Kuei C. Lee

Molecular imaging of Akt kinase activity.

The serine/threonine kinase Akt mediates mitogenic and anti-apoptotic responses that result from activation of multiple signaling cascades. It is considered a key determinant of tumor aggressiveness and is a major target for anticancer drug development. Here, we describe a new reporter molecule whose bioluminescence activity within live cells and in mice can be used to measure Akt activity. Akt activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to activation or inhibition of receptor tyrosine kinase, inhibition of phosphoinositide 3-kinase, or direct inhibition of Akt. The results provide unique insights into the pharmacokinetics and pharmacodynamics of agents that modulate Akt activity, revealing the usefulness of this reporter for rapid dose and schedule optimization in the drug development process.

Prospective Early Response Imaging Biomarker for Neoadjuvant Breast Cancer Chemotherapy

Purpose: The American Cancer Society estimates that in 2006, 212,920 women will be diagnosed with breast cancer and that 40,970 women will die from the disease. The development of more efficacious chemotherapies has improved outcomes, but the rapid assessment of clinical benefit from these agents remains challenging. In breast cancer patients receiving neoadjuvant chemotherapy, treatment response is traditionally assessed by physical examination and volumetric-based measurements, which are subjective and require macroscopic changes in tumor morphology. In this study, we evaluate the feasibility of using diffusion magnetic resonance imaging (MRI) as a reliable and quantitative measure for the early assessment of response in a breast cancer model. Experimental Design: Mice implanted with human breast cancer (MX-1) were treated with cyclophosphamide and evaluated using diffusion MRI and growth kinetics. Histologic analyses using terminal nucleotidyl transferase–mediated nick end labeling and H&E were done on tumor samples for correlation with imaging results. Results: Cyclophosphamide treatment resulted in a significant reduction in tumor volumes compared with controls. The mean apparent diffusion change for treated tumors at days 4 and 7 posttreatment was 44 ± 5% and 94 ± 7%, respectively, which was statistically greater (P < 0.05) than the control tumors at the same time intervals. The median time-to-progression for control and treated groups was 11 and 32 days, respectively (P < 0.05). Conclusion: Diffusion MRI was shown to detect early changes in the tumor microenvironment, which correlated with standard measures of tumor response as well as overall outcome. Moreover, these findings show the feasibility of using diffusion MRI for assessing treatment response of a breast tumor model in a neoadjuvant setting.

An Imaging Biomarker of Early Treatment Response in Prostate Cancer that Has Metastasized to the Bone

Prostate cancer ranks as the most common lethal malignancy diagnosed and the second leading cause of cancer mortality in American men. Although high response rates are achieved using androgen blockade as first-line therapy, most men progress toward hormone-refractory prostate cancer. Systemic chemotherapies have been shown to improve clinical outcome in hormone refractory prostate cancer patients; however, they are not curative. Due to the high incidence of bone involvement in hormone-refractory prostate cancer, assessment of treatment response in metastatic prostate cancer to the bone remains a major clinical need. In this current study, we investigated the feasibility of using the functional diffusion map (fDM) as an imaging biomarker for assessing early treatment response in a preclinical model of metastatic prostate cancer. The fDM biomarker requires a pretreatment and midtreatment magnetic resonance imaging diffusion map, which is used to quantify spatially distinct therapeutic-induced changes in the Brownian motion (or diffusion) of water within tumor tissue. Because water within tumor cells is in a restricted environment relative to extracellular water, loss of cell membrane integrity and cellular density during therapy will be detected by fDM as an increase in diffusion. Regions of significantly increased diffusion values were detected early using fDM in docetaxel-treated versus untreated metastatic prostate bone tumors at 7 days post treatment initiation (P < 0.05), indicating loss of tumor cell viability. Validation of fDM results was accomplished by histologic analysis of excised tissue. Results from this study show the capability of fDM as a biomarker for detection of bone cancer treatment efficacy, thus warranting clinical evaluation.

Synergy between anti-CCL2 and docetaxel as determined by DW-MRI in a metastatic bone cancer model.

Metastatic prostate cancer continues to be the second leading cause of cancer death in American men with an estimated 28,660 deaths in 2008. Recently, monocyte chemoattractant protein‐1 (MCP‐1, CCL2) has been identified as an important factor in the regulation of prostate metastasis. CCL2, shown to attract macrophages to the tumor site, has a direct promotional effect on tumor cell proliferation, migration, and survival. Previous studies have shown that anti‐CCL2 antibodies given in combination with docetaxel were able to induce tumor regression in a pre‐clinical prostate cancer model. A limitation for evaluating new treatments for metastatic prostate cancer to bone is the inability of imaging to objectively assess response to treatment. Diffusion‐weighted MRI (DW‐MRI) assesses response to anticancer therapies by quantifying the random (i.e., Brownian) motion of water molecules within the tumor mass, thus identifying cells undergoing apoptosis. We sought to measure the treatment response of prostate cancer in an osseous site to docetaxel, an anti‐CCL2 agent, and combination treatments using DW‐MRI. Measurements of tumor apparent diffusion coefficient (ADC) values were accomplished over time during a 14‐day treatment period and compared to response as measured by bioluminescence imaging and survival studies. The diffusion data provided early predictive evidence of the most effective therapy, with survival data results correlating with the DW‐MRI findings. DW‐MRI is under active investigation in the pre‐clinical and clinical settings to provide a sensitive and quantifiable means for early assessment of cancer treatment outcome. J. Cell. Biochem. 107: 58–64, 2009.

Dynamic Imaging of Emerging Resistance during Cancer Therapy

One of the greatest challenges in developing therapeutic regimens is the inability to rapidly and objectively assess tumor response due to treatment. Moreover, tumor response to therapeutic intervention in many cases is transient, and progressive alterations within the tumor may mask the effectiveness of an initially successful therapy. The ability to detect these changes as they occur would allow timely initiation of alternative approaches, maximizing therapeutic outcome. We investigated the ability of diffusion magnetic resonance imaging (MRI) to provide a sensitive measure of tumor response throughout the course of treatment, possibly identifying changes in sensitivity to the therapy. Orthotopic 9L gliomas were subjected to two separate therapeutic regimens, with one group receiving a single 5-day cycle (1omega) of low-dose 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and a second group receiving two cycles at the same dose, bisected with 2 days of rest (2omega). Apparent diffusion coefficient maps were acquired before and throughout treatment to observe changes in water mobility, and these observations were correlated to standard measures of therapeutic response and outcome. Our results showed that diffusion MRI was indeed able to detect the emergence of a drug-resistant tumor subpopulation subsequent to an initially successful cycle of BCNU therapy, leading to minimal gains from a second cycle. These diffusion MRI findings were highly correlated with tumor growth delay, animal survival, and ex vivo growth inhibition assays showing emerging resistance in excised tumors. Overall, this study highlights the ability of diffusion MRI to provide sensitive dynamic assessment of therapy-induced response, allowing early opportunities for optimization of therapeutic protocols.

Noninvasive Molecular Imaging Sheds Light on the Synergy between 5-Fluorouracil and TRAIL/Apo2L for Cancer Therapy

Purpose: In a previous report, a recombinant luciferase reporter, activated during apoptosis via caspase-3 cleavage, was developed for imaging of apoptosis using bioluminescence. The ability to noninvasively image apoptosis in vivo could dramatically benefit the preclinical development of therapeutics targeting the apoptotic pathway. In this study, we examined the use of 5-fluorouracil (5-FU) for sensitizing D54 tumors to tumor necrosis factor α–related apoptosis–inducing ligand (TRAIL) therapy by monitoring apoptotic activity in vivo using bioluminescence imaging. Experimental Design: Using our apoptosis imaging platform and diffusion magnetic resonance imaging (MRI), we monitored the antitumor effects of 5-FU, TRAIL, and 5-FU + TRAIL using D54 xenografts. Additionally, volumetric and histologic analyses were done for correlation with findings from bioluminescence imaging and diffusion MRI. Results: Bioluminescence imaging showed that therapy with TRAIL alone produced an initial 400% increase in apoptotic activity that rapidly diminished during the 10-day treatment period despite continued therapy. In contrast, concomitant 5-FU and TRAIL therapy elicited an apoptotic response that was sustained throughout the entire therapeutic course. Using diffusion MRI, an enhanced tumor response was detected when concomitant therapy was given versus TRAIL-alone therapy. Last, concomitant therapy resulted in a prolonged growth delay (∼9 days) compared with TRAIL alone (∼4 days). Conclusion: We showed that concomitant 5-FU and TRAIL therapy indeed enhanced apoptotic activity in vivo, which translated into greater tumor control. Moreover, this technique sheds light on the synergy of 5-FU and TRAIL as evidenced by differences in the temporal activation of caspase-3 resulting from the different therapeutic regimens.

Fusion of the HSV-1 tegument protein vp22 to cytosine deaminase confers enhanced bystander effect and increased therapeutic benefit

A major limitation in cancer gene therapy, specifically gene-dependent enzyme prodrug therapy (GDEPT), is inefficient gene delivery and expression. The suicide gene cytosine deaminase (CD) and its substrate, 5-fluorocytosine (5-FC), have been extensively explored due to the inherent ‘bystander’ effect achieved through diffusion of the toxic metabolite 5-fluorouracil (5-FU). In this study, we aimed to enhance this ‘bystander’ effect by fusing the Saccharomyces cerevisiae CD to the HSV-1 tegument protein vp22, a novel translocating protein. Two constructs were created: one with vp22 fused to CD (vp22CD) and a second wherein a truncated vp22, lacking the necessary residues for trafficking, fused to CD (delvp22CD). The generated 9L stable lines exhibited similar growth rates, enzyme expression, CD activity, and sensitivity to 5-FC and 5-FU. However, mixed population colony formation assays demonstrated greater bystander effect with the vp22CD fusion as compared to delvp22CD. This enhancement was maintained in vivo where 9L tumors expressing 20 or 50% vp22CD exhibited increased growth delay compared to the respective delvp22CD tumors. Moreover, adenoviral transduction of established wild-type 9L tumors showed increased growth delay with vp22CD (Ad-EF_vp22CD) as compared to equivalent CD (Ad-EF_CD) transduced tumors. Finally, confirming the increased efficacy, 19F magnetic resonance spectroscopy (MRS) of vp22CD-expressing tumors demonstrated increased 5-FU levels as compared to tumors expressing the nontranslocating CD. These results together demonstrated that fusion of vp22 to CD resulted in CD translocation, which in turn amplified conversion of 5-FC to 5-FU in vivo and enhanced the therapeutic benefit of this GDEPT strategy.

Evaluation of Treatment-Associated Inflammatory Response on Diffusion-Weighted Magnetic Resonance Imaging and 2-[18F]-Fluoro-2-Deoxy-d-Glucose-Positron Emission Tomography Imaging Biomarkers

Purpose: Functional imaging biomarkers of cancer treatment response offer the potential for early determination of outcome through the assessment of biochemical, physiologic, and microenvironmental readouts. Cell death may result in an immunologic response, thus complicating the interpretation of biomarker readouts. This study evaluated the temporal effect of treatment-associated inflammatory activity on diffusion magnetic resonance imaging and 2-[18F]-fluoro-2-deoxy-d-glucose-positron emission tomography imaging (FDG-PET) biomarkers to delineate the effects of the inflammatory response on imaging readouts. Experimental Design: Rats with intracerebral 9L gliosarcomas were separated into four groups consisting of control, an immunosuppressive agent dexamethasone (Dex), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and BCNU+Dex. Animals were imaged using diffusion-weighted magnetic resonance imaging and FDG-PET at 0, 3, and 7 days posttreatment. Results: In the BCNU- and BCNU+Dex-treated animal groups, diffusion values increased progressively over the 7-day study period to ∼23% over baseline. The FDG percentage change of standard uptake value decreased at day 3 (−30.9%) but increased over baseline levels at day 7 (+20.1%). FDG-PET of BCNU+Dex-treated animals were found to have percentage of standard uptake value reductions of −31.4% and −24.7% at days 3 and 7, respectively, following treatment. Activated macrophages were observed on day 7 in the BCNU treatment group with much fewer found in the BCNU+Dex group. Conclusions: Results revealed that treatment-associated inflammatory response following tumor therapy resulted in the accentuation of tumor diffusion response along with a corresponding increase in tumor FDG uptake due to the presence of glucose-consuming activated macrophages. The dynamics and magnitude of potential inflammatory response should be considered when interpreting imaging biomarker results. Clin Cancer Res; 16(5); 1542–52

Radiosynthesis and Evaluation of 5-( 125 I)Iodoindol-3-yl-β-D-Galactopyranoside as a β-Galactosidase Imaging Radioligand

The synthesis and investigation of 5-[125I]iodoindol-3-yl-β-d-galactopyranoside ([125I]IBDG) as a radioligand for single-photon emission computed tomography (SPECT) imaging of β-galactosidase expression are described. No-carrier-added [125I]IBDG was synthesized by a radioiododestannylation approach in > 75% overall radiochemical yield and > 99% radiochemical purity. [125I]IBDG was evaluated as a substrate using β-galactosidase-expressing (D54L) and nonexpressing (D54) human glioma cell lines. A 24-hour incubation of this substrate with cultured cells revealed a 6.5-fold greater intracellular trapping of radioactivity in D54L cells compared with D54 cells. Systemic delivery of [125I]IBDG in nude mice bearing D54L tumors failed to show significant trapping of radioactivity within these tumors by SPECT imaging. In contrast, intratumoral injection of the substrate resulted in efficient trapping of radioactivity in D54L tumors but not D54 tumors, resulting in clear SPECT visualization of the former tumor. Based on dynamic SPECT imaging and blood metabolite analysis, we conclude that although [125I]IBDG is an efficient in vivo substrate for β-galactosidase, its rapid renal clearance hampers its intratumoral availability on systemic administration.

A Transgenic Mouse for Imaging Caspase-Dependent Apoptosis within the Skin

Apoptosis is an essential process for the maintenance of normal physiology. The ability to noninvasively image apoptosis in living animals would provide unique insights into its role in normal and disease processes. Herein, a recombinant reporter consisting of beta-galactosidase gene flanked by two estrogen receptor regulatory domains and intervening Asp-Glu-Val-Glu sequences was constructed to serve as a tool for in vivo assessment of apoptotic activity. The results demonstrate that when expressed in its intact form, the hybrid reporter had undetectable beta-galactosidase activity. Caspase 3 activation in response to an apoptotic stimulus resulted in cleavage of the reporter, and thereby reconstitution of beta-galactosidase activity. Enzymatic activation of the reporter during an apoptotic event enabled noninvasive measurement of beta-galactosidase activity in living cells, which correlated with traditional measures of apoptosis in a dose- and time-dependent manner. Using a near-infrared fluorescent substrate of beta-galactosidase (9H-{1,3-dichloro-9,9-dimethylacridin-2-one-7-yl} beta-D-galactopyranoside), noninvasive in vivo imaging of apoptosis was achieved in a xenograft tumor model in response to proapoptotic therapy. Finally, a transgenic mouse model was developed expressing the ER-LACZ-ER reporter within the skin. This reporter and transgenic mouse could serve as a unique tool for the study of apoptosis in living cells and animals, especially in the context of skin biology.