The mechanism of prostate metastasis to bone marrow is a complex multistage process that is only beginning to be understood. Initial steps include down regulation of molecular binding complexes and production of enzymes such as matrix metallo-proteinases leading to the loss of cell to cell adhesion within the tumour and extravasation of tumour cells. Once in the peripheral blood the circulating prostate tumour cells bind speciﬁcally to and invade through the bone marrow endothelial barrier and basement membrane, leading to formation of bone metastases.
Traditional invasion systems are time consuming with each assay involving washing, scraping with a cotton bud to remove non-invading cells, staining, drying and manual counting. Most importantly the assay is inﬂexible and limited in that the inserts can only present the researcher with a ﬁxed end point. The BD FluoroBlokTM tumour cell invasion system is a high throughput alternative. The system is versatile and sensitive, involving no manipulation of inserts, thus reducing labour and time. It allows the researcher to do multiple real-time readings of the same inserts before and after introducing stimuli. The inserts are made of a dyed PET membrane that has a wide ﬂuorescence blocking range from 490-700 nm allowing a range of potential ﬂuorophores to be used. The system can be automated with the incorporation of the BMG LABTECH microplate reader, to increase efﬁciency and high throughput of assays for drug discovery, cell migration, motility and chemotaxis studies.
Materials & Methods
All components of the tumour cell invasion system were purchased from BD Biosciences.
- HTS FluoroBlokTM Insert 24 well / 8 μM pores
- 24 well Companion Plate for use with 9mm pores
- BD MatrigelTM Matrix 5ml
- BD BioCoatTM Tumour Cell Invasion System
- 24-Multiwell Insert Plate 8 μM pre coated MatrigelTM
- BMG LABTECH microplate reader with ﬂuorescence detection and either Gas Vent Connection or Atmospheric Control Unit (ACU) for gas control
All tissue culture reagents and SYTO 82 were purchased from Invitrogen.
The FluoroBlokTM tumour cell invasion system was prepared as shown in Figure 2. All cultures were set up using phenol red free RPMI 1640 media. FluoroBlokTM cell culture inserts were coated with 100 μl of MatrigelTM (diluted at 1:25 with RPMI 1640) and allowed to set for 2hr at 37°C (MatrigelTM pre-coated FluoroBlokTM inserts can be used).
Coated inserts were placed in the 24 well companion plate containing 1ml of RPMI 1640 / 0.1% BSA with or without chemoattractant. In the top half of the insert 2x105 GFP or SYTO 82 labelled PC-3 cells were plated in 0.25 ml of RPMI 1640 / 0.1 % BSA. Plates were then incubated at 37°C for 18 hr to allow invasion to occur after which a ﬁnal end point reading was taken on the BMG LABTECH plate reader. For real time invasion, the plate was placed in the BMG LABTECH reader pre warmed to 37°C and 5% CO2 was inﬂated either with the ACU or via Gas Vent. The BMG LABTECH reader was set to take readings every hour for 21 hr.
Results & Discussion
Initially, a comparison was made between the manual counting and the automated counting system of the FluoroBlokTMinvasion chambers. Two identical assays were set up and counted manually or by the BMG LABTECH microplate reader. Both systems gave similar results (not shown).
Initially a comparison was made between the manual counting and the automated counting system of the FluoroBlokTM invasion chambers. Two identical assays were set up and counted manually or by the BMG LABTECH microplate reader. Both systems gave similar results (not shown).
Cells expressing a ﬂuorescent protein are the ideal for use in these applications as no further manipulation is needed to be able to receive a signal when reading in the BMG LABTECH microplate reader. However, not all cells can be tranfected easily, especially primary cells. There are a number of alternatives to overcome this by either pre-labelling or post-labelling cells. Earlier investigations demonstrated that post-labelling was too unreliable hence pre-labelling was the method of choice. The problems inherent with prelabelling were the risk that the dyes could affect cell function and invasion. For this reason; the need for optimization of labelling time and side effects needed to be established beforehand. Pre-labelling is advantageous because it allows real-time invasion studies. The ﬁrst step was to test whether the BMG LABTECH plate reader was able to support cells over 21 hours to gain real-time invasion data. PC3-GFP cells were pre-labelled with SYTO 82 and plated in FluoroBlokTM invasion chambers. These were placed in the BMG LABTECH reader with either ACU or Gas Vent set at 37°C overnight inﬂ ated with 5% CO2 in air and set to take readings every hour of both GFP (488/520 nm) and SYTO 82 (530/570nm) for 21 hr. Figures 3 and 4 show that the BMG LABTECH reader supports the FluoroBlokTM invasion chamber assays over a period of 21 hr; showing similar data to an experiment where invasion chambers were left in an incubator (37°C / 5% CO2 in air) overnight and read at speciﬁc times.
Using a BMG LABTECH microplate reader with integrated Gas Vent or Atmospheric Control Unit (ACU) for reading the FluoroBlokTM tumour cell invasion system gives results comparable to the traditional invasion assay. Compared with traditional invasion systems involving washing, staining and drying steps the introduced method is fast, saving time and labour.
BMG LABTECH readers are able to take multiple readings overtime at a user deﬁned temperature and the Gas Vent or ACU enables an environment conducive to cell proliferation and migration.
FluoroBlok is a trademark of BD Falcon.