Cell lines: HMPOS cells were a kind gift from the Barroga and Fujinaga lab[22]. The original and derived cells, were tested for Mycoplasma prior, and durring the study. To confirm the authenticity of the cells, an Alkaline phosphatase assay was perfomed on all cell lines.
Generation of carboplatin resistant cell lines: The HMPOS cells were grown in media (RPMI 1640 and 10% FBS supplemented with 100 μg/mL Streptomycin and 100 units/mL Penicillin) and incubated at 37°C with 5% CO2[22]. The cells were treated with gradually increasing concentrations of carboplatin starting at 0.5 μM. Following a 72 hours incubation period, surviving cells were subsequently expanded and passaged at least once before the next treatment. If cells were at less than 70% confluency after 72 hours of treatment, the same dose was administered again for another 72 hours. If cells were at 70% confluency or greater, then the dose was increased one increment until a concentration of 10 μM was reached. The carboplatin-resistant HMPOS cells isolated at 0, 2.5 and 10 uM are referred to as HMPOS-S, HMPOS-2.5R, and HMPOS-10R, respectively.
Carboplatin proliferation assay (MTS): HMPOS cell lines were grown to confluence. Next, 10,000 cells from each cell line were plated onto a 96 well plate in triplicate in 200 ul of media. Cells were then incubated for 24 hours and subsequently serum starved for an additional 24 hours. The cells were then treated with media (vehicle control), 0.5, 1, 2.5, 4, 6, 8, and 10 μM carboplatin diluted in RPMI media for 72-hours. Next, 130 μL of the cell culture media from each well was collected and 50 μL of a mixture of RPMI 1640 + CellTiter 96™ AQueous One Solution Cell Proliferation Assay reagent, prepared per manufacturer’s instructions (Promega), was added to each well. Cells were incubated for 4 hours at 37°C. Wells were measured at 490 nm and the absorption was normalized to the control treatment.
Determination of generation time: HMPOS-S, -2.5R, and -10R cells were plated in 6-well plates at a density of 100,000 cells per well in triplicate. After a 48-hour incubation period cells were counted using a hemocytometer. The generation time of these cells was calculated according to the following formula where H = time elapsed between plating and counting, c2 = count of cells 48 hours after plating, and c1 = count of cells upon plating (100,000)[23]. The generation time values for each triplicate were averaged together and repeated for a total of three independent replicates.
Cell cycle phase analysis: Isolated cells were fixed in 70% EtOH overnight then 25 μL of RNase A (Thermo Scientific) and 10 μL of propidium iodide (final concentration 0.6 μg/mL) were added. Cells were interrogated using a Cytoflex FACS (BD Coulter). Software analysis was performed using the CytExpert software (BD Coulter).
Exosome isolation, quantification, and validation: HMPOS cell lines were grown as previously described[24]. Once cells reached 60-70% confluency, their media was changed to contain RPMI 1640, 100 μg/mL Streptomycin and 100 units/mL Penicillin, and 10% exosome depleted FBS (ThermoFisher) at a total volume of 15 mLs. Cells were grown for 24-48 hours, after which the media was collected and stored at -20°C. Pooled media was thawed at 37°C and spun at 10,000 rpm for 30 minutes on a high-speed centrifuge (Eppendorf 5804R, rotor F34-6-38). The supernatant was collected and transferred into 10.4 mL ultracentrifuge tubes (Beckman Coulter) and centrifuged at 40,000 rpm for 70 minutes (Beckman Coulter Optima XE-90 Ultracentrifuge, rotor type 70.1 Ti, fixed angle). After removing the supernatant, the exosome pellet was re-suspended in PBS and sterile filtered through a 0.22 μM filter (Millex-GV filter, 4 mm diameter, SLGV004SL). Exosome suspensions were aliquoted and diluted by a factor of 2.5 for quantification. These dilutions were quantified relative to their protein content.
Exosomes were characterized using the NanoSight NS500 instrument (NanoSight Ltd., Amesbury, United Kingdom, NTA version 3.0 0064). Light scattered by the particles under Brownian motion were captured for 60 seconds. Samples were diluted to obtain the concentration between 50 and 150 particles per frame. Each sample was injected into the chamber (temperature: 25 °C, viscosity of PBS 0.912 - 0.913 cP) and the setting of the system were adjusted as follows: camera level: 16, slider shutter: 1300, slider gain: 512, Frames Processed: 851, Frames per Second: 14.2, Blur size: Auto; Detection Threshold: 5). The three videos for each sample were analyzed to obtain the mean, mode, and number of particles per milliliter.
Exosomal modulation of carboplatin resistance: Carboplatin-sensitive HMPOS-S cells (10,000) were plated into a 96 well plate in triplicate and incubated for 24 hours at 37C. To synchronize the cells, cultures were incubated for 24 hours in FBS free media. Cells were then pre-treated with 10 μg of HMPOS-S isolated exosomes, HMPOS-2.5R isolated exosomes, HMPOS-10R isolated exosomes, or a PBS vehicle control for an additional 24 hours. Carboplatin was then added to the wells at concentrations of 0, 2.5, and 10 μM and cells were incubated for 72 hours. At the 24hr and 48hr timepoints, relative to when carboplatin was first added, fresh exosomes and carboplatin were added as described above. After 72 hours, cells were analyzed with the MTS reagent as previously described.
Mass spectrometry: Cell lysates from HMPOS cell lines were prepared following 10 cycles of rapid freeze/thaw. The crude lysate was purified using chloroform-methanol. Exosomes were isolated and quantified as previously described. A total of 50 μg of cell lysate and exosomes (relative to protein concentration) were taken to the OSU Mass Spectrometry Center for analysis. The proteins were digested by sequencing grade modified trypsin.
Peptide analysis was achieved using an Orbitrap Fusion Lumos mass spectrometer with a Nano ESI source (Thermo Scientific) coupled with a Waters nanoAcquity UPLC system (Waters, Milford, MA). The proteolytic products were desalted and loaded on a nano Acquity UPLC 2 G Trap Column (180 μm × 20 mm, 5 μm) for 5 min with solvent 0.1% formic acid in 3% ACN at a flow rate of 5 μL/min. A nanoAcquity UPLC RPeptide BEH C18 column (100 μm × 100 mm, 1.7 μm) was applied to separate peptides following by a 120-min gradient consisting of 0.1% formic acid in H2O (mobile phase A) and 0.1% formic acid in ACN (mobile phase B), where B was increased from 3% to 10% at 3 min, 10% ® 30% at 105 min, 30% ® 90% at 108 min and held 4 min, and then decreased to 3% at 113 min and held until 120 min. The LC flow rate was set at 500 nL/min. All mass spectral data were acquired in the positive ion mode. The spray voltage was 2400 V and the ion transfer tube temperature was 300 °C. MS and MS/MS spectra were acquired by the Orbitrap analyzer (resolution 120 K at m/z 200) and Ion Trap (collision induced dissociation CID) respectively. Automatic gain control target was set to 4.0 × 105 for precursor ions and 104 for product ions. Mass tolerances were set at ± 10 ppm for precursor ions and 0.6 Da for fragment ions.
Raw data were analyzed with Thermo Scientific Proteome Discoverer 2.2 software and searched initially against the Uniprot Canis database using Sequest HT as search engine. A maximum of two missed cleavage sites was allowed. Carbamidomethylation of cysteine and oxidation of methionine were specified as static modification and dynamic modification, respectively. The overall false discovery rate (FDR) at the protein level was less than 1%. To allow for GO annotation analysis, the datasets were also searched against the Uniprot Homo sapiens protein database. Canine and human database search results were submitted to a Basic Local Alignment Search Tool (Blastp) analysis in Uniprot website (https://www.uniprot.org/blast/). Only canine proteins with more than 99% amino acid sequence similarities with its human ortholog were retained.
The fold change (FC) of a given protein was defined as the ratio of abundance between two groups (ex. HMPOS-S vs HMPOS-10R). To calculate the FC of a given protein, each peptide group ratio was first calculated as the geometric median of all combinations of ratios from all the replicates in the same group. Secondly, the protein ratio was subsequently calculated as the geometric median of the peptide group ratios. Overall, the ratio for protein X reflects the ratio of abundance of protein X in HMPOS-S controls, relative to the abundance of protein X in other samples. The protein FC between the following groups was investigated: (1) HMPOS-S vs HMPOS-2.5R vs HMPOS-10 R cell lysate and (2) HMPOS-S vs HMPOS-2.5R vs HMPOS-10R exosomes.
Immunoblotting: Osteosarcoma HMPOS cells were isolated and lysed in RIPA buffer (Bio-Rad). Quantified lysate protein (10 ug) was mixed with LDS Sample Buffer (Invitrogen). and loaded on to pre-cast 4-12% Bis-Tris gels. Electrophoresis of gels was at 100V for 1-2 hours (Life Technologies, Eugene, OR). Proteins were transferred to nitrocellulose membranes and membranes blocked for one hour in Blocking Buffer (LI-COR, Lincoln, NE). Membranes were then incubated with the following: rabbit anti-canine non-phosphorylated (Active) β-catenin (Ser45) (Cell Signaling Technologies), rabbit anti-canine phospho-β-catenin (Ser33/37/Thr41) (Cell Signaling Technologies), and β-Actin Mouse Antibody (Santa Cruz Biotechnologies). After washing with buffer (TBS, 10% Tween), membranes were incubated with the following secondary antibodies: goat anti-mouse IgG-HRP (Santa Cruz Biotechnologies), and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnologies). Membranes were developed using the Supersignal West Femto Maximum Sensitivity Chemiluminescent Substrate (Thermo Fisher).
Patient sera collection : Patient sera was obtained from historical samples stored at the Oregon State University Biorepository. All samples collected to the biorepository at Oregon State University are in line with the Institutional Animal Care and Use Committee guidelines. Serum was taken prior to therapy from adult (>1 year) medium- and large-breed dogs (>20 kg) with pathology-verified appendicular osteosarcoma. All Patients underwent standard of care treatment which included an amputation of the affected limb, and carboplatin chemotherapy (270-300 mg/m2 administered every 3 weeks for a total of 4 doses).
Statistical Analyses: All statistical analysis was performed on GraphPad Prism Software. MTS assay validation of carboplatin resistance, generation time data, luminometry data, and quantitative RT-PCR data were all analyzed using a one-way ANOVA analysis, followed by Dunnett’s Multiple Comparisons test to analyze significance relative to appropriate controls. MTS assay data from treatment of sensitive HMPOS cells with carboplatin and exosomes was subjected to a two-way ANOVA analysis, followed by Bonferroni post-tests to compare all potential significant interactions. Statistical significance was assigned based on p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).
To generate graphical representations of the canine patient serum exosome data, protein abundance values were normalized to the sum of each column, and all values were subjected to a t-test. The heatmap rows are ordered by t-score between the sample groups. Red coloration represents higher expression relative to the average of the first five columns (the patient cohort that responded well to chemotherapy), and blue represents lowered expression.