Antitumor Effect of Bleomycin Nanoaerosol in Murine Carcinoma Model.

Bleomycin, which is widely used as an antitumor agent, possesses serious adverse effects such as pulmonary toxicity. Local nanoaerosol deposition for lung cancer treatment is a promising alternative to drug delivery to lung lesions. The aim of this work is to test the hypothesis that bleomycin nanoaerosol can be effectively used to treat multiple lung metastases. To obtain bleomycin nanoaerosol, an aerosol generator based on electrospray of a solution of a nonvolatile substance with gas-phase neutralization of charged aerosol particles was used. Lung metastases in murine Lewis lung carcinoma and B16 melanoma animal models were counted. The effect of inhaled bleomycin nanoparticles on the number and volume of metastases, as well as pulmonary side effects, was investigated. Using a mouse exposure chamber, the dose-dependent effect of inhaled bleomycin on tumor volume was evaluated in comparison with intraperitoneal administration. Bleomycin nanoaerosol reduced the volume of metastases and produced a higher antitumor effect at much lower doses. It has been established that long-term exposure to nanoaerosol with a low dose of bleomycin is capable of suppressing cancer cell growth. The treatment was well tolerated. In the lungs, minor changes were found in the form of focal-diffuse infiltration of the lung parenchyma.

Cancer-Retina Antigens in the Urine of Bladder and Prostate Cancer Patients.

It has recently been shown that combination of arrestin and recoverin can serve as an effective urinary biomarker for renal cell carcinoma with sensitivity and specificity of over 92%. In this work, we studied the possibility of detecting these antigens in the urine in other urological oncological diseases - bladder cancer (BC) and prostate cancer (PCa). Urine samples from 40 BC patients and 40 PCa patients were analyzed using an ultrasensitive microarray immunoassay with a detection limit of 0.1 pg/ml. It was shown that in BC the sensitivity of determining combination of arrestin with recoverin is 58% (AUC 0.76, 95% CI 0.66-0.86), while in PCa it is 60% (AUC 0.7, 95% CI 0.68-0.88). It has been established that in patients with bladder and prostate cancer who had a positive test, these antigens are not detected in 90% of cases after removal of the tumor. In the future, the obtained results could become the basis for developing new approaches for timely detection of relapses of such diseases and treatment control, as well as for the development of new diagnostic methods.

Non-Invasive Diagnostics of Renal Cell Carcinoma Using Ultrasensitive Immunodetection of Cancer-Retina Antigens.

Renal cell carcinoma (RCC) is the most common urological malignancy with a high mortality and low detection rate. One of the approaches to improving its diagnostics may be the search for new non-invasive biomarkers in liquid biopsy and development of more sensitive methods for their detection. Cancer-retina antigens, which are known to be aberrantly expressed in malignant tumors, are present in liquid biopsy at extremely low concentrations. Using the developed multiplex immunoassay with a detection limit of 0.1 pg/ml, urine and serum samples of 89 patients with RCC and 50 non-cancer patients were examined for the presence of cancer-retina antigens (arrestin, recoverin, rhodopsin kinase, and transducin); the difference between the RCC and control groups was evaluated with the χ2 test. The results showed high diagnostic efficiency of a combination of arrestin and recoverin: at a threshold of 0.1 pg/ml, the sensitivity was 96%, specificity 92%, and AUC = 0.96 (95% confidence interval, 0.93-0.99). Seven days after nephrectomy, the concentration of the antigens returned to the level characteristic of the control group. Therefore, arrestin in a combination with recoverin can serve as a diagnostic non-invasive urinary biomarker of RCC.

Amyloid Aggregates of Smooth-Muscle Titin Impair Cell Adhesion.

Various amyloid aggregates, in particular, aggregates of amyloid ß-proteins, demonstrate in vitro and in vivo cytotoxic effects associated with impairment of cell adhesion. We investigated the effect of amyloid aggregates of smooth-muscle titin on smooth-muscle-cell cultures. The aggregates were shown to impair cell adhesion, which was accompanied by disorganization of the actin cytoskeleton, formation of filopodia, lamellipodia, and stress fibers. Cells died after a 72-h contact with the amyloid aggregates. To understand the causes of impairment, we studied the effect of the microtopology of a titin-amyloid-aggregate-coated surface on fibroblast adhesion by atomic force microscopy. The calculated surface roughness values varied from 2.7 to 4.9 nm, which can be a cause of highly antiadhesive properties of this surface. As all amyloids have the similar structure and properties, it is quite likely that the antiadhesive effect is also intrinsic to amyloid aggregates of other proteins. These results are important for understanding the mechanisms of the negative effect of amyloids on cell adhesion.

Improving Immunoassay Performance with Cleavable Blocking of Microarrays.

Among the key issues that are commonly associated with the development of microarray-based assays are nonspecific binding and diffusion constraints. Here we present a novel strategy addressing both of these challenges simultaneously. The essence of the method consists in blocking the microarray surface with a blocking agent containing a perfluoroalkyl chain and a disulfide linker. The resulting surface is hydrophobic, and no immiscible liquid layer remains on it upon cyclically draining and replenishing the sample solution, ensuring an efficient mass transfer of an analyte onto a microarray. Prior to the signal detection procedure, disulfide bonds are chemically cleaved, and the perfluoroalkyl chains are removed from the microarray surface along with nonspecifically adsorbed proteins, resulting in extremely low background. Using conventional fluorescent detection, we show a 30-fold increase in signal/background ratio compared to a common epoxy-modified glass substrate. The combination of this technique with magnetic beads detection results in a simple and ultrasensitive cholera toxin (CT) immunoassay. The limit of detection (LOD) is 1 fM, which is achieved with an analyte binding time of 1 h. Efficient mass transfer provides highly sensitive detection of whole virus particles despite their low diffusion coefficient. The achieved LOD for vaccinia virus is 104 particles in 1 mL of sample. Finally, we have performed for the first time the simultaneous detection of whole virus and CT protein biomarker in a single assay. The developed technique can be used for multiplex detection of trace amounts of pathogens of various natures.

Can new immunoassay techniques improve bladder cancer diagnostics With protein biomarkers?

The search for new diagnostic tests for cancer or ways to improve existing tests is primarily driven by the desire to identify the disease as early as possible. In this report, we summarize the current knowledge of the most promising diagnostic protein bladder cancer (BC) markers reported over the last decade. Unfortunately, analysis of published data suggests that a reliable, highly sensitive biomarker test-system based on ELISA for detecting BC has not yet been developed. The use of more sensitive assays to detect ultra-low concentrations of biomarkers not available for ELISA, could be very beneficial. Based on the literature and pilot experimental data, we conclude that a highly sensitive immunoassay using microarrays and magnetic labels, could be an effective and cheap technique suitable for the detection of diagnostically relevant BC biomarkers.