189P - Image-guided labeling of live cells in tertiary lymphoid structures for single-cell transcriptomics

Background

Tertiary lymphoid structures (TLSs) are lymphocyte aggregates that correlate with better prognosis in most cancers. TLSs possess anti-tumor potentials but their compositions and functions vary across cancer types and stages, so our understanding of TLS remains largely descriptive. Current studies are limited to fixed pathological samples for immunohistochemistry or spatial transcriptomics, yielding restricted mRNA content in rare cell types. To study the anti-tumor role of TLS, we choose prostate cancer, cause of one of the highest cancer-related deaths in U.S, as our disease model. Castration-resistant prostate cancer (CRPC) is a devastating disease state with high mortality and limited treatment options, including immune checkpoint therapy (ICT). Due to the positive correlation between TLS and ICT response, we want to promote the anti-tumor potentials of TLS in CRPC.

Methods

We aim to develop a region-specific in situ labeling methodology to capture live TLS cells in CRPC mice for characterizations. Since loss of PTEN is a common mutation in prostate cancer, we employed PB-Cre⁺;Pten^loxp/loxp mice to elucidate TLS composition and function in CRPC. Through photocatalytic labeling in tissue sections, we covalently tagged biotin to live TLS cells, followed by biotin⁺ TLS isolation for single-cell RNA sequencing (scRNA-seq) analysis.

Results

Our region-specific labeling method is validated in cell lines and tissue sections, including germinal center (GC) enrichment. Our study identified key clusters within CRPC TLS, including CD8⁺T, follicular helper T (Tfh), GC B, Mki67⁺ T/B cells and Naaa⁺ dendritic cells (DCs), among others. TLS also enriched for certain clones in TCR/BCR repertoires.

Conclusions

Through photocatalytic covalent labeling with biotin, we achieved image-guided labeling of live TLS cells for scRNA-seq and identified TLS-enriched cells with potential functions in antigen presentation, cytotoxicity and antibody secretion. We anticipate detecting changes in TLS signatures and functions following PI3K drug treatment, enabling a comprehensive analysis of TLS. Moreover, we plan to refine our methodology to expand the scope of application in other crucial biomedical research areas.

Legal entity responsible for the study

Peking University.

Funding

Peking University; Shenzhen Bay Laboratory.

Disclosure

All authors have declared no conflicts of interest.