Name: coculture assays to study macrophage and microglia stimulation of glioblastoma invasion
Uploaded: 2016-10-20T12:30:00
Description: Watch this Scientific Journal Video about Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion at JoVE.com

We would like to thank Dr. Konstantin Dobrenis for providing murine microglia for these studies.

1. Fluorescent Labeling of Cells
NOTE: Label glioblastoma cell lines and microglia with fluorescent dyes 9. Alternatively, generate cell lines that constitutively express fluorescent proteins such as GFP/RFP as described in 14.
<ol>
	<li>Plate cells on a 6 well plate such that they will be 70 - 80% confluent on day of staining. For the murine glioblastoma cell line GL261 and human glioblastoma cell line U87, plate 1 x 106 and 1.5 x 106 cells, respectively on a 6 cm dis...

<ol>
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J., 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=Microglial+stimulation+of+glioblastoma+invasion+involves+epidermal+growth+factor+receptor+(EGFR)+and+colony+stimulating+factor+1+receptor+(CSF-1R)+signaling.">Microglial stimulation of glioblastoma invasion involves epidermal growth factor receptor (EGFR) and colony stimulating factor 1 receptor (CSF-1R) signaling.</a> Mol Med. 18, 519-527 (2012).</li><li>Miller, I. S., 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=Semapimod+sensitizes+glioblastoma+tumors+to+ionizing+radiation+by+targeting+microglia.">Semapimod sensitizes glioblastoma tumors to ionizing radiation by targeting microglia.</a> PLoS One. 9 (5), (2014).</li><li>Goswami, S., 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=Macrophages+promote+the+invasion+of+breast+carcinoma+cells+via+a+colony-stimulating+factor-1/epidermal+growth+factor+paracrine+loop.">Macrophages promote the invasion of breast carcinoma cells via a colony-stimulating factor-1/epidermal growth factor paracrine loop.</a> Cancer Res. 65 (12), 5278-5283 (2005).</li><li>House, R. P., 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=Two+functional+S100A4+monomers+are+necessary+for+regulating+nonmuscle+myosin-IIA+and+HCT116+cell+invasion.">Two functional S100A4 monomers are necessary for regulating nonmuscle myosin-IIA and HCT116 cell invasion.</a> Biochemistry. 50 (32), 6920-6932 (2011).</li><li>Wang, W., 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+activity+status+of+cofilin+is+directly+related+to+invasion,+intravasation,+and+metastasis+of+mammary+tumors.">The activity status of cofilin is directly related to invasion, intravasation, and metastasis of mammary tumors.</a> J Cell Biol. 173 (3), 395-404 (2006).</li><li>Zagzag, D. 1., Miller, D. C., Chiriboga, L., Yee, H., Newcomb, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+immunohistochemistry+as+a+novel+experimental+tool+for+the+detection+of+glioma+cell+invasion+in+vivo.">Green fluorescent protein immunohistochemistry as a novel experimental tool for the detection of glioma cell invasion in vivo.</a> Brain Pathol. 13 (1), 34-37 (2003).</li><li>Tsuchiya, S., 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=Establishment+and+characterization+of+a+human+acute+monocytic+leukemia+cell+line+(THP-1).">Establishment and characterization of a human acute monocytic leukemia cell line (THP-1).</a> Int. J. Cancer. 26 (2), 171-176 (1980).</li><li>Dobrenis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+cell+culture+and+in+transplantation+therapy+for+central+nervous+system+disease.">Microglia in cell culture and in transplantation therapy for central nervous system disease.</a> Methods. 16 (3), 320-344 (1998).</li><li>Coniglio, S. J., Segall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+of+microglia+stimulated+glioblastoma+invasion.">Molecular mechanism of microglia stimulated glioblastoma invasion.</a> Matrix Biol. 32 (7-8), 372-380 (2013).</li><li>See, A. P., Parker, J. 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The highly invasive nature of high grade astrocytomas and glioblastoma make these brain cancers very deadly. It is therefore of paramount importance to understand the molecular and cellular mechanisms of glioblastoma invasion. Much has been learned about the process of glioblastoma invasion already 17. Using the assay formats detailed in this paper, our laboratory has shown in both mouse and human models that tumor associated macrophages can stimulate glioma cell invasion by 5 - 10 fold. This coculture model f...

Glioblastoma multiforme is an aggressive human brain cancer with a median survival of approximately 12 months from the time of diagnosis 1,2. Glioblastoma is one of the most deadly and clinically challenging cancers as it is refractory to standard chemotherapy and surgical resection. The diffuse nature of glioblastoma enables tumor cells to spread throughout the normal brain making the advanced tumor practically impossible to surgically resect completely. This highly invasive aspect is a hallmark feature of glioblastoma and other advanced astrocytomas. Therefore, the focus of much research has been on the molecular mechanism of glioblastoma cell invasion. The glioblastoma tumor microenvironment plays vital roles in establishing malignancy 3-6. Tumor associated macrophages/microglia were shown to be responsible for promoting glioblastoma invasion 7,8. Most of these studies however measured the effect of macrophages/microglia using assays which physically separate them from the glioblastoma cells. Our laboratory has set out to generate improved assays which allow us to study how glioblastoma invasion is dependent on macrophages/microglia in cocultures and enable us to image the physical interaction between them during invasion.
Classic assays to measure cell invasion include the &#34;standard&#34; Boyden chamber chemotaxis and chemoinvasion formats. Here the cells to be studied are plated in a plastic chamber which contains a polycarbonate filter on the bottom that has pores of a specified size (generally between 0.4 and 8 &#181;M in diameter). The process of cell invasion involves a physical barrier, usually composed of extracellular matrix protein. In the chemoinvasion assay, the preferred matrix used is Matrigel (hereafter referred to as &#34;matrix&#34;), an extracellular matrix protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and consists largely of collagen type IV and laminin. The chambers are then placed in a tissue culture well which contains cell culture media with or without growth factors that are suspected to stimulate invasion. Cells which have a higher invasive capacity will invade through the extracellular matrix coated filter at a higher frequency and adhere to the underside of the filter. We have modified this assay in order to assess the role of microglia and tumor associated macrophages on glioblastoma cell invasion.
We have been able to determine using coculture assays described within this paper that microglia can stimulate the invasion of two glioblastoma cell lines by 5 - 10 fold 9,10. This reflects what is observed in animal models of glioblastoma. Furthermore, we developed a three dimensional invasion assay where the interactions between glioblastoma cells and macrophages/microglia can be examined more directly. The extent of glioblastoma cell invasion stimulated by macrophages/microglia in the 3D assay is comparable to what is seen using the matrix coated chamber approach. Similar assays were previously developed to study breast carcinoma interactions with macrophages during invasion 11-13. Both methods described in this paper should aid in the ability to dissect the molecular mechanism(s) of macrophage/microglia-stimulated invasion of glioblastoma cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Corning BioCoat Matrigel Invasion Chamber: With BD Matrigel Matrix</td>
			<td>Corning/Fisher Scientific</td>
			<td>Cat: 354481</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophage Serum Free Media (MSFM) (500 ml)</td>
			<td>Life Technologies</td>
			<td>12065-074</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Red CMTPX Dye</td>
			<td>Life Technologies/Molecular Probes</td>
			<td>C34552</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Green CMFDA Dye</td>
			<td>Life Technologies/Molecular Probes</td>
			<td>C2925</td>
			<td></td>
		</tr>
		<tr>
			<td>GL261 cell line</td>
			<td>National Cancer Institute (NCI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U87 cell line</td>
			<td>American Tissue Type Culture Collection</td>
			<td>HTB-14</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 cell line</td>
			<td>American Tissue Type Culture Collection</td>
			<td>ATCC TIB-202</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium (500 ml)</td>
			<td>Life Technologies/Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma Aldrich</td>
			<td>F1635</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Transwell polycarbonate membrane cell culture inserts (8 &#181;M pore) 48 per pack.</td>
			<td>Corning</td>
			<td>CLS3422</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex 3-D Culture Matrix Reduced Growth Factor Basement Membrane Extract, PathClear</td>
			<td>Trevigen</td>
			<td>3445-005-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>Cat: 10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, Fraction V, Heat Shock Treated</td>
			<td>Fisherscientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5M EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma Aldrich</td>
			<td>P8139-1MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

coculture assays to study macrophage and microglia stimulation of glioblastoma invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Using the methods outlined here, we have shown that microglia and macrophages can substantially stimulate glioblastoma cell invasion. Two different invasion assays are employed and are depicted in Figure 1. In Figure 2, GL261 cells that constitutively express the fluorescent protein mCherry were plated on pre-coated chambers with and without microglia for 48 hr. GL261 cells were minimally invasive on their own however when cultured with microglia the inva...

Watch this Scientific Journal Video about Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion at JoVE.com

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

The overall goal of this experiment is to model the interaction of glioblastoma cells with microglia and macrophages during invasion in an in vitro assay. So this method can help answer key questions in the tumor microenvironment field, such as which compounds or treatments can interfere with cancer interaction with macrophages, a cell type of the microenvironment during the progression to malignancy. One of the main advantages of this technique is that it's relatively easy to perform and can be completed at a reasonable time and seems to accurately model glioblastoma invasion in vivo.Begin by differentiating the cell line of interest using phorbol myristate acetate, as follows. First, plate two to three times 10 to the fifth THP-1 cells in 1.5 milliliters of medium in a six well culture plate. Immediately after plating, add 100 nanomolar of PMA and incubate at 37 degrees Celsius and 5%CO2 for 48 hours.After 48 hours, remove the PMA by aspirating the media, wash once with one-X PBS and add fresh media. Incubate for another 48 hours until cells are ready to use in the assay. Then equilibrate matrix pre-coated chambers by placing them in a well containing 500 microliters of media without serum and then adding 500 microliters of serum free media to the top.Incubate the chambers for two hours at 37 degrees Celsius and 5%CO2. Next, gently aspirate the media from the culture vessels containing the fluorescently-labeled glioblastoma cell lines and the microglia and macrophages. Then add 500 microliters of two millimolar EDTA and PBS and incubate for five to 10 minutes in the tissue culture incubator.Collect the cells in five milliliters of media containing 0.3%bovine serum albumin and centrifuge for five minutes at 120 times G.Following the centrifugation, aspirate the supernatant and resuspend the cell pellet in appropriate media to a concentration of one times 10 to the sixth cells per milliliter. Use the hemocytometer to count the cells. Next, add 500 microliters of the appropriate media containing 0.3%BSA to the wells of a 24 well plate.Then remove the media from the equilibrated chambers and place the chambers in the wells of the 24 well plate. If inhibitors are being used to study their effect on macrophage or microglia stimulated glioma invasion, add the appropriate concentration to both the bottom and top portions of the chamber. Depending on the cell line used, mix glioblastoma cells and mouse microglia or glioma cells, and macrophage cells according to the details in the written portion of the protocol.Add the cell mixture to the top chamber and incubate for 24 to 48 hours, depending on cell type. After the incubation, remove the cells from the top of the chamber by gentle aspiration. Then place the chambers in 3.7%formaldehyde in PBS to fix.Allow the chambers to remain in fixative for 15 minutes at room temperature. Finally, remove the fixative by gentle aspiration and replace with 500 microliters of one-X PBS. First, thaw the matrix mix at four degrees Celsius overnight.Next, prepare a 10 milligrams per milliliter concentration of matrix in the appropriate media on ice. Then, harvest the labeled cells for suspension in matrix as previously shown. Then pipette 1.5 times 10 to the fifth GL261 cells in a 150 microliter volume plus five times 10 to the fourth microglia cells in 50 microliters into a 1.5 milliliter microfuge tube.Add two times 10 to the fifth GL261 cells in a 200 microliter volume into a separate 1.5 milliliter microfuge tube. Spin the cells in a microfuge for five minutes at 120 times G.Then, resuspend the cells in 200 microliters of cold matrix on ice. For this step it is crucial to have ice tray ready in the hood, keep tubes on ice, tips in the refrigerator, make sure everything is chilled in order to prevent polymerization of the mature gel prematurely.After resuspension, plate 50, 000 cells in a 50 microliter volume onto the center of the top compartment of an eight micron pore size chamber insert. Then incubate it at 37 degrees Celsius for 30 minutes to allow polymerization to occur. After 30 minutes, add 200 microliters of serum free medium to the upper chamber and 700 microliters of serum containing cell growth medium to the lower well.After incubating for 48 hours, remove the cells from the top part of the chamber by gentle aspiration. Fix the chambers as before and replace the fixative with 500 microliters of one-X PBS before imaging the cells. This representative image shows invading mCherry expressing GL261 cells in the absence of microglia.The cells were plated on matrix coated chambers and incubated for 48 hours. The images were taken using a 10X objective and the scale bar represents 400 microns. Here the GL261 cells were combined with microglia.The number of GL261 invasive cells that cross the filter were quantified. As seen here, coculturing the mouse glioblastoma cell line GL261 with microglia enhances its invasion by up to 10 fold in the 2D assay. This image shows the effect of pharmacological inhibitors on microglia stimulated GL261 invasion compared to DMSO control.Data shown are from six independent experiments. This assay was successfully employed using human cell lines as well. U87 cells plated on matrix coated chambers and stained with CMFDA green are shown after a 24 hour incubation.Here the U87 cells were plated with differentiated THP1 macrophages. This image shows quantitation of the assay counting only invasive U87 cells. The human macrophage cell line THP1 stimulated U87 glioblastoma cell invasion.Data shown are the mean values from 10 independent experiments. This image from a 3D invasion assay of GL261 and microglia coculture is a composite from a Z-series of images and shows GL261 cells stained with CMFDA green embedded in matrix alone. Here the GL261 cells were cocultured with murine microglia labeled with CMPTX red.Here GL261 cells on the underside of the filter, which are determined to be invasive, are shown. The total number of invading GL261 cells was determined and normalized to that of GL261 cells in monoculture. Data shown represent the mean of three independent experiments performed in duplicate.So once mastered, this technique can be completed from start to finish in two hours or so. And the final results from the experiment can be obtained after 48 hours. So while performing this technique, it's very important to have everything chilled and at four degrees including the tubes and the pipettes.After watching this video, you should have a good understanding of how to establish glioblastoma in macrophage microglia coculture invasion assays.

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Research

JoVE Journal

Cancer Research

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

CAM-Delam Assay to Score Metastatic Properties by Quantifying Delamination and Invasion Capacity of Cancer Cells

The major cause of cancer-related deaths is metastasis formation (i.e., when cancer cells spread from the primary tumor to distant organs and form secondary tumors). Delamination, defined as the degradation of the basal lamina and basement membrane, is the initial process that facilitates the transmigration and spread of cancer cells to other tissues and organs. Scoring the delamination capacity of cancer cells would indicate the metastatic potential of these cells.
We have developed a standardized method, the ex ovo CAM-Delam assay, to visualize and quantify the ability of cancer cells to delaminate and invade, thereby being able to assess metastatic aggressiveness. Briefly, the CAM-Delam method includes seeding cancer cells in silicone rings on the chick chorioallantoic membrane (CAM) at embryonic day 10, followed by incubation from hours to a few days. The CAM-Delam assay includes the use of an internal humidified chamber during chick embryo incubation. This novel approach increased embryo survival from 10%-50% to 80%-90%, which resolved previous technical problems with low embryo survival rates in different CAM assays.
Next, the CAM samples with associated cancer cell clusters were isolated, fixed, and frozen. Finally, cryostat-sectioned samples were visualized and analyzed for basement membrane damage and cancer cell invasion using immunohistochemistry. By evaluating various known metastatic and non-metastatic cancer cell lines designed to express green fluorescent protein (GFP), the CAM-Delam quantitative results showed that the delamination capacity patterns reflect metastatic aggressiveness and can be scored into four categories. Future use of this assay, apart from quantifying delamination capacity as an indication of metastatic aggressiveness, is to unravel the molecular mechanisms that control delamination, invasion, the formation of micrometastases, and changes in the tumor microenvironment.

The CAM-Delam assay to evaluate the metastatic capacity of cancer cells is relatively fast, easy, and cheap. The method can be used for unraveling the molecular mechanisms regulating metastasis formation and for drug screening. An optimized assay for analyzing human tumor samples could be a clinical method for personalized cancer treatment.

The major cause of cancer-related deaths is metastasis formation (i.e., when cancer cells spread from the primary tumor to distant organs and form ...

Translational Orthotopic Models of Glioblastoma Multiforme

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors&#160;displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain ...