A three-dimensional co-culture system to investigate macrophage-dependent tumor cell invasion
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Keywords
macrophage, motility, 3D co-culture, invasion, mammosphere
Abstract
Macrophages infiltrate cancers and promote progression to invasion and metastasis. To directly examine tumor-associated macrophages (TAMs) and tumor cells interacting and co-migrating in a three-dimensional (3D) environment, we have developed a co-culture model that uses a PyVmT mouse mammary tumor-derived cell line and mouse bone marrow-derived macrophages (BMM). The Py8119 cell line was cloned from a spontaneous mammary tumor in a Tg(MMTV:LTR-PyVmT) C57Bl/6 mouse and these cells form 3-dimensional (3D) spheroids under conditions of low adhesion. Co-cultured BMM infiltrate the Py8119 mammospheres and embedding of the infiltrated mammospheres in Matrigel leads to subsequent invasion of both cell types into the surrounding matrix. This physiologically relevant co-culture model enables examination of two critical steps in the promotion of invasion and metastasis by BMM: 1) macrophage infiltration into the mammosphere and, 2) subsequent invasion of macrophages and tumor cells into the matrix. Our methodology allows for quantification of BMM infiltration rates into Py8119 mammospheres and demonstrates that subsequent tumor cell invasion is dependent upon the presence of infiltrated macrophages. This method is also effective for screening macrophage motility inhibitors. Thus, we have developed a robust 3D in vitro co-culture assay that demonstrates a central role for macrophage motility in the promotion of tumor cell invasion.
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2. Petersen OW, Rønnov-Jessen L, Howlett AR, Bissell MJ. Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proceedings of the National Academy of Sciences. 1992;89(19):9064-8.
3. Lin RZ, Chang HY. Recent advances in three‐dimensional multicellular spheroid culture for biomedical research. Biotechnol J. 2008;3(9‐10):1172-84.
4. Bissell MJ, Radisky DC, Rizki A, Weaver VM, Petersen OW. The organizing principle: microenvironmental influences in the normal and malignant breast. Differentiation. 2002;70(9‐10):537-46.
5. Seton-Rogers SE, Lu Y, Hines LM, Koundinya M, LaBaer J, Muthuswamy SK, et al. Cooperation of the ErbB2 receptor and transforming growth factor β in induction of migration and invasion in mammary epithelial cells. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(5):1257-62.
6. Guiet R, Van Goethem E, Cougoule C, Balor S, Valette A, Al Saati T, et al. The process of macrophage migration promotes matrix metalloproteinase-independent invasion by tumor cells. J Immunol. 2011;187(7):3806-14.
7. Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer. 2004;4(1):71-8.
8. Noy R, Pollard JW. Tumor-associated macrophages: from mechanisms to therapy. Immunity. 2014;41(1):49-61.
9. Wynn TA, Chawla A, Pollard JW. Macrophage biology in development, homeostasis and disease. Nature. 2013;496(7446):445-55.
10. Goswami S, Sahai E, Wyckoff JB, Cammer M, Cox D, Pixley FJ, et al. Macrophages promote the invasion of breast carcinoma cells via a colony-stimulating factor-1/epidermal growth factor paracrine loop. Cancer research. 2005;65(12):5278-83.
11. Wyckoff J, Wang W, Lin EY, Wang Y, Pixley F, Stanley ER, et al. A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer Res. 2004;64(19):7022-9.
12. Pyonteck SM, Akkari L, Schuhmacher AJ, Bowman RL, Sevenich L, Quail DF, et al. CSF-1R inhibition alters macrophage polarization and blocks glioma progression. Nature Med. 2013;19(10):1264-72.
13. Strachan DC, Ruffell B, Oei Y, Bissell MJ, Coussens LM, Pryer N, et al. CSF1R inhibition delays cervical and mammary tumor growth in murine models by attenuating the turnover of tumor-associated macrophages and enhancing infiltration by CD8+ T cells. Oncoimmunology. 2013;2(12):e26968.
14. Mouchemore KA, Sampaio NG, Murrey MW, Stanley ER, Lannutti BJ, Pixley FJ. Specific inhibition of PI3K p110δ inhibits CSF‐1‐induced macrophage spreading and invasive capacity. FEBS Journal. 2013;280(21):5228-36.
15. Sampaio NG, Yu W, Cox D, Wyckoff J, Condeelis J, Stanley ER, et al. Phosphorylation of CSF-1R Y721 mediates its association with PI3K to regulate macrophage motility and enhancement of tumor cell invasion. J Cell Sci. 2011;124(12):2021-31.
16. Dwyer AR, Mouchemore KA, Steer JH, Sunderland AJ, Sampaio NG, Greenland EL, et al. Src family kinase expression and subcellular localization in macrophages: implications for their role in CSF-1-induced macrophage migration. Journal of Leukocyte Biology. 2016:jlb. 2A0815-344RR.
17. Werno C, Menrad H, Weigert A, Dehne N, Goerdt S, Schledzewski K, et al. Knockout of HIF-1α in tumor-associated macrophages enhances M2 polarization and attenuates their pro-angiogenic responses. Carcinogenesis. 2010;31(10):1863-72.
18. Park H, Dovas A, Hanna S, Lastrucci C, Cougoule C, Guiet R, et al. Tyrosine Phosphorylation of Wiskott-Aldrich Syndrome Protein (WASP) by Hck Regulates Macrophage Function. Journal of Biological Chemistry. 2014;289(11):7897-906.
19. Pansa MF, Lamberti MJ, Cogno IS, Correa SG, Vittar NBR, Rivarola VA. Contribution of resident and recruited macrophages to the photodynamic intervention of colorectal tumor microenvironment. Tumor Biology. 2015:1-12.
20. Gibby K, You W-K, Kadoya K, Helgadottir H, Young LJ, Ellies LG, et al. Early vascular deficits are correlated with delayed mammary tumorigenesis in the MMTV-PyMT transgenic mouse following genetic ablation of the NG2 proteoglycan. Breast Cancer Res. 2012;14(2):R67.
21. Qian B-Z, Pollard JW. Macrophage diversity enhances tumor progression and metastasis. Cell. 2010;141(1):39-51.
22. Nozaki K, Mochizuki W, Matsumoto Y, Matsumoto T, Fukuda M, Mizutani T, et al. Co-culture with intestinal epithelial organoids allows efficient expansion and motility analysis of intraepithelial lymphocytes. Journal of gastroenterology. 2016;51(3):206-13.
23. Wang R, Xu J, Juliette L, Castilleja A, Love J, Sung S-Y, et al., editors. Three-dimensional co-culture models to study prostate cancer growth, progression, and metastasis to bone. Seminars in cancer biology; 2005: Elsevier.
24. Bertaux-Skeirik N, Centeno J, Feng R, Schumacher MA, Shivdasani RA, Zavros Y. Co-culture of Gastric Organoids and Immortalized Stomach Mesenchymal Cells. Gastrointestinal Physiology and Diseases: Methods and Protocols. 2016:23-31.
25. Boj SF, Hwang C-I, Baker LA, Chio IIC, Engle DD, Corbo V, et al. Organoid models of human and mouse ductal pancreatic cancer. Cell. 2015;160(1):324-38.
26. McCracken KW, Catá EM, Crawford CM, Sinagoga KL, Schumacher M, Rockich BE, et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature. 2014;516(7531):400-4.
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