Endothelial CXCR7 regulates breast cancer metastasis.

Atypical chemokine receptor CXCR7 (ACKR3) functions as a scavenger receptor for chemokine CXCL12, a molecule that promotes multiple steps in tumor growth and metastasis in breast cancer and multiple other malignancies. Although normal vascular endothelium expresses low levels of CXCR7, marked upregulation of CXCR7 occurs in tumor vasculature in breast cancer and other tumors. To investigate effects of endothelial CXCR7 in breast cancer, we conditionally deleted this receptor from vascular endothelium of adult mice, generating CXCR7(ΔEND/ΔEND) animals. CXCR7(ΔEND/ΔEND) mice appeared phenotypically normal, although these animals exhibited a modest 35±3% increase in plasma CXCL12 as compared with control. Using two different syngeneic, orthotopic tumor implant models of breast cancer, we discovered that CXCR7(ΔEND/ΔEND) mice had significantly greater local recurrence of cancer following resection, elevated numbers of circulating tumor cells and more spontaneous metastases. CXCR7(ΔEND/ΔEND) mice also showed greater experimental metastases following intracardiac injection of cancer cells. These results establish that endothelial CXCR7 limits breast cancer metastasis at multiple steps in the metastatic cascade, advancing understanding of CXCL12 pathways in tumor environments and informing ongoing drug development targeting CXCR7 in cancer.

Comparative study reveals better far-red fluorescent protein for whole body imaging.

Genetically encoded far-red and near-infrared fluorescent proteins enable efficient imaging in studies of tumorigenesis, embryogenesis, and inflammation in model animals. Here we report comparative testing of available GFP-like far-red fluorescent proteins along with a modified protein, named Katushka2S, and near-infrared bacterial phytochrome-based markers. We compare fluorescence signal and signal-to-noise ratio at various excitation wavelength and emission filter combinations using transiently transfected cell implants in mice, providing a basis for rational choice of optimal marker(s) for in vivo imaging studies. We demonstrate that the signals of various far-red fluorescent proteins can be spectrally unmixed based on different signal-to-noise ratios in different channels, providing the straightforward possibility of multiplexed imaging with standard equipment. Katushka2S produced the brightest and fastest maturing fluorescence in all experimental setups. At the same time, signal-to-noise ratios for Katushka2S and near-infrared bacterial phytochrome, iRFP720 were comparable in their optimal channels. Distinct spectral and genetic characteristics suggest this pair of a far-red and a near-infrared fluorescent protein as an optimal combination for dual color, whole body imaging studies in model animals.

CXCL12-γ in primary tumors drives breast cancer metastasis.

Compelling evidence shows that chemokine C-X-C motif chemokine ligand 12 (CXCL12) drives metastasis in multiple malignancies. Similar to other key cytokines in cancer, CXCL12 exists as several isoforms with distinct biophysical properties that may alter signaling and functional outputs. However, effects of CXCL12 isoforms in cancer remain unknown. CXCL12-α, -ß and -γ showed cell-type-specific differences in activating signaling through G protein-dependent pathways in cell-based assays, while CXCL12-γ had greatest effects on recruitment of the adapter protein ß-arrestin 2. CXCL12-ß and -γ also stimulated endothelial tube formation to a greater extent than CXCL12-α. To investigate the effects of CXCL12 isoforms on tumor growth and metastasis, we used a mouse xenograft model of metastatic human breast cancer combining CXCR4+ breast cancer cells and mammary fibroblasts secreting an isoform of CXCL12. Altough all CXCL12 isoforms produced comparable growth of mammary tumors, CXCL12-γ significantly increased metastasis to bone marrow and other sites. Breast cancer cells originating from tumors with CXCL12-γ fibroblasts upregulated RANKL (receptor activator of nuclear factor-κB ligand), contributing to bone marrow tropism of metastatic cancer cells. CXCL12-γ was expressed in metastatic tissues in mice, and we also detected CXCL12-γ in malignant pleural effusions from patients with breast cancer. In our mouse model, mammary fibroblasts disseminated to sites of breast cancer metastases, providing another mechanism to increase levels of CXCL12 in metastatic environments. These studies identify CXCL12-γ as a potent pro-metastatic molecule with important implications for cancer biology and effective therapeutic targeting of CXCL12 pathways.

Microfluidic source-sink model reveals effects of biophysically distinct CXCL12 isoforms in breast cancer chemotaxis.

Chemokines critically regulate chemotaxis in normal and pathologic states, but there is limited understanding of how multicellular interactions generate gradients needed for cell migration. Previous studies of chemotaxis of CXCR4+ cells toward chemokine CXCL12 suggest the requirement of cells expressing scavenger receptor CXCR7 in a source-sink system. We leveraged an established microfluidic device to discover that chemotaxis of CXCR4 cells toward distinct isoforms of CXCL12 required CXCR7 scavenging only under conditions with higher than optimal levels of CXCL12. Chemotaxis toward CXCL12-ß and -γ isoforms, which have greater binding to extracellular molecules and have been largely overlooked, was less dependent on CXCR7 than the more commonly studied CXCL12-α. Chemotaxis of CXCR4+ cells toward even low levels of CXCL12-γ and CXCL12-ß still occurred during treatment with a FDA-approved inhibitor of CXCR4. We also detected CXCL12-γ only in breast cancers from patients with advanced disease. Physiological gradient formation within the device facilitated interrogation of key differences in chemotaxis among CXCL12 isoforms and suggests CXCL12-γ as a biomarker for metastatic cancer.

Scavenging of CXCL12 by CXCR7 promotes tumor growth and metastasis of CXCR4-positive breast cancer cells.

Chemokine CXCL12 and receptor CXCR4 control multiple steps in primary tumor growth and metastasis in breast cancer and more than 20 other human malignancies. Mechanisms that regulate availability of CXCL12 in tumor microenvironments will substantially impact cancer progression and ongoing efforts to target the CXCL12-CXCR4 pathway for cancer chemotherapy. We used dual luciferase imaging to investigate CXCR7-dependent scavenging of CXCL12 in breast tumors in vivo and quantify effects of CXCR7 on tumor growth and metastasis of a separate population of CXCR4+ breast cancer cells. In a mouse xenograft model of human breast cancer, in vivo imaging showed that malignant cells expressing CXCR7 reduced bioluminescent CXCL12 secreted in the primary tumor microenvironment. Capitalizing on sensitive detection of bioluminescent CXCL12, we also demonstrated that CXCR7+ cells reduced amounts of chemokine released from orthotopic tumors into the circulation. Immunofluorescence staining of human primary breast cancers showed expression of CXCR4 and CXCR7 on malignant cells in ≈30% of cases. In most cases, CXCR4 and CXCR7 predominantly were expressed on separate populations of malignant cells in a tumor. We modeled these cases of human breast cancer by co-implanting tumor xenografts with CXCR4+ breast cancer cells, human mammary fibroblasts secreting CXCL12, and CXCR7+ or control breast cancer cells. Bioluminescence imaging showed that CXCR7+ breast cancer cells enhanced proliferation of CXCR4+ breast cancer cells in orthotopic tumors and spontaneous metastases. Treatment with a small-molecule inhibitor of CXCR7 chemokine limited the growth of CXCR4+ breast cancer cells in tumors that also contained malignant CXCR7+ cells. These studies establish a new in vivo imaging method to quantify chemokine scavenging by CXCR7 in the tumor microenvironment and identify that CXCR7+ cells promote growth and metastasis of CXCR4+ breast cancer cells.

Constitutive and chemokine-dependent internalization and recycling of CXCR7 in breast cancer cells to degrade chemokine ligands.

CXCR7 is a receptor for chemokines including CXCL12 (stromal-derived factor-1), a molecule that promotes tumor growth and metastasis in breast cancer and other malignancies. Building on the recent observation that CXCR7 sequesters CXCL12, we investigated mechanisms for CXCR7-dependent uptake of chemokines. Breast cancer cells expressing CXCR7 accumulated chemokines CXCL12 and CXC11 present at concentrations <1 ng/ml, unlike cells expressing CXCR4. CXCR7-dependent accumulation of chemokines was reduced by inhibitors of clathrin-mediated endocytosis. After CXCR7-mediated internalization, CXCL12 trafficked to lysosomes and was degraded, although levels of CXCR7 remained stable. CXCR7 reduced CXCL12 in the extracellular space, limiting the amounts of chemokine available to acutely stimulate signaling through CXCR4. CXCR7 constitutively internalized and recycled to the cell membrane even in the absence of ligand, and addition of chemokines did not significantly enhance receptor internalization. Chemokines at concentrations less than the Kd values for ligand-receptor binding did not alter levels of CXCR7 at the cell surface. Higher concentrations of chemokine ligands reduced the total cell surface expression of CXCR7 without affecting receptor internalization, indicating that receptor recycling was inhibited. CXCR7-dependent uptake of chemokines and receptor trafficking were regulated by beta-arrestin 2. These studies establish mechanisms through which CXCR7 regulates the availability of chemokine ligands in the extracellular space.

Nanolitre liquid patterning in aqueous environments for spatially defined reagent delivery to mammalian cells.

Microscale biopatterning enables regulation of cell-material interactions and cell shape, and enables multiplexed high-throughput studies in a cell- and reagent-efficient manner. The majority of available techniques rely on physical contact of a stamp, pin, or mask with mainly a dry surface. Inkjet and piezoelectric printing is carried out in a non-contact manner but still requires a substantially dry substrate to ensure fidelity of printed patterns. These existing methods, therefore, are limited for patterning onto delicate surfaces of living cells because physical contact or substantially dry conditions are damaging to them. Microfluidic patterning with laminar streams does enable non-contact patterning in fully aqueous environments but with limited throughput and reagent diffusion across interfacial flows. Here, we describe a polymeric aqueous two-phase system that enables patterning nanolitres of a reagent-containing aqueous phase, in arbitrary shapes, within a second aqueous phase covering a cell monolayer. With the appropriate medium formulation, reagents of interest remain confined to the patterned phase without significant diffusion. The fully aqueous environment ensures high reagent activity and cell viability. The utility of this strategy is demonstrated with patterned delivery of genetic materials to mammalian cells for phenotypic screening of gene expression and gene silencing.

MDR1 P-glycoprotein reduces influx of substrates without affecting membrane potential.

MDR1 (multidrug resistance) P-glycoprotein (Pgp; ABCB1) decreases intracellular concentrations of structurally diverse drugs. Although Pgp is generally thought to be an efflux transporter, the mechanism of action remains elusive. To determine whether Pgp confers drug resistance through changes in transmembrane potential (E(m)) or ion conductance, we studied electrical currents and drug transport in Pgp-negative MCF-7 cells and MCF-7/MDR1 stable transfectants that were established and maintained without chemotherapeutic drugs. Although E(m) and total membrane conductance did not differ between MCF-7 and MCF-7/MDR1 cells, Pgp reduced unidirectional influx and steady-state cellular content of Tc-Sestamibi, a substrate for MDR1 Pgp, without affecting unidirectional efflux of substrate from cells. Depolarization of membrane potentials with various concentrations of extracellular K(+) in the presence of valinomycin did not inhibit the ability of Pgp to reduce intracellular concentration of Tc-Sestamibi, strongly suggesting that the drug transport activity of MDR1 Pgp is independent of changes in E(m) or total ion conductance. Tetraphenyl borate, a lipophilic anion, enhanced unidirectional influx of Tc-Sestamibi to a greater extent in MCF-7/MDR1 cells than in control cells, suggesting that Pgp may, directly or indirectly, increase the positive dipole potential within the plasma membrane bilayer. Overall, these data demonstrate that changes in E(m) or macroscopic conductance are not coupled with function of Pgp in multidrug resistance. The dominant effect of MDR1 Pgp in this system is reduction of drug influx, possibly through an increase in intramembranous dipole potential.

Overexpression of IRF9 confers resistance to antimicrotubule agents in breast cancer cells.

IRF9/p48/ISGF3gamma (IRF9) is an IFN regulatory factor that mediates signaling by type I IFNs (IFNalpha and IFNbeta). After single-step selection of breast adenocarcinoma cells in paclitaxel, differential display and single gene analysis demonstrated that transcriptional activation of IRF9 and other IFN-responsive genes, independent of IFN, corresponded with resistance to antimicrotubule agents. Transient overexpression of IRF9 reproduced the drug-resistance phenotype and induced expression of IFN-responsive genes. However, drug resistance was not induced by overexpression of Stat1 or Stat2, or treatment with IFNalpha per se. Using a donor-matched array of cDNA prepared from human tumor and normal tissue from a variety of organs, we observed overexpression of IRF9 in approximately one-half of breast and uterine tumors, which indicated that IRF9 may be important in signaling in these tumor types. These data identify a novel IFN-independent role for IRF9 in the development of resistance to antimicrotubule agents in breast tumor cells and may link downstream mediators of IFN signaling to drug resistance in human cancers.

Effects of MDR1 and MDR3 P-glycoproteins, MRP1, and BCRP/MXR/ABCP on the transport of (99m)Tc-tetrofosmin.

Multidrug resistance (MDR1) P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP1), and breast cancer resistance protein (BCRP/MXR/ABCP) are members of the ATP-binding-cassette (ABC) superfamily of membrane transporters and are thought to function as energy-dependent efflux pumps of a variety of structurally diverse chemotherapeutic agents. We herein report the characterization of (99m)Tc-Tetrofosmin, a candidate radiopharmaceutical substrate of ABC transporters. (99m)Tc-Tetrofosmin showed high membrane potential-dependent accumulation in drug-sensitive KB 3-1 cells and low antagonist-reversible accumulation in MDR KB 8-5 and KB 8-5-11 cells in proportion to levels of MDR1 Pgp expression. In KB 8-5 cells, EC(50) values of the potent MDR antagonists N-(4-[2-(1,2,3, 4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9, 10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), (2R)-anti-5-¿3-[4-(10, 11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2 -hydroxypropoxy ¿quinoline trihydrochloride (LY335979), and (3'-keto-Bmt')-[Val(2)]-cyclosporin A (PSC 833) were 40, 66, and 986 nM, respectively. Furthermore, only baculoviruses carrying human MDR1, but not MDR3, conferred both a decrease in accumulation of (99m)Tc-Tetrofosmin in host Spodoptera frugiperda (Sf9) cells and a GF120918-induced enhancement. Transport studies with a variety of stably transfected and drug-selected tumor cell lines were performed with (99m)Tc-Tetrofosmin and compared with (99m)Tc-Sestamibi, a previously validated MDR imaging agent. MDR1 Pgp readily transported each agent. To a lesser extent, MRP1 also transported each agent, likely as co-transport substrates with GSH; neither agent was a substrate for the BCRP/MXR/ABCP half-transporter. In mdr1a(-/-) and mdr1a/1b(-/-) mice, (99m)Tc-Tetrofosmin showed approximately 3. 5-fold greater brain uptake and retention compared with wild-type, with no net change in blood pharmacokinetics, consistent with transport in vivo by Pgp expressed at the capillary blood-brain barrier. Molecular imaging of the functional transport activity of ABC transporters in vivo with (99m)Tc-Tetrofosmin and related radiopharmaceuticals may enable non-invasive monitoring of chemotherapeutic and MDR gene therapy protocols.

Structure and inhibition of plasmepsin II, a hemoglobin-degrading enzyme from Plasmodium falciparum.

Plasmodium falciparum is the major causative agent of malaria, a disease of worldwide importance. Resistance to current drugs such as chloroquine and mefloquine is spreading at an alarming rate, and our antimalarial armamentarium is almost depleted. The malarial parasite encodes two homologous aspartic proteases, plasmepsins I and II, which are essential components of its hemoglobin-degradation pathway and are novel targets for antimalarial drug development. We have determined the crystal structure of recombinant plasmepsin II complexed with pepstatin A. This represents the first reported crystal structure of a protein from P. falciparum. The crystals contain molecules in two different conformations, revealing a remarkable degree of interdomain flexibility of the enzyme. The structure was used to design a series of selective low molecular weight compounds that inhibit both plasmepsin II and the growth of P. falciparum in culture.

Kinetic analysis of plasmepsins I and II aspartic proteases of the Plasmodium falciparum digestive vacuole.

Plasmepsins I and II are Plasmodium falciparum aspartic proteases implicated in hemoglobin degradation. Using a synthetic fluorogenic peptide substrate based on the initial hemoglobin cleavage site, we have analyzed kinetic parameters of the two enzymes in native and recombinant forms. Both native plasmepsins cleave the model substrate well. Recombinant plasmepsin II behaves similarly to native enzyme, substantiating its usefulness for inhibition and structural studies. In contrast, recombinant plasmepsin I does not resemble its native homolog kinetically. A hybrid molecule, in which the polyproline loop of plasmepsin I has been replaced by the homologous sequence from plasmepsin II, still maintains the specificity/kinetics of plasmepsin II. This suggests that the polyproline loop, important for substrate recognition in the mammalian aspartic protease renin, does not play a similar role in the plasmepsins.

Tracheal cytotoxin structural requirements for respiratory epithelial damage in pertussis.

The respiratory epithelial pathology of pertussis (whooping cough) can be reproduced by tracheal cytotoxin (TCT), a disaccharide-tetrapeptide released by Bordetella pertussis. TCT is a muramyl peptide, a class of peptidoglycan-derived compounds which have many biological activities including adjuvanticity, somnogenicity, pyrogenicity, and cytotoxicity. The structural requirements for muramyl peptides to produce some of these biological effects have been partially characterized. Using in vitro assays with respiratory epithelial cells and tissue, we have previously determined that the disaccharide moiety of TCT is not involved in toxicity and that the side-chain functional groups of diaminopimelic acid (A2pm) are crucial for toxicity. In this study, we determine the importance of every amino acid, functional group and chiral centre in the peptide portion of TCT. Although lactyl tetrapeptides are the most toxic of the TCT fragments, producing dose-response curves identical to TCT, the smallest analogues of TCT which are active in our assay are of the form X-gamma-(D)-Glu-meso-A2pm, where X may be an amino acid or a blocking group. Within this active substructure, main-chain chirality and all functional groups are essential for toxicity. This definition of the core region of TCT indicates that the TCT interaction site is unlike almost all other muramyl peptide interaction sites for which structure-activity data are available.

Bordetella pertussis tracheal cytotoxin and other muramyl peptides: distinct structure-activity relationships for respiratory epithelial cytopathology.

Tracheal cytotoxin (TCT) is a disaccharide-tetrapeptide released by Bordetella pertussis, the causative agent of pertussis (whooping cough). We have previously determined the structure of TCT to be GlcNAc-1,6-anhydro-MurNAc-L-Ala-gamma-D-Glu-meso-A2pm-D-Ala, where MurNAc = N-acetylmuramic acid and A2pm = diaminopimelic acid. Purified TCT reproduces the respiratory cytopathology observed during pertussis, including ciliostasis and extrusion of ciliated cells. We have tested structural analogs of TCT for their ability to reproduce native TCT toxicity in explanted hamster tracheal tissue and hamster trachea epithelial (HTE) cell cultures. Other investigators have evaluated many of these analogs, which are muramyl or desmuramyl peptides, for muramyl peptide activities such as immunopotentiation, induction of slow-wave sleep, and pyrogenicity. Four desmuramyl peptides were produced in our laboratory from B. pertussis peptidoglycan or by chemical synthesis, including unusual peptides containing alpha-aminopimelic acid in place of A2pm. Based on the relative ability of compounds to inhibit DNA synthesis in HTE cells, truncated analogs lacking A2pm entirely or lacking only the side-chain amine or carboxyl group of A2pm were less active than TCT by a factor of at least 1000. All active analogs included a native or near-native peptide moiety, independent of the presence, absence, or substitution of the sugar moiety. We conclude that the structural requirements for TCT toxicity differ considerably from those for most other muramyl peptide activities, in that the disaccharide moiety is irrelevant for toxicity and both the free amino and carboxyl groups of the A2pm side chain are required for activity.