Stage IV, the point in tumor progression in which cancer spreads beyond the primary site and regional lymph nodes and is found in other organs, is the cancer stage that most often leads to patient mortality [
1]. The tumor’s microenvironment plays a critical role in tumor growth and the development of metastasis where the interaction between tumor cells and the associated stroma and cellular components modulates the tumor’s progression and patient prognosis. Recently, the acellular 4D lung model has successfully mimicked the development of metastasis [
2]. It is named the 4D model because of its perfusion of tumor nodules that allows it to change over time and grow in the 3D space. Findings from the 4D model suggest that the only component of tumor microenvironment that is important to show tumor progression is an intact natural matrix [
2].
The acellular 4D lung model is created by removing all of the cells from a rat heart and lung block [
3,
4]. This natural lung matrix maintains its three-dimensional architecture, including perfusable vascular beds and preserved airways. The matrix is composed of collagen, proteoglycans, and elastic fibers that preserve the architecture of airways and capillaries. A unique feature of the matrix is that this composition is preserved among species in the distal airways [
5]. Furthermore, the basement membranes of the alveolar septa are preserved after decellularization in this model [
3]. The acellular 4D lung model shows that when tumor cells are placed into the trachea, they form perusable nodules in the lung matrix [
6]. Moreover, the model allows tumor cells to secrete proteins that are more similar those found in lung cancer patients than the same tumor cells grown on a petri dish [
7]. The acellular 4D lung model mimics metastasis, with the placement of all tumor cells in the left lung lobes and perfusion of the model in the bioreactor through the pulmonary artery. In order for the tumor cells to enter the right lung, the cells would need to leave the epithelial space in the left side, enter the vasculature, and enter the other epithelial space on the right side. Over time, this process occurred as metastatic lesions formed in the right lung and grew over time in the 4D model [
2]. There are significant differences in the spatial organization of the tumor cells where the primary tumor grew in a pattern along the airway and the metastatic lesion formed in a distribution that is consistent with cancer distributed along the vasculature. The model’s unique vascular channel allowed dead cells as well as live circulating tumor cells (CTC) to enter the vasculature. The CTC showed differences in behavior and gene expression compared to those cells initially placed in the model. The CTC took longer to attach to the petri dish than the parental cells placed in the model and they stayed alive in supernatant with decreased expression of integrin beta 4 (ITGB4) [
8]. In addition, CTCs were resistant to chemotherapy [
9]. There is no difference in the number of live CTC from the 4D model when they are placed in the petri dish, with or without Cisplatin, while the same dose for the parental cells (2D) placed in the model showed a significant reduction of live cells [
9]. Previous studies also show that the CTC form metastatic lesions in the 4D model [
2].
A major drawback of this acellular 4D model has been the lack of cellular components that are found in a patient’s tumor microenvironment. These studies suggest that the natural matrix architecture is the only component necessary for complex perfusable nodules to form, the creation of CTC, and ultimately metastatic lesion formation. However, one could argue that this phenomenon is simply due to the artificial creation of an acellular environment. Thus, in this study, we show that the ex vivo 4D lung model can mimic the metastatic process in a normal cellular environment. We postulate that non-small cell and small cell lung cancer cell lines as well as breast cancer cell lines will grow in the model and form a primary tumor, CTC, and metastatic lesions.