Establishment of orthotopic osteosarcoma animal models in immunocompetent rats through muti-rounds of in-vivo selection

BMC Cancer Yao et al. BMC Cancer (2024) 24:703 https://doi.org/10.1186/s12885-024-12361-z Open Access RESEARCH Establishment of orthotopic osteosarcoma animal models in immunocompetent rats through muti-rounds of in-vivo selection Mengyu Yao1,2†, Zehua Lei1,2†, Feng Peng1,2, Donghui Wang3*, Mei Li1,2, Guoqing Zhong1,2, Hongwei Shao1,3, Jielong Zhou1,2, Chang Du4* and Yu Zhang1,2* Abstract Immunodeficient murine models are usually used as the preclinical models of osteosarcoma. Such models do not effectively simulate the process of tumorigenesis and metastasis. Establishing a suitable animal model for understanding the mechanism of osteosarcoma and the clinical translation is indispensable. The UMR106 cell suspension was injected into the marrow cavity of Balb/C nude mice. Tumor masses were harvested from nude mice and sectioned. The tumor fragments were transplanted into the marrow cavities of SD rats immunosuppressed with cyclosporine A. Through muti-rounds selection in SD rats, we constructed orthotopic osteosarcoma animal models using rats with intact immune systems. The primary tumor cells were cultured invitro to obtain the immune-tolerant cell line. VX2 tumor fragments were transplanted into the distal femur and parosteal radius of New Zealand white rabbit to construct orthotopic osteosarcoma animal models in rabbits. The rate of tumor formation in SD rats (P1 generation) was 30%. After four rounds of selection and six rounds of acclimatization in SD rats with intact immune systems, we obtained immune-tolerant cell lines and established the orthotopic osteosarcoma model of the distal femur in SD rats. Micro-CT images confirmed tumor-driven osteolysis and the bone destruction process. Moreover, the orthotopic model was also established in New Zealand white rabbits by implanting VX2 tumor fragments into rabbit radii and femurs. We constructed orthotopic osteosarcoma animal models in rats with intact immune systems through muti-rounds in-vivo selection and the rabbit osteosarcoma model. Keywords Animal model, Osteosarcoma, Orthotopic transplantation, Cell line, Immune system † Mengyu Yao and Zehua Lei contribute equally to this work. *Correspondence: Donghui Wang Chang Du Yu Zhang 1 Department of Orthopedics, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou 510080, China 2 GuangDong Engineering Technology Research Center of Functional Repair of Bone Defects and Biomaterials, Guangzhou 510080, China 3 Hebei Key Laboratory of Biomaterials and Smart Theranostics, School of Health Sciences and Biomedical Engineering, Hebei University of Technology, Tianjin 300130, China 4 Department of Biomedical Engineering, School of Materials Science and Engineering, South China University of Technology, Guangzhou 510006, China © The Author(s) 2024. 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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yao et al. BMC Cancer (2024) 24:703 Introduction Osteosarcoma, the most common primary malignant tumor of the bone, is often occurs in adolescents. Osteosarcoma primarily occurs in the metaphyses of long bones, including the distal femur, proximal tibia, and humerus [1, 2]. The five-year survival rate of all the patients at all the stages of osteosarcoma is approximately 60-70% [3]. In particular, osteosarcoma patients with lung metastasis maintain even poorer prognoses. Although neoadjuvant chemotherapy has enhanced the management of osteosarcoma by facilitating preoperative planning and potentially modifying postoperative treatment, the prognosis and five-year survival rate of osteosarcoma patients have not markedly improved over the past few decades [4]. One of the main factors that hinder the development of osteosarcoma treatment is the lack of a full understanding of the cellular and molecular mechanisms underlying the development of this condition. A suitable animal model can help in promoting the investigation of cellular and molecular mechanisms related to osteosarcoma [5]. The current in-vivo osteosarcoma models include spontaneous, inducible, and transplantation osteosarcoma models and other genetically engineered models [6]. Among them, transplantation models, including homografts and xenografts, are the most important ones. Xenografts have been developed by implanting human osteosarcoma cells or tissues into immunodeficient murine hosts, such as athymic nude mice, which possess a greater tumor-forming efficiency and can be manipulated and evaluated easily [7]. In 1993, the first successful xenogeneic tumor model was constricted by Berlin. He developed a spontaneous metastasis model in athymic mice by utilizing the v-Ki-ras-oncogene-transformed human osteosarcoma cell line (KRIB), which was orthotopically implanted into the tibial bones of nude mice [8]. Despite the numerous advantages of xenogeneic models, the loss of immune system in immunodeficient murine models does not allow proper tumor-host interactions and hinders the investigation of tumor initiation and metastasis and the function of immune cells in antitumor immune reactions or tumor immune evasion in the tumor environment [9–11]. In contrast, homografts of osteosarcoma (including orthotopically transplanted homografts and heterotopically transplanted homografts) have several advantages in biologically relevant host microenvironments and are currently the most extensively used method for the establishment of osteosarcoma tumor models. Heterotopically transplanted models can be established in the subcutis or musculature. For example, Chen et al. used the subcutaneous model of Balb/C mice to study the in-vivo photothermal antitumor effects of three-dimensional (3D), printed Wesselsite [SrCuSi4O10] nanosheets integrated with polycaprolactone (SC/PCL) Page 2 of 11 composite scaffolds [12]. However, without the participation of normal bone stromal cells and matrix proteins and cytokines, the subcutaneous and intramuscular models cannot simula (...truncated)