Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Summary

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Abstract

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Introduction

Investigating the fundamental and biomedical characteristics of cancer cell invasion/migration and subsequent metastasis establishment is the subject of an intense research^1,2. Metastasis is the ultimate stage of cancer and its clinical management remains elusive. A better understanding of metastasis at the cellular and molecular levels will enable the development of more efficient therapies³.

Several properties of metastatic cells can be explored in vitro⁴ including their stemness and potential to acquire a transition state (e.g., epithelioid-mesenchymal transition) to migrate and invade within and from the primary tumor⁵. However, the in vitro assessment of invasion/metastasis processes has been a challenge since it virtually excludes the contribution of the blood/lymphatic circulation. Organotypic cultures that embed tumor fragments in collagen gels have previously been used to monitor cancer aggressiveness. Although the complexity of tumors is preserved (e.g., the presence of non-cancerous cells), tumor fragments are exposed to limited medium diffusion, to sampling variation, and to an overgrowth of stromal cells⁶. An alternative method consists in growing cancer cells within components of the extracellular matrix (ECM), which mimics the three-dimensional (3D) cell environment. The proliferation of breast cancer cell lines in a collagen gel and/or a basement membrane-derived matrix is amongst the best-characterized examples of 3D cell culture. By using specific 3D cell culture environments, the disorganized assembly observed for breast cancer cells grown under standard conditions can be reversed to the spontaneous formation of mammary acini and tubular structures^7-10. Furthermore, the formation of multicellular tumor spheroids derived from adenocarcinoma cancer cells congregated using different techniques (e.g., hanging drops, floating spheroids, agar embedment) now constitutes the most commonly used 3D cell culture assay^11-13. However, this assay is limited by the restricted set of cancer cell lines that can form spheroids and by the short period available to study cells in these conditions.

In this visualized technique, we herein introduce a sophisticated 3D cell culture assay where cancer cells of interest are embedded in a collagen gel to allow the in vitro formation of a pseudo-primary tumor that can be alternatively coated with a basement membrane-derived matrix. Once formed, the pseudo-primary tumor is then sandwiched in an acellular matrix (fibrin gel in the present case), which allows the cancer cells to cross the interface between the two matrix compartments (see Figure 1). Interestingly, secondary tumor-like structures originating from the pseudo-primary tumor along with aggressive cancer cells appear in the fibrin gel. Such a 3D culture system offers the flexibility required to investigate, for example, anticancer drugs, gene expression and cell-cell and/or cell-ECM interactions^14-16.

Figure 1: Overview of the Method. Schematic summary of the method to generate the 3D cell culture system as a model for cancer studies. Please click here to view a larger version of this figure.

Protocol

NOTE: No ethics consideration since animal and human cancer cells were purchased or kindly provided to us.

1. Making Collagen Plugs (Pseudo-primary Tumor)

1. Prepare a collagen dispersion. Type I collagen from rat tail tendons (RTT) can be either extracted and sterilized as previously reported¹⁷, or purchased. Disperse freeze-dried RTT collagen (3.25-3.50 mg/ml in 0.02 N acetic acid) using a blender (high-speed setting; five 2 min runs) for a uniform mixing.
2. Harvest (trypsin-EDTA, usually) and use trypan blue exclusion for counting viable cells using a hemocytometer. Adjust to the desired cell density (5 x 10⁴ cells per plug).
3. Prepare all solutions (NaOH, fetal bovine serum, DMEM 5x, NaHCO₃) separately (Table 1) under sterile conditions and keep chilled on ice. Note: The order of addition of the various solutions is important to prevent osmotic or acidic shocks in cells.
4. Perform cell dispersion (1.25 x 10⁶ cells) into the final collagen solution (5 ml) as quickly as possible. Mix well (by pipetting up and down) while avoiding air bubbles, and then quickly distribute 200 µl of the ready-to-use solution in each well of a 96-well plate. Gently strike the multi-well plate on the work area surface of the cell culture hood to remove air bubbles and to spread the solution evenly inside the wells.
5. After filling up all the wells (this step takes about 15-20 min per 96-well plate), store it into the incubator.
6. Incubate the plate at 37 °C from 2 hr to overnight. Collagen gelation (i.e., fibrillogenesis) occurs within 30 min. Add culture medium (100 µl/well) to the culture to perform an overnight incubation.

2. First Layer of Fibrin Gel

1. Fibrinogen Solution Preparation.
   NOTE: The same batch of fibrin gel should ideally be used for more reproducible results, as fibrin gel formation may vary between different batches of commercial freeze-dried fibrinogen.
   1. Always use a freshly prepared fibrinogen solution. Bring the freeze-dried fibrinogen to room temperature before opening the vial to avoid hydrate crystal formation.
   2. Progressively dissolve the fibrinogen in pre-warmed (37 °C) Hank's Balanced Salt Solution (HBSS) with Ca²⁺/Mg²⁺ at a working concentration of 3 mg/ml (consider preparing a 15% excess of the minimum final volume required: e.g., 17.25 mg in 5.75 ml for a 5 ml solution).
      1. Add pre-warmed HBSS dropwise at first to solubilize fibrinogen fragments. Break down larger fragments with a spatula in the beaker. Agitate the beaker from time to time to facilitate mixing. Do not use a stirrer plate during the procedure. Dissolve the remaining powder by pipetting the suspension up and down.
   3. Keep the fibrinogen solution lukewarm while sterilizing the solution by passing it through a 0.22 µm filter. Note: If the HBSS is not warm enough or the fibrinogen not fully dissolved, the solution may clog the filter. If applicable, replace filter once or twice, which may decrease the fibrinogen concentration, and thus fibrin clot stiffness.
   4. Suspend the cells of interest (e.g., endothelial cells) into ready-to-use fibrinogen solution while adjusting its final volume, as an alternative procedure.
2. Preparing the Thrombin Solutions.
   1. Prepare a stock solution in ddH₂O (50 NIH units/ml), then sterilize it using a 0.22 µm filter.
   2. Use a fibrinogen/thrombin ratio ≥1:0.0075 (v/v) in order to generate the fibrin gel.
3. Generating the Fibrin Gel.
   1. Keep the sterile fibrinogen and thrombin stock solutions on ice during all the next steps. The fibrin gels are allowed to form in 24-well plates.
   2. Promptly overlay the surface of each well with the fibrinogen solution (200 µl/well) while avoiding air bubble formation. Process 6 wells at a time.
   3. Once the fibrinogen solution completely covers the surface of the wells, tilt the plate at a 45° angle and add 1.5 µl of thrombin solution to the first well by dropping the thrombin into the center of the well, and then gently swirl the plate horizontally for 1-2 sec.
      1. Leave the plate in a stable position under the laminar flow hood (5-10 min) until the gelation/clotting process has completed (N.B.: the polymerization process must not be disturbed, e.g., by transporting the plate to the incubator).
   4. Once the first six wells have polymerized, repeat the same sequence (i.e. the 3 previous steps) for the next six wells until all wells have been processed.

3. Second Layer of Fibrin Gel and Sandwiched Collagen Plug

1. Option A: (Using the Collagen Plug Immediately).
   1. Make sure that the first layer of fibrin gel has polymerized in all wells by delicately tilting the plate. Place the 96-well plate containing the collagen gel plugs side by side with the 24-well plate (containing the fibrin gels) to ease transfer of the collagen plugs.
   2. Add one drop of HBSS into each well of the plate containing the collagen plugs.
   3. Remove each collagen plug from the well with a thin needle mounted on a syringe (used as a handle) or using a micro-spoon (see video). Transfer each collagen plug onto the first fibrin gel layer using one or two micro-spoons, while making sure that the collagen plug is well centered into the well and that sterility is well maintained.
   4. Overlay the previously formed fibrin gel with the second layer of fibrinogen solution (300 µl/well) and introduce the thrombin as described in 2.3, keeping a minimal 1:0.0075 ratio and a sequence of six wells at a time.
2. Option B (Coating the Collagen Plug with a Thin Layer of Growth Factor-reduced Basement Membrane (GFRBM)).
   1. Cool all the prepared solutions and instruments beforehand and keep them at 4 °C or on ice (e.g., pipettes, tips, test tubes) during handling since frozen aliquots of GFRBM are very sensitive to excessive heating rate during thawing (follow the manufacturer's instructions).
   2. Following removal from the plate wells, soak each collagen plug for 2 min in a 1.5-ml centrifuge tube on ice containing 100 µl of a pure GRFBM solution.
   3. Transfer each coated plug onto the first fibrin layer while ensuring it is well-centered, as described earlier. Incubate the plug-containing plates at 37 °C for 5 min to allow the GRFBM to form a gel. Add the second fibrin layer as in step 3.1.4.

4. Cell Culture Medium Conditions

1. Fill each well with culture medium (400 µl). The culture media and supplements will be selected based on the cell line and experimental conditions.
2. Add aprotinin, an antifibrinolytic agent, to culture medium at a final concentration of 100 kallikrein inhibitor units (KIU)/ml.
   NOTE: Store the plates in a cell culture incubator under the conditions used for the cell line tested.
3. Replenish cultures with fresh medium every other day or according to the experimental schedule, and add aprotinin. Before adding fresh medium, slightly tilt the plate (at a 30-35° angle) and incline the pipet against the side of the well while carefully suctioning the conditioned medium under constant observation.

Results

As previously mentioned, an interesting feature of this 3D cell culture assay is that cancer cells can not only migrate from the collagen plug to the adjacent fibrin gel, but also establish secondary tumors (e.g., satellite tumor-like structures). This can be directly observed with an inverted phase contrast microscope at low and high magnifications through the gel thickness, especially with a long working distance condenser (Figure 2). Using this 3D cell culture...

Discussion

As an important technical footnote, it is essential that no gap is present at the interface between the central and the peripheral gels. Otherwise, it might reduce the capacity of the cells to migrate/invade the fibrin gel. A space between the collagen and the fibrin gels may form during the first 24 hr of culture if thrombin has not been appropriately diluted. It is also possible that the cell line tested might lead the collagen gel to contract during culture, thereby causing a relatively large space to form between bot...

Materials

Name	Company	Catalog Number	Comments
Freeze-dried collagen	Sigma-Aldrich	C7661	from rat tail tendon (soluble dispersion) or home-made (see Rajan et al., ref.#14)
Fibrinogen (freeze-dried)	Sigma-Aldrich	F8630	Type I-S, 65 - 85% protein with ≥ 75% of protein is clottable
Thrombin	EMD Chemicals Inc.	605157	Gibbstown, NJ; NIH units/mg dry weight
Growth factor-reduced Matrigel	Corning	356234	Previously from BD Biosciences
Aprotinin	Sigma-Aldrich	A6279	solution at 5 - 10T IU/ml (Trypsin Inhibitor Unit)
Micro-spoons	Fisher Scientific	2140115	Fisherbrand Handi-Hold Microspatula
96 well plate, round base	Sarstedt	3925500
24 well plate	Sarstedt	3922
Dulbecco's modified Eagle's Medium	Sigma Chemical, Co.	D5546	DMEM
Fetal Bovine Serum	VWR	CAA15-701	FBS, Canadian origin.
Trypsin-EDTA	Sigma Chemical, Co.	T4049
Hank’s Balanced Salt Solution	Sigma Chemical, Co.	H8264	HBSS