Characterization of a novel microfluidic platform for the isolation of rare single cells to enable CTC analysis from head and neck squamous cell carcinoma patients

Detailed examination of tumor components is leading‐edge to establish personalized cancer therapy. Accompanying research on cell‐free DNA, the cell count of circulating tumor cells (CTCs) in patient blood is seen as a crucial prognostic factor. The potential of CTC analysis is further not limited to the determination of the overall survival rate but sheds light on understanding inter‐ and intratumoral heterogeneity. In this regard, commercial CTC isolation devices combining an efficient enrichment of rare cells with a droplet deposition of single cells for downstream analysis are highly appreciated. The Liquid biopsy platform CTCelect was developed to realize a fully‐automated enrichment and single cell dispensing of CTCs from whole blood without pre‐processing. We characterized each process step with two different carcinoma cell lines demonstrating up to 87 % enrichment (n = 10) with EpCAM coupled immunomagnetic beads, 73 % optical detection and dispensing efficiency (n = 5). 40 to 56.7 % of cells were recovered after complete isolation from 7.5 ml untreated whole blood (n = 6). In this study, CTCelect enabled automated dispensing of single circulating tumor cells from HNSCC patient samples, qPCR‐based confirmation of tumor‐related biomarkers and immunostaining. Finally, the platform was compared to commercial CTC isolation technologies to highlight advantages and limitations of CTCelect. This system offers new possibilities for single cell screening in cancer diagnostics, individual therapy approaches and real‐time monitoring.     

Synergistic effect of Abraxane that combines human IL15 fused with an albumin‐binding domain on murine models of pancreatic ductal adenocarcinoma

Nab‐paclitaxel (Abraxane), which is a nanoparticle form of albumin‐bound paclitaxel, is one of the standard chemotherapies for pancreatic ductal adenocarcinoma (PDAC). This study determined the effect of Abraxane in combination with a fusion protein, hIL15‐ABD, on subcutaneous Panc02 and orthotopic KPC C57BL/6 murine PDAC models. Abraxane combined with hIL15‐ABD best suppressed tumour growth and produced a 40%–60% reduction in the tumour size for Panc02 and KPC, compared to the vehicle group. In the combination group, the active form of interferon‐γ (IFN‐γ)‐secreting CD8⁺ T cells and CD11b⁺CD86⁺ M1 macrophages in tumour infiltrating lymphocytes (TILs) were increased. In the tumour drainage lymph nodes (TDLNs) of the combination group, there was a 18% reduction in CD8⁺IFN‐γ⁺ T cells and a 0.47% reduction in CD4⁺CD25⁺FOXP3⁺ regulatory T cells, as opposed to 5.0% and 5.1% reductions, respectively, for the control group. Superior suppression of CD11b⁺GR‐1⁺ myeloid‐derived suppressor cells (MDSCs) and the induction of M1 macrophages in the spleen and bone marrow of mice were found in the combination group. Abraxane and hIL15‐ABD effectively suppressed NF‐κB‐mediated immune suppressive markers, including indoleamine 2,3‐dioxygenase (IDO), Foxp3 and VEGF. In conclusion, Abraxane combined with hIL15‐ABD stimulates the anticancer activity of effector cells, inhibits immunosuppressive cells within the tumour microenvironment (TME) of PDAC, and produces a greater inhibitory effect than individual monotherapies.     

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

The p14^ARF protein is a well‐known regulator of p53‐dependent and p53‐independent tumor‐suppressive activities. In unstressed cells, p14^ARF is predominantly sequestered in the nucleoli, bound to its nucleolar interaction partner NPM. Upon genotoxic stress, p14^ARF undergoes an immediate redistribution to the nucleo‐ and cytoplasm, where it promotes activation of cell cycle arrest and apoptosis. Here, we identify p14^ARF 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 p14^ARF. In the absence of cellular stress, these arginines are crucial for nucleolar localization of p14^ARF. Genotoxic stress causes augmented interaction between PRMT1 and p14^ARF, accompanied by arginine methylation of p14^ARF. PRMT1‐dependent NLS/NoLS methylation promotes the release of p14^ARF from NPM and nucleolar sequestration, subsequently leading to p53‐independent apoptosis. This PRMT1‐p14^ARF 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 p14^ARF’s stress‐induced tumor‐suppressive function.     

Increased SPON1 promotes pancreatic ductal adenocarcinoma progression by enhancing IL‐6 trans‐signalling

ObjectivesThis study investigated the specific molecular mechanism and the roles of extracellular matrix protein Spondin 1 (SPON1) in the development of pancreatic ductal adenocarcinoma (PDAC).Materials and MethodsThe expression pattern and clinical relevance of SPON1 was determined in GEO, Ren Ji and TCGA datasets, further validated by immunohistochemical staining and Kaplan‐Meier analysis. Loss and gain of function experiments were employed to investigate the cellular function of SPON1 in vitro. Gene set enrichment analysis, luciferase assay, immunofluorescence and Western blot and immunoprecipitation were applied to reveal the underlying molecular mechanisms. Subcutaneous xenograft model was used to test the role of SPON1 in tumour growth and maintenance in vivo.ResultsSPON1 is significantly upregulated in PDAC tumour tissues and correlated with progression of PDAC. Loss and gain of function experiments showed that SPON1 promotes the growth and colony formation ability of pancreatic cancer cells. Combining bioinformatics assays and experimental signalling evidences, we found that SPON1 can enhance the IL‐6/JAK/STAT3 signalling. Mechanistically, SPON1 exerts its oncogenic roles in pancreatic cancer by maintaining IL‐6R trans‐signalling through stabilizing the interaction of soluble IL‐6R (sIL‐6R) and glycoprotein‐130 (gp130) in PDAC cells. Furthermore, SPON1 depletion greatly reduced the tumour burden, exerted positive effect with gemcitabine, prolonging PDAC mice overall survival.ConclusionsOur data indicate that SPON1 expression is dramatically increased in PDAC and that SPON1 promotes tumorigenicity by activating the sIL‐6R/gp130/STAT3 axis. Collectively, our current work suggests SPON1 may be a potential therapy target for PDAC patient.

Extracellular matrix protein spondin 1 is significantly upregulated in PDAC tumour cell, which exerts its oncogenic roles in pancreatic cancer by maintaining IL6R trans‐signalling through stabilizing the interaction of sIL6R and GP130 in PDAC cell, resulting in STAT3 signalling activating and tumour cell growth.     

RNA sensing via the RIG‐I‐like receptor LGP2 is essential for the induction of a type I IFN response in ADAR1 deficiency

RNA editing by the adenosine deaminase ADAR1 prevents innate immune responses to endogenous RNAs. In ADAR1‐deficient cells, unedited self RNAs form base‐paired structures that resemble viral RNAs and inadvertently activate the cytosolic RIG‐I‐like receptor (RLR) MDA5, leading to an antiviral type I interferon (IFN) response. Mutations in ADAR1 cause Aicardi‐Goutières Syndrome (AGS), an autoinflammatory syndrome characterized by chronic type I IFN production. Conversely, ADAR1 loss and the consequent type I IFN production restricts tumor growth and potentiates the activity of some chemotherapeutics. Here, we show that another RIG‐I‐like receptor, LGP2, also has an essential role in the induction of a type I IFN response in ADAR1‐deficient human cells. This requires the canonical function of LGP2 as an RNA sensor and facilitator of MDA5‐dependent signaling. Furthermore, we show that the sensitivity of tumor cells to ADAR1 loss requires LGP2 expression. Finally, type I IFN induction in tumor cells depleted of ADAR1 and treated with some chemotherapeutics fully depends on LGP2 expression. These findings highlight a central role for LGP2 in self RNA sensing with important clinical implications.     

Single‐cell transcriptome analysis reveals defective decidua stromal niche attributes to recurrent spontaneous abortion

ObjectivesSuccessful pregnancy involves the homeostasis between maternal decidua and fetoplacental units, whose disruption contributes to compromised pregnancy outcomes, including recurrent spontaneous abortion (RSA). The role of cell heterogeneity of maternal decidua in RSA is yet to be illustrated.Materials and methodsA total of 66,078 single cells from decidua samples isolated from patients with RSA and healthy controls were analysed by unbiased single‐cell RNA sequencing (scRNA‐seq).ResultsOur scRNA‐seq results revealed that stromal cells are the most abundant cell type in decidua during early pregnancy. RSA samples are accompanied by aberrant decidualization and obviously obstructed communication between stromal cells and other cell types, such as abnormal activation of macrophages and NK cells. In addition, the over‐activated TNF superfamily member 12 (TNFSF12, TWEAK) and FASLG in RSA are closely related to stromal cell demise and pregnancy failure.ConclusionsOur research reveals that the cell composition and communications in normal and RSA decidua at early pregnancy and provides insightful information for the pathology of RSA and will pave the way for pregnancy loss prevention.

Recurrent spontaneous abortion (RSA), characterized by pregnancy loss before 20 weeks of gestation more than twice, is an intricated pregnancy complication with enigmatic underlying mechanism ascribes to its complex pathogenesis. The homeostasis between the developing foetus and maternal decidua is critical for pregnancy maintenance. By exploring the cell heterogeneity in normal and RSA decidua utilizing scRNA‐Seq, we unravel the discrepancies in cell composition and communications in these two distinct deciduae. Our investigations uncover that stromal cells are the most abundant cell populations in the decidua, with three different subpopulations at various decidualization stages and two fibroblasts. There are two separated trajectories of stromal cell decidualization marked by PLA2G2A and WNT4. As the most abundant cell population in the decidua, the stromal cells dominate the communications with other cell types, including endothelial cells, macrophages, uNK cells and perivascular cells. Compared with normal decidua, decidualized stromal cells are overtly decreased in RSA decidua with augmented macrophages. In addition, we present some previously unappreciated signaling pathways among different cells types in decidua and also depict the remarkably changed communications between normal and RSA decidual. The aberrant activated TWEAK and FASLG in RSA are considered to be potential reasons for stromal cells demise and pregnancy failure. Our research reveals the cell composition and communications in normal and RSA decidua at early pregnancy and provides insightful information for the pathology of RSA and will pave the way for pregnancy loss prevention.     

Single‐cell dynamics of chromatin activity during cell lineage differentiation in Caenorhabditis elegans embryos

Elucidating the chromatin dynamics that orchestrate embryogenesis is a fundamental question in developmental biology. Here, we exploit position effects on expression as an indicator of chromatin activity and infer the chromatin activity landscape in every lineaged cell during Caenorhabditis elegans early embryogenesis. Systems‐level analyses reveal that chromatin activity distinguishes cellular states and correlates with fate patterning in the early embryos. As cell lineage unfolds, chromatin activity diversifies in a lineage‐dependent manner, with switch‐like changes accompanying anterior–posterior fate asymmetry and characteristic landscapes being established in different cell lineages. Upon tissue differentiation, cellular chromatin from distinct lineages converges according to tissue types but retains stable memories of lineage history, contributing to intra‐tissue cell heterogeneity. However, the chromatin landscapes of cells organized in a left–right symmetric pattern are predetermined to be analogous in early progenitors so as to pre‐set equivalent states. Finally, genome‐wide analysis identifies many regions exhibiting concordant chromatin activity changes that mediate the co‐regulation of functionally related genes during differentiation. Collectively, our study reveals the developmental and genomic dynamics of chromatin activity at the single‐cell level.     

Influence of interferon-α on the expression of the cancer stem cell markers in pancreatic carcinoma cells

The cytokine interferon-α (IFNα) belongs to the group of type I interferons already used in cancer therapy. This drug possesses radio- and chemo-sensitizing, and shows anti-angiogenic properties. Cancer stem cells (CSC) are a unique population of tumor cells that initiate secondary tumors, and are responsible for metastasis formation. Patients with pancreatic ductal adenocarcinoma (PDAC) have an especially poor prognosis, with 5-year survival rates of only ~1% and median survival of 4–6 months. PDAC is characterized by the presence of CSC. In this work we demonstrate for the first time that IFNα up-regulates the expression of the CSC markers CD24, CD44 and CD133 in in vitro and in vivo models of PDAC. We showed the IFNα effects on the migration and invasion of PDAC cells, which is associated with the level of the CSC marker expression. In vivo, this drug inhibits tumor growth but promotes metastasis formation in the early stage of tumor growth. We propose that IFNα may enhance the enrichment of CSC in PDAC tumors. Additionally we also suggest that in combination therapy of solid tumors with IFNα, this drug should be given to patients prior to chemotherapy to achieve the CSC activation.     

Targeted therapies in pancreatic cancer: Promises and failures

Pancreatic ductal adenocarcinoma (PDAC) is an incidence rate nearly equal to its mortality rate. The poor prognosis of the disease can be explained by the absence of effective biomarkers for screening and early detection, together with the aggressive behavior and resistance to the currently available chemotherapy. The therapeutic failure can also be attributed to the inter-/intratumor genetic heterogeneity and the abundance of tumor stroma that occupies the majority of the tumor mass. Gemcitabine is used in the treatment of PDAC; however, the response rate is less than 12%. A recent phase III trial revealed that the combination of oxaliplatin, irinotecan, fluorouracil, and leucovorin could be an option for the treatment of metastatic PDAC patients with good performance status, although these approaches can result in high toxicity level. Further investigations are required to develop innovative anticancer agents that either improve gemcitabine activity, within novel combinatorial approaches or acts with a better efficacy than gemcitabine. The aim of the current review is to give an overview of preclinical and clinical studies targeting key dysregulated signaling pathways in PDAC.     

Prognostic value and immune cell infiltration of hypoxic phenotype‐related gene signatures in glioblastoma microenvironment

Glioblastoma (GBM) is a malignant intracranial tumour with the highest proportion and lethality. It is characterized by invasiveness and heterogeneity. However, the currently available therapies are not curative. As an essential environmental cue that maintains glioma stem cells, hypoxia is considered the cause of tumour resistance to chemotherapy and radiation. Growing evidence shows that immunotherapy focusing on the tumour microenvironment is an effective treatment for GBM; however, the current clinicopathological features cannot predict the response to immunotherapy and provide accurate guidance for immunotherapy. Based on the ESTIMATE algorithm, GBM cases of The Cancer Genome Atlas (TCGA) data set were classified into high‐ and low‐immune/stromal score groups, and a four‐gene tumour environment‐related model was constructed. This model exhibited good efficiency at forecasting short‐ and long‐term prognosis and could also act as an independent prognostic biomarker. Additionally, this model and four of its genes (CLECL5A, SERPING1, CHI3L1 and C1R) were found to be associated with immune cell infiltration, and further study demonstrated that these four genes might drive the hypoxic phenotype of perinecrotic GBM, which affects hypoxia‐induced glioma stemness. Therefore, these might be important candidates for immunotherapy of GBM and deserve further exploration.     

Neutrophil-Derived Proteases in the Microenvironment of Pancreatic Cancer -Active Players in Tumor Progression

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the fibro-inflammatory microenvironment, consisting of activated pancreatic stellate cells, extracellular matrix proteins, and a variety of inflammatory cells, such as T cells, macrophages, or neutrophils. Tumor-infiltrating immune cells, which are found in nearly all cancers, including PDAC, often fail to eliminate the tumor, but conversely can promote its progression by altering the tumor microenvironment. Pancreatic cancer cells are able to attract polymorphonuclear neutrophils (PMN) via tumor secreted chemokines and in human PDAC, PMN infiltrates can be observed in the vicinity of tumor cells and in the desmoplastic tumor stroma, which correlate with undifferentiated tumor growth and poor prognosis. The behavior of tumor-infiltrating neutrophils in the tumor micromilieu is not yet understood at a mechanistic level. It has been shown that PMN have the potential to kill tumor cells, either directly or by antibody-dependent cell-mediated cytotoxicity, but on the other side various adverse effects of PMN, such as promotion of aggressive tumor growth with epithelial-to-mesenchymal transition and increased metastatic potential, have been described. Recent therapeutic approaches for PDAC focus not only the tumor cell itself, but also elements of the tumor microenvironment. Therefore, the role of PMN and their derived products (e.g. cytokines, proteases) as a new vein for a therapeutic target should be critically evaluated in this context. This review summarizes the current understanding of the interplay between proteases of tumor-infiltrating neutrophils and pancreatic tumor cells and elements of the desmoplastic stroma.     

Functional heterogeneity of cytotoxic T cells and tumor resistance to cytotoxic hits limit anti‐tumor activity in vivo

Cytotoxic T cells (CTLs) can eliminate tumor cells through the delivery of lethal hits, but the actual efficiency of this process in the tumor microenvironment is unclear. Here, we visualized the capacity of single CTLs to attack tumor cells in vitro and in vivo using genetically encoded reporters that monitor cell damage and apoptosis. Using two distinct malignant B‐cell lines, we found that the majority of cytotoxic hits delivered by CTLs in vitro were sublethal despite proper immunological synapse formation, and associated with reversible calcium elevation and membrane damage in the targets. Through intravital imaging in the bone marrow, we established that the majority of CTL interactions with lymphoma B cells were either unproductive or sublethal. Functional heterogeneity of CTLs contributed to diverse outcomes during CTL–tumor contacts in vivo. In the therapeutic settings of anti‐CD19 CAR T cells, the majority of CAR T cell–tumor interactions were also not associated with lethal hit delivery. Thus, differences in CTL lytic potential together with tumor cell resistance to cytotoxic hits represent two important bottlenecks for anti‐tumor responses in vivo.     

ALIX and ceramide differentially control polarized small extracellular vesicle release from epithelial cells

Exosomes, important players in cell–cell communication, are small extracellular vesicles of endocytic origin. Although single cells are known to release various kinds of exosomes (referred to as exosomal heterogeneity), very little is known about the mechanisms by which they are produced and released. Here, we established methods of studying exosomal heterogeneity by using polarized epithelial cells and showed that distinct types of small extracellular vesicles (more specifically CD9‐ and CD63‐positive, Annexin I‐negative small extracellular vesicles, which we refer to as exosomes herein) are differentially secreted from the apical and basolateral sides of polarized epithelial cells. We also identify GPRC5C (G protein‐coupled receptor class C group 5 member C) as an apical exosome‐specific protein. We further demonstrate that basolateral exosome release depends on ceramide, whereas ALIX, an ESCRT (endosomal sorting complexes required for transport)‐related protein, not the ESCRT machinery itself, is required for apical exosome release. Thus, two independent machineries, the ALIX–Syntenin1–Syndecan1 machinery (apical side) and the sphingomyelinase‐dependent ceramide production machinery (basolateral side), are likely to be responsible for the polarized exosome release from epithelial cells.