Multimodal Somatostatin Receptor Theranostics Using [(64)Cu]Cu-/[(177)Lu]Lu-DOTA-(Tyr(3))octreotate and AN-238 in a Mouse Pheochromocytoma Model

Abstract

Pheochromocytomas and extra-adrenal paragangliomas (PHEO/PGLs) are rare catecholamine-producing chromaffin cell tumors. For metastatic disease, no effective therapy is available. Overexpression of somatostatin type 2 receptors (SSTR2) in PHEO/PGLs promotes interest in applying therapies using somatostatin analogs linked to radionuclides and/or cytotoxic compounds, such as [(177)Lu]Lu-DOTA-(Tyr(3))octreotate (DOTATATE) and AN-238. Systematic evaluation of such therapies for the treatment of PHEO/PGLs requires sophisticated animal models. In this study, the mouse pheochromocytoma (MPC)-mCherry allograft model showed high tumor densities of murine SSTR2 (mSSTR2) and high tumor uptake of [(64)Cu]Cu-DOTATATE. Using tumor sections, we assessed mSSTR2-specific binding of DOTATATE, AN-238, and somatostatin-14. Therapeutic studies showed substantial reduction of tumor growth and tumor-related renal monoamine excretion in tumor-bearing mice after treatment with [(177)Lu]Lu-DOTATATE compared to AN-238 and doxorubicin. Analyses did not show agonist-dependent receptor downregulation after single mSSTR2-targeting therapies. This study demonstrates that the MPC-mCherry model is a uniquely powerful tool for the preclinical evaluation of SSTR2-targeting theranostic applications in vivo. Our findings highlight the therapeutic potential of somatostatin analogs, especially of [(177)Lu]Lu-DOTATATE, for the treatment of metastatic PHEO/PGLs. Repeated treatment cycles, fractionated combinations of SSTR2-targeting radionuclide and cytotoxic therapies, and other adjuvant compounds addressing additional mechanisms may further enhance therapeutic outcome.

Keywords: DOTATATE; PET; SPECT; catecholamines; doxorubicin.; metanephrines; neuroendocrine tumors; optical in vivo imaging.

Figure 1

mSSTR2 status of murine pheochromocytoma cells and tumors. (A) Western blot analyses of murine organs, tumors and cell cultures; (B) immunocytochemistry in MPC-mCherry cell cultures in vitro; bars: 30 µm; (C) immunohistochemistry on tissue sections of MPC-mCherry tumors; (left) overview of a tumor 4 weeks after cell injection, arrows indicate the absence of mSSTR2 in necrotic regions; bars: 1 mm; (right) membranous distribution of mSSTR2 in tumors; bars: 10 µm; (M) molecular weight of proteins in kDa; (nc) negative control in presence of blocking peptide (human SSTR2 fragment).

Figure 2

(A-D) Binding of somatostatin analogs to mSSTR2 on MPC-mCherry tumor sections. (A) mSSTR2 saturation binding analysis at increasing concentrations of the radioligand [⁶⁴Cu]Cu-DOTATATE; non-specific binding was determined in presence of 5 µmol/L non-labeled DOTATATE; (B) Scatchard analysis of radioligand binding; (C) time-dependence of radioligand binding; (D) competition of radioligand with increasing concentrations of various SSTR2 agonists; (E-F) cellular uptake of [⁶⁴Cu]Cu-DOTATATE in MPC-mCherry cell cultures at various temperatures; inhibition of radioligand uptake by co-incubation with various somatostatin agonists at 1 µmol/L; data are presented as means ± SEM; significance of differences was tested as compared to control; † p < 0.01; ‡ p < 0.001.

Figure 3

Distribution of the [⁶⁴Cu]Cu-DOTATATE in mice bearing MPC-mCherry tumors; radiotracer accumulation was blocked by co-injection of octreotide, [^natLu]Lu-DOTATATE and AN-238 at 0.2 µmol/kg; (A) FLI of tumors; (B) PLI of radiotracer distribution; (C) PET maximum intensity projections of radiotracer distribution; (D) radiotracer accumulation in tumors as determined in PLI studies; (E) radiotracer accumulation in tumors as determined in PET studies; (F) radiotracer accumulation in organs and tumors as determined in ex vivo radiotracer distribution studies; (tu) tumor; (bl) bladder; data are presented as means ± SEM; significance of differences was tested as compared to control; * p < 0.05; † p < 0.01; ‡ p < 0.001.

Figure 4

Progression of MPC-mCherry tumors in mice undergoing doxorubicin (6.9 µmol/kg), AN-238 (0.2 µmol/kg), and [¹⁷⁷Lu]Lu-DOTATATE (80 MBq/animal) treatment; (A) mCherry FLI/X-ray overlays of tumor-bearing mice after 15 and 30 days pci; (B) monitoring of tumor volume; (C) monitoring of ¹⁷⁷Lu activity in tumors after [¹⁷⁷Lu]Lu-DOTATATE treatment as determined by PLI; (¹⁷⁷Lu_eff/corr) effective and decay-corrected ¹⁷⁷Lu activity; (D) [¹⁷⁷Lu]Lu-DOTATATE PLI after 16 days pci; (E) [¹⁷⁷Lu]Lu-DOTATATE SPECT after 16 days pci; (↓) treatment start; data are presented as means ± SEM; significance of differences was tested as compared to control after 30 days pci; * p < 0.05; † p < 0.01; ‡ p < 0.001.

Figure 5

Renal monoamine excretion in MPC-mCherry tumor-bearing mice undergoing doxorubicin (6.9 µmol/kg), AN-238 (0.2 µmol/kg), and [¹⁷⁷Lu]Lu-DOTATATE (80 MBq/animal) treatment; (↓) treatment start; (DA) dopamine; (NE) norepinephrine; (MTY) 3-methoxytyramine; (NMN) normetanephrine; (EPI) epinephrine; (MN) metanephrine; data are presented as means ± SEM; significance of differences was tested as compared to renal monoamine excretion before cell injection (0 days pci); † p < 0.01; ‡ p < 0.001.

Figure 6

mSSTR2 status of MPC-mCherry tumors after AN-238 (0.2 µmol/kg) and [¹⁷⁷Lu]Lu-DOTATATE (80 MBq/animal) treatment; (A) mSSTR2 immunohistochemistry on MPC-mCherry tumor sections; bars (200x) 50 µm, bars (1000x): 10 µm; (B-C) Densitometric evaluation of relative mSSTR2 levels per chromogranin A-positive tumor cells; (nc) negative control; (M) molecular weight of proteins in kDa; data are presented as means ± SEM; significance of differences was tested as compared to control.

Figure 7

Adverse effects of doxorubicin (6.9 µmol/kg), AN-238 (0.2 µmol/kg), and [¹⁷⁷Lu]Lu-DOTATATE (80 MBq/animal) treatment in mice; (A) monitoring of body weight, (B) white blood cell count after 30 days pci; (↓) treatment start; data are presented as means ± SEM; significance of differences was tested as compared to control; * p < 0.05.