This work was supported by grant R15CA169978 from the National Institutes of Health. Additional funding came from Villanova University.

All experiments and methods were performed as authorized by Villanova University Institutional Animal Care and Use Committee (IACUC).
1. Gene Expression in Cultured Mouse Mammary Tumor Cells
<ol>
	<li>First, prepare the cell lines to be tested.</li>
	<li>Use a breeding colony of BALB/cV mice. These mice carry the BALB/cV strain of mouse mammary tumor virus, transmitted to pups in milk6,7. Fifty percent of breeding females develop mammary tumors by 10 months of age.
	<ol>
		<li>To establish a tumor cell line, sacrifice a tumor-bearing dam using an IACUC-approved protocol. Soak the abdominal skin in 70% EtOH and remove the tumor under sterile conditions in a laminar flow hood. Tumors are subcutaneous, so take care to avoid puncturing the peritoneum.</li>
		<li>Place tumor fragments from non-necrotic areas in a petri dish with a small amount of sterile Hanks Balanced Saline Solution (HBSS) and mince very finely with a sterile razor blade.</li>
		<li>Transfer the dispersed cell clumps to a T25 cell culture flask containing 5 mL Dulbecco&#39;s Modified Eagle Medium or DMEM (4.5 g/L glucose, phenol red, and L-glutamine) supplemented with 50% Fetal Bovine Serum or FBS and 1% antibiotic/antimycotic solution. Grow cells in treated cell culture flasks in an incubator with 5% CO2 and 100% humidity at 37 &#176;C.</li>
		<li>Wash off non-adherent cells after 24-48 h and replace the DMEM with 50% FBS and 1% antibiotic/antimycotic solution. Return the cells to the incubator.</li>
		<li>Replace liquid culture media every 3 days.</li>
		<li>Split or passage the cells when they are 90% confluent. Remove culture media and wash adherent cells with HBSS (without calcium or magnesium). Incubate cells with 0.25% Trypsin-EDTA solution for 1-5 min at 37 &#176;C until cells are rounded and detached. Dilute cells 1:10 in fresh culture media and add to a new flask. A T-25 cell culture flask requires 5-8 mL culture media, 5 mL of HBSS to wash, and 1 mL of trypsin-EDTA to passage. Return the flask to the incubator.</li>
		<li>Reduce the FBS concentration to 40% after the first passage, then 30% after the second passage, then 20% after the third passage, and finally 10% for all subsequent passages. The process of establishing growth in standard DMEM medium with 10% FBS requires four passages and 2 - 8 months.</li>
	</ol>
	</li>
	<li>Once the cell line(s) are established, alter the expression of suspected oncogenes by transfection of shRNA targeting mRNA or CRISPR for non-essential genes.
	<ol>
		<li>Establish antibiotic-resistant stable cell lines with individual clones that are verified by western blot and/or RT-qPCR for consistent results, however, transient transfections can work as well since the assay only requires 22 h. Control cell lines with non-specific target sequences are also required.</li>
	</ol>
	</li>
</ol>
2. In Vitro Invasion Assay
<ol>
	<li>Grow adherent mouse mammary tumor cells in a T25 flask until 70-90% confluent for Day 1 of the experiment. 
	NOTE: Other cell lines or experimental conditions may require different cell culture media. For example, if hormone-responsive breast cancer cells are being tested, charcoal-filtered serum may be needed to remove compounds that activate estrogen or other hormone receptors.</li>
	<li>Prepare the Boyden chamber inserts by warming them to room temperature (from -20 &#176;C storage) for about 20 min in a cell culture hood. Avoid freeze-thaw cycles for unused inserts. Use three inserts (replicates) to generate statistically valid data for each cell line for each experiment.
	<ol>
		<li>Add pre-warmed 37 &#176;C serum-free DMEM media to the well and then the insert. For inserts designed for 24-well dishes, add 500 &#181;L of serum-free DMEM to the well and then 500 &#181;L of serum-free DMEM to the insert so the porous membrane and gel are hydrated on both sides. 
		NOTE: For larger inserts designed for a 6-well dish, use 2 mL of serum-free DMEM on both sides.</li>
	</ol>
	</li>
	<li>Place the dish with inserts into a 37 &#176;C cell culture incubator with 5% CO2 and 100% humidity for at least 2 h to thoroughly hydrate and acclimate.</li>
	<li>Prepare cells by removing the growth media and rinsing the cells with 5 mL HBSS.
	<ol>
		<li>Remove the HBSS and add 1 mL 0.25% trypsin-EDTA solution for 1 - 5 min at 37 &#176;C or until cells appear rounded or show signs of detachment from the flask.</li>
		<li>Gently tap the flask to detach all the cells. Resuspend the cells in 5 mL of DMEM + 10% FBS and transfer to a sterile 15 mL centrifuge tube.</li>
		<li>Centrifuge the cells at 1,000 x g for 5 min to gently pellet the cells. Remove the media and replace with 5 mL serum-free DMEM to resuspend the cells.</li>
		<li>Repeat the centrifugation and suspension in serum-free media twice more for a total of 3 washes.</li>
		<li>Thoroughly resuspend the cells in the final 5 mL of serum-free DMEM and ensure there are no clumps of cells.</li>
	</ol>
	</li>
	<li>Determine the cell concentration using a hemocytometer. Count only viable cells. Dilute the cell suspension to 5 x 104 cells/mL in 5 mL of serum-free DMEM. 
	NOTE: This concentration yields 2,500 cells per insert in a 24-well dish. This number of cells is enough for aggressive cancer cells with high rates of migration and invasion. Less aggressive cell lines may require more cells, up to 10,000 cells per insert for observable invading cells. If reduced cell invasion is expected, consider maximizing invading cells by increasing cell number. If increased cell invasion is expected, begin with fewer cells.</li>
	<li>Remove the cell culture dish from the incubator and gently aspirate the media from the insert. Lift the insert and aspirate the media from the well. Working quickly, add the chemoattractant to the lower chamber. For a 24-well dish, add 750 &#181;L of DMEM with 5% FBS.
	<ol>
		<li>Place the insert in the well and add the cells to the insert. For a 24-well dish, use 500 &#181;L of cell suspension. For a 6-well dish insert, use 2.5 mL of chemoattract and 2 mL of cells. Ensure that no bubbles of air are present on either side of the membrane.</li>
		<li>Return the dish to the cell culture incubator for 22 h.</li>
	</ol>
	</li>
	<li>After 22 h, fix and stain the cells. Prepare a solution of 1% paraformaldehyde in 1x phosphate buffered saline (PBS) for fixation.
	<ol>
		<li>In a clean 24-well cell culture dish, add 1 mL of fixative to individual wells so there is one well for each insert.</li>
		<li>Prepare the staining solution (freshly made) of 0.1% crystal violet (w/v) in a solution of PBS with 10% ethanol (v/v). Similarly, add 1 mL of staining solution to a clean well in a 24-well culture dish for each insert.</li>
		<li>Remove each insert one at a time with forceps and place a sterile cotton swab inside the insert and swab the upper side of the membrane to remove unmigrated cells.</li>
		<li>Repeat with a second cotton swab. The membrane is rather strong, so gentle pressure while swabbing does not compromise the integrity of the membrane.</li>
		<li>Remove any remaining media from the inside of the insert and add 750 &#181;L of PBS to wash away detached cells.</li>
		<li>Remove the PBS and repeat the wash. Place the insert into a well containing fixative to fix the migrated cells on the underside of the membrane. Repeat for all inserts.</li>
		<li>Fix the inserts for 15 min at room temperature.</li>
		<li>Following fixation remove the insert. Wash the insert again with 750 &#181;L PBS.</li>
		<li>Place the insert into the well with the staining solution to stain all migrated and fixed cells. Stain the cells for 15 min at room temperature. 
		NOTE: For 6-well dish inserts, use 2 mL of fixative solution, 2 mL staining solution, and 2 mL of PBS wash.</li>
	</ol>
	</li>
	<li>Use a beaker with distilled water to destain the inserts. Remove the inserts and dip into the distilled water until the water running off the insert is clear.
	<ol>
		<li>Remove any excess water droplets and place the inserts onto a filter paper sidewise to air dry (usually overnight).</li>
	</ol>
	</li>
	<li>Prepare the membrane for imaging by labeling a clean glass microscope slide for each insert and place a small drop of microscope immersion oil in the center of the slide.
	<ol>
		<li>Detach the membrane from the insert using a scalpel to cut around the perimeter of the membrane on the inside of the plastic insert.</li>
		<li>Remove the membrane using forceps and place on the top of the oil drop on the slide to hold it in place. 
		NOTE: The membranes are very thin and very light, so they can easily be lost by gentle air flow.</li>
	</ol>
	</li>
</ol>
3. Imaging and Analysis
<ol>
	<li>Since the porous membranes are clear and staining cells with crystal violet give them contrast, use a compound light microscope to view the cells. Use a camera and/or software to quantify for many samples but is not necessary. View cells at 5x, 10x, or 20x magnification. For quantification, use multiple, non-overlapping images at 10x magnification. Alternatively, count all migrated cells on the membrane at 10x magnification.</li>
	<li>Determine the total number of invading cells or cells per area for all samples. For each experiment, perform each condition in the assay with three replicate inserts and repeat multiple times for statistically useful results.</li>
	<li>When comparing different cell lines with different growth rates and migration rates, do a parallel experiment to compare cells that invade through a protein matrix and compare to a Boyden Chamber assembly without protein matrix (migrated cells only). Calculate percent invasion for each cell line (Number of invaded cells/number of migrated cells x 100%). 
	NOTE: This approach can help compare invasion rate to migration rates. If comparing different conditions applied to the same cell line, calculating invasion alone is more relevant calculations. The outer edge of the membrane sometimes has a higher number of cells than the central areas and is, therefore, less accurate. If this occurs, exclude those cells from the quantification and ensure that all cells are removed by a cotton swab in a repeat experiment.</li>
</ol>

<ol>
	<li>He, X., Lee, B., Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-ECM+Interactions+in+Tumor+Invasion.">Cell-ECM Interactions in Tumor Invasion.</a> Advances in Experimental Medicine and Biology. 936, 73-91 (2016).</li><li>Albini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+in+vitro+assay+for+quantitating+the+invasive+potential+of+tumor+cells.">A rapid in vitro assay for quantitating the invasive potential of tumor cells.</a> Cancer Research. 47 (12), 3239-3245 (1987).</li><li>Simon, N., Noel, A., Foidart, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+in+vitro+reconstituted+basement+membrane+assay+to+assess+the+invasiveness+of+tumor+cells.">Evaluation of in vitro reconstituted basement membrane assay to assess the invasiveness of tumor cells.</a> Invasion and Metastasis. 12 (3-4), 156-167 (1992).</li><li>Benton, G., Arnaoutova, I., George, J., Kleinman, H. K., Koblinski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+from+discovery+and+ECM+mimicry+to+assays+and+models+for+cancer+research.">Matrigel: from discovery and ECM mimicry to assays and models for cancer research.</a> Advanced Drug Delivery Reviews. 79-80, 3-18 (2014).</li><li>Kleinman, H. K., Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+basement+membrane+matrix+with+biological+activity.">Matrigel: basement membrane matrix with biological activity.</a> Seminars in Cancer Biology. 15 (5), 378-386 (2005).</li><li>Kang, J. J., Schwegel, T., Knepper, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+similarity+between+the+long+terminal+repeat+coding+regions+of+mammary-tumorigenic+BALB/cV+and+renal-tumorigenic+C3H-K+strains+of+mouse+mammary+tumor+virus.">Sequence similarity between the long terminal repeat coding regions of mammary-tumorigenic BALB/cV and renal-tumorigenic C3H-K strains of mouse mammary tumor virus.</a> Virology. 196 (1), 303-308 (1993).</li><li>Slagle, B. L., Lanford, R. E., Medina, D., Butel, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+mammary+tumor+virus+proteins+in+preneoplastic+outgrowth+lines+and+mammary+tumors+of+BALB/cV+mice.">Expression of mammary tumor virus proteins in preneoplastic outgrowth lines and mammary tumors of BALB/cV mice.</a> Cancer Research. 44 (5), 2155-2162 (1984).</li><li>Schmidt, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+oncogenic+phenotype+by+the+nuclear+body+protein+ZC3H8.">Regulation of the oncogenic phenotype by the nuclear body protein ZC3H8.</a> BMC Cancer. 18 (1), 759 (2018).</li><li>Neophytou, C., Boutsikos, P., Papageorgis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+and+Emerging+Therapeutic+Targets+of+Triple-Negative+Breast+Cancer+Metastasis.">Molecular Mechanisms and Emerging Therapeutic Targets of Triple-Negative Breast Cancer Metastasis.</a> Frontiers in Oncology. 8, 31 (2018).</li><li>Al-Alwan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fascin+is+a+key+regulator+of+breast+cancer+invasion+that+acts+via+the+modification+of+metastasis-associated+molecules.">Fascin is a key regulator of breast cancer invasion that acts via the modification of metastasis-associated molecules.</a> PloS One. 6 (11), e27339 (2011).</li><li>Wang, M. J., Zhang, H., Li, J., Zhao, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA-98+inhibits+the+proliferation,+invasion,+migration+and+promotes+apoptosis+of+breast+cancer+cells+by+binding+to+HMGA2.">microRNA-98 inhibits the proliferation, invasion, migration and promotes apoptosis of breast cancer cells by binding to HMGA2.</a> Bioscience Reports. 38 (5), (2018).</li><li>Zheng, Y. F., Luo, J., Gan, G. 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The authors have nothing to disclose.

The in vitro invasion assay is an inexpensive, rapid, quantitative, and straightforward method for studying the factors promoting cancer cell invasion. Breast cancer is the most commonly diagnosed cancer among women. Of the three major subtypes of breast cancer, triple negative, (or ER-, PR-, HER2/neu-), is the most aggressive, most likely to metastasize, and most deadly9. Therefore, understanding the genes and expression that result in metastasis can help find new therapeutic targets and...

The in vitro invasion assay can be a useful tool when measuring a cell&#39;s ability to migrate through a protein-coated membrane, analogous to the first steps in metastasis. A key feature of malignant cancer cells is their ability to migrate through and invade nearby tissues. Cancer that has spread or metastasized poses more treatment challenges and has lower rates of long-term survival, while localized tumors are easier to treat and have higher rates of long-term survival. In order to metastasize, cancer cells must leave the primary tumor and migrate into the circulatory or lymphatic system, a process which requires passing through the extracellular matrix and basement membrane1. In the process called the epithelial mesenchymal transition (EMT), the tumor cells must break cell-cell contacts, migrate directionally, and invade nearby blood or lymph vessels. The initial steps of this metastasis cascade are of great interest since these steps are what can make cancer deadlier. The genetic and epigenetic factors involved in the early steps of metastasis are the focus of a great amount of research, but accurate and reliable experimental tools are needed to test these early steps both in vivo and in vitro.
Tools to measure changes in cell migration such as wound healing (scratch) assays or growth in 3D environments such as soft agar assays can partially address the need for experimental methods of measuring early steps of metastasis, but an assay to measure invasion is more challenging since the process occurs in the body within a complex tumor microenvironment. For the purposes of screening drugs or gene alterations to determine important factors in invasion and metastasis, a system that can be used in vitro with cultured cells and mimic the challenges faced by metastatic cells in vivo is the invasion assay2,3. Breast cancer is the most commonly diagnosed type of cancer in women and the second leading cause of cancer death in women, so understanding the genes responsible for breast cancer cell invasion and metastasis is critically important for public health. Moreover, mouse cells are a useful model system for studying breast cancer and its progression.
The in vitro invasion assay is based on the Boyden Chamber assembly where two chambers of growth media are separated by a porous membrane3. To mimic the tumor microenvironment, a protein-rich gel is also included to separate cells in one chamber from a chemoattractant in the other and act as a basement membrane barrier. In order to migrate towards the chemoattractant, cells must first pass through the protein-rich barrier then pass through the porous membrane - a process analogous to how metastatic cells migrate through stroma. The protein-rich gel can be altered based on the needs of the experiment, but usually consists of collagen, or basement membrane extract (e.g., Matrigel)4. It is a complex mixture of proteins, proteoglycans and growth factors, but mostly consists of laminins and collagen IV 4,5. Cells must then pass through a porous membrane typically made of polycarbonate, polyester, or polytetrafluoroethylene (PTFE). Membranes may be purchased commercially with or without a protein gel (typically collagens), or the gel may be purchased separately and added. The pore size can be adjusted based on the cell size. While pore sizes are available from 0.4 - 8.0 &#181;m, only pores from 3.0 - 8.0 &#181;m are large enough for cell migration. The invasion assay has been used to determine the effectiveness of inhibitors on the ability of cells to migrate and invade. While lacking the exact tumor microenvironment that is present in vivo, the in vitro invasion assay is beneficial at screening many conditions in a short time while minimizing the need for animal models. The goal of these experiments is to compare gene expression of suspected oncogenes and determine the effects on cancer cell behavior and disease aggressiveness using the in vitro invasion assay and other tests. Overall, the invasion assay provides consistent, quantitative, and rapid results for determining metastatic potential while also being a relatively inexpensive, straightforward, and adaptable method.

<table><tbody><tr><td>24-well plates</td><td>Corning</td><td>353504</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>ThermoFisher</td><td>15240062</td><td></td></tr><tr><td>BALB/c mice</td><td></td><td></td><td></td></tr><tr><td>Cell Culture Incubator</td><td></td><td></td><td></td></tr><tr><td>Cell Culture Treated Flasks</td><td></td><td></td><td></td></tr><tr><td>Clinical cenrifuge</td><td></td><td></td><td></td></tr><tr><td>Cotton swab</td><td>Puritan</td><td>25-806</td><td></td></tr><tr><td>Crystal Violet</td><td>Sigma Aldrich</td><td>C0775</td><td></td></tr><tr><td>Distilled water</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td>ThermoFisher</td><td>10566-016</td><td>high glucose, GlutaMAX</td></tr><tr><td>Ethanol</td><td></td><td></td><td></td></tr><tr><td>FBS</td><td>Sigma Aldrich</td><td>F2442-500ML</td><td></td></tr><tr><td>Forcepts</td><td></td><td></td><td></td></tr><tr><td>Glass Slide</td><td>VWR</td><td>16004-422</td><td></td></tr><tr><td>HBSS</td><td>ThermoFisher</td><td>14025076</td><td>no calcium, no magnesium</td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>Imersion oil</td><td></td><td></td><td></td></tr><tr><td>Invasion Chambers (24-well)</td><td>Corning</td><td>354480</td><td>Cat. #354481 for 6-well</td></tr><tr><td>Light Microscope</td><td></td><td></td><td></td></tr><tr><td>Lipofectamine Transfection Reagent</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td></td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>Scalpel, disposable</td><td></td><td></td><td>#11</td></tr><tr><td>shRNA</td><td></td><td></td><td></td></tr><tr><td>Sterile Transfer pipet</td><td></td><td></td><td></td></tr><tr><td>Trypsin-EDTA</td><td>ThermoFisher</td><td>25200056</td><td></td></tr></tbody></table>

the use of mouse mammary tumor cells in an in vitro invasion assay as a measure of oncogenic cell behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

This method of in vitro invasion through a protein matrix was used to assess the aggressive phenotypes and oncogenic cell behaviors of mouse mammary tumor cells with altered expression of the zinc finger protein ZC3H88. In conjunction with other approaches that also examine cell migration and growth in 3D environments, it was found that higher levels of expression of Zc3h8 in tumor cell lines, or by promoter-mediated expression from a plasmid, resulted in rapid rat...

Watch this Scientific Journal Video about The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior at JoVE.com

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

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Research

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Genetics

5.1K Views.  Villanova University. The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

Video: The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

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

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Cancer Research

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

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 ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

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 ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.