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.
We have recently reported a potential alternative tumor suppressor function for p16 relating to its capacity to regulate oxidative stress and observed that oxidative dysregulation in p16-depleted cells was most profound in melanocytes, compared to keratinocytes or fibroblasts.,Moreover, in the absence of p16 depletion or exogenous oxidative insult, melanocytes exhibited significantly higher basal levels of reactive oxygen species (ROS) than these other epidermal cell types.,Given the role of oxidative stress in melanoma development, we speculated that this increased susceptibility of melanocytes to oxidative stress (and greater reliance on p16 for suppression of ROS) may explain why genetic compromise of p16 is more commonly associated with predisposition to melanoma rather than other cancers.,Here we show that the presence of melanin accounts for this differential oxidative stress in normal and p16-depleted melanocytes.,Thus the presence of melanin in the skin appears to be a double-edged sword: it protects melanocytes as well as neighboring keratinocytes in the skin through its capacity to absorb UV radiation, but its synthesis in melanocytes results in higher levels of intracellular ROS that may increase melanoma susceptibility.