The establishment of kidney cancer organoid line in drug testing.

INTRODUCTION: Kidney cancer is a common urological malignancy worldwide with an increasing incidence in recent years. Among all subtypes, renal cell carcinoma (RCC) represents the most predominant malignancy in kidney. Clinicians faced a major challenge to select the most effective and suitable treatment regime for patients from a wide range of modalities, despite improved understanding and diagnosis of RCC. OBJECTIVE: Recently, organoid culture gained more interest as the 3D model is shown to be highly patient specific which is hypothetically beneficial to the investigation of precision medicine. Nonetheless, the development and application of organotypic culture in RCC is still immature, therefore, the primary objective of this study was to establish an organoid model for RCC. MATERIALS AND METHODS: Patients diagnosed with renal tumor and underwent surgical intervention were recruited. RCC specimen was collected and derived into organoids. Derived organoids were validated by histological examminations, sequencing and xenograft. Drug response of organoids were compared with resistance cell line and patients' clinical outcomes. RESULTS: Our results demonstrated that organoids could be successfully derived from renal tumor and they exhibited high concordance in terms of immunoexpressional patterns. Sequencing results also depicted concordant mutations of driver genes in both organoids and parental tumor tissues. Critical and novel growth factors were discovered during the establishment of organoid model. Besides, organoids derived from renal tumor exhibited tumorigenic properties in vivo. In addition, organoids recapitulated patient's in vivo drug resistance and served as a platform to predict responsiveness of other therapeutic agents. CONCLUSION: Our RCC organoid model recaptiluated histological and genetic features observed in primary tumors. It also served as a potential platform in drug screening for RCC patients, though future studies are necessary before translating the outcomes into clinical practices.

Quantitative comparison of the renal pelvic urine and bladder urine to examine modifications of the urine proteome by the lower urinary tract.

PURPOSE: Urine proteome is a valuable reservoir of biomarkers for disease diagnosis and monitoring. Following formation as the plasma filtrate in the kidney, urine is progressively modified by the active reabsorption and secretion of the urinary tract. However, little is known about how the urine proteome changes as it passes along the urinary tract. EXPERIMENTAL DESIGN: To investigate this, we compared the proteome composition of the renal pelvis urine (RPU) and individually self-voided bladder urine (BU) collected from seven unilateral urinary tract obstruction male patients by LC-MS/MS screening. To our knowledge, this is the first proteomic comparison of RPU and BU samples from the same individual. RESULTS: Overall, RPU and BU proteomes did not exhibit proteins that were exclusively present in all samples of one urine type while in none of the other type. Nonetheless, BU had more overrepresented proteins that were observed at a higher frequency than RPU. Label-free quantitative analyses revealed BU-RPU differential proteins that are enriched in exosomes and extracellular proteins. However, the differences were not significant after corrections for multiple testing. Interestingly, we observed a significant increase of collagen peptides with hydroxyproline modifications in the BU samples, suggesting differences in protein modifications. CONCLUSIONS AND CLINICAL RELEVANCE: Our study revealed no substantial differences at the protein level between the BU and RPU samples. Future investigations with expanded cohorts would provide more insights about the urothelial-urinary interactions.

In vitro cytotoxicity of human urine and its potential toxic parameters towards bladder cancer cells.

BACKGROUND: Bladder cancer (CaB) has a high recurrence rate despite surgery. As bladder is constantly filled with urine, it is worthwhile to investigate whether it could have any detrimental effects on bladder cancer cells. METHODS: We investigated the cytotoxicity of urine samples from CaB patients and normal controls on four CaB cell lines and tested the percentage of cell death, proliferation, adhesion, invasion and colonies formation ability. In order to identify the potential components involving in urine cytotoxicity, we evaluated some basic physiochemical parameters of urines, such as pH, osmolarity, creatinine (Cr), sodium (Na), potassium (K), chloride (Cl), calcium (Ca) and phosphate (PO4). We further compared the pH values of urine samples between CaB who developed recurrence versus those who did not. A more in-depth analysis on inflammatory markers was performed for two representative urine samples which demonstrated opposite cytoxic effects. RESULTS: 23 CaB patients and 20 normal controls were recruited into this study. According to in vitro experiments, both CaB and non-CaB urines had comparable effect on cell toxicity, proliferation, adhesion, invasion and colonies formation ability in four cell lines, HTB9, RT4, T24 and UMUC3, while RT4 was the most sensitive to urine toxicity. After evaluating the relationship between basic physiochemical parameters and cytotoxicity, we found out that there were strong negative correlations between pH value and 24 hours death rate for the 4 CaB cell lines (HTB9 r = -0.6651, p<0.001; RT4 r = -0.8335, p<0.001; T24 r = -0.4924, p<0.001; UMUC3 r = -0.7066, p<0.001). Osmolarity, urine Cr and PO4 all had weakly or moderately positive correlations with CaB cells on 24 hours death rate. CaB patients who developed recurrence had more alkaline urine than those who did not develop recurrence. In the urine sample with the highest cytoxicity, high concentrations of IL-6 and IFN-gamma were found. CONCLUSIONS: Our study confirmed that there was not statistically significant difference in cytotoxicity between CaB and non-CaB urines. However, we identified some parameters that could have an impact on cytotoxicity towards CaB cells. Modifying certain urine characteristics peri-operatively may induce cytotoxicity, avoid tumour re-implantation, and reduce the chance of cancer recurrence.

Oral antibiotics perturbation on gut microbiota after prostate biopsy.

Introduction: The use of antibiotics may induce the changes in gut microbiota. Previous studies have shown conflicting results on whether the changed gut microbiota by antibiotics can be recovered. Our study aims to investigate whether the gut microbiota could be recovered after a single dose of oral co-amoxiclav before transrectal ultrasound-guided transperineal prostate biopsy (TPPBx) in 5 weeks' time. Methods: Fifteen patients with elevated serum prostate-specific antigen (PSA) were recruited to provide pre-antibiotic and post-antibiotic fecal samples. The V4 region of 16S rRNA was sequenced. Analysis was performed by QIIME2. Alpha- and beta-diversities were analyzed, as well as the differential enrichment by Linear discriminant analysis Effect Size (LEfSe) analysis. Results: Both the alpha- and beta-diversities of the pre- and post-antibiotic fecal samples were significantly different. Genera that are associated with alleviation of inflammation were enriched in the pre-antibiotic fecal samples, while the inflammation-associated genera were more enriched in the post-antibiotic fecal samples. Conclusion: A single dose of oral co-amoxiclav before TPPBx could have led to a change of gut microbiota that cannot be recovered in 5 weeks' time. Microbiome studies on prostate cancer patients should be cautioned on the use of post-prostate biopsy fecal sampling. Further studies should be conducted for the impact on gut microbiome for TPPBx alone.

The Association between Spermidine/Spermine N1-Acetyltransferase (SSAT) and Human Malignancies.

Spermidine/spermine N1-acetyltransferase (SSAT) functions as a critical enzyme in maintaining the homeostasis of polyamines, including spermine, spermidine, and putrescine, in mammalian cells. SSAT is a catalytic enzyme that indirectly regulates cellular physiologies and pathways through interaction with endogenous and exogenous polyamines. Normally, SSAT exhibits only at a low cellular level, but upon tumorigenesis, the expression, protein level, and activities of SSAT are altered. The alterations induce cellular damages, including oxidative stress, cell cycle arrest, DNA dynamics, and proliferation by influencing cellular mechanisms and signaling pathways. The expression of SSAT has been reported in various studies to be altered in different cancers, and it has been correlated with tumor development and progression. Tumor grades and stages are associated with the expression levels of SSAT. SSAT can be utilized as a target for substrate binding, and excreted metabolites may be used as a novel cancer biomarker. There is also potential for SSAT to be developed as a therapeutic target. Polyamine analogs could increase SSAT expression and increase the cytotoxicity of chemotherapy to tumor cells. Drugs targeting polyamines and SSAT expression have the potential to be developed into new cancer treatments in the future.

Identification of piRNA Targets in Urinary Extracellular Vesicles for the Diagnosis of Prostate Cancer.

Emerging studies demonstrate that PIWI-interacting RNAs (piRNAs) are associated with various human cancers. This study aimed to evaluate the urinary extracellular vesicles (EVs) piRNAs as non-invasive biomarkers for prostate cancer (PCa) diagnosis. RNA was extracted from urinary EVs from five PCa patients and five healthy controls (HC), and the piRNAs were analyzed by small RNA sequencing. Dysregulated piRNAs were identified and then validated in another 30 PCa patients and 10 HC by reverse-transcription polymerase chain reaction (RT-qPCR). The expressions of novel_pir349843, novel_pir382289, novel_pir158533, and hsa_piR_002468 in urinary EVs were significantly increased in the PCa group compared with the HC group. The area under the curve (AUC) of novel_pir158533, novel_pir349843, novel_pir382289, hsa_piR_002468, and the combination of the four piRNA in PCa diagnosis was 0.723, 0.757, 0.777, 0.783, and 0.853, respectively. After the RNAhybrid program analysis, all four piRNAs had multiple potential binding sites with key mRNAs in PTEN/PI3K/Akt, Wnt/beta-catenin, or androgen receptor pathway, which are critical in PCa development and progression. In conclusion, our findings indicate that specific piRNAs in urinary EVs may serve as non-invasive diagnostic biomarkers for PCa.

Urinary Cell-Free DNA in Bladder Cancer Detection.

Urinary bladder cancer is a common urological cancer. Although flexible cystoscopy is widely employed in bladder cancer detection, it is expensive, invasive, and uncomfortable to the patients. Recently, urinary cell-free DNA (ucfDNA) isolated from urine supernatant has been shown to have great potential in bladder cancer detection and surveillance. Molecular features, such as integrity and concentration of ucfDNA, have been shown to be useful for differentiating bladder cancer patients from healthy controls. Besides, bladder cancer also exhibits unique genetic features that can be identified from sequencing and expression of ucfDNA. Apart from bladder cancer detection, ucfDNA is also useful for molecular classification. For example, ucfDNA exhibits significant differences, both molecularly and genetically, in non-muscle-invasive and muscle-invasive bladder cancers. There is no doubt that ucfDNA is a very promising tool for future applications in the field of bladder cancer.