Drugs
All drugs were available from commercial sources including: Phorbol 12-myristate 13-acetate (PMA) from Cayman Chemical Company (Ann Arbor, MI); recombinant human IL-10 cytokine (rhIL-10) and macrophage colony-stimulating factor (rhM-CSF) from Sino Biological (Beijing, China); Lenalidomide (LEN) and dexamethasone (DEX) from Selleck Chemicals (Houston, TX); Anti-human CD210 (IL-10R, clone 3F9) blocking antibody from BioLegend (San Diego, CA).
Ethics statement for human tissues and animal experiments
MM patient and healthy donor BM aspirates were obtained from Siteman Cancer Center at Washington University in St. Louis School of Medicine. Informed consent was obtained from all patients with an approval from the Washington University Medical School Institutional Review Board committee (protocol number: 201102270) and in accordance with the Declaration of Helsinki.
All animal studies were conducted according to guidelines established and approved by the Ethical Committee for Animal Experiments at Washington University in St. Louis School of Medicine.
Luminex multiplex cytokine screen
C57BL/KaLwRij (6–11 weeks old) immunocompetent mice were spontaneously generated and utilized for in vivo IL-10 measurement. Mice were injected with 5TGM1-GFP murine myeloma cells (1x106/mouse, i.v.) to establishing an aggressive form of MM, as previously described49. Naïve mice were used as controls (n = 5 MM-bearing, n = 5 naïve, age-matched). Mice were euthanized 28 days post tumor innoculation and BM was harvested and supernatants were collected.
BM supernatants were analyzed with Luminex immunoassay at the Bursky Center for Human Immunology and Immunotherapy Programs at Washington University. The bead assay consisted of a custom ThermoFisher Procartaplex 15-plex Panel having Th1/Th2 Cytokine 11-plex (EPX110-20820-901) beads with sRANKL (EPX01A-26037-901), IL-10 (EPX01A-20614-901), M-CSF (EPX01A-26039-901), and VEGF (EPX01A-20619-901) Simplex beads added to the beads mix. Samples were analyzed using a FLEXMAP3D machine (Luminex Corp, Austin, TX), and data was analyzed using Milliplex Analyst 5.1.0 software (EMD Millipore, Billerica, MA) using a 5-parameter log curve fit algorithm.
Cell culture
MM.1S cell line was purchased from the American Type Culture Collection (ATCC, Rockville, MD); THP-1 was kindly gifted from Dr. Lori Setton’s Lab (Washington University in St. Louis); MM.1S-CBR-GFP and THP-CBR-GFP cell lines were kindly gifted by Dr. John DiPersio’s Lab (Washington University School of Medicine). All cell lines were cultured with RPMI-1640 media (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies, Grand Island, NY), 1% L-Glutamine, and 1% Penicillin-Streptomycin (Corning, Tewksbury, MA). Cells were cultured at 37°C and in 5% CO2 in a NuAire water jacket incubator (NuAire, Plymouth, MN). Media were refreshed every 3–4 days.
Primary human samples
BM aspirates from healthy donors and MM patients were centrifuged and supernatant was collected; red blood cells were removed from BM mononuclear cells (BMMCs) using red blood cell lysis buffer (Invitrogen; Waltham, MA), as previously described50.
Primary monocytes were obtained from freshly isolated PBMCs, which were plated in MEMα medium supplemented with 10% FBS, 1% L-Glutamine, 1% Penicillin-Streptomycin, and 25ng/ml rhM-CSF; on tissue culture treated flasks. Non adherent cells were gently removed 2 days after initial plating, and the remaining monocytes were lifted from the flasks using 0.05% Trypsin and further used.
Human IL-10 cytokine level
IL-10 concentration in BM supernatant was performed using the MAX Human IL-10 enzyme-linked immunosorbent assay (ELISA) kit (BioLegend, San Diego, CA), according to manufacturer’s instructions. Absorbance was read at 450nm (signal) and 570nm (background) with SpectraMax i3 multimode microplate spectrophotometer (Molecular Devices, San Jose, CA). IL-10 level was determined based on standard curve ran in the same experiment.
Additionally, cytokine secretion by MM.1S cells was detected by Human Cytokine Antibody Array (Abcam, Cambridge, UK). Patient BM Negative Fraction (NF) were cultured with or without MM.1S for 2 days. The supernatants were collected, and undiluted samples were analyzed according to manufacturer’s protocol. Secreted cytokines were detected by cytokine array chemiluminescence, and signal intensities were analyzed by ImageJ, background corrected, and normalized against medium controls.
3DTEBM culture
3D-Tissue Engineered BM (3DTEBM) cultures were established and digested as previously described23, 24. Cell line or primary patient samples were subjected to 3DTEBM cultures, in the presence of antibody and/or drug treatments. The 3DTEBM scaffolds were supplemented with media on top and incubated at 37°C. At the end of the experiment, 3D scaffolds were digested and cells were retrieved, and subjected to flow cytometry analysis.
Patient and cell line macrophage M2 polarization
For studies involving patient BM or humanized mice BM, samples were first fixed and premetallized using the Fix/Perm Kit (BD, Franklin Lakes, NJ), then macrophages were identified with anti-human or mouse CD68-FITC. M1 phenotype was determined with CD80-APC and M2 phenotype with CD163-BV421. M2 polarization was represented as ratio of M1 and M2 mean fluorescence intensities.
For studies THP macrophages in vitro, THP macrophages were differentiated from THP-1-CBR-GFP monocyte cell line, differentiated in complete RPMI medium supplemented with 50ng/ml PMA in 6-well plates at 0.5x106 cells per well. THP macrophages were removed from wells with 0.25% Trypsin. For effect of MM on THP polarization, GFP + THP macrophages were co-cultured with or without human MM.1S cells in 3DTEBM for 3 days. For effect of IL-10 on THP polarization, GFP + THP macrophages were cultured with or without 100ng/ml human recombinant IL-10 in the 3DTEBM for 3 days. 3D gels were digested, cells were retrieved, and M2/M1 polarization was determined on GFP + THP cells with M1 and M2 markers described above.
In vivo macrophage M2 polarization
huCD34-NCG (strain 695, female, 21–31 weeks old, single donor) mice were purchased from Charles River Laboratories (Wilmington, MA). Mice (n = 3) were inoculated with MM.1S-CBR-GFP cells (2x106/mouse, i.v.) and allowed to grow for 3 weeks until sufficient tumor burden was detected by bioluminescence imaging, carried out with IVIS 50 bioluminescence imaging system (PerkinElmer, Waltham, MA) as described before51. Naïve mice without MM inoculation were used as control (n = 3). Mice were sacrificed, BM was flushed from femurs, and were stained for macrophages and their M1/M2 phenotype, as described above.
Effect of α-IL-10R antibody on macrophage repolarization in vitro and in vivo
Patient BM derived 3DTEBM cultures, or MM.1S co-culture with THP macrophages, were treated with or without anti-IL-10R antibody (5ug/ml) for 4 days. The resulting macrophage phenotype was determined as described above. Additionally, huCD34-NCG mice inoculated with human MM.1S were treated with or without anti-IL-10R antibody treatment (n = 3 for each; i.p., 100ug/mouse), twice a week for two weeks. Mice were sacrificed, BM was flushed from femurs, and were stained for macrophages and their phenotype, as described above.
Western blotting
For STAT3 signaling, THP-1 macrophages were stimulated with untreated control, rhIL-10 (at 10ng/ml or 100ng/ml) or U266 MM conditioned media (CM, at 50% or 100% CM). For conditions involving IL-10R inhibition, THP were pre-treated with anti-IL-10R antibody for 4 hours before stimulation. After 30min, media was washed off and THP cells were lifted for western blotting. Western blotting was performed, as previously described 44.
For proliferation and cell cycle signaling in MM cells, THP macrophages were pretreated with or without 10µg/ml of α-IL-10R for 4 hours, followed by addition of MM.1S (5x106 cells/well; final ratio THP1:MM was 1:5) for 24 hours. MM.1S cells were harvested for western blotting,
Blotting was performed for anti-pSTAT3 (Cat#9145), anti-pAKT (Cat#4060), anti-pS6R (Cat#4858), anti-pRB (Cat #9308), anti-rabbit IgG HRP secondary antibody (Cat#7074P2) (Cell Signaling Technology, MA), and bands were quantified using ImageJ Software and normalized to GAPDH.
Cell survival by flow cytometry
For proliferation and drug resistance experiments, MM.1S-CBR-GFP cells were cultured alone or co-cultured with THP macrophages in the 3DTEBM. Treatments were anti-IL-10R antibodies (5ug/ml), lenalidomide (1µM), or dexamethasone (1µM) supplemented on top of the 3DTEBM gels. After 4 days of treatment, 3D gels were digested, and cell survival was determined as number of GFP + MM cells, normalized by counting beads (Invitrogen, Carlsbad, CA).
In vivo efficacy
NSG-SGM3 (013062, female, 7 weeks old) were purchased from Jackson Laboratories (Bar Harbor, Maine). Mice were inoculated with MM.1S-CBR-GFP cells (i.v., 2x106/mouse), and 3 days post-inoculation, human monocytes were injected (i.v., 1x106/mouse). Treatments began at 14 days post-inoculation when tumors were sufficiently measurable.
For the first experiment, 14 mice were randomized to 2 groups (n = 7) and treated with (1) vehicle control or (2) α-IL-10R mAbs (i.p., 5mg/kg, twice/week), for a total of 21 days. In a second experiment, another 14 mice were randomized to 2 groups (n = 7) after sufficient tumor inoculation, and treated with (1) lenalidomide alone (oral, 5mg/kg, daily) or (2) lenalidomide and α-IL-10R mAb (i.p., 2.5mg/kg), for a total of 21 days.
For both experiments, tumor burden was tracked bi-weekly with bioluminescence imaging at day 14, day 28, and day 42, using IVIS-50 (PerkinElmer, Waltham, MA). Images were analyzed with Living Image program (PerkinElmer).
Statistical analysis
Experiments were carried out in quadruplicates and repeated at least three times. Statistical significance is determined by student’s t-test, unless otherwise stated. P-value < 0.05 represents statistically significant.