Small-animal SPECT and SPECT/CT: important tools for preclinical investigation.

The need to study dynamic biologic processes in intact small-animal models of disease has stimulated the development of high-resolution nuclear imaging methods. These methods are capable of clarifying molecular interactions important in the onset and progression of disease, assessing the biologic relevance of drug candidates and potential imaging agents, and monitoring therapeutic effectiveness of pharmaceuticals serially within a single-model system. Single-photon-emitting radionuclides have many advantages in these applications, and SPECT can provide 3-dimensional spatial distributions of gamma- (and x-) ray-emitting radionuclide imaging agents or therapeutics. Furthermore, combining SPECT with CT in a SPECT/CT system can assist in defining the anatomic context of biochemical processes and improve the quantitative accuracy of the SPECT data. Over the past decade, dedicated small-animal SPECT and SPECT/CT systems have been developed in academia and industry. Although significant progress in this arena has been realized through system development and biologic application, further innovation continues to address challenges in camera sensitivity, spatial resolution, and image reconstruction and quantification. The innumerable applications of small-animal SPECT and SPECT/CT in drug development, cardiology, neurology, and oncology are stimulating further investment in education, research, and development of these dedicated small-animal imaging modalities.

Technological development and advances in single-photon emission computed tomography/computed tomography.

Single-photon emission computed tomography/computed tomography (SPECT/CT) has emerged during the past decade as a means of correlating anatomical information from CT with functional information from SPECT. The integration of SPECT and CT in a single imaging device facilitates anatomical localization of the radiopharmaceutical to differentiate physiological uptake from that associated with disease and patient-specific attenuation correction to improve the visual quality and quantitative accuracy of the SPECT image. The first clinically available SPECT/CT systems performed emission-transmission imaging using a dual-headed SPECT camera and a low-power x-ray CT subsystem. Newer SPECT/CT systems are available with high-power CT subsystems suitable for detailed anatomical diagnosis, including CT coronary angiography and coronary calcification that can be correlated with myocardial perfusion measurements. The high-performance CT capabilities also offer the potential to improve compensation of partial volume errors for more accurate quantitation of radionuclide measurement of myocardial blood flow and other physiological processes and for radiation dosimetry for radionuclide therapy. In addition, new SPECT technologies are being developed that significantly improve the detection efficiency and spatial resolution for radionuclide imaging of small organs including the heart, brain, and breast, and therefore may provide new capabilities for SPECT/CT imaging in these important clinical applications.

(18)F-FDG-PET/CT evaluation of response to treatment in lymphoma: when is the optimal time for the first re-evaluation scan?

Assessing the response to treatment as soon after treatment initiation is one of the key reasons for imaging lymphoma patients. The optimal time after initiating treatment for assessing response to treatment has yet to be determined. Therefore, we were prompted to review our experience with serial (18)F-FDG PET/CT in patients undergoing treatment for Hodgkin's disease (HD) and non Hodgkin's lymphoma (NHL). This is a retrospective study (Feb 2003 - Oct 2004) of 20 patients, 11 men and 9 women, with age range of 7-75 years with diagnosis of HD (10) and NHL (10), who had PET/CT at our institution prior, during and at the completion of therapy. Restaging PET/CT was done after 2 cycles of chemotherapy in 10 patients (group A) and after 4 cycles of chemotherapy in 10 pts (group B). A total of 60 scans were reviewed. The DeltaSUV from baseline to first PET/CT was on average 67.6% in group A and 75.1% in group B. This had no statistical significance (P value: 0.31). The DeltaSUV from baseline to post-therapy PET/CT was on average 72.9% in group A and 79.8% in group B. This difference also had no statistical significance (P value: 0.24). The correlation coefficient was 0.98 in group A and 0.80 in group B. Results of PET/CT after 2 cycles of chemotherapy did not statistically differ from the results of PET/CT after 4 cycles of chemotherapy. These results need to be confirmed in larger, prospective, randomized trials.

Adipose-derived adult stromal cells heal critical-size mouse calvarial defects.

In adults and children over two years of age, large cranial defects do not reossify successfully, posing a substantial biomedical burden. The osteogenic potential of bone marrow stromal (BMS) cells has been documented. This study investigates the in vivo osteogenic capability of adipose-derived adult stromal (ADAS) cells, BMS cells, calvarial-derived osteoblasts and dura mater cells to heal critical-size mouse calvarial defects. Implanted, apatite-coated, PLGA scaffolds seeded with ADAS or BMS cells produced significant intramembranous bone formation by 2 weeks and areas of complete bony bridging by 12 weeks as shown by X-ray analysis, histology and live micromolecular imaging. The contribution of implanted cells to new bone formation was 84-99% by chromosomal detection. These data show that ADAS cells heal critical-size skeletal defects without genetic manipulation or the addition of exogenous growth factors.

Bone morphogenetic protein 2 and retinoic acid accelerate in vivo bone formation, osteoclast recruitment, and bone turnover.

Reconstruction of craniofacial defects presents a substantial biomedical burden, and requires complex surgery. Interestingly, children after age 2 years and adults are unable to heal large skull defects. This nonhealing paradigm provides an excellent model system for craniofacial skeletal tissueengineering strategies. Previous studies have documented the in vivo osteogenic potential of adipose-derived stromal (ADS) cells and bone marrow-derived stromal (BMS) cells. This study investigates the ability to accelerate in vivo osteogenesis on ex vivo recombinant human bone morphogenetic protein 2 (BMP-2) and retinoic acid stimulation. Mouse osteoblasts, ADS cells, and BMS cells were seeded onto apatite-coated PLGA scaffolds, stimulated with rhBMP-2 and retinoic acid ex vivo for 4 weeks, and subsequently implanted into critically sized (4 mm) calvarial defects. Samples were harvested after 2, 4, 8, and 12 weeks. Areas of complete bony bridging were noted as early as 2 weeks in vivo; however, osteoclasts were attracted to the scaffold as identified by calcitonin receptor staining and tartrate-resistant acid phosphatase activity staining. Although the optimal method of in vitro osteogenic priming for mesenchymal cells remains unknown, these results provide evidence that BMP-2 and retinoic acid stimulation of multipotent cells ex vivo can subsequently induce significant quantities of bone formation within a short time period in vivo.

The role of a positron- and high-energy gamma photon probe in intraoperative localization of recurrent melanoma.

PURPOSE: This preliminary study retrospectively evaluated the ability of intraoperative localization of recurrent melanoma using F-18 fluorodeoxyglucose (FDG) and a probe sensitive to both high-energy gamma rays and positrons to enable complete tumor resection and improved patient outcome. MATERIALS AND METHODS: Three hours before surgery for resection of recurrent melanoma, 5 patients (mean age, 52 +/- 22 years) with a history of local surgery, radiation therapy, and/or large habitus received 14.6 +/- 3.2 mCi of F-18 FDG. Intraoperative tumor localization was performed with a radiation probe (PET-Probe; IntraMedical Imaging LLC, Los Angeles, CA). Intraoperative tumor tissue activities, background tissue activities, pathology results, and patient follow up (clinical/imaging) were recorded. RESULTS: Eight of the 19 surgical specimens were identified by the probe as having increased FDG uptake when compared with the surrounding tissues before resection. All 8 specimens contained melanoma. Of the 11 specimens that were not identified using the probe, one contained melanoma, yielding a sensitivity of 89% (8 of 9) and a specificity of 100% (10 of 10). In 3 of the 5 cases, the probe allowed the identification of nonvisualized and nonpalpable tumor foci that were later confirmed pathologic. At an average follow up of 210 days (range, 30-515 days), 2 of 5 patients had no evidence of recurrent melanoma by clinical or radiographic evaluations. CONCLUSION: In the setting of recurrent melanoma, there appear to be potential benefits to intraoperative detection with FDG and a positron-detecting probe, particularly in cases with challenging or altered anatomy.

Tumor imaging using a standardized radiolabeled adapter protein docked to vascular endothelial growth factor.

UNLABELLED: Direct radiolabeling of proteins can result in the loss of targeting activity, requires highly customized procedures, and yields heterogeneous products. Here we describe a novel imaging complex comprised of a standardized (99m)Tc-radiolabeled adapter protein noncovalently bound to a "Docking tag" fused to a "Targeting protein". The assembly of this complex is based on interactions between human 109-amino acid (HuS) and 15-amino acid (Hu-tag) fragments of ribonuclease I, which serve as an "Adapter protein" and a Docking tag, respectively. METHODS: HuS modified with hydrazinonicotinamide (HYNIC) was radiolabeled using (99m)Tc-tricine to a specific activity of 3.4-7.4 MBq/microg. Protein complexes were then formed by mixing (99m)Tc-HuS with equimolar amounts of either Hu-tagged VEGF(121) (Hu-VEGF [vascular endothelial growth factor]) or Hu-tagged anti-VEGFR-2 single-chain antibody (Hu-P4G7) and incubating on ice for 15 min. 4T1 luc/gfp luciferase-expressing murine mammary adenocarcinoma cells (1 x 10(4)) were implanted subcutaneously or injected intravenously into BALB/c mice. Bioluminescent imaging (BLI) was performed 10 d later. Immediately after BLI visualization of tumor, 18.5-37 MBq of tracer (5-10 microg of protein) were injected via tail vein. One hour later planar or SPECT images were obtained, followed by killing the mice. RESULTS: There was significantly (P = 0.0128) increased uptake of (99m)Tc-HuS/Hu-VEGF (n = 10) within subcutaneous tumor as compared with (99m)Tc-HuS/Hu-P4G7 (n = 5) at biodistribution assay (2.68 +/- 0.75 vs. 1.8 +/- 0.21; tumor-to-subcutaneous tissue [ratio of specific activities], respectively), despite similar molecular weights. The focal (99m)Tc-HuS/Hu-VEGF uptake seen on planar images (3.44 +/- 1.16 [tumor to soft-tissue background]) corresponded directly to the locations of tumor observed by BLI. Region of interest analyses of SPECT images revealed a significant increase of (99m)Tc-HuS/Hu-VEGF (n = 5) within the lungs with BLI-detectable pulmonary tumor nodules as compared with controls (n = 4) (right: 4.47 +/- 2.07 vs. 1.79 +/- 0.56; left: 3.66 +/- 1.65 vs. 1.62 +/- 0.45, tumor lung [counts/pixel]/normal lung [counts/pixel], respectively). CONCLUSION: (99m)Tc-HuS/Hu-VEGF complex is stable for at least 1 h in vivo and can be effectively used to image mouse tumor neovasculature in lesions as small as several millimeters in soft tissue. We expect that a similar approach can be adapted for in vivo delivery of other targeting proteins of interest without affecting their bioactivity.

The use of (99m)technetium-labeled MCP-1 to assess graft coronary artery disease in rat cardiac allografts.

BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) is associated with the development of graft coronary artery disease (GCAD) following cardiac transplantation. This study assessed whether technetium 99m ((99m)Tc)-labeled MCP-1 binds its receptors in chronic cardiac transplants and thereby provides a potential modality to assess GCAD. METHODS: Allogeneic (PVG-->ACI, n = 9) and syngeneic (ACI-->ACI, n = 9) rat heterotopic heart transplants were performed. Allograft recipients were treated with 7.5 mg/kg per day of Cyclosporin A for 10 days until tolerance was achieved. After 90 days, animals were injected intravenously with (99m)Tc-MCP-1 and killed after 1 hour. Radioactivity of heart tissues was measured and standardized to uptake in the overall blood pool. Two-dimensional (99m)Tc-MCP-1 uptake (autoradiographs) was imaged by exposing 50-microm sections on a phosphoimager overnight. ED-1 staining of monocyte/macrophages was performed on serial sections. Additional sections were stained with elastin von Gieson and hematoxylin. Hearts were scored for luminal narrowing and intima/media ratio (I/M) with computerized image analysis. RESULTS: Allografts exhibited significantly more luminal narrowing (22.5 +/- 10.7% vs 2.6 +/- 4.6, p = 0.0005) and higher I/M (0.173 +/- 0.151 vs 0.015 +/- 0.029, p = 0.0088) than isografts. The ratio of (99m)Tc-MCP-1 uptake in allografts (1.04 +/- 0.4) was greater than that of isograft controls (0.72 +/- 0.11, p = 0.03). Pixel counts of autoradiographs and ED-1-stained sections demonstrated a modest correlation between the two (R(2) = 0.50). No significant differences were seen in acute rejection scores. CONCLUSION: (99m)Tc-MCP-1 uptake was higher in allografts vs isografts and was consistent with a greater degree of GCAD. These data demonstrating increased radiopharmaceutical uptake in hearts with GCAD provide a foundation for the development of a potentially non-invasive imaging assay of this disease process in heart transplantation.

Zinc chloride-mediated reduction of apoptosis as an adjunct immunosuppressive modality in cardiac transplantation.

BACKGROUND: Zinc (Zn) blocks caspase-3 activation in cardiac allografts and therefore may synergistically decrease apoptosis along with cyclosporine (CsA), which inhibits mitochondrial release of cytochrome c. Simultaneous treatment of rat recipients of heterotopic heart transplants with zinc chloride (ZnCl(2)) thus may allow lower doses of CsA for immunosuppression. METHODS: PVG (RT1(c)) rat hearts were transplanted heterotopically into the abdomen of ACI (RT1(a)) rats. Group 1 (n = 15) rats received no treatment. Group 2 rats (n = 8) received 2 mg/kg/day CsA (sub-therapeutic dose) by oral gavage. Group 3 rats (n = 9) received 2 mg/kg/day oral CsA in addition to 1 mg/kg/day sub-cutaneous ZnCl(2) delivered by osmotic pump. All rats were imaged using Annexin V-bound (99m)Technetium ((99m)Tc-Annexin V) on post-operative Day 4 and subsequently killed. Annexin V avidly binds apoptotic cells in vivo. Region of interest per whole body (WB) data were calculated using the images. The allograft survival study was conducted with n = 11, 6, and 5 in control, CsA, and CsA+Zn groups, respectively. Finally, percentages of allografts that reached tolerance were measured in both CsA-only and CsA+Zn groups (n = 8 each). RESULTS: Zinc chloride had an additive effect with CsA on apoptotic blockade and graft survival. The regions of interest per WB uptake of (99m)Tc-Annexin V were 2.43% +/- 0.37%, 2.08% +/- 0.52%, and 1.49% +/- 0.29%*, and acute survivals were 6.4 +/- 1.7, 7.2 +/- 2.1, and 11.2 +/- 2.5* days for control, CsA, and CsA+Zn groups, respectively (*p < 0.001 vs controls). In addition, 87.5% of allografts became tolerant and survived for 90 days in the CsA+Zn group compared with only 37.5% in the CsA-only group (p = 0.049). CONCLUSION: Zinc-mediated reduction of apoptosis served as an effective adjunct immunosuppressive therapy to CsA in a rat model of cardiac transplantation.

Development of radiocontrast agents for vascular imaging: progress to date.

The revolution in molecular imaging techniques is profoundly changing the understanding of the pathophysiology and treatment of atherosclerosis. With these rapid changes there is an increasing demand for development of sensitive and well tolerated novel imaging agents that can be rapidly translated from small animal models into patients with atherosclerosis. Nuclear medicine and positron emission tomography techniques have the ability to detect and serially monitor a variety of biologic and pathophysiologic processes usually with tracer quantities of radiolabeled peptides, drugs, and other molecules at dosages free of pharmacologic adverse effects unlike the current generation of intravenous agents required for magnetic resonance imaging (MRI) and computed axial tomography (CT) scanning. A representative sampling of the wide array of radiopharmaceuticals developed specifically for radionuclide imaging of atherosclerosis, that have been approved for clinical use and those in pre-clinical trials, have been reviewed in this article. The presence of an inflammatory stimulus increases expression of CC (cysteine-cysteine motif) chemokine receptor (CCR)-2 on monocytes and macrophages, and somatostatin receptors on T lymphocytes. Radiolabeled monocyte chemoattractant protein (MCP)-1 binds with high affinity to CCR-2 and can be used to detect subacute and chronic inflammatory lesions. Similarly, radiolabeled octreotide or depreotide can be used to detect activated T lymphocytes which may identify the vulnerable plaque. Animal models indicate that (99m)Tc-annexin V, (125)I-MCP-1 and [(18)F]-fluoro-2-deoxyglucose are effective in identifying apoptotic cell death, macrophage infiltration and metabolic activity in atheromatous lesions, respectively. Expression of alpha(v)beta(3) integrin is increased in activated endothelial cells and vascular smooth muscle cells after vascular injury, and alpha(v)beta(3) integrin is minimally expressed on smooth muscle cells and is not expressed on quiescent epithelial cells. Radiolabeled high-affinity peptides can be used to target the alpha(v)beta(3) integrin and visualize areas of vascular damage. Advances in technology such as the micro-single photon emission computed tomography (microSPECT) have the potential to overcome the drawbacks of older CT and MRI methodologies, such as lack of biologically relevant ligands and compatible blood pool contrast agents for imaging. Despite these advances in imaging technology, the small size of atheromatous lesions makes it difficult to detect using external imaging techniques. Therefore, recently there has been renewed interest in the use of intravascular catheter-based radiation detectors.

Apoptosis in a rodent model of cranial suture fusion: in situ imaging and gene expression analysis.

Craniosynostosis, the premature fusion of cranial sutures, is one of the most common craniofacial anomalies, with a reported incidence of up to one in 2500 live births. Despite its prevalence, the cause of craniosynostosis remains unknown. Previously, apoptosis has been postulated to be a contributing factor in the pathogenesis of craniosynostosis, although the role of programmed cell death in cranial sutures is poorly understood. To address this problem, the authors used an established rodent model of posterior-frontal suture fusion and sagittal suture patency to globally examine apoptosis in cranial sutures. Apoptosis was evaluated by systemically coinjecting Sprague-Dawley rats with both fluorescent and technetium-99m-labeled annexin V at time points before, during, and after the period of predicted posterior-frontal suture fusion to determine the magnitude and time course of overall apoptotic activity in both fusing and patent sutures. Using these novel in situ imaging techniques, the authors observed a significant increase in the overall levels of apoptosis in both the posterior-frontal and sagittal suture complexes during the period of predicted posterior-frontal suture fusion. To further explore this increase in apoptotic activity, they used microarray technology to study apoptosis-related genes within the suture complex. Interestingly, there was activation of distinct apoptotic pathways in the posterior-frontal and sagittal sutures during the period of predicted posterior-frontal suture fusion. Whereas increased transcription of genes associated with the mitochondria-mediated apoptotic pathway occurred in the posterior-frontal suture during fusion, activation of genes associated with the death receptor-mediated apoptotic pathway predominated in the patent sagittal suture during the same time period. These data suggest that although overall apoptotic activity in rat patent and fusing sutures is similar, the pathways mediating apoptosis within each suture are distinct.

Pattern of 18F-FDG uptake in the spinal cord in patients with non-central nervous system malignancy.

STUDY DESIGN: Retrospective review. OBJECTIVE: To (1) propose a standard method to quantitate 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG) uptake in the spinal cord and (2) use this methodology to retrospectively characterize the pattern of uptake within the entire spinal cord using whole-body positron emission tomography/computed tomography (PET/CT) imaging. SUMMARY OF BACKGROUND DATA: A physiologic understanding of glucose metabolism within the spinal cord may provide insight regarding infectious, inflammatory, vascular, and neoplastic spinal cord diseases. METHODS: Institutional review board approval was obtained. A total of 131 consecutive whole-body PET/CT studies from July to August 2004 were reviewed, and using exclusionary criteria of: (1) severe spinal arthropathy or curvature, (2) motion artifact, (3) canal hardware, (4) spinal tumor, and (5) marrow hyperplasia, 92 studies of neurologically intact patients (49 men and 43 women) were selected for a retrospective review of spinal cord 18F-FDG activity. The transaxial CT was used to define the canal and circular regions of interests were placed within the canal at the level of the vertebral body midpoint from C1 to L3. Region of interest total count, area, and maximum standardized uptake value (SUVmax) were recorded. Measurements at L5 served as an internal control. For comparative analysis, the cord-to-background (CTB) ratio was defined as spinal cord SUVmax to L5 SUVmax. RESULTS: Mean CTB decreased along each spinal level from cranial to caudal (P < 0.001). Significant relative increases were observed at the T11-T12 vertebral body levels (P < 0.001). Although insignificant, a relative increase was observed at C4. No significant interactions of age or sex on CTB were observed. CONCLUSION: The pattern of 18F-FDG uptake within the spinal cord, observed in patients with non-central nervous system malignancy, may be helpful in understanding glucose physiology of spinal cord diseases and warrants further research.

Incidental PET/CT Detection of Breast Cancer in a Patient with Negative Mammogram and Breast Sonogram.

We present the case of a 57-year-old woman with known Hodgkins lymphoma who presented with PET/CT findings of a breast mass and diseased axillary lymph node despite negative results from a recent routine mammogram and ultrasound. Needle biopsy of the mass and lymph node confirmed a diagnosis of infiltrating ductal carcinoma of the breast with nodal metastases.

Intravascular radiation detectors for the detection of vulnerable atheroma.

An intravascular catheter was developed to identify inflammation in coronary atheroma. Inflammation in atheroma is associated with large numbers of macrophages. These cells have increased metabolism, increased expression of chemotactic receptors, and a high frequency of apoptosis-associated phosphatidylserine expression. Each of these parameters can be identified in vivo using specific radiolabeled agents: metabolism can be identified with 18F fluorodeoxyglucose (FDG), receptor expression with 99mTc monocyte chemotactic peptide-1, and apoptosis with 99mTc annexin V. The locally increased concentration of these tracers is readily demonstrable in experimental lesions by ex vivo autoradiography; however, the small lesion size makes it difficult to identify atheroma in the coronaries with conventional imaging equipment. In contrast, with a radiation-sensitive catheter, optimized to sense charged particle rather than gamma or x-radiation, specific lesions could be identified and localized. Charged particle radiation is emitted as a byproduct of nearly all radioactive decay but is typically most abundant in radionuclides that decay by beta emission (either positrons or negatrons). Prototype catheters, using a plastic scintillator mated to an optical fiber, have been tested in the laboratory with the positron-emitting radiopharmaceutical 18FDG. The catheter had sufficient sensitivity to detect lesions concentrating nanocurie concentrations of 18FDG. Ex vivo experiments in apo-e-/- mice confirmed the ability of the catheter to detect 18FDG in aortic lesions. These feasibility studies demonstrate the sensitivity of a beta-sensitive catheter system. Additional mechanical refinements are needed to optimize the system in anticipation of in vivo animal studies.

Detection of focal hypoxic-ischemic injury and neuronal stress in a rodent model of unilateral MCA occlusion/reperfusion using radiolabeled annexin V.

In this study we wished to determine whether technetium-99m annexin V, an in vivo marker of cellular injury and death, could be used to noninvasively monitor neuronal injury following focal middle cerebral artery (MCA) occlusion/reperfusion injury. Sixteen adult male Sprague-Dawley rats (along with four controls) underwent left (unilateral) MCA intraluminal beaded thread occlusion for 2 h followed by reperfusion. One hour following tail vein injection of 5-10 mCi of (99m)Tc-annexin V, animals underwent either single-photon emission computerized tomography (SPECT) or autoradiography followed by immunohistochemical analyses. There was abnormal, bilateral, multifocal uptake of (99m)Tc-annexin V in each cerebral hemisphere as seen by both SPECT and autoradiography at 4 h and 1, 3, and 7 days after initiation of occlusion. The average maximal annexin V uptake at 4 h was 310%+/-85% and 365%+/-151% above control values (P<0.006) within the right and left hemispheres, respectively, peaking on day 3 with values of 925%+/-734% and 1,194%+/-643% (P<0.03) that decreased by day 7 to 489%+/-233% and 785%+/-225% (P<0.01). Total lesional volume of the left hemisphere was 226%, 261%, and 451% ( P<0.03) larger than the right at 4, 24, and 72 h after injury, respectively. Annexin V localized to the cytoplasm of injured neurons ipsilateral to the site of injury as well as to otherwise normal-appearing neurons of the contralateral hemisphere as confirmed by dual fluorescent microscopy. It is concluded that there is abnormal bilateral, multifocal annexin V uptake, greater on the left than on the right side, within 4 h of unilateral left MCA ischemic injury and that the uptake peaks at 3 days and decreases by 7 days after injury. This pattern suggests that neuronal stress may play a role in the response of the brain to focal injury and be responsible for annexin V uptake outside the region of ischemic insult.

Multi-modality imaging identifies key times for annexin V imaging as an early predictor of therapeutic outcome.

Radiolabeled annexin V may provide an early indication of the success or failure of anticancer therapy on a patient-by-patient basis as an in vivo marker of tumor cell killing. An important question that remains is when, after initiation of treatment, should annexin V imaging be performed. To address this issue, we obtained simultaneous in vivo measurements of tumor burden and uptake of radiolabeled annexin V in the syngeneic orthotopic murine BCL1 lymphoma model using in vivo bioluminescence imaging (BLI) and small animal single-photon emission computed tomography (SPECT). BCL1 cells labeled for fluorescence and bioluminescence assays (BCL1-gfp/luc) were injected into mice at a dose that leads to progressive disease within two to three weeks. Tumor response was followed by BLI and SPECT before and after treatment with a single dose of 10 mg/kg doxorubicin. Biodistribution analyses revealed a biphasic increase of annexin V uptake within the tumor-bearing tissues of mice. An early peak occurring before actual tumor cells loss was observed between 1 and 5 hr after treatment, and a second longer sustained rise from 9 to 24 hr after therapy, which heralds the onset of tumor cell loss as confirmed by BLI. Multimodality imaging revealed the temporal patterns of tumor cell loss and annexin V uptake revealing a better understanding of the timing of radiolabeled annexin V uptake for its development as a marker of therapeutic efficacy.