Improving Antibody Labeling of Spheroids Embedded in Thick Intact 3D Matrices via Microwave Irradiation

Introduction:: Three-dimensional hydrogel cell cultures have progressively been used to create in vitro models that preserve morphological and biochemical tissue features. Immunofluorescence labeling is paramount to understanding biomolecular interactions and cellular activity within these complex models. To preserve the 3D structure and label cells in their environment, the whole sample can be stained with antibodies, however, the limited passive diffusion of antibodies through thick 3D samples results in prolonged staining and washing steps which can reach several days of processing time. Microwave irradiation has been shown to facilitate fixative penetration in a variety of soft and hard tissues by increasing the diffusion of molecules within the sample [1, 2]. Utilizing a microwave processing system with a temperature-controlled surface and vacuum chamber, uniform irradiation of the sample can be achieved with control of microwave power, temperature, and the pressure surrounding the sample. The objective of this study is to determine if microwave irradiation could provide greater depth penetration of antibodies over a short period of time within 3D in vitro tumor models consisting of cancerous and non-cancerous breast epithelial spheroids embedded in thick collagen hydrogels.

Materials and Methods:: Non-cancerous (MCF10A) and cancerous (MCF7) breast epithelial spheroids were created by seeding cells in low adhesion 96-well plates at starting densities of 3,000 or 1,000 cells/mL to create larger or smaller spheroids. After 3 days, spheroids were placed within collagen hydrogels (2.7 mg/mL) in 8-well chambered slides (Fig. 1A). After 24 hours, spheroids were fixed with a solution of 4% PFA and 1% TritonX-100 for 45 min followed by a wash with 1X PBS^+/+ for 10 min at room temperature. Following the wash, samples underwent a methanol dehydration and rehydration series in the microwave (Pelco Biowave® Pro+ with Pelco Coldspot® temperature-controlled surface and vacuum chamber). Samples were then washed and blocked with 0.2% TritonX-100 and 1% BSA for 10 min. The antibody staining solution (anti-β-Catenin-AF647, Abcam) was added to the samples and then microwave irradiated for a specified amount of time with power cycling of 5 min irradiation on and 2 min off. Slides were placed in a large dish containing 0.2% TritonX-100 in 1X PBS^+/+ to wash the unbound antibodies for 20 min, followed by DAPI staining using the microwave for 30 min and another final wash for 30 min in the microwave. All microwave processes were performed at 250 W under vacuum. After staining was complete, samples were imaged via an inverted confocal laser scanning microscope (Zeiss, LSM880) to image antibody fluorescence as well as second harmonic generation (SHG) imaging of the collagen matrix.

Results and Discussions:: A primary conjugated antibody, anti-β-Catenin-AF647, labeling cell-cell adhesions, was chosen for initial studies as it had high specificity to both spheroid types as determined by staining spheroids in absence of hydrogel matrix. Spheroids that were ~250 µm in diameter were embedded in collagen hydrogels with 500 µm of collagen above them which served as the effective barrier for antibody diffusion (Fig. 1A). Microwave-assisted staining was performed with 90 min or 150 min of irradiation during the antibody labeling step and showed incomplete staining of the spheroid center (Fig. 1B-C, F-G). By increasing the antibody staining time by 1 hour, we observed an increased radial depth penetration of ~40 µm as determined by the β-Catenin imaging (Fig. 1E, C, and G). In contrast, smaller spheroids (~125 µm diameter) embedded in collagen had labeling throughout with 90 minutes of microwave irradiation time during the antibody labeling step (Fig. 1D, H). When following the same staining protocol on the benchtop without microwave irradiation, it was observed that there was decreased antibody labeling of the spheroids and increased non-specific background signal compared to using the microwave. SHG imaging of the collagen matrix before and after microwave irradiation showed no apparent changes in the matrix architecture.

Conclusion: Microwave-assisted staining of breast epithelial spheroids embedded in thick 3D matrices improved depth penetration of antibody staining and enabled labeling of ~125 µm embedded spheroids in shorter time (~8 hours in total) when compared to conventional benchtop techniques (greater than 24 hours). Using microwave irradiation in combination with temperature control and vacuum showed no alteration or degradation of the samples. By manipulating the microwave irradiation time, increased depth penetration can be achieved when labeling large spheroid structures while still preserving sample integrity. Our results showcase a technique to utilize microwave irradiation to facilitate efficient immunofluorescent labeling of cells within thick, intact 3D in vitro models in a reduced time period. Ongoing and future work will optimize this technique for complete labeling of large spheroids and to apply this method to other 3D in vitro models of different cell types and hydrogel matrices.

References (Optional): :
1. Boon, M.E. and L.P. Kok, Microwaves for immunohistochemistry. Micron, 1994. 25(2): p. 151-170.
2. Jamur, M.C., et al., Microwave fixation improves antigenicity of glutaraldehyde-sensitive antigens while preserving ultrastructural detail. J Histochem Cytochem, 1995. 43(3): p. 307-11.