Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes—besides other forms—a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. Here we deconstruct the larval mnemonic organization after aversive olfactory conditioning. We show that after odor-high salt conditioning larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3’5’-monophosphate (cAMP) signaling and synapsin gene function. In addition, we show for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution.

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50 Years after the generation of the most studied human breast cancer cell line, MCF-7, many research results fundamentally changed patient outcomes for the better. The mortality of breast cancer in women has decreased significantly, especially in the last 30 years. But the total number of deaths is rising again in the last ten years. Considering that breast cancer is the most common cancer in women and the second most common cancer overall, with incidences of 24,2% and 11,6%, respectively, total numbers of new cases are in the millions every year. First line drugs for the treatment of breast cancer often target microtubules (e.g. Vinblastine and Paclitaxel) or induce DNA damage (e.g. Carboplatin and Cisplatin (CisPt)). In addition to these classic therapeutic drugs new compounds that specifically target cancer-driving molecular alterations have been developed. Among these is the group of BH3-mimetics. The efficacy of several BH3-mimetics is investigated in numerous clinical trials while the BCL-2 specific inhibitor ABT-199 (Venetoclax) is already approved for clinical application in chronic lymphatic leukemia (CLL), acute myeloid leukemia (AML) and small lymphocytic lymphoma (SLL). BH3-mimetics target the apoptosis machinery by interacting with anti-apoptotic Bcl-2 proteins, mimicking the function of pro-apoptotic BH3-only proteins. In turn, the pro-apoptotic potential of pore-forming BCL-2 effectors BAX, BAK and BOK is released inducing mitochondrial outer membrane permeabilization and cell death. Recently we reported synergistic cell death induction by ABT-199 in combination with the proteasome inhibitor Bortezomib (BOZ) in soft tissue sarcoma (STS) cells. ABT-199 and BOZ induce accumulation of BOK and the BH3-only protein NOXA (Muenchow et al. 2020). Here, we combined ABT-199, CisPt and Nutlin-3 in breast cancer cell lines that differ in expression of the estrogen receptor (ER) and p53 activity. Similar to the results in STS cells synergistic induction of apoptotic cell death was observed by FACS analysis of the mitochondrial membrane potential (TMRM) and exposure of phosphatidyl serine (Annexin-V). Western blot analysis revealed that ABT-199 induced accumulation of NOXA and MCL-1, irrespective of the ER status or p53 mutation. Simultaneous treatment with ABT-199 and CisPt or Nutlin-3 further increased expression of NOXA and MCL-1. Interestingly, qRT-PCR revealed enhanced expression of NOXA and MCL-1 mRNA in ABT-199 treated cells indicating transcriptional regulation. However, transcriptional induction of NOXA was also detected independent of p53 activity. Thus, combined therapeutic approaches using CisPt and ABT-199 should be effective irrespective of ER and p53 expression. Nevertheless, in p53 proficient tumors Nutlins might increase therapeutic efficacy. Citation Format: Benjamin Schaefer, Sandra Weller, Tobias Beigl, Kathrin Boepple, Reiner Hoppe, Klaus Schulze-Osthoff, Hans-Georg Kopp, Walter E. Aulitzky, Frank Essmann. Synergistic cell death induction in breast cancer cell lines by combining ABT-199 and cisplatin irrespective of estrogen receptor and p53 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1946.

Cultivation of tumor tissue slices provides an ex vivo model capturing both tumor heterogeneity and its native microenvironment. Slices are commonly cultured either free-floating in medium or filter-supported. These conditions lead both to culture-dependent stress (free-floating culture condition) and intra-slice gradients regarding proliferation, marker expression and oxygen supply (filter-supported culture condition) (Davies et al Sci Rep (2015) 10.1038/srep17178). To facilitate homogenous supply with nutrients and oxygen, we developed a new method to culture tumor tissue slices. The precision-cut tissue slices (150µm to 300µm thickness) are kept in-between two organotypic supports and fixed in a special chamber allowing continuous perfusion with medium and drugs. The chamber is settled vertically inside of a 50 ml tube with air exchange capacity and connected with a syringe pump via a silicon tube. The whole system is cultured inside the cell culture incubator. Several different types of mouse xenografts (MCF-7, H1437) and primary human tumor (lung and ovarian cancer) tissue slices have been cultured with this new system and compared with the commonly used filter-support culture. Both breast and lung xenograft tissues slices showed a gradient of proliferation, HIF-1α and hormone receptor (ER) expression in the filter-supported culture condition but not with the new perfusion air culture system. The same results were obtained with primary tumor samples. Primary lung tumor and ovarian cancer tissue slices also showed a gradient of HIF-1α expression after cultivation in the filter-supported system but not in the new perfusion air culture system when cotton membranes are used as scaffold. In addition, the choice of the material of the organotypic support allows a variety of biological studies. Scaffolds from de-cellularized porcine intestine provide niches for migrating cells and are suited for studying tumor invasiveness. When used as organotypic support, primary ovarian cancer can be cultured up to 7 days with good tissue morphology and structure and migrating cells into the scaffold can be counted as a measure of invasiveness. When the tissues are sandwiched between polycarbonate membranes (pore size: 12 µm), oxygen gradients can be generated similar to gradients observed around vessels in vivo. Our perfusion air culture system facilitates the cultivation of tumor tissue slices due to its flexibility and adjustability of all culture conditions such as oxygen, scaffolds and flow rate parameters. It allows studying tumor slices under conditions closely resembling the in vivo situation. Citation Format: Kathrin Boepple, Meng Dong, Emma Davis, Julia Schueler, Heike Walles, John Hickman, Walter E. Aulitzky, Heiko van der Kuip. Perfusion air culture of tissue slices: A new method to cultivate tumor tissue with minimal culture-dependent tissue stress [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 5040.

Ovarian cancer is one of the five cancer types with highest incidence of death in women. Due to a lack of diagnostic options, timely detection of ovarian cancer is difficult and 75% of patients are diagnosed at a late stage. The standard therapy for ovarian cancer is resection of the affected tissue, followed by chemotherapy. However, patients frequently suffer from relapse of the disease, as some tumor cells persist chemotherapy and grow into a new, more aggressive tumor. To offer therapeutic approaches that prevent relapse by eradicating persister cells we aimed to identify the molecular mechanisms and determinants that are characteristic for persister cells. As initial cellular model system OVCAR-3 cells were used to select ovarian cancer persister cells. Selection was performed analogously to serum levels of chemotherapy patients, i.e. OVCAR-3 cells were incubated twice in the presence of 13 µM Cisplatin for 4 hours. Cisplatin-selection identified clones were collected and in further analyses compared to randomly collected untreated control clones. In order to account the native tumor microenvironment and heterogeneity within the tumor and among patients we cultured precision-cut tissue slices from primary ovarian tumor tissue with 280 µm thickness. Tumor tissue slices were cultured either under control conditions or exposed to 13 µM Cisplatin. Slices were further processed and cultured in Matrigel producing tumor-derived organoids, i.e. tumoroids. In contrast to OVCAR-3 control clones, the Cisplatin-selected OVCAR-3 persister cells showed filopodia-like structures. A scratch assay demonstrated enhanced motility of OVCAR-3-persister cells. Interestingly, cell death analysis (Annexin V) of Cisplatin exposed control and persister clonal cell lines showed the presence of surviving cells in both, persister and control groups. To identify the molecular determinants specific for persister cells we performed RNA sequencing. Data showed that, in line with initial results, the motility associated genes Snail and Vimentin are up regulated in persister clones. We successfully generated tumoroids from patient-derived ovarian cancer tissue slices. Tumoroids were cultured for more than two months and displayed a high viability. After exposure to Cisplatin less tumoroids generated from tumor slices were found. OVCAR-3-derived persister clones show morphological alterations (filopodia-like structures), enhanced motility and expression of motility associated genes. We expect the identification of genetic determinants for persister cell characteristics from comparison of RNAseq results of OVCAR-3 persister clones and tumor-derived organoids. The identification of genetic determinants will direct the development of therapeutics approaches targeting persister cells in ovarian cancer to prevent recurrence of tumors. Citation Format: Kathrin Boepple, Meng Dong, Andrea Gaissler, Bernd Winkler, Markus Kleih, Frank Essmann, Walter E. Aulitzky. Ovarian cancer persister cells: 2D and 3D in-depth characterization and analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 653.