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 dish 24 hr before staining.</li>
	<li>Prepare fluorescent cell stain dye solution in DMSO and add 5 &#181;M dye to media, either Roswell Park Memorial Institute medium (RPMI) or Macrophage Serum Free Medium (MSFM), vortex well.</li>
	<li>Incubate cells with dye for 30 min at 37 &#176;C and 5% CO2.</li>
	<li>Remove dye containing media and add fresh media (RPMI or MSFM) to the cells.</li>
	<li>Incubate cells for 30 min. Note: Cells are now stained and ready to be used in experiment.</li>
</ol>
2. Pre-coated Matrix Chamber Coculture Invasion Assay
<ol>
	<li>Differentiate THP-1 cells using phorbol myristate acetate (PMA)15.
	<ol>
		<li>Plate 2 - 3 x 105 THP-1 cells in 1.5 ml of RPMI/10% FBS in a 6 well culture plate. Immediately after plating add 100 nM PMA and incubate at 37 &#176;C, 5% CO2 for 48 hr. 
		NOTE: After 48 hr, THP-1 cells that have successfully undergone differentiation will be adherent and spread to the bottom of the well taking on a round morphology.</li>
		<li>Remove PMA by washing once with 1x PBS and replace with fresh RPMI/10% FBS. Wait another 48 hr until cells are ready to use in assay.</li>
	</ol>
	</li>
	<li>Equilibrate matrix pre-coated chambers by placing them in a well containing 500 &#181;l of media alone (no serum) and adding 500 &#181;l of media alone to the top. Incubate chambers for 2 hr at 37 &#176;C and 5% CO2. 
	NOTE: Pre-coated chambers were purchased from the manufacturer; see Table of Materials/Equipment.</li>
	<li>To gently detach cells (fluorescently labeled glioblastoma cell lines and the microglia/macrophages), remove media from cells by aspiration. Wash cells on dish with 2 mM EDTA/PBS. Add 500 &#181;l of 2 mM EDTA/PBS and incubate for 5 - 10 min in a cell incubator at 37 &#176;C and 5% CO2.</li>
	<li>Resuspend cells in 5 ml of media containing 0.3% Bovine Serum Albumin (BSA).</li>
	<li>Centrifuge for 5 min at 120 x g.</li>
	<li>Aspirate supernatant and resuspend pellet in MSFM/0.3% BSA media (for GL261 cells and microglia) or RPMI/0.3% BSA (for U87 and THP-1 cells) such that the concentration of cells is equal to 1 x 106 cells/ml. Use a hemocytometer to count the cells.</li>
	<li>Add 500 &#181;l media/0.3% BSA per well of 24 well plate.</li>
	<li>Remove media from chambers that have been equilibrated for at least 2 hr (step 2.2). Place chambers in well containing 500 &#181;l media/0.3% BSA (step 2.7).</li>
	<li>If inhibitors are being used to study their effect on macrophage/microglia stimulated glioma invasion, add the appropriate concentration to both the bottom and top portions of the chamber. 
	NOTE: The concentrations of CSF-1R inhibitor used to fully inhibit microglia stimulated GL261 glioblastoma invasion can be found in reference 9.</li>
	<li>Depending on the cell lines used, add cells to the top chamber as described in the next two steps: mouse glioblastoma (2.10.1) and human glioblastoma (2.10.2).
	<ol>
		<li>Mix 1.5 x 105 GL261 (glioblastoma) cells (150 &#181;l) and 5 x 104 mouse microglia (50 &#181;l) (see step 2.6 for media details). Bring volume to 500 &#181;l by adding 300 &#181;l MSFM/0.3% BSA. Add cell mixture to the top chamber and incubate at 37 &#176;C , 5% CO2 for 48 hr. 
		&#8203;NOTE: Murine microglia are isolated from neonatal mice as described in 16.</li>
	</ol>
	</li>
</ol>
Mix 7.5 x 104 U87 (glioblastoma) cells (75 &#181;l) and 2.5 x 104 THP1 (macrophage) cells (25 &#181;l). Bring volume in 500 &#181;l by adding 400 &#181;l RPMI/0.3% BSA. Add the cell mixture to the top chamber. Incubate cells at 37 &#176;C, 5% CO2 for 24 hr.
<ol>
	<li>After incubation, remove cells from top of chamber by gentle aspiration and fix chambers by placing in 3.7% formaldehyde in PBS. Allow chambers to remain in fixative for 15 min at room temperature (or overnight at 4 &#176;C). Remove fixative by gentle aspiration and replace with 500 &#181;l 1x PBS. Proceed to section 4 for image analysis. 
	&#8203;NOTE: Experiment can be paused at this moment and saved for imaging in 1x PBS. Fluorescent dye signals can be detected for up to 2 weeks after fixation.</li>
</ol>
3. Invasion Assay &#34;3D&#34;-embedding Glioma and Macrophages/Microglia in Matrix
<ol>
	<li>Thaw matrix mix at 4 &#176;C overnight. Perform all further manipulations using matrix on ice in the hood.</li>
	<li>Prepare 10 mg/ml concentration of matrix in media (MSFM or RPMI) with 0.3% BSA on ice. 
	NOTE: Stock concentration of matrix is typically around 15 mg/ml.</li>
	<li>To prepare cells (labelled in step 1) for suspension in matrix, remove media from cells by aspiration. Wash cells on dish with 2 mM EDTA/PBS. Add 500 &#181;l of 2 mM EDTA/PBS to cells and incubate for 5 - 10 min in incubator at 37 &#176;C and 5% CO2.</li>
	<li>Resuspend cells in 5 ml of media containing 0.3% BSA.</li>
	<li>Centrifuge for 5 min at 120 x g.</li>
	<li>Aspirate supernatant from pellet. Resuspend pellet in media/0.3% BSA such that the concentration of cells is equal to 1 x 106 cells/ml. Use a hemocytometer to count the cells.</li>
	<li>Add 1.5 x 105 GL261 cells (in 150 &#181;l) + 5 x 104 microglia cells (in 50 &#181;l) into a 1.5 ml microfuge tube. Add 2 x 105 GL261 cells (200 &#181;l) into a separate 1.5 ml microfuge tube. 
	NOTE: GL261 cells alone without microglia serves as a control.</li>
	<li>Spin cells in microfuge for 5 min at 120 x g.</li>
	<li>Resuspend cells in 200 &#181;l cold matrix on ice.</li>
	<li>Plate 50 &#181;l (50,000 cells) onto the center of the top compartment of 8 &#181;M pore size chamber insert.</li>
	<li>Incubate at 37 &#176;C for 30 min for polymerization to occur.</li>
	<li>Add 200 &#181;l serum-free medium to the upper chamber and 700 &#181;l serum containing cell growth medium to the lower well.</li>
	<li>Incubate at 37 &#176;C, 5% CO2 for 48 hr.</li>
	<li>After incubation, remove cells from top of chamber by gentle aspiration and fix chambers by placing in 3.7% formaldehyde in PBS. Allow chambers to remain in fixative for 15 min at room temperature (or overnight at 4 &#176;C). Remove fixative by gentle aspiration and replace with 500 &#181;l 1x PBS. Proceed to section 4 for image analysis.</li>
</ol>
4. Imaging Assays
<ol>
	<li>Remove chambers from 3.7% formaldehyde and wash in well containing fresh 500 &#181;l 1x PBS. 
	NOTE: Do not allow chambers to dry.</li>
	<li>Using a cotton tipped applicator, gently clean the non-invasive cells from the top portion of the filter (side facing the inside of the chamber) by scraping the filter surface. Start gently and increase pressure gradually. Do this at least 3 times per chamber.</li>
	<li>Place chambers in either a glass-bottomed dish or leave in a 24 well plate.</li>
	<li>Place sample on the stage of an epifluorescent or laser confocal fluorescent microscope equipped with camera and image capture software 9,10.</li>
	<li>Take several 10X or 20X images of representative fields.</li>
	<li>Using an image analysis software, such as ImageJ, count the number of glioblastoma cells that have crossed the filter to the underside 9,10.</li>
	<li>If imaging the &#34;3D&#34; invasion assay (described in section 3), generate a Z stack series using a fluorescent laser confocal microscope 5-6. To image the invasive cell population on the underside of the filter, follow the protocol steps 4.2 -&#160; 4.6 described above.</li>
</ol>

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The authors have nothing to disclose.

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 ...

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13.9K Views.  Kean University. 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.

Video: Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

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 ...

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 ...

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.