Characterization of tumor-associated macrophages heterogeneity, their complex interactions with the tumor microenvironment and their spatial distribution

Tumor-associated macrophages (TAMs) represent the most abundant immune cell component of the tumor microenvironment (TME), and they can promote tumor progression and metastasis. To analyse TAMs in tumor and correlate subset-specific TAM markers with patient prognosis, we developed high-throughput computational imaging techniques. We exploited the possibility to work on whole slide images and perform automate image analysis using several digital tools. First, through computational analysis of classical immunohistochemistry stained slides, we studied the correlation between TAM markers and patient’s outcome. We found that high levels of MS4A4A correlates with poor prognosis of patients. We also considered MS4A4A+ macrophages (Mφ) morphology and their localization within tissue, since in previous studies these features have been shown to correlate with patients’ outcome. So, we divided MS4A4A+ Mφ into "single" and "grouped", based on their morphology, size and density to evaluate a possible correlation between the presence of these two types of Mφ and patients prognosis. Second, we focused on tumor tissue and structure, by computational analysis of H&E-stained slides. We were able to quantify tumor heterogeneity in terms of cell composition and distribution within tissue. Moreover, the use of OpalTM allowed the discovery and the quantification of four macrophage subpopulations that are very heterogeneous among patients. We thought that it could be very interesting to focus on the correlation between their heterogeneity and patients prognosis. Finally, to better understand the role of TAMs in tumor, we considered their complex interactions with other cellular components of TME and their spatial distribution. So, we performed a deeper investigation of the immune contexture, taking advantage of the HyperionTM platform. It allows the simultaneous investigation of up to 37 protein markers in tissues preserving their architecture and cell morphology information by using metals instead of fluorophores.