The term vasculogenic mimicry (VM) refers to the capacity of certain cancer cells to form fluid-conducting structures within a tumor in an endothelial cell (EC)-free manner. Ever since its first report by Maniotis in 1999, the existence of VM has been an extremely contentious issue. The overwhelming consensus of the literature suggests that VM is frequently observed in highly aggressive tumors and correlates to lower patient survival. While the presence of VM in vivo in animal and patient tumors are claimed upon the strong positive staining for glycoproteins (Periodic Acid Schiff, PAS), it is by no means universally accepted. More controversial still is the existence of an in vitro model of VM that principally divides the scientific community. Original reports demonstrated that channels or tubes occur in cancer cell monolayers in vitro when cultured in matrigel and that these structures may support fluid movement. However, several years later many papers emerged stating that connections formed between cancer cells grown on matrigel represented VM. We speculate that this became accepted by the cancer research community and now the vast majority of the scientific literature reports both presence and mechanisms of VM based on intercellular connections, not the presence of fluid conducting tubes. In this opinion paper, we call upon evidence from an exhaustive review of the literature and original data to argue that the majority of in vitro studies presented as VM do not correspond to this phenomenon. Furthermore, we raise doubts on the validity of concluding the presence of VM in patient samples and animal models based solely on the presence of PAS+ staining. We outline the requirement for new biomarkers of VM and present criteria by which VM should be defined in vitro and in vivo .

Introduction: Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite the strong correlation with reduced patient survival, the mechanisms by which a tumor can create this self-generated irrigation system are still not fully understood. The process of VM in vitro can only occur in Matrigel; however, the protein component and signaling pathways involved in this process are unknown. Methods: Using an established in vitro model of VM, of ovarian and breast cancer cells (HEY and MDA-MB-231, respectively) on Matrigel coating, we utilized pharmacological inhibitors, gene silencing and blocking antibodies to elucidate the signaling pathways involved in the process of VM. Immunofluorescence and sirius red staining were used to determine the glycoprotein-rich component lining the lumen of the tubular structures. Results: Differently to what observed in the presence of Matrigel, VM did not occur when cancer cells were cultivated on plastic, glass or heat denatured Matrigel (10 mins at 65°C). Using exclusively Collagen I or Laminin 111 to mimic the extracellular matrix we observed than only in the presence of Laminin 111 could VM formation occur. Laminin is secreted and deposited by HEY cells and constitutes a part of the luminal lining. Silencing of integrin β1, but not β3, by siRNA and antibody blocking prevents this process. Chemical inhibition of PI3K pathway and metalloproteases (MMP) activation demonstrate that these pathways are also essential. RNAseq analysis suggests that this process has minimal dependence on de novo transcriptional activity. Discussion and conclusion: We have shown that VM only occurs when cells are seeded on Matrigel but not on plastic, glass or heat denatured Matrigel, suggesting that this phenomenon is susceptible to substrate/matrix rigidity. Furthermore, we identified Laminin as the essential matrix protein secreted and deposited by cancer cells to allow for VM assembly. Its interaction with integrin β1, and the consequent regulation of the activity of MMP leads to the remodeling of the ECM to favor the connection of the VM channels to the microcirculation system. This pathway is not heavily dependent on transcription but requires the PI3K pathway. As VM is strongly associated with poor patient survival, understanding the formation of this alternative irrigation system may deliver new druggable targets. Citation Format: Gabriel Mingo, Andres Valdivia, Varina Aldana, Javiera Pradenas, Nicole Babbitt, Pamela Gonzalez, Francisco Nualart, Jorge Díaz, Lisette Leyton, Cristina Bertocchi, Gareth Owen. A characterization of cancer vasculogenic mimicry: Extracellular matrix induced cellular signaling to lumen formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3150.

Introduction: The presence of an endothelial cell-free perfusion pathway, known as Vasculogenic Mimicry (VM), strongly correlates with reduced patient survival, however the mechanisms by which a tumor can create a self-generated irrigation system remain unknown. Currently there is no mechanism of VM formation reported in fluid conducting tubular structures. Herein, using a standardized in vitro model, we sought to understand the pathways required for VM formation. Methods: We utilized confocal microscopy and Imaris reconstruction to demonstrate that cancer cells can create an internal tubular network in cultured cancer cell lines, cancer spheres and primary cancer cultures of ovarian cancer. Matrigel and 3D matrices containing specific protein components allowed the deciphering of the extracellular matrix proteins involved in VM formation. Intercellular pathways were resolved using chemical inhibitors. Results: VM only occurs when cells are grown in 3D culture and only 30% of primary cancer cultures undergo this process. The presence of laminin 1 is sufficient to trigger the formation of tubular structures and this may involve the alpha2beta1 integrin and metalloproteases. Downstream FAK and PI3K/AKT intercellular pathways are required to form functional tubular networks. Microarray data elucidated the presence of the mRNA transcripts that were increased in cells that undergo VM. Discussion: We demonstrate that Laminin 1, a component of the extracellular matrix (ECM), can promote the process of VM, which further involves the downstream signaling of the potentially druggable targets of the PI3K pathway. As VM is associated with reduced patient survival, elucidation of the mechanisms of formation may deliver new cancer therapies. Funding: FONDECYT 1180241, CONICYT FONDAP 15130011, IMII P09/016F Citation Format: Andres Valdivia, Gabriel Mingo, Varina Aldana, Alejandra Sandoval, Alejandro Corvalan, Francisco Nualart, Gareth I. Owen. Extracellular signaling linked to PI3K/AKT triggers the formation of vasculogenic mimcry in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 191.