To identify potential unique and specific binding sites, we performed epitope mapping of the FZD7 protein. To ensure the selection of antibodies specific to the FZD7 receptor, four epitopes with minimal homology to other class frizzled (class F) G-protein-coupled receptors, consisting of 10 frizzled receptors (FZD (1–10)) and smoothened (SMO) (Schulte, 2010), were selected spanning throughout the FZD7 protein (the N-terminal region and C-terminal region), and peptides were generated accordingly (Scheme 1). Peptide 1 is a 14-amino acid (aa)-long sequence directed at a target antigen within the cysteine-rich domain (CRD) (Saldanha et al., 1998) and located at the N′ terminal region of the protein (aa76-aa89). Peptide 2, 16 aa long, also targets an antigen at the N′ terminal region (aa167-aa182). Peptide 3, 16 aa long, was directed at an antigen in close proximity to the transmembrane domain of the N′ terminal region of the protein (aa 195–aa 210). Finally, peptide 4, 16 aa long, was directed at an antigen located at the C′ terminal, intracellular region of the protein (aa559–aa574).

These peptides were injected into BALB/C mice (80 µg peptide/mouse) to generate specific antibodies according to standard protocols (Yokoyama, 2001). For each specific peptide, several antibodies were produced. Peptide matching was performed by ELISA to detect the specific target antigen of each antibody clone.

To screen for potent monoclonal antibodies (mAbs), the effects of the supernatants of the hybridoma clones were first verified by ELISA on FZD7-expressing WT cells. Primary tumor cells obtained from various primary WT donors, along with cells derived from WT xenografts in immunodeficient mice, were analyzed for FZD7 expression levels using flow cytometry. FZD7 levels were monitored in the different tumors and remained steady throughout the experiments (Figures 1A,B).

Clones were analyzed by applying medium containing the secreted antibodies from the hybridoma cell culture to the assayed cells. After a 48-h treatment course, we analyzed the control and treated cells for viability.

Overall, 4 antibody-producing hybridomas generated from different immunizing peptides (Supplementary Table S1) demonstrated significantly reduced proliferation in WT cells (Figure 1C; *p < 0.05; **p < 0.01). Surprisingly, clones 288.1, 288.2, and 288.5 generated by immunization with the intracellular peptide 4 significantly inhibited WT cell proliferation. Two of those antibodies were chosen for further analysis: 288.1 and 288.5.

Direct protein–antibody interactions were verified by a protein immunoprecipitation (P-IP) assay in SK-MEL28 cells, a human melanoma cell line that expresses an average of 15% FZD7 (Supplementary Figure S1). The results showed that P-IP of the FZD7 protein was successful when using αFZD7-288.1 but not 288.5 (Figure 2A). Since specific direct interactions were observed only for αFZD7-288.1, these interactions were further tested via functional in vitro experiments.

To test the specificity of αFZD7-288.1, we compared our generated αFZD7-288.1 antibody to a commercial anti-FZD7 Ab (Merck Millipore). Western blot (WB) analysis revealed a similar pattern for identifying the predicted 64 kDa protein, as well as its modified forms by phosphorylation, glycosylation, or both (Supplementary Figure S2). The target antigen of the selected antibody, αFZD7-288.1, was confirmed to be antigen 4 by blocking the antibody-receptor interaction following incubation with the immunizing peptide (Supplementary Figure S3).

Specificity of the combinations of the various concentrations of αFZD7-288.1 was evaluated in HEK293T cells overexpressing the full-length FZD7 (Figure 2B) and in WT cells in which FZD7 was knocked down by shRNA (Figure 2C). We found specific binding to the FZD7 receptor, as shown in 2B. Flow cytometry analysis of live WT cells stained for both αFZD7-288.1 and the αFZD7-APC antibody (R&D) revealed a range of overlapping subpopulations. Interestingly, variability was noted for both antibodies; however, cells stained positive for αFZD7-288.1 were mostly observed as a subpopulation within the cells detected by the commercial antibody (Figure 2D).

Since αFZD7-288.1 was designed to target FZD7, a receptor for Wnt signaling, we aimed to examine its effect on this pathway to determine the underlying mechanism. WT cells were treated for 48 h with 5 μg/mL αFZD7-288.1, after which the protein levels of active β-catenin were measured via protein analysis. Active β-catenin and FZD7 protein levels were decreased in the cell lysates of treated WT cells (Figures 3A,B), suggesting that the whole signaling pathway is indeed downregulated following treatment with αFZD7-288.1. To further investigate the changes in β-catenin translocation following treatment, immunofluorescence staining of treated and untreated WT cells was performed. β-catenin is a key mediator of the canonical Wnt signaling pathway. In response to a Wnt stimulus or specific gene mutations, β-catenin is stabilized and translocated from the cytoplasm to the nucleus, where it binds to TCF/LEF-1 transcription factors to transactivate genes that drive tumor formation (Jamieson et al., 2014). Immunofluorescence staining demonstrated strong nuclear localization of β-catenin and weak cytoplasmic staining in the untreated cells, whereas the cytoplasmic localization of β-catenin was mostly observed in the treated WT cells (Figure 3C). These results suggest that αFZD7-288.1 treatment leads to inhibition of the Wnt/β-catenin pathway.

To explore the mechanistic impact of αFZD7-288.1-induced inhibition of Wnt signaling, we performed gene expression analysis of canonical Wnt pathway-related genes via real-time PCR. Our results demonstrate a decrease in the mRNA expression levels of CTNNB1 (encodes β-catenin) and FZD7. Additionally, C-MYC, a known oncogene (He et al., 1998) and AXIN2, known to mediate tumor cell dedifferentiation and tissue-invasive activity (Yook et al., 2006) were also expressed at lower levels following the inhibition of Wnt signaling. Furthermore, Cyclin D1 (CCND1), a Wnt target gene that was previously reported to be linked to cell cycle arrest, were correspondingly expressed at lower levels. This fact further links Wnt inhibition to tumor proliferation arrest (Tetsu and McCormick, 1999). Finally, the Wnt pathway inhibitors DKK1 (Chamorro et al., 2005) and SFRP1 were also significantly decreased in WT cells (Figures 3D–F). As a control for specificity within the Wnt signaling pathway, αFZD7-288.5, which does not specifically interact with FZD7 (Figure 2A), was utilized. The mRNA levels of Wnt pathway-related genes remained unaffected by αFZD7-288.5 (Supplementary Figure S5).

We further examined whether Wnt inhibition affects WT cell viability. Since the Wnt pathway is known to regulate cell proliferation and survival in various cancer types (Bilir et al., 2013; Sinnberg et al., 2011; King et al., 2012) and because of the highly proliferative nature of FZD7-expressing cells (Pode-Shakked et al., 2011), we first tested whether Wnt pathway inhibition via αFZD7-288.1 could lead to changes in cell proliferation. To evaluate proliferation, we calculated the cell growth rate (defined as the number of cell divisions per time unit). Doubling time was determined using a verified online calculator (R.V., 2006) based on measurements of cell numbers at 4 different time points (Figure 4A). Compared with untreated cells, αFZD7-288.1-treated WT cells had a significantly longer doubling time (approximately 49 h), which typically demonstrated a doubling time of 28 h and the significantly lower growth rate (defined as the number of cell divisions per day (Figure 4B). Reduced proliferation in cells treated with αFZD7-288.1 was also observed by MTS assay as shown in (Figure 4C). Further analysis of WT cell proliferation by immunofluorescence staining for Ki67 revealed reduced proliferation in treated cells, with only 13% Ki67-positive cells compared to 29% in control-treated cells (Figure 4D), consistent with their relatively decreased growth rate. Since Ab-mediated blockade of FZD7 was shown to induce cell death, we next examined whether treatment with our novel antibody, αFZD7-288.1, could result in cancer cell death. Flow cytometry analysis via annexin V staining of cancer cells treated with 5 μg/mL αFZD7-288.1 increased the percentage of preapoptotic and apoptotic cells (Figure 4E). Accordingly, using the trypan blue cell viability assay, we found that the percentage of cell death was significantly greater in the WT cells than in the controls (Figure 4F).

A common property of most cancers is their ability to migrate (Wirtz et al., 2011). Therefore, we assessed whether canonical Wnt pathway inhibition affects the migration potential of treated cells. We tested changes in the ability of WT cells to migrate through a scrape made in the cell monolayer in the serum-free medium to minimize proliferation and isolate migration effects. Inhibition of canonical Wnt signaling resulted in a reduced migratory capacity of the αFZD7-288.1-treated cells compared to the untreated cells (Figure 5A). An additional defining property of cancer stem cells (CSCs) is their ability to form cell spheres (Wang et al., 2018). Since FZD7 was previously shown to be a potential cancer stem cell marker in WT plants, we next asked whether treatment with αFZD7-288.1 affects this function. A sphere formation assay showed that a significantly lower number of spheres formed from treated cells than from control cells. Furthermore, the spheres that formed from the treated cells were smaller and less condensed (Figure 5B).

To determine the cellular compartment to which αFZD7-288.1 localizes, immunofluorescence (IF) staining was performed with antibodies against FZD7 and NCAM1, a WT CSC membrane-associated marker (Shukrun et al., 2014a). Cells were co-stained with fluorophore-conjugated antibodies against FZD7 (red) and NCAM1 (green), and merged images showed prominent yellow signals, indicating spatial membrane proximity (Figure 6A). Further, to evaluate the functional impact of FZD7 targeting, WT cells were treated with αFZD7-288.1 for 48 h. In line with previous reports identifying ALDH1⁺ and NCAM1⁺ cells as tumor-propagating populations with Wnt/β-catenin pathway activity and poor prognosis (Shukrun et al., 2014b; Raved et al., 2019), αFZD7-288.1 treatment led to significant downregulation of key CSC markers, including NCAM1, SIX2, CITED1, and ALDH1, as measured by qRT-PCR (Figure 6B). Notably, these markers overlap with populations resembling fetal nephron progenitors (SIX2⁺CITED1⁺) known to promote WT progression via ITGβ1–FAK–ERK signaling (Petrosyan et al., 2023). Together, these findings indicate that αFZD7-288.1 disrupts the stem-like compartment in WT, supporting a role for FZD7 in sustaining CSC properties. This underscores the embryonic plasticity of WT CSCs and highlights FZD7 as a potential therapeutic target to overcome resistance and limit tumor propagation.

Having shown the effect of αFZD7-288.1 on primary WT cells, specifically on CSC population, we explored whether αFZD7-288.1 has the potential to affect canonical Wnt signaling in other tumor cells. To achieve this goal, the melanoma SK-MEL28 and cervical cancer HeLa cell lines, previously linked to Wnt pathway aberrations (Deng et al., 2015a; Deng et al., 2015b; Tiwary and Xu, 2016; Spr et al., 2015; Anastas et al., 2014), were evaluated for FZD7 expression and treated with αFZD7-288.1. Treatment with αFZD7-288.1 inhibited the canonical Wnt pathway in both cell lines (Supplementary Figures S6, S7, respectively). These results support our hypothesis that αFZD7-288.1 may be a promising therapeutic option for a broad range of FZD7 canonical Wnt pathway-related tumors.

Next, we sought to determine the effect of αFZD7-288.1 on WT growth via a xenograft model. In our previous study and in several other studies, treatment with the tubulin inhibitor paclitaxal (PTX) induced dramatic and favorable responses toward WT growth. Therefore, we compared the antitumor effect of αFZD7-288.1 to that of PTX (Markovsky et al., 2017; Ramanathan et al., 2000).

The tumor injection protocol, tumor growth (volume) curves and body weight changes of each group are shown in Figure 7. Treatment with 10 mg/kg αFZD7-288.1 significantly inhibited tumor growth throughout the experiment starting after the second injection (p < 0.01). (Figure 7D). Three of the treated tumors were found to have a reduced volume compared to the initial tumor size (further in the text referred to as group A), while the other two tumors continued to grow but more slowly than did the control mice (further in the text referred to as group B) (Figures 7E,F). Like in the αFZD7-288.1-treated group, in the PTX-treated group, tumor growth was substantially inhibited after the second dose, followed by a decrease in tumor volume. Although PTX had a stronger and more rapid effect than did αFZD7-288.1 on all the tumors (n = 3; Figure 7F), the treated mice experienced weight loss, suggesting strong toxicity, which was not detected with the FZD antibody. This body weight loss of more than 20% following the fifth injection compelled us to stop the experiment.

Treatment of tumors larger than 500 mm³ with αFZD7-288.1 had similar results, with a delayed effect. A decrease in tumor volume was recorded in the treated tumors after the fifth injection (Supplementary Figure S8A). As mentioned above, since significant weight loss and death of three mice was recorded within the PTX-treated group, the experiment was terminated 10 days after the first inoculation (Supplementary Figure S8B).

Flow cytometry analysis of the tumors processed from the single-cell cohort demonstrated a significant decrease (fold change of 0.445) in the percentage of FZD7-expressing cells in the αFZD7-288.1-treated tumors compared to that in the control group (Figure 7G) and Supplementary Figure S8C. The expression of the canonical Wnt pathway genes of FZD7, β-CATENIN, AXIN2, CCND1, C-MYC, DKK1, and sFRP1 decreased in the treated tumors as measured by qRT-PCR (Figure 7H).

To further evaluate changes within the tumors, we performed immunohistological analysis of the tumors treated with saline, PTX and αFZD7-288.1. Two separate tumors representing tumors with reduced volumes (type A) and tumors with growth inhibition (type B) from the αFZD7-288.1-treated group were stained. Blocking Wnt signaling activity with αFZD7-288.1 resulted in reduced cell proliferation, as indicated by Ki67 staining, and induced cell death, as shown by extensive Caspase3 staining. Type A tumors exhibited minimal proliferation according to Ki67 staining and extensive Caspase3 staining, demonstrating increased apoptosis. These results coincide with the reduction in tumor volume observed during the in vivo experiment. Type B IHC staining revealed lower proliferation levels than did the control groups, and type B IHC staining was accompanied by lower apoptosis than was observed in type A tumors. This can account for the reduced response to αFZD7-288.1 treatment, as indicated by the inhibition of tumor growth. As expected, a similar effect was observed for the chemotherapy-treated tumors (Figures 8A,B).

Monoclonal antibodies are raised against peptides corresponding to sequences specific to FZD7. Peptides used for antibody generation were synthesized by Sigma-Aldrich using solid-phase peptide synthesis (SPPS) with standard Fmoc (9-fluorenylmethoxycarbonyl) chemistry. Synthesis was performed on a resin support with stepwise addition of amino acids from the C-terminus to the N-terminus. Following chain assembly, peptides were cleaved from the resin and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) to a purity of >80%. Peptide identity and purity were verified by analytical HPLC and mass spectrometry (MS). For immunization, the peptides were conjugated to KLH and injected into BALB/C mice (RRID:IMSR_ENV:HSD-162) (80 µg peptide/mouse) to generate specific antibodies according to standard protocols (Yokoyama, 2001). Upon clone selection, the clones were stabilized, produced in a bioreactor, and purified on a protein A-agarose column using AktaPrime (Pharmacia).

The primary WT sample was obtained from the patient within 1 h of surgery. Informed consent was given by the legal guardians of the patients according to the Declaration of Helsinki. The tumors were designated W002-W041, 12 of which were used in this study (Supplementary Table S3).

Animal experiments were performed in adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with institutional Animal Care and Use Committee approval. In this work, 5- to 8-week-old female nonobese diabetic severe combined immunodeficient NOD/SCID (RRID:IMSR_HAR:170) mice were used. WT xenografts (WT-Xn) were generated from immunodeficient mice as previously described (Pode-Shakked et al., 2013; Dekel et al., 2006).

1. Cell lines: All cell lines were obtained from ATCC and tested negative for mycoplasma contamination.SK-MEL28 cells (RRID:CVCL_0526) were cultured in EMEM (Sigma‒Aldrich), and HEK293 (BCRJ Cat# 0009, RRID:CVCL_0045) and HeLa (TKG Cat# TKG 0331, RRID:CVCL_0030) cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Biological Industries). All media were supplemented with 10% fetal bovine serum (FBS; Invitrogen), 1% penicillin streptomycin (Pen-strep) 100 M, and 1% L-glutamine (both from Biological Industries).

1. For preliminary experiments, cells were cultured on 96-well plates at 4,000 cells/well. Twenty-four hours after seeding, the cells were treated for an additional 48 h with spent medium from the different hybridoma clones containing the secreted antibodies.

10⁵ cells were suspended in FACS buffer supplemented with 0.5% BSA (Sigma‒Aldrich) and 2 mM EDTA in Dulbecco’s phosphate-buffered saline (PBS) (both from Biological Industries). The cells were then blocked with a mixture of FcR blocking reagent (Miltenyi Biotec) and human serum (1:1). The cells were stained with an isotype control (R and D Systems Cat# IC006A, RRID:AB_357254) or a primary antibody against FZD7 (R and D Systems Cat# FAB1981A, RRID:AB_2232278). Cell viability was tested using 7AAD viability staining solution (eBioscience). Cell labeling was detected using a FACSCalibur (RRID:SCR_000401). FACS results were analyzed using FlowJo (RRID:SCR_008520) analysis software. Viable cells were defined by their forward scatter/side scatter (FSC/SSC) profiles and lack of 7AAD staining. For analysis of p-WT and WT-Xn, freshly removed tumors were dissociated into single-cell suspensions as previously described and immediately analyzed. Cultured cells were detached using nonenzymatic cell dissociation solution (Sigma‒Aldrich) before staining.

1. Protein isolation Cultured cells were treated as described above. Total protein was extracted using lysis buffer (1% Triton-X100, 20 mM Tris-HCl, 120 mM NaCl) and a tablet of protease inhibitor (Complete Mini Protease Inhibitor Cocktail; Roche). The mixture was incubated for 1 h on ice and centrifuged at 16,100 × g for 20 min.

A sequence verification Sigma Mission viral vector (pLKO.1-puro)-based short hairpin RNA library (five clones targeting different sequences in the coding regions of the FZD7 gene, denoted as sh8343, sh8344, sh8345, sh8346, and sh8347; Supplementary Table S5) against FZD7 (FZD7-shRNA) was purchased from Sigma‒Aldrich. For transfections, the calcium phosphate protocol was used on HEK293 (BCRJ Cat# 0009, RRID:CVCL_0045) cells. 1 × 10⁶ cells were co-transfected with 10 µg of shRNA plasmid DNA vector, 5 µg of the packaging vector psPAX2 (RRID:Addgene_12260), and 2.5 µg of the envelope vector pMD2.G (RRID:Addgene_12259). The transfection efficiency of this procedure was determined by qPCR and reached 60%–70% at 72 h. Viral supernatants were collected 72 h post-transfection, filtered through a 0.45 μm PES filter, and concentrated by ultracentrifugation at 25,000 rpm for 2 h at 4 °C or using PEG-it Virus Precipitation Solution (System Biosciences), according to the manufacturer’s instructions. Viral pellets were resuspended in PBS and stored at −80 °C until use. Lentiviral titers were determined by qPCR-based titration (Lenti-X qRT-PCR Titration Kit, Takara Bio) following the manufacturer’s protocol, and the multiplicity of infection (MOI) was calculated based on these titers. For infections, target cells were seeded at ∼50% confluence and infected in the presence of 8 μg/mL polybrene (Sigma-Aldrich). Infections were performed at an MOI of 5. Transduced cells were selected with 2 μg/mL puromycin for 5 days starting 48 h post-infection. As a negative control, cells were infected with a MISSION non-targeting pLKO.1-puro lentiviral control vector (RRID:Addgene_8453).

Total RNA was isolated using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. cDNA was synthesized using a High Capacity cDNA reverse transcription kit (Applied Biosystems, ABI). qPCR was performed using the StepOnePlus Real-Time PCR System (ABI) and the specific TaqMan Gene Expression assays for the relevant genes in the presence of TaqMan Fast Universal PCR Master Mix (both from ABI). hHPRT1 and hGAPDH were used as endogenous controls (Supplementary Table S6). The results were analyzed using StepOnePlus Real-time Software in the ΔΔCT method, which determines the fold change in gene expression relative to a comparator sample. Statistical analysis was performed using a non‐paired two‐tailed t‐test. A p value < 0.05 was considered to indicate statistical significance. (Livak and Schmittgen, 2001; Schmittgen and Livak, 2008).

The samples were formalin-fixed and paraffin-embedded. Sections were pretreated using OmniPrep solution (Zytomed Systems) and blocked using Cas‐Block solution (Invitrogen) according to the manufacturer’s protocol. After 1 h of incubation at room temperature (RT), the sections were incubated with primary antibodies (Supplementary Table S7). The samples were then washed and incubated with secondary antibodies.

1. For immunohistochemistry (IHC), sections were incubated with HRP-conjugated goat anti-mouse (Vector Laboratories Cat# MP-7452, RRID:AB_2744550) and goat anti-rabbit (Vector Laboratories Cat# MP-7451, RRID:AB_2631198) antibodies for 30 min at RT. Staining was detected using an ImmPACT DAB kit (Vector) according to the manufacturer’s protocol. Hematoxylin was used for counterstaining.

The results are expressed as the mean values ± SEMs, unless otherwise indicated. Significant differences in xenograft size, cell viability (MTS assay), gene expression (qPCR) and surface marker expression (flow cytometry) were evaluated using a non‐paired two‐tailed t‐test. In vivo experiment was a pilot study; a formal power calculation was not applied. Statistical differences in the in vivo experiments were determined using Student’s t-test in Excel software assuming unequal variances. For all the statistical analyses, the level of significance was set as *p < 0.05 and **p < 0.01.

The anti-Frizzled 7 288.1 antibody shows promise by inducing cell death, inhibiting proliferation, and suppressing Wnt signaling, demonstrating efficacy in Wilms tumor xenograft studies.