Name: mammary epithelial and endothelial cell spheroids as a potential functional in vitro model for breast cancer research
Uploaded: 2021-07-12T15:00:00
Description: Watch this Scientific Journal Video about Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research at JoVE.com

This research was supported by Cancer Research Foundation / Swiss Cancer League grant KFS-4125-02-2017 to RKD and National Institute of Health grant DK079307 to EKJ.

1. Cell culture
NOTE: Conduct cell handling under sterile conditions.
<ol>
	<li>Human Umbilical Vein Endothelial Cells (HUVECs) subculture
	<ol>
		<li>Coat 75 cm2 flasks with collagen (5 &#181;g/cm2) (rat-tail) overnight (ON) at room temperature (RT) or 2-3 h at 37 &#176;C, rinse with water, and allow the flask to dry.</li>
		<li>Grow HUVECs in growth medium (EBM-2, Endothelial Basal Medium-2) supplemented with glutamine (1x = 2 mM), antibiotic-antimycotic solution (AA;...

Compared to 2D cell cultures, revolutionary 3D spheroid culture technology is a better and more powerful tool to reconstruct an organ&#39;s microenvironment, cell-cell interactions, and drug responses in vitro. This is the first protocol describing the formation of spheroids from multicellular (epithelial and endothelial) cell lines for breast cancer research. This protocol ensures spheroidal 3D growth of spheroids for up to 5 days, and spheroids can be examined after paraffin embedding, sectioning, and histolog...

The use of animals for experiments has limitations. Animal studies cannot accurately mimic disease progression in humans, and animals and humans do not have identical responses to pathogens. Additionally, restrictions in animal experimentation due to concerns for animal suffering and ethical problems1,2 increasingly constrain research programs. In vitro systems have been widely developed to circumvent the use of animals; moreover, the use of human cells has made in vitro models more relevant for the pathophysiological and therapeutic investigation. Conventional monolayer (2D) cell cultures are widely used because they mimic human tissues to some degree. However, 2D monocultures fail to mimic human organs, and 2D monocultures are unable to simulate the complex microenvironment of the original organs and mimic the in vivo situation3,4,5,6. Additionally, in monolayer cell cultures, drug treatments could easily destroy/damage all of the cells. Importantly, some of these limitations can be overcome by switching to multilayer three-dimensional (3D) cell cultures7,8. In fact, 3D culture models have been shown to better reflect the cellular structure, layout, and function of cells in primary tissues. These 3D cultures can be formed using a variety of cell lines, similar to a functional organ. Indeed, there are two models of 3D cultures. One model produces spheroids of aggregated cells that form clusters and reorganize them into spheres (scaffold-free models). The second yields organoids, which have a more complex structure and consist of combinations of multiple organ-specific cells, which are considered as a miniature version of organs9,10. Due to this, 3D culture systems represent an innovative technology with many biological and clinical applications. Thus, spheroids and organoids have numerous applications for disease modeling and studies related to regenerative medicine, drug screening, and toxicological studies6,11,12,13,14,15. Carcinogenic spheroids, derived from 3D technology, recreate the morphology and phenotype of relevant cell types, mimic the in vivo tumor microenvironment, and model cell communications and signaling pathways that are operational during tumor development16,17,18. In addition, to improve cancer biology understanding, tumor spheroids/organoids can also be used to identify a potential patient-specific anti-cancer therapy (personalized) and assess its efficacy, toxicity, and long-term effects19,20,21,22. Spheroids have opened prominent opportunities to investigate pathophysiology, disease modeling and drug screening because of their ability to preserve cellular and three-dimensional tissue architecture, the ability to mimic the in vivo situation, and the cell-cell interactions. However, one must also be aware of the limitations of this system, such as the lack of vascular/systemic component, functional immune or nervous system, and the system represents a reductionist approach as compared to animal models. Indeed, in contrast to animal models, 3D structures provide only an approximation of the biology within a human body. Understanding the limitations of the 3D method may help researchers to design more refined and valid processes for producing spheroids that better represent an organ at a larger scale23,24,25.
Cancer is the leading cause of death worldwide, and breast cancer is the most common cancer in women26,27,28. To mimic the complex microenvironment of breast cancer, breast cancer spheroids should be cultured using cells that play a prominent role in breast tumors, i.e., epithelial cells, endothelial cells, fibroblasts, and/or immune cells. Moreover, for a spheroid representing breast cancer, the expression of female hormone receptors (estrogen/progesterone receptors), ability to conserve the patient tumor histological status, and ability to mimic response to therapy should also be considered. Studies have shown that 3D co-culture systems have a cellular organization similar to that of the primary tissue in vivo, have the capability to react in real-time to stimuli, and have functional androgen receptors29,30,31,32. Hence, a similar approach could be useful to mimic a breast tumor in vitro. The purpose of the current protocol is to establish a new method of generating breast cancer spheroids. This method utilizes estrogen receptor-positive MCF-7 cells (an immortalized human cell line of epithelial cells) and vascular endothelial cells (HUVECs) or lymphatic endothelial cells (HMVEC-DNeo) to create a model that mimics or closely reflects the interactions between these cells within a tumor. Although MCF-7 (estrogen-responsive) and endothelial cells have been used to develop spheroids in the present study, other cells such as fibroblasts which represent ~80% of the breast tumor mass, could also be combined in the future to better represent and mimic breast tumor.
There are several methods to form spheroids, such as: 1) the hanging droplet method that employs gravity33,34; 2) the magnetic levitation method that uses magnetic nanoparticles exposed to an external magnet35, and 3) the spheroid microplate method that is performed by seeding cells on low-attachment plates36,37. On the basis of the existing methods, which use only one cell type, the present protocol has been optimized using epithelial and endothelial cells to better mimic the growth conditions of breast cancer tumors in vivo38,39,40,41. This method can be easily achieved in the laboratory at a low cost and with minimal equipment requirements. Based on the need/goals of the lab, different approaches were used to form spheroids and gain relevant cellular material from these spheroids. In this context, for DNA, RNA, or protein analysis, the 3D spheroids are produced by co-culturing endothelial and epithelial cells with the hanging drop method. However, for functional studies, for example, to monitor cell growth after short interfering (siRNA) transfection and/or hormone treatment, the spheroids are generated using U-bottom plates.
The purpose of this technical protocol is to provide a detailed step-by-step description for 1) forming breast cancer multicellular-type spheroids, 2) preparing samples for histological staining, and 3) collection of cells for extraction of RNA, DNA, and proteins. Both the inexpensive hanging drop method and the more expensive U-bottom plates are used to form spheroids. Here, a protocol for preparing (fixing) spheroids for sectioning and subsequent immunostaining with markers to assess cell proliferation, apoptosis, and distribution of epithelial and endothelial cells within a spheroid is provided. Additionally, this protocol shows a complete step-by-step analysis of histological data using ImageJ software. Interpretation of biological data varies depending on the type of experiment and the antibodies used. Sectioning of fixed spheroids and subsequent staining of the sections was performed by a routine pathology lab (Sophistolab: info@sophistolab.ch)

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm &#215; 20 mm tissue-culture treated culture dishes</td>
			<td>Corning</td>
			<td>CLS430167</td>
			<td></td>
		</tr>
		<tr>
			<td>149MULTI0C1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35 x 10 mm Tissue Culture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Serological Pipet, Polystyrene, Individually Packed, Sterile</td>
			<td>Falcon</td>
			<td>357543</td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Photoshop</td>
			<td></td>
			<td></td>
			<td>Version: 13.0.1 (64-bit)</td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Solution (100&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein-AM</td>
			<td>Sigma-Aldrich</td>
			<td>17783</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 (cluster of differentiation 31)</td>
			<td>Cell Marque</td>
			<td>131M-95</td>
			<td>monoclonal mouse ab clone JC70</td>
		</tr>
		<tr>
			<td>CellTracker Green CMFDA (5-chloromethylfluorescein diacetate)</td>
			<td>Invitrogen</td>
			<td>C7025</td>
			<td></td>
		</tr>
		<tr>
			<td>CK8/18 (cytokeratins 8 and 18)</td>
			<td>DBS</td>
			<td>Mob189-05</td>
			<td>monoclonal mouse ab, clone 5D3</td>
		</tr>
		<tr>
			<td>CKX41 Inverted Microscope</td>
			<td>Olympus Life Science</td>
			<td></td>
			<td>Olympus DP27 digital camera</td>
		</tr>
		<tr>
			<td>Cleaved Caspase 3</td>
			<td>Cell Signaling</td>
			<td>9661L</td>
			<td>polyclonal rabbit ab</td>
		</tr>
		<tr>
			<td>Collagen (rat tail)</td>
			<td>Roche</td>
			<td>11 179 179 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Coulter ISOTON II Diluent</td>
			<td>Beckman Coulter</td>
			<td>844 80 11</td>
			<td>Diluent II</td>
		</tr>
		<tr>
			<td>Coulter Z1, Cell Counter</td>
			<td>Coulter Electronics, Luton, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dehydrated Culture Media: Noble Agar</td>
			<td>BD Difco</td>
			<td>BD 214220</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium/Nutrient Mixture F-12 Ham</td>
			<td>Sigma-Aldrich</td>
			<td>D6434</td>
			<td></td>
		</tr>
		<tr>
			<td>EBM-2 Endothelial Cell Growth Basal Medium-2</td>
			<td>Lonza</td>
			<td>190860</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium homodimer</td>
			<td>Sigma-Aldrich</td>
			<td>46043</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FCS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum Charcoal Stripped (FCS sf)</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH3006803</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence stereo microscopes Leica M205 FA</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement (100x)</td>
			<td>Gibco</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red</td>
			<td>Gibco</td>
			<td>14175053</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Life Technologies</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC &#8211; Human Umbilical Vein Endothelial Cells</td>
			<td>Lonza</td>
			<td>CC-2517</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institute of Health, USA</td>
			<td></td>
			<td>Wayne Rasband 
			Version: 1.52a (64-bit)</td>
		</tr>
		<tr>
			<td>Ki67</td>
			<td>Cell Marque</td>
			<td>275R-16</td>
			<td>monoclonal rabbit ab, clone SP6</td>
		</tr>
		<tr>
			<td>Leica Histocore Multicut Rotary Microtome</td>
			<td></td>
			<td>149MULTI0C1</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Serum Growth Supplement Kit (LSGS Kit)</td>
			<td>Gibco</td>
			<td>S003K</td>
			<td></td>
		</tr>
		<tr>
			<td>MCF-7 cells &#8211; human breast adenocarcinoma cell line</td>
			<td>Clinic for Gynecology, University Hospital Zurich</td>
			<td></td>
			<td>Provived from Dr Andr&#232; Fedier obtained from ATCC</td>
		</tr>
		<tr>
			<td>Nunclon Sphera 96U-well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>174925</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) 1x</td>
			<td>Gibco</td>
			<td>10010015</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Lysis Buffer</td>
			<td>Cell Signaling, Danvers, USA</td>
			<td>9803</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick-RNA Miniprep Kit</td>
			<td>Zymo Research</td>
			<td>R1055</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Lysis Buffer</td>
			<td>Zymo Research</td>
			<td>R1060-1-100</td>
			<td>Contents in Quick-RNA Miniprep Kit</td>
		</tr>
		<tr>
			<td>Rotina 46R Centrifuge</td>
			<td>Hettich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Bottom Polystyrene Tubes, 5 mL</td>
			<td>Falcon</td>
			<td>352003</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Bandelin electronics, Berlin, DE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tecan Infinite series M200 microplate reader</td>
			<td>Tecan, Salzburg, AU</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Flasks 75</td>
			<td>TPP</td>
			<td>90076</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T3924</td>
			<td></td>
		</tr>
	</tbody>
</table>

mammary epithelial and endothelial cell spheroids as a potential functional in vitro model for breast cancer research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

The spheroids model using epithelial and endothelial co-cultures is required to closely mimic in vivo conditions of breast tumors for in vitro experiments. The scheme in Figure 1 depicts the protocol to form spheroids with breast cancer epithelial cells and vascular or lymphatic endothelial cells (Figure 1). Each cell type is seeded separately in a 3.5 cm round dish and treated with growth stimulators/inhibitors or transfected with oligonucleot...

Watch this Scientific Journal Video about Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research at JoVE.com

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

The breast cancer cell spheroids described herein will allow researchers to investigate molecular mechanisms of cancer cell endothelial cell interactions that are involved in proliferation and metastasis of cancer cells. The main advantage of this 3D model is that it provides a more realistic cellular arrangement that improves the reliability of inferences regarding the pathophysiology and treatment of breast cancer. Demonstrating the procedure will be Giovanna Azzarito, a PhD student from my laboratory.Begin by treating or transfecting the plated cells for 24 hours, or as desired. For identifying cell distribution in spheroid, wash the cells with one milliliter of Hanks'Balanced Salt Solution or HBSS, containing calcium and magnesium and stay in HUVECs using 0.5 micrograms per milliliter blue dye. And MCF-7 cells using 0.5 micromolar green dye diluted in the respect of growth medium.Place the plate into a 37 degree Celsius and five percent CO2 incubator for 30 to 40 minutes. Wash the cells with one milliliter of HBSS without calcium and magnesium. Then add one milliliter of enzymatic detachment re-agent, which is 0.25%trypsin for HUVEC and 0.5%trips in four MCF-7 to each brown dish using a P1000 pipette.Elect each cell type separately in a five milliliter round bottom polystyrene tube, and add one milliliter of 10%Fetal Calf Serum or FCS medium using a P1000 pipette to stop the enzymatic reaction. Then centrifuge the cell suspensions at 250 times G for five minutes. Carefully aspirate the supernatant and suspend the cells in one milliliter of steroid free medium.Use 100 microliters of each cell suspension diluted with 10 milliliters of Diluent 2 to determine the total cell number. Turn on the machine and flush by pressing the function and start buttons, with Diluent 2 solution in a cell counter vial. Use set up as four and press start to measure the blank with fresh Diluent 2 solution.Change the scintillation vial with 100 microliters of cell suspension in 10 milliliters of Diluent 2 solution. Use set up as four and press start to measure the samples. Note the number of cells per milliliter on the digital display, Then prepare five milliliters of each cell suspension and mix them to get a final volume of 10 milliliters in a one to one ratio, using manual repeating pipette to pipette out 100 microliters of the cell suspension into each well of the 96-well U-bottom plate.Place the plate into an incubator under standard tissue culture conditions and check for a spheroids formation after 48 hours. Take pictures of this spheroids at 40 X magnification using a phase contrast or fluorescence stereo microscope. For preparing the hanging drop culture mix the cell suspensions in a one-to-one ratio to get a final volume of two milliliters.Use a P20 pipette to see the cell mixture in the form of 15 microliter drops on the inverted lead of a 10 centimeter Petri dish. Invert the lid with the drops and add five milliliters of EBM-2 base medium at the bottom of the dish to avoid evaporation of the drops. Collect approximately 50 spheroids in a 1.5 milliliter tube with a gut tip of a P200 microliter pipette.Allow them to settle to the bottom of the tube by gravity and then discard the supernatant. Fix this spheroids with 500 microliters of 4%PFA for one hour at room temperature. Boil 2%Nobel Algor Solution in PBS using a hot plate with a magnetic stir for three to five minutes to dissolve the augurous powder completely and cooled down to around at 60 degrees Celsius.Remove the PFA with a P1000 pipette, wash this spheroids with 500 microliters of PBS and allow them to sediment. Once settled discard the supernatant carefully pipette 600 microliters of Agora solution into the 1.5 milliliter tube with spheroids and placed the tube immediately in a centrifuge with a horizontal rotor at 177 times G for two minutes. Add a short string in the middle of the Agro Solution to easily remove the plug from the tube.Solidify the Agros plug on ice, or at four degrees Celsius add 500 microliters of PBS into the 1.5 milliliter tube to avoid drying out the pellet. Acquire images of spheroid sections using a stereo microscope. Calculate the amount of solution necessary to add a 10 microliters per well of a mixture of calcium Aam and ethidium homodimers diluted using EBM-2 in a one to 50 ratio.Add the staining mixture to the spheroids and place the plate in the incubator under the standard, the tissue culture conditions for 30 to 60 minutes. Acquire pictures at 40 X magnification using the fluorescence stereo microscope. In U-bottom plates the spheroids formation was observed 48 hours after seeding of MCF seven cells.However, the spheroids formation was not observed in the case of endothelial cells or lymphatic cells. Seeding the MCF seven plus endothelial cells or MCF seven plus lymphatic cells at one-to-one ratio also resulted in spheroid formation. Time dependent sequential growth of MCF seven spheroid was recorded ranging from 24 to 120 hours.The spheroids size increased from around 100 micrometers to around 200 micrometers over at five days. With an increased growth rate observed in the spheroids treated with the growth stimulator. The structure of the spheroids and the shape of their cells showed no abnormalities following transfection with control oligonucleotides in the presence of Lipofectamine.Cells expressing their proliferative marker Ki-67 were distributed homogeneously in this spheroids. Cleave to caspase three staining of this spheroids showed apathetic cells after four days of culture. Studying of the cells in this spheroids with CD-31 and Ki-67 revealed that 55%of the cells are epithelial 45%are endothelial and 25%of cells are proliferating.To assess the percentage of live and dead cells four day old spheroids were stained with calcium Aam and ethidium-homodimer. A freshly thought spheroid also stain for dead and live cells revealed that spheroids are very sensitive to freezing conditions. When following his protocol, make sure that healthy cells are used to mix aphrodite Another thing to remember would be to avoid damaging these aphrodite during DP cap to obtain a good sections also ensurer to palate the aphrodite before the across polymerizes.This model could be further improved by including other cells, such as fibroblasts involved in tumor progression and using it to assess the efficacy of therapeutic agents targeting a cancer growth.

mammary-epithelial-endothelial-cell-spheroids-as-potential-functional

Research

JoVE Journal

Cancer Research

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

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

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

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

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

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

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast 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.