The work described was supported by the South Dakota Governors&#39; Office of Economic Development, the South Dakota Board of Regents Competitive Research Grant Program (SD-BOR-CRGP), and the Department of Pharmaceutical Sciences at South Dakota State University for their support.

1. 2D cell culture
<ol>
	<li>Grow PANC-1 cells in Dulbecco&#39;s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) under sterile conditions. Grow up to 70%-80% confluency before passages, and do not use beyond the 20th passage. Refer to the process described in step 4.2.1. 
	NOTE: As a reference point, 1 x 106&#160;cells in 20 mL of media need about 2-3 days to reach 70-80% confluency.</li>
	<li>Grow HPaSteC cells in stellate cell media supplemented with 2% FBS, 1% PenStrep, and 1% Stellate Cell Growth Supplement (SteCGS) using the kit supplied by the manufacturer under sterile conditions. Grow according to instructions from the manufacturer (with some modifications) as shown in step 4.2.2 in a Poly-L-Lysine coated flask.
	<ol>
		<li>Skip the FBS neutralization step during trypsinization from the manufacturer&#39;s protocol since trypsin neutralization solution (TNS) is used. Neutralize the cell suspension with an overall volume of 10 mL of TNS as described in step 4.2.2.2. 
		NOTE: As a reference point, 0.5 x 106&#160;cells and 1 x 106&#160;cells in 20 mL of media need 3 days and 2 days, respectively, to attain 90% confluency.</li>
		<li>Harvest cells at 90% confluency. Do not use cells beyond Passage 7.</li>
	</ol>
	</li>
	<li>Maintain cells at 37 &#176;C, with 5% CO2, in sterile incubators with relative humidity at 90%-95%. Perform all cell culture studies in a T-75 flask.</li>
</ol>
2. Poly(2 -hydroxyethyl methacrylate (poly-HEMA) solution coating for 96 well plate
<ol>
	<li>Prepare poly-HEMA solution at 5 mg/mL using 95% ethanol by allowing it to be mixed overnight using a heated stirrer at 37 &#176;C. (e.g., 500 mL of solution needs 2.5 g of poly-HEMA)
	<ol>
		<li>Mark the start volume location before adding the stirrer. Use 95% ethanol to make up for any loss in the solution the next day.</li>
	</ol>
	</li>
	<li>Filter the final solution in a sterile hood using a 0.22-&#181;m filter and store it in the fridge at 4 &#176;C. 
	NOTE: Poly-HEMA is used to increase the hydrophobicity of a surface, which serves as a deterrent for cells to attach. As this study requires the cells not to attach to the wells and grow as a monolayer, poly-HEMA is used as an additional deterrent to the natural low attachment feature of the plates, thereby making them ultra-low attachment plates.</li>
	<li>Add 50 &#181;L of the cold solution to each well of a 96-well round polystyrene bottom microwell plate inside a cell culture hood to coat the plate.</li>
	<li>Leave the wells with the lid on in a hot air oven at 37 &#176;C for 3 days to ensure the plates are completely dry.</li>
	<li>UV sterilize the plates for 30 min in the cell culture hood prior to cell seeding. Store in a tightly closed zip lock for long-term storage and sterilize before use.</li>
</ol>
3. 2D cell culture planning
<ol>
	<li>Time the experiment so that PANC-1 cells are available at 70%-80% confluency on the same day the HPaSteC cells reach 90%. 
	NOTE: Vials containing about 1 million cells of either PANC-1 or HPaSteC, when raised from liquid nitrogen on the same day, with some variation, require 8 days of PANC-1 culture and 6-7 days of HPaSteC culture to have both cell lines ready on the same day.</li>
</ol>
4. 3D culture growth
<ol>
	<li>Refer to the overview of the entire process in Figure 1 before starting the process. 
	CAUTION: Both PANC-1 and HPaSteC are of biological origin. Handle PANC-1 (cancerous cell line) with care.</li>
	<li>Trypsinzing and counting cells
	<ol>
		<li>Trypsinze and count PANC-1 cells:
		<ol>
			<li>Discard the supernatant and wash the PANC-1 cells with 9 mL of HBSS (3 mL at a time). Discard used reagents in the hood in a 20-25% V/V bleach solution to ensure complete death of living/viable material.</li>
			<li>Trypsinize the cells with 2 mL of trypsin. After nearly 10 min (may need more time), when all the cells are detached, neutralize the suspension with 10 mL of DMEM + 10% FBS. Begin with 3 mL and follow this with subsequent wash steps using 1 mL per wash (7 washes remaining) to ensure maximum collection of trypsinized cells.</li>
			<li>Pool each neutralized aliquot into the same 15 mL tube. The final volume in the tube will be close to 12 mL (~2 mL of trypsin that has been neutralized with 10 mL media).</li>
			<li>Centrifuge the neutralized cell suspension at 700 x g for 2 min and 30 s. Discard the supernatant and resuspend the pellet obtained in 1 mL of DMEM +10% FBS. Take an aliquot from this for cell counting and label the value obtained from this section as &#34;Count A&#34;.</li>
		</ol>
		</li>
		<li>Trypsinze and count HPaSteC cells.
		<ol>
			<li>Discard the supernatant and wash the HPaSteC cells with 9 mL of HBSS (3 mL at a time).</li>
			<li>Trypsinize the cells with 2 mL of trypsin. After nearly 10 min, when all the cells are detached, neutralize the suspension with 10 mL of trypsin neutralization solution. Begin with 3 mL and follow this with subsequent wash steps using 1 mL per wash (7 washes remaining) to ensure maximum collection of trypsinized cells.</li>
			<li>Pool each neutralized aliquot into the same 15 mL tube. The final volume in the tube will be close to 12 mL (~2 mL of trypsin that has been neutralized with 10 mL of neutralization solution).</li>
			<li>Centrifuge the neutralized cell suspension at 700 x g for 2 min and 30 s. Discard the supernatant and resuspend the pellet obtained in 1 mL of the supplemented stellate cell media. Take an aliquot from this for cell counting and label the value obtained from this section as &#34;Count B&#34;.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Diluting initial cell suspensions
	<ol>
		<li>Pause at this step if needed (not over 0.5 h) for a short while after obtaining either both counts A and B or after trypsinizing and neutralizing both PANC-1 and HPaSteC. Maintain unused culture media at 37 &#176;C and cell suspensions at room temperature (RT; 25 &#177; 2 &#176;C).</li>
		<li>Use Count A and Count B obtained previously, to take aliquots and make respective dilutions (in separate tubes) so that the final volume is 1 mL (diluent: DMEM + 10% FBS for both vials) and the final count is between 110,000-140,000 cells for each cell type. Once this dilution is made, verify the cell counts for each dilution to give Count C (PANC-1) and Count D (HPaSteC).</li>
		<li>Pause at this step if needed (not over 15-30 min) after obtaining Count C and Count D. Do not stop once the final dilutions are made in step 4.3.3.2; experiment to avoid cell settling. Maintain unused culture media at 37 &#176;C and cell suspensions at RT (25 &#177; 2 &#176;C).
		<ol>
			<li>Maintain the final number of cells needed per well at HPaSteC: PANC-1: 60:120. Use 110 wells for the calculation to account for excess. Calculate with a base value of 100 &#181;L of cell suspension per well (total suspension volume is 11 mL for 110 wells with HPaSteC: PANC-1: 60:120). Make this suspension in a sterile 50 mL centrifuge tube for ease of mixing.</li>
			<li>Use Count C and Count D, and spike enough cell suspension into a DMEM + 10% FBS solution to have HPaSteC: PANC-1: 60:120 in a final volume of 11 mL (6,600 HPaSteC cells and 13,200 PANC-1 cells in 11 mL). Mix the solution thoroughly using a 1 mL pipette by stirring and pipetting up and down. Avoid froth formation.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Seeding
	<ol>
		<li>Use a 200 &#181;L or 100 &#181;L pipette to mix the final suspension (stir by moving the pipette tip around combined with pipetting up and down) at each step prior to adding 100 &#181;L of the suspension per well. Add the 100 &#181;L gently along the corner of the well.
		<ol>
			<li>Re-use the same tip for multiple wells unless it falls off. Mix back and forth and count to 10 or 15 (not seconds) between additions to each well for the first half (roughly the first 43 wells) of the plate. Lower the mixing count to 5-10 between each well for the second half (remaining 43 wells) since the suspension volume is lesser and requires less mixing duration.</li>
			<li>Do not use a repeating pipettor since the hydrophobic poly-HEMA coating will cause the suspension to bounce off the plate, leading to uneven distribution.</li>
		</ol>
		</li>
		<li>Discard any excess suspension after all wells are filled.</li>
	</ol>
	</li>
	<li>Centrifugation
	<ol>
		<li>Centrifuge the final plate containing the cell suspension to bring all the cells together as a pellet. Carefully wrap the plate with parafilm around the border of the plate to prevent contamination during this step. Post wrapping, centrifuge the plate with a counterbalance at 1000 x g for 2 min.</li>
		<li>Take the plate back into the cell culture hood (use ethanol-sprayed gloves to handle), remove the parafilm, and store the plates in the incubator at 37 &#176;C, with 5% CO2. Do not spray the plate with ethanol-water mix at any point.</li>
	</ol>
	</li>
	<li>Culture maintenance
	<ol>
		<li>View cells coming together to form spheroids under a light microscope at 4x-5x magnification by day 3. Follow the culture maintenance protocol as described below.
		<ol>
			<li>On day 3, measure out 5-6 mL of DMEM + 10% FBS and add 50 &#181;L of it to each well along the side of the well.
			<ol>
				<li>Do not mix or agitate the media in any way during addition, as this will damage the spheroid or lead to accidental spheroid removal. The final volume per well will be ~150 &#181;L.</li>
			</ol>
			</li>
			<li>Beyond day 3, refer to Figure 2 to become familiar with the structure of the well and the location of the light reflection (between the cylindrical and curved portion of the well) seen on the media referred to as the halo. Perform operations close to the halo with caution to ensure the spheroids remain undamaged.</li>
			<li>On day 6, measure 10-11 mL of DMEM + 10% FBS. Use a 1 mL pipette to pull out media in bulk from multiple wells by pulling out media until the halo. Do this for the entire plate, followed by media replacement. Collect supernatant in bulk and discard, or discard it as it is pulled out.</li>
			<li>Replace the discarded media with fresh media by adding 100 &#181;L of media along the side of each well. The final volume in the well will be close to 200 &#181;L.
			<ol>
				<li>Do not pull media beyond the halo, as it increases the risk of accidentally pulling out the spheroid.</li>
			</ol>
			</li>
			<li>On day 10/11, measure 10-11 mL of DMEM + 10% FBS. Use a 1 mL pipette to pull out media in bulk from multiple wells by pulling out media until the halo. Do this for the entire plate, followed by media replacement. Collect supernatant in bulk and discard, or discard it as it is pulled out.</li>
			<li>Replace the discarded media with fresh media by adding 100 &#181;L of media along the side of each well. The final volume in the well will be close to 200 &#181;L.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Final day assessment
	<ol>
		<li>Perform drug treatment on the spheroids on day 14. Image the spheroids prior to use using a brightfield inverted microscope under a 4x objective to determine the start volume of the spheroid. Calculate the Volume (V) using the following formula: 
		&#8203;V = 0.5&#160;&#215; L&#160;&#215; W2</li>
		<li>Follow the equation to calculate spheroid volumes is described above. Here L is the length of the major axis, and W is the width (longest line perpendicular to the major axis). Convert all original units from micrometers to millimeters to obtain a volume in cubic millimeter.</li>
		<li>The start volume in each well on day 14 is between 150-200 &#181;L due to evaporation. For drug treatments, remove media until the halo is reached from each well and replace it with 100 &#181;L of the drug solution at 2x strength, coupled with some gentle mixing (mix media gently back and forth while counting to 2 between each well).
		<ol>
			<li>Use 2x strength solutions to allow the final drug solution to be at 1x and to avoid completely removing old media from the wells, failing which the spheroids may be disturbed. Use the spheroids within 3 days of this time frame.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Spheroid collection
	<ol>
		<li>Measure spheroid volumes prior to collection as described in step 4.7.2.</li>
		<li>Collect the spheroids on day 14 or 17, depending on the experiment&#39;s needs, by removing media from each well until the halo is reached.</li>
		<li>Use a 1 mL pipette to remove media and collect the supernatant in a 15 mL conical centrifuge tube from multiple wells.</li>
		<li>Once all the wells have media at the halo level, reduce the setting on the 1 mL pipette to between 200-300 &#181;L.</li>
		<li>Align the plate against the background of the hood or dark background (e.g., a tube rack) to see the spheroids settled at the bottom.</li>
		<li>Gently insert the tip near the spheroid (go below the halo) and pipette a small amount of media (e.g., a visual estimate of close to 50 &#181;L) back and forth to disturb the spheroid. Do not put the tip directly over the spheroid, as this can damage its structure.
		<ol>
			<li>Do not pull all the media out of the well at this step. Control the thumb movement firmly on the piston of the pipette.</li>
		</ol>
		</li>
		<li>Displace media until the spheroid no longer remains at the center and can be seen moving against the background. Pull up enough media to suck out the spheroid and move it to the desired location (e.g., pool it with other spheroids in a well that is part of the same treatment set).</li>
	</ol>
	</li>
	<li>Discarding waste
	<ol>
		<li>Discard all liquid samples once neutralized with bleach according to protocols from the respective labs&#39; Environment and Health Safety Department. Autoclave and discard all used pipettes and flasks with biohazardous waste.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66625/66625fig01.jpg" /> 
Figure 1: Overview of the process to grow 3D desmoplastic pancreatic cancer spheroids (Generated using BioRender). The figure gives an overview of the basic processes involved; namely, trypsinzing cells, using the initial cell count to make dilute cell suspensions, preparing a co-culture using the diluted cell suspensions, adding cell suspensions to each well, incubating the cultures, performing media maintenance and final spheroid formation as expected on day 14. <a href="https://www.jove.com/files/ftp_upload/66625/66625fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66625/66625fig02.jpg" /> 
Figure 2: Structure of the U bottom well. The right image is an exaggerated shape to demonstrate the &#34;halo&#34; portion of the well. The figure aims to define where the &#34;halo&#34; portion of the well is as working above the halo is critical to growing the spheroids and avoiding accidental loss. <a href="https://www.jove.com/files/ftp_upload/66625/66625fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Evaluation of ECM components and confocal microscopy
<ol>
	<li>Use immunostaining to evaluate the response of the ECM to drug therapy according to the protocol described by Durymanov et al.38.
	<ol>
		<li>Pool the spheroids together and wash twice with 100 &#181;L of PBS. Embed in optimal cutting temperature (OCT) tissue embedding medium, freeze, and maintain at -80 &#176;C.</li>
		<li>Cut the frozen tumor blocks into 10 &#181;m sections, fix the sections in acetone-methanol (1:1) mixture for 15 min, and allow them to air-dry at RT.</li>
	</ol>
	</li>
	<li>To determine ECM components, immunostain the cryosections with rabbit monoclonal anti-fibronectin primary antibodies, rabbit polyclonal anti-type I collagen antibodies, rabbit polyclonal anti-laminin antibodies, and sheep polyclonal anti-hyaluronic acid antibodies.</li>
	<li>Use goat antirabbit IgG labeled with Alexa Fluor 488 or donkey anti-sheep conjugated with Alexa Fluor 568 as secondary antibodies. 
	NOTE: Primary and secondary antibodies were diluted at 1:200 and 1:300, respectively.</li>
	<li>Acquire images of all spheroid sections using a confocal laser scanning microscope equipped with 20x/0.45 objective lens.</li>
</ol>

3D-PDAC Model Capturing Desmoplasia for Therapeutic Evaluation

The authors have nothing to disclose.

The duration and cell ratios chosen to grow the spheroids were based on studies as reported previously38. When attempting to optimize these studies by substituting NIH3T3 cells for HPaSteC cells, spheroid volumes and apoptosis patterns were found to closely resemble the reported optimized parameters (reported for PANC-1: NIH3T3:: 120:12) when PANC-1: HPaSteC ratios were at 120: 60. Although these studies only measure apoptosis until day 14, the protocol described in this process continues to use s...

Pancreatic cancer&#39;s poor prognosis is associated with a variety of reasons, among which is its lack of easily detectable biomarkers leading to a late detection. Another major reason is the thick stroma surrounding the tissue, which leads to reduced blood supply. The deposition of large amounts of extracellular matrix (ECM), cell-cell interaction, endothelial cells, various immune cells, pericytes, proliferating myofibroblast, and fibroblast population, and the presence of non-neoplastic cells (together constituting the desmoplastic reaction)1, constitute the thick stroma that is responsible for PDAC&#39;s chemo and radiotherapeutic resistance2. Cancer and stromal cells have a complex, dynamic, and bidirectional interaction. Although some elements either attenuate or accelerate disease progression, most processes are adaptive during the tumor&#39;s development1. This provides an environment rich with growth factors, proangiogenic factors, proteases, and adhesion molecules. These factors promote angiogenesis, cell proliferation, metastasis, and invasion3,4. Together, they are immune and drug-privileged sanctuary for the tumor, resulting in drug resistance.
The desmoplasia is a complex mixture which consists of various ECM proteins, along with immune cells and pancreatic stellate cells (PSC). Together, these tend to form a scaffold for the cells to grow. PSCs are one of the largest components of the stromal compartment5. Their ability to produce enzymes like matrix metalloproteases (MMP), tissue inhibitors of matrix metalloproteases (TIMP) and cancer associated fibroblasts (CAF)6&#160;imply they are likely to play a critical role in development of the desmoplastic reaction. The ECM, cancer-associated fibroblasts (CAF), and vasculature are the cardinal aspects of PDAC. Among CAFs, myofibroblast and inflammatory CAFs are speculated to be involved in active crosstalk responsible for pro-tumor properties7. The more extensive the fibroblastic formations on the tumor, the poorer the prognosis8,9,10.
Monolayer cell culture through established cell lines continues to remain a useful tool for analyzing drug toxicity and is a good starting point for proof of concept and discovery studies. Established cell culture lines, however, lack germline DNA and clinical relatability11. Since they are grown on flat surfaces, they undergo different in vitro selection criteria compared to when they are a part of the tumor, divide abnormally and lose their differentiated phenotype12. Overall, single cell cultures limit tumor heterogeneity and therefore lose clinical relevance. They are unable to accurately represent the complexity of the tumor&#39;s microenvironment (for e.g., the ECM). 3D culture can more closely replicate the complex tumor microenvironment.
3D culture was introduced in the 1970&#39;s for healthy cells and their neoplastic counterparts13. Several techniques have been used to study the morphology and architecture of malignant tissues through spheroids14. Co-cultures with stromal cells can model TME signals. An upregulation of EMT markers was seen when cells were co-cultured with stellate cells15. PDAC spheroids and their interaction with the stroma can be modelled by co-culturing with ECM components. Co-culturing specifically with PSCs have been reported to produce clinically relevant drug cytotoxicity data16,17,18. PSCs also aid drug resistance by evading apoptosis and stimulating proliferation of cancer cells through various paracrine factors19&#160;and by inducing EMT transition. It, therefore, becomes critical to include the PSCs from an early stage in the criteria used to evaluate the success of a drug or drug delivery system. The PSC&#39;s ability to enhance proliferation and support faster growth in combination, compared to pancreatic cancer cells alone, has also been seen in vivo when subcutaneous flank injections of the two cell lines were evaluated in immunocompromised mice20.
The ability of a cell type to interact with ECM components is also critical to consider when growing co-cultured spheroids. BxPC-3 and PANC-1 have been reported to have equal affinities in binding to collagen. The two cell lines also bind equivalently to laminin, although there have been reports that BxPC-3 binds better21,22,23,24,25. In terms of migration, Stahle et al.26&#160;demonstrated a 5x faster motility for PANC-1 cells as compared to BxPC-3. PANC-1 cells were also reported to migrate primarily as single cells, whereas BxPC-3 cells migrate as a tightly packed sheet. The choice of the cells also affects the size of the tumor25. BxPC-3 tumors were shown to be larger27,28&#160;than those obtained from PANC-1, whereas one study demonstrated the opposite29&#160;case. Despite their differences in size and motility, both cells have been reported to need long periods of latency to form tumors in mice. This duration can be especially long for BxPC-3 ranging from 4 weeks to 4 months25. However, there is also literature where BxPC-328&#160;or BxPC-3 cancer stem cells30&#160;have formed visible tumors quicker, implying there could be variation seen in tumor growth durations. The durations stated here should, therefore, only serve as an initial guideline for tumor growth rates.
BxPC-3 cells form spheroids with loose cells on the surface and dense cores, whereas PANC-1 cells have been reported to form both porous but robust spheroids31 as well as compact spheroids. PANC-1 cells have also been reported to be less differentiated and more aggressive32. Keeping the aggressive nature32 at the forefront, combined with the PANC-1 cells&#39; higher motility, ability to form compact spheroids, and ability to interact with ECM components, PANC-1 cells were chosen for spheroid studies.
In the last few years, spheroid culture has seen a lot of success in demonstrating an advantage in its clinical relevancy compared to two-dimensional (2D) cultures. Its relevance has been leveraged in using this technique as a substitute to animal studies and to better understand the tumors&#39; biology. The clinical relevance of spheroids, especially when co-cultured with PSC&#39;s has enabled their use to study various functions of the spheroid such as stiffness33, expression of TGF-&#946;34,35,36,37,38, E-cadherin, F-actin18,34,36,37, &#945;-SMA34,35,37,38, lactate dehydrogenase (LDHA)32, HIF-1&#945;35,39, drug resistance16,37,40, cell migration41, cell invasion37, fibrosis35, radiation resistance42, phenotypical changes18, heterogeneity36, cellular levels of interactions39 and demonstrate ECM components37,38,39. Many of the protocols that were used to obtain the data described rely on Matrigel, the hanging drop method, printed molds, or other scaffolds to help support spheroid and ECM growth. The studies also usually involve the use of either non-human fibroblastic cells or freshly isolated stellate cells from patients. While using stellate cells is critical for the tumors to resemble in-vivo conditions, the inter-patient variability associated with fresh extractions makes these studies difficult to replicate.
This protocol aims to demonstrate a model that is easy to develop, reproducible, clinically relevant, and free of scaffolding, thereby relying exclusively on the co-cultures&#39; abilities to naturally generate the ECM. To do this, a simple co-culture method involving a mix of PANC-1 cells (due to their natural tendency to migrate as single cells) along with human pancreatic stellate cells (HPaSteC) was chosen, due to their ability to behave like stem cells and be highly drug-resistant. Using the studies by Durymanov et al.38as a baseline, the protocol detailed below was established after further optimizing parameters such as cell ratios and durations between media changes. The spheroids resulting from this protocol can be used as a model system for new drug candidate evaluation40.
Additionally, for users not familiar with the spheroid culture, the work Peirsman et al.43discussing the development of the MISpheroID knowledgebase may be helpful. It establishes some minimum information guidelines that could help cope with heterogeneity between lab protocols. Although with some limitations, the work demonstrated that the choice of culture media, cell lines, spheroid formation method, and the final spheroid size are critical in determining the phenotypic properties of spheroids.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Axio Observer inverted microscope</td>
			<td>Carl Zeiss</td>
			<td>0450-354</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer Auto T4</td>
			<td>Nexcelom Bioscience LLC</td>
			<td>Auto-T4</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, powder, high glucose</td>
			<td>Gibco</td>
			<td>12100046</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-sheep conjugated with Alexa Fluor 568</td>
			<td>Abcam</td>
			<td>ab175712</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Cytiva</td>
			<td>SH3091003HI</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat antirabbit IgG labeled with Alexa Fluor 488&#160;</td>
			<td>Abcam</td>
			<td>ab150077</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution (HBSS)&#160;</td>
			<td>Gibco</td>
			<td>14175145</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Pancreatic Stellate Cells (HPaSteC)</td>
			<td>ScienCell</td>
			<td>3830</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Nikon</td>
			<td>Nikon</td>
			<td>Eclipse Ts 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc 96-Well Polystyrene Round Bottom Microwell Plates</td>
			<td>Thermo Scientific</td>
			<td>12-565-331</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Fluoview FV1200 confocal laser</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td>Discontinued product</td>
		</tr>
		<tr>
			<td>PANC-1</td>
			<td>ATCC</td>
			<td>CRL-1469</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-HEMA</td>
			<td>Sigma</td>
			<td>P3932</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-laminin antibodies&#160;</td>
			<td>Abcam</td>
			<td>ab11575</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-type I collagen antibodies&#160;</td>
			<td>Abcam</td>
			<td>ab34710</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep polyclonal anti-hyaluronic acid antibodies</td>
			<td>Abcam</td>
			<td>ab53842</td>
			<td></td>
		</tr>
		<tr>
			<td>Stellate cell media complete kit&#160;</td>
			<td>ScienCell</td>
			<td>5301</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>MP Biomedicals, LLC</td>
			<td>153571</td>
			<td>Trypsin solution prepared according to manufacturers protocol and used at 0.25%w/v&#160;</td>
		</tr>
		<tr>
			<td>Trypsin Neutralization Solution (TNS)</td>
			<td>ScienCell</td>
			<td>103</td>
			<td></td>
		</tr>
	</tbody>
</table>

growing desmoplastic three-dimensional pancreatic cancer spheroids from co-culture

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of &#60;12%. The biggest barrier to therapy is the dense desmoplastic extracellular matrix (ECM) that surrounds the tumor and reduces vascularization, generally termed desmoplasia. A variety of drug combinations and formulations have been tested to treat the cancer, and although many of them show success pre-clinically, they fail clinically. It, therefore, becomes important to have a clinically relevant model available that can predict the response of the tumor to therapy. This model has been previously validated against resected clinical tumors. Here a simple protocol to grow desmoplastic three-dimensional (3D)-coculture spheroids is described that can naturally generating a robust ECM and do not require any external matrix sources or scaffold to support their growth.
Briefly human pancreatic stellate cells (HPaSteC) and PANC-1 cells are used to prepare a suspension containing the cells in a 1:2 ratio, respectively. The cells are plated in a poly-HEMA coated, 96-well low attachment U-well plate. The plate is centrifuged to allow the cells to form an initial pellet. The plate is stored in the incubator at 37 &#176;C with 5% CO2, and media is replaced every 3 days. Plates can be imaged at designated intervals to measure spheroid volume. Following 14 days of culture, mature desmoplastic spheroids are formed (i.e. average volume of 0.048 + 0.012 mm3&#160;(451 &#181;m x 462.84 &#181;m)) and can be utilized for experimental therapy assessment. Mature ECM components include collagen-I, hyaluronic acid, fibronectin, and laminin.

Three of the most critical steps involved in growing the spheroids are the initial cell count, the mixing steps while seeding the spheroids, and performing timely media changes to allow the spheroids to grow (Figure 1). Additionally, being familiar with Figure 2&#160;on media changes after day 3 is critical to allow for effective media changes due to the increased media volume per well. When all these steps are performed according to the directi...

Author Spotlight: Validating Cancer Therapy Responses with Desmoplastic Spheroid Models

Pancreatic cancer remains one of the toughest cancers to treat. Therefore, it is critical that pre-clinical models evaluating treatment efficacy are reproducible and clinically relevant. This protocol describes a simple co-culture procedure to generate reproducible, clinically relevant desmoplastic spheroids.

Growing Desmoplastic Three-Dimensional Pancreatic Cancer Spheroids from Co-Culture

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of &#60;12%. The biggest barrier to therapy is the ...

growing-desmoplastic-three-dimensional-pancreatic-cancer-spheroids

Research

JoVE Journal

Cancer Research

1.2K Views.  South Dakota State University. Pancreatic cancer remains one of the toughest cancers to treat. Therefore, it is critical that pre-clinical models evaluating treatment efficacy are reproducible and clinically relevant. This protocol describes a simple co-culture procedure to generate reproducible, clinically relevant desmoplastic spheroids.

Video: Author Spotlight: Validating Cancer Therapy Responses with Desmoplastic Spheroid Models

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

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.

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 ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...