Spatial mapping of the hepatocellular carcinoma landscape identifies unique intratumoural perivascular-immune neighbourhoods

Background & Aims Hepatocellular carcinoma (HCC) develops in the context of chronic inflammation, however, the opposing roles the immune system plays in both the development and control of tumours is not fully understood. Mapping immune cell interactions across the distinct tissue regions could provide greater insight into the role individual immune populations have within tumours. Approach & Results Using highly multiplexed imaging mass cytometry, we have mapped the immune landscape of tumour, invasive margin, and adjacent non-tumour regions across sixteen resected tumours comprising of 144 regions of interest and over 10,000 parameters. We show immune cell densities remain largely consistent across these three regions, except for subsets of monocyte-derived macrophages which are enriched within the tumours. Mapping cellular interactions across these regions in an unbiased manner identifies immune neighbourhoods comprised of tissue-resident T cells, dendritic cells, and various macrophage populations around perivascular spaces. Importantly, we identify multiple immune cells within these neighbourhoods interacting with VEGF+ perivascular macrophages. Conclusions Cellular interactions, but not cell densities, differ between intratumoural and adjacent non-tumour regions in HCC. Unique intratumoural immune neighbourhoods around the perivascular space points to an altered landscape within tumours. Enrichment of VEGF+ perivascular macrophages within these tumours could play a key role in angiogenesis and vascular permeability. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was funded by the Joan Krefft bequest to the A.W. Morrow Gastroenterology and Liver Centre (Royal Prince Alfred Hospital) and Cancer Institute NSW (2021/ATRG2028). Felix Marsh-Wakefield is supported by the International Society for the Advancement of Cytometry (ISAC) Marylou Ingram Scholars Program. Cositha Santhakumar is supported by the Sydney Cancer Partners PhD scholarship. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Institutional human research ethics was obtained by the Sydney Local Health District Ethics Review Committee (HREC 2020/ETH02093). Research was conducted in accordance with both the Declarations of Helsinki and Istanbul, with written consent given in writing by all subjects. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. * HCC : Hepatocellular carcinoma VEGF : Vascular endothelial growth factor TME : Tumour immune microenvironment IMC : Imaging mass cytometry FFPE : Formalin-fixed paraffin embedded MVI : Microvascular invasion H&E : Haematoxylin and eosin ROI : Region of interest TIL : Tumour-infiltrating lymphocyte TMA : Tissue microarray IHC : Immunohistochemistry RT : Room temperature mIHC : Multiplex immunohistochemistry TSA : Tyramide signalling amplification PCA : Principal component analysis sPLS-DA : Sparse partial least squares-discriminant analysis TNM : Tumour-node-metastasis PERMANOVA : Permutational multivariate analysis of variance PVM : perivascular macrophage TGF-β : transforming growth factor-beta TAM : tumour-associated macrophage MRS : myeloid response score MAIT : cells mucosal-associated invariant T cells