Abstract B027: Molecular imaging on Claudin-4 expression in ovarian cancer models using targeted iron oxide nanoprobes

Abstract Introduction: There is an unmet clinical need for screening of early-stage asymptomatic ovarian cancer (OvCa), with no reliable circulating biomarkers and low sensitivity of transvaginal ultrasound for small serous tubal intraepithelial carcinoma (STIC) lesions. Claudin-4 (CLDN4) is expressed in 80% of all human OvCa and upregulated 3.2-fold in STIC lesions compared to normal fallopian tube (p<0.0001). Our team has developed highly innovative antibody-iron oxide nanoworms (CLIO NW) with high affinity to the CLDN4 protein. Here, we report on the use of quantitative magnetic resonance imaging (MRI) for the non-invasive detection of CLDN4+ OvCa cells in vitro and in vivo. Experimental Procedures: A panel of OvCa cells (OVCAR8, OVCAR3) with different levels of CLDN4 expression (confirmed by immunoblots) were incubated with targeted and non-targeted CLIO NW (2 mg/L iron) for 1 hour. Quantitative T2-weighted MRI maps (qT2-MRI) with 16 echo times were acquired on cell pellets to assess iron oxide accumulation as a function of decreased T2 relaxation times (T2-rt). Next, flank xenograft models were introduced using female nu/nu mice (n=5) bearing OVCAR8 CLDN4+ (right tumor, RT) and OVCAR8 CLDN4- (left tumor, LT) tumors (each mouse served as its own negative control). Volumetric MRI and qT2-MRI were obtained prior (baseline, BL) and 24, 48, 72 hrs after IV injection of 5 mg/kg CLIO NWs. The harvested tumors were analyzed by histopathology and immunofluorescence to correlate the NW uptake with in vivo changes in T2-rt. Results: In vitro results demonstrate a highly efficient 98% uptake of CLIO NW into CLDN4+ positive cells after 1 hr of incubation. This uptake correlated with a drastic decrease of T2-rt on qT2-MRI on cell pellets, but only in CLDN4+ OVCAR8 cells (26.6+/-1.3ms in CLDN4+ versus 178+/-16ms in CLDN4- cells). Next, we have established a unique mouse model bearing both CLDN4+ (RT) and CLDN4- (LT) flank xenografts. The MRI experiments were performed when the subcutaneous tumors reached at least 30 mm3 (as assessed by structural MRI). The average CLDN4+ tumor volumes were significantly higher than CLDN4- tumors 25 days after inoculation (RT-to-LT volume rations 1.8+/-1.4). There were no differences in the T2-rt for RT vs LT prior to CLIO NW injection at the BL. A greater decrease in T2-rt was observed in CLDN4+ RT tumors 24 hrs (-13% of BL), 48 hrs (-12%) and 72 hrs (-10%) when compared to CLDN4- LT (-6%, p<0.023) in the same animal. The ex vivo tissue analysis showed enhanced uptake of CLIO NW by cytokeratin+ tumor cells and CD45+ immune cells, consistent with the decreased T2-rt from the MRI analysis. Summary: Non-targeted iron oxide nanoparticles (such as Ferumoxytol) are already in animal and human use as T2 MRI contrast agents in vascular and hepatic MRI applications since they drastically decrease T2-rt, producing a darkening in T2-MRI. Here, we successfully introduced a smart molecular iron oxide nanoprobe (CLIO NW) for targeted molecular imaging of CLDN4 expression as an attempt for early detection of OvCa by advanced MRI screening. Citation Format: Natalie J. Serkova, Leah Roseberry, Jenna Steiner, Vadym Zaberezhnyy, Hanmant Gaikwad, Aislinn Warmka, Tomomi Yamamoto, Elizabeth Woodruff, Kian Behbakht, Benjamin G. Bitler, Dmitri Simberg. Molecular imaging on Claudin-4 expression in ovarian cancer models using targeted iron oxide nanoprobes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr B027.