Macropinocytosis Inhibitor Research Articles

CXCL12-induced macropinocytosis modulates two distinct pathways to activate mTORC1 in macrophages.

Although growth factors and chemokines elicit different overall effects on cells-growth and chemotaxis, respectively-and activate distinct classes of cell-surface receptors, nonetheless, they trigger similar cellular activities and signaling pathways. The growth factor M-CSF and the chemokine CXCL12 both stimulate the endocytic process of macropinocytosis, and both activate the mechanistic target of rapamycin complex 1 (mTORC1), a protein complex that regulates cell metabolism. Recent studies of signaling by M-CSF in macrophages identified a role for macropinocytosis in the activation of mTORC1, in which delivery of extracellular amino acids into lysosomes via macropinocytosis was required for activation of mTORC1. Here, we analyzed the regulation of macropinosome (MP) formation in response to CXCL12 and identified 2 roles for macropinocytosis in the activation of mTORC1. Within 5 min of adding CXCL12, murine macrophages increased ruffling, macropinocytosis and amino acid-dependent activation of mTORC1. Inhibitors of macropinocytosis blocked activation of mTORC1, and various isoform-specific inhibitors of type 1 PI3K and protein kinase C (PKC) showed similar patterns of inhibition of macropinocytosis and mTORC1 activity. However, unlike the response to M-CSF, Akt phosphorylation (pAkt) in response to CXCL12 required the actin cytoskeleton and the formation of macropinocytic cups. Quantitative fluorescence microscopy showed that phosphatidylinositol (3,4,5)-trisphosphate (PIP3), a product of PI3K and an upstream activator of Akt, localized to macropinocytic cups and that pAkt occurred primarily in cups. These results indicate that CXCL12 activates mTORC1 via 2 mechanisms: 1) that the macropinocytic cup localizes Akt signaling and 2) that MPs convey extracellular nutrients to lysosomes.

Vascular permeability in the RG2 glioma model can be mediated by macropinocytosis and be independent of the opening of the tight junction

This study evaluates the extravasation pathways of circulating macromolecules in a rat glioma model (RG2) which was observed by both magnetic resonance imaging using ultrasmall superparamagnetic iron oxide and electron microscopy. Although magnetic resonance imaging signal enhancement was observed as soon as 10 min after injection (9.4% 2 h after injection), electron microscopy showed that endothelial cells were still tightly sealed. However, circulating immunoglobulin G and ultrasmall superparamagnetic iron oxide were found in large membrane compartments of endothelial cells, in the basal lamina (7.4 ± 1.2 gold particles/µm2 in the tumor versus 0.38 ± 0.17 in healthy tissue, p = 1.4.10−5) and between tumoral cells. Altogether, this strongly suggests an active transport mediated by macropinocytosis. To challenge this transport mechanism, additional rats were treated with amiloride, an inhibitor of macropinocytosis, leading to a reduction of membrane protrusions (66%) and of macropinosomes. Amiloride however also opened tumoral tight junctions allowing a larger extravasation of ultrasmall superparamagnetic iron oxide (magnetic resonance imaging signal enhancement of 35.7% 2 h after injection). Altogether, these results suggest that ultrasmall superparamagnetic iron oxide and immunoglobulin G in the RG2 glioma model follow an active extravasation pathway mediated by a macropinocytosis process. Amiloride also appears as a potential strategy to facilitate the extravasation of chemotherapeutic drugs in glioma.

Abstract 3276: The tissue and cellular destination of therapeutic IgGs in glioblastoma

Abstract Most patients with recurrent glioblastoma (GBM) are treated with bevacizumab, a humanized monoclonal antibody (mAb) that binds VEGF-A and inhibits its binding to VEGFR. Approximately 30% of GBM patients are non-responsive to bevacizumab and the underlying mechanism for the lack of response is not known. It has been assumed that bevacizumab solely targets circulating VEGF-A in blood. We hypothesized that bevacizumab and human IgGs in general gain access to the perivascular niche that contains cancer stem cells (CSCs) in GBM. We found that bevacizumab gains access to the perivascular tumor area through leaky blood vessels and was internalized by tumor cells in an orthotopic xenograft mouse model of GBM. In vitro, CSCs (CD133+) from GBM rapidly internalized either bevacizumab or human IgG into membrane protrusions that contained actin and internalization was significantly inhibited by a macropinocytosis inhibitor (EIPA), suggesting CSCs internalize bevacizumab or human IgG via macropinocytosis. Furthermore, bevacizumab or human IgG was largely detected in the Rab4+ “fast” recycling compartment at 5 min, and both were largely detected in the LAMP1+ compartment (late endosome/lysosome) at 3 hr in the CSCs. CSCs (CD133+) from GBM do not express the neonatal Fc receptor, the canonical pathway for recycling of IgG. Administration of bevacizumab to an orthotopic xenograft mouse model of established GBM showed that bevacizumab was partially co-localized with Rab4+ or with LAMP1+ in perivascular tumor cells, consistent with our in vitro findings. Taken together, our data show that in GBM, humanized IgG, including bevacizumab, gains access to the perivascular tumor space and is then macropinocytosed by CSCs and trafficked to a recycling compartment or to the late endosome/lysosome. These data suggest that alterations in endocytosis or recycling in the CSCs could impact the fate of therapeutic IgGs like bevacizumab and ultimately influence a patients’ response to GBM therapy. Citation Format: Gaelle Muller-Greven, Cathleen Carlin, Steven Toms, Manmeet Ahluwalia, Markus Bredel, Justin Lathia, Jeremy Rich, Petra Hamerlik, Candece L. Gladson. The tissue and cellular destination of therapeutic IgGs in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3276.

Abstract 3001: Accurins are endocytosed by KRAS-mutant cells via macropinocytosis

Abstract Background: BIND-014 is a novel prostate-specific membrane antigen (PSMA)-targeted Accurin (polymeric nanoparticle) encapsulating docetaxel. In a Phase 2 non-small cell lung cancer (NSCLC) trial, BIND-014 demonstrated promising anti-tumor activity in patients with tumors expressing KRAS mutations (mutated Kirsten ras oncogene homolog). KRAS mutations in NSCLC are generally associated with poor response to currently available drug therapy regimens, including docetaxel. Mutant KRAS stimulates an endocytic process known as macropinocytosis, in which extracellular fluid and its constituents are internalized non-selectively into cells. We hypothesized that Accurins preferentially accumulate in KRAS-mutant cells via macropinocytosis, potentially contributing to anti-tumor activity of BIND-014 in KRAS-mutant cancer. Methods: To determine whether BIND-014 is internalized in cells with high levels of macropinocytosis, we treated KRAS-mutant, macropinocytosis-high human NSCLC A549 cells and human bladder cancer T24 cells with PSMA targeted Accurins in which docetaxel was replaced by a fluorescent label, and visualized co-localization with fluorescently labeled markers of macropinocytosis, dextran and BSA, by confocal microscopy. In addition, we quantified the levels of labeled Accurin uptake in A549 cells by fluorescence-activated cell sorting (FACS) and compared them to labeled Accurin internalization in macropinocytosis-low, KRAS-wild-type BxPC3 cells. We further tested the macropinocytosis-dependence of labeled Accurin uptake by treating A549 and BxPC3 cells with the macropinocytosis inhibitor EIPA and measuring changes in Accurin internalization by FACS. Results: Confocal microscopy analysis of A549 and T24 cells co-treated with labeled Accurin and dextran or BSA showed substantial accumulation of Accurin in dextran- and BSA-positive macropinosomes. Moreover, FACS analysis of A549 cells treated with 0.1 ug and 1.0 ug of labeled Accurin revealed 60.4% and 81.8% uptake, respectively, that could be reduced to 3.6% and 3.7% with 50 uM EIPA. By contrast, BxPC3 cells showed 1.8% and 1.5% uptake of 0.1 ug and 1.0 ug labeled Accurin that was reduced to 1.3% and 0.9%, respectively, with 50 uM EIPA by FACS analysis. Conclusions: These data demonstrate that Accurins preferentially enter human cancer cells that display high levels of macropinocytosis and co-localize with several markers of macropinosomes. Moreover, Accurin internalization can be inhibited in macropinocytosis-positive cells with an established chemical inhibitor of macropinocytosis. BIND-014 may therefore preferentially accumulate in KRAS-mutant NSCLC tumors, thereby contributing to the clinical activity seen to date in this tumor type. Citation Format: Jane E. Cullis, Laura Taylor, Susan Low, Dafna Bar-Sagi. Accurins are endocytosed by KRAS-mutant cells via macropinocytosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3001.

A Bispecific Antibody Promotes Aggregation of Ricin Toxin on Cell Surfaces and Alters Dynamics of Toxin Internalization and Trafficking.

JJX12 is an engineered bispecific antibody against ricin, a member of the medically important A-B family of toxins that exploits retrograde transport as means to gain entry into the cytosol of target cells. JJX12 consists of RTA-D10, a camelid single variable domain (VHH) antibody directed against an epitope on ricin’s enzymatic subunit (RTA), linked via a 15-mer peptide to RTB-B7, a VHH against ricin’s bivalent galactose binding subunit (RTB). We previously reported that JJX12, but not an equimolar mixture of RTA-D10 and RTB-B7 monomers, was able to passively protect mice against a lethal dose ricin challenge, demonstrating that physically linking RTB-B7 and RTA-D10 is critical for toxin-neutralizing activity in vivo. We also reported that JJX12 promotes aggregation of ricin in solution, presumably through the formation of intermolecular crosslinking. In the current study, we now present evidence that JJX12 affects the dynamics of ricin uptake and trafficking in human epithelial cells. Confocal microscopy, as well as live cell imaging coupled with endocytosis pathway-specific inhibitors, revealed that JJX12-toxin complexes are formed on the surfaces of mammalian cells and internalized via a pathway sensitive to amiloride, a known inhibitor of macropinocytosis. Moreover, in the presence of JJX12, retrograde transport of ricin to the trans-Golgi network was significantly reduced, while accumulation of the toxin in late endosomes was significantly enhanced. In summary, we propose that JJX12, by virtue of its ability to crosslink ricin toxin, alters the route of toxin uptake and trafficking within cells.

The Pro-inflammatory Cytokine Interleukin-6 Regulates Nanoparticle Transport Across Model Follicle-Associated Epithelium Cells

The aim of this study was to investigate whether the pro-inflammatory cytokines improved the function of the cell monolayer model of the human follicle-associated epithelium (FAE) of co-culture of Caco-2 cells on permeable filters with Raji B-cells underneath from the viewpoint of particle transport. Exposure to tumor necrosis factor-α resulted in an almost maintained epithelial integrity/paracellular permeability combined with an increased nanoparticle transport in a dose-dependent manner while the effects of interleukin (IL)-1β were limited. Exposure to IL-6 significantly enhanced the nanoparticle transport with the limited disruption of the cell monolayer integrity. The addition of IL-6 or tumor necrosis factor-α to Caco-2 monolayers without Raji B-cells did not enhance nanoparticle transport. In our IL-6 treated FAE model, the nanoparticle transport almost disappeared at 4°C or after the addition of 5-(N-ethyl-N-isopropyl) amiloride, an inhibitor of macropinocytosis. Furthermore, IgA binding, presumably by a secretory IgA receptor, a marker of M-cells was observed on the apical side of our model FAE. These results indicate that the combined effect of IL-6 with unknown factors from Raji-B cells made the FAE model more functional with regard to nanoparticle transport. The IL-6 enhanced FAE model will be a useful platform for nanoparticle drug delivery research across the intestinal epithelium.

Characteristics of Mitochondrial Transformation into Human Cells.

Mitochondria can be incorporated into mammalian cells by simple co-incubation of isolated mitochondria with cells, without the need of transfection reagents or any other type of intervention. This phenomenon was termed mitochondrial transformation, and although it was discovered in 1982, currently little is known regarding its mechanism(s). Here we demonstrate that mitochondria can be transformed into recipient cells very quickly, and co-localize with endogenous mitochondria. The isolated mitochondria interact directly with cells, which engulf the mitochondria with cellular extensions in a way, which may suggest the involvement of macropinocytosis or macropinocytosis-like mechanisms in mitochondrial transformation. Indeed, macropinocytosis inhibitors but not clathrin-mediated endocytosis inhibition-treatments, blocks mitochondria transformation. The integrity of the mitochondrial outer membrane and its proteins is essential for the transformation of the mitochondria into cells; cells can distinguish mitochondria from similar particles and transform only intact mitochondria. Mitochondrial transformation is blocked in the presence of the heparan sulfate molecules pentosan polysulfate and heparin, which indicate crucial involvement of cellular heparan sulfate proteoglycans in the mitochondrial transformation process.

Large-Scale Screening and Identification of Novel Ebola Virus and Marburg Virus Entry Inhibitors.

Filoviruses are highly infectious, and no FDA-approved drug therapy for filovirus infection is available. Most work to find a treatment has involved only a few strains of Ebola virus and testing of relatively small drug libraries or compounds that have shown efficacy against other virus types. Here we report the findings of a high-throughput screening of 319,855 small molecules from the Molecular Libraries Small Molecule Repository library for their activities against Marburg virus and Ebola virus. Nine of the most potent, novel compounds that blocked infection by both viruses were analyzed in detail for their mechanisms of action. The compounds inhibited known key steps in the Ebola virus infection mechanism by blocking either cell surface attachment, macropinocytosis-mediated uptake, or endosomal trafficking. To date, very few specific inhibitors of macropinocytosis have been reported. The 2 novel macropinocytosis inhibitors are more potent inhibitors of Ebola virus infection and less toxic than ethylisopropylamiloride, one commonly accepted macropinocytosis inhibitor. Each compound blocked infection of primary human macrophages, indicating their potential to be developed as new antifiloviral therapies.

Enzymatically Modified Low-Density Lipoprotein Promotes Foam Cell Formation in Smooth Muscle Cells via Macropinocytosis and Enhances Receptor-Mediated Uptake of Oxidized Low-Density Lipoprotein.

Enzyme-modified nonoxidized low-density lipoprotein (ELDL) is present in human atherosclerotic lesions. Our objective is to understand the mechanisms of ELDL uptake and its effects on vascular smooth muscle cells (SMC). Transformation of murine aortic SMCs into foam cells in response to ELDL was analyzed. ELDL, but not acetylated or oxidized LDL, was potent in inducing SMC foam cell formation. Inhibitors of macropinocytosis (LY294002, wortmannin, amiloride) attenuated ELDL uptake. In contrast, inhibitors of receptor-mediated endocytosis (dynasore, sucrose) and inhibitor of caveolae-/lipid raft-mediated endocytosis (filipin) had no effect on ELDL uptake in SMC, suggesting that macropinocytosis is the main mechanism of ELDL uptake by SMC. Receptor for advanced glycation end products (RAGE) is not obligatory for ELDL-induced SMC foam cell formation, but primes SMC for the uptake of oxidized LDL in a RAGE-dependent manner. ELDL increased intracellular reactive oxygen species, cytosolic calcium, and expression of lectin-like oxidized LDL receptor-1 in wild-type SMC but not in RAGE(-/-) SMC. The macropinocytotic uptake of ELDL is regulated predominantly by intracellular calcium because ELDL uptake was completely inhibited by pretreatment with the calcium channel inhibitor lacidipine in wild-type and RAGE(-/-) SMC. This is in contrast to pretreatment with PI3 kinase inhibitors which completely prevented ELDL uptake in RAGE(-/-) SMC, but only partially in wild-type SMC. ELDL is highly potent in inducing foam cells in murine SMC. ELDL endocytosis is mediated by calcium-dependent macropinocytosis. Priming SMC with ELDL enhances the uptake of oxidized LDL.

Effect of Aminated Mesoporous Bioactive Glass Nanoparticles on the Differentiation of Dental Pulp Stem Cells.

Mesoporous bioactive nanoparticles (MBNs) have been developed as promising additives to various types of bone or dentin regenerative material. However, biofunctionality of MBNs as dentin regenerative additive to dental materials have rarely been studied. We investigated the uptake efficiency of MBNs-NH2 with their endocytosis pathway and the role of MBNs-NH2 in odontogenic differentiation to clarify inherent biofunctionality. MBNs were fabricated by sol-gel synthesis, and 3% APTES was used to aminate these nanoparticles (MBNs-NH2) to reverse their charge from negative to positive. To characterize the MBNs-NH2, TEM, XRD, FTIR, zeta(ξ)-potential measurements, and Brunauer–Emmett–Teller analysis were performed. After primary cultured rat dental pulp stem cells (rDPSCs) were incubated with various concentrations of MBNs-NH2, stem cell viability (24 hours) with or without differentiated media, internalization of MBNs-NH2 in rDPSCs (~4 hours) via specific endocytosis pathway, intra or extracellular ion concentration and odontoblastic differentiation (~28 days) were investigated. Incubation with up to 50 μg/mL of MBNs-NH2 had no effect on rDPSCs viability with differentiated media (p>0.05). The internalization of MBNs-NH2 in rDPSCs was determined about 92% after 4 hours of incubation. Uptake was significantly decreased with ATP depletion and after 1 hour of pre-treatment with the inhibitor of macropinocytosis (p<0.05). There was significant increase of intracellular Ca and Si ion concentration in MBNs-NH2 treated cells compared to no-treated counterpart (p<0.05). The expression of odontogenic-related genes (BSP, COL1A, DMP-1, DSPP, and OCN) and the capacity for biomineralization (based on alkaline phosphatase activity and alizarin red staining) were significantly upregulated with MBNs-NH2. These results indicate that MBNs-NH2 induce odontogenic differentiation of rDPSCs and may serve as a potential dentin regenerative additive to dental material for promoting odontoblast differentiation.

Faint electric treatment-induced rapid and efficient delivery of extraneous hydrophilic molecules into the cytoplasm

Effective delivery of extraneous molecules into the cytoplasm of the target cells is important for several drug therapies. Previously, we showed effective in vivo transdermal delivery of naked siRNA into skin cells induced by faint electric treatment (ET) iontophoresis, and significant suppression of target mRNA levels (Kigasawa et al., Int. J. Pharm., 2010). This result indicates that electricity promoted the delivery of siRNA into cytoplasm. In the present study, we analyzed the intracellular delivery of naked anti-luciferase siRNA by faint ET, and found that the luciferase activity of cells expressing luciferase was reduced by in vitro ET like in vivo iontophoresis. Cellular uptake of fluorescent-label siRNA was increased by ET, while low temperature exposure, macropinocytosis inhibitor amiloride and caveolae-mediated endocytosis inhibitor filipin significantly prevented siRNA uptake. These results indicate that the cellular uptake mechanism involved endocytosis. In addition, voltage sensitive fluorescent dye DiBAC4 (3) penetration was increased by ET, and the transient receptor potential channel inhibitor SKF96365 reduced siRNA uptake, suggesting that faint ET reduced membrane potentials by changing intracellular ion levels. Moreover, to analyze cytoplasmic delivery, we used in-stem molecular beacon (ISMB), which fluoresces upon binding to target mRNA in the cytoplasm. Surprisingly, cytoplasmic ISMB fluorescence appeared rapidly and homogeneously after ET, indicating that cytoplasmic delivery is markedly enhanced by ET. In conclusion, we demonstrated for the first time that faint ET can enhance cellular uptake and cytoplasmic delivery of extraneous molecules.

Abstract 5226: Glioma stem cells internalize perivascular bevacizumab via a non-canonical pathway and target it for recycling or degradation

Abstract Antiangiogenic therapy shows great promise for treatment of cancer. Bevacizumab is a humanized mAb that blocks VEGF-A, thereby inhibiting angiogenesis. It has received FDA approval for patients with recurrent glioblastoma (GBM); however, approximately 30% of patients are non-responsive and the underlying mechanism for the lack of response is not known. It has been assumed that Bevacizumab solely targets circulating VEGF-A; however, VEGF-A found in the perivascular space of GBM tumors could also be an important target. We hypothesized that Bevacizumab is transcytosed across brain endothelial cells (ECs), gains access to the perivascular niche containing glioma stem cells (GSCs), and that differences in GSC endocytosis and trafficking of Bevacizumab to either a recycling or degradation compartment determines a patients’ response. We found that Bevacizumab is internalized by normal and tumor-isolated brain ECs in a time-dependent manner. ECs transcytose 20% of Bevacizumab after 30 minutes, and 40% after 120 minutes. Importantly, we found that 95% of GSCs internalize Bevacizumab within 30 minutes. Internalized Bevacizumab in GSCs can be recycled to the extracellular space via a Rab4+ fast recycling compartment, or degraded via trafficking to the LAMP1+ compartment (late endosome/lysosome). Furthermore, Bevacizumab internalization in GSCs was partially inhibited by a macropinocytosis inhibitor (EIPA) suggesting macropinocytosis is one mechanism of internalization. GSCs do not internalize Bevacizumab via the canonical IgG internalization pathway since they do not express the neonatal Fc Receptor (FcRn). Supporting our in vitro data, we found a gradient of Bevacizumab extending from the vessel into the tumor in an orthotopic xenograft mouse model of GBM administered Bevacizumab, and Bevacizumab was readily detected within perivascular tumor cells. Our data suggest that the transcytosis of Bevacizumab by brain and tumor-associated ECs, and the endocytosis and trafficking of Bevacizumab by perivascular GSCs influences Bevacizumab effectiveness in patients with GBM. Citation Format: Gaelle Muller-Greven, Cathleen Carlin, Justin Lathia, Richard Prayson, Manmeet Ahluwalia, Steven Toms, Markus Bredel, Jeremy Rich, Petra Hamerlik, Candece L. Gladson. Glioma stem cells internalize perivascular bevacizumab via a non-canonical pathway and target it for recycling or degradation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5226. doi:10.1158/1538-7445.AM2015-5226

Abstract 4399: Utilizing cobalt coordination chemistry as a traceless prodrug strategy in targeted drug delivery

Abstract We recently reported the synthesis of albumin nanoparticles using cobalt as a traceless, reductively labile crosslinker. Here, we report in vitro characterization of particle uptake for fluorescently labeled cobalt crosslinked albumin nanoparticles (Co-Alb-FITC NPs) via image-based flow cytometry as well as preliminary toxicity data. Uptake of Co-Alb-FITC NPs by gastric carcinoma cells (SNU-5) was rapid, but could be dramatically reduced through the addition of an inhibitor of macropinocytosis. High levels of non-specific uptake of other particle types were also observed suggesting SNU-5 cells possess high macropinocytic activity. Cancers harboring mutated Ras (&amp;gt;20% of all cancers) are known to take up protein via macropinocytosis to satisfy metabolic demands. Because SNU-5 cells are not known harbor mutated Ras, our findings may indicate that other cancers rely on this mechanism as well, which would have important implications for observing selectivity in active nanoparticle targeting strategies. The Co-Alb NPs themselves were found to exhibit no inherent toxicity. Based on these initial findings, Co-Alb NPs were loaded with the active metabolite of camptothecin, SN-38, to explore their potential as a delivery vector for hydrophobic drugs. An albumin-topotecan prodrug conjugate was also synthesized by crosslinking the amine group in topotecan with lysine residues in the protein, which should be applicable to other amine-containing therapeutics. Characterization of drug loading and release will be discussed along with preliminary in vitro characterization. Citation Format: Robby A. Petros, Duong T. Nguyen, Ronaldo J. Cavazos, Clifford S. Morrison, Jana B. Lampe, Alesha N. Harris, Brian K. McFarlin. Utilizing cobalt coordination chemistry as a traceless prodrug strategy in targeted drug delivery. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4399. doi:10.1158/1538-7445.AM2015-4399

Abstract 5462: Extracellular ATP promotes cancer cell drug resistance to tyrosine kinase inhibitors by competing for the ATP-binding site of tyrosine kinase

Abstract ATP plays important roles in cellular processes in cancer cells, which are a heterogeneous population in tumors regarding their genetic background and oxygen and energy supplies. The fact that cancer cells undergo rapid proliferation suggests that they must possess mechanisms, which may not be present in normal cells, to meet their high energy needs. Studies by others revealed that extracellular ATP levels of various cancer types are 10^3 to 10^4 times higher than those in their corresponding normal tissues (1). We recently reported that extracellular ATP can be taken up directly by cancer cells by macropinocytosis (2). Based on these results, we hypothesized that the internalized extracellular ATP not only promotes cancer cell growth and survival, but also drug resistance. In this study, human lung cancer A549 and breast cancer MCF7 cells were used as cell models to determine the effects of extracellular ATP on drug resistance to tyrosine kinase inhibitors (TKIs) in cancer cells. Cancer cell viability and intracellular ATP measurement results showed that extracellular ATP substantially increased intracellular ATP levels and promoted cancer cell drug resistance to TKIs. Viability of TKI treated cancer cells was significantly increased in the presence of extracellular ATP, compared to cancer cells with TKI treatment alone. Administration of macropinocytosis inhibitors significantly reduced the drug resistance induced by extracellular ATP, suggesting that extracellular ATP promoted drug resistance by contributing to the intracellular ATP pool. Protein and protein phosphorylation analyses results suggested that the increased intracellular ATP reversed the inhibition of TKIs by competing with the inhibitor for the ATP-binding site of platelet-derived growth factor receptor (PDGFR) and enhancing Akt and MAPK signaling pathways. These findings identify, for the first time, new biological functions of extracellular ATP in tumors and may lead to novel anti-cancer strategies combating drug resistance in cancers. 1. Pellegatti P, et al. PLoS ONE 2008;3:e2599. 2. Qian Y, et al. Cancer Lett 2014;351:242-51. Citation Format: Xuan Wang, Yanrong Qian, Yunsheng Li, Xiaozhuo Chen. Extracellular ATP promotes cancer cell drug resistance to tyrosine kinase inhibitors by competing for the ATP-binding site of tyrosine kinase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5462. doi:10.1158/1538-7445.AM2015-5462

Active phagocytosis of Mycobacterium tuberculosis (H37Ra) by T lymphocytes (Jurkat cells)

This study aimed to co-culture Jurkat T lymphocytes with inactivated Mycobacterium tuberculosis (Mtb H37Ra), explore whether T lymphocytes could phagocytose H37Ra cells, and determine the underlying mechanism. Jurkat T lymphocytes were co-cultured with H37Ra cells, and confocal laser scanning microscopy, electron microscopy, and flow cytometry techniques were used to identify phagocytosis and elucidate its mechanism. After Jurkat T lymphocytes phagocytosed H37Ra cells, the cell body became larger, with abundant cytoplasm, the portion of the nucleus closest to the bacterium deformed, long and short pseudopodia were extended, and the folds of the cell membrane formed depressions that created phagocytic vesicles surrounding the bacterium. The macropinocytosis inhibitor amiloride and the cytoskeletal inhibitor cytochalasin D were found to inhibit phagocytic efficacy; serum complements might enhance phagocytosis through opsonization. Jurkat T lymphocytes could actively phagocytose inactivated Mtb via the macropinocytotic mechanism. Actin remodeling played an important role in the macropinocytotic process. Serum complements may regulate phagocytosis.

Abstract 143: Macropinocytosis Mediates Enzymatically Modified LDL (ELDL)-Induced Murine Smooth Muscle Cell Formation: A Role for RAGE In ELDL Endocytosis and Upregulation of Scavenger Receptor LOX1

Background: ELDL is present in human atherosclerotic lesions and promotes foam cell formation in cultured macrophages and vascular smooth muscle cells (SMC). Here we study mechanism of ELDL uptake and its effects on SMCs. Methods and Results: Incubation of wild type murine aortic SMCs with 10 μg/ml ELDL (trypsin, cholesterol esterase modified) results in enhanced foam cell formation (analyzed by Oil Red O, lipid measurement) compared to SMCs incubated with acetylated LDL (500 μg/ml; -50%, p&lt;0.01) and oxidized LDL (200 μg/ml; -75%, p&lt;0.01). Inhibitors of macropinocytosis (50 μM LY294006, 2 μM wortmannin, and 3 mM amiloride) attenuated ELDL uptake (-50%, -50%, -100% respectively). In contrast, inhibitors of receptor mediated endocytosis (100 μM dynasore, 0.1 M Sucrose), and inhibitors of caveolae /lipid raft mediated endocytosis (5mM MBCD, 5 μM filipin) had no effect on ELDL uptake in SMCs. Moreover, ELDL incubation led to increased expression of scavenger receptor LOX1 (+ 3 fold, p&lt;0.01) in wild type SMC’s, but not in SMC deficient in Receptor for AGE (RAGE-/-), while CD36 and SRA1 remained unchanged in both the SMCs. Importantly, RAGE-/- SMCs upon pretreatment with PI3K inhibitors that only partially inhibited macropinocytosis of ELDL in wild type SMCs, completely prevented ELDL uptake in RAGE-/- SMCs. Mechanistically, ELDL upregulates ROS (detected using H2DCFDA) and down regulates PIP3 (detected by pAkt immunoblotting) in wild type, but not in RAGE-/- SMCs. Since ROS is known to regulate macropinocytosis via increased Ca2+ levels, we tested Ca2+ channel inhibitor lacidipine (30 μM), and found complete inhibition of ELDL uptake in both, wild type and RAGE-/- SMCs. Lastly, we speculate that the fused structure of LDL in the ELDL preparation is preferentially activating RAGE, since oligomerization of ligands are known to increase RAGE signaling, and FPLC analysis demonstrated that ELDL consists mostly of fused LDL particles. Conclusions: ELDL is highly potent in inducing foam cells in aortic SMCs. ELDL endocytosis is mediated by RAGE-regulated, Ca2+ dependent macropinocytosis.

Simian hemorrhagic fever virus cell entry is dependent on CD163 and uses a clathrin-mediated endocytosis-like pathway.

Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques but is thought to be nonpathogenic for humans. To date, the SHFV life cycle is almost completely uncharacterized on the molecular level. Here, we describe the first steps of the SHFV life cycle. Our experiments indicate that SHFV enters target cells by low-pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-β-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knockout study and electron microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, α-chymotrypsin, and trypsin) abrogated entry, indicating that the SHFV cell surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component. Simian hemorrhagic fever virus (SHFV) causes highly lethal disease in Asian macaques resembling human illness caused by Ebola or Lassa virus. However, little is known about SHFV's ecology and molecular biology and the mechanism by which it causes disease. The results of this study shed light on how SHFV enters its target cells. Using electron microscopy and inhibitors for various cellular pathways, we demonstrate that SHFV invades cells by low-pH-dependent, actin-independent endocytosis, likely with the help of a cellular surface protein.

Abstract 5257: Cancer cells have not only sweet tooth but also large mouths: Evidence for uptake of ATP into KRas oncogene-expressing cancer cells by macropinocytosis

Abstract Cancer cells are known to exhibit reprogrammed metabolism and prefer to use glycolygsis for their ATP synthesis even when oxygen is abundant, a phenomenon called the Warburg effect. However, cancer cells are also known for more rapid growth and proliferation, therefore requiring more ATP than normal cells of the same tissues. How cancer cells manage to use low efficiency glycolysis but still meet their higher ATP demands is enigmatic. In our previous study, we found that extracellular ATP induced large intracellular ATP increase and drug resistance to tyrosine kinase inhibitors (TKIs) that function as ATP analogs / competitors in cancer cells. The induced ATP increase was from neither OXPHOS nor glycolysis, and was not dependent on AMPK, the master ATP regulator. Furthermore, the increase did not involve new gene expression or new protein synthesis. Recently, it was reported that cells transformed with an oncogenic form of KRas gene (KRas+) exhibit a phenotype of macropinocytosis, a type of endocytosis. KRas-induced macropinocytosis nonspecifically takes up extracellular proteins. All these led us to hypothesize that the increased intracellular ATP induced by extracellular ATP is, at least in part, not produced intracellularly but is transported into cancer cells. To test this hypothesis, high molecular weight fluorescent dextran was use to determine if KRas+ human non-small cell lung cancer A549 cells exhibit macropinocytosis. Nonhydrolyzable fluorescent ATP analog was used to visualize if the ATP analog can be transported into A549 cells along with dextran. Fluorescence microscopy studies revealed that A549 cells exhibit the phenotype of macropinocytosis, which can be reduced by a macropinocytosis inhibitor EIPA. HMW dextran and the nonhydrolysable fluorescent ATP analog were found co-localized inside A549 cells, demonstrating that they were transported into A549 cells via macropinosomes. It was also found that the ATP increase cannot be explained by purinergic receptor signaling alone. In comparison, KRas- human breast cancer MCF7 cells do not take up HMW dextran, indicating that they do not exhibit macropinocytosis. All these provide strong and visual evidence that ATP can be transported into KRas+ A549 cancer cells by macropinocytosis. Other experiments using radioactive ATP or purinergic receptor inhibitors also strongly support this conclusion. This is the first time that ATP (ATP analog) is shown to be taken into cancer cells. This finding will have major impact on how we view cancer cells in terms of their energy exchange with other cancer or noncancer cells in the tumor and on the Warburg effect. It will also have profound implications on how to more effectively inhibiting KRas+ or KRas- cancer cells by targeting ATP transport and ATP metabolism. Citation Format: Yanrong Qian, Xuan Wang, Yi Liu, Yunsheng Li, Robert A. Colvin, Xiaozhuo Chen. Cancer cells have not only sweet tooth but also large mouths: Evidence for uptake of ATP into KRas oncogene-expressing cancer cells by macropinocytosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5257. doi:10.1158/1538-7445.AM2014-5257

Recombinant adeno-associated virus utilizes cell-specific infectious entry mechanisms.

Understanding the entry and trafficking mechanism(s) of recombinant adeno-associated virus (rAAV) into host cells can lead to evolution in capsid and vector design and delivery methods, resulting in enhanced transduction and therapeutic gene expression. Variability of findings regarding the early entry pathway of rAAV supports the possibility that rAAV, like other viruses, can utilize more than one infectious entry pathway. We tested whether inhibition of macropinocytosis impacted rAAV transduction of HeLa cells compared to hepatocellular carcinoma cell lines. We found that macropinocytosis inhibitor cytochalasin D blocked rAAV transduction of HeLa cells (>2-fold) but enhanced (10-fold) transduction in HepG2 and Huh7 lines. Similar results were obtained with another macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl) amiloride (EIPA). The augmented transduction was due to neither viral binding nor promoter activity, affected multiple rAAV serotypes (rAAV2, rAAV2-R585E, and rAAV8), and influenced single-stranded and self-complementary virions to comparable extents. Follow-up studies using CDC42 inhibitor ML141 and p21-activated kinase 1 (PAK1) siRNA knockdown also resulted in enhanced HepG2 transduction. Microscopy revealed that macropinocytosis inhibition correlated with expedited nuclear entry of the rAAV virions into HepG2 cells. Enhancement of hepatocellular rAAV transduction extended to the mouse liver in vivo (4-fold enhancement) but inversely blocked heart tissue transduction (13-fold). This evidence of host cell-specific rAAV entry pathways confers a potent means for controlling and enhancing vector delivery and could help unify the divergent accounts of rAAV cellular entry mechanisms. There is a recognized need for improved rAAV vector targeting strategies that result in delivery of fewer total particles, averting untoward toxicity and/or an immune response against the vector. A critical step in rAAV transduction is entry and early trafficking through the host cellular machinery, the mechanisms of which are under continued study. However, should the early entry and trafficking mechanisms of rAAV differ across virus serotype or be dependent on host cell environment, this could expand our ability to target particular cells and tissue for selective transduction. Thus, the observation that inhibiting macropinocytosis leads to cell-specific enhancement or inhibition of rAAV transduction that extends to the organismic level exposes a new means of modulating vector targeting.

Endocytosis of collagen by hepatic stellate cells regulates extracellular matrix dynamics.

Hepatic stellate cells (HSCs) generate matrix, which in turn may also regulate HSCs function during liver fibrosis. We hypothesized that HSCs may endocytose matrix proteins to sense and respond to changes in microenvironment. Primary human HSCs, LX2, or mouse embryonic fibroblasts (MEFs) [wild-type; c-abl(-/-); or Yes, Src, and Fyn knockout mice (YSF(-/-))] were incubated with fluorescent-labeled collagen or gelatin. Fluorescence-activated cell sorting analysis and confocal microscopy were used for measuring cellular internalization of matrix proteins. Targeted PCR array and quantitative real-time PCR were used to evaluate gene expression changes. HSCs and LX2 cells endocytose collagens in a concentration- and time-dependent manner. Endocytosed collagen colocalized with Dextran 10K, a marker of macropinocytosis, and 5-ethylisopropyl amiloride, an inhibitor of macropinocytosis, reduced collagen internalization by 46%. Cytochalasin D and ML7 blocked collagen internalization by 47% and 45%, respectively, indicating that actin and myosin are critical for collagen endocytosis. Wortmannin and AKT inhibitor blocked collagen internalization by 70% and 89%, respectively, indicating that matrix macropinocytosis requires phosphoinositide-3-kinase (PI3K)/AKT signaling. Overexpression of dominant-negative dynamin-2 K44A blocked matrix internalization by 77%, indicating a role for dynamin-2 in matrix macropinocytosis. Whereas c-abl(-/-) MEF showed impaired matrix endocytosis, YSF(-/-) MEF surprisingly showed increased matrix endocytosis. It was also associated with complex gene regulations that related with matrix dynamics, including increased matrix metalloproteinase 9 (MMP-9) mRNA levels and zymographic activity. HSCs endocytose matrix proteins through macropinocytosis that requires a signaling network composed of PI3K/AKT, dynamin-2, and c-abl. Interaction with extracellular matrix regulates matrix dynamics through modulating multiple gene expressions including MMP-9.