Regulation of folate transport at the mouse arachnoid barrier.

BACKGROUND: Folates are a family of B9 vitamins essential for normal growth and development in the central nervous system (CNS). Transport of folates is mediated by three major transport proteins: folate receptor alpha (FRα), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Brain folate uptake occurs at the choroid plexus (CP) epithelium through coordinated actions of FRα and PCFT, or directly into brain parenchyma at the vascular blood-brain barrier (BBB), mediated by RFC. Impaired folate transport can occur due to loss of function mutations in FRα or PCFT, resulting in suboptimal CSF folate levels. Our previous reports have demonstrated RFC upregulation by nuclear respiratory factor-1 (NRF-1) once activated by the natural compound pyrroloquinoline quinone (PQQ). More recently, we have identified folate transporter localization at the arachnoid barrier (AB). The purpose of the present study was to further characterize folate transporters localization and function in AB cells, as well as their regulation by NRF-1/PGC-1α signaling and folate deficiency. METHODS: In immortalized mouse AB cells, polarized localization of RFC and PCFT was assessed by immunocytochemical analysis, with RFC and PCFT functionality examined with transport assays. The effects of PQQ treatment on changes in RFC functional expression were also investigated. Mouse AB cells grown in folate-deficient conditions were assessed for changes in gene expression of the folate transporters, and other key transporters and tight junction proteins. RESULTS: Immunocytochemical analysis revealed apical localization of RFC at the mouse AB epithelium, with PCFT localized on the basolateral side and within intracellular compartments. PQQ led to significant increases in RFC functional expression, mediated by activation of the NRF-1/PGC-1α signalling cascade. Folate deficiency led to significant increases in expression of RFC, MRP3, P-gp, GLUT1 and the tight junction protein claudin-5. CONCLUSION: These results uncover the polarized expression of RFC and PCFT at the AB, with induction of RFC functional expression by activation of the NRF-1/PGC-1α signalling pathway and folate deficiency. These results suggest that the AB may contribute to the flow of folates into the CSF, representing an additional pathway when folate transport at the CP is impaired.

Fabrication of folic acid-conjugated pyrimidine-2(5H)-thione-encapsulated curdlan gum-PEGamine nanoparticles for folate receptor targeting breast cancer cells.

In this study 5-((2-((3-methoxy benzylidene)-amino)-phenyl)-diazenyl)-4,6-diphenyl pyrimidine-2(5H)-thione was synthesized. The pharmacological applications of pyrimidine analogs are restricted due to their poor pharmacokinetic properties. As a solution, a microbial exopolysaccharide (curdlan gum) was used to synthesize folic acid-conjugated pyrimidine-2(5H)-thione-encapsulated curdlan gum-PEGamine nanoparticles (FA-Py-CG-PEGamine NPs). The results of physicochemical properties revealed that the fabricated FA-Py-CG-PEGamine NPs were between 100 and 400 nm in size with a majorly spherical shaped, crystalline nature, and the encapsulation efficiency and loading capacity were 79.04 ± 0.79 %, and 8.12 ± 0.39 % respectively. The drug release rate was significantly higher at pH 5.4 (80.14 ± 0.79 %) compared to pH 7.2. The cytotoxic potential of FA-Py-CG-PEGamine NPs against MCF-7 cells potentially reduced the number of cells after 24 h with 42.27 µg × mL-1 as IC50 value. The higher intracellular accumulation of pyrimidine-2(5H)-thione in MCF-7 cells leads to apoptosis, observed by AO/EBr staining and flow cytometry analysis. The highest pyrimidine-2(5H)-thione internalization in MCF-7 cells may be due to folate conjugated on the surface of curdlan gum nanoparticles. Further, internalized pyrimidine-2(5H)-thione increases the intracellular ROS level, leading to apoptosis and inducing the decalin in mitochondrial membrane potential. These outcomes demonstrated that the FA-Py-CG-PEGamine NPs were specificity-targeting folate receptors on the plasma membranes of MCF-7 Cells.

Folate Receptor ß (FRß) Expression on Myeloid Cells and the Impact of Reticuloendothelial System on Folate-Functionalized Nanoparticles' Biodistribution in Cancer.

Folate uptake is largely mediated by folate receptor (FR)ß, encoded by FOLR2 gene, in myeloid immune cells such as granulocytes, monocytes, and especially in macrophages that constitute the reticuloendothelial system (RES) and infiltrate the tumor microenvironment. Since the myeloid immune compartment dynamically changes during tumorigenesis, it is critical to assess the infiltration status of the tumors by FRß-expressing myeloid cells to better define the targeting efficacy of folate-functionalized drug delivery systems. On the other hand, clearance by RES is a major limitation for the targeting efficacy of nanoparticles decorated with folate. Therefore, the aims of this study are (i) to determine the amount and subtypes of FRß+ myeloid cells infiltrating the tumors at different stages, (ii) to compare the amount and subtype of FRß+ myeloid cells in distinct organs of tumor-bearing and healthy animals, (iii) to test if the cancer-targeting efficacy and biodistribution of a prototypic folate-functionalized nanoparticle associates with the density of FRß+ myeloid cells. Here, we report that myeloid cell infiltration was enhanced and FRß was upregulated at distinct stages of tumorigenesis in a mouse breast cancer model. The CD206+ subset of macrophages highly expressed FRß, prominently both in tumor-bearing and healthy mice. In tumor-bearing mice, the amount of all myeloid cells, but particularly granulocytes, was remarkably increased in the tumor, liver, lungs, spleen, kidneys, lymph nodes, peritoneal cavity, bone marrow, heart, and brain. Compared with macrophages, the level of FRß was moderate in granulocytes and monocytes. The density of FRß+ immune cells in the tumor microenvironment was not directly associated with the tumor-targeting efficacy of the folate-functionalized cyclodextrin nanoparticles. The lung was determined as a preferential site of accumulation for folate-functionalized nanoparticles, wherein FRß+CD206+ macrophages significantly engulfed cyclodextrin nanoparticles. In conclusion, our results demonstrate that the tumor formation augments the FR levels and alters the infiltration and distribution of myeloid immune cells in all organs which should be considered as a major factor influencing the targeting efficacy of nanoparticles for drug delivery.

Vaccination with folate receptor-alpha peptides in patients with ovarian cancer following response to platinum-based therapy: A randomized, multicenter clinical trial.

OBJECTIVE: Folate receptor alpha (FRα) is overexpressed on >90% of high-grade epithelial ovarian cancers (EOC). Targeting FRα with antibody-drug conjugates has proven utility in the platinum-resistant setting. It is also a potential therapeutic target for immuno-oncologic agents, such as peptide vaccines that work primarily via adaptive and humoral immunity. We tested the hypothesis that FRα peptide immunization could improve outcomes in patients with EOC following response to platinum-based therapy. METHODS: We conducted a randomized, double-blind, multicenter, phase II study to evaluate the safety and efficacy of TPIV200 (a multi-epitope FRα peptide vaccine admixed with GM-CSF) versus GM-CSF alone in 120 women who did not have disease progression after at least 4 cycles of first-line platinum-based therapy. Patients were vaccinated intradermally once every 4 weeks up to 6 times, followed by a boosting period of 6 vaccinations at 12-week intervals. Primary endpoints included safety, tolerability, and progression free survival (PFS). RESULTS: At study termination with a median follow-up of 15.2 months (range 1.2-28.4 months), 68 of 119 intention-to-treat patients had disease progression (55% in TPIV200 + GM-CSF arm and 59% in GM-CSF alone arm). The median PFS was 11.1 months (95% CI 8.3-16.6 months) with no significant difference between the treatment groups (10.9 months with TPIV200 + GM-CSF versus 11.1 months with GM-CSF, HR, 0.85; upper 90% CI 1.17]. No patient experienced a ≥ grade 3 drug-related adverse event. CONCLUSION: TPIV200 was well tolerated but was not associated with improved PFS. Additional studies are required to uncover potential synergies using multiepitope vaccines targeting FRα. Trial Registration NLM/NCBI Registry, NCT02978222, https://clinicaltrials.gov/search?term=NCT02978222.

Maintenance with mirvetuximab soravtansine plus bevacizumab vs bevacizumab in FRα-high platinum-sensitive ovarian cancer.

At first recurrence, platinum-sensitive ovarian cancer (PSOC) is frequently treated with platinum-based chemotherapy doublets plus bevacizumab, then single-agent bevacizumab. Most patients' disease progresses within a year after chemotherapy, emphasizing the need for novel strategies. Mirvetuximab soravtansine-gynx (MIRV), an antibody-drug conjugate, comprises a folate receptor alpha (FRα)-binding antibody and tubulin-targeting payload (maytansinoid DM4). In FRα-high PSOC, MIRV plus bevacizumab previously showed promising efficacy (objective response rate, 69% [95% CI: 41-89]; median progression-free survival, 13.3 months [95% CI: 8.3-18.3]; median duration of response, 12.9 months [95% CI: 6.5-15.7]) and safety. The Phase III randomized GLORIOSA trial will evaluate MIRV plus bevacizumab vs. bevacizumab alone as maintenance therapy in patients with FRα-high PSOC who did not have disease progression following second-line platinum-based doublet chemotherapy plus bevacizumab.Clinical Trial Registration: ClinicalTrials.gov ID: NCT05445778; GOG.org ID: GOG-3078; ENGOT.ESGO.org ID: ENGOT-ov76.

Most patients with ovarian cancer are initially treated with platinum-based chemotherapy. If the cancer reappears/recurs after more than 6 months following this therapy, it is called platinum-sensitive ovarian cancer (PSOC). Patients with PSOC usually receive additional platinum-based chemotherapy along with bevacizumab, a drug that reduces tumor growth by decreasing its blood supply. If patients improve or are stable on this therapy, they are usually kept on bevacizumab alone for 'maintenance therapy'. Unfortunately, this maintenance therapy does not work long-term in all patients, so better long-term treatments are needed. The GLORIOSA (NCT05445778) clinical trial will compare maintenance therapy with bevacizumab alone to maintenance therapy with bevacizumab plus a drug called mirvetuximab soravtansine-gynx (MIRV) to determine which therapy leads to better results in patients with PSOC. MIRV is made up of an antibody that binds to a specific protein (folate receptor alpha [FRα]) on cancer cells to directly deliver a cancer-killing drug. MIRV received US FDA approval to be used as a therapy for patients with ovarian cancer who are resistant to platinum-based chemotherapy and express high levels of FRα. The GLORIOSA trial will study maintenance therapy with MIRV plus bevacizumab in patients with PSOC who have not had cancer progression after second-line platinum-based chemotherapy plus bevacizumab, and whose cancer expresses high amounts of FRα. The main purpose of this trial is to determine if MIRV plus bevacizumab leads to better patient survival and decreases cancer growth and spread when compared with bevacizumab alone.

Synthesis and Preclinical Evaluation of [18F]AlF-NOTA-Asp2-PEG2-Folate as a Novel Folate-Receptor-Targeted Tracer for PET Imaging.

Recently, the folate receptor (FR) has become an exciting target for the diagnosis of FR-positive malignancies. Nevertheless, suboptimal in vivo pharmacokinetic properties, particularly high uptake in the renal and hepatobiliary systems, are important limiting factors for the clinical translation of most FR-based radiotracers. In this study, we developed a novel 18F-labeled FR-targeted positron emission tomography (PET) tracer [18F]AlF-NOTA-Asp2-PEG2-Folate modified with a hydrophilic linker (-Asp2-PEG2) to optimize its pharmacokinetic properties and conducted a comprehensive preclinical assessment. The [18F]AlF-NOTA-Asp2-PEG2-Folate was manually synthesized within 30 min with a non-decay-corrected radiochemical yield of 16.3 ± 2.0% (n = 5). Among KB cells, [18F]AlF-NOTA-Asp2-PEG2-Folate exhibited high specificity and affinity for FR. PET/CT imaging and biodistribution experiments in KB tumor-bearing mice showed decent tumor uptake (1.7 ± 0.3% ID/g) and significantly decreased uptake in kidneys and liver (22.2 ± 2.1 and 0.3 ± 0.1% ID/g at 60 min p.i., respectively) of [18F]AlF-NOTA-Asp2-PEG2-Folate, compared to the known tracer [18F]AlF-NOTA-Folate (78.6 ± 5.1 and 5.3 ± 0.5 % ID/g at 90 min p.i., respectively). The favorable properties of [18F]AlF-NOTA-Asp2-PEG2-Folate, including its efficient synthesis, decent tumor uptake, relatively low renal uptake, and rapid clearance from most normal organs, portray it as a promising PET tracer for FR-positive tumors.

New hopes and promises in the treatment of ovarian cancer focusing on targeted treatment-a narrative review.

Unfortunately, ovarian cancer is still diagnosed most often only in an advanced stage and is also the most lethal gynecological cancer. Another problem is the fact that treated patients have a high risk of disease recurrence. Moreover, ovarian cancer is very diverse in terms of molecular, histological features and mutations. Many patients may also develop platinum resistance, resulting in poor response to subsequent lines of treatment. To improve the prognosis of patients with ovarian cancer, it is expected to make better existing and implement new, promising treatment methods. Targeted therapies seem very promising. Currently, bevacizumab - a VEGF inhibitor and therapy with olaparib - a polyADP-ribose polymerase inhibitor are approved. Other methods worth considering in the future include: folate receptor α, immune checkpoints or other immunotherapy methods. To improve the treatment of ovarian cancer, it is also important to ameliorate the determination of molecular features to describe and understand which group of patients will benefit most from a given treatment method. This is important because a larger group of patients treated for ovarian cancer can have a greater chance of surviving longer without recurrence.

Targeted Liposomal Co-delivery of an Immunogenic Cell Death Inducer and a Toll-Like Receptor 4 Agonist for Enhanced Cancer Chemo-immunotherapy.

Anticancer chemo-immunotherapy has gained considerable attention across various scientific domains as a prospective approach for the comprehensive eradication of malignant tumors. Recent research has particularly been focused on traditional anthracycline chemo drugs, such as doxorubicin and mitoxantrone. These compounds trigger apoptosis in tumor cells and evoke immunogenic cell death (ICD). ICD is a pivotal initiator of the cancer-immunity cycle by facilitating the release of damage-associated molecular patterns (DAMPs). The resultant DAMPs released from cancer cells effectively activate the immune system, resulting in an increase in tumor-infiltrating T cells. In this study, we have innovated a co-delivery strategy involving folate-modified liposomes to deliver doxorubicin and monophosphoryl lipid A (MPLA) simultaneously to tumor tissue. The engineered liposomes exploit the overexpression of folate receptors within the tumor tissues. Delivered doxorubicin initiates ICD at the tumor cells, further enhancing the immunogenic stimulus. Additionally, MPLA helps T cell priming by activating antigen-presenting cells. This intricate interplay culminates in a synergistic effect, ultimately resulting in an augmented and potentiated anticancer chemo-immunotherapeutic liposomal treatment.

Targeted Chemo-Phototherapy in Red Light with Novel Doxorubicin and Iron(III) Complex-Functionalized Gold Nanoconjugate (Dox-Fe@FA-AuNPs).

The anticancer efficacy of doxorubicin, an anthracycline-based and FDA-approved chemotherapeutic drug, is significantly hindered by acquired chemoresistance and severe side effects despite its potent anticancer properties. To overcome these challenges, we developed an innovative therapeutic formulation that integrates targeted chemotherapy and phototherapy within a single platform using gold nanoparticles (AuNPs). This novel nanoconjugate, designated as Dox-Fe@FA-AuNPs, is co-functionalized with folic acid, doxorubicin, and an iron(III)-phenolate/carboxylate complex, enabling cancer-specific drug activation. Here, we report the synthesis, characterization, and comprehensive physico-chemical and biological evaluations of Dox-Fe@FA-AuNPs. The nanoconjugate exhibited excellent solubility, stability, and enhanced cellular uptake in folate receptor-positive cancer cells. The nanoconjugate was potently cytotoxic against HeLa and MDA-MB-231 cancer cells (HeLa: 105.5±16.52 µg mL-1; MDA-MB-231: 112.0±12.31 µg mL-1; MDA-MB-231 (3D): 156.31±19.35 µg mL-1) while less cytotoxic to the folate(-) cancer cells (MCF-7, A549 and HepG2). The cytotoxicity was attributed to the pH-dependent release of doxorubicin, which preferentially occurs in the acidic tumor microenvironment. Additionally, under red light irradiation, the nanoconjugate generated ROS, inducing caspase-3/7-dependent apoptosis with a photo-index (PI) >50, and inhibited cancer cell migration. Our findings underscore the potential of Dox-Fe@FA-AuNPs as a highly effective and sustainable platform for targeted chemo-phototherapy.

Folate receptor alpha expression in low-grade serous ovarian cancer: Exploring new therapeutic possibilities.

OBJECTIVE: Mirvetuximab soravtansine may be a potentially effective therapeutic option for ovarian low-grade serous carcinoma (LGSC), but the prevalence of folate receptor alpha (FRα) overexpression in this tumor type is unknown. We sought to characterize FRα expression in LGSC and its association with clinical and molecular features. METHODS: FRα immunohistochemistry was performed on a tissue microarray comprised of 89 LGSCs and 42 ovarian serous borderline tumors (SBTs). Clinical tumor-normal panel-based sequencing was performed on 78 LGSCs. Associations between FRα-high status and clinicopathologic characteristics and survival outcomes were examined. RESULTS: Of 89 LGSCs, 36 (40%) were FRα-high (≥75% of viable tumor cells exhibiting moderate-to-strong membranous expression). Of 9 patients with LGSC and samples from different timepoints, 4 (44%) had discordant results, with conversion from FRα-negative to FRα-high in 3 (33%) cases. There was no association between FRα-high status with age, race, or progression-free/overall survival. A MAPK pathway genetic alteration, most commonly involving KRAS (n = 23), was present in 45 (58%) LGSCs. Those lacking MAPK pathway alterations were more likely to be FRα-high compared to MAPK-altered LGSCs (61% vs 20%, p < 0.001). In SBTs, FRα-high expression was associated with high-risk (micropapillary) histology and/or subsequent LGSC recurrence compared to conventional SBTs without malignant recurrence (53% vs 9%, p = 0.008). CONCLUSIONS: Future studies of FRα-directed therapy in patients with LGSC are warranted. Discordant FRα status at recurrence suggests potential benefit for retesting. A biomarker-driven approach to direct treatment selection in LGSC is recommended. As high FRα expression is more common amongst tumors lacking MAPK pathway genetic alterations, FRα testing to determine eligibility for mirvetuximab soravtansine therapy is particularly recommended for this subgroup.

Folate Receptor Targeting Mn(II) Complex Encapsulated Porous Silica Nanoparticle as an MRI Contrast Agent for Early-State Detection of Cancer.

Cancer is recognized as one of the major causes of mortality, however, early-stage detection can increase the survival chance greatly. It is recognized that folate receptors are gradually overexpressed in the cellular membrane with the progress of cancer from stage 1 to stage 4. Utilizing the fact, herein, developed a porous silica nanoparticle system C1@SiO2-FA-NP; A) impregnated with thermodynamically stable Mn(II) complex (1) molecules within the core of the nanoparticle, and B) surface functionalized with folate units. It exhibited a high longitudinal relaxivity value r1 = 21.45 mM-1s-1 that substantially increased to r1 = 40.97 mM-1s-1 in the presence of 0.67 mM concentration of BSA under the physiological condition. The in vitro fluorescent images after surface conjugation of C1@SiO2-FA-NP with FITC (fluorescein isothiocyanate) buttressed the inclusion of the nanoparticle exclusively within the cancerous HeLa cells than that of healthy HEK293 cells. The importance of the surface-bound folate unit in the nanoparticle is further established by comparing the fluorescent images of HeLa cells in the absence of the group. Finally, the applicability of C1@SiO2-FA-NP as the T1-weighted MRI contrast agent for early-stage cancer diagnosis is established within C57BL/6 mice after infecting the mice with HeLa cells.

Nonclinical pharmacodynamics of boron neutron capture therapy using direct intratumoral administration of a folate receptor targeting novel boron carrier.

Background: Boron neutron capture therapy (BNCT) is a precise particle radiation therapy known for its unique cellular targeting ability. The development of innovative boron carriers is crucial for the advancement of BNCT technologies. Our previous study demonstrated the potential of PBC-IP administered via convection-enhanced delivery (CED) in an F98 rat glioma model. This approach significantly extended rat survival in neutron irradiation experiments, with half achieving long-term survival, akin to a cure, in a rat brain tumor model. Our commitment to clinical applicability has spurred additional nonclinical pharmacodynamic research, including an investigation into the effects of cannula position and the time elapsed post-CED administration. Methods: In comprehensive in vivo experiments conducted on an F98 rat brain tumor model, we meticulously examined the boron distribution and neutron irradiation experiments at various sites and multiple time intervals following CED administration. Results: The PBC-IP showed substantial efficacy for BNCT, revealing minimal differences in tumor boron concentration between central and peripheral CED administration, although a gradual decline in intratumoral boron concentration post-administration was observed. Therapeutic efficacy remained robust, particularly when employing cannula insertion at the tumor margin, compared to central injections. Even delayed neutron irradiation showed notable effectiveness, albeit with a slightly reduced survival period. These findings underscore the robust clinical potential of CED-administered PBC-IP in the treatment of malignant gliomas, offering adaptability across an array of treatment protocols. Conclusions: This study represents a significant leap forward in the quest to enhance BNCT for the management of malignant gliomas, opening promising avenues for clinical translation.

Fullerene C60 Conjugate with Folic Acid and Polyvinylpyrrolidone for Targeted Delivery to Tumor Cells.

The use of targeted drug delivery systems, including those based on selective absorption by certain receptors on the surface of the target cell, can lead to a decrease in the minimum effective dose and the accompanying toxicity of the drug, as well as an increase in therapeutic efficacy. A fullerene C60 conjugate (FA-PVP-C60) with polyvinylpyrrolidone (PVP) as a biocompatible spacer and folic acid (FA) as a targeting ligand for tumor cells with increased expression of folate receptors (FR) was obtained. Using 13C NMR spectroscopy, FT-IR, UV-Vis spectrometry, fluorometry and thermal analysis, the formation of the conjugate was confirmed and the nature of the binding of its components was established. The average particle sizes of the conjugate in aqueous solutions and cell culture medium were determined using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). The FA-PVP-C60 showed antiradical activity against •DPPH, •OH and O2•-, but at the same time, it was shown to generate 1O2. It was found that the conjugate in the studied concentration range (up to 200 µg/mL) is non-toxic in vitro and does not affect the cell cycle. To confirm the ability of the conjugate to selectively accumulate through folate-mediated endocytosis, its uptake into cells was analyzed by flow cytometry and confocal microscopy. It was shown that the conjugate is less absorbed by A549 cells with low FR expression than by HeLa, which has a high level of expression of this receptor.

Folic acid-mediated enhancement of the diagnostic potential of luminescent europium-doped hydroxyapatite nanocrystals for cancer biomaging.

Early and accurate cancer diagnosis is crucial for improving patient survival rates. Luminescent nanoparticles have emerged as a promising tool in fluorescence bioimaging for cancer diagnosis. To enhance diagnostic accuracy, ligands promoting endocytosis into cancer cells are commonly incorporated onto nanoparticle surfaces. Folic acid (FA) is one such ligand, known to specifically bind to folate receptors (FR) overexpressed in various cancer cells such as cervical and ovarian carcinoma. Therefore, surface modification of luminescent nanoparticles with FA can enhance both luminescence efficiency and diagnostic accuracy. In this study, luminescent europium-doped hydroxyapatite (EuHAp) nanocrystals were prepared via hydrothermal method and subsequently modified with (3-Aminopropyl)triethoxysilane (APTES) followed by FA to target FR-positive human cervical adenocarcinoma cell line (HeLa) cells. The sequential grafting of APTES and then FA formed a robust covalent linkage between the nanocrystals and FA. Rod-shaped FA-modified EuHAp nanocrystals, approximately 100 nm in size, exhibited emission peaks at 589, 615, and 650 nm upon excitation at 397 nm. Despite a reduction in photoluminescence intensity following FA modification, fluorescence microscopy revealed a remarkable 120-fold increase in intensity compared to unmodified EuHAp, attributed to the enhanced uptake of FA-modified EuHAp. Additionally, confocal microscope observations confirmed the specificity and the internalization of FA-modified EuHAp nanocrystals in HeLa cells. In conclusion, the modification of EuHAp nanocrystals with FA presents a promising strategy to enhance the diagnostic potential of cancer bioimaging probes.

Corrigendum: Therapeutic strategies targeting folate receptor α for ovarian cancer.

[This corrects the article DOI: 10.3389/fimmu.2023.1254532.].

In Vivo Labeling and Detection of Circulating Tumor Cells in Mice Using OTL38.

PURPOSE: We recently developed an optical instrument to non-invasively detect fluorescently labeled circulating tumor cells (CTCs) in mice called 'Diffuse in vivo Flow Cytometry' (DiFC). OTL38 is a folate receptor (FR) targeted near-infrared (NIR) contrast agent that is FDA approved for use in fluorescence guided surgery of ovarian and lung cancer. In this work, we investigated the use OTL38 for in vivo labeling and detection of FR + CTCs with DiFC. PROCEDURES: We tested OTL38 labeling of FR + cancer cell lines (IGROV-1 and L1210A) as well as FR- MM.1S cells in suspensions of Human Peripheral Blood Mononuclear cells (PBMCs) in vitro. We also tested OTL38 labeling and NIR-DIFC detection of FR + L1210A cells in blood circulation in nude mice in vivo. RESULTS: 62% of IGROV-1 and 83% of L1210A were labeled above non-specific background levels in suspensions of PBMCs in vitro compared to only 2% of FR- MM.1S cells. L1210A cells could be labeled with OTL38 directly in circulation in vivo and externally detected using NIR-DiFC in mice with low false positive detection rates. CONCLUSIONS: This work shows the feasibility of labeling CTCs in vivo with OTL38 and detection with DiFC. Although further refinement of the DiFC instrument and signal processing algorithms and testing with other animal models is needed, this work may eventually pave the way for human use of DiFC.

Folate receptor alpha protein expression in ovarian serous cystadenocarcinoma tumors of The Cancer Genome Atlas: exploration beyond single-agent therapy.

Epithelial ovarian cancer (EOC) can be highly lethal, with limited therapeutic options for patients with non-homologous recombination deficient (HRD) disease. Folate receptor alpha (FOLR1/FRα)-targeting agents have shown promise both alone and in combination with available therapies, but the relationship of FRα to other treatment-driving biomarkers is unknown. The Cancer Genome Atlas (TCGA) was queried to assess protein and mRNA expression and mutational burden in patients with differential FRα protein-expressing ovarian tumors, and the results referenced against the standard 324 mutations currently tested through FoundationOne Companion Diagnostics to identify targets of interest. Of 585 samples within TCGA, 121 patients with serous ovarian tumors for whom FRα protein expression was quantified were identified. FRα protein expression significantly correlated with FOLR1 mRNA expression (p=7.19×1014). Progression free survival (PFS) for the FRα-high group (Q1) was 20.7 months, compared to 16.6 months for the FRα-low group (Q4, Logrank, p=0.886). Overall survival (OS) was 54.1 months versus 36.3 months, respectively; however, this result was not significant (Q1 vs. Q4, Logrank, p=0.200). Mutations more commonly encountered in patients with high FRα-expressing tumors included PIK3CA and FGF family proteins. Combinations of FRα-targeting agents with PI3K, mTOR, FGF(R) and VEGF inhibitors warrant investigation to evaluate their therapeutic potential.

Phase 1b study of mirvetuximab soravtansine, a folate receptor alpha (FRα)-targeting antibody-drug conjugate, in combination with carboplatin and bevacizumab in patients with platinum-sensitive ovarian cancer.

OBJECTIVE: Evaluate the antitumor activity and safety profile of the triplet combination of mirvetuximab soravtansine (MIRV), carboplatin, and bevacizumab in recurrent, platinum-sensitive ovarian cancer. METHODS: Participants with recurrent, platinum-sensitive epithelial ovarian, fallopian tube, or primary peritoneal cancer (1-2 prior lines of therapy) received MIRV (6 mg/kg adjusted ideal body weight), carboplatin (AUC5), and bevacizumab (15 mg/kg) once every 3 weeks. Carboplatin could be discontinued after 6 cycles per investigator discretion; continuation of MIRV+bevacizumab as maintenance therapy was permitted. Eligibility included folate receptor alpha (FRα) expression by immunohistochemistry (≥50% of cells with ≥2+ intensity; PS2+ scoring); prior bevacizumab was allowed. Tumor response, duration of response (DOR), progression-free survival (PFS), and adverse events (AEs) were assessed. RESULTS: Forty-one participants received triplet therapy, with a median of 6, 12, and 13 cycles of carboplatin, MIRV, and bevacizumab, respectively. The confirmed objective response rate was 83% (9 complete and 25 partial responses). The median DOR was 10.9 months; median PFS was 13.5 months. AEs (any grade) occurred as expected, based on each agent's safety profile; most common were diarrhea (83%), nausea (76%), fatigue (73%), thrombocytopenia (71%), and blurred vision (68%). Most cases were mild to moderate (grade ≤2), except for thrombocytopenia, for which most drug-related discontinuations occurred, and neutropenia. CONCLUSIONS: This triplet regimen (MIRV+carboplatin+bevacizumab) was highly active, with a tolerable AE profile in participants with recurrent, platinum-sensitive, FRα-expressing ovarian cancer. Thrombocytopenia was the primary cause of dose modifications. These outcomes compare favorably to historical data reported for platinum-based chemotherapy plus bevacizumab regimens in similar patient populations.

Tris-assisted one-step fabrication of functional carbon dots for specific folate receptor positive-expressed cancer cell imaging.

Specific staining of cancer cells is momentous for cancer research. Nanoprobe with multivalent recognition is emerging as powerful tools for bioimaging, but the nonspecific cell uptake and complex functional modification procedures are still obstacles for specific detection and convenient synthesis. Carbon dots (CDs) with an intrinsic targeting ability, excellent optical properties and biocompatibility acquired from an efficient one-step fabrication procedure were urgently desired in specific cancer cells visualization. Herein, inspired by the interrelationships between interface and biomolecular mechanisms, we suggested that it was possible to construct CDs with the desired characteristics for folate receptor (FR) positive-expressed cancer cell imaging via rich hydroxyl groups Tris-assisted one-step hydrothermal treatment of folate acid (FA) and l-Arginine (L-Arg) precursors. The prepared small-sized F-CDs were equipped with abundant hydroxyl, pterin and negative charge surface, and possessed environmental friendliness, outstanding photostability and biocompatibility. Moreover, F-CDs had an intrinsic FR positive-expressed cancer cell targeting ability without any post-modification of the ligands. Rich hydroxyl groups play a vital role in endowing the optical properties and biological effects of F-CDs. F-CDs could be used as a promising candidate for FR-expressed cancer cell labeling and tracking. In addition, the caveolae-mediated endocytosis pathway of F-CDs was ascertained. More importantly, experimental results confirmed that the combination of physicochemical properties may provide an efficient strategy to overcome non-specific cell uptake interactions for cell labeling. Our strategy put forward a promising alternative to design fluorescent CDs for extensive chemical and biomedical applications.

Clinical significance of postoperative folate receptor-positive circulating tumor cells (FR + CTCs) for long-term prognosis in patients with invasive adenocarcinoma (IAC) of the lung.

BACKGROUND: The aim of the study was to evaluate the prognostic value of postoperative folate receptor-positive circulating tumor cell (FR + CTC) detection in patients with stage I-III invasive adenocarcinoma (IAC) treated with surgery. METHODS: Patients with lung adenocarcinoma (LUAD) who underwent surgical resection in Peking University Cancer Hospital and received postoperative FR + CTC analysis from July 2016 to January 2021 were retrospectively collected. Comparisons between or among groups were made using the Kruskal-Wallis or Mann-Whitney U tests. Survival curves were estimated using the Kaplan-Meier method and compared using the log-rank test. Cox proportional hazard regression analyses were performed to explore the factors predicting recurrence and survival. RESULTS: There were significant differences between the high and low groups in terms of age (p = 0.002), postoperative CA199 (p = 0.038), and postoperative SCC (p = 0.024). There were no significant differences in the other indicators (all p>0.05). N stage 1, N stage 2, and neoadjuvant therapy (NAT) were independent risk factors for disease recurrence and death; pleural invasion (PI), and nerve invasion were independent risk factors for death. The Kaplan-Meier curve showed a notable trend for a worse disease-free survival (DFS) or overall survival (OS) for patients with high levels of FR + CTCs in our study, but none of these were statistically significant. CONCLUSION: The detection of FR + CTCs postoperatively was an independent predictor of recurrence in patients treated for stage I-III IAC. Standardized detection methods and optimal time points for assessment should be established in future studies.