Folate Receptor Expression Research Articles

Abstract 5418: Tumor-targeted nanoparticle delivery of HuR-RNAi suppresses lung cancer cell proliferation and cell migration

Abstract Objective. HuR is an mRNA-binding protein that specifically binds to AU rich (ARE) sites at the 3′end of the mRNA of several growth factors, cell-cycle regulators, and transcription-regulating proteins and transports the mRNA to the cytoplasm for protein translation. HuR overexpression has been demonstrated to be a poor prognostic marker in a spectrum of human cancers and correlate with chemotherapy resistance. On the basis of these reports we hypothesized HuR is a druggable target for cancer therapy and its inhibition will down-regulate multiple oncoproteins resulting in efficient tumor cell killing. To test our hypothesis, we developed and tested the efficacy of a nanoparticle (NP) containing HuR-specific siRNA and targeted towards folate receptor (Fr) overexpressing lung cancer cells in vitro. Methods. Human lung cancer (H1299, A549) and normal fibroblast (MRC-9) cell lines were used in the present study. Folate receptor (Fr) overexpressing KB cell line was used as positive control. Tumor targeted nanoparticle was synthesized by mixing cationic lipid (DOTAP) with a neutral lipid (cholesterol) in equimolar ratio. The NPs thus formed were used to encapsulate fluorescently labeled siRNA for determining transfection efficiency; scrambled (control) siRNA or HuR-specific siRNA (100 nm) for efficacy studies. The control- and HuR-siRNA-containing NPs were subsequently decorated with DSPE-PEG5000-folate and labeled as C-FNP and HuR-FNP respectively. Transfection efficiency, NP specificity, cell viability, cell migration and HuR knock down studies were performed using standard laboratory assay protocols. Results. HuR protein expression in H1299 and A549 was higher than in MRC-9 cell line. Folate receptor (Fr) expression from highest to lowest was in the following order KB> H1299>MRC9. Fr expression was not detected in A549 cells. Transfection efficiency study showed FNP uptake was highest in KB cells followed by H1299 cells with lowest uptake by A549 cells. Addition of exogenous folic acid resulted in reduced FNP uptake by H1299 cells demonstrating NP specificity towards Fr. Treatment of H1299 cells with HuR-FNP significantly reduced cell viability at 48h compared to C-FNP-treatment and correlated with a marked reduction in HuR mRNA and protein expression levels. Analysis for proteins whose mRNAs are targets for and regulated by HuR (Bcl2, Cyclin D1, HIF-1α) showed diminished expression in HuR-FNP-treated cells. Finally, cell migration was significantly inhibited in HuR-FNP-treated H1299 cells compared to C-FNP treatment. Conclusions. Tumor-targeted nanoparticle delivery of HuR-RNAi in lung cancer cells selectively inhibited HuR and HuR-regulated oncoproteins resulting in diminished cell proliferation and cell migration in vitro. Acknowledgement. This study was funded by a grant (R01CA167516-01) from the National Cancer Institute. Citation Format: Ranganayaki Muralidharan, Anish Babu, Kanthesh Basalingappa, Anupama Munshi, Rajagopal Ramesh. Tumor-targeted nanoparticle delivery of HuR-RNAi suppresses lung cancer cell proliferation and cell migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5418. doi:10.1158/1538-7445.AM2014-5418

Abstract 1677: Preclinical activity of Vintafolide/MK-8109 monotherapy and in combination with standard of care therapy in triple-negative breast cancer models

Abstract Vintafolide (also known as EC145 or MK-8109), is a small molecule drug conjugate for the treatment of cancers expressing high affinity folate receptor (FR). Vintafolide consists of the anti-mitotic vinca alkaloid desacetylvinblastine monohydrazide (DAVLBH) chemically linked to folic acid. Binding of vintafolide to FR located on the cell surface delivers the chemotherapy payload directly to the tumor cell. Clinical investigations of vintafolide are underway in ovarian and lung cancer, indications with a high prevalence of FR expression. Triple negative breast cancer (TNBC) may represent an additional indication for vintafolide due to the prevalence of FR expression (∼30%), sensitivity to vinca alkaloids, and unmet medical need. Vintafolide and/or DAVLBH were evaluated in preclinical models of TNBC as monotherapies and in combination with taxanes (paclitaxel or docetaxel). Taxanes represent a commonly used standard of care therapy for TNBC. A panel of TNBC cell lines was generally sensitive to DAVLBH, with IC50s ranging from 4-67 nM. In the majority of cell lines tested, DAVLBH in combination with paclitaxel provided combination benefit and induced more cell death than either single agent. Vintafolide and DAVLBH were further evaluated in vivo in the FR-high MDA-MB-231 and FR-low CAL51 TNBC xenograft models. Vintafolide was dosed at its MTD of 9.6 mg/kg three times per week (TIW), and at 1.5 mg/kg TIW. DAVLBH was dosed at its MTD of 0.77 mg/kg TIW. Mice were dosed for 3 weeks followed by a 6 week follow-up. Both vintafolide doses resulted in marked MDA-MB-231 tumor regressions of 56-78% at the end of therapy (Day 21) and 75% cures (6 of 8 mice) over the 6 week follow-up period. In the CAL51 model, vintafolide produced 8% and 76% tumor growth inhibition (TGI), respectively, at the 1.5 and 9.6 mg/kg doses, and no cures. DAVLBH was less efficacious compared to vintafolide, giving 96% TGI in the MDA-MB-231 model and 46% TGI in the CAL51 model. In mechanism of action studies in the MDA-MB-231 xenograft model, a dose of vintafolide that gave tumor regressions/cures was associated with increased phospho-histone H3 staining, indicative of a mitotic block of tumor cells, and consistent with the mechanism of action of vinca alkaloids. In combination therapy experiments in vivo, docetaxel (20 mg/kg weekly) monotherapy achieved near tumor stasis (98% TGI) of MDA-MB-231 tumors over 3 weeks of therapy, but all tumors re-grew upon cessation of treatment. Since vintafolide monotherapy at 1.5 mg/kg TIW gave significant regressions and cures, a combination benefit with docetaxel could not be determined. However, DAVLBH and docetaxel combination therapy delayed tumor re-growth over single agents upon follow-up, suggesting benefit of a vinca alkaloid / taxane combination. Taken together, these preclinical data support further investigation of vintafolide monotherapy and in combination with taxane therapy in TNBC. Citation Format: Brian B. Haines, Jennifer O'Neil, Marlene C. Hinton, Christopher Ware, Tammie C. Yeh, Tianxiao Sun, Kristen L. Picard, Theresa Zhang, Emmett V. Schmidt, Isabelle Dussault. Preclinical activity of Vintafolide/MK-8109 monotherapy and in combination with standard of care therapy in triple-negative breast cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1677. doi:10.1158/1538-7445.AM2014-1677

Surface Orchestration of Gold Nanoparticles Using Cysteamine as Linker and Folate as Navigating Molecule for Synaphic Delivery of Doxorubicin

We report synthesis, characteristics and in vitro tests of folate-conjugated gold nanoparticles (AuNPs) and doxorubicin, having Cysteamine as linker. AuNPs were chemically synthesized using sodium citrate. Characterization was done using UV-visible absorption spectroscopy, Field Emission Gun Scanning Electron Microscopy (FEG-SEM), Fourier Transform Infra Red (FTIR) spectroscopy, Particle size Analyzer. Charge distribution was studied by recording Zeta potential. AuNPs were used for attaching folic acid through a linker cysteamine-HCl and finally preparing AuNP/Cysteamine-HCl/Folic acid/Doxorubicin complex. The cell cytotoxicity was studied in MDCK cells (normal cells) and HeLa cells (cancerous cells) having high level of folate receptor expression. In-vitro drug release kinetics was calculated using standard statistical models and was found to follow Hixson-Crowell drug release kinetics model.

Evaluation of antitumor effect of zoledronic acid entrapped in folate-linked liposome for targeting to tumor-associated macrophages

In this study, we investigated the possibility of whether depletion of tumor-associated macrophages (TAMs) by zoledronic acid (ZOL) entrapped in folate-linked liposome could inhibit tumor angiogenesis and consequently tumor growth. Abundant expression of folate receptor β (FRβ) mRNA was detected in tumor tissues from subcutaneously implanted FR-negative tumor cells in mice, indicating that FRβ mRNA was expressed in TAMs, not in tumor cells. When fluorescent-labeled folic acid was intravenously injected into mice bearing FR-positive human nasopharyngeal tumor KB, and FR-negative human prostate tumor PC-3 and mouse colon adenocarcinoma Colon 26, fluorescence was strongly detected in KB and Colon 26 tumors, and moderately in PC-3 tumor, indicating that folic acid was taken up by TAMs via FRβ in PC-3 and Colon 26 tumors. For evaluation of ZOL delivery to TAMs, folate-linked liposomal ZOL (FL-ZOL) for TAM targeting and non-folate-linked liposomal ZOL (L-ZOL) as a control were prepared by incorporation with 0.5 mol% folate-PEG2000-DSPE and 0.5 mol% PEG2000-DSPE, respectively, into the liposomal formulation of egg phosphatidylcholine and cholesterol. FL-ZOL showed high cytotoxicity for KB and murine macrophage RAW264.7 cells, but not for Colon 26 cells, suggested that FL-ZOL was selectively taken up via FR-mediated endocytosis. However, injections of FL-ZOL and L-ZOL did not induce antitumor activities for KB and Colon 26 tumor-bearing mice, and had a lethal effect by high toxicity. From these findings, the severe in vivo toxicity of liposomal ZOL limited its utility for in vivo TAM targeting, although FL-ZOL could selectively induce in vitro cytotoxicity via FR.

Target: a Randomized, Phase Ii Trial Comparing Vintafolide Versus Vintafolide Plus Docetaxel, Versus Docetaxel Alone in Second-Line Treatment of Folate-Receptor-Positive Non-Small Cell Lung Cancer (Nsclc) Patients

ABSTRACT Aim: The folate receptor (FR) is expressed in many epithelial cancers, including NSCLC, and may be a useful biomarker for therapy selection. Vintafolide, a folate-vinca alkaloid drug conjugate, is a FR-targeted drug. Its companion imaging agent, 99mTc-etarfolatide, enables non-invasive imaging of FR expression. The TARGET study (NCT01577654) assessed the benefit of FR-targeted therapy in 199 second-line NSCLC patients with all target lesions expressing FR [FR(100%)]. Methods: Patients were randomized 1:1:1 to vintafolide, vintafolide + docetaxel (DTX), or DTX alone. Vintafolide (2.5 mg) was administered on D1, 4, 8, and 11 and DTX (75 mg/m2) on D1 of a 3-week cycle. The primary endpoint was PFS; secondary endpoints included OS and ORR. The significance level of each PFS and OS analysis was one-sided alpha = 0.10 with no multiple testing adjustments. Results: See table. Vintafolide Vinta + DTX DTX All patients ORR Median PFS (months, 95% Cl) PFS HR (vs. DTX; 95% Cl)* 1-sided p-value Median OS (months, 95% Cl) OS HR (vs. DTX; 95% Cl)* 1-sided p-value N = 63 6% 1.6 (1.4; 3.2) 1.35 (0.92; 1.96) 0.9421 8.4 (5.6; 12.3) 1.05 (0.68; 1.61) 0.5818 N = 68 22% 4.2 (2.8; 5.4) 0.75 (0.52; 1.09) 0.0696 11.5 (7.3; 13.4) 0.88 (0.58; 1.36) 0.2874 N = 68 13% 3.3 (1.72; 4.2) - - 8.8 (5.4; 12.6) - - Adenocarcinoma patients ORR Median PFS (months, 95% Cl) PFS HR (vs. DTX; 95% Cl)* 1-sided p-value Median OS (months, 95% Cl) OR HR (vs. DTX; 95% Cl)* 1-sided p-value N = 41 5% 1.6 (1.4; 3.3) 1.40 (0.88; 2.24) 0.9247 8.4 (5.1; 12.5) 1.00 (0.59; 1.71 0.4957 N = 43 21% 4.2 (2.6; 5.4) 0.73 (0.46; 1.16) 0.0899 12.5 (8.1; - ) 0.70 (0.40; 1.22) 0.1018 N = 49 14% 3.0 (1.6; 4.2) - - 6.6 (4.8; 12.9) - - * Unstratified Cox model. With the pre-specified stratified analysis adjusting for baseline factors (time since last chemotherapy, best response and stage), the OS HR for vintafolide + DTX vs. DTX were 0.75 (1-sided p = 0.1066) for all patients, and 0.51 (1-sided p = 0.0147) for the predefined adenocarcinoma patient subgroup. Conclusions: Vintafolide + DTX showed clinically meaningful improvement across all efficacy endpoints (ORR, PFS and OS) over single-agent DTX, with the largest benefit observed in the adenocarcinoma subgroup. Disclosure: J. Symanowski: Other substantive relationships: Consultant for Endocyte, Inc.; W. Perez: Other substantive relationships: Employee of Endocyte, Inc.; W. Harb: Corporate-sponsored research: Endocyte sponsored participation in EC1456 study. All other authors have declared no conflicts of interest.

Trial in progress: A randomized double-blind phase 3 trial comparing vintafolide + pegylated liposomal doxorubicin (PLD) versus PLD + placebo in patients with platinum-resistant ovarian cancer (PROCEED)

Objectives: Folate receptor (FR) is expressed on the majority of epithelial ovarian cancers and FR expression appears to be a negative prognostic factor in this setting. Vintafolide (EC145) is a small-molecule drug-conjugate of folate designed to selectively deliver its cytotoxic payload, desacetylvinblastine monohydrazide (DAVLBH), to FR-expressing cells. 99mTc-Etarfolatide (EC20) is a small-molecular-weight folate-targeted companion imaging agent that can be used for the non-invasive, real-time assessment of functionally active FRs.

Imaging the Folate Receptor on Cancer Cells with 99mTc-Etarfolatide: Properties, Clinical Use, and Future Potential of Folate Receptor Imaging

Folate receptor (FR) can be used as a therapeutic target because of its expression on different epithelial cancers, such as ovarian, non-small cell lung, endometrial, and breast cancer. Assessing FR expression in tumors may help to identify patients who can benefit from FR-targeted therapeutics, such as vintafolide and farletuzumab. Different methods exist to detect FR expression. Tissue sampling has limited clinical utility, mainly because it requires an invasive procedure. (99m)Tc-etarfolatide, a (99m)Tc-labeled folate conjugate, is in late-phase trials in Europe and the United States. It allows noninvasive, whole-body imaging of the FR. This review focuses on this FR-imaging agent and how it may be used to direct FR-targeted therapy.

Synthesis and Cellular Uptake of Folic Acid-Conjugated Cellulose Nanocrystals for Cancer Targeting

Elongated nanoparticles have recently been shown to have distinct advantages over spherical ones in targeted drug delivery applications. In addition to their oblong geometry, their lack of cytotoxicity and numerous surface hydroxyl groups make cellulose nanocrystals (CNCs) promising drug delivery vectors. Herein we report the synthesis of folic acid-conjugated CNCs for the targeted delivery of chemotherapeutic agents to folate receptor-positive cancer cells. Folate receptor-mediated cellular binding/uptake of the conjugate was demonstrated on human (DBTRG-05MG, H4) and rat (C6) brain tumor cells. Folate receptor expression of the cells was verified by immunofluorescence staining. Cellular binding/uptake of the conjugate by DBTRG-05MG, H4, and C6 cells was 1452, 975, and 46 times higher, respectively, than that of nontargeted CNCs. The uptake mechanism was determined by preincubation of the cells with the uptake inhibitors chlorpromazine or genistein. DBTRG-05MG and C6 cells internalized the conjugate primarily via caveolae-mediated endocytosis, whereas H4 cells internalized the conjugate primarily via clathrin-mediated endocytosis.

Preparation, characterization, and in vitro antitumor activity of folate conjugated chitosan coated EGCG nanoparticles

Folate conjugated chitosan coated EGCG nanoparticles (FCS-EGCG-NPs) were prepared using the ionic gelation method with folic acid modified carboxymethyl chitosan (FA-CMC) and chitosan hydrochloride as carriers of catechin EGCG. Characteristics of FCS-EGCG-NPs were determined using transmission electron microscopy (TEM) and fourier transform infrared spectroscopy (FTIR). Synthesized FCS-EGCG-NPs were spherical in shape with a mean diameter of 400 nm. The maximum encapsulation rate of nanoparticles loaded with EGCG was 75%. FTIR spectra suggested formation of an amide linkage between carboxyl groups of FA-CMC and the amine groups of chitosan hydrochloride. FCS-EGCG-NPs demonstrated sustained release of EGCG in buffer solutions of different pH values. The antitumor activity of FCS-EGCG-NPs towards different cancer cells was also investigated. FCS-EGCG-NPs had a greater tumor inhibition effect on cancer cells having a large expression of folic acid receptors on the surface than cancer cells with lesser expression.

Folate receptor alpha in human plasma: relationship to folate status and effect of supplementation in healthy reproductive aged females (827.17)

Little research has been conducted to determine the effect of chronic folic acid (FA) intake on folate receptors (FR). We aimed to determine if long‐term supplementation with FA (140 or 400 μg/d) changes expression of FRα in blood. Samples from a 40‐wk randomized placebo controlled trial in 144 reproductive aged females were used. Plasma FRα was determined by ELISA, plasma folate vitamer concentrations by LC‐MS/MS, and red blood cell (RBC) folate by microbiologic assay. The geometric mean (SD) FRα concentration was 12.4 (1.7) nmol/L at baseline and 13.2 (1.6) nmol/L at 40‐wks. There were no significant correlations between FRα and any blood folate indices at baseline, however, after 40‐wks, FRα was significantly correlated with RBC folate (Spearman’s r = 0.217, P = 0.021). In a multivariable model, FRα at baseline significantly predicted FRα at 40‐wks (Ratio 2.41, 95% CI 2.23 – 2.60, P < 0.001). While there was no overall effect of treatment (P = 0.124), exploratory subgroup analysis revealed a borderline association of 400 μg/d FA with FRα at 40‐wks (Ratio 1.10, 95% CI 1.00‐1.20, P = 0.043), yet no effect of 140 μg/d FA (Ratio 1.04, 95% CI 0.95‐1.13, P = 0.414). This suggests that FRα in plasma, which may reflect FRα from decomposed RBCs and neutrophils, may be slightly increased by long‐term FA supplementation. Larger studies are needed to determine effects of chronic FA exposure on FR expression, including in other tissues.Grant Funding Source: Supported by University of Otago Research Grant

Targeted poly (l-γ-glutamyl glutamine) nanoparticles of docetaxel against folate over-expressed breast cancer cells

A novel folate (FA) conjugated poly(l-γ-glutamyl glutamine) (PGG) nanoparticle loaded with docetaxel (DTX) was prepared to take advantage of both targeted drug delivery in breast cancer and reducing the overall side effects due to the adjuvant free formulation in comparison with Taxotere(®). Nanoprecipitation method was employed to prepare nanoparticles (NPs). The chemical structure of PGG synthesized polymers and PGG-FA conjugates and polymeric nanoparticles were characterized by H NMR, FTIR spectroscopy, field emission scanning electron microscopy, and laser scanning confocal microscopy. The average size of optimized nanoparticles with the aid of Box-Behnken experimental design was 131.96 ± 5.34(nm) with polydispersity of 0.089 ± 0.019, zeta potential of -25.8 ± 2.21(mV), and entrapment efficiency of 67.83 ± 3.29(%). In vitro cytotoxicity of the designed NPs was investigated by MTT assay against three chosen cell lines of MCF7, 4T1, and A549 based on their folate receptor expression capacity and was compared with Taxotere(®). Moreover, PGG-FOL NPs were loaded with 6-coumarin for cellular uptake investigation. In order to assess the antitumor efficacy and biodistribution of targeted NPs, 4T1 murine breast tumors were established on the balb/c mice and in vivo studies were performed. The obtained results showed that the novel designed system was highly effective against tumor cells and successfully localized in the tumor site.

In vivo optical imaging of folate receptor‐β in head and neck squamous cell carcinoma

Folate receptor (FR) expression, although known to be elevated in many types of cancer and inflammatory cells, has not been well characterized in head and neck squamous cell carcinoma (HNSCC). We hypothesized that tumor infiltrating inflammatory cells expressing FR-β could allow fluorescent visualization of HNSCC tumors using folate conjugated dyes even when FR expression in cancer cells is low. Retrospective review of clinical pathologic specimens and in vivo animal study. A tissue microarray with tumor and tumor-free tissue from 22 patients with HNSCC was stained with antibodies to FR-α and FR-β. We characterized FR-β(+) cells by examining CD45, CD68, CD206, and transforming growth factor (TGF)-β expression. To investigate fluorescent imaging, mice with orthotopic tumor xenografts were imaged in vivo after intravenous injections of folate conjugated fluorescein isothiocyanate (folate-FITC) and were histologically evaluated ex vivo. All tumor samples demonstrated significant FR-β staining and negligible FR-α staining. FR-β(+) cells found in tumors coexpressed CD68 and had increased expression of CD206 and TGF-β characteristic of tumor-associated macrophages. In the xenograft models, tumors showed strong in vivo fluorescence after folate-FITC injection in contrast to surrounding normal tissues. Histologic examination of the xenograft tissue similarly showed folate-FITC uptake in areas of inflammatory cellular infiltrate. Although HNSCC tumor cells do not express FR, HNSCC tumors contain a significant population of FR-β-expressing macrophages. Folate conjugated fluorescent dye is able to specifically target and label tumor xenografts to permit macroscopic fluorescence imaging due to FR-β expression on the infiltrating inflammatory cells.

Folic acid-coupled nano-paclitaxel liposome reverses drug resistance in SKOV3/TAX ovarian cancer cells

Chemotherapy could be used as an effective treatment for ovarian cancer and subsequent peritoneal metastasis. Administration of chemoagents in a targeted manner may bring the advantage of higher efficiency and lower drug resistance. In the present study, folate receptor (FR)-targeted nano-paclitaxel formulations were generated and tested for cytotoxicity in a peritoneal xenograft model of paclitaxel-resistant ovarian cancer and SKOV3/TAX cell lines. Immunocytochemical staining confirmed the expression of FR in both SKOV3 and SKOV3/TAX cells. The enrichment of the folic acid-coupled PEGylated nano-paclitaxel liposome (FA-NP) in FR-positive cells was visualized with fluorescence. The uptake of the FA-NP peaked at 4 h and was more robust than nontargeted PEGylated nano-paclitaxel liposome (NP). FA-NP but not NP markedly inhibited the growth of ovarian cancer cells and induced a two-fold increase in the doubling time. The cytotoxic effects of FA-NP were more potent than NP in both SKOV3 cells [50% of inhibition concentration (IC50), 5.67 vs. 50.2 μg/ml, FA-NP vs. NP] and SKOV3/TAX cells (IC50, 0.38 vs. >200 μg/ml, FA-NP vs. NP). FA-NP caused more G2-M cell cycle arrest and apoptotic changes in ovarian cancer cells than NP or regular paclitaxel. However, these effects were blunted in the presence of free FA, which competitively inhibited the receptor-mediated uptake of FA-NP particles. Intraperitoneal (i.p.) administration of FA-NP but not regular paclitaxel, NP, or vehicle significantly prolonged the survival and reduced tumor nodule number (2.9±0.3) in BALB/c nude mice. FA-NP also markedly enhanced the percentage of apoptotic cells in peritoneal xenografts of paclitaxel-resistant ovarian cancer cells (44.6±8.5 vs. 3.2±1.1% for vehicle, 22.4±5.9% for regular paclitaxel, and 35.2±7.7% for NP; P<0.05). However, intravenous administration of FA-NP at the same dose failed to induce apoptosis (20.1±6.2%; P<0.05) and inhibit tumor nodule number to the same extent as intraperitoneal administration. FA-NP reversed the drug resistance in paclitaxel-resistant SKOV3/TAX ovarian cancer cells both in vitro and in vivo. Localized and targeted administration of the FR-targeted chemoagents might prolong the survival time in patients with drug-resistant ovarian cancer.

Profile of vintafolide (EC145) and its use in the treatment of platinum-resistant ovarian cancer.

ObjectiveOur aim was to review the profile of vintafolide (EC145) and its rationale for use in platinum-resistant ovarian cancer. First we investigated the folate receptors (FRs), folate’s pathway into cells, and its expression in normal and cancerous cells, before detailing the mechanism of action of vintafolide, its clinical applications, and the results of different study phases.Materials and methodsA literature search was conducted through PubMed/Medline, Google, ClinicalTrials.gov and websites of pharmaceutical companies. Only articles in English were selected. All articles investigating folate receptor expression in ovarian cancer were selected first, than articles reviewing platinum resistance. Papers about vintafolide were collected, while those talking about synthesis and biochemistry concerns were excluded. The different Phase I and II studies were read, and an update on the website of pharmaceuticals companies were added.ResultsFR is a bundle-membrane receptor that is expressed normally in some normal tissues on the apical surface of cells, but highly expressed in ovarian cancer cells (>80%). It collects folate through endocytosis. Chemotherapy does not modify its expression in ovarian cancer cells, and its expression appears to be mostly associated with a poor prognosis and platinum resistance. Vintafolide is a folate-desacetylvinblastine monohydrazide conjugate, allowing a liberation of the drug into the cytoplasm of cancerous cells via the FR-α (FRα) and endocytosis, with high specificity. Phase I studies showed a 2.5 mg bolus dose to be nontoxic, with moderately adverse events. Phase II clinical trials for the first time demonstrated a statistically significant improvement in disease-free survival in patients with platinum-resistant ovarian cancer, and in those with a very poor prognosis who had already received three to four lines of systemic chemotherapy. The greater benefits were observed in patients with highly expressed FRα.ConclusionVintafolide is a promising targeted agent for recurrent platinum-resistant ovarian cancer, first, thanks to its mechanism of action and the characteristics of FRα in ovarian cancer, and, second, because of the favorable results observed in the first clinical trials on platinum-resistant ovarian cancer. Phase III clinical trials are currently ongoing and are expected to confirm these results.

Small Molecule-Initiated Light-Activated Semiconducting Polymer Dots: An Integrated Nanoplatform for Targeted Photodynamic Therapy and Imaging of Cancer Cells

Photodynamic therapy (PDT) is a noninvasive and light-activated method for cancer treatment. Two of the vital parameters that govern the efficiency of PDT are the light irradiation to the photosensitizer and visual detection of the selective accumulation of the photosensitizer in malignant cells. Herein, we prepared an integrated nanoplatform for targeted PDT and imaging of cancer cells using folic acid and horseradish peroxidase (HRP)-bifunctionalized semiconducting polymer dots (FH-Pdots). In the FH-Pdots, meta-tetra(hydroxyphenyl)-chlorin (m-THPC) was used as photosensitizer to produce cytotoxic reactive oxygen species (ROS); fluorescent semiconducting polymer poly[2-methoxy-5-((2-ethylhexyl)oxy)-p-phenylenevinylene] was used as light antenna and hydrophobic matrix for incorporating m-THPC, and amphiphilic Janus dendrimer was used as a surface functionalization agent to conjugate HRP and aminated folic acid onto the surface of FH-Pdots. Results indicated that the doped m-THPC can be simultaneously excited by the on-site luminol-H2O2-HRP chemiluminescence system through two paths. One is directly through chemiluminescence resonance energy transfer (CRET), and the other is through CRET and subsequent fluorescence resonance energy transfer. In vitro PDT and specificity studies of FH-Pdots using a standard transcriptional and translational assay against MCF-7 breast cancer cells, C6 glioma cells, and NIH 3T3 fibroblast cells demonstrated that cell viability decreased with increasing concentration of FH-Pdots. At the same concentration of FH-Pdots, the decrease in cell viability was positively relevant with increasing folate receptor expression. Results from in vitro fluorescence imaging exhibited that more FH-Pdots were internalized by cancerous MCF-7 and C6 cells than by noncancerous NIH 3T3 cells. All the results demonstrate that the designed semiconducting FH-Pdots can be used as an integrated nanoplatform for targeted PDT and on-site imaging of cancer cells.

A Quantitative Relaxometric Version of the ELISA Test for the Measurement of Cell Surface Biomarkers

Quantitative measurement of marker expression in diseased cells is still a topic of considerable interest and different methodologies are currently under intense scrutiny. This work aims at developing an in vitro diagnostic method based on the release of paramagnetic species from relaxometrically “silent” liposomes operated by the action of a phospholipase A2 (PLA2) previously targeted to the epitope of interest. The released paramagnetic species causes an increase of the longitudinal water proton relaxation rate proportional to the number of PLA2 bound to the cell outer surface. The sensitivity of the herein proposed method, named R-ELISA, was attempted in the detection of folate receptor expression on human ovarian cancer cells by functionalizing PLA2 with folic acid. Receptor/cell number of 8.3×105 has been measured on IGROV-1 cells. The R-ELISA assay can detect nanomolar cell suspension receptor concentrations and has been validated by well-established spectrofluorimetric procedures.

A Quantitative Relaxometric Version of the ELISA Test for the Measurement of Cell Surface Biomarkers

Quantitative measurement of marker expression in diseased cells is still a topic of considerable interest and different methodologies are currently under intense scrutiny. This work aims at developing an in vitro diagnostic method based on the release of paramagnetic species from relaxometrically "silent" liposomes operated by the action of a phospholipase A2 (PLA2) previously targeted to the epitope of interest. The released paramagnetic species causes an increase of the longitudinal water proton relaxation rate proportional to the number of PLA2 bound to the cell outer surface. The sensitivity of the herein proposed method, named R-ELISA, was attempted in the detection of folate receptor expression on human ovarian cancer cells by functionalizing PLA2 with folic acid. Receptor/cell number of 8.3×10(5) has been measured on IGROV-1 cells. The R-ELISA assay can detect nanomolar cell suspension receptor concentrations and has been validated by well-established spectrofluorimetric procedures.

Assessment of folate receptor expression and folate uptake in multicentric lymphomas in dogs

To determine expression of folate receptors (FRs) and folate uptake in multicentric lymphomas in dogs. 10 dogs with histopathologically confirmed multicentric lymphoma and 20 archival lymph node biopsy specimens from dogs with multicentric lymphoma. Multicentric lymphomas in 10 dogs were prospectively evaluated for FR expression by use of immunohistochemical analysis and for in vivo folate uptake by use of nuclear scintigraphy. Dogs with FR-expressing tumors were eligible for FR-targeted chemotherapy. Twenty archival lymphoma biopsy specimens were also evaluated with immunohistochemical analysis. FRs were not detected with immunohistochemical analysis in lymph node samples obtained from the 10 dogs or in archival biopsy specimens. However, nuclear scintigraphy revealed uptake of radioactive tracer in 6 of 10 dogs. Five of these 6 dogs were treated with an FR-targeted chemotherapeutic agent; results of treatment were complete remission in 1 dog, stable disease in 2 dogs, and progressive disease in 2 dogs. Treatment-related toxicoses generally were mild. This study provided strong evidence for folate uptake in a substantial portion of multicentric lymphomas of dogs and indicated the antitumor activity of FR-targeted chemotherapeutics for these cancers. Use of FR-targeted chemotherapeutics may be promising for the treatment of FR-expressing multicentric lymphomas in dogs. Further studies are needed to determine reasons for lack of immunoreactivity to currently identified anti-FR antibodies and to develop improved methods for detecting FRs in lymphomas of dogs.

Enhanced Receptor-Mediated Endocytosis and Cytotoxicity of a Folic Acid-Desacetylvinblastine Monohydrazide Conjugate in a Pemetrexed-Resistant Cell Line Lacking Folate-Specific Facilitative Carriers but with Increased Folate Receptor Expression

The reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptors (FR) are folate-specific transporters. Antifolates currently in the clinic, such as pemetrexed, methotrexate, and pralatrexate, are transported into tumor cells primarily via RFC. Folic acid conjugated to cytotoxics, a new class of antineoplastics, are transported into cells via FR-mediated endocytosis. To better define the role of PCFT in antifolate resistance, a methotrexate-resistant cell line, M160-8, was selected from a HeLa subline in which the RFC gene was deleted and PCFT was highly overexpressed. These cells were cross-resistant to pemetrexed. PCFT function and the PCFT mRNA level in M160-8 cells were barely detectable, and FR-α function and mRNA level were increased as compared with the parent cells. While pemetrexed rapidly associated with FR and was internalized within endosomes in M160-8 cells, consistent with FR-mediated transport, subsequent pemetrexed and (6S)-5-formyltetrahydrofolate export into the cytosol was markedly impaired. In contrast, M160-8 cells were collaterally sensitive to EC0905, a folic acid-desacetylvinblastine monohydrazide conjugate also transported by FR-mediated endocytosis. However, in this case a sulfhydryl bond is cleaved to release the lipophilic cytotoxic moiety into the endosome, which passively diffuses out of the endosome into the cytosol. Hence, resistance to pemetrexed in M160-8 cells was due to entrapment of the drug within the endosome due to the absence of PCFT under conditions in which the FR cycling function was intact.

Role Of Folate Receptor Targeted Therapy In Multiple Myeloma

In multiple myeloma (MM) the interaction of plasma cells, bone marrow stromal cells and tumor associated macrophages (TAMs) plays a significant role in conferring resistance to therapy and in the maintenance of residual disease. Folate receptors (FR) are specifically expressed on metabolically active malignant cells and TAMs and their expression in normal tissues and resting macrophages is limited. FR mediates folate uptake by receptor-mediated endocytosis. This qualifies the receptor to be exploited for drug delivery of folate conjugated cancer therapeutics. Our primary goal in this study is to evaluate the expression of functional FR in MM cells and TAMs with a view to exploit this for folate conjugated targeted therapy for MM. First we evaluated the presence of TAMs in paraffin embedded bone marrow slides of newly diagnosed MM patients with CD68 (pan-Macrophage marker) and CD163 antibodies (specific marker of TAMs) and found extensive infiltration of macrophages in bone marrow from MM patients. Next to evaluate expression of FR in MM cells, we employed an FR antibody and evaluated MM cell lines with immunoblot, flow cytometry and confocal microscopy. In a panel of ten MM cell lines, we found that out of the three FR isoforms (α, β and γ), FR beta (FR-β) is expressed by all of them. Next to test whether this expressed receptor is indeed functional, we incubated the cells with folate deficient media, added different concentrations of EC17 (folate conjugated to FITC, Endocyte Inc.) to the medium and looked for the uptake by flow cytometry after washing off the drug at different time points (after 10, 20, 40 and 60 min).We observed that the uptake begins in 10 min and is saturated at 1 hour with 100nM Folate-FITC. With confocal imaging, Folate-FITC was found in the cytoplasm of MM cells suggesting internalization of Folate-FITC and localization in the cytoplasm. In addition to myeloma cell lines, we also confirmed the uptake of Folate-FITC in CD138+ plasma cells of a newly diagnosed myeloma patient by flow cytometry. This strongly suggests that MM cells express functional FR-β. To test the specificity of this FR mediated uptake, we pre-incubated cells with 0.1mM folic acid in medium for 30 min and then added EC17. This maneuver blocked the activity mediated by FR and no uptake was observed , which proves that the Folate-FITC is internalized only through the FR. To evaluate the expression of FR in-vivo samples, we stained paraffin embedded bone marrow slides of newly diagnosed MM patients with FR-β antibody and TAM specific markers. We observe that FR is expressed on both MM cells as well as TAMs. To assess the endurance of cytotoxic effect of folate conjugated chemotherapeutic agents, we treated MM cell lines with folate conjugated vinka alkaloids and compared them to unconjugated drug and found no significant difference in their action suggesting conjugation with folate does not alter its efficacy. To assess potential toxicity of folate conjugated therapeutics, we obtained CD34+ cells and looked for the uptake of Folate-FITC with flow cytometry. We found no uptake and this is in line with previous reports suggesting that CD34 positive cells express nonfunctional FR. So we propose that FR qualify as potential targets for cancer treatment. Folate targeted therapy using folate-conjugated drugs which can selectively act against both MM cells and supporting TAMs has the potential of specific anti-MM tumoricidal activity. This therapeutic approach would broaden the use of drugs that could be conjugated with folate for MM therapy. Additionally assessment of TAMs in bone marrow sections of MM patients would add another feature for grading, classifying and prognosticating MM. Disclosures: Fonseca: Cylene: Research Funding; AMGEN: Consultancy; Millennium: Consultancy; Binding Site: Consultancy; Onyx: Consultancy, Research Funding; Lilly: Consultancy; BMS: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Medtronic: Consultancy; Otsuka: Consultancy; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents &amp; Royalties.