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.

Epidermal growth factor receptor (EGFR) has been well characterized as an important target for cancer therapy. Immunotherapy based on EGFR-specific antibodies (e.g., panitumumab and cetuximab) has shown great clinical promise. However, increasing evidence has indicated that only a subgroup of patients receiving these antibodies will benefit from them, and even patients who do initially experience a major response may eventually develop therapeutic resistance. In this study, we investigated whether panitumumab and cetuximab can serve as delivery vehicles for tumor-targeted radionuclide therapy in a preclinical tumor model that did not respond to immunotherapy. The in vitro toxicity and cell binding properties of panitumumab and cetuximab were characterized. Both antibodies were conjugated with 1,4,7,10-tetraazadodecane-N,N′,N″,N‴-tetraacetic acid (DOTA) and radiolabeled with 177Lu. Small-animal SPECT/CT, biodistribution, and radioimmunotherapy (RIT) studies of 177Lu-DOTA–panitumumab (177Lu-Pan) and 177Lu-DOTA–cetuximab (177Lu-Cet) were performed in the UM-SCC-22B tumor model. Both 177Lu-Pan and 177Lu-Cet exhibited favorable tumor targeting efficacy. The tumor uptake was 20.92 ± 4.45, 26.10 ± 5.18, and 13.27 ± 1.94% ID/g for 177Lu-Pan, and 15.67 ± 3.84, 15.72 ± 3.49, and 7.82 ± 2.36% ID/g for 177Lu-Cet at 24, 72, and 120 h p.i., respectively. RIT with a single dose of 14.8 MBq of 177Lu-Pan or 177Lu-Cet significantly delayed tumor growth. 177Lu-Pan induced more effective tumor growth inhibition due to a higher tumor uptake. Our results suggest that panitumumab and cetuximab can function as effective carriers for tumor-targeted delivery of radiation, and that RIT is promising for targeted therapy of EGFR-positive tumors, especially for those tumors that are resistant to antibody-based immunotherapy.Keywords: antibody; epidermal growth factor receptor; small-animal imaging; targeted therapy;

Optical imaging is emerging as a powerful tool for the noninvasive imaging of the biological processes in living subjects. This study aimed to investigate whether optical imaging of integrin αvβ3 and vascular endothelial growth factor (VEGF) expression can serve as sensitive biomarkers for tumor early response to antiangiogenic therapy.We synthesized two near-infrared fluorescence (NIRF) imaging agents, CF680R-3PRGD2 and CF750-BevF(ab')2, which were designed to specifically bind to integrin αvβ3 and VEGF, respectively. The ability of optical imaging using the two imaging agents for early monitoring the antiangiogenic effect of sunitinib was evaluated.CF680R-3PRGD2 and CF750-BevF(ab')2 specifically bound to their respective targets in vitro and in HT-29 tumor-bearing nude mice. Sunitinib treatment led to significantly decreased tumor uptake of CF680R-3PRGD2 (e.g., 7.47 ± 1.62 % vs. 4.24 ± 0.16 % on day 4; P < 0.05) and CF750-BevF(ab')2 (e.g., 7.43 ± 2.43 % vs. 4.04 ± 1.39 % on day 2; P < 0.05) in vivo. Immunofluorescence staining and an enzyme-linked immunosorbent assay confirmed that sunitinib-induced changes in tumor uptake of CF680R-3PRGD2 and CF750-BevF(ab')2 were correlated with changes in the levels of integrin αvβ3 and VEGF. Radiobiodistribution of 99mTc-3PRGD2 and 125I-BevF(ab')2, the radiocounterparts of CF680R-3PRGD2 and CF750-BevF(ab')2, respectively, also validated optical imaging results.Longitudinal monitoring of tumor integrin αvβ3 and VEGF expression could be used as early biomarkers for tumor response to antiangiogenic therapy. This strategy may facilitate the development of new antiangiogenic drugs, and be used for elucidation of the underlying mechanisms of therapies involving the integrin and the VEGF signaling pathway.

Since their discovery in 2006, induced pluripotent stem cells (iPSCs) have gained increasing interest for tissue regeneration and transplantation therapies. However, teratoma formation after iPSC transplantation is one of the most serious drawbacks that may limit their further clinical application. We investigated here whether human iPSC-derived teratomas could be detected by an integrin-targeting agent 99mTc-PEG4-E[PEG4-c(RGDfK)]2 (99mTc-3PRGD2).Human-induced pluripotent stem cells (hiPSCs) were generated and characterized. In vitro integrin αvβ3 expression levels of hiPSC- and hiPSC-derived teratoma cells were determined by flow cytometry. 99mTc-3PRGD2 was prepared, and planar gamma imaging and biodistribution studies were carried out in teratoma-bearing severe combined immunodeficient (SCID) mice. Positron emission tomography (PET) imaging of teratomas with 2-deoxy-2-[18F]fluoro-d-glucose (18F-FDG) was also performed for comparison. Integrin αvβ3 expression in teratoma tissues was determined by immunofluorescence staining.99mTc-3PRGD2 showed high (2.82 ± 0.21 and 2.69 ± 0.73%ID/g at 0.5 and 1 h pi, respectively) and specific (teratoma uptake decreased from 2.69 ± 0.73 to 0.53 ± 0.26%ID/g after blocking with cold 3PRGD2) uptake in teratoma tissues, and planar gamma imaging demonstrated the feasibility of noninvasively detecting the teratoma formation with 99mTc-3PRGD2. 18F-FDG showed low teratoma uptake and thus failed to detect the teratomas. Ex vivo immunofluorescence staining validated the integrin αvβ3 expression in the vasculature during teratoma formation.Gamma imaging with 99mTc-3PRGD2 is a promising approach for the noninvasive monitoring of tumorigenicity after hiPSCs transplantation.

We recently designed and synthesized a Glu-c(RGDyK)-bombesin (RGD-BBN) heterodimeric peptide exhibiting a dual integrin αvβ3 and gastrin-releasing peptide receptor (GRPR) targeting property. In this study, we investigated whether 99mTc-labeled RGD-BBN peptide could be used for the noninvasive detection of lung carcinoma by using small-animal single-photon emission computed tomography (SPECT)/CT. RGD-BBN peptide was conjugated with 6-hydrazinonicotinyl (HYNIC) and then radiolabeled with 99mTc using tricine and TPPTS as the coligands (TPPTS = trisodium triphenylphosphine-3,3′,3″-trisulfonate). The biodistribution, planar gamma imaging, and small-animal SPECT/CT studies of 99mTc-HYNIC(tricine)(TPPTS)-RGD-BBN (99mTc-RGD-BBN) were performed in C57/BL6 mice bearing Lewis lung carcinoma (LLC) or bearing both inflammation and LLC. HYNIC-RGD-BBN possessed a dual integrin αvβ3 and GRPR binding capacity. 99mTc-RGD-BBN was prepared with a high radiochemical purity (>98%), and it exhibited specific tumor imaging with high contrast to the contralateral background. 99mTc-RGD-BBN was superior to 18F-FDG for distinguishing lung carcinoma from inflammation. The uptake of 99mTc-RGD-BBN in LLC xenografts was 2.69 ± 0.66% ID/g at 1 h postinjection (p.i.) and was decreased to 1.99 ± 0.61% ID/g at 2 h p.i. The inflammation uptake of 99mTc-RGD-BBN was 1.20 ± 0.32% ID/g at 1 h and 0.56 ± 0.17% ID/g at 2 h p.i., respectively. High pancreas uptake (25.76 ± 5.49%ID/g and 19.56 ± 6.78% ID/g at 1 and 2 h p.i., respectively) was also found due to the high GRPR expression of this organ. Small-animal SPECT/CT using 99mTc-RGD-BBN can specifically detect the LLC pulmonary metastases. Our results suggested that SPECT/CT with 99mTc-RGD-BBN would provide an effective approach for the noninvasive detection of lung cancer.Keywords: GRPR; integrin αvβ3; lung carcinoma; RGD-bombesin; single-photon emission computed tomography (SPECT);

Tumor-associated macrophages (TAMs) play essential roles in tumor invasion and metastasis, and contribute to drug resistance. Clinical evidence suggests that TAM levels are correlated with local tumor relapse, distant metastasis, and poor prognosis in patients. In this study, we synthesized a TAM-targeted probe (IRD-αCD206) by conjugating a monoclonal anti-CD206 antibody with a near-infrared phthalocyanine dye. We then investigated the potential application of the IRD-αCD206 probe to near-infrared fluorescence (NIRF) imaging and photoimmunotherapy (PIT) of tumors resistant to treatment with the kinase inhibitor sorafenib. Sorafenib treatment had no effect on tumor growth in a 4T1 mouse model of breast cancer, but induced M2 macrophage polarization in tumors. M2 macrophage recruitment by sorafenib-treated 4T1 tumors was noninvasively visualized by in vivo NIRF imaging of IRD-αCD206. Small-animal single-photon emission computed tomography (SPECT)/CT and intratumoral microdistribution analysis indicated TAM-specific localization of the IRD-αCD206 probe in 4T1 tumors after several rounds of sorafenib treatment. Upon light irradiation, IRD-αCD206 suppressed the growth of sorafenib-resistant tumors. In vivo CT imaging and ex vivo histological analysis confirmed the inhibition of lung metastasis in mice by IRD-αCD206 PIT. These results demonstrate the utility of the IRD-αCD206 probe for TAM-targeted diagnostic imaging and treatment of tumors that are resistant to conventional therapeutics.