Background Harnessing tissue-resident immunity for tumour clearance requires approaches that rescue local effector CD8+ T cells from intrinsic dysfunction, while overcoming extrinsic suppression by regulatory immune counterparts within the tumour niche. Thus, sampling immune cells sequestered in tumours is critical to the discovery of targetable regulatory pathways and subsets to enhance immunotherapy responses. In hepatocellular carcinoma (HCC), diagnostic core biopsies often contain a mixture of immune cells from tumour and surrounding liver, and their invasive nature has restricted their application. In recent years, our group demonstrated the utility of fine needle aspiration (FNA) for immune surveillance in chronic liver disease.¹ Here, we evaluated the hypothesis that FNA sampling of liver tumours would offer a minimally invasive approach to monitoring distinct tissue-compartmentalised immune responses to dissect primary and secondary mechanisms of tumour immune escape and immunotherapy resistance.

Methods Matched blood, FNA and core biopsy samples were obtained concurrently from patients with advanced HCC prior to undergoing systemic therapy. FNA was performed using a 22-gauge spinal needle, while the biopsy was obtained using an 18-gauge needle via a 17-gauge co-axial approach. Peripheral blood mononuclear cells (PBMC) from blood (n=25) and tumour infiltrating leukocytes (TIL) from FNA (n= 25) and biopsy (n=15) were isolated for ex-vivo spectral multi-parameter (36-colour) flow cytometric characterisation of effector and regulatory immune populations.

Results FNA reproducibly yielded a broad range of viable immune subsets local to the tumour microenvironment (TME). Crucially, FNA were able to extract tissue-resident CD8⁺ T cells (T_RM, CD69⁺CD103⁺CD8) that could not be sampled in blood. The majority of tumour CD8⁺ T_RM sampled before immunotherapy expressed inhibitory immune checkpoints including PD-1, Tim-3 and 2B4, accounting for significantly higher expression of these therapeutic targets on the global CD8⁺ T cells extracted from FNA or biopsies than from blood. Within the myeloid compartment, dendritic cells were reduced whereas granulocytic/polymorphonuclear myeloid derived suppressor cells (g-MDSC/PMN-MDSC) were significantly expanded in tumour FNA compared to blood, allowing characterisation of their dominant homing and immunoregulatory mechanisms to define novel therapeutic targets.

Conclusions We show that minimally invasive FNA are capable of comprehensively sampling the distinct immune landscape of HCC compartmentalised at the site of disease, including the expansion of key immunotherapeutic targets such as T-cell checkpoints and g-MDSCs. The resulting data have enabled characterisation of these immunotherapeutic targets and hold potential for future personalised refinement of targeted immunotherapy to overcome tumour immune escape.

Reference

1. Gill US, Pallett LJ, Thomas N, Burton AR, Patel AA, Yona S, et al. Fine needle aspirates comprehensively sample intrahepatic immunity. Gut 2019;68:1493–503.

Ethics Approval This study was granted ethical approval to obtain patient blood and tumour samples by the UK West Midlands -Solihull Research Ethics Committee (REC reference 21/WM/0205).