LAUSR

Simple Summary Many cancer types are reported to have high lymphangiogenic receptor Neuropilin-2 (Nrp2) expression, including colorectal cancer (CRC). Nrp2 is shown to be associated with tumor progression in vivo and poor prognosis in CRC patients. Although the role of Nrp2 is well established in lymphangiogenesis, the tumor cell-intrinsic role of Nrp2 remains elusive. Here, we employed murine CRC tumor-derived mesenchymal-like organoids to induce Nrp2 depletion. We demonstrate that Nrp2 deletion in CRC organoids results in a drastically altered phenotype that is characterized by mesenchymal-to-epithelial transition (MET), and an acquired dependency on IR signaling and autophagy. This phenotype is preserved in subcutaneous tumors generated by CRC organoids. We conclude that there is a complex interaction between Nrp2 and alternative pro-survival mechanisms in aggressive CRC, which could be therapeutically exploited. Abstract Neuropilin-2 (Nrp2), an important regulator of lymphangiogenesis and lymphatic metastasis, has been associated with progression in colorectal cancer (CRC). However, the tumor cell-intrinsic role of Nrp2 in cancer progression is incompletely understood. To address this question, we employed CRISPR-Cas9 technology to generate Nrp2-knockout organoids derived from murine CRC tumors with a mesenchymal phenotype. Transcriptome profiling and tumor tissue analysis showed that Nrp2 loss resulted in mesenchymal-to-epithelial transition (MET), which was accompanied with restored polarity and tight junction stabilization. Signaling pathway analysis revealed that Nrp2-knockout organoids acquire de novo dependency on insulin receptor (IR) signaling and autophagy as alternative survival mechanisms. Combined inhibition of IR signaling and autophagy prevented the stabilization of cell-cell junctions, reduced metabolic activity, and caused profound cell death in Nrp2-knockout organoids. Collectively, the data demonstrate a key role for Nrp2 in maintaining the aggressive phenotype and survival of tumor-derived CRC organoids. The identified connection between Nrp2, insulin receptor signaling and autophagy may guide the development of novel combination-treatment strategies for aggressive CRC.