NanoCellAnnotator: Formalizing Expert Cell Type Annotation with Large Language Models

Motivation: Cell-type annotation in spatial transcriptomics is challenging due to sparse gene panels, spatial heterogeneity, and limited availability of tissue-matched reference atlases. Recent approaches have explored large language models (LLMs) for integrating biological knowledge during annotation, but unconstrained inference can produce biologically unsupported predictions and hallucinated cell types. In addition, many LLM-based pipelines rely on large cloud-hosted models that limit reproducibility and deployment in privacy-sensitive environments. Results: We introduce NanoCellAnnotator, a biologically constrained and confidence-aware framework for automated cell-type annotation in spatial transcriptomics. The framework de-couples spatial structure discovery, deterministic biological evidence construction, and language model-based semantic inference. Spatial clusters are identified using hybrid spatially regularized non-negative matrix factorization (hSNMF), after which cluster-level marker genes are abstracted into ontology-derived functional programs using Gene Ontology enrichment and GO-slim projection. A lightweight locally executable language model performs constrained label selection within a curated admissible label space derived from PanglaoDB and CellMarker. Annotation confidence is estimated independently using marker support strength and lineage separation, enabling ambiguous or heterogeneous clusters to be explicitly flagged. We evaluate NanoCellAnnotator on Xenium spatial transcriptomics data from intrahepatic cholangiocarci-noma and an independent breast cancer spatial transcriptomics dataset. The framework recovers canonical cell populations with high confidence while identifying heterogeneous or transitional spatial domains as ambiguous. Agreement with manual annotations was evaluated using accuracy and adjusted Rand index. Availability: Code available at https://github.com/ishtyaqmahmud/NanoCellAnnotator.