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).

<ol><li>Rich, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+stem+cells%3A+understanding+tumor+hierarchy+and+heterogeneity%5BTitle%5D)+AND+%22Medicine+%28Baltimore%29%22%5BJournal%5D)">Cancer stem cells: understanding tumor hierarchy and heterogeneity.</a> Medicine (Baltimore). 95 (1), Suppl 1 2-7 (2016).</li>
<li>Wang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+tumor+microenvironment+in+tumorigenesis%5BTitle%5D)+AND+%22J+Cancer%22%5BJournal%5D)">Role of tumor microenvironment in tumorigenesis.</a> J Cancer. 8 (5), 761-773 (2017).</li>
<li>Quail, D. F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microenvironmental+regulation+of+tumor+progression+and+metastasis%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Microenvironmental regulation of tumor progression and metastasis.</a> Nat Med. 19 (11), 1423-1437 (2013).</li>
<li>Sica, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macrophage+polarization+in+tumour+progression%5BTitle%5D)+AND+%22Semin+Cancer+Biol%22%5BJournal%5D)">Macrophage polarization in tumour progression.</a> Semin Cancer Biol. 18 (5), 349-355 (2008).</li>
<li>Mantovani, A., Marchesi, F., Malesci, A., Laghi, L., Allavena, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumour-associated+macrophages+as+treatment+targets+in+oncology%5BTitle%5D)+AND+%22Nat+Rev+Clin+Oncol%22%5BJournal%5D)">Tumour-associated macrophages as treatment targets in oncology.</a> Nat Rev Clin Oncol. , (2017).</li>
<li>Petersen, O. W., Ronnov-Jessen, L., Howlett, A. R., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+with+basement+membrane+serves+to+rapidly+distinguish+growth+and+differentiation+pattern+of+normal+and+malignant+human+breast+epithelial+cells%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells.</a> Proc Natl Acad Sci U S A. 89 (19), 9064-9068 (1992).</li>
<li>Debnath, J., Muthuswamy, S. K., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphogenesis+and+oncogenesis+of+MCF-10A+mammary+epithelial+acini+grown+in+three-dimensional+basement+membrane+cultures%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.</a> Methods. 30 (3), 256-268 (2003).</li>
<li>Debnath, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+apoptosis+in+creating+and+maintaining+luminal+space+within+normal+and+oncogene-expressing+mammary+acini%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini.</a> Cell. 111 (1), 29-40 (2002).</li>
<li>Muthuswamy, S. K., Li, D., Lelievre, S., Bissell, M. J., Brugge, J. S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/11533657">ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini.</a> Nat Cell Biol. 3 (9), 785-792 (2001).</li>
<li>Sameni, M., Dosescu, J., Moin, K., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+imaging+of+proteolysis%3A+stromal+and+inflammatory+cells+increase+tumor+proteolysis%5BTitle%5D)+AND+%22Mol+Imaging%22%5BJournal%5D)">Functional imaging of proteolysis: stromal and inflammatory cells increase tumor proteolysis.</a> Mol Imaging. 2 (3), 159-175 (2003).</li>
<li>Sameni, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MAME+models+for+4D+live-cell+imaging+of+tumor%3A+microenvironment+interactions+that+impact+malignant+progression%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">MAME models for 4D live-cell imaging of tumor: microenvironment interactions that impact malignant progression.</a> J Vis Exp. (60), (2012).</li>
<li>Jedeszko, C., Sameni, M., Olive, M. B., Moin, K., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+protease+activity+in+living+cells%3A+from+two+dimensions+to+four+dimensions%5BTitle%5D)+AND+%22Curr+Protoc+Cell+Biol%22%5BJournal%5D)">Visualizing protease activity in living cells: from two dimensions to four dimensions.</a> Curr Protoc Cell Biol. 4, Chapter 4 Unit 4 20(2008).</li>
<li>Chimal-Ramirez, G. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MMP1%2C+MMP9%2C+and+COX2+expressions+in+promonocytes+are+induced+by+breast+cancer+cells+and+correlate+with+collagen+degradation%2C+transformation-like+morphological+changes+in+MCF-10A+acini%2C+and+tumor+aggressiveness%5BTitle%5D)+AND+%22Biomed+Res+Int%22%5BJournal%5D)">MMP1, MMP9, and COX2 expressions in promonocytes are induced by breast cancer cells and correlate with collagen degradation, transformation-like morphological changes in MCF-10A acini, and tumor aggressiveness.</a> Biomed Res Int. 2013, 279505(2013).</li>
<li>Espinoza-Sanchez, N. A., Chimal-Ramirez, G. K., Mantilla, A., Fuentes-Panana, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IL-1beta%2C+IL-8%2C+and+Matrix+Metalloproteinases-1%2C+-2%2C+and+-10+Are+Enriched+upon+Monocyte-Breast+Cancer+Cell+Cocultivation+in+a+Matrigel-Based+Three-Dimensional+System%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">IL-1beta, IL-8, and Matrix Metalloproteinases-1, -2, and -10 Are Enriched upon Monocyte-Breast Cancer Cell Cocultivation in a Matrigel-Based Three-Dimensional System.</a> Front Immunol. 8, 205(2017).</li>
<li>Li, Y., Wang, L., Pappan, L., Galliher-Beckley, A., Shi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IL-1beta+promotes+stemness+and+invasiveness+of+colon+cancer+cells+through+Zeb1+activation%5BTitle%5D)+AND+%22Mol+Cancer%22%5BJournal%5D)">IL-1beta promotes stemness and invasiveness of colon cancer cells through Zeb1 activation.</a> Mol Cancer. 11, 87(2012).</li>
<li>Gallagher, S., Winston, S. E., Fuller, S. A., Hurrell, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunoblotting+and+immunodetection%5BTitle%5D)+AND+%22Curr+Protoc+Mol+Biol%22%5BJournal%5D)">Immunoblotting and immunodetection.</a> Curr Protoc Mol Biol. 10, Chapter 10 Unit 10 18(2008).</li>
<li>Troeberg, L., Nagase, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zymography+of+metalloproteinases%5BTitle%5D)+AND+%22Curr+Protoc+Protein+Sci%22%5BJournal%5D)">Zymography of metalloproteinases.</a> Curr Protoc Protein Sci. 21, Chapter 21 Unit 21 15(2004).</li>
<li>Arevalo-Romero, H., Meza, I., Vallejo-Flores, G., Fuentes-Panana, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Helicobacter+pylori+CagA+and+IL-1beta+Promote+the+Epithelial-to-Mesenchymal+Transition+in+a+Nontransformed+Epithelial+Cell+Model%5BTitle%5D)+AND+%22Gastroenterol+Res+Pract%22%5BJournal%5D)">Helicobacter pylori CagA and IL-1beta Promote the Epithelial-to-Mesenchymal Transition in a Nontransformed Epithelial Cell Model.</a> Gastroenterol Res Pract. 2016, 4969163(2016).</li>
<li>Reed, J. R., Leon, R. P., Hall, M. K., Schwertfeger, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin-1beta+and+fibroblast+growth+factor+receptor+1+cooperate+to+induce+cyclooxygenase-2+during+early+mammary+tumourigenesis%5BTitle%5D)+AND+%22Breast+Cancer+Res%22%5BJournal%5D)">Interleukin-1beta and fibroblast growth factor receptor 1 cooperate to induce cyclooxygenase-2 during early mammary tumourigenesis.</a> Breast Cancer Res. 11 (2), 21(2009).</li>
<li>Ma, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidermal+growth+factor+%28EGF%29+and+interleukin+%28IL%29-1beta+synergistically+promote+ERK1%2F2-mediated+invasive+breast+ductal+cancer+cell+migration+and+invasion%5BTitle%5D)+AND+%22Mol+Cancer%22%5BJournal%5D)">Epidermal growth factor (EGF) and interleukin (IL)-1beta synergistically promote ERK1/2-mediated invasive breast ductal cancer cell migration and invasion.</a> Mol Cancer. 11, 79(2012).</li>
<li>Grande, S. M., Bannish, G., Fuentes-Panana, E. M., Katz, E., Monroe, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tonic+B-cell+and+viral+ITAM+signaling%3A+context+is+everything%5BTitle%5D)+AND+%22Immunol+Rev%22%5BJournal%5D)">Tonic B-cell and viral ITAM signaling: context is everything.</a> Immunol Rev. 218, 214-234 (2007).</li>
<li>Benton, G., Arnaoutova, I., George, J., Kleinman, H. K., Koblinski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrigel%3A+from+discovery+and+ECM+mimicry+to+assays+and+models+for+cancer+research%5BTitle%5D)+AND+%22Adv+Drug+Deliv+Rev%22%5BJournal%5D)">Matrigel: from discovery and ECM mimicry to assays and models for cancer research.</a> Adv Drug Deliv Rev. 79-80, 3-18 (2014).</li>
<li>Worthington, P., Pochan, D. J., Langhans, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peptide+Hydrogels+-+Versatile+Matrices+for+3D+Cell+Culture+in+Cancer+Medicine%5BTitle%5D)+AND+%22Front+Oncol%22%5BJournal%5D)">Peptide Hydrogels - Versatile Matrices for 3D Cell Culture in Cancer Medicine.</a> Front Oncol. 5, 92(2015).</li>
<li>Tibbitt, M. W., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrogels+as+extracellular+matrix+mimics+for+3D+cell+culture%5BTitle%5D)+AND+%22Biotechnol+Bioeng%22%5BJournal%5D)">Hydrogels as extracellular matrix mimics for 3D cell culture.</a> Biotechnol Bioeng. 103 (4), 655-663 (2009).</li>
<li>Schlaermann, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+human+gastric+primary+cell+culture+system+for+modelling+Helicobacter+pylori+infection+in+vitro%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro.</a> Gut. 65 (2), 202-213 (2016).</li>
<li>Pompaiah, M., Bartfeld, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gastric+Organoids%3A+An+Emerging+Model+System+to+Study+Helicobacter+pylori+Pathogenesis%5BTitle%5D)+AND+%22Curr+Top+Microbiol+Immunol%22%5BJournal%5D)">Gastric Organoids: An Emerging Model System to Study Helicobacter pylori Pathogenesis.</a> Curr Top Microbiol Immunol. 400, 149-168 (2017).</li>
<li>Takebe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascularized+and+functional+human+liver+from+an+iPSC-derived+organ+bud+transplant%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Vascularized and functional human liver from an iPSC-derived organ bud transplant.</a> Nature. 499 (7459), 481-484 (2013).</li>
<li>Takasato, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directing+human+embryonic+stem+cell+differentiation+towards+a+renal+lineage+generates+a+self-organizing+kidney%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney.</a> Nat Cell Biol. 16 (1), 118-126 (2014).</li>
</ol>

The authors declare that they do not have any conflict of interest.

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, etc. Importantly, not only the interaction between tumor cells and ECM are essential for the progression of cancer, but also the communication established between tumor and non-tumor cells, such as immune cells. The protocols provided here facilitate the understanding of the interactions between immune cells and cancer cells within the environment provided by the ECM, the inflammatory response promoted by these interactions, and how this inflammatory condition create a positive loop fostering chemoattraction of monocytes and more invasion of cancer cells.
A great advantage of using ECME is that it provides a biological scaffold suitable to perform a variety of experimental 3D models22. It is enriched with ECM proteins such as laminin, collagen IV, entactin, heparan sulfate proteoglycan, and growth factors, which in vivo would be interacting with epithelial cells. A disadvantage is that because it comes from lysates of murine sarcomas, the concentration of their components varies between different lots. So, it is often necessary to characterize several lots to obtain more homogeneous data. In the laboratory, each new lot is tested in two ways: 1) by evaluating the correct acini formation of MCF-10A cells, and 2) by assessing the invasiveness of BrC cell lines of well-established invasive capacity. Another option is to work with other scaffolding products, such as Hydrogel, which is a synthetic nanofiber peptide scaffold. The stiffness of the 3D culture can be controlled by adjusting the concentration of Hydrogel23,24. Importantly, this system could be applied to study the interaction between any type of neoplastic cell and hematopoietic or non-hematopoietic cells. It could even be possible to design more complex systems using more than two different cell lineages. One important advantage of the 3D models is that they allow study of the cell morphology and cell organization shaped by ECM interactions, which are altered during oncogenic transformation. Likewise, one can study oncogenic features such as proteolytic degradation, and whether cells initially placed in different layers of the culture signal to each other to promote direct interaction/communication. Moreover, different cell lineages can be labeled with different fluorochromes so that they can be isolated after the experiment to address specific questions to each individual cell type, using, for example RNA and/or protein expression analysis13. Here, IL-1&#946; secretion to the medium in primary BrC cells/PM co-cultures is shown, supporting a possible pivotal role of this cytokine in the indirect communication between tumor cells and monocytes that triggers malignant progression.
Another essential experimental protocol in the laboratory is the analysis of acini formation in 3D cultures of non-transformed MCF-10A cells. This assay was used to test for viral and cellular oncogene activity, and to test how the tumor microenvironment influences feature related to aggressive cancers. For instance, during the normal development of the acini, luminal central cells lose contact with the basal membrane proteins (provided by ECME) triggering mechanisms of cell death, known as anoikis. It was observed that both the supernatants from primary BrC cells or recombinant IL-1&#946; promote MCF-10A cells resistance to anoikis. Because a high concentration of IL-1&#946; is promoted upon BrC cell-monocyte interaction, this observation supports that the tumor stroma is an integral component of tumor progression to highly aggressive stages.
The migration and invasion protocols described here constitute useful 3D complementary assays in which we can support the capability of cells to migrate or invade in response to stimuli derived from the tumor stroma. It was shown that chemokines (GM-CSF and MCP-1) found in elevated concentrations in 3D cultures of primary BrC cells can promote migration of U937, THP-1, and PMs, supporting the ability of aggressive tumor cells to promote a pro-inflammatory microenvironment. Furthermore, IL-8 that is also found in increased concentration in the supernatants from co-cultures of BrC cells/monocytes, promoted increased invasion of both commercial aggressive BrC cells (HS578T and MDA-MB-231) and primary BrC cells.
Regarding the condition of cells, it is important to work with primary BrC cells before they reach ten passages to take advantage of their proliferation peak and avoid potential post-isolation genetic and epigenetic changes resulting from in vitro culture. Another essential aspect when working with primary cells is to confirm their identity after isolation. We used a panel of biomarkers to determine their epithelial lineage identity, which includes PanCK, Muc-1, and EpCAM (specified in step 2.5). Similarly, it is preferable to use fresh PMs, of which each batch is tested assuring that they are at the same stage of differentiation. Each new batch used presented the phenotype CD34neg CD11bpos CD14pos CD64pos CD68neg CD16neg, which corresponds to non-activated immature monocytes. Importantly, we do not mix monocytes from different donors, as mixing results in monocyte activation. Instead, we independently tested BrC co-cultures using monocytes from at least two different donors.
3D culture systems still have the limitation that only a few elements of tumor biology can be reliably modeled. A new alternative is the more complex 3D models of organoids, which are based on the expansion and differentiation of stem cells in vitro. Organoids also develop highly organized epithelial structures. Examples include the gastric 25,26, liver 27, and kidney organoids28. However, organoid models for every human organ remain to be achieved; furthermore, they require much more complex and expensive experimental conditions.
In conclusion, here we describe in detail a workflow of assays: starting from isolation of tumor cells from BrC patients, testing their inflammatory capacity in an ECME-based 3D culture model, testing how that inflammatory microenvironment serves them to recruit additional inflammatory cells, such as monocytes, to finally analyzing the communication between both types of cells, and how that communication further fosters an inflammatory microenvironment that promotes progression to more aggressive cancer stages. Additionally, we describe the morphology and phenotype of primary BrC cells. In future studies, it will be interesting to test other inflammatory cells or even test the communication between BrC cells and multiple cells often found in the tumor stroma. These multiple-cell 3D cultures will provide a larger picture of what happens biologically in vivo during cancer progression. Comparisons between in vitro data and the patient clinical outcome will identify potential mechanisms that have a higher impact on cancer aggressiveness.

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.

<table><tbody><tr><td>U937</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>CRL-1593.2</td><td>Monocytic cell line/histiocytic lymphoma&amp;nbsp;</td></tr><tr><td>THP-1</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>TIB-202</td><td>Monocytic cell line/acute monocytic leukemia&amp;nbsp;</td></tr><tr><td>MCF-10A</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>CRL-10317</td><td>Non-transformed breast cell line</td></tr><tr><td>MCF-7</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>HTB-22</td><td>Breast Cancer Cell line</td></tr><tr><td>T47D</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>HTB-133</td><td>Breast Cancer Cell line</td></tr><tr><td>HS578T</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>HTB-126</td><td>Breast Cancer Cell line</td></tr><tr><td>MDA-MB-231</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>HTB-26</td><td>Breast Cancer Cell line</td></tr><tr><td>RPMI 1640 medium</td><td>GIBCO BRL Life Technologies</td><td>11875-093</td><td></td></tr><tr><td>DMEM High Glucose&amp;nbsp;</td><td>GIBCO BRL Life Technologies</td><td>11964-092</td><td>4.5 g/L glucose</td></tr><tr><td>DMEM/F12</td><td>GIBCO BRL Life Technologies</td><td>11039-021</td><td></td></tr><tr><td>Antibiotic/Antimycotic</td><td>GIBCO BRL Life Technologies</td><td>15240-062</td><td>100 U/mL penicillin, 100 &amp;micro;g/mL streptomycin, and 0.25 &amp;micro;g/mL Fungizone</td></tr><tr><td>Fetal Bovine Serum</td><td>GIBCO BRL Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>Horse serum&amp;nbsp;</td><td>GIBCO BRL Life Technologies</td><td>16050114</td><td></td></tr><tr><td>0.05% Trypsin-EDTA 1X</td><td>GIBCO BRL Life Technologies</td><td>25300-062</td><td></td></tr><tr><td>PBS 1X (Phosphate Buffered Saline</td><td>GIBCO BRL Life Technologies</td><td>20012-027</td><td></td></tr><tr><td>Epidermal Growth Factor (EGF)</td><td>PeproTech</td><td>AF-100-15 (1.00mg)</td><td></td></tr><tr><td>Insulin</td><td>SIGMA-ALDRICH</td><td>I1882-100MG</td><td></td></tr><tr><td>Hydrocortisone</td><td>SIGMA-ALDRICH</td><td>H088-5G</td><td></td></tr><tr><td>Cholera toxin Vibrio cholerae</td><td>SIGMA-ALDRICH</td><td>C8052-1MG</td><td></td></tr><tr><td>Matrigel&amp;nbsp;</td><td>Corning Inc</td><td>356237</td><td>Engelbreth-Holm-Swarm (EHS) mouse sarcoma, extracellular matrix extract, Store at -20&amp;deg;C until use at 4&amp;deg;C</td></tr><tr><td>GM-CSF</td><td>PeproTech</td><td>300-03</td><td></td></tr><tr><td>MCP-1</td><td>PeproTech</td><td>300-04</td><td></td></tr><tr><td>RANTES</td><td>PeproTech</td><td>300-06</td><td></td></tr><tr><td>IL-8</td><td>PeproTech</td><td>200-08</td><td></td></tr><tr><td>IL-1&amp;beta;</td><td>PeproTech</td><td>200-01B</td><td></td></tr><tr><td>Crystal-violet</td><td>Hycel M&amp;eacute;xico</td><td>541</td><td></td></tr><tr><td>Paraformaldehyde</td><td>SIGMA-ALDRICH</td><td>P6148</td><td></td></tr><tr><td>Transwell permeable supports (inserts)</td><td>Corning Inc</td><td>3422</td><td>6.5 mm diameter, 8 &amp;micro;m pore size/24-well plates&amp;nbsp;</td></tr><tr><td>Transwell permeable supports (inserts)</td><td>Thermofisher Scientific</td><td>140620</td><td>6.5 mm diameter, 0.4 &amp;micro;m pore size/24-well plates&amp;nbsp;</td></tr><tr><td>Monocyte Isolation Kit II Human&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-091-153</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>Human Cytokine/Chemokine Magnetic Bead Panel Kit 96 Well Plate Assay</td><td>EMD Millipore</td><td>HCYTOMAG-60K</td><td></td></tr><tr><td>Magpix with xPonent software (laser based fluorescent analytical test instrumentation)</td><td>Luminex Corporation</td><td>40-072</td><td></td></tr><tr><td>Lab-Tek chamber slide with cover (8 well)</td><td>Nalge Nunc International</td><td>177402</td><td></td></tr><tr><td>Glass bottom microwell 35mm petri&amp;nbsp; dishes</td><td>MatTek Corp.</td><td>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 apparent cell-cell contacts. After 5 days of culture, cells proliferated while maintaining their initial morphology. Moreover, they formed structures resembling nets without a specific structural organization, and appear piled up with no contact inhibition. A comparison with non-transformed MCF-10A cells was made, which form spherical structures resembling glandular acini. These cells display a characteristic morphology of rounded well-delimited and organized structures in homogenous sizes (of approximately 50 &#181;m). On the contrary, primary BrC cells share a much closer resemblance with aggressive BrC cells MDA-MB-231 (Figure 1).
Analysis of Cell-to-cell Direct Interactions and Collagen Degradation:
We designed a 3D co-culture model to assess the following: cell morphology, collagen degradation, cell migration, and whether there is a direct interaction between BrC cells and monocytes in the co-culture. After 5 days, both MCF-7 and MDA-MB-231 commercial BrC cells form disorganized aggregates in 3D cultures, however, they differ in their morphology. Aggressive cells MDA-MB-231 form aggregates with elongated forms with some cells tending to separate from the rest, while non-aggressive MCF-7 cells form aggregates where cells maintain rounded shapes and they appear to be more densely compacted(Figure 2A, B). DQ collagen IV was incorporated to visualize proteolytic degradation (green), with both BrC cultures exhibiting fluorescent activity. To quantitatively assess proteolytic activity, green fluorescence was measured as IOD of six 50 &#181;m2 fields. A comparison was made between individual 3D cultures of MDA-MB-231 cells and co-cultures with U937 monocytes. Statistically significant increase of proteolysis in the co-culture (Figure 2C) was observed. Also, a confocal microscopy analysis of serial transversal stacks every 10 &#181;m of the MDA-MB-231 and U937 monocytes co-culture was performed (Figure 2D). This analysis revealed that U937 monocytes migrated towards aggressive cells MDA-MB-231 with a few monocytes reaching the layer of BrC cells. However, it was evident that cell-cell direct interaction sites were not necessarily coinciding with areas of higher proteolytic activity: instead, areas of collagen degradation were uniformly spread in the culture, leading to the inference that collagen degradation was the result of indirect communication mediated by secreted factors. Therefore, the 3D co-culture model was changed to place the cells in different compartments separated by a porous membrane to allow cell-cell communication based on secreted factors, and avoid cell labeling and sorting after culture.
Analysis of IL-1&#946; in Supernatants from 3D Cultures:
Working with the indirect interaction 3D cultures, the supernatants from individual primary BrC cultures and their 5 days co-cultures with PM were harvested. These supernatants were subjected to the simultaneous analysis of 18 cytokines and 5 MMPs using commercial kits and an instrument specifically designed for this analysis. Of all analytes tested, significantly increased levels of IL-1&#946; and IL-8 were observed in the primary BrC cell-monocyte co-cultures, both crucial inflammatory cytokines that have been previously associated with malignant progression15,18,19,20 (Figure 3; results for IL-1&#946;). This example is another type of analysis that 3D cultures allow to mimic the communication network that shapes the tumor microenvironment.
Characterization of Supernatants and Acini Structure:
On the basis of the 3D experimental system developed by Debnath, Muthuswamy, and Brugge7, where MCF-10A were established as a model of the mechanisms associated with oncogenesis, evaluation of whether the secreted factors from the primary BrC cell supernatants influenced the formation of the MCF-10A 3D acini-like structures, similar to the activity observed after transduction of viral and cellular oncogenes7,21 was performed. MCF-10A cells were cultivated with two different supernatants from two primary BrC cell lines to observe lumen formation (as a measure of resistance to anoikis). At day 14, the spheroids were evaluated by confocal microscopy transversal cuts at 0, 25, 50, 75, and 100% depth, and in both cases, it was found that MCF-10A cells evaded anoikis (measured by counting the number of central (luminal) cells in the acini (Figure 4B, C)). Thus, MCF-10A cell acini that are formed with the conditioned media from BrC cells, lack a well-formed hollow lumen, unlike MCF-10A that are grown with its standard culture medium (as described in step 1.2)(Figure 4A). In the 3D co-culture system, IL-1&#946; was highly secreted; therefore, the MCF-10A cells were also cultivated with human recombinant IL-1&#946;18. Figure 4D shows a confocal microscopy transversal cut at 50% depth of the acini in the presence of IL-1&#946; (lower panels), and reveals that this cytokine also confers resistance to anoikis. Thus, soluble factors secreted by primary BrC cells, such as IL-1&#946;, promote the survival of non-transformed MCF-10A cells that lose ECM interactions, a characteristic of invasive cancers.
3D Co-cultures Promote an Inflammatory Response Associated with Migration of Monocytes and Invasion of Cancer Cells:
We previously demonstrated that primary BrC cells in 3D culture secrete high basal levels of pro-inflammatory chemokines known to attract monocytes14. Thus, we analyzed the migratory capacities of monocytes in response to the chemokines found enriched in the conditioned media of the BrC cells. The migratory capacity of commercial monocytes U937 and THP-1 and fresh PMs was evaluated in response to three different chemokines: GM-CSF, MCP-1, and RANTES. U937 and THP-1 monocytes were capable of migrating in response to GM-CSF and MCP-1, with U937 as the most responsive, while both monocytes had a null response to RANTES. Importantly, PMs showed a high basal migratory activity and the most potent response to MCP-1 (Figure 5). IL-8 was also enriched after the 3D co-culture of primary BrC cells and monocytes. Thus, we analyzed whether IL-8 modifies the capacity of invasion of commercial and primary BrC cell lines. The commercial aggressive BrC cell lines (HS578T and MDA-MB-231) invaded in response to IL-8, while the non-aggressive (MCF-7 and T47D) did not invade. Surprisingly, primary BrC cells were more invasive than commercial aggressive BrC cell lines in response to IL-8 (Figure 6A, right panels). In some cases, cells invade as clusters and therefore IOD analysis was necessary (Figure 6B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56589/56589fig1.jpg" /> 
Figure 1: Representative images of BrC cells in 3D cultures. From left to right: control of non-transformed MCF-10A cells forming characteristic glandular acini, with rounded morphology, well-delimited cell contacts, and organized structures of homogenous sizes (approximately 50 &#181;m on average, optical magnification of 200X). Middle panels show primary BrC cells cultured at low density (2 days) and high density (5 days). Cells adhere to the ECME and maintain a low density at early time points. It is evident that cells present an elongated spindle-like shape, some with two or more long cytoplasmic projections, and with no apparent cell-cell adhesion. On the other hand, high-density cells at later points formed structures resembling nets without a specific structural organization. These cells appeared piled up showing no contact inhibition. A control of commercial MDA-MB-231 cells after 5 days in 3D culture is shown; these aggressive BrC cells share a much closer resemblance with primary BrC cultures (optical magnification of 100X). Scale bar represents 50 &#181;m. 800 cells were seeded at the beginning of the experiment. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56589/56589fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56589/56589fig2.jpg" /> 
Figure 2: Analysis of ECM degradation. Non-aggressive MCF-7 cells stained with a yellow fluorochrome (A) and aggressive MDA-MB-231 cells stained with a red fluorochrome (B) cultivated for 5 days in 3D conditions; 0.5% of green fluorescence-labeled collagen IV was incorporated to visualize ECM degradation. In A and B, the upper left pictures represent the levels of collagen degradation, the upper right panels represent the optical pictures, the bottom left represent the BrC cells, and the bottom right panels show the merge of fluorescent and optical images. For A and B, the optical magnification of 200X and scale bar of 100 &#181;m are presented. (C) Representative images of ECM proteolysis (green) in 3D culture of individual MDA-MB-231 cells as control, compared with ECM proteolysis of 3D co-culture of MDA-MB-231 with U937 monocytes. Scale bars represent 10 &#181;m. The IOD (integrated optical density) of six 50 &#181;m2 fields from each condition was measured. The mean and the standard error of the mean (SEM) from the quantifications are presented; two asterisks represent a statistically significant difference with p &#60; 0.01 (Mann-Whitney test). (D) Direct interaction of MDA-MB-231 cells (gray shade mass) with fluorescently stained U937 monocytes (orange); green fluorescence corresponds to proteolysis. Serial slices were generated every 10 &#181;m by confocal microscopy to address the localization and direct interactions between both cell lines. Optical magnification of 200X; scale bars represent 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56589/56589fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56589/56589fig3.jpg" /> 
Figure 3:&#160;IL-1&#946; concentration is increased in 3D co-cultures. One representative result of IL-1&#946; levels (pg/mL) in supernatants from eight (PC1-8) primary BrC cells, cultured individually in 3D (3D), and compared against their 3D co-cultures with PM (CC). A control of PM in individual 3D culture was included for comparison (PM). Results were extracted from a larger multiplexing analysis, where several cytokines were simultaneously detected in each sample by means of an immunodetection-based technique available as a commercial kit. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56589/56589fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56589/56589fig4.jpg" /> 
Figure 4: Secreted factors from primary BrC cells induce resistance to anoikis in non-transformed MCF-10A cells. MCF-10A cells were grown in 3D conditions with standard culture media (column A and upper panels of D), in which it can be observed that the acini present typical hollow lumens (25-75% depth highlighted with a red square); when MCF-10A cells were grown with two different supernatants from two primary BrC cultures (columns B and C), lumens are not hollow but filled with cells denoting resistance to anoikis. Similarly, when MCF-10A cells were cultured in 3D conditions adding human recombinant IL-1&#946; (20 ng/mL), no hollow lumen can be appreciated at 50% depth confocal microscopy sections of the acini (lower panels of D). The right oval caricature represents the confocal microscopy sections that were made at 0, 25, 50, 75 and 100% depth of the acini. Images show nuclei stained with DAPI (blue) and E-cadherin in green. Optical magnification of 200X, scale bars represent 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56589/56589fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56589/56589fig5.jpg" /> 
Figure 5: Migratory capacity of monocytes. Representative images of the migratory capacity of U937, THP-1 and PM in response to GM-CSF, MCP-1 and RANTES. Controls without chemokine are included (first column from left to right). The numbers below the images indicate the number of migratory cells in each condition; U937 and THP-1 monocytes were mainly responsive to GM-CSF and MCP-1, while PM showed a high migratory capacity per se, and were the most responsive cells to MCP-1. Optical magnification of 100X; scale bars represents 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56589/56589fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56589/56589fig6.jpg" /> 
Figure 6: Aggressive and primary BrC cells invade in response to IL-8.&#160;A. Invasion assays with two non-aggressive (MCF-7 and T47D), two highly-aggressive (HS578T and MDA-MB-231) BrC cell lines and one primary BrC culture in response to IL-8 used as chemoattractant. Upper panels correspond to controls without chemokine showing no invasion, lower panels show the response to IL-8, with highly-aggressive BrC cells and primary BrC cells invading through the membrane of the cell culture insert. B. Example of cells that invade in clusters; in these cases, measurement of invasion is done with an image analysis program to determine invasion in terms of IOD (integrated optical density) per area. The numbers below the images represent the number of invading cells in each condition. Optical magnification of 100X; scale bars represent 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56589/56589fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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, ...

analyzing-communication-between-monocytes-primary-breast-cancer-cells

Nanyang Technological University

Research

JoVE Journal

Cancer Research

11.1K Views.  Hospital Infantil de México Federico Gómez. Hier beschrijven we een driedimensionale cultuur-methode voor het analyseren van de morfologie van primaire borstkankercellen, alsmede over de studie van hun directe/indirecte interacties met monocyten en de resultaten zoals de aantasting van het collageen, immuun cel aanwerving, cel invasie, en bevordering van kanker-gerelateerde ontsteking.

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

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 ...

Medicine

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 fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

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 ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Bioengineering

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

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 ...

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.

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 ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

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

Samenvatting

Meer video's verkennen