Objectives: Despite significant advancements in targeted treatments, including recent FDA approval of Pluvicto® ([¹⁷⁷Lu]-Lu-PSMA-617), a PSMA-RPT (Prostate Specific Membrane Antigen – Radiopharmaceutical Therapy), metastatic castration-resistant prostate cancer (mCRPC) remains incurable. While patients initially respond to treatment, the response is not durable and PSMA expression appears to decrease in recurrent tumors [1]. The efficacy of PSMA-RPTs on tumors with heterogeneous PSMA expression is unclear, and initial studies show heterogeneity may contribute to treatment resistance and recurrence [2][3]. Despite its importance in treatment, the impact of heterogeneous PSMA expression on treatment outcomes remains largely unexplored. To address this gap, we are developing clinically relevant disease models for monitoring PSMA heterogeneity at the cellular level and understanding its effects on treatment outcomes with PSMA-RPTs.

Methods: To achieve this objective, we selected the castration/Enzalutamide-resistant human cell line CWR22RV1 (22RV1) with relatively low PSMA expression. Cell subpopulations were sorted based on PSMA expression levels (high and low) and maintained separately, with stable PSMA expression levels confirmed through multiple passages.  Flow cytometry, immunoblotting, and radioligand binding assays were employed to validate PSMA expression. Cell doubling rate and radioligand uptake over time was established. Tumors comprising mixed ratios of high and low PSMA-expressing cells were established, and tumor growth dynamics were assessed using calipers and bioluminescence. PET imaging with [⁶⁸Ga]-Ga-PSMA-11 in tumor-bearing mice was utilized to quantify relative PSMA levels, with expression further confirmed by immunohistochemical (IHC) staining. Additionally, hypoxia status of tumors was confirmed through IHC staining for HIF-1α and CD31 markers.

Results: Subpopulations of 22RV1 cells with high and low PSMA levels maintained stable expression levels over numerous passages. Radioligand binding indicated 250 times higher PSMA expression in PSMA-high cells compared to PSMA-low cells, with total uptake stabilized at 4 hours after radioligand application. Tumor growth rates were 2.5 times higher in PSMA-high tumors compared to PSMA-low. [⁶⁸Ga]-Ga-PSMA-11 PET imaging in mice bearing PSMA high, heterogeneous (1:1 high:low), and low expressing tumors revealed uptake levels of 4.9%, 1.9% and 0.3% ID/g respectively. This PSMA expression in tumors was confirmed by IHC. Hypoxia status, as indicated by CD31 and HIF-1α IHC staining, was similar amongst PSMA-low, heterogeneous, and high expressing tumors.

Conclusions: We have developed a novel preclinical model with heterogeneous PSMA expression. Our model has high clinical relevance and provides a robust platform to study the influence of tumor heterogeneity on the outcomes of PSMA-RPT treatment.

Acknowledgments: Partial funding provided by NIH/NCI grant R01 CA262675 (NP), R35CA232130 (JSL), P30 CA008748, and DoD PCRP grant W81XWH-19-1-0536 is acknowledged.

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Image/Figure Caption:

PSMA expression of a heterogeneous model is traceable in vivo and ex vivo. A. NSG mice were subcutaneously shoulder xenografted with 5×10⁶ mixed 50% PSMA-low and 50% PSMA-high cells (50/50, n=3). An additional control cohort was given bilateral shoulder xenografts of 100% PSMA-high and 100% PSMA-low cells (H, L, n=3). B. Tumor growth measurements, measured with calipers twice weekly, showed enhanced tumor growth with increasing PSMA content. C. [⁶⁸Ga]Ga-PSMA-11 PET imaging was performed 25 days post-xenograft. 50/50 cohort had ~40% PSMA expression relative to PSMA-high (see quantification in E). D. PSMA immunohistochemistry at 28 days post-xenograft confirmed 50/50 cohort had intermediate PSMA expression compared to high and low controls (see quantification in F). E/F. PSMA heterogeneous tumors have statistically significant, differentiable PSMA expression compared to high and low homogeneous controls.

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