# Chapter 15

# Analysis of Breast Cancer Cell Invasion Using an Organotypic Culture System

# **Romana E. Ranftl and Fernando Calvo**

# **Abstract**

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.

**Key words** Organotypic, Invasion, Metastasis, Breast cancer, Cancer-associated fibroblasts

## **1 Introduction**

Metastatic dissemination is the major clinical complication in most types of cancer and the cause of 90% of cancer-related deaths [1]. Invasion of cancer cells into the peritumoral stroma is a key step in metastasis [2, 3]. Thus, understanding the mechanisms regulating the invasive abilities of cancer cells is imperative to inform the development of therapeutic modalities to minimize cancer dissemination and improve patient survival [4]. Local cancer cell invasion is a multifunctional and dynamic process occurring in three dimensions that is actively modulated by the peritumoral stroma [5–7]. Cancer-associated fibroblasts (CAFs) are particularly relevant as they can produce soluble factors that promote cancer cell invasion [8]. In addition, CAF-dependent matrix remodeling via focalized proteolysis activity [9] or actomyosindependent force generation [10, 11] can lead to the formation of tracks through the extracellular matrix (ECM) that enable

Zuzana Koledova (ed.), *3D Cell Culture: Methods and Protocols*, Methods in Molecular Biology, vol. 1612, DOI 10.1007/978-1-4939-7021-6\_15, © The Author(s) 2017

subsequent cancer cell invasion. Accordingly, three-dimensional (3D) models of cancer cell invasion that incorporate stromal components such as fibroblasts and physiologically relevant ECMs recapitulate more closely the in vivo situation. These models provide a platform for investigating the complex interactions between tumor and its microenvironment that are more likely to lead to novel insights of clinical relevance.

Here, we describe a 3D organotypic invasion assay adapted for the robust and accurate assessment of breast cancer cell invasion induced by CAFs [11–13]. This approach was originally developed as a 3D coculture model by Fusenig and colleagues [14], and further developed by other groups to study squamous cell carcinoma invasion [10, 15]. Briefly, CAFs are embedded in a dense gel composed of fibrillar collagen I and basement membrane matrix (termed Matrigel®, Cultrex®, or Engelbroth-Holm-Swarm matrix), which contains laminins, collagen IV, proteoglycans, and a broad spectrum of growth factors (*see* Fig. 1). A thin layer of gel is used to cover the breast cancer cells seeded on the surface of the CAFcontaining gel to mimic the physiological condition of breast tissue. Gels are subsequently laid on a grid and maintained partially immersed in cell medium. Organotypic gels are then fixed and processed by standard histopathological procedures followed by quantitative and qualitative analysis of breast cancer cell invasion using standard image processing and analysis software.

**Fig. 1** Schematic representation of the workflow of an organotypic invasion assay. Step 1: Embed fibroblasts in gel and seed in 24-well dish. Step 2: Cancer cells are seeded in a single-cell suspension on top of the gel. Step 3: Once the cells have adhered, remove the medium and lift the remodeled gel onto gel-coated nylon filter on a metal bridge. Coat the cancer cells with a thin layer of gel. Step 4: Feed with complete medium up to the nylon filter. Incubate at 37 °C, 5% CO2 for 5 days to allow for cancer cell invasion. Step 5: Terminate assay by fixing organotypic gels. Process gels for H&E staining

## **2 Materials**

1. Cancer-associated fibroblasts (CAFs): (a) For the mouse model: CAFs from breast carcinomas from the FVB/n MMTV-PyMT murine model [11, 16]. (b) For the human model: CAFs from a resection of a human breast carcinoma. 2. Normal fibroblasts (NFs): (a) For the mouse model: NFs from mammary glands of FVB/n wild-type siblings [11, 16]. (b) For the human model: NFs from a resection of a reduction mammoplasty. 3. Breast cancer cells: (a) For the mouse model: 410.4 or 4T1 cells (ATTC®- CRL-2539™) (*see* **Note 1**). (b) For the human model: MDA-MB-231 (ATCC®-HTB-26) (*see* **Note 2**). 4. Fibroblast culture medium: 10% fetal bovine serum (FBS), 1× GlutaMax™ (Gibco®) and 1× insulin–transferrin–selenium (ITS, Gibco®) in Dulbecco's modified Eagle's medium (DMEM). Store at 4 °C. Warm up before use. 5. Cancer cell culture medium: 10% FBS and 1× GlutaMax™ in DMEM (*see* **Note 3**). Store at 4 °C. Warm up before use. 6. Sterile phosphate buffered saline (PBS): 3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, pH 7.4. Warm up before use. 7. Sterile 0.05% trypsin–0.02% EDTA. Store at 4 °C. Warm up before use. 8. If a Killing Assay is performed: selective compounds such as puromycin (puromycin dihydrochloride from *Streptomyces alboniger*, Sigma) (*see* **Note 4**). 1. 5× DMEM: 5% αDMEM powder (Gibco®), 2% NaHCO3 (0.24 M NaHCO3), 0.1 M Hepes pH 7.5. Store at 4 °C (*see* **Note 5**). 2. Fibroblast culture medium. 3. FBS. 4. Rat-tail collagen type I, high concentration (BD Biosciences). Store at 4 °C (*see* **Note 6**). 5. Matrigel® (BD Biosciences). Store at −80 °C in 1 mL aliquots (*see* **Note 7**). *2.1 Tissue Culture 2.2 Gel Preparation*

**Fig. 2** Gel preparation and processing steps. (**a**) Metal bridges with approximate dimensions indicated. (**b**) Useful tools for handling the organotypic cultures, nylon filters and metal bridges. (**c**) Cell-free or fibroblastcontaining gels are plated in a 24-well plate on ice. (**d**) The ability of fibroblasts to contract the collagen:Matrigel gel (a measure of their matrix remodeling capacity) can be documented prior to further processing. Images show duplicate samples of organotypic cultures free of cells and with different human CAFs that have low ("l"), medium ("m"), and high ("h") contractility, as indicated. (**e**, **f**) Nylon filters are soaked in gel (**e**) and then separated in a culture dish for setting and fixing (**f**). (**g**–**k**) Setting up the organotypic gel on the metal bridges. Sterile bridges are placed in a 6-well plate (**g**) and covered by a nylon filter (**h**). The organotypic cultures are lifted from the 24-well plate and placed over the filter:bridge using a spatula (**i**); a thin gel layer is added on the top to cover the cancer cell monolayer (**j**); and complete medium is added to the 6-well dish until soaking the nylon filter underneath the organotypic gel (**k**)



# **3 Methods**

This protocol is an adapted version of a previously described method for SCC12 carcinoma cells [10] (*see* Fig. 1 for a schematic representation). We have optimized the organotypic invasion assay for murine and human breast cancer cells (410.4/4T1 and MDA-MB-231) in combination with murine or human fibroblasts, respectively (*see* **Note 8**). On a general basis, cancer cells are not invasive in this setting and rely on CAF activities to invade. It is recommended to test the behavior of alternative cancer cell types in the assay by plating them on top of a fibroblast-free gel matrix. Heterogeneity is also observed in CAFs in terms of their ability to contract and remodel gel matrices. The amount of fibroblasts and cancer cells to be used, as well as the length of the protocol, need to be optimized if alternative models are used (*see* **Note 9**).

#### 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*






#### 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*


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*



	- 2. Fix the gels in 2 mL of gel fixing solution at 4 °C overnight.
	- 3. Next day, wash the gels with PBS (2 mL) for 10 min (×3).
	- 4. Using the forceps and the scalpel cut the gel in two halves and store one of them in 70% ethanol at 4 °C (*see* **Note 23**).
	- 5. Embed the other half of the gel in paraffin blocks and perform standard H&E staining on sections (*see* **Notes 24** and **25**).

#### 1. 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*


#### 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. *3.7 Killing Assay*

**Fig. 3** Analysis of cancer cell invasion using carcinoma cells in an organotypic invasion assay. (**a**) Representative image of H&E staining of an organotypic invasion assay of 4T1 cells cultured in the presence of CAFs. The different parts of the organotypic gel are indicated. *Right panel* is a zoom up region that shows both collective and single cell invasion of 4T1 cells. Fibroblasts and ECM fibers are also observed. (**b**) Representative images of H&E staining of organotypic invasion assays of 4T1 murine breast cancer cells cultured in the absence and presence of either murine normal fibroblasts (NFs) or PyMT-CAFs. Invasion indexes of 4T1 cells for each

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.


**Fig. 3** (continued) experimental setup are also indicated. Scale bar, 50 μm. (**c**) Representative images of H&E staining of "killing" organotypic invasion assays of 4T1 murine breast cancer cells cultured in gels previously remodeled by murine normal fibroblasts, PyMT-CAFs or mock-remodeled (no fibroblasts). Invasion indexes of 4T1 cells for each experimental set-up are also indicated. Scale bar, 50 μm. (**d**) Representative images of H&E staining of organotypic invasion assays of MDA-MB-231 human breast cancer cells cultured in the absence and presence of human breast cancer CAFs. The invasion index is indicated. Scale bar, 50 μm. (**e**) Representative images of H&E staining of organotypic invasion assays of 410.4 murine breast cancer cells with murine NFs and CAFs. Note that 410.4 cells present a "collective" mode of invasion. Scale bar, 50 μm. (**f**) Exemplars for image analysis of a representative image of H&E staining of an organotypic invasion assay of 4T1 cells cultured in the presence of CAFs (shown in panel **a**). First, the non-invaded area (*red*) and the total area (*blue*) are measured using Image J (http://imagej.nih.gov/ij/). The invading area is then calculated by subtracting the non-invading area from the total area. The invasion index is the ratio of invaded area vs. total area. Invasion index and areas are also indicated

# **4 Notes**


# **Acknowledgments**

R.R. and F.C. are funded by The Institute of Cancer Research (UK). F.C. is also supported by Worldwide Cancer Research (Grant 15-0273). We thank Dr. Erik Sahai and Steven Hooper for contributing to develop the technique described here. We also thank lab members for help and advice, and for critically reading the manuscript.

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