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

Summary

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.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of <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 °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 (451 µm x 462.84 µm)) and can be utilized for experimental therapy assessment. Mature ECM components include collagen-I, hyaluronic acid, fibronectin, and laminin.

Introduction

Pancreatic cancer'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's chemo and radiotherapeutic resistan....

Protocol

1. 2D cell culture

1. Grow PANC-1 cells in Dulbecco'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 20^th passage. Refer to the process described in step 4.2.1.
   NOTE: As a reference point, 1 x 10⁶ cells in 20 mL of media need about 2-3 days to reach 70-80% confluency.
2. Grow HPaSteC cells in stellate cell media supplemented.......

Discussion

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.......

Materials

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