Characterization of CD147, CA9, and CD70 as Tumor-Specific Markers on Extracellular Vesicles in Clear Cell Renal Cell Carcinoma

Extracellular vesicles (EVs) are secreted by healthy and tumor cells and are involved in cell–cell communication. Tumor-released EVs could represent a new class of biomarkers from liquid biopsies. The aim of this study was to identify tumor-specific EV markers in clear cell renal carcinoma (ccRCC) using cell lines and patient-derived tissue samples. EVs from ccRCC cell lines (786-O, RCC53, Caki1, and Caki2) and patient tissues were isolated via ultracentrifugation. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting using exosome and putative tumor markers (epithelial cell adhesion molecule (EpCAM), carbonic anhydrase 9 (CA9), CD70, CD147). The tumor markers were verified using immunohistochemistry. CA9 was expressed in Caki2 cells and EVs, and CD147 was found in the cells and EVs of all tested ccRCC cell lines. In tumor tissues, we found an increased expression of CA9, CD70, and CD147 were increased in cell lysates and EV fractions compared to normal tissues. In contrast, EpCAM was heterogeneously expressed in tumor samples and positive in normal tissue. To conclude, we developed an effective technique to isolate EVs directly from human tissue samples with high purity and high concentration. In contrast to EpCAM, CA9, CD70, and CD147 could represent promising markers to identify tumor-specific EVs in ccRCC

The Potential Use of Electrochemotherapy in the Treatment of Uveal Melanoma: In Vitro Results in 3D Tumor Cultures and In Vivo Results in a Chick Embryo Model

Uveal melanoma (UM) is the most common primary intraocular tumor that arises from neoplastic melanocytes in the choroid, iris, and ciliary body. Electrochemotherapy (ECT) has been successfully established for the treatment of skin and soft tissue metastatic lesions, deep-seated tumors of the liver, bone metastases, and unresectable pancreas lesions. The aim of this study was to evaluate the effect of ECT in vitro in 3D spheroid culture systems in primary and metastatic UM cell lines. We also investigated the chick embryo chorioallantoic membrane (CAM) as an in vivo model system for the growth and treatment of UM tumors using ECT. The cytotoxic effect of ECT in 3D spheroids was analyzed seven days following treatment by assessment of the size and MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction] assay. The cytotoxicity of ECT after intratumoral or intraarterial administration was evaluated histologically. In vitro and in vivo ECT caused a significant reduction in tumor size and viability compared to electroporation or chemotherapy in both sections of our study. The current results underline the effectiveness of ECT in the treatment of UM and prepare the way for further investigation of its potential application in UM

The metastatic potential of seminomatous germ cell tumours is associated with a specific microRNA pattern

Background Seminomatous germ cell tumours (SGCT) are the most frequent malignancy in young men. Reliable prognostic biomarkers for the prediction of metastasis at diagnosis and the risk of relapse in clinical stage I (CSI) are lacking. Adjuvant therapies carry a risk of overtreatment, whereas salvage therapies have a risk of high toxicities. Thus, the identification of reliable prognostic biomarkers is highly desirable to identify patients who will benefit from early adjuvant treatment. MicroRNAs (miRNAs) regulate tumour development and progression, and their potential as biomarkers has already been proven in a variety of malignancies. Objectives The aim of our study was to define a specific miRNA expression pattern that discriminates metastatic from non‐metastatic primary SGCT. Materials and methods Total RNA was isolated from 24 formalin‐fixed paraffin‐embedded (FFPE) primary SGCT tumours (10 non‐metastatic, five metachronously and nine synchronously metastatic) and from 10 normal testicular tissue samples. Microarray analysis was performed for global miRNA expression profiling. The results were validated by quantitative real‐time polymerase chain reaction (qRT‐PCR). Statistical analysis was performed using SPSS. Results Microarray analyses revealed a specific miRNA pattern that distinguishes metastatic from non‐metastatic SGCT. Sixty‐three miRNAs were differentially expressed in metastatic compared to non‐metastatic tumours (P < .01). Microarray results were confirmed by qRT‐PCR for three out of five selected miRNAs (miR‐29c‐5p, miR‐506‐3p and miR‐371a‐5p; P < .05). All five miRNAs (miR‐29c‐5p, miR‐506‐3p, miR‐1307‐5p, miR‐371a‐5p and miR‐371a‐3p) showed differential expression between tumour and normal tissues (P < .05). Conclusion Metastatic primary SGCTs are characterized by a specific miRNA expression pattern. Therefore, specific miRNAs could represent a new tool to predict the metastatic potential in SGCT patients

miRNA Expression Characterizes Histological Subtypes and Metastasis in Penile Squamous Cell Carcinoma

Although microRNAs are described as promising biomarkers in many tumor types, little is known about their role in PSCC. Thus, we attempted to identify miRNAs involved in tumor development and metastasis in distinct histological subtypes considering the impact of HPV infection. In a first step, microarray analyses were performed on RNA from formalin-fixed, paraffin-embedded tumor (22), and normal (8) tissue samples. Microarray data were validated for selected miRNAs by qRT-PCR on an enlarged cohort, including 27 tumor and 18 normal tissues. We found 876 significantly differentially expressed miRNAs (p ≤ 0.01) between HPV-positive and HPV-negative tumor samples by microarray analysis. Although no significant differences were detected between normal and tumor tissue in the whole cohort, specific expression patterns occurred in distinct histological subtypes, such as HPV-negative usual PSCC (95 differentially expressed miRNAs, p ≤ 0.05) and HPV-positive basaloid/warty subtypes (247 differentially expressed miRNAs, p ≤ 0.05). Selected miRNAs were confirmed by qRT-PCR. Furthermore, microarray data revealed 118 miRNAs (p ≤ 0.01) that were significantly differentially expressed in metastatic versus non-metastatic usual PSCC. The lower expression levels for miR-137 and miR-328-3p in metastatic usual PSCC were validated by qRT-PCR. The results of this study confirmed that specific miRNAs could serve as potential diagnostic and prognostic markers in single PSCC subtypes and are associated with HPV-dependent pathways

Characterization of CD147, CA9, and CD70 as Tumor-Specific Markers on Extracellular Vesicles in Clear Cell Renal Cell Carcinoma

Extracellular vesicles (EVs) are secreted by healthy and tumor cells and are involved in cell&ndash;cell communication. Tumor-released EVs could represent a new class of biomarkers from liquid biopsies. The aim of this study was to identify tumor-specific EV markers in clear cell renal carcinoma (ccRCC) using cell lines and patient-derived tissue samples. EVs from ccRCC cell lines (786-O, RCC53, Caki1, and Caki2) and patient tissues were isolated via ultracentrifugation. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting using exosome and putative tumor markers (epithelial cell adhesion molecule (EpCAM), carbonic anhydrase 9 (CA9), CD70, CD147). The tumor markers were verified using immunohistochemistry. CA9 was expressed in Caki2 cells and EVs, and CD147 was found in the cells and EVs of all tested ccRCC cell lines. In tumor tissues, we found an increased expression of CA9, CD70, and CD147 were increased in cell lysates and EV fractions compared to normal tissues. In contrast, EpCAM was heterogeneously expressed in tumor samples and positive in normal tissue. To conclude, we developed an effective technique to isolate EVs directly from human tissue samples with high purity and high concentration. In contrast to EpCAM, CA9, CD70, and CD147 could represent promising markers to identify tumor-specific EVs in ccRCC

Allogeneic Limbal Transplants Integrate into the Corneal Surface and Lead to an Improved Visual Acuity

Limbal stem cell deficiency (LSCD) severely impairs vision and can lead to blindness. LSCD causes include chemical burns, infections, multiple previous operations and congenital malformations. Allogeneic limbal transplantation is a procedure for treating LSCD where prepared limbal tissue is attached using a double running suture during allogeneic penetrating keratoplasty (PKP). A total of 22 patients underwent ALT surgery between February 2019 and June 2022 at the University Hospital Halle (Saale). Regular follow-up was performed postoperatively every three months and included visual acuity testing, pressure measurement, slit lamp microscopic examination, fundoscopy, corneal topography and anterior segment optical coherence tomography (AS-OCT). The mean patient age was 69.5 years, and the mean follow-up was 19 months. All included patients had LSCD and multiple previous surgeries. Patient LSCD etiology was 59% infectious and 41% traumatic. ALTs integrated into corneal surfaces in all patients, demonstrated on AS-OCT. Since most patients initially received allogeneic limbal transplants, none of the operated eyes had surgical complications. Overall, visual acuity improved postoperatively from an initial 2.06 to 1.44 logarithm of the minimum angle of resolution (logMAR). Allogeneic limbal transplantation can be used to treat LSCD and its integration into the surrounding corneal tissue can be observed on AS-OCT

The metastatic potential of seminomatous germ cell tumours is associated with a specific microRNA pattern

Background Seminomatous germ cell tumours (SGCT) are the most frequent malignancy in young men. Reliable prognostic biomarkers for the prediction of metastasis at diagnosis and the risk of relapse in clinical stage I (CSI) are lacking. Adjuvant therapies carry a risk of overtreatment, whereas salvage therapies have a risk of high toxicities. Thus, the identification of reliable prognostic biomarkers is highly desirable to identify patients who will benefit from early adjuvant treatment. MicroRNAs (miRNAs) regulate tumour development and progression, and their potential as biomarkers has already been proven in a variety of malignancies. Objectives The aim of our study was to define a specific miRNA expression pattern that discriminates metastatic from non‐metastatic primary SGCT. Materials and methods Total RNA was isolated from 24 formalin‐fixed paraffin‐embedded (FFPE) primary SGCT tumours (10 non‐metastatic, five metachronously and nine synchronously metastatic) and from 10 normal testicular tissue samples. Microarray analysis was performed for global miRNA expression profiling. The results were validated by quantitative real‐time polymerase chain reaction (qRT‐PCR). Statistical analysis was performed using SPSS. Results Microarray analyses revealed a specific miRNA pattern that distinguishes metastatic from non‐metastatic SGCT. Sixty‐three miRNAs were differentially expressed in metastatic compared to non‐metastatic tumours (P < .01). Microarray results were confirmed by qRT‐PCR for three out of five selected miRNAs (miR‐29c‐5p, miR‐506‐3p and miR‐371a‐5p; P < .05). All five miRNAs (miR‐29c‐5p, miR‐506‐3p, miR‐1307‐5p, miR‐371a‐5p and miR‐371a‐3p) showed differential expression between tumour and normal tissues (P < .05). Conclusion Metastatic primary SGCTs are characterized by a specific miRNA expression pattern. Therefore, specific miRNAs could represent a new tool to predict the metastatic potential in SGCT patients

In situ Gelling Amphotericin B Nanofibers: A New Option for the Treatment of Keratomycosis

The purpose of our research was the development of Amphotericin B-loaded in situ gelling nanofibers for the treatment of keratomycosis. Different formulation strategies were applied to increase the drug load of the sparingly water-soluble Amphotericin B in electrospun Gellan Gum/Pullulan fibers. These include bile salt addition, encapsulation in poly(lactic-co-glycolic acid) (PLGA) nanoparticles and formation of a polymeric Amphotericin B polyelectrolyte complex. The Amphotericin B polyelectrolyte complex (AmpB-Eu L) performed best and was very effective against the fungal strain Issatchenkia orientalis in vitro. The complex was characterized in detail by attenuated total reflection infrared spectroscopy, X-ray powder diffraction, and differential scanning calorimetry. A heat induced stress test was carried out to ensure the stability of the polyelectrolyte complex. To gain information about the cellular tolerance of the developed polyelectrolyte complex a new, innovative multilayered-stratified human cornea cell model was used for determination of the cellular toxicity in vitro. For a safe therapy, the applied ophthalmic drug delivery system has to be sterile. Sterilization by electron irradiation caused not degradation of pure Amphotericin B and also for the bile salt complex. Furthermore, the developed Amphotericin B polyelectrolyte complex was not degraded by the irradiation process. In conclusion, a new polyelectrolyte Amphotericin B complex has been found which retains the antifungal activity of the drug with sufficient stability against irradiation-sterilization induced drug degradation. Furthermore, in comparison with the conventional used eye drop formulation, the new AmpB-complex loaded nanofibers were less toxic to cornea cells in vitro. Electrospinning of the Amphotericin B polyelectrolyte complex with Gellan Gum/ Pullulan leads to the formation of nanofibers with in situ gelling properties, which is a new and promising option for the treatment of keratomycosis. © Copyright © 2020 Göttel, Lucas, Syrowatka, Knolle, Kuntsche, Heinzelmann, Viestenz and Mäder