ISREC-SCCL Symposium 2023

Investigation of the tumor microenvironment (TME) by multiplex immunofluorescence (mIF) has expanded our knowledge of the spatial immune context. mIF has proven a powerful technique for identifying new biomarkers and therapeutical targets. However, state-of-the-art mIF protocols remain technically challenging. Manual execution and use of dedicated reagents render them lengthy and costly. Their reproducibility and transferability between different tissue types are often questioned. 

Here, we show the validation of a new Immuno-Oncology (IO) Core Panel of 13 clinically relevant biomarkers to enable a spatial analysis of the TME on the COMET™ platform across various tissue types. 

Formalin-fixed paraffin-embedded human tissue sections from tonsils and a 24-core multi-organ tissue microarray (TMA) were stained using the IO Core Panel on the COMET™ platform by fully automated sequential immunofluorescence (seqIF™), which consists of cycles of staining, imaging, and elution. The panel allows for simultaneous detection of CD3, CD4, CD8, CD45, FOXP3, PD-1, PD-L1, CD11c, CD20, CD56, CD68, αSMA and Ki-67 by indirect immunofluorescence using unlabeled primary antibodies and Alexa Fluor™ Plus secondary antibodies. The 13-plex IO Core panel was developed and validated as positive control tissue on tonsils. To compare seqIF™ and immunohistochemistry (IHC) staining patterns, the sections retrieved from COMET™ after seqIF™ were stained by a histology facility with standard IHC established for routine pathological diagnosis. All markers demonstrate accurate detection with specific seqIF™ staining, comparable to gold standard IHC counterparts. Subsequently, the panel was transferred on a multi-organ TMA, including several tumoral and non-tumoral specimens, showing robust performance across multiple tissue types. The protocol was optimized to achieve high staining quality for all 13 markers regarding signal specificity, sensitivity, ratio to the background, and dynamic range. The repeatability and reproducibility of the automated IO Core Panel on the COMET™ platform were verified by day-to-day tests on one platform and among multiple platforms. 

Investigation of the tumor microenvironment (TME) by multiplex immunofluorescence (mIF) has expanded our knowledge of the spatial immune context. mIF has proven a powerful technique for identifying new biomarkers and therapeutical targets. However, state-of-the-art mIF protocols remain technically challenging. Manual execution and use of dedicated reagents render them lengthy and costly. Their reproducibility and transferability between different tissue types are often questioned. 

Here, we show the validation of a new Immuno-Oncology (IO) Core Panel of 13 clinically relevant biomarkers to enable a spatial analysis of the TME on the COMET™ platform across various tissue types. 

Formalin-fixed paraffin-embedded human tissue sections from tonsils and a 24-core multi-organ tissue microarray (TMA) were stained using the IO Core Panel on the COMET™ platform by fully automated sequential immunofluorescence (seqIF™), which consists of cycles of staining, imaging, and elution. The panel allows for simultaneous detection of CD3, CD4, CD8, CD45, FOXP3, PD-1, PD-L1, CD11c, CD20, CD56, CD68, αSMA and Ki-67 by indirect immunofluorescence using unlabeled primary antibodies and Alexa Fluor™ Plus secondary antibodies. The 13-plex IO Core panel was developed and validated as positive control tissue on tonsils. To compare seqIF™ and immunohistochemistry (IHC) staining patterns, the sections retrieved from COMET™ after seqIF™ were stained by a histology facility with standard IHC established for routine pathological diagnosis. All markers demonstrate accurate detection with specific seqIF™ staining, comparable to gold standard IHC counterparts. Subsequently, the panel was transferred on a multi-organ TMA, including several tumoral and non-tumoral specimens, showing robust performance across multiple tissue types. The protocol was optimized to achieve high staining quality for all 13 markers regarding signal specificity, sensitivity, ratio to the background, and dynamic range. The repeatability and reproducibility of the automated IO Core Panel on the COMET™ platform were verified by day-to-day tests on one platform and among multiple platforms. 

The concept of precision oncology emerged to match targeted therapies to the molecular profile of each tumor. However, the cellular composition and architecture of tumor tissues are additional parameters that influence response to therapies. Non–Hodgkin lymphomas are a very heterogenous group of tumors arising from lymphocytes at different stages of differentiation. In order to reliably anticipate responses to treatment, lymphoma models must preserve the spatial organization and the functional interdependency between the malignant and non-malignant cells. However, modeling lymphoma ex vivo is challenging due its highly heterogeneous nature, its intrinsic cellular composition, and the complexity of translating organoid technology from other cancer types into lymphoma. To address these challenges, we have established a tissue explant culture system for lymphoma tissues using murine models. FACS analyses, single-cell RNA sequencing, multiplex IHF, and spatial transcriptomics confirm that our system is able to support tumor growth and tissue architecture. As the tissue explants retain histological, cellular, and molecular characteristics distinctive of the original tissue, we called them lymphomoids . To anticipate sensitivity to anti-cancer therapies, we tested response to targeted therapies on lymphomoids obtained from 19 human primary lymphomas. Histopathological and spatial biology studies using spatial transcriptomics (Visium) and 34-plex immunofluorescence stainings (Lunaphore) followed by computational quantitation analyses showed patient–specific sensitivity to particular compounds and revealed features on the tumor tissue composition associated with resistance or response to therapies. Importantly, in four cases the response to therapy observed in the lymphomoids anticipated the patient’s clinical outcome. Altogether, lymphomoids represent an innovative tool to assess therapy response in lymphoma patients and uncover novel aspects of lymphoma biology.