Patient-Derived Tumor Xenografts Are Susceptible to Formation of Human Lymphocytic Tumors

AbstractPatient-derived xenograft (PDX) tumor models have emerged as a new approach to evaluate the effects of cancer drugs on patients’ personalized tumor grafts enabling to select the best treatment for the cancer patient and providing a new tool for oncology drug developers. Here, we report that human tumors engrafted in immunodeficient mice are susceptible to formation of B-and T-cell PDX tumors. We xenografted human primary and metastatic tumor samples into immunodeficient mice and found that a fraction of PDX tumors generated from patients’ samples of breast, colon, pancreatic, bladder and renal cancer were histologically similar to lymphocytic neoplasms. Moreover, we found that the first passage of breast and pancreatic cancer PDX tumors after initial transplantation of the tumor pieces from the same human tumor graft could grow as a lymphocytic tumor in one mouse and as an adenocarcinoma in another mouse. Whereas subcutaneous PDX tumors resembling human adenocarcinoma histology were slow growing and non-metastatic, we found that subcutaneous PDX lymphocytic tumors were fast growing and formed large metastatic lesions in mouse lymph nodes, liver, lungs, and spleen. PDX lymphocytic tumors were comprised of B-cells which were Epstein-Barr virus positive and expressed CD45 and CD20. Because B-cells are typically present in malignant solid tumors, formation of B-cell tumor may evolve in a wide range of PDX tumor models. Although PDX tumor models show great promise in the development of personalized therapy for cancer patients, our results suggest that confidence in any given PDX tumor model requires careful screening of lymphocytic markers

Identification of a New Epitope in uPAR as a Target for the Cancer Therapeutic Monoclonal Antibody ATN-658, a Structural Homolog of the uPAR Binding Integrin CD11b (αM)

The urokinase plasminogen activator receptor (uPAR) plays a role in tumor progression and has been proposed as a target for the treatment of cancer. We recently described the development of a novel humanized monoclonal antibody that targets uPAR and has anti-tumor activity in multiple xenograft animal tumor models. This antibody, ATN-658, does not inhibit ligand binding (i.e. uPA and vitronectin) to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-tumor activity of ATN-658, we set out to identify the epitope on uPAR to which ATN-658 binds. Guided by comparisons between primate and human uPAR, epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and alanine scanning mutagenesis identified the region of aa 268–275 of uPAR as the epitope for ATN-658. No known function has previously been attributed to this epitope Structural insights into epitope recognition were obtained from structural studies of the Fab fragment of ATN-658 bound to uPAR. The structure shows that the ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the epitope mapping results. Intriguingly, when bound to uPAR, the complementarity determining region (CDR) regions of ATN-658 closely mimic the binding regions of the integrin CD11b (αM), a previously identified uPAR ligand thought to be involved in leukocyte rolling, migration and complement fixation with no known role in tumor progression of solid tumors. These studies reveal a new functional epitope on uPAR involved in tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR

Atrial natriuretic peptide attenuates LPS-induced lung vascular leak: role of PAK1

Increased levels of atrial natriuretic peptide (ANP) in the models of sepsis, pulmonary edema, and acute respiratory distress syndrome (ARDS) suggest its potential role in the modulation of acute lung injury. We have recently described ANP-protective effects against thrombin-induced barrier dysfunction in pulmonary endothelial cells (EC). The current study examined involvement of the Rac effector p21-activated kinase (PAK1) in ANP-protective effects in the model of lung vascular permeability induced by bacterial wall LPS. C57BL/6J mice or ANP knockout mice (Nppa−/−) were treated with LPS (0.63 mg/kg intratracheal) with or without ANP (2 μg/kg iv). Lung injury was monitored by measurements of bronchoalveolar lavage protein content, cell count, Evans blue extravasation, and lung histology. Endothelial barrier properties were assessed by morphological analysis and measurements of transendothelial electrical resistance. ANP treatment stimulated Rac-dependent PAK1 phosphorylation, attenuated endothelial permeability caused by LPS, TNF-α, and IL-6, decreased LPS-induced cell and protein accumulation in bronchoalveolar lavage fluid, and suppressed Evans blue extravasation in the murine model of acute lung injury. More severe LPS-induced lung injury and vascular leak were observed in ANP knockout mice. In rescue experiments, ANP injection significantly reduced lung injury in Nppa−/− mice caused by LPS. Molecular inhibition of PAK1 suppressed the protective effects of ANP treatment against LPS-induced lung injury and endothelial barrier dysfunction. This study shows that the protective effects of ANP against LPS-induced vascular leak are mediated at least in part by PAK1-dependent signaling leading to EC barrier enhancement. Our data suggest a direct role for ANP in endothelial barrier regulation via modulation of small GTPase signaling

Gβ phosphorylation is critical for efficient chemotropism in yeast

Mating yeast cells interpret complex pheromone gradients and polarize their growth in the direction of the closest partner. Chemotropic growth depends on both the pheromone receptor and its associated G-protein. Upon activation by the receptor, Gα dissociates from Gβγ and Gβ is subsequently phosphorylated. Free Gβγ signals to the nucleus via a MAPK cascade and recruits Far1-Cdc24 to the incipient growth site. It is not clear how the cell establishes and stabilizes the axis of polarity, but this process is thought to require local signal amplification via the Gβγ-Far1-Cdc24 chemotropic complex, as well as communication between this complex and the activated receptor. Here we show that a mutant form of Gβ that cannot be phosphorylated confers defects in directional sensing and chemotropic growth. Our data suggest that phosphorylation of Gβ plays a role in localized signal amplification and in the dynamic communication between the receptor and the chemotropic complex, which underlie growth site selection and maintenance.</jats:p

Using Tumor Explants for Imaging Mass Spectrometry Visualization of Unlabeled Peptides and Small Molecules

Matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) imaging mass spectrometry has emerged as a powerful, label-free technique to visualize penetration of small molecules <i>in vivo</i> and <i>in vitro</i>, including in 3D cell culture spheroids; however, some spheroids do not grow sufficiently large to provide enough area for imaging mass spectrometry. Here, we describe an <i>ex vivo</i> method for visualizing unlabeled peptides and small molecules in tumor explants, which can be divided into pieces of desired size, thus circumventing the size limitations of many spheroids. As proof-of-concept, a small molecule drug (4-hydroxytamoxifen), as well as a peptide drug (cyclosporin A) and peptide chemical probe, can be visualized after <i>in vitro</i> incubation with tumor explants so that this technique may provide a solution to robing cell penetration by unlabeled peptides

Metabolomic analysis of a selective ABCA1 inducer in obesogenic challenge provides a rationale for therapeutic development

BACKGROUND: Therapeutic agents with novel mechanisms of action are needed to combat the growing epidemic of type 2 diabetes (T2D) and related metabolic syndromes. Liver X receptor (LXR) agonists possess preclinical efficacy yet produce side effects due to excessive lipogenesis. Anticipating that many beneficial and detrimental effects of LXR agonists are mediated by ABCA1 and SREPB1c expression, respectively, we hypothesized that a phenotypic optimization strategy prioritizing selective ABCA1 induction would identify an efficacious lead compound with an improved side effect profile over existing LXRβ agonists. METHODS: We synthesized and characterized a novel small molecule for selective induction of ABCA1 vs. SREBP1c in vitro. This compound was evaluated in both wild-type mice and a high-fat diet (HFD) mouse model of obesity-driven diabetes through functional, biochemical, and metabolomic analysis. FINDINGS: Six weeks of oral administration of our lead compound attenuated weight gain, glucose intolerance, insulin signaling deficits, and adiposity. Global metabolomics revealed suppression of gluconeogenesis, free fatty acids, and pro-inflammatory metabolites. Target identification linked these beneficial effects to selective LXRβ agonism and PPAR/RXR antagonism. INTERPRETATION: Our observations in the HFD model, combined with the absence of lipogenesis and neutropenia in WT mice, support this novel approach to therapeutic development for T2D and related conditions