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Abstract Current treatment outcome of patients with glioblastoma (GBM) remains poor. Following standard therapy, recurrence is universal with limited survival. Tumors from 173 GBM patients are analysed for somatic mutations to generate a personalized peptide vaccine targeting tumor-specific neoantigens. All patients were treated within the scope of an individual healing attempt. Among all vaccinated patients, including 70 treated prior to progression (primary) and 103 treated after progression (recurrent), the median overall survival from first diagnosis is 31.9 months (95% CI: 25.0–36.5). Adverse events are infrequent and are predominantly grade 1 or 2. A vaccine-induced immune response to at least one of the vaccinated peptides is detected in blood samples of 87 of 97 (90%) monitored patients. Vaccine-specific T-cell responses are durable in most patients. Significantly prolonged survival is observed for patients with multiple vaccine-induced T-cell responses (53 months) compared to those with no/low induced responses (27 months; P = 0.03). Altogether, our results highlight that the application of personalized neoantigen-targeting peptide vaccine is feasible and represents a promising potential treatment option for GBM patients.

This study aims to highlight the importance of choosing the appropriate co-polymer or co-polymer mixed combinations in order to design value-added nasal dosage forms. Local therapy of upper respiratory tract-related infections, such as nasal rhinosinusitis is of paramount importance, thus advanced local therapeutic options are required. Dexamethasone was encapsulated into three different polymeric micelle formulations: Soluplus or TPGS-only and their mixed combinations. Dynamic light scattering measurements proved that the particles have a micelle size less than 100 nm in monodisperse distribution, with high encapsulation efficiency above 80% and an at least 7-fold water solubility increase. Tobramycin, as an antimicrobial agent, was co-formulated into the in situ gelling systems which were optimized based on gelation time and gelation temperature. The sol–gel transition takes place between 32–35 °C, which is optimally below the temperature of the nasal cavity in a quick manner below 5 min, a suitable strategic criterion against the mucociliary clearance. In vitro drug release and permeability studies confirmed a rapid kinetics in the case of the encapsulated dexamethasone accompanied with a sustained release of tobramycin, as the hydrophilic drug.

IntroductionNeuroendocrine neoplasms (NEN) are a rare and heterogenous group of tumors arising from neuroendocrine cells in multiple organs. Neuroendocrine tumors (NET) G3 encompass a small subgroup accounting for less than 10% of all neuroendocrine neoplasms. In contrast to NET G1 and G2 as well as neuroendocrine carcinomas (NEC), in NET G3 data on treatment and patient outcomes are still limited. Especially in a metastasized tumor stage, the role of surgery, peptide receptor radionucleotide therapy (PRRT), and systemic chemotherapy is not clearly defined.MethodsIn this real-life cohort, we consecutively analyzed clinical outcome in NET G3 patients receiving different diagnostic and treatment.Results and discussionWe found that even metastasized NET G3 patients undergoing surgery, or receiving radiation, somatostatin analogues (SSA), and PRRT showed a clear survival benefit. Interestingly, all treatment regimen were superior to classical chemotherapeutic agents. In addition, somatostatin receptor (SSTR) PET-CT, FDG PET-CT, and repetitive biopsies were shown to be useful diagnostic and prognostic tools in NET G3. Our study demonstrates that patients with highly proliferative NET G3 might benefit from less aggressive treatment modalities commonly used in low proliferative NEN.

The current research aims to develop thermosensitive polymeric micelles loaded with risperidone for nasal administration, emphasizing the added benefits of their thermosensitive behavior under nasal conditions. An initial risk assessment facilitated the advanced development process, confirming that the key indicators of thermosensitivity were suitable for nasal application. The polymeric micelles exhibited an average size of 118.4 ± 3.1 nm at ambient temperature and a size of 20.47 ± 1.2 nm at 36.5 °C, in both cases in monodisperse distribution. Factors such as pH and viscosity did not significantly impact these parameters, demonstrating appropriate nasal applicability. The model formulations showed a rapid, burst-like drug release profile in vitro, accompanied by a quick and high permeation rate at nasal conditions. Overall, the Quality by Design-based risk assessment process led to the development of an advanced drug delivery system capable of administering risperidone through the nasal cavity.

Network targeting of disease-specific nodes represents a useful principle for designing combination cancer therapy. In this case of a patient with relapsed leptomeningeal glioblastoma, comprehensive molecular diagnosis led to the identification of a disease network characterized by multiple disease-specific synthetic lethal vulnerabilities involving DNA repair, REDOX homeostasis, and impaired autophagy which suggested a novel network-targeting combination therapy (NTCT). A treatment regimen consisting of lomustine, olaparib, digoxin, metformin, and high dose intravenous ascorbate was employed using the principle of intra-patient dose escalation to deliver the treatment with adequate safety measures to achieve a definitive clinical result.

Bovine serum albumin (BSA) has been used extensively as a suitable carrier system for alternative drug delivery routes, such as nasal administration. However, the optimization of BSA nanoparticles with respect to their nasal applicability has not been widely studied. The present study focuses on the characterization of BSA nanoparticles prepared using the desolvation method, followed by a gelation process to facilitate intranasal drug delivery. The results demonstrated that the ratio of BSA and the desolvating agent, ethanol, played a critical role in the nanoparticle characteristics of the BSA nanogel matrices (BSA-NGs). Based on the gelling properties, the formulations of BSA-NG 2, BSA-NG 4, and BSA-NG 6 were selected for further investigation. The Raman spectra confirmed that there were no specific changes to the secondary structures of the BSA. The mucoadhesion studies revealed moderately high mucoadhesive properties, with a mucin binding efficiency (MBE) value of around 67%, allowing the dose to avoid elimination due to rapid mucociliary clearance of the nasal passage. Via studying the nexus of the carrier system, BSA-NGs loaded with dexamethasone as a model drug were prepared and evaluated by differential scanning calorimetry (DSC) and thermal gravimetry (TG), ascertaining that no ethanol remained in the samples after the freeze-drying process. Furthermore, the viscosity measurements exhibited moderate viscosity, which is suitable for nasal liquid preparations. The in vitro release studies performed with a simulated nasal electrolyte solution (SNES) medium showed 88.15–95.47% drug release within 4 h. In conclusion, BSA nanoparticle gelling matrices can offer potential, value-added drug delivery carriers for improved nasal drug administration.

In this present formulation study, vinpocetine-loaded nano-spray-dried polymeric micelles were developed via nano-spray-drying. Three different mucoadhesive excipients were applied in the studies, namely chitosan, hyaluronic acid and hydroxypropyl methylcellulose. In all cases, the formulations had a proper particle size and drug content after drying with spherical morphology and amorphous structure. After rapid dissolution in water, the polymeric micelles had a particle size around 100–130 nm, in monodisperse size distribution. The high encapsulation efficiency (>80%) and high solubilization (approx. 300-fold increase in thermodynamic solubility) contributed to rapid drug release (>80% in the first 15 min) and fast passive diffusion at simulated nasal conditions. The formulated prototype preparations fulfilled the demands of a low-viscosity, moderately mucoadhesive nasal drug delivery system, which may be capable of increasing the overall bioavailability of drugs administered via the auspicious nasal drug delivery route.

The current research is aimed at investigating the relationship between the formulation components and conditions in the case of a binary drug delivery system, where antidiabetic drugs are co-formulated into polymeric micelles embedded in sodium alginate. Compared to chemical modifications of polymers with alginate, our development provides a simpler and scalable formulation process. Our results prove that a multi-level factorial design-based approach can ensure the development of a value-added polymeric micelle formulation with an average micelle size of 123.6 ± 3.1 nm and a monodisperse size distribution, showing a polydispersity index value of 0.215 ± 0.021. The proper nanoparticles were co-formulated with sodium alginate as a biologically decomposing and safe-to-administer biopolymer. The Box–Behnken factorial design ensured proper design space development, where the optimal sodium alginate bead formulation had a uniform, extended-release drug release mechanism similar to commercially available tablet preparations. The main conclusion is that the rapid-burst-like drug release can be hindered via the embedment of nanocarriers into biopolymeric matrices. The thermally stable formulation also holds the benefit of uniform active substance distribution after freeze-drying.

Abstract Healing of gastrointestinal ulcers after Hemospray application was reported in literature. The pathophysiological mechanism of action of hemostatic powders is not elucidated so far. A prospective animal model was performed to evaluate the effect of Hemospray application on the healing process of artificially induced ulcers of the upper and lower gastrointestinal tract. In 10 pigs, 20 ulcers were created in each the upper and the lower gastrointestinal tract by endoscopic mucosal resection. 50% of the pigs were immediately treated with Hemospray application, the others were not treated. Ulcer size was measured endoscopically on day 0, 2, and 7. On day 7 the ulcers were histopathological evaluated for capillary ingrowth and the thickness of the collagen layer. After 7 days the sizes of the ulcers decreased significantly (stomach: − 22.8% with Hemospray application, − 19% without Hemospray application; rectum: − 50.8% with Hemospray application, − 49.5% without Hemospray application; p = 0.005–0.037), but without significant difference between both groups. This study shows no significant effect of the hemostatic powder Hemospray on ulcer healing in the upper and lower gastrointestinal tract compared with untreated controls, neither harmful nor beneficial. However, some trends merit further trials in patients and may indicate a possible mechanism of accelerated mucosal healing.

Abstract The prostate‐specific membrane antigen (PSMA) has been demonstrated in numerous studies to be expressed specifically on prostate carcinoma cells and on the neovasculature of several other cancer entities. However, the simultaneous expression of PSMA on both, tumor cells as well as tumor vessels remains unclear, even if such “dual” expression would constitute an important asset to facilitate sufficient influx of effector cells to a given tumor site. We report here on the generation of a PSMA antibody, termed 10B3, which exerts superior dual reactivity on sections of prostate carcinoma and squamous cell carcinoma of the lung. 10B3 was used for the construction of T‐cell recruiting bispecific PSMAxCD3 antibodies in Fab‐ and IgG‐based formats, designated Fabsc and IgGsc, respectively. In vitro, both molecules exhibited comparable activity. In contrast, only the larger IgGsc molecule induced complete and durable elimination of established tumors in humanized mice due to favorable pharmacokinetic properties. Upon treatment of three patients with metastasized prostate carcinoma with the IgGsc reagent, marked activation of T cells and rapid reduction of elevated PSA levels were observed.

Abstract Background Oncolytic virotherapy is an upcoming treatment option for many tumor entities. But so far, a first oncolytic virus only was approved for advanced stages of malignant melanomas. Neuroendocrine tumors (NETs) constitute a heterogenous group of tumors arising from the neuroendocrine system at diverse anatomic sites. Due to often slow growth rates and (in most cases) endocrine non-functionality, NETs are often detected only in a progressed metastatic situation, where therapy options are still severely limited. So far, immunotherapies and especially immunovirotherapies are not established as novel treatment modalities for NETs. Methods In this immunovirotherapy study, pancreatic NET (BON-1, QGP-1), lung NET (H727, UMC-11), as well as neuroendocrine carcinoma (NEC) cell lines (HROC-57, NEC-DUE1) were employed. The well characterized genetically engineered vaccinia virus GLV-1 h68, which has already been investigated in various clinical trials, was chosen as virotherapeutical treatment modality. Results Profound oncolytic efficiencies were found for NET/NEC tumor cells. Besides, NET/NEC tumor cell bound expression of GLV-1 h68-encoded marker genes was observed also. Furthermore, a highly efficient production of viral progenies was detected by sequential virus quantifications. Moreover, the mTOR inhibitor everolimus, licensed for treatment of metastatic NETs, was not found to interfere with GLV-1 h68 replication, making a combinatorial treatment of both feasible. Conclusions In summary, the oncolytic vaccinia virus GLV-1 h68 was found to exhibit promising antitumoral activities, replication capacities and a potential for future combinatorial approaches in cell lines originating from neuroendocrine neoplasms. Based on these preliminary findings, virotherapeutic effects now have to be further evaluated in animal models for treatment of Neuroendocrine neoplasms (NENs).

Hemoconcentration during exercise is a well-known phenomenon, however, the extent to which dehydration is involved is unclear. In our study, the effect of dehydration on exercise-induced hemoconcentration was examined in 12 elite Hungarian kayak-canoe athletes. The changes of blood markers were examined during acute maximal workload in hydrated and dehydrated states. Dehydration was achieved by exercise, during a 120-minute extensive-aerobic preload. Our research is one of the first studies in which the changes in blood components were examined with a higher time resolution and a wider range of the measured parameters. Hydration status had no effect on the dynamics of hemoconcentration during both the hydrated (HS) and dehydrated (DHS) load, although lower maximal power output were measured after the 120-minute preload [HS Hemoglobin(Hgb)Max median 17.4 (q1 17.03; q3 17.9) g/dl vs. DHS HgbMax median 16.9 (q1 16.43; q3 17.6) g/dl (n.s); HS Hematocrit(Hct)Max 53.50 (q1 52.28; q3 54.8) % vs. DHS HctMax 51.90 (q1 50.35; q3 53.93) % (n.s)]. Thirty minutes after the maximal loading, complete hemodilution was confirmed in both exercises. Dehydration had no effect on hemoconcentration or hemodilution in the recovery period [HS HgbR30' 15.7 (q1 15.15; q3 16.05) g/dl (n.s.) vs. DHS HgbR30' 15.75 (q1 15.48; q3 16.13) g/dl (n.s.), HS HctR30' 48.15 (q1 46.5; q3 49.2) % vs. DHS HctR30' 48.25 (q1 47.48; q3 49.45) % (n.s.)], however, plasma osmolality did not follow a corresponding decrease in hemoglobin and hematocrit in the dehydrated group. Based on our data, metabolic products (glucose, lactate, sodium, potassium, chloride, bicarbonate ion, blood urea nitrogen) induced osmolality may not play a major role in the regulation of hemoconcentration and post-exercise hemodilution. From our results, we can conclude that hemoconcentration depends mainly on the intensity of the exercise.

Albumin nanocarrier research and development is a challenging area in the field of personalized medicine and in providing advanced therapeutic solutions. Albumin as a biocompatible, nonimmunogenic, and non-toxic protein carrier that can be exploited to conjugate drugs with poor bioavailability to improve on this feature. With many different perspectives and desired target profiles, a systematic structural approach must be used in nanoparticle development. The extended Research and Development (R&D) Quality by Design thinking and methodology proved to be useful in case of specific nanoparticle development processes before. However, the coacervation method is the most frequently applied preparation method for HSA nanoparticles; there is a lack of existing research work which has directly determined the influence of process parameters, control strategy, or design space. With a quality-management-driven strategy, a knowledge space was developed for these versatile nanoparticles and an initial risk assessment was conducted on the quality-affecting factors regarding the coacervation method, followed by an optimization process via Plackett–Burman and Box–Behnken experimental design. As a result of screening the effect of process variables on the fabrication of HSA nanoparticles, an optimized colloidal drug delivery system was engineered with desired nanoparticulate properties.

BackgroundTreatment options for poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinoma are unsatisfactory and prognosis is generally poor. Lenvatinib (LEN), a multi-tyrosine kinase inhibitor targeting fibroblast growth factor receptors (FGFR) 1-4 is approved for advanced radioiodine refractory thyroid carcinoma, but response to single agent is poor in ATC. Recent reports of combining LEN with PD-1 inhibitor pembrolizumab (PEM) are promising.Materials and MethodsPrimary ATC (n=93) and PDTC (n=47) tissue samples diagnosed 1997-2019 at five German tertiary care centers were assessed for PD-L1 expression by immunohistochemistry using Tumor Proportion Score (TPS). FGFR 1-4 mRNA was quantified in 31 ATC and 14 PDTC with RNAscope in-situ hybridization. Normal thyroid tissue (NT) and papillary thyroid carcinoma (PTC) served as controls. Disease specific survival (DSS) was the primary outcome variable.ResultsPD-L1 TPS≥50% was observed in 42% of ATC and 26% of PDTC specimens. Mean PD-L1 expression was significantly higher in ATC (TPS 30%) than in PDTC (5%; p

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterised by extensive matrix deposition that has been implicated in impaired drug delivery and therapeutic resistance. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates collagen deposition and is highly upregulated in the activated stroma subtype with poor prognosis in PDAC patients. Methods: KrasG12D;p48-Cre;SPARC−/− (KC-SPARC−/−) and KrasG12D;p48-Cre;SPARCWT (KC-SPARCWT) were generated and analysed at different stages of carcinogenesis by histological grading, immunohistochemistry for epithelial and stromal markers, survival and preclinical analysis. Pharmacokinetic and pharmacodynamic studies were conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunohistochemistry following gemcitabine treatment (100 mg/kg) in vivo. Findings: Global genetic ablation of SPARC in a KrasG12D driven mouse model resulted in significantly reduced overall and mature collagen deposition around early and advanced pancreatic intraepithelial neoplasia (PanIN) lesions and in invasive PDAC (p

Liposomal formulations, as versatile nanocarrier systems suitable for targeted delivery, have a highly focused role in the therapy development of unmet clinical needs and diagnostic imaging techniques. Formulating nanomedicine with suitable zeta potential is an essential but challenging task. Formulations with a minimum ±30 mV zeta potential are considered stable. The charge of the phospholipid bilayer can be adjusted with membrane additives. The present Quality by Design-derived study aimed to optimise liposomal formulations prepared via the thin-film hydration technique by applying stearylamine (SA) or dicetyl phosphate (DCP) as charge imparting agents. This 32 fractional factorial design-based study determined phosphatidylcholine, cholesterol, and SA/DCP molar ratios for liposomes with characteristics meeting the formulation requirements. The polynomials describing the effects on the zeta potential were calculated. The optimal molar ratios of the lipids were given as 12.0:5.0:5.0 for the SA-PBS pH 5.6 (optimised sample containing stearylamine) and 8.5:4.5:6.5 for the DCP-PBS pH 5.6 (optimised sample containing dicetyl phosphate) particles hydrated with phosphate-buffered saline pH 5.6. The SA-PBS pH 5.6 liposomes had a vesicle size of 108 ± 15 nm, 0.20 ± 0.04 polydispersity index, and +30.1 ± 1.2 mV zeta potential, while these values were given as 88 ± 14 nm, 0.21 ± 0.02, and −36.7 ± 3.3 mV for the DCP-PBS pH 5.6 vesicles. The prepared liposomes acquired the requirements of the zeta potential for stable formulations.

This work aimed to develop lomustine (LOM) and n-propyl gallate (PG)-loaded liposomes suitable for targeting glioblastoma multiforme (GBM) via the auspicious nose-to-brain drug delivery pathway. The therapeutical effect of LOM, as a nitrosourea compound, can be potentiated by PG suitable for enhanced anti-cancer therapy. Nose-to-brain delivery of PG and LOM combined in liposomes can overcome the poor water solubility, absorption properties, and toxicity issues in the systemic circulation. Optimization and characterization of the liposomal carrier with binary drug contents were carried out in order to achieve adequate encapsulation efficiency, loading capacity, drug release, and ex vivo permeation. The optimized liposome co-encapsulated with both drugs showed suitable Z-average (127 ± 6.9 nm), size distribution (polydispersity index of 0.142 ± 0.009), zeta potential (−34 ± 1.7 mV), and high encapsulation efficacy (63.57 ± 1.3% of PG and 73.45 ± 2.2% of LOM, respectively) meeting the acceptance criteria of nose-to-brain transport for both drugs. MTT assays of PG-LOM formulations were also conducted on NIH/3T3 (murine embryonic fibroblast), U87 (glioblastoma), and A2780 (ovarian cancer) cell lines indicating reduced an antiproliferative effect on all types of cells. Our results supported the use of this novel combination of LOM and PG in a liposomal formulation as a promising carrier for glioblastoma targeting via the intranasal route.

Summary: The cellular origin of sporadic pancreatic neuroendocrine tumors (PNETs) is obscure. Hormone expression suggests that these tumors arise from glucagon-producing alpha cells or insulin-producing β cells, but instability in hormone expression prevents linage determination. We utilize loss of hepatic glucagon receptor (GCGR) signaling to drive alpha cell hyperproliferation and tumor formation to identify a cell of origin and dissect mechanisms that drive progression. Using a combination of genetically engineered Gcgr knockout mice and GCGR-inhibiting antibodies, we show that elevated plasma amino acids drive the appearance of a proliferative population of SLC38A5+ embryonic progenitor-like alpha cells in mice. Further, we characterize tumors from patients with rare bi-allelic germline GCGR loss-of-function variants and find prominent tumor-cell-associated expression of the SLC38A5 paralog SLC7A8 as well as markers of active mTOR signaling. Thus, progenitor cells arise from adult alpha cells in response to metabolic signals and, when inductive signals are chronically present, drive tumor initiation.

This study aimed to produce thermosensitive liposomes (TSL) by applying the quality by design (QbD) concept. In this paper, our research group collected and studied the parameters that significantly impact the quality of the liposomal product. Thermosensitive liposomes are vesicles used as drug delivery systems that release the active pharmaceutical ingredient in a targeted way at ~40–42 °C, i.e., in local hyperthermia. This study aimed to manufacture thermosensitive liposomes with a diameter of approximately 100 nm. The first TSLs were made from DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine) and DSPC (1,2-dioctadecanoyl-sn-glycero-3-phosphocholine) phospholipids. Studies showed that the application of different types and ratios of lipids influences the thermal properties of liposomes. In this research, we made thermosensitive liposomes using a PEGylated lipid besides the previously mentioned phospholipids with the thin-film hydration method.

In this study, the effect of Cremophor® RH 40 (CR 40) classic micelles and Soluplus® (SP) polymeric micelles were investigated on a novel granule-type drug-delivery system containing megestrolacetate (MGA). Using a risk assessment-based approach on the formulation via melt technology resulted in the formation of these granules, presented as the dosage, with proper particle size and flow characteristics. Due to the application of a eutectic carrier base composition, gentle process conditions were reached, retaining the crystalline structure of the carrier system and allowing for the proper distribution of MGA in the granules. The increased water solubility (0.111 mg/mL to 2.154 mg/mL), and the decreased nano particle size (102.27 nm) with uniform distribution (polydispersity index of 0.259) and colloid stability (zeta potential of −12.99 mV) resulted in SP polymeric micelles prevailing over CR 40 micelles in this gastric dissolution study, performed in biorelevant fasted and fed state drug-release media. Mathematical characterization and kinetic model fitting supported the fast drug-release mechanism of polymeric micelles over micelles. The value-added polymeric micelle-containing formulation developed can be successfully administered perorally and the enhanced drug release offers the possibility of greater drug absorption in the gastrointestinal tract.

The objective of the present study was to develop n-propyl gallate-loaded solid lipid nanoparticles (PG-SLNs) in a hydrogel (HG) formulation using Transcutol-P (TC-P) as a permeation enhancer. Modified solvent injection technique was applied to produce optimized PG-SLNs via the Quality by Design approach and central composite design. The in vitro mucoadhesion, scavenging activity, drug release, permeation studies of PG from PG-SLNs-loaded HG were evaluated under simulated nasal conditions. Compared with in vitro release behavior of PG from SLNs, the drug release from the PG-SLNs-loaded HG showed a lower burst effect and sustained release profile. The cumulative permeation of PG from PG-SLNs-loaded HG with TC-P was 600 μg/cm2 within 60 min, which is 3–60-fold higher than PG-SLNs and native PG, respectively. Raman mapping showed that the distribution of PG-SLNs was more concentrated in HG having lower concentrations of hyaluronic acid. The scavenging assay demonstrated increased antioxidant activity at higher concentrations of HG. Due to enhanced stability and mucoadhesive properties, the developed HG-based SLNs can improve nasal absorption by increasing residence time on nasal mucosa. This study provides in vitro proof of the potential of combining the advantages of SLNs and HG for the intranasal delivery of antioxidants.

Introduction: Outcome after postoperative radiochemotherapy (RT-CT) for patients with advanced head and neck squamous cell carcinomas (HNSCC) remains unsatisfactory, especially among those with HPV negative tumours. Therefore, new biomarkers are needed to further define subgroups for individualised therapeutic approaches. Preclinical and first clinical observations showed that the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12) play an important role in tumour cell proliferation, survival, cancer progression, metastasis and treatment resistance. However, the data on the prognostic value of SDF-1/CXCR4 expression for HNSCC are conflicting. The aim of our hypothesis-generating study was to retrospectively explore the prognostic potential of SDF-1/CXCR4 in a well-defined cohort of HNSCC patients collected within the multicenter biomarker study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG). Material and methods: Patients with stage III and IVA HNSCC of the oral cavity, oropharynx and hypopharynx were treated with resection and adjuvant radiotherapy (RT) with ≥60 Gy and concurrent cisplatin-based chemotherapy (CT). Tissue micro-arrays (TMAs) from a total of 221 patients were generated from surgical specimens, 201 evaluated for the SDF-1 and CXCR4 expression by immunofluorescence and correlated with clinico-pathological and outcome data. Results: In univariate and multivariate analyses intracellular SDF-1 expression was associated with lower loco-regional control (LRC) in the entire patient group as well as in the HPV16 DNA negative subgroup. CXCR4 expression showed a trend for lower LRC in the univariate analysis which was not confirmed in the multivariate analysis. Neither for SDF-1 nor CXCR4 expression associations with distant metastasis free or overall survival were found. Conclusions: Our exploratory data support the hypothesis that overexpression of intracellular SDF-1 is an independent negative prognostic biomarker for LRC after postoperative RT-CT in high-risk HNSCC. Prospective validation is warranted and further exploration of SDF-1/CXCR4 as a potential therapeutic target to overcome treatment resistance in HNSCC appears promising.

Background: Radiofrequency ablation (RFA) is an established treatment option for malignancies located in the liver. RFA-induced irreversible coagulation necrosis leads to the release of danger signals and cellular content. Hence, RFA may constitute an endogenous in situ tumor vaccination, stimulating innate and adaptive immune responses, including tumor-antigen specific T cells. This may explain a phenomenon termed abscopal effect, namely tumor regression in untreated lesions evidenced after distant thermal ablation or irradiation. In this study, we therefore assessed systemic and local immune responses in individual patients treated with RFA.Methods: For this prospective clinical trial, patients with liver metastasis from colorectal carcinoma (mCRC) receiving RFA and undergoing metachronous liver surgery for another lesion were recruited (n = 9) during a 5-year period. Tumor and non-malignant liver tissue samples from six patients were investigated by whole transcriptome sequencing and tandem-mass spectrometry, characterizing naturally presented HLA ligands. Tumor antigen-derived HLA-restricted peptides were selected by different predefined approaches. Further, candidate HLA ligands were manually curated. Peripheral blood mononuclear cells were stimulated in vitro with epitope candidate peptides, and functional T cell responses were assessed by intracellular cytokine staining. Immunohistochemical markers were additionally investigated in surgically resected mCRC from patients treated with (n = 9) or without RFA (n = 7).Results: In all six investigated patients, either induced immune responses and/or pre-existing T cell immunity against the selected targets were observed. Multi-cytokine responses were inter alia directed against known tumor antigens such as cyclin D1 but also against a (predicted) mutation contained in ERBB3. Immunohistochemistry did not show a relevant influx of immune cells into distant malignant lesions after RFA treatment (n = 9) as compared to the surgery only mCRC group (n = 7).Conclusions: Using an individualized approach for target selection, RFA induced and/or boosted T cell responses specific for individual tumor antigens were more frequently detectable as compared to previously published observations with well-characterized tumor antigens. However, the witnessed modest RFA-induced immunological effects alone may not be sufficient for the rejection of established tumors. Therefore, these findings warrant further clinical investigation including the assessment of RFA combination therapies e.g., with immune stimulatory agents, cancer vaccination, and/or immune checkpoint inhibitors.

Nanoparticle research and development for pharmaceuticals is a challenging task in the era of personalized medicine. Specialized and increased patient expectations and requirements for proper therapy adherence, as well as sustainable environment safety and toxicology topics raise the necessity of well designed, advanced and smart drug delivery systems on the market. These stakeholder expectations and social responsibility of pharma sector open the space and call new methods on the floor for new strategic development tools, like Quality by Design (QbD) thinking. The extended model, namely the R&D QbD proved to be useful in case of complex and/or high risk/expectations containing or aiming developments. This is the case when we formulate polymeric micelles as promising nanotherapeutics; the risk assessment and knowledge-based quality targeted QbD approach provides a promising tool to support the development process. Based on risk assessment, many factors pose great risk in the manufacturing process and affect the quality, efficacy and safety profile. The quality-driven strategic development pathway, based on deep prior knowledge and an involving iterative risk estimation and management phases has proven to be an adequate tool, being able to handle their sensitive stability issues and make them efficient therapeutic aids in case of several diseases.

The aim of this study was to develop an intranasal in situ thermo-gelling meloxicam-human serum albumin (MEL-HSA) nanoparticulate formulation applying poloxamer 407 (P407), which can be administered in liquid state into the nostril, and to increase the resistance of the formulation against mucociliary clearance by sol-gel transition on the nasal mucosa, as well as to improve drug absorption. Nanoparticle characterization showed that formulations containing 12–15% w/w P407 met the requirements of intranasal administration. The Z-average (in the range of 180–304 nm), the narrow polydispersity index (PdI, from 0.193 to 0.328), the zeta potential (between −9.4 and −7.0 mV) and the hypotonic osmolality (200–278 mOsmol/L) of MEL-HSA nanoparticles predict enhanced drug absorption through the nasal mucosa. Based on the rheological, muco-adhesion, drug release and permeability studies, the 14% w/w P407 containing formulation (MEL-HSA-P14%) was considered as the optimized formulation, which allows enhanced permeability of MEL through blood–brain barrier-specific lipid fraction. Cell line studies showed no cell damage after 1-h treatment with MEL-HSA-P14% on RPMI 2650 human endothelial cells’ moreover, enhanced permeation (four-fold) of MEL from MEL-HSA-P14% was observed in comparison to pure MEL. Overall, MEL-HSA-P14% can be promising for overcoming the challenges of nasal drug delivery.

Our study aimed to develop an “ex tempore” reconstitutable, viscosity enhancer- and preservative-free meloxicam (MEL)-loaded polymeric micelle formulation, via Quality by Design (QbD) approach, exploiting the nose-to-brain pathway, as a suitable tool in the treatment of neuroinflammation. The anti-neuroinflammatory effect of nose-to-brain NSAID polymeric micelles was not studied previously, therefore its investigation is promising. Critical product parameters, encapsulation efficiency (89.4%), Z-average (101.22 ± 2.8 nm) and polydispersity index (0.149 ± 0.7) and zeta potential (−25.2 ± 0.4 mV) met the requirements of the intranasal drug delivery system (nanoDDS) and the targeted profile liquid formulation was transformed into a solid preservative-free product by freeze-drying. The viscosity (32.5 ± 0.28 mPas) and hypotonic osmolality (240 mOsmol/L) of the reconstituted formulation provides proper and enhanced absorption and probably guarantees the administration of the liquid dosage form (nasal drop and spray). The developed formulation resulted in more than 20 times faster MEL dissolution rate and five-fold higher nasal permeability compared to starting MEL. The prediction of IVIVC confirmed the great potential for in vivo brain distribution of MEL. The nose-to-brain delivery of NSAIDs such as MEL by means of nanoDDS as polymeric micelles offers an innovative opportunity to treat neuroinflammation more effectively.

In order to foster the systematic identification of novel genes with important functional roles in pancreatic cancer, we have devised a multi-stage screening strategy to provide a rational basis for the selection of highly relevant novel candidate genes based on the results of functional high-content analyses. The workflow comprised three consecutive stages: 1) serial gene expression profiling analyses of primary human pancreatic tissues as well as a number of in vivo and in vitro models of tumor-relevant characteristics in order to identify genes with conspicuous expression patterns; 2) use of 'reverse transfection array' technology for large-scale parallelized functional analyses of potential candidate genes in cell-based assays; and 3) selection of individual candidate genes for further in-depth examination of their cellular roles. A total of 14 genes, among them 8 from "druggable" gene families, were classified as high priority candidates for individual functional characterization. As an example to demonstrate the validity of the approach, comprehensive functional data on candidate gene ADRBK1/GRK2, which has previously not been implicated in pancreatic cancer, is presented.

Nrf2 and TGF-β1 both affect tumorigenesis in a dual fashion, either by preventing carcinogen induced carcinogenesis and suppressing tumor growth, respectively, or by conferring cytoprotection and invasiveness to tumor cells during malignant transformation. Given the involvement of Nrf2 and TGF-β1 in the adaptation of epithelial cells to persistent inflammatory stress, e.g. of the pancreatic duct epithelium during chronic pancreatitis, a crosstalk between Nrf2 and TGF-β1 can be envisaged. By using premalignant human pancreatic duct cells (HPDE) and the pancreatic ductal adenocarcinoma cell line Colo357, we could show that Nrf2 and TGF-β1 independently but additively conferred an invasive phenotype to HPDE cells, whereas acting synergistically in Colo357 cells. This was accompanied by differential regulation of EMT markers like vimentin, Slug, L1CAM and E-cadherin. Nrf2 activation suppressed E-cadherin expression through an as yet unidentified ARE related site in the E-cadherin promoter, attenuated TGF-β1 induced Smad2/3-activity and enhanced JNK-signaling. In Colo357 cells, TGF-β1 itself was capable of inducing Nrf2 whereas in HPDE cells TGF-β1 per-se did not affect Nrf2 activity, but enhanced Nrf2 induction by tBHQ. In Colo357, but not in HPDE cells, the effects of TGF-β1 on invasion were sensitive to Nrf2 knock-down. In both cell lines, E-cadherin re-expression inhibited the proinvasive effect of Nrf2. Thus, the increased invasion of both cell lines relates to the Nrf2-dependent downregulation of E-cadherin expression. In line, immunohistochemistry analysis of human pancreatic intraepithelial neoplasias in pancreatic tissues from chronic pancreatitis patients revealed strong Nrf2 activity already in premalignant epithelial duct cells, accompanied by partial loss of E-cadherin expression. Our findings indicate that Nrf2 and TGF-β1 both contribute to malignant transformation through distinct EMT related mechanisms accounting for an invasive phenotype. Provided a crosstalk between both pathways, Nrf2 and TGF-β1 mutually promote their tumorigenic potential, a condition manifesting already at an early stage during inflammation induced carcinogenesis of the pancreas.

Pancreatic ductal adenocarcinoma (PDAC) remains an important cause of malignancy-related death and is the eighth most common cancer with the lowest overall 5-year relative survival rate. To identify new molecular markers and candidates for new therapeutic regimens, we investigated the gene expression profile of microdissected cells from 11 normal pancreatic ducts, 14 samples of PDAC, and 4 well-characterized pancreatic cancer cell lines using the Affymetrix U133 GeneChip set. RNA was extracted from microdissected samples and cell lines, amplified, and labeled using a repetitive in vitro transcription protocol. Differentially expressed genes were identified using the significance analysis of microarrays program. We found 616 differentially expressed genes. Within these, 140 were also identified in PDAC by others, such as Galectin-1, Galectin-3, and MT-SP2. We validated the differential expression of several genes (e.g., CENPF, MCM2, MCM7, RAMP, IRAK1, and PTTG1) in PDAC by immunohistochemistry and reverse transcription polymerase chain reaction. We present a whole genome expression study of microdissected tissues from PDAC, from microdissected normal ductal pancreatic cells and pancreatic cancer cell lines using highdensity microarrays. Within the panel of genes, we identified novel differentially expressed genes, which have not been associated with the pathogenesis of PDAC before.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive stroma being also present in chronic pancreatitis (CP). Using immunohistochemistry, the stroma of CP and PDAC was comprehensively analyzed and correlated with epithelial/carcinoma-related alterations and clinicopathological patient characteristics. While there were no significant differences between CP and PDAC regarding the distribution of CD3+ T cells and α-SMA+ fibroblasts, proportions of CD4+ and CD8+ T cells were significantly lower and numbers of CD25+(CD4+) and FoxP3+(CD4+) regulatory T cells were greater in PDAC compared with CP. Macrophages were more prevalent in CP, but localized more closely to carcinoma cells in PDAC, as were γδ-T cells. Duct-related FoxP3 and L1CAM expression increased from CP to PDAC, while vimentin expression was similarly abundant in both diseases. Moreover, stromal and epithelial compartments of well-differentiated tumors and CPs shared considerable similarities, while moderately and poorly differentiated tumors significantly differed from CP tissues. Analysis of 27 parameters within each pancreatic disease revealed a significant correlation of i) CD4+ and FoxP3+CD4+ T cells with FoxP3 expression in PDAC cells, ii) α-SMA+ fibroblasts with L1CAM expression and proliferation in PDAC cells, iii) CD3 and CD8 expression with γδ-TCR expression in both pancreatic diseases and iv) CD68+ and CD163+ macrophages with vimentin expression in PDAC cells. High expression of FoxP3, vimentin and L1CAM in PDAC cells as well as a tumor-related localization of macrophages each tended to correlate with higher tumor grade. Multivariate survival analysis revealed a younger age at time of surgery as a positive prognostic marker for PDAC patients with the most frequently operated disease stage T3N1M0. Overall this study identified several interrelationships between stroma and epithelial/carcinoma cells in PDACs but also in CP, which in light of previous experimental data strongly support the view that the inflammatory stroma contributes to malignancy-associated alterations already in precursor cells during CP.

BACKGROUND AND AIMS: High frequency electrosurgery has a key role in the broadening application of liver surgery. Its molecular signature, i.e. the metabolites evolving from electrocauterization which may inhibit hepatic wound healing, have not been systematically studied. METHODS: Human liver samples were thus obtained during surgery before and after electrosurgical dissection and subjected to a two-stage metabolomic screening experiment (discovery sample: N = 18, replication sample: N = 20) using gas chromatography/mass spectrometry. RESULTS: In a set of 208 chemically defined metabolites, electrosurgical dissection lead to a distinct metabolic signature resulting in a separation in the first two dimensions of a principal components analysis. Six metabolites including glycolic acid, azelaic acid, 2-n-pentylfuran, dihydroactinidiolide, 2-butenal and n-pentanal were consistently increased after electrosurgery meeting the discovery (p

Current treatment of paediatric hepatocellular carcinoma (HCC) is often inefficient due to advanced disease at diagnosis and resistance to common drugs. The aim of this study was to generate a cell line derived from a paediatric HCC in order to expand research in this field. We established the HC-AFW1 cell line from a liver neoplasm of a 4-year-old boy through culturing of primary tumor specimens. The cell line has been stable for over one year of culturing and has a doubling time of 40 h. The tumour cells have an epithelial histology and express HCC-associated proteins such as Alpha-fetoprotein (AFP), Glypican 3, E-cadherin, CD10, CD326, HepPar1 and Vimentin. Forty-nine amino acids in exon 3 of β-Catenin that involve the phosphorylation sites of GSK3 were absent and β-Catenin is detectable in the cell nuclei. Cytogenetic analysis revealed large anomalies in the chromosomal map. Several alterations of gene copy numbers were detected by genome-wide SNP array. Among the different drugs tested, cisplatin and irinotecan showed effective inhibition of tumour cell growth in a proliferation assay at concentrations below 5 µg/ml. Subcutaneous xenotransplantation of HC-AFW1 cells into NOD/SCID mice resulted in fast growing dedifferentiated tumours with high levels of serum AFP. Histological analyses of the primary tumour and xenografts included national and international expert pathological review. Consensus reading characterised the primary tumour and the HC-AFW1-derived tumours as HCC. HC-AFW1 is the first cell line derived from a paediatric HCC without a background of viral hepatitis or cirrhosis and represents a valuable tool for investigating the biology of and therapeutic strategies for childhood HCC.

BACKGROUND: Erythropoietin (Epo) administration has been reported to have tumor-promoting effects in anemic cancer patients. We investigated the prognostic impact of endogenous Epo in patients with pancreatic ductal adenocarcinoma (PDAC). METHODOLOGY: The clinico-pathological relevance of hemoglobin (Hb, n = 150), serum Epo (sEpo, n = 87) and tissue expression of Epo/Epo receptor (EpoR, n = 104) was analyzed in patients with PDAC. Epo/EpoR expression, signaling, growth, invasion and chemoresistance were studied in Epo-exposed PDAC cell lines. RESULTS: Compared to donors, median preoperative Hb levels were reduced by 15% in both chronic pancreatitis (CP, p

BackgroundPancreatic ductal adenocarcinoma (PDAC) remains an important cause of cancer death. Changes in apoptosis signaling in pancreatic cancer result in chemotherapy resistance and aggressive growth and metastasizing. The aim of this study was to characterize the apoptosis pathway in pancreatic cancer computationally by evaluation of experimental data from high-throughput technologies and public data bases. Therefore, gene expression analysis of microdissected pancreatic tumor tissue was implemented in a model of the apoptosis pathway obtained by computational protein interaction prediction.Methodology/principal findingsApoptosis pathway related genes were assembled from electronic databases. To assess expression of these genes we constructed a virtual subarray from a whole genome analysis from microdissected native tumor tissue. To obtain a model of the apoptosis pathway, interactions of members of the apoptosis pathway were analysed using public databases and computational prediction of protein interactions. Gene expression data were implemented in the apoptosis pathway model. 19 genes were found differentially expressed and 12 genes had an already known pathophysiological role in PDAC, such as Survivin/BIRC5, BNIP3 and TNF-R1. Furthermore we validated differential expression of IL1R2 and Livin/BIRC7 by RT-PCR and immunohistochemistry. Implementation of the gene expression data in the apoptosis pathway map suggested two higher level defects of the pathway at the level of cell death receptors and within the intrinsic signaling cascade consistent with references on apoptosis in PDAC. Protein interaction prediction further showed possible new interactions between the single pathway members, which demonstrate the complexity of the apoptosis pathway.Conclusions/significanceOur data shows that by computational evaluation of public accessible data an acceptable virtual image of the apoptosis pathway might be given. By this approach we could identify two higher level defects of the apoptosis pathway in PDAC. We could further for the first time identify IL1R2 as possible candidate gene in PDAC.

BACKGROUND: The Ras and Notch signaling pathways are frequently activated during development to control many diverse cellular processes and are often dysregulated during tumorigenesis. To study the role of Notch and oncogenic Kras signaling in a progenitor cell population, Pdx1-Cre mice were utilized to generate conditional oncogenic Kras(G12D) mice with ablation of Notch1 and/or Notch2. METHODOLOGY/PRINCIPAL FINDINGS: Surprisingly, mice with activated Kras(G12D) and Notch1 but not Notch2 ablation developed skin papillomas progressing to squamous cell carcinoma providing evidence for Pdx1 expression in the skin. Immunostaining and lineage tracing experiments indicate that PDX1 is present predominantly in the suprabasal layers of the epidermis and rarely in the basal layer. Further analysis of keratinocytes in vitro revealed differentiation-dependent expression of PDX1 in terminally differentiated keratinocytes. PDX1 expression was also increased during wound healing. Further analysis revealed that loss of Notch1 but not Notch2 is critical for skin tumor development. Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and β-catenin signaling inhibition capabilities. CONCLUSIONS/SIGNIFICANCE: Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function. In addition, this finding may be relevant for research using Pdx1-Cre transgenic strains. Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors.