Three-Dimensional Imaging for Multiplex Phenotypic Analysis of Pancreatic Microtumors Grown on a Minipillar Array Chip

Abstract

Three-dimensional (3D) culture of tumor spheroids (TSs) within the extracellular matrix (ECM) represents a microtumor model that recapitulates human solid tumors in vivo, and is useful for 3D multiplex phenotypic analysis. However, the low efficiency of 3D culture and limited 3D visualization of microtumor specimens impose technical hurdles for the evaluation of TS-based phenotypic analysis. Here, we report a 3D microtumor culture-to-3D visualization system using a minipillar array chip combined with a tissue optical clearing (TOC) method for high-content phenotypic analysis of microtumors. To prove the utility of this method, phenotypic changes in TSs of human pancreatic cancer cells were determined by co-culture with cancer-associated fibroblasts and M2-type tumor-associated macrophages. Significant improvement was achieved in immunostaining and optical transmission in each TS as well as the entire microtumor specimen, enabling optimization in image-based analysis of the morphology, structural organization, and protein expression in cancer cells and the ECM. Changes in the invasive phenotype, including cellular morphology and expression of epithelial–mesenchymal transition-related proteins and drug-induced apoptosis under stromal cell co-culture were also successfully analyzed. Overall, our study demonstrates that a minipillar array chip combined with TOC offers a novel system for 3D culture-to-3D visualization of microtumors to facilitate high-content phenotypic analysis.

Anti-Cancer Activity Profiling of Chemotherapeutic Agents in 3D Co-Cultures of Pancreatic Tumor Spheroids with Cancer-Associated Fibroblasts and Macrophages

“…Our 3D co-culture model using microfluidic channel chips simulated indirect cancer-stroma interaction and allowed the measurement of cell-type specific changes without additional separation processes. Moreover, our model allows the analysis of 3D spatial and temporal heterogeneity in the culture to track the subpopulations with phenotypic changes [ 24 , 32 ]. Due to low cell number cultured in small size of channel dimension, our model has low suitability for molecular techniques, such as western blotting.…”

3D Coculture between Cancer Cells and Macrophages: From Conception to Experimentation

“…This is the preferred matrix for reproducing desmoplastic tumors characterized by the abnormal deposition of collagen-rich ECM, such as those of breast, pancreatic, and colon cancers. Collagen has already been used in the 3D culture of breast (MDA-MB-231 and PyMT), ,− pancreatic (BxPC3, Panc-1, and Capan-1) ,,, colon (HCT116), lung (HCC), ovarian (SKOV3), cartilage (patient-derived cells), skin (YUMM1.7), and soft tissue (patient-derived cells) cancer cells. This biomaterial is characterized by its versatility, weak antigenicity, robust biocompatibility, and ability to be physically and chemically modified. , Among the different isoforms, the most used is collagen I, which can account for up to 90% of the ECM in many tissues (synthetic or natural, usually from rat tails).…”

“…Regarding human lines, the most used is the monocytic cell line THP-1 derived from an acute monocytic leukemia patient. This cell line, which resembles primary monocytes and macrophages in terms of morphology and functional properties, has been widely used to study immune response as these cells are in both a monocytic state and a macrophage-like state. ,,,,,,,,,,, The second most frequently used immortalized human cell line is U937, which is derived from a malignant lymphoma cell and exhibits monocyte morphology. This cell line (derived from more mature monocytes than THP-1) has been widely used to study the mechanisms underlying monocyte and macrophage differentiation. ,,,,, Finally, a third line, the MM6 monocyte line (Mono-Mac-6), which was isolated from a patient suffering from monoblastic leukemia, is another possibility.…”

“…First, flow cytometry is used to quantify and certify either the macrophagic phenotype present in the coculture (CD14, CD68, CD163, CD206, PD-L1, CD11b/c, CD45, CD64, CD16, CD36, CD86, CD83, HLA-DR, CD200R, CD300E, and CD276) or the presence of certain proteins involved in their interactions (CCR2, MCSFR, TNF-α, IFN-γ, IL-10, and IL-6). ,,,,,, To this end, cells can be recovered by matrix dissociation using collagenase for scaffold and microfluidic models or by tumoroid dissociation using Trypsin EDTA for organotypic models. Finally, slide staining techniques, such as immunofluorescence or immunohistochemistry, can also be used to characterize the cell types in the coculture and attest to the presence of various markers (TAMs: PD-L1, CD80, CD204, CD68, CD163, CD14, CD68, CD2O6; others: COX-2, MMP2/9, TGF-β1, α-SMA, cleaved caspases 3, β-catenin, and E-cadherin). ,,,,,,,,,, Scaffold models can be directly fixed with their matrix and incubated overnight with antibodies, without preventing antibody penetration. Organotypic models, on the other hand, are fixed and cut to be labeled, which highlights the antibody penetration problem in denser models.…”

Modeling the Role of Cancer-Associated Fibroblasts in Tumor Cell Invasion

“…The Kuh lab has developed a mini-pillar chip where multicellular spheroids are generated on the tips of each pillar before being inverted over a 96-well plate filled with culture media ( Figure 2 D). This platform is unique in that spheroids can be subjected to culture conditions and treatments of interest, and then while remaining on the same pillar chip, be embedded either in Optical Cutting Temperature (OCT) compound for cryo-sectioning or Histogel to be processed into a paraffin block for sectioning and staining [ 93 , 94 , 95 ]. They further applied this model by seeding a layer of collagen embedded with PSCs at the bottom of the wells ( Figure 2 D(i)).…”