Lineage Plasticity in SCLC Generates Non-Neuroendocrine Cells Primed for Vasculogenic Mimicry

Introduction Vasculogenic mimicry (VM), the process of tumor cell trans-differentiation to endow endothelial-like characteristics supporting de novo vessel formation, is associated with poor prognosis in several tumor types, including small cell lung cancer (SCLC). In genetically engineered mouse models (GEMMs) of SCLC, NOTCH and MYC co-operate to drive a neuroendocrine (NE) to non-NE phenotypic switch and co-operation between NE and non-NE cells is required for metastasis. Here, we define the phenotype of VM-competent cells and molecular mechanisms underpinning SCLC VM using circulating tumor cell-derived explant (CDX) models and GEMMs. Methods We analysed perfusion within VM vessels and their association with NE and non-NE phenotypes using multiplex immunohistochemistry in CDX and GEMMs. VM-proficient cell subpopulations in ex vivo cultures were molecularly profiled by RNA sequencing and mass spectrometry. We evaluated their 3D structure and defined collagen-integrin interactions. Results We show that VM vessels are present in 23/25 CDX models and in 2 GEMMs. Perfused VM vessels support tumor growth and only Notch-active non-NE cells are VM-competent in vivo and ex vivo, expressing pseudohypoxia, blood vessel development and extracellular matrix (ECM) organization signatures. On Matrigel, VM-primed non-NE cells re-model ECM into hollow tubules in an integrin beta 1-dependent process. Conclusions We identify VM as an exemplar of functional heterogeneity and plasticity in SCLC and these findings take significant steps towards understanding the molecular events that enable VM. These results support therapeutic co-targeting of both NE and non-NE cells to curtail SCLC progression and to improve SCLC patient outcomes in future. ### Competing Interest Statement C.D has received research funding from AstraZeneca, Astex Pharaceuticals, Bioven, Amgen, Carrick Therapeutics, Merck AG, Taiho Oncology, Clearbridge Biomedics, Angle PLC, Menarini Diagnostics, GSK, Bayer, Boehringer Ingelheim, Roche, BMS, Novartis, Celgene, Epigene Therapeutics Inc and Thermo Fisher Scientific. C.D has received consultancy fees/honoraria and advisory board from AstraZeneca, Biocartis, Merck AG. J.S. has received research funding from Stemcentrx/Abbvie and Pfizer and licensed a patent to Forty Seven Inc/Gilead on the use of CD47 blocking strategies in SCLC. G.H and I.C have filed a patent covering use of FOXC2 and FOXC2-regulated gene sets as diagnostics and as a route toward development of VM inhibitors.