Browsing by Author "Kaimakliotis, Hristos"
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Item A novel, safe, fast and efficient treatment for Her2-positive and negative bladder cancer utilizing an EGF-anthrax toxin chimera(Wiley, 2020) Jack, Sherwin; Madhivanan, Kayalvizhi; Ramadesika, Swetha; Subramanian, Sneha; Edwards, Daniel F., II; Elzey, Bennett D.; Dhawan, Deepika; McCluskey, Andrew; Kischuk, Erin M.; Loftis, Alexander R.; Truex, Nicholas; Santos, Michael; Lu, Mike; Rabideau, Amy; Pentelute, Bradley; Collier, John; Kaimakliotis, Hristos; Koch, Michael; Ratliff, Timothy L.; Knapp, Deborah W.; Aguilar, Ruben C.; Urology, School of MedicineBladder cancer is the sixth most common cancer in the United States, and it exhibits an alarming 70% recurrence rate. Thus, the development of more efficient antibladder cancer approaches is a high priority. Accordingly, this work provides the basis for a transformative anticancer strategy that takes advantage of the unique characteristics of the bladder. Unlike mucin-shielded normal bladder cells, cancer cells are exposed to the bladder lumen and overexpress EGFR. Therefore, we used an EGF-conjugated anthrax toxin that after targeting EGFR was internalized and triggered apoptosis in exposed bladder cancer cells. This unique agent presented advantages over other EGF-based technologies and other toxin-derivatives. In contrast to known agents, this EGF-toxin conjugate promoted its own uptake via receptor microclustering even in the presence of Her2 and induced cell death with a LC50 < 1 nM. Furthermore, our data showed that exposures as short as ≈3 min were enough to commit human (T24), mouse (MB49) and canine (primary) bladder cancer cells to apoptosis. Exposure of tumor-free mice and dogs with the agent resulted in no toxicity. In addition, the EGF-toxin was able to eliminate cells from human patient tumor samples. Importantly, the administration of EGF-toxin to dogs with spontaneous bladder cancer, who had failed or were not eligible for other therapies, resulted in ~30% average tumor reduction after one treatment cycle. Because of its in vitro and in vivo high efficiency, fast action (reducing treatment time from hours to minutes) and safety, we propose that this EGF–anthrax toxin conjugate provides the basis for new, transformative approaches against bladder cancer.Item Bottom-Up Fabrication of Plasmonic Nanoantenna-Based High-throughput Multiplexing Biosensors for Ultrasensitive Detection of microRNAs Directly from Cancer Patients’ Plasma(ACS, 2020) Masterson, Adrianna N.; Liyanage, Thakshila; Kaimakliotis, Hristos; Derami, Hamed Gholami; Deiss, Frédérique; Sardar, Rajesh; Chemistry and Chemical Biology, School of ScienceThere is an unmet need in clinical point-of-care (POC) cancer diagnostics for early state disease detection, which would greatly increase patient survival rates. Currently available analytical techniques for early stage cancer diagnosis do not meet the requirements for POC of a clinical setting. They are unable to provide the high demand of multiplexing, high-throughput, and ultrasensitive detection of biomarkers directly from low volume patient samples (“liquid biopsy”). To overcome these current technological bottle-necks, herein we present, for the first time, a bottom-up fabrication strategy to develop plasmonic nanoantenna-based sensors that utilize the unique localized surface plasmon resonance (LSPR) properties of chemically synthesized gold nanostructures, gold triangular nanoprisms (Au TNPs), gold nanorods (Au NRs), and gold spherical nanoparticles (Au SNPs). Our Au TNPs, NRs, and SNPs display refractive index unit (RIU) sensitivities of 318, 225, and 135 nm/RIU respectively. Based on the RIU results, we developed plasmonic nanoantenna-based multiplexing and high-throughput biosensors for the ultrasensitive assay of microRNAs. MicroRNAs are directly linked with cancer development, progression, and metastasis, thus they hold promise as next generation biomarkers for cancer diagnosis and prognosis. The developed biosensors are capable of assaying five different types of microRNAs at an attomolar detection limit. These sets of microRNAs include both oncogenic and tumor suppressor microRNAs. To demonstrate the efficiency as a POC cancer diagnostic tool, we analyzed the plasma of 20-bladder cancer patients without any sample processing steps. Importantly, our liquid biopsy-based biosensing approach is capable of differentiating healthy from early (“non-metastatic”) and late (“metastatic”) stage cancer with a p value <0.0001. Further, receiver operating characteristic analysis shows that our biosensing approach is highly specific, with an area under the curve of 1.0. Additionally, our plasmonic nanoantenna-based biosensors are regenerative, allowing multiple measurements using the same biosensors, which is essential in low- and middle-income countries. Taken together, our multiplexing and high-throughput biosensors have the unmatched potential to advance POC diagnostics and meet global needs for early stage detection of cancer and other diseases (e.g., infectious, autoimmune, and neurogenerative diseases).Item Durability of Response to Primary Chemoablation of Low-Grade Upper Tract Urothelial Carcinoma Using UGN-101, a Mitomycin-Containing Reverse Thermal Gel: OLYMPUS Trial Final Report(AUA, 2022-04) Matin, Surena F.; Pierorazio, Phillip M.; Kleinmann, Nir; Gore, John L.; Shabsigh, Ahmad; Hu, Brian; Chamie, Karim; Godoy, Guilherme; Hubosky, Scott G.; Rivera, Marcelino; O'Donnell, Michael; Quek, Marcus; Raman, Jay D.; Knoedler, John J.; Scherr, Douglas; Weight, Christopher; Weizer, Alon; Woods, Michael; Kaimakliotis, Hristos; Smith, Angela B.; Linehan, Jennifer; Coleman, Jonathan; Humphreys, Mitchell R.; Pak, Raymond; Lifshitz, David; Verni, Michael; Klein, Ifat; Konorty, Marina; Strauss-Ayali, Dalit; Hakim, Gil; Seltzer, Elyse; Schoenberg, Mark; Lerner, Seth P.; Urology, School of MedicinePurpose: Our goal was to evaluate long-term safety and durability of response to UGN-101, a mitomycin-containing reverse thermal gel, as primary chemoablative treatment for low-grade upper tract urothelial carcinoma. Materials and Methods: In this open-label, single-arm, multicenter, phase 3 trial (NCT02793128), patients ≥18 years of age with primary or recurrent biopsy-proven low-grade upper tract urothelial carcinoma received 6 once-weekly instillations of UGN-101 via retrograde catheter to the renal pelvis and calyces. Those with complete response (defined as negative ureteroscopic evaluation, negative cytology and negative for-cause biopsy) 4–6 weeks after the last instillation were eligible for up to 11 monthly maintenance instillations and were followed for ≥12 months with quarterly evaluation of response durability. Durability of complete response was determined by ureteroscopic evaluation; duration of response was estimated by the Kaplan-Meier method. Treatment-emergent adverse events (TEAEs) were monitored. Results: Of 71 patients who initiated treatment, 41 (58%) had complete response to induction therapy and consented to long-term followup; 23/41 patients (56%) remained in complete response after 12 months (95% CI 40, 72), comprising 6/12 (50%) who did not receive any maintenance instillations and 17/29 (59%) who received ≥1 maintenance instillation. Kaplan-Meier analysis of durability was estimated as 82% (95% CI 66, 91) at 12 months. Ureteric stenosis was the most frequently reported TEAE (31/71, 44%); an increasing number of instillations appeared to be associated with increased incidence of urinary TEAEs. Conclusions: Durability of response to UGN-101 with or without maintenance treatment is clinically meaningful, offering a kidney-sparing therapeutic alternative for patients with low-grade disease.Item Early and Midterm Complications of the Continent Catheterizable Indiana Pouch Urinary Diversion: A 7-year Experience(Elsevier, 2022-09) Burns, Ramzy; Speir, Ryan; Kern, Sean Q.; Jarvis, Hannah; Schmidt, Jonathan; Cary, Clint; Masterson, Timothy; Gardner, Thomas; Bihrle, Richard; Koch, Michael; Kaimakliotis, Hristos; Urology, School of MedicineObjectives To describe the most recent 7 year experience with 137 Indiana pouch patients at a single institution and provide data on complications with this type of urinary diversion during the first postoperative year. Methods We queried our bladder cancer database to identify all patients who underwent cystectomy with continent catheterizable urinary reservoir between 2012 and 2018. Pre-, intra-, and postoperative data were collected. Complications were stratified into early (within 90 days) and midterm (90-365 days). The primary outcomes were postoperative complications, and overall and cancer-specific mortality. Results A total of 137 patients underwent open cystectomy with Indiana pouch creation. Of these, 93% were radical cystectomies. On average, the operation took 422 minutes. There were 53 (39%) patients who experienced any type of complication during the first postoperative year (Clavien II-V). Twenty-five patients (18.2%) readmitted in the early postoperative period vs 18 (13.1%) patients midterm. There were 10 (7.3%) patients that required early reoperation and 11 (8%) in the midterm period. The overall mortality rate was 1.5% early and 3.7% midterm, with the majority of the mortality rate attributed to cancer progression (85.7%). Conclusion Patients undergoing continent catheterizable reservoir urinary diversion appear to have comparable complication rates to other urinary diversions published in the literature. At high-volume urologic institutions, Indiana Pouch creation is a suitable option for select patients desiring a continent diversion.Item A Novel Liquid Biopsy-Based Approach for Highly Specific Cancer Diagnostics: Mitigating False Responses in Assaying Patient Plasma-Derived Circulating microRNAs through Combined SERS and Plasmon-Enhanced Fluorescence Analyses(RSC, 2020) Masterson, Adrianna N.; Liyanage, Thakshila; Berman, Claire; Kaimakliotis, Hristos; Johnson, Merrell; Sardar, Rajesh; Chemistry and Chemical Biology, School of ScienceStudies have shown that microRNAs, which are small noncoding RNAs, hold tremendous promise as next-generation circulating biomarkers for early cancer detection via liquid biopsies. A novel, solid-state nanoplasmonic sensor capable of assaying circulating microRNAs through a combined surface-enhanced Raman scattering (SERS) and plasmon-enhanced fluorescence (PEF) approach has been developed. Here, the unique localized surface plasmon resonance properties of chemically-synthesized gold triangular nanoprisms (Au TNPs) are utilized to create large SERS and PEF enhancements. With careful modification to the surface of Au TNPs, this sensing approach is capable of quantifying circulating microRNAs at femtogram/microliter concentrations. Uniquely, the multimodal analytical methods mitigate both false positive and false negative responses and demonstrate the high stability of our sensors within bodily fluids. As a proof of concept, microRNA-10b and microRNA-96 were directly assayed from the plasma of six bladder cancer patients. Results show potential for a highly specific liquid biopsy method that could be used in point-of-care clinical diagnostics to increase early cancer detection or any other diseases including SARS-CoV-2 in which RNAs can be used as biomarkers.Item One-Pot Analytical Pipeline for Efficient and Sensitive Proteomic Analysis of Extracellular Vesicles(American Chemical Society, 2023) Liu, Yi-Kai; Wu, Xiaofeng; Hadisurya, Marco; Li, Li; Kaimakliotis, Hristos; Iliuk, Anton; Tao, W. Andy; Urology, School of MedicineExtracellular vesicle (EV) proteomics emerges as an effective tool for discovering potential biomarkers for disease diagnosis, monitoring, and therapeutics. However, the current workflow of mass spectrometry-based EV proteome analysis is not fully compatible in a clinical setting due to inefficient EV isolation methods and a tedious sample preparation process. To streamline and improve the efficiency of EV proteome analysis, here we introduce a one-pot analytical pipeline integrating a robust EV isolation approach, EV total recovery and purification (EVtrap), with in situ protein sample preparation, to detect urinary EV proteome. By incorporating solvent-driven protein capture and fast on-bead digestion, the one-pot pipeline enabled the whole EV proteome analysis to be completed within one day. In comparison with the existing workflow, the one-pot pipeline was able to obtain better peptide yield and identify the equivalent number of unique EV proteins from 1 mL of urine. Finally, we applied the one-pot pipeline to profile proteomes in urinary EVs of bladder cancer patients. A total of 2774 unique proteins were identified in 53 urine samples using a 15 min gradient library-free data-independent acquisition method. Taken altogether, our novel one-pot analytical pipeline demonstrated its potential for routine and robust EV proteomics in biomedical applications.Item Plasmoelectronic-Based Ultrasensitive Assay of Tumor Suppressor microRNAs Directly in Patient Plasma: Design of Highly Specific Early Cancer Diagnostic Technology(ACS, 2019) Liyanage, Thakshila; Masterson, Adrianna N.; Oyem, Hector H.; Kaimakliotis, Hristos; Nguyen, Hang; Sardar, Rajesh; Chemistry and Chemical Biology, School of ScienceIt is becoming understood that microRNAs hold great promise for noninvasive liquid biopsies for screening for different types of cancer, but current state-of-the-art RT-PCR and microarray techniques have sensitivity limitations that currently restrict their use. Herein, we report a new transduction mechanism involving delocalization of photoexcited conduction electrons wave function of gold triangular nanoprism (Au TNP) in the presence of -ssDNA/microRNA duplexes. This plasmoelectronic effect increases the electronic dimension of Au TNPs and substantially affects their localized surface plasmon resonance (LSPR) properties that together allow us to achieve a sensitivity for microRNA assay as low as 140 zeptomolar concentrations for our nanoplasmonic sensors. We show that the position of a single base-pair mismatch in the -ssDNA/microRNA duplex dramatically alters the LSPR properties and detection sensitivity. The unprecedentedly high sensitivity of nanoplasmonic sensors has allowed us to assay four different microRNAs (microRNA-10b, -182, -143, and -145) from bladder cancer patient plasma (50 μL/sample). For the first time, we demonstrate the utility of a label-free, nanoplasmonic sensor in quantification of tumor suppressor microRNAs, the level of tumor suppressor microRNAs goes down in a cancer patient as compared to normal healthy individuals, in metastatic and nonmetastatic bladder cancer patient plasma. Our statistical analysis of patient samples unequivocally suggests that the tumor suppressor microRNAs are more specific biomarkers (p-value of <0.0001) than oncogenic microRNAs for differentiation between metastatic and nonmetastatic bladder cancer, and nonmetastatic cancer from healthy individuals. This work demonstrating the electron wave functions delocalization dependent ultrasensitive LSPR properties of noble metal nanoparticles has a great potential for fabrication of miniaturized and extremely powerful sensors to investigate microRNA properties in other cancers (for example breast, lung, and pancreatic) through liquid biopsy.Item A Prospective Program to Reduce the Clinical Incidence of Clostridium Difficile Colitis Infection after Cystectomy(Elsevier, 2018) Calaway, Adam C.; Jacob, Joseph M.; Tong, Yan; Shumaker, Luke; Kitley, Weston; Boris, Ronald S.; Cary, Clint; Kaimakliotis, Hristos; Masterson, Timothy A.; Bihrle, Richard; Koch, Michael O.; Urology, School of MedicinePurpose The development of Clostridium difficile infection after cystectomy is associated with significant morbidity and mortality. We implemented a prospective screening program to identify asymptomatic carriers of Clostridium difficile and assessed its impact on clinical Clostridium difficile infection rates compared to historical matched controls. Materials and Methods Prospective Clostridium Difficile screening prior to cystectomy began in March 2015. The 380 consecutive patients undergoing cystectomy prior to initiation of screening (control cohort) were matched based on 5 clinical factors with the 386 patients who underwent cystectomy from March 2015 to December 2017 (trial cohort). Screened positive patients were placed in contact isolation and treated prophylactically with Metronidazole. Multivariable models were built on an intention-to-screen and an effectiveness of screening basis to determine if screening reduced the rates of symptomatic Clostridium Difficile infections postoperatively. Results With the implementation of the screening protocol, Clostridium difficile infections rates declined from 9.4 to 5.5% (OR 0.52, p=0.0268) on an intention-to-screen protocol and from 9.2 to 4.9% on an effectiveness of screening protocol (OR 0.46, p=0.0174). Conclusions Clostridium difficile screening prior to cystectomy is associated with a significant decrease in rates of clinically symptomatic infections postoperatively. These results should be confirmed in a randomized controlled trial.Item Targeted elastin-like polypeptide fusion protein for near-infrared imaging of human and canine urothelial carcinoma(Impact Journals, 2022-09-06) Aayush, Aayush; Darji, Saloni; Dhawan, Deepika; Enstrom, Alexander; Broman, Meaghan M.; Idrees, Muhammad T.; Kaimakliotis, Hristos; Ratliff, Timothy; Knapp, Deborah; Thompson, David; Pathology and Laboratory Medicine, School of MedicineCystoscopic visualization of bladder cancer is an essential method for initial bladder cancer detection and diagnosis, transurethral resection, and monitoring for recurrence. We sought to develop a new intravesical imaging agent that is more specific and sensitive using a polypeptide based NIR (near-infrared) probe designed to detect cells bearing epidermal growth factor receptors (EGFR) that are overexpressed in 80% of urothelial carcinoma (UC) cases. The NIR imaging agent consisted of an elastin like polypeptide (ELP) fused with epidermal growth factor (EGF) and conjugated to Cy5.5 to give Cy5.5-N24-EGF as a NIR contrast agent. In addition to evaluation in human cells and tissues, the agent was tested in canine cell lines and tissue samples with naturally occurring invasive UC. Flow cytometry and confocal microscopy were used to test cell-associated fluorescence of the probe in T24 human UC cells, and in K9TCC-SH (high EGFR expression) and K9TCC-Original (low EGF expression) canine cell lines. The probe specifically engages these cells through EGFR within 15 min of incubation and reached saturation within a clinically relevant 1 h timeframe. Furthermore, ex vivo studies with resected canine and human bladder tissues showed minimal signal from normal adjacent tissue and significant NIR fluorescence labeling of tumor tissue, in good agreement with our in vitro findings. Differential expression of EGFR ex vivo was revealed by our probe and confirmed by anti-EGFR immunohistochemical staining. Taken together, our data suggests Cy5.5-ELP-EGF is a NIR probe with improved sensitivity and selectivity towards BC that shows excellent potential for clinical translation.