Introduction: Detachment from the extracellular matrix (ECM) is the first step of the metastatic cascade. It is a regulated process involving interaction between tumor cells and tumor microenvironment (TME). Iron is a key micronutrient within the TME. Here, we explored the role of iron in the ability of ovarian cancer cells to successfully detach from the ECM. Methods: HEY and PEO1 ovarian cancer cells were grown in 3D conditions. To mimic an iron rich TME, culture media were supplemented with 100 μM Fe³⁺. Cell mortality was evaluated by cytofluorimetric assay. The invasive potential of tumor spheroids was performed in Matrigel and documented with images and time-lapses. Iron metabolism was assessed by analyzing the expression of CD71 and FtH1, and by quantifying the intracellular labile iron pool (LIP) through Calcein-AM cytofluorimetric assay. Ferroptosis was assessed by quantifying mitochondrial reactive oxygen species (ROS) and lipid peroxidation through MitoSOX and BODIPY-C11 cytofluorimetric assays, respectively. Ferroptosis markers GPX4 and VDAC2 were measured by Western blot. FtH1 knockdown was performed by using siRNA. Results: To generate spheroids, HEY and PEO1 cells prevent LIP accumulation by upregulating FtH1. 3D HEY moderately increases FtH1, and LIP is only slightly reduced. 3D PEO1upregulate FtH1 and LIP results significantly diminished. HEY tumor spheroids prevent iron import downregulating CD71, while PEO1 cells strongly enhance it. Intracellular ROS drop down during the 2D to 3D transition in both cell lines, but more significantly in PEO1 cells. Upon iron supplementation, PEO1 cells continue to enhance CD71 and FtH1 without accumulating the LIP and ROS and do not undergo ferroptosis. HEY, instead, accumulate LIP, undergo ferroptosis and attenuate their sphere-forming ability and invasiveness. FtH1 knockdown significantly reduces the generation of PEO1 tumor spheroids, although without sensitizing them to ferroptosis. Discussion: Iron metabolism reprogramming is a key event in the tumor spheroid generation of ovarian cancer cells. An iron-rich environment impairs the sphere-forming ability and causes cell death only in ferroptosis sensitive cells. A better understanding of ferroptosis sensitivity could be useful to develop effective treatments to kill ECM-detached ovarian cancer cells.

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