Background: Integrins are cell adhesion receptors consisting of 18α and 8β subunits. A subset of integrins (αvβ3) recognizes Arg-Gly-Asp (RGD) peptide motifs which are involved in the neovascularization and progression of various cancers. The aim of the study was to investigate if αvβ3 can serve as a molecular target for the treatment of thyroid cancer (TC) with a novel radiolabeled RGD analog 177Lu-EB-RGD. Methods: Integrin αvβ3 mRNA and/or protein expression was evaluated in 496 TC included in The Cancer Genome Atlas, tissue microarray including 70 TC and 10 normal thyroid samples, and 14 TC cell lines. BRAF-like or RAS-like expression profile was determined through standard BRS scores ranging from -1 to 0 for BRAF-like and 0 to 1 for RAS-like TC. The association between BRS and the αvβ3 expression was tested by the Spearman correlation (r). Nude mice xenografts developing αvβ3 expressing TC after subcutaneous injection of 5*10^6 TC cells were subjected to monotherapy with 0.5 mCi 177Lu-EB-RGD (177Lu) or in combination with Lenvatinib (177Lu+L). The therapeutic efficacy of 177Lu-EB-RGD was compared with standard-of-care Lenvatinib alone (L) and placebo (P). The continuous data were presented as medians with [25-75% interquartile ranges] and compared using Kruskal-Wallis test. The mixed-effects models were used for longitudinal data analysis with adjusted p≤ 0.05 considered statistically significant. Results: We found a moderate negative correlation between BRS and αv (r=-0.5, p<0.001), and β3 (r=-0.27, p<0.001), revealing that BRAF-like tumors have a higher mRNA expression of αvβ3 integrins. Consistently, the highest αvβ3 mRNA and/or protein expression was found in the BRAF-like TC cell lines OCUT2 (BRS=-0.56), TPC1 (BRS=-0.4), K1 (BRS=-0.29), and Hurthle TC cell line XTC1 (BRS=-0.46). The immunostaining revealed a higher αvβ3 expression in papillary TC compared with follicular TC (p=0.002), and in normal thyroid (p<0.001). Poorly differentiated TC had a similar αvβ3 expression to papillary TC (p=0.14). In the thyroid cancer xenograft mouse model, all treatment modalities were more effective than the placebo in decreasing tumor size as early as 5 days after therapy initiation. A significant difference in growth curve and tumor volume was maintained at the study endpoint (L 0.584 cm3 [0.196-0.984] vs P 0.911 cm3 [0.183-1.68], p=0.001; 177Lu 0.259 cm3 [0.103-0.376] vs P, p<0.001; and 177Lu+L 0.274 cm3 [0.108-0.406] vs P, p<0.001). The combination therapy (177Lu+L) resulted in decreased tumor volume as compared with monotherapy with Lenvatinib (p=0.05) but had a similar effect as compared with 177Lu-EB-RGD monotherapy (p=0.99). Conclusions: The radiolabeled αvβ3 analog 177Lu-EB-RGD has potent growth inhibitory effects in TC characterized by a high integrins expression. The αvβ3 integrin could potentially serve as a molecular target for therapy with radiolabeled RGD analogs in TC. Citation Format: Sonam Kumari, Zhantong Wang, Shilpa Thakur, Laura Abaandou, Oksana Gavrilova, Huiyan Lu, Lixin Lang, Dale Kiesewetter, Vasyl Vasko, Joanna Klubo-Gwiezdzinska. Radiolabeled αvβ3 analog 177Lu-EB-RGD is an effective therapeutic agent in thyroid cancer xenograft mouse model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3946.

Supplemental figure 4: VAC treatment did not improve the uptake of SST analogs in the tumors of the mice models. (A) Representative PET images comparing the uptake of 86Y-DOTA-EB-TATE between the control and VAC treated FTC133 subcutaneous mice model. The bar graph shows no significant difference in the uptake of 68Ga-DOTA-TATE, 68Ga-DOTA-JR11 and 86Y-DOTA-EB-TATE between the control (n=5) and VAC (n=5) treated mice. The representative immunohistochemistry images show an increased cytoplasmic expression of SSTR2 in the tumors of the VAC treated mice in comparison to the tumors of the control mice (Supplemental Table 2). (B) Representative PET images comparing the uptake of 86Y-DOTA-EB-TATE between the control and VAC treated FTC133 metastatic mice model. The bar graph shows no significant difference in the uptake of 68Ga-DOTA-TATE, 68Ga-DOTA-JR11 and 86Y-DOTA-EB-TATE between the control (n=8-12) and VAC (n=8-12) treated mice. The immunohistochemistry images show no difference in the expression of SSTR2 in the metastatic tumors of the control and VAC treated FTC133 mice (Supplemental Table 2). (C) Representative PET images comparing the uptake of 86Y-DOTA-EB-TATE between the control and VAC treated TT subcutaneous mice model. The bar graph shows no significant difference in the uptake of 68Ga-DOTA-TATE, 68Ga-DOTA-JR11 and 86Y-DOTA-EB-TATE between the control (n=5) and VAC (n=5) treated mice. The representative immunohistochemistry images show no difference in the expression of SSTR2 within the tumors of the control and VAC treated mice (Supplemental Table 2). (D) Representative PET images comparing the uptake of 86Y-DOTA-EB-TATE between the control and VAC treated AR42J subcutaneous mice model. The bar graph shows no significant difference in the uptake of 68Ga-DOTA-TATE, 68Ga-DOTA-JR11 and 86Y-DOTA-EB-TATE between the control (n=4) and VAC (n=5) treated mice. The representative immunohistochemistry images show an increased expression of SSTR2 in the tumors of the VAC treated mice in comparison to the tumors of the control mice (Supplemental Table 2). Tumors (Tu) and metastasis (Met) are indicated by white arrows. The SUV scales range from 0 to 3 for 68Ga-DOTA-TATE and 68Ga-DOTA-JR11 and 0 to 15 for 86Y-DOTA-EB-TATE. Data are presented as mean{plus minus}SD.

Supplemental figure 1: SSTR2 is overexpressed in thyroid cancer tissues in comparison to normal thyroid tissues. (A) SSTR2 expression in different stages of PTC. (B) SSTR2 expression in histological subtypes of PTC. (C) Comparison of SSTR2 mRNA expression in different TC cell lines. (D) TT cells had significantly higher expression than BCPAP and FTC133 cells at the mRNA level. *p<0.05, ***p<0.001.

Abstract Purpose: The goal of this study was to analyze the role of somatostatin receptor type 2 (SSTR2) as a molecular target for the imaging and treatment of thyroid cancer through analysis of SSTR2 expression and its epigenetic modulation and testing tumor uptake of different radiolabeled SSTR2 analogues. Experimental Design: We analyzed SSTR2 expression by immunostaining of 92 thyroid cancer tissue samples and quantified standard uptake values (SUVmax) of SSTR2 analogue, 68Ga-DOTA-TATE, by PET/CT imaging in 25 patients with metastatic thyroid cancer. We utilized human thyroid cancer cell lines characterized by differential SSTR2 expression (TT, BCPAP, and FTC133) and rat pancreatic cell line (AR42J) with intrinsically high SSTR2 expression for functional in vitro studies. SSTR2-high (AR42J) and SSTR2-low (FTC133) xenograft mouse models were used to test the uptake of radiolabeled SSTR2 analogues and their therapeutic efficacy in vivo. Results: Thyroid cancer had a higher SSTR2 expression than normal thyroid. Hurthle cell thyroid cancer was characterized by the highest 68Ga-DOTA-TATE uptake [median SUVmax, 16.5 (7.9–29)] than other types of thyroid cancers. In vivo studies demonstrated that radiolabeled DOTA-EB-TATE is characterized by significantly higher tumor uptake than DOTA-TATE (P < 0.001) and DOTA-JR11 (P < 0.001). Treatment with 177Lu-DOTA-EB-TATE extended survival and reduced tumor size in a mouse model characterized by high somatostatin (SST) analogues uptake (SUVmax, 15.16 ± 4.34), but had no effects in a model with low SST analogues uptake (SUVmax, 4.8 ± 0.27). Conclusions: A novel SST analogue, 177Lu-DOTA-EB-TATE, has the potential to be translated from bench to bedside for the targeted therapy of patients characterized by high uptake of SST analogues in metastatic lesions.

Background: Differentiated thyroid cancer (DTC) and medullary thyroid cancer (MTC) are characterized by the overexpression of somatostatin receptor type 2 (SSTR2). There are limited data on the use of SSTR2 as a molecular target for imaging and treatment of DTC and MTC. The goal of our study was to compare the diagnostic accuracy of three different radiolabeled SSTR2 analogs in DTC and MTC mouse models: JR11, an antagonist; DOTATATE, an agonist; and EB-TATE, a novel modified agonist characterized by a longer half-life in the blood. Methods: SSTR2 expression was analyzed in two DTC cell lines (BCPAP and FTC133) and one MTC cell line (TT) by RT-PCR and immunoblotting. A rat pancreatic cell line (AR42J), characterized by intrinsically high SSTR2 expression, was used as a positive control. A metastatic (MT) mouse model was generated using immunocompromised mice which develop lung and liver metastases after tail vein injection of FTC133 and BCPAP cells. Subcutaneous (SC) xenograft mice models were generated by injecting FTC133, BCPAP, TT, and AR42J cells. The fast-growing FTC133 and AR42J cells formed soft, sizable tumors in 2 weeks, while BCPAP and TT cells formed very solid tumors after 4 and 8 weeks, respectively. Maximum standard uptake value of 68Ga-DOTATATE, 86Y-EB-TATE, and 68Ga-DOTA-JR11 (IPSEN®) was compared in each mouse in MT and SC models by PET/CT. Results: Among thyroid cancer cell lines, TT cells were characterized by the highest SSTR2 expression at the mRNA level compared with BCPAP (p<0.001) and FTC133 (p<0.001). At the protein level, BCPAP and TT cells were characterized by similar SSTR2 expression and FTC133 cells had the lowest expression. Among examined radiolabeled SSTR2 analogs, 86Y-EB-TATE showed the highest tumor uptake in all MT and SC mouse models in comparison to 68Ga-DOTATATE (p< 0.001) and 68Ga-DOTA-JR11, (p<0.001). 68Ga-DOTA-JR11 was characterized by higher tumor uptake than 68Ga-DOTATATE in only the SC FTC133 mouse model (p=0.01). The AR42J mouse model (positive control) had the highest tumor uptake of all three radioactive analogs in comparison to DTC and MTC mice models (p<0.001). Among thyroid cancer mice models, aggressively growing FTC133 tumors were characterized by the highest uptake of 68Ga-DOTA-JR11 and 86Y-EB-TATE compared with slow-growing BCPAP (p<0.01 & p<0.001, respectively) and TT tumors (p<0.05 & p<0.001, respectively) in SC mice models. There was no significant difference in the 68Ga-DOTATATE uptake between FTC133 and BCPAP mice models (p= 0.97), which were characterized by significantly higher uptake than the TT mouse model (p= 0.01 & 0.03, respectively). Conclusion: We show for the first time that a novel SSTR2 analog 86Y-EB-TATE is characterized by superior tumor uptake compared to 68Ga-DOTATATE and 68Ga-DOTA-JR11 in TC mice models. Uptake of the radiolabeled analogs not only depends on SSTR2 expression levels, but also on the tumor aggressiveness.