Applications - Application Notes
Cellular Analysis of 3D Spheroid-Based Tumor Invasion Assays
10-Oct-14Cytation 3, Cytation 5
Authors: Brad Larson, Principal Scientist, Applications Department, BioTek Instruments, Inc., Winooski, VT Hilary Sherman, Corning Life Sciences, Corning, NY
Metastasis is the main cause of death in cancer patients and one of the most complex biological processes in human diseases1. The development of therapies designed to forestall the metastatic activity of tumors has been met with multiple challenges. The first is the initial focus on single target remedies. As many types of cancers develop multiple mutations during tumor progression2, individual cancers are often little affected by this type of drug. The advancement of novel methods that allow for the discernment of the effect a potential therapy has on the invasive phenotype of a particular type of cancer has proven invaluable to circumventing these early failures. A second hurdle is the choice of an appropriate cell model. Tumors in vivo exist as a three-dimensional (3D) mass of multiple cell types, including cancer and stromal cells3. Therefore, incorporating a 3D spheroid-type cellular structure that includes co-cultured cell types forming a tumoroid, provides a more predictive model than the use of individual cancer cells cultured on the bottom of a well in traditional two-dimensional (2D) format. A final hindrance which is necessary to overcome is the proper analysis of microscopic images captured of the tumor invasion process. The ability to observe changes in tumoroid size, as well as individual cell movement through the matrix in a quantitative way, is critical.
Here we demonstrate a method for the generation of 3D spheroidal tumoroid structures, creation of a suitable invasion matrix, image-based monitoring of tumor invasion, and cellular analysis of captured images. Corning Spheroid Microplates were incorporated for tumoroid formation, and performance of the invasion process. Tumor invasion tracking was performed by the Cytation 3 using digital microscopy. Cellular analysis of captured images was carried out with Gen5 Data Analysis Software. Multiple analysis methods using either brightfield or fluorescence microscopy will be presented.
Materials and Methods
MDA-MB-231 RFP cells (Catalog No. AKR-251) were purchased from Cell Biolabs, Inc. (San Diego, CA, USA). Human neonatal dermal fibroblasts (Catalog No. cAP-0008RFP) were purchased from Angio-Proteomie (Boston, MA, USA). Both cell types were propagated in Advanced DMEM Medium (Catalog No. 12491-015) plus Fetal Bovine Serum (FBS), 10% (Catalog No. 10437-028) and Pen-Strep-Glutamine, 1x (Catalog No. 10378-016) each from Life Technologies (Carlsbad, CA, USA).
Corning® Matrigel® Basement Membrane Matrix (Catalog No. 354234) was purchased from Corning Life Sciences (Corning, NY). Recombinant Human CXCL12/SDF-1 alpha (Catalog No. 350-NS-010) was purchased from R&D Systems (Minneapolis, MN).
Cytation 3 combines automated digital microscopy and conventional multi-mode microplate detection providing rich phenotypic cellular information and well-based quantitative data. With special emphasis on live-cell assays, Cytation 3 features temperature control to 45 °C (37 ± 0.5 °C), CO2/O2 gas control and dual injectors for kinetic assays. The instrument was used to image spheroids, as well as individual cell invasion through the Matrigel matrix.
Gen5 software controls the operation of the Cytation 3 for both automated digital microscopy and PMT-based microplate reading. Image acquisition is completely automated from sample translation, focusing and exposure control. Cellular analysis allows counting of individual cells breaking away from the tumoroid, as well as examination of the tumoroid as a single object.
Corning Spheroid Microplates
Corning® 96-well black, clear-bottom spheroid microplates (Catalog No. 4520) are coated with the Ultra Low Attachment surface which is a non-cytotoxic, and biologically inert covalently bonded hydrogel that prevents cell attachment. Novel well geometry aids spheroid formation in the center of each well. Each microplate contains an optically clear round bottom, which is ideal for cellular imaging, as well as a black, opaque body, which prevents cross talk.
Cell Preparation and Spheroid Formation
MDA-MB-231 and fibroblast cells were harvested and diluted to a concentration of 5.0x104 cells/ mL in complete medium. The two volumes were combined to create final concentrations of 2.5x104 cells/mL for each cell type. 100 μL of cell suspension was then pipetted to appropriate wells. Following dispensing, the plate was placed at 37 °C/5% CO2.
Image-Based Spheroid Formation Monitoring
Spheroid formation was monitored every 24 hours. The plate was placed into the Cytation 3, previously set to 37 °C/5% CO2 using Gen5 as well as a gas control module. Focusing was performed using the brightfield channel. The typical cell aggregation period was 48 hours.
Invasion Matrix Preparation
Upon spheroid formation completion, 70 μL of complete medium was removed from each well, and replaced by 70 μL of serum-free medium. This was repeated on a daily basis to create a serum starvation period of 48 hours. Matrigel matrix was then thawed on ice. When just thawed, the spheroid plate was placed on ice in a refrigerator for 5 minutes to cool the wells. With the plate still on ice, 100 μL of Matrigel was then added to each well. The plate was centrifuged at 300 x g for 5 minutes in a swinging bucket centrifuge that had been previously set to 4 oC. This positions the spheroids in the middle of the well. The plate was then transferred to a 37 °C/5% CO2 incubator for one hour to initiate gel formation.
Tumor Invasion Assay Performance
Following the one hour gel formation incubation period, 100 μL of 300 ng/mL (3x) CXCL12 was added to appropriate wells. The plate was then placed into the Cytation 3 which had been set to 37 °C/5% CO2. Using a 4x objectives, and the brightfield channel, a single focal height was selected that brought the spheroid into focus. Using the selected focal height, exposure settings were then optimized for the brightfield and RFP channels. This information was then entered into the imaging read step of the procedure. After the optimization process, automated imaging was performed, and continued every 24 hours pursuant, to track tumor invasion over a 5 day period.
Determination of Tumor Invasion using Cellular Analysis
Cellular analysis was performed with captured 4x images. Multiple methods were employed using either brightfield or RFP channels, which allowed the Gen5 software to examine changes in the main tumoroid structure, invadopodia projections, as well as individual cells invading into the matrix.
Results and Discussion
Analysis of Cell Migration
Images of a well containing 2.5x103 MDA-MB-231 RFP cells, and 2.5x103 fibroblasts captured after 0, 3, 4, and 5 days of incubation with 100 ng/mL CXCL12 (Figure 1). Brightfield, as well as fluorescence imaging using the RFP channel were carried out to fully visualize the extent of invasion on each day. Brightfield and RFP images were then overlaid to create the final composite images.
Figure 1. Image-based Monitoring of MDA-MB-231/Fibroblast Tumor Invasion. Overlaid brightfield and RFP images taken over a total 120 hour incubation period using a 4x objective.
Changes in tumoroid size and shape are observed throughout the incubation period. Invadopodia and individual cell invasion can also be visualized after 72 hours. The extent of each exhibit a steady increase from 72 to 120 hours of exposure to CXCL12.
Cellular Analysis Method #1: Individual MDA-MB-231 Cell Count
The goal of the first cellular analysis method employed was to count the number of MDA-MB-231 cells breaking away from the original tumoroid structure. Images captured with the RFP channel were used, in order to easily distinguish cells from background. Minimum and maximum object size limits were set so that only individual cells were identified (Table 1). The “Split touching objects” capability was also unchecked so that cells within the tumoroid would not be counted.
Table 1. Tumor Invasion Analysis Method #1 Criteria.
Figure 2. Cellular Analysis Method #1 Images and Results. (A.) 4x RFP images of objects counted using Table 1 criteria. (B.) Graph of individual MDA-MB-231 cells invading away from the tumoroid structure.
The graph in Figure 2B demonstrates that the number of MDA-MB-231 cells invading into the matrix, in response to CXCL12, increases steadily over the 120 hour incubation period.
Cellular Analysis Method #2: Invadopodia Measurement
The second cellular analysis method was designed to measure invadopodia extending away from the tumoroid and into the extracellular matrix (ECM). The role of these protrusions is the breakdown of ECM, thus aiding the invasion process. Brightfield images were used as the ECM and invadopodia are only visible with this channel. Minimum and maximum object size limits were increased, and “Split touching objects” was unchecked to be able to detect small and large protrusions (Table 2). Image smoothing also improved the ability to correctly identify each extension away from the tumoroid.
Table 2. Tumor Invasion Analysis Method #2 Criteria.
Figure 3. Cellular Analysis Method #2 Images and Results. (A.) 4x brightfield images showing invadopodia objects counted using Table 2 criteria. (B.) Graph of total area and perimeter covered by invadopodia per image.
The images in Figure 3A clearly illustrate that the activity of invadopodia from in vitro tumoroid structures is much the same as that referenced for tumors in vivo. These protrusions can also be identified, and their area and perimeter properly quantified, as seen in Figure 3B.
Cellular Analysis Method #3: Tumoroid Analysis using RFP Signal
The third cellular analysis method tracks changes in the original tumoroid as cells invade outward, through the use of the MDA-MB-231 cell RFP signal. The RFU threshold helps to discriminate between cell signal and background fluorescence. The larger minimum and maximum object sizes also ensure that the software “sees” the tumoroid as a single object instead of a group of small cells.
Table 3. Tumor Invasion Analysis Method #3 Criteria.
Figure 4. Cellular Analysis Method #3 Images and Results. (A.) 4x RFP images displaying object masks drawn around tumoroid structures using Table 3 criteria. Tumoroid size (B.), area (C.), and perimeter (D.), as well as mean RFP signal within each object mask (E.) graphed using results from identified objects.
Using the described parameters, cellular analysis information is taken solely from the individual identified objects (Figure 4A), while fluorescent signal from all other areas of the image is ignored. This allows any changes to the tumoroid to be tracked. The graphs in Figure 4B, C and E illustrate how the size, area, and RFP signal emanating from the tumoroid decrease as invasion proceeds. This can be explained by the fact that an increasing number of cells are moving away from the originally formed spheroid. Therefore the remaining structure shrinks in total size, in addition to the average fluorescent signal from within the object decreasing as well. The perimeter of the object mask, in contrast, increases proportionately during the incubation period (Figure 4D). This may seem erroneous at first. However, when examining the tumoroid structures and subsequently drawn object masks closely, it can be observed that the outer edge of the tumoroid becomes increasingly irregular as more cells continue to break away. This serves to increase the perimeter, even while the area is decreasing.
Cellular Analysis Method #4: Tumoroid and Invadopodia Analysis using Brightfield Signal
In the final cellular analysis method, the parameters set to monitor invadopodia are slightly modified to allow the tracking of both tumoroid changes and invadopodia development as one object. In this way all changes to the original structure are tracked as a whole. This is accomplished by an increase in the threshold RFU and minimum object size values, which enables Gen5™ to also include the tumoroid in the object mask.
Table 4. Tumor Invasion Analysis Method #4 Criteria.
Figure 5. Cellular Analysis Method #4 Images and Results. (A.) 4x brightfield images displaying object masks drawn around tumoroid and invadopodia structures using Table 4 criteria. Object size (B.), and area (C.), as well as total brightfield signal within each object mask (D.) graphed using results from identified objects.
The images in Figure 5A illustrate how the adjusted parameters allow for object masks to be drawn around all portions of the tumoroid and invadopodia. Using this method, not only can total changes in size and area of the original tumoroid be tracked (Figure 5B and C), but also the change in total brightfield signal from within the complete object as it changes during the invasion process (Figure 5D).
Cellular Analysis Summary
While all analysis methods shown above represent accurate, analytical approaches to measure tumor invasion, the relative change, and therefore potential ease of comparison from varying experimental conditions, differs depending on the evaluated effect. Tracking the original tumoroid structure, which represents a negative change, yields a relatively small shift in values when compared to data captured from the original structure (Figure 6A). Assessment of invasion, in comparison, yields larger relative increases over time. When monitoring cell movement away from the original tumoroid, a portion of the entire invasion process, an 8-fold change in values is seen over the incubation period (Figure 6B). Tracking the signal from individual invadopodia also yields a sizable increase in values (Figure 6C). Therefore, when the entire invasion process is surveyed (Figure 6D), as with Cellular Analysis Method #4, a substantial (and easily visualized) relative change is observed.
Figure 6. Cellular Analysis Method Relative Change Results. Data comparisons relative to time 0 values. (A.) Comparison of mean RFP signal captured within object masks drawn around tumoroid structure. (B.) Ratio of cells invading away from tumoroid. (C.) Brightfield signal captured from identified invadopodia over time. (D.) Ratio of brightfield signal captured within tumoroid and invadopodia.
3D tumor invasion assays are an important addition in the quest to generate relevant in vitro phenotypic data with potential new anti-metastatic drugs. Through the incorporation of Corning’s Spheroid Microplate, the generation of tumoroid structures and performance of the invasion assay is easily accomplished in the same microplate. Monitoring of the invasion process is completed in an automated fashion via imaging using the Cytation™ 3. Finally, with the addition of the Gen5™ Data Analysis Software, multiple cellular analysis methods become available to accurately quantify the invasive characteristics of cells, and their response to test molecules.
- Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144: 646-674. doi:10.1016/j. cell.2011.02.013. PubMed: 21376230.
- Wood LD, Parsons DW, Jones S, Lin J, Sjöblom T et al. (2007) The genomic landscapes of human breast and colorectal cancers. Science 318: 1108-1113. doi:10.1126/ science.1145720. PubMed: 17932254.
- Mao Y, Keller E, Garfield D, Shen K, Wang J. Stromal cells in tumor microenvironment and breast cancer. Cancer and Metastasis Reviews. 2013 32: 303-315.