The discovery of immune checkpoint proteins such as PD-1/PDL-1 and CTLA-4 represents a significant breakthrough in the field of cancer immunotherapy. Therefore, humanized monoclonal antibodies, targeting these immune checkpoint proteins have been utilized successfully in patients with metastatic melanoma, renal cell carcinoma, head and neck cancers and non-small lung cancer. The US FDA has successfully approved three different categories of immune checkpoint inhibitors (ICIs) such as PD-1 inhibitors (Nivolumab, Pembrolizumab, and Cemiplimab), PDL-1 inhibitors (Atezolimumab, Durvalumab and Avelumab), and CTLA-4 inhibitor (Ipilimumab). Unfortunately, not all patients respond favourably to these drugs, highlighting the role of biomarkers such as Tumour mutation burden (TMB), PDL-1 expression, microbiome, hypoxia, interferon-γ, and ECM in predicting responses to ICIs-based immunotherapy. The current study aims to review the literature and updates on ICIs in cancer therapy.

Background: Recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) generally has a poor prognosis for patients with limited treatment options. While incorporating immune checkpoint inhibitors (ICIs) has now become the standard of care, the efficacy is variable, with only a subset of patients responding. The complexity of the tumor microenvironment (TME) and the role of tertiary lymphoid structures (TLS) have emerged as critical determinants for immunotherapeutic response. Methods: In this study, we analyzed two independently collected R/M HNSCC patient tissue cohorts to better understand the role of TLS in response to ICIs. Utilizing a multi-omics approach, we first performed targeted proteomic profiling using the Nanostring GeoMx Digital Spatial Profiler to quantify immune-related protein expression with spatial resolution. This was further characterized by spatially resolved whole transcriptome profiling of TLSs and germinal centers (GCs). Deeper single-cell resolved proteomic profiling of the TLSs was performed using the Akoya Biosciences Phenocycler Fusion platform. Results: Our proteomic analysis revealed the presence of T lymphocyte markers, including CD3, CD45, and CD8, expressing cells and upregulation of immune checkpoint marker PD-L1 within tumor compartments of patients responsive to ICIs, indicative of ‘hot tumor’ phenotypes. We also observed the presence of antigen-presenting cells marked by expression of CD40, CD68, CD11c, and CD163 with upregulation of antigen-presentation marker HLA-DR, in patients responding to ICIs. Transcriptome analysis of TLS and GCs uncovered a marked elevation in the expression of genes related to immune modulation, diverse immune cell recruitment, and a potent interferon response within the TLS structure. Notably, the distribution of TLS-tumor distance was found to be significantly different across response groups (H = 9.28, p = 0.026). The proximity of TLSs to tumor cells was found to be a critical indicator of ICI response, implying that patients with TLSs located further from tumor cells have worse outcomes. Conclusion: The study underscores the multifaceted role of TLSs in modulating the immunogenic landscape of the TME in R/M HNSCC, likely influencing the efficacy of ICIs. Spatially resolved multi-omics approaches offer valuable insights into potential biomarkers for ICI response and highlight the importance of profiling the TME complexity when developing therapeutic strategies and patient stratification.

Cancer immunotherapy has been rejuvenated by the growing understanding of the immune system's role in tumor activity over the past two decades. During cancer initiation and progression, tumor cells employ various mechanisms that resemble peripheral immune tolerance to evade the antitumor responses of the immune system. Immune checkpoint molecules are the major mechanism of immune resistance that are exploited by tumor cells to inhibit T‐cell activation and suppress immune responses. The targeting of immune checkpoint pathways has led to substantial improvements in survival rates in a number of solid cancers. However, a lack of understanding of the heterogeneity of the tumor microenvironment (TME) has resulted in inefficient therapy responses. A greater understanding of the TME is needed to identify patients likely to respond, and those that will have resistance to immune checkpoint inhibitors (ICIs). Advancement in spatial single‐cell technologies has allowed deeper insight into the phenotypic and functional diversities of cells in the TME. In this review, we provide an overview of ICI biomarkers and highlight how high‐dimensional spatially resolved, single‐cell approaches provide deep molecular insights into the TME and allow for the discovery of biomarkers of clinical benefit.

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