Replication Study: Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET
- Knockout and Tumor Model Center, Stanford Cancer Institute, United States
- System Biosciences LLC, United States
- The Institute for Stem Cell Biology and Regenerative Medicine, United States
- Stanford School of Medicine, United States
Figures
Figure 1 with 1 supplement
Characterization of shMet B16-F10 cells and exosomes.
B16-F10 cells engineered to express shScr or shMet were used to purify exosomes. (A) Representative Western blots of exosomes and B16-F10 cells expressing the indicated shRNA were probed with …
Figure 1—figure supplement 1
Multiplicity of infection (MOI) ratios tested for stable cell line generation.
B16-F10 cells infected with various MOI ratios were characterized for shMet, Met, and Met expression. (A) Relative expression levels of shMet (normalized to U6) was determined by qRT-PCR for each …
Figure 2 with 1 supplement
Primary tumor growth and metastatic burden of mice injected with exosomes.
Female C57BL/6 mice were subcutaneously injected with B16-F10-luciferase cells after 4 weeks of intravenous injections (3 times a week) of shScr exosomes, shMet exosomes, or synthetic liposomes. (A) …
Figure 2—figure supplement 1
Alternative visualizations of tumor growth and metastatic burden.
This is the same experiment as Figure 2. (A-C) Line graphs (y-axis is natural log scale) of primary tumor volume plotted for each animal rather than averages. Number of mice same as Figure 2A. (D) …
Figure 3
Meta-analyses of each effect.
Effect size (Cliff’s delta) and 95% confidence interval are presented for Peinado et al., 2012, this replication attempt (RP:CB), and a meta-analysis to combine the two effects. Cliff’s delta is a …
Tables
Table 1
Nanosight analysis of exosomes.
Summary of exosome number and size distribution, with or without the finite track length adjustment (FTLA) algorithm, quantified by NanoSight analysis. All values (mean, SD, median, span, …
| Mean | Median | Sd | Span | Particles/mL | ||
|---|---|---|---|---|---|---|
| shMet | FTLA size distribution | 91.07 | 72.9 | 62.89 | 1.253 | 6.253e+11 |
| size distribution | 91 | 75 | 65.44 | 1.334 | 6.253e+11 | |
| shScr | FTLA size distribution | 88.47 | 71.8 | 56.04 | 1.211 | 9.477e+11 |
| size distribution | 88.67 | 73.67 | 61.8 | 1.321 | 9.477e+11 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus, C57BL/6, female) | C57BL/6 | Charles River | Strain code: 027; RRID: IMSR_CRL:27 | |
| Cell line (M. musculus) | B16-F10 | doi:10.1038/nm.2753 | RRID: CVCL_0159 | |
| Cell line (M. musculus) | B16-F10-luc | doi:10.1038/nm.2753 | expresses firefly luciferase | |
| Other | synthetic uniloamellar 100 nm liposomes | Encapsula NanoSciences | 13 mg/ml L-a-Phosplatydilcholine, 2.78 mg/ml cholesterol (7:3 molar ratio P:C) | |
| Sequence-based reagent | pGIPZ non-silencing shRNA lentiviral control particles | Dharmacon/GE Life Sciences | cat# RHS4348 | sense sequence: 5’-ATCTCGCTTGGGCGAGAGTAAG-3’ |
| Sequence-based reagent | pGIPZ mouse Met shRNA lentiviral particles | Dharmacon/ GE Life Sciences | cat# VGM5520- 200377256; clone ID: V3LMM_ 456078 | sense sequence: 5’-CCAGACTTTTCATACAAGA-3’ |
| Antibody | mouse anti-Hsc70 | Enzo Life Sciences | cat# ALX-804–067; RRID: AB_10538284 | 1:500 dilution in SuperBlock, O/N at 4 ˚C |
| Antibody | mouse anti-Tsg101 | Santa Cruz Biotechnology | cat# sc-7964; RRID: AB_671392 | 1:500 dilution in SuperBlock, O/N at 4 ˚C |
| Antibody | rabbit anti-Cd63 | System Biosciences | cat# EXOAB-CD63A-1; RRID:AB_2561274 | 1:1000 dilution in SuperBlock, O/N at 4 ˚C |
| Antibody | mouse anti-Met | Cell Signaling Technology | cat# 3127; RRID:AB_331361 | 1:1000 dilution in SuperBlock, O/N at 4 ˚C |
| Antibody | rabbit anti-pMet (Tyr 1234/5) | Cell Signaling Technology | cat# 3077; RRID:AB_2315156 | 1:1000 dilution in SuperBlock, O/N at 4 ˚C |
| Antibody | rabbit anti-Gapdh | Santa Cruz Biotechnology | cat# sc-25778; RRID:AB_10167668 | 1:1000 dilution in SuperBlock, O/N at 4 ˚C |
| Antibody | rabbit anti-pMet (Tyr 1234/5) | Thermo Fisher Scientific | cat# MA5-15083; RRID:AB_10983015 | 1:1000 dilution in SuperBlock, O/N at 4 ˚C |
| Antibody | HRP-conjugated sheep anti-mouse | GE Healthcare | cat# NA931; RRID:AB_772210 | 1:15,000 dilution in SuperBlock, 1 hr at RT |
| Antibody | HRP-conjugated donkey anti-rabbit | GE Healthcare | cat# NA934; RRID:AB_772206 | 1:15,000 dilution in SuperBlock, 1 hr at RT |
| Sequence-based reagent | shMet primer | this paper | Forward: 5’-CCAGACTTT TCATACAAGAATA-3’ | |
| Sequence-based reagent | universal reverse primer | QuantiMir Kit, System Biosciences | cat# RA420A-1 | |
| Sequence-based reagent | U6 primer | LncRNA Profiler qPCR Array Kit, System Biosciences | cat# RA900A-1 | |
| Sequence-based reagent | Met primer | this paper | Forward: 5’-CTGGACA GATTGTGGGAGTAAG −3’ | |
| Sequence-based reagent | Gapdh primer | LncRNA Profiler qPCR Array Kit, System Biosciences | cat# RA900A-1 | |
| Software, algorithm | ImageJ | doi:10.1038/nmeth.2089 | RRID:SCR_003070 | version 1.49 u |
| Software, algorithm | SDS | Applied Biosystems | RRID:SCR_015806 | version 2.4 |
| Software, algorithm | Nanoparticle tracking Analysis (NTA) | Nanosight | RRID:SCR_014239 | version 2.3 |
| Software, algorithm | Living Image | Caliper Life Sciences | RRID:SCR_014247 | version 4.4 |
| Software, algorithm | R Project for statistical computing | https://www.r-project.org | RRID:SCR_001905 | version 3.5.1 |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.39944.008
-
Reporting standard 1
The ARRIVE guidelines checklist.
- https://doi.org/10.7554/eLife.39944.009
