FOLR1 Human

What is the normal tissue distribution pattern of FOLR1 in humans?

FOLR1 exhibits a highly restricted expression pattern in normal human tissues. It is predominantly expressed on the luminal (apical) surface of polarized epithelia, including proximal kidney tubules, type 1 and 2 pneumocytes in the lungs, choroid plexus, ovary, fallopian tube, uterus, cervix, epididymis, submandibular salivary gland, bronchial glands, and trophoblasts in the placenta. This limited distribution pattern is important when considering FOLR1 as a potential therapeutic target, as it suggests minimal off-target effects in normal tissues. In several specialized tissues, FOLR1 functions in the transcytosis of folates across cellular barriers, such as transporting folates across the blood-brain barrier via the choroid plexus, transferring folates from mother to fetus in the placenta, and reabsorbing folates from pre-urine in the kidney .

How does FOLR1 transport folates into cells compared to other folate transporters?

FOLR1 is one of three major types of folate transporters, but its folate transport efficiency differs significantly from other transporters like the reduced folate carrier (RFC). In cancer cell line studies, even when FOLR1 is overexpressed, it generally transports less folate into cells than RFC. For example, in five ovarian cancer cell lines, FOLR1 only contributes approximately 20% of the uptake of serum folate 5-methyl-THF, while RFC is responsible for about 70% of the uptake. Interestingly, FOLR1 has a much higher affinity for the synthetic, non-natural folic acid (FA) than for naturally-occurring reduced folates—approximately 14-fold higher affinity for FA than for 5-methyl-THF. This contrasts with RFC, which transports FA one-to-two orders of magnitude less efficiently than reduced folates. These differential affinities have important methodological implications when designing experiments, as using FA versus reduced folates can shift cellular responses toward FOLR1-dependent or RFC-dependent mechanisms .

What experimental methods are most reliable for detecting FOLR1 protein expression in human tissues?

For reliable detection of FOLR1 protein expression in human tissues, a combination of approaches is recommended. Immunohistochemistry using validated antibodies such as the anti-FOLR1 monoclonal antibody LK26 provides good tissue-level detection. For increased specificity assessment, multiplexing imaging systems can be employed with anti-FOLR1 scFv-Fc fusion proteins compared against established antibodies. This approach allows for the analysis of staining profiles across multiple tissue types simultaneously and helps distinguish true FOLR1 expression from background or non-specific staining. When evaluating FOLR1 expression in cancer specimens, it's essential to include appropriate controls, including FOLR1-knockout cell lines (e.g., OV-90 FOLR1 KO) to validate antibody specificity. For broader tissue cross-reactivity experiments, an automated high-plex imaging approach analyzing multiple tissue types (including adrenal gland, ovary, pancreas, thyroid, cerebellum, cerebrum, lung, and others) provides comprehensive expression data .

In which cancer types is FOLR1 most frequently overexpressed?

FOLR1 demonstrates variable overexpression across different cancer types, with the highest prevalence observed in ovarian cancers. In primary ovarian tumors, 72% express high levels of FOLR1, with even higher rates in specific subtypes: 82-100% of serous carcinomas, 22-100% of endometrioid carcinomas, and 63-80% of clear cell carcinomas express high FOLR1. Metastatic ovarian cancers show particularly high FOLR1 expression rates of 90-100%. Beyond ovarian cancer, FOLR1 is overexpressed in several other malignancies, including spindle cell type liposarcoma (100%), fibrosarcoma (70%), uterine metastatic endometrial carcinoma (100%), cervical squamous cell carcinoma (41%), malignant melanoma (40%), papillary thyroid carcinoma (30%), follicular thyroid carcinoma (22%), and to lesser extents in gastric adenocarcinoma (11%) and esophageal squamous cell carcinoma (10%). This differential expression pattern across cancer types provides valuable information for researchers focusing on FOLR1 as a potential biomarker or therapeutic target .

How does FOLR1 expression compare between primary tumors and metastatic lesions?

Research data indicates that FOLR1 expression frequently increases during cancer progression from primary tumors to metastatic lesions. In ovarian cancer, while primary tumors show 72% high FOLR1 expression, metastatic lesions demonstrate significantly higher rates of 90-100%. This upregulation is accompanied by a substantial increase in FOLR1 protein levels, with metastatic ovarian cancer showing a 30.1-fold increase in FOLR1 protein compared to normal ovarian tissue (versus 22.3-fold in primary serous carcinoma). Similarly, in endometrial cancer, 100% of metastatic lesions express FOLR1 compared to 20% of primary tumors, with an 8.6-fold increase in FOLR1 protein in metastatic lesions versus 9.8-fold in primary tumors (compared to normal tissue). These findings suggest that FOLR1 may play a functional role in the metastatic process or be selected for during metastatic progression. For researchers investigating the mechanisms of metastasis, these expression patterns make FOLR1 an interesting candidate for study, particularly in gynecologic malignancies .

What evidence supports non-canonical signaling roles for FOLR1 independent of folate transport?

Several lines of evidence support non-canonical signaling roles for FOLR1 independent of its folate transport function. First, contrasting effects have been observed between FOLR1 and RFC in cancer cell behavior despite both being folate transporters. In SKOV-3 ovarian cancer cells, FOLR1 knockdown reduced cell proliferation, migration, and invasiveness, while RFC overexpression produced similar effects, suggesting that FOLR1's cancer-promoting properties are not simply due to increased folate uptake. Second, anti-FOLR1 antibodies that block folate binding have been shown to stimulate erythropoietic cell proliferation, indicating a folate-independent signaling role. Third, rapid activation of signaling pathways (within 5-30 minutes after folate addition) has been observed in multiple cell types, including increased STAT3 phosphorylation in HeLa cells within 5 minutes of folic acid addition. Finally, physical interactions between FOLR1 and signaling molecules have been demonstrated through co-immunoprecipitation experiments, including interactions with gp130 (in JAK-STAT signaling), DGCR8 and Drosha (in miRNA processing), and CAF-1. These multiple lines of evidence provide a strong case for non-canonical signaling functions of FOLR1 beyond its role in folate transport .

What roles does FOLR1 play in gene regulation and transcriptional control?

Recent evidence suggests that FOLR1 may function directly in gene regulation and transcriptional control. In O9-1 neural crest cells, FOLR1 has been shown to regulate pluripotency genes through multiple mechanisms. Chromatin immunoprecipitation (ChIP) experiments demonstrate FOLR1 binding to the regulatory DNA regions of Oct4, Sox2, and Klf4 within 15-30 minutes after folic acid treatment (0.23 μM). This is accompanied by increased expression of these pluripotency factors and Trim71 within 12-24 hours, an effect that requires FOLR1. Additionally, FOLR1 appears to regulate microRNA expression, decreasing miR-let-7 and miR-138 after 6 hours of treatment. The mechanism likely involves physical interactions with microRNA processing machinery, as co-immunoprecipitation studies show FOLR1 interacting with DGCR8 and Drosha. In differentiated glial cells, the 38 kD isoform of FOLR1 translocates to the nucleus within 30 minutes of 5-methyl-THF treatment (0.022 μM) and interacts with CAF-1, a chromatin assembly factor. This nuclear localization of FOLR1 is required for 5-methyl-THF to dedifferentiate glial cells, suggesting a direct role in chromatin regulation and cell fate determination .

What considerations are important when designing experiments to investigate FOLR1's non-canonical functions?

When designing experiments to investigate FOLR1's non-canonical functions, several critical considerations should be addressed. First, the type of folate used is crucial—researchers must specify whether they are using reduced folates (involved in one-carbon metabolism) or folic acid (FA, a synthetic, non-natural form). FOLR1 has approximately 14-fold higher affinity for FA than for the serum folate 5-methyl-THF, while RFC transports FA 10-100 times less efficiently than reduced folates. Therefore, using FA shifts cellular responses toward FOLR1-dependent mechanisms, while reduced folates may engage both FOLR1 and RFC. Second, the concentration of folate is important—physiological concentrations versus supraphysiological doses can yield different results. Third, timing of measurements is critical—non-canonical signaling events typically occur rapidly (within 2-10 minutes), while metabolic effects may take longer. Fourth, appropriate controls should include FOLR1 knockdown/knockout and RFC manipulation to distinguish transport-dependent from signaling-dependent effects. Fifth, researchers should perform physical interaction studies (co-immunoprecipitation) to demonstrate FOLR1 association with signaling components, while recognizing that such associations may be direct or indirect via intermediary proteins. Finally, basal folate levels in experimental media must be considered, as they can affect cellular responses to added folates .

What methods are most effective for validating FOLR1-specific binding of therapeutic candidates?

For validating FOLR1-specific binding of therapeutic candidates, a multi-step workflow incorporating several complementary techniques is most effective. First, flow cytometry provides essential preliminary screening, comparing binding to FOLR1-expressing cells versus FOLR1 knockout controls. This approach allows for rapid assessment of binding specificity and affinity. Second, cross-reactivity testing against related proteins (such as FOLR2, FOLR3, FOLR4, and their murine counterparts) is crucial due to the high degree of similarity within the folate receptor family. Third, advanced imaging techniques, particularly multiplexing imaging systems using anti-FOLR1 candidates alongside established reference antibodies (like LK26), provide deeper insights into binding characteristics. Fourth, comprehensive tissue cross-reactivity testing across multiple normal tissues (adrenal gland, ovary, pancreas, thyroid, cerebellum, cerebrum, lung, spleen, uterus, cervix, breast, placenta, heart, skin, skeletal muscle, kidney, stomach, small intestine, liver, and salivary gland) and malignant tissues helps identify potential off-tumor binding. Finally, functional validation through high-throughput co-culture assays with target cells (in the case of CAR T cells) under challenging conditions (unfavorable effector-to-target ratios and repetitive target cell exposure) ensures that specificity translates to functional efficacy. This comprehensive approach provides robust validation of FOLR1-specific binding .

How can researchers effectively differentiate between FOLR1's metabolic and signaling functions in experimental settings?

Differentiating between FOLR1's metabolic and signaling functions requires carefully designed experimental approaches that can distinguish these interrelated processes. First, time-course experiments are essential—signaling events typically occur within minutes (2-10 minutes after folate stimulation), while metabolic changes generally require longer time frames. Second, using FOLR1 mutants that maintain structural integrity but lack folate-binding ability can help separate binding-dependent from structure-dependent functions. Third, comparing the effects of anti-FOLR1 antibodies that block folate binding with direct folate administration can reveal folate-independent signaling roles, as demonstrated in erythropoietic cells where anti-FOLR1 antibodies stimulated proliferation despite preventing folate binding. Fourth, simultaneous manipulation of FOLR1 and other folate transporters (particularly RFC) helps distinguish transporter-specific effects from general folate metabolism effects. If FOLR1 knockdown and RFC overexpression produce opposite effects despite both affecting folate transport, this suggests non-metabolic roles for FOLR1. Fifth, direct measurement of one-carbon metabolism outputs (nucleotide synthesis, methionine cycle metabolites) alongside signaling events can establish temporal relationships between these processes. Finally, protein-protein interaction studies through techniques like co-immunoprecipitation, proximity ligation assays, or FRET can reveal physical associations between FOLR1 and signaling components that would not be expected in a purely metabolic role .

What are the most promising therapeutic approaches targeting FOLR1 in cancer?

Several therapeutic approaches targeting FOLR1 in cancer show promising potential based on current research. CAR T-cell therapy represents one of the most advanced approaches, with researchers developing fully human FOLR1-directed CAR binding domains that target both human and murine FOLR1. This approach allows for better translation between preclinical mouse models and human applications. The effectiveness of FOLR1-targeting CAR T cells has been demonstrated in various ovarian cancer cell lines, including OV-90, OVCAR-3, and SKOV-3, with specificity validated using FOLR1 knockout controls. Another promising approach involves the development of antibody-drug conjugates (ADCs) targeting FOLR1, leveraging its high expression in certain cancers and limited expression in normal tissues. Companion diagnostics, such as the VENTANA FOLR1 Assay, have been developed to identify patients most likely to benefit from FOLR1-targeted therapies, particularly in epithelial ovarian cancer. Additionally, small molecule inhibitors and folate-conjugated drugs that exploit FOLR1's role in cellular uptake represent alternative strategies. The development of these various therapeutic modalities is supported by FOLR1's restricted normal tissue expression, high prevalence in certain cancer types, and emerging understanding of its roles in cancer progression beyond folate transport .

How does FOLR1 expression in Xenopus development inform potential developmental toxicities of FOLR1-targeted therapies?

Research on FOLR1 in Xenopus laevis (African clawed frog) development provides important insights that may inform potential developmental toxicities of FOLR1-targeted therapies. In Xenopus embryos, FOLR1 localizes to the apical surface of the neural plate and plays a crucial role in neural tube formation by facilitating the constriction required to shape the neural tube. This function appears to be independent of folate transport, as FOLR1 physically interacts with C-cadherin and β-catenin, components of adherens junctions, and is required for the endocytosis of C-cadherin from the apical surface to facilitate constriction. This non-canonical role in regulating adherens junctions during embryonic development suggests potential risks of FOLR1-targeted therapies during pregnancy. Since FOLR1 is expressed in trophoblasts in the human placenta and is involved in transporting folates from mother to fetus, FOLR1-targeted therapies could potentially interfere with both folate transport to the developing embryo and structural developmental processes mediated by FOLR1's non-canonical functions. These concerns are particularly relevant when considering the use of FOLR1-targeted therapies in pregnant women or women of childbearing potential. Preclinical developmental toxicity studies should therefore assess not only folate transport disruption but also potential interference with FOLR1's structural roles in embryonic development .

What criteria should be used when developing and validating FOLR1 companion diagnostics for patient selection?

When developing and validating FOLR1 companion diagnostics for patient selection, several critical criteria should be considered. First, analytical validation studies must establish the assay's specificity, sensitivity, reproducibility, and robustness using industry-standard protocols. The VENTANA FOLR1 Assay exemplifies this approach, undergoing rigorous analytical verification before clinical implementation. Second, appropriate controls are essential—positive controls should include tissues known to express FOLR1 (such as specific ovarian cancer subtypes), while negative controls should include FOLR1 knockout tissues or cell lines and tissues known not to express FOLR1. Third, the assay should distinguish between different levels of FOLR1 expression, not just positive versus negative status, as expression levels may correlate with therapeutic response. Fourth, clinical validation should correlate FOLR1 expression patterns with response to FOLR1-targeted therapies in clinical trials, establishing clinically meaningful cutoff values. Fifth, the companion diagnostic should be evaluated across multiple cancer types where FOLR1-targeted therapies might be applied, as expression patterns and prognostic significance vary between cancers (e.g., breast cancer versus ovarian cancer). Finally, the diagnostic should ideally detect functionally relevant FOLR1 rather than just protein presence—this may involve assessing membrane localization or specific isoforms associated with therapeutic response. These comprehensive criteria ensure that FOLR1 companion diagnostics accurately identify patients most likely to benefit from FOLR1-targeted therapies .