Development of spatial B cell antigen mapping to resolve TLS function in pancreatic cancer

Abstract public domain (NIH RePORTER)

PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers, with a five-year survival rate under 15% and limited therapeutic options. A striking but unresolved feature of PDAC is the presence of tertiary lymphoid structures (TLSs), ectopic immune aggregates that resemble germinal centers and have been linked to improved prognosis and responsiveness to immunotherapy in many other cancers. In PDAC, however, TLSs emerge early in precursor pancreatic intraepithelial neoplasias (PanINs) and persist into invasive disease, yet they appear ineffective in promoting durable antitumor immunity. Why TLSs sometimes promote tumor control while in other cases permit immune escape remains a fundamental and unanswered question. A central barrier to progress is the inability to determine B cell antigen specificity in situ. Current spatial transcriptomic platforms can recover B cell receptor (BCR) sequences and clonal architecture, but they cannot identify cognate antigens. Dissociative methods can link BCRs to antigens, but the spatial and organizational context of TLSs is lost in the process. Without technologies that preserve both antigen specificity and tissue context, it is impossible to define whether TLSs mount tumor-specific immune responses, react to bystander antigens, or facilitate immune evasion In Aim 1, we will engineer DNA-barcoded antigen libraries compatible with spatial transcriptomic arrays, validating the system in the well-established HEL/MD4 mouse model before expanding to a PDAC-focused antigen panel. Animal models are required for these studies because the HEL/MD4 system provides a controlled setting with defined B cell antigen specificity in tissues that cannot be replicated in vitro, enabling validation of antigen detection accuracy and sensitivity prior to application in human tissues. In Aim 2, we will apply spatial BCR sequencing and transcriptome profiling to frozen PanIN and PDAC specimens to define B cell clonal evolution and TLS organization, and, where feasible, integrate antigen mapping to determine whether TLSs harbor tumor-specific, bystander, or immuneevasive responses. Importantly, even without antigen mapping, these analyses will yield high-value insights into TLS clonal architecture, maturation states, and their relationship to disease progression. The significance of this work is twofold. First, it will establish a generalizable platform capable of resolving B cell antigen specificity, clonal identity, transcriptional program, and tissue location in intact tissue. Second, it will apply this technology to PDAC, a cancer of urgent clinical need, thereby uncovering mechanisms that may explain why TLSs support tumor control in some settings but fail in others. By combining technological innovation with application to a pressing biological problem, this exploratory R21 project is positioned to generate transformative insights into the role of TLSs in cancer progression.

Public health relevance public domain (NIH RePORTER)

PROJECT NARRATIVE Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers, in part because the immune system fails to mount effective antitumor responses. This project will develop a spatial transcriptomics platform to directly map B cell antigen specificity in tissue, enabling discovery of how tertiary lymphoid structures influence PDAC progression. Insights from this work may inform new therapeutic strategies to reprogram immune responses and improve outcomes for patients with pancreatic cancer and beyond.