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 therapy^2,3. Therefore, understanding the molecular pathways that trigger invasion is essential to better characterize the risk of invasive progression and to develop therapeutic interventions which can prevent invasive progression.

Tumor invasive progression occurs in a complex three-dimensional (3-D) environment and involves tumor cell interaction with other tumor cells, stroma, basement membrane, and other types of cells including immune cells, fibroblasts, muscle cells and vascular endothelial cells. Permeable support (e.g., Transwell) assay systems are commonly employed to quantitate cancer cell invasion⁴, but these systems are limited because they do not allow microscopic monitoring of the invasion process in real-time and the retrieval of samples for further staining and molecular analysis is challenging. Development of a 3-D bladder tumor spheroid system to study invasion is desirable because it allows the incorporation of defined microenvironmental components with the convenience of in vitro systems.

In this protocol, we describe a system to interrogate the invasive processes of human bladder cancer cells using a 3-D spheroid invasion assay incorporating collagen-based gel matrices and confocal microscopy to allow investigators to monitor cell motility and invasion in real-time (Figure 1A). This system is versatile and can be modified to interrogate various stromal/tumor settings. It can incorporate most bladder cancer cell lines or primary bladder tumors and additional stromal cells such as cancer associated fibroblasts and immune cells^5,6,7. This protocol describes a matrix composed of type-1 collagen, but can be modified to incorporate other molecules such as fibronectin, laminin, or other collagen proteins. Invasive processes can be followed for 72 h or longer depending on the capability of the microscope and system used. Fixation and immunofluorescence staining of the tumor embedded in the 3-D matrix before, during, and after invasion allows the interrogation of proteins upregulated in invasive cells, thus providing crucial information that usually absent or difficult to gather using other 3-D culture models. This system can also be utilized to screen compounds which block invasion, and to delineate signaling pathways affected by such compounds.

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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.
   2. One day prior to the start of the experiment, trypsinize (by using 0.25% Trypsin-EDTA) cells, quantify cell concentration and distribute 1 x 10⁶ cells in 3 mL culture media in each well of a 6-well ultra-low attachment plate.
   3. Incubate cells in low attachment conditions at 37 °C for ≥16 h. This should allow adequate time for cells to aggregate into spheroids for most bladder cancer cell lines.
      NOTE: The formation of spheroids can be observed by inverted bright field microscope. The optimal size and number of spheroids may depend on individual experiments, but we find that spheroids with diameters from 50 µm to 150 µm are generally suitable for time-lapse imaging using a 20X objective lens.
   4. To incorporate additional cell types, such as fibroblasts or immune cells into the spheroids, mix the desired cells with cancer cells at desired ratio (1:1, 1:10, etc.) in an ultra-low attachment plate and incubate at 37 °C for ≥16 h to allow formation of mixed cell type spheroids.
2. Growing from primary tumors
   NOTE: Tumor spheroids can also be derived from primary bladder tumor sources such as tumors developed from carcinogen-induced bladder tumor models⁸. For example, bladder tumors generated from mice fed with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), can be harvested and minced into small pieces (about 0.5 mm³) using sterile surgical instruments. Digested primary samples of human bladder cancers can also be studied in this system.
   1. Collect and wash 0.5 mm³ tumor pieces in 5 mL ice-cold phosphate-buffered saline (PBS). Centrifugation at 200 x g for 5 min at 4 °C. Repeat this washing step once.
   2. Collect tumor pieces by centrifugation at 200 x g for 10 min at 4 °C. Remove the supernatant and add 5 mL of DMEM containing 10% FBS. Transfer tumor spheroid/media mixture to a 6-well ultra-low attachment plate. Incubate at 37 °C for ≥16 h before embedding into collagen matrix (see step 2.3).

2. Preparing the 3-D culture chamber

1. Dilute type-1 collagen (derived from rat tail) in DMEM containing 10% FBS to make a 2 mg/mL mixture according to manufacturer's instructions.
   1. In short, mix collagen with appropriate amount of 1 N NaOH solution and DMEM by gentle pipetting based upon the manufacturer's instructions to achieve physiologic pH.
   2. After mixing collagen and DMEM, quickly coat the wells of the chamber slide (for most applications, chamber slides with a number 1.5 cover-glass is optimal) with the collagen-DMEM mixture prior to solidification (200 µL/well).Allow the coated chamber slide to be stationary at room temperature for 15 min.
      NOTE: The composition of collagen-based matrix can be modified by adding other extracellular matrix ingredients, such as fibronectin or laminin, according to the purposes of experiment.
2. Gently pipet 500 µL of media containing spheroids (or tumor pieces, if primary tumor sample is used) from the 6-well ultra-low attachment plate into an empty 1.5 mL microcentrifuge tube. Wait for 2 min to let the spheroids settle to the bottom of the tube.
3. Carefully remove the supernatant from the tube. Prepare another tube of type-1 collagen (2 mg/mL) mixed with DMEM containing 10% FBS and quickly add 500 µL of this mixture to the spheroids. Gently mix the spheroids and collagen by slow pipetting.
4. Add 250 µL of spheroids/collagen mixture to a well of collagen-pre-coated chamber slide. Allow the collagen matrix containing spheroids to solidify completely (about 30 min at 37 °C).
5. After the collagen matrix is solidified, add 1 mL of DMEM containing 10% FBS to each well (Figure 1B). Incubate the chamber slide in a 37 °C incubator supplied with 5% CO₂ until ready for imaging.

3. Live Cell Time-lapse Imaging

1. Turn on the confocal microscope following the manufacturer's instructions and ensure that the climate chamber reaches 37 °C and is supplied with 5% CO₂.
   NOTE: Our microscope and chamber usually require 1 h to reach system equilibrium.
2. Carefully transfer the chamber slide to the slide adaptor attached to the microscope.
3. Locate the spheroids of interest using a low power objective (e.g., 5X) and start imaging with the higher power objective (in current protocol, 20X objective is used for most of the live cell imaging for its better image quality).
   NOTE: For primary bladder tumor samples, 5X and 10X objectives may be needed in order to cover larger imaging area. For our time-lapse imaging, the imaging interval is set as 30 min and a Z stack is used to image the whole spheroid. Typically the distance between Z-slices is set to 4 µm for the 20X objective, and the total distance of Z axis is around 200 µm. Images can be obtained using DIC and with fluorescence if cells/tissues harbor fluorescent protein markers.
4. Perform time-lapse imaging for 24–72 h.
   NOTE: The duration is determined by the cell type and the needs of the experiment.

4. Preparation of Sample Block Containing Cancer Spheroids for Frozen Tissue Sectioning

1. Carefully lift the block of collagen gel and spheroids from the chamber slides by using small forceps. Place the collagen gel block in a plastic histology mold.
2. Rinse the collagen gel with 1x PBS briefly, and then fix it with 4% paraformaldehyde (PFA) in PBS for 30 min at room temperature.
   CAUTION: PFA is potential carcinogen and should be handled with care.
3. Wash the collagen gel with 1.5 mL of 1x PBS on a shaker for 15 min. Replace the PBS and repeat the previous washing step 3x.
4. Apply a thin layer (about 3 mm) of optimal cutting temperature (OCT) compound to cover the bottom of a new plastic histology mold. Place the fixed and washed collagen gel on top of the OCT compound, then carefully embed the whole gel by filling the mold with OCT compound while avoiding the formation of any air bubbles in the OCT.
5. Leave the mold at 4 °C for 1 h.
6. Place the mold containing the sample and OCT compound on a 100 mm Petri dish floating on liquid nitrogen. Allow the sample to flash-freeze completely.
7. Store the frozen sample block at -80 °C for future use.

5. Immunofluorescence Imaging for Frozen Sectioned Cancer Spheroids

1. Transfer the frozen sample block from the -80 °C freezer to the -20 °C chamber of a cryostat.
2. Perform conventional frozen sectioning by setting the section interval to 7 µm. Let sectioned samples attach to the glass slide without wrinkles or trapped air.
   1. Store the slides at -80 °C before performing further staining.
      NOTE: Different intervals may be used to fit experimental needs.
3. Air dry the slides for 1 h at room temperature.
4. Permeabilize the samples with PBS containing 0.5% Triton X-100 for 15 min.
5. Wash samples 3x with PBS for 10 min per wash.
6. Encircle the samples on the slide with hydrophobic barriers by using a hydrophobic barrier pen.
7. Treat the samples with blocking solution (1x PBS containing 5% bovine serum albumin (BSA)), for 1 h at room temperature.
8. Apply 40 µL of primary antibody-containing solution (1x PBS containing 5% BSA with 1:100 dilution of primary antibody) to each sample on the slide.
9. Incubate the slides in primary antibody at 37 °C for 1 h or at room temperature overnight (the incubation time and conditions vary depending on primary antibody).
10. Wash samples 3x with PBS for 15 min (per wash).
11. Apply 40 µL of secondary antibody-containing solution (1x PBS containing 5% of BSA with 1:300 secondary antibody dilution) to each sample on the slide.
12. Wash samples 3x with PBS for 15 min (per wash).
13. Stain the samples with Hoechst 33342 solution (1 µg/mL, diluted in PBS) at room temperature for 10 min. Then wash samples with PBS for 5 min.
14. Mount the samples with mounting medium and cover them with appropriately sized cover slips. Leave the slides in dark at room temperature for 24 h and then perform confocal microscopy.

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Results

Successful creation of invasive bladder cancer tumor spheroid requires the formation of appropriately sized tumor spheroids from cell lines or primary tumors. Figure 2A shows appropriately sized spheroids developed from four human bladder cancer cell lines (UM-UC9, UM-UC13, UM-UC14, 253J, and UM-UC18). Figure 2B shows a tumor spheroid from a BBN-generated mouse bladder tumor embedded in collagen ...

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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...

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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