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We assume that athletic success is associated with certain beliefs that on the one hand promote performance-enhancing behavior (training volume), but on the other hand can also be detrimental to health (sports addiction). These beliefs are succinctly characterized by the title of the 9-item "Mind-over-Body" scale presented here. They are the three beliefs that 1) athletic performance requires a high level of effort, 2) that willpower plays an important role in athletic success, and 3) that athletic success requires pain tolerance. A total of six web-survey-based studies with a total of 1,121 participants (approximately gender parity), including individuals with different levels of athletic performance (no competition; amateur sport; regional, national, or international competition), examined the psychometric network and construct and criterion validity of the MoB scale. Exploratory graph analyses, which included the studies with the largest sample sizes, showed that the three belief components (effort, willpower, pain) form separable communities within the MoB network and that the MoB items form communities distinct from self-control and self-efficacy. Meta-analyzed correlations across all six studies showed low positive correlations with self-control and self-efficacy. In terms of criterion validity, MoB beliefs were positively correlated with training volume and exercise addiction. We discuss MoBs as "on the edge of unhealthy" and place MOBs within a framework of related but distinct concepts.

Human-relevant systems that mimic the 3D tumor microenvironment (TME), particularly the complex mechanisms of immuno-modulation in the tumor stroma, in a reproducible and scalable format are of high interest for the drug discovery industry. Here, we describe a novel 3D in vitro tumor panel comprising 30 distinct PDX models covering a range of histotypes and molecular subtypes and cocultured with fibroblasts and PBMCs in planar (flat) extracellular matrix hydrogels to reflect the three compartments of the TME—tumor, stroma, and immune cells. The panel was constructed in a 96-well plate format and assayed tumor size, tumor killing, and T-cell infiltration using high-content image analysis after 4 days of treatment. We screened the panel first against the chemotherapy drug Cisplatin to demonstrate feasibility and robustness, and subsequently assayed immuno-oncology agents Solitomab (CD3/EpCAM bispecific T-cell engager) and the immune checkpoint inhibitors (ICIs) Atezolizumab (anti-PDL1), Nivolumab (anti-PD1) and Ipilimumab (anti-CTLA4). Solitomab displayed a strong response across many PDX models in terms of tumor reduction and killing, allowing for its subsequent use as a positive control for ICIs. Interestingly, Atezolizumab and Nivolumab demonstrated a mild response compared to Ipilimumab in a subset of models from the panel. We later determined that PBMC spatial proximity in the assay setup was important for the PD1 inhibitor, hypothesizing that both duration and concentration of antigen exposure may be critical. The described 30-model panel represents a significant advancement toward screening in vitro models of the tumor microenvironment that include tumor, fibroblast, and immune cell populations in an extracellular matrix hydrogel, with robust and standardized high content image analysis in a planar hydrogel. The platform is aimed at rapidly screening various combinations and novel agents and forming a critical conduit to the clinic, thus accelerating drug discovery for the next generation of therapeutics.

Cancer remains the leading cause of disease-related death in children. For the many children who experience relapses of their malignant solid tumors, usually after very intensive first-line therapy, curative treatment options are scarce. Preclinical drug testing to identify promising treatment elements that match the molecular make-up of the tumor is hampered by the fact that (i) molecular genetic data on pediatric solid tumors from relapsed patients and thus our understanding of tumor evolution and therapy resistance are very limited to date and (ii) for many of the high-risk entities, no appropriate and molecularly well-characterized patient-derived models and/or genetic mouse models are currently available. However, recent regulatory changes enacted by the European Medicines Agency (class waiver changes) and the maturation of the RACE for Children act with the FDA, will require a significant increase in preclinical pediatric cancer research and clinical development must occur. We detail the outcome of a pediatric cancer international multistakeholder meeting whose output aims at defining an international consensus on minimum preclinical testing requirements for the development of innovative therapies for children and adolescents with cancer. Recommendations based on the experience of the NCI funded PPTP/C (www.ncipptc.org) and the EU funded ITCC-P4 public private partnership (https://www.itccp4.eu/) are provided for the use of cell-based and mouse models for pediatric solid malignancies, as well as guidance on the scope and content of preclinical proof-of-concept data packages to inform clinical development dependent on clinical urgency. These recommendations can serve as a minimal guidance necessary to jumpstart preclinical pediatric research globally.

Supplementary Data from International Consensus on Minimum Preclinical Testing Requirements for the Development of Innovative Therapies For Children and Adolescents with Cancer

Figure S1: PRS-343 specificity for 4-1BB within TNF receptor superfamily. Seven recombinant human TNF receptor superfamily proteins were purchased from Sino Biological (4-1BB: 10041-H08H, RANK: 16078-H08H, GITR: 13643-H08H, Ox40: 10481-H08H) or R&D Systems (CD30: 6126-CD, TNF-RII: 1089-R2, TNF-RI: 636-R1) and used for determination of PRS-343 selectivity for 4-1BB. Proteins were coated to an ELISA plate, PRS-343 was added in a dilution series and detected via HRP-labeled anti-human IgG Fc antibody. Within the set of tested TNF receptor family proteins, PRS-343 binds exclusively to 4-1BB. Figure S2: Characterization of multiple bispecific formats of a 4-1BB targeting Anticalin protein recombinantly fused to an anti-HER2 antibody (A). ELISA based binding properties of all formats were compared to parental building blocks (C) while the ability to activate T-cells (IL2 induction) was assessed in a co-culture assay. Figure S3: Cytokine release assay with PRS-343. PBMC were isolated from the blood of twelve healthy donors and incubated for 72 hours with PRS-343 either air dried, in soluble form, or wet coated. Four concentrations of PRS-343 in a volume of 50 µl were tested in each setting as indicated in the figure. The anti-CD3 monoclonal antibody OKT3 at three different concentrations served as the positive control, and an IgG4 isotype antibody was the negative control. Supernatant levels of ten cytokines (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, GM-CSF, IFN-γ and TNF-α) were analyzed. The figure shows the average response for the ten donors that displayed a significant response to OKT3, and for a selection of the most relevant cytokines. Figure S4: Dose-dependent 4-1BB activation of a 4-1BB over-expressing Jurkat Nf-kB reporter cell line induced by PRS-343 with ON preincubation of the drug in the presence of NCI-N87 (HER2 high), MKN45 (HER2 low) and HepG2 (HER2 null) cell lines, or without tumor cells. Briefly, cancer cells were seeded onto tissue culture plates with PRS-343 (at 10, 1, 0.1 and 0,01 nM) and incubated ON. All plates were then washed twice with PBS. 4-1BB over-expressing Jurkat Nf-kB reporter (at 3:1 ratio) were added to each well. Following a 6 hours incubation, Bio-Glow luciferase reagent was added to each well and luminescence was measured. Figure S5: Fold increase changes of a panel of cytokines induced by human T-cells co-stimulated by PRS-343 in the presence of SKBR-3 (HER2 high) or MCF-7 (HER2 low). Figure S6: h-4-1BB expression in T-cells during co-culture assay. Using a similar set up as described in M&M, purified Pan T-cells (from healthy donors) and SK-BR3 high Her2 expressing cells were co-incubated in the presence of coated anti-CD3 antibody. After 24, 48 and 72 hours of co-incubation, Pan T-cells were collected and 4-1BB expression on CD3 positive cells was assessed by flow cytometry. (A) shows the % of 4-1BB positive T-cells for two donors and (B) shows histogram of h-4-1BB expression on CD3 positive T-cells from two donors (red 24 hours; blue 48 hours; brown 72 hours). Table S1: Relative HER2 cell surface expression on a panel of cell lines. Expression was experimentally determined using a specific anti-HER2-antibody binding capacity (sABC [HER2]) and quantitative indirect immunofluorescence in flow cytometry (QIFIKIT). HER2 surface levels are also provided relative to the level on SKBR3 cells which were chosen as a reference cell line.

Supplementary Legend

Purpose:4-1BB (CD137) is a key costimulatory immunoreceptor and promising therapeutic target in cancer. To overcome limitations of current 4-1BB–targeting antibodies, we have developed PRS-343, a 4-1BB/HER2 bispecific molecule. PRS-343 is designed to facilitate T-cell costimulation by tumor-localized, HER2-dependent 4-1BB clustering and activation.Experimental Design:PRS-343 was generated by the genetic fusion of 4-1BB–specific Anticalin proteins to a variant of trastuzumab with an engineered IgG4 isotype. Its activity was characterized using a panel of in vitro assays and humanized mouse models. The safety was assessed using ex vivo human cell assays and a toxicity study in cynomolgus monkeys.Results:PRS-343 targets 4-1BB and HER2 with high affinity and binds both targets simultaneously. 4-1BB–expressing T cells are efficiently costimulated when incubated with PRS-343 in the presence of cancer cells expressing HER2, as evidenced by increased production of proinflammatory cytokines (IL2, GM-CSF, TNFα, and IFNγ). In a humanized mouse model engrafted with HER2-positive SK-OV-3 tumor cells and human peripheral blood mononuclear cells, PRS-343 leads to tumor growth inhibition and a dose-dependent increase of tumor-infiltrating lymphocytes. In IND-enabling studies, PRS-343 was found to be well tolerated, with no overt toxicity and no relevant drug-related toxicologic findings.Conclusions:PRS-343 facilitates tumor-localized targeting of T cells by bispecific engagement of HER2 and 4-1BB. This approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity compared with current monospecific approaches. The reported data led to initiation of a phase I clinical trial with this first-in-class molecule.See related commentary by Su et al., p. 5732

Supplementary Information: Supplementary Figures 1-11, Supplementary Figure Legends, Supplementary Tables 1-4

Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3–1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3–1 and RIG-I mRNA was also observed during decitabine treatment in vivo in serially sorted peripheral blood AML blasts. In patient-derived monosomal karyotype AML murine xenografts, decitabine treatment resulted in superior survival rates compared with cytarabine. Collectively, these data demonstrate preferential gene derepression and ERV reactivation in AML with chromosomal deletions, providing a mechanistic explanation that supports the clinical observation of superiority of HMA over cytarabine in this difficult-to-treat patient group.Significance:These findings unravel the molecular mechanism underlying the intriguing clinical activity of HMAs in AML/MDS patients with chromosome 7 deletions and other monosomal karyotypes.See related commentary by O'Hagan et al., p. 813

Mutation-targeting therapies have become a transformative technology and in many ways a new paradigm for cancer treatment for patients. The early development of Erlotinib for non-small cell lung cancer paved the way for EGFR treatment, irrespective of tumor indication subtype. And the more recent launch of Sotorasib for KRASG12C mutation has marked a turning point for the once undruggable mutation. The advent of gene specific-treatments requires a reconfiguration of preclinical models which more effectively represent the said mutation in populations of diverse backgrounds and tumor indications. 3D models which have the ability to recreate the key hallmarks of cancer - such as growth, invasion, immune infiltration and suppression, stromal transformation, drug diffusion, as well as gene mutational signatures - are of exceptional interest to drive rapid and scalable compound efficacy screening. Here, we developed four 3D in vitro tumor panels carrying mutations in one of the following genes - KRAS, EGFR, BRCA1, and BRAF - as well as wildtype models for comparison. The tumors were grown in a hydrogel matrix in 96-well plates with stromal fibroblasts and screened with the standard of care (e.g. Sotorasib for KRASG12C, Erlotinib for EGFRmut, the PARP inhibitor Olaparib for BRCAmut, and Vemurafenib for BRAFV600E) and other known therapeutic compounds that target their respective mutations. The utility of a 3D hydrogel matrix and fibroblasts in the panels’ models were key in recreating the stromal compartment of the tumor microenvironment. Endpoints were determined within four days of treatment, including tumor size reduction and tumor killing, via high-content image analysis. The Panels’ robustness as demonstrated by the reproducible results in independent experiments suggests they can be utilized at in vitro pharmacology stages and earlier in Hit and Lead finding screens. Citation Format: Bin Xue, Julia Schüler, Christopher Harrod, Zoji Bomya, Kolin C. Hribar. KRAS, BRAF, BRCA1 and EGFR mutation-specific panels using hydrogel-based 3D in vitro tumor models [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 1722.

Many teachers experience high levels of work-related strain due to time pressure, which over time can lead to various health problems, such as emotional exhaustion. However, there is growing evidence that this could be a reciprocal effect. Moreover, it is known that perceived social support can buffer the negative effects of stress, such as time pressure, on health outcomes. Less is known about buffering effects of received social support. Based on longitudinal data of n=1071 Swiss primary and secondary school teachers over the course of one school year, the present study examined the reciprocal relationship between teachers��� perceived time pressure and emotional exhaustion and whether received social support from the school principal buffers this relationship. Results of a random intercept cross-lagged panel model show a strong relationship between teachers��� perceived time pressure and emotional exhaustion at the between-person level, but no effects at the within-person level. Further, received social support was directly related to less perceived time pressure and less emotional exhaustion. The results showed neither evidence for reciprocal effects between perceived time pressure and emotional exhaustion nor for a buffering effect of received social support from the school principal. Concluding, present findings indicate that the receipt of social support from the school principal is a central job resource that beneficially relates to teachers��� experience of time pressure and emotional exhaustion., +repphzhbib2021F

Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3–1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3–1 and RIG-I mRNA was also observed during decitabine treatment in vivo in serially sorted peripheral blood AML blasts. In patient-derived monosomal karyotype AML murine xenografts, decitabine treatment resulted in superior survival rates compared with cytarabine. Collectively, these data demonstrate preferential gene derepression and ERV reactivation in AML with chromosomal deletions, providing a mechanistic explanation that supports the clinical observation of superiority of HMA over cytarabine in this difficult-to-treat patient group. Significance: These findings unravel the molecular mechanism underlying the intriguing clinical activity of HMAs in AML/MDS patients with chromosome 7 deletions and other monosomal karyotypes. See related commentary by O'Hagan et al., p. 813

Sport and exercise can be boring. In the general population, thinking of sports as boring has been linked to exercising less. However, less is known about the role of boredom in people who participate in ultra-endurance competitions: Do these athletes also associate their sports with boredom, and does boredom pose a self-regulatory challenge that predicts if they encounter a crisis during an ultra-endurance competition? Here, we investigate these questions with a sample of N = 113 (n = 34 female) competitors of a 24 h hour running competition, aged M = 37.6 ± 13.8 years. In this study, n = 23 very extreme athletes competed as single starters or in a relay team of 2, and n = 84 less extreme athletes competed in relay teams of 4 or 6. Before the run, athletes completed self-report measures on sport-specific trait boredom, as well as the degree to which they expected boredom, pain, effort, and willpower to constitute self-regulatory challenges they would have to cope with. After the run, athletes reported the degree to which they actually had to deal with these self-regulatory challenges and if they had faced an action crisis during the competition. Analyses revealed that very extreme athletes displayed a significantly lower sport-specific trait boredom than less extreme athletes (p = 0.024, d=−0.48). With respect to self-regulatory challenges, willpower, pain, and effort were expected and reported at a much higher rate than boredom. However, only boredom was as a significant predictor of experiencing a crisis during the competition (odds ratio = 12.5, p = 0.02). Our results show that boredom also matters for highly active athletes. The fact that the experience of boredom—and not more prototypical competition-induced challenges, such as pain or effort—were linked to having an action crisis highlights the relevance of incorporating boredom into the preparation for a race and to the performance management during competition.

BACKGROUND Previous research shows that providing social support in socio-evaluative stress situations reduces participants´ stress responses. This stress-buffer effect, however, does not hold for everybody and some studies even found a stress-amplifying effect of social support. Motive disposition research suggests that social motives (affiliation and power) lead to differential and sometimes even opposing affective, and physiological responses to interpersonal interaction processes. We here integrate both lines of research and hypothesize that participants with strong affiliation motives benefit whereas participants with strong power motives do not benefit from social support in terms of psychobiological responses to a given stressor. Further, participants with strong affiliation and power motives are expected to respond to social support with an arousal of motive-specific affects and reproductive hormone responses (affiliation: progesterone, power: estradiol, testosterone). In addition, we test sex differences in the response to social support and in strengths of social motives. OBJECTIVE The main objective of the present study is to test whether social motives and participants´ sex moderate the effects of social support in stressful situations. METHODS We aim to collect data of 308 participants recruited at the local university of the authors. Participants´ social motives are assessed using a standardized measure in motive research (Picture Story Exercise). Then, the Trier Social Stress designed for groups is used to experimentally induce psychosocial stress. One group of participants receives social support from a confederate of the experimenter whereas the control group does not. Stress responses will be assessed by a modified version of the state anxiety scale of the State – Trait Anxiety Inventory (Spielberger, 1970) and by physiological indicators of stress (cortisol, alpha-amylase gained from saliva samples) at seven points of measurement. Reproductive hormones will be analyzed from four out of these seven saliva samples. Heart rate and heart rate variability will be assessed continuously. We additionally measure participants´ performance in the interview (part of TSST) using a self-developed categorization system. RESULTS The ethics committee of the University of Constance approved the application to conduct the study on December 18, 2018. Furthermore, the study was retrospectively registered in the German Clinical Trials Registry (DKRS) on March 09, 2022, under the following number: DRKS00028503. The start of the experiment was planned for the beginning of 2019 but was postponed to spring and summer 2022 due to Covid-19. Publication of the first results is planned for spring 2023. CONCLUSIONS Our theory-driven integration of social motives in social support research, and the precise analysis of sex differences might disentangle inconsistent findings in TSST- research. The more faceted view on individual differences has direct implications for applied contexts as it provides a framework for tailored conceptualizations of social support programs. CLINICALTRIAL German Clinical Trials Registry (DKRS) on March 09, 2022, DRKS00028503. https://www.drks.de/drks_web/navigate.do;jsessionid=24B169C7FD816B830EB40A8F24B8B2FD?navigationId=resultsExt

Self-control is a highly adaptive human capacity and research on self-control is booming. To further facilitate self-control research, especially in conditions where time-constraints might render the use of multi-item measures of self-control problematic, a validated time-efficient single item measure would be an asset. However, such a measure has not yet been developed and tested. Here, we address this gap by reporting the psychometric properties of a single item measure of self-control and by assessing its localization within a larger theorized psychometric network consisting of self-control, boredom and if-then planning. In a high-powered (N = 1553) study with paid online workers from the US (gender: 47.3% female, 51.7% male, 1% other; age: 40.36 ± 12.65 years), we found evidence for the convergent validity (Brief Self-Control Scale), divergent validity (Short Boredom Proneness Scale and If-Then Planning Scale), and criterion validity (objective and subjective socio-economic status) of the single item measure of self-control (“How much self-control do you have?”). Network psychometrics further revealed that the single item was part of the self-control subnetwork and clearly distinguishable from boredom and if-then planning, which together with self-control form a larger psychometric network of psychological dispositions that are relevant for orienting goal directed behavior. Thus, the present findings indicate that self-control can be adequately captured with the single item measure presented here, thereby extending the methodological toolbox of self-control researchers by a highly-time efficient measure.

Glioblastoma (GBM) is characterized by a particularly invasive phenotype, supported by oncogenic signals from the fibroblast growth factor (FGF)/ FGF receptor (FGFR) network. However, a possible role of FGFR4 remained elusive so far. Several transcriptomic glioma datasets were analyzed. An extended panel of primary surgical specimen-derived and immortalized GBM (stem)cell models and original tumor tissues were screened for FGFR4 expression. GBM models engineered for wild-type and dominant-negative FGFR4 overexpression were investigated regarding aggressiveness and xenograft formation. Gene set enrichment analyses of FGFR4-modulated GBM models were compared to patient-derived datasets. Despite widely absent in adult brain, FGFR4 mRNA was distinctly expressed in embryonic neural stem cells and significantly upregulated in glioblastoma. Pronounced FGFR4 overexpression defined a distinct GBM patient subgroup with dismal prognosis. Expression levels of FGFR4 and its specific ligands FGF19/FGF23 correlated both in vitro and in vivo and were progressively upregulated in the vast majority of recurrent tumors. Based on overexpression/blockade experiments in respective GBM models, a central pro-oncogenic function of FGFR4 concerning viability, adhesion, migration, and clonogenicity was identified. Expression of dominant-negative FGFR4 resulted in diminished (subcutaneous) or blocked (orthotopic) GBM xenograft formation in the mouse and reduced invasiveness in zebrafish xenotransplantation models. In vitro and in vivo data consistently revealed distinct FGFR4 and integrin/extracellular matrix interactions. Accordingly, FGFR4 blockade profoundly sensitized FGFR4-overexpressing GBM models towards integrin/focal adhesion kinase inhibitors. Collectively, FGFR4 overexpression contributes to the malignant phenotype of a highly aggressive GBM subgroup and is associated with integrin-related therapeutic vulnerabilities.

Pancreatic cancer (PDAC) is a highly aggressive malignancy for which the identification of novel therapies is urgently needed. Here, we establish a human PDAC organoid biobank from 31 genetically distinct lines, covering a representative range of tumor subtypes, and demonstrate that these reflect the molecular and phenotypic heterogeneity of primary PDAC tissue. We use CRISPR-Cas9 genome editing and drug screening to characterize drug-gene interactions with ARID1A and BRCA2. We find that missense- but not frameshift mutations in the PDAC driver gene ARID1A are associated with increased sensitivity to the kinase inhibitors dasatinib (p < 0.0001) and VE-821 (p < 0.0001). We conduct an automated drug-repurposing screen with 1,172 FDA-approved compounds, identifying 26 compounds that effectively kill PDAC organoids, including 19 chemotherapy drugs currently approved for other cancer types. We validate the activity of these compounds in vitro and in vivo. The in vivo validated hits include emetine and ouabain, compounds which are approved for non-cancer indications and which perturb the ability of PDAC organoids to respond to hypoxia. Our study provides proof-of-concept for advancing precision oncology and identifying candidates for drug repurposing via genome editing and drug screening in tumor organoid biobanks.

Treatment with anaplastic lymphoma kinase (ALK) inhibitors significantly improves outcome for non-small-cell lung cancer (NSCLC) patients with ALK-rearranged tumors. However, clinical resistance typically develops over time and, in the majority of cases, resistance mechanisms are ALK-independent. We generated tumor cell cultures from multiple regions of an ALK-rearranged clinical tumor specimen and deployed functional drug screens to identify modulators of ALK-inhibitor response. This identified a role for PI3Kβ and EGFR inhibition in sensitizing the response regulating resistance to ALK inhibition. Inhibition of ALK elicited activation of EGFR, and subsequent MAPK and PI3K-AKT pathway reactivation. Sensitivity to ALK targeting was enhanced by inhibition or knockdown of PI3Kβ. In ALK-rearranged primary cultures, the combined inhibition of ALK and PI3Kβ prevented the EGFR-mediated ALK-inhibitor resistance, and selectively targeted the cancer cells. The combinatorial effect was seen also in the background of TP53 mutations and in epithelial-to-mesenchymal transformed cells. In conclusion, combinatorial ALK- and PI3Kβ-inhibitor treatment carries promise as a treatment for ALK-rearranged NSCLC. Treatment with anaplastic lymphoma kinase (ALK) inhibitors significantly improves outcome for non-small-cell lung cancer (NSCLC) patients with ALK-rearranged tumors. However, clinical resistance typically develops over time and, in the majority of cases, resistance mechanisms are ALK-independent. We generated tumor cell cultures from multiple regions of an ALK-rearranged clinical tumor specimen and deployed functional drug screens to identify modulators of ALK-inhibitor response. This identified a role for PI3K beta and EGFR inhibition in sensitizing the response regulating resistance to ALK inhibition. Inhibition of ALK elicited activation of EGFR, and subsequent MAPK and PI3K-AKT pathway reactivation. Sensitivity to ALK targeting was enhanced by inhibition or knockdown of PI3K beta. In ALK-rearranged primary cultures, the combined inhibition of ALK and PI3K beta prevented the EGFR-mediated ALK-inhibitor resistance, and selectively targeted the cancer cells. The combinatorial effect was seen also in the background of TP53 mutations and in epithelial-to-mesenchymal transformed cells. In conclusion, combinatorial ALK- and PI3K beta-inhibitor treatment carries promise as a treatment for ALK-rearranged NSCLC.

Epithelial-to-mesenchymal transition (EMT) is discussed to be centrally involved in invasion, stemness, and drug resistance. Experimental models to evaluate this process in its biological complexity are limited. To shed light on EMT impact and test drug response more reliably, we use a lung tumor test system based on a decellularized intestinal matrix showing more in vivo-like proliferation levels and enhanced expression of clinical markers and carcinogenesis-related genes. In our models, we found evidence for a correlation of EMT with drug resistance in primary and secondary resistant cells harboring KRAS\(^{G12C}\) or EGFR mutations, which was simulated in silico based on an optimized signaling network topology. Notably, drug resistance did not correlate with EMT status in KRAS-mutated patient-derived xenograft (PDX) cell lines, and drug efficacy was not affected by EMT induction via TGF-β. To investigate further determinants of drug response, we tested several drugs in combination with a KRAS\(^{G12C}\) inhibitor in KRAS\(^{G12C}\) mutant HCC44 models, which, besides EMT, display mutations in P53, LKB1, KEAP1, and high c-MYC expression. We identified an aurora-kinase A (AURKA) inhibitor as the most promising candidate. In our network, AURKA is a centrally linked hub to EMT, proliferation, apoptosis, LKB1, and c-MYC. This exemplifies our systemic analysis approach for clinical translation of biomarker signatures.

Basic psychological need satisfaction is essential for the wellbeing of teachers and motivation at work. Transformational leadership contributes to the development and maintenance of a respectful, constructive atmosphere, a supportive working climate, and has been suggested to be a crucial factor for the satisfaction of the need for relatedness of employees. Transformational leadership is also considered as an ideal leadership style in the school setting, but most studies did not distinguish between individual and team effects of this leadership behavior. In the present study, we applied the dual-focused model of transformational leadership and focused on social processes to address the question of whether individual- and group-focused transformational leadership behavior contribute differently to satisfaction of the need for relatedness of teachers. Based on longitudinal data with three measurement points across one school year of n=1,217 teachers, results of structural equational models supported the notion of the dual effects of transformational leadership: Individual-focused transformational leadership was directly positively related to change in satisfaction of the need for relatedness of teachers. The relationship between group-focused transformational leadership and change in satisfaction of the need for relatedness was mediated by received social support from team members. These findings emphasize the importance of school leadership behavior of principals for satisfaction of the need for relatedness of teachers. The satisfaction of the need for relatedness is, therefore, not only satisfied through the direct interaction between the school principal and the individual teacher but also through interactions of the school principal with the whole team. Our results confirm that school principals should focus their leadership behavior both on individual need satisfaction of teachers as well as on team development., +repphzhbib2021F

BackgroundCancer immunotherapy represents a burgeoning new direction for oncology therapeutic innovation, with the principal thesis of activating one’s own immune system to irradicate cancer as opposed to the injection of foreign cytotoxic agents like chemotherapy. The first generation of checkpoint inhibitors unleash cytotoxic T cells to locate and kill their tumor target, however, only a small subset of patients (e.g. ~30% of NSCLC patients1) respond favorably. In order to advance the next generation of immunotherapy to the clinic, we critically need models which more accurately represent the immunosuppressive tumor microenvironment (TME).MethodsHere, we describe a novel 3D in vitro tumor model platform which engineers the tumor, stromal, and immune cell compartments of the TME in an extracellular matrix hydrogel (VersaGel2) and a high throughput 96-well format. The system has been extensively tested across multiple solid tumor indications, such as colorectal, lung, pancreatic, breast, and others. Specifically for this study, we utilized NSCLC PDX cells from the Charles River compendium and cocultured with human dermal fibroblasts (HDF) and PBMCs to study the effects of checkpoint inhibitor monoclonal antibodies, Pembrolizumab (anti-PD1) and Atezolizumab (anti-PDL1), on T cell infiltration and T cell-mediated killing using high content imaging. Supernatants were analyzed for cytokines and the 3D models were subsequently digested for flow cytometry.ResultsThe 3D models demonstrated varying degrees of T cell infiltration and killing capacity across PDX in a dose-dependent manner to checkpoint inhibitors, and the inclusion of fibroblasts played a critical role in further modulating response. Moreover, the data revealed clinically-relevant levels of CD3+, CD4+, and CD8+ T cell subpopulations and cytokine secretions such as IFN-gamma.ConclusionsThese data suggest a novel 3D model platform for assaying immunotherapeutic efficacy as well as its mechanism of action in the context of the TME. Future studies will include applying this novel platform to additional tumor models and screening multiple forms of immunotherapy – such as small molecules, biologics, and cell and gene therapy – in drug discovery, preclinical testing, and precision medicine.ReferencesHaslam A, Prasad V. Estimation of the percentage of US patients with cancer who are eligible for and respond to checkpoint inhibitor immunotherapy drugs. JAMA Netw 2019;2(5):e192535.Hribar KC, Wheeler CJ, Bazarov A, Varshneya K, Yamada R, Buckley P, Patil CG. A simple three-dimensional hydrogel platform enables ex vivo cell culture of patient and PDX tumors for assaying their response to clinically relevant therapies. Mol Canc Ther 2019;18(3):718–725.

BackgroundIdentifying how to optimally combine immunotherapies with other available anti-cancer therapies is a major challenge in oncology. A systematic method to screen many potential combination therapies ideally in vivo has remained elusive. We have utilized an implantable microdevice (IMD) performing cassette microdosing that measures intratumor drug responses and anti-tumor immunity for 20 agents in parallel. For each of the agents, local tumor response is measured by cyclical immunofluorescence for deep cellular response phenotyping. This approach is combined with systemic administration of checkpoint inhibitors to examine whether local immunogenic cell death (ICD) induced by a given drug microdose potentiates the immunotherapy’s anti-tumor effect.MethodsThe measurements were performed in a humanized mouse model of renal cancer, patient derived xenograft (PDX) RXF488. The PDX is derived from a 68 year old male patient suffering from clear cell renal carcinoma. RXF488 was implanted subcutaneously in 30 NSG mice. Animals were stratified into 6 groups with n= 4–6. Humanization was performed by the intravenous injection of 5x10e6 human peripheral blood mononuclear cells (PBMC) prior to the first treatment. Systemic anti-PD1 treatment was applied in the presence and absence of the microdevice loaded with eleven different drugs. Control groups received the microdevice in the presence or absence of PBMC. Beside the histological examination of the tumor tissue, flow cytometry (FC) was performed on bone marrow, spleen and tumor tissue to determine infiltration of human immune cells.ResultsFC analyses revealed no influence of the treatment on the human immune cells in bone marrow and spleen. The anti-PD1 treatment induced an increase in huCD45+ cells specifically in the tumor tissue and a decrease of the CD4/CD8 ratio in these cells only 48h after treatment. Our combination screen identified LXH254, Sorafenib and Doxorubicin exhibiting the highest increase in apoptosis induction when combined with checkpoint inhibitors. The increased efficacy from immunotherapy administration coincided with increased induction of ICD. We were able to verify the results of the screening experiment in a conventional setting with systemic combination treatment in the same PDX model.ConclusionsOur results demonstrate that local tumor response signatures of ICD can be used to systemically identify synergistic combinations of a range of drugs with immunotherapy on a tumor specific basis. The approach may represent a new paradigm for efficient in vivo screening of novel combinations, particularly with combinations involving immunotherapies.

Implicit motive research has shown that implicit motives are important predictors of behavior and well-being. However, little is known about the interrelationship between the different implicit motives measures frequently applied. We aimed to shed light on the convergent validity of three implicit motive measures and wanted to test their assumed statistical independence from three explicit motive measures. Therefore, we administered the picture story exercise (PSE), the operant motive test (OMT), and the multi-motive grid in one and the same study. As explicit measures, we used the personality research form, the motive enactment test, and a goal questionnaire. We investigated the statistical overlaps between all these measures (sample: 202 undergraduate students) and found that the implicit motive measures showed either no or only little correlation with each other. Furthermore, they also partly correlated with explicit motive measures. Supplementary analyses showed that the lack of statistical overlap between PSE and OMT can partly be ascribed to their different scoring systems. published

Endurance sports pose a plethora of mental demands that exercisers have to deal with. Unfortunately, investigations of exercise-specific demands and strategies to deal with them are insufficiently researched, leading to a gap in knowledge about athletic requirements and strategies used to deal with them. Here, we investigated which obstacles exercisers experience during an anaerobic (Wingate test) and an aerobic cycling test (incremental exercise test), as well as the strategies they considered helpful for dealing with these obstacles (qualitative analysis). In addition, we examined whether thinking of these obstacles and strategies in terms of if-then plans (or implementation intentions; i.e., “If I encounter obstacle O, then I will apply strategy S!”) improves performance over merely setting performance goals (i.e., goal intentions; quantitative analysis). N = 59 participants (age: M = 23.9 ± 6.5 years) performed both tests twice in a 2-within (Experimental session: 1 vs. 2) × 2-between (Condition: goal vs. implementation intention) design. Exercisers’ obstacles and strategies were assessed using structured interviews in Session 1 and subjected to thematic analysis. In both tests, feelings of exertion were the most frequently stated obstacle. Motivation to do well, self-encouragement, and focus on the body and on cycling were frequently stated strategies in both tests. There were also test-specific obstacles, such as boredom reported in the aerobic test. For session 2, the obstacles and strategies elicited in Session 1 were used to specify if-then plans. Bayesian mixed-factor ANOVA suggests, however, that if-then plans did not help exercisers to improve their performance. These findings shed novel light into the mental processes accompanying endurance exercise and the limits they pose on performance.

We propose a deep learning workflow for the classification of hematoxylin and eosin stained histological whole-slide images of non-small-cell lung cancer. The workflow includes automatic extraction of meta-features for the characterization of the tumor. We show that the tissue-classification produces state-of-the-art results with an average F1-score of 83%. Manual supervision indicates that experts, in practice, accept a far higher percentage of predictions. Furthermore, the extracted meta-features are validated via visualization revealing relevant biomedical relations between the different tissue classes. In a hypothetical decision-support scenario, these meta-features can be used to discriminate the tumor response with regard to available treatment options with an estimated accuracy of 84%. This workflow supports large-scale analysis of tissue obtained in preclinical animal experiments, enables reproducible quantification of tissue classes and immune system markers, and paves the way towards discovery of novel features predicting response in translational immune-oncology research.

Key Points mRNA decapping gene EDC4 is a novel fusion partner of MLL in AML. Genes functioning in mRNA decapping may compose a distinct group of MLL fusion partners that links MLL function with mRNA decapping in AML.

Objectives Successful sprint starts require self-control: Athletes need to avoid a false start (impulse control) and at the same time need to start as fast as possible (action initiation). Research from cognitive neuroscience shows that such self-control acts hinge on activity in areas in the lateral Prefrontal Cortex (lPFC). We are harnessing these findings in order to accurately analyze and better understand the neural basis of self-controlled sprint start performance. Design In a within-subject experimental design, participants executed three different sprint start sequences (Ready-Set-Go) for ten times each. In the no-start condition, participants only had to avoid producing a false start (impulse control) and in the experimental conditions - either with fixed or with supposedly variable set-start intervals - they additionally had to execute a fast start (impulse control + action initiation). Methods We used functional near-infrared spectroscopy (fNIRS) to assess cerebral oxygenation in the lPFC during sprint start in 33 male participants. Results Results show that cerebral oxygenation increased after the set-signal and this increase was particularly pronounced in the fixed and supposedly-variable start conditions. Post-hoc analyses further indicated that oxygenation differences between no-start and the two start conditions were particularly pronounced in anterior parts of the LPFC. Discussion This is the first study to reveal oxygenation changes in self-control relevant cortical areas during sprint start performance. This substantiates the claim that sprint starts impose self-control demands and provides a much called for application of neuroscience findings to the sport context.

Background: Systemic treatment options for metastatic renal cell carcinoma (RCC) have significantly expanded in recent years. However, patients refractory to tyrosine kinase and immune checkpoint inhibitors still have limited treatment options and patient-individualized approaches are largely missing. Patients and Methods: In vitro drug screening of tumor-derived short-term cultures obtained from seven patients with clear cell RCC was performed. For one patient, a patient-derived xenograft (PDX) mouse model was established for in vivo validation experiments. Drug effects were further investigated in established RCC cell lines. Results: The proteasome inhibitor carfilzomib was among the top hits identified in three of four patients in which an in vitro drug screening could be performed successfully. Carfilzomib also showed significant acute and long-term cytotoxicity in established RCC cell lines. The in vivo antitumoral activity of carfilzomib was confirmed in a same-patient PDX model. The cytotoxicity of carfilzomib was found to correlate with the level of accumulation of ubiquitinated proteins. Conclusions: In this proof-of-concept study, we show that patient-individualized in vitro drug screening and preclinical validation is feasible. However, the fact that carfilzomib failed to deliver a clinical benefit in RCC patients in a recent phase II trial unrelated to the present study underscores the complexities and limitations of this strategy.

Colorectal cancer (CRC) accounts for about 10% of cancer deaths worldwide. Colon carcinogenesis is critically influenced by the tumor microenvironment. Cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs) represent the major components of the tumor microenvironment. TAMs promote tumor progression, angiogenesis and tissue remodeling. However, the impact of the molecular crosstalk of tumor cells (TCs) with CAFs and macrophages on monocyte recruitment and their phenotypic conversion is not known in detail so far. In a 3D human organotypic CRC model, we show that CAFs and normal colonic fibroblasts are critically involved in monocyte recruitment and for the establishment of a macrophage phenotype, characterized by high CD163 expression. This is in line with the steady recruitment and differentiation of monocytes to immunosuppressive macrophages in the normal colon. Cytokine profiling revealed that CAFs produce M-CSF, and IL6, IL8, HGF and CCL2 secretion was specifically induced by CAFs in co-cultures with macrophages. Moreover, macrophage/CAF/TCs co-cultures increased TC invasion. We demonstrate that CAFs and macrophages are the major producers of CCL2 and, upon co-culture, increase their CCL2 production twofold and 40-fold, respectively. CAFs and macrophages expressing high CCL2 were also found in vivo in CRC, strongly supporting our findings. CCL2, CCR2, CSF1R and CD163 expression in macrophages was dependent on active MCSFR signaling as shown by M-CSFR inhibition. These results indicate that colon fibroblasts and not TCs are the major cellular component, recruiting and dictating the fate of infiltrated monocytes towards a specific macrophage population, characterized by high CD163 expression and CCL2 production.

Targeting mitosis by taxanes is one of the most common chemotherapeutic approaches in various malignant solid tumors, but cancer cells may survive antimitotic treatment with attainable in vivo concentrations due to mitotic slippage with a residual activity of the ubiquitin ligase anaphase-promoting complex (APC/C) and a continuous slow ubiquitin-proteasome-dependent cyclin B-degradation leading to mitotic exit. Therefore, blocking cyclin B-proteolysis via additional proteasome (PI) or APC/C-inhibition may have the potential to enhance tumor cell eradication by inducing a more robust mitotic block and mitotic cell death. Here, we analyzed this approach in different cell lines and more physiological patient-derived xenografts (PDX) from lung and breast cancer. The sequential combination of paclitaxel with the PI bortezomib enhanced cell death, but in contrast to the hypothesis during interphase and not in mitosis in both lung and breast cancer. APC/C-inhibition alone or in sequential combination with paclitaxel led to strong mitotic cell death in lung cancer. But in breast cancer, with high expression of the anti-apoptotic regulator Mcl-1, cell death in interphase was induced. Here, combined APC/C- and Mcl-1-inhibition with or without paclitaxel was highly lethal but still resulted in interphase cell death. Taken together, the combination of antimitotic agents with a clinically approved PI or inhibitors of the APC/C and Mcl-1 is a promising approach to improve treatment response in different solid tumors, even though they act entity-dependent at different cell cycle phases.

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules., There is an unmet clinical need to identify therapeutic options for the treatment of pancreatic cancer (PDAC). Here the authors present a systematic screening approach for the identification of potential PDAC cell surface target candidates for CAR-T cell based immunotherapy, followed by their functional validation in preclinical models.

(1) Background: A large body of research has examined the positive effects of physical activity on physical and mental health. However, for some, excessive exercise can develop into an addiction that is detrimental to their health. In the present study, we examine potential personal (self-control, self-concordance) and social (social support) resources that we assume to be related to exercise dependence. (2) Methods: One hundred and forty athletes from different endurance sports participated in an online survey. Exercise dependence, self-control, self-concordance, and social support were assessed using questionnaires that are well-established in health and sport psychology. Additionally, further sport-relevant and demographic variables were assessed. (3) Results: Correlational analyses supported our hypotheses that exercise dependence is negatively correlated with the personal resources trait, state self-control, and self-concordance. Social support, however, was not significantly correlated with exercise dependence. Furthermore, the results of a mediation analysis revealed that the relationship between both personal traits (self-control, self-concordance) and exercise dependence was mediated by state self-control. (4) Conclusions: Our results indicate that trait self-control and self-concordance might be important personal resources that protect against exercise dependence by making state self-control available.

Physical activity counteracts some of the negative consequences associated with chronic neurological diseases. Here, we describe the levels of physical activity (PA) and sports activity (Sport) in patients with multiple sclerosis (pMS, n = 59) and chronic stroke (pStroke, n = 67) and test compliance with the recommendation for health-promoting physical activity of the World-Health Organization (WHO). Secondly, we tested for differences between the groups of patients, and thirdly, we examined relationships between PA and Sport with psychological indicators of perceived energy (fatigue and vitality) and self-beliefs (self-efficacy and self-control). Psychological constructs were assessed with validated measures from different disciplines in Psychology. A statistical aim was to describe interpretations gained by (non-) parametric Bayesian and Null-Hypothesis-Significance Testing statistics (NHST) on the example of the conducted tests for differences and relationships. Descriptive analyses revealed that pMS and pStroke complied with recommendations of the WHO, but with large variance indicating that patient groups are not homogenous. Tests for differences showed that the PA difference between pMS and pStroke can be attributed to the higher proportion of women in the pMS sample as they engage more in household chores (important part of PA). Tests for relationships showed that for pStroke, vitality, self-control, and self-efficacy were positively related to the level of sports activity. Furthermore, pStroke who were sport active had lower fatigue and higher self-control and self-efficacy scores than sport inactive people. Although they address slightly different questions, the Bayesian and the NHST approach led to similar general conclusions.

Identifying how to optimally combine immunotherapies with other available anti-cancer therapies is a major challenge in oncology. We have utilized an implantable microdevice performing cassette microdosing that measures intratumor drug responses and anti-tumor immunity for eleven agents in parallel. Local tumor response is measured by cyclical immunofluorescence for deep cellular response phenotyping. This approach is combined with the systemic administration of checkpoint inhibitors to examine whether local immunogenic cell death (ICD) induced by a given drug microdose potentiates the immunotherapy’s anti-tumor effect. The measurements were performed in a humanized mouse model of clear cell renal cancer, patient derived xenograft (PDX) RXF488. RXF488 was implanted subcutaneously in 30 NSG mice. Animals were stratified into 6 groups with n= 4-6. Humanization was performed by the intravenous injection of 5x10e6 human peripheral blood mononuclear cells prior to the first treatment. Systemic anti-PD1 treatment was applied in the presence and absence of the microdevice loaded with eleven different drugs. Control groups received the empty microdevice in the presence or absence of PBMC. Beside the histological examination of the tumor tissue, flow cytometry (FC) was performed on bone marrow, spleen and tumor tissue to determine infiltration of human immune cells. FC analyses revealed no influence of the treatment on the human immune cells in bone marrow and spleen. The anti-PD1 treatment induced an increase in huCD45+ cells specifically in the tumor tissue only 48h after treatment. Several agents showed a significant increase in apoptosis induction when nivolumab was added: The largest increase was observed for the panRAF inhibitor LXH254, Sorafenib, Oxaliplatin and Doxorubicin. The increased efficacy from immunotherapy administration has a strong positive correlation with increased induction of ICD in the tumor microenvironment determined by CD11b, ICAM-1 and MHC-II expression: drugs that showed the highest increase in apoptosis when combined with aPD-1 showed an increased likelihood for markers associated with ICD, namely Oxaliplatin and LXH254. We were able to verify the results of the screening experiment in a conventional setting with systemic combination treatment arms in the same PDX model. Our results demonstrate that local tumor response signatures of ICD can be used to systemically identify synergistic combinations of a range of drugs with immunotherapy on a tumor specific basis. The approach may represent a new paradigm for efficient in vivo screening of novel combinations, particularly with combinations involving immunotherapies. Citation Format: Oliver Jonas, Eva Oswald, Kanstantsin Lashuk, Sebastian Ahn, Julia Schüler. A controlled drug release device facilitates the identification of favorable combinations of immune-oncology drugs with targeted or cytotoxic drugs in a patient-derived humanized mouse model of renal cancer [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 4196.

With epigenetic therapy emerging as a potential therapy for solid tumors and recent reports that indicate an influence of hypomethylating agents on the PD-1/PD-L1 expression in tumor tissue, epigenetic therapy in combination with immune-modulatory compounds might open-up new avenues for cancer treatment. To investigate the influence of hypomethylating agents on the PD-1/PD-L1 signaling cascade in breast cancer and its implication on possible treatment options, we determined the effect of 5-Azacytidine (5-AZA), decitabine (DAC) and anti-PD1 in mono- and combined therapy on the mouse breast cancer model 4T1 in vitro and in vivo. In a life cell imaging based 2D system we evaluated the influence of the different treatment arms on tumor growth, methylation status and PD-L1 expression. Furthermore, we determined the effect of 5-AZA and DAC on autologous T cells in the presence and absence of 4T1 cancer cells. The combination of 5-AZA and anti-PD1 treatment was further evaluated in an in vivo setting. 24 4T1 bearing balb/c mice were treated with either an isotype control, 5-AZA, anti-PD1 or a combination of the latter. Tumor growth was monitored by caliper measurement. At the end of the experiment tumor and hematopoietic organs were analyzed by flow cytometry. A panel of 36 murine cytokines was determined in the murine serum at different time points. Low dose 5-AZA (IC10) and DAC (IC2.5) induced a down regulation of PD-L1 on 4T1 cells in vitro. Interestingly, this effect was most prominent at the dose level and time point when the hypomethylation effect of the respective compound was most pronounced (day 7). 5-AZA and DAC induced an upregulation of PD-1 on autologous CD4+ as well as CD8+ T cells. Of note, 4T1 cells reduced the expression of PD-1 on autologous CD4+ and CD8+ T cells. Antitumoral activity in vitro was determined in all treatment arms. An additive effect of the combination was specifically determined when combining 5-AZA and anti PD-1. Hence, 5-AZA and anti PD-1 were investigated in vivo more in detail. 4T1 is characterized by an immune suppressive tumor microenvironment as indicated by the in vitro results: we determined a reduced CD4/CD8 ratio and a high percentage of MDSC. These features were reverted by the applied treatments. Along the same line, 5-AZA in mono- and in combination therapy with anti-PDL1 specifically increased the G-CSF levels in murine serum. In addition, the PDL-1 downregulation on the tumor cells was reproduced in vivo. Finally, the combination arm displayed the most prominent antitumoral activity. In summary, we have strong evidence that low dose hypomethylating agents influence the immune landscape of breast cancer via the modulation of PD-1/PD-L1 signaling. The combination with checkpoint inhibitors was superior to the monotherapy and might lead to new treatment options for breast cancer patients. Citation Format: Julia Schüler, Agon Kolica, Hung Ngyuen, Anna Edinger, Kanstantsin Lashuk, Eva Oswald. Hypomethylating agents modulate the PD-1/PD-L1 signaling in a murine breast cancer model and show additive effects in the combination with PD-1 blockage in vitro and in vivo [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 1349.

High throughput screening offers tangible benefits towards rapidly testing various permutations of novel or existing therapeutic agents. In particular, tumor panels that cover a range of histotypes and molecular subtypes have been previously developed, such as the NCI-60, however they utilize cell lines and, in some cases, a 2D cell culture format, which limit their translatability to preclinical and clinical trials. Moreover, the biological complexity of the tumor microenvironment (TME) has revealed a need for more translatable 3D in vitro tumor models that reflect the in vivo physiological outcome to therapies, particularly with the explosion of immunotherapy programs in drug discovery which target the immune compartment of the TME. Here, we describe for the first time a 3D in vitro PDX panel comprising 30 distinct PDX models in coculture with fibroblasts and PBMCs in engineered extracellular matrix hydrogels that display distinct similarities to the three compartments of the TME - tumor, stroma, and immune cells. Histotypes in the panel include, amongst others, Non-small cell lung, colorectal, breast, pancreatic, gastric, melanoma, and renal cancers. The panel is constructed in a high throughput 96-well format and rapidly assays tumor growth delay and other endpoints such as apoptosis in a dose-dependent manner across various drug modalities such as small molecules, biologics and cell therapy. The panel has been tested against targeted therapy (Docetaxel, Cisplatin) and immunomodulatory agents (Pembrolizumab, Atezolizumab) and the results correlate to the corresponding PDX in vivo data. Interestingly, cisplatin treatment displayed an average size reduction of >30% for melanoma but a varied response in NSCLC tumors. And, several NSCLC responded more favorably to checkpoint inhibitors, however, the lung epidermoid PDX demonstrated no significant response. Moreover, subsequent cytokine analysis and immunofluorescence staining of several models revealed protein signatures of cancer-associated fibroblasts and CD3+ sequestration in the tumor stroma in some 3D models, suggesting the fibroblasts’ critical role in regulating the immune response. In short, the 30-PDX Panel described here represents a large step forward towards achieving translatable efficacy data at the earliest stages of drug discovery where little is known about the mechanism of action for a particular therapeutic agent or combination of agents. Citation Format: Kolin C. Hribar, Bin Xue, Christopher Harrod, Timothy Jensen, Julia Schüler. High throughput screening of 30 PDX cell lines in a 3D ECM hydrogel platform, incorporating tumor, stroma and immune components to demonstrate simultaneous investigation of multiple anti-tumor modalities [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 1880.

Metastatic disease in solid cancer in general and specifically in colorectal cancer (CRC) is still the main reason for a poor prognosis underlining the need for new therapeutic strategies. Furthermore, a dysregulated secretion is one major driver of cancer progression and invasion and therefore holds promise as a general therapeutic target for the treatment of metastases. The overall aim of this study is to gain a quantitative understanding of the CRC secretome to elucidate the influence of the microenvironment on selected players and vice versa the influence of the secretome in vitro and in vivo. In a first attempt to elucidate the role of secreted factors in colon cancer progression, we cultured 20 CRC cell lines (five PDX derived and 15 commercial lines) in 3D spheroid cultures and compared the secreted cytokine profile with the 2D culture conditions of the same CRC cell line panel. We analyzed 45 cytokines in duplicates by using a human specific magnetic bead-based assay. Based on the cluster analysis of those cytokine profiles we categorized the CRC lines into low metastatic, intermediate and highly metastatic lines. To validate this clustering in vivo we implanted one representative cell line of each cluster orthotopically in immune-compromised NSG mice (n=10 per line in two independent experiments) and followed tumor growth by optical imaging twice a week over the course of up to 50 days. Tumor growth and metastases were confirmed by histological examination and terminal ex vivo imaging of the organs. The murine and human cytokine profile of different organs and the serum of tumor bearing mice was determined as well. The predicted highly metastatic cell line HCT116 displayed a latency of 24 days post implantation: after that time tumor cells were detected via optical imaging not only at the implantation site, but as well in kidney, spleen, liver and lungs in 100% of the examined animals. The intermediate cell line HT29 had a latency of 38 days and showed tumor growth in colon and liver of all animals and lung metastases on 80% of the implanted mice. Mice bearing the PDX derived cell line 269, representing the low metastatic phenotype, had to be sacrificed 50 days post implant. Tumor cells were detected at the implantation site. Micro-metastases in liver and lung were detected in only 75% of the animals. The secretome pattern specific for an individual cell line was predictive for the metastatic potential in vivo in an orthotopic mouse model. Of note, none of the mentioned cell lines metastasized when implanted subcutaneously. The detailed analysis of the mouse and human cytokine profiles will help to elucidate the complex crosstalk between tumor cells and the tumor microenvironment. The modulation of individual signaling cascades in vitro and in vivo will help to gain a deeper understanding of the metastatic process thereby enabling the development of possible new therapies. Citation Format: Jacqueline Bersano, Dorothee Lenhard, Kanstantsin Lashuk, Eva Oswald, Julia Schüler. The secretome of colorectal cancer cell lines cultured in vitro is predictive for their growth and metastatic behavior in an orthotopic in vivo mouse model [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 3126.

Brain and spinal cord tumors are the second most common group of cancers in children. For children who experience relapses of their tumors, usually after very intensive first-line therapy, curative treatment options are scarce. Thus, the need for predictive preclinical platforms explicitly for pediatric brain tumors is an urgent need. In the framework of the ITCC-P4 public-private partnership, we thus far fully established 23 pediatric brain tumor-derived PDX models. A selection of four high grade glioma, one medulloblastoma and one ependymoma models were used to validate a protocol for fluorescence-based optical imaging. Tumor growth characteristics, latency and histopathology were evaluated for the un-transduced and iRFP713-transduced models side by side. In case of iRFP713-transduced PDX models, tumor load was determined twice a week with the Pearl trilogy system (LiCor, Germany). All animals were examined for neurological symptoms daily and body weight was examined twice a week. The latency of the tumor models ranged from 29 days to 180 days. The take rate was 100% across all the models with n=12 NSG mice per setting. The transduction did not influence the take rate, but 30 - 40% more donor material was needed due to viability loss during the overnight transduction. It was not possible to determine the transduction efficiency for iRFP713 in the overnight culture as the signal is getting upregulated only 48h - 72h post transduction, e.g., when the cells are already implanted. The signal was stable for up to 180 days in the slowest tumor model ependymoma HN0579. The fastest model, high grade glioma HG0068, reached termination criteria within 24 days. Histopathological examination was strictly correlated with the tumor load determined by optical imaging in situ and ex vivo (organ imaging). The histopathological investigation of the mouse brains displayed no differences in tumor localization, size, and invasiveness between the transduced and the un-transduced lines. The proliferation rate determined by Ki-67 staining was not influenced by the modification of the cells. Further molecular and phenotypic characterization of the transduced vs the un-transduced PDX will increase the utility of this platform for the development of new drugs and the identification of innovative drug targets. Citation Format: Eva Oswald, Kanstantsin Lashuk, Johannes Gojo, Dorothee Lenhard, Norman Mack, Sonja Krausert, Daniela Lötsch, David Jones, Marcel Kool, Till Milde, Walter Berger, Stefan M. Pfister, Julia Schüler. Establishment and characterization of pediatric brain tumor models in an orthotopic mouse model [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 1673.

The advent of anti-HER2 therapy has prominently prolonged the time of disease progression and survival for metastatic breast cancer patients, where a decent proportion is suffering from a HER2+ tumor. Beside classical approaches via antibodies against HER2, the use of Chimeric Antigen Receptor T (CAR-T) cells also in a solid cancer context is getting more and more attention. In our study, we evaluated the efficacy of Trastuzumab versus HER2 targeting CAR-T cells in a panel of human cancer cell lines (SK-OV3, Hs578T and JIMT-1) with different HER2 expression levels in vitro in 2D as well as 3D and in vivo in immunocompromised mice. In vitro, the tumor growth and invasion of the CAR-T cells was measured via fluorescence-based live cell imaging. On the last experiment day, a metabolic read-out (CellTiter-Glo, CTG assay) was performed. In vivo, we measured tumor growth inhibition (TGI) via caliper measurement and tumor tissue and hematopoietic organs were analyzed by flow cytometry (FC), Immunohistochemistry (IHC) and multiplex cytokine analysis. The HER2 targeting CAR-T cells eradicated the 3D spheroids of the HER2+ SK-OV3 as well as JIMT-1 in a dose-dependent manner. The untransduced control T cells did not influence the tumor growth in vitro. Trastuzumab displayed efficacy in 2D and 3D in SKOV3. As expected, JIMT-1 cells were resistant to trastuzumab treatment despite their positive HER2 status. The HER2- cell line Hs578T served as a negative control and proved to be resistant to any treatment in this study. The HER2-targeting CAR-T cells were applied in vivo in two different doses to JIMT-1 tumor bearing NSG mice (n=8/group) and tumor volume was measured over time. Again, the CAR-T cells were able to induce a complete remission. In contrast, the tumors displayed progressive disease under therapy with the untransduced T cells or Trastuzumab. Interestingly, the CAR-T cells induced a tumor swelling between eight- and twelve-days post injection very similar to the situation in the patient. Four weeks post injection the human T cells could be detected in spleen, peripheral blood, liver and bone marrow of the JIMT- bearing mice. The implication of this finding for possible side effects of the HER2 CAR´s remains to be elucidated. A PK/PD study is currently ongoing for this purpose. Taken together the 3D live cell imaging platform proved to be a feasible tool for efficacy testing of biologics as well as cellular therapies. Our in house developed HER2 CAR-T cells proved to be specific and effective in eradicating the targeted cancer cells in vitro and in vivo. The mechanism behind the modulated sensitivity of the HER2+ JIMT-1 cells against HER2-targeted treatment will help to shed some light in possible resistance mechanism and hopefully have some translational value for patients suffering from this disease. Citation Format: Eva Oswald, Gemma Moiset, Kanstantsin Lashuk, Sophie Vermond, Monique Hazenoot, Julia Schüler. Allogenic HER2-CAR T cells overcome intrinsic trastuzumab resistance in vitro and in vivo in a preclinical model of breast cancer [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 574.

Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3-1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3-1 and RIG-I mRNA was also observed during decitabine treatment