This work was supported by grants from the Canadian Breast Cancer Foundation-Ontario Region, the Canada Foundation for Innovation (No. 13199), and donor support from John and Donna Bristol through the London Health Sciences Foundation (to A.L.A.). Studentship and fellowship support were provided by the Ontario Graduate Scholarship program (Province of Ontario, to G.M.P. and J.E.C.), the Canada Graduate Scholarship-Master's program (to M.M.P), the Canadian Institutes of Health Research (CIHR)-Strategic Training Program (to M.M.P., G.M.P and J.E.C.) and the Pamela Greenaway-Kohlmeier Translational Breast Cancer Research Unit at the London Regional Cancer Program (to M.M.P., G.M.P., J.E.C. and Y.X.). A.L.A. is supported by a CIHR New Investigator Award and an Early Researcher Award from the Ontario Ministry of Research and Innovation.

All animal studies were conducted in accordance with the recommendations of the Canadian Council on Animal Care, under protocols approved by the Western University Animal Use Subcommittee.
1. Organ Isolation (Lung, Brain, Bone, Lymph Node)
<ol>
	<li>Prepare four sterile 50 ml conical tubes (one for each organ to be isolated) containing approximately 30 ml of sterile phosphate-buffered saline (PBS). Pre-weigh each tube of PBS using an electronic balance.</li>
	<li>Euthanize 6-12 week old mouse by CO2 inhalation. Mice should be left in the CO2 chamber for approximately 1 - 2 min&#160;or until the mouse stops moving and breathing. Successful euthanasia can be further confirmed by a lack of heartbeat when checked manually with a finger. Avoid cervical dislocation as this method may rupture blood vessels of the neck leading to difficulty removing axillary lymph nodes. 
	Note: Previous work has specifically used healthy female nude mice, Hsd:Athymic Nude-Foxn1nu13.</li>
	<li>In a sterile tissue culture hood, place the mouse on its back on a polystyrene foam pad, spread the limbs and use pins to keep them in place.</li>
	<li>Using sterile forceps and scissors, cut the abdominal skin at the midline at the genitalia and cut upwards toward the mouth. Gently pull back the abdominal skin from the abdominal muscles and pin in place on the polystyrene foam pad.</li>
	<li>Locate the axillary, brachial, and inguinal lymph nodes. 
	Note: Lymph nodes are usually surrounded by fatty tissue. The inguinal lymph nodes are the easiest to locate as they are found superficially at the junction of two blood vessels on the pulled back abdominal skin. Axillary and brachial lymph nodes are located deeper within the tissue and require gentle maneuvering of tissue.
	<ol>
		<li>After you have located the lymph nodes, use the scissors to gently and carefully cut the lymph nodes away from the skin, fat and vessels and remove them from the mouse. To confirm proper dissection, roll the forceps over the removed tissue. If a hard lump exists when rolling the forceps over the tissue, then a lymph node has likely been removed successfully.</li>
		<li>Place removed lymph nodes in ice cold PBS.</li>
	</ol>
	</li>
	<li>Using the forceps and scissors, open the abdominal cavity by cutting through the exposed abdominal wall in an upward motion towards the chest. Carefully cut through the sternum, exposing the thoracic cavity.</li>
	<li>Locate the diaphragm below the lungs and cut the diaphragm. It should pull towards the ribs due to tension.</li>
	<li>Lift the lungs from underneath and cut the underlying tissue towards the trachea. This allows the lungs to be removed freely from the thoracic cavity. Remove the heart and lungs en bloc and place in ice cold PBS. The heart can be removed from the lungs here or just before weighing in Step 2.</li>
	<li>Remove the pins, keeping the mouse in place on the polystyrene foam pad. Turn the mouse over and cut the skin of the lower back all the way across from flank to flank.</li>
	<li>Using a sterile piece of gauze to hold the torso of the mouse, peel the back skin of the mouse over the legs and feet of the mouse.</li>
	<li>Using the same piece of sterile gauze, hold the lower leg in place, carefully break the ankle joint of the mouse foot and peel the skin over the joint proximally towards the knee joint.</li>
	<li>Using scissors, remove the tibia free from the knee joint and place in ice cold PBS.</li>
	<li>Repeat steps 1.11) to 1.12) with opposite limb.</li>
	<li>Using forceps, hold the femur in place, cut away surrounding muscle tissue using the scissors and remove the femur, placing it in ice cold PBS.</li>
	<li>Repeat step 1.14) with opposite limb.</li>
	<li>Using a new piece of sterile gauze, hold the head of the mouse in place. Using forceps and scissors, gently remove the skin to expose the skull. Using scissors, carefully cut the occipital skull from the top center in a straight and downward line to expose the posterior brain.</li>
	<li>Using forceps, scoop underneath the brain towards the anterior and remove the whole brain. Place the brain in ice cold PBS.</li>
	<li>Repeat steps 1.1) to 1.17) for at least four mice.</li>
</ol>
2. Organ Weighing
<ol>
	<li>Following organ isolation, weigh each PBS tube containing lung and brain tissue using an electronic balance.</li>
	<li>Calculate the weight difference by subtracting the pre-isolation weight (PBS only) from the weight of the PBS tube + organs (lung and brain).</li>
	<li>Determine the amount of media needed to resuspend tissue fragments from the calculated weight difference. 
	Note: Lung and brain tissue are weight normalized by resuspension in a 4:1 media:tissue ratio (vol/wt).</li>
</ol>
3. Generation of Lung- and Brain- Conditioned Media
<ol>
	<li>In a sterile tissue culture hood, invert PBS tubes containing lung or brain three times to remove residual blood from organs and aspirate PBS containing blood. Repeat with fresh cold PBS until solution appears clear with no evidence of blood.</li>
	<li>Place lungs and brains in separate 60 mm2 glass petri dishes. Using two sterile scalpel blades, mince lungs or brains by repeatedly slicing back and forth until tissue fragments are approximately ~ 1 mm3 in size.</li>
	<li>Resuspend tissue fragments in appropriate volume (determined previously in step 2.3) of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM):F12 media supplemented with 1x&#160;concentrated mitogen supplement and penicillin (50 &#181;g/ml)/streptomycin (50 &#181;g/ml).</li>
	<li>Add resuspended lung or brain tissue fragments to one well of a 6-well plate.</li>
	<li>Incubate tissue fragments in media for 24 hr at 37 &#176;C and 5% CO2. Following incubation, collect conditioned media for each tissue and further dilute by adding three equivalent volumes of fresh media in a 50 ml conical tube.</li>
	<li>Centrifuge at 1,000 x g in diluted conditioned media for each organ at 4 &#176;C for 15 min to remove large tissue debris. Collect media supernatant and filter through a 0.22 &#181;m syringe filter.</li>
	<li>Pool conditioned media from each organ (i.e., lung with lung and brain with brain) from multiple mice to account for mouse-to-mouse variability. Aliquot and store conditioned media at -80 &#176;C until use.</li>
</ol>
4. Generation of Bone Marrow-conditioned Media
<ol>
	<li>In a sterile tissue culture hood, trim excess tissue from the bone and remove epiphyses (end pieces) from the bones with scissors.</li>
	<li>Using a 27 &#189; G needle, flush medullary cavity of bones by pushing 1 ml of PBS through the center of each bone. This will allow you to collect bone marrow stromal cells (BMSC)into a fresh tube containing PBS.</li>
	<li>Centrifuge at 1,000 x g for 5 min at 4 &#176;C and wash BMSC twice with PBS. Resuspend bone marrow stromal cells in 20 ml of bone stromal cell growth media (DMEM:F12 media supplemented with 1x concentrated mitogen supplement, penicillin (50 &#181;g/ml)/streptomycin (50 &#181;g/ml) and 10% fetal boven serum (FBS)).</li>
	<li>Plate 10 ml of resuspended bone marrow stromal cells in one T75 flask. 
	Note: Combine and plate cells from every 2 mice in each T75 flask; for four mice, two T75 flasks are needed (approximately 1 x 107 cells/flask).</li>
	<li>Incubate bone marrow stromal cells in bone stromal cell growth media for 24 hr at 37 &#176;C and 5% CO2. Following incubation, remove media from both T75 flasks and put into new T75 flask, label this flask as &#34;Floater Flask&#34;. Add fresh bone stromal cell growth media to previous 2 flasks and incubate all 3 flasks at 37 &#176;C and 5% CO2.</li>
	<li>After cells reach approximately 70% confluency (approximately 5 - 7 days) passage cells. To do this, remove medium and wash cells twice with PBS (3 ml each wash). Remove PBS and add 3 ml of trypsin/EDTA solution, ensuring that trypsin covers the entire surface of the flask. After cells lift off the flask (~ 2 - 3 min), stop trypsinization reaction by adding 3 ml bone stromal cell growth media). Centrifuge 900 x g for 5 min at 4 &#176;C, discard media, and resuspend cells in 10 ml of fresh bone stromal cell growth media.</li>
	<li>Pool cells from all flasks and passage 1:5 into three new T75 flasks and incubate at 37 &#176;C and 5% CO2. After cells once again reach 70%, repeat step 4.6 and pool and passage all adherent cells a second time. Re-plate all cells to four T75 flasks and incubate 37 &#176;C and 5% CO2. Bone stromal cell growth media should be used for all steps.</li>
	<li>Allow cells to reach confluence, wash cells three times with PBS and add bone stromal cell collection media (DMEM:F12 media + 1x concentrated mitogen supplement + penicillin (50 &#181;g/ml)/streptomycin (50 &#181;g/ml); 10 ml/T75), making sure that this media is free of FBS. Collect bone marrow-conditioned media 72 hr later, filter through a 0.22 &#181;m filter, pool, aliquot, and store at -80 &#176;C until use.</li>
	<li>If desired, confirm the phenotype of the isolated bone marrow stromal cells (BMSC) using antibodies against mouse Sca-1, CD105, CD29, and CD73, CD44, using standard flow cytometry techniques as described previously13.</li>
</ol>
5. Generation of Lymph Node-conditioned Media
<ol>
	<li>In a sterile tissue culture hood, invert PBS tube containing lymph nodes three times to remove residual blood from lymph nodes and aspirate bloody PBS. Repeat with fresh cold PBS until solution appears clear with no evidence of blood.</li>
	<li>Place lymph nodes in 60 mm2 glass petri dishes. Using two sterile scalpel blades, mince lymph nodes by repeatedly slicing back and forth until tissue fragments are approximately 1 mm3 in size.</li>
	<li>In a conical tube, resuspend tissue fragments in 10 ml of Roswell Park Memorial Institute 1640 (RPMI1640) media supplemented with penicillin (50 &#181;g/ml)/streptomycin (50 &#181;g/ml), 5 x 10-5 M sterile &#946;-mercaptoethanol (1.75 &#181;l/500 ml media) and 10% FBS.</li>
	<li>Centrifuge at 900 x g for 5 min at 4 &#176;C and resuspend all cells in 30 ml media. Add 5 ml media/well in a 6-well plate and incubate for 7 days at 37 &#176;C and 5% CO2.</li>
	<li>Following the 7 day incubation, discard media, wash adherent cells with 5 ml warm PBS and add 5 ml fresh media to each well. Allow cells to grow to confluency (approximately 5 - 7 days), trypsinize, pool all cells, and passage as described in step 4.6. Re-plate cells to 6-well plate. Repeat this step three times.</li>
	<li>After three passages, allow cells to grow to confluency, wash wells three times with PBS and add 2 ml/well of DMEM:F12 + 1x concentrated mitogen supplement and penicillin (50 &#181;g /ml)/streptomycin (50 &#181;g/ml), ensuring that this media is free of FBS. Collect lymph node-conditioned media after 72 hr, pool, aliquot, and store at -80 &#176;C until use.</li>
	<li>If desired, confirm the phenotype of the isolated lymph node stromal cells (LNSC) using antibodies against mouse CD45 and gp38, using standard flow cytometry techniques as described previously13.</li>
</ol>
6. Use of Organ-conditioned Media for Downstream Assays Related to Metastatic Behavior of Cancer Cells
<ol>
	<li>Once the organ-conditioned media has been generated as described in steps 1 - 5, use it to carry out different downstream cell and molecular biology assays in order to determine the influence of soluble organ-derived factors on the metastatic behavior of cancer cells. Examples of standard assays (described elsewhere in the literature) include protein arrays13, cellular growth assays13, cellular migration assays13, tumorsphere formation14, and RT-PCR15. The original base media used to generate the organ-conditioned media (i.e., unconditioned DMEM:F12 media supplemented with 1x concentrated mitogen supplement and penicillin (50 &#181;g/ml)/streptomycin (50 &#181;g/ml) should be used as a negative control.</li>
</ol>

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2015.">Cancer statistics, 2015.</a> CA Cancer J Clin. 65 (1), 5-29 (2015).</li><li>Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organ+microenvironment+and+cancer+metastasis.">The organ microenvironment and cancer metastasis.</a> Differentiation. 70 (9-10), 498-505 (2002).</li><li>Chambers, A. F., Groom, A. C., MacDonald, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+and+growth+of+cancer+cells+in+metastatic+sites.">Dissemination and growth of cancer cells in metastatic sites.</a> Nat Rev Cancer. 2 (8), 563-572 (2002).</li><li>Kennecke, H., 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=Metastatic+behavior+of+breast+cancer+subtypes.">Metastatic behavior of breast cancer subtypes.</a> J Clin Oncol. 28 (20), 3271-3277 (2010).</li><li>Nguyen, D. X., Bos, P. D., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+from+dissemination+to+organ-specific+colonization.">Metastasis: from dissemination to organ-specific colonization.</a> Nat Rev Cancer. 9 (4), 274-284 (2009).</li><li>Ewing, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+Diseases:+A+Treatise+on+Tumors.">Neoplastic Diseases: A Treatise on Tumors.</a> Am J Med Sci. 176 (2), 278 (1928).</li><li>Paget, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+secondary+growths+in+cancer+of+the+breast.+1889.">The distribution of secondary growths in cancer of the breast. 1889.</a> Cancer Metastasis Rev. 8 (2), 98-101 (1989).</li><li>Weiss, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comments+on+hematogenous+metastatic+patterns+in+humans+as+revealed+by+autopsy.">Comments on hematogenous metastatic patterns in humans as revealed by autopsy.</a> Clin Exp Metastasis. 10 (3), 191-199 (1992).</li><li>Bos, P. D., 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=Genes+that+mediate+breast+cancer+metastasis+to+the+brain.">Genes that mediate breast cancer metastasis to the brain.</a> Nature. 459 (7249), 1005-1009 (2009).</li><li>Kang, Y., 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+multigenic+program+mediating+breast+cancer+metastasis+to+bone.">A multigenic program mediating breast cancer metastasis to bone.</a> Cancer Cell. 3 (6), 537-549 (2003).</li><li>Minn, A. J., 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=Genes+that+mediate+breast+cancer+metastasis+to+lung.">Genes that mediate breast cancer metastasis to lung.</a> Nature. 436 (7050), 518-524 (2005).</li><li>Chu, J. E., 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=Lung-Derived+Factors+Mediate+Breast+Cancer+Cell+Migration+through+CD44+Receptor-Ligand+Interactions+in+a+Novel+Ex+Vivo+System+for+Analysis+of+Organ-Specific+Soluble+Proteins.">Lung-Derived Factors Mediate Breast Cancer Cell Migration through CD44 Receptor-Ligand Interactions in a Novel Ex Vivo System for Analysis of Organ-Specific Soluble Proteins.</a> Neoplasia. 16 (2), (2014).</li><li>Deepak, S., 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=Real-Time+PCR:+Revolutionizing+Detection+and+Expression+Analysis+of+Genes.">Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes.</a> Curr Genomics. 8 (4), 234-251 (2007).</li><li>Hammerschmidt, S. I., 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=Stromal+mesenteric+lymph+node+cells+are+essential+for+the+generation+of+gut-homing+T+cells+in+vivo.">Stromal mesenteric lymph node cells are essential for the generation of gut-homing T cells in vivo.</a> J Exp Med. 205 (11), 2483-2490 (2008).</li><li>Dominici, 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=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Baddoo, 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=Characterization+of+mesenchymal+stem+cells+isolated+from+murine+bone+marrow+by+negative+selection.">Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection.</a> J Cell Biochem. 89 (6), 1235-1249 (2003).</li><li>Furger, K. A., Menon, R. K., Tuck, A. B., Bramwell, V. H., Chambers, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+and+clinical+roles+of+osteopontin+in+cancer+and+metastasis.">The functional and clinical roles of osteopontin in cancer and metastasis.</a> Curr Mol Med. 1 (5), 621-632 (2001).</li><li>Radisky, E. S., Radisky, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases+as+breast+cancer+drivers+and+therapeutic+targets.">Matrix metalloproteinases as breast cancer drivers and therapeutic targets.</a> Front Biosci (Landmark Ed). 20, 1144-1163 (2015).</li><li>Radisky, E. S., Radisky, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase-induced+epithelial-mesenchymal+transition+in+breast+cancer.">Matrix metalloproteinase-induced epithelial-mesenchymal transition in breast cancer.</a> J Mammary Gland Biol Neoplasia. 15 (2), 201-212 (2010).</li><li>Radisky, E. S., Radisky, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+induction+of+breast+cancer:+inflammation+and+invasion.">Stromal induction of breast cancer: inflammation and invasion.</a> Rev Endocr Metab Disord. 8 (3), 279-287 (2007).</li><li>Kakinuma, T., Hwang, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines,+chemokine+receptors,+and+cancer+metastasis.">Chemokines, chemokine receptors, and cancer metastasis.</a> J Leukoc Biol. 79 (4), 639-651 (2006).</li><li>Zlotnik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+and+cancer.">Chemokines and cancer.</a> Int J Cancer. 119 (9), 2026-2029 (2006).</li><li>Schlesinger, M., Bendas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+cell+adhesion+molecule-1+(VCAM-1)--an+increasing+insight+into+its+role+in+tumorigenicity+and+metastasis.">Vascular cell adhesion molecule-1 (VCAM-1)--an increasing insight into its role in tumorigenicity and metastasis.</a> Int J Cancer. 136 (11), 2504-2514 (2015).</li><li>Cook, K. L., Shajahan, A. N., Clarke, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+endocrine+resistance+in+breast+cancer.">Autophagy and endocrine resistance in breast cancer.</a> Expert Rev Anticancer Ther. 11 (8), 1283-1294 (2011).</li><li>Singh, P., Alex, J. M., Bast, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+receptor+(IR)+and+insulin-like+growth+factor+receptor+1+(IGF-1R)+signaling+systems:+novel+treatment+strategies+for+cancer.">Insulin receptor (IR) and insulin-like growth factor receptor 1 (IGF-1R) signaling systems: novel treatment strategies for cancer.</a> Med Oncol. 31 (1), 805 (2014).</li><li>Lee, S. H., Jeong, D., Han, Y. S., Baek, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pivotal+role+of+vascular+endothelial+growth+factor+pathway+in+tumor+angiogenesis.">Pivotal role of vascular endothelial growth factor pathway in tumor angiogenesis.</a> Ann Surg Treat Res. 89 (1), 1-8 (2015).</li><li>Erdmann, R. B., Gartner, J. G., Leonard, W. J., Ellison, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+functional+TSLP+receptors+mitigates+Th2+polarization+and+the+establishment+and+growth+of+4T1+primary+breast+tumours+but+has+different+effects+on+tumour+quantities+in+the+lung+and+brain.">Lack of functional TSLP receptors mitigates Th2 polarization and the establishment and growth of 4T1 primary breast tumours but has different effects on tumour quantities in the lung and brain.</a> Scand J Immunol. 78 (5), 408-418 (2013).</li><li>Chambers, A. F., 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=Steps+in+tumor+metastasis:+new+concepts+from+intravital+videomicroscopy.">Steps in tumor metastasis: new concepts from intravital videomicroscopy.</a> Cancer Metastasis Rev. 14 (4), 279-301 (1995).</li><li>Chiang, A. C., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+metastasis.">Molecular basis of metastasis.</a> N Engl J Med. 359 (26), 2814-2823 (2008).</li><li>Gupta, G. P., 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=Identifying+site-specific+metastasis+genes+and+functions.">Identifying site-specific metastasis genes and functions.</a> Cold Spring Harb Symp Quant Biol. 70, 149-158 (2005).</li><li>Frantz, C., Stewart, K. M., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+at+a+glance.">The extracellular matrix at a glance.</a> J Cell Sci. 123, 4195-4200 (2010).</li><li>Price, A. P., England, K. A., Matson, A. M., Blazar, B. R., Panoskaltsis-Mortari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+decellularized+lung+bioreactor+system+for+bioengineering+the+lung:+the+matrix+reloaded.">Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded.</a> Tissue Eng Part A. 16 (8), 2581-2591 (2010).</li><li>Bonnans, C., Chou, J., 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=Remodelling+the+extracellular+matrix+in+development+and+disease.">Remodelling the extracellular matrix in development and disease.</a> Nat Rev Mol Cell Biol. 15 (12), 786-801 (2014).</li><li>Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix:+not+just+pretty+fibrils.">The extracellular matrix: not just pretty fibrils.</a> Science. 326 (5957), 1216-1219 (2009).</li><li>Psaila, B., Lyden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metastatic+niche:+adapting+the+foreign+soil.">The metastatic niche: adapting the foreign soil.</a> Nat Rev Cancer. 9 (4), 285-293 (2009).</li><li>Lee, R. H., Oh, J. Y., Choi, H., Bazhanov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+factors+secreted+by+mesenchymal+stromal+cells+and+tissue+repair.">Therapeutic factors secreted by mesenchymal stromal cells and tissue repair.</a> J Cell Biochem. 112 (11), 3073-3078 (2011).</li></ol>

The authors declare that they have no competing financial interests.

Metastasis is a complex process by which a series of cellular events are ultimately responsible for tissue invasion and distant tumor establishment4,30,31. The ex vivo model system presented here can be utilized to study two important aspects of metastatic progression: cancer cell homing or migration to a specific organ (&#34;getting there&#34;) and growth in that organ (&#34;growing there&#34;). Many studies have previously focused on identifying key molecular characteristics associated with the canc...

Breast cancer is the most frequently diagnosed cancer in women and the second leading cause of cancer-related deaths1. Breast cancer&#39;s high mortality rate is mainly due to the failure of conventional therapy to mitigate and eliminate metastatic disease; approximately 90% of cancer-related deaths are due to metastasis2. Understanding the underlying molecular mechanisms of the metastatic cascade is paramount to the development of therapeutics effective in both early and late-stage breast cancer.
Past research has helped elucidate the multistep nature of breast cancer metastasis and it is hypothesized that the outcome of both cancer progression and metastasis is largely dependent upon interactions between cancer cells and the host environment3. Clinical observations indicate that many cancers display organ tropism, i.e., the tendency to preferentially metastasize to specific organs.In the case of breast cancer, a patient&#39;s disease typically spreads or metastasizes to 5 main sites, including the bone, lungs, lymph node, liver, and brain4-6. Many theories have been developed to explain this process, but only a few have withstood the test of time. Ewing&#39;s theory of metastasis, proposed in the 1920s, hypothesized thatthe distribution of metastasis was strictly due to mechanical factors; whereby tumor cells are carried throughout the body by normal defined physiological blood flow patterns and simply arrest in the first capillary bed they encounter7. In contrast, Stephen Paget&#39;s 1889 &#34;seed and soil&#34; hypothesis suggested that additional molecular interactions were responsible for survival and growth of metastases, whereby cancer cells (&#34;seeds&#34;) can only establish themselves and proliferatein organ microenvironments that produce appropriate molecular factors (&#34;soil&#34;)8. Almost a century later, Leonard Weiss undertook a meta-analysis of previously published autopsy data and confirmed Ewing&#39;s prediction that many metastatic tumors detected at the time of autopsy were found in the anticipated proportions that would be expected if metastatic organ tropism was determined by blood flow patterns alone. However, in manyinstances there were fewer or more metastases formed at certain sites then would be expected by Ewing&#39;s proposed mechanical factors9. These accounts and theories suggest that specific organ microenvironments play a critical role in the dissemination patterns and subsequent growth and survival of many cancers, including breast cancer.
Past research efforts have mainly focused on tumor-cell derived factors and their contribution to the organ tropism observed in breast cancer metastasis10-12, however little research has explored factors derived from the organ microenvironment that may provide a favorable niche for the establishment of breast cancer metastases. This is largely attributable to the technical challenges of studying components of the organ microenvironment in vitro. 
The current article describes a comprehensive ex vivo model system for studying the influence of soluble components of the lymph node, bone, lung, and brain on the metastatic behavior of human breast cancer cells. Previous studies have validated this model system by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential13. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and to evaluate the role of organ-derived soluble factors in driving the process of cancer metastasis.

<table><tbody><tr><td>50 ml conical tubes</td><td>Thermo Scientific (Nunc)</td><td>339652</td><td>Keep sterile</td></tr><tr><td>1x Phosphate-buffered saline</td><td>ThermoFisher Scientific</td><td>10010-023</td><td>Keep sterile</td></tr><tr><td>Nude mice</td><td>Harlan Laboratories</td><td>Hsd:Athymic Nude-Foxn1nu</td><td>Use at 6 - 12 weeks of age</td></tr><tr><td>Polystyrene foam pad</td><td>N/A</td><td>N/A</td><td>The discarded lid (~ 4 x 8 inches or larger) of a polystyrene foam shipping container can be used for this purpose. Sterilize by wiping with ethanol.</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11050-10</td><td>Keep sterile</td></tr><tr><td>Scissors</td><td>Fine Science Tools</td><td>14058-11</td><td>Keep sterile</td></tr><tr><td>Gauze pads</td><td>Fisher Scientific</td><td>22-246069</td><td>Keep sterile</td></tr><tr><td>60 mm&lt;sup&gt;2&lt;/sup&gt; glass petri dishes</td><td>Sigma-Aldrich</td><td>CLS7016560</td><td>Keep sterile</td></tr><tr><td>Scalpel blades</td><td>Fisher Scientific</td><td>S95937A</td><td>Keep sterile</td></tr><tr><td>DMEM:F12</td><td>Life Technologies</td><td>21331-020</td><td>Warm in 37 &amp;deg;C water bath before use, keep sterile&amp;nbsp;</td></tr><tr><td>1x Mito + Serum Extender</td><td>BD Biosciences</td><td>355006</td><td>Referred to as &quot;concentrated mitogen supplement&quot; in the manuscript. Keep sterile</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td>Keep sterile</td></tr><tr><td>Rosewell Park Memorial Institute 1640 (RPMI 1640)</td><td>Life Technologies</td><td>11875-093</td><td>Warm in 37 &amp;deg;C water bath before use, keep sterile&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F1051-500ML</td><td>Keep sterile</td></tr><tr><td>Trypsin/EDTA solution</td><td>ThermoFisher Scientific</td><td>R-001-100</td><td>Warm in 37 &amp;deg;C water bath before use, keep sterile&amp;nbsp;</td></tr><tr><td>6-well tissue culture plates</td><td>Thermo Scientific (Nunc)</td><td>140675</td><td>Keep sterile</td></tr><tr><td>0.22 &amp;mu;m syringe filters</td><td>Sigma-Aldrich</td><td>Z359904</td><td>Keep sterile</td></tr><tr><td>T75 tissue culture flasks</td><td>Thermo Scientific (Nunc)</td><td>178905</td><td>Keep sterile</td></tr><tr><td>Transwells</td><td>Sigma-Aldrich</td><td>CLS3464</td><td>Keep sterile, use for migration assays</td></tr><tr><td>Anti-mouse Sca-1</td><td>R&amp;amp;D Systems</td><td>FAB1226P</td><td>use at 10 &amp;micro;l/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Anti-mouse CD105</td><td>R&amp;amp;D Systems</td><td>FAB1320P</td><td>use at 10 &amp;micro;l/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Anti-mouse CD29</td><td>R&amp;amp;D Systems</td><td>FAB2405P-025</td><td>use at 10 &amp;micro;l/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Anti-mouse CD73</td><td>R&amp;amp;D Systems</td><td>FAB4488P</td><td>use at 10 &amp;micro;l/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Anti-mouse CD44</td><td>R&amp;amp;D Systems</td><td>MAB6127-SP</td><td>use at 0.25 &amp;micro;g/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Anti-mouse CD45</td><td>eBioscience</td><td>11-0451-81</td><td>use at 5 &amp;micro;l/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Anti-mouse gp38</td><td>eBioscience</td><td>12-5381-80</td><td>use at 10 &amp;micro;l/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>&amp;beta;-mercaptoethanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M6250&amp;nbsp;</td><td>Keep sterile</td></tr><tr><td>Protein arrays</td><td>RayBiotech Inc.</td><td>AAM-BLM-1-2</td><td>Use 1 array per media condition (including negative control), in triplicate</td></tr></tbody></table>

generation of organ-conditioned media and applications for studying organ-specific influences on breast cancer metastatic behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

Generation of Organ-conditioned Media
An overview diagram/schematic of the process of organ isolation and generation of conditioned media is presented in Figure 1, with representative photographic images of the procedure shown in Figure 2. It should be noted that when this protocol was first under development, liver was included in our analysis because it is a common site of bre...

Watch this Scientific Journal Video about Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior at JoVE.com

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

generation-organ-conditioned-media-applications-for-studying-organ

Research

JoVE Journal

Medicine

8.4K Views.  London Health Sciences Centre. This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells. 

Video: Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Genetics

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Cancer Research

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Developmental Biology

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

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

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

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

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

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.

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 ...

Bioengineering

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Summary

Explore More Videos

Chapters in this video

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Establishment and Culture of Patient-Derived Breast Organoids

Generation of Multicellular Human Primary Endometrial Organoids

Generation of Mosaic Mammary Organoids by Differential Trypsinization

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

Growth and Characterization of Irradiated Organoids from Mammary Glands

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres