Cooperative cell invasion: matrix metalloproteinase-mediated incorporation between cells

Progression to metastatic disease is a leading cause of cancer death. Tumors are a complex mixture of cell types, both genetically heterogeneous malignant cells and associated nonmalignant cells. Models mimicking this heterogeneous cell environment have revealed that invasive cell populations can induce dissemination by otherwise poorly/noninvasive tumor cells, known as cooperative invasion. Neuroblastoma tumors arise in children and are characterized by mixed cellular populations in vivo, consisting chiefly of neuronal (N)-type and substrate (S)-type cells. The S-type cells have all the hallmarks of invasive leader cell populations and have been coisolated with N-type cells from metastatic bone lesions, but to date their ability to induce cooperative invasion has not been investigated. Therefore, in the present study, we analyzed the invasive behavior of mixed N-type and S-type multicellular spheroids embedded in three-dimensional collagen gels. Our analyses show that S-type cells induce invasion of either single cells or small cell clusters of N-type cells. In contrast to other reports of cooperative invasion in which mixed cultures exhibit a follow-the-leader mechanism, we show coincident emergence of S-and N-type cells from mixed spheroids. Our data suggest mutual effects between the two cell types. Thus, whereas coculture with S-type cells induces N-type invasion, coculture with N-type cells slows S-type invasion. Using matrix metalloproteinase (MMP) inhibitors and cell incorporation assays, we demonstrate that MMP activity is required for S-type cells to insert into layers of N-type cells. Our study therefore highlights an important role for S-type neuroblastoma cells in the invasion process and reveals a new mechanism of cooperative invasion.