We would like to thank Dr. Avital Sarusi-Portuguez for help in data analysis and Dr. Dror Kolodkin-Gal for assistance in establishing the protocol in a previous study. We thank all past and present members of the Parnas lab. We thank Dr. Gillian Kay and Dr. Michael Kanovsky for their help in editing. This project has received funding from the Israel Science Foundation grant (No. 526/18 O.P.), the Alex U. Soyka Program, and a grant from the Israel Cancer Research Fund (Research Career Development Award).

The joint ethics committee (Institutional Animal Care and Use Committee) of the Hebrew University (Jerusalem, Israel) and Hadassah Medical Center (Jerusalem, Israel) approved the study protocol for animal welfare (MD-18-15417-5 &#34;Tissue dynamics in pancreatic cancer in mice&#34;), and the protocol presented here complied with all relevant ethical regulations for animal testing and research. The Hebrew University is an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited institute.
NOTE: The mouse strain stock #007908, stock #019378, and stock #008179 were obtained from Jackson&#39;s laboratory. PRT (Kras+/LSL-G12D; Ptf1a-CreER; Rosa26LSL-tdTomato) mice were created by crossing the above strains. Mice from both genders, between 6 weeks and 15 months of age, were used for the study. Tamoxifen was prepared by dissolving the powder in corn oil. Adult mice (6-8 weeks of age, females and males), were injected with tamoxifen subcutaneously on days 0 and 2 at a dose of 400 mg/kg and examined twice a week following the injection. It was not possible to measure tumors as they were internally located; therefore, euthanasia was performed if abnormal clinical signs were observed according to the ethical protocol. Mice were euthanized at different time points post-tamoxifen induction, using isoflurane and cervical dislocation.
1. Pancreatic dissection
NOTE: For optimal yield during extraction and to ensure good cell viability, rapid dissection is critical. To shorten the time required for pancreas isolation, all instruments and equipment must be ready on ice before euthanizing the mouse.
<ol>
	<li>Euthanize the mouse by CO2&#160;asphyxiation and verify using cervical dislocation. From this step on, all procedures must be performed with sterile dissecting instruments.</li>
	<li>Fix the mouse and spray the abdomen with 70% ethanol. Make a V-shaped incision of 2.5 cm in the genital area with scissors and forceps and proceed upward to fully open the abdominal cavity.</li>
	<li>Locate the stomach on the left side of the mouse. Locate the pancreas, which is near the spleen. Separate the pancreas from the stomach and duodenum using two forceps (without tearing). Continue and separate the pancreas from the small intestine, jejunum, and ileum.</li>
	<li>Move the pancreas to the right side of the mouse. Separate the remaining connections between the pancreas and the thoracic cavity with forceps to completely detach the pancreas and attached spleen.</li>
	<li>Remove the pancreas and spread it out for examination in a Petri dish on ice. 
	NOTE: Care must be taken during this step to remove only the pancreas, and not remove mesenteric fat tissue or other adjacent tissue along with the pancreas, to avoid cellular contamination.</li>
</ol>
2. Enzymatic and mechanical dissociation of the pancreas
<ol>
	<li>Prepare the following buffers in advance.
	<ol>
		<li>Dissociation buffer 1: 4 mL of trypsin C + 6 mL of phosphate buffered saline (PBS) (see Table 1) for each sample.</li>
		<li>Dissociation buffer 2: 9 mL of Hanks&#8242; balanced salt solution (HBSS) 1x; 4% bovine serum albumin (BSA); 1 mL of collagenase P (10 mg/mL); 200 &#181;L of trypsin inhibitor (10 mg/mL); and 200 &#181;L of DNase I (10 mg/mL) (see Table 1).</li>
		<li>Wash buffer: 50 mL of HBSS 1x; 2 g of BSA; 1 mL of trypsin inhibitor (10 mg/mL); and 1 mL of DNase 1 (10 mg/mL).</li>
		<li>Enzyme activity stop solution: HBSS 1x containing 5% fetal bovine serum (FBS) and 150 g of DNase I (0.2 mg/mL).</li>
	</ol>
	</li>
	<li>Place the pancreas in a 50 mL tube on ice. Rinse the pancreas in 10% FBS in HBSS 1x. The fatty tissue will float and the pancreas will sink. This is an easy way to visualize and quickly remove the contaminating white adipose tissue still attached to the pancreas.</li>
	<li>Transfer the mouse pancreatic tissue to a sterile Petri dish containing 5 mL of HBSS 1x on ice. Cut the pancreas into small pieces of 1 to 3 mm3 using Noyes scissors and a scalpel (Figure 2A). In case of more than one sample, the samples should be kept on ice in 10% FBS/HBSS 1x.</li>
	<li>Transfer the tissues to a centrifuge tube. Centrifuge at 350 x g at 4 &#176;C for 5 min. Aspirate and discard the supernatant to remove cell fragments and blood cells.</li>
	<li>Resuspend the pieces in dissociation buffer 1 containing 0.02% trypsin C-0.05% EDTA for 10 min at 37 &#176;C with agitation (180 rpm). Immediately wash with 10% FBS/Dulbecco&#39;s modified Eagle&#39;s medium (DMEM). Centrifuge for 5 min at 350 g at 4 &#176;C.</li>
	<li>Wash again by resuspending the pellet in 10 mL of wash buffer and centrifuging at 350 x g for 5 min at 4 &#176;C before the next dissociation step.</li>
	<li>Incubate the pancreas in dissociation buffer 2 for 15 min at 37 &#176;C with agitation (180 rpm).</li>
	<li>After 15 min, perform mechanical dissociation by vigorously pipetting the pancreatic fragments up and down in sterile pipettes of decreasing size (25, 10, and 5 mL serological pipettes) 10 times and bring back to 37 &#176;C.
	<ol>
		<li>After an additional 5 min, repeat the mechanical dissociation and use light microscopy to monitor the dissociation according to the amount of single-cell suspension. Usually if at this stage, less than 90% of the suspension consists of isolated single cells, a longer incubation time is needed. Use trypan blue to monitor cell viability.</li>
		<li>Continue with the incubation and take samples to detect the dissociation every 5 min. 
		NOTE: The total incubation time depends on the tissue and can vary between samples. The incubation and tissue dissociation should end when 90% of the cells are separated into single cells or when a reduction in viability is detected.</li>
	</ol>
	</li>
	<li>After the pancreatic tissue is well dissociated (corresponding to the disappearance of pancreatic fragments and the increasing turbidity of the solution) (Figure 2), stop the enzymatic reaction by washing twice with enzyme activity stop solution for 5 min at 4 &#176;C. From this step, keep the cell suspension on ice.</li>
	<li>Pass the cell suspension through a 70&#181;m nylon mesh and check the cell viability under the microscope. Smaller sizes of nylon mesh may reduce cell viability.</li>
	<li>Resuspend and wash the pellet with 5-10 mL of ice-cold buffered wash solution. Count the cells.</li>
	<li>If several red blood cells are observed, treat the cells with a red blood cell lysis buffer for 2 min at room temperature. In cases where clumps are observed, the sample should be treated again with trypsin, as described in step 2.5. Viability is detected with trypan blue under the microscope, and if viability is less than 80%, live cells should be isolated using the magnetic-activated cell sorting (MACS) dead cells removal kit with MACS MS columns (see Table 1).</li>
</ol>

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2019.">Cancer statistics, 2019.</a> CA: a Cancer Journal for Clinicians. 69 (1), 7-34 (2019).</li><li>Yachida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distant+metastasis+occurs+late+during+the+genetic+evolution+of+pancreatic+cancer.">Distant metastasis occurs late during the genetic evolution of pancreatic cancer.</a> Nature. 467 (7319), 1114-1117 (2010).</li><li>Peng, 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=Author+correction:+single-cell+RNA-seq+highlights+intra-tumoral+heterogeneity+and+malignant+progression+in+pancreatic+ductal+adenocarcinoma.">Author correction: single-cell RNA-seq highlights intra-tumoral heterogeneity and malignant progression in pancreatic ductal adenocarcinoma.</a> Cell Research. 29 (9), 777 (2019).</li><li>Hruban, R. H., Wilentz, R. E., 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=Genetic+progression+in+the+pancreatic+ducts.">Genetic progression in the pancreatic ducts.</a> The American Journal of Pathology. 156 (6), 1821-1825 (2000).</li><li>Schlesinger, 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=Single-cell+transcriptomes+of+pancreatic+preinvasive+lesions+and+cancer+reveal+acinar+metaplastic+cells'+heterogeneity.">Single-cell transcriptomes of pancreatic preinvasive lesions and cancer reveal acinar metaplastic cells' heterogeneity.</a> Nature Communications. 11 (1), 4516 (2020).</li><li>Kolodkin-Gal, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senolytic+elimination+of+Cox2-expressing+senescent+cells+inhibits+the+growth+of+premalignant+pancreatic+lesions.">Senolytic elimination of Cox2-expressing senescent cells inhibits the growth of premalignant pancreatic lesions.</a> Gut. 71 (2), 345-355 (2021).</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>Elyada, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-species+single-cell+analysis+of+pancreatic+ductal+adenocarcinoma+reveals+antigen-presenting+cancer-associated+fibroblasts.">Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts.</a> Cancer Discovery. 9 (8), 1102-1123 (2019).</li><li>Bernard, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomics+of+pancreatic+cancer+precursors+demonstrates+epithelial+and+microenvironmental+heterogeneity+as+an+early+event+in+neoplastic+progression.">Single-cell transcriptomics of pancreatic cancer precursors demonstrates epithelial and microenvironmental heterogeneity as an early event in neoplastic progression.</a> Clinical Cancer Research. 25 (7), 2194-2205 (2019).</li><li>Moncada, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+microarray-based+spatial+transcriptomics+and+single-cell+RNA-seq+reveals+tissue+architecture+in+pancreatic+ductal+adenocarcinomas.">Integrating microarray-based spatial transcriptomics and single-cell RNA-seq reveals tissue architecture in pancreatic ductal adenocarcinomas.</a> Nature Biotechnology. 38 (3), 333-342 (2020).</li><li>Hwang, W. 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=Single-nucleus+and+spatial+transcriptome+profiling+of+pancreatic+cancer+identifies+multicellular+dynamics+associated+with+neoadjuvant+treatment.">Single-nucleus and spatial transcriptome profiling of pancreatic cancer identifies multicellular dynamics associated with neoadjuvant treatment.</a> Nature Genetics. 54 (8), 1178-1191 (2022).</li><li>Cui Zhou, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+restricted+drivers+and+transitional+cell+populations+cooperate+with+the+microenvironment+in+untreated+and+chemo-resistant+pancreatic+cancer.">Spatially restricted drivers and transitional cell populations cooperate with the microenvironment in untreated and chemo-resistant pancreatic cancer.</a> Nature Genetics. 54 (9), 1390-1405 (2022).</li><li>Habib, 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=Massively+parallel+single-nucleus+RNA-seq+with+DroNc-seq.">Massively parallel single-nucleus RNA-seq with DroNc-seq.</a> Nature Methods. 14 (10), 955-958 (2017).</li></ol>

The authors declare no competing interests.

In this article, we present a protocol for pancreatic tissue dissociation. The protocol is simple, easy to use, and provides a tool to isolate viable single cells from pancreatic tissue at different stages during the malignancy process, including solid tumors. In previous studies, different types of collagenases were used to digest the pancreas8,9. Using a very potent collagenase, such as collagenase D, results in a large population of immune cells and a lower pe...

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancer types1. Clinical evidence supports the notion that PDAC develops from exocrine-system cells, including acinar cells, over many years, driven by mutations in the KRAS proto-oncogene2.
Pancreatic tumors include many different cell types, and it has been demonstrated that malignant cells count for only 20%-50% of the tumor mass3. Different cell types interact with the epithelial cells, support their transformation, and enhance tumor formation and growth. Early events cause acinar metaplasia, which gives rise to microscopic lesions called pancreatic intraepithelial neoplasia (PanINs), which can in some cases develop into PDAC4.
There is a critical need to investigate these interactions and target pivotal signals. Single-cell RNA-sequencing (scRNA-seq) is a powerful method that reveals gene expression at a single-cell resolution, thereby tracking the changes that epithelial cells undergo, thus enabling the exploration of pancreatic cancer development. 
Tissue dissection and digestion to single cells is the first stage in a scRNA-seq experiment. Several factors make pancreatic tissue digestion especially challenging: i) acinar cells account for more than 90% of the pancreas and acinar cells contain large amounts of digestive enzymes, including proteases and RNases that reduce the quality of RNA-based libraries; (ii) acinar cells are very sensitive and may lyse if standard protocols are used; (iii) acinar cells express a small number of genes at very high levels. Therefore, if these cells are lysed during the experiment, this can contaminate the observed gene expression profile of other cells; (iv) pancreatic tissue recovered from tumors is desmoplastic, making it hard to dissect without damaging the cells. Thus, even though maintaining high viability of all the cell types is required, the large number and sensitivity of acinar cells add additional complexity. These factors impose difficulties in achieving a single-cell suspension that is more than 80% viable and has no clumps, as is required for scRNA-seq experiments. 
Here, we developed a protocol using trypsin C and collagenase P, along with frequent tissue monitoring. This supports dissociation to single cells while retaining high viability to support the success of scRNA-seq experiments5,6.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent or Resource</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m nylon mesh&#160;</td>
			<td>Corning</td>
			<td>cat##431751</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>cat# A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase P</td>
			<td>Roche</td>
			<td>cat# 11213857001</td>
			<td></td>
		</tr>
		<tr>
			<td>Critical Commercial Assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma Aldrich</td>
			<td>cat#MBD0015</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Roche</td>
			<td>cat# 10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Experimental Models: Organisms/Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum South American</td>
			<td>ThermoFisher</td>
			<td>Cat#10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution</td>
			<td>Biological industries</td>
			<td>cat#02-018-1A&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>KRASLSL-G12D mice</td>
			<td>Jackson Laboratory</td>
			<td>JAX008179</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS dead cells removal kit</td>
			<td>Milteny Biotec</td>
			<td>cat#130-090-101</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biological industries</td>
			<td>cat#02-023-1A&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ptf1a-CreER mice</td>
			<td>Jackson Laboratory</td>
			<td>JAX019378</td>
			<td></td>
		</tr>
		<tr>
			<td>Ptf1a-CreER; Rosa26LSL-tdTomato mice</td>
			<td>Jackson Laboratory</td>
			<td>JAX007908</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin C-EDTA 0.05%</td>
			<td>Biological industries</td>
			<td>cat# 03-053-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Roche</td>
			<td>cat#T6522</td>
			<td></td>
		</tr>
	</tbody>
</table>

pancreatic tissue dissection to isolate viable single cells

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for approximately 90% of the pancreas and includes cells that produce and secrete digestive enzymes. The digestive enzymes are produced in the pancreatic acinar cells, stored in vesicles called zymogens, and are then released into the duodenum via the pancreatic duct to initiate metabolic processes. The enzymes produced by the acinar cells can kill cells or degrade cell-free RNA. In addition, acinar cells are fragile, and common dissociation protocols result in a large number of dead cells and cell-free proteases and RNases. Therefore, one of the biggest challenges in pancreatic tissue digestion is recovering intact and viable cells, especially acinar cells. The protocol presented in this article shows a two-step method that we developed to meet this need. The protocol can be used to digest normal pancreata, pancreata that include pre-malignant lesions, or pancreatic tumors that include a large number of stromal and immune cells.

In a recently published work5, we applied the protocol described above to explore the early stages of PDAC development using a mouse model. The mouse was genetically engineered to include the cassettes&#160;Ptf1a-CreER, LSL-Kras-G12D, LSL-tdTomato7, which allow the expression of constitutively active KRAS in acinar cells after tamoxifen injection.
After cervical dislocation (according to the mouse ethi...

Watch this Scientific Journal Video about Pancreatic Tissue Dissection to Isolate Viable Single Cells at JoVE.com

Pancreatic Tissue Dissection to Isolate Viable Single Cells

Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing.

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for ...

pancreatic-tissue-dissection-to-isolate-viable-single-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

2.4K Views.  The Hebrew University of Jerusalem. Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing. 

Video: Pancreatic Tissue Dissection to Isolate Viable Single Cells

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

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 ...

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

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 ...

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.

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 ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...