ESPU Meeting on Wednesday 19, April 2023, 13:15 - 14:15
13:15 - 13:18
S01-1 (OP)
Nao IGUCHI 1, Duncan WILCOX 2, Anna MALYKHINA 3 and Dan WOOD 2
1) University of Colorado Denver, Surgery, Aurora, USA - 2) Children's Hospital Colorado, Aurora, USA - 3) University of Colorado Denver, Aurora, USA
PURPOSE
To investigate long term outcome of voiding dysfunction during early childhood using a murine model. Despite the association between childhood dysfunctional voiding and lifelong bladder dysfunction, the underlying mechanism remains poorly understood. Neonatal maternal separation (NMS) disturbs the physiological voiding cycle in mouse pups; which is normally maintained by perigenital-bladder reflex via their mother licking the perineum.
MATERIAL AND METHODS
Newborn mouse pups were divided into control and NMS groups after birth. NMS pups were removed from their dam and housed individually (6h/day) from postnatal day 2 to 14. The control group stayed with their dam all the time. Long-term effects of early life voiding dysfunction on lower urinary tract (LUT) were assessed in vivo by void spot assay at 3-, 6-, and 12-month-old, and in vitro by detrusor physiology studies using bladder strips at 12-month-old.
RESULTS
NMS male mice showed urinary frequency with small voids by 1.6 to 2.2-fold compared to the control mice (p<0.05) through middle-age (12month-old ≈ 40yr-old human). A similar trend towards increased number of small voids was observed in female mice experienced NMS compared to those in control group, while the change was but statistically insignificant (1.2 to 1.5-fold, p>0.05). No differences in the number of large voids and total volume of void were detected between NMS and the control mice. A significant increase in nerve-mediated and purinergic agonist-induced detrusor contractility was observed in NMS group compared to the control group (1.4-fold, p<0.05). There were no changes in contractility in response to carbachol or substance-P (SP) between the two groups. However, preincubation of the bladder strips with SP induced a 1.6-fold larger decrease in nerve-mediated contractility in NMS group than that in the control group (p<0.005).
CONCLUSIONS
Our results provide evidence that the early life disturbance of normal voiding cycle had prolonged impacts on LUT function accompanied with an enhanced involvement of purinergic and peptidergic neurons/signaling in detrusor contractility even into later of life.
13:18 - 13:21
S01-2 (OP)
Nao IGUCHI 1, Duncan WILCOX 2, Anna MALYKHINA 3 and Nicholas COST 2
1) University of Colorado Denver, Surgery, Aurora, USA - 2) Children's Hospital of Colorado, Aurora, USA - 3) University of Colorado Denver, Aurora, USA
PURPOSE
The aim of this study was to investigate the effects on lower urinary tract (LUT) function of systemic vincristine (VCR) exposure during childhood using a murine model. Despite well-known VCR-induced peripheral neuropathy (VIPN), the impact of VIPN on LUT function remains to be elucidated. Our recent study showed childhood cancer survivors who received VCR and/or doxorubicin reported higher rates of LUT dysfunction (LUTD) than control cohort, warrant further investigation.
MATERIAL AND METHODS
With IACUC approval, male CD-1 mice (3.5-wk-old) received an ip injection of 750µg/kg of VCR twice per week for 4 weeks. Control mice received saline. At 1 month after the last treatment, LUT and detrusor function were evaluated by in vivo, awake cystometry and by an in vitro physiological recording using bladder strips, bladder histology and gene expression pattern analysis by qPCR.
RESULTS
VCR exposure induced a 6-fold increase in the number of non-void contractions compared to the control group (p=0.01). A decrease in the detrusor contractility triggered by depolarization of either detrusor muscle or nerves besides an increased responses to carbachol and ATP were observed in VCR group, suggesting that VCR induced a sensitization of cholinergic and purinergic pathway in the bladder. Bladder histology revealed an accumulation of mast cells in VCR group, which is in agreement with the qPCR data, an upregulation of genes related to mast cell activation, CD117 and TrpV2 (2.3- and1.7-fold, p<0.05) and a downregulation of TGFβ1 (0.17-fold, p=0.013) which suppresses mast cell activation.
CONCLUSIONS
Systemic VCR exposure in juvenile mice induced LUTD alongside an increased activity of cholinergic and purinergic pathways in the bladder. Our data also suggests an involvement of mast cells in VCR-induced LUTD. Our results indicate that systemic VCR exposure impacts LUT function and therefore follow-up urological assessment would benefit these children who receive VCR as part of their anti-cancer treatment.
13:21 - 13:24
S01-3 (OP)
Hortense ALLIOT 1, Marc-David LECLAIR 2, Stéphane BIRKLE 3, Pauline THOMAS 3 and Sébastien FARAJ 2
1) CHU de Nantes Hôpital Mere Enfant, Pediatric surgery, Nantes, FRANCE - 2) CHU de Nantes Hôpital Mere Enfant, Pediatric Surgery Department, Nantes, FRANCE - 3) INSERM U892, Centre de Recherche en Cancérologie de Nantes-Angers, Institut de Recherche en Santé de l'Université de Nantes, Nantes, FRANCE
PURPOSE
To generate OAcGD2-specific CAR Vγ9Vδ2T cells and assess their anti-tumor potency in pre-clinical neuroblastoma (NB) models.
MATERIAL AND METHODS
Flow cytometry analysis (FCM) of primary patient-derived NB cells and NB cell lines were performed to study OAcGD2. Peripheral Blood Mononuclear Cells from healthy donors were stimulated using zoledronic-acid to expand γδ T cells that were then retrovirally transduced to express a second-generation OAcGD2-specific CAR (8B6-ScFv-huIgG1Fc-CD28-CD3ζ). Control CAR γδ T cells were generated expressing a CD3ζ-signaling domain depleted CAR. CAR expression was analyzed using FCM. CAR γδ T cell activation was assessed by a CD107a degranulation assay. Specific cytotoxicity of the CAR γδ T on IMR5 target cells was calculated using a chromium-51 release assay. GFP-expressing IMR5 cells cultured in 3D system, referred as spheroïds, were used to study CAR γδ T's long-term anti-NB activity using video-microscopy analysis. CAR γδ T's phenotype and cytokine secretion were analyzed using FCM.
RESULTS
OAcGD2 was expressed in the primary patient-derived and NB cell lines tested. Vγ9Vδ2T cells were expanded from PBMC and reached > 80 % purity. CAR transduction efficacy was >75 %. OAcGD2-specific γδ T cells were effective against OAcGD2+ tumor cell spheroïds, showing CD107a cell surface expression, cytokine production and direct target cell killing.
CONCLUSIONS
OAcGD2-directed Vγ9Vδ2T cell production is feasible and lead to pure and efficient effector cells. OAcGD2-specific Vγ9Vδ2 T cells may provide a promising addition for HRNB therapy and potentially for other OcGD2-expressing pediatric solid tumors.
13:24 - 13:27
S01-4 (OP)
Grégoire SCHNEIDER 1, Cécile PICARD 2, Marie CASTETS 3, Frédérique DIJOUD 2, Pierre-Yves MURE 1 and Ingrid PLOTTON 4
1) Hôpital Femme Mère Enfant, Hospices Civils de Lyon, Department of Pediatric Surgery, Lyon, FRANCE - 2) Groupement Hospitalier Est, Hospics Civils de Lyon, Department of Pathology, Lyon, FRANCE - 3) Centre Léon Bérard, Centre de Recherche en Cancérologie de Lyon (CRCL), Childhood Cancer & Cell Death, Lyon, FRANCE - 4) Groupement Hospitalier Est, Hospics Civils de Lyon, Hormonology, molecular and rare diseases endocrinology, Lyon, FRANCE
PURPOSE
Pediatric ACT are rare. Despite pediatric histoprognostic scores, distinction between benign and malignant tumors remain challenging. Prognostic is poor in malignant tumors when complete surgical resection is not feasible.
Appropriate care for these patients needs better prognostic stratification and better biology understanding to develop new treatments. We aimed to address these issues through transcriptomic analysis.
MATERIAL AND METHODS
We performed RNAseq on FFPE (Formol Fixed and Paraffine Embedded) samples on a monocentric cohort from 2000 to 2021.
Medical charts were reviewed to allow a categorization as benign or malignant tumor. Comparison of transcriptomic data between these 2 groups was done using R software and DESeq2 package in order to identify differentially expressed genes. Databases (Gene Ontology, MutSigDB and KEGG) were consulted to perform Gene Set Enrichment Analysis (GSEA), aiming to reveal biological processes and pathways probably involved in malignancy.
RESULTS
RNAseq data of 13 tumors were obtained (6 categorized as malignant, 7 as benign).
Using log2 fold-change of 1.5 as a treshold for differentially expressed genes, we found 206 overexpressed genes and 165 underexpressed genes in the malignant group, compared to benign.
In the overexpressed list, GSEA identified significant representation of cell cycle genes and epithelial-to-mesenchymal-transition genes.
Metabolic disorders could also be involved in malignancy, as solute carriers (SLCs) genes were found underexpressed and significantly represented in malignant tumors, besides significantly overexpressing phosphoglycerate dehydrogenase (PHGDH).
Cell cycle deregulation has already been described, but the possible involvement of epithelial-to-mesenchymal transition and metabolic disorders was not found in the literature on pediatric ACT.
CONCLUSIONS
Profiles and mecanisms of malignancy in pediatric ACT seem to involve cell cycle deregulation, epithelial-to-mesenchymal transition and metabolic disorders. This should be confirmed. Prognostic and therapeutic tools could derived from these data and subsequent studies.
13:45 - 13:48
S01-5 (OP)
Yutao LU 1, Jens C. DJURHUUS 2, Yazan F. H. RAWASHDEH 3, Rikke NØRREGAARD 2 and L. Henning OLSEN 4
1) Aarhus University Hospital-Skeiby, Urology, Aarhus, DENMARK - 2) Department of Clinical Medicine, Aarhus, DENMARK - 3) Department of Clinical Medicine, Aarhus University & 2Department of Urology-Paediatric Urology, Aarhus University Hospital, Aarhus, DENMARK - 4) 1Department of Clinical Medicine, Aarhus University, Aarhus, DENMARK
PURPOSE
Progression of bladder fibrosis leads to changes in bladder storage and voiding function which may ultimately lead to damaged kidney function. We previously described the protocols and applications of precision cut human bladder slices (PCBS). Here we investigate the value of PCBS for screening effective anti-fibrotic compounds.
MATERIAL AND METHODS
Human PCBS were exposed to transforming growth factor-β(TGF-β) pathway inhibitors with established anti-fibrotic efficacy. Fibrosis was evaluated on collagen type I (Col-1), fibronectin and connective tissue growth factor (CTGF) gene levels using qPCR. Viability was evaluated by measuring ATP levels. IRB approval was obtained from all patients.
RESULTS
Bladder biopsies of 20 patients operated for non-fibrotic causes were obtained. Each biopsy of 1x1 cm was divided into a median 32 slices. PCBS were treated with TGF-β for 48 hours to induce fibrosis. In slices of every biopsy, the efficacy of anti-fibrotic compounds; Pirfenidon (PFD), Relaxin-2 (RLN) and Bone morphogenetic protein 7 (BMP-7) were tested in combination with TGF-β treatment. The biopsy of each patient served thereby as its own control. Pirfenidon suppressed the upregulation of the fibrosis markers Col-1 and CTGF after stimulation with TGF-β.
CONCLUSIONS
Cultured whole bladder wall slices can be used for screening effective compounds before clinical trials. Pirfenidon might be a candidate for further studies on bladder fibrosis.
13:48 - 13:51
S01-6 (OP)
Dafni PLANTA 1, Tim GERWINN 1, Souzan SALEMI 2, Maya HORST 1 and Daniel EBERLI 2
1) University Children's Hospital Zürich, Urology, Zürich, SWITZERLAND - 2) Laboratory for Urologic Oncology and Stem Cell Therapy, Urology, Zürich, SWITZERLAND
PURPOSE
Bladder tissue engineering providing implants with autologous cells might represent a solution for treatment of children with end stage bladder diseases. Unfortunately, diseased smooth muscle cells (SMCs) retain tissue specific impairment in conventional 2D cell culture. It has been found that SMCs cultured in spheroids (3D SMCs) exhibit a more promising contractile phenotype. However, their clinical translation has not been established yet. In this study we aim to investigate whether 3D SMCs continue to show phenotypic improvements after being seeded into a carrier matrix, which then could be used for reconstructive surgery.
MATERIAL AND METHODS
Rat detrusor SMCs were isolated, expanded as 2D SMCs and formed into spheroids. Collagen hydrogels serving as carrier matrices were seeded with either 2D SMCs or 3D SMCs and incubated for two weeks in vitro. Expression of the SMC specific differentiation and contractile markers Calponin, Smoothelin and MyH11 was quantified on the genetic expression level and protein level. SMC outgrowth was assessed.
RESULTS
3D SMCs in collagen hydrogel presented a significantly higher genetic expression of SMC specific contractile markers than 2D SMCs. These results were confirmed on protein level. 3D SMCs showed an even distribution within the hydrogel after two weeks and their density was similar to the one of 2D SMCs.
CONCLUSIONS
3D SMCs colonizing a collagen hydrogel form a dense network and continue to show a superior contractile phenotype compared to 2D SMCs, thus showing promise for clinical translation and future bladder tissue engineering applications.
13:51 - 13:54
S01-7 (OP)
Zhongzhong CHEN 1, Bo ZHU 2, Fuying LAN 1, Jing WANG 3, Yu DING 1 and Fang CHEN 1
1) Shanghai Children's Hospital，School of Medicine，Shanghai Jiao Tong University, Department of Urology, Shanghai, CHINA - 2) School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai Yangtze River Delta Eco-Environmental Change and Management Observation and Research Station, Shanghai, CHINA - 3) Shanghai Children's Hospital，School of Medicine，Shanghai Jiao Tong University, Shanghai, CHINA
PURPOSE
Hypospadias is mediated by androgen receptor (AR) signaling during the neonatal period. Despite that genetic mutations play a principal role in the etiology of hypospadias, an increasing evidence has demonstrated that the alterations of the most prevalent adenosine-to-inosine (A-to-I) RNA editing is associated with progression of various diseases. However, how and to what extent A-to-I editing impact patients with hypospadias are unknown.
MATERIAL AND METHODS
To comprehensively investigate the potential role of A-to-I RNA editing sites in hypospadias, we parsed the transcriptomes and translatomes of eight patients with hypospadias and five non hypospadias samples. Common SNPs that are thought to introduce false-positive detections of RNA editing sites were removed from the analyses to reduce the extent of misleadingly substitutions by bedtools. RNA editing sites were annotated using SNPEff. The exclusively RNA editing sites in hypospadias that we obtained were further subjected to functional enrichment analysis to elucidate their role in the development of hypospadias.
RESULTS
458,152 A-to-I editing sites with 28,061 genes were identified in patients with hypospadias. 66% of editing events exist in intron and intergenic region. 55 A-to-I editing sites with 25 genes were exclusively identified in patients with hypospadias. Among these genes, UBE3A, SH3RF2 and RNF141 were significantly enriched in protein autoubiquitination (p<0.01). UBE3A, in which RNA editing events located in splice region, is involved in the hormonal regulation of male sexual development as an androgen receptor coactivator.
CONCLUSIONS
The A-to-I RNA editing events are very common and may dysregulate the androgen signaling in hypospadias. These findings provide new insights into the current molecular mechanism in the development of hypospadias.
13:54 - 13:57
S01-8 (OP)
Astgik PETROSYAN 1, Paola AGUIARI 1, Qi ZHANG 1, Xiaogang HOU 1, Candace JOHNSON 1, Roger DE FILIPPO 1, Kevin LEMLEY 2, Eun Ji CHUNG 3, Stefano DA SACCO 4 and Laura PERIN 1
1) Children's Hospital Los Angeles, Urology, Los Angeles, USA - 2) Children's Hospital Los Angeles, Nephrology, Los Angeles, USA - 3) University of Southern California, Los Angeles, USA - 4) Children's Hospital Los Angeles, Los Angeles, USA
PURPOSE
Significant molecular and functional changes within the glomerulus, and specifically in podocytes, the cells in charge of the ultrafiltration, are responsible for initiation and progression of renal damage. Our data indicate that elevation of miR-193a plays a key role in regulating podocyte biology by controlling cell cycle phases. Using different tools (glomerulus-on-a-chip: GOAC, human COL4-defective podocytes, FUCCI mice, nanoparticles, and spatial transcriptomics), we have identified miR-193a as a possible disease target in our model of CKD, Alport Syndrome (AS).
MATERIAL AND METHODS
GOAC was seeded with human glomerular endothelial cells and amniotic fluid podocytes of AS and healthy patients to recapitulate the function and structure of the glomerular filtration barrier. miR-193a studies were performed using mimics and inhibitors. Nanoparticles (micelle) containing miR-193a inhibitor were designed to specifically target podocytes. AS FUCCI mice (cell cycle indicator mouse model) were used to track podocyte cell cycle modulation. Biopsies of Alport patients were used to confirm miR-193a expression by in situ hybridization and to perform Digital Spatial Profiling (DSP) using Nanostring technology.
RESULTS
Spatial transcriptomics identified in human AS glomeruli altered gene expression for podocyte structure and GEC phenotype, matrix turnover proteins, miR-193a targets, and cell cycle. Generated AS-GOAC presented impaired permselectivity, and proteomics revealed a distinctive AS signature. In AS-GOAC, miR-193a inhibition, delivered with innovative nanoparticles designed targeting podocytes, restored to normal the altered podocyte cell cycle, and regulated downstream miR-193a targets (WT1, ItgαVβ3/osteopontin, and VEGF) necessary for podocyte homeostasis.
CONCLUSIONS
We show that upregulating of miR-193a induces changes in gene expression and alteration of the cell cycle phases specifically in AS podocytes. Inhibiting miR-193a using micelle technology may re-establish glomerular function by modulating important molecular pathways responsible for podocyte survival representing a therapeutic target in AS settings.