Selective but not pan-CDK inhibition abrogates 5-FU-driven tissue factor upregulation in colon cancer.

Thromboembolic events are complications in cancer patients and hypercoagulability has been linked to the tissue factor (TF) pathway, making this an attractive target. Here, we investigated the effects of chemotherapeutics and CDK inhibitors (CDKI) abemaciclib/palbociclib (CDK4/6), THZ-1 (CDK7/12/13), and dinaciclib (CDK1/2/5/9) alone and in combination regimens on TF abundance and coagulation. The human colorectal cancer (CRC) cell line HROC173 was treated with 5-FU or gemcitabine to stimulate TF expression. TF+ cells were sorted, recultured, and re-analyzed. The effect of treatment alone or in combination was assessed by functional assays. Low-dose chemotherapy induced a hypercoagulable state and significantly upregulated TF, even after reculture without treatment. Cells exhibited characteristics of epithelial-mesenchymal transition, including high expression of vimentin and mucin. Dinaciclib and THZ-1 also upregulated TF, while abemaciclib and palbociclib downregulated it. Similar results were observed in coagulation assays. The same anticoagulant activity of abemaciclib was seen after incubation with peripheral immune cells from healthy donors and CRC patients. Abemaciclib reversed 5-FU-induced TF upregulation and prolonged clotting times in second-line treatment. Effects were independent of cytotoxicity, senescence, and p27kip1 induction. TF-antibody blocking experiments confirmed the importance of TF in plasma coagulation, with Factor XII playing a minor role. Short-term abemaciclib counteracts 5-FU-induced hypercoagulation and eventually even prevents thromboembolic events.

The varying extent of humoral and cellular immune responses to either vector- or RNA-based SARS-CoV-2 vaccines persists for at least 18 months and is independent of infection.

The corona virus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome corona-virus 2 (SARS-CoV-2) spurred a worldwide race for the development of an efficient vaccine. Various strategies were pursued; however, the first vaccines to be licensed presented the SARS-CoV-2 spike protein either in the context of a non-replicating adenoviral vector or as an mRNA construct. While short-term efficacies have extensively been characterized, the duration of protection, the need for repeated boosting, and reasonable vaccination intervals have yet to be defined. We here describe the adaptive immune response resulting from homologous and heterologous vaccination regimen at 18 months after primary vaccination. To that extent, we monitored 176 healthcare workers, the majority of whom had recovered from previous SARS-CoV-2 infection. In summary, we find that differences depending on primary immunization continue to exist 18 months after the first vaccination and these findings hold true irrespective of previous infection with the virus. Homologous primary immunization with BNT162b2 was repeatedly shown to produce higher antibody levels and slower antibody decline, leading to more effective in vitro neutralization capacities. Likewise, cellular responses resulting from in vitro re-stimulation were more pronounced after primary immunization involving BNT162b2. In contrast, IL-2 producing memory T helper and cytotoxic T cells appeared independent from the primary vaccination regimen. Despite these differences, comparable infection rates among all vaccination groups suggest comparable real-life protection.IMPORTANCEVaccination against the severe acute respiratory syndrome corona-virus 2 (SARS-CoV-2) was shown to avert severe courses of corona virus disease 2019 (COVID-19) and to mitigate spreading of the virus. However, the duration of protection and need for repeated boosting have yet to be defined. Monitoring and comparing the immune responses resulting from various vaccine strategies are therefore important to fill knowledge gaps and prepare for future pandemics.

CCR2 macrophage response determines the functional outcome following cardiomyocyte transplantation.

BACKGROUND: The immune response is a crucial factor for mediating the benefit of cardiac cell therapies. Our previous research showed that cardiomyocyte transplantation alters the cardiac immune response and, when combined with short-term pharmacological CCR2 inhibition, resulted in diminished functional benefit. However, the specific role of innate immune cells, especially CCR2 macrophages on the outcome of cardiomyocyte transplantation, is unclear. METHODS: We compared the cellular, molecular, and functional outcome following cardiomyocyte transplantation in wildtype and T cell- and B cell-deficient Rag2del mice. The cardiac inflammatory response was assessed using flow cytometry. Gene expression profile was assessed using single-cell and bulk RNA sequencing. Cardiac function and morphology were determined using magnetic resonance tomography and immunohistochemistry respectively. RESULTS: Compared to wildtype mice, Rag2del mice show an increased innate immune response at steady state and disparate macrophage response after MI. Subsequent single-cell analyses after MI showed differences in macrophage development and a lower prevalence of CCR2 expressing macrophages. Cardiomyocyte transplantation increased NK cells and monocytes, while reducing CCR2-MHC-IIlo macrophages. Consequently, it led to increased mRNA levels of genes involved in extracellular remodelling, poor graft survival, and no functional improvement. Using machine learning-based feature selection, Mfge8 and Ccl7 were identified as the primary targets underlying these effects in the heart. CONCLUSIONS: Our results demonstrate that the improved functional outcome following cardiomyocyte transplantation is dependent on a specific CCR2 macrophage response. This work highlights the need to study the role of the immune response for cardiomyocyte cell therapy for successful clinical translation.

Transcriptome alterations in peripheral blood B cells of patients with multiple sclerosis receiving immune reconstitution therapy.

BACKGROUND: Multiple sclerosis (MS) is a chronic, inflammatory and neurodegenerative disease that leads to irreversible damage to the brain and spinal cord. The goal of so-called "immune reconstitution therapies" (IRTs) is to achieve long-term disease remission by eliminating a pathogenic immune repertoire through intense short-term immune cell depletion. B cells are major targets for effective immunotherapy in MS. OBJECTIVES: The aim of this study was to analyze the gene expression pattern of B cells before and during IRT (i.e., before B-cell depletion and after B-cell repopulation) to better understand the therapeutic effects and to identify biomarker candidates of the clinical response to therapy. METHODS: B cells were obtained from blood samples of patients with relapsing-remitting MS (n = 50), patients with primary progressive MS (n = 13) as well as healthy controls (n = 28). The patients with relapsing MS received either monthly infusions of natalizumab (n = 29) or a pulsed IRT with alemtuzumab (n = 15) or cladribine (n = 6). B-cell subpopulation frequencies were determined by flow cytometry, and transcriptome profiling was performed using Clariom D arrays. Differentially expressed genes (DEGs) between the patient groups and controls were examined with regard to their functions and interactions. We also tested for differences in gene expression between patients with and without relapse following alemtuzumab administration. RESULTS: Patients treated with alemtuzumab or cladribine showed on average a > 20% lower proportion of memory B cells as compared to before IRT. This was paralleled by profound transcriptome shifts, with > 6000 significant DEGs after adjustment for multiple comparisons. The top DEGs were found to regulate apoptosis, cell adhesion and RNA processing, and the most highly connected nodes in the network of encoded proteins were ESR2, PHB and RC3H1. Higher mRNA levels of BCL2, IL13RA1 and SLC38A11 were seen in patients with relapse despite IRT, though these differences did not pass the false discovery rate correction. CONCLUSIONS: We show that B cells circulating in the blood of patients with MS undergoing IRT present a distinct gene expression signature, and we delineated the associated biological processes and gene interactions. Moreover, we identified genes whose expression may be an indicator of relapse risk, but further studies are needed to verify their potential value as biomarkers.

Short-term immune-checkpoint inhibition partially rescues perturbed bone marrow hematopoiesis in mismatch-repair deficient tumors.

Wide-spread cancer-related immunosuppression often curtails immune-mediated antitumoral responses. Immune-checkpoint inhibitors (ICIs) have become a state-of-the-art treatment modality for mismatch repair-deficient (dMMR) tumors. Still, the impact of ICI-treatment on bone marrow perturbations is largely unknown. Using anti-PD1 and anti-LAG-3 ICI treatments, we here investigated the effect of bone marrow hematopoiesis in tumor-bearing Msh2loxP/loxP;TgTg(Vil1-cre) mice. The OS under anti-PD1 antibody treatment was 7.0 weeks (vs. 3.3 weeks and 5.0 weeks, control and isotype, respectively). In the anti-LAG-3 antibody group, OS was 13.3 weeks and thus even longer than in the anti-PD1 group (p = 0.13). Both ICIs induced a stable disease and reduced circulating and splenic regulatory T cells. In the bone marrow, a perturbed hematopoiesis was identified in tumor-bearing control mice, which was partially rescued by ICI treatment. In particular, B cell precursors and innate lymphoid progenitors were significantly increased upon anti-LAG-3 therapy to levels seen in tumor-free control mice. Additional normalizing effects of ICI treatment were observed for lin-c-Kit+IRF8+ hematopoietic stem cells, which function as a "master" negative regulator of the formation of polymorphonuclear-myeloid-derived suppressor cell generation. Accompanying immunofluorescence on the TME revealed significantly reduced numbers of CD206+F4/80+ and CD163+ tumor-associated M2 macrophages and CD11b+Gr1+ myeloid-derived suppressor cells especially upon anti-LAG-3 treatment. This study confirms the perturbed hematopoiesis in solid cancer. Anti-LAG-3 treatment partially restores normal hematopoiesis. The interference of anti-LAG-3 with suppressor cell populations in otherwise inaccessible niches renders this ICI very promising for subsequent clinical application.

The addition of arginine deiminase potentiates Mithramycin A-induced cell death in patient-derived glioblastoma cells via ATF4 and cytochrome C.

BACKGROUND: Arginine auxotrophy constitutes a shortcoming for ~ 30% of glioblastoma multiforme (GBM). Indeed, arginine-depleting therapy using arginine deiminase from Streptococcus pyogenes (SpyADI) has proven activity against GBM in preclinical studies. The good safety profile of SpyADI renders this agent an ideal combination partner for cytostatic therapy. METHODS: In this study, we combined the antineoplastic antibiotic Mithramycin A (MitA) with SpyADI to boost single-agent activity and analyzed underlying response mechanisms in-depth. RESULTS: MitA monotherapy induced a time- and dose-dependent cytotoxicity in eight patient-derived GBM cell lines and had a radiosensitizing effect in all but one cell line. Combination treatment boosted the effects of the monotherapy in 2D- and 3D models. The simultaneous approach was superior to the sequential application and significantly impaired colony formation after repetitive treatment. MitA monotherapy significantly inhibited GBM invasiveness. However, this effect was not enhanced in the combination. Functional analysis identified SpyADI-triggered senescence induction accompanied by increased mitochondrial membrane polarization upon mono- and combination therapy. In HROG63, induction of lysosomes was seen after both monotherapies, indicative of autophagy. These cells seemed swollen and had a more pronounced cortically formed cytoskeleton. Also, cytochrome C and endoplasmatic reticulum-stress-associated proteins ATF4 and Calnexin were enhanced in the combination, contributing to apoptosis. Notably, no significant increases in glioma-stemness marker were seen. CONCLUSIONS: Therapeutic utilization of a metabolic defect in GBM along with cytostatic therapy provides a novel combination approach. Whether this SpyADI/MitA regimen will provide a safe alternative to combat GBM, will have to be addressed in subsequent (pre-)clinical trials.

Functional Genomic Screening During Somatic Cell Reprogramming Identifies DKK3 as a Roadblock of Organ Regeneration.

Somatic cell reprogramming and tissue repair share relevant factors and molecular programs. Here, Dickkopf-3 (DKK3) is identified as novel factor for organ regeneration using combined transcription-factor-induced reprogramming and RNA-interference techniques. Loss of Dkk3 enhances the generation of induced pluripotent stem cells but does not affect de novo derivation of embryonic stem cells, three-germ-layer differentiation or colony formation capacity of liver and pancreatic organoids. However, DKK3 expression levels in wildtype animals and serum levels in human patients are elevated upon injury. Accordingly, Dkk3-null mice display less liver damage upon acute and chronic failure mediated by increased proliferation in hepatocytes and LGR5+ liver progenitor cell population, respectively. Similarly, recovery from experimental pancreatitis is accelerated. Regeneration onset occurs in the acinar compartment accompanied by virtually abolished canonical-Wnt-signaling in Dkk3-null animals. This results in reduced expression of the Hedgehog repressor Gli3 and increased Hedgehog-signaling activity upon Dkk3 loss. Collectively, these data reveal Dkk3 as a key regulator of organ regeneration via a direct, previously unacknowledged link between DKK3, canonical-Wnt-, and Hedgehog-signaling.

Inflammatory Joint Disease Is a Risk Factor for Streptococcal Sepsis and Septic Arthritis in Mice.

Septic arthritis is a medical emergency associated with high morbidity and mortality, yet hardly any novel advances exist for its clinical management. Despite septic arthritis being a global health burden, experimental data uncovering its etiopathogenesis remain scarce. In particular, any interplay between septic arthritis and preceding joint diseases are unknown as is the contribution of the synovial membrane to the onset of inflammation. Using C57BL/6 mice as a model to study sepsis, we discovered that Group A Streptococcus (GAS) - an important pathogen causing septic arthritis - was able to invade the articular microenvironment. Bacterial invasion resulted in the infiltration of immune cells and detrimental inflammation. In vitro infected fibroblast-like synoviocytes induced the expression of chemokines (Ccl2, Cxcl2), inflammatory cytokines (Tnf, Il6), and integrin ligands (ICAM-1, VCAM-1). Apart from orchestrating immune cell attraction and retention, synoviocytes also upregulated mediators impacting on bone remodeling (Rankl) and cartilage integrity (Mmp13). Using collagen-induced arthritis in DBA/1 × B10.Q F1 mice, we could show that an inflammatory joint disease exacerbated subsequent septic arthritis which was associated with an excessive release of cytokines and eicosanoids. Importantly, the severity of joint inflammation controlled the extent of bone erosions during septic arthritis. In order to ameliorate septic arthritis, our results suggest that targeting synoviocytes might be a promising approach when treating patients with inflammatory joint disease for sepsis.

Tbx3 fosters pancreatic cancer growth by increased angiogenesis and activin/nodal-dependent induction of stemness.

Cell fate decisions and pluripotency, but also malignancy depend on networks of key transcriptional regulators. The T-box transcription factor TBX3 has been implicated in the regulation of embryonic stem cell self-renewal and cardiogenesis. We have recently discovered that forced TBX3 expression in embryonic stem cells promotes mesendoderm specification directly by activating key lineage specification factors and indirectly by enhancing paracrine NODAL signalling. Interestingly, aberrant TBX3 expression is associated with breast cancer and melanoma formation. In other cancers, loss of TBX3 expression is associated with a more aggressive phenotype e.g. in gastric and cervical cancer. The precise function of TBX3 in pancreatic ductal adenocarcinoma remains to be determined. In the current study we provide conclusive evidence for TBX3 overexpression in pancreatic cancer samples as compared to healthy tissue. While proliferation remains unaltered, forced TBX3 expression strongly increases migration and invasion, but also angiogenesis in vitro and in vivo. Finally, we describe the TBX3-dependency of cancer stem cells that perpetuate themselves through an autocrine TBX3-ACTIVIN/NODAL signalling loop to sustain stemness. Thus, TBX3 is a new key player among pluripotency-related genes driving cancer formation.

A Dynamic Role of TBX3 in the Pluripotency Circuitry.

Pluripotency represents a cell state comprising a fine-tuned pattern of transcription factor activity required for embryonic stem cell (ESC) self-renewal. TBX3 is the earliest expressed member of the T-box transcription factor family and is involved in maintenance and induction of pluripotency. Hence, TBX3 is believed to be a key member of the pluripotency circuitry, with loss of TBX3 coinciding with loss of pluripotency. We report a dynamic expression of TBX3 in vitro and in vivo using genetic reporter tools tracking TBX3 expression in mouse ESCs (mESCs). Low TBX3 levels are associated with reduced pluripotency, resembling the more mature epiblast. Notably, TBX3-low cells maintain the intrinsic capability to switch to a TBX3-high state and vice versa. Additionally, we show TBX3 to be dispensable for induction and maintenance of naive pluripotency as well as for germ cell development. These data highlight novel facets of TBX3 action in mESCs.

A time frame permissive for Protein Kinase D2 activity to direct angiogenesis in mouse embryonic stem cells.

The protein kinase D isoenzymes PKD1/2/3 are prominent downstream targets of PKCs (Protein Kinase Cs) and phospholipase D in various biological systems. Recently, we identified PKD isoforms as novel mediators of tumour cell-endothelial cell communication, tumour cell motility and metastasis. Although PKD isoforms have been implicated in physiological/tumour angiogenesis, a role of PKDs during embryonic development, vasculogenesis and angiogenesis still remains elusive. We investigated the role of PKDs in germ layer segregation and subsequent vasculogenesis and angiogenesis using mouse embryonic stem cells (ESCs). We show that mouse ESCs predominantly express PKD2 followed by PKD3 while PKD1 displays negligible levels. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated at the time of germ layer segregation. Time-restricted PKD2-activation limits mesendoderm formation and subsequent cardiovasculogenesis during early differentiation while leading to branching angiogenesis during late differentiation. In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer. Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs. Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination.