mteb/CovidQA huggingface dataset:
Abstract: The vacuolar-type H(+)-ATPase (v-ATPase) is the major proton pump that acidifies intracellular compartments of eukaryotic cells. Since the inhibition of v-ATPase resulted in anti-tumor and anti-metastatic effects in different tumor models, this enzyme has emerged as promising strategy against cancer. Here, we used the well-established v-ATPase inhibitor archazolid, a natural product first isolated from the myxobacterium Archangium gephyra, to study the consequences of v-ATPase inhibition in endothelial cells (ECs), in particular on the interaction between ECs and cancer cells, which has been neglected so far. Human endothelial cells treated with archazolid showed an increased adhesion of tumor cells, whereas the transendothelial migration of tumor cells was reduced. The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the
integrin β1 subunit reversed this process. Tumor cells preferentially adhered to the β1-integrin ligand collagen and archazolid led to an increase in the amount of collagen on the surface of ECs. The accumulation of collagen was accompanied by a strong decrease of the expression and activity of the protease cathepsin B. Overexpression of cathepsin B in ECs prevented the capability of archazolid to increase the adhesion of tumor cells onto ECs. Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound.
Text: The vacuolar-type H + -ATPase (v-ATPase) is the major proton pump responsible for acidification of intracellular compartments in eukaryotic cells [1] . The enzyme consists of two multi-subunit complexes, the soluble V 1 transmembrane V o subcomplex required for the proton transport across membranes [1, 2] . In most cell types v-ATPases are only expressed in the endomembrane system to regulate and maintain the acidic pH of intracellular compartments such as lysosomes, endosomes, the Golgi apparatus, secretory granules and coated vesicles [3] . The function of v-ATPases is essential for cellular processes such as vesicular trafficking, receptor-mediated endocytosis and protein degradation and processing. In specialized cell types including osteoclasts and renal epithelial cells, v-ATPases can also be expressed on the plasma membrane, where they pump protons into the extracellular space [2] [3] [4] . In cancer cells v-ATPases are expressed on the plasma membrane in order to
eliminate toxic cytosolic H + . Most importantly, v-ATPases contribute to the acidic tumor microenvironment, which leads to the activation of proteases, thus facilitating tumor cell migration, invasion and angiogenesis [5] [6] [7] . Since the inhibition of v-ATPase was shown to reduce the invasiveness of cancer cells and metastasis formation [8, 9] , this enzyme has emerged as a promising drug target in the recent years. Archazolid A and B are highly potent and specific inhibitors of v-ATPases [10] . They were first isolated from the myxobacterium Archangium gephyra [11] . These compounds inhibit v-ATPase at low nanomolar concentrations [10, 12] by binding to the subunit c of the V o complex. As their biological activity is comparable to the v-ATPase inhibitors bafilomycin and concanamycin [10, 11] , archazolids are natural compounds of high interest that can be used both as a tool to study the consequences of v-ATPase inhibition and as a lead for drug development. Archazolids can be
either produced by fermentation [11] or by total synthesis [13, 14] .
In the field of cancer research several studies reported on interesting pharmacological effects of archazolid: It reduced the migration of different invasive tumor cells in vitro and cancer cell metastasis in vivo in a breast tumor mouse model [15] . Furthermore, archazolid activated pathways of cellular stress response and apoptosis in highly invasive tumor cells [16] . In classically activated macrophages, archazolid selectively induced the generation of tumor necrosis factor α (TNFα), which may indirectly promote tumor suppression [17] .
Up to now, the role of v-ATPases in endothelial cells has only rarely been investigated. The endothelium plays a crucial role in the pathogenesis and progression of cancer: The metastatic cascade includes local angiogenesis at the site of the primary tumor and adhesion of tumor cells at the site of metastasis [18] . Angiogenesis, the development of new blood vessels out of existing ones, depends on the proliferation, migration and differentiation of endothelial cells [19] . This process ensures the nutrient supply of the tumor and its growth [20] . Circulating cancer cells can adhere to the endothelium at distant sites. This adhesive interaction is mediated by receptors and corresponding ligands expressed on tumor and endothelial cells [18, 21] . V-ATPases have been reported to regulate intracellular pH and cell migration in microvascular endothelial cells [22, 23] . A recent study showed that the inhibition of v-ATPase by concanamycin prevented proliferation, reduced migration and
impaired angiogenesis-related signaling in endothelial cells [24] . So far, there are no investigations on the role of endothelial v-ATPases for the process of tumor cell adhesion onto the endothelium. Thus, we were interested in the consequences of the inhibition of endothelial v-ATPase by archazolid on the interaction between endothelial and cancer cells. Various cell adhesion molecules on the endothelium, such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion protein (VCAM-1), E-selectin or N-cadherin [21] as well as integrins expressed on cancer cells have been reported to mediate cell adhesion of cancer cells onto endothelial cells [25] [26] [27] . Accordingly, we focused on these cell adhesion molecules and integrins. For the first time, our study revealed a link between the function of v-ATPases and the adhesion and transmigration properties of endothelial cells.
CellTiter-Blue Cell Viability Assay (Promega, Mannheim, Germany) was performed according to the manufacturer's protocol for determining the cell viability of cells after treatment with archazolid. This assay is based on the ability of metabolically active cells to reduce resazurin which results in fluorescent resorufin. The CellTiter-Blue Reagent was added to the cells 4 h before the endpoint of treatment. Fluorescence was measured with an Infinite F200 pro microplate reader (Tecan, Männedorf, Switzerland) at 560 nm (excitation) and 590 nm (emission).
CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) was performed according to the manufacturer's instructions for determining the lactate dehydrogenase (LDH) release after treatment with archazolid. Lysis buffer was added to untreated cells 45 min before the end of treatment to induce the release of this enzyme. LDH is a cytosolic enzyme that is released by leaky cells. Released LDH catalyzes the enzymatic conversion of lactate to pyruvate which provides NADH for the conversion of iodonitrotetrazolium violet into a red formazan product in the presence of diaphorase. The absorbance was measured with a Varioskan Flash microplate reader (Thermo Fisher Scientific) at 490 nm.
LysoTracker Red DND-99 (Life Technologies, Thermo Fisher Scientific) is a dye to measure pH values in viable cells. HUVECs were cultured to confluence on collagen G-coated μ-slides (80826, ibidi, Martinsried, Germany) before they were treated with archazolid for 24 h. 1 μg/ ml Hoechst 33342 (Sigma-Aldrich, Munich, Germany) was used to visualize the nuclei and 50 nM LysoTracker Red DND-99 was used to visualize the acidic compartments which correspond to the lysosomes. Both dyes were incubated for 10 min at 37˚C before acquisition of single images by a Leica DMI6000 B fluorescence microscope (Leica Microsystems, Wetzlar, Germany).
HUVECs were seeded in collagen G-coated 24-well plates and grown to confluence for two days before treatment. The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye (5 μM in serum-free DMEM, 37˚C) for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37˚C. Non-adherent tumor cells were washed off three times with PBS containing Ca 2+ and Mg 2+ . Tumor cell adhesion was determined by fluorescence measurements with an Infinite F200 pro microplate reader (Tecan) at 485 nm (excitation) and 535 nm (emission).
For blocking the integrin β1 subunit on MDA-MB-231 or PC-3 cells, CellTracker Greenlabeled MDA-MB-231 or PC-3 cells were incubated with an anti-integrin β1 antibody (P5D2, ab24693, Abcam, Cambridge, United Kingdom) at a concentration of 1 μg antibody per one million cells in 1 ml DMEM. Before adding to archazolid-treated HUVECs, MDA-MB-231 or PC-3 cells were washed once with DMEM. For blocking the integrin β1 subunit on HUVECs, the cells were incubated with the anti-integrin β1 antibody (0.1 μg/well in ECGM). HUVECs were washed once with ECGM before untreated MDA-MB-231 or PC-3 cells were added to HUVECs.
For the adhesion of MDA-MB-231 or PC-3 cells onto extracellular matrix (ECM) components 24-well plates were coated with collagen G (10 μg/ml in PBS), human plasma fibronectin (10 μg/ml PBS) or laminin-411 (10 μg/ml in Dulbecco's PBS [DPBS] containing Ca 2+ and Mg 2+ ) at 4˚C overnight. The adhesion of MDA-MB-231 and PC-3 cells onto these three most prominent ECM components was carried out as described above (10 min adhesion at 37˚C).
HUVECs were grown on a porous filter membrane (Transwell insert, polycarbonate membrane, 8 μm pores; Corning, New York, USA) for 48 h and were treated as indicated. Untreated MDA-MB-231 cells were labeled with CellTracker Green CMFDA Dye (as described in the section cell adhesion assay) and resuspended in medium 199 (PAN-Biotech) containing 0.1% BSA. HUVECs were washed twice with medium 199 containing 0.1% BSA before MDA-MB-231 cells were allowed to transmigrate through the endothelial monolayer for 24 h. Medium 199 containing 0.1% BSA was used as negative control and medium 199 containing 20% FCS was used as chemoattractant for transmigration in the lower compartment. Non-migrated cells remaining in the upper compartment were carefully removed using a cotton swab. Transmigrated cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and transmigration was quantified by measuring the fluorescence signal at 485 nm (excitation) and 535 nm (emission).
HUVECs were grown to confluence on 6-well plates before they were treated with archazolid for 12 h. The cells were induced to upregulate the gene expression of cell adhesion molecules by TNFα. RNA was isolated using the RNeasy Mini Kit from Qiagen (Hilden, Germany) according to the manufacturer's protocol. On-column DNase digestion was performed to remove genomic DNA. RNA was transcribed into cDNA by Superscript II (Life Technologies, Thermo Fisher Scientific). qPCR experiments were performed using a StepOnePlus System (Applied Biosystems, Thermo Fisher Scientific) and data was analyzed by the StepOne and Ste-pOnePlus Software v2.3. Power SYBR Green PCR Master Mix (Life Technologies) and the comparative C T quantitation method (2 -ΔΔCT ) were used.
HUVECs were grown to confluence on 12-well plates before they were treated with archazolid for 24 h. Cells were treated with TNFα for 24 h to induce the expression of cell adhesion molecules. Subsequently, the cells were detached with HyClone HyQTase (GE Healthcare, Freiburg, Germany). In the case of ICAM-1 the detached cells were fixed with 4% formaldehyde (Polysciences, Hirschberg an der Bergstraße, Germany) in PBS for 10 min and washed once with PBS before incubating with the fluorescein isothiocyanate (FITC)-labeled anti-human CD54 (mouse, ICAM-1) antibody (MCA1615F, Biozol, Eching, Germany) at room temperature for 45 min. For all other proteins, the cells were not fixed and washed once with PBS before incubating with the antibodies phycoerythrin (PE)-labeled anti-human CD106 (mouse, VCAM-1), PE-labeled anti-human CD62E (mouse, E-selectin) and PE-labeled anti-human CD325 (mouse, N-cadherin) from Becton Dickinson on ice for 45 min. These antibodies were diluted in PBS containing
0.2% BSA. The surface expression of cell adhesion molecules was measured by flow cytometry (FACSVerse, Becton Dickinson, Heidelberg, Germany).
To stain the surface collagen on HUVECs, cells were incubated with an anti-human collagen antibody (rabbit, 1:40, ab36064, Abcam) on ice for 30 min. The staining procedure was performed on ice to ensure that surface proteins or antibodies are not endocytosed. The cells were washed once with PBS containing Ca 2+ and Mg 2+ before they were fixed with Roti-Histofix (Carl Roth). Alexa Fluor 488-conjugated anti-rabbit antibody (goat, 1:400, A11008, Life Technologies) was used as secondary antibody and Hoechst 33342 (1 μg/ml, Sigma-Aldrich) was used to visualize nuclei.
Confluent HUVECs in 6-well plates were treated as indicated. Cells were washed with ice-cold PBS and lysed with RIPA buffer supplemented with protease inhibitors (Complete Mini EDTA-free; Roche, Mannheim, Germany), sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride, β-glycerophosphate, sodium pyrophosphate and H 2 O 2 . Protein determination was performed using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of proteins (10-20 μg) were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad Laboratories, Munich, Germany). Separated proteins were transferred onto polyvinylidene difluoride membranes by tank blotting (Bio-Rad Laboratories) for immunodetection. Membranes were blocked with 5% boltinggrade milk powder (Carl Roth) in TBS containing 0.1% Tween 20 (Sigma-Aldrich). The following antibodies were used: mouse anti-human cathepsin B antibody (IM27L, Merck) (1:500), mouse anti-β-actin-peroxidase antibody
(A3854, Sigma-Aldrich) (1:100,000) and antimouse IgG horse radish peroxidase (HRP)-linked antibody (7076, Cell Signaling, Frankfurt, Germany) (1:5,000). ImageJ version 1.49m was used for densitometric analysis.
Cathepsin B activity assay was performed as described in the publication by Kubisch et al. [28] . Confluent HUVECs or HMEC-1 seeded in 6-well plates were treated as indicated. Cells were washed with PBS and lysed with the non-denaturating M-PER mammalian protein extraction reagent (78501, Thermo Fisher Scientific) supplemented with protease inhibitors (Complete Mini EDTA-free, Roche), sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride. The fluorogenic cathepsin B substrate Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride (C5429, Sigma-Aldrich) was added to 30 μg of the cell lysate diluted in assay buffer supplemented with 2 mM L-cysteine (C7880, Sigma-Aldrich) and incubated for 30 min at 40˚C. Fluorescence was measured at 348 nm (excitation) and 440 nm (emission) with a microplate reader (Varioskan Flash, Thermo Fisher Scientific). The intensity of the fluorescence signal corresponded to the cathepsin B enzyme activity. For background subtraction the cathepsin B
inhibitor CA-074Me (Enzo Life Sciences, Lörrach, Germany) was added to an additional reaction.
The HUVEC Nucleofector Kit (Lonza, Cologne, Germany) was used to transfect HUVECs. The transfection was performed according to the manufacturer's protocol using 2.5 μg plasmid DNA for 500,000 cells (Nucleofector 2b Device, Lonza). 48 h after transfection the cells were treated for further experiments. The addgene plasmid #11249 hCathepsin B was kindly provided by Hyeryun Choe [29] . hCathepsin B was digested with PmeI and XbaI and the linear DNA fragment not corresponding to the human CTSB gene was religated to generate the empty pcDNA3.1 (-) delta MCS plasmid that was used for control transfections. The original backbone of hCathepsin B is the pcDNA3.1 (-) from Thermo Fisher Scientific. The control vector pcDNA3.1 (-) delta MCS used for our transfections was cloned on the basis of hCathepsin B and is therefore lacking almost the whole part of the multiple cloning site of the pcDNA3.1 (-).
Statistical analyses were performed using GraphPad Prism 5.0 (San Diego, USA). One-way ANOVA followed by Tukey's post-hoc test or unpaired t-test was used for the evaluation of a minimum of three independent experiments. The numbers of independently performed experiments (n) are stated in the corresponding figure legends. p 0.05 was considered as statistically significant. Data are expressed as mean ± standard error of the mean (SEM).
Since the v-ATPase inhibitor archazolid has never been used for studies in endothelial cells, we first performed cytotoxicity assays. We treated confluent HUVECs with up to 1 nM archazolid for 24 and 48 h and observed that this treatment has neither an influence on the metabolic activity nor on the release of LDH after 24 h (Fig 1A and 1B, left panels) . The metabolic activity and the release of LDH were only slightly affected by the highest concentration of archazolid after 48 h (Fig 1A and 1B, right panels) . Consequently, the following experiments were all carried out after 24 h (or less) of archazolid treatment in order to exclude any cytotoxic effects of archazolid within our experimental settings.
Microscopic analysis revealed that also the integrity of the endothelial monolayer was not affected by archazolid, but the cells showed a slightly different morphology (Fig 2A) : Archazolid-treated cells were swollen compared to control cells, which was not unexpected, as vacuolation of the endoplasmic reticulum (ER) has been described for other cell types and is typical for v-ATPase inhibitors [11, 16, 24, 30] . This effect was obvious both in subconfluent and in confluent cells (Fig 2A) . Inhibition of v-ATPase prevents the acidification of lysosomes [1, 31] . Using the cell-permeable dye LysoTracker Red DND-99, it is possible to label the acidic lysosomes in living cells. Thus, this dye can serve as an indicator of v-ATPase inhibition. To proof that archazolid is also functionally active as a v-ATPase inhibitor in HUVECs, cells were treated with 1 nM archazolid before they were incubated with LysoTracker Red DND-99 and Hoechst 33342. As shown in Fig 2B, the red vesicular staining
corresponding to acidified lysosomes in control cells disappeared completely after treatment with archazolid. In summary, archazolid treatment for 24 h was not cytotoxic to quiescent HUVECs, but inhibited the functionality of the v-ATPase.
We analyzed the adhesion of MDA-MB-231 cells onto HUVECs. Confluent HUVECs were treated with up to 1 nM archazolid for 24 h. Untreated MDA-MB-231 cells were labeled with Cell-Tracker Green CMFDA Dye. Interestingly, v-ATPase inhibition strongly increased the attachment of the metastatic breast carcinoma cell line MDA-MB-231 onto HUVECs after 10 and 120 min of adhesion (Fig 3A and 3B) . We also investigated the influence of archazolid on the transendothelial migration of MDA-MB-231 cells. HUVECs seeded in a Boyden chamber were treated with 1 nM archazolid for 24 h. CellTracker Green-labeled MDA-MB-231 cells (not treated with archazolid) were allowed to transmigrate through the endothelial monolayer for 24 h. As shown in Fig 3C, archazolid significantly decreased the transendothelial migration of MDA-MB-231 cells.
The influence of archazolid on tumor cell adhesion was not only studied in HUVECs, which represent macrovascular endothelial cells, but also in microvascular HMEC-1 cells. Moreover, besides the breast cancer cell line MDA-MB-231, also PC-3 prostate cancer cells were used as a second metastatic cancer cell line. Archazolid treatment of endothelial cells increased the attachment of MDA-MB-231 cells onto the HMEC-1 monolayer after 120 min of adhesion ( Fig 4A) and increased the attachment of PC-3 cells onto the HUVEC monolayer after 30 and 60 min of adhesion (Fig 4B) . Of note, the adhesion of non-metastatic Jurkat cells, an acute T cell leukemia cell line, remained unaffected after treatment of HUVECs with archazolid (S1A Fig). Taken together, archazolid treatment augmented the adhesive properties of both micro-and macrovascular endothelial cells for metastatic tumor cells, but not for non-metastatic ones. Of note, cancer cell adhesion onto archazolid-activated endothelial cells
increased with the time of adhesion.
The adhesion of tumor cells onto the endothelium is in principle similar to that of leukocytes, but slightly differs in the molecules that mediate the adhesion process. Ligands for the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin were found to be expressed on tumor cells and to mediate tumor-endothelial cell interaction [21] . Inhibition of the v-ATPase might affect the expression of endothelial cell adhesion molecules on mRNA or protein levels. To determine the mRNA expression of ICAM-1, VCAM-1, E-selectin and Ncadherin, HUVECs were treated with archazolid for 12 h. TNFα is known to upregulate the expression of ICAM-1, VCAM-1 and E-selectin [32] and, thus, served as positive control. Quantitative real-time PCR showed that v-ATPase inhibition in HUVECs did not alter the mRNA levels of ICAM-1, VCAM-1, E-selectin and N-cadherin (Fig 5A) . The protein expression of these adhesion molecules on the surface of endothelial cells was analyzed by flow
cytometry. Archazolid (1 nM, 24 h) did not affect the cell surface expression of ICAM-1, VCAM-1, E-selectin and N-cadherin (Fig 5B) .
Besides ICAM-1, VCAM-1, E-selectin and N-cadherin, also integrins are able to mediate the process of cell adhesion [33] [34] [35] . Since none of the cell adhesion molecules expressed on HUVECs were regulated upon archazolid treatment, we considered integrins as potential interaction partners. Within this protein family β1-integrins have been reported to mediate tumor cell adhesion onto quiescent endothelial cells [25] . In order to elucidate the role of β1-integrins for the archazolid-induced tumor cell adhesion, the integrin β1-subunit was blocked either on MDA-MB-231 cells, PC-3 cells or on HUVECs. (Of note, as in all experiments throughout this study, only endothelial cells were treated with archazolid.) After blocking β1-integrins on MDA-MB-231 or PC-3 cells, the archazolid-induced tumor cell adhesion was reduced almost to control level (Fig 6A and 6B , left panels), whereas blocking of β1-integrins on HUVECs had no significant effect on the increase of tumor cell adhesion by
v-ATPase inhibition (Fig 6A and 6B , right panels).
Depending on their α subunit, β1-integrins have a variety of ligands including extracellular matrix (ECM) components such as collagen, fibronectin and laminin [35] . Therefore, we hypothesized that archazolid treatment of endothelial cells might lead to an upregulation of these components. MDA-MB-231 and PC-3 cells were allowed to adhere onto plastic that was coated with these ECM components. This cell adhesion assay revealed that MDA-MB-231 as well as PC-3 cells favor the interaction with the ECM component collagen, as the adhesion onto collagen is much higher than onto the uncoated plastic control (Fig 7A) . MDA-MB-231 and PC-3 cells also adhered to fibronectin-coated plastic, but to a much lesser extent compared to the collagen coating. Therefore, we focused on the interaction between these two tumor cell lines and collagen. Blocking of the integrin β1 subunit on MDA-MB-231 and PC-3 cells clearly abolished the interaction with collagen (Fig 7B) , indicating that the attachment of
these tumor cells to collagen is mediated by β1-integrins.