PO-389 The role of immune microenvironment in determining the organ-specific homing of metastases

S Garcia Mulero, JM Piulats, V Moreno, R Sanz-Pamplona
2018 Tumour Immunology   unpublished
Introduction Metastasis to different organs or tissues may require distinct sets of regulators which may influence the homing and growth of tumour cells to specific secondary sites. Under the hypothesis that the immune microenvironment of the different niches may play an important role in this process, we have categorised metastatic samples from different primary tumours based on their immune profile. Material and methods Gene expression data from metastatic samples with different primary
more » ... origin (n=342) were downloaded from open repository GEO. Samples were scored using different gene expression profiles and characterised on the basis of their immune and stromal infiltration and activation of immune response pathways (Immunophenoscore, MCPcounter, ESTIMATE; among others). Resultant scores were analysed for statistical differences with ANOVA test. Multivariate analysis was used for clustering the samples based in their immune-features. Results and discussions As expected, significant differences were found between the immune profiles of samples metastasizing in distinct organs. For instance, breast cancer metastasis in lung showed a much higher immunogenic score than breast metastasis in brain (p=5e-4), suggesting a different immune microenvironment modulation. Also in breast, significant differences have been found in cell lineages infiltration, lung metastasis being the ones with the highest T cell component (p=0.002) and liver metastasis the ones with the lowest infiltration of endothelial cells (p=0.005). Moreover, in other cancer types like melanoma, samples showed differences among different metastatic locations. Interestingly, when comparing metastatic samples originating from different primary tumour, a high concordance among secondary tumours in immune scores were found; specifically in brain metastasis. These results suggest that cells needs to share similar molecular profiles to evade the immune surveillance and growth in a specific niche, independently of their origin. Conclusion Metastases from the same primary tumour growing in different organs show differences in their immune profile. However, those samples from different primary origin but growing in the same secondary organ shared a characteristic immune profile. These results suggest that immune system plays a role in determining the organ-specific homing of metastasis. Introduction Syngeneic mouse tumour models are widely used to analyse tumour immunology due to their fully competent immune-repertoire and have paved the way for novel immunotherapy agents in multiple tumour entities. However, the amount and composition of tumour infiltrating leukocytes (TIL) is highly variable. This complicates targeted analysis, in particular for small leukocyte subpopulations that may not be analysed properly or lost in the background noise. When working with large cohort sizes, immune-phenotyping by flow cytometry is time consuming and highly work intensive. We have developed improved workflows combining automated tissue dissociation with novel TIL specific isolation reagents to allow for more accurate and faster analysis of TILs and TIL subpopulations. Material and methods We have developed novel TIL specific enrichment reagents for the magnetic cell sorting (MACS) based isolation directly from dissociated tumour tissue. The composition of these populations before and after separation was analysed by flow cytometry. The developed tools were combined with optimised tissue dissociation and flow panels to establish comprehensive workflows. Results and discussions We have established workflows combining optimised and automated tissue dissociation using the gen-tleMACS platform with TIL specific isolation to improve and accelerate downstream analysis. Isolation of TIL was improved by developing new CD45, CD4+, CD8+, and pan T cell enrichment reagents for MACS-based isolation directly from dissociated tumour tissue. Applying this workflow, CD45 +TIL were enriched to purities above 80%-90% with high yields (>70%) from divers syngeneic mouse tumours. This allowed for rapid and reliable analysis even of small TIL subpopulations. Moreover, by using the newly developed T cell reagents, T cells were enriched to purities higher than 90%, allowing for a reliable analysis of rare T cell subpopulations. Importantly, while TIL or T cell enrichment significantly reduced analysis time and reagent costs in immune subset analysis, the composition of infiltrating cells was not affected, excluding the risk of introducing a bias by this method. Conclusion We have developed improved workflows for the isolation of generic TIL and T cells from mouse tumours reducing time and costs of downstream analysis while standardising and enhancing the detection and quantification of immune cell subpopulations. CD45 +TILs, pan-, CD4 + -or CD8 + -T cells can directly be isolated from dissociated mouse tumours and analysed for subpopulations.
doi:10.1136/esmoopen-2018-eacr25.901 fatcat:n2nx4ulswbfajk76pqrzqzi6ga