The authors thank Chalid Ghadban for his excellent technical assistance, Andreas Wodarz and Stephan Heermann for their technical advice regarding confocal and time-lapse microscopy. There is no conflict of interests for any of the authors. This work is funded by the German Research Council (DFG) in Project 2 of Forschergruppe 942 (FOR942 BI 703/3-1), by the Dres. Bayer-Stiftung (Baden W&#252;rttembergischer Krebspreis, Germany) and by the Research Program of the Faculty of Medicine, Georg-August-University G&#246;ttingen, Germany.

This new model is an adaptation of a previously published organotypic hippocampal brain slice approach9-12. Modifications and additions were introduced to optimize the cancer cell-brain tissue interactions and to guarantee reproducibility. The study was reviewed and approved by the local ethics committee. Animals were treated carefully according to the guidelines for animal care at the University Medicine G&#246;ttingen. The organotypic brain slice coculture can be subdivided into two steps. Step one is the preparation of the organotypic brain slice. Step two comprises the tumor cell preparation and deposition in the coculture model.
1. Organotypic Brain Slice
<ol>
	<li>Prepare the dissection medium consisting of minimum essential medium (MEM), supplemented with 0.2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 4.5 mg/ml of glucose.</li>
	<li>Decapitate the mice from any mouse strain between postnatal day six and eight (P6-8).</li>
	<li>Remove the brain rapidly from the skull under aseptic conditions and transfer it to ice-cold dissection medium.</li>
	<li>Remove the frontal pole and the cerebellum from the whole brain section.</li>
	<li>Fix and stabilize the brain on a stage with cryo glue and 5% agarose.</li>
	<li>Slice the brain sections horizontally to a 350 &#956;m thickness by using a vibratome.</li>
	<li>Collect four to six whole brain slices from a single mouse brain, depending on the species and age.</li>
	<li>Prepare the culture medium consisting of 50% MEM, 25% Hanks&#39; balanced salt solution (HBSS), 25% normal horse serum (NHS), 0.2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin (Sigma, Munich, Germany), and 4.5 mg/ml glucose.</li>
	<li>Put each organotypic brain slice on a 0.4 &#956;m polycarbonate transwell membrane insert in a six-well plate with 1 ml of cultivation medium in the lower well.</li>
	<li>Culture organotypic brain slices overnight in a humidified atmosphere with 5% CO2 at 37 &#176;C incubator.</li>
</ol>
2. The Slice Coculture Model
<ol>
	<li>Embed 105 of GFP-transfected tumor or other cells (e.g. MCF-7-GFP cells) in 20 &#956;l gel matrix, consisting of 15% RPMI medium and 85% ECM gel.</li>
	<li>Place the MCF-7- gel matrix mix into a sterile metallic spacer (3.8 mm diameter) directly adjacent to the cortical region of the organotypic brain slice and incubate for 2 hr.</li>
	<li>Remove the spacer and allow the 3D tumor spheroid to coculture with the organotypic slice for 24-96 hr.</li>
	<li>Change the culture medium every other day.</li>
</ol>
3. Immunofluorescence Staining of Astrocytes and Microglial in the Organotypic Brain Slice Coculture
<ol>
	<li>Fix the organotypic brain slice coculture with 4% paraformaldehyde for 8 hr at 4 &#176;C.</li>
	<li>Wash the slice coculture with PBST (PBS with 0.5% Triton X-100) for 5 min.</li>
	<li>Block the samples with normal goat serum in PBST (1:20) at room temperature for 1 hr.</li>
	<li>Stain the astrocytes by incubating the brain slice coculture with anti-glial fibrillary acidic protein monoclonal antibody (GFAP, 1:200 in PBST) for 36 hr at 4 &#176;C followed by goat anti-mouse-TRITC (1:100 in PBST) staining for 1 hr at room temperature.</li>
	<li>Wash the samples with PBST three times for 5 min. </li>
	<li>Stain the microglial cells with ILB4-Alexa Fluor 647 (1:100 in PBST) for 1 hr at room temperature.</li>
	<li>Counterstain the brain slice coculture with DAPI (1:1,000) for 3 min at room temperature.</li>
	<li>Mount and coverslip the brain slice coculture with DAKO fluorescent mounting medium.</li>
	<li>Evaluate the grade of tumor invasion based on the following scoring system: 0 = none of the cells; + &#60; 1/3; ++ = 1/3 - 2/3; +++ &#8805; 2/3 of the invaded cells (first measure the length of the contact section between the tumor plug and slice, then measure the fraction of contact detectable by the invading cells).</li>
</ol>
4. Live Imaging of the Interaction between Glial and Tumor Cells
<ol>
	<li>Perform the experiment under a Leica inverted DMI 6000B microscope at 10X magnification lens and a Leica DFC 350 FX CCD camera.</li>
</ol>

<ol>
	<li>Langley, R. R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+seed+and+soil+hypothesis+revisited--the+role+of+tumor-stroma+interactions+in+metastasis+to+different+organs.">The seed and soil hypothesis revisited--the role of tumor-stroma interactions in metastasis to different organs.</a> Int. J. Cancer. 128, 2527-2535 (2011).</li><li>Joyce, J. A., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+metastasis.">Microenvironmental regulation of metastasis.</a> Nat. Rev. Cancer. 9, 239-252 (2009).</li><li>Mantovani, A., Allavena, P., Sica, A., Balkwill, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-related+inflammation.">Cancer-related inflammation.</a> Nature. 454, 436-444 (2008).</li><li>Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+organ+microenvironment+in+brain+metastasis.">The role of the organ microenvironment in brain metastasis.</a> Semin. Cancer Biol. 21, 107-112 (2011).</li><li>Rath, B. H., Fair, J. M., Jamal, M., Camphausen, K., Tofilon, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+enhance+the+invasion+potential+of+glioblastoma+stem-like+cells.">Astrocytes enhance the invasion potential of glioblastoma stem-like cells.</a> PloS one. 8, e54752 (2013).</li><li>Steeg, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+metastasis:+mechanistic+insights+and+clinical+challenges.">Tumor metastasis: mechanistic insights and clinical challenges.</a> Nat. Med. 12, 895-904 (2006).</li><li>Eichler, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+brain+metastases-translation+to+new+therapies.">The biology of brain metastases-translation to new therapies.</a> Nat. Rev. Clin. Oncol. 8, 344-356 (2011).</li><li>Winkler, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+glioma+cell+invasion+in+vivo+reveals+mechanisms+of+dissemination+and+peritumoral+angiogenesis.">Imaging glioma cell invasion in vivo reveals mechanisms of dissemination and peritumoral angiogenesis.</a> Glia. 57, 1306-1315 (2009).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J. Neurosci. Methods. 37, 173-182 (1991).</li><li>Kreutz, S., Koch, M., Ghadban, C., Korf, H. W., Dehghani, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+and+neuronal+damage:+differential+effects+of+THC,+AEA+and+2-AG+on+activated+microglial+cells+and+degenerating+neurons+in+excitotoxically+lesioned+rat+organotypic+hippocampal+slice+cultures.">Cannabinoids and neuronal damage: differential effects of THC, AEA and 2-AG on activated microglial cells and degenerating neurons in excitotoxically lesioned rat organotypic hippocampal slice cultures.</a> Exp. Neurol. 203, 246-257 (2007).</li><li>De Simoni, A., Yu, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+organotypic+hippocampal+slice+cultures:+interface+method.">Preparation of organotypic hippocampal slice cultures: interface method.</a> Nat. Protoc. 1, 1439-1445 (2006).</li><li>Fuller, L., Dailey, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rodent+hippocampal+slice+cultures.">Preparation of rodent hippocampal slice cultures.</a> CSH Protoc. 2007, pdb prot4848 (2007).</li><li>Lorger, M., Felding-Habermann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+changes+in+the+brain+microenvironment+during+initial+steps+of+breast+cancer+brain+metastasis.">Capturing changes in the brain microenvironment during initial steps of breast cancer brain metastasis.</a> Am. J. Pathol. 176, 2958-2971 (2010).</li><li>Brana, C., Biggs, T. E., Mann, D. A., Sundstrom, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+macrophage+hippocampal+slice+co-culture+system:+application+to+the+study+of+HIV-induced+brain+damage.">A macrophage hippocampal slice co-culture system: application to the study of HIV-induced brain damage.</a> J. Neurosci. Methods. 90, 7-11 (1999).</li><li>Kim, J. A., Yamada, M. K., Nishiyama, N., Matsuki, N., Ikegaya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mossy+fiber+pathfinding+in+multilayer+organotypic+cultures+of+rat+hippocampal+slices.">Mossy fiber pathfinding in multilayer organotypic cultures of rat hippocampal slices.</a> Cell. Mol. Neurobiol. 23, 115-119 (2003).</li><li>Matsumura, H., Ohnishi, T., Kanemura, Y., Maruno, M., Yoshimine, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+glioma+cell+invasion+by+confocal+laser+scanning+microscopy+in+a+novel+brain+slice+model.">Quantitative analysis of glioma cell invasion by confocal laser scanning microscopy in a novel brain slice model.</a> Biochem. Biophys. Res. Commun. 269, 513-520 (2000).</li><li>Charles, N. A., Holland, E. C., Gilbertson, R., Glass, R., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+tumor+microenvironment.">The brain tumor microenvironment.</a> Glia. , (2011).</li><li>Mizutani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fractalkine+receptor+but+not+CCR2+is+present+on+microglia+from+embryonic+development+throughout+adulthood.">The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood.</a> J. Immunol. 188, 29-36 (2012).</li><li>Kettenmann, H., Hanisch, U. K., Noda, M., Verkhratsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+microglia.">Physiology of microglia.</a> Physiol. Rev. 91, 461-553 (2011).</li><li>Markovic, D. S., Glass, R., Synowitz, M., Rooijen, N., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+stimulate+the+invasiveness+of+glioma+cells+by+increasing+the+activity+of+metalloprotease-2.">Microglia stimulate the invasiveness of glioma cells by increasing the activity of metalloprotease-2.</a> J. Neuropathol. Exp. Neurol. 64, 754-762 (2005).</li><li>Baumert, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pathology-based+substrate+for+target+definition+in+radiosurgery+of+brain+metastases.">A pathology-based substrate for target definition in radiosurgery of brain metastases.</a> Int. J. Radiat. Oncol. Biol. Phys. 66, 187-194 (2006).</li><li>Palmieri, D., Chambers, A. F., Felding-Habermann, B., Huang, S., Steeg, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+metastasis+to+a+sanctuary+site.">The biology of metastasis to a sanctuary site.</a> Clin. Cancer Res. 13, 1656-1662 (2007).</li><li>Pukrop, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+promote+colonization+of+brain+tissue+by+breast+cancer+cells+in+a+Wnt-dependent+way.">Microglia promote colonization of brain tissue by breast cancer cells in a Wnt-dependent way.</a> Glia. 58, 1477-1489 (2010).</li><li>Bos, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+that+mediate+breast+cancer+metastasis+to+the+brain.">Genes that mediate breast cancer metastasis to the brain.</a> Nature. 459, 1005-1009 (2009).</li></ol>

We have nothing to disclose.

Previous histological investigations of cerebral metastasis demonstrated rapid and drastic changes of the resident glial cells, especially of astrocytes and microglia13. To study these changes and interactions with the carcinoma cells, this novel coculture system is well-suited. Other research fields already have long-standing experience with organotypic hippocampal brain slices. One advantage is that the organotypic hippocampal brain slice cultures are viable and effectively preserved for days to weeks, making it suitable for long-term experiments. Since Stoppini&#39;s introduction of the organotypic hippocampal slice system in 1991, it has widely been used, for example in research of degenerative diseases. Thus, this innovate coculture system represents a modification of a well-established approach with a range of applications in tumor biology9,11. The modification offered us a reproducible model to evaluate the grade of tumor invasion afterwards and cultures grown by the interface method are ideally suited for experiments that require a three-dimensional structure. Several techniques have been used to coculture organotypic hippocampal slices with other cells. These include an indirect system between macrophage cells and the organotypic brain slice14, and a direct coculture of two different slices from the hippocampal region15. Glioma aggregates have also been cocultured with brain slices16. These models can be used to analyze cellular and molecular events in the brain slices but do not allow direct, physiological contact between tumor cells, microglia and the brain parenchyma. Furthermore, this method allows the observation of microglia without contamination of bone marrow-derived peripheral monocytes/macrophages. The use of CCR2 and CX3CR1 transgenic mouse model is a critical improvement due to the fact that it is difficult to distinguish the resident microglia from invading monocytes based on their similar properties17,18,19. Though intracerebral injection of carcinoma cells allows investigating tumor progression, it cannot tell much as to whether the surrounded macrophage-like cells originate from the brain-resident microglia population or from bone marrow-derived peripheral monocytes/macrophages17. The team of Kettenmann introduced an organotypic brain slice model that involved inoculating glioma cells into brain slices with a micromanipulator 20. However, primary malignant gliomas differ in many regards from metastatic carcinomas. First, malignant gliomas are of mesenchymal origin, do not metastasize outside the nervous system, and migrate/invade as single cells with no border between tumor and brain tissue. By contrast, infiltrative growth is a typical pathological characteristic, and such carcinomas usually migrate/invade as cohorts. Second, carcinomas often try to rebuild epithelial structures in the brain, while glial cells try to separate the tumor from the brain tissue by a (pseudo) capsule. Considering biological and morphological features, malignant glioma and metastasis of carcinomas are not really comparable. For these reasons, we modified and developed a coculture system where we do not inject but coculture a carcinoma cell plug adjacent to the brain slice. Furthermore, we observed microglial and astrocytic accumulation at the border of the tumor plug, meaning that microglia enter the tumor plug and could be easily detected by bright field microscopy and confirmed afterwards by confocal microscopy. The cancer cells invade into the brain slice, which is comparable to the real in vivo situation and to an observation made by Baumert and colleagues, who found an infiltration zone in 63% of autopsy cases with brain metastases21.
Because of the missing blood perfusion, there are only resident macrophages/microglia in this culture. Moreover, because of the missing T cells and, therefore, absent allo-reactivity, human carcinoma cells could be used for coculture even with brain slices of immunocompetent mice or rats (NMRI, B6, or Wistar). This could serve as an alternative to the nude mouse model. Since four to five slices can be obtained from each mouse, significantly reduced numbers of animals are required, in comparison to the existing injection models. In addition, the animals do not suffer for a long period of metastatic disease and they do not undergo operative procedures repetitively22.
With this coculture system, we have demonstrated the activation of microglia by cancer cells and the capacity of promoting cancer cell invasion. Additionally, this is the first time, to our knowledge, microglia were found to actively transport carcinoma cells23.
Despite all these advantages, the coculture system has, indeed, also limitations. It is still an in vitro model missing the steps of metastasis prior to colonization. Because of the lack of perfusion, it is not possible to study the extravasation. Thus, the alternative way to study the extravasation is to use either the in vivo injection model or the modified Boyden chamber system with extracellular matrix, HUVEC and astrocytes to mimic the blood-brain barrier22,24. The brain slice coculture method is yet a reliable and reproducible model with many advantages and a potential for a wide variety of applications, such as analysis of colonization, in particular with a focus on the role of the resident cells. The combination with other established techniques (such as immunohistochemistry, confocal microscopy and time-lapse microscopy) supports the investigation of direct cell-to-cell interactions. It is an easy alternative and complementation of other techniques and offers access to the investigation of the cues and effects as imposed by the metastatic microenvironment.

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBSS)</td>
			<td>Gibco, Darmstadt,Germany</td>
			<td>24020</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum essential medium (MEM)</td>
			<td>Gibco, Darmstadt , Germany</td>
			<td>32360</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>PAA Laboratories Inc., C&#246;lbe, Germany</td>
			<td>E15-840</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (PBS)</td>
			<td>Pan Biotech, Aidenbach, Germany</td>
			<td>P04-36500</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal horse serum (NHS)</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>10091148</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>B. Braum, Melsungen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine-Penicillin-Streptomycin solution</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>G1146</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>Leica VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>Leica SM 2000R</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membrane</td>
			<td>BD Falcon, Heidelberg,Germany</td>
			<td>353090</td>
			<td>transwell membrane insert (0.4 &#956;m pore size)</td>
		</tr>
		<tr>
			<td>ECM gel</td>
			<td>Trevigen, R&#38;D, Wiesbaden-Nordenstadt, Germany</td>
			<td>3432-005-01</td>
			<td>Basement membrane extract</td>
		</tr>
		<tr>
			<td>Metallic spacer</td>
			<td>Kig GmbG, Kirkel, Germany</td>
			<td>DIN 433</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss, G&#246;ttingen, Germany</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
		<tr>
			<td>Time-lapse microscope and camera</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>DMI 6000B microscope and a DFC 350 FX CCD camera</td>
			<td></td>
		</tr>
		<tr>
			<td>Anatomy microscope</td>
			<td>Zeiss, G&#246;ttingen, Germany</td>
			<td>Stemi SV11</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1.04005.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>G3893</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG, F(ab&#39;)2- TRITC</td>
			<td>Santa Cruz, Heidelberg, Germany</td>
			<td>SC3796</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4 from Griffonia simplicifolia, Alexa Fluor 647 conjugate</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>132450</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6&#39;-diamidino-2-phenylindole dihyfrochloride)</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>D8417</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent mounting medium</td>
			<td>DAKO, Glostrup, Denmark</td>
			<td>S3023</td>
			<td></td>
		</tr>
	</tbody>
</table>

coculture system with an organotypic brain slice and 3d spheroid of carcinoma cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

First, all used carcinoma cells (human: MCF-7 and murine: 410.4) invaded the organotypic mouse brain slice. The invasion was therefore species-independent (Figure 1A), which indicates a wide range of applications in a species-independent manner. Additionally, microglia as well as astrocytes accumulated at the interface as previously described in vivo and in patient samples13. Time-lapse imaging over an extended period of time not only revealed the viability of the slice but also offered a good platform to observe the cellular interactions. By time-lapse experiments, microglial cells were recorded to enter the 3D cell sphere while, in turn, cancer cells also invaded the brain slice (Figures 1B1-B2). Moreover, we have previously shown the ability of microglia to transport carcinoma cells by a still enigmatic mechanism to thereby assist in the carcinoma invasion. Using immunofluorescence labeling techniques, we described co-localizations of tumor cells and stromal cells (e.g. microglia and astrocytes), both in the brain tissue and in the tumor cell plug (Figures 1C-D), suggesting a tight interaction between these cells during the invasion process. Comparable results were obtained when mouse brain slices were cocultured either with human (Figures 1C1-C3) or murine (Figures 1D1-D3) carcinoma cells - showing the wide range of applications for studying cells from different species, genotypes and strains.
<img alt="Figure 1" src="/files/ftp_upload/50881/50881fig1.jpg" fo:content-width="4.75in" fo:src="/files/ftp_upload/50881/50881fig1highres.jpg" /> 
Figure 1. Brain slice coculture model with a 3D spheroid of mouse and human carcinoma cells. A) Quantification of cancer cell invasion in organotypic whole brain slice cocultures with a mouse breast cancer cell line 410.4 or a human breast cancer cell line MCF-7. Data represent the percentage of the degree of cell invasion into brain slice in each group with n &#8805; 38. There was no significant difference between these two groups (Kruskal-Wallis test). B) Time-lapse images of organotypic whole brain slice cocultures with MCF-7-GFP cells and representative images from day2 (B1) and day 5 (B2) were shown. C-D) Confocal microscopy images showed cohorts of carcinomas, astrocytes and microglia. Double staining of astrocytes (anti-GFAP-TRITC, red) and microglia (ILB4-Alexa Fluor 647, violet) of the whole coculture with the 350 &#956;m thick organotypic brain slice and the GFP-transfected tumor cell plug (green): 3D-MCF-7-GFP (C1-C3) and 3D-410.4-GFP (D1-D3). White dashes showed the edge of the brain slice and the tumor invasion front. Scale bars represent 50 &#956;m. Microglia, astrocytes and tumors colocalizations (C1 and D1), astrocytes-tumors colocalizations (C2 and D2) and microglia-tumor colocalizations (C3 and D3) in the slice coculture. <a href="https://www.jove.com/files/ftp_upload/50881/50881fig1highres.jpg" target="_blank">Click here to view larger image</a>

Watch this Scientific Journal Video about Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells at JoVE.com

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

coculture-system-with-an-organotypic-brain-slice-3d-spheroid

Research

JoVE Journal

Medicine

20.5K Views.  University of Göttingen. The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Video: Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

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.

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. &#160;

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena described in those patients for whom a brain autopsy has been requested. Although the advancements of biomedical sciences and neuroimaging have revolutionized the neuropsychiatric field, they have also generated the misleading idea that brain autopsies have only a confirmatory value. This false idea created a drastic reduction of autopsy rates and, consequently, a reduced possibility to perform more detailed and extensive neuropathological investigations, which are necessary to comprehend numerous normal and pathological aspects yet unknown of the human brain. The traditional inferential method of correlation between observed neuropsychiatric phenomena and corresponding localization/characterization of their possible neurohistological correlates continues to have an undeniable value. In the context of neuropsychiatric diseases, the traditional clinicopathological method is still the best possible methodology (and often the only available) to link unique neuropsychiatric features to their corresponding neuropathological substrates, since it relies specifically upon the direct physical assessment of brain tissues. The assessment of postmortem brains is based on brain cutting procedures that vary across different neuropathology centers. Brain cuttings are performed in a relatively extensive and systematic way based on the various clinical and academic contingencies present in each institution. A more anatomically inclusive and symmetric bi-hemispheric brain cutting methodology should at least be used for research purposes in human neuropathology to coherently investigate, in depth, normal and pathological conditions with the peculiarities of the human brain (i.e., hemispheric specialization and lateralization for specific functions). Such a method would provide a more comprehensive collection of neuropathologically well-characterized brains available for current and future biotechnological and neuroimaging techniques. We describe a symmetric bi-hemispheric brain cutting procedure for the investigation of hemispheric differences in human brain pathologies and for use with current as well as future biomolecular/neuroimaging techniques.

Organized brain cutting procedures are necessary to correlate specific neuropsychiatric phenomena with definitive neuropathologic diagnoses. Brain cuttings are performed differently based on various clinico-academic contingencies. This protocol describes a symmetric bihemispheric brain cutting procedure to investigate hemispheric differences in human brain pathologies and to maximize current and future biomolecular/neuroimaging techniques.

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing.
This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 &#181;m thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling.
OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.