This study was supported by Yeditepe University. All data and figures used in this article were previously published34.

Written informed consent of the patients was obtained after the approval from the Institutional Ethics Committee.
1. DPSC Isolation and Culture
<ol>
	<li>Transfer wisdom teeth obtained from young adults aged between 17 and 20 to 15 mL tubes containing complete Dulbecco&#39;s Modified Eagle Medium (DMEM) [low glucose DMEM media, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/amphotericin (PSA) solution], within 8 h after resection. Keep the tissue material cold (4 &#176;C) during transfer to avoid potential cell death.</li>
	<li>Remove the pulp tissue by sterile extraction forceps from the center of the tooth carefully, place the pulp tissue in the cold complete DMEM medium in 10 cm tissue culture dishes, and mince them into small pieces (2-3 mm) by scalpel. 
	NOTE: All experimental procedures should be carried out under sterile conditions in laminar flow hood. This non-enzymatic technique has been previously used21,22,23.</li>
	<li>Place small pulp tissues inside the tissue culture treated 6-well plates and add 200 &#181;L of complete DMEM media to cover each small pulp tissue pieces.</li>
	<li>Incubate the tissue culture wells at 37 &#176;C in a humidified air atmosphere (80% humidity) with 5% CO2 for 2 h to provide tissue attachment. 
	NOTE: This step could be prolonged to 3-4 h by controlling the evaporation of media.</li>
	<li>Add appropriate volume (2-2.5 mL) of complete DMEM medium to the wells and incubate at 37 &#176;C in a humidified air atmosphere with 5% CO2 for cells to spread from the tissue. 
	NOTE: Cells become visible after approximately 4 days and reach confluency after 8-9 days.</li>
	<li>When cells reach 80% confluency, remove media from 6-well plates, wash with 2 mL of phosphate buffered saline (PBS), and add 2 mL of trypsin. Incubate for 2 min in an incubator at 37 &#176;C with humidified air atmosphere and 5% CO2. Then add 2 mL of complete DMEM medium followed by 2 min incubation to inhibit trypsin. Centrifuge cells at 300 x g for 5 min to pellet cells.</li>
	<li>Passage cells to the flasks in complete DMEM media and store for further experiments. Add 15 mL of complete DMEM media to the T-75 flasks and transfer cells from two wells of the 6-well plate to one T-75 flasks and incubate at 37 &#176;C with humidified air atmosphere and 5% CO2.</li>
</ol>
2. Characterization of DPSCs
<ol>
	<li>Perform morphological analyses.
	<ol>
		<li>Seed cells (step 1.6) in tissue culture coated flasks (or 6-well plates) in complete DMEM medium for at least 8 passages to observe cell morphology.</li>
		<li>Visualize cells by light microscope and define fibroblast-like cell morphology. Cells should attach to the culture dishes and have spindle-like cell morphology. 
		NOTE: Alternatively, cells can be cultured as single cells for up to 14 days in well-plates to observe colony formation capacity that is a specific characteristic of fibroblasts and MSCs.</li>
	</ol>
	</li>
	<li>Perform surface marker analyses.
	<ol>
		<li>Trypsinize the cells from step 1.7. Remove media from the T-75 tissue culture flask, wash with 2 mL of PBS, and add 2 mL of trypsin. Incubate for 2 min in an incubator at 37 &#176;C with humidified air atmosphere and 5% CO2. Then add 2 mL of complete DMEM medium followed by 2 min incubation to inhibit trypsin. Centrifuge cells at 300 x g for 5 min to pellet cells.</li>
		<li>Fix the cells with 4% paraformaldehyde for 20 min at room temperature in 1.5 mL tubes and then wash them with 500 &#181;L of PBS 3 times to remove paraformaldehyde.</li>
		<li>Incubate fixed cells with the antibodies against CD29, CD34, CD14, CD45, CD90, CD105, CD166, and CD73 for 1 h at 4 &#176;C in 100 &#181;L of PBS. 
		NOTE: The concentration of antibody used is 0.5 &#181;g/mL. CD34, CD14, and CD45 are used as negative markers, while CD29, CD90, CD105, CD166, and CD73 are used as positive cell surface markers for MSCs.</li>
		<li>Wash cells 3 times with PBS and use respective secondary antibodies such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), etc. for labelling. Incubate cells with 1:500 diluted secondary antibodies in 100 &#181;L of PBS for 30 min at 4 &#176;C and wash 3 times with PBS.</li>
		<li>Keep samples in the dark for flow cytometry analysis and detect positive and negative staining by flow cytometry. 
		NOTE: Use unstained control cells to arrange forward and side scatter. Arrange gating of positively stained populations by excluding dead cells, debris, and un-stained population. Use 100 &#181;m nozzle with 45 psi sheath pressure and collect 10,000 events to determine positive DPSCs by arranging channels.</li>
	</ol>
	</li>
	<li>Perform differentiation of DPSCs.
	<ol>
		<li>Seed 1 &#215; 104 cells onto 24-well plates in complete DMEM media and incubate for 24 h at 37 &#176;C in a humidified air atmosphere with 5% CO2.</li>
		<li>Formulate differentiation media using compete DMEM medium as base media. Prepare osteogenic media by mixing 100 nM dexamethasone,10 mM &#946;-glycerophosphate, and 0.2 mM ascorbic acid. Prepare chondrogenic media by mixing 1&#215; insulin-transferrin-selenium (ITS-G), 100 nM dexamethasone, 100 ng/mL transforming growth factor beta (TGF-&#946;), 14 &#956;g/mL ascorbic acid, and 1 mg/mL bovine serum albumin (BSA). Prepare adipogenic media by mixing 100 nM dexamethasone, 5 &#956;g/mL insulin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 60 &#956;M indomethacin. 
		NOTE: Differentiation media can be kept at 4 &#176;C for at least one week.</li>
		<li>Change growth media to osteo-, chondro-, or adipo-genic differentiation media and refresh media twice a week for two weeks.</li>
		<li>Confirm differentiation by von Kossa and Alcian blue staining, enzyme activity (alkaline phosphatase activity), immunocytochemistry, and quantitative polymerase chain reaction (qPCR) analyses according to the protocols previously described21.
		<ol>
			<li>Perform von Kossa and Alcian blue stainings on cells that are fixed with 4% paraformaldehyde at room temperature for 10 min. Wash the fixed cells with PBS and stain them with the vonKossa kit according to the manufacturer&#8217;s recommendations to observe calcium deposits.</li>
			<li>Prepare the Alcian blue staining solution by dissolving 1.00 g of Alcian blue dye in 100 mL of 3% (v/v) acetic acid for further staining. Wash the fixed cells with PBS and stain cells for 30 min with Alcian blue solution. Visualize the stained samples by a light microscope.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Preparation of Condition Medium (CM)
<ol>
	<li>Replace media of cells from step 1.7 with fresh complete DMEM 24 h before CM collection. 
	NOTE: Passage 2-4 is recommended.</li>
	<li>Collect condition medium (CM) from cultured DPSCs when cells reach 80% confluency. Centrifuge collected media at 300 x g for 5 min to remove waste tissue material and cell debris. 
	NOTE: Alternatively, use 0.2 &#181;m syringe filters to remove debris from the condition medium.</li>
	<li>Collect supernatant and store at -20 &#176;C for further experiments. 
	NOTE: Keep the supernatant at -80 &#176;C for long-term storage.</li>
</ol>
4. Treatment of Cancer Cells with CM
<ol>
	<li>Perform cell viability analyses.
	<ol>
		<li>Seed PC-3 cells (human prostate cancer cells) onto 96-well plates at a cell density of 5 &#215; 103 cells/well in complete DMEM and incubate in a humidified chamber at 37 &#176;C and 5% CO2 for 24 h.</li>
		<li>Treat cells with 10, 20, 30, 40, and 50% of CM (v/v) mixed with complete DMEM for 24 h.</li>
		<li>Measure cell viability by using 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS)-assay as described previously24.</li>
	</ol>
	</li>
	<li>Perform terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses.
	<ol>
		<li>Seed PC-3 cells onto 6-well cell culture plates at a cell density of 2 &#215; 105 cells/well and incubate in a humidified chamber at 37 &#176;C and 5% CO2 overnight.</li>
		<li>Mix 20% CM (v/v) with complete DMEM medium and apply to the cells for 24 h.</li>
		<li>Trypsinize cells and suspend in 50 &#956;L of TUNEL reaction mixture (labeling solution + enzyme solution, supplied with the kit) and incubate at 37 &#176;C for 60 min in a humidified and 5% CO2 atmosphere. 
		NOTE: For trypsinization, remove media from 6-well cell culture plates, wash with 1 mL of PBS and add 500 &#181;L of trypsin. Incubate for 2 min in an incubator at 37 &#176;C with humidified air atmosphere and 5% CO2. Add 1 mL of complete DMEM medium followed by 2 min incubation to inhibit trypsin. Centrifuge cells at 300 x g for 5 min to pellet cells.</li>
		<li>Rinse with PBS and analyze cells in PBS by using flow cytometry.</li>
	</ol>
	</li>
	<li>Perform qPCR analyses.
	<ol>
		<li>Seed PC-3 cells onto 6-well cell culture plates at a cell density of 2 &#215; 105 cells/well and incubate in a humidified incubator at 37 &#176;C and 5% CO2 overnight.</li>
		<li>Mix 20% CM (v/v) with complete DMEM medium and apply to the cells for 24 h.</li>
		<li>Trypsinize cells and collect cell pellet for RNA isolation and cDNA synthesis. 
		NOTE: Remove media from 6-well cell culture plates, wash with 1 mL of PBS, and add 500 &#181;L of trypsin. Incubate 2 min in an incubator at 37 &#176;C with humidified air atmosphere and 5% CO2. Add 1 mL of complete DMEM medium followed by 2 min incubation to inhibit trypsin. Centrifuge cells at 300 x g for 5 min to pellet cells.</li>
		<li>Perform qPCR experiments according to the previously described protocol25.</li>
	</ol>
	</li>
	<li>Perform cell migration of cancer cells.
	<ol>
		<li>Seed 1 &#215; 105 PC-3 cells onto 12-well plates and incubate in a humidified incubator overnight at 37 &#176;C and 5% CO2.</li>
		<li>Scratch cells with a sterile 200 &#956;L tip and change medium immediately with fresh medium containing various concentrations of CM [e.g., 10, 20, 30, 40, and 50% of CM (v/v) mixed with complete DMEM].</li>
		<li>Observe scratches under an inverted microscope and take pictures at different time intervals (0 and 24 h).</li>
		<li>Measure the scratch closure by using Image J software using the formula: 
		<img alt="Equation" src="/files/ftp_upload/58825/58825eq1.jpg" /> 
		NOTE: Open the scratch image with Image J software. Draw a line that has the same magnitude as the scale bar that already exists in the image. Click analyze, set scale, and observe distance in pixels as the magnification of the drawn line. Write the size of the scale bar to the known distance part, arrange unit (pixels, cm, etc.), and click ok. Go to the analyze section again and click on measurements. This will first give the size of the scale bar as the selected unit. Click on one edge of scratch and drag until reaching the other end of the scratch. Note the value for each time point (0 h and 24 h). Plug these values into the formula above and calculate the scratch closure.</li>
	</ol>
	</li>
</ol>
5. Cell Migration by Indirect Contact of Cancer Cells and DPSCs
<ol>
	<li>Seed 3 &#215; 104 DPSCs onto 24-well plate inserts with 0.4 &#956;m pore and incubate in a humidified incubator overnight at 37 &#176;C.</li>
	<li>Seed PC-3 cells onto 24-well plates at a cell density of 5 &#215; 104 and incubate in a humidified incubator overnight at 37 &#176;C and 5% CO2.</li>
	<li>Scratch PC-3 cells with a sterile 200 &#956;L tip, change medium with fresh medium, and place inserts carrying DPSCs onto PC-3 cells.</li>
	<li>Observe cells under an inverted microscope and take pictures at different time intervals (0 and 24 h) to analyze cell migration.</li>
</ol>
6. Co-culture Assay and Flow Cytometry Analysis
<ol>
	<li>Label PC-3 cells and DPSCs by using PKH67 (green) and PKH26 (red) fluorescent cell linker dyes, respectively26.</li>
	<li>Trypsinize PC-3 and DPSCs cells, respectively. Remove media from T-75 tissue culture flask, wash with 2 mL of PBS, and add 2 mL of trypsin. Incubate for 2 min in an incubator at 37 &#176;C with humidified air atmosphere and 5% CO2. Add 2 mL of complete DMEM medium followed by 2 min incubation to inhibit trypsin.</li>
	<li>Centrifuge cells at 300 x g for 5 min, discard supernatant, and resuspend cell pellets in the dye solution prepared in diluent-C buffer supplied by the kit (see Table of Materials).</li>
	<li>Incubate cells in the dye solution for 10 min and terminate the staining reaction by adding 100 &#181;L of FBS. Centrifuge cells at 300 x g for 5 min, discard the supernatant, and wash the cells with complete growth medium before co-culturing.</li>
	<li>Plate labeled cells (5 &#215; 104/well) onto 6-well plates at 1:1 ratio (DPSCs:PC3). Maintain co-cultured cells in complete DMEM.</li>
	<li>Collect cells after 24 h or 48 h incubation periods by centrifugation of cells at 300 x g for 5 min and washing with PBS.</li>
	<li>Resuspend cells in 300 &#181;L of fluorescence-activated cell sorting (FACS) buffer in 5 mL round bottom flow cytometry tubes. Vortex to disperse cell aggregates right before the sample analysis.</li>
	<li>Use a 100 &#181;m nozzle with 45 psi sheath pressure. 
	NOTE: Extremely high flow rate might decrease the sensitivity of fluorescence detection.</li>
	<li>Use unstained control cells and single colored cells to adjust appropriate forward and side scatter laser voltage for cell types and compensation as mentioned previously27. 
	NOTE: Use gating for live cells to exclude cell debris, dead cells, or aggregates.</li>
	<li>Collect 10,000 events (100,000 is preferable) to determine percent positive green DPSCs and red PC-3 cells by arranging FL-1 (green) and FL-2 (red) channels.</li>
</ol>

<ol>
	<li>Camberlain, G., Fox, J., Ashton, B., Middleton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells:+their+phenotype,+differentiation+capacity,+immunological+features,+and+potential+for+homing.">Mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing.</a> Stem Cells. 25 (11), 2739-2749 (2007).</li><li>Demirci, S., Do&#287;an, A., &#350;ahin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dental Stem Cells. , 109-124 (2016).</li><li>Fox, J. M., Chamberlain, G., Ashton, B. A., Middleton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+into+the+understanding+of+mesenchymal+stem+cell+trafficking.">Recent advances into the understanding of mesenchymal stem cell trafficking.</a> British journal of haematology. 137 (6), 491-502 (2007).</li><li>Chang, A. I., Schwertschkow, A. H., Nolta, J. A., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+mesenchymal+stem+cells+in+cancer+progression+and+metastases.">Involvement of mesenchymal stem cells in cancer progression and metastases.</a> Current cancer drug targets. 15 (2), 88-98 (2015).</li><li>Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumors:+wounds+that+do+not+heal.">Tumors: wounds that do not heal.</a> New England Journal of Medicine. 315 (26), 1650-1659 (1986).</li><li>Lu, Y. -. 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=The+growth+inhibitory+effect+of+mesenchymal+stem+cells+on+tumor+cells+in+vitro+and+in+vivo.">The growth inhibitory effect of mesenchymal stem cells on tumor cells in vitro and in vivo.</a> Cancer biology &amp; therapy. 7 (2), 245-251 (2008).</li><li>Secchiero, 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=Human+bone+marrow+mesenchymal+stem+cells+display+anti-cancer+activity+in+SCID+mice+bearing+disseminated+non-Hodgkin's+lymphoma+xenografts.">Human bone marrow mesenchymal stem cells display anti-cancer activity in SCID mice bearing disseminated non-Hodgkin's lymphoma xenografts.</a> PloS one. 5 (6), e11140 (2010).</li><li>Hong, I. -. S., Lee, H. -. Y., Kang, K. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+and+cancer:+friends+or+enemies?.">Mesenchymal stem cells and cancer: friends or enemies?.</a> Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 768, 98-106 (2014).</li><li>Brennen, W. N., Chen, S., Denmeade, S. R., Isaacs, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Mesenchymal+Stem+Cells+(MSCs)+at+sites+of+human+prostate+cancer.">Quantification of Mesenchymal Stem Cells (MSCs) at sites of human prostate cancer.</a> Oncotarget. 4 (1), 106 (2013).</li><li>Klopp, A. H., Gupta, A., Spaeth, E., Andreeff, M., Marini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+dissecting+a+discrepancy+in+the+literature:+do+mesenchymal+stem+cells+support+or+suppress+tumor+growth.">Concise review: dissecting a discrepancy in the literature: do mesenchymal stem cells support or suppress tumor growth.</a> Stem cells. 29 (1), 11-19 (2011).</li><li>Albini, A., Sporn, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumour+microenvironment+as+a+target+for+chemoprevention.">The tumour microenvironment as a target for chemoprevention.</a> Nature Reviews Cancer. 7 (2), 139 (2007).</li><li>Quante, 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=Bone+marrow-derived+myofibroblasts+contribute+to+the+mesenchymal+stem+cell+niche+and+promote+tumor+growth.">Bone marrow-derived myofibroblasts contribute to the mesenchymal stem cell niche and promote tumor growth.</a> Cancer cell. 19 (2), 257-272 (2011).</li><li>Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., Shi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+human+dental+pulp+stem+cells+(DPSCs)+in+vitro+and+in+vivo.">Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo.</a> Proceedings of the National Academy of Sciences. 97 (25), 13625-13630 (2000).</li><li>Mori, G., 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=Dental+pulp+stem+cells:+osteogenic+differentiation+and+gene+expression.">Dental pulp stem cells: osteogenic differentiation and gene expression.</a> Annals of the new York Academy of Sciences. 1237 (1), 47-52 (2011).</li><li>Mori, G., 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=Osteogenic+properties+of+human+dental+pulp+stem+cells.">Osteogenic properties of human dental pulp stem cells.</a> Journal of biological regulators and homeostatic agents. 24 (2), 167-175 (2010).</li><li>Kerkis, 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=Isolation+and+characterization+of+a+population+of+immature+dental+pulp+stem+cells+expressing+OCT-4+and+other+embryonic+stem+cell+markers.">Isolation and characterization of a population of immature dental pulp stem cells expressing OCT-4 and other embryonic stem cell markers.</a> Cells Tissues Organs. 184 (3-4), 105-116 (2006).</li><li>Potdar, P. D., Jethmalani, Y. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dental+pulp+stem+cells:+applications+in+future+regenerative+medicine.">Human dental pulp stem cells: applications in future regenerative medicine.</a> World journal of stem cells. 7 (5), 839 (2015).</li><li>Ahmed, N. E. -. M. B., Murakami, M., Hirose, Y., Nakashima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+dental+pulp+stem+cell+secretome+for+Alzheimer's+disease+treatment:+an+in+vitro+study.">Therapeutic potential of dental pulp stem cell secretome for Alzheimer's disease treatment: an in vitro study.</a> Stem cells international. 2016, (2016).</li><li>Gorin, C., 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=Priming+dental+pulp+stem+cells+with+fibroblast+growth+factor-2+increases+angiogenesis+of+implanted+tissue-engineered+constructs+through+hepatocyte+growth+factor+and+vascular+endothelial+growth+factor+secretion.">Priming dental pulp stem cells with fibroblast growth factor-2 increases angiogenesis of implanted tissue-engineered constructs through hepatocyte growth factor and vascular endothelial growth factor secretion.</a> Stem cells translational medicine. 5 (3), 392-404 (2016).</li><li>Wakayama, 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=Factors+secreted+from+dental+pulp+stem+cells+show+multifaceted+benefits+for+treating+acute+lung+injury+in+mice.">Factors secreted from dental pulp stem cells show multifaceted benefits for treating acute lung injury in mice.</a> Cytotherapy. 17 (8), 1119-1129 (2015).</li><li>Do&#287;an, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+stem+cells+is+promoted+by+amphiphilic+pluronic+block+copolymers.">Differentiation of human stem cells is promoted by amphiphilic pluronic block copolymers.</a> International Journal of Nanomedicine. 7, 4849 (2012).</li><li>Ta&#351;l&#305;, P. N., Do&#287;an, A., Demirci, S., &#350;ahin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boron+enhances+odontogenic+and+osteogenic+differentiation+of+human+tooth+germ+stem+cells+(hTGSCs)+in+vitro.">Boron enhances odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs) in vitro.</a> Biological trace element research. 153 (1-3), 419-427 (2013).</li><li>Yalvac, 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=Isolation+and+characterization+of+stem+cells+derived+from+human+third+molar+tooth+germs+of+young+adults:+implications+in+neo-vascularization,+osteo-,+adipo-and+neurogenesis.">Isolation and characterization of stem cells derived from human third molar tooth germs of young adults: implications in neo-vascularization, osteo-, adipo-and neurogenesis.</a> The pharmacogenomics journal. 10 (2), 105 (2010).</li><li>Do&#287;an, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+pentaborate+pentahydrate+and+pluronic+containing+hydrogel+increases+cutaneous+wound+healing+in+vitro+and+in+vivo.">Sodium pentaborate pentahydrate and pluronic containing hydrogel increases cutaneous wound healing in vitro and in vivo.</a> Biological trace element research. 162 (1-3), 72-79 (2014).</li><li>Do&#287;an, A., Yalva&#231;, M. E., Y&#305;lmaz, A., Rizvanov, A., &#350;ahin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+F68+on+cryopreservation+of+mesenchymal+stem+cells+derived+from+human+tooth+germ.">Effect of F68 on cryopreservation of mesenchymal stem cells derived from human tooth germ.</a> Applied biochemistry and biotechnology. 171 (7), 1819-1831 (2013).</li><li>Rizvanov, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+and+self-organization+of+human+mesenchymal+stem+cells+and+neuro-blastoma+SH-SY5Y+cells+under+co-culture+conditions:+A+novel+system+for+modeling+cancer+cell+micro-environment.">Interaction and self-organization of human mesenchymal stem cells and neuro-blastoma SH-SY5Y cells under co-culture conditions: A novel system for modeling cancer cell micro-environment.</a> European Journal of Pharmaceutics and Biopharmaceutics. 76 (2), 253-259 (2010).</li><li>Troiano, L., 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=Multiparametric+analysis+of+cells+with+different+mitochondrial+membrane+potential+during+apoptosis+by+polychromatic+flow+cytometry.">Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry.</a> Nature protocols. 2 (11), 2719 (2007).</li><li>Melzer, C., von der Ohe, J., Lehnert, H., Ungefroren, H., Hass, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cell+niche+models+and+contribution+by+mesenchymal+stroma/stem+cells.">Cancer stem cell niche models and contribution by mesenchymal stroma/stem cells.</a> Molecular cancer. 16 (1), 28 (2017).</li><li>Aguirre, A., Planell, J., Engel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+bone+marrow-derived+endothelial+progenitor+cell/mesenchymal+stem+cell+interaction+in+co-culture+and+its+implications+in+angiogenesis.">Dynamics of bone marrow-derived endothelial progenitor cell/mesenchymal stem cell interaction in co-culture and its implications in angiogenesis.</a> Biochemical and biophysical research communications. 400 (2), 284-291 (2010).</li><li>Bogdanowicz, D. R., Lu, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomimetics and Stem Cells. , 29-36 (2013).</li><li>Plotnikov, 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=Cell-to-cell+cross-talk+between+mesenchymal+stem+cells+and+cardiomyocytes+in+co-culture.">Cell-to-cell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture.</a> Journal of cellular and molecular. 12 (5a), 1622-1631 (2008).</li><li>Bogdanowicz, D. R., Lu, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+cell-cell+communication+in+co-culture.">Studying cell-cell communication in co-culture.</a> Biotechnology journal. 8 (4), 395-396 (2013).</li><li>Brunetti, G., 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=High+expression+of+TRAIL+by+osteoblastic+differentiated+dental+pulp+stem+cells+affects+myeloma+cell+viability.">High expression of TRAIL by osteoblastic differentiated dental pulp stem cells affects myeloma cell viability.</a> Oncology reports. 39 (4), 2031-2039 (2018).</li><li>Do&#287;an, A., Demirci, S., Apdik, H., Apdik, E. A., &#350;ahin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+stem+cells+(DPSCs)+increase+prostate+cancer+cell+proliferation+and+migration+under+in+vitro+conditions.">Dental pulp stem cells (DPSCs) increase prostate cancer cell proliferation and migration under in vitro conditions.</a> Tissue and Cell. 49 (6), 711-718 (2017).</li></ol>

The authors have nothing to disclose.

Contribution of MSCs to tumor environment is regulated by several interactions including hybrid cell generation via cell fusions, entosis or cytokine and chemokine activities between stem cells and cancer cells28. Structural organization, cell-cell interactions, and secreted factors determine cancer cell behavior in terms of tumor promotion, progression, and metastasis to surrounding tissue. Proper ex vivo model systems to investigate the mechanisms behind the interactions of res...

Mesenchymal stem cells (MSCs), with&#160;the ability of differentiation and contribution to regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and adipose, have been isolated from almost all tissues in the adult body1,2. Other than providing tissue homeostasis by producing resident cells in case of chronic inflammation or an injury, they produce vital cytokines and growth factors to orchestrate angiogenesis, immune system, and tissue remodeling3. The interaction of MSCs with cancer tissue is not well-understood, but accumulating evidence suggests that MSCs might promote tumor initiation, progression, and metastasis4.
The homing ability of MSCs to the injured or chronically inflamed area makes them a valuable candidate for stem cell-based therapies. However, cancer tissues, &#34;never healing wounds&#34;, also release inflammatory cytokines, pro-angiogenic molecules, and vital growth factors, which attract MSCs to the cancerogenous area5. While there are limited reports showing inhibitory effects of MSCs on cancer growth6,7, their cancer progression and metastasis promoting effects have been extensively reported8. MSCs directly or indirectly affect carcinogenesis in different ways including suppressing immune cells, secreting growth factors/cytokines that support cancer cell proliferation and migration, enhancing angiogenic activity, and regulating epithelial-mesenchymal transition (EMT)9,10. Tumor environment consists of several cell types including cancer-associated fibroblasts (CAFs) and/or myofibroblasts, endothelial cells, adipocytes, and immune cells11. Of those, CAFs are the most abundant cell type in the tumor area that secrete various chemokines promoting cancer growth and metastasis8. It has been shown that bone marrow-derived MSCs can differentiate into CAFs in the tumor stroma12.
Dental pulp stem cells (DPSCs), characterized as the first dental tissue-derived MSCs by Gronthos et al.13 in 2000 and then widely investigated by others14,15, express pluripotency markers such as Oct4, Sox2, and Nanog16 and can differentiate into various cell linages17. Gene and protein expression analysis proved that DPSCs produce comparable levels of growth factors/cytokines with other MSCs such as vascular endothelial growth factor (VEGF), angiogenin, fibroblast growth factor 2 (FGF2), interleukin-4 (IL-4), IL-6, IL-10, and stem cell factor (SCF), as well as fms-like tyrosine kinase-3 ligand (Flt-3L) that might promote angiogenesis, modulate immune cells, and support cancer cell proliferation and migration18,19,20. While the interactions of MSCs with cancer environment have been well-documented in the literature, the relationship between DPSCs and cancer cells has not been evaluated yet. In the present study, we established co-culture and condition medium treatment strategies for a highly metastatic prostate cancer cell line, PC-3, and DPSCs to propose potential action of mechanism of dental MSCs in cancer progression and metastasis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11885084</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Invitrogen</td>
			<td>16000044</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>PSA</td>
			<td>Lonza</td>
			<td>17-745E</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Invitrogen</td>
			<td>25200056</td>
			<td>For cell dissociation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>10010023</td>
			<td>For washes</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>Component of differentiation media</td>
		</tr>
		<tr>
			<td>&#946;-Glycerophosphate</td>
			<td>Sigma</td>
			<td>G9422</td>
			<td>Component of osteogenic differentiation medium</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544</td>
			<td>Component of osteo- and chondro-genic differentiation medium</td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS &#8722;G)</td>
			<td>Invitrogen</td>
			<td>41400045</td>
			<td>Component of chondrogenic differentiation medium</td>
		</tr>
		<tr>
			<td>TGF-&#946;</td>
			<td>Sigma</td>
			<td>SRP3171</td>
			<td>Component of chondrogenic differentiation medium</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I6634</td>
			<td>Component of adipogenic differentiation medium</td>
		</tr>
		<tr>
			<td>Isobutyl-1-methylxanthine (IBMX)</td>
			<td>Sigma</td>
			<td>I7018</td>
			<td>Component of adipogenic differentiation medium</td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma</td>
			<td>I7378</td>
			<td>Component of adipogenic differentiation medium</td>
		</tr>
		<tr>
			<td>MTS Reagent</td>
			<td>Promega</td>
			<td>G3582</td>
			<td>Cell viability analyses</td>
		</tr>
		<tr>
			<td>TUNEL Assay</td>
			<td>Sigma</td>
			<td>11684795910</td>
			<td>Apoptotic analyses</td>
		</tr>
		<tr>
			<td>24-well plate inserts</td>
			<td>Corning</td>
			<td>3396</td>
			<td>For trans-well migration assay</td>
		</tr>
		<tr>
			<td>PKH67</td>
			<td>Sigma</td>
			<td>PKH67GL</td>
			<td>For co-culture cell staining</td>
		</tr>
		<tr>
			<td>PKH26</td>
			<td>Sigma</td>
			<td>PKH26GL</td>
			<td>For co-culture cell staining</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>For cell fixation</td>
		</tr>
		<tr>
			<td>von Kossa Kit</td>
			<td>BioOptica</td>
			<td>04-170801.A</td>
			<td>For cell staining (differentiation)</td>
		</tr>
		<tr>
			<td>Alcian blue</td>
			<td>Sigma</td>
			<td>A2899</td>
			<td>For cell staining (differentiation)</td>
		</tr>
	</tbody>
</table>

mesenchymal stem cell isolation from pulp tissue and co-culture with cancer cells to study their interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

Figure 1 depicts the general MSC characteristics of DPSCs under culture conditions. DPSCs exert fibroblast-like cell morphology after plating (Figure 1B). MSC surface antigens (CD29, CD73, CD90, CD105, and CD166) are highly expressed while hematopoietic markers (CD34, CD45, and CD14) are negative (Figure 1C). Changes at the morphological and molecular level related to osteo-, chondro-, and adipo-geni...

Watch this Scientific Journal Video about Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions at JoVE.com

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

mesenchymal-stem-cell-isolation-from-pulp-tissue-co-culture-with

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JoVE Journal

Cancer Research

10.2K Views.  Yeditepe University. We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Video: Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

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 ...

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide clues to overcoming radioresistance. Voltage-gated calcium channel &#945;2&#948;1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in non-small cell lung cancer (NSCLC) cell lines. Using calcium channel &#945;2&#948;1 subunit as an example of a CSC marker, methods to study the radiosensitivity of CSCs in NSCLC cell lines are presented. CSCs are sorted with putative markers by flow cytometry, and the self-renewal capacity of sorted cells is evaluated by sphere formation assay. Colony formation assay, which determines how many cells lose the ability to generate descendants forming the colony after a certain dose of radiation, is then performed to assess the radiosensitivity of sorted cells. This manuscript provides initial steps for studying the radiosensitivity of CSCs, which establishes the basis for further understanding of the underlying mechanisms.

The presence of cancer stem cells have been associated with relapse or poor outcomes after radiotherapy. This manuscript describes the methods to study the radiosensitivity of cancer stem cells in lung cancer cell lines.

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma micro-environment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the&#160;in vivo&#160;situation compared to traditional 2D cultures.&#160;Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.&#160;

This protocol presents a 3D biomimetic model with accompanying fibrotic stromal compartment. Prepared with physiologically relevant hydrogels in ratios mimicking the bio-physical properties of the stromal extracellular matrix, an active mediator of cellular interactions, tumor growth and metastasis.

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

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Chapters in this video

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Studying TGF-β Signaling and TGF-β-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions