Pitch-tunable pillar arrays for high-throughput culture and immunohistological analysis of tumor spheroids

Abstract

Tumor spheroids are multicellular, three-dimensional (3D) cell culture models closely mimicking the microenvironments of human tumors in vivo, thereby providing enhanced predictability, clinical relevancy of drug efficacy and the mechanism of action. Conventional confocal microscopic imaging remains inappropriate for immunohistological analysis due to current technical limits in immunostaining using antibodies and imaging cells grown in 3D multicellular contexts. Preparation of microsections of these spheroids represents a best alternative, yet their sub-millimeter size and fragility make it less practical for high-throughput screening. To address these problems, we developed a pitch-tunable 5 × 5 mini-pillar array chip for culturing and sectioning tumor spheroids in a high throughput manner. Tumor spheroids were 3D cultured in an alginate matrix using a twenty-five mini-pillar array which aligns to a 96-well. At least a few tens of spheroids per pillar were cultured and as many as 25 different treatment conditions per chip were evaluated, which indicated the high throughput manner of the 5 × 5 pillar array chip. The twenty-five mini-pillars were then rearranged to a transferring pitch so that spheroid-containing gel caps from all pillars can be embedded into a specimen block. Tissue array sections were then prepared and stained for immunohistological examination. The utility of this pitch-tunable pillar array was demonstrated by evaluating drug distribution and expression levels of several proteins following drug treatment in 3D tumor spheroids. Overall, our mini-pillar array provides a novel platform that can be useful for culturing tumor spheroids as well as for immunohistological analysis in a multiplexed and high throughput manner.

Fig. 1. Schematic illustration of the assembly of a 5 × 5 pillar array for tumor spheroid culture and cryosection. (A) Assembly of 5 × 5 pillars at 9 mm distance for 3D cell cultures in 96-well format. Cross-sectional view of the pillars placed into the 96-well plate is also shown. (B) Assembly of 5 × 5 pillars at 3.2 mm distance on the pillar holder for embedding and cryosectioning. Each pillar has a diameter of 2 mm.

Fig. 2. Experimental procedures used for 5 × 5 pillar arrays for culturing and cryosectioning tumor spheroids. (A) Loading 1 μL human cell–alginate mixture at the tip of the pillars. (B) Culturing the cells in 3D for 4 to 6 days. (C) Exposing 3D-cultured tumor spheroids to drugs by immersing the pillars into 96-wells containing drug solutions. (D) Freezing the spheroid-loaded pillar array over vapor-phase liquid nitrogen. (E) Rearranging pillars to the center of the array for micro-section on a single microscope slide. (F) Embedding the pillar array in OCT and freezing at −20 °C to prepare the OCT/spheroids block. (G) Removing the embedding jig and the pillars. (H) Sectioning the OCT/spheroids block with a cryo-microtome. (I) Staining cryosections on a microscope slide, followed by image acquisition.

Fig. 3. Cross sectional images of tumor spheroids cultured on the 5 × 5 pillar array. HT-29, DLD-1 and AsPC-1 cells were cultured in alginate for 4 to 6 days to form spheroids on pillar tips and stained with DAPI. (A) Hematoxylin staining of HT-29 spheroids was done on a cryosections of 5 × 5 pillar array. The enlarged image shows the morphology of spheroids. Scale bar, 50 μm. (B) Sectional images and size distribution of tumor spheroids of HT-29, DLD-1, AsPC-1 cells. DAPI staining was performed either on cryosections (HT-29) or on pillar tips (DLD-1 and AsPC-1) followed by optical sectioning. Scale bar, 100 μm. Size distribution, (mode and median).

Fig. 4. Uptake of DOX in HT-29 spheroids grown on 5 × 5 pillar array. (A) Composite fluorescence images of 5 × 5 array of HT-29 cell spheroids exposed to DOX. HT-29 spheroids cultured for 5 days were exposed to DOX for 30 min (5 replicates per concentration) and cryosectioned for confocal imaging. Representative images of HT-29 spheroids exposed to 0, 25, 50, 75, or 100 μM DOX are shown in the right panel. Scale bar, 50 μm. (B) Linear relationship between average fluorescence intensity and DOX concentration, showing concentration-dependent uptake of DOX into HT-29 spheroids.

Fig. 5. Uptake of TSLs containing DOX in HT-29 spheroids grown on a 5 × 5 pillar array. HT-29 spheroids cultured for 5 days were exposed to TSLs containing DOX for 2 hours at 40 °C and cryosectioned for image acquisition. (A) Image of the 5 × 5 array of HT-29 spheroids exposed to temperature-sensitive liposomes with different transition temperatures (40.5 °C for TSL1; 41.07 °C for TSL2) or NTSL in triplicate per concentration. (B) Average fluorescence intensity determined from triplicate pillar spots loaded with HT-29 spheroids following drug exposure.

Fig. 6. Analysis of drug-induced cell death in HT-29 spheroids grown on the pillar array. Spheroids cultured for 6 days were exposed to 5-FU, TPZ, GEM, OX, and PA for 2 days, and subjected to cryosection and immunocytochemical staining. (A) Composite image of the 5 × 5 spheroid array, showing a concentration-dependent increase in c-PARP-1 expression (red) with DAPI (blue) for nucleus. (B) Relative cell viability and c-PARP-1 expression as a function of drug concentration. The fluorescent intensities were obtained from the array image in (A). (C) Relative c-PARP-1 expression obtained from the pillar array at 33 μM dosage of the five compounds. (D) Relative c-PARP-1 expression obtained from Western blots at 33 μM for the five compounds.

Fig. 7. Immunofluorescence detection of intracellular and matricellular proteins in HT-29 cells grown as tumor spheroids on pillar arrays. (A) Representative sectional images of a single HT-29 spheroid immunologically stained for several proteins including type I collagen, laminin, E-cadherin, CTGF, and TGF-β. Scale bar, 50 μm. (B) Changes in type I collagen and CTGF expression was evaluated in HT-29 spheroids following exposure to a panel of anticancer drugs. Multiple spheroids were shown on the cross-sectional images of one pillar spot in (B).