FGF2-FGFR1 signaling regulates release of Leukemia-Protective exosomes from bone marrow stromal cells
- Jacqueline Martinez
- Isabel English
- Sunil K Joshi
- Renata Scopim-Ribiero
- David K Edwards V
- Anupriya Agarwal
- Danielle Jorgens
- Jeffrey W Tyner
- Brian J Druker
- Elie Traer
- Oregon Health & Science University, United States
- Howard Hughes Medical Institute, United States
Figures
Figure 1 with 3 supplements
Extracellular vesicles (ECVs) secreted by HS-5 cells are internalized by MOLM14 and K562 cells and protect from treatment with AC220 or imatinib, respectively.
HS-5 conditioned media (CM) was collected and separated by ultracentrifugation at 100,000 g into a supernatant (S100) and pellet (P100) fraction containing ECVs. These fractions were incubated with …
Figure 1—figure supplement 1
Comparison of protection from recombinant FGF2, HS-5 ECVs, and CM after ECV depletion (-ECV) in both K562 and MOLM14 cells.
HS-5 conditioned media (CM) was collected and separated by ultracentrifugation at 100,000 g into ECVs and CM without ECVs (-ECV). These fractions and RPMI media ± 10 ng/ml recombinant FGF2 were …
Figure 1—video 1
HS-5 ECVs were stained with DiI (red) and K562 cells were stained with DiO (green) and incubated for 24 hr at 37°C as described in Materials and methods.
Cells were washed, placed on Poly-D-Lysine coated chamber slides, and DAPI-stained. Z-stack imaging was performed on an Olympus IX71 inverted microscope.
Figure 1—video 2
HS-5 ECVs were stained with DiI (red) and MOLM14 cells were stained with DiO (green) and incubated for 24 hr at 37°C as described in Materials and methods.
Cells were washed, placed on Poly-D-Lysine coated chamber slides, and DAPI-stained. Z-stack imaging was performed on an Olympus IX71 inverted microscope.
Figure 2
FGF2 is enriched in exosomes from HS-5 bone marrow stromal cells.
(A) Immunoblot of FGFR1, FGF2 and actin in HS-5 and HS-27 whole cell lysates. (B) HS-5 and HS-27 CM were ultracentrifuged at 100,000 g for 2 hr at four degrees C. CM, soluble protein (S100), and ECV …
Figure 3 with 1 supplement
HS-5 cells secrete more exosomes than HS-27 cells.
Equal numbers of HS-5 and HS-27 cells were plated in RPMI with exosome-depleted FBS for 24 hr. The ECVs were pelleted by ultracentrifugation at 100,000 g for 2 hr at four degrees C and resuspended …
Figure 3—figure supplement 1
Methods for exosome quantification and further evaluation of microvesicle populations.
(A) CM fractionated by sucrose density gradient assessed for microvesicles by nanoparticle tracking analysis (Nanosight). (B) Fractions assessed by flow cytometry optimized for virus particles …
Figure 4 with 3 supplements
FGF2 is an autocrine growth factor in bone marrow stromal cells, and FGFR inhibition attenuates growth.
(A) HS-5 cells were cultured in media ± 250 nM PD173074 for one week and then equal numbers of cells were replated for comparison. After adhesion, the cells cultured in PD173074 were washed and …
Figure 4—figure supplement 1
Pre-treatment of HS-5 stromal cells with FGFR inhibitor reduces protective properties of HS-5 CM when added to K562 cells exposed to imatinib.
(A) HS-5 cells were cultured in media ± 250 nM PD173074 for one week and then equal numbers of cells were replated for comparison. After adhesion, the cells cultured in PD173074 were washed and …
Figure 4—figure supplement 2
Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2.
Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, …
Figure 4—figure supplement 3
Cultured primary human bone marrow stroma exhibits trilineage differentiation.
Primary mesenchymal stromal cells at passage four were differentiated into adipocytes, osteocytes, and chondrocytes using the Human Mesenchymal Stem Cell Functional Identification Kit (R and D …
Figure 5 with 2 supplements
FGFR inhibition decreases exosome production in FGF2-expressing stroma.
HS-5 cells were exposed to a gradient of the FGFR inhibitors (A) PD173074 and (B) BGJ-398 for 48 hr prior to collecting CM. ECVs were pelleted by ultracentrifugation at 100,000 g and quantified by …
Figure 5—figure supplement 1
FGFR inhibition reduces HS-5 cell exosome secretion.
(A) HS-5 cells were exposed to 500 nM PD173074 (PD) for 2, 4 and 6 hr before collection of CM and ECVs as previously described. ECVs were quantified by Virocyt. Results obtained in triplicate. Error …
Figure 5—figure supplement 2
Scanning electron microscopy of HS-5 cells shows altered membrane dynamics after FGFR inhibition.
Cells grown on coverslips were fixed with 2% paraformaldehyde and 1% glutaraldehyde (Ted Pella Inc, Redding, CA, USA) in phosphate buffered saline for at least one hour. Following three rinses in …
Figure 6 with 2 supplements
Genetic silencing of FGFR1 or deletion of FGF2 attenuates exosome secretion.
A doxycycline-inducible lentiviral shRNA targeting FGFR1 was used to create a stable HS-5 cell line. The cells were then treated with doxycycline to induce FGFR1 silencing and compared to a GIPZ …
Figure 6—figure supplement 1
Genetic silencing of FGFR1 by siRNA reduces exosome secretion and protection capacity of HS-5 stromal cells.
FGFR1 siRNA pool was purchased from Thermo Fisher Scientific Dharmacon RNAi Technologies (Waltham, MA, USA). HS-5 cells were transfected with siRNAs using Lipofectamine 2000 reagent purchased from …
Figure 6—figure supplement 2
Genetic silencing of FGFR1 by CRISP/CAS9 reduces exosome secretion and protection capacity of HS-5 stromal cells.
(A) FGFR1 and FGF2 genes were knocked out in HS-5 cells by lentiviral CRISPR-Cas9 genome editing. Each gene was targeted with two single guide RNA sequences (labeled 1 or 2). However, once FGF2 and …
Figure 7 with 3 supplements
Fgf2 -/- mice survive significantly longer with TKI therapy in a murine BCR-ABL leukemia model.
Fgf2 +/+ bone marrow was removed from donor mice and spinoculated with pMIG BCR-ABL retrovirus containing an IRES-GFP marker. The transfected bone marrow was then transplanted into lethally …
Figure 7—figure supplement 1
Fgf2 +/+ and -/- mice demonstrate engraftment of leukemia by immunohistochemistry (IHC) of GFP.
Fgf2 +/+ marrow was removed from donor mice and spinoculated with pMIG BCR-ABL retrovirus containing an IRES-GFP marker. The transfected marrow was then transplanted into lethally irradiated Fgf2 …
Figure 7—video 1
ECVs collected from Fgf2 +/+ primary marrow stromal cell cultures were stained with DiI (red) and lineage-depleted hematopoietic cells from Fgf2 +/+ marrow were stained with DiO (green) and incubated for 24 hr at 37°C as described in Materials and methods.
Cells were washed, placed on Poly-D-Lysine coated chamber slides, and DAPI-stained. Z-stack imaging was performed on an Olympus IX71 inverted microscope.
Figure 7—video 2
ECVs collected from Fgf2 -/- primary marrow stromal cell cultures were stained with DiI (red) and lineage-depleted hematopoietic cells from Fgf2 +/+ marrow were stained with DiO (green) and incubated for 24 hr at 37°C as described in Materials and methods.
Cells were washed, placed on Poly-D-Lysine coated chamber slides, and DAPI-stained. Z-stack imaging was performed on an Olympus IX71 inverted microscope.
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (homo sapiens) | FGF2 | NA | ||
| Gene (mus musculus) | Fgf2 | NA | ||
| Gene (homo sapiens) | FGFR1 | NA | ||
| Gene (mus musculus) | Fgfr1 | NA | ||
| Strain, strain background (mus musculus) | Fgf2tm1Doe/J Fgf2 +/+ and -/- mice | Jackson Laboratory | RRID:MGI:2679603 | |
| Genetic reagent (homo sapiens) | FGF2 | Thermo Fisher Scientific | shRNA in TRIPZ lentiviral vector | |
| Genetic reagent (homo sapiens) | FGFR1 | Thermo Fisher Scientific | shRNA in TRIPZ lentiviral vector | |
| Genetic reagent (homo sapiens) | AddGene | GeCKO lentiCRISPRv2 hSpCas9 and guide RNA | ||
| Genetic reagent (homo sapiens) | FGF2-1 | GenScript | CRISPR/Cas nine guide RNA design | |
| Genetic reagent (homo sapiens) | FGF2-2 | GenScript | CRISPR/Cas nine guide RNA design | |
| Genetic reagent (homo sapiens) | FGFR1-1 | GenScript | CRISPR/Cas nine guide RNA design | |
| Genetic reagent (homo sapiens) | FGFR1-2 | GenScript | CRISPR/Cas nine guide RNA design | |
| Genetic reagent (mus musculus) | pMIG with BCR-ABL and GFP | murine retrovirus | ||
| Cell line (homo sapiens) | MOLM14 | Dr. Yoshinobu Matsuo | RRID:CVCL_7916 | |
| Cell line (homo sapiens) | K562 | American Type Culture Collection | RRID:CVCL_0004 | |
| Cell line (homo sapiens) | HS-5 | Dr. Beverly Torok-Storb | RRID:CVCL_3720 | |
| Cell line (homo sapiens) | HS-27 | Dr. Beverly Torok-Storb | RRID:CVCL_0335 | |
| Antibody | Mouse monoclonal anti-FGFR1 | Cell Signaling | 9740 | Dilution 1:1000 |
| Antibody | Rabbit polyclonal anti-FGF2 | Santa Cruz | Sc-79 | Dilution 1:500 |
| Antibody | Rabbit monoclonal anti-CD63 | ABCAM | ab134045 | Dilution 1:1000 |
| Antibody | Rabbit polyclonal anti-CD9 | Santa Cruz | Sc-9148 | Dilution 1:200 |
| Antibody | Mouse monoclonal anti-tsg-101 | Santa Cruz | Sc-7964 | Dilution 1:200 |
| Antibody | Mouse monoclonal anti-actin | Millipore | MAB1501 | Dilution 1:5000 |
| Peptide, recombinant protein | FGF2 (human) | Peprotech | ||
| Commercial assay or kit | Thermo Scientific lentiviral transfection kit | |||
| Chemical compound, drug | quizartinib (AC220) | LC labs | ||
| Chemical compound, drug | imatinib | LC labs | ||
| Chemical compound, drug | nilotinib | SelleckChem | ||
| Chemical compound, drug | PD173074 | SelleckChem | ||
| Chemical compound, drug | BGJ-398 | SelleckChem | ||
| Chemical compound, drug | doxycycline | Fisher | ||
| Software, algorithm | CellProfiler | Cell area |
Additional files
-
Supplementary file 1
Additional information on antibodies used in paper.
- https://doi.org/10.7554/eLife.40033.024
-
Transparent reporting form
- https://doi.org/10.7554/eLife.40033.025
