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Replication Study: Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET

  1. Jeewon Kim
  2. Amirali Afshari
  3. Ranjita Sengupta
  4. Vittorio Sebastiano
  5. Archana Gupta
  6. Young H Kim
  7. Reproducibility Project: Cancer Biology  Is a corresponding author
  1. Knockout and Tumor Model Center, Stanford Cancer Institute, United States
  2. System Biosciences LLC, United States
  3. The Institute for Stem Cell Biology and Regenerative Medicine, United States
  4. Stanford School of Medicine, United States
Replication Study
Cite this article as: eLife 2018;7:e39944 doi: 10.7554/eLife.39944
3 figures, 2 tables, 1 data set and 2 additional files

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 antibodies specific for total Met (top panel) and Gapdh (bottom panel). Membranes were cut at ~75 kDa so that Met and Gapdh could be probed in parallel. Repeat indicates the number of independently isolated exosome and cell lysate preparations from the same batch of infected cells. The fourth lane, labeled ‘Cells’ are lysate from B16-F10 cells expressing shScr. (B) Representative Western blots of exosomes and B16-F10 cells expressing the indicated shRNA were probed with antibodies specific for phosphorylated (Tyr 1234/1235) Met (pMet) (top panel) and Gapdh (bottom panel). Membranes were cut at ~75 kDa so that pMet and Gapdh could be probed in parallel. Repeat indicates the number of independently isolated exosome preparations from the same batch of infected cells. (C) Western blot bands were quantified for cells. Met or pMet levels were normalized to Gapdh, and protein expression presented relative to shScr conditions. Expression level of shScr condition was assigned a value of 1. Means reported and error bars represent SD. Results are from 3 independent biological repeats for Met expression and 4 independent biological repeats for pMet expression. Exploratory analysis: one-sample t-test on Met levels (Met/Gapdh) in shMet cells compared to a constant of 1 (shScr cells): t(2) = 8.41, p = 0.014, Bonferroni corrected p = 0.028, Cohen’s d = 4.85, 95% CI [0.55, 9.43]; one-sample t-test on pMet levels (Met/Gapdh) in shMet cells compared to a constant of 1 (shScr cells): t(3) = 8.94, p = 0.003, Bonferroni corrected p = 0.006, Cohen’s d = 4.47, 95% CI [1.02, 8.01]. (D) Representative Western blot of exosomes isolated from cells expressing the indicated shRNA probed with exosome markers Hsc70, Tsg101, and Cd63 specific antibodies. Experiment performed on 3 independent biological repeats for each condition from the same batch of infected cells. Additional details for this experiment can be found at https://osf.io/aqm2m/.

https://doi.org/10.7554/eLife.39944.002
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 cell line. For each MOI tested, fold change in shMet expression in shMet cells relative to shScr cells was determined. Expression level of shScr cells was assigned a value of 1, which is indicated by the dashed line. Means reported from one biological repeat. (B) Relative expression levels of Met (normalized to Gapdh) was determined by qRT-PCR for each cell line. For each MOI tested, fold change in Met expression in shMet cells relative to shScr cells was determined. Expression levels of shScr cells was assigned a value of 1, which is indicated by the dashed line. Means reported from one biological repeat. (C) Western blots using anti-Met (top panel) and anti-Gapdh (bottom panel) antibodies. Membranes were cut at ~75 kDa so that Met and Gapdh could be probed in parallel. Repeat indicates the number of independently isolated cell lysate preparations from the same batch of infected cells. Additional details for this experiment can be found at https://osf.io/aqm2m/.

https://doi.org/10.7554/eLife.39944.003
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) Following primary tumor detection caliper measurements were taken 3 times a week and used to calculate tumor volume. Line graph of tumor volume (y-axis is natural log scale) with means reported and error bars representing s.e.m. Number of mice monitored per group: synthetic liposomes = 7, shScr = 7, shMet = 7. Of note, not all mice had detectable primary tumors at the first measurement, one of the mice injected with synthetic liposomes never formed a detectable primary tumor despite the presence of metastatic burden, and two mice (one injected with B16-F19 shScr exosomes and one injected with synthetic liposomes) were found dead before the end of the study (day 17). Individual mouse tumor volume data is reported in Figure 2 - figure supplement 1. (B) At the end of the experiment (day 18), primary tumors were excised and weighed. Dot plot with means reported as crossbars and error bars represent s.e.m. Number of primary tumor weights per group: synthetic liposomes = 5, shScr = 6, shMet = 7. Kruskal-Wallis test on all three groups: H(2) = 2.85, p = 0.24. Planned Wilcoxon-Mann-Whitney comparison between shScr and synthetic liposomes: U = 22, p = 0.247, Cliff’s d = 0.47, 95% CI [-0.28, 0.86]. Planned Wilcoxon-Mann-Whitney comparison between shScr and shMet: U = 32, p = 0.138, Cliff’s d = 0.52, 95% CI [-0.10, 0.85]. (C) Metastatic burden in lungs quantified by luciferin photon flux at 18 days after B16-F10-luc tumor cell injections. Box and whisker plot (y-axis is natural log scale) with median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Individual data points represented as dots. Number of mice per group: synthetic liposomes = 6, shScr = 6, shMet = 7. One-way ANOVA on the luciferin photon flux values (natural log-transformed); F(2,16) = 0.226, uncorrected p = 0.800 with a priori alpha level of 0.025, Bonferroni corrected p > 0.99. Planned contrast between shScr and synthetic liposomes; Fisher’s LSD test; t(16) = 0.543, p = 0.594 with a priori alpha level of 0.025, Bonferroni corrected p > 0.99, Cohen’s d = 0.31, 95% CI [-0.83, 1.45]. Planned contrast between shScr and shMet; Fisher’s LSD test; t(16) = 0.620, p = 0.544 with a priori alpha level of 0.025, Bonferroni corrected p > 0.99, Cohen’s d = 0.34, 95% CI [-0.76, 1.44]. (D) Metastatic burden in femurs quantified by luciferin photon flux at 18 days after B16-F10-luc tumor cell injections. Box and whisker plot (y-axis is natural log scale) with median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Individual data points represented as dots. Number of mice per group: synthetic liposomes = 6, shScr = 6, shMet = 7. One-way ANOVA on the luciferin photon flux values (natural log-transformed); F(2,16) = 0.190, uncorrected p = 0.829 with a priori alpha level of 0.025, Bonferroni corrected p > 0.99. Planned contrast between shScr and synthetic liposomes; Fisher’s LSD test; t(16) = 0.573, p = 0.575 with a priori alpha level of 0.025, Bonferroni corrected p > 0.99, Cohen’s d = 0.33, 95% CI [-0.82, 1.46]. Planned contrast between shScr and shMet; Fisher’s LSD test; t(16) = 0.105, p = 0.918 with a priori alpha level of 0.025, Bonferroni corrected p > 0.99, Cohen’s d = 0.06, 95% CI [-1.03, 1.15]. Additional details for this experiment can be found at https://osf.io/mzywk/.

https://doi.org/10.7554/eLife.39944.005
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) Line graph of primary tumor volume data plotted on a linear scale with means reported and error bars representing s.e.m. Number of mice same as Figure 2A. (E) Lung metastatic burden data or (F) femur metastatic burden data with means reported and error bars representing s.e.m. Number of mice same as Figure 2C,D. Of note, although the data are not normal on a linear scale, which means presenting mean and s.e.m. is not appropriate, these box plots are presented to allow a direct comparison to how the original study data were presented. Additional details for this experiment can be found at https://osf.io/mzywk/.

https://doi.org/10.7554/eLife.39944.006
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 standardized measure of how often a value in one group is larger than the values from another group, with a larger positive value indicating a decrease in metastatic burden in animals injected with either shMet exosomes or liposomes compared to animals injected with shScr exosomes. Random effects meta-analysis: lung metastatic burden (shScr v. liposome, p = 0.149; shScr v. shMet, p = 0.226) and femur metastatic burden (shScr v. liposome, p = 0.264; shScr v. shMet, p = 0.317). Sample sizes used in Peinado et al., 2012 and this replication attempt are reported under the study name. Additional details for these meta-analyses can be found at https://osf.io/c69jx/.

https://doi.org/10.7554/eLife.39944.007

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, concentration (particles/ml)) are given as averages for preps generated during this study (n = 15 per cell line).

https://doi.org/10.7554/eLife.39944.004
MeanMedianSdSpanParticles/mL
shMetFTLA size distribution91.0772.962.891.2536.253e+11
size distribution917565.441.3346.253e+11
shScrFTLA size distribution88.4771.856.041.2119.477e+11
size distribution88.6773.6761.81.3219.477e+11
Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain
background
(Mus musculus,
C57BL/6, female)
C57BL/6Charles RiverStrain code:
027; RRID:
IMSR_CRL:27
Cell line
(M. musculus)
B16-F10doi:10.1038/nm.2753RRID:
CVCL_0159
Cell line
(M. musculus)
B16-F10-lucdoi:10.1038/nm.2753expresses
firefly
luciferase
Othersynthetic
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 Sciencescat# RHS4348sense 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’
Antibodymouse
anti-Hsc70
Enzo Life
Sciences
cat#
ALX-804–067; RRID:
AB_10538284
1:500 dilution in
SuperBlock, O/N at 4 ˚C
Antibodymouse
anti-Tsg101
Santa Cruz
Biotechnology
cat# sc-7964; RRID:
AB_671392
1:500 dilution
in SuperBlock,
O/N at 4 ˚C
Antibodyrabbit
anti-Cd63
System
Biosciences
cat#
EXOAB-CD63A-1;
RRID:AB_2561274
1:1000 dilution in
SuperBlock, O/N at 4 ˚C
Antibodymouse anti-MetCell Signaling
Technology
cat# 3127;
RRID:AB_331361
1:1000 dilution in
SuperBlock, O/N at 4 ˚C
Antibodyrabbit anti-pMet
(Tyr 1234/5)
Cell Signaling
Technology
cat# 3077;
RRID:AB_2315156
1:1000 dilution in
SuperBlock, O/N at 4 ˚C
Antibodyrabbit anti-GapdhSanta Cruz
Biotechnology
cat# sc-25778;
RRID:AB_10167668
1:1000 dilution in
SuperBlock, O/N at 4 ˚C
Antibodyrabbit anti-pMet
(Tyr 1234/5)
Thermo Fisher
Scientific
cat# MA5-15083;
RRID:AB_10983015
1:1000 dilution
in SuperBlock,
O/N at 4 ˚C
AntibodyHRP-conjugated
sheep anti-mouse
GE Healthcarecat# NA931;
RRID:AB_772210
1:15,000 dilution in
SuperBlock, 1 hr at RT
AntibodyHRP-conjugated donkey anti-rabbitGE Healthcarecat# NA934;
RRID:AB_772206
1:15,000 dilution in
SuperBlock, 1 hr at RT
Sequence-based
reagent
shMet primerthis paperForward: 5’-CCAGACTTT
TCATACAAGAATA-3’
Sequence-based
reagent
universal
reverse primer
QuantiMir Kit,
System Biosciences
cat# RA420A-1
Sequence-based reagentU6 primerLncRNA Profiler
qPCR Array Kit,
System Biosciences
cat# RA900A-1
Sequence-based
reagent
Met primerthis paperForward: 5’-CTGGACA
GATTGTGGGAGTAAG −3’
Sequence-based
reagent
Gapdh primerLncRNA Profiler qPCR Array Kit, System Biosciencescat# RA900A-1
Software,
algorithm
ImageJdoi:10.1038/nmeth.2089RRID:SCR_003070version 1.49 u
Software,
algorithm
SDSApplied
Biosystems
RRID:SCR_015806version 2.4
Software,
algorithm
Nanoparticle
tracking Analysis
(NTA)
NanosightRRID:SCR_014239version 2.3
Software,
algorithm
Living ImageCaliper Life SciencesRRID:SCR_014247version 4.4
Software,
algorithm
R Project for
statistical
computing
https://www.r-project.orgRRID:SCR_001905version 3.5.1

Data availability

Additional detailed experimental notes, data, and analysis are available on OSF (RRID:SCR_003238) (https://osf.io/ewqzf/; Kim et al., 2018). This includes the R Markdown file (https://osf.io/hz3k7/) that was used to compose this manuscript, which is a reproducible document linking the results in the article directly to the data and code that produced them (Hartgerink, 2017).

The following data sets were generated
  1. 1

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