Speaker
Description
Introduction: Bone marrow is one of the most preferable sites for metastasis, but the complicated in vivo metastatic niches make it challenging to study cancer cell colonization. We took a novel approach to establish an in vitro complex bone marrow environment on a dynamic 3D culture system, Bone marrow (BM)-on-chip. With a specific focus on prostate cancer, we designed, fabricated, and performed an in vitro on-chip cell culture to compare the tumorigenic potential of PTEN-negative PC3 prostate cancer cells with or without intact hemidesmosomes (HD).
Methodology: The BM-on-chip is designed in a standardized 96-well microplate format with a lumen structure, yielding a high throughput platform that allows co-culture and real-time observation of the colonization process. The 3D microenvironment of the bone marrow niche is created by sequentially loading tdTomato-expressing MC3T3 osteoblasts and GFP-positive PC3 cells into collagen I-coated microchannels at days 0 and 7 respectively. The cells are supplemented by the bi-directional flow of medium through the microchannels. Using Leica SP8 Falcon confocal microscope in a live-cell chamber, the assessment of osteoblast and PC3 cells co-culture was done first on day 14, followed by the addition of 0.5 nM or 1 nM DTX into the culture medium. The next imaging was on day 21 to analyze the effect of DTX on cell survival. The areas of MC3T3 cells were compared by analyzing the image data using IMARIS x64 9.2.1 software.
Results: HD-deficient PC3 cells were generated by knocking out the expression of α6-integrin subunit (α6-KO) using CRISPR/Cas9-mediated gene editing. We found that PC3 cells attached on top of fibrillar-shaped osteoblast and formed relatively small foci while α6-KO cells formed much larger cell clusters that were tightly integrated into the osteoblastic structures. Interestingly, co-culture of PC3 cells with osteoblast caused the reduction of MC3T3 osteoblasts whereas this effect was not observed in co-cultures containing α6-KO cells. Next, the cells were treated with docetaxel (DTX) which is a drug commonly used in prostate cancer treatment. Comparative analysis revealed dose-dependent reduction of PC3 cells area and volume after 7 days of incubation with DTX. In contrast, PC3 α6-KO cells appeared relatively resistant to DTX treatment, possibly due to their tight integration into MC3T3 osteoblasts. These observations are in line with our mouse model and 2D cells analysis results showing increased metastasis and DTX-resistance of HD-deficient α6-KO PC3 cells.
Conclusion: Our data shows that the BM-on-chip model can be successfully used for functional analysis of osteoblasts-prostate cancer cells co-culture. It reveals that PC3 α6-KO cells readily colonize osteoblast niches where they show robust resistance to DTX treatment when compared with control PC3 cells.
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