Cells
Cells were cultured in a humidified incubator at 37ºC with 5% CO2 and atmospheric oxygen. All cells used in this study were consistently confirmed to be free from mycoplasma by using a MycoFluor™ Mycoplasma Detection Kit (ThermoFisher, M7006). 293FT cells from Clontech (632180) were maintained in DMEM medium supplemented with 10% (v/v) fetal bovine serum (FBS, ThermoFisher, 10437-036). Human iPSCs from ALSTEM (iPS11) were grown in mTeSR1 medium (StemCell Technologies, 85850). Human iPSC-derived monocytes and macrophages were maintained in X-VIVOTM 15 medium (Lonza, 04-418Q). Human U937 cells from ATCC (CRL-1593.2TM) were maintained in RPMI 1640 medium with 10% (v/v) FBS. Bone-marrow derived macrophages (BMDMs) were derived from mice according to an established protocol81 and cultured in RPMI 1640 medium with 10% (v/v) FBS. Human GSCs (CCF-3264 and CCF-DI315) were derived from human primary GBMs as previously described72,82,83 and maintained in Neurobasal medium (Invitrogen, 12349015) supplemented with B-27 (Invitrogen, 12587010), glutamine (2 mM, ThermoFisher, 35050061), non-essential amino acids (ThermoFisher, 11140050), sodium pyruvate (1 mM, ThermoFisher, 11360070), epidermal growth factor (EGF, 20 ng/mL, Goldbio, 1150-04-100), and basic fibroblast growth factor (bFGF, 20 ng/mL, R&D Systems, 4114-TC-01M). Unless otherwise indicated, the Gibco® antibiotic-antimycotic (ThermoFisher, 15240062) was used to prevent contamination in all the media.
Human GBM Surgical Specimens
Surgical specimens of human GBMs were collected from the Brain Tumor and Neuro-Oncology Center of the Cleveland Clinic according to a protocol approved by the Cleveland Clinic Institutional Review Broad. GBM surgical specimens were used for isolation of GSCs and immunofluorescent analyses. Specifically, the human GBM GSCs CCF-DI315 and CCF-3264 were derived from a 49-year old male patient and a 65-year female patient, respectively. CCF-4321 primary GBM specimen was collected from a 74-year old male patient. CCF-DI257 GBM specimen was collected from a 55-year old male patient. CCF-3303 GBM specimen was collected from a 36-year old female patient. CW-1797 GBM specimen was collected from a 57-year old male patient. CW-1798 GBM specimen was collected from a 47-year old male patient. CCF-2445 GBM specimen was collected from a 50-year old male patient.
Mice
All animal experiments were performed in accordance with protocols approved by the IACUC at the Cleveland Clinic Lerner Research Institute. Both male and female NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) were used for establishing GBM xenografts (PDXs) for the in vivo studies. Mice were maintained in a 12-hour light/12-hour dark cycle, and provided with sterilized water and food ad libitum at the Biological Resource Unit of the Cleveland Clinic Lerner Research Institute.
Chemicals and Reagents
MK-8931 was purchased from Selleckchem (S8173) and Medkoo (331024), dissolved at a concentration of 100 mg/mL in dimethyl sulfoxide (DMSO, Santa Cruz, sc-358801) as a stock solution, and stored at -20ºC until use. D-Luciferin was bought from GoldBio (LUCK-10G), prepared at a concentration of 15 mg/mL in sterile water as a stock solution, and stored at -20ºC until use. Etoposide (sc-3512B) was from Santa Cruz, prepared at a concentration of 1 mM in DSMO, stored at -20ºC until use. 32% Paraformaldehyde (PFA, 15714) was from Electron Microscopy Sciences and diluted to 4% with PBS before use. Protease (04693159001) and phosphatase inhibitors (04906837001) tablets were from Roche. Recombinant Human SCF (300-07), VEGF (100-20), IL4 (200-04), IL6 (200-06), IL10 (200-10), sIL6R (200-06RC), and TGFβ (200-21) were from Peprotech. Recombinant Human M-CSF (574806) and IL3 (578006) were from Biolegend. Recombinant Human bFGF was from R&D Systems (4114-TC-01M). Recombinant Human EGF was from GoldBio (1150-04-100). According to the manufacturer’s instructions, all the recombinant proteins were prepared at a 1,000× concentration as stock solutions and stored at -80ºC until use. All other chemicals and reagents were purchased from Sigma-Aldrich.
Derivation of Monocytes and Macrophages from Human iPS Cells (iPSCs)
Human iPSC-derived monocytes and macrophages were prepared in accordance with an established protocol11,84. To generate GFP+ iPSCs, human iPSCs were seeded in a 10-cm tissue culture dish and transduced with GFP through lentiviral infection for 12 hours. Two days post infection, GFP+ iPSCs were selected and maintained in the mTeSR1 medium (Lonza) for differentiation to monocytes and macrophages. To induce the formation of embryoid bodies (EBs), GFP+ iPSCs were seeded on an ultra-low attachment plate (Costar, 7007) in 100 μL of mTeSRTM1 medium supplemented with BMP4 (50 ng/mL, Abcam, ab87063), SCF (20 ng/mL, Peprotech, 300-07), Y27632 (50 μM, SellckChem, S1049), and VEGF (20 ng/mL, Peprotech, 100-20). The 96-well ultra-low attachment plate was centrifuged at 800 rpm for three minutes and the plate was placed into an incubator and left for four days. At day 2, 50 μL of culture medium in the well was aspirated and replaced by 50 μL of fresh mTeSRTM1 medium containing the above inducers. To derive monocytes, ten EBs were transferred to each well of a six-well tissue culture plate and cultured in X-VIVOTM 15 medium (Lonza, 04-418Q) supplemented with IL3 (25 ng/mL, Biolegend, 578006), M-CSF (100 ng/mL, Biolegend, 574806), glutamine (2 mM, ThermoFisher, 35050061), and β-mercaptoethanol (0.055 M, ThermoFisher, 21985023) for two weeks. The medium was changed every five days. Once monocytes were visible in the supernatant of the cultures, non-adherent monocytes were harvested. To produce macrophages, the iPSC-derived monocytes (1.5 × 105) were plated in each well of 6-well plates and cultured in X-VIVOTM 15 medium with M-CSF (100 ng/mL, Biolegend, 574806) for six days. To generate M2-like macrophages, the iPSC-derived monocytes were seeded in each well of 6-well plates and primed by treatment with phorbol 12-myristate 13-acetate (PMA, 5 nM) for two days to produce M0 macrophages, which were further treated with IL4 (20 ng/mL, Peprotech, 200-04), IL10 (20 ng/mL, Peprotech, 200-10), and TGFβ (20 ng/mL, Peprotech, 200-21) for three days to generate M2-like macrophages.
Preparation of Bone Marrow-derived Monocytes (BMDMs)
BMDMs were isolated and cultured according to an established protocol81. In brief, mouse bone marrow cells were collected by flushing the femurs and tibias with sterile PBS and then treated by red blood cell lysis buffer to remove red blood cells. The cells were re-suspended and cultured in RPMI 1640 medium with 10% FBS and M-CSF (100 ng/mL) for seven days to differentiate into BMDMs.
Identification of Potential Drugs to Activate Macrophage Phagocytosis
To screen for potential small molecules that activate phagocytosis of iPSC-derived macrophages against cancer cells, GFP+ iPSC-derived macrophages (5 × 104 cells) were seeded in each well of 24-well plates and treated with small molecules from an inhibitor library (SellckChem) or existing drugs that had been used in clinical trials for other diseases for two days. After washing, the cells were maintained in RPMI 1640 medium for two hours. Next, tdTomato-expressing glioma stem cells (GSCs: CCF-3264) were added to each well and co-incubated with the macrophages in RPMI 1640 medium with 10% FBS for another two hours. After incubation, the co-cultures were washed three times with warm RPMI 1640 medium to remove free cancer cells, and fluorescent and phase images were captured with a fluorescent microscope. Phagocytosis was detected and measured as inclusion bodies of cancer cells (in red) within macrophages (in green) according to a published study39.
To detect the MK-8931-activated phagocytosis of bone marrow-derived macrophages (BMDMs) against cancer cells, BMDMs were treated with DMSO (Control) or MK-8931 (50 μg/mL) for two days and pre-stained with CellTracker™ Green CMFDA Dye (1 μM, ThermoFisher, C2925). After washing, the BMDMs were maintained in RPMI 1640 medium for two hours. Next, tdTomato-expressing CCF-3264 GSCs (2 × 105 cells) were added to each well and co-incubated with the labelled BMDMs in RPMI 1640 medium with 10% FBS for another two hours. After incubation, the co-cultures were washed several times with warm RPMI 1640 medium to remove free cancer cells, and the fluorescent images were captured with a fluorescent microscope.
Plasmids for Overexpression or Knockdown
To construct the pCDH-tdTomato vector, full-length tdTomato was amplified from the pCDH-EF1-Luc2-P2A-tdTomato (Addgene, 72486) vector with the following primers: Forward: 5'-GCT AGC CCA ATC ATT TAA ATA TAA CTT-3', Reverse: 5'-GCG GCC GCT TAC TTG TAC AGC TCG TCC ATG C-3', and then cloned into the pCDH-CMV-MCS-EF1α-Neo vector at the Nhe1 and Not1 sites. The sequence of inserted tdTomato was confirmed by DNA sequencing. The constitutively active STAT3 (STAT3-C-Flag) and pCDH-luciferase constructs were generated in our lab10,73. ShRNAs against BACE1 (shBACE1) or a non-targeting sequence (shNT) were purchased from Sigma-Aldrich. The lentivirus packaging vectors (ps-PAX2 and pCI-VSVG) were from Addgene.
Production of Lentiviruses
Lentiviruses for expression of shRNAs (shBACE1 or shNT) or overexpression of an ectopic protein (STAT3-C) were produced in 293FT cells and prepared as previously described11,72,78,83. Briefly, 293FT cells (Clontech, 632180) were co-transduced with targeting plasmids and packaging vectors pCI-VSVG (Addgene, 1733) and ps-PAX2 (Addgene, 12260) by using calcium phosphate. Four days after transfection, the supernatants were harvested and virus titers were determined as described previously73. For infection, cells were treated with lentivirus at a multiplicity of infection (MOI) of 1.
Generation of Stable Cell Lines
To generate tdTomato-expressing stable glioma cells, GSCs (CCF-3264) were transduced with tdTomato by means of lentiviral infection for 12 hours. Two days post-infection, the cells were treated with neomycin (500 μg/mL, Santa Cruz, sc-29065A) for seven days to select stable clones. The expression of tdTomato was confirmed by fluorescence microscopy.
To generate luciferase-expressing stable glioma cells, GSCs (CCF-3264 or CCF-DI315) were transduced with firefly luciferase by means of lentiviral infection for 12 hours. Two days post-infection, the cells were treated with puromycin (2 μg/mL, Fisher Scientific, BP2956100) for seven days to select stable clones. The luciferase activity was confirmed by using the Luciferase Assay System (Promega, E1500).
To establish stable U937 cells expressing shBACE1 or shNT, the cells were transduced with shBACE1 or shNT by means of lentiviral infection. Two days post-infection, the cells were treated with puromycin (2 μg/mL) for seven days to select stable clones. Immunoblotting was used to test the knockdown efficacy of BACE1 in the U937 stable cells.
Derivation of pTAM-like M2 Macrophages from U937 Cells
U937-derived pTAM-like M2 macrophages were prepared according to an established protocol11,12. In brief, cells grown in a 10-cm tissue culture dish were primed with PMA (5 nM) for two days to produce M0 macrophages, which were further induced by IL4 (20 ng/mL, Peprotech, 200-04), IL10 (20 ng/mL, Peprotech, 200-10), and TGFβ (20 ng/mL, Peprotech, 200-21) for three days to generate pTAM-like M2 macrophages.
RNA Isolation and RT-PCR
Total RNA was isolated from cells by using the PureLinkTM RNA Kit (ThermoFisher, 12183020) and reverse-transcribed into cDNA with M-MLV reverse transcriptase (Promega, PR-M1701). Real-time PCR (qPCR) was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems) by using the SYBR-green qPCR Kit (Alkali Scientific, QS2050). Expression values were normalized to GAPDH. Gene-specific primers includes: BACE1 (forward): 5'-GCA GGG CTA CTA CGT GGA GA-3', BACE1 (reverse): 5'-GTA TCC ACC AGG ATG TTG AGC-3'; GAPDH (forward): 5'-AAG GTG AAG GTC GGA GTC AA C-3', GAPDH (reverse) 5'-GGG GTC ATT GAT GGC AAC AAT A-3'.
Establishment of Glioblastoma Xenografts (PDXs) and Drug Treatment in vivo
To establish xenografts for in vivo studies, intracranial transplantation of GSCs into the brains of NSG mice was performed as described previously10-12,22,83. Briefly, GSCs (CCF-3264 or CCF-DI315) expressing luciferase were injected into the right cerebral cortex at a depth of 3.5 mm. For the co-transplantation experiments, human glioma stem cells (GSCs, CCF-DI315) expressing luciferase in combination with or without the monocyte-derived macrophages expressing shBACE1 (shBACE1: #1 or #2) or non-targeting control (shNT) were co-injected into NSG mouse brains through intracranial injection (GSCs:Macrophages = 1:2). In vivo bioluminescent imaging (IVIS) was performed twice per week to monitor the tumor growth, using the Spectrum CT Imaging System (PerkinElmer), before and after treatment. For drug treatment, a stock solution of MK-8931 at 100 mg/mL in DMSO was diluted in 0.5% (w/v) methylcellulose (Sigma-Aldrich, M0512) to 6 mg/mL34. Mice bearing the xenografts were treated with MK-8931 (30 mg/kg) or the control (DMSO) once daily by oral gavage for two weeks or until the humane endpoint was reached. To collect mouse brains bearing tumors, cardiac perfusion with PBS and 4% PFA (Electron Microscopy Sciences, 15714) was performed. The brains were fixed and sectioned for further immunofluorescent, histochemical and histological analyses.
Irradiation on Intracranial GBM Xenografts (PDXs)
Irradiation (IR) was performed with a Pantek X-ray irradiator (once per week) at a low dose (2 Gy). To protect the mice and limit the side effects of irradiation, anesthetized mice were covered with a lead plate and only the tumor implantation sites were exposed to the fractioned radiation. Mice were sacrificed at the indicated time points or upon the appearance of neurological signs. Mouse brains bearing the tumors were collected for further analyses through cardiac perfusion with PBS and 4% PFA (Electron Microscopy Sciences, 15714). Only animals who suffered accidental death (for example, due to infection or intracranial injection) were excluded from the data analysis.
Immunofluorescent Analysis
Immunofluorescent staining of tumor tissues or cells was performed as described in our previous publications10-12,22,83. In brief, tumor sections or cells were fixed with 4% PFA for ten minutes, washed three times with cold PBS for five minutes each, permeabilized by 0.5 % (v/v) triton X-100 (Bio-Rad, 1610407) for ten minutes, and blocked with 3% (w/v) BSA (Sigma-Aldrich, A7906) in PBS for one hour at room temperature. For Ki67 and pSTAT3 staining, antigen retrieval was performed by incubating the sections in boiled antigen retrieval buffer (Vector Laboratories, H-3300) for 15 minutes before permeabilization. Primary antibodies were added to the sections or cells and incubated overnight at 4ºC. Primary antibodies used for immunofluorescence in this study were diluted as described below: anti-BACE1 (Abcam, ab2077, 1:50; Thermo Fisher Scientific, MA1-177, 1:50), anti-IBA1 (Abcam, ab5076, 1:200; Wako chemicals, 019-19741, 1:200), anti-CD11b (Bio-Rad, MCA711GT, 1:100), anti-FIZZ1 (Abcam, ab39626, 1:100), anti-CD163 (Santa Cruz, sc-33560 and sc-33715, 1:100), anti-ARG1 (BD Biosciences, 610708, 1:300), anti-HLA-DR (Biolegend, 307602, 1:100), anti-CD11c (BD Pharmingen, 558079, 1:100), anti-TRA-1-85 (RD System, MAB3195, 1:300), anti-CD31 (Dako, M082301, 1:100), anti-cleaved caspase 3 (Cell signaling, 9661, 1:100), anti-Ki67 (Abcam, ab15580, 1:100), anti-SOX2 (Bethyl laboratory, A301-739A, 1:200), and anti-pSTAT3 (Tyr705) (Cell signaling, 9131, 1:100). After the incubation of the primary antibodies, the sections or cells were washed three times with cold PBS for five minutes each and then incubated with the secondary antibodies for one hour at room temperature. The secondary antibodies used in this study included Alexa Fluor® 488 Donkey Anti-Mouse IgG (Invitrogen, A-21202, 1:200), Alexa Fluor® 568 Donkey Anti-Mouse IgG (Invitrogen, A-10037, 1:200), Alexa Fluor® 488 Donkey Anti-Rabbit IgG (Invitrogen, A-21206, 1:200), Alexa Fluor® 568 Donkey Anti-Rabbit IgG (Invitrogen, A-10042, 1:200), Alexa Fluor® 488 Donkey Anti-Goat IgG (Invitrogen, A-11055, 1:200), Alexa Fluor® 568 Donkey Anti-Goat IgG (Invitrogen, A-11057, 1:200), Alexa Fluor® 594 Donkey Anti-rat IgG (Invitrogen, A-21209, 1:200), Alexa Fluor® 568 Goat Anti-Armenian Hamster (Abcam, ab175716, 1:200), and Alexa Fluor® 488 Goat Anti-Rabbit (Invitrogen, A-11008, 1:200). After washing three times with cold PBS for five minutes each, the sections or cells were counterstained by DAPI (Cell Signaling, 4083, 1:5000) and sealed with mounting medium (Sigma-Aldrich, F4680). Finally, images were captured by a fluorescence microscopy (Leica DM4000) and further analyzed with ImageJ software (https://imagej.nih.gov/).
Immunoblot Analysis
Immunoblot analysis was performed as previously described10-12,22,83,85. Briefly, cells were lysed with RIPA buffer [50 mM TrisHCl (pH7.4), 150 mM NaCl, 2 mM EDTA, 1% (v/v) NP-40, 0.1% (w/v) SDS, protease inhibitor (one tablet per 10 mL of RIPA buffer, Roche)] for 20 minutes on ice. For the blots of phosphorylated protein, phosphatase inhibitor (one tablet per 10 mL RIPA buffer, Roche) was used. The cell lysates or conditioned medium (some experiments) were collected and subjected to SDS-PAGE and blotted onto PVDF membranes (ASI, XR730). After blockade with 5% (w/v) non-fat milk (RPI, M17200) in TBST, the membranes were incubated with primary antibodies overnight at 4°C. After the incubation of the first antibodies, the membranes were washed three times with TBST for ten minutes each. Then, the membranes were incubated with the second HRP-linked antibodies in the 5% milk for one hour at room temperature. The second HRP-linked antibodies were anti-mouse IgG (Cell signaling, 7076, 1:5000), anti-rabbit IgG (Cell signaling, 7074, 1:5000), and anti-goat IgG (Santa Cruz, sc-2354, 1:5000). After washing three times with TBST for ten minutes each, signals on the membranes were developed in the ECL HRP substrates (Advansta, K-12045) and images were acquired by a molecular imager (Bio-Rad, Universal Hood II) and analyzed by the Image Lab software (Bio-Rad).
Statistical Analysis
All bar graphs represent mean ± SEM unless otherwise indicated. For the survival analysis and correlation between gene expressions in glioblastoma patients, the data were provide by TCGA and downloaded from GlioVis. For the survival analysis of glioblastoma patients, the patients were divided into Bace1high and Bace1low groups with GlioVis (http://gliovis.bioinfo.cnio.es/) and Kaplan-Meier survival curves were generated. The log-rank survival analysis was performed with GraphPad Prism 5 software (https://www.graphpad.com/) to compare significance among different groups. All quantitative data presented were mean ± SEM from at least 3 repeats or samples per data point. Experimental details such as number of animals or cells and experimental replication were provided in the figure legends. Data inclusion/exclusion criteria was not applied in this study. Significant differences were determined between two groups using the Student’s t test or among multiple groups using one-way ANOVA and statistical significance was set at p < 0.05.