Ex vivo modelling of the immune response in cutaneous squamous cell carcinoma

Abstract

Cutaneous squamous cell carcinoma (cSCC) is one of the commonest cancers worldwide and mortality from advanced and metastatic disease is increasing year-on-year. Anti-programmed cell death-1 (PD-1) therapy is now licensed for the therapy of advanced and metastatic cSCC, but complete responses to this immunotherapy are seen in fewer than 15% of patients. There is an urgent need to better understand cSCC immunology to ensure that the investigation and development of novel therapies and combinations of therapies is targeted appropriately. While the PD-1 checkpoint axis is key in the management of advanced cSCC, our understanding of its effect on the tumour-immune microenvironment is limited and it is essential to build on this to identify factors that might either predict or improve treatment outcomes.

To enable ex vivo investigation of immunotherapeutic molecules in human cSCC, a patient-derived explant model of cSCC was developed using a tissue slice culture (TSC) system. Using this TSC system with freshly excised primary human cSCCs, tissue slices remained viable in culture over several days, maintaining tumour microarchitecture and producing consistent, reproducible, dose-dependent immune responses to anti-CD3 stimulation and inhibition with ciclosporin A. Treatment of cSCC in TSC with anti-programmed death-ligand 1 (PD-L1) therapy potentiated T cell immune responses, as did treatment with an anti-CD28 agonist (TGN1412), although this potentiation was not seen with anti-4-1BB agonist therapy. Combining anti-PD-L1 with anti-CD28 treatment demonstrated an additive effect, highlighting a potential avenue for potentiating treatment of the PD-1 checkpoint axis.

One of the key determinants of response to to PD-1 therapy in many cancers is the presence of intra- and peritumorual lymphoid aggregates known as tertiary lymphoid structures (TLSs). They are known to be present in cSCC, but their influence on tumour biology is poorly understood. Tumours that contain TLSs can be identified through gene signatures in their transcriptomes. A systematic search of the literature identified nine independent gene signatures validated for the identification of TLS-containing tumours in RNA seq data across a range of cancers. To investigate the presence of these signatures in cSCC, RNA seq data from eleven publicly available datasets comprising 180 cSCC and 85 normal skin samples was integrated and uniformly processed. Gene Set Variation Analysis (GSVA) using these gene sets enabled samples to be clustered into TLS-high and TLS-low groups and cSCCs were significantly more likely to be present in the TLS-high cluster. TLS-high tumours were positively associated with prognostic scores that suggested a tendency to well-differentiated tumours, lower risk of metastasis, and better response to immunotherapy.

Having validated this cSCC RNA seq dataset in the identification of TLSs, it was used to identify evidence of adenosinergic signalling in cSCC. The adenosinergic pathway is known to induce a pro-tumoural immune response and is a potentially promising therapeutic target in immuno-oncology. In cSCC, intratumoural adenosine concentrations are high, and CD39 (a key marker of the adenosinergic pathway) is associated with poor outcome. Therefore, it was deemed sensible to characterise the influence of the adenosinergic pathway in cSCC. The combination of immunohistochemistry, immunofluorescence and flow cytometry demonstrated that CD73 and CD39 were expressed on various cells, including subsets of lymphocytes and endothelial cells, within the tumour microenvironment. At the RNA level, CD39 expression was higher in T cells from cSCC than from normal skin. A high gene signature of adenosinergic signalling strongly correlated with the TLS signature and associated with increased expression of genes and gene modules associated with immune response, angiogenesis, and hypoxia.

In conclusion, this thesis reports on the development an effective ex vivo TSC system to investigate cSCC immunology and immunotherapy and highlights a promising combination therapy targeting anti-PD-L1/anti-CD28 combination. Additionally, it characterises key roles for both TLSs and the adenosinergic pathway that influence tumour immunity and prognosis. Taken together, this identifies potential future treatment targets that could improve therapeutic responses in cSCC.

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Identifiers

ORCID for George Stewart Coltart: orcid.org/0000-0001-7648-8741

ORCID for Eugene Healy: orcid.org/0000-0001-5591-6970

ORCID for Matthew Rose-Zerilli: orcid.org/0000-0002-1064-5350

ORCID for Aymen Al-Shamkhani: orcid.org/0000-0003-0727-4189

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