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Efficacy of recombinant Bacillus Calmette-Guerin containing dltA in in vivo three-dimensional bio-printed bladder cancer-on-a-chip and ex vivo orthotopic mouse model.

ABSTRACT:

Purpose

We investigated the efficacy and optimal dosage of recombinant Bacillus Calmette-Guérin-dltA (rBCG-dltA) in a high-throughput 3D bio-printed bladder cancer-on-a-chip (BCOC) and orthotopic bladder cancer mouse model.

Materials and methods

We fabricated high-throughput BCOC with microfluidic systems, enabling efficient drug screening. The efficacy of rBCG-dltA was evaluated using BCOC by the cell viability assay, monocyte migration assay, and measuring cytokine levels. The anti-tumor effect was compared using the orthotopic bladder cancer mouse model.

Results

The cell proliferation rates of T24 and 253J bladder cancer cell lines (mean±standard error) were measured at three days after treatment. In T24 cell line, there was significantly decreased T24 cells compared to control at rBCG 1 multiplicity of infection (MOI) and 10 MOI (30 MOI: 63.1±6.4, 10 MOI: 47.4±5.2, 1 MOI: 50.5±7.5, control: 100.0±14.5, p<0.05). In 253J cell line, a statistically significant decrease in 253J cell count compared to control and mock BCG 30 MOI (30 MOI: 11.2±1.3, 10 MOI: 22.5±2.3, 1 MOI: 39.4±4.7, Mock: 54.9±10.8, control: 100.0±5.6, p<0.05). The migration rates of THP-1 cells showed increased patterns after rBCG-dltA treatment in BCOC. The concentration of tumor necrosis factor-α and interleukin-6 after rBCG-dltA 30 MOI treatment was higher than control in T24 and 253J cell line.

Conclusions

In conclusion, rBCG-dltA has the potential to have better anti-tumor activity and immunomodulatory effects than BCG. Furthermore, high-throughput BCOCs have potential to reflect the bladder cancer microenvironment.

SUBMITTER: Choi J

PROVIDER: S-EPMC10172047 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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Efficacy of recombinant Bacillus Calmette-Guérin containing dltA in <i>in vivo</i> three-dimensional bio-printed bladder cancer-on-a-chip and <i>ex vivo</i> orthotopic mouse model.

Choi Joongwon J Jung Tae Young TY Kim Jung Hoon JH Maeng Sejung S Kang Su Jeong SJ Kim Mirinae M Choi Young Wook YW Choi Se Young SY Kim Sung-Hwan SH Chang In Ho IH

Investigative and clinical urology 20230501 3

<h4>Purpose</h4>We investigated the efficacy and optimal dosage of recombinant Bacillus Calmette-Guérin-dltA (rBCG-dltA) in a high-throughput 3D bio-printed bladder cancer-on-a-chip (BCOC) and orthotopic bladder cancer mouse model.<h4>Materials and methods</h4>We fabricated high-throughput BCOC with microfluidic systems, enabling efficient drug screening. The efficacy of rBCG-dltA was evaluated using BCOC by the cell viability assay, monocyte migration assay, and measuring cytokine levels. The a ...[more]

PMID: 37341010

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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.