The C-type lectin DCIR contributes to the immune response and pathogenesis of colorectal cancer.

Development and progression of malignancies are accompanied and influenced by alterations in the surrounding immune microenvironment. Understanding the cellular and molecular interactions between immune cells and cancer cells has not only provided important fundamental insights into the disease, but has also led to the development of new immunotherapies. The C-type lectin Dendritic Cell ImmunoReceptor (DCIR) is primarily expressed by myeloid cells and is an important regulator of immune homeostasis, as demonstrated in various autoimmune, infectious and inflammatory contexts. Yet, the impact of DCIR on cancer development remains largely unknown. Analysis of available transcriptomic data of colorectal cancer (CRC) patients revealed that high DCIR gene expression is associated with improved patients' survival, immunologically "hot" tumors and high immunologic constant of rejection, thus arguing for a protective and immunoregulatory role of DCIR in CRC. In line with these correlative data, we found that deficiency of DCIR1, the murine homologue of human DCIR, leads to the development of significantly larger tumors in an orthotopic murine model of CRC. This phenotype is accompanied by an altered phenotype of tumor-associated macrophages (TAMs) and a reduction in the percentage of activated effector CD4+ and CD8+ T cells in CRC tumors of DCIR1-deficient mice. Overall, our results show that DCIR promotes antitumor immunity in CRC, making it an attractive target for the future development of immunotherapies to fight the second deadliest cancer in the world.

A Paradoxical Role for Regulatory T Cells in the Tumor Microenvironment of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is considered to be a poorly immunogenic cancer type that combines a low mutation burden with a strong immunosuppressive tumor microenvironment. Regulatory T cells (Tregs) are major drivers of immune suppression but their prognostic role, particularly in gastrointestinal malignancies, remains controversial. Lymphocytic infiltration in 122 PDAC samples was assessed by multispectral immunofluorescence with anti-Keratin, -CD3, -CD8, -FOXP3 and -CD163 antibodies. Differential infiltration by Tregs was analyzed in the context of transcriptomic profiles that were available for 65 tumors. High infiltration of CD3+CD8- (mainly CD4+) T cells and, especially, of the subset expressing FOXP3 (Tregs) was associated with improved patient survival, whilst cytotoxic CD3+CD8+ T cell infiltration did not have an impact on overall survival. Transcriptomic analysis revealed three signatures in PDAC tumors comprising of epithelial-mesenchymal transition (EMT)/stromal, metabolic, and secretory/pancreatic signature. However, none of these signatures explained differences in Treg infiltration. We show that Tregs associate with improved overall survival in PDAC patients. This effect was independent of cytotoxic T cell infiltration and the transcriptomic profiles of their respective tumors. These findings provide a new layer of complexity in the study of PDAC tumor microenvironment that must be considered when developing immunotherapeutic interventions for this disease.

Local and systemic immune profiles of human pancreatic ductal adenocarcinoma revealed by single-cell mass cytometry.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy in need of effective (immuno)therapeutic treatment strategies. For the optimal application and development of cancer immunotherapies, a comprehensive understanding of local and systemic immune profiles in patients with PDAC is required. Here, our goal was to decipher the interplay between local and systemic immune profiles in treatment-naïve patients with PDAC. METHODS: The immune composition of PDAC, matched non-malignant pancreatic tissue, regional lymph nodes, spleen, portal vein blood, and peripheral blood samples (collected before and after surgery) from 11 patients with PDAC was assessed by measuring 41 immune cell markers by single-cell mass cytometry. Furthermore, the activation potential of tumor-infiltrating lymphocytes as determined by their ability to produce cytokines was investigated by flow cytometry. In addition, the spatial localization of tumor-infiltrating innate lymphocytes in the tumor microenvironment was confirmed by multispectral immunofluorescence. RESULTS: We found that CD103+CD8+ T cells with cytotoxic potential are infrequent in the PDAC immune microenvironment and lack the expression of activation markers and checkpoint blockade molecule programmed cell death protein-1 (PD-1). In contrast, PDAC tissues showed a remarkable increased relative frequency of B cells and regulatory T cells as compared with non-malignant pancreatic tissues. Besides, a previously unappreciated innate lymphocyte cell (ILC) population (CD127-CD103+CD39+CD45RO+ ILC1-like) was discovered in PDAC tissues. Strikingly, the increased relative frequency of B cells and regulatory T cells in pancreatic cancer samples was reflected in matched portal vein blood samples but not in peripheral blood, suggesting a regional enrichment of immune cells that infiltrate the PDAC microenvironment. After surgery, decreased frequencies of myeloid dendritic cells were found in peripheral blood. CONCLUSIONS: Our work demonstrates an immunosuppressive landscape in PDAC tissues, generally deprived of cytotoxic T cells and enriched in regulatory T cells and B cells. The antitumor potential of ILC1-like cells in PDAC may be exploited in a therapeutic setting. Importantly, immune profiles detected in blood isolated from the portal vein reflected the immune cell composition of the PDAC microenvironment, suggesting that this anatomical location could be a source of tumor-associated immune cell subsets.

Mesh, flap or combined repair of perineal hernia after abdominoperineal resection - A systematic review and meta-analysis.

AIM: The aim of this systematic review was to analyse recurrence rates after different surgical techniques for perineal hernia repair. METHOD: All original studies (n ≥ 2 patients) reporting recurrence rates after perineal hernia repair after abdominoperineal resection (APR) were included. The electronic database PubMed was last searched in December 2021. The primary outcome was recurrent perineal hernia. A weighted average of the logit proportions was determined by the use of the generic inverse variance method and random effects model. RESULTS: A total of 19 studies involving 172 patients were included. The mean age of patients was 64 ± 5.6 years and the indication for APR was predominantly cancer (99%, 170/172). The pooled percentage of recurrent perineal hernia was 39% (95% CI: 27%-52%) after biological mesh closure, 29% (95% CI: 21%-39%) after synthetic mesh closure, 37% (95% CI: 14%-67%) after tissue flap reconstruction only and 9% (95% CI: 1%-45%) after tissue flap reconstruction combined with mesh. CONCLUSION: Recurrence rates after mesh repair of perineal hernia are high, without a clear difference between biological and synthetic meshes. The addition of a tissue flap to mesh repair seemed to have a favourable outcome, which warrants further investigation.

The identification of the anthracycline aclarubicin as an effective cytotoxic agent for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer, mainly due to its delayed diagnosis and lack of effective therapeutic options. Therefore, it is imperative to find novel treatment options for PDAC. Here, we tested a series of conventional chemotherapeutics together with anthracycline compounds as single agents or in combination, determining their effectivity against established commercial and patient-derived, low-passage PDAC cell lines. Proliferation and colony formation assays were performed to determine the anticancer activity of anthracyclines; aclarubicin and doxorubicin, on commercial and patient-derived, low-passage PDAC cell lines. In addition, the effect of standard-of-care drugs gemcitabine and individual components of FOLFIRINOX were also investigated. To evaluate which mechanisms of cell death were involved in drug response, cleavage of poly(ADP-ribose)polymerase was evaluated by western blot. Aclarubicin showed superior antitumor activity compared to other anthracyclines and standard of care drugs (gemcitabine and individual components of FOLFIRINOX) in a patient-derived, low-passage PDAC cell line and in commercial cell lines. Importantly, the combination of gemcitabine and aclarubicin showed a synergistic effect at a dose range where the single agents by themselves were ineffective. In parallel, evaluation of the antitumor activity of aclarubicin demonstrated an apoptotic effect in all PDAC cell lines. Aclarubicin is cytotoxic for commercial and patient-derived low-passage PDAC cell lines, at doses lower than peak serum concentrations for patient treatment. Our findings support a (re)consideration of aclarubicin as a backbone of new combination regimens for pancreatic cancer patients.

Complex coacervates as extraction media.

Various solvents such as ionic liquids, deep eutectic solvents, and aqueous two phase systems have been suggested as greener alternatives to existing extraction processes. We propose to add macroscopic complex coacervates to this list. Complex coacervates are liquid-like forms of polyion condensates and consist of a complex of oppositely charged polyions and water. Previous research focussing on the biological significance of these polyion-rich phases has shown that polyion condensates have the ability to extract certain solutes from water and back-extract them by changing parameters such as ionic strength and pH. In this study, we present the distribution coefficients of five commonly used industrial chemicals, namely lactic acid, butanol, and three types of lipase enzymes in poly(ethylenimine)/poly(acrylic acid) complex coacervates. It was found that the distribution coefficients can vary strongly upon variation of tunable parameters such as polyion ratio, ionic strength, polyion and compound concentrations, and temperature. Distribution coefficients ranged from approximately 2 to 50 depending on the tuning of the system parameters. It was also demonstrated that a temperature-swing extraction is possible, with back-extraction of butanol from complex coacervates with a recovery of 21.1%, demonstrating their potential as extraction media.

Recent Developments in Solvent-Based Fluid Separations.

The most important developments in solvent-based fluid separations, separations involving at least one fluid phase, are reviewed. After a brief introduction and discussion on general solvent trends observed in all fields of application, several specific fields are discussed. Important solvent trends include replacement of traditional molecular solvents by ionic liquids and deep eutectic solvents and, more recently, increasing discussion around bio-based solvents in some application fields. Furthermore, stimuli-responsive systems are discussed; the most significant developments in this field are seen for CO2-switchable and redox-responsive solvents. Discussed fields of application include hydrocarbons separations, carbon capture, biorefineries, and metals separations. For all but the hydrocarbons separations, newly reported electrochemically mediated separations seem to offer exciting new windows of opportunities.

Immunotherapy for pancreatic cancer: chasing the light at the end of the tunnel.

BACKGROUND: Checkpoint blockade immunotherapy has had a significant impact on the survival of a subset of patients with advanced cancers. It has been particularly effective in immunogenic cancer types that present large numbers of somatic mutations in their genomes. To date, all conventional immunotherapies have failed to produce significant clinical benefits for patients diagnosed with pancreatic cancer, probably due to its poor immunogenic properties, including low numbers of neoantigens and highly immune-suppressive microenvironments. CONCLUSIONS: Herein, we discuss advances that have recently been made in cancer immunotherapy and the potential of this field to deliver effective treatment options for pancreatic cancer patients. Preclinical investigations, combining different types of therapies, highlight possibilities to enhance anti-tumor immunity and to generate meaningful clinical responses in pancreatic cancer patients. Results from completed and ongoing (pre)clinical trials are discussed.

A critical role for linker DNA in higher-order folding of chromatin fibers.

Nucleosome-nucleosome interactions drive the folding of nucleosomal arrays into dense chromatin fibers. A better physical account of the folding of chromatin fibers is necessary to understand the role of chromatin in regulating DNA transactions. Here, we studied the unfolding pathway of regular chromatin fibers as a function of single base pair increments in linker length, using both rigid base-pair Monte Carlo simulations and single-molecule force spectroscopy. Both computational and experimental results reveal a periodic variation of the folding energies due to the limited flexibility of the linker DNA. We show that twist is more restrictive for nucleosome stacking than bend, and find the most stable stacking interactions for linker lengths of multiples of 10 bp. We analyzed nucleosomes stacking in both 1- and 2-start topologies and show that stacking preferences are determined by the length of the linker DNA. Moreover, we present evidence that the sequence of the linker DNA also modulates nucleosome stacking and that the effect of the deletion of the H4 tail depends on the linker length. Importantly, these results imply that nucleosome positioning in vivo not only affects the phasing of nucleosomes relative to DNA but also directs the higher-order structure of chromatin.

Mechanical and structural properties of archaeal hypernucleosomes.

Many archaea express histones, which organize the genome and play a key role in gene regulation. The structure and function of archaeal histone-DNA complexes remain however largely unclear. Recent studies show formation of hypernucleosomes consisting of DNA wrapped around an 'endless' histone-protein core. However, if and how such a hypernucleosome structure assembles on a long DNA substrate and which interactions provide for its stability, remains unclear. Here, we describe micromanipulation studies of complexes of the histones HMfA and HMfB with DNA. Our experiments show hypernucleosome assembly which results from cooperative binding of histones to DNA, facilitated by weak stacking interactions between neighboring histone dimers. Furthermore, rotational force spectroscopy demonstrates that the HMfB-DNA complex has a left-handed chirality, but that torque can drive it in a right-handed conformation. The structure of the hypernucleosome thus depends on stacking interactions, torque, and force. In vivo, such modulation of the archaeal hypernucleosome structure may play an important role in transcription regulation in response to environmental changes.

Multiplexed Nanometric 3D Tracking of Microbeads Using an FFT-Phasor Algorithm.

Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional (3D) position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorenz-Mie scattering theory yields the bead's position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method, such as comparison with previously measured diffraction patterns, known as look-up tables. Here, we introduce an alternative 3D phasor algorithm that provides robust bead tracking with nanometric localization accuracy in a z range of over 10 µm under nonoptimal imaging conditions. The algorithm is based on a two-dimensional cross correlation using fast Fourier transforms with computer-generated reference images, yielding a processing rate of up to 10,000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real time on a generic CPU. The accuracy of 3D phasor tracking was extensively tested and compared to a look-up table approach using Lorenz-Mie simulations, avoiding experimental uncertainties. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead-tracking applications, especially when high throughput is required and image artifacts are difficult to avoid.

Correction to: Molecular and pharmacological modulators of the tumor immune contexture revealed by deconvolution of RNA-seq data.

It was highlighted that the original article [1] contained a typesetting mistake in the name of Noel Filipe da Cunha Carvalho de Miranda. This was incorrectly captured as Noel Filipe da Cunha Carvahlo de Miranda. It was also highlighted that in Fig. 3C the left panels Y-axis were cropped and in Fig. 5C, CD8 bar was cropped. This Correction article shows the correct Figs. 3 and 5. The original article has been updated.

Molecular and pharmacological modulators of the tumor immune contexture revealed by deconvolution of RNA-seq data.

We introduce quanTIseq, a method to quantify the fractions of ten immune cell types from bulk RNA-sequencing data. quanTIseq was extensively validated in blood and tumor samples using simulated, flow cytometry, and immunohistochemistry data.quanTIseq analysis of 8000 tumor samples revealed that cytotoxic T cell infiltration is more strongly associated with the activation of the CXCR3/CXCL9 axis than with mutational load and that deconvolution-based cell scores have prognostic value in several solid cancers. Finally, we used quanTIseq to show how kinase inhibitors modulate the immune contexture and to reveal immune-cell types that underlie differential patients' responses to checkpoint blockers.Availability: quanTIseq is available at http://icbi.at/quantiseq .

Feasibility of subcutaneous implantable cardioverter-defibrillator implantation with opioid sparing truncal plane blocks and deep sedation.

INTRODUCTION: The subcutaneous implantable cardioverter-defibrillator (S-ICD) is most commonly implanted under general anesthesia (GA), due to the intraoperative discomfort associated with tunneling and dissection. Postoperative pain can be substantial and is often managed with opioids. There is a growing interest in transitioning away from the routine use of GA during S-ICD implantation, while also controlling perioperative discomfort without the use of narcotics. As such, we assessed the feasibility of a multimodal analgesia regimen that included regional anesthesia techniques in patients undergoing S-ICD implantation. METHODS AND RESULTS: Twenty patients received truncal plane block (TBL) immediately before S-ICD implantation. The first 10 patients were implanted under general anesthesia (GA + TBL), and the next 10 patients were implanted under deep sedation (DS + TBL). Additionally, the DS + TBL patients were also prescribed a structured regimen of nonopioid analgesics in the perioperative period. Opioid consumption was calculated as milligram morphine equivalents (MME). In-hospital opioid consumption was significantly lower in the patients implanted with DS + TBL (MME = 0) as compared with patients receiving GA + TBL (MME = 60; P = 0.004). CONCLUSIONS: Subcutaneous ICD implantation with anesthesia-delivered DS and a multimodal anesthetic regimen that includes TBL is feasible and associated with significantly less perioperative opioid consumption.

Cancer immunophenotyping by seven-colour multispectral imaging without tyramide signal amplification.

Checkpoint blockade immunotherapies have revolutionised cancer treatment in the last decade. Nevertheless, these are only beneficial for a small proportion of cancer patients. Important prognosticators for response to immunotherapy are the mutation burden of tumours as well as the quality and quantity of tumour-infiltrating immune cells. High-throughput multiplex immunophenotyping technologies have a central role in deciphering the complexity of anti-tumour immune responses. Current techniques for the immunophenotyping of solid tumours are held back by the lack of spatial context, limitations in the number of targets that can be visualised simultaneously, and/or cumbersome protocols. We developed a tyramide signal amplification-free method for the simultaneous detection of seven cellular targets by immunofluorescence. This method overcomes limitations posed by most widespread techniques and provides a unique tool for extensive phenotyping by multispectral fluorescence microscopy. Furthermore, it can be easily implemented as a high-throughput technology for validation of discovery sets generated by RNA sequencing or mass cytometry and may serve in the future as a complementary diagnostic tool.

Rigid Basepair Monte Carlo Simulations of One-Start and Two-Start Chromatin Fiber Unfolding by Force.

The organization of chromatin in 30 nm fibers remains a topic of debate. Here, we quantify the mechanical properties of the linker DNA and evaluate the impact of these properties on chromatin fiber folding. We extended a rigid basepair DNA model to include (un)wrapping of nucleosomal DNA and (un)stacking of nucleosomes in one-start and two-start chromatin fibers. Monte Carlo simulations that mimic single-molecule force spectroscopy experiments of folded nucleosomal arrays reveal different stages of unfolding as a function of force and are largely consistent with a two-start folding for 167 and one-start folding for 197 nucleosome repeat length fibers. The major insight is that nucleosome unstacking and subsequent unwrapping is not necessary to obtain quantitative agreement with experimental force extension curves up to the overstretching plateau of folded chromatin fibers at 3-5 pN. Nucleosome stacking appears better accommodated in one-start than in two-start conformations, and we suggest that this difference can compensate the increased energy for bending the linker DNA. Overall, these simulations capture the dynamic structure of chromatin fibers while maintaining realistic physical properties of the linker DNA.

Unraveling DNA Organization with Single-Molecule Force Spectroscopy Using Magnetic Tweezers.

Genomes carry the genetic blueprint of all living organisms. Their organization requires strong condensation as well as carefully regulated accessibility to specific genes for proper functioning of their hosts. The study of the structure and dynamics of the proteins that organize the genome has benefited tremendously from the development of single-molecule force spectroscopy techniques that allow for real-time, nanometer accuracy measurements of the compaction of DNA and manipulation with pico-Newton scale forces. Magnetic tweezers in particular have the unique ability to complement such force spectroscopy with the control over the linking number of the DNA molecule, which plays an important role when DNA organizing proteins form or release wraps, loops, and bends in DNA. Here, we describe all the necessary steps to prepare DNA substrates for magnetic tweezers experiments, assemble flow cells, tether DNA to magnetics bead inside flow cell, and manipulate and record the extension of such DNA tethers. Furthermore, we explain how mechanical parameters of nucleo-protein filaments can be extracted from the data.

Probing Chromatin Structure with Magnetic Tweezers.

Magnetic tweezers form a unique tool to study the topology and mechanical properties of chromatin fibers. Chromatin is a complex of DNA and proteins that folds the DNA in such a way that meter-long stretches of DNA fit into the micron-sized cell nucleus. Moreover, it regulates accessibility of the genome to the cellular replication, transcription, and repair machinery. However, the structure and mechanisms that govern chromatin folding remain poorly understood, despite recent spectacular improvements in high-resolution imaging techniques. Single-molecule force spectroscopy techniques can directly measure both the extension of individual chromatin fragments with nanometer accuracy and the forces involved in the (un)folding of single chromatin fibers. Here, we report detailed methods that allow one to successfully prepare in vitro reconstituted chromatin fibers for use in magnetic tweezers-based force spectroscopy. The higher-order structure of different chromatin fibers can be inferred from fitting a statistical mechanics model to the force-extension data. These methods for quantifying chromatin folding can be extended to study many other processes involving chromatin, such as the epigenetic regulation of transcription.

Autologous iPSC-Based Vaccines Elicit Anti-tumor Responses In Vivo.

Cancer cells and embryonic tissues share a number of cellular and molecular properties, suggesting that induced pluripotent stem cells (iPSCs) may be harnessed to elicit anti-tumor responses in cancer vaccines. RNA sequencing revealed that human and murine iPSCs express tumor-associated antigens, and we show here a proof of principle for using irradiated iPSCs in autologous anti-tumor vaccines. In a prophylactic setting, iPSC vaccines prevent tumor growth in syngeneic murine breast cancer, mesothelioma, and melanoma models. As an adjuvant, the iPSC vaccine inhibited melanoma recurrence at the resection site and reduced metastatic tumor load, which was associated with fewer Th17 cells and increased CD11b+GR1hi myeloid cells. Adoptive transfer of T cells isolated from vaccine-treated tumor-bearing mice inhibited tumor growth in unvaccinated recipients, indicating that the iPSC vaccine promotes an antigen-specific anti-tumor T cell response. Our data suggest an easy, generalizable strategy for multiple types of cancer that could prove highly valuable in clinical immunotherapy.

Toehold-enhanced LNA probes for selective pull down and single-molecule analysis of native chromatin.

The organization of DNA into chromatin is thought to regulate gene expression in eukaryotes. To study its structure in vitro, there is a need for techniques that can isolate specific chromosomal loci of natively assembled chromatin. Current purification methods often involve chemical cross-linking to preserve the chromatin composition. However, such cross-linking may affect the native structure. It also impedes single molecule force spectroscopy experiments, which have been instrumental to probe chromatin folding. Here we present a method for the incorporation of affinity tags, such as biotin, into native nucleoprotein fragments based on their DNA sequence, and subsequent single molecule analysis by magnetic tweezers. DNA oligos with several Locked Nucleic Acid (LNA) nucleotides are shown to selectively bind to target DNA at room temperature, mediated by a toehold end in the target, allowing for selective purification of DNA fragments. The stability of the probe-target hybrid is sufficient to withstand over 65 pN of force. We employ these probes to obtain force-extension curves of native chromatin fragments of the 18S ribosomal DNA from the yeast Saccharomyces cerevisiae. These experiments yield valuable insights in the heterogeneity in structure and composition of natively assembled chromatin at the single-molecule level.