Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Summary

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

Abstract

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Introduction

Ovarian cancer is the deadliest gynecologic malignancy¹. The majority of patients are diagnosed after the cancer has disseminated throughout the peritoneal cavity. Once the cancer has spread throughout the peritoneal cavity, cytoreductive surgery and chemotherapy are often not sufficient treatment to prevent cancer recurrence and chemoresistance, resulting in a less than 30% 5-year survival rate. Ovarian cancer metastasis is predominantly limited to the peritoneal cavity, and several other cancer types, including gastric, pancreatic, and colon cancers, metastasize to the same anatomic sites in the peritoneal cavity. In general, ovarian cancer cells detach from the in situ carcinoma in the fallopian tube or the primary ovarian tumor, travel in peritoneal fluid as single cells or spheroids, and attach to mesothelium-lined surfaces of the omentum, bowel, and abdominal wall².

The tumor microenvironment plays an important role in disease progression and chemoresistance in many cancers^3-6. The peritoneal cavity is a unique microenvironment, with a mesothelial cell monolayer covering the majority of surfaces (Figure 1A)⁷. The mesothelial lining acts as a barrier that creates a low-friction surface, which tends to be protective against cancer cell adhesion⁸. Immediately underneath this mesothelial-lined surface is a layer made predominantly of fibroblasts and extracellular matrix (ECM), which promote cancer cell adhesion and invasion⁸. Ovarian cancer cells secrete factors that induce changes in the mesothelial cell lining that enhance ovarian cancer cell adhesion, invasion, and metastasis^9,10. Ovarian cancer cells adhere to the mesothelial surface via integrin and CD44-mediated mechanisms (Figure 1B)^11-16.

Historically, several 3D models have been developed to investigate ovarian cancer interactions with the microenvironment. Some of the first models studied ovarian cancer-ECM interface^17-21, ovarian cancer-mesothelial cell communication^13,14,21-24, or both²⁵ (reviewed by us ²⁶). Niedbala et al. discovered that ovarian cancer cells display a quicker and firmer adhesion to ECM than to mesothelial cells or to plastic alone²⁵. However, these models did not histologically resemble the peritoneal microenvironment. Therefore, we established a 3D organotypic model to more thoroughly replicate the ovarian cancer microenvironment. In order to better understand the role of the microenvironment and the interaction between cancer and peritoneal cells in the peritoneal dissemination of ovarian cancer, we have developed a 3D organotypic in vitro culture model of the peritoneal cavity lining (schematic in Figure 1C). The proposed model is composed of primary human fibroblasts and ECM, covered with a layer of primary human mesothelial cells-each cell type is isolated from human omentum. Histologically, this model resembles the normal peritoneal or omental lining, and provides a surface on which we can study the tumor microenvironment, the interaction between cancer cells and normal tissue, and the processes of cancer cell adhesion, invasion, and proliferation⁸.

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Protocol

All research protocols described have been reviewed by the University of Chicago Institutional Review Board (IRB). Informed consent was obtained from each patient before surgery and the study was approved by the University of Chicago IRB. A biological safety cabinet type 2 and gloves should be used when handling human tissue for protection and to reduce risk of contaminating cells.

1. Isolation and Culture of Primary Untransformed Stromal Cells

1. Human tissue collection and preparation.
   1. Obtain specimens of human omentum, 2 cm³, removed during an abdominal surgery, and immediately immerse tissue in RT phosphate-buffered saline (PBS) (Figure 2A). A piece of omentum 3 cm x 2 cm typically yields 1 million primary human mesothelial cells (HPMC) and 200,000 primary human fibroblasts or normal omental fibroblasts (NOF).
   2. Spin down the PBS the tissue was immersed in at 0.5 x g for 3 min as soon as possible after collection (< 2 hr in PBS) and transfer the tissue to fresh 20 ml PBS in a 50 ml conical tube. Cut up tissue into 5mm³ pieces by mincing with scalpels in a 15 cm diameter, sterile culture dish (Figure 2B).
2. Primary human mesothelial cell isolation⁸
   1. To isolate HPMC, transfer the minced tissue into 50 ml conical tubes and wait 1 min for the solid pieces to float to the top (Figure 2C & D). HPMC and red blood cells (RBC) will be present in the liquid on the bottom. Use a pipet to remove the liquid from the bottom of the tube and into a new conical tube. Spin the liquid down at 0.5 x g for 3 min and aspirate the supernatant from the HPMC/RBC pellet.
      1. Repeat the process three times: add 20 ml PBS to the minced tissue, allow tissue to rise, remove liquid from the bottom with a pipette and into the HPMC/RBC tube, spin down, and remove the supernatant. After all spins are completed and the supernatant is aspirated, the pellet will contain HPMC and RBC.
   2. Plate all cells from the pellet into a 75 cm² flask in 15 ml of full growth media (DMEM with 10% fetal bovine serum [FBS],1% MEM vitamins [93 mg/L], 1% MEM nonessential amino acids [81.4 mg/L], 1% penicillin streptomycin [Pen-Strep, 100 U/ml penicillin and 100 µg/ml streptomycin]).
   3. Perform a secondary PBS wash to isolate additional HPMCs.
      1. For the secondary PBS wash, shake the remaining solid tissue at 200 rpm, 37 °C for 10 min in 20 ml of PBS, then wait 1 min for the tissue to float to the top. Remove the liquid from the bottom of the tube into a new tube, centrifuge at 0.5 x g for 3 min, and aspirate the supernatant.
      2. Plate all the HPMC/RBC from the secondary PBS wash in a separate 75 cm² flask in 15 ml of full growth media.
   4. To isolate any remaining HPMC, shake the minced tissue at 200 rpm, 37 °C degrees for 10 min in 20 ml of a 1:1 0.25% trypsin 25 mM EDTA:PBS solution by volume. Allow the tissue to rise, collect the liquid at the bottom of the tube into a new 50 ml tube, and spin down at 0.5 g x 3 min.
      1. Aspirate the supernatant and plate all the HPMC/RBC in a separate 75 cm² flask in 15 ml of full growth media.
   5. Culture the plated cells at 37°C and 5% CO₂ in a humidified environment. Feed plated cells by adding 15 ml of full growth media on days 3 and 5 without removing the spent media. HPMC can be cultured for 5-7 days before splitting.
      1. Use low-passage HPMC (up to passage 2) in all experiments to minimize dedifferentiation and modification of original phenotype. Use a wide-field microscope preferably with a 20x objective with plastic differential interference contrast capabilities or 4-20x objective with phase contrast capabilities (phase contrast filters on microscope) for taking all images of cells, and a 10x objective ()in bright field to analyze immunohistochemical 3,3'-Diaminobenzidine (DAB) staining.
   6. Confirm that the HPMC are cuboidal and express cytokeratin 8 and vimentin by performing immunohistochemistry⁸ (Figure 3A, C, E).
3. NOF isolation⁸
   1. Prepare 10 ml of a stock of collagenase type III solution (10x) by combining a 1:1:1 mixture of 100x Pen-Strep: 1,500 units activity/ml of collagenase type III: 714 units activity/ml of hyaluronidase in PBS.
   2. Shake the minced omentum tissue that remains after HPMC isolation at 200 rpm, 37 °C for 6 hr in 10 ml of the 10x collagenase type III solution diluted in serum-free media (DMEM with 1% MEM vitamins [93 mg/L], 1% MEM nonessential amino acids [81.4 mg/L], 1% penicillin streptomycin [Pen-Strep, 100 U/ml penicillin and 100 µg/ml streptomycin]). Digested tissue will look opaque and may have some fibrous debris (Figure 2E).
   3. Centrifuge the solution containing NOF cells in suspension at 0.5 x g for 3 min at RT, aspirate the supernatant and plate the pellet in a 75 cm² flask in 13 ml of full growth media.
   4. Remove old media and replace with 15 ml of fresh full growth media after 24 hr. NOF can be cultured for 1-3 days before splitting. Note proliferation of the NOF will cease when the cells reach confluency.
   5. Confirm that the primary human fibroblasts are flat, elongated cells and express vimentin but not cytokeratin 8 by performing immunohistochemistry⁸ (Figure 3B, D, F). Use low-passage NOF for experiments (ideally before passage 3) to minimize dedifferentiation and modification of original phenotype. Use a wide-field microscope with a 20x objective with plastic DIC capabilities for taking all phase images of cells, and a 10x objective in bright field to analyze immunohistochemical DAB staining.

2. Plating the Organotypic Culture⁸

1. Release NOF from the 75 cm culture flask by rinsing with 10 ml of PBS followed by 3 ml of 0.25% trypsin/25 mM EDTA for no more than 5 min. Neutralize trypsin with at least 3 times the volume of full growth media.
2. Transfer trypsinized cells into a 50 ml conical tube and spin down at 0.5 g x 3 min. Remove supernatant and bring cells back up in 5 ml of full growth media. Use a cell counter to count the cells recovered from the culture flask.
3. Dilute cells in the appropriate volume of full growth media to plate 2,000-4,000 NOF per 100 µl onto a black, clear-bottomed, 96-well plate. Add 0.5 µg/100 µl of rat-tail collagen I to the cell mixture before plating. Let cells sit at 37 °C in 5% CO₂ in a humidified environment for at least 4 hr, or until the cells to adhere to the plate surface.
4. Release HPMC from the culture flask using the same methods described in steps 2.1 and 2.2. Dilute cells in the appropriate amount of full growth media to plate 10,000-20,000 HPMC per 50 µl on top of the NOF already plated with collagen I in 96-well plates. Let the cells sit at 37 °C in 5% CO₂ in a humidified environment for at least 18 hr before beginning experiments.
5. Culture green fluorescent protein (GFP)-expressing HeyA8 ovarian cancer cells in full growth media. HeyA8 ovarian cancer cells are fluorescently labeled by expression of a lentiviral vector expressing copepod cGFP (pCDH-CMV-MCS-EF1). HeyA8 ovarian cancer cells should be 80-90% confluent on the day of use (logarithmic growth phase). Release cells from the culture plate and count using the same methods described in steps 2.1-2.2 for primary cells.
6. Allow ovarian cancer cells to recover in full growth media for 15-20 min at 37°C in a conical tube with the lid loosely sealed.

3. Adhesion Assay⁸

1. After recovery, pellet the ovarian cancer cells by spinning for 3 min at 0.5 x g and then remove the supernatant and dilute the cells in the appropriate volume of serum-free media to achieve a concentration of 50,000 cells/100 µl.
2. Invert the 96-well plates with the HPMC/NOF organotypic cultures to remove spent media, and add 100 µl of the cancer cell suspension in serum-free media to each well. Incubate the plate at 37 °C in 5% CO₂ in a humidified environment for 30 min to 4 hr.
3. Invert the 96-well plate to remove media and non-adherent cells. Use a multichannel pipet at low power to carefully and gently pipet 100 µl of PBS into each well, and invert plate again. Repeat the PBS wash once more.
4. Invert to remove PBS and add 100 µl 4% paraformaldehyde to each well. Allow the plate to sit for 20 min to fix the cells. After 20 min, invert the plate and add 100 µl PBS.
5. Note that the total fluorescence of wells can be reported or the number of cells can be quantified. For quantification, first plate a standard curve in duplicate using HeyA8 cells at a concentration of 1 million cells/ml.
   1. Make the standard curve by spinning down 1 million cells, removing media, and allowing them to sit in 1 ml 4% paraformaldehyde for 20 min.
   2. Spin cells again and bring up in 1 ml PBS (recount cells to confirm 1 million/ml concentration). Plate 50,000, 40,000, 30,000, 20,000, 10,000, 5000, 1,000, and 0 cells into each well in duplicate, and add PBS for a final total volume of 50 µl in each well.
6. Bottom read the culture plate on a fluorescent plate reader (excitation = 488 nm, emission = 528 nm), scaling high wells (50,000 on standard curve). Use the standard curve to calculate how many ovarian cancer cells adhered to the organotypic culture in each well (Figure 4A-C).

4. Proliferation Assay¹⁰

1. After the ovarian cancer cells recover (see steps 2.5 and 2.6 above), pellet the cells by spinning for 3 min at 0.5 x g and then dilute the cells in the appropriate volume of 1% FBS media (DMEM with 1% FBS, 1% MEM vitamins [93 mg/L], 1% MEM nonessential amino acids [81.4 mg/L], 1% penicillin streptomycin [Pen-Strep, 100 U/ml penicillin and 100 µg/ml streptomycin]) to achieve a concentration of 4,000 cells/150 µl.
2. Invert the 96-well plate with the HPMC/NOF organotypic cultures to remove spent media, and add 150 µl of the cancer cell suspension in 1% FBS media to each well. Incubate the plate at 37°C in 5% CO₂ in a humidified environment for 72 hr.
3. Bottom read the culture plate on a fluorescent plate reader once a day (excitation = 488 nm, emission = 528 nm) to evaluate the relative proliferation of cells. Continue the assay for 96 hr, changing the media at 48 hr (Figure 5A-C). Be careful not to disturb the culture when changing media (inverting the plate is preferable).

5. Invasion Assay⁸

1. Before plating the 3D culture, add 7.5 µg rat-tail collagen I in 200 µl PBS to a 24-well culture plate insert (8 µm pore size). Let the plate incubate O/N, then carefully remove the PBS with a pipet immediately before plating the HPMC/NOF organotypic culture onto the collagen-coated inserts (step 2, above).
2. Prepare the ovarian cancer cells as in step 3.1. To plate the ovarian cancer cells onto the organotypic cultures on the inserts, carefully use a pipet to remove excess media from the top of the inserts. Add 100 µl of the ovarian cancer cell suspension in serum-free media to each well.
3. Add 750 µl of full growth media into the well below the insert to induce cancer cell invasion into the organotypic cultures. Incubate the plate at 37 °C in 5% CO₂ in a humidified environment for 24 hr.
   Note: Ovarian cancer cells that invade the organotypic culture will cross the insert and remain on the bottom side of the insert.
4. After 24 hr, grasp the insert with tongs and briefly rinse it in a beaker of PBS, then move the insert into an empty well. Scrub the top of each insert with both sides of a cotton swab for a total of 1 min. Quickly place the insert into a plate containing 750 µl 4% paraformaldehyde in each well. Allow each insert to sit in paraformaldehyde for 10 min before transferring the inserts into wells filled with 750 µl PBS.
5. View the invaded cells (adhered and fixed to the bottom of inserts) with a microscope at 2.5x magnification. When the entire well is within the field of view, adjust the magnification to 10x and take 5 pictures, one near the center and 1 in each quadrant of the well, avoiding taking images that are too close to the edges. Follow the same pattern for each well.
6. Use the manufacturer's program to count the number of cells in each image to get an average number of cells invaded per field in each well (Figure 6A-C) according to their protocol.

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Results

The organotypic culture was assembled by first mixing primary human fibroblasts with collagen type I and then overlaying this culture with 5 times the number of mesothelial cells. The culture was incubated for at least 18 hr before ovarian cancer cells were added to study adhesion, invasion or proliferation. Each assay was repeated with multiple (n=3-5) 3D cultures obtained from different patients and numerous wells were tested in each condition for the adhesion (n=5), proliferation (n=5) and invasion assays (n=3). ...

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Discussion

An organotypic model of the peritoneal microenvironment was established to assess the individual and collective function(s) of both the cellular and innate components of the microenvironment in ovarian cancer dissemination. The specific protocols for plating and customizing the 3D organotypic culture to investigate ovarian cancer cell adhesion, proliferation, and invasion are provided. Primary human omental mesothelial cells and fibroblasts were isolated from patients and used at an early passage to preserve normal morph...

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Materials

Name	Company	Catalog Number	Comments
1. Isolation and culture of primary cells
PBS	Fisher Scientific	SH3001304
Single-edged razor blades	Fisher Scientific	12-640
15 cm culture dishes	BD Biosciences	353025
Glass flask	?	?
Fetal Bovine Serum (FBS)	Life Technologies	16000044_3616914956
DMEM with L-Glutamine	Corning	10-013-CV
MEM Vitamins	Corning	25-020-Cl
MEM Nonessential amino acids	Corning	25-025-CI
Penicillin-Streptomycin	Corning	30-002-CI
Shaker	Thermo-Fisher	MaxQ 4450
Centrifuge	Eppendorf	5702
Incubator	Thermo-Fisher	Forma Series II Water Jacketed CO2 Incubator Model 3100
Trypsin EDTA, 1x (0.25%)	Corning	25-053-CI
Hyaluronidase	Worthington Biochemical	LS002592
T-75 Flasks	BD Biosciences	353136
T-175 Flasks	BD Biosciences	353112
Pipet tips	Rainin	P2, P10, P20, P200 and P1000
Pipet tips	Corning	Filtered tips P2, P10, P20, P200 and P1000
Name of Reagent/ Equipment	Company	Catalog Number	Comments/Description
2. Plating 3D culture
Cell Counter	Invitrogen	Countess
Countess Cell Counting Chamber Slides	Invitrogen	C10313
Trypan Blue Stain (0.4%)	Gibco	15250-061
Collagen Type I (Rat Tail)	BD Biosciences	354236
96 well plate, clear bottom, black	BD Biosciences	353219
Name of Reagent/ Equipment	Company	Catalog Number	Comments/Description
3. Adhesion assay
Multichannel pipet	Eppendorf	Xplorer 300
Paraformaldehyde solution 4% in PBS	Santa Cruz Biotechnology	sc-281692
Plate reader	Molecular Devices	Minimax
Name of Reagent/ Equipment	Company	Catalog Number	Comments/Description
5. Invasion assay
Cell Culture Inserts (8um, 24-well)	BD Biosciences	353097
Cotton swabs	Q-tips	cotton swabs
Microscope	Zeiss	Axiovert 200m
Cell Profiler	public domain
24 well plate	BD Biosciences	353047
Name of Reagent/ Equipment	Company	Catalog Number	Comments/Description
6. Antibodies
Anti-Integrin αVβ3 Antibody, clone LM609	EMD Millipore	MAB1976
Beta 1	Oncosynergy	OS2966
Alpha 5 [CD49e]	ID Pharmingen	555615
Beta 4 [CD104]	EMD Millipore	MAB 2058