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

Summary

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.

Abstract

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.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors, and soon to become the second leading cause of death^1,2,3. 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. Furthermo....

Protocol

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 pancre....

Results

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.......

Discussion

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 blood^5,6,7, urine⁸, and saliva⁹, have shown promising results in disc.......

Materials

List of materials used in this article

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