Breast Cancer Stem Cells Upregulate IRF6 in Stromal Fibroblasts to Induce Stromagenesis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. BCSC/Fibroblast Co-Culture
2.3. Lentiviral Production and Transduction
2.4. Flow Cytometry
2.5. Rapid MACE-Seq
2.6. RNA Preparation and qPCR
2.7. Immunofluorescence Staining
2.8. Immunohistochemistry
2.9. Protein Isolation and Western Blotting
2.10. Orthotopic Breast Cancer Xenografts
2.11. Statistical Analysis
3. Results
3.1. Fibroblasts Densely Aggregate along the Periphery of BCSC Clusters
3.2. CF Population Creates an ECM-Rich Region around BCSC Clusters
3.3. Gene Expression Profiling of CF Population Reveals Upregulation of CAF Characteristics and Genes Regulating Fibrosis
3.4. IRF6 Knockdown Fibroblasts Fail to Aggregate at the BCSC Cluster Periphery
3.5. BCSC KD CF Show Reduced Fibronectin Deposition
3.6. IRF6 in Stromal Fibroblasts of Xenograft Models Regulate Stromal Development and Necrosis
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
BCSC | Breast cancer stem cells |
Fi | Fibroblast |
BCSC + Fi | BCSC/fibroblast co-culture |
CAF | Cancer-associated fibroblasts |
CSC | Cancer stem cells |
COL1A1 | Collagen type I alpha 1 chain |
ECM | Extracellular matrix |
FN | Fibronectin |
DEG | Differentially expressed gene |
GO | Gene ontology |
IRF6 | Interferon regulatory factor 6 |
CF | Cluster fibroblasts |
NCF | Non-cluster fibroblasts |
Co Fi | Control fibroblasts |
KD Fi | Knockdown fibroblasts |
OE Fi | Overexpression fibroblasts |
Scr CF | Scrambled cluster fibroblasts |
KD CF | Knockdown cluster fibroblasts |
OE CF | Overexpression cluster fibroblasts |
SPE | Stromal phenotype of encapsulation |
TACS | Tumor-associated collagen signatures |
TME | Tumor microenvironment |
TNBC | Triple-negative breast cancer |
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No. | Gene | Description | BCSC1 CF (FC) | BCSC1 CF (p-Value) | BCSC2 CF (FC) | BCSC2 CF (p-Value) |
---|---|---|---|---|---|---|
1. | AP1M2 | Adaptor Related Protein Complex 1 Subunit Mu 2 | 6.4 | 2.91 × 10−5 | 10.7 | 2.08 × 10−14 |
2. | BST2 | Bone Marrow Stromal Cell Antigen 2 | 8.4 | 3.85 × 10−11 | 10.2 | 3.17 × 10−16 |
3. | C6orf223 | Long Intergenic Non-Protein Coding RNA 3040 | 5.9 | 4.90 × 10−8 | 8.5 | 2.06 × 10−16 |
4. | CA2 | Carbonic Anhydrase 2 | 7.7 | 1.73 × 10−7 | 9.8 | 3.47 × 10−12 |
5. | CBLC | CBL Proto-Oncogene C | 7.4 | 6.48 × 10−6 | 7.7 | 9.73 × 10−7 |
6. | CCL5 | C-C Motif Chemokine Ligand 5 | 5.9 | 4.17 × 10−5 | 7.4 | 6.59 × 10−8 |
7. | CCL20 | C-C Motif Chemokine Ligand 20 | 6.2 | 1.36 × 10−5 | 6.1 | 1.28 × 10−5 |
8. | CD70 | Cluster of Differentiation 70 | 6.7 | 1.35 × 10−5 | 7.9 | 1.24 × 10−7 |
9. | COBL | Cordon-Bleu WH2 Repeat Protein | 6.3 | 1.29 × 10−4 | 9.0 | 4.00 × 10−9 |
10. | CPVL | Carboxypeptidase Vitellogenic Like | 5.4 | 8.33 × 10−4 | 9.1 | 6.53 × 10−10 |
11. | CRB3 | Crumbs Cell Polarity Complex Component | 7.3 | 1.92 × 10−6 | 8.9 | 1.77 × 10−9 |
12. | CXCL11 | C-X-C Motif Chemokine Ligand 11 | 6.7 | 2.97 × 10−6 | 6.2 | 1.21 × 10−5 |
13. | DEPP1 | DEPP Autophagy Regulator 1 | 6.7 | 3.05 × 10−5 | 10.5 | 2.97 × 10−12 |
14. | DSC3 | Desmocollin 3 | 6.4 | 3.05 × 10−5 | 9.3 | 1.35 × 10−10 |
15. | ELF3 | E74 Like ETS Transcription Factor 3 | 8.5 | 1.05 × 10−13 | 11.4 | 1.33 × 10−24 |
16. | EPPK1 | Epiplakin | 7.2 | 1.11 × 10−5 | 7.5 | 2.39 × 10−6 |
17. | FGFBP1 | Fibroblast Growth Factor Binding Protein 1 | 8.1 | 2.36 × 10−6 | 9.9 | 1.89 × 10−9 |
18. | FOXA1 | Forkhead box A1 | 6.1 | 1.35 × 10−4 | 9.4 | 1.47 × 10−10 |
19. | FXYD3 | FXYD Domain Containing Ion Transport Regulator 3 | 9.4 | 9.70 × 10−10 | 9.5 | 9.85 × 10−10 |
20. | GJB2 | Gap Junction Protein Beta 2 | 6.7 | 1.35 × 10−5 | 9.5 | 7.78 × 10−11 |
21. | GRHL2 | Grainyhead Like Transcription Factor 2 | 6.8 | 2.21 × 10−5 | 9.3 | 7.91 × 10−10 |
22. | IGSF3 | Immunoglobulin Superfamily Member 3 | 6.7 | 7.13 × 10−5 | 8.9 | 2.57 × 10−8 |
23. | IQANK1 | IQ Motif and Ankyrin Repeat Containing 1 | 6.1 | 8.73 × 10−5 | 8.7 | 2.41 × 10−9 |
24. | IRF6 | Interferon Regulatory Factor 6 | 7.0 | 3.40 × 10−12 | 6.2 | 1.32 × 10−9 |
25. | KDF1 | Keratinocyte Differentiation Factor 1 | 6.8 | 1.04 × 10−5 | 9.7 | 2.44 × 10−11 |
26. | KRT7 | Keratin 7 | 5.6 | 1.00 × 10−12 | 7.9 | 3.37 × 10−25 |
27. | LAD1 | Ladinin 1 | 8.8 | 2.14 × 10−8 | 10.2 | 3.78 × 10−11 |
28. | LCN2 | Lipocalin 2 | 8.8 | 8.42 × 10−9 | 13.2 | 4.43 × 10−19 |
29. | LTB | Lymphotoxin Beta | 6.1 | 9.54 × 10−5 | 9.8 | 1.35 × 10−11 |
30. | MIR205HG | MIR205 host gene | 9.9 | 1.39 × 10−10 | 6.8 | 2.35 × 10−5 |
31. | MISP | Mitotic Spindle Positioning | 6.7 | 5.26 × 10−5 | 8.0 | 2.91 × 10−7 |
32. | MPZL2 | Myelin Protein Zero Like 2 | 6.9 | 1.55 × 10−5 | 9.5 | 2.39 × 10−10 |
33. | MST1R | Macrophage Stimulating 1 Receptor | 5.9 | 1.71 × 10−4 | 8.5 | 6.05 × 10−9 |
34. | NECTIN4 | Nectin Cell Adhesion Molecule 4 | 5.7 | 3.82 × 10−5 | 7.5 | 9.49 × 10−9 |
35. | NMU | Neuromedin U | 7.5 | 2.97 × 10−6 | 7.5 | 2.46 × 10−6 |
36. | OAS1 | 2′-5′-Oligoadenylate Synthase 1 | 9.8 | 2.04 × 10−16 | 7.9 | 1.29 × 10−10 |
37. | OASL | 2′-5′-Oligoadenylate Synthase Like | 6.8 | 9.78 × 10−9 | 6.9 | 4.08 × 10−9 |
38. | PI3 | Peptidase Inhibitor 3 | 6.0 | 2.48 × 10−11 | 5.1 | 2.49 × 10−8 |
39. | PROSER2 | Proline And Serine Rich 2 | 6.5 | 5.60 × 10−5 | 7.4 | 2.49 × 10−6 |
40. | PRSS8 | Serine Protease 8 | 6.2 | 2.51 × 10−4 | 9.6 | 7.05 × 10−10 |
41. | RAB25 | Member RAS Oncogene Family | 7.6 | 4.76 × 10−5 | 10.5 | 2.52 × 10−9 |
42. | S100A14 | S100 Calcium Binding Protein A14 | 7.7 | 2.76 × 10−11 | 10.0 | 1.12 × 10−18 |
43. | S100A9 | S100 Calcium Binding Protein A9 | 7.2 | 1.15 × 10−6 | 10.0 | 9.77 × 10−13 |
44. | SAA2 | Serum Amyloid A2 | 6.7 | 2.09 × 10−5 | 8.45 | 1.55 × 10−8 |
45. | SCNN1A | Sodium Channel Epithelial 1 Subunit Alpha | 7.0 | 1.64 × 10−7 | 7.3 | 3.46 × 10−8 |
46. | SFN | Stratifin | 8.4 | 1.15 × 10−9 | 9.5 | 3.75 × 10−12 |
47. | SLPI | Secretory Leucocyte Peptidase Inhibitor | 7.7 | 7.67 × 10−7 | 11.1 | 1.17 × 10−13 |
48. | SMOC1 | SPARC Related Modular Calcium Binding 1 | 7.6 | 5.85 × 10−7 | 9.0 | 1.07 × 10−9 |
49. | SNX10 | Sorting Nexin 10 | 6.8 | 1.96 × 10−5 | 6.7 | 1.53 × 10−5 |
50. | SPINT2 | Serine Peptidase Inhibitor, Kunitz Type 2 | 5.4 | 1.06 × 10−11 | 8.5 | 2.45 × 10−28 |
51. | TACSTD2 | Tumor Associated Calcium Signal Transducer 2 | 6.2 | 4.29 × 10−12 | 8.5 | 2.06 × 10−22 |
52. | TGFA | Transforming Growth Factor Alpha | 7.0 | 4.50 × 10−6 | 9.6 | 2.93 × 10−11 |
53. | TINAGL1 | Tubulointerstitial Nephritis Antigen Like 1 | 9.8 | 4.63 × 10−11 | 10.8 | 2.77 × 10−13 |
54. | WFDC2 | WAP Four-Disulphide Core Domain 2 | 6.1 | 5.80 × 10−5 | 6.3 | 1.81 × 10−5 |
55. | XIST | X Inactive Specific Transcript | 6.2 | 1.36 × 10−4 | 8.3 | 4.68 × 10−8 |
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Muralidharan, H.; Hansen, T.; Steinle, A.; Schumacher, D.; Stickeler, E.; Maurer, J. Breast Cancer Stem Cells Upregulate IRF6 in Stromal Fibroblasts to Induce Stromagenesis. Cells 2024, 13, 1466. https://doi.org/10.3390/cells13171466
Muralidharan H, Hansen T, Steinle A, Schumacher D, Stickeler E, Maurer J. Breast Cancer Stem Cells Upregulate IRF6 in Stromal Fibroblasts to Induce Stromagenesis. Cells. 2024; 13(17):1466. https://doi.org/10.3390/cells13171466
Chicago/Turabian StyleMuralidharan, Harshini, Thomas Hansen, Anja Steinle, David Schumacher, Elmar Stickeler, and Jochen Maurer. 2024. "Breast Cancer Stem Cells Upregulate IRF6 in Stromal Fibroblasts to Induce Stromagenesis" Cells 13, no. 17: 1466. https://doi.org/10.3390/cells13171466