In Vitro invasive pattern of hepatocellular carcinoma cell line HCCLM9 based on three-dimensional cell culture and quantum dots molecular imaging

Abstract

This study aimed to establish a new in vitro three-dimensional (3D) cell culture and use quantum dots (QDs) molecular imaging to examine the invasive behaviors of hepatocellular carcinoma (HCC) cells. Each well of the 24-well cell culture plate was cover-slipped. Matrigel diluted with serum-free DMEM was added and HCCLM9 cells were cultured on the Matrigel. The cell morphological and cell growth characteristics were observed by inverted microscopy and laser confocal microscopy at different culture time. Cell invasive features were monitored by QDs-based real-time molecular imaging techniques. The results showed that on this 3D cell culture platform, HCCLM9 cells exhibited typical multi-step invasive behaviors, including reversion of cell senescence, active focal proliferation and dominant clones invasion. During the process, cells under 3D cell culture showed biological behaviors of spatio-temporal characteristics. Cells first merged on the surface of matrix, then gradually infiltrated and migrated into deep part of matrix, presenting polygonal morphology with stretched protrusions, forming tubular, annular and even network structure, which suggested that HCC cells have the morphological basis for vasculogenic mimicry. In addition, small cell clones with their edges well-circumscribed in early stage, progressed into a large irregular clone with ill-defined edge, while the other cells developed invadopodia. And QDs probing showed MT1-MMP was strongly expressed in the invadopodia. These findings indicate that a novel 3D cell culture platform has been successfully established, which can mimic the in vivo tumor microenvironment, and when combined with QDs-based molecular imaging, it can help to better investigate the invasive behaviors of HCC cells.

Keywords

3D cell culture / tumor microenvironment / tumor invasion / quantum dots

References

[1] JemalA, BrayF, CenterMM, et al.. Global cancer statistics. CA Cancer J Clin, 2011, 61(2): 69-90

[2] TangZY, YeSL, LiuYK, et al.. A decade’s studies on metastasis of hepatocellular carcinoma. J Cancer Res Clin Oncol, 2004, 130(4): 187-196

[3] ChafferCL, WeinbergRA. A perspective on cancer cell metastasis. Science, 2011, 331(6024): 1559-1564

[4] YamadaKM, CukiermanE. Modeling tissue morphogenesis and cancer in 3D. Cell, 2007, 130(4): 601-610

[5] HorningJL, SahooSK, VijayaraghavaluS, et al.. 3-D tumor model for in vitro evaluation of anticancer drugs. Mol Pharm, 2008, 5(5): 849-862

[6] KimJB. Three-dimensional tissue culture models in cancer biology. Semin Cancer Biol, 2005, 15(5): 365-77

[7] GriffithLG, SwartzMA. Capturing complex 3D tissue physiology in vitro. Nat Rev Mol Cell Biol, 2006, 7(3): 211-224

[8] EgebladM, WerbZ. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer, 2002, 2(3): 161-174

[9] Kraning-RushCM, CareySP, CalifanoJP, et al.. The role of the cytoskeleton in cellular force generation in 2D and 3D environments. Phys Biol, 2011, 8(1): 015009

[10] ChenC, XiaHS, GongYP, et al.. The quantitative detection of total HER2 load by quantum dots and the identification of a new subtype of breast cancer with different 5-year prognosis. Biomaterials, 2010, 31(33): 8818-8825

[11] PengCW, TianQ, YangGF, et al.. Quantum-dots based simultaneous detection of multiple biomarkers of tumor stromal features to predict clinical outcomes in gastric cancer. Biomaterials, 2012, 33(23): 5742-5752

[12] HoonDS, KitagoM, KimJ, et al.. Molecular mechanisms of metastasis. Cancer Metastasis Rev, 2006, 25(2): 203-220

[13] WeaverVM, HowlettAR, Langton-WebsterB, et al.. The development of a functionally relevant cell culture model of progressive human breast cancer. Semin Cancer Biol, 1995, 6(3): 175-184

[14] WangW, LinP, SunBC, et al.. Role of vasculogenic mimicry and endothelium-dependent vessel in metastasis of laryngeal cancer. Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi(Chinese), 2012, 47(5): 400-405

[15] ChunTH, SabehF, OtaI, et al.. MT1-MMP-dependent neovessel formation within the confines of the three-dimensional extracellular matrix. J Cell Biol, 2004, 167(4): 757-767

[16] Page-McCawA, EwaldAJ, WerbZ. Matrix metallo-proteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Bio, 2007, 8(3): 221-233

[17] MaJL, HanSX, ZhuQ, et al.. Role of Twist in vasculogenic mimicry formation in hypoxic hepatocellular carcinoma cells in vitro. Biochem Biophys Res Commun, 2011, 408(4): 686-691