A Scaffold-Assisted 3D Cancer Cell Model for Surface-Enhanced Raman Scattering-Based Real-Time Sensing and Imaging

Abstract

Despite recent advances in the development of scaffold-based three-dimensional (3D) cell models, challenges persist in imaging and monitoring cell behavior within these complex structures due to their heterogeneous cell distribution and geometries. Incorporating sensors into 3D scaffolds provides a potential solution for real-time, in situ sensing and imaging of biological processes such as cell growth and disease development. We introduce a 3D printed hydrogel-based scaffold capable of supporting both surface-enhanced Raman scattering (SERS) biosensing and imaging of 3D breast cancer cell models. The scaffold incorporates plasmonic nanoparticles and SERS tags, for sensing and imaging, respectively. We demonstrate the scaffold's adaptability and modularity in supporting breast cancer spheroids, thereby enabling spatial and temporal monitoring of tumor evolution.

Keywords: 3D cell models; 3D printing; SERS; bioimaging; biosensing; tumor microenvironment.

Figure 1

(A) SEM image showing a top view 3D-printed scaffold (1 cm × 1 cm). A top-view photograph is shown as an inset. (B) SEM image showing a cross-section of the scaffold. A side-view photograph is shown as an inset. (C) SERS maps based on the intensity of the MBA peak at 1084 cm^–1, showing details of the XY plane (focused on the second layer of the scaffold) and corresponding XZ and YZ planes, defined by green lines (scale bars: 400 μm).

Figure 2

(A,B) 3D confocal fluorescence microscopy reconstructions of (GFP-expressing) MCF7 cells cultured on scaffolds coated with fibronectin and collagen, after 4 (A) and 21 (B) days in vitro (DIV) (Scale bars: 500 μm). (C) SERS spectra of AuNR@2NAT in the scaffold and AuNS@4BPT in labeled MCF7 cells. (D) 3D SERS reconstruction of MCF7 cells labeled with AuNS@4BPT (blue) on a scaffold labeled with AuNR@2NAT (red) after 5 DIV. The cube dimensions are 2 × 2 × 1 mm³ (scale bars: 500 μm).

Figure 3

Monitoring of spheroidal growth (MCF7 in red, HDF in green) in a PEGDA scaffold over time. (A) Schematic view of the preparation of the scaffold containing AuNR@2NAT, optimized ECM, and SERS-labeled MCF7 and HDF cells and spheroids. (B) MIP from Z-stack confocal fluorescence imaging of spheroid growth after 1 DIV. Scale bar: 1000 μm. (C) Confocal microscopy images at different times of the spheroid found in the yellow square in (B). Scale bars: 50 μm. (D) Merged XY SERS maps showing spheroid growth within a scaffold labeled with AuNR@2NAT (cyan), MCF7 cells labeled with AuNS@BT (red), and HDF cells labeled with AuNS@4BPT (green). Scale bars: 500 μm.

Figure 4

Equivalent fluorescence and SERS imaging of cell aggregates or spheroids (RFP-expressing MCF7 in red, GFP-expressing HDF in green) inside the scaffold. (A) Confocal fluorescence image showing cell organization in the area marked with a purple frame (inset) after 4 DIV. (B) High-resolution SERS map of the same region after 4 DIV. (C) High-resolution SERS map of the same region overlapping the optical image from the scaffold. SERS images show HDF cells labeled with AuNS@4BPT (in green) and MCF7 cells with AuNS@BT (in red). Dotted square frames in A and B correspond to the same regions in the sample.

Figure 5

(A) Scheme of the scaffold with optimized ECM-containing spheroids made of MCF7 and HDF cells, together with ligand-free AuNRs. (B) SERS peak intensity maps (150 μm × 150 μm) for those peaks identified in PCA analysis around a cell spheroid (green dashed line in the optical image) after 4 DIV (Scale bars = 50 μm). (C) Uniform Manifold Approximation and Projection (UMAP) of the whole spectra collected over the course of the cell culture experiment at 4, 6, and 8 DIV (data points are color-coded by the DIV, as labeled). (D) SERS spectra of the different clusters identified in (C). (E) Percentage of the distribution of spectra at different DIV across the three clusters.