Differential Expression of CKLF-like MARVEL Transmembrane Domain-Containing Protein 6 and Programmed Cell Death Ligand 1 as Prognostic Biomarkers in Upper Tract Urothelial Carcinoma.

Upper tract urothelial carcinoma (UTUC) accounts for 5-10% of all UCs. Immune checkpoint inhibitors (ICIs) have been established for UCs. The prognostic and predictive potential of programmed cell death ligand 1 (PD-L1) expression to stratify patients benefiting from ICIs is not fully understood, and additional markers influencing the impact of PD-L1-mediated ICI response are needed. Previously, the chemokine-like MARVEL transmembrane domain-containing protein 6 (CMTM6) was identified as a positive regulator of PD-L1. Our aim was to investigate the expression profiles and impact of PD-L1 and CMTM6 protein status on the prognostic parameters and survival of UTUC patients. In this retrospective study, the combined positive score (CPS), tumor proportion score (TPS), and immune cell score (ICS) for PD-L1 and CMTM6 were determined. High PD-L1 CPS, ICS, and TPS were found in 77.4%, 58.3%, and 45.2% of cases, and high CMTM6 CPS, ICS, and TPS were seen in 52.5%, 51.5%, and 55.5% of cases, respectively. The scores of both markers had a significant positive correlation. High PD-L1 and CMTM6 expression was coupled with higher pT status, WHO grade, necrosis, and metastasis (p < 0.05, respectively). In the univariate survival analysis, patients with a PD-L1 ICS high and higher degree of intratumoral inflammation showed significantly longer overall survival. Compared to other studies on UC, our study shows a substantially higher rate of PD-L1-positive tumors. CMTM6 was associated with more aggressive tumors.

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.

Cyclin-dependent kinase inhibitors in head and neck cancer and glioblastoma-backbone or add-on in immune-oncology?

Cyclin-dependent kinases (CDK) control the cell cycle and play a crucial role in oncogenesis. Pharmacologic inhibition of CDK has contributed to the recent clinical approval of dual CDK4/6 inhibitors for the treatment of breast and small cell lung cancer. While the anticancer cell effects of CDK inhibitors are well-established, preclinical and early clinical studies describe additional mechanisms of action such as chemo- and radiosensitization or immune stimulation. The latter offers great potential to incorporate CDK inhibitors in immune-based treatments. However, dosing schedules and accurate timing of each combination partner need to be respected to prevent immune escape and resistance. In this review, we provide a detailed summary of CDK inhibitors in the two solid cancer types head and neck cancer and glioblastoma multiforme; it describes the molecular mechanisms of response vs. resistance and covers strategies to avoid resistance by the combination of immunotherapy or targeted therapy.

PEGylation increases antitumoral activity of arginine deiminase of Streptococcus pyogenes.

Arginine auxotrophy is a metabolic defect that renders tumor cells vulnerable towards arginine-depleting substances, such as arginine deiminase (ADI) from Streptococcus pyogenes (SpyADI). Previously, we confirmed SpyADI susceptibility on patient-derived glioblastoma multiforme (GBM) models in vitro and in vivo. For application in patients, serum half-life of the enzyme has to be increased and immunogenicity needs to be reduced. For this purpose, we conjugated the S. pyogenes-derived SpyADI with 20 kDa polyethylene glycol (PEG20) moieties, achieving a PEGylation of seven to eight of the 26 accessible primary amines of the SpyADI. The PEGylation reduced the overall activity of the enzyme by about 50% without affecting the Michaelis constant for arginine. PEGylation did not increase serum stability of SpyADI in vitro, but led to a longer-lasting reduction of plasma arginine levels in mice. Furthermore, SpyADI-PEG20 showed a higher antitumoral capacity towards GBM cells in vitro than the native enzyme. KEY POINTS: • PEGylation has no effect on the affinity of SpyADI for arginine • PEGylation increases the antitumoral effects of SpyADI on GBM in vitro • PEGylation prolongs plasma arginine depletion by SpyADI in mice.

Combined vaccine-immune-checkpoint inhibition constitutes a promising strategy for treatment of dMMR tumors.

BACKGROUND: Mlh1-knock-out-driven mismatch-repair-deficient (dMMR) tumors can be targeted immunologically. By applying therapeutic tumor vaccination, tumor growth is delayed but escape mechanisms evolve, including upregulation of immune-checkpoint molecules (LAG-3, PD-L1). To counteract immune escape, we investigated the therapeutic activity of a combined tumor vaccine-immune-checkpoint inhibitor therapy using α-PD-L1. DESIGN: In this trial, Mlh1-knock-out mice with established gastrointestinal tumors received single or thrice injections of α-PD-L1 monoclonal antibody clone 6E11 (2.5 mg/kg bw, q2w, i.v.) either alone or in combination with the vaccine. Longitudinal flow cytometry and PET/CT imaging studies were followed by ex vivo functional immunological and gene expression assays. RESULTS: 6E11 monotherapy slightly increased median overall survival (mOS: 6.0 weeks vs. control 4.0 weeks). Increasing the number of injections (n = 3) improved therapy outcome (mOS: 9.2 weeks) and was significantly boosted by combining 6E11 with the vaccine (mOS: 19.4 weeks vs. 10.2 weeks vaccine monotherapy). Accompanying PET/CT imaging confirmed treatment-induced tumor growth control, with the strongest inhibition in the combination group. Three mice (30%) achieved a complete remission and showed long-term survival. Decreased levels of circulating splenic and intratumoral myeloid-derived suppressor cells (MDSC) and decreased numbers of immune-checkpoint-expressing splenic T cells (LAG-3, CTLA-4) accompanied therapeutic effects. Gene expression and protein analysis of residual tumors revealed downregulation of PI3K/Akt/Wnt-and TGF-signaling, leading to T cell infiltration, reduced numbers of macrophages, neutrophils and MDSC. CONCLUSIONS: By successful uncoupling of the PD-1/PD-L1 axis, we provide further evidence for the safe and successful application of immunotherapies to combat dMMR-driven malignancies that warrants further investigation.

Combined Gemcitabine and Immune-Checkpoint Inhibition Conquers Anti-PD-L1 Resistance in Low-Immunogenic Mismatch Repair-Deficient Tumors.

Tumors arising in the context of Lynch Syndrome or constitutional mismatch repair deficiency are hypermutated and have a good response towards immune-checkpoint inhibitors (ICIs), including α-PD-L1 antibodies. However, in most cases, resistance mechanisms evolve. To improve outcomes and prevent resistance development, combination approaches are warranted. Herein, we applied a combined regimen with an α-PD-L1 antibody and gemcitabine in a preclinical tumor model to activate endogenous antitumor immune responses. Mlh1-/- mice with established gastrointestinal tumors received the α-PD-L1 antibody (clone 6E11; 2.5 mg/kg bw, i.v., q2wx3) and gemcitabine (100 mg/kg bw, i.p., q4wx3) in mono- or combination therapy. Survival and tumor growth were recorded. Immunological changes in the blood were routinely examined via multi-color flow cytometry and complemented by ex vivo frameshift mutation analysis to identify alterations in Mlh1-/--tumor-associated target genes. The combined therapy of α-PD-L1 and gemcitabine prolonged median overall survival of Mlh1-/- mice from four weeks in the untreated control group to 12 weeks, accompanied by therapy-induced tumor growth inhibition, as measured by [18F]-FDG PET/CT. Plasma cytokine levels of IL13, TNFα, and MIP1ß were increased and also higher than in mice receiving either monotherapy. Circulating splenic and intratumoral myeloid-derived suppressor cells (MDSCs), as well as M2 macrophages, were markedly reduced. Besides, residual tumor specimens from combi-treated mice had increased numbers of infiltrating cytotoxic T-cells. Frameshift mutations in APC, Tmem60, and Casc3 were no longer detectable upon treatment, likely because of the successful eradication of single mutated cell clones. By contrast, novel mutations appeared. Collectively, we herein confirm the safe application of combined chemo-immunotherapy by long-term tumor growth control to prevent the development of resistance mechanisms.

NSG mice as hosts for oncological precision medicine.

Patient-derived xenograft (PDX) models have been rediscovered as meaningful research tool. By using severely immunodeficient mice, high-engraftment rates can be theoretically achieved, permitting clinical stratification strategies. Apart from engraftment efficacy, tolerability towards certain cytostatic drugs varies among individual mouse strains thus impeding large-scale screenings. Here, we aimed at optimizing an in vivo treatment schedule using the widely applied cytostatic drug 5-fluoruracil (5-FU) for exemplary response prediction in colorectal cancer (CRC) PDX models. Four different individual CRC PDX models were engrafted into NOD.Cg-PrkdcscidIl2rgtm1Wjl (NSG) mice. Mice with established PDX were allocated to different treatment groups, receiving 5-FU, the oral prodrug Capecitabine, or 5-FU/leucovorin (LV) at different doses. Body weight, tumor size, and general behavior were assessed during therapy. Ex vivo analyses were done from blood samples, liver, as well as tumor resection specimen. Engraftment efficacy was high as expected in NSG mice, yielding stable PDX growth for therapy stratification. However, overall tolerability towards 5-FU was unexpectedly low, whereas the prodrug Capecitabine as well as the combination of 5-FU/LV at low doses were well tolerated. Accompanying plasma level determination of DYPD, the rate-limiting enzyme for 5-FU-mediated toxicity, revealed reduced activity in NSG mice compared with other common laboratory mouse strains, offering a likely explanation for the drug incompatibility. Also, the De Ritis quotient was highly elevated in treated mice, reflecting overall organ injury even at low doses. Summarizing these findings, NSG mice are ideal hosts for in vivo engraftment studies. However, the complex immunodeficiency reduces tolerance to certain drugs, thus making those mice especially sensitive. Consequently, such dose finding and tolerance tests constitute a necessity for similar cancer precision medicine approaches.

High mutational burden in colorectal carcinomas with monoallelic POLE mutations: absence of allelic loss and gene promoter methylation.

Hypermutator-type colorectal carcinomas are microsatellite-stable and have point mutations of the exonuclease domain of the DNA polymerase ε or δ genes (POLE and POLD1, respectively), and an ultrahigh tumor mutational burden (TMB). These tumors may be associated with enhanced antitumor immunity and preferentially afflict younger patients, but this notion awaits validation by accrual of further cases for detailed correlative phenotypic and molecular study. We performed POLE and POLD1 exonuclease domain Sanger sequencing of 271 unselected colorectal carcinomas. We identified two microsatellite-stable tumors with somatic POLE p.P286R variants, both with ultrahigh TMBs as demonstrated by whole exome sequencing. A POLE p.V411L was found in another two microsatellite-stable tumors with ultrahigh TMBs. Two of these four tumors were from young patients (<50 years old, nonsyndromic), and there was seen a prominent T-cell infiltration in three of them. Furthermore, a somatic POLE p.A465T was found in a Lynch-associated tumor, which, hypothetically, might have enhanced TMB (which was the highest of all). In two tumors, a somatic POLE p.V411L and a POLD1 p.E279K, respectively, were found only focally, and TMBs were low. It is commonly assumed that compromise of one allele is sufficient, but this has not been specifically addressed. Therefore, resequencing of the POLE or POLD1 mutations was done with DNA from tumor cells isolated by laser-capture microdissection. This demonstrated that the mutations were monoallelic, and there was no evidence of a "second hit", neither by allelic loss (allelotyping with polymorphic microsatellite markers), nor by promoter methylation (Pyromark CpG assays). Taken together, including at least the more common DNA polymerase mutations in NGS panels allows for straightforward identification of hypermutator-type colorectal carcinomas which often may be "immunoreactive". This is important at least in young patients or when a metastasizing stage of disease has been reached and immune-checkpoint therapy enters deliberation.

In vivo vaccination with cell line-derived whole tumor lysates: neoantigen quality, not quantity matters.

BACKGROUND: Cancer vaccines provide a complex source of neoantigens. Still, increasing evidence reveals that the neoantigen quality rather than the quantity is predictive for treatment outcome. METHODS: Using the preclinical Mlh1-/- tumor model, we performed a side-by side comparison of two autologous cell-line derived tumor lysates (namely 328 and A7450 T1 M1) harboring different tumor mutational burden (TMB; i.e. ultra-high: 328; moderate-high: A7450 T1 M1). Mice received repetitive prophylactic or therapeutic applications of the vaccine. Tumor incidence, immune responses and tumor microenvironment was examined. RESULTS: Both tumor cell lysates delayed tumor formation in the prophylactic setting, with the A7450 T1 M1 lysate being more effective in decelerating tumor growth than the 328 lysate (median overall survival: 37 vs. 25 weeks). Comparable results were achieved in therapeutic setting and could be traced back to antigen-driven immune stimulation. Reactive T cells isolated from A7450 T1 M1-treated mice recognized autologous Mlh1-/- tumor cells in IFNγ ELISpot, but likewise YAC-1 cells, indicative for stimulation of both arms of the immune system. By deciphering local effects, vaccines shaped the tumor microenvironment differently. While A7450 T1 M1 prophylactically vaccinated tumors harbored low numbers of myeloid-derived suppressor cells (MDSC) and elevated CD8-T cell infiltrates, vaccination with the 328 lysate evoked MDSC infiltration. Similar effects were seen in the therapeutic setting with stable disease induction only upon A7450 T1 M1 vaccination. Untangling individual response profiles revealed strong infiltration with LAG3+ and PD-L1+ immune cells when treatments failed, but almost complete exclusion of checkpoint-expressing lymphocytes in long-term survivors. CONCLUSIONS: By applying two tumor cell lysates we demonstrate that neoantigen quality outranks quantity. This should be considered prior to designing cancer vaccine-based combination approaches.

The contact system proteases play disparate roles in streptococcal sepsis.

Sepsis causes an activation of the human contact system, an inflammatory response mechanism against foreign surfaces, proteins and pathogens. The serine proteases of the contact system, factor XII and plasma kallikrein, are decreased in plasma of septic patients, which was previously associated with an unfavorable outcome. However, the precise mechanisms and roles of contact system factors in bacterial sepsis are poorly understood. We, therefore, studied the physiological relevance of factor XII and plasma kallikrein in a mouse model of experimental sepsis. We show that decreased plasma kallikrein concentration in septic mice is a result of reduced mRNA expression plasma prekallikrein gene, indicating that plasma kallikrein belong to negative acute phase proteins. Investigations regarding the pathophysiological function of contact system proteases during sepsis revealed different roles for factor XII and plasma kallikrein. In vitro, factor XII decelerated bacteria induced fibrinolysis, whereas plasma kallikrein supported it. Remarkably, depletion of plasma kallikrein (but not factor XII) by treatment with antisense-oligonucleotides, dampens bacterial dissemination and growth in multiple organs in the mouse sepsis model. These findings identify plasma kallikrein as a novel host pathogenicity factor in Streptococcus pyogenes sepsis.

Suspected Hereditary Cancer Syndromes in Young Patients: Heterogeneous Clinical and Genetic Presentation of Colorectal Cancers.

Colorectal cancer (CRC) is rare in young patients without a confirmed family history of cancer. Reports of an increased prevalence of POLD1/POLE mutations in young patients with colorectal cancer have raised awareness and support routine genetic testing for patients with early-onset tumors. In cases of CRC without proven MMR-germline mutation, molecular analyses are warranted to confirm or rule out other familial CRC syndromes. This article describes the cases of two young male patients, who presented with locally advanced and metastatic CRC, and reports the results of the germline mutational analyses done for both patients. These cases demonstrate the importance of special care and molecular diagnostic procedures for young patients with CRC. KEY POINTS: Patients with colorectal cancer who are younger than 50 years at initial diagnosis (early onset) should routinely undergo genetic testing.Early- and very-early-onset patients (younger than 40 years) with absence of microsatellite instability should be considered for tumor mutation burden testing and/or DNA polymerase proofreading mutation.The mutational signature of HSP110 within mismatch repair deficiency-related tumors may help to identify patients likely to benefit from 5-fluorouracil-based chemotherapy.Intensified, maintained, and specific surveillance may help to reduce secondary tumor progression.

Arginine-Depleting Enzymes - An Increasingly Recognized Treatment Strategy for Therapy-Refractory Malignancies.

Arginine auxotrophy occurs in certain tumor types and is usually caused by the silencing of argininosuccinate synthetase 1 or arginine lyase genes. Such tumors are often associated with an intrinsic chemoresistance and thus a poor prognosis. Arginine auxotrophy however renders these tumors vulnerable to treatment with arginine-degrading enzymes. Among the most frequently applied arginine-degrading agents are bacterial arginine deiminases (ADI). The anti-cancerous effects of ADI derived from different bacteria were extensively studied in numerous preclinical cell culture and xenograft models. Mycoplasma-derived ADI-PEG20 is most commonly used and is currently under clinical investigation as a single agent therapeutic as well as in combination with different antineoplastic compounds. Mechanistically, ADI is capable of reducing metabolic activity in tumor cells, contributing to autophagy, senescence and apoptosis in arginine auxotrophic cells. Although clinical trials are promising, the resistance development upon initial treatment response is an increasing challenge. Furthermore, interference of ADI with the tumor microenvironment is poorly understood. In the present review, we outline recent experimental ADI-based treatment approaches and their translation into the clinic. Furthermore, we summarize new insights into the molecular mechanisms underlying the anti-cancer effects of ADI that might facilitate the refinement of ADI-based combination therapy approaches.

Frameshift mutational target gene analysis identifies similarities and differences in constitutional mismatch repair-deficiency and Lynch syndrome.

Mismatch-repair deficient (MMR-D) malignancies include Lynch Syndrome (LS), which is secondary to germline mutations in one of the MMR genes, and the rare childhood-form of constitutional mismatch repair-deficiency (CMMR-D); caused by bi-allelic MMR gene mutations. A hallmark of LS-associated cancers is microsatellite instability (MSI), characterized by coding frameshift mutations (cFSM) in target genes. By contrast, tumors arising in CMMR-D patients are thought to display a somatic mutation pattern differing from LS. This study has the main goal to identify cFSM in MSI target genes relevant in CMMR-D and to compare the spectrum of common somatic mutations, including alterations in DNA polymerases POLE and D1 between LS and CMMR-D. CMMR-D-associated tumors harbored more somatic mutations compared to LS cases, especially in the TP53 gene and in POLE and POLD1, where novel mutations were additionally identified. Strikingly, MSI in classical mononucleotide markers BAT40 and CAT25 was frequent in CMMR-D cases. MSI-target gene analysis revealed mutations in CMMR-D-associated tumors, some of them known to be frequently hit in LS, such as RNaseT2, HT001, and TGFßR2. Our results imply a general role for these cFSM as potential new drivers of MMR-D tumorigenesis.

p16 overexpression identifies oncogenic high-risk HPV infection in non-oropharyngeal squamous cell carcinoma of the head and neck.

BACKGROUND: Human papillomavirus (HPV) is an increasing risk factor for cancer. HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) is associated with a favorable outcome. Blockstaining for p16 is a surrogate marker for HPV+ OPSCC. In oral and laryngeal squamous cell carcinoma (OSCC/LSCC), the relevance of p16 immunohistochemistry, alone or in combination with other cell cycle-related proteins, to identify HPV-driven non-OPSCC is less well understood. METHODS: We stained for p16, pRb, cyclin D1, and p53 in 327 HNSCC. In 310 OPSCC, HPV-status was assessed by HPV DNA PCR. In 119 non-OPSCC, RNA in situ hybridization was additionally performed. HPV-status was correlated with staining patterns, p53 and clinical data. RESULTS: The OPSCC showed blockstaining for p16 in 36%, 8% were equivocal. Of these, HPV-testing was performed in 57%, and 53% were positive for HPV DNA. HPV-association correlated with absence of pRb and cyclin D1 and favorable outcome. In non-OPSCC, 18% showed p16-blockstaining, and 13% showed E6/E7 RNA. Six of seven HPV+ OSCC and 8/8 LSCC lost pRb and cyclin D1. Compared to HPV-negative counterparts, patients with HPV+ cancers had lower rates of alcohol consumption and keratinizing morphology. HPV-positive OSCC had a longer overall survival (p < 0.05). HPV subtype 16 was the most common. CONCLUSIONS: We conclude that HPV-positive non-OPSCC are associated with p16 overexpression and low levels of pRb and cyclin D1. High expression of pRb and cyclin D1 indicates HPV-negativity.

A glutamatergic biomarker panel enables differentiating Grade 4 gliomas/astrocytomas from brain metastases.

Background: The differentiation of high-grade glioma and brain tumors of an extracranial origin is eminent for the decision on subsequent treatment regimens. While in high-grade glioma, a surgical resection of the tumor mass is a fundamental part of current standard regimens, in brain metastasis, the burden of the primary tumor must be considered. However, without a cancer history, the differentiation remains challenging in the imaging. Hence, biopsies are common that may help to identify the tumor origin. An additional tool to support the differentiation may be of great help. For this purpose, we aimed to identify a biomarker panel based on the expression analysis of a small sample of tissue to support the pathological analysis of surgery resection specimens. Given that an aberrant glutamate signaling was identified to drive glioblastoma progression, we focused on glutamate receptors and key players of glutamate homeostasis. Methods: Based on surgically resected samples from 55 brain tumors, the expression of ionotropic and metabotropic glutamate receptors and key players of glutamate homeostasis were analyzed by RT-PCR. Subsequently, a receiver operating characteristic (ROC) analysis was performed to identify genes whose expression levels may be associated with either glioblastoma or brain metastasis. Results: Out of a total of 29 glutamatergic genes analyzed, nine genes presented a significantly different expression level between high-grade gliomas and brain metastases. Of those, seven were identified as potential biomarker candidates including genes encoding for AMPA receptors GRIA1, GRIA2, kainate receptors GRIK1 and GRIK4, metabotropic receptor GRM3, transaminase BCAT1 and the glutamine synthetase (encoded by GLUL). Overall, the biomarker panel achieved an accuracy of 88% (95% CI: 87.1, 90.8) in predicting the tumor entity. Gene expression data, however, could not discriminate between patients with seizures from those without. Conclusion: We have identified a panel of seven genes whose expression may serve as a biomarker panel to discriminate glioblastomas and brain metastases at the molecular level. After further validation, our biomarker signatures could be of great use in the decision making on subsequent treatment regimens after diagnosis.

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.

Alteration of DSS-mediated immune cell redistribution in murine colitis by oral colostral immunoglobulin.

BACKGROUND: Oral bovine colostrum prophylaxis accelerates the recovery of dextran sulfate sodium (DSS)-induced colitis. In the present study the beneficial effects on acute intestinal inflammation of two major colostral components, secretory immunoglobulin A and lactoferrin, were investigated. Outbred NMRI mice received whole bovine colostrum (BC, 20 mg/kg body weight), colostral bovine lactoferrin (bLf, 150 mg/kg), or secretory immunoglobulin A (sIgA, 1-2 mg/kg body weight) daily by oral gavage, either two weeks before induction of colitis (prophylaxis) or after disease establishment (therapy). Bovine serum albumin (BSA, 150 mg/kg body weight) and immunoglobulin G (IgG, 1 and 2 mg/kg body weight) served as protein controls. Colitis was induced by providing 5% DSS solution ad libitum for seven days. RESULTS: Compared to BSA, BC therapy improved occult blood, stool consistency, and clinical recovery from colitis but did not prevent initial weight loss. In contrast, administration of bLf did not influence the course of colitis in either the prophylactic or the therapeutic setting. Therapeutic application of sIgA promoted weight gain in the recovery phase of colitis but failed to improve other clinical parameters. Prophylactically-fed sIgA influenced immune cell redistribution, normalized peripheral blood CD11c⁺CD83⁺ mature dendritic cells, modulated colonic immune cell infiltration, and altered the numbers of both DSS-induced regulatory γδ TCR⁺ T cells and CD11b⁺Gr-1⁺ myeloid suppressor cells in the lymph nodes and spleens of mice. CONCLUSIONS: These data demonstrated the potential of colostrum in disease recovery and epithelial homeostasis following intestinal injury. Colostral sIgA failed to improve acute disease activity but promoted weight gain and modulated immune cell responses that are involved in the genesis of colitis.

Mistletoe lectin has a shiga toxin-like structure and should be combined with other Toll-like receptor ligands in cancer therapy.

Mistletoe extract (ME) is applied as an adjuvant treatment in cancer therapy in thousands of patients each year in Europe. The main immunostimulating component of mistletoe extract, mistletoe lectin, recently has been shown to be a pattern recognition receptor ligand and hence is binding to an important class of pathogen-sensing receptors. Pattern recognition receptor ligands are potent activators of dendritic cells. This activation is a prerequisite for a full-blown T-cell response against cancer cells. Pattern recognition receptor ligands are increasingly recognized as important players in cancer immunotherapy. We collect evidence from case studies on spontaneous regression, from epidemiology, from experiments in a mouse cancer model, and from protein structure comparisons to argue that a combination of mistletoe therapy with other pattern recognition receptor ligand substances leads to an increased immune stimulatory effect. We show that mistletoe lectin is a plant protein of bacterial origin with a 3D structure very similar to shiga toxin from Shigella dysenteriae, which explains the remarkable immunogenicity of mistletoe lectin. Secondly, we show that a combination of pattern recognition receptor ligands applied metronomically in a cancer mouse model leads to complete remission, while single pattern recognition receptor ligands slowed tumor growth. Taken together, we propose to combine mistletoe drugs with other pattern recognition receptor ligand drugs to increase its efficacy in adjuvant or even primary cancer therapy.

Combinations of TLR ligands: a promising approach in cancer immunotherapy.

Toll-like receptors (TLRs), a family of pattern recognition receptors recognizing molecules expressed by pathogens, are typically expressed by immune cells. However, several recent studies revealed functional TLR expression also on tumor cells. Their expression is a two-sided coin for tumor cells. Not only tumor-promoting effects of TLR ligands are described but also direct oncopathic and immunostimulatory effects. To clarify TLRs' role in colorectal cancer (CRC), we tested the impact of the TLR ligands LPS, Poly I:C, R848, and Taxol on primary human CRC cell lines (HROC40, HROC60, and HROC69) in vitro and in vivo (CT26). Taxol, not only a potent tumor-apoptosis-inducing, but also TLR4-activating chemotherapeutic compound, inhibited growth and viability of all cell lines, whereas the remaining TLR ligands had only marginal effects (R848 > LPS > Poly I:C). Combinations of the substances here did not improve the results, whereas antitumoral effects were dramatically boosted when human lymphocytes were added. Here, combining the TLR ligands often diminished antitumoral effects. In vivo, best tumor growth control was achieved by the combination of Taxol and R848. However, when combined with LPS, Taxol accelerated tumor growth. These data generally prove the potential of TLR ligands to control tumor growth and activate immune cells, but they also demonstrate the importance of choosing the right combinations.

Knockdown of coagulation factor VII has potential anticoagulant and anti-inflammatory effects in a mouse model of group A streptococcal sepsis.

Infections originating from subcutaneous tissues are among the most common invasive infections caused by group A streptococcus (GAS) and associated with systemic coagulation activation. The role of intrinsic coagulation factors on GAS virulence has recently been determined, but the role of the extrinsic coagulation factor VII is unknown. Using a mouse model, in which GAS-sepsis emerges from a subcutaneous infection, we show that FVII is a negative acute phase protein. F7 knockdown using antisense oligonucleotides resulted in an attenuated systemic coagulation activation and inflammatory response in septic animals. The findings indicate FVII's ability to modify the host response.