Current clinical classification of pancreatic ductal adenocarcinoma (PDAC) is unable to predict prognosis or response to chemo- or immunotherapy and does not take into account the host reaction to PDAC cells. Our aim is to classify PDAC according to host- and tumor-related factors into clinically/biologically relevant subtypes by integrating molecular and microenvironmental findings. A well-characterized PDAC cohort ( = 110) underwent next-generation sequencing with a hot spot cancer panel while next-generation tissue microarrays were immunostained for CD3, CD4, CD8, CD20, PD-L1, p63, hyaluronan-mediated motility receptor (RHAMM), and DNA mismatch repair proteins. Previous data on FOXP3 were integrated. Immune cell counts and protein expression were correlated with tumor-derived driver mutations, clinicopathologic features (TNM 8th edition, 2017), survival, and epithelial-mesenchymal transition (EMT)-like tumor budding. Three PDAC subtypes were identified: the "immune escape" (54%), poor in T and B cells and enriched in FOXP3 regulatory T cells (Treg), with high-grade budding, frequent ,, and mutations, and poor outcome; the "immune rich" (35%), rich in T and B cells and poorer in FOXP3 Tregs, with infrequent budding, lower and mutation rate, and better outcome and a subpopulation with tertiary lymphoid tissue (TLT), mutations in DNA damage response genes ( and ), and the best outcome; and the "immune exhausted" (11%), with immunogenic microenvironment and two subpopulations-one with PD-L1 expression and a high mutation rate and a microsatellite-unstable subpopulation with a high prevalence of mutations. The combination of low budding, low stromal FOXP3 counts, presence of TLTs, and absence of mutations confers significant survival advantage in patients with PDAC. Immune host responses correlate with tumor characteristics, leading to morphologically recognizable PDAC subtypes with prognostic/predictive significance. .

Highlights d Map of PDAC dependencies using RNA-seq, ChIP-seq, and genome-wide CRISPR screening d Expression and direct utilization of cytokine and immune signals in PDAC stem cells d Nuclear hormone receptor RORg regulates mouse and human pancreatic cancer d Pharmacologic blockade of RORg reduces tumor burden and improves survival

BackgroundSexual selection has initially been thought to occur exclusively at the precopulatory stage in terms of contests among males and female mate choice, but research over the last four decades revealed that it often continues after copulation through sperm competition and cryptic female choice. However, studying these postcopulatory processes remains challenging because they occur internally and therefore are often difficult to observe. In the transparent free-living flatworm Macrostomum lignano, a recently established transgenic line that expresses green fluorescent protein (GFP) in all cell types, including sperm, offers a unique opportunity to non-invasively visualise and quantify the sperm of a GFP-expressing donor inside the reproductive tract of wild-type recipients in vivo. We here test several aspects of the reproductive performance of the transgenic individuals and the accuracy of the techniques involved in assessing the GFP-expressing worms and their sperm. We then show the usefulness of these methods in a study on sperm displacement.ResultsGFP-expressing worms do not differ from wild-type worms in terms of morphology, mating rate and reproductive success. In addition, we show that the GFP signal is reliably and unequivocally expressed by all GFP-expressing individuals observed under epifluorescence illumination. However, the intensity of the GFP signal emitted by sperm of GFP expressing donors can vary (which we show to be at least in part due to sperm ageing) and the GFP marker is inherited according to Mendel’s laws in most, but not all, of the individuals. Nevertheless, we argue these two issues can be addressed with an appropriate experimental design. Finally, we demonstrate the value of the GFP-techniques by comparing the number of GFP-expressing sperm in a wild-type recipient before and after mating with a competing sperm donor, providing clear experimental evidence for sperm displacement in M. lignano. This result suggests that sperm donors can displace previously stored sperm and replace it with their own.ConclusionThe availability of the GFP-techniques in a transparent organism provide unique opportunities to visualise and quantify internal processes in the female reproductive tract after mating, which opens new avenues in the study of sexual selection.

BackgroundIn colorectal cancer, CDX2 expression is lost in approximately 20% of cases and associated with poor outcome. Here, we aim to validate the clinical impact of CDX2 and investigate the role of promoter methylation and histone deacetylation in CDX2 repression and restoration.MethodsCDX2 immunohistochemistry was performed on multi-punch tissue microarrays (n = 637 patients). Promoter methylation and protein expression investigated on 11 colorectal cancer cell lines identified two CDX2 low expressors (SW620, COLO205) for treatment with decitabine (DNA methyltransferase inhibitor), trichostatin A (TSA) (general HDAC inhibitor), and LMK-235 (specific HDAC4 and HDAC5 inhibitor). RNA and protein levels were assessed. HDAC5 recruitment to the CDX2 gene promoter region was tested by chromatin immunoprecipitation.ResultsSixty percent of tumors showed focal CDX2 loss; 5% were negative. Reduced CDX2 was associated with lymph node metastasis (p = 0.0167), distant metastasis (p = 0.0123), and unfavorable survival (multivariate analysis: p = 0.0008; HR (95%CI) 0.922 (0.988–0.997)) as well as BRAFV600E, mismatch repair deficiency, and CpG island methylator phenotype. Decitabine treatment alone induced CDX2 RNA and protein with values from 2- to 25-fold. TSA treatment ± decitabine also led to successful restoration of RNA and/or protein. Treatment with LMK-235 alone had marked effects on RNA and protein levels, mainly in COLO205 cells that responded less to decitabine. Lastly, decitabine co-treatment was more effective than LMK-235 alone at restoring CDX2.ConclusionCDX2 loss is an adverse prognostic factor and linked to molecular features of the serrated pathway. RNA/protein expression is restored in CDX2 low-expressing CRC cell lines by demethylation and HDAC inhibition. Importantly, our data underline HDAC4 and HDAC5 as new epigenetic CDX2 regulators that warrant further investigation.Electronic supplementary materialThe online version of this article (10.1186/s13148-018-0548-2) contains supplementary material, which is available to authorized users.

Aims The tumour–node–metastasis classification system is used for prognostication purposes and to guide patient management. However, in colorectal cancer (CRC), additional markers are needed to stratify prognostic subgroups. Two promising markers have emerged from large bodies of research: tumour budding and T cell host response (CD3, CD8 and CD45RO infiltrates). However, attempts to combine these two parameters have been sparse. The aim of this study was to perform an assessment of potential protagonists that could be used in a combined score (budding/T cell score, BTS). Methods and results This descriptive, retrospective study was performed on a multipunch tissue microarray containing material from 345 patients with stages I–IV CRC. Areas from tumour centre, front and microenvironment were stained for pancytokeratin/CD3, pancytokeratin/CD8 and pancytokeratin/CD45RO. Tumour buds were scored manually and T cell infiltrates digitally using open‐source software. Tumour buds, T cell counts and combined BTS were associated with clinicopathological features and overall survival (OS). A higher combined BTS score (buds/CD8, tumour centre) performed better than budding or CD8/CD3 alone in predicting nodal metastases (P < 0.0001, OR = 1.466, 95% CI = 1.115–1.928). Only higher BTS (buds/CD3) were significantly associated with poorer OS on multivariate analysis (P = 0.012, hazard ratio = 1.218, 95% confidence interval = 1.044–1.419). Conclusions Although CD8+/CD3+ T cells are predictive of tumour biology in CRC, we found a combined BTS to be stronger in predicting survival and certain features with high clinical relevance, such as nodal metastases, in comparison to budding or T cells alone. Further studies combining T cell infiltrates and tumour budding are necessary to optimise risk assessment of CRC.