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>

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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><tbody><tr><td>Hank&amp;#039;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&amp;ouml;lbe, Germany</td><td>E15-840</td><td></td></tr><tr><td>Dulbecco&amp;#039;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 &amp;mu;m pore size)</td></tr><tr><td>ECM gel</td><td>Trevigen, R&amp;amp;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&amp;ouml;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&amp;ouml;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&amp;#039;)&lt;sub&gt;2&lt;/sub&gt;- 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&amp;#039;,6&amp;#039;-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://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50881/50881fig1highres.jpg" target="_blank">Click here to view larger image</a>

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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 ...

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Research

JoVE Journal

Medicine

20.6K 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

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Cancer Research

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

Developmental Biology

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 ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

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 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 ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Neuroscience

Establishing a Brain Tumor in an Organotypic Slice Co-culture Model

Source: Chadwick, E. J. et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53304">A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies.</a> J. Vis. Exp. (2015).
This video demonstrates the development of a 3D co-culture system. Mouse brain slices are placed on a membrane, and fluorescently labeled human brain tumor cells are allowed to grow on it. Once the system is ready, it is stained for imaging.

Ustalenie guza mózgu w organotypowym modelu kokultury warstw

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Plating the Slices onto the ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

Generating and Maintaining Slice Cultures from Human Glioblastoma Tissue

Source: Parker, J. J., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53557">A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs.</a> J. Vis. Exp. (2017).
The video demonstrates the generation and maintenance of organotypic slice cultures from glioblastoma tissue. The isolated tissue is embedded in an agarose matrix, and thin sections are obtained using a vibratome. These sections are then placed on permeable membrane inserts within a microplate and cultured in a slice culture maintenance media for long-term maintenance.

Generowanie i utrzymywanie kultur plastrów z ludzkiej tkanki glejaka wielopostaciowego

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Pre-slicing Preparation 

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