RCAN1.4 regulates tumor cell engraftment and invasion in a thyroid cancer to lung metastasis-on-a-chip microphysiological system

Progressive metastasis is the primary cause of cancer-related deaths. It has been recognized that many cancers are characterized by long periods of stability followed by subsequent progression. Genes termed metastasis progression suppressors (MPS) are functional gatekeepers of this process, and their loss leads to late-stage progression. Previously, we identified regulator of calcineurin 1, isoform 4 (RCAN1.4) as a functional MPS for several cancers, including thyroid cancer, a tumor type prone to metastatic dormancy. RCAN1.4 knockdown increases expression of the cancer-promoting transcription factor NFE2-like bZIP transcription factor (NFE2L3), and through this mechanism increases cancer cell proliferation and invasion in<i>in vitro</i>and<i>in vivo</i>and promotes metastatic potential to lungs in tail vein models. However, the mechanisms by which RCAN 1.4 regulates specific metastatic steps is incompletely characterized. Studies of the metastatic cascade are limited in mouse systems due to high cost and long duration. Here, we have shown the creation of a thyroid-to-lung metastasis-on-a-chip (MOC) model to address these limitations, allowing invasion analysis and quantification on a single cell level. We then deployed the platform to investigate RCAN1.4 knockdown in fluorescently tagged hTh74 and FTC236 thyroid cancer cell lines. Cells were circulated through microfluidic channels, running parallel to lung hydrogel constructs allowing tumor cell-lung tissue interactions. Similar to studies in mouse models, RCAN1.4 knockdown increased NFE2L3 expression, globally increased invasion distance into lung constructs and had cell line and clonally dependent variations on bulk metastatic burden. In line with previous<i>in vivo</i>observations, RCAN1.4 knockdown had a greater impact on hTh74 metastatic propensity than FTC236. In summary, we have developed and validated a novel MOC system evaluate and quantify RCAN1.4-regulated thyroid cancer cell lung adherence and invasion. This system creates opportunities for more detailed and rapid mechanistic studies the metastatic cascade and creates opportunities for translational assay development.