Investigating the Role of Tertiary Lymphoid Structures in Tumour-Mediated Immunosuppression in pancreaticobiliary cancer employing spatial biology characterisation.

Dr Nigel Jamieson, Dr Peter Bankhead, Professor John Le Quesne

PDAC has one of the worst outcomes of any cancer, with a 5-year survival of less than 5%. It persists as the 4th, and, due to lack of progress, will soon be the 2nd leading cause of cancer death in our society. Current standard of care therapeutic options are and the platinum-based combination FOLFIRINOX, which extend median survival, to 8.5 and 11months respectively. Immunotherapeutic strategies are showing efficacy in several other cancer types including melanoma and lung cancer, but progress in PDAC remains slow with poor efficacy of single agent regimens. However, our previous work, and the work of others has revealed that understanding active immune avoidance mechanisms specific to PDAC, and targeting these using combination therapies can lead to meaningful responses (Steele et al, 2016).

Generally, for pancreaticobiliary malignancy, distant metastasis is the primary cause of cancer-related death. To colonize distant tissues, cancer cells must migrate while evading elimination by the immune system. Evidence suggests that key steps in the induction process of immune tolerance occur early in the metastatic cascade, located at regional lymph nodes. However, the nature of the interactions between tumour and immune cells remains poorly understood, particularly for those occurring within the lymph nodes. Despite lymph node involvement being commonly assessed in pancreatic cancer patients to determine disease stage and hence treatment plan, they are understudied in the context of metastatic progression. More recently it has become apparent that Tertiary lymphoid structures (TLSs) are lymphoid formations found in nonlymphoid tissues. TLS develop in inflamed tissues associated with chronic inflammatory disorders, autoimmunity, and in multiple cancer types including pancreaticobiliary.

In the context of tumours, TLSs facilitate the influx of immune cells to cancer and have therefore attracted interest as a means of improving anticancer immunity and favourable treatment response in patients. Our inhouse data demonstrates significantly improved outcomes for patients with pancreatic cancer who have TLS present within the resected tumours (Figure 1). Furthermore, the presence of TLS following neoadjuvant chemoradiotherapy therapy is not uniform and is associated with outcome.

To study the immunosuppressive role of lymph node metastases and TLS in human pancreaticobiliary cancer we will analyse a variety of retrospective and prospective tissue cohorts which will enable discovery and validation. This project will leverage the newly established techniques of spatial-omics, thereby retaining the spatial information which is usually lost when using tissue extraction and homogenisation techniques and non-spatially selective approaches such as bulk transcriptomic analysis. We propose three inter-connected approaches that focus our scientific theme via different platforms:

1) High-dimensional in-situ imaging

Whole section analysis (N=200) but also custom tissue microarrays of treatment naive (N=100) and neoadjuvantly treated pancreaticobiliary cancers (N=100) focussed on lymph nodes and TLS will be interrogated using Hi-plex (N=50) discovery protein assessment (CODEX) (Figure 2) and Lower plex (N=8) analysis across larger specimens.

2) Spatial transcriptomic (ST) analysis

We will undertake broad ST discovery using VISIUM and focussed regional and cell population ST analysis using the Nanostring GeoMx (Figure 3).

3) Single-cell RNASeq

In parallel, tissue collected as part of the Spatial-Panc prospective tissue resource will enable ScRNA-seq analysis of lymph node metastasis/ TLS from a subgroup of resections (performed by Nigel Jamieson).

These efforts will yield highly multiplexed, multi-scale datasets which will be analyzed by novel bio-computational methods and image analysis strategies to reconstruct intraregional (cellular neighbourhoods) and intercellular molecular interaction networks to identify, critical mediators of tumour immunosuppression. Our ultimate objective is to advance understanding of the systemic consequences of lymph node metastases and identify new therapeutic approaches to immunotherapy.

This strategy will be applicable across multiple cancer types being studied within the CRUK Scotland Centre, inform pre-clinical models including functional imaging, and enhance Clinical trial datasets including Precision-Panc.

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