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Additional file 2. Supplementary methods, and supplementary figures Fig S1-S6.

Simple Summary Neuroendocrine tumors of the gastrointestinal tract (GEP-NEN) are a rare type of tumor with considerable variability in the course of disease, which makes clinical management particularly challenging. Biomarkers that are able to guide personalized treatment decisions would be of importance, but are not yet available. In this study, we demonstrate that tissue expression, as well as circulating levels of the cytokine Flt3L in advanced and aggressive tumors, predict survival and time to progression. Increased tumoral Flt3L was also associated with upregulation of genes related to immune activation, suggesting Flt3L as a surrogate marker of host anti-tumor immunity. Therefore, Flt3L measurements in serum may hold promise as a biomarker of disease outcome that could support personalized treatment decisions in GEP-NEN patients, potentially guiding researchers towards viable immunotherapies. Abstract Background: The clinical management of high-grade gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) is challenging due to disease heterogeneity, illustrating the need for reliable biomarkers facilitating patient stratification and guiding treatment decisions. FMS-like tyrosine kinase 3 ligand (Flt3L) is emerging as a prognostic or predictive surrogate marker of host tumoral immune response and might enable the stratification of patients with otherwise comparable tumor features. Methods: We evaluated Flt3L gene expression in tumor tissue as well as circulating Flt3L levels as potential biomarkers in a cohort of 54 patients with GEP-NEN. Results: We detected a prominent induction of Flt3L gene expression in individual G2 and G3 NEN, but not in G1 neuroendocrine tumors (NET). Flt3L mRNA expression levels in tumor tissue predicted the disease-related survival of patients with highly proliferative G2 and G3 NEN more accurately than the conventional criteria of grading or NEC/NET differentiation. High level Flt3L mRNA expression was associated with the increased expression of genes related to immunogenic cell death, lymphocyte effector function and dendritic cell maturation, suggesting a less tolerogenic (more proinflammatory) phenotype of tumors with Flt3L induction. Importantly, circulating levels of Flt3L were also elevated in high grade NEN and correlated with patients’ progression-free and disease-related survival, thereby reflecting the results observed in tumor tissue. Conclusions: We propose Flt3L as a prognostic biomarker for high grade GEP-NEN, harnessing its potential as a marker of an inflammatory tumor microenvironment. Flt3L measurements in serum, which can be easily be incorporated into clinical routine, should be further evaluated to guide patient stratification and treatment decisions.

Transgenic mice are often generated by Random Integration (RI) of the transgene into the genome of murine Embryonic Stem (mES) cells. Since the chromosomal integration site of the gene of interest has a strong impact on expression patterns in the animal (ôposition effectö), generation of transgenic mouse models is hindered by high screening efforts to identify appropriate loci. Strategies devised to circumvent the problems of RI, e.g. site specific integration by homologous recombination, suffer from various limitations like the small number of suitable integration sites currently available. Hence, the aim of this work was to screen for chromosomal loci in mES cells that allow for high and well regulated transgene expression in mice and to render them reusable via Recombinase Mediated Cassette Exchange (RMCE). The screening cassette applied for this purpose contained an autoregulatory positive feedback loop driven by the tetracycline dependent system. As autoregulated transgene expression has not yet been examined in detail on transgenic mouse level, evaluation of this mode of regulated expression was a major issue of this work. Luciferase reporter levels from two random loci and the ROSA26 reference locus revealed strong variations that seem to be specific to autoregulation. In vivo bioluminescence analyses of transgenic mice derived from the two above mentioned clones showed a strong heterogeneity of expression between individuals of the same line with some animals completely shutting off expression. As expected for the autoregulated design, luciferase expression in one transgenic mouse line could only be induced by doxycycline if the animal had previously shown detectable basal levels. For both transgenic lines activity of the cassette was restricted to certain tissues. In conclusion, proof of principle for the envisioned "tag-and-target" strategy has been provided by this work and the tools and methods have been established to start large scale screening., Transgene Mäuse werden oft durch Integration des Transgens in zufällige chromosomale Loci muriner embryonaler Stammzellen (mES Zellen) generiert. Der jeweilige Integrationsort hat einen starken Einfluss auf das Expressionsverhalten, was aufwändiges Screening nach geeigneten Loci erforderlich macht. Es gibt mehrere Möglichkeiten diesen Positionseffekt zu umgehen, z.B. durch spezifische Integration des Transgens in einen definierten Locus durch homologe Rekombination. Diese Strategien unterliegen aber einigen Beschränkungen, u. a. der geringen Anzahl zur Zeit verwendbarer Loci. Deshalb befasst sich diese Arbeit mit dem Screening nach chromosomalen Loci in mES Zellen, welche eine hohe und gut regulierte Transgenexpression erlauben. Diese Integrationsorte werden über Rekombinase vermittelten Kassetten Austausch (RMCE) wieder verwendbar gemacht. Dazu wurde eine Screening Kassette mit einem autoregulierten, Tetracyclin (Tet) abhängigen positiven Feedback Loop eingesetzt. Die Charakterisierung dieses autoregulierten, Tet abhängigen Expressionsmodus in transgenen Mäusen war ein wichtiger Aspekt dieser Arbeit. Die Luziferase Reporter Expression von zwei zufälligen Loci und vom ROSA26 Referenz Locus in mES Zellen schwankte stark, was vermutlich eine spezifische Eigenschaft der Autoregulation ist. Aus den ersten beiden Klonen wurden transgene Mäuse generiert. In vivo Biolumineszenz Analysen dieser Tiere zeigten ein sehr heterogenes Expressionsverhalten in beiden Linien; einige Mäuse unterdrückten die Expression komplett. Wie für das autoregulierte System erwartet konnte die Expression durch Gabe von Doxycyclin in einer Linie induziert werden sofern das jeweilige Tier detektierbare Basalwerte aufwies. In beiden transgenen Linien war die Aktivität der Kassette auf bestimmte Gewebe beschränkt. Zusammenfassend konnte die Funktionalität der verwendeten „tag and target“ Strategie nachgewiesen werden und die notwendigen Methoden für ein groß angelegtes Screening etabliert werden.