Bone metastasis is a common complication in advanced cancer patients, and bone is the third most common site of cancer metastasis. The occurrence of bone metastases always indicates poor prognosis. Non-small cell lung cancer is the third most susceptible tumor to bone metastasis, with 30–40% of patients developing bone metastasis during the course of the disease [1], [2]. In addition to the damage to the body caused by tumor cells, tumor bone metastasis can also lead to a series of adverse events called skeletal-related events (SREs), including pain, pathologic fractures, spinal cord compression, malignant hypercalcemia and disability, resulting in a rapid decline in patient survival and quality of life [3].

Although remarkable achievements have been made in the early diagnosis and treatment of lung cancer, there is a lack of effective treatments for patients with bone metastasis. The main treatment for such patients is still primary tumor treatment and palliative therapy [4]. Tumor bone metastasis is a complex biological process involving multiple steps and factors [5]. The unclear mechanism is the most important factor restricting the development of effective treatments for tumor bone metastasis. Currently, there are several in vivo animal models available for studying bone metastasis. Intracardiac (IC) injection is the standard technique for studying the circulation extravasation and colonization of tumor cells. However, the technique is difficult to perform, and mice are prone to immediate mortality and metastasis of vital organs [6]. Tail vein injection (IV) is simple to perform, but tumor cells are more likely to remain in the lungs, while the incidence of bone metastasis is low [7], [8], [9]. In situ injection (e.g., within the fat pad of the breast) may improve all stages of the cascade from primary tumor growth to distant metastasis, but metastasis after in situ injection is cell line-dependent and varies considerably in the frequency and location of secondary tumor growth [10], [11]. In situ injection in the femur or tibia allows some tumor cells to remain localized in the bone, but a larger proportion of cells also enter the lungs [12], [13]. Farhoodi et al. [14] used murine-derived breast cancer cells injected via the caudal artery (CA) to create a bone metastasis model that could be consistently detected after 2 weeks. Kuchimaru et al. [15] injected multiple human tumor cells (1×106) through the CA injection, and bone metastasis could be detected later. Tumor cells injected in the CA are forced to move upstream against the arterial blood flow for a short period before entering the femoral artery through the common iliac artery, eventually depositing and forming tumors in the hind limb bone but are not limited to bone tissue. Farhoodi et al. [14] showed that the metastasis rate of vital organs was 4.301% in the first week and 21.74% in the second week after caudal artery injection. The above modeling approaches demonstrate common limitations. First, the location of bone metastasis is not fixed, and there is a high risk of vital organ metastasis, which makes the stability and consistency between models poor, further affecting the reliability of subsequent experiments such as drug or immunotherapy. Second, human-derived cell lines or other cell lines with relatively slow metastatic development have low tumorigenic rates and long tumorigenic times using CA, which reduce the efficiency of follow-up studies and result in a short window for effective treatment.

The key to these problems lies in the low concentration of tumor cells at the target site, which might be overcome by developing a strategy to manipulate tumor cells to remain and grow precisely at the corresponding site by reducing the dissemination of tumor cell in other organs, which will limit immune clearance and promote robust colonization. Currently, micro/nanomotors and magnetic control strategies provide a solution to this challenge. Methods for manipulating the active delivery of micro/nanomotors show great potential in biomedicine. A variety of micromotor platforms have been developed thus far, including micromotors driven by external stimuli and biohybrid micromotors that combine cells, bacteria or algae with micro/nanocarriers [16], [17], [18], [19], [20]. They can be used for targeted drug/gene delivery, microsurgery, biopsy, tissue repair targeted tumor treatment, etc. [21], [22], [23], [24], [25]. Among them, cell-based biohybrid micromotors can be driven by magnetic [26], [27], [28], [29], optical [30], acoustic [31], [32], and electric fields [33] to move toward the target location based on a specified path, enabling targeted control.

Among various types of micromotors, magnetic fields and magnetic nanoparticles are commonly used in medical applications because of their clinical safety and tissue permeability [34]. Iron oxide nanoparticles such as maghemite (Fe2O3) and magnetite (Fe3O4) have been approved by the U.S. Food and Drug Administration (FDA) for diagnosis and treatment [35], [36]. In addition, magnetically driven cells have garnered attention owing to their enhanced retention at the target site under magnetic field actuation. Wang et al. [37] showed that external magnetism increased the retention of super paramagnetic iron oxide nanoparticle (SPION)-labeled adipose-derived stem cells in the urethral sphincter and promoted the recovery of sphincter structure and function in a rat model of stress urinary incontinence. Similarly, Farshid Qiyami Hour et al. [38] demonstrated that the magnetic targeted cell delivery technique can effectively retain SPIONS-labeled human Wharton's jelly derived mesenchymal stem cells (MSCs) in the hippocampus of alzheimer's disease (AD) rats. Previous work by our team [13] has demonstrated that magnetically empowered luciferase-MSCs can move along the bone marrow cavity toward the magnet in the femur. Therefore, we developed a simple but effective strategy to achieve precise tumor formation in bone metastasis models using the magnetic micro-living-motor (MLM) system combined with an intra-bone marrow microinjector containing a syringe and a needle core invented by our team (patent no. 201620090904.2), mainly involving two steps (Fig. 1). First, the magnetic nanomotors magnetize the tumor cells to convert them into MLM, and then a precise magnetic field is used to navigate the MLM to promote targeted cellular colonization and proliferation in the bone marrow cavity, thus enabling precise modeling in bone.

The CA injection method is the latest method for constructing bone metastasis models that develop bone metastases predominantly in the hind limbs. In this study, we compared the tumor formation speed, bone metastasis rate, consistency and stability of the magnetic MLM system with CA by constructing a lung cancer bone metastasis model, as well as further evaluation its role in drug screening, mechanistic research, etc.