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.

Whole-exome sequencing reveals a comprehensive germline mutation landscape and identifies twelve novel predisposition genes in Chinese prostate cancer patients.

Prostate cancer is the most inheritable cancer with approximately 42% of disease risk attributed to inherited factors by studies of twins, indicating the importance of additional genetic screening to identify predisposition variants. However, only DNA damage repair (DDR) genes have been investigated thoroughly in prostate cancer. To determine the comprehensive germline mutation landscape in Chinese prostate cancer patients, we performed whole exome sequencing in 100 Han Chinese patients with prostate cancer in Hong Kong and identified deleterious germline mutations. A total of 36 deleterious germline variants in 25 genes were identified in 29% patients. Variants were found in eight pathways, including DNA methylation, DDR, and tyrosine-protein kinase. These findings were validated in an independent Chinese cohort of 167 patients with prostate cancer in Shanghai. Seven common deleterious-variant-containing genes were found in discovery cohort (7/25, 28%) and validation cohort (7/28, 25%) with three genes not described before (LDLR, MYH7 and SUGCT) and four genes previously reported (FANCI, ITGA6, PABPC1 and RAD54B). When comparing with that of a cohort of East Asian healthy individuals, 12 non-DDR novel potential predisposition genes (ADGRG1, CHD4, DNMT3A, ERBB3, GRHL1, HMBS, LDLR, MYH7, MYO6, NT5C2, NUP98 and SUGCT) were identified using the discovery and validation cohorts, which have not been previously reported in prostate cancer patients in all ethnic groups. Taken together, this study reveals a comprehensive germline mutation landscape in Chinese prostate cancer patients and discovers 12 novel non-DDR predisposition genes to lay the groundwork for the optimization of genetic screening.

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.

The Potential Role of Spermine and Its Acetylated Derivative in Human Malignancies.

Polyamines are essential biomolecules for normal cellular metabolism in humans. The roles of polyamines in cancer development have been widely discussed in recent years. Among all, spermine alongside with its acetylated derivative, N1, N12-Diacetylspermine, demonstrate a relationship with the diagnosis and staging of various cancers, including lung, breast, liver, colorectal and urogenital. Numerous studies have reported the level of spermine in different body fluids and organ tissues in patients with different types of cancers. Currently, the role and the underlying mechanisms of spermine in cancer development and progression are still under investigation. This review summarized the roles of spermine in cancer development and as a diagnostic, prognostic and therapeutic tool in various cancers.

In vitro assessment of intra-operative and post-operative environment in reducing bladder cancer recurrence.

Urinary bladder cancer is a common cancer worldwide. Currently, the modality of treating and monitoring bladder cancer is wide. Nonetheless, the high recurrence rate of non-muscle-invasive bladder cancer after surgical resection is still unsatisfactory. Hereby, our study demonstrated whether the intra-operative and post-operative environments will affect bladder cancer recurrence utilizing in vitro cell line model. Bladder cancer cell lines were submerged in four different irrigating fluids for assessing their tumorigenic properties. Our results showed that sterile water performed the best in terms of the magnitude of cytotoxicity to cell lines. Besides, we also investigated cytotoxic effects of the four irrigating agents as well as mitomycin C (MMC) in normothermic and hyperthermic conditions. We observed that sterile water and MMC had an increased cytotoxic effect to bladder cancer cell lines in hyperthermic conditions. Altogether, our results could be translated into clinical practice in the future by manipulating the intra-operative and post-operative conditions in order to lower the chance of residual cancer cells reimplant onto the bladder, which in turns, reducing the recurrence rate of bladder cancers.

Endothelial dysfunction after androgen deprivation therapy and the possible underlying mechanisms.

INTRODUCTION: Androgen deprivation therapy (ADT) is a key treatment modality in the management of prostate cancer (PCa), especially for patients with metastatic disease. Increasing evidences suggest that patients who received ADT have increased incidence of diabetes, myocardial infarction, stroke, and even mortality. It is important to understand the pathophysiological mechanisms on how ADT increases cardiovascular risk and induces cardiovascular events, which would provide important information for potential implementation of preventive measures. METHODS: Twenty-six 12-week-old male SD rats were divided into four groups for different types of ADTs including: the bilateral orchidectomy group (Orx), LHRH agonist group (leuprolide), LHRH antagonist group (degarelix), and control group. After treated with drug or adjuvant injection every 3 weeks for 24 weeks, all rats were sacrificed and total blood were collected. Aorta, renal arteries, and kidney were preserved for functional assay, immunohistochemistry, western blot, and quantitative reverse-transcription polymerase chain reaction. RESULTS: In vascular reactivity assays, aorta, intrarenal, and coronary arteries of all three ADT groups showed endothelial dysfunction. AT1R and related molecules at protein and messenger RNA (mRNA) level were tested, and AT1R pathway was shown to be activated and played a role in endothelial dysfunction. Both ACE and AT1R mRNA levels were doubled in the aorta in the leuprolide group while Orx and degarelix groups showed upregulation of AT1R in the kidney tissues. By immunohistochemistry, our result showed higher expression of AT1R in the intrarenal arteries of leuprolide and degarelix groups. The role of reactive oxygen species in endothelial dysfunction was confirmed by DHE fluorescence, nitrotyrosine overexpression, and upregulation of NOX2 in the different ADT treatment groups. CONCLUSION: ADT causes endothelial dysfunction in male rats. GnRH receptor agonist compared to GnRH receptor antagonist, showed more impairment of endothelial function in the aorta and intrarenal arteries. Such change might be associated with upregulation and activation of AngII-AT1R-NOX2 induced oxidative stress in the vasculature. These results help to explain the different cardiovascular risks and outcomes related to different modalities of ADT treatment.

Current status of organoid culture in urological malignancy.

Urological cancers are common malignancies worldwide. Several conventional models, for example, two-dimensional cell culture and animal models have been used for decades to study tumor genetics. Nonetheless, these methods have limitations in reflecting the real tumor microenvironment in vivo, thereby hindering the development of anti-cancer therapeutic agents. Recently, three-dimensional culture models have gained attention because they can overcome the drawbacks of traditional methods. Above all, three-dimensional organoid models are able to mimic the tumor microenvironment in human bodies more closely as they are able to demonstrate the interactions between cells and extracellular matrix. This type of model has therefore extended our understanding of urological cancers. Tumor cells in organoid models can also be co-cultured with other cellular components, such as peripheral blood lymphocytes, and allow further understanding of the effect of tumor microenvironments on tumor growth. Furthermore, organoid models allow a prolonged culturing period, therefore, tumor evolution, progression and maintenance can also be assessed. Organoid models can be derived from each specific patient, and this facilitates investigation of individual cancer-specific mutations and their subtypes. As a result, the development of personalized medication targeting the signaling pathways or biomolecules of interest will be possible. In the present review, we summarize the development and applications of three-dimensional organoid cultures in urological cancers, mainly focusing on prostate, urinary bladder and kidney cancers, and assess the future prospects of this model.

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.

The Emerging Clinical Role of Spermine in Prostate Cancer.

Spermine, a member of polyamines, exists in all organisms and is essential for normal cell growth and function. It is highly expressed in the prostate compared with other organs and is detectable in urine, tissue, expressed prostatic secretions, and erythrocyte. A significant reduction of spermine level was observed in prostate cancer (PCa) tissue compared with benign prostate tissue, and the level of urinary spermine was also significantly lower in men with PCa. Decreased spermine level may be used as an indicator of malignant phenotype transformation from normal to malignant tissue in prostate. Studies targeting polyamines and key rate-limiting enzymes associated with spermine metabolism as a tool for PCa therapy and chemoprevention have been conducted with various polyamine biosynthesis inhibitors and polyamine analogues. The mechanism between spermine and PCa development are possibly related to the regulation of polyamine metabolism, cancer-driving pathways, oxidative stress, anticancer immunosurveillance, and apoptosis regulation. Although the specific mechanism of spermine in PCa development is still unclear, ongoing research in spermine metabolism and its association with PCa pathophysiology opens up new opportunities in the diagnostic and therapeutic roles of spermine in PCa management.

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.