3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Summary

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

Abstract

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

Introduction

Invasion is a critical step in cancer progression, which is required for metastasis, and is associated with lower survival and poor prognosis in patients. In human bladder cancer, the most common malignancy of the urinary tract which causes about 165,000 deaths per year worldwide, cancer stage, treatment and prognosis are directly related to the presence or absence of invasion¹. Around 75% of the cases of bladder cancer are non-muscle invasive and are managed with local resection. In contrast, muscle-invasive bladder cancers (about 25% of all cases) are aggressive tumors with high metastatic rates and are treated with aggressive multimodality t....

Protocol

1. Growing Cancer Spheroids

1. Growing from cell lines
   1. Culture human bladder cancer cells under conventional adherent cell culture conditions and maintain in a 37 °C incubator supplied with 5% CO₂. Maintain cells at <90% confluency.
      NOTE: Culture media used is Dulbecco's modified minimum essential media (DMEM) containing 4.5 g/L D-glucose, L-Glutamine, 110 mg/L sodium pyruvate, and supplied with 10% fetal bovine serum (FBS) throughout this protocol.

Discussion

Here we describe a 3-D tumor spheroid model that allows real-time observation of bladder cancer invasion which is critical for cancer progression and metastasis. This system is amenable to the incorporation of various stromal and cellular components to allow investigators to better recapitulate the tissue microenvironment where bladder cancer invasion takes place. Bladder cancer spheroids can be generated from various sources such as cell lines (including genetically modified cell lines useful for the examination of sign.......

Materials

Name	Company	Catalog Number	Comments
Human bladder cancer cell lines UM-UC9, UM-UC13, UM-UC14, UM-UC18, 253J
DMEM cell culture medium	Thermo Fisher Scientific	11995065
Fetal bovine serum	Thermo Fisher Scientific	26140079
Antibiotic-Antimycotic (100X)	Thermo Fisher Scientific	15240062
Trypsin-EDTA (0.25%), phenol red	Thermo Fisher Scientific	25200056
Bovine serum albumin (BSA)	Sigma-Aldrich	A3803
Phosphate-buffered saline (PBS), pH 7.4	Thermo Fisher Scientific	10010023
Costar Ultral-low attachment 6-well cluster	Corning	3471
Conventional inverted microscope	Carl Zeiss	491206-0001-000	General use for cell culture and checking spheroids
Collagen type 1 from rat tail, high concentration	Corning	354249
Nunc Lab-Tek II Chambered Coverglass	Thermo Fisher Scientific	155382
Confocal microscope	Carl Zeiss	LSM800	A confocal miscoscope with climate chamber, multi-location imaging, and Z-stack scanning function
Cryostat micromtome	Leica Biosystems	CM3050 S
Zen 2 Image processing software	Carl Zeiss
Paraformaldehyde solution	Electron Microscopy Sciences	15710
ImmEdge Hydrophobic Barrier PAP Pen	Vector Laboratories	H4000
O.C.T compound	Thermo Fisher Scientific	23730571
Hoechst 33342 solution	Thermo Fisher Scientific	62249
Anti-ATDC (Trim29) antibody	Sigma-Aldrich	HPA020053
Anti-Cytokeratin 14 antibody	Abcam	ab7800
Anti-Vimentin antibody	Abcam	ab24525
ProLong Diamond			Mounting medium