Experimental study on the establishment of Aromatase inhibitor associated bone loss model after premenopausal breast cancer and the mechanism of bone loss

Abstract

Purpose/Aims

To construct a nude mouse model of aromatase inhibitor-associated bone loss (AIBL) after premenopausal breast cancer surgery, and to explore the possible mechanism of letrozole-induced bone loss.

Rationale/Background

At present, clinical and experimental research on AIBL mainly focuses on postmenopausal breast cancer patients, ignoring the premenopausal population of AIs combined with Ovarian Function Suppression. The mechanism of AIBL is not only the well-known sharp decline of estrogen, but also the lack of exploration of the cellular mechanism and factors related to bone metabolism. H-type blood vessels contribute to angiogenesis and bone formation in the bone microenvironment. It is a sensitive indicator for evaluating bone mass andSlit guided ligand 3 (SLIT3) is a type of angiogenic factor secreted by osteoblasts. Knocking out SLIT3 will lead to the reduction of H-type vascular endothelial cells in bone and resulting in a decrease in bone mass. Based on this, it will be helpful to establish AIBL animal model and explore the mechanism of bone loss, which will help optimize the endocrine therapy regimen.

Methods

The postoperative AIBL model of premenopausal breast cancer was established by inoculation and resection of breast cancer xenografts, bilateral ovariectomy and letrozole gavage. BALB/c nude mice were randomly divided into 5 groups: Control group (Control group), postoperative group (MX group), castration group (MX+OVX group), model group A (MX+OVX+Le group), model group B (OVX+Le group). The eyeball blood of mice was collected to detect the related bone metabolism and bone-related hormones by ELISA. The bone mineral density and trabecular microstructure of the femur and tibia were evaluated by mirco-CT, the bone tissue was evaluated by HE staining, the activity of osteoblasts was evaluated by OCN immunohistochemistry, and the activity of osteoclasts was evaluated by TRAP immunohistochemistry. Immunofluorescence staining of type H blood vessel (CD31hiEmcnhi) was used to explore the potential mechanism and related targets of AIBL.

Results

Compared with the control group, there were significant differences in serum E2, P1NP, CTX-1, GH and SLIT3 in model A and model B groups (P< 0.05). Bone mineral density was significantly reduced by mirco-CT (P< 0.05), and the decrease in model group A was more significant. In HE staining, the number of bone trabeculae in the model A group was significantly reduced. In addition, TRAP and OCN immunohistochemical staining showed that the trabeculae of model A group were surrounded by more osteoclasts and fewer osteoblasts. Compared to the control group, H-type blood vessels in model A group were smaller under immunofluorescence.

Implications

Model group A is more suitable as an AIBL animal model after premenopausal breast cancer surgery. Mirco- CT combined with pathological staining is helpful in optimizing and evaluating changes in bone mineral density and bone microstructure in animal models.