Use of the inhibitor of ALK fusion onco-protein, crizotinib (PF02341066), has achieved impressive clinical efficacy in patients with ALK-positive non-small cell lung cancer. Nevertheless, acquired resistance to this drug occurs inevitably in approximately a year, limiting the therapeutic benefits of this novel targeted therapy. In this study, we found that autophagy was induced in crizonitib-resistant lung cancer cells and contributed to drug resistance. We observed that ALK was downregulated in the crizotinib-resistant lung cancer cell line, H3122CR-1, and this was causally associated with autophagy induction. The degree of crizotinib resistance correlated with autophagic activity. Activation of autophagy in crizotinib-resistant H3122CR-1 cells involved alteration of the Akt/mTOR signaling pathway. Furthermore, we demonstrated that chloroquine, an inhibitor of autophagy, could restore sensitivity of H3122CR-1 to crizotinib and enhance its efficacy against drug-resistant lung cancer. Thus, modulating autophagy may be worth exploring as a new strategy to overcome acquired crizonitib resistance in ALK-positive lung cancer.

This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2) as fluorescent probes for in vitro assays of integrin αvβ3/αvβ5 expression in tumor tissues. FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were prepared, and their integrin αvβ3/αvβ5 binding affinity was determined using the displacement assay against 125I-echistatin bound to U87MG glioma cells. IC50 values of FITC-Galacto-RGD2, FITC-3P-RGD2, and FITC-RGD2 were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin αvβ3/αvβ5 binding affinity followed a general trend: FITC-Galacto-RGD2 ∼ FITC-3P-RGD2 > FITC-RGD2. The xenografted tumor-bearing models were established by subcutaneous injection of 5 × 106 tumor cells into shoulder flank (U87MG, A549, HT29, and PC-3) or mammary fat pad (MDA-MB-435) of each athymic nude mouse. Three to six weeks after inoculation, the tumor size was 0.1–0.3 g. Tumors were harvested for integrin αvβ3/αvβ5 staining, as well as hematoxylin and eosin (H&E) staining. Six human carcinoma tissues (colon cancer, pancreatic cancer, lung adenocarcinoma, squamous cell lung cancer, gastric cancer, and esophageal cancer) were obtained from recently diagnosed cancer patients. Human carcinoma slides were deparaffinized in xylene, rehydrated with ethanol, and then used for integrin αvβ3/αvβ5 staining, as well as H&E staining. It was found that the tumor staining procedures with FITC-conjugated cyclic RGD peptides were much simpler than those with the fluorescence-labeled integrin αvβ3 antibodies. Since FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were able to co-localize with the fluorescence-labeled integrin β3 antibody, their tumor localization and tumor cell binding are integrin αvβ3-specific. Quantification of the fluorescent intensity in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3) and six human carcinoma tissues revealed an excellent linear relationship between the relative integrin αvβ3/αvβ5 expression levels determined with FITC-Galacto-RGD2 and those obtained with the fluorescence-labeled anti-human integrin β3 antibody. There was also an excellent linear relationship between the tumor uptake (%ID/g) of 99mTc-3P-RGD2 (an integrin αvβ3/αvβ5-targeted radiotracer) and the relative integrin αvβ3/αvβ5 expression levels from the quantification of fluorescent intensity in the tumor tissues stained with FITC-Galacto-RGD2. These results suggest that FITC-conjugated cyclic RGD peptides might be useful to correlate the in vitro findings with the in vivo imaging data from an integrin αvβ3/αvβ5-targeted radiotracer. The results from this study clearly showed that the FITC-conjugated cyclic RGD peptides (particularly FITC-3P-RGD2 and FITC-Galacto-RGD2) are useful fluorescent probes for assaying relative integrin αvβ3/αvβ5 expression levels in tumor tissues.

This study sought to evaluate [99mTc(HYNIC-Galacto-RGD2)(tricine)(TPPTS)] (99mTc-Galacto-RGD2: HYNIC = 6-hydrazinonicotinyl; Galacto-RGD2 = Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(SAA-PEG2-(1,2,3-triazole)-1-yl-4-methylamide)]]2 (SAA = 7-amino-L-glycero-L-galacto-2,6-anhydro-7-deoxy-heptanamide, and PEG2 = 3,6-dioxaoctanoic acid); and TPPTS = trisodium triphenylphosphine-3,3',3"-trisulfonate) as a new radiotracer for tumor imaging. Galacto-RGD2 was prepared via the copper (I)-catalyzed 1,3-dipolar azide–alkyne Huisgen cycloaddition. HYNIC-Galacto-RGD2 was prepared by reacting Galacto-RGD2 with sodium succinimidyl 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinate (HYNIC-OSu) in the presence of diisopropylethylamine, and was evaluated for its integrin αvβ3 binding affinity against 125I-echistatin bound to U87MG glioma cells. The IC50 value for HYNIC-Galacto-RGD2 was determined to be 20 ± 2 nM. 99mTc-Galacto-RGD2 was prepared in high specific activity (~185 GBq/µmol) and high radiochemical purity (>95%), and was evaluated in athymic nude mice bearing U87MG glioma xenografts for its tumor-targeting capability and biodistribution. The tumor uptake of 99mTc-Galacto-RGD2 was 10.30 ± 1.67, 8.37 ± 2.13, 6.86 ± 1.33 and 5.61 ± 1.52 %ID/g at 5, 30, 60 and 120 min p.i., respectively, which was in agreement with high integrin αvβ3 expression on glioma cells and neovasculature. Its lower uptake in intestines, lungs and spleen suggests that 99mTc-Galacto-RGD2 has advantages over 99mTc-3P–RGD2 ([99mTc(HYNIC-3P–RGD2)(tricine)(TPPTS)]: 3P–RGD2 = PEG4-E[PEG4-c(RGDfK)]2; PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) for imaging tumors in the chest and abdominal regions. U87MG tumors were readily detected by SPECT and the tumor uptake of 99mTc-Galacto-RGD2 was integrin αvβ3-specific. 99mTc-Galacto-RGD2 also had very high metabolic stability. On the basis of results from this study, it was concluded that 99mTc-Galacto-RGD2 is an excellent radiotracer for imaging integrin αvβ3-positive tumors and related metastases.

This study sought to evaluate the impact of multiple negative charges on blood clearance kinetics and biodistribution properties of 99mTc-labeled RGD peptide dimers. Bioconjugates HYNIC-P6G-RGD2 and HYNIC-P6D-RGD2 were prepared by reacting P6G-RGD2 and P6D-RGD2, respectively, with excess HYNIC-OSu in the presence of diisopropylethylamine. Their IC50 values were determined to be 31 ± 5 and 41 ± 6 nM, respectively, against 125I-echistatin bound to U87MG glioma cells in a whole-cell displacement assay. Complexes [99mTc(HYNIC-P6G-RGD2)(tricine)(TPPTS)] (99mTc-P6G-RGD2) and [99mTc(HYNIC-P6D-RGD2)(tricine)(TPPTS)] (99mTc-P6D-RGD2) were prepared in high radiochemical purity (RCP > 95%) and specific activity (37–110 GBq/μmol). They were evaluated in athymic nude mice bearing U87MG glioma xenografts for their biodistribution. The most significant difference between 99mTc-P6D-RGD2 and 99mTc-P6G-RGD2 was their blood radioactivity levels and tumor uptake. The initial blood radioactivity level for 99mTc-P6D-RGD2 (4.71 ± 1.00%ID/g) was ∼5× higher than that of 99mTc-P6G-RGD2 (0.88 ± 0.05%ID/g), but this difference disappeared at 60 min p.i. 99mTc-P6D-RGD2 had much lower tumor uptake (2.20–3.11%ID/g) than 99mTc-P6G-RGD2 (7.82–9.27%ID/g) over a 2 h period. Since HYNIC-P6D-RGD2 and HYNIC-P6G-RGD2 shared a similar integrin αvβ3 binding affinity (41 ± 6 nM versus 31 ± 5 nM), the difference in their blood activity and tumor uptake is most likely related to the nine negative charges and high protein binding of 99mTc-P6D-RGD2. Despite its low uptake in U87MG tumors, the tumor uptake of 99mTc-P6D-RGD2 was integrin αvβ3-specific. SPECT/CT studies were performed using 99mTc-P6G-RGD2 in athymic nude mice bearing U87MG glioma and MDA-MB-231 breast cancer xenografts. The SPECT/CT data demonstrated the tumor-targeting capability of 99mTc-P6G-RGD2, and its tumor uptake depends on the integrin αvβ3 expression levels on tumor cells and neovasculature. It was concluded that the multiple negative charges have a significant impact on the blood clearance kinetics and tumor uptake of 99mTc-labeled dimeric cyclic RGD peptides.

Introduction 99mTc-Teboroxime ([99mTcCl(CDO)(CDOH)2BMe]) is a member of the BATO (boronic acid adducts of technetium dioximes) class of 99mTc(III) complexes. This study sought to explore the impact of co-ligands on solution stability, heart uptake and myocardial retention of [99mTc(L)(CDO)(CDOH)2BMe] (99mTc-Teboroxime: L = Cl; 99mTc-Teboroxime(F): L = F; 99mTc-Teboroxime(SCN): L = SCN; and 99mTc-Teboroxime(N3): L = N3). Methods Radiotracers 99mTc-Teboroxime(L) (L = F, SCN and N3) were prepared by reacting 99mTc-Teboroxime with NaF, NaSCN and NaN3, respectively. Biodistribution and imaging studies were carried out in Sprague-Dawley rats. Image quantification was performed to compare their heart retention and liver clearance kinetics. Results Complexes 99mTc-Teboroxime(L) (L = F, SCN and N3) were prepared in high yield with high radiochemical purity. All new radiotracers were stable for >6 h in the kit matrix. In its HPLC chromatogram, 99mTc-Teboroxime showed one peak at ~15.5 min, which was shorter than that of 99mTc-Teboroxime(F) (~16.4 min). There were two peaks for 99mTc-Teboroxime(SCN) at 16.5 and 18.3 min. 99mTc-Teboroxime(N3) appeared as a single peak at 18.4 min. Their heart retention and liver clearance curves were best fitted to the bi-exponential decay function. The half-times of fast/slow components were 1.6 ± 0.4/60.7±8.9 min for 99mTc-Teboroxime, 0.8±0.2/101.7±20.7 min for 99mTc-Teboroxime(F), 1.2±0.3/84.8±16.6 min for 99mTc-Teboroxime(SCN), and 2.9±0.9/51.6±5.0 min for 99mTc-Teboroxime(N3). The 2-min heart uptake followed the order of 99mTc-Teboroxime (3.00±0.37%ID/g) > 99mTc-Teboroxime(N3) (2.66±0.01 %ID/g) ≈ 99mTc-Sestamibi (2.55±0.46 %ID/g) > 99mTcN-MPO (2.38±0.15 %ID/g). 99mTc-Teboroxime remains the best in first-pass extraction. The best image acquisition window is 0 – 5 min for 99mTc-Teboroximine and 0 – 15 min for 99mTc-Teboroximine(N3). Conclusion Co-ligands had significant impact on the heart uptake and myocardial retention of complexes [99mTc(L)(CDO)(CDOH)2BMe] (L = Cl, F, SCN and N3). Future studies should be directed towards minimizing the liver uptake and radioactivity accumulation in the blood vessels while maintaining their high heart uptake.

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This study sought to evaluate K(HYNIC)2 (K = lysine and HYNIC = 6-hydrazinonicotinyl) as a bifunctional chelator for 99mTc-labeling of biomolecule. In this study, four K(HYNIC)2–conjugated cyclic RGD peptides, K(HYNIC)2-RGD2 (RGD2 = E[c(RGDfK)]2), K(HYNIC)2-3G-RGD2 (3G-RGD2 = Gly-Gly-Gly-E[Gly-Gly-Gly-c(RGDfK)]2), K(HYNIC)2-2P-RGD2 (2P-RGD2 = E[PEG4-c(RGDfK)]2, and PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid), and K(HYNIC)2-3P-RGD2 (3P-RGD2 = PEG4-E[PEG4-c(RGDfK)]2) were prepared, and evaluated for their integrin αvβ3 binding affinity. IC50 values were determined to be 47 ± 2, 35 ± 2, 37 ± 2, 85 ± 2 and 422 ± 15 nM for K(HYNIC)2-2P-RGD2, K(HYNIC)2-3P-RGD2, K(HYNIC)2-3G-RGD2, K(HYNIC)2-RGD2 and c(RGDyK), respectively, against 125I-echistatin bound to U87MG cells. Macrocyclic complexes [99mTc(K(HYNIC)2-RGD2)(tricine)] (1), [99mTc(K(HYNIC)2-3G-RGD2)(tricine)] (2), [99mTc(K(HYNIC)2-2P-RGD2)(tricine)] (3), and [99mTc(K(HYNIC)2-3P-RGD2)(tricine)] (4) were prepared, and evaluated in athymic nude mice bearing U87MG glioma xenografts for their tumor targeting capability and biodistribution. It was found that 1 – 4 all had high solution stability and more than two isomers, as evidenced by the presence of multiple radiometric peaks in their radio-HPLC chromatograms. The tumor uptake of 1 – 4 was 3.78 ± 0.81, 7.46 ± 1.68, 9.74 ± 1.65 and 8.59 ± 1.52 %ID/g, respectively, which was completely consistent with trend of integrin αvβ3 binding affinity for cyclic RGD peptides. Replacing [99mTc(HYNIC)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3′,3″-trisulfonate) with [99mTc(K(HYNIC)2)(tricine)] had little impact on radiotracer tumor uptake; but it had significant effect on the uptake of radiotracer in kidneys, lungs and spleen. The tumor was clearly visualized by SPECT/CT with excellent contrast in a glioma-bearing mouse administered with 4. K(HYNIC)2 would be particularly useful for 99mTc-labeling of small biomolecules with one or more disulfide linkages.

Purpose: 99mTc-3P-RGD2 is a 99mTc-labeled dimeric cyclic RGD peptide that binds to integrin αvβ3 with high affinity and specificity. The purpose of this study was to demonstrate the utility of 99mTc-3P-RGD2 SPECT/CT (single photon emission computed tomography/computed tomography) as a molecular imaging tool for noninvasive monitoring breast tumor early response to antiangiogenesis therapy with linifanib, and to illustrate its limitations in monitoring the efficacy of anti-αvβ3 treatment.Methods: To support SPECT/CT imaging, biodistribution and therapy studies, the xenografted breast cancer model was established by subcutaneous injection of 5 × 106 MDA-MB-435 cells into the fat pad of each athymic nude mouse. Linifanib (ABT-869) was used as antiangiogenesis agent. The tumor volume was 180 ± 90 mm3 on the day (-1 day) before baseline SPECT/CT. Each animal was treated twice daily with vehicle or 12.5 mg/kg linifanib. Longitudinal 99mTc-3P-RGD2 SPECT/CT imaging was performed on days -1, 1, 4 and 11. Tumors were harvested at each time point for pathological analysis of hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Tumor uptake of 99mTc-3P-RGD2 was calculated from SPECT/CT quantification. When cyclic peptide E[c(RGDfK)]2 (RGD2) was used as the anti-αvβ3 agent, SPECT/CT images were obtained only at 7 and 21 days after last RGD2 dose.Results: The tumor uptake of 99mTc-3P-RGD2 from SPECT/CT quantification was almost identical to that from biodistribution. There was a dramatic reduction in both %ID and %ID/cm3 tumor uptake of 99mTc-3P-RGD2 during the first 24 hours of linifanib therapy. The therapeutic effect of linifanib was on both tumor cells and vasculature, as determined by IHC analysis of integrin αvβ3 and CD31. Changes in tumor vasculature were further confirmed by pathological H&E analysis of tumor tissues. While its %ID tumor uptake increased steadily in vehicle-treated group, the %ID tumor uptake of 99mTc-3P-RGD2 decreased in linifanib-treated group slowly over the 11-day study period. The degree of tumor response to linifanib therapy correlated well to the integrin αvβ3 expression levels before linifanib therapy.Conclusion: 99mTc-3P-RGD2 is an excellent radiotracer for monitoring integrin αvβ3 expression during and after linifanib therapy. 99mTc-3P-RGD2 SPECT/CT is an useful molecular imaging tool for patient selection before antiangiogenic and anti-αvβ3 therapy; but it would be difficult to use 99mTc-3P-RGD2 for accurate and noninvasive monitoring of early tumor response to anti-αvβ3 therapy.