GPR124 regulates murine brain embryonic angiogenesis and BBB formation by an intracellular domain-independent mechanism.

The GPR124/RECK/WNT7 pathway is an essential regulator of CNS angiogenesis and blood-brain barrier (BBB) function. GPR124, a brain endothelial adhesion seven-pass transmembrane protein, associates with RECK, which binds and stabilizes newly synthesized WNT7 that is transferred to frizzled (FZD) to initiate canonical ß-catenin signaling. GPR124 remains enigmatic: although its extracellular domain (ECD) is essential, the poorly conserved intracellular domain (ICD) appears to be variably required in mammals versus zebrafish, potentially via adaptor protein bridging of GPR124 and FZD ICDs. GPR124 ICD deletion impairs zebrafish angiogenesis, but paradoxically retains WNT7 signaling upon mammalian transfection. We thus investigated GPR124 ICD function using the mouse deletion mutant Gpr124ΔC. Despite inefficiently expressed GPR124ΔC protein, Gpr124ΔC/ΔC mice could be born with normal cerebral cortex angiogenesis, in comparison with Gpr124-/- embryonic lethality, forebrain avascularity and hemorrhage. Gpr124ΔC/ΔC vascular phenotypes were restricted to sporadic ganglionic eminence angiogenic defects, attributable to impaired GPR124ΔC protein expression. Furthermore, Gpr124ΔC and the recombinant GPR124 ECD rescued WNT7 signaling in culture upon brain endothelial Gpr124 knockdown. Thus, in mice, GPR124-regulated CNS forebrain angiogenesis and BBB function are exerted by ICD-independent functionality, extending the signaling mechanisms used by adhesion seven-pass transmembrane receptors.

Microdissection tools to generate organoids for modeling the tumor immune microenvironment.

Patient-derived tumor organoids have emerged as promising models for predicting personalized drug responses in cancer therapy, but they typically lack immune components. Preserving the in vivo association between tumor cells and endogenous immune cells is critical for accurate testing of cancer immunotherapies. Mechanical dissection of tumor specimens into tumor fragments, as opposed to enzymatic digestion into single cells, is essential for maintaining these native tumor-immune cell spatial relationships. However, conventional mechanical dissection relying on manual mincing is time-consuming and irreproducible. This study describes two microdissection devices, the µDicer and µGrater, to facilitate the generation of intact tumor fragments from mouse B16 melanoma, a common model of human melanoma. The µDicer- and µGrater-cut tumor fragments were used to generate air‒liquid interface (ALI) organoids that copreserve tumor cells with infiltrating immune subsets without artificial reconstitution. The µDicer, consisting of a hexagonal array of silicon microblades, was employed to investigate the effect of organoid size. The viability of ALI organoid immune cells appeared insensitive to organoid sizes exceeding ~400 µm but diminished in organoids ~200 µm in size. The µGrater, consisting of an array of submillimeter holes in stainless steel, was employed to accelerate dissection. For the samples studied, the µGrater was 4.5 times faster than manual mincing. Compared with those generated by manual mincing, ALI organoids generated by the µGrater demonstrated similar viability, immune cell composition, and responses to anti-PD-1 immunotherapy. With further optimization, the µGrater holds potential for integration into clinical workflows to support the advancement of personalized cancer immunotherapy.