We thank Dr. Laura L. Bronsart for providing GFP and dTomato-labeled RAW 264.7 macrophages. This research was financially supported by NIH grant #R00CA201304.

Animal studies were performed in accordance with institutional guidelines and protocols approved by the Vanderbilt University Institutional Animal Care and Use Committee.
1. Preparation of mice and cell acquisition (adapted from Nguyen-Ngoc et al.11)
<ol>
	<li>Sacrifice athymic Nu/Nu mice (8-10 weeks old) using CO&#173;2 asphyxiation followed by cervical dislocation. Clean the skin using 70% ethanol.</li>
	<li>Resect abdominal and inguinal mammary glands fr...

<ol>
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R., 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=Twist1-induced+dissemination+preserves+epithelial+identity+and+requires+E-cadherin.">Twist1-induced dissemination preserves epithelial identity and requires E-cadherin.</a> Journal of Cell Biology. 204 (5), 839-856 (2014).</li><li>Ewald, A. J., Brenot, A., Duong, M., Chan, B. S., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+Epithelial+Migration+and+Cell+Rearrangements+Drive+Mammary+Branching+Morphogenesis.">Collective Epithelial Migration and Cell Rearrangements Drive Mammary Branching Morphogenesis.</a> Developmental Cell. 14, 570-581 (2008).</li><li>Nguyen-Ngoc, K. -. V., 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=ECM+microenvironment+regulates+collective+migration+and+local+dissemination+in+normal+and+malignant+mammary+epithelium.">ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium.</a> Proceedings of the National Academy of Sciences. 89 (19), E2595-E2604 (2012).</li><li>Nguyen-Ngoc, K. -. V., Shamir, E. R., Huebner, R. J., Beck, J. N., Cheung, K. J., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Culture+Assays+of+Murine+Mammary+Branching+Morphogenesis+and+Epithelial+Invasion.">3D Culture Assays of Murine Mammary Branching Morphogenesis and Epithelial Invasion.</a> Tissue Morphogenesis: Methods and Protocols. 1189, 135-162 (2015).</li><li>Ewald, A. 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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=An+atlas+of+mouse+mammary+gland+development.">An atlas of mouse mammary gland development.</a> Journal of Mammary Gland Biology and Neoplasia. 5 (2), 227-241 (2000).</li><li>Maier, P., Hartmann, L., Wenz, F., Herskind, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pathways+in+response+to+ionizing+radiation+and+their+targetability+for+tumor+radiosensitization.">Cellular pathways in response to ionizing radiation and their targetability for tumor radiosensitization.</a> International Journal of Molecular Sciences. 17 (1), (2016).</li><li>LaBarge, M. A., Garbe, J. C., Stampfer, M. 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In this protocol, we have developed a method for reproducible growth and characterization of irradiated mammary organoids (Figure 1). An irradiation dose of 20 Gy was applied to mirror previous in vivo models of tumor cell recruitment5. Irradiation of mammary glands ex vivo prior to organoid formation allowed for isolation of radiation damage effects without a corresponding infiltration of immune cells. The development of an in vitro irradiated normal tissue model ena...

Approximately 60% of the triple negative breast cancer (TNBC) patients choose breast-conserving therapy (BCT) as a form of treatment1. In this treatment modality, the tumor containing part of the breast tissue is removed, and the surrounding normal tissue is exposed to ionizing radiation to kill any residual tumor cells. Treatment reduces recurrence in much of the breast cancer population; however, approximately 13.5% of treated patients with TNBC experience locoregional recurrences2. Therefore, studying how radiation may recruit circulating tumor cells (CTCs) will lead to important insights into local recurrence3,4.
Previous work has shown that radiation of the normal tissue increases recruitment of various cell types5. In pre-clinical models of TNBC, irradiation of normal tissue increased macrophage and subsequently tumor cell recruitment to normal tissues5. Immune status influenced tumor cell recruitment to irradiated sites, with tumor cell migration observed in immunocompromised subjects. Recapitulating these interactions using organoids derived from mammary glands will allow the observation of cell migration and cell-stromal interactions in real time with microscopy and live cell imaging to determine the role of radiation damage in altering tumor cell behavior.
Mouse mammary organoids have helped elucidate key steps in the development of the mammary gland. A mammary organoid is a multicellular, three dimensional construct of isolated mammary epithelium that is larger than 50 &#956;m6,7,8,9,10. Using primary epithelial organoids, Simian et al. evaluated necessary factors for branching in the mammary gland7. Shamir et al. discovered that dissemination can occur without an epithelial to mesenchymal transition, providing insight into the metastatic cascade8. Methods for generating and characterizing organoids from mammary gland tissue are well established6,11,12,13. However, to our knowledge, methods for growing irradiated organoids from mammary glands have not been reported. A protocol for growing and characterizing irradiated organoids would be a critical step in recapitulating radiation-induced immune and tumor cell recruitment.
In this paper, we report a method for growing and characterizing irradiated mammary epithelial organoids in low adhesion microplates coated with a hydrophilic polymer that supports the formation of spheroids. These organoids were co-cultured with macrophages to examine immune cell infiltration kinetics. This work can be extended to include co-culturing organoids with adipose cells to recapitulate mammary characteristics, breast cancer cells to visualize tumor cell recruitment, and CD8+ T cells to study tumor-immune cell interactions. Previously established protocols may be used to evaluate irradiated organoids. Earlier models co-culturing mammary organoids and immune cells have shed light on mechanisms of metastasis and dissemination. DeNardo et al. found that CD4+ T cell regulation of tumor associated macrophages enhanced a metastatic phenotype of mammary adenocarcinomas14. Co-culture models have also been used to elucidate mechanisms of biological development. Plaks et al. clarified the role of CD4+ T cells as down-regulators of mammary organogenesis15. However, our group is the first to establish a procedure of visualizing how normal tissue irradiation influences immune cell behavior. Because normal tissue irradiation has been shown to enhance tumor cell recruitment5, this protocol can be further developed to analyze how tumor cell behavior is altered by irradiation of normal tissue and cells, leading to a greater understanding of cancer recurrence.

<table>
	<tbody>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>VWR</td>
			<td>16004-128</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-cytokeratin 14</td>
			<td>abcam</td>
			<td>ab181595</td>
			<td>Lot: GR3200524-3</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A1933-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium Histolyticum, Type VIII</td>
			<td>Sigma</td>
			<td>C2139</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Gibco</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermofisher</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Fisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>E-Cadherin</td>
			<td>Cell Signaling</td>
			<td>24E10</td>
			<td>Lot: 13</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma</td>
			<td>F0926</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit secondary</td>
			<td>abcam</td>
			<td>ab150077</td>
			<td>green 
			Lot: GR3203000-1</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit secondary</td>
			<td>abcam</td>
			<td>ab150080</td>
			<td>red 
			Lot: GR3192711-1</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Fisher</td>
			<td>62249</td>
			<td>Lot: TG2611041</td>
		</tr>
		<tr>
			<td>Insulin (10 mg/mL)</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium, 100x</td>
			<td>Gibco</td>
			<td>51500-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane (basement membrane extracted from Engelbreth-Holm-Swarm mouse sarcoma)</td>
			<td>Corning</td>
			<td>356237</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Vector Laboratories</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclon Sphera 96 well plates</td>
			<td>Thermo</td>
			<td>174927</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>VWR</td>
			<td>10128-856</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/strep</td>
			<td>Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>abcam</td>
			<td>ab176757</td>
			<td>Lot: GR3214582-16</td>
		</tr>
		<tr>
			<td>Tight Junction Protein 1</td>
			<td>Novus</td>
			<td>NBP1-85047</td>
			<td>Lot: C115428</td>
		</tr>
		<tr>
			<td>Triton X-100 (4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol)</td>
			<td>Sigma</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>27250-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20 (Polyethylene glycol sorbitan monolaurate)</td>
			<td>Sigma</td>
			<td>P1379-100ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

growth and characterization of irradiated organoids from mammary glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Irradiated epithelial mammary organoids were successfully obtained from mouse mammary glands, processed, and cultured on low-adhesion plates (Figure 1). Organoid yield was tested by seeding in different growth environments (Figure 2A-G). Seeding cells directly onto tissue culture treated 10 cm cell plates yielded an overgrowth of fibroblast cells. Fibroblasts were identified under phase contrast microscopy in or near the same plane of focus as organoids, and they quickly gre...

Watch this Scientific Journal Video about Growth and Characterization of Irradiated Organoids from Mammary Glands at JoVE.com

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

This protocol can be used and further developed to analyze how ionizing radiation affects tumor cell recruitment, leading to a greater understanding of cancer recurrence. Mammary organoids are powerful models. They mimic basic in vivo characteristics, but they allow for easy isolation of biological variables.Use of low-adhesion plates negates work-flow challenges associated with protein matrices. Triple-negative breast cancer patients experience local regional recurrence at higher rates following therapy. Studying how radiation influences tumor and immune cell behavior will lead to important insights into recurrence mechanisms.Demonstrating confocal microscopy of the organoids is Javier Gomez, a graduate student in the Silvera Batista lab. Begin this procedure with removal of abdominal and inguinal mammary glands from sacrificed mice as detailed in the text protocol. 45 minutes after irradiation of the samples as described in the text protocol, place mammary glands in a 35-millimeter sterile cell plate and mince with scalpels.Mince with approximately 40 strokes until the tissue relaxes and pieces are obtained that are no larger than approximately one square millimeter in area. Now transfer the tissue to collagenase solution in a 50-milliliter centrifuge tube. Use 10 milliliters of collagenase solution per mouse.Place the sample tubes in a water bath for 30 to 60 minutes at 37 degrees Celsius, vortexing for five seconds every 10 minutes. Digestion is complete when the collagenase solution is cloudy. Spin down the digested solution at 450-times-G for 10 minutes at room temperature.Now three layers are observed. The supernatant is composed of fat, the middle layer is an aqueous solution, and the bottom is a pellet. The pellet appears red, as it is a mixture of epithelial cells, individual stromal cells, and red blood cells.Pre-coat all pipettes, pipette tips, and centrifuge tubes with BSA solution prior to contact by addition and removal of BSA solution. BSA solution can be reused, although it should sterile-filtered before each experiment. For additional recovery, transfer the supernatant to a fresh BSA-coated 15-milliliter tube.Pipette up and down vigorously to disperse the fat layer. Centrifuge the sample at 450-times-G for 10 minutes at room temperature. Aspirate the supernatant, leaving a small amount of media in the tube to avoid aspirating the cell pellet.Now aspirate the aqueous layer from the tube with the original pellet. Add 10 milliliters of DMEM/F12 to the tube with the original pellet, and transfer to the second tube. Pipette vigorously to combine and resuspend the two pellets.After centrifuging at 450-times-G for 10 minutes at room temperature, aspirate the supernatant and add four milliliters of DMEM/F12 to the tube. Add 40 microliters of deoxyribonuclease to the suspension and gently shake by hand for two to five minutes at room temperature. Add six milliliters of DMEM/F12 and pipette thoroughly.After centrifuging the tube at 450-times-G for 10 minutes at room temperature, aspirate the supernatant to the 0.5-milliliter mark. Resuspend the pellet in 10 milliliters of DMEM/F12 and pipette thoroughly. Now centrifuge the tube by pulsing to 450-times-G and stopping four seconds after reaching that speed.Repeat the washing steps three more times to purify organoids via centrifugal differentiation. The pellet should now be an off-white color consisting of only epithelial organoids. Resuspend the pellet in 10 milliliters of DMEM/F12.Pipette thoroughly to create a homogenous solution. Transfer 50 microliters to a 30-millimeter Petri dish and view under a phase-contrast microscope at 20X. Count the number of organoids with a tally counter.Pipette 50 microliters of organoids into each well of a low-adhesion plate. Add 150 microliters of organoid medium to bring the total working volume to 200 microliters. Carefully replace the medium every two days.Maintain GFP or D-tomato-labeled raw 264.7 macrophages in DMEM media supplemented with 10%FPS and 1%penicillin-streptomycin. Seed the cells into the organoid medium at the desired density. Use live cell phase contrast and fluorescent imaging to monitor macrophage infiltration over time.Remove organoid medium from the wells by carefully aspirating. Fix the samples with 10%neutral-buffered formalin for 15 minutes at room temperature. Wash the fixed organoids three times for five minutes in 1X PBS.If desired, fixed samples can be stored at four degrees Celsius for one week for further staining. Following wash, permeabilize the fixed organoids for five minutes. Block the fixed organoids with 5%normal goat serum in 0.1%PBST for one hour at room temperature before washing three times for five minutes with PBS.Incubate the fixed organoids with anti-cytokeratin-14, E-cadherin, or tight-junction protein one, in 1%NGS in PBST for one hour at room temperature. Then wash three times for five minutes in PBST. Now incubate the organoids with goat anti-rabbit secondary antibody with 1%NGS PBST for one hour at room temperature.Cover with foil to avoid light exposure. After washing as before, use the nuclear dye to stain nuclei for approximately a half-hour. Wash the stained organoids for five minutes in PBS three times.If using a chamber slide, mount with a cover slip. Store the organoids wrapped in foil at four degrees Celsius for up to two weeks. Irradiated organoids could be cultured in low-adhesion plates, or within basement membrane, but the most rapid growth occurred in low-adhesion plates.Organoids recapitulated mammary gland characteristics. Constructs that are morphologically similar to ducts and lobes were observed. Growth trends indicated that when organoids were immediately seeded after digestion and sorting, non-irradiated organoids grew faster than irradiated organoids, most likely due to cell growth arrest resulting from mechanisms of DNA damage repair.Organoids expressed epithelial characteristics, which were evaluated through immunofluorescent staining. Irradiated organoids expressed epithelial markers. Cytokeratin-14, a marker of myoepithelium, was expressed strongly on the surface of irradiated organoids.Additionally, E-cadherin and tight-junction protein one were expressed within cellular junctions of organoids. These proteins are essential for proper cell adhesion. After irradiation, organoids continued to retain their epithelial characteristics.Fluorescent staining of organoids could be visualized within low-adhesion plates using fluorescence microscopy. However, the clearest visualization was obtained via confocal microscopy. Corrected total fluorescence intensity was calculated by subtracting the background, and normalizing by organoid area.Growing organoids in the 96-well low-adhesion plates also simplified co-culture experiments. When seeded at concentrations typical in the mammary gland, macrophage colocalization increased with irradiated organoids. When performing this procedure, the most important things to remember are to not over-digest the organoids, and to appropriately purify the centrifugal differentiation.Following this procedure, additional co-culture experiments can be performed. Organoids can be co-cultured with tumor cells, other immune cells, and stromal cells to recreate the irradiated microenvironment associated with recurrence. This method will be important for evaluating how normal tissue damage specifically influences immune cell dynamics, and will eventually be used to understand tumor cell recruitment mechanisms.Mammary organoids have elucidated mechanisms of mammary gland development, and have robustly recapitulated breast cancer in vitro. We hope that our model provides insights about radiation-induced tumor cell recruitment.

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Research

JoVE Journal

Bioengineering

8.0K visualizações.  Vanderbilt University. Este protocolo pode ser usado e desenvolvido para analisar como a radiação ionizante afeta o recrutamento de células tumorais, levando a uma maior compreensão da recidiva do câncer. Organoides mamários são modelos poderosos. Eles imitam características básicas in vivo, mas permitem o fácil isolamento das variáveis biológicas.O uso de placas de baixa adesão nega os desafios de fluxo de trabalho associados às matrizes proteicas. Pacientes com câncer de mama triplo negativo...