Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice.
Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.
Recent cancer research has made significant advancements in identifying genetic mutations, molecular changes and possible treatments for a variety of brain tumors. Despite this progress, brain tumors remain one of the top causes of cancer-related mortality for adults and children. Limiting factors in brain tumor research include the restricted availability of primary patient samples and cell lines and the difficulty in replicating the unique and heterogeneous brain microenvironment in accessible experimental systems. For many brain tumors the conditions required to maintain tumor cells over time are not yet known. Even for brain tumors that can be grown in cell suspension as neurospheres, culture conditions may affect the tumor cells1,2. Indeed, the addition of basic fibroblast growth factor or epidermal growth factor to encourage proliferation and inhibit differentiation may alter gene expression1. Other methods for tumor cell growth such as tumor propagation in mice via orthotopic or subcutaneous xenograft of tumor cells are valuable assays, but are limited by factors such as time of tumor development (especially for low grade tumors), cost, and the number of tumor cells that can be injected and studied. Thus current methods for growing human brain tumor cells are inadequate for maintaining certain tumor types, and often provide artificial environments that do not closely mimic in vivo tumor environments.
Distinct types of pediatric brain tumors grow in highly specialized locations within the brain[3, 4] and this is likely to reflect distinct microenvironmental requirements for tumor growth[5]. This protocol describes a novel system where cells that are difficult to propagate in normal culture conditions can be grown in an organotypic brain microenvironment which mimics in vivo tumor growth conditions. In this quantitative assay, fluorescently labeled brain tumor cells are plated on juvenile mouse brain organotypic slices and monitored over time. This assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies in a clinically relevant brain microenvironment.
Ethics Statement: The following procedure involving animal subjects were done in accordance with the National Institutes of Health guidelines and were approved by the Dana-Farber Cancer Institutional Animal Care and Use Committee. All human subjects work was reviewed by the Institutional Review Board Committees of the Brigham and Women's Hospital and Dana-Farber Cancer Institute, and by Stanford University for appropriate use, that informed consent was obtained from all subjects when required, and appropriate waiver of consent requirements was obtained for minimal risk studies.
Timeline of Slice Culture Protocol:
Figure 1. Timeline of Brain Tumor/ Organotypic Slice Co-culture Protocol. This figure depicts the timeline of the slice culture procedure encompassing all eight days of the experiment and major steps of the procedure. The timeline is relative to Day 0 when the cells are plated onto the slice in order to highlight the importance of beginning the procedure days before plating cells. Please click here to view a larger version of this figure.
1. Dissection Buffer
2. Slice Culture Media
4. Coat the Slice Culture Inserts with Laminin
Note: The following procedure can be done up to one day before starting the dissection.
5. Preparation for Dissection
6. Dissection
Note: Process pups one at a time.
Figure 2. Dissection Cuts. These images show the necessary dissection cuts to remove the skull from the brain of a P6 mouse. Cuts are shown as dotted lines. (A) Cut 1 is shown. Cuts 1 and 2 are made from the brainstem/posterior bilaterally connecting to the eye socket on each side.. (B) Cuts 3 and 4 are shown. Cut 3 is made from one eye socket to the other connecting Cuts 1 and 2. Cut 4 begins at the midline of cut 3 and continues towards the tip of the nose dividing the skull between the olfactory bulbs (Scale bar = 4.4 mm). Please click here to view a larger version of this figure.
7. Embedding the Brains in Agarose
8. Slicing with the Vibratome
9. Plating the Slices onto the Inserts
10. Changing the Slice Culture Media
11. Plating Tumor Cells on the Slice
12. Imaging and Fixing
Note: a Nikon Eclipse Ni C2si upright confocal was used to take a large image scan of the whole sagittal slice at 4X with red and green fluorescent channels. If scanning feature is not available, take multiple images sequentially across the slice and later stitch the images together in Photoshop (using the location of microspheres and the edge of the slice to navigate).
13. Quantification of Images (Using ImageJ)
Note: ImageJ settings may need to be adjusted and optimized to account for image and microscope quality as well as tumor cell size.
14. Staining
15. Mounting the Slices
This section exemplifies the type of results to be expected from utilizing the brain tumor/organotypic slice co-culture to investigate regional microenvironment preference as well as to test new therapies. We show that the assay is designed to replicate the microenvironment for brain tumors, as the tissue organization and proliferative state of the slice is maintained (Figure 3). We also demonstrate that an increase in the number of tumor cells on the slice over time may partially be due to the migration of cells onto the brain slice microenvironment (Figure 4). We have also shown that this co-culture can be used as a quantitative assay by calculating the fold change in cell number over the week in culture for specific regions of the slice, or for the entire brain slice (Figure 6) Preliminary results from multiple tumor cell types have shown that the number of tumor cells can be quantified by confocal imaging of the 200 µm thick slice due to the fact that the cells only migrate to a depth of 20-50 µm into the slice.
We have found that green fluorescent microspheres are an effective control for changes in slice topography because they show no movement or change in number throughout the time in culture (Figure 5). After fixing the co-culture, staining can be done to examine tumor cells in a brain microenvironment and the effects of drugs on tumor cell proliferation, cell death, or changes in protein expression (Figure 7). We have demonstrated that this assay can be used to test drug therapies through a comparison of mouse medulloblastoma cells treated with a Smo (Smoothened) antagonist LDE225 (Sonidegib) or with a vehicle control. Fold increase in cell number on the slice over one week in culture was quantified and represented graphically (cell number on Day 7/cell number on Day 1) These data indicate that LDE225 greatly decreases tumor cell number in comparison to the control6. This effect can also be seen in the representative images included (Figure 8). We also show images of human medulloblastoma cells grown in the slice culture assay (Figure 9).
Figure 3. Brain Tumor/ Organotypic Slice Co-culture Assay Design. (A) Organotypic sagittal brain slice from a P6 mouse is placed directly on a semi-porous membrane and medium is added to the bottom of the culture dish. The culture is immersed in the medium on one side and is accessible to oxygen from the other side. Labeled tumor cells are overlaid onto the slice and cell number can be followed over time. (B) In culture, the slice maintains a tissue organization that closely resembles that observed in vivo. Preservation of the brain microenvironment is demonstrated by EdU labeling of proliferative granule neuron precursors in the external granule layer (EGL) of the cerebellum within the slice culture. In the slice culture, these precursor cells incorporate the thymidine analog EdU, as would be observed in vivo at this developmental stage (P6) (white arrow indicates EGL). Please click here to view a larger version of this figure.
Figure 4. Human Astrocytoma Cells in Brain Tumor/ Organotypic Slice Co-culture Assay. An increase in human brain tumor cells on the slice may reflect relocalization of tumor cells from the membrane to the slice culture. (A) An area on the edge of a slice at Day 1 (top) and Day 7 (bottom) where cells may migrate from the membrane onto the brain slice. (B) A second example of possible cell migration onto the brain at the edge of the slice. Day 1 (top) and Day 7 (bottom). Please click here to view a larger version of this figure.
Figure 5. Microsphere Control Beads. Day 1 (red) and Day 7 (green) control images of fluorescent microspheres are overlaid (yellow) to reveal no movement of the microspheres over a week in culture. Day 1 and Day 7 images were taken at the same position within the slice (Scale bar = 45 µm). Please click here to view a larger version of this figure.
Figure 6. Quantification in ImageJ. (A) The image of a slice is opened in ImageJ and the whole slice and/or regions are outlined for quantification. (B) The region of interest is selected and a duplicate image is made. (C) The background fluorescence is subtracted out of the image. (D) The threshold is set for cell size and shape, determining what will be counted. (E) Analyze particles to count the cell number within the region of interest. Fold change in cell number can then be calculated by dividing the number of cells on Day 7 by the number of cells on Day 1 for each region of interest or the entire area of the slice. Please click here to view a larger version of this figure.
Figure 7. Staining for Proliferation. Image exemplifies how staining (after fixing the slice in PFA) can successfully be done on the slice after one week in culture. Image shows DAPInuclear staining (blue), red fluorescently labeled mouse medulloblastoma cells, and green labeling for incorporation of Thymidine analogue into DNA as a marker for proliferation. EDU was included in the slice culture media (10 µM) for one week (white arrows indicates cells positive for EDU and yellow arrows indicate cells negative for EDU) (Scale bar = 50 µm). Please click here to view a larger version of this figure.
Figure 8. Mouse Medulloblastoma Cells Treated with LDE225. This figure demonstrates drug treatment of tumor cells in the slice overlay assay system. (A) Graphical representation of data collected from a mouse medulloblastoma experiment. DMSO was the control condition (CTL) and LDE225 (Sonidegib) (1 µM) (LDE) was the treatment condition. Fold increase in cell number on the slice over one week in culture was quantified (cell number on Day 7/cell number on Day 1), (n = 12 CTL, n = 13 LDE, Error bars = SEM). (B) Representative images of vehicle control treated slices on Day 1 (top) and Day 7 (bottom). (C) Representative images of LDE treated slices on Day 1 (top) and Day 7 (bottom). Images were taken at 4X magnification. Please click here to view a larger version of this figure.
Figure 9. Human Medulloblastoma Cells in Brain Tumor/Organotypic Slice Co-culture. Human medulloblastoma cells were obtained from James M. Olson at the University of Washington, and were grown for one week in the slice culture assay. (A) Day 1 image of human medulloblastoma cells grown on the mouse brain slice. (B) An image of the same mouse brain slice with human medulloblastoma cells after one week in culture (Day 7). Images were taken at 4X magnification. Please click here to view a larger version of this figure.
This protocol describes how brain tumor cells can be fluorescently labeled and plated on a sagittal brain section of a P6 mouse and then monitored for one week in culture. This brain tumor/organotypic slice co-culture assay can be used to determine the effect of regional microenvironment on tumor cell number and may also be used as a system for measuring the efficacy of new drug treatments on human tumor growth. Previous studies have used a similar strategy to assess the role of brain micro-environment on neural precursor proliferation7,8.
This assay in which brain tumor cells are seeded in an organotypic slice culture9 provides a system for growing human brain tumor cells that are difficult to propagate in normal cell culture conditions. One critical aspect of this procedure is the importance of maintaining the health of the slice and the tumor cells. The length of time that the slices sit in buffer during the dissection and at RT during imaging should be limited, to ensure that the slices and cells remain as healthy as possible. If primary human brain tumor cells are being used in this assay, the cells should be plated on the slices as soon as possible after biopsy. Therefore the slices should be prepared before surgery. To prevent any differential effect of imaging time among slices, the amount of time spent out of the incubator should be short and consistent for all slice cultures in an experiment. The microsphere controls are important as they provide spatially consistent marks over time. In addition, distortions in the slice culture due to shrinkage, tearing, or folding are readily apparent by imaging the microsphere beads.
One of the limitations of this protocol is that the cells can only be propagated in this slice culture system for approximately one week. Nonetheless, for some types of brain tumors this is a significant improvement over the current state of affairs. A second limitation is that the blood-brain-barrier is eliminated, which is an important consideration in evaluating drugs that may be used for treating brain tumors. A third consideration is that this is a low throughput assay that cannot be used for testing a library of compounds.
This assay is versatile and easily modified to study different types of tumor cells and a variety of investigative questions. This protocol can be used as a quantitative assay to examine the effects of novel therapies on tumor cell survival and growth (Sun et al., personal communication). A comparison of the fold change in tumor cell number in distinct regions of the brain provides a method for deciphering the effects of microenvironment on tumor growth.
This brain tumor/organotypic slice co-culture system also provides the possibility to investigate the relationship between brain tumor cells and specific brain microenvironments. Many types of brain tumors show a distinct pattern of developing in specific brain regions and microenvironments3,4. By growing labeled human tumor cells on a sagittal mouse brain slice, cells can be monitored for regional preference which may be consistent with the in vivo region of growth. Further examination of the microenvironment that the tumor cells prefer could lead to the identification of potential factors that support tumor cell growth. This assay may aid in improving in vitro cell culture conditions and may also provide a system to study how tumor initiation can be prevented or more effectively treated.
There are specific types of brain tumors which can not be propagated in normal cell culture conditions or by mouse orthotopic or subcutaneous xenografts. For these tumor types it is difficult to test new drug therapies on human tumor cells. This protocol demonstrates that human brain tumor cells can be grown in the slice culture assay and drug treatments can be assessed quantitatively. Following drug treatment, co-staining of the tumor cells can provide further evaluation of the drug’s effect on tumor cell proliferation and pathway inhibition. Recent studies have shown that there may be a drastic difference between a drugs effect on cancer cells in a normal monolayer cell culture vs. a 3D heterogeneous cell culture environment10. Similarly adult normal and tumor epithelial cells, which have a short lifespan in vitro, have been shown to conditionally reprogram to a proliferative state when grown on fibroblast feeder cells in combination with a Rho kinase inhibitor11. These studies further support the importance of maintaining tumor cells in an organotypic, 3D, clinically relevant microenvironment, and the relevance of this cell culture system to current cancer research. Therefore, this is a valuable assay for testing drugs on human tumor cells in a clinically relevant manner.
The authors have nothing to disclose.
This work is supported by grants from the NIH (P01CA142536 to RAS, T32CA009361 to DPY) and the Pediatric Low Grade Astrocytoma foundation.
HEPES | Invitrogen | 17504044 | |||
Glucose | Invitrogen | 17502048 | |||
Pennicillin Streptomycin | Life Technologies | 15140-122 | |||
HBSS | Life Technologies | 14185-052 | |||
B-27 | Life Technologies | 17504-044 | |||
N2 | Life Technologies | 17502-048 | |||
Glutamax | Life Technologies | 35050061 | |||
Neurobasal-A- Medium minus phenol red | Invitrogen | 12349015 | |||
Low Melting Point Agarose | Promega | V2111 | |||
Slice Culture Inserts | Milipore | PICM0RG50 | |||
laminin | Invitrogen | 23017015 | |||
Cm-DiI | Invitrogen | V22888 | |||
EDU (Labeling and Detection) | Life Technologies | c10337 | |||
Microspheres | Life Technologies | F-21010 | |||
Vibratome | Leica | N/A | |||
Confocal Microscope | Nikon Eclipse Ni C2si | N/A | |||
Image J software | N/A | N/A | |||
5mm Cover Glasses | Fisher Scientific | 64-0700 (CS-5R) |