We thank Roberta Migliore for technical assistance. The research leading to these results has received funding from Associazione Italiana per la ricerca sul cancro (AIRC) under IG2016-ID.18443 project &#8211; P.I. Marchesi Federica. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

For all patients enrolled, peripheral blood and pancreatic juice were collected at the time of surgery according to protocols approved by the Ethical Committee of the Institution. All the patients were enrolled in the study after signed informed consent including collection of biological specimens and clinical data. The study was approved by the Ethical Committee of the Institution (protocol number ICH-595, approval issued on May 2009). Procedures involving mice and their care were conformed to EU and Institutional Guidelines (protocol ID 121/2016-PR).
1. Isolation of pancreatic juice
NOTE: The withdrawal of pancreatic juice is executed in the context of an open procedure of pancreatic resection (e.g., pancreaticoduodenectomy, total pancreatectomy, distal pancreatectomy) by an equipe of expert pancreatic surgeons.
<ol>
	<li>Patient selection
	<ol>
		<li>Consider for the procedure any patient scheduled for an open pancreatic resection.</li>
		<li>Confirm inclusion if main pancreatic duct size is deemed sufficient to allow pancreatic juice retrieval. The minimum limit in diameter of the main pancreatic duct is considered 2 mm at contrast-enhanced CT imaging.</li>
	</ol>
	</li>
	<li>Pre-operative study of pancreatic duct at contrast-enhanced CT imaging to help the planning of pancreatic juice retrieval
	<ol>
		<li>Tridimensionally localize the main pancreatic duct within the gland at the level of the pancreatic neck: measure the distance of the main pancreatic duct from the anterior, superior and inferior pancreatic margin on cross-sectional slides, and coronal and sagittal renderings. Once in the operating theatre, use these measurements to approximate the correct place where to puncture the pancreas to cannulate the Wirsung duct and to retrieve pancreatic juice.</li>
	</ol>
	</li>
	<li>Preparation of material
	<ol>
		<li>Sterile material: Open the sterile envelope of one 25 G needle and one 3 mL syringe, and position them in the sterile field with the cooperation of the scrub nurse.</li>
		<li>Unsterile material: Keep a 3 mL K2EDTA vacuum test tube ready at hand in the operating room for the storage of the fluid.</li>
	</ol>
	</li>
	<li>Preparation of the patient
	<ol>
		<li>Position the patient on the operating room bed. Induce mixed general anesthesia, using Remifentanil, Sevorane and Rocuronium, then intubate and start ventilating the patient. Position the patient in supine decubitus with the right arm tucked to the body and the left arm abducted to 90&#176; degrees secured on an armboard.</li>
		<li>Disinfect the skin of the abdomen at the site of the incision. Create and maintain a sterile field on the abdomen draping the patient.</li>
	</ol>
	</li>
	<li>Surgical procedure
	<ol>
		<li>Perform a subcostal incision and gain access to the abdominal cavity. Position a Rochard abdominal retraction for organ&#160;exposure.</li>
		<li>Expose and mobilize the pancreas through Kocher maneuver, opening of the gastrocolic ligament, incision of the retroperitoneal tissue along superior and inferior border of the pancreas creating a dissection plane between pancreatic neck and superior mesenteric vein located posteriorly.</li>
		<li>Once the pancreas is mobilized and exposed, proceed to pancreatic juice withdrawal before sectioning of pancreatic neck.</li>
	</ol>
	</li>
	<li>Identification and localization of the pancreatic duct
	<ol>
		<li>Estimate the location of the pancreatic duct using the measurement taken at imaging and then palpate the anterior surface of the pancreas to identify its precise location.</li>
	</ol>
	</li>
	<li>Collection of pancreatic juice
	<ol>
		<li>Hold the pancreatic head and the duodenum from underneath and elevate it with the left hand, marking the location of the pancreatic duct with the first digit.</li>
		<li>Take hold with the right hand of the 3 mL syringe with the 25 G needle mounted on.</li>
		<li>Use the right hand to insert the needle in the pancreas just distal to the left thumb. Decide the depth of penetration and the degree of inclination of the needle based on preoperative measurements and on the perception of having penetrated the duct wall.</li>
		<li>Withdraw the juice with the syringe. If it is not possible to retrieve the juice, relocate the needle in the four directions trying to cannulate the pancreatic duct.</li>
		<li>Once the pancreatic juice is retrieved, move it outside the sterile field and transfer it to the 3 mL K2EDTA vacuum test tube. Keep at 4 &#176;C until the sample is transferred to the lab and proceed to further processing as early as possible. 
		NOTE: The volume of pancreatic juice that can be recovered with this procedure varies greatly, ranging approximately from 0.2 mL to 3 mL in our experience. The amount of juice retrieved is highly dependent on the patient: the dimension of the Wirsung duct and the functional status of the pancreas (functioning versus atrophic gland). In our experience there is no expedient that can be used to increase the amount of pancreatic juice retrieved.</li>
	</ol>
	</li>
</ol>
2. Processing of pancreatic juice
<ol>
	<li>Centrifuge pancreatic juice at 400 x g for 10 min at 4 &#176;C to remove any cells or debris. 
	NOTE: Pancreatic juice should be clear and transparent in color before centrifugation. Blood contamination during surgery can sometimes occur, making the sample appear murkier and redder in color. Consider excluding such samples from further analyses.</li>
	<li>Recover the supernatant, aliquot and store at -80 &#176;C until further analyses.</li>
</ol>
3. Induction of subcutaneous tumors
NOTE: The murine Panc02 and DT6606 cell lines were obtained from Prof. Lorenzo Piemonti (San Raffaele Diabetes Institute, Milan, Italy) and Prof. Francesco Novelli (Center for Experimental Research and Medical Studies, Torino, Italy) respectively, as previously described12.
<ol>
	<li>Growth of tumor cells
	<ol>
		<li>Culture Panc02 and DT6606 cells in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS), 2mM L-Glutamine and 1% penicillin-streptomycin antibiotic.</li>
		<li>Thaw frozen cells 1-2 weeks before tumor injection, according to the growth rate of the cell line.</li>
		<li>Grow cells at 37 &#176;C with 5% CO2 and 95% humidity in sterile conditions.</li>
		<li>Detach cells with 0.025% Trypsin/EDTA solution for 5 min at 37 &#176;C when they reach 80% confluency and eliminate trypsin by centrifugation. 
		NOTE: DT6606 are primary, not immortalized, cells, derived from the LSL-KrasG12D-Pdx1-Cre mouse, and should not be passaged more than 3 times before injection in vivo in order to maintain their original characteristics. It is recommended to thaw DT6606 cells 7-10 days prior to injection.</li>
	</ol>
	</li>
	<li>Injection of tumor cells in vivo
	<ol>
		<li>Trypsinize cells (see step 3.1.4) and wash them once with Phosphate-Buffered Saline (PBS). Eliminate PBS by centrifugation and resuspend cells in fresh PBS before counting.</li>
		<li>Count the cells and resuspend them in PBS at a concentration of 0.5-1 x 107 cells/mL in order to have a final concentration of 0.5-1 x 106 cells/100 &#956;L to inject in each mouse. Prepare the cells in excess. Keep the cells at 4 &#176;C or on ice until the end of the procedure.</li>
		<li>Group the animals (8-week old female C57BL/6J mice) in different cages according to different cell lines or treatments.</li>
		<li>Restrain the animals manually and anaesthetize them using a mixture of Ketamine (80 mg/kg) and Xylazine (10 mg/kg) or according to locally-approved procedures.</li>
		<li>Shave the site of injection, usually a flank above a leg, with an electric shaver and carefully clean the site of injection with alcohol.</li>
		<li>Pipette up and down the cell suspension with a 1 mL syringe, removing any air bubbles by moving the piston up and down. Attach a 25 G needle to the syringe and push the piston upwards until the cell suspension reaches the needle opening.</li>
		<li>Pinch the skin of the flank with flat-tipped forceps and carefully insert the needle at the base of skin fold between the forceps without puncturing the peritoneal cavity or the musculature. To verify the correct position of the needle, gently try to move the tip of the needle sideways under the skin. The needle should move freely.</li>
		<li>Slowly inject 100 &#956;L of cell suspension (containing 0.5-1 x 106 cells), clamp gently the site of injection for a few seconds and slowly withdraw the needle without any sideways movement.</li>
		<li>Return the mouse back to its cage and monitor the recovery from anesthesia.</li>
		<li>Check tumor growth using a caliper for 3-4 weeks. Euthanize the animals when tumors reach approximately 0.5-1 cm3 using CO2 or according to locally-approved procedures.</li>
	</ol>
	</li>
</ol>
4. Isolation of tumor interstitial fluid (TIF)
<ol>
	<li>Excision of subcutaneous tumors
	<ol>
		<li>Block the limbs of the animals with paper tape and clean the skin with alcohol. Cut the skin open on the abdomen in order to separate it from the peritoneum and go on up to the limbs. Excise the tumor grown under the skin of the flank with the help of&#160;scissors, clamps and eventually a scalpel.</li>
		<li>Weigh the tumor and keep it in a clean tube on ice until following isolation of TIF.</li>
	</ol>
	</li>
	<li>Isolation of TIF by centrifugation
	<ol>
		<li>Cut the tumor in half, rinse the two parts quickly in PBS and blot them gently on filter paper to remove excess of PBS. Carry out these steps as fast as possible to avoid evaporation from the tumor.</li>
		<li>Immediately transfer the tumor into a 20 &#956;m nylon cell strainer affixed atop a 50 mL conical tube.</li>
		<li>Centrifuge the tube at 400 x g for 10 min at 4 &#176;C. 
		NOTE: This low speed centrifugation preserves cell integrity avoiding contamination of TIF by intracellular compartment. Intracellular content leakage can be tested in downstream applications for example by assessing the presence of intracellular housekeeping proteins, such as ribosomal proteins25.</li>
		<li>Recover the TIF from the bottom of the tube, eventually aliquot it and immediately freeze it on dry ice and store at -80 &#176;C until further analysis. 
		Optional: Based on the downstream proteomic analysis to be performed, dilute the sample in PBS with a protease inhibitor cocktail to avoid degradation of specific molecules. 
		NOTE: Depending on the composition of the tumor, in some cases very small tumors do not yield any fluid.</li>
	</ol>
	</li>
	<li>Isolation of TIF by elution
	<ol>
		<li>Cut the tumor into small pieces (&#8776;1-3 mm3) with scissors or a scalpel and rinse carefully with cold PBS. 
		NOTE: In this step it is very important to work fast and perform minimum manipulation to avoid cell damage.</li>
		<li>Transfer the tumor pieces into a 1.5 mL tube and add 500 &#956;L of PBS with a protease inhibitor cocktail to avoid degradation of analytes. Incubate for 1 hour at 37 &#176;C and 5% CO2.</li>
		<li>Recover the supernatant and transfer it to a new 1.5 mL tube. Centrifuge at 1,000 x g for 5 min at 4 &#176;C to remove any cells from the sample.</li>
		<li>Transfer the supernatant to a new tube and centrifuge again at 2,000 x g for 8 min at 4 &#176;C.</li>
		<li>Transfer the supernatant to a new tube and centrifuge again at 20,000 x g for 30 min at 4 &#176;C to remove any debris. Recover the supernatant. Immediately aliquot and freeze TIF sample on dry ice and store at -80 &#176;C until further analysis.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

In this study we have described the technique to intraoperatively collect pancreatic juice, a largely unexplored fluid biopsy. We have recently shown that pancreatic juice can be exploited as a source of metabolic markers of disease12. Metabolomic analysis on other liquid biopsies, such as blood5,6,7, urine8, and saliva9, have shown promising results in disc...

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors, and soon to become the second leading cause of death1,2,3. It is well-known for its immunosuppressive microenvironment and for its unresponsiveness to immunotherapy protocols4. Currently, surgical resection is still the only curative option for PDAC, yet there is a high frequency of early relapses and postsurgical complications. The lack of specific symptoms until an advanced stage does not allow for an early diagnosis, contributing to the deadliness of the disease. Furthermore, the overlap of symptoms between PDAC and other benign pancreatic pathologies can hamper the achievement of a prompt and reliable diagnosis with the current diagnostic strategies. The identification of variables associated to specific pancreatic pathologies could facilitate the surgical decision-making process and improve patient profiling.
Promising results in biomarker discovery have been achieved using easily accessible body fluids, such as blood5,6,7, urine8, saliva9 and pancreatic juice10,11,12. Many studies have exploited comprehensive &#8220;omics&#8221; approaches, such as genomic, proteomic and metabolomic techniques, to identify candidate molecules or signatures that could discriminate between PDAC and other benign pancreatic afflictions. We recently demonstrated that pancreatic juice, a relatively unexplored body fluid, can be used to identify metabolic signatures of patients with distinct clinical profiles12. Pancreatic juice is a protein-rich fluid, which accumulates the secretome of pancreatic ductal cells and flows to the main pancreatic duct, and then to the main common bile duct. Due to its proximity to the pancreas, it could be strongly affected by microenvironmental perturbations induced by the tumor mass (Figure 1), and therefore more informative than blood or urine, or tissue-based profiling. Several studies have explored the potential of pancreatic juice to identify novel biomarkers of disease using various approaches, including cytologic analysis13, proteomic analysis performed by mass-spectrometry14,15, assessment of genetic and epigenetic markers such as K-ras and p53 mutations16,17, alterations in DNA methylation18, and miRNAs19. Technically, pancreatic juice can be collected intraoperatively or with minimally invasive procedures, such as endoscopic ultrasound, retrograde cholangio-pancreatography, or by endoscopic collection of duodenal juice secretion20. It is not yet clear to what extent pancreatic juice composition is affected by the collection technique used. We describe here the intraoperative collection procedure and show that pancreatic juice can represent a precious source for PDAC biomarkers.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61687/61687fig01.jpg" /> 
Figure 1: Schematic representation of pancreatic juice collection.&#160;(A) Schematic representation depicting the secretion of pancreatic juice into the pancreatic duct and its collection during surgery. The inset shows a close-up of the tumor microenvironment: pancreatic juice collects molecules released by tumor and stromal cells in the pancreatic ducts. <a href="https://www.jove.com/files/ftp_upload/61687/61687fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The collection of pancreatic juice in genetic and orthotopic mouse models of PDAC would be appreciated in the perspective to exploit this biofluid in preclinical mechanistic studies; however, this procedure can be technically very challenging and is not feasible for simpler models such as subcutaneous tumors. For this reason, we identified tumor interstitial fluid (TIF) as an alternative source to pancreatic juice, for its similar characteristic of acting as an indicator of surrounding perturbations. Interstitial fluid (IF) is the extracellular liquid, found outside blood and lymphatic vessels, which bathes tissue cells21. IF composition is affected by both blood circulation to the organ and local secretion; in fact, surrounding cells actively produce and secrete proteins in the IF21. The interstitium reflects microenvironmental changes of surrounding tissues and could therefore represent a valuable source for biomarker discovery in several pathological contexts, such as tumors. The high concentration of locally secreted proteins in TIF can be used to identify candidate molecules to be tested as prognostic or diagnostic biomarkers in plasma22,23,24. Several studies have proven TIF to be a suitable sample for high-throughput proteomic approaches, such as mass spectrometry techniques23,24,25, as well as multiplex ELISA approaches26, and microRNA profiling27.
Several approaches have been proposed for the isolation of IF in tumors, which can be broadly categorized as in vivo (capillary ultrafiltration28,29,30,31 and microdialysis32,33,34,35) and ex vivo methods (tissue centrifugation22,36,37,38 and tissue elution39,40,41,42). These techniques have been reviewed in extensive detail43,44. The choice of the appropriate method should take into account issues such as the downstream analyses and applications and the volume recovered. We recently used this approach as a proof of principle to demonstrate the different metabolic activity of tumors from two murine pancreatic adenocarcinoma cell lines12. Based on literature24,38, we chose to use the low speed centrifugation method to avoid cell breakage and dilution from intracellular content. Both the amount of glucose and lactate in TIF reflected the different glycolytic characteristics of the two different cell lines. Here we describe in detail the protocol for the two most commonly used methods for the isolation of TIF: tissue centrifugation and tissue elution (Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61687/61687fig02.jpg" /> 
Figure 2: Schematic representation of tumor interstitial fluid isolation methods.&#160;Schematic illustration of the techniques described in detail in the protocol, namely tissue centrifugation (A) and tissue elution (B). <a href="https://www.jove.com/files/ftp_upload/61687/61687fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>BD Biosciences</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tube</td>
			<td>Greiner BioOne</td>
			<td>GR616201</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;m nylon cell strainer</td>
			<td>pluriSelect</td>
			<td>43-50020-03</td>
			<td></td>
		</tr>
		<tr>
			<td>25G needle</td>
			<td>BD Biosciences</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL K2EDTA vacutainer</td>
			<td>BD Biosciences</td>
			<td>366473</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>BD Biosciences</td>
			<td>309656</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>Corning</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamps</td>
			<td>Medicon</td>
			<td>06.20.12</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable scalpel</td>
			<td>Medicom</td>
			<td>9000-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Microtech</td>
			<td>MG10432</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat-tipped forceps</td>
			<td>Medicon</td>
			<td>06.00.10</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Lonza</td>
			<td>ECB3001D</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>34044100</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium</td>
			<td>Euroclone</td>
			<td>ECB9006L</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Medicon</td>
			<td>02.04.09</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA 1x</td>
			<td>Lonza</td>
			<td>BE17-161F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraglutamine 100x</td>
			<td>Lonza</td>
			<td>BE17-605E/U1</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

We followed the procedure described above to obtain pancreatic juice from patients with PDAC (n=31) and other benign pancreatic afflictions (non-PDAC, n=9), including pancreatitis (n=2), papillary-ampulla tumors (n=4), neuroendocrine tumors (n=2), intraductal papillary mucinous neoplasia (IPMN; n=1)12. The pancreatic juice samples were then subjected to metabolomic analysis using nuclear magnetic resonance (1H-NMR)12. By filtering the broad NMR signa...

Watch this Scientific Journal Video about Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma at JoVE.com

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

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 ...

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Cancer Research

3.1K Views.  Humanitas Clinical and Research Center-IRCCS. 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.

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

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the 3-dimensional (3D) modeling of this disease have been described. Patient-derived organoid (PDO) models can be isolated from both surgical specimens as well as small biopsies and form rapidly in culture. Importantly, organoid models preserve the pathogenic genetic alterations detected in the patient&#39;s tumor and are predictive of the patient&#39;s treatment response, thus enabling translational studies. Here, we provide comprehensive protocols for adapting tissue culture workflow to study 3D, matrix embedded, organoid models. We detail methods and considerations for isolating and propagating primary PDAC organoids. Furthermore, we describe how bespoke organoid media is prepared and quality controlled in the laboratory. Finally, we describe assays for downstream characterization of the organoid models such as isolation of nucleic acids (DNA and RNA), and drug testing. Importantly we provide critical considerations for implementing organoid methodology in a research laboratory.

Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these models.

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations. To characterize proteome-level variation and tumor heterogeneity at scale, high-throughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (H&#38;E)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.

This protocol describes a high-throughput workflow for artificial intelligence-driven segmentation of pathology-confirmed regions of interest from stained, thin tissue section images for enrichment of histology-resolved cell populations using laser microdissection. This strategy includes a novel algorithm enabling the transfer of demarcations denoting cell populations of interest directly to laser microscopes.

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Mapping the Tumor Immune Landscape: An In-Depth Multiplex IHC Analysis Protocol

Multiplex Immunohistochemical Analysis of the Spatial Immune Cell Landscape of the Tumor Microenvironment

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. Multiplex immunohistochemistry is a valuable tool to visualize and identify different types of immune cells in tumor tissues while retaining its spatial information. Here we provide detailed protocols to analyze lymphocyte, myeloid, and dendritic cell populations in tissue sections. Starting from cutting formalin-fixed paraffin-embedded sections, automatic multiplex staining procedures on an automated platform, scanning of the slides on a multispectral imaging microscope, to the analysis of images using an in-house-developed machine learning algorithm ImmuNet. These protocols can be applied to a variety of tumor specimens by simply switching tumor markers to analyze immune cells in different compartments of the sample (tumor versus invasive margin) and apply nearest-neighbor analysis. This analysis is not limited to tumor samples but can also be applied to other (non-)pathogenic tissues. Improvements to the equipment and workflow over the past few years have significantly shortened throughput times, which facilitates the future application of this procedure in the diagnostic setting.

Author Spotlight: Unlocking Insights into the Immune Cell Landscape of Tumors

This protocol describes in detail how immune cell characterization of the tumor microenvironment using multiplex immunohistochemistry is carried out.

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. ...

Murine Pancreatic Imaging Using Implanted Stabilized Window

Stabilized Window for Intravital Imaging of the Murine Pancreas

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

Author Spotlight: Revolutionizing Pancreatic Disease Understanding Through Advanced Intravital Imaging 

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

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