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Prostate cancer is the third leading cause of cancer-related death in men in the United States. Taxane chemotherapy is a staple therapy for men with metastatic prostate cancer, yet the median survival is less than 2 years in this setting. New strategies are needed to overcome taxane resistance to improve patient survival. Fatty acid synthase (FASN) is overexpressed in many types of cancer, and several inhibitors have been designed in the past 30 years. Previously, we showed that the FASN inhibitor orlistat was able to synergize with taxanes in two established taxane-resistant (TxR) cell lines. In the current study, we investigated five FASN inhibitors-cerulenin, orlistat, triclosan, thiophenopyrimidine fasnall, and pyrazole derivative TVB-3166 for their potential to synergize with docetaxel (a microtubule stabilizer) and vinblastine (a microtubule destabilizer) in TxR cell lines. Orlistat, TVB-3166, and fasnall synergistically inhibited cell viability when combined with docetaxel and vinblastine in PC3-TxR and DU145-TxR cells. Confocal microscopy and immunoblot with an antidetyrosinated tubulin antibody demonstrated that enhanced microtubule stability was induced by the combined treatment of FASN inhibitors and docetaxel compared with docetaxel alone, while combinations of FASN inhibitors with vinblastine diminished microtubule stability compared to vinblastine alone., Competing Interests: The authors declare the following competing financial interest(s): AMM has intellectual property related to hyaluronic acid formulations of FASN inhibitors., (© 2023 American Chemical Society.)

SERS immunoassay biosensors hold immense potential for clinical diagnostics due to their high sensitivity and growing interest in multi-marker panels. However, their development has been hindered by difficulties in designing compatible extrinsic Raman labels. Prior studies have largely focused on spectroscopic characteristics in selecting Raman reporter molecules (RRMs) for multiplexing since the presence of well-differentiated spectra is essential for simultaneous detection. However, these candidates often induce aggregation of the gold nanoparticles used as SERS nanotags despite their similarity to other effective RRMs. Thus, an improved understanding of factors affecting the aggregation of RRM-coated gold nanoparticles is needed. Substituent electronic effects on particle stability were investigated using various para-substituted thiophenols. The inductive and resonant effects of functional group modifications were strongly correlated with nanoparticle surface charge and hence their stability. Treatment with thiophenols diminished the negative surface charge of citrate-stabilized gold nanoparticles, but electron-withdrawing substituents limited the magnitude of this diminishment. It is proposed that this phenomenon arises by affecting the interplay of competing sulfur binding modes. This has wide-reaching implications for the design of biosensors using thiol-modified gold surfaces. A proof-of-concept multiplexed SERS biosensor was designed according to these findings using the two thiophenol compounds with the most electron-withdrawing substitutions: NO 2 and CN.

The diagnosis and monitoring of cancer have been facilitated by discovering tumor "biomarkers" and methods to detect their presence. Yet, for certain cancers, we still lack sensitive and specific biomarkers or the means to quantify subtle concentration changes successfully. The identification of new biomarkers of disease and improving the sensitivity of detection will remain key to changing clinical outcomes. Patient liquid biopsies (serum and plasma) are the most easily obtained sources for noninvasive analysis of proteins that tumor cells release directly and via extracellular microvesicles and tumor shedding. Therefore, an emphasis on creating reliable assays using serum/plasma and " direct, in-solution " ELISA approaches has built an industry centered on patient protein biomarker analysis. A need for improved dynamic range and automation has resulted in the application of ELISA principles to paramagnetic beads with chemiluminescent or fluorescent detection. In the clinical testing lab, chemiluminescent paramagnetic assays are run on automated machines that test a single analyte, minimize technical variation, and are not limited by serum sample volumes. This differs slightly from the R&D setting, where serum samples are often limiting; therefore, multiplexing antibodies to test multiple biomarkers in low serum volumes may be preferred. This review summarizes the development of historical biomarker "standards", paramagnetic particle assay principles, chemiluminescent or fluorescent biomarker detection advancements, and multiplexing for sensitive detection of novel serum biomarkers., Competing Interests: A.R.S., M.A.H., and S.K.B have an equity interest in Sanguine Diagnostics and Therapeutics. N.C.J., J.J.S., and W.M.J. are employed by Sanguine Diagnostics and Therapeutics. All other authors declare no competing interests., (Copyright © 2021 Nicole C. Japp et al.)

The presence of positive surgical margins confers an increased risk of biochemical relapse and need for salvage therapy in men undergoing radical prostatectomy. Image-guided surgery using near-infrared (NIR) fluorescent contrast agents is a potential method to detect remaining cancerous tissue. The objective of this study was to evaluate three hyaluronic acid (HA) nanoparticle (NP) formulations loaded with NIR fluorophore for their ability to contrast-enhance prostate cancer. HA was modified by conjugation with the hydrophobic ligand, aminopropyl-1-pyrenebutanamide to drive nanoparticle self-assembly. Indocyanine green (ICG) was physicochemically entrapped in the HA-NP, termed NanoICG. Alternatively, Cy7.5 was directly conjugated to amphiphilic HA, termed NanoCy7.5. NanoCy7.5 was synthesized with two HA molecular weights to determine the HA size contribution to delivery to PC3 prostate tumor xenografts. Contrast-enhancement of the tumors and relative biodistribution were assessed by a series of fluorescence imaging, image-guided surgery with spectroscopy, and microscopic techniques. Intravenously administered NanoICG improved tumor signal-to-noise ratio (SNR) at 24 h over ICG by 2.9-fold. NanoCy7.5 with 10 kDa and 100 kDa HA improved tumor SNR by 6.6- and 3.1-fold over Cy7.5 alone, respectively. The PC3 xenograft was clearly identified with the image-guided system providing increased contrast enhancement compared to surrounding tissue for NanoICG and NanoCy7.5 with 10 kDa HA. NIR fluorescence microscopy showed that Cy7.5 in NPs with 10 kDa HA were distributed throughout the tumor, while NanoCy7.5 with 100 kDa HA or NanoICG delivered dye mainly to the edge of the tumor. CD31 staining suggested that PC3 tumors are poorly vascularized. These studies demonstrate the efficacy of a panel of HA-derived NPs in identifying prostate tumors in vivo, and suggest that by tuning the structural properties of these NPs, optimized delivery can be achieved to poorly vascularized tumors., Statement of Significance: We have demonstrated the potential of a panel of near-infrared fluorescent (NIRF) nanoparticles (NPs) for image-guided surgery in a prostate cancer xenograft model. Image-guided surgery and imaging of organs ex vivo showed greater tumor signal and contrast when mice were administered NIRF dyes that were covalently conjugated to (NanoCy7.5 10k-PBA ) or physicochemically entrapped in (NanoICG PBA ) hyaluronic acid (HA) NPs, compared to free dyes. These results show the potential to use these NPs as tools to detect the margins of tumors and to differentiate healthy and tumor tissue intraoperatively. Moreover, this project provides insight into selecting optimal formulation strategies for poorly vascularized tumors., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Pancreatic ductal adenocarcinoma is highly lethal and surgical resection is the only potential curative treatment for the disease. In this study, hyaluronic acid derived nanoparticles with physico-chemically entrapped indocyanine green, termed NanoICG, were utilized for intraoperative near infrared fluorescence detection of pancreatic cancer. NanoICG was not cytotoxic to healthy pancreatic epithelial cells and did not induce chemotaxis or phagocytosis, it accumulated significantly within the pancreas in an orthotopic pancreatic ductal adenocarcinoma model, and demonstrated contrast-enhancement for pancreatic lesions relative to non-diseased portions of the pancreas. Fluorescence microscopy showed higher fluorescence intensity in pancreatic lesions and splenic metastases due to NanoICG compared to ICG alone. The in vivo safety profile of NanoICG, including, biochemical, hematological, and pathological analysis of NanoICG-treated healthy mice, indicates negligible toxicity. These results suggest that NanoICG is a promising contrast agent for intraoperative detection of pancreatic tumors., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Taxane-based therapy provides a survival benefit in patients with metastatic prostate cancer, yet the median survival is less than 20 months in this setting due in part to taxane-associated resistance. Innovative strategies are required to overcome chemoresistance for improved patient survival. Here, NanoOrl, a new experimental nanoparticle formulation of the FDA-approved drug, orlistat, was investigated for its cytotoxicity in taxane-resistant prostate cancer utilizing two established taxane-resistant (TxR) cell lines. Orlistat is a weight loss drug that inhibits gastric lipases, but is also a potent inhibitor of fatty acid synthase (FASN), which is overexpressed in many types of cancer. NanoOrl was also investigated for its potential to synergize with taxanes in TxR cell lines. Both orlistat and NanoOrl synergistically inhibited cell viability when combined with paclitaxel, docetaxel, and cabazitaxel in PC3-TxR and DU145-TxR cells, yet these combinations were also additive in parental lines. We observed synergistic levels of apoptosis in TxR cells treated with NanoOrl and docetaxel in combination. Mechanistically, the synergy between orlistat and taxanes was independent of effects on the P-glycoprotein multidrug resistance protein, as determined by an efflux activity assay. On the other hand, immunoblot and immunofluorescence staining with an anti-detyrosinated tubulin antibody demonstrated that enhanced microtubule stability was induced by combined NanoOrl and docetaxel treatment in TxR cells. Furthermore, TxR cells exhibited higher lipid synthesis, as demonstrated by 14 C-choline incorporation that was abrogated by NanoOrl. These results provide a strong rationale to assess the translational potential of NanoOrl to overcome taxane resistance. Mol Cancer Ther; 16(9); 1819-30. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Despite recent advances in radiotherapy, a majority of patients diagnosed with pancreatic cancer (PC) do not achieve objective responses due to the existence of intrinsic and acquired radioresistance. Identification of molecular mechanisms that compromise the efficacy of radiation therapy and targeting these pathways is paramount for improving radiation response in PC patients. In this review, we have summarized molecular mechanisms associated with the radio-resistant phenotype of PC. Briefly, we discuss the reversible and irreversible biological consequences of radiotherapy, such as DNA damage and DNA repair, mechanisms of cancer cell survival and radiation-induced apoptosis following radiotherapy. We further describe various small molecule inhibitors and molecular targeting agents currently being tested in preclinical and clinical studies as potential radiosensitizers for PC. Notably, we draw attention towards the confounding effects of cancer stem cells, immune system, and the tumor microenvironment in the context of PC radioresistance and radiosensitization. Finally, we discuss the need for examining selective radioprotectors in light of the emerging evidence on radiation toxicity to non-target tissue associated with PC radiotherapy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

CD44 is a widely-distributed type I transmembrane glycoprotein that binds hyaluronic acid (HA) in most cell types, including primary tumor cells and cancer-initiating cells and has roles in cell migration, cell-cell, and cell-matrix adhesion. HA-derived conjugates and nanoparticles that target the CD44 receptor on cells have been reported for targeted delivery of therapeutics and imaging agents. Altering crucial interactions of HA with CD44 active sites holds significant importance in modulating targeting ability of hyaluronic acid to other cancer types that do not express the CD44 receptor or minimizing the interaction with CD44 + cells that are not target cells. The approach adopted here was deacetylation of the N-acetyl group and selective sulfation on the C6-OH on the HA polymer, which form critical interactions with the CD44 active site. Major interactions identified by molecular modeling were confirmed to be hydrogen bonding of the C6-OH with Tyr109 and hydrophobic interaction of the N-acetyl group with Tyr46, 83 and Ile 92. Modified HA was synthesized and characterized and its interactions were assessed by in vitro and molecular modeling approaches. In vitro techniques included flow cytometry and fluorescence polarization, while in silico approaches included docking and binding calculations by a MM-PBSA approach. These studies indicated that while both deacetylation and sulfation of HA individually decrease CD44 interaction, both chemical modifications are required to minimize interaction with CD44 + cells. The results of this study represent the first step to effective retargeting of HA-derived NPs for imaging and drug delivery.

Tumor tissue that remains undetected at the primary surgical site can cause tumor recurrence, repeat surgery, and treatment strategy alterations that impose a significant patient and healthcare burden. Intraoperative near infrared fluorescence (NIRF) imaging is one potential method to identify remaining tumor by visualization of NIR fluorophores that are preferentially localized to the tumor. This requires development of fluorophores that consistently identify tumor tissue in different patients and tumor types. In this study we examined a panel of NIRF contrast agents consisting of polymeric nanoparticle (NP) formulations derived from hyaluronic acid (HA), with either physically entrapped indocyanine green (ICG) or covalently conjugated Cy7.5. Using orthotopic human breast cancer MDA-MB-231 xenografts in nude mice we identified two lead formulations. One, NanoICG PBA , with physicochemically entrapped ICG, showed 2.3-fold greater tumor contrast than ICG alone at 24 h (p < 0.01), and another, NanoCy7.5 100-H , with covalently conjugated Cy7.5, showed 74-fold greater tumor contrast than Cy7.5 alone at 24 h (p < 0.0001). These two lead formulations were then tested in immune competent BALB/c mice bearing orthotopic 4T1 breast cancer tumors. NanoICG PBA showed 2.2-fold greater contrast than ICG alone (p < 0.0001), and NanoCy7.5 100-H showed 14.8-fold greater contrast than Cy7.5 alone (p < 0.0001). Furthermore, both NanoICG PBA and NanoCy7.5 100-H provided strong tumor enhancement using image-guided surgery in mice bearing 4T1 tumors. These studies demonstrate the efficacy of a panel of HA-derived NPs in delineating tumors in vivo , and identifies promising formulations that can be used for future in vivo tumor removal efficacy studies., Competing Interests: The authors have declared that no competing interest exists.

Background: Despite its promise as a highly useful therapy for pancreatic cancer (PC), the addition of external beam radiation therapy to PC treatment has shown varying success in clinical trials. Understanding PC radioresistance and discovery of methods to sensitise PC to radiation will increase patient survival and improve quality of life. In this study, we identified PC radioresistance-associated pathways using global, unbiased techniques., Methods: Radioresistant cells were generated by sequential irradiation and recovery, and global genome cDNA microarray analysis was performed to identify differentially expressed genes in radiosensitive and radioresistant cells. Ingenuity pathway analysis was performed to discover cellular pathways and functions associated with differential radioresponse and identify potential small-molecule inhibitors for radiosensitisation. The expression of FDPS, one of the most differentially expressed genes, was determined in human PC tissues by IHC and the impact of its pharmacological inhibition with zoledronic acid (ZOL, Zometa) on radiosensitivity was determined by colony-forming assays. The radiosensitising effect of Zol in vivo was determined using allograft transplantation mouse model., Results: Microarray analysis indicated that 11 genes (FDPS, ACAT2, AG2, CLDN7, DHCR7, ELFN2, FASN, SC4MOL, SIX6, SLC12A2, and SQLE) were consistently associated with radioresistance in the cell lines, a majority of which are involved in cholesterol biosynthesis. We demonstrated that knockdown of farnesyl diphosphate synthase (FDPS), a branchpoint enzyme of the cholesterol synthesis pathway, radiosensitised PC cells. FDPS was significantly overexpressed in human PC tumour tissues compared with healthy pancreas samples. Also, pharmacologic inhibition of FDPS by ZOL radiosensitised PC cell lines, with a radiation enhancement ratio between 1.26 and 1.5. Further, ZOL treatment resulted in radiosensitisation of PC tumours in an allograft mouse model., Conclusions: Unbiased pathway analysis of radioresistance allowed for the discovery of novel pathways associated with resistance to ionising radiation in PC. Specifically, our analysis indicates the importance of the cholesterol synthesis pathway in PC radioresistance. Further, a novel radiosensitiser, ZOL, showed promising results and warrants further study into the universality of these findings in PC, as well as the true potential of this drug as a clinical radiosensitiser.

Pancreatic cancer (PC) remains one of the most lethal human malignancies with poor prognosis. Despite all advances in preclinical research, there have not been significant translation of novel therapies into the clinics. The development of genetically engineered mouse (GEM) models that produce spontaneous pancreatic adenocarcinoma (PDAC) have increased our understanding of the pathogenesis of the disease. Although these PDAC mouse models are ideal for studying potential therapies and specific genetic mutations, there is a need for developing syngeneic cell lines from these models. In this study, we describe the successful establishment and characterization of three cell lines derived from two (PDAC) mouse models. The cell line UN-KC-6141 was derived from a pancreatic tumor of a Kras(G12D);Pdx1-Cre (KC) mouse at 50 weeks of age, whereas UN-KPC-960 and UN-KPC-961 cell lines were derived from pancreatic tumors of Kras(G12D);Trp53(R172H);Pdx1-Cre (KPC) mice at 17 weeks of age. The cancer mutations of these parent mice carried over to the daughter cell lines (i.e. Kras(G12D) mutation was observed in all three cell lines while Trp53 mutation was observed only in KPC cell lines). The cell lines showed typical cobblestone epithelial morphology in culture, and unlike the previously established mouse PDAC cell line Panc02, expressed the ductal marker CK19. Furthermore, these cell lines expressed the epithelial-mesenchymal markers E-cadherin and N-cadherin, and also, Muc1 and Muc4 mucins. In addition, these cell lines were resistant to the chemotherapeutic drug Gemcitabine. Their implantation in vivo produced subcutaneous as well as tumors in the pancreas (orthotopic). The genetic mutations in these cell lines mimic the genetic compendium of human PDAC, which make them valuable models with a high potential of translational relevance for examining diagnostic markers and therapeutic drugs.

There is an urgent need to develop alternative therapies against lethal pancreatic cancer (PC). Ocimum sanctum ("Holy Basil") has been used for thousands of years in traditional Indian medicine, but its anti-tumorigenic effect remains largely unexplored. Here, we show that extracts of O. sanctum leaves inhibit the proliferation, migration, invasion, and induce apoptosis of PC cells in vitro. The expression of genes that promote the proliferation, migration and invasion of PC cells including activated ERK-1/2, FAK, and p65 (subunit of NF-κB), was downregulated in PC cells after O. sanctum treatment. Intraperitoneal injections of the aqueous extract significantly inhibited the growth of orthotopically transplanted PC cells in vivo (p<0.05). Genes that inhibit metastasis (E-cadherin) and induce apoptosis (BAD) were significantly upregulated in tumors isolated from mice treated with O. sanctum extracts, while genes that promote survival (Bcl-2 and Bcl-xL) and chemo/radiation resistance (AURKA, Chk1 and Survivin) were downregulated. Overall, our study suggests that leaves of O. sanctum could be a potential source of novel anticancer compounds in the future., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Ossifying fibromyxoid tumor (OFMTs) of soft parts is a rare soft tissue and bone tumor of borderline malignancy displaying an uncertain line of differentiation. The existence of fully malignant OFMT is controversial. To better understand the natural history and line of differentiation taken by OFMT, we studied 46 cases by light microscopic, immunohistochemical (IHC), genomic, proteomic, and fluorescence in situ hybridization (FISH) methods. Cases were classified according to the 2003 Folpe and Weiss system. Clinical and follow-up information was obtained. IHC for S-100 protein, desmin, epithelial membrane antigen (EMA), cytokeratins, smooth muscle actin (SMA), INI-1, neurofilament protein (NFP), CD56d excitatory amino acid transporter-4 (EAAT4), and MUC4 was performed on formalin-fixed, paraffin-embedded (FFPE) tissues. Gene expression profiling and proteomic studies were conducted on FFPE tissues from 13 and 5 cases, respectively. FISH for INI-1 was performed on 10 cases. The 46 tumors arose in 29 men and 17 women (median age, 52 y; range 39 to 63 y) and involved the proximal (N=17) and distal extremities (N=13), head and neck (N=9), and trunk (N=5). Median tumor size was 5.4 cm (range, 1.0 to 21.0 cm). Cases were classified as typical OFMT (26 of 46, 57%), atypical OFMT (5 of 46, 11%), and malignant OFMT (15 of 46 cases, 32%). Clinical follow-up (27 cases, median 55 months' duration) showed all patients with typical and atypical OFMT to be alive without disease. Adverse events, including 3 local recurrences, 3 metastases, and 3 deaths, were seen only in malignant OFMT. IHC results were as follows: S-100 protein (30 of 41, 73%), desmin (15 of 39, 38%), cytokeratin (4 of 35, 11%), EMA (5 of 32, 16%), SMA (2 of 34, 6%), INI-1 (lost in mosaic pattern in 14 of 19, 74%), EAAT4 (31 of 39, 80%), MUC4 (3 of 14, 21%), NFP (8 of 10, 80%) and CD56 (6 of 14, 43%). Gene expression profiling showed typical and malignant OFMTs to cluster together, distinct from schwannian tumors. Proteomic study showed expression of various collagens, S-100 protein, and neuron-related proteins. FISH showed INI-1 deletion in 5 of 7 (71%) cases. We conclude that malignant OFMTs exist and may be recognized by the previously proposed criteria of Folpe and Weiss. Expression of neuron-related markers, in addition to Schwann cell and cartilage-associated markers, suggests a "scrambled" phenotype in OFMTs. Loss of INI-1 or other genes on 22q is likely important in the pathogenesis of these rare tumors.