Name: analyzing the communication between monocytes and primary breast cancer cells in an extracellular matrix extract (ecme)-based three-dimensional system
Uploaded: 2018-01-08T13:00:00
Description: Watch this Scientific Journal Video about Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System at JoVE.c

This work was supported by CONACyT FONSEC SSA/IMSS/ISSSTE Project No. 233061 to Ezequiel M. Fuentes-Panan&#225; and by Fondo de Apoyo a la Investigaci&#243;n, Hospital Infantil de M&#233;xico Federico G&#243;mez (project number HIM-2014-053). Espinoza-S&#225;nchez NA is a doctoral student from Programa de Doctorado en Ciencias Biom&#233;dicas, Universidad Nacional Aut&#243;noma de M&#233;xico (UNAM) and received fellowship 231663 from CONACYT. E-S NA, also acknowledge the financial support provided by the Mexican Institute of Social Security (IMSS).

Samples from BrC patients were obtained from the tissue bank of the Unidad de Investigaci&#243;n en Virolog&#237;a y C&#225;ncer, Hospital Infantil de M&#233;xico Federico G&#243;mez. This study was approved by the scientific, ethical, and biosecurity review boards of the Hospital Infantil de M&#233;xico Federico G&#243;mez: Comit&#233; de Investigaci&#243;n, Comit&#233; de &#201;tica en Investigaci&#243;n and Comit&#233; de Bioseguridad. All patients were prospectively enrolled and were informed about the nature of the ...

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Epithelial cells grow in a 3D spatial conformation and their interaction with the ECM proteins is pivotal for tissue homeostasis. Many cancer studies have been based on cells grown in monolayers (2D) and although they have been critical to understanding many aspects of tumor formation and progression, monolayers do not recapitulate the characteristics that the ECM imposes on cells, for instance: limiting proliferation, adhesion-dependent cell survival, apical-basolateral polarity, ECM remodeling, cell differentiation, <e...

Tumor biology is far more intricate than previously thought. Mounting evidence shows that tumor cells are more than a mere bulk of uncontrolled proliferating cells; rather, different neoplastic cells seem to perform different functions displaying high organization and hierarchy within the tumor1. Tumor cells are also in intimate communication with non-transformed cells: macrophages, fibroblasts, lymphocytes, adipocytes, endothelial cells, among other cells are all immersed in the scaffold proteins and polysaccharides that constitute the ECM. Numerous direct and indirect interactions are established between transformed and non-transformed cells and with the ECM, which exerts a powerful influence over the disease outcome2,3. In the specific case of BrC, it is particularly important to dissect the communication of BrC cells with TAMs, considering that TAMs have been found to play a critical role in the evolution of the tumor, increasing the risk of metastasis and of disease recurrence4,5.
To analyze intra-tumoral interactions and their possible outcomes, new 3D in vitro approaches have been developed based on the use of ECM extracts that provide a much more complex microenvironment, closer to the reality of tumor biology, in comparison to the conventional monolayer cell cultures in which the cells grow attached to plastic. Petersen and Bissell6 provided the first model of nonmalignant and malignant mammary epithelial cells cultured on a laminin-rich basement membrane and were the first to describe the 3D organotypic structures that discriminate nonmalignant human breast epithelial cells from their malignant counterparts. A decade later, the model developed by Debnath, Muthuswamy, and Brugge7,8,9 provided a valuable tool to elucidate the biological pathways compromised during malignant transformation of glandular acini, such as large acini formation due to uncontrolled proliferation, delocalization of tight junction proteins as evidence of impaired cell polarization, and loss of acini lumen as a result of cell resistance to anoikis, a type of programmed cell death that occurs in anchorage-dependent cells when they detach from the surrounding ECM. The models of Sameni, Jedeszko, and Sloane have focused on imaging proteolytic activity by cells, which is closely related to invasiveness, another crucial trait of tumor malignancy10,11,12. These models rely on protein matrices mixed with different fluorescence-quenched protein substrates (DQ-gelatin, DQ-collagen I, and DQ-collagen IV), in which fluorescent signals are indicative of the proteolytic degradation of collagen. 3D models are also used to study stem cell properties of both non-transformed and tumor cells, in which cell aggregates, also termed spheroids, can be cultured in suspension or in ECM-like proteins interrogating for mechanisms of cell differentiation, asymmetric cell division, cell-to-cell adherence, and cell motility13,14. Invasion assays allow testing of the intrinsic aggressiveness of the tumor and the identification of the molecules that serve as chemoattractants during the invasive process15. Overall, 3D models represent an affordable diversification of in vitro cell culture that more closely reflect normal and oncogenic tissue morphogenesis.
We have designed a 3D cell co-culture system based on the aforementioned models7,10,11, using both human commercial BrC cell lines of known aggressive potential (luminal and triple-negative types) and primary cells explanted from BrC patients. We first developed a model where either non-aggressive (MCF-7) or aggressive (MDA-MB-231) BrC cells were co-cultured with U937 monocytes in an extracellular matrix extract (ECME)-based 3D system that allowed direct cell-cell interactions. These co-cultures were used to determine how the communication between these two cell lineages influenced the transcription of a set of genes related to cancer aggressive behavior. A significant increase of cyclooxygenase-2 (COX-2) transcript was observed that coincided with an increased production of one of its products, prostaglandin E2 (PGE2), a finding that highlighted the role of inflammation in cancer progression. Increased transcription of MMP was also observed that correlated with greater collagen proteolysis when aggressive MDA-MB-231 cells were co-cultured with U937 monocytes in DQ-Collagen IV-containing cultures. Of note, our co-cultures did not support the assumption that cell-cell interaction mechanisms are needed for collagen degradation. It rather suggested that communication between the two cell lineages was mediated by secreted molecules. Furthermore, the supernatants harvested from these co-culture assays contained soluble factors that disorganized glandular acini formed by non-transformed MCF-10A cells13. It was found that aggressive and primary BrC cells secreted elevated levels of monocyte chemotactic molecules MCP-1, GM-CSF, and RANTES. Thus, we outlined a 3D culture in which cells were separated in cell culture inserts to prevent cell-cell interactions. These cultures were used to address the indirect communication between BrC cells and monocytes. For these assays, non-aggressive and aggressive commercial BrC cell lines and primary BrC cells, and three different types of human monocytes: commercial U937 and THP-1 cells, and primary monocytes (PMs) isolated from peripheral blood of healthy donors (all monocytes were used in a non-activated state) were used. Increased concentrations of inflammatory cytokines IL-1&#946; and IL-8 were observed to be enriched in co-culture. Similarly, it was found that MMP-1, MMP-2, and MMP-10 were also increased in the BrC cells-monocyte co-cultures, and thus reinforcing previous findings14. In this manuscript, a point-by-point workflow of primary BrC cells isolation and testing in 3D cultures is presented along with representative results. This work constitutes a good example of the great potential that in vitro 3D cell systems provide to interrogate specific aspects of tumor biology.

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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">U937</td>
			<td style="width:260px;">American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">CRL-1593.2</td>
			<td style="width:463px;">Monocytic cell line/histiocytic lymphoma&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">THP-1</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">TIB-202</td>
			<td>Monocytic cell line/acute monocytic leukemia&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">MCF-10A</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td>CRL-10317</td>
			<td>Non-transformed breast cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">MCF-7</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">HTB-22</td>
			<td>Breast Cancer Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">T47D</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">HTB-133</td>
			<td>Breast Cancer Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">HS578T</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">HTB-126</td>
			<td>Breast Cancer Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">MDA-MB-231</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">HTB-26</td>
			<td>Breast Cancer Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">RPMI 1640 medium</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DMEM High Glucose&#160;</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">11964-092</td>
			<td>4.5 g/L glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DMEM/F12</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">11039-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Antibiotic/Antimycotic</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">15240-062</td>
			<td>100 U/mL penicillin, 100 &#181;g/mL streptomycin, and 0.25 &#181;g/mL Fungizone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Fetal Bovine Serum</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Horse serum&#160;</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td>16050114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">0.05% Trypsin-EDTA 1X</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">25300-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS 1X (Phosphate Buffered Saline</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal Growth Factor (EGF)</td>
			<td>PeproTech</td>
			<td>AF-100-15 (1.00mg)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin</td>
			<td>SIGMA-ALDRICH</td>
			<td>I1882-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrocortisone</td>
			<td>SIGMA-ALDRICH</td>
			<td>H088-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholera toxin Vibrio cholerae</td>
			<td>SIGMA-ALDRICH</td>
			<td>C8052-1MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Matrigel&#160;</td>
			<td>Corning Inc</td>
			<td>356237</td>
			<td style="width:463px;">Engelbreth-Holm-Swarm (EHS) mouse sarcoma, extracellular matrix extract, Store at -20&#176;C until use at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">GM-CSF</td>
			<td>PeproTech</td>
			<td>300-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">MCP-1</td>
			<td>PeproTech</td>
			<td>300-04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">RANTES</td>
			<td>PeproTech</td>
			<td>300-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">IL-8</td>
			<td>PeproTech</td>
			<td>200-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">IL-1&#946;</td>
			<td>PeproTech</td>
			<td>200-01B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Crystal-violet</td>
			<td>Hycel M&#233;xico</td>
			<td>541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Paraformaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Transwell permeable supports (inserts)</td>
			<td>Corning Inc</td>
			<td style="width:143px;">3422</td>
			<td>6.5 mm diameter, 8 &#181;m pore size/24-well plates&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Transwell permeable supports (inserts)</td>
			<td>Thermofisher Scientific</td>
			<td style="width:143px;">140620</td>
			<td>6.5 mm diameter, 0.4 &#181;m pore size/24-well plates&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Monocyte Isolation Kit II Human&#160;</td>
			<td style="width:260px;">Miltenyi Biotec</td>
			<td style="width:143px;">130-091-153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">LS Columns</td>
			<td style="width:260px;">Miltenyi Biotec</td>
			<td style="width:143px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">Human Cytokine/Chemokine Magnetic Bead Panel Kit 96 Well Plate Assay</td>
			<td style="width:260px;">EMD Millipore</td>
			<td style="width:143px;">HCYTOMAG-60K</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">Magpix with xPonent software (laser based fluorescent analytical test instrumentation)</td>
			<td style="width:260px;">Luminex Corporation</td>
			<td style="width:143px;">40-072</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Lab-Tek chamber slide with cover (8 well)</td>
			<td style="width:260px;">Nalge Nunc International</td>
			<td style="width:143px;">177402</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Glass bottom microwell 35mm petri&#160; dishes</td>
			<td style="width:260px;">MatTek Corp.</td>
			<td style="width:143px;">P35G-1.5-14-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing the communication between monocytes and primary breast cancer cells in an extracellular matrix extract (ecme)-based three-dimensional system

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Morphological Analysis of BrC Cells in 3D Cultures:
The morphology of primary BrC cells growing in 3D cultures at low and high densities was studied over 5 days. During the first 48 h, cells adhere to the ECME and maintain a low density. At this time point, it can clearly be appreciated that cells present an elongated spindle-like shape, some with two or more long cytoplasmic projections and without showing appa...

Watch this Scientific Journal Video about Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System at JoVE.c

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

The overall goal of this protocol is to study the direct and indirect communication between primary breast cancer cells and monocytes in a three-dimensional co-culture system. This method can help answer key questions in inflammatory microenvironment of breast cancer field such as how immune cells are regraded and activated, how these cells help to sustain the tumor inflammatory microenvironment and how these microenvironment facilitates tumor cell invasion. The main advantage of this technique is that it provides an affordable alternative to study intra-tumor communication and illustrates the potential of in vitro, 3-D cell systems for interrogating specific features of tumor biology.Particularly those related to tumor aggression. To begin, cultivate two times 10 to the sixth U937 and THP1 monocytes in 10 milliliters of RPMI1640 medium, supplemented with 10 percent FBS and one percent antibiotic antimycotic. Also cultivate fresh PMs in supplemented DMEMF12 medium.Incubate the cells at 37 degrees Celsius in a humidified five percent carbon dioxide environment. Plate four time 10 to the fifth monocytes in one milliliter per well of their corresponding medium. Place an insert in each well and add 900 microliters of the four times 10 to the fifth BrC cell suspension in the corresponding medium.Incubate the cultures at 37 degrees Celsius in a humidified five percent carbon dioxide environment for five days and recover the supernatants. Retrieve the cells by trypsinization if subsequent analysis is performed. Include controls of individual cell cultures with the respective media.To label BrC cells and monocytes with commercially available cumerine and rhodamine before cell culture, prepare a monolayer of two times 10 to the sixth BrC cells of interest and replace the standard medium with sufficient, pre-warmed working solution of the fluorescent dye. Incubate the cells at 37 degrees Celsius in a humidified five percent carbon dioxide environment for 30 minutes. Then aspirate the dye solution and use sufficient one XPBS to gently rinse the cells.Aspirate the PBS and add the standard medium. To trypsinize the cells, add two milliliters of 05 percent trypsin and 0.48 millimolar EDTA to the flask and incubate the cells at 37 degrees Celsius for five to 10 minutes. Add 200 microliters of FBS to stop the trypsinization and seven milliliters of the corresponding medium without supplements.Re-suspend the cells by pipetting, then transfer 10 microliters of the cells to a Nuebauer chamber and count them. Next, pellet the cells at 430 times g and room temperature for five minutes, then discard the supernatant and use three milliliters of pre-warmed fluorescent dye working solution to re-suspend the cells. Incubate the cell suspension at 37 degrees Celsius in a humidified five percent carbon dioxide environment for 30 minutes.After pelleting the cells again, discard the dye working solution and use five to seven milliliters of one XPBS to gently re-suspend the cells. Then, after pelleting the cells once more and discarding the PBS, re-suspend the cells in five to seven milliliters of standard medium. Count 10 microliters of the cell suspension.Set up a co-culture by spreading enough ECME at the bottom of the well to form an even layer in each well of a four well chamber slide system. Plate 20 microliters of a single cell suspension containing five time 10 to the fifth labeled BrC cells per well. After 15 to 20 minutes at 37 degrees celsius, add a suspension of 2.5 times 10 to the fifth labeled monocytes in 80 microliters of assay medium supplemented with 60 percent ECME.Allow the ECME to solidify at 37 degrees Celsius for 15 to 20 minutes. Now add one milliliter of a one to one mixture of the BrC cells and monocyte culture medium. Incubate the co-cultures at 37 degrees Celsius in a humidified five percent carbon dioxide environment for 24 hours, 48 hours, or five days to track changes at different time points.To degrade the ECM proteins and recover the cells from the cultures, aspirate and discard the medium, then add 0.5 milliliters of one XPBS with 0.1 percent trypsin and 0.25 percent EDTA to the culture. Incubate the cells at 37 degrees Celsius for three hours. Following the incubation, add 0.5 milliliters of one XPBS with 10 percent FBS to neutralize the trypsin and re-suspend the cells by vigorously pipetting to attain a single cell suspension.After pelleting the cells, discard the supernatant and re-suspend the cells in five milliliters of one XPBS with 10 percent FBS. Repeat this step once. Subject the cell suspension to fax with the appropriate instrument.Ensure that the final populations are at least 95 percent pure. In each well of an eight well chamber slide system spread a 40 microliter base of ECME and let it solidify at 37 degrees Celsius for 30 minutes. Seed 800 MCF10 A cells per well in 400 microliters of supplemented DMEM F12 culture medium according to the text protocol.Then add 400 microliters of conditioned medium or supplemented DMEM F12 culture medium with 20 nanograms per milliliter of human recombinant IL1 beta. Place the chamber slide into a glass petri dish containing a 35 milliliter petri dish filled with two milliliters of PBS. The MCF 10A cells should be seeded in each well very slowly to avoid damage of the base of ECME and to prevent cells from settling down and forming a monoly.Using an optical microscope record glandular acini formation every 24 hours for 14 days. After 14 days of culture, add 100 microliters of 100 nanomolar dapy in one XPBS and incubate the samples at room temperature with constant stirring for 25 minutes. Rinse the samples with one XPBS at room temperature, three times for five minutes each with constant stirring.Use mounting medium specific for fluorescent staining to mount the preparations. Then seal the mounted samples with transparent polish and maintain the slides, protected from light at four degrees Celsius. Demonstrating the procedure in the confocal microscope will be Vadim Perez, a researcher from The National Laboratory of Advanced Microscoping.Analyze the acini with a confocal microscope taking images of transversal stacks at different depths of the acini. Visualize different cellular proteins in the acini with proper staining protocols. For example, caheren was stained here to assess cell to cell adhesion, cell polarity, and oralimen formation.The morphology of primary BrC cells growing in 3D cultures at low and high densities was studied over five days. During the first 48 hours, cells adhere to the ECME with an elongated spindle shape and form knit-like structures at five days. After five days in 3D co-cultures, both MCF7 and MDA-MB-231 commercial BrC cells formed disorganized aggregates.However, aggressive MDA-MB-231 cells for aggregates with elongated forms, while non-aggressive MCF7 cells for aggregates with rounded shapes that are more densely compacted then MDA-MB-231 cells. The supernatants from indirect interaction 3D primary BrC cultures and their five day co-cultures with primary monocytes were harvested. Significantly increased levels of IL1 beta and IL8 were observed in the primary BrC cell monocyte co-cultures.Both crucial and flammatory cytokines that have been previously associated with malignant progression. MCF10A cells were cultivated with two different supernatants from two primary BrC cell lines to observe lumen formation as a measure of resistance to anoykis. Unlike MCF10A cells grown under standard conditions, bi-confocal microscopy at day 14, transversal cuts of the spheroids show that MCF10A cells evaded anoykis as measured by counting the number of central cells in the acini.Following this procedure, other methods like medical analysis can be performed in order to answer additional questions. Test the signaling pathways in both cancer migration. After its development this technique paved the way for researchers in the field of tumor micro-environments to explore the communications of breast cancer cells with mass cells, fibroblasts, neutrofils, or even different clones of tumor cells in 3D co-culture systems.After watching this video, you should have a good understanding of how to establish a 3D culture system to model the tumor microenvironment.

Research

JoVE Journal

Cancer Research

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

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