Abstract
The presence and interaction of immune cells in the tumor microenvironment is of significant importance and has a great impact on disease progression and response to therapy. Hence, their identification is of high interest for prognosis and treatment decisions. Besides detailed phenotypic analyses of immune, as well as tumor cells, spatial analyses is an important parameter in the complex interplay of neoplastic and immune cells—especially when moving into focus efforts to develop and validate new therapeutic strategies. Ex vivo analysis of tumor samples by immunohistochemistry staining methods conserves spatial information is restricted to single markers, while flow cytometry (disrupting tissue into single cell suspensions) provides access to markers in larger numbers. Nevertheless, this comes at the cost of scarifying morphological information regarding tissue localization and cell–cell contacts. Further detrimental effects incurred by, for example, tissue digestion include staining artifacts. Consequently, ongoing efforts are directed towards methods that preserve, completely or in part, spatial information, while increasing the number of markers that can potentially be interrogated to the level of conventional flow cytometric methods. Progression in multiplex immunohistochemistry in the last ten years overcame the limitation to 1–2 markers in classical staining methods using DAB with counter stains or even pure chemical staining methods. In this study, we compared the multiplex method Chipcytometry to flow cytometry and classical IHC-P using DAB and hematoxylin. Chipcytometry uses frozen or paraffin-embedded tissue sections stained with readily available commercial fluorophore-labeled antibodies in repetitive cycles of staining and bleaching. The iterative staining approach enables sequential analysis of a virtually unlimited number of markers on the same sample, thereby identifying immune cell subpopulations in the tumor microenvironment in the present study in a humanized mouse melanoma model.
Highlights
The tumor microenvironment (TME) is a complex network of interactions between immune cell populations, cancer cells, and vascular and stromal components, which play a critical role in cancer cell growth, disease, prognosis, and therapeutic approaches
Anti-tumor responses can be assessed in these systems, xenoreactivity, and onset of graft-versus-host disease (GvHD) conceal which cellular component or immunological event mediates observed effects
Expression of human major histocompatibility complex (MHC) class I and/or II molecules on murine cells possibly reduce xenoreactivity and delay onset of GvHD mediated by human leukocyte antigen (HLA)-matched CD8+ and CD4+ T cells
Summary
We used inoculation of immunodeficient mice with a melanoma cell line to establish in situ immunomonitoring with the future goal to validate the effects of different therapeutic approaches in vivo. Several groups used immunodeficient mice transplanted with human tumors and subsequent transfer of human immune cells and therapeutic mAb [5,6,7]. Advanced humanized mouse systems, including transgenic expression of relevant human molecules in immunodeficient mice, are a possible solution to improve the robustness of these models [8]. Expression of human major histocompatibility complex (MHC) class I and/or II molecules on murine cells possibly reduce xenoreactivity and delay onset of GvHD mediated by human leukocyte antigen (HLA)-matched CD8+ and CD4+ T cells
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