Podocalyxin is a CD34-related sialomucin that is expressed at high levels by podocytes, and also by mesothelial cells, vascular endothelia, platelets, and hematopoietic stem cells. To elucidate the function of podocalyxin, we generated podocalyxin-deficient (podxl −/−) mice by homologous recombination. Null mice exhibit profound defects in kidney development and die within 24 hours of birth with anuric renal failure. Although podocytes are present in the glomeruli of the podxl −/− mice, they fail to form foot processes and slit diaphragms and instead exhibit cell–cell junctional complexes (tight and adherens junctions). The corresponding reduction in permeable, glomerular filtration surface area presumably leads to the observed block in urine production. In addition, podxl −/− mice frequently display herniation of the gut (omphalocele), suggesting that podocalyxin may be required for retraction of the gut from the umbilical cord during development. Hematopoietic and vascular endothelial cells develop normally in the podocalyxin-deficient mice, possibly through functional compensation by other sialomucins (such as CD34). Our results provide the first example of an essential role for a sialomucin in development and suggest that defects in podocalyxin could play a role in podocyte dysfunction in renal failure and omphalocele in humans.

Asthma is a pulmonary inflammatory disease dependent on eosinophil and mast cell infiltration into the lung. CD34 is a sialomucin expressed by both of these cell types, and we have used CD34 Ϫ/Ϫ mice and a standard mouse model of asthma to evaluate the importance of CD34 expression on disease development. In comparison with wild-type (wt) mice, CD34 Ϫ/Ϫ mice exhibited a dramatic reduction in all hallmarks of allergic asthma, including lowered airway inflammatory cell infiltration, airway hyperresponsiveness, and mast-cell recruitment. Bone marrow transplantation experiments confirmed that these defects are due to CD34 expression by bone marrow-derived cells. This was not, however, due to an inability to respond to antigen as, on a per cell basis, wt and CD34 Ϫ/Ϫ inflammatory cells exhibit identical responses in cytokine production. We found a striking reduction in mobility of CD34 Ϫ/Ϫ eosinophils in vitro, the major component of inflammatory infiltrates, which was consistent with proposed models for CD34 as an inhibitor of cell-cell adhesion. In summary, our data suggest that CD34 enhances mast-cell and eosinophil invasiveness and that its expression by these cells is a prerequisite for development of allergic asthma. (Blood.

We designed and evaluated a novel albuminbinder-conjugated 177 Lu-PSMA-617 derivative, 177 Lu-HTK01169, with an extended blood retention time to maximize the radiation dose delivered to prostate tumors expressing prostate-specific membrane antigen (PSMA). PSMA-617 and HTK01169 that contained N-[4-(p-iodophenyl)butanoyl]-Glu as an albuminbinding motif were synthesized using the solid-phase approach. Binding affinity to PSMA was determined by in vitro competition-binding assay. 177 Lu labeling was performed in acetate buffer (pH 4.5) at 90 °C for 15 min. SPECT/CT imaging, biodistribution, and endoradiotherapy studies were conducted in mice bearing PSMA-expressing LNCaP tumor xenografts. Radiation dosimetry was calculated using OLINDA software. Lu-PSMA-617 and Lu-HTK01169-bound PSMA with high affinity (K i values = 0.24 and 0.04 nM, respectively). SPECT imaging and biodistribution studies showed that 177 Lu-PSMA-617 and 177 Lu-HTK01169 were excreted mainly via the renal pathway. With fast blood clearance (0.68%ID/g at 1 h postinjection), the tumor uptake of 177 Lu-PSMA-617 peaked at 1 h postinjection (15.1%ID/g) and gradually decreased to 7.91%ID/g at 120 h postinjection. With extended blood retention (16.6 and 2.10%ID/g at 1 and 24 h, respectively), the tumor uptake of 177 Lu-HTK01169 peaked at 24 h postinjection (55.9%ID/g) and remained at the same level by the end of the study (120 h). Based on dosimetry calculations, 177 Lu-HTK01169 delivered an 8.3-fold higher radiation dose than 177 Lu-PSMA-617 to LNCaP tumor xenografts. For the endoradiotherapy study, the mice treated with 177 Lu-PSMA-617 (18.5 MBq) all reached humane end point (tumor volume >1000 mm 3 ) by Day 73 with a median survival of 58 days. Mice treated with 18.5, 9.3, 4.6, or 2.3 MBq of 177 Lu-HTK01169 had a median survival of >120, 103, 61, and 28 days, respectively. With greatly enhanced tumor uptake and treatment efficacy compared to 177 Lu-PSMA-617 in preclinical studies, 177 Lu-HTK01169 warrants further investigation for endoradiotherapy of prostate cancer.

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The cystine transporter (system xC−) is an antiporter of cystine and glutamate. It has relatively low basal expression in most tissues and becomes upregulated in cells under oxidative stress (OS) as one of the genes expressed in response to the antioxidant response element (ARE) promoter. We have developed 18F-5-fluoro-aminosuberic acid (FASu), a Positron Emission Tomography (PET) tracer that targets system xC−. The goal of this study was to evaluate 18F-FASu as a specific gauge for system xC− activity in vivo and its potential for breast cancer imaging. Methods 18F-FASu specificity towards system xC− was studied by cell inhibition assay, cellular uptake following OS induction with diethyl maleate (DEM), with and without anti-xCT siRNA knockdown, in vitro uptake studies and in vivo uptake in a system xC− transduced xenograft model. In addition, radiotracer uptake was evaluated in three separate breast cancer models MDA-MB-231, MCF-7 and ZR-75-1. Results Reactive oxygen species (ROS)-inducing DEM increased glutathione levels and 18F-FASu uptake, while gene knockdown with anti-xCT siRNA led to decreased tracer uptake. 18F-FASu uptake was robustly inhibited by system xC− inhibitors or substrates, while the uptake was significantly higher in transduced cells and tumors expressing xCT compared to the wild type HEK293T cells and tumors (p<0.0001 for cells, p=0.0086 for tumors). 18F-FASu demonstrated tumor uptake in all three breast cancer cell lines studied. Among them, triple negative breast cancer MDA-MB-231 had the highest tracer uptake (p=0.0058 when compared with MCF-7; p<0.0001 when compared with ZR-75-1), which also has the highest xCT mRNA level. Conclusions 18F-FASu as a system xC− substrate is a specific PET tracer for functional monitoring of system xC− and OS imaging. By enabling non-invasive analysis of xC− responses in vivo, this biomarker may serve as a valuable target for the diagnosis and treatment monitoring of certain breast cancers.

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Ga-PSMA-11 is currently the most popular prostate-specific membrane antigen (PSMA) radioligand used in the clinic to detect prostate cancer and metastases. However, the high uptake of Ga-PSMA-11 in kidneys can create halo-artifacts resulting in lower detection sensitivity for lesions adjacent to the kidneys. In this study, we developed twoGa-labeled PSMA-targeted tracers, Ga-HTK01166 andGa-HTK01167, based on Ga-PSMA-617 with the goal of improving tumor-to-kidney ratio compared toGa-PSMA-11. The 2-naphthylalanine (2-Nal) in PSMA-617 was replaced with 2-indanylglycine (Igl) or 3,3-diphenylalanine (Dip) to synthesize HTK01166 and HTK01167, respectively. Binding affinities ( K) of Ga-PSMA-11, Ga-PSMA-617, Ga-HTK01166, and Ga-HTK01167 to PSMA were 3.13 ± 0.40, 1.23 ± 0.08, 5.74 ± 2.48, and 25.7 ± 9.84 nM, respectively, as determined by in vitro competition binding assays. Ga labeling was performed in HEPES buffer with microwave heating, andGa-labeled PSMA-11, PSMA-617, HTK01166, and HTK01167 were obtained in 46-69% average decay-corrected radiochemical yield with >99% radiochemical purity and 62.9-152 GBq/μmol average specific activity. PET imaging and biodistribution studies were performed in mice bearing PSMA-expressing LNCap prostate cancer xenografts. All tracers enabled clear visualization of tumors in PET images with excellent tumor-to-background contrast. The uptake values (%ID/g) for tumor and kidneys at 1 h postinjection were 8.91 ± 0.86 and 204 ± 70.6 for Ga-PSMA-11, 16.7 ± 2.30 and 29.2 ± 5.14 forGa-PSMA-617, 14.1 ± 4.40 and 147 ± 59.6 for Ga-HTK01166, and 7.79 ± 1.65 and 4.30 ± 1.80 forGa-HTK01167. The tumor-to-kidney ratios for Ga-labeled PSMA-11, PSMA-617, HTK01166, and HTK01167 were 0.05 ± 0.02, 0.63 ± 0.10, 0.10 ± 0.02, and 1.98 ± 0.63, respectively. Compared withGa-PSMA-617, Ga-HTK01166 showed comparable tumor uptake and almost 5-fold higher kidney uptake, whereasGa-HTK01167 exhibited lower tumor and kidney uptake. Compared with Ga-PSMA-11,Ga-HTK01167 had similar tumor uptake and tumor-to-blood contrast ratio (23.8 ± 6.71 vs 20.4 ± 4.98) but higher tumor-to-background contrast ratios for other background organs especially for kidneys. Our data indicate that substitution of 2-Nal in PSMA-617 with other lipophilic amino acid can modulate PSMA binding affinity and their pharmacokinetics in vivo.