The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.
Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.
Despite significant improvements in cancer immunotherapy over the past decade, our mechanistic understanding of sensitivity and resistance to treatments are still fairly poor1. It is well-established that tumors display substantial heterogeneity, and that the dynamic interactions of the tumor cells with their microenvironment as well as with the immune cells, impact tumor cell death, invasive behavior and response to treatments that include immunotherapy1,2,3. As one arm of the adaptive immune system, T-cells execute cell-specific cytotoxicity. The analysis of T-cell recognition and response to cancer cells provides mechanistic insights into resistance and sensitivity to immune modulatory treatments.
In vitro modeling and monitoring interactions between cancer and T-cells in an appropriate environment has been challenging and so far, resulted in limited mechanistic insights. Most cell-based assays rely on a two-dimensional (2D) environment, that lacks key features that are critical for recapitulating the three-dimensional (3D) in vivo physiology4,5,6, namely spatial cell-cell interactions, contact with the extracellular matrix (ECM)7, dynamic metabolic demand, increased hypoxia due to mass growth8, and effects of the tumor microenvironment (TME)9. On the other hand, there are still a number of shortcomings with the currently used three-dimensional (3D) co-culture and invasion assay systems: (1) the time consuming nature of spheroid generation and harvest5,10, (2) the lack of control over spheroid size, shape and cell density11,12, (3) the low-throughput type assays, (4) the requirement for special equipment13,14, (5) the need to transfer the co-culture into distinct environments for different assays15,16,17. In particular, transferring of a co-culture assay often leads to disruption of spheroids and loss of the co-culture integrity. This applies especially for “loose” spheroids with reduced cell-cell adhesion. For example, most 3D invasion assays require that spheroids are harvested after their initial formation and then resuspended in ECM14,15,16. This resuspension step results in a loss of control over the distance between spheroids. Since distance between tumor spheroids impacts their invasive behavior, this loss of control introduces high inter-assay variance and reduces the reproducibility. Furthermore, the application of cell fractionation assays by consecutive centrifugation steps for assessment of the peripheral and tumor spheroid infiltrating immune cells is limited to tumor cell populations that generate more stable spheroids17.
Concept and approach
Our approach addresses the above-mentioned deficiencies using an “All-in-One”—3D spheroid co-culture model, which does not require the transfer of spheroids for subsequent assays. We adapted a spheroid formation device (see Table of Materials) to generate an assay for simultaneously monitoring invasive behavior of cancer cells and cytotoxicity of co-cultured T-cells. This method is user-friendly, inexpensive and allows for quick and easy handling in a relatively high-throughput 3D setting. Dependent on the type of device used, up to 81 large uniformly-sized spheroids can be generated in a single pipetting step with control over the individual spheroid size by modifying the number of cells seeded. Spheroid formation is forced by enhanced cell-cell interactions in scaffold-free agarose multi-well casts with U-shaped bottoms. We adapted this 3D system for dynamic cell-based functional studies as well as endpoint molecular and biochemical assays that include fluorescence activated cell sorting (FACS), immunofluorescence (IF) or immunohistochemistry (IHC) staining as well as gene expression analysis of the intact 3D co-culture.
For functional studies, embedding spheroids in type I collagen within the agarose casts results in invasion of cancer cells from equidistant spheroids and permits monitoring essential cell line-specific features, such as single cell vs. collective cell migration18,19. Furthermore, the collagen matrix is easily separated from the agarose cast, resulting in a 1‒2 mm thick patch containing multiple spheroids, which can be further processed for IF-staining and imaging by confocal microscopy. This can reveal distinct cell invasion and cell-matrix interactions in a high-throughput screening. Also, cells in the collagen matrix can be isolated after collagen digestion and single cell dissociation for subsequent cell cultivation or analysis.
For IHC analysis of spheroids, after fixation and sectioning of the agarose cast, proteins or other molecules of interest are detectable whilst maintaining the geographic positions of the spheroids. In the approach described here, spheroids are directly embedded in Hydroxyethyl agarose processing gel within the agarose cast and the gel serves as a “lid” to retain the spheroids at the bottom of the microwells. After paraffin embedding of the agarose cast20, serial horizontal sectioning is performed with the bottom of the cast serving as the starting point.
This approach contrasts with conventional IHC sectioning of spheroids that requires harvesting of cells before embedding in Hydroxyethyl agarose processing gel21 and risks disruption of spheroids thus losing the spatial arrangement of cells. Also, cell fractionation by centrifugation for assessing whether immune cells infiltrated or remained peripheral to tumor spheroids17 is avoided by direct embedding.
Furthermore, 3D co-culture can be performed by admixing tumor, stromal or immune cells, and thus studying tumor cell crosstalk or recapitulating different tumor microenvironments for analyzing cell-cell interactions including co-cultures with endothelial cells16.
This 3D spheroid co-culture setting can be used to perform co-culture of different cell types present in the tumor microenvironment and to assess the effects of altered ECM elements. Besides type I collagen, other ECM components (e.g., matrigel, matrigel/collagen mixtures, fibronectin), can be used since tumor cell invasion is impacted by the abundance of different substrates22. Also, the microwells of the agarose cast are suitable for spheroid formation of primary cell lines and for cells with low cell-cell adhesion.
A list and explanation of some frequently used words throughout the protocol can be found in Supplementary File 1.
1. Generation of spheroids
2. Co-culture with T-cells
3. Embedding of 3D co-culture into type I collagen matrix
4. Cytotoxicity assay
5. Hydroxyethyl agarose processing gel embedding for IHC sectioning
NOTE: Here it is critical to avoid using low-melting agarose for generating the agarose casts.
6. Monitoring and analyzing spheroid invasion in co-culture
NOTE: The time-point of imaging spheroid invasion into the collagen I matrix is to be decided by the investigator. Acquire cell culture images using an inverted microscope with 10x magnification. The ideal time-point is dependent on the cell line being tested, as well as the ECM component. More invasive cell lines will begin to spread into the collagen within a few hours after adding the collagen. Since the T-cells in the co-culture might prevent a full view on the egress from the spheroids at very early time-points, generally images are taken at 0 h (as reference), 24 h and 48 h after adding the collagen.
7. Immunofluorescence staining
8. Isolation of cells from the collagen matrix
9. RNA extraction from the collagen matrix
The 3D co-culture model allows for different assays shown in Figure 1A, which can be combined or modified as needed. In our established experimental setup, tumor and T-cells are co-cultured for 2 days followed by initiation of the invasion assay for selection of invasive and/or resistant tumor cells (Figure 1B). On day 4 the quantitation of invasion is performed and “survivor” cells are isolated from the collagen matrix or directly processed for RNA23 or DNA extraction from matrix (Figure 1B). Embedding the 3D culture in type I collagen within the microwells of the agarose cast allows monitoring and analysis of invasion by using Image J to first demarcate the total area using the software’s freehand draw tool and then calculate the ratio over the demarcated spheroid area (Figure 2A), and/or by counting the number of “spikes” leaving the spheroid. Using two primary murine pancreatic cancer cell lines (cell line 1 and cell line 2), different spheroid shapes and invasive behavior were observed and quantified accordingly (Figure 2B). Cell line 1 shows a more compact spheroid formation and “spiky” invasion, comparable to single cell invasion, whereas cell line 2 forms more loose spheroids and shows a collective invasion pattern (Figure 2C). Co-culture was performed with two different tumor clonal cell lines seeded at the same time (Figure 3A‒E) to follow their interactions during subsequent assays, and with tumor cells and T-cells upon tumor spheroid formation (Figure 3F‒J). For detailed assessment of the invasive behavior, immunofluorescent staining was performed (Figure 4). After separation of the collagen matrix from the agarose cast, IF staining, and transfer to a glass slide (Figure 4A‒B), confocal imaging was performed in a high-throughput manner (Figure 4C‒J). The size and consistence of the agarose cast allow embedding of the whole 3D culture system in paraffin for serial sectioning and immunohistochemistry (IHC) staining for quantifying the spatial relation between tumor and T-cells (Figure 5). T-cells present in the section can be identified and further characterized by cell surface marker staining exemplified here for CD8 (Figure 5D,E). T-cells that have infiltrated the tumor spheroid can be counted relative to those that remained in the periphery of the spheroid. Figure 5D,E shows examples of distinct infiltration of T-cells into tumor spheroids grown from tumor cell line 1 (D) and cell line 2 (E). Table 1 shows typical experimental setups for the assays, and the yield of representative and analyzable samples at the end of the experiment for each protocol.
Figure 1: Workflow, analyses and timeline of experiments. (A) An 81-microwell and 35-microwell rubber mold are filled with 2% agarose in 1x PBS to generate an agarose cast with multiple microwells. The diameter of a rubber mold is 3.5 cm. The size of the 35-microwell agarose cast is 13 mm x 8 mm, and of the 81-microwell cast is 13 mm x 13 mm. Spheroids are formed upon cell seeding into the chambers of the agarose cast in a single pipetting step. Co-culture with T-cells is performed within the same cast. Functional monitoring and potential assays are shown. (B) Timeline of experiments. Tumor cells are co-cultured with autologous T-cells for 2 days allowing for a maximum interaction between both cell types. The invasion assay is initiated after two days. Endpoint analyses are performed after further two days to monitor the invasive and survival phenotype of tumor cells as well as the proliferation and survival of T-cells. Please click here to view a larger version of this figure.
Figure 2: Quantification of invasion. Invasion can be quantified by image analysis, e.g., using Image J software and counting the number of “spikes” per spheroid. (A) Calculation of the total invasion area as a ratio of total area to the spheroid area. Scale bar: 300 µm. (B) Examples of two different primary murine pancreatic cancer cell lines (cell line 1 and 2) in spheroid formation at different magnifications and invasion into type I collagen. (C) Analysis of invasion is performed by counting the number of spikes per spheroid (left diagram; error bars: 2.63 for cell line 1, 1.47 for cell line 2) and calculate the invasion area as described (right diagram; error bars: 0.36 for cell line 1, 1.28 for cell line 2). Please click here to view a larger version of this figure.
Figure 3: Co-culture with dye-labeled tumor and T-cells. (A‒E) Mix of two differently dye-labeled primary murine pancreatic cancer clonal cell lines (green and red) and magnified view of one representative microwell (B‒E). (F‒J) Co-culture of pre-labeled tumor (green) and T-cells (red). (G‒J) Magnified view of one representative microwell shows one tumor–T-cell co-culture upon tumor spheroid formation. Please click here to view a larger version of this figure.
Figure 4: Immunofluorescence staining. Immunofluorescence (IF) staining was performed after separating the collagen matrix from the agarose cast. (A‒B) After IF staining, the collagen patches are transferred to a glass slide and covered with glass coverslips. (C‒F) Example of an IF-stained collagen patch including tumor spheroids with (C) Hoechst, (D) keratin 8, (E) phalloidin and (F) overlay. Panels (G‒J) Show the respective magnified view of a single spheroid. Scale bar = 300 µm. Please click here to view a larger version of this figure.
Figure 5: Immunohistochemistry sectioning. The agarose cast with the 3D culture immersed in hydroxyethyl agarose processing gel, was embedded in paraffin, sectioned and processed for immunohistochemistry (IHC) staining. (A) Paraffin block used for horizontal sectioning that starts from the bottom of the agarose cast to obtain serial sections of multiple tumor cell/T-cell co-cultures within a single cast; scale bar = 5 mm. (B) Hematoxylin & eosin-stained section of an agarose cast containing 3D co-culture of tumor and T-cells. Scale bar: 1 mm. (C) Magnified view of an H&E-stained co-culture within the agarose cast. CD8 staining of T-cells co-cultured with cell line 1 (D) and cell line 2 (E). Scale bars in C‒E = 200 µm. Please click here to view a larger version of this figure.
protocol | typical setup | cells seeded (per cast) | typical yield |
cytotoxicity assay | 2x 81-microwell casts | 81,000 | 100,000 cells |
IHC assay | 1x 81-microwell cast | 81,000 | 40 spheroids |
Invasion assay | 2x 35-microwell casts | 35,000 | 50 spheroids |
IF assay | 2x 35-microwell casts | 35,000 | 50 spheroids |
Cell isolation from collagen I | 2x 35-microwell casts | 35,000 | 50,000 cells |
RNA extraction from collagen I | 12x 81-microwell casts | 243,000 | 400-600 ng/µl |
Table 1: Typical experimental setup and yield for the protocols. The table shows the typical experimental setup for each protocol and the typical yield of analyzable samples (number of cells, spheroids or RNA concentration) at the end of the experiment, respectively. IHC= immunohistochemistry; IF= immunofluorescence.
Supplementary File 1. Please click here to download this file.
The method presented here describes 3D tumor spheroid generation, which allows co-culture with T-cells, cell-based functional and molecular assays, as well as a variety of monitoring and analysis possibilities using a single device. The major advantage of our approach is that it does not necessitate transfer of the 3D culture to a separate assay and maintains the integrity of the 3D culture throughout the assays.
The workflow presented here can be modified as needed. The incubation times for spheroid formation, T-cell co-culture or cytotoxicity assay, may need to be altered for different experimental conditions or cell lines.
There are a few steps throughout the assays, which require close adherence to the protocol. These are in general: removing the cell culture medium before adding a second cell line for co-culture, as well as embedding the 3D culture in ECM or hydroxyethyl agarose processing gel within the agarose casts. It is absolutely critical to slowly and carefully remove the medium of the seeding chamber by tilting the well-plate and targeting one corner of the chamber with a micropipette. As long as the agarose casts contain cells, we recommend to always remove any medium in the well with a micropipette, instead of using a pipettor. Adding a second cell line and embedding in ECM or hydroxyethyl agarose processing gel needs to be performed slowly and drop-wise in order to prevent flushing out the cells in the microwells. The most critical step of the collagen invasion assay is the incubation time before inverting the casts in the well-plate with the wells. Reducing the time might cause the collagen to drop out from the cast, and exceeding the time might cause the culture to be pressed down to the bottom of the microwells, resulting in unevenly distributed spheroid invasion. Inverting the cast enables cells in the microwells to completely submerge in the liquid collagen before it polymerizes and solidifies. The surface tension between the agarose cast and the plastic bottom of the well-plate, generates a “hanging-drop”15,24 during the 3D spheroid invasion.
It is important to note that the incubation time before inverting the casts has been established for embedding in type I collagen. With other ECM components, this step must be adjusted accordingly. Of note, complete removal of residual media should not be attempted to avoid an inadvertent loss of cells present in the microwells. This residual media results in a slight dilution of the added ECM. This needs to be taken into consideration for the analysis and adaptation from other assay systems.
The co-culture described here allows for a maximum tumor/T-cell interaction before the invasion of tumor cells is assayed: Tumor and T-cells are co-cultured for 2 days before embedding in type I collagen and then identifying surviving and invasive tumor cells over the next two days (Figure 1B). Of note, when the co-culture was initiated while T-cells were resuspended in collagen I, T-cells failed to show an impact on tumor cell invasion and cytotoxicity. This effect might be due to the T-cells being more distributed in collagen and thus less concentrated around the spheroids. This suggests that the direct interaction between tumor and T-cells during the two days of co-culture prior to the invasion assay is critical for assessment of T-cell mediated effects on the tumor cells.
Nevertheless, some of the advantages of this 3D model come with disadvantages. This high-throughput setting allows easy and quick seeding of cells into the agarose cast in one pipetting step, resulting in the generation of a multitude of uniformly sized spheroids within one cast. However, since the spheroids are all located within the same cast, they can also be easily removed, e.g., during removal of the cell culture medium within the seeding chamber and while adding T-cells for co-culture. Therefore, the amount of yield for each protocol is strongly dependent on the technical skills and experience of the investigator, but also on the type of assay being performed. As Table 1 suggests, a possible loss of spheroids of about 50% at the end of an experiment must be taken into consideration. Moreover, during the seeding step, cells might not equally distribute over the area of the seeding chamber, resulting in more or less representative spheroids within the agarose cast. From our experience, this effect increases with the size of the agarose cast. Accordingly, replicates must be considered while planning out the experiment.
The spatiotemporal interaction of cells within the 3D system can be assessed by time lapse imaging, which presents another monitoring option in this model. Furthermore, the small diameter of the microwells and the single pipetting step to seed cells, are suitable for performing single cell cloning. Lastly, patient’s samples (e.g. from biopsies) can be analyzed in the assay due to the small number of cells required for the microwells and the high-throughput feature of the 3D system. The inclusion of stimulating or blocking drugs (e.g. anti-PD-1 or anti-PD-L1) to probe the tumor cell/T-cell interaction is a logical extension of the assay.
In conclusion, the 3D spheroid co-culture model presented here provides a flexible framework for monitoring cancer cell invasion and cytotoxicity of co-cultured T-cells in a biologically relevant setting. The resulting crosstalk can be visualized while maintaining the integrity of the 3D culture and thus provide mechanistic insights into tumor cell – T-cell interactions.
The authors have nothing to disclose.
We thank Virginie Ory, PhD for helpful discussions and advice on the approach of the 3D co-culture model. We also thank Elizabeth Jones for excellent technical assistance with the IHC sectioning. This study was supported by grants from the DFG (Deutsche Forschungsgemeinschaft) to YL (LI 2547/4-1) and the National Institutes of Health to AW (R01 CA231291), to ATR (R01 CA205632), to GWP (R01 CA218670), and the Core Grant of the Cancer Center (P30 CA51008).
3D Petri Dishes | Microtissues Inc | Z764019 & Z764051 | referred to as "rubber molds" in the protocols; 81-microwell & 35-microwell molds |
8-well Chamber Slides | Lab-Tek | 154534 | |
Agarose Type I, low EEO | Sigma-Aldrich | A6013 | |
anti-rabbit-HRP conjugated secondary antibody | Agilent | K4003 | ready to use |
Collagen Type I, Rat Tail, 100 mg | Millipore | 08-115 | |
Collagenase Type 4, 1 g | Worthington | LS004188 | |
DMEM, fetal bovine serum | ThermoFisher | 11965092, 16000044 | referred to as "cell culture medium" in the protocols |
Harris hematoxylin | ThermoFisher | SH30-500D | |
HistoGel | ThermoFisher | HG-4000-012 | referred to as "Hydroxyethyl agarose processing gel" in the protocols |
Hoechst | Life Technologies | H1399 | 1/1000 dilution |
Phalloidin 546 | Invitrogen | 486624 | 1/200 dilution |
rabbit anti-CD8 antibody | Cell Signaling | 98941 | 1/25 dilution |
rat anti-keratin 8 | DSHB | TROMA-I AB_531826 | 1/500 dilution |
RNeasy Mini Kit | Qiagen | 74104 | referred to as "RNA extraction kit" in the protocols |
RPMI | ThermoFisher | 11875093 | for T-cell culture medium |
Triton X-100 | BioRad | 1610407 | referred to as "Octoxynol" in the protocols |
Trizol | ThermoFisher | 15596026 | referred to as "guanidinium thiocyanate with phenol" in the protocols |
Tween 20 | Sigma-Aldrich | P1379 | referred to as "polysorbate 20" in the protocols |
TypLE | ThermoFisher | 12604013 | referred to as "cell dissociation enzymes solution" in the protocols |