Salivary excretion of systemically injected [18F]DCFPyL in prostate cancer patients undergoing PSMA scans.

Introduction: Prostate-specific membrane antigen (PSMA) is present in high amounts in salivary glands, but it is unclear whether labeled binders of PSMA are excreted in the saliva. Methods: Ten patients with prostate cancer underwent whole-body [18F]DCFPyL PET/CT (NCT03181867), and saliva samples were collected between 0-120 minutes post-injection. [18F]DCFPyL salivary excretion was measured over 120 minutes and expressed as %ID/g. Protein-associated binding was estimated by the percentage of [18F]DCFPyL versus parent radiotracer. Results: All PET scans of 10 patients (69 ± 8 years) with histologically confirmed prostate cancer (PSA= 2.4 ± 2.4, and Gleason Grade = 6-9) showed high uptake of [18F]-DCFPyL in salivary glands while 8 patients demonstrated high uptake in the saliva at 45 minutes. The intact [18F]-DCFPyL (98%) was also confirmed in the saliva samples at 120 min with increasing salivary radioactivity between 30-120 min. Conclusion: Systemically injected [18F]DCFPyL shows salivary gland uptake, an increasing amount of which is secreted in saliva over time and is not maximized by 120 minutes post-injection. Although probably insignificant for diagnostic studies, patients undergoing PSMA-targeted therapies should be aware of radioactivity in saliva.

Exploration of Imaging Biomarkers for Metabolically-Targeted Osteosarcoma Therapy in a Murine Xenograft Model.

Background: Osteosarcoma (OS) is an aggressive pediatric cancer with unmet therapeutic needs. Glutaminase 1 (GLS1) inhibition, alone and in combination with metformin, disrupts the bioenergetic demands of tumor progression and metastasis, showing promise for clinical translation. Materials and Methods: Three positron emission tomography (PET) clinical imaging agents, [18F]fluoro-2-deoxy-2-D-glucose ([18F]FDG), 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT), and (2S, 4R)-4-[18F]fluoroglutamine ([18F]GLN), were evaluated in the MG63.3 human OS xenograft mouse model, as companion imaging biomarkers after treatment for 7 d with a selective GLS1 inhibitor (CB-839, telaglenastat) and metformin, alone and in combination. Imaging and biodistribution data were collected from tumors and reference tissues before and after treatment. Results: Drug treatment altered tumor uptake of all three PET agents. Relative [18F]FDG uptake decreased significantly after telaglenastat treatment, but not within control and metformin-only groups. [18F]FLT tumor uptake appears to be negatively affected by tumor size. Evidence of a flare effect was seen with [18F]FLT imaging after treatment. Telaglenastat had a broad influence on [18F]GLN uptake in tumor and normal tissues. Conclusions: Image-based tumor volume quantification is recommended for this paratibial tumor model. The performance of [18F]FLT and [18F]GLN was affected by tumor size. [18F]FDG may be useful in detecting telaglenastat's impact on glycolysis. Exploration of kinetic tracer uptake protocols is needed to define clinically relevant patterns of [18F]GLN uptake in patients receiving telaglenastat.

Comparative Outcome of Hybrid External Fixator Versus Primary Ilizarov Fixator in the Treatment of Open Distal Tibia Extra-Articular Fractures.

Introduction: Because one-third of the tibia is subcutaneous throughout most of its length and its location, it is more prone to open fractures. Open distal tibia fractures are mostly due to RTA and sports injuries. The goal of treatment is to obtain a healed, well-aligned fracture; pain-free weight-bearing; and functional range of motion of the knee and ankle. Materials and Methods: 33 patients of the 18-60-year age group with open distal tibia extra-articular fractures (without vascular injury), less than 3 weeks old trauma were included in the prospective study for 1 year period (1st June 2019 to 31st May 2020). 17 cases were treated with the Hybrid external fixator (HEF) and 16 cases were treated with the Ilizarov fixator (IF). Results: Significantly (P < 0.05), the mean duration of surgery was less in the HEF group (67.6 min), faster union of open type-II fractures in the HEF group (16.4 weeks), and also a higher AOFAS score at 6 months in open type-II fractures in the HEF group (84.4). There were two cases of equinus deformity in the IF group and one case of valgus deformity in the HEF group. Conclusion: HEF and IF are both equally effective in the treatment of open distal tibia extra-articular fractures with the advantage of stable fracture fixation, early weight-bearing, preserving soft tissue, minimal periosteal stripping, and providing one-staged definitive intervention. However, HEF is preferred over IF in terms of less operating time, faster union, and a better functional outcome with minimal complications.

Direct epitranscriptomic regulation of mammalian translation initiation through N4-acetylcytidine.

mRNA function is influenced by modifications that modulate canonical nucleobase behavior. We show that a single modification mediates distinct impacts on mRNA translation in a position-dependent manner. Although cytidine acetylation (ac4C) within protein-coding sequences stimulates translation, ac4C within 5' UTRs impacts protein synthesis at the level of initiation. 5' UTR acetylation promotes initiation at upstream sequences, competitively inhibiting annotated start codons. Acetylation further directly impedes initiation at optimal AUG contexts: ac4C within AUG-flanking Kozak sequences reduced initiation in base-resolved transcriptome-wide HeLa results and in vitro utilizing substrates with site-specific ac4C incorporation. Cryo-EM of mammalian 80S initiation complexes revealed that ac4C in the -1 position adjacent to an AUG start codon disrupts an interaction between C and hypermodified t6A at nucleotide 37 of the initiator tRNA. These findings demonstrate the impact of RNA modifications on nucleobase function at a molecular level and introduce mRNA acetylation as a factor regulating translation in a location-specific manner.

Fluorine-18 Labeled Urea-Based Ligands Targeting Prostate-Specific Membrane Antigen (PSMA) with Increased Tumor and Decreased Renal Uptake.

High expression of prostate-specific membrane antigen (PSMA) in prostate cancers prompted the development of the PSMA-targeted PET-imaging agent [18F]DCFPyL, which was recently approved by the FDA. Fluorine-18-labeled Lys-Urea-Glu-based oxime derivatives of [18F]DCFPyL were prepared for the comparison of their in vitro and in vivo properties to potentially improve kidney clearance and tumor targeting. The oxime radiotracers were produced by condensation of an aminooxy functionalized PSMA-inhibitor Lys-Urea-Glu scaffold with fluorine-18-labeled aldehydes. The radiochemical yields were between 15-42% (decay uncorrected) in 50-60 min. In vitro saturation and competition binding assays with human prostate cancer cells transfected with PSMA, PC3(+), indicated similar high nM binding affinities to PSMA for all radiotracers. In vivo biodistribution studies with positive control PC3(+) tumor xenografts showed that the kidneys had the highest uptake followed by tumors at 60 min. The PC3(+) tumor uptake was blocked with non-radioactive DCFPyL, and PC3(-) tumor xenograft (negative control) tumor uptake was negligible indicating that PSMA targeting was preserved. The most lipophilic tracer, [18F]2a, displayed comparable tumor-targeting to [18F]DCFPyL and a desirable alteration in pharmacokinetics and metabolism, resulting in significantly lower kidney uptake with a shift towards hepatobiliary clearance and increased liver uptake.

Competitive blocking of salivary gland [18F]DCFPyL uptake via localized, retrograde ductal injection of non-radioactive DCFPyL: a preclinical study.

BACKGROUND: PSMA-targeted radionuclide therapy (TRT) is a promising treatment for prostate cancer (PCa), but dose-limiting xerostomia can severely limit its clinical adaptation, especially when using alpha-emitting radionuclides. With [18F]DCFPyL as a surrogate for PSMA-TRT, we report a novel method to selectively reduce salivary gland (SG) uptake of systemically administered [18F]DCFPyL by immediate prior infusion of non-radioactive standard of [18F]DCFPyL (DCFPyL) directly into the SG via retrograde cannulation. METHODS: A dose-finding cohort using athymic nude mice demonstrated proof of principle that SG uptake can be selectively blocked by DCFPyL administered either locally via cannulation (CAN group) or systemically (SYS group). The experiments were repeated in a validation cohort of 22RV1 tumor-bearing mice. Submandibular glands (SMG) of CAN mice were locally blocked with either saline or DCFPyL (dose range: 0.01× to 1000× molar equivalent of the radioactive [18F]DCFPyL dose). The radioactive dose of [18F]DCFPyL was administered systemically 10 min later and the mice euthanized after 1 h for biodistribution studies. Toxicity studies were done at up to 1000× dose. RESULTS: In the dose-finding cohort, the SYS group showed a dose-dependent 12-40% decrease in both the SMG T/B and the kidney (tumor surrogate). Mild blocking was observed at 0.01× , with maximal blocking reached at 1× with no additional blocking up to 1000× . In the CAN group, blocking at the 0.1× and 1× dose levels resulted in a similar 42-53% decrease, but without the corresponding decrease in kidney uptake as seen in the SYS group. Some evidence of "leakage" of DCFPyL from the salivary gland into the systemic circulation was observed. However, experiments in 22RV1 tumor-bearing mice at the 0.1× and 1× dose levels confirm that, at the appropriate blocking dose, SG uptake of [18F]DCFPyL can be selectively reduced without affecting tumor uptake and with no toxicity. CONCLUSION: Our results suggest that direct retrograde instillation of DCFPyL into the SG could predictably and selectively decrease salivary uptake of systemically administered [18F]DCFPyL without altering tumor uptake, if given at the appropriate dose. This novel approach is easily translatable to clinical practice and has the potential to mitigate xerostomia, without compromising the therapeutic efficacy of the PSMA-TRT.

In Vitro and In Vivo Comparison of 3,2-HOPO Versus Deferoxamine-Based Chelation of Zirconium-89 to the Antimesothelin Antibody Anetumab.

Introduction: [227Th]Th-3,2-HOPO-MSLN-mAb, a mesothelin (MSLN)-targeted thorium-227 therapeutic conjugate, is currently in phase I clinical trial; however, direct PET imaging using this conjugate is technically challenging. Thus, using the same MSLN antibody, we synthesized 3,2-HOPO and deferoxamine (DFO)-based zirconium-89 antibody conjugates, [89Zr]Zr-3,2-HOPO-MSLN-mAb and [89Zr]Zr-DFO-MSLN-mAb, respectively, and compared them in vitro and in vivo. Methods: [89Zr]Zr-3,2-HOPO-MSLN-mAb and [89Zr]Zr-DFO-MSLN-mAb were evaluated in vitro to determine binding affinity and immunoreactivity in HT29-MSLN and PDX (NCI-Meso16, NCI-Meso21) cells. For both the zirconium-89 conjugates, in vivo studies (biodistribution/imaging) were performed at days 1, 3, and 6, from which tissue uptake was determined. Results: Both the conjugates demonstrated a low nanomolar binding affinity for MSLN and >95% immunoreactivity. In all the three tumor types, biodistribution of [89Zr]Zr-DFO-MSLN-mAb resulted in higher tumor uptake(15.88-28-33%ID/g) at all time points compared with [89Zr]Zr-3,2-HOPO-MSLN-mAb(7-13.07%ID/g). [89Zr]Zr-3,2-HOPO-MSLN-mAb femur uptake was always higher than [89Zr]Zr-DFO-MSLN-mAb, and imaging results concurred with the biodistribution studies. Conclusions: Even though the conjugates exhibited a high binding affinity for MSLN, [89Zr]Zr-DFO-MSLN-mAb showed a higher tumor and lower femur uptake than [89Zr]Zr-3,2-HOPO-MSLN-mAb. Nevertheless, [89Zr]Zr-3,2-HOPO-MSLN-mAb could be used to study organ distribution and lesion uptake with the caveat of detecting MSLN-positive bone lesions. Clinical trial (NCT03507452).

Monitoring PSMA Responses to ADT in Prostate Cancer Patient-Derived Xenograft Mouse Models Using [18F]DCFPyL PET Imaging.

PURPOSE: PSMA overexpression has been associated with aggressive prostate cancer (PCa). However, PSMA PET imaging has revealed highly variable changes in PSMA expression in response to ADT treatment ranging from increases to moderate decreases. To better understand these PSMA responses and potential relationship to progressive PCa, the PET imaging agent, [18F]DCFPyL, was used to assess changes in PSMA expression in response to ADT using genomically characterized LuCaP patient-derived xenograft mouse models (LuCaP-PDXs) which were found to be sensitive to ADT (LuCaP73 and LuCaP136;CS) or resistant (LuCaP167;CR). METHODS: [18F]DCFPyL (2-(3-{1-carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid) was used to assess PSMA in vitro (saturation assays) in LuCaP tumor membrane homogenates and in vivo (imaging/biodistribution) in LuCaP-PDXs. Control and ADT-treated LuCaPs were imaged before ADT (0 days) and 2-, 7-, 14-, and 21-days post-ADT from which tumor:muscle ratios (T:Ms) were determined and concurrently tumor volumes were measured (caliper). After the 21-day imaging, biodistributions and histologic/genomic (PSMA, AR) analysis were done. RESULTS: [18F]DCFPyL exhibited high affinity for PSMA and distinguished different levels of PSMA in LuCaP tumors. Post-ADT CS LuCaP73 and LuCaP136 tumor volumes significantly decreased at day 7 or 14 respectively vs controls, whereas the CR LuCaP167 tumor volumes were minimally changed. [18F]DCFPyL imaging T:Ms were increased 3-5-fold in treated LuCaP73 tumors vs controls, while treated LuCaP136 T:Ms remained unchanged which was confirmed by day 21 biodistribution results. For treated LuCaP167, T:Ms were decreased (~ 45 %) vs controls but due to low T:M values (<2) may not be indicative of PSMA level changes. LuCaP73 tumor PSMA histologic/genomic results were comparable to imaging/biodistribution results, whereas the results for other tumor types varied. CONCLUSION: Tumor responses to ADT varied from sensitive to resistant among these LuCaP PDXs, while only the high PSMA expressing LuCaP model exhibited an increase in PSMA levels in response to ADT. These models may be useful in understanding the clinical relevance of PSMA PET responses to ADT and potentially the relationship to disease progression as it may relate to the genomic signature.

Sodium Fluoride-18 and Radium-223 Dichloride Uptake Colocalize in Osteoblastic Mouse Xenograft Tumors.

Background: Patients with osteoblastic bone metastases are candidates for radium-223 (223RaCl2) therapy and may undergo sodium fluoride-18 (18F-NaF) positron emission tomography-computed tomography imaging to identify bone lesions. 18F-NaF has been shown to predict 223RaCl2 uptake, but intratumor distributions of these two agents remain unclear. In this study, the authors evaluate the spatial distribution and relative uptakes of 18F-NaF and 223RaCl2 in Hu09-H3 human osteosarcoma mouse xenograft tumors at macroscopic and microscopic levels to better quantify their correlation. Materials and Methods: 18F-NaF and 223RaCl2 were co-injected into Hu09-H3 xenograft tumor severe combined immunodeficient mice. Tumor content was determined from in vivo biodistributions and visualized by PET, single photon emission computed tomography, and CT imaging. Intratumor distributions were visualized by quantitative autoradiography of tumor tissue sections and compared to histology of the same or adjacent sections. Results: 18F and 223Ra accumulated in proportional amounts in whole Hu09-H3 tumors (r2 = 0.82) and in microcalcified regions within these tumors (r2 = 0.87). Intratumor distributions of 18F and 223Ra were spatially congruent in these microcalcified regions. Conclusions: 18F-NaF and 223RaCl2 uptake are strongly correlated in heterogeneously distributed microcalcified regions of Hu09-H3 xenograft tumors, and thus, tumor accumulation of 18F is predictive of 223Ra accumulation. Hu09-H3 xenograft tumors appear to possess certain histopathological features found in patients with metastatic bone disease and may be useful in clarifying the relationship between administered 223Ra dose and therapeutic effect.

Targeted inhibition of PI3 kinase/mTOR specifically in fibrotic lung fibroblasts suppresses pulmonary fibrosis in experimental models.

Idiopathic pulmonary fibrosis (IPF) is a lethal disease with an average life expectancy of 3 to 5 years. IPF is characterized by progressive stiffening of the lung parenchyma due to excessive deposition of collagen, leading to gradual failure of gas exchange. Although two therapeutic agents have been approved from the FDA for IPF, they only slow disease progression with little impact on outcome. To develop a more effective therapy, we have exploited the fact that collagen-producing myofibroblasts express a membrane-spanning protein, fibroblast activation protein (FAP), that exhibits limited if any expression on other cell types. Because collagen-producing myofibroblasts are only found in fibrotic tissues, solid tumors, and healing wounds, FAP constitutes an excellent marker for targeted delivery of drugs to tissues undergoing pathologic fibrosis. We demonstrate here that a low-molecular weight FAP ligand can be used to deliver imaging and therapeutic agents selectively to FAP-expressing cells. Because induction of collagen synthesis is associated with phosphatidylinositol 3-kinase (PI3K) activation, we designed a FAP-targeted PI3K inhibitor that selectively targets FAP-expressing human IPF lung fibroblasts and potently inhibited collagen synthesis. Moreover, we showed that administration of the inhibitor in a mouse model of IPF inhibited PI3K activation in fibrotic lungs, suppressed production of hydroxyproline (major building block of collagen), reduced collagen deposition, and increased mouse survival. Collectively, these studies suggest that a FAP-targeted PI3K inhibitor might be promising for treating IPF.

Design and validation of fibroblast activation protein alpha targeted imaging and therapeutic agents.

Background: Cancer-associated fibroblasts (CAFs) comprise a major cell type in the tumor microenvironment where they support tumor growth and survival by producing extracellular matrix, secreting immunosuppressive cytokines, releasing growth factors, and facilitating metastases. Because tumors with elevated CAFs are characterized by poorer prognosis, considerable effort is focused on developing methods to quantitate, suppress and/or eliminate CAFs. We exploit the elevated expression of fibroblast activation protein (FAP) on CAFs to target imaging and therapeutic agents selectively to these fibroblasts in solid tumors. Methods: FAP-targeted optical imaging, radioimaging, and chemotherapeutic agents were synthesized by conjugating FAP ligand (FL) to either a fluorescent dye, technetium-99m, or tubulysin B hydrazide. In vitro and in vivo studies were performed to determine the specificity and selectivity of each conjugate for FAP in vitro and in vivo. Results: FAP-targeted imaging and therapeutic conjugates showed high binding specificity and affinity in the low nanomolar range. Injection of FAP-targeted 99mTc into tumor-bearing mice enabled facile detection of tumor xenografts with little off-target uptake. Optical imaging of malignant lesions was also readily achieved following intravenous injection of FAP-targeted near-infrared fluorescent dye. Finally, systemic administration of a tubulysin B conjugate of FL promoted complete eradication of solid tumors with no evidence of gross toxicity to the animals. Conclusion: In view of the near absence of FAP on healthy cells, we conclude that targeting of FAP on cancer-associated fibroblasts can enable highly specific imaging and therapy of solid tumors.

Comparison of Prostate-Specific Membrane Antigen Expression Levels in Human Salivary Glands to Non-Human Primates and Rodents.

Background: Prostate-specific membrane antigen (PSMA) has emerged as a promising target for developing radionuclide therapy (RNT) in prostate cancer; however, accumulation of PSMA-RNT in salivary glands can result in irreversible xerostomia. Methods to prevent PSMA-RNT-related xerostomia could be clinically useful; however, little is known about PSMA expression in salivary glands of preclinical animal models. Using [18F]DCFPyL autoradiography/biodistribution, PSMA expression levels were determined in salivary glands of various preclinical monkey and rodent species and compared with humans. Methods: Binding affinities (Kd) and PSMA levels (Bmax) were determined by in vitro [18F]DCFPyL autoradiography studies. In vivo rodent tissue uptakes (%ID/g) were determined from [18F]DCFPyL biodistributions. Results: [18F]DCFPyL exhibited low nanomolar Kd for submandibular gland (SMG) PSMA across all the species. PSMA levels in human SMG (Bmax = 60.91 nM) were approximately two-fold lower compared with baboon SMG but were two- to three-fold higher than SMG PSMA levels of cynomolgus and rhesus. Rodents had the lowest SMG PSMA levels, with the mouse being 10-fold higher than the rat. In vivo rodent biodistribution studies confirmed these results. Conclusions: SMG of monkeys exhibited comparable PSMA expression to human SMG whereas rodents were lower. However, the results suggest that mice are relatively a better small animal preclinical model than rats for PSMA salivary gland studies.

A Study of 219Rn Outgassing and 211Pb Contamination from 223Ra In dry, Liquid, and Murine Tissue Samples.

INTRODUCTION: A study of Pb contamination caused by the outgassing of Rn from Ra in dry, liquid, and murine tissues samples has been made to help design proper handling procedures for Ra in preclinical biodistribution work. MATERIALS AND METHODS: Pb activity levels were measured from Ra in dry, liquid, and tissue samples using aspiration and autoradiography techniques. RESULTS: Using aspiration techniques on dry samples of Ra, an average Rn outgassing rate of 51% ± 21% was measured with one measurement reaching as high as 81%. 31% ± 4% Pb contamination was measured within a 4.3 cm radius of a dry Ra source placed inside a 10-cm-diameter petri dish where the lip of the petri dish contained the Rn dissemination. Without the containment of the petri dish, Rn can reach as far as 7.8 cm from the source with trace levels spreading further. Using aspiration techniques on liquid samples of Ra, outgassing rates of Rn were 0.9% ± 0.3%. The outgassing levels in harvested organs from a biodistribution were as high as 10.1% ± 0.4% for an intraperitoneally injected mouse and 0.204% ± 0.006% for an intravenously injected mouse. The outgassing of the intravenously injected mouse carcass was less than 0.1%. CONCLUSION: In dry form, the high levels of Rn outgassing from a Ra source necessitate the use of ventilated biohoods when handling or preparing dry Ra from source vials. The very low levels of Rn outgassing from Ra liquid sources reduces exposure to Rn by a factor of 50. Rn exposure from murine organ tissue reaches levels of 10% when handling organs from an intraperitoneal injection and less than 0.2% for an intravenous injection.

PIWI-interacting RNA 39980 promotes tumor progression and reduces drug sensitivity in neuroblastoma cells.

Neuroblastoma (NB) is the leading pediatric cancer known for its heterogeneity and clinical aggressiveness leading to chemoresistance. Recent evidence in small RNA research has led to the discovery of PIWI-interacting RNAs (piRNAs) which work in an orchestrated fashion to modulate gene expression both in homeostatic conditions and abnormalities like cancer including NB. This study aims to decipher the possible role of a repeat-derived piRNA, piR-39980 (identified from our previous piRNA profiling study in human NB cell lines) in tumorigenesis of NB cells. piR-39980, overexpressed in NB cells act as an oncopiR and promotes tumor progression, while its inhibition resulted in reduced viability, invasion as well as the migration of IMR-32 cells. Interestingly, we observed that inhibition of piRNA induces senescence of NB cells without affecting the classical apoptosis pathway by modulating the expression of JAK3 through target binding. In addition, piR-39980 was found to desensitize the effect of doxorubicin and inhibit drug-induced apoptosis. Overall, we report piR-39980, as the first oncopiR which might serve as a novel therapeutic target for this malignancy.

Single nucleotide polymorphisms in piRNA-pathway genes: an insight into genetic determinants of human diseases.

With the development of advanced high-throughput genotyping technologies, there has been a dramatic improvement in identifying millions of single nucleotide polymorphisms (SNPs) across the human genome. SNPs located within the genes involved in biogenesis and function of small regulatory RNAs such as PIWI-interacting RNAs (piRNAs) can alter physiological processes by affecting gene expression. The genetic variations within PIWI genes and their associated factors such as TDRDs, EIFs, and KIF17 etc. have shown significant association with dreadful human diseases such as Alzheimer's disease, cancer, and schizophrenia. In this review, we have attempted to survey and summarize the association of all the genetic variants reported in different piRNA-pathway genes with diseases and discern their potential in clinical manifestations which will serve as a cornerstone for subsequent studies to decrypt the molecular mechanisms of SNPs in developing diseases.

miR-197-5p inhibits sarcomagenesis and induces cellular senescence via repression of KIAA0101.

The abnormal expressions of microRNAs (miRNAs) are known to be associated with various pathophysiological processes that lead to the development of a plethora of diseases including cancer. Among several miRNAs studied so far, miR-197 has been reported to play a vital role either as an oncogene or tumor suppressor in different cancers. However, its role in carcinogenesis of fibrosarcoma has not yet been elucidated. Therefore, the current study investigated the role of miR-197-5p, which is significantly downregulated in HT1080 fibrosarcoma cells compared to IMR90-tert fibroblast cells. The transient overexpression of miR-197-5p causes a significant decrease in viability and proliferation of fibrosarcoma cells in both concentration- and time-dependent manners. Interestingly, we did not observe any significant changes in cell cycle pattern or apoptotic cell populations, but rather noticed cellular senescence of fibrosarcoma cells upon overexpression of miR-197-5p. Further, this miRNA suppresses the metastatic properties, such as migration, invasion, and anchorage-independent growth of fibrosarcoma possibly through targeting KIAA0101, which is a proliferating cell nuclear antigen-associated factor and overexpressed in the malignancy. In nutshell, our result revealed that miR-197-5p acts as an oncosuppressor miRNA in fibrosarcoma through target regulation of KIAA0101, which can be exploited for developing RNA-based therapeutic strategies for the cure of this malignancy.

Small molecule targeted NIR dye conjugate for imaging LHRH receptor positive cancers.

Overexpression of Luteinizing Hormone Releasing Hormone Receptor (LHRH-R) in various cancers and restricted expression of the receptor in healthy cells qualifies it as a valuable cancer biomarker. Previously, LHRH-R targeted peptides have been utilized to deliver attached payloads to LHRH-R expressing cancers. We report here for the first time the utilization of a small molecule non-peptidic ligand (BOEPL) of LHRH-R to deliver attached payloads to LHRH-R positive tumors. For this purpose, we linked the BOEPL ligand to a near infrared dye via various linkers. In vitro, these conjugates demonstrated low nanomolar binding affinity and in vivo they exhibited receptor-mediated uptake specifically in tumor tissue. Moreover, tumor uptake could be blocked by administration of excess unlabeled conjugate, and time course experiments showed retention of the dye conjugate in the tumor up to 12 h post injection. Because uptake of BOEPL-targeted NIR dye conjugates by nonmalignant organs/tissues was negligible and since the transient presence of targeted NIR dye in the kidneys was a result of clearance mechanism, we suggest that a BOEPL-targeted NIR dye might constitute a useful agent for fluorescence-guided surgery of LHRH-R positive cancers. Moreover, our results also provide proof of concept that BOEPL can be successfully used to deliver attached payloads to LHRH-R positive tumors in vivo.

The Distribution Volume of 18F-Albumin as a Potential Biomarker of Antiangiogenic Treatment Efficacy.

Objective: 18F-albumin, a vascular imaging agent, may have potential to assess tumor responses to anti-angiogenic therapies. In these studies tumor distribution volume of 18F-albumin were first determined in various human tumor xenografts from biodistribtuion measurments and then one of the tumor type was used to evaluate changes in 18F-albumin uptake in anti-angiognic tumor model. Method: 18F-albumin was synthesized via conjugation of 6-[18F]fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester, [18F]F-Py-TFP, with rat albumin. From the biodistribution of 18F-albumin in various human tumor xenografts tumor distribution volumes (DVs; tumor%ID/g:blood%ID/g) were first determined at various time points. Then, the ability of 18F-albumin to detect tumor angiogenic inhibition in one of these tumor types (U87MG) following treatment with sunitinib was evaluated by position emission tomography (PET) imaging at 0, 7, 14, and 21 days post treatment. Caliper measurements of tumor dimensions were also made at these same times. At Day 21, following imaging, biodistributions, autoradiography of tumor tissues and tumor blood vessel counts (CD31 IHC) were performed. Results: 18F-albumin retention in various tumors steadily increased over time with U87MG tumor exhibiting the highest uptake (DV) at all times. Significant decreases in 18F-albumin DVs were observed one week post-treatement (-39%) vs. controls whereas tumor caliper volumes were not significantly decreased until days 14 and 21. At day 21 the significant decrease in DVs in the treatment group (-44%) paralleled biodistribution DV measurements and was consistent with autoradiography and CD31 IHC findings. Conclusion: These data suggest that 18F-albumin DVs obtained by imaging may serve as an early biomarker of the effectiveness of anti-angiogenic therapy and thus aid in patient management and treatment planning.

Attenuation of neuroblastoma cell growth by nisin is mediated by modulation of phase behavior and enhanced cell membrane fluidity.

Antimicrobial peptides have been attracting significant attention as potential anti-cancer therapeutic agents in recent times. Yet most antimicrobial peptides seem to possess cytotoxic effects on non-cancerous cells. Nisin, an antimicrobial peptide and FDA approved food preservative, has recently been found to induce selective apoptotic cell death and reduced cell proliferation in different cancer cell lines. However, the mechanism of nisin interaction with cancer cell membranes remains unexplored. Using potentiometric dye-based fluorescence and monolayer surface pressure-area isotherms we find that nisin interaction enhances the fluidity and reduces the dipole potential of a neuroblastoma cell membrane model. The quantified compressibility modulus suggests that the changes in fluidity are predominantly driven by the nisin interaction with the non-raft like regions. However, the measured positive Gibbs free energy of mixing and enthalpy hints that nisin, owing to its unfavorable mixing with cholesterol, might significantly disrupt the raft-like domains.