Tumor Cells

Cancerous cells have altered cellular functions as compared to the normally functioning, non-malignant cells from which they are derived.

Cell morphology and signaling pathway studies in vitro that incorporate the use of 3D culture systems can give insights into the effects of mis-regulated or mis-expressed proteins, as exemplified by human mammary carcinoma cells (T4-2)2 (Figure 1). The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cell migration can be studied using either BD Falcon™ Cell Culture Inserts or BD FluoroBlok™ Cell Culture Inserts for moderate to high-throughput screening (Figure 2).

Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. Invasion through BD Matrigel™ Matrix-coated Cell Culture Inserts has become the gold standard for quantitative and qualitative measurement of the metastatic potential of a cell1, 3-9. This matrix provides a true barrier to non-invasive cells while presenting the appropriate protein structure for penetration of invading cells.

The BD BioCoat™ Matrigel Invasion Chambers and BD BioCoat Tumor Invasion Systems are optimized systems that utilize standardized coating procedures to ensure even coating of BD Matrigel™ Matrix for reproducible results (Figure 3). The BD BioCoat Tumor Invasion System provides a unique, quantitative platform that can be used to determine the effects of anti-metastatic compounds on invasive cell types (Figure 4). For in vivo studies, BD Matrigel Matrix can be used to help support tumor cell engraftment in mice10-12. These tools allow researchers to dissect various areas of tumor biology, from analysis of signaling pathways in vitro to in vivo tumor formation.

Figure 1. Adherance of HEK-293 Cells to BD BioCoat Poly-D-Lysine Cultureware

Figure 2. Adherance of HEK-293 Cells to BD BioCoat Poly-D-Lysine Cultureware

HEK-293 cells have enhanced attachment to BD BioCoat Poly-D-Lysine Cultureware as compared to BD Falcon Tissue Culture-treated Cultureware. An equal number of cells were plated on BD BioCoat Poly-D-Lysine 384-well black/clear (right) and BD Falcon Tissue Culture-treated 384-well Black/Clear Plates (left) and grown under serum-free conditions. Before washing (top), there were a similar number of cells in the BD BioCoat Poly-D-Lysine coated wells and the BD Falcon Tissue Culture-treated wells. After washing, using a Skatron Washer (Molecular Devices) (middle), the cells remained attached to the BD BioCoat Poly-D-Lysine wells while few cells remained attached to the BD Falcon Tissue Culturetreated wells. Post-wash, the cells were visualized using Calcein AM (bottom).

Figure 2. HT-1080 Migration

Figure 2. HT-1080 Migration

Migration of Calcein AM (A) and DiIC12(3) (B) labeled human fibrosarcoma cells (HT-1080) through BD Falcon FluoroBlok 96-Multiwell Inserts, 8 μm pore size. DMEM with 5% FCS was used as a chemoattractant in the lower wells, while DMEM/0.1% BSA was added to the control wells. The plates were incubated for four hours at 37°C, after which fluorescence of cells which had migrated through the microporous membrane was measured on the Applied Biosystems CytoFluor® 4000 and PerkinElmer HTS 7000 Plus fluorescent plate readers using excitation/emission wavelengths of 485/530 nm for Calcein AM or 530/590 nm for DiIC12(3). Values represent the mean of 8 wells ± S.D. Migration from as few as 4,000 input cells can be detected.


 
Figure 3. Comparison of Mean Percent Invasion

Figure 3. Comparison of Mean Percent Invasion

Multiple lots of BD BioCoat 96-Multiwell Tumor Invasion System (A) and BD BioCoat 24-Multiwell Tumor Invasion System (B) were assayed to show reproducibility with these systems. Fluorescently labeled cells residing on the bottom of the insert membrane were measured post-invasion with either a Victor2™ plate reader (BD BioCoat 96-Multiwell Tumor Invasion System) or a CytoFluor®>/sup> plate reader (BD BioCoat 24-Multiwell Tumor Invasion System). Mean percent invasion of NIH-3T3 and HT-1080 cells were compared. Cells were labeled post-invasion using BD Calcein AM.

Figure 4. Inhibition of PC3 Migration and Invasion by Doxycycline

Figure 4. Inhibition of PC3 Migration and Invasion by Doxycycline

PC3 invasion is inhibited by doxycycline. PC3 cell invasion was measured using BD BioCoat 24- Multiwell Tumor Invasion System, which is based on the fluorescence blocking BD FluoroBlok™ PET microporous membrane, and migration was measured using BD Falcon HTS FluoroBlok 24- Multiwell Insert System. At the end of the assay, cells were stained with BD Calcein AM.

References

  1. Kong D, Li Y, Wang Z, Banerjee S, Sarkar FH. (2007) Inhibition of angiogenesis and invasion by 3,3’-diindolylmethane is mediated by the NF-κB downstream target genes MMP-9 and uPA that regulated bioavailability of VEGF in prostate cancer. Cancer Res. 67(7):3310.

  2. Itoh M, Nelson CM, Myers CA, Bissell MJ. (2007) Rap1 integrates tissue polarity, lumen formation, and tumorigenic potential in human breast epithelial cells. Cancer Res. 67(10):47

  3. Albini A and Benelli R. (2007) The chemoinvasion assay: a method to assess tumor and endothelial cell invasion and its modulation. Nat Protocols. 2(3):505.

  4. Oxelmark E, Roth JM, Brooks PC, Braunstein SE, Schneider RJ, Garabedian MJ. (2006) The cochaperone p23 differentially regulates estrogen receptor target genes and promotes tumor cell adhesion and invasion. Mol Cell Biol. 26(14):5205.

  5. Duxbury MA, Ito H, Zinner MJ, Ashley SW, Whang EE. (2004) EphA2: a determinant of malignant cellular behavior and a potential therapeutic target in pancreatic adenocarcinoma. Oncogene. 23:1448.

  6. Seton-Regers SE, Lu Y, Hines LM, Koundinya M, LaBaer J, Muthuswamy SK, Brugge JS. (2004) Cooperation of the ErbB2 receptor and transforming growth factor β in induction of migration and invasion in mammary epithelial cells. Proc Natl Acad Sci. 101(5):1257.

  7. Singh A, Singh UP, Grizzle WE, Lillard Jr JW. (2004) CXCL12- CXCR4 interactions modulate prostate cancer cell migration, metalloproteinase expression and invasion. Lab Invest. 84:1666.

  8. Takada Y, Kobayashi Y, Aggarwal BB. (2005) Evodiamine abolishes constitutive and inducible NF-κB activation by inhibiting IκBα kinase activation, thereby suppressing NF-κB-regulated antiapoptotic and metastatic gene expression, up-regulating apoptosis, and inhibiting invasion. J Biol Chem. 280(17):17203.

  9. Ichikawa H, Takada Y, Murakami A, Aggarwal BB. (2005) Identification of a novel blocker of I kappa B alpha kinase that enhances cellular apoptosis and inhibits cellular invasion through suppression of NFkappa B-regulated gene products. J Immunol. 174(11):7383.

  10. Cho RW, Wang X, Diehn M, Shedden K, Chen GY, Sherlock G, Gurney A, Lewicki J, Clarke MF. (2008) Isolation and molecular characterization of cancer stem cells in MMTV-Wnt-1 murine breast tumors. Stem Cells. 26(2):364.

  11. Purhonen S, Palm J, Rossi D, Kaskenpaa N, Rajantie I, Yla-Herttuala S, Alitalo K, Weissman IL, Salven P. (2008) Bone marrow-derived circulating endothelial precursors do not contribute to vascular endothelium and are not needed for tumor growth. Proc Natl Acad Sci. 105(18):6620.

  12. Feldmann G, Dhara S, Fendrich V, Bedja D, Beaty R, Mullendore M, Karikari C, Alvarez H, Iacobuzio-Donahue C, Jimeno A, Gabrielson KL, Matsui W, Maitra A. (2007) Blockade of hedgehog signaling inhibits pancreatic cancer invasion and metastases: a new paradigm for combination therapy in solid cancers. Cancer Res. 67:2187.

AlexaFluor is a trademark of Invitrogen Corporation.
CytoFlour is a trademark of Applied Biosystems.
MetaMorph is a registered trademark of Universal Imaging Corporation (UIC).
mTeSR1 and WiCell is the property of WiCell Research Institute.
PuraMatrix is a registered trademark of 3DM, Inc.
Victor2 is a trademark of PerkinElmer, Inc.


For Research Use Only. Not for use in diagnostic or therapeutic procedures.