Supported by NIH grants P30 CA051008 and T32 CA009686 (ATR).

1. Generation of Spheroids
<ol>
	<li>Prepare single-cell suspension for hanging drop cultures by detaching adherent cancer cell cultures of ~70% confluence using a PBS wash followed by exposure to 0.05% trypsin-EDTA solution.
	<ol>
		<li>Neutralize the trypsin solution with cell culture media and count the cells using an aliquot of the cell suspension. Note: The specific cell culture media will depend on the cell line being tested. Follow ATCC media recommendations, where the cell culture media is typically DMEM + 10% FBS. More information can be found in the Materials Table.</li>
		<li>Perform a dilution to allow for the seeding of 500-1,000 cells per 20 &#181;l drop of cell culture media.</li>
	</ol>
	</li>
	<li>Acquire 10 cm dish and add 5 ml of sterile PBS to the bottom. Note: This step protects the hanging drops from evaporation. Lower-cost &#8220;bacteriological grade&#8221; dishes can be used in place of cell culture grade dishes, as cells will not contact the culture surface.</li>
	<li>Use a multi-channel pipet to transfer 20 &#181;l droplets of the diluted cell suspension to the inner surface of the lid.
	<ol>
		<li>Pipet 40 drops (5 rows of 8 drops) onto the lid of the 10 cm dish.</li>
	</ol>
	</li>
	<li>Invert the lid and place over the culture dish. Incubate the hanging drop cultures at 37 &#176;C for 72 hr to generate spheroids.
	<ol>
		<li>Flip the lid in a confident, yet controlled manner. Note: Inverting the lid too fast or too slow may cause the droplets to shift. Some cell lines may form spheroids in 48 hr or less, while others may require more than 72 hr to produce compact aggregates.</li>
	</ol>
	</li>
</ol>
2. Embedding of Spheroids into 3D Matrix
<ol>
	<li>Thaw an aliquot of growth factor-reduced basement membrane materials at 4 &#176;C overnight before embedding the spheroids.
	<ol>
		<li>Optional: Layer wells with ECM in advance to prevent potential spheroid interaction with the tissue culture surface.
		<ol>
			<li>Pipet 200 &#181;l of ECM per well on a 24-well plate.</li>
			<li>Tilt the plate to ensure that the ECM covers the entire well surface.</li>
			<li>Carefully remove excess ECM with a pipet.</li>
			<li>Incubate the plate until the wells are dry: 3 hr at room temperature or overnight at 4 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Collect the spheroids by tilting the lid of the tissue culture dish and pooling the media. Transfer the media with the spheroids into a 1.5 ml microcentrifuge tube.</li>
	<li>Allow 10 min for the spheroids to settle at the bottom of the microcentrifuge tube. Spheroids should be visible by eye.</li>
	<li>Mix 100 &#181;l of the basement membrane materials with 100 &#181;l of cold (4 &#176;C) type I collagen in a separate pre-chilled tube. Keep the mixture at 4 &#176;C to prevent either gel from solidifying prematurely.
	<ol>
		<li>Use pre-chilled pipet tips if transferring small volumes.</li>
		<li>Give care when mixing the basement membrane materials and collagen type I to prevent air bubble formation. Note: Air bubbles may hinder imaging later in the protocol if they become embedded in the gel.</li>
	</ol>
	</li>
	<li>Aspirate the spheroids from the 40 &#181;l bottom portion of the microcentrifuge tube, and combine with the basement membrane materials/type I collagen mixture. Be careful to prevent air bubble formation when mixing. Note: The solution containing of 100 &#181;l basement membrane materials, 100 &#181;l collagen type I, and 40 &#181;l of the spheroids will create enough material for 4 independent 3D cultures. This can be scaled up or down.</li>
	<li>Pipet 40 &#181;l drops of the viscous mixture into the centers of wells on a 24-well plate. Keep the plate level to prevent the mixture from running into the side of the well. Note: A single column on the 24-well plate &#8211; 4 wells &#8211; is recommended for each condition to be tested.</li>
	<li>Place the plate into a 37 &#176;C incubator and leave undisturbed for 30 min. The 3D culture will polymerize during this period.</li>
	<li>Slowly submerge the 3D cultures in 1 ml of cell culture media.
	<ol>
		<li>Ensure that the media is warm when adding it into the wells to further promote gel polymerization.</li>
		<li>CRITICAL STEP: Gently add the media to the 3D cultures. Pipetting the media too quickly into the wells can lead to detachment of the 3D cultures from the tissue culture surface.</li>
	</ol>
	</li>
</ol>
3. Monitoring and Analyzing Spheroid Invasion
<ol>
	<li>Image invasion from the spheroids into the surrounding 3D matrix at time-points decided by the investigator. Acquire photographs using an inverted microscope with the 20x objective lens. Note: Ideal time-points will differ depending on the cell line being tested. More invasive cell lines will begin their egress from the spheroids shortly after plating and, therefore, take initial photographs within a couple of hours after plating. Generally, photographs are taken at 0 hr (after plating), 24 hr, and 48 hr. Invasion typically concludes 24 to 48 hr after plating.</li>
	<li>Quantitate invasive ability using image analysis software like Image J.
	<ol>
		<li>Analyze invasion as the cell distance from the edge of the spheroid.
		<ol>
			<li>Use the Straight Draw Tool to mark the radius and/or maximal invasive distance.</li>
			<li>Click &#8220;Analyze&#8221; in the top menu, and then click &#8220;Measure&#8221; to display the length measurement.</li>
		</ol>
		</li>
		<li>Analyze invasion as the total invasive area outside the spheroid.
		<ol>
			<li>Use the Freehand Draw Tool to trace the border of the spheroid and/or total invasive area.</li>
			<li>Click &#8220;Analyze&#8221; in the top menu, and then click &#8220;Measure&#8221; to display the area measurement.</li>
		</ol>
		</li>
		<li>Use a plugin like ROI Manager Tools to create measurements for a stack of images.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

This study evaluated the performance of a panel of cancer cell lines in a spheroid invasion assay (Table 1). Generally, we find that spheroid formation is enhanced in the more epithelial-looking cell lines, where the presence of cell-cell junctions promotes the formation of a spheroid-like architecture. Known cell lines that have undergone an epithelial-mesenchymal transition, like MDA-MB-231 cells do not form spheroids in the hanging drop culture most likely due to their reduced E-, N-, and P-cadherin e...

It is established that cancer cell motility is predictive of metastatic potential, given that such behavior facilitates invasion through the basement membrane and entry into the circulatory system1. Most research on motility has focused on how cells behave in a two-dimensional (2D) setting, though it is becoming widely recognized that the movement of cells in a three-dimensional (3D) matrix is more representative of how these same cells will actually behave in vivo2. 3D culture systems are increasingly used to study cellular behaviors that range from cell morphology, to growth kinetics and drug sensitivity3. The desire to monitor cancer cell invasion within the context of a 3D milieu has led to the synthesis of previously established techniques involving the generation of 3D cell aggregates (spheroids) via the hanging drop culture method4, followed by embedding these spheroids into a 3D extracellular matrix (ECM) composed of collagen and basement membrane materials5. This method seeks to improve upon these previously established techniques by providing a streamlined approach that can be easily utilized to compare invasion under a variety of experimental conditions.
More traditional ways to assess cell motility in vitro are the scratch-wound assay and the transwell assay6. The former assay depicts cell motility in a 2D setting, and is therefore independent of a variety of features critical for in vivo invasion, e.g. protease activity7. The transwell assay can better model cell invasion when the well inserts are coated with ECM substrates but, only a single parameter, i.e. the appearance of cells on the opposite membrane surface is measured, and many nuances of cell invasion are thus not readily observable. In contrast to these techniques, the cancer cell spheroid invasion assay (Figure 1) allows for the real-time monitoring of cell invasion in a setting that is not only physiologically relevant, but also permits important cell line-specific features to be visualized, such as individual vs. collective cell migration8. This method also affords advantages over standard 3D culture growth assays. The generation of cellular aggregates via the hanging drop method initially constrains cell movement, so that cells will be incentivized to invade after this constraint is lifted. Furthermore, once that constraint is lifted, cell egress will proceed in a uniform direction that can then be conveniently quantitated.
The most popular ECM materials used for cancer cell spheroids assays are Matrigel and type I collagen, where each of these components has important and distinct roles in influencing metastatic behavior. Matrigel is a secreted mixture of proteins produced by Engelbreth-Holm Swarm mouse sarcoma cells, and is enriched in basement membrane proteins such as laminin, entactin, and type IV collagen9. For this reason Matrigel is henceforth referred to as &#8220;basement membrane materials.&#8221; These basement membrane materials provide essential ligands needed for integrin adhesion during cancer cell invasion10 in addition to many other proteins that exert a range of effects on cell behavior11. In comparison, type I collagen, commonly prepared from acid digests of tendons and other dense collagenous structures12, is a much simpler matrix material that serves as a major structural element of the connective tissue and stroma supporting tissues and organs of the body. It has been demonstrated that the physical characteristics of collagen can regulate a number of features of cell motility; for example, the alignment of collagen fibrils at the tumor-stromal interface permits cancer cells to subsequently migrate along those fibrils when invading into the stroma13. In the assay presented here, both type I collagen and basement membrane materials are utilized as tools to study 3D cancer cell-stroma interactions.
The effect of inhibition or stimulation of pathways that control invasion can be monitored after the cells have been embedded in the 3D matrix. Cells can be pretreated during growth in the hanging drops or upon transfer to the 3D culture, depending on whether a lengthy treatment will be required to modulate invasion. For shorter treatments, it is recommended that the drug be mixed with the spheroid suspension after collection, as well as the media that will surround the 3D cultures, to facilitate adequate drug exposure to the cells. Next, normal or tumor-associated stromal cells can be admixed with the matrix material to evaluate their role in modulating tumor cell invasion, or to determine how paracrine and autocrine signaling influences cell behavior. This idea was shown in a study where the coculture of colon cancer and endothelial cells in hanging drops led to a vascular network within the spheroids14. Finally, the ECM constituents can also be altered, as cancer cell invasion is impacted by different substrates15. The method presented below will thus provide a framework for assessing cancer cell invasion under a variety of conditions. In general it was found that not all cell lines will create spheroids in the hanging drops and epithelial-looking cell lines typically form regular spheres.

<table><tbody><tr><td>Matrigel Growth Factor Reduced</td><td>Corning</td><td>CB-40234</td><td></td></tr><tr><td>Collagen Type I, Rat Tail, 100 mg</td><td>Millipore</td><td>08-115</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11995-065</td><td></td></tr><tr><td>RPMI 1640 Medium&amp;nbsp;</td><td>Life Technologies</td><td>11875-093</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Fetal Bovine Serum, Heat Inactivated</td><td>Omega Scientific</td><td>FB-12</td><td></td></tr><tr><td>100 mm TC-treated Dishes</td><td>Corning Incorporated</td><td>430167</td><td></td></tr><tr><td>24-well TC-treated Plates</td><td>NEST Biotechnlology</td><td>702001</td><td></td></tr><tr><td>Olympus IX-71 Inverted Microscope</td><td></td><td></td><td></td></tr><tr><td>Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and LNCaP cells, which were mantained in RPMI + 10% FBS.</td><td></td><td></td><td></td></tr></tbody></table>

a cancer cell spheroid assay to assess invasion in a 3d setting

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

Using the spheroid invasion assay (Figure 1), a panel of cancer cell lines was tested for their ability to form spheroids, as well as for the amount of cell egress exhibited after implantation in a 3D matrix consisting of basement membrane materials and type I collagen (Table 1). These results demonstrate that not every cancer cell line will create well-formed spheroids, where cell lines possessing an epithelial morphology in vitro tended to produce regular and smooth aggregates...

Watch this Scientific Journal Video about A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting at JoVE.com

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

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

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

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32.4K Views.  Georgetown University. This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored. 

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

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Cancer Research

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

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 ...

3D Models in Cancer Biology: Invasion Dynamics in Heterospheroids

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) ...

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 ...

Examining Macrophage-Tumor Spheroid Interactions in a 3D Collagen Matrix Setup

A Macrophage-Tumor Spheroid Co-Invasion Assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

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 ...

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users&#8217; needs.

Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion.

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major ...

Biology

Generation of a Three-Dimensional Spheroid Model of Tumor Cell Invasion

<a href='https://app.jove.com/v/60998/a-3d-spheroid-model-for-glioblastoma'>Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma.&#160;J. Vis. Exp.&#160;(2020)</a>The video demonstrates the generation of a three-dimensional spheroid model of tumor cell invasion. Spheroids are formed from human brain tumor cells and embedded in a collagen matrix. The tumor cells at the spheroid periphery adopt an invasive phenotype, secreting proteases to degrade the collagen and advancing to invade the surrounding matrix.

Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma.&#160;J. Vis. Exp.&#160;(2020)The video demonstrates the generation of a three-dimensional ...