Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity
- Department of Pharmacology, Northwestern University, United States
- Driskill Graduate Program in Life Science, Feinberg School of Medicine, Northwestern University, United States
- Biological Sciences Division, Pacific Northwest National Laboratory, United States
- Department of Electrical and Computer Engineering, TEES-AgriLife Center for Bioinformatics and Genomic Systems Engineering, Texas A&M University, United States
- Department of Toxicology and Cancer Biology, University of Kentucky, United States
- Quantitative Data Science Core, Center for Genetic Medicine, Northwestern University Feinberg School of Medicine, United States
- Department of Medicine, Hematology/Oncology Division, Feinberg School of Medicine, Northwestern University, United States
- Pathology Core Facility, Northwestern University, United States
- Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, United States
Figures
Figure 1 with 2 supplements
CD81 interacts with CD44 on the membrane and promotes mammosphere formation of triple negative breast cancer (TNBC) cells.
Representative images (A) and bar graphs (B) of the mammospheres of MDA-MB-231 cell groups (WT, CD44KO, CD81KO, dKO pool populations), 17 days after seeded at 2000 cells per well (6-well plate) in …
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Figure 1—source data 1
Uncropped blots associated with Figure 1F.
- https://cdn.elifesciences.org/articles/82669/elife-82669-fig1-data1-v2.pdf
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Figure 1—source data 2
Uncropped blots associated with Figure 1H.
- https://cdn.elifesciences.org/articles/82669/elife-82669-fig1-data2-v2.pdf
Figure 1—figure supplement 1
Characterization of CD44KO and CD81KO breast cancer cell lines.
(A) Schematic of mass spectrometry workflow. TN1 patient-derived xenograft (PDX) tumor was dissociated and sorted based on CD44 and CD24 expression and analyzed by mass spec. Four hundred and …
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Figure 1—figure supplement 1—source data 1
Uncropped blots associated with Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/82669/elife-82669-fig1-figsupp1-data1-v2.pdf
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Figure 1—figure supplement 1—source data 2
Uncropped blots associated with Figure 1—figure supplement 1C.
- https://cdn.elifesciences.org/articles/82669/elife-82669-fig1-figsupp1-data2-v2.pdf
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Figure 1—figure supplement 1—source data 3
Uncropped blots associated with Figure 1—figure supplement 1D.
- https://cdn.elifesciences.org/articles/82669/elife-82669-fig1-figsupp1-data3-v2.pdf
Figure 1—figure supplement 2
CD81 colocalizes with CD44 on cellular membrane in breast cancer cell line.
(A, B) IncuCyte images and curve analyses of cell confluence of human MDA-MB-231 WT, CD44KO, and CD81KO cells (C) and mouse 4T1 WT and Cd81KO cells (D) over time. Scale bar is 300 µm. N= 3. Error …
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Figure 1—figure supplement 2—source data 1
Uncropped blots associated with Figure 1—figure supplement 2C.
- https://cdn.elifesciences.org/articles/82669/elife-82669-fig1-figsupp2-data1-v2.pdf
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Figure 1—figure supplement 2—source data 2
Uncropped blots associated with Figure 1—figure supplement 2E.
- https://cdn.elifesciences.org/articles/82669/elife-82669-fig1-figsupp2-data2-v2.pdf
Figure 2 with 3 supplements
Global mass spectrometry and phosphoproteomic profiling of MDA-MB-231 cells with siCD81 and siCD44 KDs.
Global mass spectrometry heatmap (A) and KEGG pathway analysis (B) of altered top pathways with significantly expressed proteome within six different clusters (G1–G6) in siControl, siCD81, and siCD44…
Figure 2—figure supplement 1
SEMA7A increased in CD81-depleted cells.
RNA-sequencing-based transcriptome analysis shows siCD81-altered pathways (A) and heatmap (B), including down- and upregulated genes with ≥1.5-fold changes in MDA-MB-231 cells compared to the siRNA …
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Figure 2—figure supplement 1—source data 1
Uncropped blots associated with Figure 2—figure supplement 1C.
- https://cdn.elifesciences.org/articles/82669/elife-82669-fig2-figsupp1-data1-v2.pdf
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Figure 2—figure supplement 1—source data 2
Uncropped blots associated with Figure 2—figure supplement 1F.
- https://cdn.elifesciences.org/articles/82669/elife-82669-fig2-figsupp1-data2-v2.pdf
Figure 2—figure supplement 2
Global and phosphoproteomic analyses in CD81- and CD44-depleted cells.
(A) The Volcano plots of different abundance in global proteome and phosphoproteome between adherent and clustered tumor cells. (B) Immunoblots and bar graphs of CD44 and CD81 in MDA-MB-231 cells …
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Figure 2—figure supplement 2—source data 1
Uncropped blots associated with Figure 2—figure supplement 2B.
- https://cdn.elifesciences.org/articles/82669/elife-82669-fig2-figsupp2-data1-v2.pdf
Figure 2—figure supplement 3
The kinase reactome networks in CD81- and CD44-depleted cells.
The MaxQuant kinase reactome networks based on the downregulated (A) and upregulated (B) phosphorylation sites in both siCD81- and siCD44-transfected cells and known curation of kinase–substrate …
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Figure 2—figure supplement 3—source data 1
Uncropped blots associated with Figure 2—figure supplement 3C.
- https://cdn.elifesciences.org/articles/82669/elife-82669-fig2-figsupp3-data1-v2.pdf
Figure 3 with 3 supplements
CD81 and CD44 are required for exosome-induced cancer stemness.
Transmission electron microscopy images (A) of WT, 44KO, and 81KO MDA-MB-231 cells and number of vacuoles observed per cell (B). Yellow arrows point to vacuoles or early endosomes/endocytic …
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Figure 3—source data 1
Uncropped blots associated with Figure 3E.
- https://cdn.elifesciences.org/articles/82669/elife-82669-fig3-data1-v2.pdf
Figure 3—figure supplement 1
Effects of CD44/CD81 depletion on cellular pathways related to extracellular vesicles (EVs).
(A) GO Localization analysis of downregulated proteins in pooled CD44KO cells compared to WT MDA-MB-231 cells (N = 3 replicates, p < 0.05). (B) GO Processes analyses of global mass …
Figure 3—figure supplement 2
Proteomic pathway analysis of evCD44 derived from CD44KO cells.
(A) Proteomic pathways altered in the extracellular vesicles (EVs) derived from CD44KO cells (ev44KO) compared to evWT from WT cells. (B–F) GO analyses of proteins down- or upregulated in the ev44KO …
Figure 3—figure supplement 3
CD81 is required for exosome-induced effects on stemness phenotype.
(A) Immunoblot analyses for OCT 4, STAT3, phosphoSTAT3 (pSTAT3), FAK, pFAK, CD44, and β-actin using CD81KO MDA-MB-231 cells educated with phosphate-buffered saline (PBS) or extracellular vesicles …
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Figure 3—figure supplement 3—source data 1
Uncropped blots associated with Figure 3—figure supplement 3.
- https://cdn.elifesciences.org/articles/82669/elife-82669-fig3-figsupp3-data1-v2.pdf
Figure 4 with 5 supplements
CD81 is associated with patient survival and enriched in circulating tumor cells (CTCs) promoting tumor cell aggregation.
Kaplan–Meier plots of CD81 protein expression in patients with triple negative breast cancer (TNBC) correlate with an unfavorable overall survival (A), relapse-free survival (B), and …
Figure 4—figure supplement 1
Kaplan–Meier plots of patient survival based on CD81 protein expression.
Kaplan–Meier plots of patient outcomes with triple negative breast cancer (TNBC), stratified by CD81 expression levels that correlate with an unfavorable overall survival (OS), relapse-free survival …
Figure 4—figure supplement 2
Tissue microarray (TMA) clinical characteristics.
(A) Table of tumor TMA breast cancer patient characteristics. (B) Immunohistochemical staining of CD81 and CD44 expression (in brown) and hemotoxylin (in blue) in TMA of breast tumors (N = 77 with …
Figure 4—figure supplement 3
Gating strategy of circulating tumor cells (CTCs) by flow cytometry and the RNA expression level of CD81 in single versus clustered CTCs.
(A) Forward scatter channel (FSC)-gated singles and clusters of WT and CD44KO MDA-MB-231 cells in suspension (n = 3 replicates). N= 3. Error bars represent standard deviation. T-Test p = 0.0038. (B) …
Figure 4—figure supplement 4
Cluster formation of tumor cells (MDA-MB-231, 4T1, and patient-derived xenograft [PDX] models) in CD81 and CD44 modified cells.
IncuCyte images (right) and quantified tumor cell aggregation curves (left) of MDA-MB-231 WT/CD81KO cells (*p = 0.02) (A), 4T1 WT/Cd81KO cells (******p < 5.8E−08) (B), and TN2 PDX siCon/siCD81 …
Figure 4—figure supplement 5
CD81 KD inhibits breast cancer cell migration and invasion.
(A) Cell migration of MDA-MB-231 WT, CD44KO, and CD81KO cells to close scratch wound, analyzed by Incucyte time-lapse imaging in every 2 hr. CD44KO and CD81KO groups took longer time to fill the …
Figure 5
CD81 promotes tumorigenesis of triple negative breast cancer (TNBC) cells.
(A) Table of serial dilutions of tumorigenic results with CD81+ and CD81−TN1 PDX, CD81 WT/KO 4T1 cells, and CD81 WT/KO/KO + CD81 overexpression (OE) MDA-MB-231. One-tailed Student T-test. Pictures …
Figure 6 with 2 supplements
CD81 deficiency abrogates lung metastasis in breast cancer.
(A) Photos of 4T1 orthotopic tumors (left panels) grown from implantations of comparable (1000) WT and (6000) Cd81KO cells into the L4/R4 mammary fat pads (N = 5 Balb/c mice with 10 injections) and …
Figure 6—figure supplement 1
CD81 KD inhibits breast cancer cell metastasis.
(A) HE images of lung metastasis colonies (arrow pointed region) observed in the mice injected with WT cells and absent with 81KO MDA-MB-231 cells, harvested on day 26 after tail vein injection. …
Figure 6—figure supplement 2
Schematic summary.
Schematic summary of CD81 in interacting with CD44 on the cytoplasmic membrane of tumor-initiating cells (TIC), facilitating the exosome cargo packaging with CD44 and CD81, promoting exosome-induced …
Author response image 1
Full blots of Suppl.
Figure 8A. Full Immunoblot analyses for OCT4, STAT3, phosphoSTAT3 (pSTAT3), FAK, pFAK, CD44 and β Actin using CD81KO MDA-MB-231 cells educated with PBS or EVs derived from WT, CD81KO, and CD44KO, …
Author response image 2
Repeated Figure 6M.
BLI images of dissected lungs on day 1 following the tail vein injections of 4T1-WT and Cd81KO cells into Babl/c mice (n=4) * P = 0.03. Two-tailed student T-test was used.
Author response image 3
CD81 is required for EV-induced effects on stemness signature proteins and mammosphere formation.
Representative images (left two panels) and quantifications (right panel) of mammospheres (Day 5) derived from 500 MDA-MB-231 cells in suspension after transfections with scrambled (Scr) RNAs and …
Tables
Author response table 1
| EV-producing cell | EV counts/mL | |
|---|---|---|
| NTA | MFV | |
| WT | 1.24e+09 +/- 9.44e+07 | 0.96e+09 +/- 2e+08 |
| CD44KO | 1.57e+09 +/- 2.80e+07 | 1.56e+09 +/- 2e+08 |
| CD81KO | 1.62e+09 +/- 1.68e+07 | 1.75e+09 +/- 9e+07 |
Additional files
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Supplementary file 1
Overlapping list of 38 proteins in CD44 and siCD44.
- https://cdn.elifesciences.org/articles/82669/elife-82669-supp1-v2.xlsx
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Supplementary file 2
Down or up-regulated genes without cutoffs.
- https://cdn.elifesciences.org/articles/82669/elife-82669-supp2-v2.xlsx
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Supplementary file 3
RNAseq data: down- and up-regulated pathways with 1.5 fold change.
- https://cdn.elifesciences.org/articles/82669/elife-82669-supp3-v2.xlsx
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Supplementary file 4
Global and phosphoproteomics data in cluster versus adherent cells.
- https://cdn.elifesciences.org/articles/82669/elife-82669-supp4-v2.xlsx
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Supplementary file 5
KEGG Pathways of global proteome clusters (G1 to G6) and phosphoproteome cluster (P1 to P6).
- https://cdn.elifesciences.org/articles/82669/elife-82669-supp5-v2.xlsx
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Supplementary file 6
Heatmap for siCD81 and siCD44 global proteomics in clusters.
- https://cdn.elifesciences.org/articles/82669/elife-82669-supp6-v2.xlsx
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Supplementary file 7
Phosphoproteomics data for heatmap in siCD81 and siCD44.
- https://cdn.elifesciences.org/articles/82669/elife-82669-supp7-v2.xlsx
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Supplementary file 8
Differentially Expressed Proteins in MDA-MB-231 WT versus CD44KO cell by mass spectrometry.
- https://cdn.elifesciences.org/articles/82669/elife-82669-supp8-v2.xlsx
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Supplementary file 9
Altered proteins in EVs of MDA-MB-231 WT versus CD44KO by mass spectrometry.
- https://cdn.elifesciences.org/articles/82669/elife-82669-supp9-v2.xlsx
