Mice, cell lines and reagents
All mice used were 6-8 week old C57Bl/6 females (Jackson Laboratory, Bar Harbor, ME). All experimental protocols were approved by the animal ethic committee of the Lady Davis Institute of McGill University. The mouse T-cell lymphoma cell line EG.7 (EL4 cells transfected to express chicken ovalbumin), human medulloblastoma cell line Daoy, and the human myeloma cell line U266 were purchased from ATCC (VA, USA) and propagated according to manufacturer's instructions. The murine medulloblastoma cell line PS125 was established from medulloblastomas derived from the Smo/Smo transgenic mouse  and propagated in DMEM/F-12 supplemented with 10% FBS, 1% L-glutamine, 1% MEM non-essential amino acids, and 1% N-2 at 37°C in 5% CO2. Antibodies for CD19, CD44, CD45, CD73, CD105 and CD138 were purchased from BD Biosciences (San Diego, CA). Mouse recombinant CCL2 protein (CCL2 1-76), ELISA kits for mouse CCL2 and human IL6, anti-human CCR2 antibody, CCR2 primers, and Annexin-V/PI detection kits were purchased from R&D systems (Minneapolis, USA). Antibody for α-tubulin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for BAX, pSTAT3 and total STAT3 were purchased from Cell Signalling Technology (Danvers, MA). RNA extraction kit was purchased from Qiagen (Mississauga, ON, CANADA). Contigen was purchased from Bard Urological Division (Covington, GA, USA). The 5-76 variant (CCL2 5-76) of murine CCL2 was synthesized from Genecust (Dudelange, Luxembourg). The monocyte enrichment kit was purchased from StemCell Technologies (Vancouver, Canada).
Isolation and characterization of mouse mesenchymal stromal cells
Whole bone marrow was harvested by flushing femurs and tibias bones of female CCL2-/- C57BL/6 mice with DMEM. Collected cells were plated and cultured until the appearance of a homogeneous polyclonal population of mesenchymal stromal cells (MSC). The plasticity of isolated MSCs was tested as previously reported . For CCR2 expression on MSC, RT-PCR was performed on extracted RNA using purchased primers.
Engineering CCL2-/-MSC to express GMME1
We have previously demonstrated that wild-type MSCs could generate in a paracrine fashion truncated CCL2 (5-76) capable of antagonizing CCR2-expressing cells . Therefore, CCL2-/-MSCs were used in this study to avoid any confounding effects arising from endogenous MSC production of CCL2 and derivatives. The generation and concentration of green fluorescent protein (GFP) or GMME1 retroparticles using the bicistronic AP2 vector were generated as previously reported . The level of GMME1 expression was analyzed through the assessment of GFP by flow cytometry, while GMME1 secretion level in the harvested DMEM conditioned medium was quantified using a CCL2 ELISA kit. Alternatively, GMME1 protein was purified from the conditioned medium with an affinity column (Pierce Biotechnology, Rockford, USA, Kit # 26148) loaded with anti-mouse GM-CSF antibodies (R&D, Minneapolis, USA) by following the instruction described in the kit. The purified GMME1 protein was dialyzed with fresh DMEM medium, and concentrated for use.
To test the proliferative property of GMME1, the mouse lymphoma EG7 or human multiple myeloma U266 cell lines were plated at a density of 105 cells/well in a 96-well plate and treated with increasing concentrations of cytokines for 48 hours. The reaction was read at 570 nm after adding 20 μL of 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). For Apoptosis analysis, the mouse EG7 or human U266 cell lines were cultured for 48 hrs with equimolar concentrations (1.5 pmol) of CCL2 (1-76), CCL2 (5-76), or GMME1 then analyzed by PI/Annexin-V. WT and CCR2-/- monocytes were enriched to 90% purity using negative selection following the bone marrow flush of femurs and tibias. Purified cells were then cultured for 48 hrs in control or GMME1 supernatant. A cell-killing assay was also performed on two medulloblastoma cell lines PS125 (mouse-derived) and Daoy (human-derived), treated with or without GMME1 for 48 hrs and cell death measured by flow-cytometry using PI and Annexin-V. Alternatively, Daoy cells were also treated with GMME1 in conditioned medium or affinity-purified GMME1 protein, and the cell growth was assessed by MTT assay. Western blot was performed on the lysate derived from treated cell lines probed with anti-BAX antibodies, or anti-pSTAT3 or anti-STAT3 antibodies. IL-6 secretion by U266 was quantified with ELISA, following the different cytokine treatments. For signalling analysis, a sandwich ELISA for mouse/human STAT3 was performed.
Cancer induction and treatments
To study the locoregional effect of GMME1 on tumor development, 2 × 106 MSC-GFP were co-implanted with 106 EG7 cells subcutaneously (sc) in immunocompetent C57Bl/6. For systemic efficacy of the fusokine, 106 EG7 cells were injected sc in immunocompetent C57Bl/6 mice on one side, and an sc implant of contigen-embedded gene-engineered MSCs (2 × 106 cells per implant) was injected on the opposite flank as previously described . Tumor appearance and volume were assessed every 48 hrs. To investigate the levels of circulating GMME1 in treated mice, the sera were collected at week 3 post-implantation of the neo-organoid and screened by CCL2 ELISA to detect the CCL2 moiety of the fusokine according to manufacturer's instructions.
GMME1-induced apoptosis of primary myeloma cells from patients
Bone marrow aspirates from consenting myeloma patients (n = 5) were processed as previously described . The IRB protocol was approved by Emory University. White blood cells in the marrow were isolated with lymphocyte separation medium (Mediatech Inc., Manassas, VA), and stained with anti-human CD38 (PE), CD45 (APC-Cy7), CD138 (FITC) (BD Bioscience, San Jose, CA), or CCR2 (PerCP) (Biolegend, San Diego, CA) antibodies or isotype controls (1:100 dilution). CD38+CD45-CD138+ cells were considered as myeloma cells . Alternatively, white blood cells (106 cells/ml) were cultured in RPMI 1640 medium with 10% FBS in presence of GMME1 (20 ng/ml). Condition medium without GMME1 served as control. After 48-hour culture, the cells were harvested and stained with anti-human CD38, CD45 antibodies, and Annexin. FACS analysis was performed on a BD FACSCanto II flow cytometer. Data were analyzed with FlowJo 9.1 software.
P values were calculated using the ANOVA test, or paired t-test in Figure Six F.