Optical Coherence Tomography Detects Necrotic Regions and Volumetrically Quantifies Multicellular Tumor Spheroids

Abstract

Three-dimensional (3D) tumor spheroid models have gained increased recognition as important tools in cancer research and anticancer drug development. However, currently available imaging approaches used in high-throughput screening drug discovery platforms, for example, bright-field, phase contrast, and fluorescence microscopies, are unable to resolve 3D structures deep inside (>50 μm) tumor spheroids. In this study, we established a label-free, noninvasive optical coherence tomography (OCT) imaging platform to characterize 3D morphologic and physiologic information of multicellular tumor spheroids (MCTS) growing from approximately 250 to 600 μm in height over 21 days. In particular, tumor spheroids of two cell lines, glioblastoma (U-87MG) and colorectal carcinoma (HCT116), exhibited distinctive evolutions in their geometric shapes at late growth stages. Volumes of MCTS were accurately quantified using a voxel-based approach without presumptions of their geometries. In contrast, conventional diameter-based volume calculations assuming perfect spherical shape resulted in large quantification errors. Furthermore, we successfully detected necrotic regions within these tumor spheroids based on increased intrinsic optical attenuation, suggesting a promising alternative of label-free viability tests in tumor spheroids. Therefore, OCT can serve as a promising imaging modality to characterize morphologic and physiologic features of MCTS, showing great potential for high-throughput drug screening. Cancer Res; 77(21); 6011-20. ©2017 AACR.

Figure 1

Illustration of a custom spectral domain optical coherence imaging (SD-OCT) platform for 3D tumor spheroids imaging. (A) A schematic diagram of the SD-OCT platform. (B) 3D tumor spheroids were cultured in a round-bottom, ultra-low-attachment 96 wells plate. (C) From depth-resolved images of a single 3D tumor spheroid, the diameter, height, volume and distribution of necrotic tissue can be measured.

Figure 2

Growth dynamics of 3D tumor spheroids measured with OCT. (A) Timeline for 3D tumor spheroid preparation and imaging. At each time point, OCT imaging was performed on all spheroids, prior to the viability test, H&E and TUNEL staining on 1–2 randomly selected spheroids. (B–D) Sequential en face, cross-section and 3D rendered OCT images to illustrate the growth dynamics of a U-87 MG tumor spheroids over 21 days. The U-87 MG spheroid remained tightly clustered throughout the 21-day development. (E–G) Growth dynamics of an HCT 116 tumor spheroid over 21 days. Distinct evolution of its geometric shape was observed after Day 11, where the spheroid became disrupted and flattened. Scale Bars: 100 µm.

Figure 3

Quantitative analysis of the growth kinetics of 3D tumor spheroids in terms of size (diameter and height) and volume. (A–F) Two tumor spheroids from U-87 MG and HCT 116 cell lines at Day 18 were shown as examples to illustrate how average diameter and height were measured from en face and cross-sectional OCT images. Growth curves of U-87 MG (G) and HCT 116 (H) tumor spheroids were shown. While similar growth trends of average diameter and height were revealed for U-87 MG spheroids in (G), discrepancy between average diameter and height of HCT 116 tumor spheroids after Day 11 was observed in (H), in consistence with our finding of heterogeneous geometry in OCT images (D–F). Comparisons of growth curves of the volume of U-87 MG (I) and HCT 116 (J) tumor spheroids between voxel-based measurements and diameter-based measurements were shown. While volumes obtained from both methods were similar for U-87 MG tumor spheroids throughout 21 days, volumes of HCT 116 tumor spheroids were significantly over-estimated with diameter-based measurements compared to voxel-based measurements, especially after Day 11. Scale Bars: 100 µm.

Figure 4

Determination of necrotic regions of HCT 116 tumor spheroids on Days 4, 14, and 18 based on intrinsic optical attenuation contrast. The backscattered signals in cross-sectional OCT images (A, E, I) were used to derive intensity profiles along each axial scan line (B, F, J). High attenuation regions (indicated in red lines in F, J) could be clearly observed in the intensity profiles of the tumor spheroids on Days 14 and 18 (F, J), but not on the tumor spheroid on Day 4 (B). Further analyses of optical attenuation coefficient histograms (C, G, K) were performed to determine the threshold to separate low and high attenuation regions (i.e. 0.48 mm⁻¹), which is calculated as the median of the two peak values (P₁=0.36 mm⁻¹, P₂=0.60 mm⁻¹). High-attenuation regions above the threshold highlighted in red (H, L) were detected as the necrotic cores in the tumor spheroids. The region of necrotic tissue clearly increased as the spheroid developed. Scale Bars: 100 µm.

Figure 5

Comparison of necrotic regions identified based on optical attenuation contrast with fluorescent, histology and immunohistochemistry (IHC) results. OCT attenuation image of a HCT 116 tumor spheroid on Day 14 (A) was compared with the corresponding fluorescent image (B). The spheroid in fluorescent image was stained with calcein AM (live cells, green) and propidium iodide (dead cells, red). A good match between necrotic regions identified from OCT attenuation image (A: high attenuation region in red) and fluorescent image (B: PI staining in red) was observed. OCT attenuation images of two HCT 116 tumor spheroids on Day 4 (C) and Day 14 (H) were compared with corresponding H&E (D, I) and TUNEL stained (E, J) slices, respectively. No necrotic core was observed in either OCT, H&E or TUNEL images on Day 4. In contrast, the tumor spheroid on Day 14 clearly exhibited a necrotic region in all images (H–L). The highlighted high attenuation region in OCT image (H) matched well with the necrotic region (I) and apoptotic region (J) identified by H&E and TUNEL staining, respectively. Zoom-in views of gray square-highlighted areas in H&E and TUNEL images (D, E, I, J) were shown in (F, G, K, L), respectively. The contours of high attenuation region in OCT image were indicated in black dash lines in H&E and TUNEL images (B, I–L). Scale Bars: 100 µm.