Orthotopic pituitary tumors generated by stereotaxic GC cell injection in immunocompetent rats

Abstract

Innovative treatment strategies for pituitary tumors are necessary to limit the disease burden and to improve survival in cases of carcinomas. The paucity and inaccuracy of available preclinical models substantially hamper pituitary research and drug discovery. Hence, we describe a novel method to generate orthotopic pituitary tumors via stereotaxic injection of somatotroph GC cells into the pituitaries of immunocompetent Wistar Furth rats. Tumor growth was monitored by repeated 7 Tesla magnetic resonance imaging. The procedure consistently led to rapidly expanding intra- and suprasellar growth hormone-secreting tumors within their native anatomical environment. The generated tumors faithfully reproduced the microarchitecture of human somatotroph pituitary adenomas, including the immune infiltrates and other typical components of their microenvironment, which is a prerequisite for testing immunomodulating agents. This orthotopic model of proliferative pituitary tumors developed in immunocompetent hosts therefore unlocks new opportunities for preclinical studies.

Keywords: Immunocompetent rat; Pituitary adenoma; Pituitary research; Preclinical model; Tumor microenvironment.

Fig. 1

Illustration of the tumor implantation surgery procedure generating orthotopic pituitary tumors. (A) Schematic representation of the stereotactic trajectory for reaching the pituitary lobes on a coronal plane (white dots on image A). (B) 3D representation of the procedure leading to orthotopically grafted pituitary tumors. (C) Early-stage organized conglomerates of tumoral GC-cells within the pituitary 9 days after surgery, identified by immunohistochemistry staining of Pit-1 showing sparsely distributed GC cells within the pituitary, with one spot of aggregated tumoral cells (zoom x10). (D): Close-up image of the aggregated tumoral cells (zoom x60)

Fig. 2

Radiological monitoring of tumor growth kinetics using 7 Tesla magnetic resonance imaging (MRI). T2-weighted sequences in coronal sections of three rats (rows) at three different time points (columns) illustrating large sellar and suprasellar tumors responsible for acute obstructive hydrocephalus (yellow arrow) and premature death of the animals (crossed red images)

Fig. 3

Growth hormone (GH) secretion and bioactivity. (A) Graphical representation showing the increase in serum GH concentration over time in orthotopically grafted animals. (B) Photograph of an acrogigantic rat after subcutaneous injection of GC cells (right) compared to her same-age sister control injected with vehicle (left). (C) Graphical representation of weigh gain during the study across different groups (GC SPI = GC cells stereotactic pituitary injection; GC SC = GC cells subcutaneous injection, SHAM = control animals). (D) Intraoperative photograph of the spinal metastasis (green arrow) compressing the thoracic spinal cord (black arrow) of a subcutaneously injected animal

Fig. 4

Ex vivo characterization of the tumors. (A) Immunohistochemistry of Pit-1 expression showing massive nuclear staining. (B) Immunohistochemistry of growth hormone (GH) showing significant cytoplasmic staining. (C) Immunohistochemistry of Ki-67 expression showing significant nuclear staining. (D) Hematoxylin-Eosin-Saffron staining showing several mitoses (yellow arrows). (E) Immunohistochemistry of CD34 expression showing endothelial cells staining of neovessels. (F) Immunohistochemistry of S-100B staining intra-tumoral folliculostellate cells. (G) Immunohistochemistry of CD3 illustrating intra-tumoral T-cells. (H) Immunohistochemistry of CD68 illustrating intra-tumoral macrophages