TITLE: Analysis of Breast Cancer Cell Invasion Using an Organotypic Culture System
AUTHORS: Romana E. Ranftl, Fernando   Calvo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Metastasis is the main cause of cancer patient mortality. Local tumor invasion is a key step in metastatic dissemination whereby cancer cells dislodge from primary tumors, migrate through the peritumoral stroma and reach the circulation. This is a highly dynamic process occurring in three dimensions that involves interactions between tumor, stromal cells, and the extracellular matrix. Here we describe the organotypic culture system and its utility to study breast cancer cell invasion induced by cancer-associated fibroblasts. This is a three-dimensional model that reproduces the biochemical and physiological properties of real tissue and allows for investigating the molecular and cellular mechanisms involving tumor and its microenvironment, and their contribution to cancer cell invasion. This system provides a robust, accurate, and reproducible method for measuring cancer cell invasion and represents a valuable tool to improve the mechanistic understanding of the initial steps in metastasis.</div></div>

[STEPS]
?. [3.1 Gel Preparation]
All components of the gel mix must be kept on ice. Here we describe the protocol for 1 mL gel mix containing fibroblasts or without fibroblasts. This is the volume required for one sample in one well of a 24-well plate. The total volume of gel mix needs to be scaled up according to sample size. Generally, it is recommended to prepare gel in excess to avoid pipetting errors due to the viscous nature of the components.
?. [3.1 Gel Preparation]
Mix ,  , and .
[FBS]
[5× DMEM]
[fibroblast culture medium]
?. [3.1 Gel Preparation]
Add  and  (see Notes 10 and 11). Keep the mixture  (see Note 12).
[ Matrigel®]
[collagen I]
on ice
?. [3.1 Gel Preparation]
Trypsinize fibroblasts of interest (NFs/CAFs) from a monolayer to a single cell suspension. Count the cells, centrifuge , and resuspend the pellet at a concentration of 107 cells/mL in fibroblast culture medium (see Notes 13 and 14).
Centrifuge: 400 34, 5 min
?. [3.1 Gel Preparation]
Avoiding bubble formation, add  to the gel mix. If a gel without fibroblasts is required, add  instead of cell suspension (see Note 15).
[cell suspension]
[fibroblast culture medium]
?. [3.1 Gel Preparation]
Add  of the mixture in a 24 well-plate well (Fig. 2c) (see Note 16).
900 µl
?. [3.1 Gel Preparation]
Place the plate at  and 5% CO2 for .
37 °C
?. [3.1 Gel Preparation]
When the gel is set, add  of appropriate medium on top and incubate  at and 5% CO2 (see Note 17).
1 mL
37 °C
?. [3.2 Breast Cancer Cell Preparation]
Depending on the number and matrix-remodeling ability of the fibroblasts, some of the gels may have already contracted at this point. The plate can be scanned to document this, as gel contraction can serve as an additional read-out of CAF function (Fig. 2d).
?. [3.2 Breast Cancer Cell Preparation]
Aspirate the medium carefully from the 24-well plate containing the gels.
?. [3.2 Breast Cancer Cell Preparation]
Apply  on the top of each of the gels (see Notes 18 and 19).
[cancer cell suspension]
?. [3.2 Breast Cancer Cell Preparation]
Incubate at  and 5% CO2. Leave the cells to adhere to the matrix for –.
37 °C
?. [3.3 Coating of Nylon Filters]
Given the softness of the gels, direct contact with the metal can damage the gels. Before placing the gels on the bridges, nylon filters have to be prepared and placed in-between the gels and the metal grid.
?. [3.3 Coating of Nylon Filters]
Prepare  according to the recipe described in Section 3.1 "Gel Preparation."
[gel without fibroblasts]
?. [3.3 Coating of Nylon Filters]
Coat the nylon filters using  (Fig. 2e).
[cell-free gel]
?. [3.3 Coating of Nylon Filters]
Separate the coated filters in the culture dish (Fig. 2f).
?. [3.3 Coating of Nylon Filters]
Incubate the coated nylon filters at  for .
37 °C
?. [3.3 Coating of Nylon Filters]
Fix the coated nylon, using filter fixing solution for at least  at  or  at .
37 Room temperature
4 °C
?. [3.4 Lifting the Gel and Covering the Cancer Cells]
The gels are placed on the metal bridges and medium is fed from underneath to impose a direction of invasion. The breast cancer cell layer is covered with a thin layer of gel for improved simulation of in vivo conditions (see Note 20).
?. [3.4 Lifting the Gel and Covering the Cancer Cells]
Add cancer cell culture medium to the filters and incubate for  at .
37 °C
?. [3.4 Lifting the Gel and Covering the Cancer Cells]
Place the sterile metal grid bridges in a 6-well plate with sterile forceps (Fig. 2g).
?. [3.4 Lifting the Gel and Covering the Cancer Cells]
Add a coated, washed and medium-adapted nylon filter on top of each metal bridge with sterile forceps (Fig. 2h).
?. [3.4 Lifting the Gel and Covering the Cancer Cells]
Prepare an appropriate amount of cell-free gel according to the recipe in Section "Gel Preparation" and keep the mixture  ( per organotypic gel).
on ice
100 µl
?. [3.4 Lifting the Gel and Covering the Cancer Cells]
Remove the medium on the top of the gels carefully in order not to disturb the organotypic cultures.
?. [3.4 Lifting the Gel and Covering the Cancer Cells]
Lift the gels with a sterile spatula from the 24-well and place it on a coated filter on top of a metal bridge. The cancer cell layer must be facing up (Fig. 2i, j).
?. [3.4 Lifting the Gel and Covering the Cancer Cells]
Add medium under the bridge until it is in contact with the nylon filter.  Avoid air bubbles between the interphase medium/nylon filter (Fig. 2k).
?. [3.4 Lifting the Gel and Covering the Cancer Cells]
Add  on top of the lifted gels and spread over the surface.
[cell-free gel mixture]
?. [3.4 Lifting the Gel and Covering the Cancer Cells]
Incubate the culture for 5 days (, 5% CO2) and change the medium daily (see Note 21).
37 °C
?. [3.5 Gel Fixing and Processing]
After culturing for 5 days (see Note 9), remove the medium from the plate and wash with PBS or transfer the organotypic cultures to a new 6-well plate by lifting the filter and gel. Turn the gel upside-down for fixing (cancer cell layer on the bottom, Fig. 1) (see Note 22).
?. [3.5 Gel Fixing and Processing]
Fix the gels in at .
[gel fixing solution]
4 °C
?. [3.5 Gel Fixing and Processing]
Next day, wash the gels with  for  (×3).
[PBS]
?. [3.5 Gel Fixing and Processing]
Using the forceps and the scalpel cut the gel in two halves and store one of them in 70% ethanol at  (see Note 23).
4 °C
?. [3.5 Gel Fixing and Processing]
Embed the other half of the gel in paraffin blocks and perform standard H&E staining on sections (see Notes 24 and 25).
?. [3.6 Data Analysis]
Take 5–7 pictures per gel using a bright field microscope (20×/10× magnification) (see Note 26 and Fig. 3a-e).
?. [3.6 Data Analysis]
Measure the area of noninvasive cancer cells and the total area of cancer cells (noninvasive and invasive) using Image J or a similar software (Fig. 3f).
?. [3.6 Data Analysis]
The Invasion Index is calculated by dividing the invading area (total area − invading area) by the total area (see Note 27).
?. [3.7 Killing Assay]
CAFs have been shown to form tracks in the ECM , which has been associated with increased invasiveness of cancer cells [10]. However, CAFs can also produce soluble factors that can promote cancer cell invasion [8]. The basic organotypic invasion assay described above does not allow discriminating between these two abilities.In order to specifically study the capacity of CAFs to form tracks in the gel that allow subsequent cancer cell invasion, as well as the potential of cancer cells to invade a gel previously remodeled by CAFs, a variation of the basic organotypic culture assay can be performed: the “killing” assay . Fibroblasts are seeded as described earlier and are allowed to remodel the gel matrix for 5 days before they are removed. When cancer cells are seeded on top of the gel, they will invade into the tracks formed by the fibroblasts (Fig.3c). This modified version also allows for discriminating the selective effect of chemical compounds on fibroblasts/cancer cells as both compartments are never cultured together.
?. [3.7 Killing Assay]
Incubate at , 5% CO2 for 5 days. Change the medium daily (see Notes 17 and 28).
37 °C
?. [3.7 Killing Assay]
Fibroblast removal (“killing” step): Remove the medium from the gels and add containing an appropriate selection compound (e.g., 10 μg/mL puromycin) (see Note 29).
[fibroblast culture medium]
?. [3.7 Killing Assay]
Incubate at , 5% CO2 for .
37 °C
?. [3.7 Killing Assay]
Wash the gels for  in fibroblast culture medium (×3).
?. [3.7 Killing Assay]
Continue with the procedure as described in Sections 3.2 "Breast Cancer Cell Preparation" – 3.6 "Data Analysis".
?. [3.2 Breast Cancer Cell Preparation]
Trypsinize a monolayer of breast carcinoma cells (4T1, 410.4 or MBA-MD-231), count the cells and prepare a single cell suspension at 5 × 106 cells/mL in cancer cell culture medium.
?. [3.3 Coating of Nylon Filters]
Prepare the adequate number of nylon filters (1 per organotypic condition) with a sterile forceps on top of each other in a culture dish.
?. [3.4 Lifting the Gel and Covering the Cancer Cells]
Wash the gel-coated nylon filters with sterile PBS for  (×3) to remove all traces of fixing solution.
?. [3.7 Killing Assay]
Follow the protocol as described in Section 3.1 "Gel Preparation" (steps 2–9). Once the gel is set, add  on top.
[fibroblast culture medium]
?. [3.1 Gel Preparation]
Prechill all components for the gel preparation .
on ice
?. [3.4 Lifting the Gel and Covering the Cancer Cells]
Wash the gel-coated nylon filters with sterile PBS for  (1/3)
?. [3.4 Lifting the Gel and Covering the Cancer Cells]
Wash the gel-coated nylon filters with sterile PBS for  (2/3)
?. [3.4 Lifting the Gel and Covering the Cancer Cells]
Wash the gel-coated nylon filters with sterile PBS for  (3/3)
?. [3.5 Gel Fixing and Processing]
Wash the gels with  for  (1/3)
[PBS]
?. [3.5 Gel Fixing and Processing]
Wash the gels with  for  (2/3)
[PBS]
?. [3.5 Gel Fixing and Processing]
Wash the gels with  for  (3/3)
[PBS]
?. [3.7 Killing Assay]
Incubate at , 5% CO2  in fibroblast culture medium to remove all traces of selection compound (see Note 30).
37 °C
?. [3.7 Killing Assay]
Wash the gels for  in fibroblast culture medium (1/3)
?. [3.7 Killing Assay]
Wash the gels for  in fibroblast culture medium (3/3)
?. [3.7 Killing Assay]
Wash the gels for  in fibroblast culture medium (2/3)