Doffin, A.-C. | Search bioRxiv

bioRxiv2025

Spatial tumour-immune ecosystems shape the efficacy of anti-PD1 immunotherapy in primary cutaneous melanoma

Intra-tumoral heterogeneity in melanoma arises from dynamic cancer cell plasticity and underlies various mechanisms of immune escape. Here, we combined high-plex immunofluorescence imaging with spatially resolved transcriptomics to map the architecture of melanoma cell states and their interactions with the immune microenvironment in primary cutaneous tumours prior to adjuvant anti-PD1 immune checkpoint inhibitor (ICI) treatment. Computational analyses showed that melanoma cells organise into spatially restricted patches, with a preferential organisation of undifferentiated cells associated with poor ICI efficacy. Neighbouring immune cell composition varied according to cancer cell states, with a crucial involvement of specific subsets of tumour-associated macrophages, driven by signalling pathways involving tumour-derived and microenvironmental cues such as IFN-{gamma} and hypoxia. Integrated spatial analyses further revealed tumour-immune ecosystems that stratify patient outcomes, delineating configurations either associated with ICI efficacy or metastatic relapse. These results uncover the spatial landscape of tumour ecosystems and identify signalling pathways as potential targets for improving the efficacy of ICI in melanoma. HighlightsO_LIMelanoma cell states spatial organization is associated with aPD1 therapy efficacy C_LIO_LISpatial organization of TAM subsets is a crucial determinant of ICI outcome C_LIO_LIMelanoma cells in different states co-localise with functionally distinct TAM subsets C_LIO_LIIdentification of cell-cell communication pathways that underlie tumour-TAM crosstalk C_LIO_LIA balance between tumour-immune ecosystems is associated with aPD1 therapy efficacy C_LI

cancer biology↗

bioRxiv2025

IntegrAlign: A comprehensive tool for multiimmunofluorescence panel integration through image alignment

MotivationTyramide-based multiplex-immunofluorescence (mIF) enables the simultaneous analysis of up to seven protein markers on a whole slide, providing a comprehensive approach to study the tumor microenvironment. Integrating multiple mIF panels through image alignment of serial slide significantly expands the number of cell populations analyzed in a single space. IntegrAlign was developed to optimize this integration on serial whole slides, enhancing the value and applicability of mIF for comprehensive spatial analyses and enabling biomarker discovery at scale. ResultsIntegrAlign, leveraging the SimpleITK toolkit, applies a two-step alignment using rigid and B-spline transformations to integrate serial mIF whole slides. Validation on simulated and real datasets demonstrated alignment accuracy below the diameter of a cell nucleus ([~]6 {micro}m), outperforming existing methods. This precision enhances spatial analyses by combining extended phenotypic data, supporting novel insights into tissue architecture and cellular interactions. Availability and ImplementationIntegrAlign is open-source, implemented in Python, and freely available under the MIT license at https://github.com/CAUXlab/IntegrAlign.

bioinformatics↗

bioRxiv2024

Spatial architecture of CD8+ T cells and DC subsets is critical for the response to immune checkpoint inhibitors in melanoma

BackgroundDendritic cells (DCs) are promising targets for cancer immunotherapies owing to their central role in the initiation and the control of immune responses. Their functions encompass a wide range of mechanisms mediated by different DC subsets. Several studies have identified human tumor- associated DC (TA-DC) populations through limited marker-based technologies, such as immunostaining or flow cytometry. However, tumor infiltration, spatial organization and specific functions in response to immunotherapy of each DC subset remain to be defined. MethodsHere, we implemented a multiplexed immunofluorescence analysis pipeline coupled with bio-informatic analyses to decipher the tumor DC landscape and its spatial organization within melanoma patients lesions, and its association with patients response to immune checkpoint inhibitors (ICI). For this aim, we analyze a cohort of 41 advanced melanoma patients treated with anti- PD1 alone or associated with anti-CTLA4. Distance and cell network analyses were performed to gain further insight into the spatial organization of tumor-associated DCs. A Digital Spatial Profiling analysis further characterized ecosystem of tumor-infiltrating DCs. ResultsPlasmacytoid DCs (pDCs) were the most abundant DC population, followed by conventional cDC1 and mature DCs, present in equal proportions. In contrast to CD8+ T cell frequency, and despite varying densities, all DC subsets were associated with a favorable response to ICI. Distance and cell network analyses demonstrated that tumor-infiltrating DCs were largely organized in dense areas with high homotypic connections, except for cDC1 that exhibited a more scattered distribution. We identified four patterns of ecosystems with distinct preferential interactions between DC subsets. Significantly, the proximity and interactions between CD8+ T cells and cDC1 were positively associated with patients response to ICI. ConclusionsOur study unravels the complex spatial organization of DC subsets and their interactions in melanoma patient lesions, shedding light on their pivotal role in shaping the response to ICI. Our discoveries regarding the spatial arrangement of cDC1, especially with CD8+ T cells, provide valuable clues for improving immunotherapeutic strategies in melanoma patients. What is already known on this topicDendritic cells (DCs) are promising targets for cancer immunotherapies owing to their central role in the initiation and the control of immune responses. Although conventional type 1 dendritic cells (cDC1) were proposed to contribute to immunotherapy response, their precise functions and interactions with other immune populations in human cancers are largely unknown. What this study addsThis study provides a precise characterization of the spatial distribution and organization of tumor- infiltrating DCs in a large cohort of advanced melanoma patients, and in correlation with response to immunotherapy. While DCs are organized in dense areas with high homotypic connections, cDC1 exhibit a more scattered distribution and form heterotypic aggregates with other DC subsets. More importantly, a close connection between cDC1 and CD8 T cell is uniquely correlated with the patients response to immunotherapy. How this study might affect research, practice or policyThis study improves our understanding of CD8-DC spatial organization within the tumor microenvironment and will have a broad spectrum of implications in the design of anti-tumor immune-activating compounds and the design of biomarkers of response to immunotherapy for melanoma patients.

cancer biology↗