Abstract

Despite advancements in breast cancer molecular targets, triple negative breast cancer (TNBC) treatment options remain limited. Fibroblast activation protein (FAP) is a transmembrane serine protease highly expressed in cancer associated fibroblasts of many cancer types, including TNBC, while having low expression in normal tissue. This makes FAP a potentially effective molecular target for diagnosis or therapy in TNBC. Several radiolabeled FAP inhibitors (FAPi) have recently been developed for PET imaging. However, tumor uptake, retention, and binding stability need further improvement. Our team has been developing a new FAP-targeting theragnostic probe, where the FAPi is radiolabeled with ⁶⁴Cu (for imaging) or ⁶⁷Cu (for therapy) using a sarcophagine cage chelator for better tumor retention and binding stability.  This study is aimed to characterize the uptake and retention of the new FAPi probe in animal models of TNBC.  Three TNBC models were examined, including 4T1 mouse breast cancer, MDA-MB-231 human TNBC, and MDA-MB-436 human TNBC.  Tumors were prepared either by mammary fat pad inoculation (4T1 and MDA-MB-231) or subcutaneous inoculation (MDA-MB-436). The ⁶⁴Cu labeled FAPi probe (⁶⁴Cu-Sar-FAPi) was synthesized by combining ⁶⁴Cu-acetate with the sarcophagine cage conjugated FAPi precursor and incubating for 90 min, and further purified using HPLC.  The resulting ⁶⁴Cu-Sar-FAPi had a 99.1% radiopurity and 85% radiochemical yield. Dynamic PET/CT imaging with ⁶⁴Cu-Sar-FAPi was conducted up to 48h post-injection (P.I.) in TNBC animal models. PET/CT images were analyzed using PMOD software. Standardized uptake values (SUV) in the tumor and various organs were quantified to determine biodistribution and tumor uptake at different time points. In a subgroup of animals, tumors were collected at 24 h P.I., and cryo-sectioned for autoradiography and FAP immunostaining. In a subgroup of 4T1 tumor mice, ⁶⁸Ga labeled FAPi (⁶⁸Ga-NOTA-FAPi) was administered into tumor-bearing mice. Tumor uptake and retention of ⁶⁸Ga-NOTA-FAPi were measured and compared with the performance of ⁶⁴Cu-Sar-FAPi.

Dynamic PET imaging demonstrated a rapid tumor uptake peaking around 15 min after bolus injection and slowly reduced over time.  The tumor uptake of ⁶⁴Cu-sar-FAPi was significantly high in all three TNBC models compared to surrounding tissue. The average tumor/muscle (T/M) ratio at 1h P.I. for the 4T1, MDA-MB-436, and MDA-MB-231 models was 3.13 ± 0.63, 2.25 ± 0.96 and 1.92 ± 0.39, respectively (N = 6 for each model). Compared to uptake at 1h, tumor retention was (79 ± 8.0)% at 3h, (27 ± 3.5)% at 24h, and (24 ± 2.8)% at 48h P.I. in the 4T1 model, (76 ± 6.0)% at 3h, (25 ± 7.8)% at 24h, and (20 ± 0.5)% at 48h P.I. in the MDA-MB-436 model, and (83 ± 9.8)% at 3h and (27 ± 7.6)% at 24h P.I. in the MDA-MB-231 model. In comparison, the uptake level of ⁶⁸Ga-NOTA-FAPi in 4T1 tumor was significantly lower compared to that of ⁶⁴Cu-sar-FAPi at 1hr (T/M ratio of 1.12 vs. 3.13), and tumor retention at 3h was also lower than that of ⁶⁴Cu probe (62% vs. 76%). Biodistribution studies showed that the kidneys were the major clearance organ. Liver uptake in normal mice was low with an average SUV of 0.60, 0.57, and 0.44 at 1h, 3h, and 24h, respectively, suggesting a promising radiotherapeutic agent. Comparison between ⁶⁴Cu autoradiographic images and the FAP immunostaining on the adjacent tumor sections showed high spatial correlation in probe distribution and FAP expression, confirming the high specificity of FAP targeting for the new probe.  Our results demonstrate that ⁶⁴Cu-Sar-FAPi is a promising theragnostic probe for TNBC with high tumor uptake and improved retention. Results from these imaging studies warrant a future therapeutic approach using ⁶⁷Cu-Sar-FAPi for TNBC treatment.

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Image/Figure Caption:

Figure-1. ⁶⁴Cu-Sar-FAPi PET/CT imaging in animal models of triple negative breast cancer (TNBC). High uptake of ⁶⁴Cu-Sar-FAPi in tumor (yellow arrow) is shown in three TNBC models: 4T1 mouse breast cancer, MDA-MB-436 human TNBC, and MDA-MB-231 human TNBC. Whole body biodistribution demonstrates high renal clearance and low liver uptake. Tumor retention at 3 hours post injection was at 79%, 76% and 83% for the 4T1, MDA-MB-436, and MDA-MB-231 tumors, respectively.     

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