Recent advancements in tumour microenvironment landscaping for target selection and response prediction in immune checkpoint therapies achieved through spatial protein multiplexing analysis.

Immune checkpoint therapies have significantly advanced cancer treatment. Nevertheless, the high costs and potential adverse effects associated with these therapies highlight the need for better predictive biomarkers to identify patients who are most likely to benefit from treatment. Unfortunately, the existing biomarkers are insufficient to identify such patients. New high-dimensional spatial technologies have emerged as a valuable tool for discovering novel biomarkers by analysing multiple protein markers at a single-cell resolution in tissue samples. These technologies provide a more comprehensive map of tissue composition, cell functionality, and interactions between different cell types in the tumour microenvironment. In this review, we provide an overview of how spatial protein-based multiplexing technologies have fuelled biomarker discovery and advanced the field of immunotherapy. In particular, we will focus on how these technologies contributed to (i) characterise the tumour microenvironment, (ii) understand the role of tumour heterogeneity, (iii) study the interplay of the immune microenvironment and tumour progression, (iv) discover biomarkers for immune checkpoint therapies (v) suggest novel therapeutic strategies.

Ambra1 modulates the tumor immune microenvironment and response to PD-1 blockade in melanoma.

BACKGROUND: Loss of Ambra1 (autophagy and beclin 1 regulator 1), a multifunctional scaffold protein, promotes the formation of nevi and contributes to several phases of melanoma development. The suppressive functions of Ambra1 in melanoma are mediated by negative regulation of cell proliferation and invasion; however, evidence suggests that loss of Ambra1 may also affect the melanoma microenvironment. Here, we investigate the possible impact of Ambra1 on antitumor immunity and response to immunotherapy. METHODS: This study was performed using an Ambra1-depleted BrafV600E /Pten-/ - genetically engineered mouse (GEM) model of melanoma, as well as GEM-derived allografts of BrafV600E /Pten-/ - and BrafV600E /Pten-/ -/Cdkn2a-/ - tumors with Ambra1 knockdown. The effects of Ambra1 loss on the tumor immune microenvironment (TIME) were analyzed using NanoString technology, multiplex immunohistochemistry, and flow cytometry. Transcriptome and CIBERSORT digital cytometry analyses of murine melanoma samples and human melanoma patients (The Cancer Genome Atlas) were applied to determine the immune cell populations in null or low-expressing AMBRA1 melanoma. The contribution of Ambra1 on T-cell migration was evaluated using a cytokine array and flow cytometry. Tumor growth kinetics and overall survival analysis in BrafV600E /Pten-/ -/Cdkn2a-/ - mice with Ambra1 knockdown were evaluated prior to and after administration of a programmed cell death protein-1 (PD-1) inhibitor. RESULTS: Loss of Ambra1 was associated with altered expression of a wide range of cytokines and chemokines as well as decreased infiltration of tumors by regulatory T cells, a subpopulation of T cells with potent immune-suppressive properties. These changes in TIME composition were associated with the autophagic function of Ambra1. In the BrafV600E /Pten-/ -/Cdkn2a-/ - model inherently resistant to immune checkpoint blockade, knockdown of Ambra1 led to accelerated tumor growth and reduced overall survival, but at the same time conferred sensitivity to anti-PD-1 treatment. CONCLUSIONS: This study shows that loss of Ambra1 affects the TIME and the antitumor immune response in melanoma, highlighting new functions of Ambra1 in the regulation of melanoma biology.

Multiplexed Immunohistochemistry and Digital Pathology as the Foundation for Next-Generation Pathology in Melanoma: Methodological Comparison and Future Clinical Applications.

The state-of-the-art for melanoma treatment has recently witnessed an enormous revolution, evolving from a chemotherapeutic, "one-drug-for-all" approach, to a tailored molecular- and immunological-based approach with the potential to make personalized therapy a reality. Nevertheless, methods still have to improve a lot before these can reliably characterize all the tumoral features that make each patient unique. While the clinical introduction of next-generation sequencing has made it possible to match mutational profiles to specific targeted therapies, improving response rates to immunotherapy will similarly require a deep understanding of the immune microenvironment and the specific contribution of each component in a patient-specific way. Recent advancements in artificial intelligence and single-cell profiling of resected tumor samples are paving the way for this challenging task. In this review, we provide an overview of the state-of-the-art in artificial intelligence and multiplexed immunohistochemistry in pathology, and how these bear the potential to improve diagnostics and therapy matching in melanoma. A major asset of in-situ single-cell profiling methods is that these preserve the spatial distribution of the cells in the tissue, allowing researchers to not only determine the cellular composition of the tumoral microenvironment, but also study tissue sociology, making inferences about specific cell-cell interactions and visualizing distinctive cellular architectures - all features that have an impact on anti-tumoral response rates. Despite the many advantages, the introduction of these approaches requires the digitization of tissue slides and the development of standardized analysis pipelines which pose substantial challenges that need to be addressed before these can enter clinical routine.