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Establishment of Three-Dimensional Bioprinted Bladder Cancer-on-a-Chip with a Microfluidic System Using Bacillus Calmette-Guerin.

ABSTRACT: Immunotherapy of bladder cancer is known to have favorable effects, although it is difficult to determine which patients will show a good response because of the different tumor microenvironments (TME). Here, we developed a bladder cancer-on-a-chip (BCOC) to mimic the TME using three-dimensional (3D) bioprinting and microfluidic technology. We fabricated a T24 and a 5637-cell line-based BCOC that also incorporated MRC-5, HUVEC, and THP-1 cells. We evaluated the effects of TME and assessed the immunologic reactions in response to different concentrations of Bacillus Calmette-Guérin (BCG) via live/dead assay and THP-1 monocytic migration, and concentrations of growth factors and cytokines. The results show that cell viability was maintained at 15% filling density in circle-shaped cell constructs at 20 μL/min microfluidic flow rate. A 3D co-culture increased the proliferation of BCOCs. We found that the appropriate time to evaluate the viability of BCOC, concentration of cytokines, and migration of monocytes was 6 h, 24 h, and three days after BGC treatment. Lastly, the immunotherapeutic effects of BCOC increased according to BCG dosage. To predict effects of immunotherapeutic agent in bladder cancer, we constructed a 3D bioprinted BCOC model. The BCOC was validated with BCG, which has been proven to be effective in the immunotherapy of bladder cancer.

SUBMITTER: Kim JH

PROVIDER: S-EPMC8396314 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIntravesical Bacillus Calmette-Guerin (BCG) is an effective treatment for bladder superficial carcinoma and it is being tested in interstitial cystitis patients, but its precise mechanism of action remains poorly understood. It is not clear whether BCG induces the release of a unique set of cytokines apart from its pro-inflammatory effects. Therefore, we quantified bladder inflammatory responses and alterations in urinary cytokine protein induced by intravesical BCG and compared the results to non-specific pro-inflammatory stimuli (LPS and TNF-alpha). We went further to determine whether BCG treatment alters cytokine gene expression in the urinary bladder.MethodsC57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-alpha. Morphometric analyses were conducted in bladders isolated from all groups and urine was collected for multiplex analysis of 18 cytokines. In addition, chromatin immune precipitation combined with real-time polymerase chain reaction assay (CHIP/Q-PCR) was used to test whether intravesical BCG would alter bladder cytokine gene expression.ResultsAcute BCG instillation induced edema which was progressively replaced by an inflammatory infiltrate, composed primarily of neutrophils, in response to weekly administrations. Our morphological analysis suggests that these polymorphonuclear neutrophils are of prime importance for the bladder responses to BCG. Overall, the inflammation induced by BCG was higher than LPS or TNF-alpha treatment but the major difference observed was the unique granuloma formation in response to BCG. Among the cytokines measured, this study highlighted the importance of IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-10, IL-17, GM-CSF, KC, and Rantes as discriminators between generalized inflammation and BCG-specific inflammatory responses. CHIP/Q-PCR indicates that acute BCG instillation induced an up-regulation of IL-17A, IL-17B, and IL-17RA, whereas chronic BCG induced IL-17B, IL-17RA, and IL-17RB.ConclusionTo the best of our knowledge, the present work is the first to report that BCG induces an increase in the IL-17 family genes. In addition, BCG induces a unique type of persisting bladder inflammation different from TNF-alpha, LPS, and, most likely, other classical pro-inflammatory stimuli.

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Establishment of Three-Dimensional Bioprinted Bladder Cancer-on-a-Chip with a Microfluidic System Using Bacillus Calmette-Guerin.

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