The goal of personalized medicine is to stratify individual patients to the appropriate treatment. This approach depends on extensive characterization of individual tumors and their sensitivity to therapeutics. In the context of the immunotherapy of cancer, information on the localization, abundance and activation of immune cells within individual tumors gained in importance. In this study, we showed that viable tumors from colorectal cancer patients used within our drug testing platform, exhibit different populations of infiltrating immune cells. Analysis of immune cells was conducted on disaggregated, cells from viable tumor slices. Disaggregation of precision cut cancer tissue slices was performed using the GentleMACS from Miltenyi. Immune cell subsets were analyzed by flow cytometric multiplexing of CD3, CD4, CD8 and CD45. Furthermore, we identified PD1 positive cells among the CD45+/CD3+ lymphocyte population, indicating relevance for anti-PD1 targeted therapy in colorectal cancer. As a proof of principle, precision cut cancer tissue slices were incubated for 24 hours with different concentrations of Nivolumab (anti-PD1). The read out of treatment effects was conducted in regard to cytokine secretions upon compound treatment as well as immune cell composition. Cytokines were analyzed in supernatants of tissue cultures using the proinflammatory panel from Mesoscale Discovery. The results demonstrated that immune cell compositions were stable and uniform within our precision cut cancer tissue slices both pre- and post-cultivation, and pre- and post-treatment with Nivolumab. In contrast, cytokine secretion had changed after treatment. This has been observed for different cytokines, such as INF gamma, IL-2, IL-10 and TNF alpha. A correlation to PD-1 expression on T-cells in different patients has not been seen. In order to optimize preclinical testing of immune-modulatory compounds, preclinical models, which reflect the individual tumor, as well as the individual immune components of the tumor, are mandatory. We have shown here that effects of treatments with an immune-modulatory compound (Nivolumab) were detectable in this system. Therefore, this drug testing platform represents a unique opportunity to test immune-modulatory compounds in a fully human, patient derived model that is close to in vivo situation. In the future, other immune-modulatory classes of compounds have to be tested within the system to more comprehensively elucidate the possibilities and limits of this drug testing platform in regard to immunotherapy. Citation Format: Kristina Bernoth, Florian T. Unger, Moiken Petersen, Mirja Piller, Jana Krüger, Nicole Grabinski, Hartmut Juhl, Kerstin A. David. Precision cut cancer tissues slices as human model for the testing of immuno-modulatory compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4830. doi:10.1158/1538-7445.AM2017-4830
The recent advances in immunotherapies, such as immune checkpoint modulators, bispecific antibodies, and adoptive T-cell transfer, opens new opportunities for the treatment of cancer. Having this broad spectrum of new therapeutic agents available, the demand for predictive and robust preclinical models to minimize translational failures in immuno-oncology is increasing. Indivumed has successfully implemented a model of Precision Cut Cancer Tissue slices (PCCTS) derived from viable human tumor tissue for different applications such as chemotherapeutic agents, small molecules and antibodies. In this study, we investigated the effects of OKT3® on cancer tissue slices especially in respect of gene expression changes and cytokine release. Fresh tumor pieces from three colorectal cancer patients were cut into 400 µm slices using a Krumdieck Tissue Slicer®. PCCTS were incubated for 4 h and 24 h with and without OKT3®, (Muromonab), a therapeutic antibody against CD3. After defined time points, slices were frozen, and the supernatants were collected. In addition, a second set of slices was formalin fixed and paraffin embedded for immunohistochemical analysis of T-cells. RNA isolated from frozen slices was used for library preparation with the TruSeq Stranded mRNA Library Preparation Kit followed by sequencing on a NextSeq 500 device from Illumina. The results generated by different software tools showed a background of gene expression changes caused by cultivation. On the other hand, a very specific up-regulation of genes was detectable after OKT3® treatment. Especially an induction of genes involved in immune related functions, such as IFN gamma and IL-12 signaling was seen. The analysis of cytokines in the supernatants using a ten-plex panel form Meso Scale Discovery confirmed the activation of T-cells after OKT3® treatment. In all three patients a significant increase of IFN gamma, IL-2, and TNF alpha was measured, comparable to a so called “cytokine storm” that is described in literature. The intensity of the effects correlated to the amount of tumor infiltrating T-cells, quantified by anti-CD3 staining in tissue sections. This model of PCCTS represents a unique opportunity to test immune-modulatory compounds in a fully human, patient derived model that is close to the in vivo situation. The data of this study shows that profiling of the immune response by RNA-Seq can be successfully performed. In addition to gene expression changes that occur after treatment, deconvolution based approaches to quantify tumor Infiltrating lymphocytes (TILs) from RNA-Seq data will be a key parameter to personalize the treatment of patients. Citation Format: Nicole Grabinski, Kristina Bernoth, Aljoscha Leusmann, Carolin Fleischhauer-Biermann, Dorte Wendt, Mirja Piller, Moiken Petersen, Nicole Lange, Anna Tiedemann, Monika Schoeppler, Hartmut Juhl, Gerd Helftenbein, Andrea Miegel, Frank Schnieders, Kerstin David. Immunoprofiling of precision-cut cancer tissue slices (PCCTS) as a tool for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1711.
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