Abstract
Background
Cancer-associated fibroblasts (CAFs) consist of heterogeneous cell population in terms of their differentiation potential. The functional differences in tumor progression between CAFs with mesenchymal stem/progenitor cells (MSCs/MPCs) characteristics and CAFs without MSCs/MPCs characteristics are not clarified.
Methods
CAFs and vascular adventitial fibroblasts (VAFs, which contain MSCs/MPCs) were isolated from nine primary lung cancers and were cultured in osteogenic or adipogenic medium to assess their multi-lineage differentiation. Next, we established nine single-cell-derived clones from the primary culture of CAFs and examined their differentiation potential. The effects of each single-cell-derived clone on the proliferation and migration of lung adenocarcinoma cell line, A549, were analyzed.
Results
The nine samples of VAFs and CAFs showed various degrees of osteogenic differentiation. Although the VAFs displayed the ability to undergo adipogenic differentiation, all cases of the CAFs did not. CAFs clones presented varying degrees of osteogenic differentiation. Four clones displayed comparable levels of osteogenic potential with that of the VAFs, and two clones were completely negative. As compared to the CAFs clones that possessed lower osteogenic potential, CAFs clones with higher osteogenic potential did not confer proliferative activity in A549 cells. On the contrary, these clones significantly promoted the migration of A549 cells as compared to the clones with lower osteogenic potential.
Conclusion
Our studies clearly indicate that CAFs derived from lung cancer are heterogeneous population that consists of cells with varying osteogenic potentials and that CAFs with higher osteogenic potential have a greater tumor-promoting function through the enhancement of cancer cell migration.
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Acknowledgments
This work was supported by National Cancer Center Research and Development Fund (23-A-12 and 26-A-16), the Foundation for the Promotion of Cancer Research, 3rd-Term Comprehensive 10-Year Strategy for Cancer Control, Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, and JSPS KAKENHI (24659185).
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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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Supplementary Fig. 1
Morphological appearance of non-cancer-associated fibroblasts (NCAFs), CAFs and hVAFs. Microscopic images of NCAFs (left), CAFs (center) and hVAFs (right) (TIFF 13041 kb)
Supplementary Fig. 2
Scatter plots of each specimen’s quantitative differentiation potential. a ALP and b von Kossa for osteogenic differentiation. c Oil red O for adipogenic differentiation of each specimens (TIFF 2476 kb)
Supplementary Fig. 3
Morphology of single-cell derived CAFs clones. Microscopic images of 9 single-cell derived CAFs clones (TIFF 50781 kb)
Supplementary Fig. 4
Quantitative analysis for osteogenic differentiation potential of each CAFs clones by ALP staining. Quantitative analysis of ALP-positive area of each CAFs clones. Values are the mean ± SEM (TIFF 4262 kb)
Supplementary Fig. 5
Differentiation potential of NCAFs. a The staining images of osteogenic differentiation by ALP and von Kossa methods. b Quantitative analysis of ALP and von Kossa. c The staining images of adipogenic differentiation by oil red O. d Quantitative analysis of oil red O staining. Each result was calculated from the averages of 9 specimens. Bar = 100 μm. Values are the mean ± SEM. **P < 0.01 between control vs induction (TIFF 22857 kb)
Supplementary Fig. 6
Scheme summaries. a Brief summary of WST-8 assay. The cells were treated by supernatants of CAFs clones for 2 days. After WST-8 reaction occurred for an hour, absorbance was measured at 450 nm. b Brief scheme of scratch wound healing assay. The monolayer cells were scratched and cultured in CAFs clones. Then, wound healing was observed every 6 h until 18 h (TIFF 6470 kb)
Supplementary Fig. 7
Correlation between ALP-positive area and von Kossa-positive area in single-cell-derived hVAFs clones. Clones of hVAFs were generated by single cell cloning using hTERT gene transfection. The 50 clones that induced osteogenesis were stained by ALP and von Kossa methods. This figure shows a scatter plot of quantitative analysis by ALP and von Kossa for each clone (TIFF 2009 kb)
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Suda, Y., Neri, S., Hashimoto, H. et al. Clonal heterogeneity in osteogenic potential of lung cancer-associated fibroblasts: promotional effect of osteogenic progenitor cells on cancer cell migration. J Cancer Res Clin Oncol 142, 1487–1498 (2016). https://doi.org/10.1007/s00432-016-2171-y
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DOI: https://doi.org/10.1007/s00432-016-2171-y