Multiplexed Pan Soluble Ligandome Assaying via OASIS

Area of Science:

• Cell biology
• Molecular biology
• Biotechnology

Background:

• Screening soluble protein ligands is crucial for understanding cell signaling and drug discovery.
• Current methods require arrayed formats due to non-cell-autonomous effects from ligand diffusion.
• A need exists for multiplexed pooled assaying of ligands.

Purpose of the Study:

• To develop a novel platform, Obligate Autocrine Signaling In Situ Screening (OASIS), for pooled screening of soluble protein ligands.
• To enable exclusively autocrine signaling by anchoring ligands to the expressing cell surface.
• To accelerate the functional interrogation of the human ligandome.

Main Methods:

• Developed OASIS using lentiviral delivery of genetically barcoded ligands fused to a tethering domain.
• Anchored proteins to the expressing cell's outer membrane to enforce autocrine signaling.
• Validated OASIS with IFNA2 and EGF, and performed a pan-ligandome fitness screen in hiPSCs, followed by single-cell Perturb-Seq.

Main Results:

• OASIS demonstrated uncompromised autocrine signaling with significantly reduced paracrine activity.
• A pan-ligandome screen identified potent self-renewal factors, including FGF family ligands.
• Single-cell Perturb-Seq mapped transcriptional remodeling induced by the ligandome library.

Conclusions:

• OASIS is a platform enabling pooled assaying of soluble ligands by enforcing autocrine signaling.
• The platform was validated by assaying all human ligands in hiPSCs, measuring fitness and transcriptional impacts.
• OASIS facilitates rapid interrogation of ligands and engineered binders for cell signaling studies.