Xianlei Sun

Molecular imaging of tumor-infiltrating macrophages in a preclinical mouse model of breast cancer.

Significant evidence has indicated that tumor-associated macrophages (TAMs) play a critical role in the proliferation, invasion, angiogenesis, and metastasis of a variety of human carcinomas. In this study, we investigated whether near-infrared fluorescence (NIRF) imaging using a macrophage mannose receptor (MMR; CD206)-targeting agent could be used to noninvasively visualize and quantify changes in TAMs in vivo. The CD206-targeting NIRF agent, Dye-anti-CD206, was prepared and characterized in vitro and in vivo. By using NIRF imaging, we were able to noninvasively image tumor-infiltrating macrophages in the 4T1 mouse breast cancer model. Importantly, longitudinal NIRF imaging revealed the depletion of macrophages in response to zoledronic acid (ZA) treatment. However, ZA alone did not lead to the inhibition of 4T1 tumor growth. We therefore combined anti-macrophage ZA therapy and tumor cytotoxic docetaxel (DTX) therapy in the mouse model. The results demonstrated that this combination strategy could significantly inhibit tumor growth as well as tumor metastasis to the lungs. Based on these findings, we concluded that CD206-targeted molecular imaging can sensitively detect the dynamic changes in tumor-infiltrating macrophages, and that the combination of macrophage depletion and cytotoxic therapy is a promising strategy for the effective treatment of solid tumors.

Integrin αvβ6–Targeted SPECT Imaging for Pancreatic Cancer Detection

Integrin αvβ6, a member of the integrin family, is specifically expressed in many malignancies but not in normal organs. Overexpression of integrin αvβ6 is usually correlated with malignant potential and poor prognosis. In this study, we describe the synthesis and evaluation of a 99mTc-labeled integrin αvβ6–targeting peptide as a SPECT radiotracer for the in vivo imaging of integrin αvβ6 expression. Methods: An integrin αvβ6–targeting peptide (denoted as the HK peptide) was conjugated with 6-hydrazinonicotinyl (HYNIC) and radiolabeled with 99mTc using tricine and TPPTS (trisodium triphenylphosphine-3,3′,3″-trisulfonate) as coligands. The in vitro and in vivo characteristics of 99mTc-HYNIC(tricine)(TPPTS)-HK (99mTc-HHK) were investigated in BxPC-3 (integrin αvβ6–positive) and HEK293 (integrin αvβ6–negative) models. The ability of 99mTc-HHK to detect liver metastasis of pancreatic cancer was evaluated using small-animal SPECT/CT. Results: 99mTc-HHK showed high integrin αvβ6–binding specificity both in vitro and in vivo. 99mTc-HHK was cleared rapidly from the blood and normal organs except for the kidneys. The highest uptake (0.88 ± 0.12 percentage injected dose per gram) of 99mTc-HHK in BxPC-3 tumors was observed at 0.5 h after injection. High-contrast images of integrin αvβ6–positive tumors were obtained using 99mTc-HHK. The minimum nonspecific activity accumulation in normal liver tissues rendered high-quality SPECT/CT images of metastatic lesions. Conclusion: 99mTc-HHK is a promising SPECT radiotracer for the noninvasive imaging of integrin αvβ6 expression in vivo. SPECT/CT with 99mTc-HHK could provide an effective approach for the noninvasive detection of primary and metastatic lesions of integrin αvβ6–positive tumors.

Serial in vivo imaging using a fluorescence probe allows identification of tumor early response to cetuximab immunotherapy.

Cetuximab is an antiepidermal growth factor receptor (EGFR) monoclonal antibody that has received the approval of the Food and Drug Administration (FDA) for cancer treatment. However, most clinical studies indicate that cetuximab can only elicit positive effects on a subset of cancer patients. In this study, we investigated whether near-infrared fluorescence (NIRF) imaging of tumor vascular endothelial growth factor (VEGF) expression could be a biomarker for tumor early response to cetuximab therapy in preclinical wild-type and mutant tumor models of the KRAS gene. The treatment efficacy of cetuximab was determined in both HT-29 (wild-type KRAS) and HTC-116 (mutant KRAS) human colon cancer models. A VEGF-specific optical imaging probe (Dye755-Ran) was synthesized by conjugating ranibizumab (an anti-VEGF antibody Fab fragment) with a NIRF dye. Serial optical scans with Dye755-Ran were performed in HT-29 and HTC-116 xenograft models. By using longitudinal NIRF imaging, we were able to detect early tumor response on day 3 and day 5 after initiation of cetuximab treatment in the cetuximab-responsive HT-29 tumor model. Enzyme-linked immunosorbent assay (ELISA) confirmed that cetuximab treatment inhibited human VEGF expression in the KRAS wild-type HT-29 tumor but not in the KRAS mutant HCT-116 tumor. We have demonstrated that the antitumor effect of cetuximab can be noninvasively monitored by serial fluorescence imaging using Dye755-Ran. VEGF expression detected by optical imaging could serve as a sensitive biomarker for tumor early response to drugs that directly or indirectly act on VEGF.

Early Assessment of Tumor Response to Gefitinib Treatment by Noninvasive Optical Imaging of Tumor Vascular Endothelial Growth Factor Expression in Animal Models

Epidermal growth factor receptor (EGFR) expression is upregulated in many types of tumors, and the EGFR tyrosine kinase inhibitor gefitinib has high potential as an anticancer drug. However, accumulating clinical evidence has indicated that only a subset of patients benefit from gefitinib treatment. This study aimed to determine whether optical imaging of vascular endothelial growth factor (VEGF) expression can be an early biomarker for tumor response to gefitinib therapy. Methods: A VEGF-targeting fluorescent probe Dye-BevF(ab′)2 was prepared and tested in vivo. Longitudinal optical imaging studies using Dye-BevF(ab′)2 were performed in both 22B (gefitinib-resistant) and A549 (gefitinib-responsive) tumor models at different times (days 0, 2, and 5) before and after gefitinib treatment. The imaging results were validated by ex vivo immunofluorescence staining and enzyme-linked immunosorbent assay. Results: Dye-BevF(ab′)2 exhibited high specificity for VEGF in vivo. There was no significant change in the Dye-BevF(ab′)2 uptake in gefitinib-treated 22B tumors, compared with the control group. In contrast, the A549 tumor uptake of Dye-BevF(ab′)2 in the gefitinib-treated group was significantly lower on days 2 and 5 than that in the control group and at the baseline. An in vivo gefitinib treatment study confirmed that 22B tumors were gefitinib-resistant, whereas A549 tumors were gefitinib-responsive. Immunofluorescence staining and enzyme-linked immunosorbent assay confirmed that changes in the Dye-BevF(ab′)2 uptake were correlated with VEGF expression levels in tumors. Conclusion: Optical imaging of VEGF expression with Dye-BevF(ab′)2 can be used for the early assessment of tumor response to gefitinib therapy. This approach may also be valuable for preclinical high-throughput screening of novel antiangiogenic drugs.

Molecular Imaging Reveals Trastuzumab-Induced Epidermal Growth Factor Receptor Downregulation In Vivo

Previous in vitro studies demonstrated that treating tumors expressing both epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 with trastuzumab resulted in increased EGFR homodimerization and subsequent rapid downregulation of EGFR. We investigated whether molecular imaging using near-infrared fluorescence (NIRF) imaging and PET probes could sensitively detect trastuzumab-induced EGFR downregulation in vivo. Methods: The F(ab′)2 antibody fragment PaniF(ab′)2 was generated by digesting the anti-EGFR monoclonal antibody panitumumab. PaniF(ab′)2 was labeled with either a NIRF dye or 68Ga, and optical imaging and small-animal PET imaging of Dye-PaniF(ab′)2 and 68Ga-PaniF(ab′)2, respectively, were performed in HT-29 tumor–bearing nude mice treated with trastuzumab or untreated control. Results: Longitudinal NIRF imaging studies revealed significantly reduced tumor uptake of Dye-PaniF(ab′)2 on days 5 and 7 in trastuzumab-treated HT-29 tumors, compared with control. Western blotting confirmed the downregulation of EGFR after treatment with trastuzumab. Small-animal PET on day 5 after trastuzumab treatment also demonstrated decreased 68Ga-PaniF(ab′)2 uptake in trastuzumab-treated HT-29 tumors. The tumor uptake value of 68Ga-PaniF(ab′)2 obtained from PET imaging had an excellent linear correlation with the uptake value measured using biodistribution. Conclusion: The downregulation of EGFR induced by trastuzumab treatment could be detected noninvasively using optical and PET imaging. This molecular imaging strategy could provide a dynamic readout of changes in the tumor signaling and may facilitate the noninvasive monitoring of the early tumor response to drug treatment.

Molecular Imaging of Post-Src Inhibition Tumor Signatures for Guiding Dasatinib Combination Therapy

Noninvasive, real-time, quantitative measurement of key biomarkers associated with cancer therapeutic interventions could provide a better understanding of cancer biology. We investigated in this study whether incorporating multiple molecular imaging approaches could be used to guide dasatinib anti-Src therapy and aid in the rational design of a combination therapy regimen. Methods: Bioluminescence imaging, 18F-FDG PET, integrin αvβ3–targeted SPECT/CT, and vascular endothelial growth factor–targeted near-infrared fluorescence imaging were performed before and after dasatinib treatment in a tumor mouse model. Results: There was no significant difference in the bioluminescence imaging signal or 18F-FDG tumor uptake in dasatinib-treated tumors compared with the control tumors. However, the uptake of 99mT-3PRGD2 (integrin αvβ3–specific) and DyLight755-ranibizumab (vascular endothelial growth factor–specific) in the dasatinib-treated tumors was significantly lower than that in the control tumors. In vitro studies confirmed the antiangiogenic effects of dasatinib but indicated a lack of cytotoxicity. Dasatinib plus cytotoxic docetaxel elicited marked synergistic tumor growth inhibition in vivo. Conclusion: Visualization of post-Src inhibition tumor signatures through multiple imaging approaches facilitates sensitive and quantitative measurement of cancer biomarkers in vivo, thus aiding in the rational design of dasatinib combination therapy.

Small-Animal SPECT/CT of the Progression and Recovery of Rat Liver Fibrosis by Using an Integrin αvβ3-targeting Radiotracer.

PURPOSE To assess the potential utility of an integrin αvβ3-targeting radiotracer, technetium 99m-PEG4-E[PEG4-cyclo(arginine-glycine-aspartic acid-D-phenylalanine-lysine)]2 ((99m)Tc-3PRGD2), for single photon emission computed tomography (SPECT)/computed tomography (CT) for monitoring of the progression and prognosis of liver fibrosis in a rat model. MATERIALS AND METHODS All animal experiments were performed by following the protocol approved by the institutional animal care and use committee. (99m)Tc-3PRGD2 was prepared and longitudinal SPECT/CT was performed to monitor the progression (n = 8) and recovery (n = 5) of liver fibrosis induced in a rat model by means of thioacetamide (TAA) administration. The mean liver-to-background radioactivity per unit volume ratio was analyzed for comparisons between the TAA and control (saline) groups at different stages of liver fibrosis. Data were compared by using Student t and Mann-Whitney tests. Results:of SPECT/CT were compared with those of ex vivo biodistribution analysis (n = 5). RESULTS Accumulation of (99m)Tc-3PRGD2 in the liver increased in proportion to the progression of fibrosis and TAA exposure time; accumulation levels were significantly different between the TAA and control groups as early as week 4 of TAA administration (liver-to-background ratio: 32.30 ± 3.39 vs 19.01 ± 3.31; P = .0002). Results of ex vivo immunofluorescence staining demonstrated the positive expression of integrin αvβ3 on the activated hepatic stellate cells, and the integrin αvβ3 levels in the liver corresponded to the results of SPECT/CT (R(2) = 0.75, P < .0001). (99m)Tc-3PRGD2 uptake in the fibrotic liver decreased after antifibrotic therapy with interferon α2b compared with that in the control group (relative liver-to-background ratio: 0.45 ± 0.05 vs 1.01 ± 0.05; P < .0001) or spontaneous recovery (relative liver-to-background ratio: 0.56 ± 0.06 vs 1.01 ± 0.05; P < .0001). CONCLUSION (99m)Tc-3PRGD2 SPECT/CT was successfully used to monitor the progression and recovery of liver fibrosis and shows potential applications for noninvasive diagnosis of early stage liver fibrosis.