Background

Bone metastasis is the most frequent metastasis for many cancer types, especially for those arising from prostate (90%), breast (70%), multiple myeloma (90%), lung (40%), and kidney (40%) (Coleman, 2006). The median survival of patients with bone metastasis ranges from approximately one year for lung cancer to 3-5 years for prostate cancer, breast cancer, and multiple myeloma (Campbell et al., 2012). Bone metastases often require radiotherapy and lead to bone pain, hypercalcemia, pathologic fracture, and spinal cord or nerve root compression (Coleman, 2006). Bone metastases also develop resistance to chemotherapy, as docetaxel-treated prostate cancer patients with bone metastasis still have poor prognoses (Coleman et al., 2020). Therefore, effective therapeutic strategies are still urgently needed for bone metastatic cancer patients.

Bone metastases can be classified as osteolytic or osteoclastic types according to their predominant characteristics in radiographic images. Although lysis or sclerosis may appear to predominate in some types of bone metastases, osteoclastic and osteoblastic activities are usually simultaneously involved in the formation of bone metastatic lesions. The outgrowth of cancer cells in the bone microenvironment is a process in which cancer cells communicate with osteoclasts and osteoblasts frequently via paracrine factors and receptors on their cell surfaces. Therefore, the behaviors of cancer cells in bone metastasis are distinct from those in primary tumors, thus potentially requiring different therapeutic methods.

Diverse in vivo animal models have been established to study bone metastasis. Intracardiac injection has been utilized as the gold standard to develop bone metastasis by injecting cancer cells into the left ventricle in mice, with a goal of recapitulating the bone metastasis process, including cancer cell survival in the bloodstream, extravasation, cancer cell arrest in bone, and bone metastatic tumor growth (James et al., 2015). Similarly, intracaudal arterial injection was established to provide an easy-to-use model with higher efficiency of bone metastasis (Kuchimaru et al., 2018). However, the intratibial injection model is used to mimic the scenario when cancer cells have metastasized to the bone, thus providing a better focus on the crosstalk between cancer cells and the bone microenvironment. Herein, we will focus on tibial tumorigenesis by using the intratibial injection model to mimic the setting of cancer patients who have developed bone metastasis.

In this protocol, we take the prostate cancer cell line PC-3 as an example, describe the hands-on procedures of cell preparation, intratibial injection, monitoring tibial tumor growth, and post tissue collection analyses, highlight the critical steps in cell preparation and intratibial injection, and detail how to quantitatively analyze the tibial tumors in histological samples.