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, ...

Research

JoVE Journal

Cancer Research

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

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Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

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Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

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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 ...

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...