains a pivotal issue in this field. In light of recent advances in molecular imaging, near-infrared (NIR) imaging represents a promising approach for cellular labeling and tracking of grafted SPCs. Therefore, we hypothesized that NIR labeling and in vivo tracking of SPCs is feasible and will allow to document their biological fate. Methods: Bone marrow derived meschenchymal stem cells (BMMSCs) were isolated from Yorkshire swines (n=3), and cultured for 2 weeks. Myocardial infarction was induced in all swines by 60-minute balloon occlusion of the left anterior descending artery. On day 3 post-infarction, allogenic BMMSCs (average, 20|[times]|106 cells): 1) underwent cellular labeling with the dye IR-786; 2) were reinjected in the infarct artery via intracoronary injection. Cellular labeling was assessed as follow: IR-786 labeled BMMSCs were tracked with a unique in vivo fluorescence/NIR imaging system under open chest condition. Results: BMMSCs were successfully labeled with IR-786 with near 100% efficiency. Over 90 % of BMMSCs were viable following IR-786 labeling. At baseline, the NIR signal-to-background ratio (SBR) was 1 in the infarct artery territory. Following cell injection, SBRs of the latter territory promptly increased by 3 to 5 fold (Figs 1). The SBRs remained sufficiently elevated to permit tracking of BMMSCs distributions even at the time of 60 min after the injection. These data will be discussed in conjunction with microscopic assessment of NIR labeled cells at the time of presentation. Conclusion: In vivo NIR imaging offers a new option to scrutinize reinjected cells. Cellular labeling of BMMSCs with IR786 is feasible; as well it allows both studying and appropriate tracking of the biological fate of transplanted SPCs in the setting of myocardial infarction.

Near-infrared light propagation through living tissue provides promising opportunities for the development of non-invasive imaging techniques for human care. We have developed a Fluorescence-Assisted Resection and Exploration (FLARE) imaging system for surgery. The FLARE system uses invisible near-infrared light to help the surgeon visualize critical structures intraoperatively and in real-time. We present here the continued optimization of our imaging system from a research prototype to an efficient and ergonomic tool to be used during human surgery. New, hands-free operation enables the surgeon to zoom, focus, recall and save images through a footswitch. A LabVIEW curve-fitting algorithm, in combination with stepper motor control, provides auto-focus capability. Cardiac and/or respiratory gating minimizes motion artifacts of moving objects in the surgical field, and permits in-focus imaging during long fluorescence integration times. Automated subtraction of the near-infrared fluorescence signal from background reflections minimizes the effect of ambient illumination and improves the contrast to noise ratio with only moderate effects on intensity precision. Taken together, this study improves several optical components of the FLARE system, and helps ready it for human clinical testing.

Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR) fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM), single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.

Quantum dots with a core/shell/shell structure consisting of an alloyed core of InAs(x)P(1-x), an intermediate shell of InP, and an outer shell of ZnSe were developed. The InAs(x)P(1-x) alloyed core has a graded internal composition with increasing arsenic content from the center to the edge of the dots. This compositional gradient results from two apparent effects: (1) the faster reaction kinetics of the phosphorus precursor compared to the arsenic precursor, and (2) a post-growth arsenic-phosphorus exchange reaction that increases the arsenic content. The cores have a zinc blend structure for all compositions and show tunable emission in the near-infrared (NIR) region. A first shell of InP leads to a red-shift and an increase in quantum yield. The final shell of ZnSe serves to stabilize the dots for applications in aqueous environments, including NIR biomedical fluorescence imaging. These NIR-emitting core/shell/shell InAs(x)P(1-x)/InP/ZnSe were successfully used in a sentinel lymph node mapping experiment.

Stem cells are a promising approach to cardiovascular therapeutics. Animal experiments have assessed the fate of injected stem cells through ex vivo methods on sacrificed animals. Approaches are needed for in vivo tracking of stem cells. Various imaging techniques and contrast agents for stem cell tracking will be reviewed.

Hayase, Motoya, Federica del Monte, Yoshiaki Kawase, Brian D. MacNeill, Jennifer McGregor, Ryuichi Yoneyama, Kozo Hoshino, Tsuyoshi Tsuji, Alec M. De Grand, Judith K. Gwathmey, John V. Frangioni, and Roger J. Hajjar. Catheter-based antegrade intracoronary viral gene delivery with coronary venous blockade. Am J Physiol Heart Circ Physiol 288: H2995–H3000, 2005; doi:10.1152/ajpheart.00703.2004.—The purpose of this study is to evaluate the feasibility of percutaneous antegrade myocardial gene transfer (PAMGT). A consistent and safe technique for in vivo gene transfer is required for clinical application of myocardial gene therapy. PAMGT with concomitant coronary venous blockade was performed in 12 swine. The myocardium was preconditioned with 1 min of occlusion of the left anterior descending and left circumflex arteries. The anterior interventricular vein was occluded during left anterior descending artery delivery, and the great cardiac vein at the entrance of the middle cardiac vein was occluded during left circumflex artery delivery. With arterial and venous balloons inflated (3 min) and after adenosine (25 μg) injection, PAMGT was performed by antegrade injection of an adenoviral solution (1 ml of 1011 plaque-forming units in each coronary artery) carrying β-galactosidase or saline through the center lumen of the angioplasty balloon. In one set of animals, PAMGT was performed with selective coronary vein blockade (n = 9); in another set of animals, PAMGT was performed without coronary vein blockade (n = 5). At 1 wk after gene delivery, the animals were killed. Quantitative β-galactosidase analysis was performed in the left and right ventricular walls. PAMGT was successfully performed in all animals with and without concomitant occlusion of the coronary veins. Quantitative β-galactosidase analysis showed that PAMGT with coronary blockade was superior to PAMGT without coronary blockade. β-Galactosidase activity increased significantly in the β-galactosidase group compared with the saline group: 1.34 ± 0.18 vs. 0.81 ± 0.1 ng (P ≤ 0.01) in the left ventricular wall and 0.91 ± 0.1 vs. 0.66 ± 0.07 ng (P ≤ 0.05) in the right ventricular wall. PAMGT with selective coronary venous blockade is feasible, reproducible, and safely achieved in a large-animal model.

Because many gastrointestinal (GI) tumors spread by way of lymphatics, histological assessment of the first draining lymph nodes has both prognostic and therapeutic significance. However, sentinel lymph node mapping of the GI tract by using available techniques is limited by unpredictable drainage patterns, high background signal, and the inability to image lymphatic tracers relative to surgical anatomy in real time. Our goal was to develop a method for patient-specific intraoperative sentinel lymph node mapping of the GI tract by using invisible near-infrared light. We developed an intraoperative near-infrared fluorescence imaging system that simultaneously displays surgical anatomy and otherwise invisible near-infrared fluorescence images of the surgical field. Near-infrared fluorescent quantum dots were injected intraparenchymally into the stomach, small bowel, and colon, and draining lymphatic channels and sentinel lymph nodes were visualized. Dissection was performed under real-time image guidance. In 10 adult pigs, we demonstrated that 200 pmol of quantum dots quickly and accurately map lymphatic drainage and sentinel lymph nodes. Injection into the mid jejunum and colon results in fluorescence of a single lymph node at the root of the bowel mesentery. Injection into the stomach resulted in identification of a retrogastric node. Histological analysis in all cases confirmed the presence of nodal tissue. We report the use of invisible near-infrared light for intraoperative sentinel lymph node mapping of the GI tract. This technology overcomes the limitations of currently available methods, permits patient-specific imaging of lymphatic flow and sentinel nodes, and provides highly sensitive, real-time image-guided dissection.

An efficient route to novel 1,3,5,7-tetrasubstituted derivatives of adamantane is described. This route starts from adamantane and gives the tetrafunctionalized derivative 9 in eight steps with an overall yield of 23%. These tetrahedrally shaped molecules possess three identical arms terminated by an activated carboxylic acid derivative and a protected amino function in the 1-position. We propose these tetravalent cage compounds such as 9 as scaffolds for the assembly of ligand/marker conjugates for studies of multivalent ligand receptor interactions. [reaction: see text]

Understanding the spatial and temporal drainage patterns of the pleural space could have profound impact on the treatment of lung cancer and mesothelioma. The purpose of this study was to identify the in vivo pattern of drainage from the pleural space to prognostic lymph node stations.Fifty-six rats underwent pleural space injection of a novel lymph tracer composed of recombinant human serum albumin (HSA) covalently conjugated to the near-infrared (NIR) fluorophore IRDye78 via an amide bond (HSA-78). Nodal uptake was imaged at 10, 20, 30, and 60 minutes and 4, 12, and 24 hours after injection with a custom system that simultaneously acquires color video, NIR fluorescence of HSA-78, and a merged picture of the two. Six pigs underwent the same procedure with imaging at 30 minutes, 1 hour, and 24 hours.In both the rat model and the pig model, HSA-78 drained from the pleural space to superior mediastinal lymph nodes first, followed by other intrathoracic and then extrathoracic lymph nodes over the course of 24 hours.NIR fluorescence imaging in two species shows that the superior mediastinal lymph nodes are the first to drain the pleural space. Over the course of 24 hours, the pleural space also communicates with other intrathoracic and then extrathoracic lymph nodes. This study also demonstrates an intraoperative method for identifying nodes communicating with the pleural space, with potential utility in the staging and/or resection of lung cancer and mesothelioma.

The recruitment of corepressors by DNA-bound repressors is likely to be a critical rate-limiting step in the transcriptional regulation of many genes. An excellent paradigm for such an interaction is the association of the basic helix-loop-helix zipper protein Mad1 with the corepressor mSin3A. When bound together, the Sin3 interaction domain (SID) of Mad1 forms extensive hydrophobic contacts with the four-helix bundle formed by the paired amphipathic helix 2 (PAH2) domain of mSin3A. Using the costructure to predict the principle residues required for binding, we have carried out an extensive mutational analysis to examine the Mad1 SID-mSin3A PAH2 interaction in vitro and in vivo. Bulky hydrophobic residues in the alpha1 (I308 and V311) and alpha2 (L329 and L332) helices of the PAH2 domain are necessary to accommodate the precise arrangement of bulky (L12) and short (A15 and A16) hydrophobic residues in the amphipathic Mad1 SID. We have also used phage display to derive an optimal SID, which shows an essentially identical arrangement of key residues. By manipulating these key residues, we have generated altered-specificity Mad1 SID mutants that bind only to a PAH2 domain with a reciprocal mutation, permitting us to demonstrate for the first time that these domains interact directly in vivo. We have also found that the integrity of the PAH1 domain affects the Mad1 SID-PAH2 interaction. It is conceivable that cross talk between different PAH domains and their binding partners helps to determine the subunit composition and order of assembly of mSin3A complexes.

Near-infrared (NIR) fluorescence imaging has the potential to revolutionize human cancer surgery by providing sensitive, specific, and real-time intraoperative visualization of normal and disease processes. We have previously introduced the concept of a low-cost, safe, and easy-to-use NIR fluorescence imaging system that permits the surgeon to “see” surgical anatomy and NIR fluorescence simultaneously, non-invasively, with high spatial resolution, in real-time, and without moving parts [Nakayama et al. Molecular Imaging 1, 365–377 (2002)]. In this study, we present an operational prototype designed specifically for use during large animal surgery. Such a system serves as a foundation for future clinical studies. We discuss technical considerations, and provide details of the implementation of subsystems related to excitation light, light collection, computer, and software. Using the prototype, and the clinically available NIR fluorophore indocyanine green, we demonstrate vascular imaging in 35 kg pigs. Cancer-specific applications of this imaging system include image-guided cancer resection with real-time assessment of surgical margins, image-guided sentinel lymph node mapping, intraoperative mapping of tumor and normal vasculature, image-guided avoidance of critical structures such as nerves, and intraoperative detection of occult metastases in the surgical field. Taken together, this study describes an optical imaging system engineered for eventual translation to the clinic.

At present, there is no animal model of breast cancer that forms reproducible microcalcification. The aim of this study was to develop a straightforward, reproducible model system that could be used to develop multimodality contrast agents for the identification of breast cancer microcalcification.The R3230 mammary adenocarcinoma cell line was implanted in the mammary fat pad of female Fischer 344 rats (two rats with two implanted tumors and two rats with a single implanted tumor). After growth to 1-2 cm in diameter, tumors were implanted with 100 microm hydroxyapatite crystals (positive control) or calcium oxalate crystals (negative control). Twenty-four hours after crystal implantation, rats were injected intravenously with a previously described near-infrared fluorescent bisphosphonate derivative known as Pam78, and the tumors were imaged using a reflectance optical imaging system.Tumors implanted with hydroxyapatite displayed bright, focal, near-infrared fluorescence in the area of crystal implantation. Control tumors, grown in the same animal and implanted with calcium oxalate, did not display any near-infrared fluorescence, even along the needle track used for crystal implantation.A simple and rapid animal model of focal calcification in breast cancer tumors has been developed and validated. The model used Pam78, a near-infrared fluorescent contrast agent specific for hydroxyapatite. The potential usefulness of the model for developing similar contrast agents for magnetic resonance and other imaging modalities is discussed.

Photon penetration into living tissue is highly dependent on the absorption and scattering properties of tissue components. The near-infrared region of the spectrum offers certain advantages for photon penetration, and both organic and inorganic fluorescence contrast agents are now available for chemical conjugation to targeting molecules. This review focuses on those parameters that affect image signal and background during in vivo imaging with near-infrared light and exogenous contrast agents. Recent examples of in vivo near-infrared fluorescence imaging of animals and humans are presented, including imaging of normal and diseased vasculature, tissue perfusion, protease activity, hydroxyapatite and cancer.

The selective targeting of a compound to tumors, or for that matter to any structure in the body, requires high stability of the compound in blood, biodistribution to the target site, adequate contact time with the target, adequate retention by the target, maintenance of drug potency, and adequate clearance of nontargeted compound. For over two decades, antibodies have been the scaffold of choice for delivering contrast agents and chemotherapeutics to tumor cells since they are stable in blood, typically have nanomolar affinities for their target, and, because binding and nonbinding domains are separated physically, tolerate substitution with contrast agents and chemotherapeutics. Although monoclonal antibodies (mAbs) are typically tolerant to the conjugation of chemotherapeutics, the agents themselves often lose potency in the conjugated form. Therefore, novel chemical strategies are required for releasing the cytotoxic agent, either after binding to the cancer cell surface or after endocytosis into the cell. A compelling example of this comes from the antitumor antibiotic calicheamicin. When conjugated to a tumor-targeting mAb through an amide linkage, the conjugate is accumulated by the tumor, but has no appreciable cytotoxicity. In contrast, when conjugated by using a pH-sensitive bifunctional linker that permits release of calicheamicin intracellularly (Figure 1), the conjugate shows potent antitumor activity. Indeed, gemtuzumab ozogamicin (“gem-ozo”, Mylotarg), a conjugate of a CD33-specific mAb and calicheamicin, which utilizes this bifunctional linker, is already approved for the treatment of certain acute myeloid leukemias. The taxanes are a class of potent chemotherapeutics that possess a complex chemical structure. Recently, two different strategies have been employed to conjugate taxanes to tumor-targeting monoclonal antibodies, while retaining chemotherapeutic potency. In conjugate mAb-1 (Scheme 1), an enzymatically cleavable glutarate ester bond is placed between the amide linkage to

Purpose: Small animal radioscintigraphic imaging systems aim to achieve sub-millimeter resolution. At the present time, sub-millimeter calibration sources that can be placed at will within an imaged volume are not readily available. We have developed a method for producing technetium-99m (Tc-99m) sources in less than 15 minutes with readily available reagents. Procedures: Tc-99m pertechnetate [TcO 4 ] − was incubated with 45 μm to 106 μm diameter spherical anion exchange beads, washed, and mounted as desired for instrument calibration. Results: The procedure yields spherical sources having between 6.8 μCi to 11.1 μCi of Tc-99m per source. This work shows that dual imaging of these sources using white light and radioscintigraphy permits measurement of system performance with high precision. Conclusion: Easily prepared, sub-millimeter Tc-99m spherical calibration sources are described, and it is demonstrated that such sources are useful for measuring the resolution and sensitivity of radioscintigraphic systems, such as those designed for small animal imaging. (Mol Imag Biol 2002;4:380–384)

Cardiac revascularization is presently performed without realtime visual assessment of myocardial blood flow or perfusion. Moreover, gene therapy of the heart cannot, at present, be directed to specific territories at risk for myocardial infarction. We have developed a surgical imaging system that exploits the low autofluorescence, deep tissue penetration, low tissue scatter, and invisibility of near-infrared (NIR) fluorescent light. By completely isolating visible and NIR light paths, one is able to visualize, simultaneously, the anatomy and/or function of the heart, or any desired tissue. In rat model systems, we demonstrate that the heptamethine indocyanine-type NIR fluorophores IR-786 and the carboxylic acid form of IRDye78 can be injected intravenously in the living animal to provide real-time visual assessment of myocardial blood flow or perfusion intraoperatively. This imaging system may prove useful for the refinement of revascularization techniques, and for the administration of cardiac gene therapy.

Fluorescent semiconductor nanocrystals (quantum dots [QDs]) are hypothesized to be excellent contrast agents for biomedical assays and imaging. A unique property of QDs is that their absorbance increases with increasing separation between excitation and emission wavelengths. Much of the enthusiasm for using QDs in vivo stems from this property, since photon yield should be proportional to the integral of the broadband absorption. In this study, we demonstrate that tissue scatter and absorbance can sometimes offset increasing QD absorption at bluer wavelengths, and counteract this potential advantage. By using a previously validated mathematical model, we explored the effects of tissue absorbance, tissue scatter, wavelength dependence of the scatter, water-to- hemoglobin ratio, and tissue thickness on QD performance. We conclude that when embedded in biological fluids and tissues, QD excitation wavelengths will often be quite constrained, and that excitation and emission wavelengths should be selected carefully based on the particular application. Based on our results, we produced near-infrared QDs optimized for imaging surface vasculature with white light excitation and a silicon CCD camera, and used them to image the coronary vasculature in vivo. Taken together, our data should prove useful in designing fluorescent QD contrast agents optimized for specific biomedical applications.

In vertebrates, the development and integrity of the skeleton requires hydroxyapatite (HA) deposition by osteoblasts. HA deposition is also a marker of, or a participant in, processes as diverse as cancer and atherosclerosis. At present, sites of osteoblastic activity can only be imaged in vivo using gamma-emitting radioisotopes. The scan times required are long, and the resultant radioscintigraphic images suffer from relatively low resolution. We have synthesized a near-infrared (NIR) fluorescent bisphosphonate derivative that exhibits rapid and specific binding to HA in vitro and in vivo. We demonstrate NIR light-based detection of osteoblastic activity in the living animal, and discuss how this technology can be used to study skeletal development, osteoblastic metastasis, coronary atherosclerosis, and other human diseases.

Human gene therapy approaches involving transcription factors often rely on artificial activation domains for transcriptional activation. These domains are often large (e.g., 80 amino acids for VP16), recruit multiple co-activation complexes at once, and offer no fine control over the level of transcription. In an attempt to understand the sequence and structural requirements of a minimal mammalian activator, we employed a molecular diversity approach with a peptide phage display library composed of random eight-amino acid peptides. Using the KIX domain of the mammalian co-activators p300 and CBP as target, we discovered a family of synthetic binding peptides. These peptides share significant homology with natural KIX domain ligands, and are shown to bind an overlapping, yet distinct, surface of p300/CREB-binding protein (CBP). When fused to a heterologous DNA binding domain, these synthetic peptides function as titratable, modular, and potent transcriptional activators in living cells through specific recruitment of p300/CBP, with the level of transcriptional activation proportional to the affinity of the synthetic peptide for the KIX domain. Taken together, our data demonstrate that a molecular diversity approach can be used to discover minimal, co-activator domain-specific synthetic activators, and that transcriptional activation can be modulated as desired at the level of co-activator recruitment.

We have developed a size series of unusually small, water-soluble (InAs)ZnSe (core)shell quantum dots (QDs) that emit in the near-infrared and exhibit new behavior in vivo, including multiple sequential lymph node mapping and extravasation from the vasculature. The biological utility of these fluorescent probes resulted from our intentional choice to match the semiconductor material and water-soluble ligand with a desired final hydrodynamic diameter and emission wavelength.

Top of pageAbstract Introduction: The kinetics of long term transgene expression of viral vectors is important as it directly influences the efficacy of the gene therapy. Conventional kinetic measurements require sacrificing animals at each data point and assessing the amount of transgene product in the tissue. This type of analysis is extremely laborious and contains inherent inaccuracy since it does not follow the time course of a single animal. In this study, in vivo tracking of transgene expression from adeno-aasociated virus type 1 (AAV-1) was performed using a green fluorescence protein (GFP) reporter and an specially designed optical imaging system.. Our results reveal the exact kinetics of AAV-1 expression and validate fluorescence imaging for kinetic measurements of long term expression of virally expressed genes. Materials and Method: Animals and Virus 40-day-old male hairless SKH1 mice were divided into 3 groups, each containing 3 mice. The right leg of Groups 1 and 2 were injected with 1012 and 1010 plaque forming unit (pfu) of adeno-associated virus type 1 encoding green fluorescent protain (AAV-1.GFP). respectively, in 50 ul normal saline. The left legs were injected with 50 ul normal saline as an internal control. Group 3 (control group) was injected with 50 ul normal saline into the both legs. The mice were anesthetized by intraperitoneal injection of 80 mg/kg body weight ketamine at the time of each measurement, and imaged over 13 weeks. Imaging System Selective excitation of GFP was produced by 475 nm light emission diode through a excitation filter HQ470/40x. Emitted fluorescence was collected through an emission filter HQ525/50m (Chroma Technology Corp, Rockingham, VT) on a Hamamatsu C8484 charge-coupled device camera (Hamamatsu Photonics Systems, Bridgewater, NJ). The strength of the transgene expression was measured as the ratio of the mean of fluorescence intensity of 0.5 cm2 ROI set on the right and left leg. Results and Conclusions The kinetics of GFP expression was clearly measured by assessing the fluorescence intensity of each animal. The images and graphs of representative animals are shown in the Figure. In Group 1, injected with 1012 pfu AAV-1.GFP, GFP expression reached a plateau at approximately 7 weeks, which was maintained until at least 13 weeks. In Group 2, injected with 1010 pfu AAV-1.GFP, GFP expression, reached its plateau at approximately 11 weeks. These results demonstrate the relationship between injected dose and transgene expression level, as well as the kinetics of expression.

We report the design, characterization, and validation of an optimized simultaneous color and near-infrared (NIR) fluorescence rigid endoscopic imaging system for minimally invasive surgery. This system is optimized for illumination and collection of NIR wavelengths allowing the simultaneous acquisition of both color and NIR fluorescence at frame rates higher than 6.8 fps with high sensitivity. The system employs a custom 10-mm diameter rigid endoscope optimized for NIR transmission. A dual-channel light source compatible with the constraints of an endoscope was built and includes a plasma source for white light illumination and NIR laser diodes for fluorescence excitation. A prism-based 2-CCD camera was customized for simultaneous color and NIR detection with a highly efficient filtration scheme for fluorescence imaging of both 700- and 800-nm emission dyes. The performance characterization studies indicate that the endoscope can efficiently detect fluorescence signal from both indocyanine green and methylene blue in dimethyl sulfoxide at the concentrations of 100 to 185 nM depending on the background optical properties. Finally, we performed the validation of this imaging system in vivo during a minimally invasive procedure for thoracic sentinel lymph node mapping in a porcine model.

Background TSC2-deficient cells can proliferate in the lungs, kidneys, and other organs causing devastating progressive multisystem disorders such as lymphangioleiomyomatosis (LAM) and tuberous sclerosis complex (TSC). Preclinical models utilizing LAM patient-derived cells have been difficult to establish. We developed a novel animal model system to study the molecular mechanisms of TSC/LAM pathogenesis and tumorigenesis and provide a platform for drug testing. Methods and Findings TSC2-deficient human cells, derived from the angiomyolipoma of a LAM patient, were engineered to co-express both sodium-iodide symporter (NIS) and green fluorescent protein (GFP). Cells were inoculated intraparenchymally, intravenously, or intratracheally into athymic NCr nu/nu mice and cells were tracked and quantified using single photon emission computed tomography (SPECT) and computed tomography (CT). Surprisingly, TSC2-deficient cells administered intratracheally resulted in rapid dissemination to lymph node basins throughout the body, and histopathological changes in the lung consistent with LAM. Estrogen was found to be permissive for tumor growth and dissemination. Rapamycin inhibited tumor growth, but tumors regrew after the drug treatment was withdrawn. Conclusions We generated homogeneous NIS/GFP co-expressing TSC2-deficient, patient-derived cells that can proliferate and migrate in vivo after intratracheal instillation. Although the animal model we describe has some limitations, we demonstrate that systemic tumors formed from TSC2-deficient cells can be monitored and quantified noninvasively over time using SPECT/CT, thus providing a much needed model system for in vivo drug testing and mechanistic studies of TSC2-deficient cells and their related clinical syndromes.