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 tumour 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 spe...
T cells play a central role in immunity to pathogens and tumors, but also in autoimmunity. T cell research has generated knowledge that led to multiple clinical breakthroughs, such as the generation of T cell vaccines and tumor immunotherapy approaches. However, additional work is required to fully understand T cell biology, harness their therapeutic potential and control immunopathologies. Cutting edge experimental protocols that interrogate and manipulate T cell biology often require prior purification of T cell populations. Strategies to simplify and accelerate T cell purification are highly desirable to save time, reduce bias and allow complex experiments to be performed. We established workflows combining automated tissue dissociation with T cell isolation. We developed new CD4, and CD8 T cell specific enrichment reagents that significantly accelerated the isolation protocol. T cells were isolated to purities above 90%, reducing time of downstream analysis. Importantly, culture of isolated T cells did not induce activation as measured by CD69 or CD25 upregulation, as compared to controls. Furthermore, magnetic isolation did not affect proliferation or cytokine expression following in vitro stimulation. Finally, T cell isolation could be fully automated and multiple samples could be processed in parallel. Our new workflow greatly reduces time of downstream analysis while preserving cell phenotype and functional properties. We believe these innovative tools significantly shorten time-consuming experiments and can be used to increase reproducibility and the quality of data obtained in T cell research.
Regulatory T cells (Tregs) play a pivotal role in maintaining peripheral tolerance of unspecific or excessive immune responses. Therefore, the enrichment of these cells and their cultivation is of great interest for basic and translational research. Accordingly, there is a considerable need for continually improved and reliable cell isolation protocols. We introduce here a rapid, easy, and convenient strategy for the isolation of regulatory T cells directly from anticoagulated whole blood without the need for density gradient centrifugation. The new immunomagnetic separation protocol provides an easy and fast solution for the highly specific isolation of CD4+ CD25highCD127− regulatory T cells directly from whole blood within 30 minutes ready for downstream application such as flow cytometric phenotyping, gene expression studies, cytokine analysis, suppression assays, or cell expansion. The procedure, leads to populations of CD4+ CD25highCD127− cells with high yields (2×104/mL whole blood), purities (>90%) and viability (>98%) as proven by flow cytometric analysis. In addition, enriched Tregs were capable for in vitro expansion for several weeks while remaining stable FoxP3 expression.
T cells can eliminate tumors, but tumor infiltrating (TI) T cells can become intrinsically anergic or extrinsically suppressed. Identifying the mechanisms of T cell dysfunction is necessary to improve immunotherapy efficacy. Mouse tumor models are useful tools in tumor T cell research, but analysis of TI T cells is technically challenging, time consuming and labor intensive. Their frequency can be very low and small populations may escape analysis as they get lost in the background. Importantly, TI T cells are in an antigen-rich and highly immunomodulatory cell environment, such that cell intrinsic functional analysis is hindered. It is fundamental to use new tools to streamline the workflow and generate reliable data. We established workflows combining tissue dissociation, T cell isolation and phenotyping. Tumor dissociation was automated using the gentle MACS™ and optimized for epitope preservation. Enzymatic dissociation was essential for analysis of PD1hiTim3+Lag3+CD39+ CD8 T cells in tumors. Using new T cell-specific microbeads for magnetic cell sorting we increased TI T cell purity by up to 500-fold, while maintaining activation status and phenotype. Importantly T cell isolation from the tumor’s highly immunomodulatory cellular environment was essential to assess cell-intrinsic functional properties. Finally, our data show the use of recombinant RE Afinity antibodies eliminated non-specific labeling of cells in the tumor microenvironment. We conclude that standardized processing of tumor samples and magnetic isolation of tumor infiltrating T cells greatly reduces time and cost of total workflow and downstream analysis while significantly increasing reproducibility and the quality of data obtained from TIL analysis.
Tumor-Infiltrating (TI) T cells play an important role in anti-tumor immunity, highlighted by multiple immunotherapy strategies approved by the FDA. However, the benefit of current immunotherapies is still limited, such that additional research is needed to improve efficacy. Preclinical tumor models are essential to advance immunotherapy research. However, tumor composition is highly variable and T cell numbers can be very limited. Even flow cytometry can be very time consuming. Importantly, TI T cells are embedded in a cellular environment where antigen is abundant and surrounding cells express immunomodulatory molecules, such that unbiased cell-intrinsic functional characterization is impossible. Pre-enrichment of TI T cells is highly desirable to reduce hands-on time, and generate high quality and reliable data. We developed new CD4, CD8 and Pan T cell specific enrichment reagents for magnetic cell sorting from mouse tumors. Single cell suspension was obtained by automated dissociation. T cell isolation was done either manually, or using semi or full automation. TI T cell frequencies ranged from 0.2–9%, were isolated to purities above 80% with yields ranging from 60–95%. Pan T cell isolation maintained the original CD4:CD8 ratio. CD69 was not upregulated after 18h culture, demonstrating that isolation did not result in T cell activation. Importantly, functional analysis of bulk vs. purified cells revealed significant influence of tumor and stromal environment in cytokine expression by TI T cells, which was not due to the isolation protocol. Our data, demonstrates that magnetic enrichment reduces time of analysis, increases quality of analysis and is essential for the functional characterization of TI T cells.
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