Mesenteric fibrosis in patients with small intestinal neuroendocrine tumors is associated with enrichment of alpha-smooth muscle actin-positive fibrosis and COMP-expressing stromal cells.

Neuroendocrine tumors of the small intestine (SI-NETs) often develop lymph node metastasis (LNM)-induced mesenteric fibrosis (MF). MF can cause intestinal obstruction as well as ischemia and render surgical resection technically challenging. The underlying pathomechanisms of MF are still not well understood. We examined mesenteric LNM and the surrounding stroma compartment from 24 SI-NET patients, including 11 with in situ presentation of strong MF (MF+) and 13 without MF (MF-). Differential gene expression was assessed with the HTG EdgeSeq Oncology Biomarker Panel comparing MF+ with MF- within LNM and paired stromal samples, respectively. Most interesting differentially expressed genes were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in combination with validation of associated protein levels utilizing immunohistochemistry (IHC) staining of MF+ and MF- formalin-fixed, paraffin-embedded (FFPE) patient samples. Overall, 14 genes measured with a 2549-gene expression panel were differentially expressed in MF+ patients compared to MF-. Of those, nine were differentially expressed genes in LNM and five genes in the stromal tissue (>2-fold change, p < .05). The top hits included increased COMP and COL11A1 expression in the stroma of MF+ patients compared to MF-, as well as decreased HMGA2, COL6A6, and SLC22A3 expression in LNM of MF+ patients compared to LNM of MF- patients. RT-qPCR confirmed high levels of COMP and COL11A1 in stroma samples of MF+ compared to MF- patients. IHC staining confirmed the enrichment of α-smooth muscle actin-positive fibrosis in MF+ compared to MF- patients with corresponding increase of COMP-expressing stromal cells in MF+. Since COMP is associated with the known driver for fibrosis development transforming growth factor beta and with a cancer-associated fibroblasts enriched environment, it seems to be a promising new target for MF research.

TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion.

A crucial requirement for metastasis formation in ovarian high-grade serous carcinoma (HGSC) is the disruption of the protective peritoneal mesothelium. Using co-culture systems of primary human cells, we discovered that tumor-associated NK cells induce TRAIL-dependent apoptosis in mesothelial cells via death receptors DR4 and DR5 upon encounter with activated T cells. Upregulation of TRAIL expression in NK cells concomitant with enhanced cytotoxicity toward mesothelial cells was driven predominantly by T-cell-derived TNFα, as shown by affinity proteomics-based analysis of the T cell secretome in conjunction with functional studies. Consistent with these findings, we detected apoptotic mesothelial cells in the peritoneal fluid of HGSC patients. In contrast to mesothelial cells, HGSC cells express negligible levels of both DR4 and DR5 and are TRAIL resistant, indicating cell-type-selective killing by NK cells. Our data point to a cooperative action of T and NK in breaching the mesothelial barrier in HGSC patients.

IL18 Receptor Signaling Regulates Tumor-Reactive CD8+ T-cell Exhaustion via Activation of the IL2/STAT5/mTOR Pathway in a Pancreatic Cancer Model.

Intratumoral cytotoxic CD8+ T cells (CTL) enter a dysfunctional state characterized by expression of coinhibitory receptors, loss of effector function, and changes in the transcriptional landscape. Even though several regulators of T-cell exhaustion have been identified, the molecular mechanisms inducing T-cell exhaustion remain unclear. Here, we show that IL18 receptor (IL18R) signaling induces CD8+ T-cell exhaustion in a murine pancreatic cancer model. Adoptive transfer of Il18r-/- OT-1 CD8+ CTLs resulted in enhanced rejection of subcutaneous tumors expressing ovalbumin (OVA) as a model antigen (PancOVA), compared with wild-type OT-1 CTLs. Transferred intratumoral IL18R-deficient CTLs expressed higher levels of effector cytokines TNF and IFNγ and had reduced expression of coinhibitory receptors (PD-1, TIM-3, 2B4, LAG-3) and the transcription factors Eomes and TOX. Lower expression of coinhibitory receptors and TOX on IL18R-deficient versus IL18R-sufficient CD8+ T cells were confirmed in an orthotopic KPC model. IL18R-induced T-cell exhaustion was regulated by IL2/STAT5 and AKT/mTOR pathways, as demonstrated in an in vitro exhaustion assay. Concordantly, mice deficient in NLRP3, the molecular complex activating IL18, had decreased expression of coinhibitory receptors on intratumoral T cells and similar changes in signaling pathways at the transcriptome level. Thus, molecular pathways promoting T-cell exhaustion indicate an involvement of an NLRP3-expressing tumor microenvironment, which mediates IL18 release. The Cancer Genome Atlas analysis of patients with pancreatic carcinoma showed an association between NLRP3-mediated IL18 signaling and shorter survival. These findings indicate NLRP3-mediated IL18R signaling as a regulator of intratumoral T-cell exhaustion and a possible target for immunotherapy. See related Spotlight by Stromnes, p. 400.

Pericentromeric satellite lncRNAs are induced in cancer-associated fibroblasts and regulate their functions in lung tumorigenesis.

The abnormal tumor microenvironment (TME) often dictates the therapeutic response of cancer to chemo- and immuno-therapy. Aberrant expression of pericentromeric satellite repeats has been reported for epithelial cancers, including lung cancer. However, the transcription of tandemly repetitive elements in stromal cells of the TME has been unappreciated, limiting the optimal use of satellite transcripts as biomarkers or anti-cancer targets. We found that transcription of pericentromeric satellite DNA (satDNA) in mouse and human lung adenocarcinoma was observed in cancer-associated fibroblasts (CAFs). In vivo, lung fibroblasts expressed pericentromeric satellite repeats HS2/HS3 specifically in tumors. In vitro, transcription of satDNA was induced in lung fibroblasts in response to TGFß, IL1α, matrix stiffness, direct contact with tumor cells and treatment with chemotherapeutic drugs. Single-cell transcriptome analysis of human lung adenocarcinoma confirmed that CAFs were the cell type with the highest number of satellite transcripts. Human HS2/HS3 pericentromeric transcripts were detected in the nucleus, cytoplasm, extracellularly and co-localized with extracellular vesicles in situ in human biopsies and activated fibroblasts in vitro. The transcripts were transmitted into recipient cells and entered their nuclei. Knock-down of satellite transcripts in human lung fibroblasts attenuated cellular senescence and blocked the formation of an inflammatory CAFs phenotype which resulted in the inhibition of their pro-tumorigenic functions. In sum, our data suggest that satellite long non-coding (lnc) RNAs are induced in CAFs, regulate expression of inflammatory genes and can be secreted from the cells, which potentially might present a new element of cell-cell communication in the TME.

Dissecting the role of toll-like receptor 7 in pancreatic cancer.

BACKGROUND: Toll-like receptors (TLRs) are gaining attention for their potential to influence tumor biology both on the level of the tumor cells as well as on the level of the surrounding inflammatory stroma. Previous studies resulted in partly conflicting data on the expression of TLR7 in healthy and neoplastic pancreatic tissues as well as its role in pancreatic tumor biology. METHODS: We used qRT-PCR and immunohistochemistry to asses TLR7 expression in primary patient material and cell lines. Cell viability was analyzed by MTT assay upon incubation with TLR7 agonist/antagonist. Mouse models were used to investigate the role of TLR7 in vivo. RESULTS: TLR7 is overexpressed in more than 50% of primary human pancreatic ductal adenocarcinoma (PDAC). High TLR7 expression was associated with shorter patient survival, and TLR7 inhibition in cell lines reduced viability in a dose-dependent manner. In contrast, global TLR7 deficiency did not alter survival or overall histopathological tumor features in genetic mouse models of PDAC. CONCLUSIONS: TLR7 may have opposing functions in tumor versus stroma cells. Further work is required to more precisely dissect the roles of TLR7 and its ligands in different populations of epithelial and stromal cells and to understand their relative contributions to tumor progression.

Selected commensals educate the intestinal vascular and immune system for immunocompetence.

BACKGROUND: The intestinal microbiota fundamentally guides the development of a normal intestinal physiology, the education, and functioning of the mucosal immune system. The Citrobacter rodentium-carrier model in germ-free (GF) mice is suitable to study the influence of selected microbes on an otherwise blunted immune response in the absence of intestinal commensals. RESULTS: Here, we describe that colonization of adult carrier mice with 14 selected commensal microbes (OMM12 + MC2) was sufficient to reestablish the host immune response to enteric pathogens; this conversion was facilitated by maturation and activation of the intestinal blood vessel system and the step- and timewise stimulation of innate and adaptive immunity. While the immature colon of C. rodentium-infected GF mice did not allow sufficient extravasation of neutrophils into the gut lumen, colonization with OMM12 + MC2 commensals initiated the expansion and activation of the visceral vascular system enabling granulocyte transmigration into the gut lumen for effective pathogen elimination. CONCLUSIONS: Consortium modeling revealed that the addition of two facultative anaerobes to the OMM12 community was essential to further progress the intestinal development. Moreover, this study demonstrates the therapeutic value of a defined consortium to promote intestinal maturation and immunity even in adult organisms. Video Abstract.

Partial p53 reactivation is sufficient to induce cancer regression.

BACKGROUND: Impaired p53 function is one of the central molecular features of a tumor cell and even a partial reduction in p53 activity can increase the cancer risk in mice and men. From a therapeutic perspective it is noteworthy that tumor cells often become addicted to the absence of p53 providing a rationale for developing p53 reactivating compounds to treat cancer patients. Unfortunately, many of the compounds that are currently undergoing preclinical and clinical testing fail to fully reactivate mutant p53 proteins, raising the crucial question: how much p53 activity is needed to elicit a therapeutic effect? METHODS: We have genetically modelled partial p53 reactivation using knock-in mice with inducible expression of the p53 variant E177R. This variant has a reduced ability to bind and transactivate target genes and consequently causes moderate cancer susceptibility. We have generated different syngeneically transplanted and autochthonous mouse models of p53-deficient acute myeloid leukemia and B or T cell lymphoma. After cancer manifestation we have activated E177R expression and analyzed the in vivo therapy response by bioluminescence or magnetic resonance imaging. The molecular response was further characterized in vitro by assays for gene expression, proliferation, senescence, differentiation, apoptosis and clonogenic growth. RESULTS: We report the conceptually intriguing observation that the p53 variant E177R, which promotes de novo leukemia and lymphoma formation, inhibits proliferation and viability, induces immune cell infiltration and triggers cancer regression in vivo when introduced into p53-deficient leukemia and lymphomas. p53-deficient cancer cells proved to be so addicted to the absence of p53 that even the low-level activity of E177R is detrimental to cancer growth. CONCLUSIONS: The observation that a partial loss-of-function p53 variant promotes tumorigenesis in one setting and induces regression in another, underlines the highly context-specific effects of individual p53 mutants. It further highlights the exquisite sensitivity of cancer cells to even small changes in p53 activity and reveals that changes in activity level are more important than the absolute level. As such, the study encourages ongoing research efforts into mutant p53 reactivating drugs by providing genetic proof-of-principle evidence that incomplete p53 reactivation may suffice to elicit a therapeutic response.

Cellular and soluble immune checkpoint signaling forms PD-L1 and PD-1 in renal tumor tissue and in blood.

Immune checkpoint blockade therapy is a treatment option of various metastatic cancer diseases including renal cell carcinoma (RCC). Approved antibody drugs target the co-inhibitory signaling of Programmed Cell Death Ligand-1 (PD-L1) and its receptor Programmed Cell Death-1 (PD-1). The combined evaluation of PD-L1 and PD-1 at the mRNA and protein levels in tumor tissue with differentiation of tumor and immune cells as well as of soluble forms (sPD-L1) and (sPD-1) in blood is of basic interest in assessing biomarker surrogates. Here, we demonstrate that PD-L1 determined as fraction of stained tumor cells (TPS-score) correlates with PD-L1-mRNA in tumor tissue, reflecting the predominant expression of PD-L1 in tumor cells. Conversely, PD-1 in immune cells of tumor tissue (IC-score) correlated with PD-1-mRNA tissue levels reflecting the typical PD-1 expression in immune cells. Of note, sPD-L1 in blood did not correlate with either the TPS-score of PD-L1 or with PD-L1-mRNA in tumor tissue. sPD-L1 released into the supernatant of cultured RCC cells closely followed the cellular PD-L1 expression as tested by interferon γ (IFNG) induction and siRNA knockdown of PD-L1. Further analysis in patients revealed that sPD-L1 significantly increased in blood following renal tumor resection. In addition, sPD-L1 correlated significantly with inflammation marker C-reactive protein (CRP) and with PD-L1 mRNA level in whole blood. These results indicate that the major source of sPD-L1 in blood may be peripheral blood cells and not primarily tumor tissue PD-L1.

Perfusion Patterns of Peripheral Pulmonary Granulomatous Lesions Using Contrast-Enhanced Ultrasound (CEUS) and Their Correlation with Immunohistochemically Detected Vascularization Patterns.

PURPOSE: To describe the perfusion patterns of peripheral pulmonary granulomatous lesions (PPGLs) by contrast-enhanced ultrasound (CEUS) and their correlation with vascularization patterns (VPs) represented by immunohistochemical (CD34) endothelial staining. PATIENTS AND METHODS: From January 2007 until September 2020, 10 consecutive patients with histologically confirmed PPGLs were investigated by CEUS. The time to enhancement, classified as early pulmonary-arterial (PA) pattern of enhancement versus delayed bronchial-arterial (BA) pattern of enhancement, the extent of enhancement, classified as marked or reduced, the homogeneity of enhancement, classified as homogeneous or inhomogeneous, and the decrease of enhancement, classified as rapid washout (<120 seconds) or a late washout (≥120 seconds), were analyzed retrospectively. Furthermore, the tissue samples from the study patients and as a control group, 10 samples of normal lung tissue obtained by autopsy, and 10 samples of lung tissue with acute pneumonia obtained by autopsy were immunohistochemically stained with CD34 antibody. The presence of avascular areas (AAs) and the VPs were evaluated in all tissue samples. RESULTS: On CEUS, all PPGLs showed a reduced inhomogeneous BA pattern of enhancement and a rapid washout (<120 seconds). On CD34 staining, all PPGLs showed central AAs in granulomas and a chaotic VP similar to angiogenesis in lung tumors. The lung tissue in control groups revealed on CD34 staining a regular alveolar VP. CONCLUSION: The PPGLs on CEUS show an identical perfusion pattern similar to those of malignant lesions. Furthermore, for the first time, neoangiogenesis was demonstrated as a histopathological correlate to BA pattern of enhancement on CEUS.

PRMT1 promotes the tumor suppressor function of p14ARF and is indicative for pancreatic cancer prognosis.

The p14ARF protein is a well-known regulator of p53-dependent and p53-independent tumor-suppressive activities. In unstressed cells, p14ARF is predominantly sequestered in the nucleoli, bound to its nucleolar interaction partner NPM. Upon genotoxic stress, p14ARF undergoes an immediate redistribution to the nucleo- and cytoplasm, where it promotes activation of cell cycle arrest and apoptosis. Here, we identify p14ARF as a novel interaction partner and substrate of PRMT1 (protein arginine methyltransferase 1). PRMT1 methylates several arginine residues in the C-terminal nuclear/nucleolar localization sequence (NLS/NoLS) of p14ARF . In the absence of cellular stress, these arginines are crucial for nucleolar localization of p14ARF . Genotoxic stress causes augmented interaction between PRMT1 and p14ARF , accompanied by arginine methylation of p14ARF . PRMT1-dependent NLS/NoLS methylation promotes the release of p14ARF from NPM and nucleolar sequestration, subsequently leading to p53-independent apoptosis. This PRMT1-p14ARF cooperation is cancer-relevant and indicative for PDAC (pancreatic ductal adenocarcinoma) prognosis and chemotherapy response of pancreatic tumor cells. Our data reveal that PRMT1-mediated arginine methylation is an important trigger for p14ARF 's stress-induced tumor-suppressive function.

The Hygiene Hypothesis - Learning From but Not Living in the Past.

Postulated by Strachan more than 30 years ago, the Hygiene Hypothesis has undergone many revisions and adaptations. This review journeys back to the beginnings of the Hygiene Hypothesis and describes the most important landmarks in its development considering the many aspects that have refined and generalized the Hygiene Hypothesis over time. From an epidemiological perspective, the Hygiene Hypothesis advanced to a comprehensive concept expanding beyond the initial focus on allergies. The Hygiene Hypothesis comprise immunological, microbiological and evolutionary aspects. Thus, the original postulate developed into a holistic model that explains the impact of post-modern life-style on humans, who initially evolved in close proximity to a more natural environment. Focusing on diet and the microbiome as the most prominent exogenous influences we describe these discrepancies and the resulting health outcomes and point to potential solutions to reestablish the immunological homeostasis that frequently have been lost in people living in developed societies.