This work was supported by an R01 grant (CA200572) from the NIH to PS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All animal work was approved by the Mayo Clinic IACUC.
1. Preparation of Materials, Solutions, and 3-dimensional Matrix Bases
<ol>
	<li>Cut 500 &#181;m and 105 &#181;m polypropylene meshes into 76 mm by 76 mm squares. Fold each square in half twice to create a smaller folded square. Place one 500 &#181;m and one 105 &#181;m mesh square into an autoclavable pouch.</li>
	<li>Autoclave polypropylene mesh squares, as well as two pairs of scissors and forceps.</li>
	<li>Make 100 mL of 10x Waymouth&#39;s solution. Stir 1 vial (14 g) of Waymouth&#39;s powder into 50 mL of deionized (DI) water and, when dissolved, add 30 mL of 7.5% (75 g/L) sodium bicarbonate. Bring the final volume to 100 mL using DI water and filter sterilize.
	<ol>
		<li>Store 10x Waymouth&#39;s media at 4 &#176;C and use to prepare collagen gels and 1x Waymouth&#39;s media. When precipitates form, discard.</li>
	</ol>
	</li>
	<li>Make 50 mL of 1x Waymouth&#39;s complete media per pancreas by adding 125 &#181;L of 40 mg/mL soybean trypsin inhibitor, 12.5 &#181;L of 4 mg/mL dexamethasone, and 500 &#181;L of fetal bovine serum (FBS). Sterilize by filtration and use within 48 h.</li>
	<li>Prepare 3-dimensional matrix bases using either collagen or basement membrane matrix (see Table of Materials for product information). When pipetting the base, place the plate on ice, avoid bubbles, and rotate the plate immediately after pipetting each well&#39;s volume to ensure full coverage.
	<ol>
		<li>Create collagen bases by mixing the following components on ice in a tissue culture hood: 5.5 mL of type I rat tail collagen, 550 &#181;L of 10x Waymouth&#39;s Media, and 366.6 &#181;L of 0.34 M NaOH (filtered). 
		NOTE: This is enough solution to make collagen bases for one 24-well, 12-well, or 6-well plate. One plate is sufficient for acinar isolation from one pancreas.</li>
		<li>Use the following volumes to plate the collagen base: 80 &#181;L (8-well glass slide), 200 &#181;L (24-well plate), 400 &#181;L (12-well plate), or 800 &#181;L (6-well plate).</li>
		<li>For the basement membrane matrix bases, pipet the matrix without any additional components. Use the following volumes for each well: 50 &#181;L (8-well glass slide), 120 &#181;L (24-well plate), 240 &#181;L (12-well plate), or 600 &#181;L (6-well plate).</li>
		<li>Let the bases solidify for at least 30 min in a cell culture incubator (37 &#176;C, 5% CO2) before creating a second layer of matrix with embedded cells on top of these bases (step 4).</li>
	</ol>
	</li>
	<li>In preparation for acinar isolation keep one 600 mL beaker, three 50 mL tubes, an autoclaved pair of scissors, an autoclaved pair of forceps, and an ice bucket under the hood with three weigh boats. Then, make 30 mL of HBSS with 300 &#181;L of 100x penicillin-streptomycin, putting 10 mL into each of two weigh boats and keeping the final 10 mL in a 50 mL tube (for use in steps 1.8.2 and 2.1.4).</li>
	<li>Make 40 mL of HBSS + 5% FBS, 20 mL of HBSS + 30% FBS, and 5 mL of 2 mg/mL collagenase in HBSS. Sterilize the collagenase by filtration (0.22 &#181;m pore) and keep at room temperature. Keep each of the HBSS solutions on ice.</li>
	<li>Assemble a workspace for pancreas dissection, which can be done on a lab bench.
	<ol>
		<li>Place an absorbent pad on the table, along with a polystyrene lid covered in foil. Then place a paper towel with 4 pins on top of the foil.</li>
		<li>Keep the following items within reach of the dissection workspace: an incineration bag, one set of autoclaved scissors and forceps, a spray bottle with 70% ethanol, and an ice bucket (with the 50 mL tube of HBSS containing penicillin-streptomycin from step 1.6).</li>
	</ol>
	</li>
	<li>Set a centrifuge to 4 &#176;C and a shaker to 37 &#176;C.</li>
</ol>
2. Acinar Cell Isolation
<ol>
	<li>Sacrifice the mouse via CO2 induction, and perform cervical dislocation. Immediately dissect the pancreas. To dissect the pancreas, first pin the paws of the mouse to the polystyrene lid, orient the mouse such that the tail is facing the researcher, and spray the abdomen with 70% ethanol. 
	NOTE: The mouse used in the representative results was a 12-week old non-transgenic female with C57BL/6 background. Mouse selection is further discussed in the discussion section.
	<ol>
		<li>Using a set of autoclaved scissors and forceps, lift the fur/skin with the forceps at the midline and use the scissors to make an incision through the fur and skin from the urethral opening to the diaphragm/ribcage area.</li>
		<li>Make additional incisions to the left and right such that the fur/skin is cut away to create a clear view of the abdominal cavity. Then, cut into the peritoneal lining (down the middle and to the right and left) and pull it away from the organs, as was done with the fur/skin.</li>
		<li>Lift the intestines with the forceps and put it to the left side of the mouse creating space to see the pancreas, which is light pink in color, and attached to the spleen, which is a dark red oval. The pancreatic tissue is distinguished by its soft, spongy texture. Cut out the pancreas which will run along the stomach and intertwine with the intestines.</li>
		<li>Separate the spleen from the pancreas. Then, put the pancreas in a 50 mL tube containing 10 mL of HBSS with 1x penicillin-streptomycin and bring this to the laminar flow hood. 
		NOTE: The remaining steps of the protocol should be done utilizing sterile technique in a laminar flow hood.</li>
	</ol>
	</li>
	<li>Pour the pancreas and HBSS (with penicillin-streptomycin) into an empty weigh boat and, using forceps, wash the pancreas by swirling it. Then, move the pancreas with the forceps to a second weigh boat containing HBSS (with penicillin-streptomycin). Again, wash the pancreas by swirling.</li>
	<li>Move the pancreas to the third HBSS (with penicillin-streptomycin)-containing weigh boat and begin cutting the pancreas into small pieces of 5 mm or less. Next, pour the pancreas pieces and HBSS into an empty 50 mL tube.
	<ol>
		<li>To do this, use the forceps to move the pancreas pieces into the liquid as the weigh boat is being tipped. Pour once all the pieces are no longer attached to the weigh boat. Pick up any remaining pieces with the forceps and wash the forceps in the 50 mL tube containing the pancreas in HBSS with penicillin-streptomycin.</li>
	</ol>
	</li>
	<li>Centrifuge at 931 x g for 2 min at 4 &#176;C and then remove the HBSS (and any fat that is floating) by pipetting it off with a 5 mL pipette.</li>
	<li>Add 5 mL of collagenase (diluted in HBSS in step 1.7) to the pancreas. Ensure a sealed lid by wrapping the 50 mL tube in plastic paraffin film and then place it in an incubator shaking at 220 rpm for 20 min at 37 &#176;C. 
	NOTE: The collagenase digestion time may vary, as noted in the discussion. At the end of incubation, no large pieces of tissue should remain.</li>
	<li>To stop the dissociation, place the pancreas-containing tube on ice and add 5 mL of cold HBSS + 5% FBS. Centrifuge at 931 x g for 2 min at 4 &#176;C and then pipet off the supernatant using a 5 mL pipet.</li>
	<li>Resuspend in 10 mL of HBSS + 5% FBS and centrifuge at 931 x g for 2 min at 4 &#176;C. Pipet off the supernatant using a 5 mL pipet and repeat this step with another 10 mL of HBSS + 5% FBS.</li>
	<li>Resuspend in 5 mL of HBSS + 5% FBS and, using a P1000, transfer 1 mL at a time through a 500 &#181;m mesh into a 50 mL tube. Add an additional 5 mL of HBSS + 5% FBS through the mesh with a P1000 to wash any remaining pancreatic cells through the mesh.</li>
	<li>Put the cell suspension from the 500 &#181;m mesh through the 105 &#181;m mesh by pipetting 1 mL at a time using a P1000. Next, gently pipet the cell suspension into a tube containing HBSS + 30% FBS. A layer of cell suspension will form at the top; upon centrifugation, the acinar cells will sink to form a pellet.</li>
	<li>Centrifuge at 233 x g for 2 min at 4 &#176;C. Remove the supernatant and resuspend the pellet in 1x Waymouth&#39;s complete media.
	<ol>
		<li>If proceeding directly to embedment of cells in collagen or basement membrane matrix, resuspend in a volume of 7 mL per pancreas. If proceeding to adenoviral or lentiviral infection, resuspend in 4 mL per pancreas.</li>
	</ol>
	</li>
</ol>
3. Viral Infection
<ol>
	<li>As one pancreas is sufficient for two viral infections (control and experimental, e.g., null and cre), split the cell suspension between the lids of two 35 mm plates. Using the lid of the plates allows for infection in suspension and therefore easy movement onto the final substrate later.
	<ol>
		<li>When working with virus, soak all used pipette tips in 10% bleach for at least 15 minutes. 
		NOTE: Utilization of the 35 mm plates and the 4 mL volume works well for cells from non-transgenic mice between 8 and 16 weeks old. The plate size and volume may need to be adjusted for animals with smaller or larger pancreata.</li>
	</ol>
	</li>
	<li>For adenoviral infection, add the virus (with a titer of at least 107 TU/mL) at a 1:1,000 dilution to the lid of each plate and swirl (Figure 2, GFP adenovirus). Place the plates in a cell culture incubator (37 &#176;C, 5% CO2) and swirl every 15 minutes for 1 h. Continue incubation for an additional 2 h and then proceed to embedment of cells in collagen or basement membrane matrix.</li>
	<li>After completing viral work in the hood, soak all components in the hood with 10% bleach for at least 15 minutes and then thoroughly clean the bleach off using 70% ethanol.</li>
</ol>
4. Embedment of cells in Collagen or Basement Membrane Matrix
<ol>
	<li>Make collagen gel on ice by combining 7 mL type I rat tail collagen (3.3 mg/mL), 700 &#181;L of 10x Waymouth&#39;s media, and 466.6 &#181;L of 0.34 M NaOH.</li>
	<li>Taking equal volumes of collagen gel and cell suspension, gently mix. If adding a stimulus or inhibitor, combine this with the collagen-cell suspension. Plate one well at a time (plates from step 1.5.3); keeping the plate on ice, swirl to evenly distribute the collagen-cell layer.
	<ol>
		<li>In each well, pipet the following volumes of collagen-cell suspension: 200 &#181;L (8-well glass slide), 500 &#181;L (24-well plate), 1000 &#181;L (12-well plate), or 2000 &#181;L (6-well plate). Note that ADM is induced via stimulation with TGF-&#945; (50 ng/mL) in&#160;Figure 2.</li>
	</ol>
	</li>
	<li>For embedment of cells in basement membrane matrix, mix one-part basement membrane matrix with two-parts cell suspension. As with collagen, keep the matrix-cell suspension as well as the plate on ice. Pipet one well at a time using the same volumes noted in 4.2.1 and swirl for even distribution.</li>
	<li>Place the plate in a cell culture incubator (37 &#176;C, 5% CO2) for 30 min to solidify and then add 1x Waymouth&#39;s complete media with any stimulus or inhibitor (Figure 2 uses TGF-&#945; at 50 ng/mL). Change the media (with stimulus or inhibitor) the next day and then every other day. Depending on the stimulus, ADM can be observed between day 3 and day 5.</li>
</ol>
5. Harvesting Cells from Collagen or Basement Membrane Matrix
<ol>
	<li>Collect the following materials needed for harvesting cells from collagen: 30 mL of 1 mg/mL collagenase diluted in HBSS, 40 mL of cold HBSS, 31 mL of cold PBS, two spatulas, two 50 mL tubes, and ice. Adjust the number of spatulas, tubes and amount of solutions if comparing more than two culture conditions (where each condition is &#189; of a plate). Set the centrifuge to 4 &#176;C and set the shaking incubator to 37 &#176;C.</li>
	<li>Remove the media and use spatulas to remove the collagen disks with embedded cells. For each condition, put the disks into a separate, empty 50 mL tube.</li>
	<li>Add 10 mL of 1 mg/mL collagenase in HBSS to each 50 mL tube. Wrap the tubes with plastic paraffin film and put in a 37 &#176;C shaking incubator at 225 rpm for up to 45 minutes. Monitor the digestion and remove tubes when there is no more visible collagen.</li>
	<li>Centrifuge at 233 x g at 4 &#176;C for 2 min with minimum deceleration. Take off the supernatant and resuspend in 5 mL collagenase solution per tube. Digest in a 37 &#176;C shaking incubator at 225 rpm for 10 min or until there is no visible collagen.</li>
	<li>Stop the digestion by adding 5 mL of cold HBSS and subsequently centrifuging at 233 x g for 2 min at 4 &#176;C with minimum deceleration.
	<ol>
		<li>Resuspend in 15 mL of cold HBSS and again centrifuge at 233 x g for 2 min at 4 &#176;C with minimum deceleration. Repeat.</li>
	</ol>
	</li>
	<li>Resuspend the pellet in 1 mL of PBS and move to 1.5 mL tube. Centrifuge in a swing bucket at 233 x g for 2 min at 4 &#176;C with minimum deceleration. Remove supernatant and snap-freeze in liquid nitrogen or on dry ice. 
	NOTE: The cell pellet can be stored at -70 &#176;C or used immediately in down-stream applications.</li>
	<li>To harvest basement membrane matrix-embedded cells, pipet media from each well into a 50 mL tube on ice. Then, add basement membrane matrix recovery solution to each well and scrape the cell-gel mixture into the 50 mL tube. 
	NOTE: The volume of basement membrane matrix recovery solution to add to each well is as follows: 150 &#181;L (8-well glass slide), 420 &#181;L (24-well plate), 845 &#181;L (12-well plate), and 2,000 &#181;L (6-well plate).</li>
	<li>Rinse each well twice with the basement membrane matrix recovery solution, adding the rinses to the 50 mL tube. 
	NOTE: The volume of basement membrane matrix recovery solution for each rinse is as follows: 150 &#181;L (8-well glass slide), 420 &#181;L (24-well plate), 845 &#181;L (12-well plate), and 2,000 &#181;L (6-well plate).</li>
	<li>Leave the tube on ice for 1 h or until the basement membrane matrix dissolves and follow with centrifugation at 300 x g for 5 min at 4 &#176;C. Remove the supernatant and resuspend the pellet in 1 mL of cold PBS (may transfer to 1.5 mL tube if cell pellet is desired).</li>
	<li>Centrifuge at 3,500 x g for 3 min at 4 &#176;C. Remove the supernatant and either snap freeze the cell pellet or add lysis buffer and directly proceed to downstream applications.</li>
</ol>

<ol>
	<li>Rooman, I., Real, F. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+ductal+adenocarcinoma+and+acinar+cells:+a+matter+of+differentiation+and+development.">Pancreatic ductal adenocarcinoma and acinar cells: a matter of differentiation and development.</a> Gut. 61 (3), 449-458 (2012).</li><li>Stanger, B. Z., Hebrok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+identity+in+pancreas+development+and+regeneration.">Control of cell identity in pancreas development and regeneration.</a> Gastroenterology. 144 (6), 1170-1179 (2013).</li><li>Caldas, C., Kern, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K-ras+mutation+and+pancreatic+adenocarcinoma.">K-ras mutation and pancreatic adenocarcinoma.</a> International Journal of Pancreatology. 18 (1), 1-6 (1995).</li><li>Kopp, J. 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=Identification+of+Sox9-dependent+acinar-to-ductal+reprogramming+as+the+principal+mechanism+for+initiation+of+pancreatic+ductal+adenocarcinoma.">Identification of Sox9-dependent acinar-to-ductal reprogramming as the principal mechanism for initiation of pancreatic ductal adenocarcinoma.</a> Cancer Cell. 22 (6), 737-750 (2012).</li><li>Morris, J. P. t., Cano, D. A., Sekine, S., Wang, S. C., Hebrok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-catenin+blocks+Kras-dependent+reprogramming+of+acini+into+pancreatic+cancer+precursor+lesions+in+mice.">Beta-catenin blocks Kras-dependent reprogramming of acini into pancreatic cancer precursor lesions in mice.</a> Journal of Clinical Investigation. 120 (2), 508-520 (2010).</li><li>Grippo, P. J., Nowlin, P. S., Demeure, M. J., Longnecker, D. S., Sandgren, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preinvasive+pancreatic+neoplasia+of+ductal+phenotype+induced+by+acinar+cell+targeting+of+mutant+Kras+in+transgenic+mice.">Preinvasive pancreatic neoplasia of ductal phenotype induced by acinar cell targeting of mutant Kras in transgenic mice.</a> Cancer Research. 63 (9), 2016-2019 (2003).</li><li>Sawey, E. T., Johnson, J. A., Crawford, H. 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+7+controls+pancreatic+acinar+cell+transdifferentiation+by+activating+the+Notch+signaling+pathway.">Matrix metalloproteinase 7 controls pancreatic acinar cell transdifferentiation by activating the Notch signaling pathway.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (49), 19327-19332 (2007).</li><li>Liou, G. 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=Macrophage-secreted+cytokines+drive+pancreatic+acinar-to-ductal+metaplasia+through+NF-kappaB+and+MMPs.">Macrophage-secreted cytokines drive pancreatic acinar-to-ductal metaplasia through NF-kappaB and MMPs.</a> Journal of Cell Biology. 202 (3), 563-577 (2013).</li><li>De La, O. 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=Notch+and+Kras+reprogram+pancreatic+acinar+cells+to+ductal+intraepithelial+neoplasia.">Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (48), 18907-18912 (2008).</li><li>Habbe, N., 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=Spontaneous+induction+of+murine+pancreatic+intraepithelial+neoplasia+(mPanIN)+by+acinar+cell+targeting+of+oncogenic+Kras+in+adult+mice.">Spontaneous induction of murine pancreatic intraepithelial neoplasia (mPanIN) by acinar cell targeting of oncogenic Kras in adult mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (48), 18913-18918 (2008).</li><li>Liou, G. 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=Mutant+KRas-Induced+Mitochondrial+Oxidative+Stress+in+Acinar+Cells+Upregulates+EGFR+Signaling+to+Drive+Formation+of+Pancreatic+Precancerous+Lesions.">Mutant KRas-Induced Mitochondrial Oxidative Stress in Acinar Cells Upregulates EGFR Signaling to Drive Formation of Pancreatic Precancerous Lesions.</a> Cell Report. 14 (10), 2325-2336 (2016).</li><li>Hughes, P., Marshall, D., Reid, Y., Parkes, H., Gelber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+costs+of+using+unauthenticated,+over-passaged+cell+lines:+how+much+more+data+do+we+need.">The costs of using unauthenticated, over-passaged cell lines: how much more data do we need.</a> Biotechniques. 43 (5), 577-578 (2007).</li><li>Blauer, M., Nordback, I., Sand, J., Laukkarinen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+explant+outgrowth+culture+model+for+mouse+pancreatic+acinar+cells+with+long-term+maintenance+of+secretory+phenotype.">A novel explant outgrowth culture model for mouse pancreatic acinar cells with long-term maintenance of secretory phenotype.</a> European Journal of Cell Biology. 90 (12), 1052-1060 (2011).</li><li>Gout, 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=Isolation+and+culture+of+mouse+primary+pancreatic+acinar+cells.">Isolation and culture of mouse primary pancreatic acinar cells.</a> Journal of Visualized Experiments. (78), (2013).</li><li>Artym, V. V., Matsumoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+in+three-dimensional+collagen+matrix.">Imaging cells in three-dimensional collagen matrix.</a> Current Protocols in Cell Biology. , 11-20 (2010).</li><li>Geraldo, S., Simon, A., Vignjevic, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+cytoskeletal+organization+of+invasive+cancer+cells+in+3D.">Revealing the cytoskeletal organization of invasive cancer cells in 3D.</a> Journal of Visualized Experiments. (80), e50763 (2013).</li></ol>

The authors declare that they have no competing financial interests.

The relatively short amount of time for isolation, infection, and plating primary acinar cells is an advantage of this method. In contrast, culturing acinar cells from an explant outgrowth requires little hands-on time, but it takes seven days for the outgrowth of acinar cells13. An alternative protocol for acinar isolation14 notes a very short method for obtaining acinar cells; however, EGF is an essential component in keeping acinar cells alive when isolated using that pr...

Acinar-to-ductal metaplasia (ADM) is a protective mechanism during pancreatitis and a key process driving pancreatic cancer development1 that requires further mechanistic insight. While inflammation-induced ADM is reversible2, oncogenic KRAS mutations, which are present in 90% of pancreatic cancer cases3, prevent differentiation back to an acinar phenotype4,5,6. Culturing and differentiating primary acinar cells into ductal cells in 3D culture allows for study of the molecular mechanisms regulating the ADM process, which is difficult to study in vivo. Such ex vivo studies enable real-time visualization of ADM, its drivers and its regulators. Several drivers of the ADM process, as well as mechanistic insight on their downstream signaling pathways, have been identified or verified using the here described method. These include ADM induction by TGF-&#945; (transforming growth factor alpha)-mediated expression of MMP-7 (matrix metalloproteinase-7) and activation of Notch7, as well as RANTES (regulated on activation, normal T cell expressed and secreted; also known as chemokine ligand 5 or CCL5) and TNF&#945; (tumor necrosis factor alpha)-induced ADM through activation of NF-&#954;B (nuclear factor-&#954;B)8. An additional mediator of ADM is oncogenic KRas9,10, which causes a rise in oxidative stress that enhances the ADM process through increased expression of EGFR (epidermal growth factor receptor) and its ligands, EGF (epidermal growth factor) and TGF-&#945;11.
While use of pancreatic cancer cell lines is common for in vitro studies, primary cell cultures offer many advantages. For instance, primary acinar cells from non-transgenic mice or mice harboring initiating mutations, such as KrasG12D, are the most appropriate model for studying the early event of ADM because the cells have few and controlled mutations, which are known to be present early in pancreatic cancer development. This is in contrast to many pancreatic cancer cell lines which have multiple mutations and varied expression based on passage number12. Additionally, the signaling pathways identified by such means have been verified by animal studies. One caveat of using primary acinar cells is that they cannot be transfected as typical cancer cell lines can.
The method herein details the techniques of acinar cell isolation, 3D culture that mimics ADM by adding stimulants or modulating protein expression via adenoviral or lentiviral infection, as well as re-isolation of cells at the endpoint for further analyses.

<table border="0" cellpadding="0" cellspacing="0" style="width:2181px;" width="2179">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:357px;">37 &#176;C shaking incubator</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:171px;">SHKE4000-7</td>
			<td style="width:1487px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:357px;">5% CO2, 37 &#176;C Incubator</td>
			<td style="width:167px;">NUAIRE</td>
			<td style="width:171px;">NU-5500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">50 ml tubes</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Absorbent pad, 20&#34; x 24&#34;</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">1420662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Adenovirus, Ad-GFP</td>
			<td style="width:167px;">Vector Biolabs&#160;</td>
			<td style="width:171px;">1060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Aluminum foil</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">01-213-101</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">BD Precision Glide Needle 21G x 1/2</td>
			<td style="width:167px;">Fisher Scientific&#160;</td>
			<td style="width:171px;">305167</td>
			<td>Use as pins for dissection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Beaker, 600 mL</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">FB-101-600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Bleach, 8.25%</td>
			<td style="width:167px;">Clorox</td>
			<td style="width:171px;">31009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Cell Recovery Solution</td>
			<td style="width:167px;">Corning</td>
			<td style="width:171px;">354253</td>
			<td>Referred to as &#39;basement membrane matrix recovery solution&#39; in the manuscript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Centrifuge&#160;</td>
			<td style="width:167px;">Beckman Coulter</td>
			<td style="width:171px;">Allegra X-15R Centrifuge</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Collagenase Type I, Clostridium histolyticum</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:171px;">C0130-1G</td>
			<td>Create a 100 mg/ml solution by dissolving powder in sterile molecular biology grade water. When thawing one aliquot, dilute to 10 mg/ml, filter sterilize and place 1 ml aliquots at -20 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Dexamethasone</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:171px;">D1756</td>
			<td>Create a 4 mg/ml solution by dissolving powder in methanol, aliquoting and storing at -20 &#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Ethanol, 200 proof</td>
			<td style="width:167px;">Decon Laboratories&#160;</td>
			<td style="width:171px;">2701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Fetal Bovine Serum&#160;</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:171px;">F0926-100mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Forceps&#160;</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:171px;">11002-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Forceps&#160;</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:171px;">91127-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Glass slide, 8-well</td>
			<td style="width:167px;">Lab-Tek</td>
			<td style="width:171px;">177402</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">Hank&#39;s Balanced Salt Solution (HBSS), No calcium, No magnesium, No phenol red</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:171px;">SH3048801</td>
			<td>&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Ice bucket, rectangular</td>
			<td style="width:167px;">Fisher Scientific&#160;</td>
			<td style="width:171px;">07-210-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Instant Sealing Sterilization Pouch</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">01-812-51</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">LAB GUARD specimen bags (for mouse after dissection)&#160;</td>
			<td style="width:167px;">Minigrip</td>
			<td style="width:171px;">SBL2X69S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Lentiviral Packaging Mix, Virapower</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:171px;">44-2050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Matrigel</td>
			<td style="width:167px;">Corning</td>
			<td style="width:171px;">356234</td>
			<td>Referred to as &#39;basement membrane matrix&#39; in the manuscript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Parafilm</td>
			<td style="width:167px;">Bemis</td>
			<td style="width:171px;">PM992</td>
			<td>Referred to as &#39;plastic paraffin film&#39; in the manuscript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PBS</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:171px;">SH30028.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipet tips, 10 &#181;l</td>
			<td style="width:167px;">USA Scientific</td>
			<td style="width:171px;">1110-3700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipet tips, 1000 &#181;l&#160;</td>
			<td style="width:167px;">Olympus Plastics</td>
			<td style="width:171px;">24-165RL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipet tips, 200 &#181;l&#160;</td>
			<td style="width:167px;">USA Scientific</td>
			<td style="width:171px;">1111-1700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipet-Aid</td>
			<td style="width:167px;">Drummond</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PIPETMAN Classic P10, 1-10 &#956;l</td>
			<td style="width:167px;">Gilson</td>
			<td style="width:171px;">F144802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PIPETMAN Classic P1000, 200-1000 &#956;l</td>
			<td style="width:167px;">Gilson</td>
			<td style="width:171px;">F123602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PIPETMAN Classic P20, 2-20 &#956;l</td>
			<td style="width:167px;">Gilson</td>
			<td style="width:171px;">F123600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PIPETMAN Classic P200, 20-200 &#956;l</td>
			<td style="width:167px;">Gilson</td>
			<td style="width:171px;">F123601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipettes, 10 ml&#160;</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">357551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipettes, 25 ml</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">357525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipettes, 5 ml&#160;</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">357543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Plate, 12-well</td>
			<td style="width:167px;">Corning Costar</td>
			<td style="width:171px;">3513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Plate, 24-well plate</td>
			<td style="width:167px;">Corning Costar</td>
			<td style="width:171px;">3524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Plate, 35 mm</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Plate, 6-well</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Polybrene</td>
			<td style="width:167px;">EMD Millipore</td>
			<td style="width:171px;">TR-1003-G</td>
			<td>Create a 40 mg/ml solution by dissolving powder in sterile molecular biology grade water, aliquoting and storing at -20 &#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Polypropylene Mesh, 105 &#181;m</td>
			<td style="width:167px;">Spectrum Labs</td>
			<td style="width:171px;">146436</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Polypropylene Mesh, 500 &#181;m&#160;</td>
			<td style="width:167px;">Spectrum Labs</td>
			<td style="width:171px;">146418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Scissors&#160;</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:171px;">14568-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Scissors&#160;</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:171px;">91460-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Sodium Bicarbonate (Fine White Powder)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:171px;">BP328-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Sodium Hydroxide</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:171px;">S318-500</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:357px;">Soybean Trypsin Inhibitor 8</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:171px;">17075029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Spatula</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">21-401-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Steriflip 50 ml, 0.22 micron filters&#160;</td>
			<td style="width:167px;">Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">TGF-&#945;</td>
			<td style="width:167px;">R&#38;D Systems</td>
			<td style="width:171px;">239-A-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Type I Rat Tail Collagen</td>
			<td style="width:167px;">Corning</td>
			<td style="width:171px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Waymouth MB 752/1 Medium (powder)</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:171px;">W1625-10X1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Weigh boat, hexagonal, medium&#160;</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">02-202-101</td>
			<td></td>
		</tr>
	</tbody>
</table>

mimicking and manipulating pancreatic acinar-to-ductal metaplasia in 3-dimensional cell culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Completion of the protocol herein occurs within one day and upon stimulation, ADM is seen in 3-5 days. Figure 1 depicts the sequence of the method, whereby steps 1 through 4 are completed on the first day. This includes preparation, acinar isolation, viral infection and embedment in collagen or basement membrane matrix. While the basement membrane matrix induces ADM, acinar cells within collagen I require a stimulus, such as TGF- &#945;, to undergo differenti...

Watch this Scientific Journal Video about Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture at JoVE.com

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

mimicking-manipulating-pancreatic-acinar-to-ductal-metaplasia-3

Research

JoVE Journal

Cancer Research

8.5K Views.  Mayo Clinic Florida. Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

Video: Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

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 ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

This protocol describes the key steps to generate and characterize murine oral-esophageal 3D organoids that represent normal, preneoplastic, and squamous cell carcinoma lesions induced via chemical carcinogenesis.

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. 
It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. 
The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

Murine Pancreatic Imaging Using Implanted Stabilized Window

Stabilized Window for Intravital Imaging of the Murine Pancreas

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have high morbidity and mortality. Intravital imaging (IVI) is a powerful technique enabling the high-resolution imaging of tissues in both healthy and diseased states, allowing for real-time observation of cell dynamics. IVI of the murine pancreas presents significant challenges due to the deep visceral and compliant nature of the organ, which make it highly prone to damage and motion artifacts. 
Described here is the process of implantation of the Stabilized Window for Intravital imaging of the murine Pancreas (SWIP). The SWIP allows IVI of the murine pancreas in normal healthy states, during the transformation from the healthy pancreas to acute pancreatitis induced by cerulein, and in malignant states such as pancreatic tumors. In conjunction with genetically labeled cells or the administration of fluorescent dyes, the SWIP enables the measurement of single-cell and subcellular dynamics (including single-cell and collective migration) as well as serial imaging of the same region of interest over multiple days. 
The ability to capture tumor cell migration is of particular importance as the primary cause of cancer-related mortality in PDAC is the overwhelming metastatic burden. Understanding the physiological dynamics of metastasis in PDAC is a critical unmet need and crucial for improving patient prognosis. Overall, the SWIP provides improved imaging stability and expands the application of IVI in the healthy pancreas and malignant pancreas diseases.

Author Spotlight: Revolutionizing Pancreatic Disease Understanding Through Advanced Intravital Imaging 

We present a protocol for the surgical implantation of a stabilized indwelling optical window for subcellular-resolution imaging of the murine pancreas, allowing serial and longitudinal studies of the healthy and diseased pancreas.

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have ...