We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.
Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.
Multicellular 3D spheroids have been known as a tumor model for decades, however it is only recently that they have come into more common usage as an in vitro model for many solid cancers. They are increasingly being used in high-throughput drug discovery screens as an intermediate between complex, expensive and time-consuming in vivo models and the simple, low cost 2D monolayer model 1-6. Studies in 2D culture are often unable to be replicated in vivo. Spheroid models of many types of cancer are able to mimic the growth characteristics, drug sensitivity, drug penetration, cell-cell interactions, restricted availability of oxygen and nutrients and development of necrosis that is seen in vivo in solid tumors 6-11. Spheroids develop a necrotic core, a quiescent or G1 arrested region surrounding the core, and proliferating cells at the periphery of the spheroid 7. The development of these regions may vary depending on the cell density, proliferation rate and the size of the spheroid 12. It has been hypothesized that the cellular heterogeneity seen in these different sub-regions may contribute to cancer therapy resistance 13,14. Therefore the ability to analyze cells in these regions separately is crucial to understanding tumor drug responses.
The fluorescence ubiquitination cell cycle indicator (FUCCI) system is based on the red (Kusabira Orange – KO) and green (Azami Green – AG) fluorescent tagging of Cdt1 and geminin, which are degraded in different phases of the cell cycle 15. Thus cell nuclei appear red in G1, yellow in early S and green in S/G2/M phase. We describe here two complementary methods both using FUCCI to identify the cell cycle, along with the use of imaging software or a dye diffusion flow cytometry assay to determine whether cells reside in the G1 arrested center or the outer proliferating ring, and the distance of individual cells from the edge of the spheroid. These methods were developed in our previous publication, where we demonstrated that melanoma cells in hypoxic regions in the center of the spheroid or/and in the presence of targeted therapies are able to remain in G1 arrest for extended periods of time, and can re-enter the cell cycle when more favorable conditions arise 7.
1. FUCCI Transduction and Cell Culture
2. 3D Spheroid Formation (as previously described 3,9)
3. Spheroid Vibratome Sectioning
4. Confocal Image Acquisition
5. Hoechst Dye Diffusion Assay for Flow Sorting
6. Flow Cytometry Analysis of Hoechst Stained FUCCI Spheroids
7. Image Analysis of FUCCI Spheroid Sections
There are several methods of producing tumor spheroids, this protocol uses the non-adherent growth method, where cells are cultured on agar or agarose 3,7,9. Figure 1 shows an example of a C8161 melanoma spheroid after 3 days on agar. C8161 spheroids form regular sized spheroids with a diameter of 500 – 600 μm (mean = 565, SD = 19, n = 3) after 3 days. Other melanoma cell lines that will form spheroids include: WM793, WM983C, WM983B, WM164, 1205lu (spheroids formed with this cell line are irregular and less dense 19).
In order to visualize the cell cycle of individual cells within the 3D spheroid model, C8161 melanoma cells were transduced with the FUCCI system 7,15. Due to the large size of the spheroids (up to 1 mm in diameter after 3 days on agarose and 24 hr growth in collagen for C8161), sectioning is the best option for visualizing cells in the center of the spheroid. Whole spheroids (live or fixed) may be imaged by confocal microscopy, however the confocal imaging is only able to penetrate up to approximately 150 μm, therefore a middle optical slice of the spheroid can only be obtained in spheroids with a diameter smaller than 300 μm. Figure 2A, B and C shows an example of a section through a C8161 FUCCI spheroid. A necrotic core, surrounded by G1 arrested cells, with a gradient of proliferating cells in the outer layers is evident. To identify and quantify cells based on their position in the spheroid and cell cycle status, semi-automated image analysis was performed. Figure 2D shows the FUCCI cell masks and spheroid outline, and Figure 2E shows the quantification of the numbers of red (G1) and green or yellow (S/G2/M) cells either less than 80 μm from the edge of the spheroid (outer cells) or greater than 80 μm from the spheroid edge (inner cells). This quantification shows that red cells in G1 are greatly enriched in the inner region of the spheroid, as expected.
In order to identify and potentially sort cells based on their cell cycle status and position in the spheroid, a Hoechst dye diffusion assay in combination with the FUCCI system was used. Incubation of whole spheroids with Hoechst dye results in a limited diffusion of the dye into the outer layers of the spheroid, this may be used to separate Hoechst positive outer layer cells from Hoechst negative inner spheroid cells via flow cytometry. Figure 2F shows the penetration of Hoechst up to approximately 80 μm from the spheroid edge. The Hoechst positivity distance of 80 μm from the edge was obtained by visual analysis of spheroid sections and optimization of the dye concentration and incubation time so that the Hoechst penetration closely marked the proliferating cells in the outer layers (which are largely found less than 80 μm from the edge), and did not penetrate into the G1 arrested area.The incubation time with Hoechst may be varied to obtain deeper or shallower penetration. An example of varying Hoechst penetration over time in demonstrated in Figure 3. Figure 4A shows an example of the gating for Hoechst high and low populations, while Figure 4C and D demonstrates the gating for FUCCI red, yellow and green cells. Figure 4B shows the quantification for the number of red (G1) and green or yellow (S/G2/M) cells either in the Hoechst high (outer cells) or Hoechst low (inner cells). Again this technique shows that red cells in G1 are greatly enriched in the inner region of the spheroid (cf. similarity to the image analysis in Figure 2E).
Figure 1: C8161 Spheroid. Representative phase contrast image taken at 10X magnification of a C8161 spheroid after 3 days culture on agar. Scale bar equals 100 μm. Please click here to view a larger version of this figure.
Figure 2: C8161 FUCCI Spheroid Image Analysis. Representative image of a C8161 FUCCI spheroid vibratome section taken at 20X magnification. Spheroid was cultured for three days on agarose, then a further 24 hr in collagen matrix. Confocal z-slice of (A) Azami Green, (B) Kusabira Orange2 and (C) FUCCI overlay. Scale bar = 100 μm. (D) Red and Green object masks created in Volocity, with the spheroid outline in grey. Arrow indicates 80 μm distance from the spheroid edge. (E) Quantification of the numbers of red (G1) and green/yellow (S/G2/M) cells within the inner (>80 μm from the spheroid edge) and outer (<80 μm from the spheroid edge) regions. Error bars represent the SD from 4 spheroid sections from 2 independent experiments. (F) Penetration of 10 μM Hoechst dye after 1.5 hr incubation. Scale bar = 100 μm. Please click here to view a larger version of this figure.
Figure 3: C8161 Spheroid Hoechst Dye Diffusion Time Course. Representative confocal z-slice images from the middle of whole C8161 spheroids cultured on agarose for 4 days and incubated with 10 μM Hoechst for the indicated times. Taken at 10X magnification. White bars indicate the approximate Hoechst penetration depth. Note that C8161 agarose spheroids are denser than the C8161 spheroids that have been implanted in collagen for 24 hr in Figure 2, resulting in less dye penetration at the same time point. Please click here to view a larger version of this figure.
Figure 4: C8161 FUCCI Spheroid Flow Analysis. (A) Example of Hoechst high and low gating after incubation of 20 spheroids with 10 μM Hoechst. Blue line indicates the unstained control, green line indicates a fully stained Hoechst control. (B) Quantification of the numbers of red and green/yellow cells within the inner (Hoechst low) and outer (Hoechst high) populations. Error bars represent the SD from 8 independent experiments (including both live sorting and fixed cell analysis). Example of FUCCI gating for the inner (C) and outer (D) spheroid populations. Please click here to view a larger version of this figure.
Semi-automated image analysis identified the spheroid inner G1 arrested region, and proliferating outer layers. This method may be used on live spheroids using an optical section, or in fixed spheroid sections, to identify changes in not only the cell cycle but marker expression (via immunofluorescence), cell death, or cell morphology in these different regions. Cell motility within different spheroid regions may also be quantified – if live confocal time lapse imaging along with a cell tracking image analysis step is added. Critical to image analysis is that high quality z- slice confocal images are obtained, higher resolution images allow better identification of cells. One limitation with image analysis is that it is not possible to find every cell correctly if nuclei are too dense, or if there is too much variation in the red and green intensity. FUCCI negative cells (in early G1) are not able to be found with this image analysis method, only red (G1), yellow (early S phase) and green (S/G2/M). One other drawback of the image-based analysis method described here is that it does not take into account the full 3D nature of the spheroids. Although the Volocity software can perform 3D measurements using z-stack images, confocal microscopy is unable to penetrate and image through the entire spheroid due to the large size. Multi-photon imaging may be used to obtain a z-stack of a whole spheroid for 3D measurements 7 as this technology allows penetration of up to 500 μm, although some spheroids may still be too large to image whole spheroids via this method. Another option for imaging the whole spheroid is light-sheet-based microscopy, which allows visualization of the spheroid from multiple angles and penetrates up to 200 μm inside large spheroids 20,21. Choice of imaging strategy may be influenced by the spheroid size, cell packing density and cell type.
The Hoechst dye diffusion method for separating inner and outer cells in a multicellular spheroid via flow cytometry was first described in 1982 22. This method is based on the fact that the Hoechst dye diffuses slowly into the spheroid, with the outer cells being stained first. The protocol described here combines the Hoechst method with the FUCCI system 15, which allows not only separation of inner and outer spheroid cells, but also visualization of Hoechst penetration with respect to the inner ring of G1 arrested cells. This allows accurate separation of the G1 arrested region of the spheroid. Using FUCCI alone does not allow the separation of the inner G1 arrested cells from a spheroid, given that the outer proliferating cells also contain cells in G1. A limitation is that this method only allows a crude separation of the spheroid into two regions (“outer” Hoechst positive and “inner” Hoechst negative). The benefit of this method over image analysis, is that flow cytometry allows multiple markers to be tested at once, and physical separation of live cells for further downstream analysis such as gene or protein expression, re-culturing in different environmental conditions, drug treatments or other analysis.
The FUCCI spheroid system in combination with flow cytometry and image analysis allows the identification of an inner ring of G1 arrested cells surrounding the necrotic core, and an outer layer of proliferating cells. The necrosis and G1 arrest found in the center of the spheroid is due to lack of oxygen and nutrients, and develops as the spheroid grows in size. Previously, we and others have shown co-localization of the pimonidazole hypoxia marker with the G1 arrested spheroid center 7,23. We also demonstrate that proliferation largely occurs within approximately 100 μm from the edge of the spheroid. This matches previous studies in spheroids 12,23,24, and also correlates with the distance of proliferating cells from the nearest vasculature in vivo 25,26.
An alternative method that may be used to separate cells based on their position in the spheroid for flow cytometry or other analysis is sequential trypsinization 24,27. In this method successive “shells” of the spheroid are removed by short, sequential incubation periods with trypsin at low temperatures. However using this method it is more difficult to ascertain the exact size of the outer shell (in μm thickness) that is removed compared to the Hoechst dye diffusion method, where the Hoechst penetration may be directly visualized and measured.
Separating the “inner” and “outer” cells by dye diffusion and/or image analysis can be extended to studies of FUCCI tumor xenografts in mice. However, for analysis of the cell cycle in vivo, the presence of vasculature should also be taken into account, as cells in a tumor may be able to access oxygen and nutrients from vasculature in the central regions.
The authors have nothing to disclose.
We thank Ms. Danae Sharp and Ms. Sheena Daignault for technical assistance. We thank Dr. Atsushi Miyawaki, RIKEN, Wako-city, Japan, for providing the FUCCI constructs, Dr. Meenhard Herlyn and Ms. Patricia Brafford, The Wistar Institute, Philadelphia, for providing cell lines, the Imaging and Flow Cytometry Facility at the Centenary Institute for outstanding technical support. We thank Mr. Chris Johnson and Dr. Andrew Barlow for Volocity software technical support. N.K.H. is a Cameron fellow of the Melanoma and Skin Cancer Research Institute, Australia. K.A.B. is a fellow of the Cancer Institute New South Wales (13/ECF/1-39). W.W. is a fellow of the Cancer Institute New South Wales (11/CDF/3-39). This work was supported by project grants RG 09-08 and RG 13-06 (Cancer Council New South Wales), 570778 and 1051996 (Priority-driven collaborative cancer research scheme/Cancer Australia/Cure Cancer Australia Foundation), 08/RFG/1-27 (Cancer Institute New South Wales), and APP1003637 and APP1084893 (National Health and Medical Research Council).
Hoechst 33342 | Life Technologies | H3570 | |
agarose low melting point | Life Technologies | 16520-050 | For sectioning |
noble agar | Sigma | A5431 | For making spheroids |
agarose for spheroids | Fisher Scientific | BP1356-100 | For making spheroids |
0.05% trypsin/EDTA | Life Technologies | 25300-054 | |
HBSS | Life Technologies | 14175-103 | |
10% formalin | Sigma | HT5014-1CS | CAUTION: Harmful, corrosive. Use Personal Protective Equipment, do not breath fumes (open in a fume cupboard). |
live/dead near IR | Life Technologies | L10119 | |
vibratome | Technical Products International, Inc | ||
coulture cup | Thermo-Fisher Scientific | SIE936 | Mold for sectioning spheroids |
hemocytometer | Sigma | Z359629 | |
96-well tissue culture plate | Invitro | FAL353072 | |
collagenase | Sigma | C5138 | |
confocal microscope | Leica | TCS SP5 | |
Flow cytometer analyser | Becton Dickinson | LSRFortessa | |
volocity | PerkinElmer | Imaging software | |
flowjo | Tree Star | Flow cytometry software | |
Vaccuum grease | Sigma | Z273554 | |
Mounting media | Vector Laboratories | H1000 | |
FUCCI (commercial constructs) | Life Technologies | P36238 | Transient transfection only |
Cell strainer 70 um | In Vitro | FAL352350 | |
Round bottom 5 mL tubes (sterile) | In Vitro | FAL352003 | |
Round bottom 5 mL tubes (non-sterile) | In Vitro | FAL352008 |