Acute myeloid leukemia (AML) is the most common acute leukemia and second most common form of blood cancer in children and adolescents. Despite progress in understanding the biology of AML, therapeutic intervention plans have not changed nor outcomes significantly improved over the last years. Current AML treatment consists of intensive chemotherapy followed by stem cell transplantation. While initially many patients respond well to chemotherapy, relapse rates are high and still associated with poor prognosis. Evidence suggests that chemo-resistant leukemic stem cells (LSCs) have the ability to reinitiate and sustain the disease. Therefore, novel therapies are urgently needed to improve AML treatment and well-characterized in vivo models modelling this disease are a prerequisite for improving the current state. At Reaction Biology, we have used NOG and NOD-SCID mice to establish a humanized MOLM-13 AML model concomitantly engrafted with human T cells. Luciferase positive MOLM-13_Luc AML cells were injected both in naïve mice and mice pre-treated with Cyclophosphamide. Thereafter, tumor growth was monitored by bioluminescence measurements to track disseminated tumor burden and isolated T cells were applied upon tumor growth detection (generally observed on day 8 or 15 post-tumor cell inoculation for NOG or NOD-SCID mice, respectively). For each of the inoculations, fresh T cells were obtained by negative selection from buffy coat derived PBMCs with a purity of >95 %. Following administration of human T cells, animals were monitored daily, including the collection of animal weights (3x/w), and tumor burden (2x/w) via in vivo bioluminescence imaging technique. MOLM-13_Luc tumor cells grew rapidly in all NOG mice with termination of the last animals as early as day 17. Neither Cyclophosphamide pre-treatment, nor the addition of T cells influenced tumor growth or prolonged survival. In Cyclophosphamide pre-treated NOD-SCID mice, onset of tumor growth was observed to initiate homogenously at day 13 post inoculation (with applied T cells being ineffective in reducing tumor burden), whereas tumor growth in non-pre-treated animals was heterogenous and delayed by more than 7d. The presence of T cells affected tumor growth even further. Beside T cell addition, also the application of NK cell therapies were successfully investigated (data not shown). Taken together, the MOLM-13_Luc AML tumor model is found to be suitable for the investigation of bi-specific engagers using isolated T or NK cells. Citation Format: Carla Castro, Philipp Metzger, Cynthia Obodozie, Holger Weber. Humanized MOLM-13 AML model as a versatile tool to study immune-activating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 33.

The infiltration of macrophages into the tumor microenvironment (TME) is a well-known characteristic of tumor progression and therapy resistance. Depending on the signals derived from TME, macrophage plasticity allows them to undergo a variety of polarization states. Two extremes of macrophage polarization are M1 and M2 phenotypes. M1 macrophages provide enhanced anti-tumor inflammatory reaction while M2 macrophages are usually associated with tumor progression due to their immune suppression, angiogenesis and neovascularization promoting capabilities. Modulating the macrophage differentiation or polarization has emerged as a therapeutic strategy to synergize or complement current treatments. At Reaction Biology we have established human macrophage assay pipelines to evaluate the novel macrophage polarization compounds in vitro. Both, monocyte cell lines like THP-1, and and freshly isolated monocytes from donor derived PBMCs, are of great value. Donor derived differences could become an issue during the screening phase compared to the use of cell lines. Yet, after the screening phase, the evaluation across several healthy donors is crucial to assess the impact of a compound on the general population. Monocytes from primary human peripheral blood mononuclear cell (PBMC) are isolated, differentiated into naïve macrophages, and polarized. Effects of compounds modulating the differentiation and polarization of macrophages are analyzed by flow cytometry. Moreover, the functional reprogramming of monocytes and macrophages can be analyzed by measuring the phagocytic or tumor cell killing capacity via flow cytometry or using high content screening equipment. In the addition, a wide range of secreted cytokine can be quantified by MSD ELISA. Our data shows that human primary monocytes grown under M1 polarizing conditions express high levels of CD86 and HLA-DR in contrast to those cultivated under M2 polarizing conditions, with high expression of CD163 and CD206. A pH-sensitive phagocytosis could be shown for M2 macrophages. Modulation of surface markers from polarized macrophages under different treatment conditions could be observed, as well as changes in their functionality, supporting the translational value to evaluate the efficacy of immunomodulating compounds in vitro. Citation Format: Carla Castro, Philipp Metzger, Cynthia Obodozie, Holger Weber. Exploiting macrophage differentiation and plasticity as an immunomodulating strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 671.

In the last decades, several major breakthroughs in cancer treatment were achieved through a better understanding and therapeutic targeting of the immune system. Multiparametric flow cytometry is a robust and powerful tool to evaluate the frequency and activation status of a large variety of immune cells. However, for flow cytometric analysis tumor, tissue must be digested into a single cell suspension, and thereby the information on the tumor architecture as well as the spatial distribution of immune cells is lost. The distribution of immune cells in the tumor microenvironment is often heterogeneous and a favorable immune cell distribution is crucial for an efficacious anti-tumor immunotherapy. The recent advances in multiplex immunofluorescence (mIF) allows the analysis of multiple parameters simultaneously so that the frequency as well as the spatial distribution of multiple immune cells can be analyzed. In this study, immunotherapy-responsive tumor cell lines (colon carcinoma cell lines MC38-CEA and CT26wt as well as melanoma cell line Clone M3) were implanted in the mammary fat pad (subQperiorTM), which leads to reduce ulceration rate and homogenous tumor growth compared to subcutaneous implantation. Anti-PD-1 immunotherapy reduced the tumor growth in all three tumor models significantly. Formalin-fixed paraffin-embedded tumor samples were collected from control and anti-PD-1 treated animals. Murine specific InSituPlex® (ISP) technology (Ultivue) was used to perform multiplex immune profiling of the markers CD3, CD4, CD8 and FOXP3 on mouse whole slide FFPE tumor serial sections. Slides were then imaged for high quality images of the four targets and downstream analysis performed. The evaluated T cell frequency and spatial distribution was compared to flow cytometric data of anti-PD-1 treatment in these models. In summary, flow cytometry and multiplex immunofluorescence (mIF) are valuable tools in the evaluation of immune responses in pre-clinical tumor models. Both technologies enable an improved understanding of treatment responses and facilitate the development of novel treatment modalities as well as the identification of potential predictive biomarkers. Citation Format: Philipp Metzger, Carla Castro, Cynthia Obodozie, Holger Weber. Immunophenotyping of responses to immunotherapy in syngeneic tumor models using multiplex immunofluorescence compared to multiparametric flow cytometry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4148.

CDK8 has been identified as being frequently upregulated in various types of human cancer such as colorectal cancer, certain sub-types of breast cancer and acute myeloid leukemia. In different tumor cell lines RNA interference mediated knockdown of CDK8 results in significant inhibition of tumor cell growth in vitro and in vivo. CDK8, therefore, represents an interesting target for the development of novel anti-cancer drugs. Various compounds targeting CDK8/Cyclin C with high potency in low nanomolar range have been reported. These compounds differ with respect to their binding modes, but all of them show high potency in biochemical assays, inhibition of tumor cell growth, and for many in vivo activities in different tumor cell xenograft models have been demonstrated. CDK8 binds to Cyclin C resulting in the active protein kinases complex. CDK8/Cyclin C interacts with MED12 and MED13, two proteins that regulate CDK8/Cyclin C activity and its interaction with the mediator complex. Mediator consists of 26 subunits and the interaction of the CDK8/Cyclin C/MED12/Med13 complex with mediator enables CDK8/Cyclin C to regulate gene transcription via C-terminal phosphorylation of RNA-polymerase II. This mechanism mediates an indirect regulation of transcription factors such as ß-catenin. In addition of its function via phosphorylation of RNA-polymerase II, CDK8/Cyclin C can also directly regulate the activity of transcription factors by phosphorylation. Best known examples are STAT1 and STAT5 which are phosphorylated by CDK8/Cyclin C on serine 727 and Serine 726, respectively. Recent data indicates that mutations of MED12 can result in a reduced sensitivity of CDK8/Cyclin C towards certain published CDK8/Cyclin C inhibitor in a cellular context. In this study we performed real-time quantitative PCR (qPCR) analyses to characterize the effect of different type I and type II CDK8/Cyclin C inhibitors on the cellular expression of selected genes controlled by two CDK8/Cyclin C dependent pathways, namely the WNT/ß-catenin- and the STAT-pathway. Furthermore, results of qPCR analyses of tumors derived from human xenograft in vivo models treated with type I and type II CDK8/Cyclin C inhibitors will be presented. Citation Format: Laura M. Jordt, Frank Totzke, Joachim Lauterwasser, Jan E. Ehlert, Koen Hekking, Bas Aerts, Cynthia Obodozie, Holger Weber, Gerhard Müller, Michael H. Kubbutat. Real-time quantitative PCR based analysis of transcriptional effects of CDK8/Cyclin C inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2318.

Flow cytometry is a widely applied approach for exploratory immune profiling and enables the identification of individual cells in a cell population, such as a tumor cell suspension, by staining cell markers with antibodies conjugated with a fluorophore. The markers allow for the differentiation of a large variety of immune cell populations, their change in frequency, and activation status upon treatment. Conventional flow cytometers, which use bandpass filters and need conventional compensation matrices, are limited by the number of parameters that can be simultaneously analyzed, restricting their utility. Spectral analysis technology promises a greater flexibility for panel design, a higher number of analyzed parameters, and more accurate visualization of results. Our standard all-in-one flow cytometry panel uses all possible fluorescent detectors of the flow analyzer BD Fortessa, which permits the differentiation of the main immune cell populations in the tumor, such as T cells (CD4+, CD8+, Treg), B and NK cells, macrophages (M1/M2), MDSCs (granulocytic and monocytic), and dendritic cells. However, in addition to immune cell frequency, it would also be interesting to investigate cell surface activation and memory markers like PD-1, CD40, CD44, or CD62L or intracellular activation markers like IFN-gamma, perforin, granzyme B, or Ki67. The 32-channel spectral flow analyzer SP6800 ZE from Sony allows the combination of both. Flow cytometry data of different tumor samples will be presented, as well as the comparison of the results and resolution of both flow analyzer systems. Auto-fluorescence of cells, which causes fluorescent signal contamination of other fluorescent markers in the conventional flow cytometer, can be subtracted using spectral technology to see the true fluorescent population, thereby allowing dim signal detection in rare populations. A comprehensive overview will be given regarding the pros and cons of the spectral analyzer system and difficulties switching from the one system to the other. Citation Format: Philipp Metzger, Muriel Malaisé, Cynthia Obodozie, Holger Weber. Benefit of using a spectral flow analyzer for the analysis of immune cell populations in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 629.