Metabolic Heterogeneity of Brain Tumor Cells of Proneural and Mesenchymal Origin.

Brain-tumor-initiating cells (BTICs) of proneural and mesenchymal origin contribute to the highly malignant phenotype of glioblastoma (GB) and resistance to current therapies. BTICs of different subtypes were challenged with oxidative phosphorylation (OXPHOS) inhibition with metformin to assess the differential effects of metabolic intervention on key resistance features. Whereas mesenchymal BTICs varied according to their invasiveness, they were in general more glycolytic and less responsive to metformin. Proneural BTICs were less invasive, catabolized glucose more via the pentose phosphate pathway, and responded better to metformin. Targeting glycolysis may be a promising approach to inhibit tumor cells of mesenchymal origin, whereas proneural cells are more responsive to OXPHOS inhibition. Future clinical trials exploring metabolic interventions should account for metabolic heterogeneity of brain tumors.

Alterations in the Experience of Time, Space, and Intersubjectivity and the Interaction with Pre-Existent Psychopathology during the COVID-19 Pandemic.

BACKGROUND: The COVID-19 pandemic and the measures to protect the physically vulnerable may disproportionately affect people with mental health vulnerabilities, who receive psychotherapeutic inpatient treatment, as many of these measures impact the (inter)subjective space crucial to psychotherapy. OBJECTIVE: We investigate how people with pre-existing mental health conditions and healthcare professionals experienced changes linked to the COVID-19 pandemic. METHODS: During the first COVID-19 outbreak and lockdown in spring 2020, we conducted semi-structured interviews with patients and healthcare professionals at a clinic for psychosomatic medicine in Germany and analyzed them following the principles of descriptive phenomenology focusing on social interactions, intersubjectivity, and the therapeutic space. RESULTS: We conducted a total of >30 h of interviews with 19 patients and 17 healthcare professionals. Analyses revealed that the COVID-19 pandemic and the related measures have led to a sudden loss of a sense of normality. Participants experienced changes in the perception of time, space, self, and embodied interaction with others, resulting in a profound feeling of alienation and "unhomeliness" which seemed to magnify pre-existing psychopathology. The inpatient psychotherapeutic environment provided safety by offering spatial and temporal structures and opportunities for social interaction, supporting people to find new ways to be in a changed world. CONCLUSIONS: Our study shows that despite the threat of infection, it is vital to continue to provide people with psychological vulnerabilities with a safe therapeutic space in which to regain a sense of safety in a changed world. This is particularly important, as those people seem to suffer intensely from the collateral measures of a pandemic.

Antisense Oligonucleotide in LNA-Gapmer Design Targeting TGFBR2-A Key Single Gene Target for Safe and Effective Inhibition of TGFß Signaling.

Antisense Oligonucleotides (ASOs) are an emerging drug class in gene modification. In our study we developed a safe, stable, and effective ASO drug candidate in locked nucleic acid (LNA)-gapmer design, targeting TGFß receptor II (TGFBR2) mRNA. Discovery was performed as a process using state-of-the-art library development and screening. We intended to identify a drug candidate optimized for clinical development, therefore human specificity and gymnotic delivery were favored by design. A staggered process was implemented spanning in-silico-design, in-vitro transfection, and in-vitro gymnotic delivery of small batch syntheses. Primary in-vitro and in-vivo toxicity studies and modification of pre-lead candidates were also part of this selection process. The resulting lead compound NVP-13 unites human specificity and highest efficacy with lowest toxicity. We particularly focused at attenuation of TGFß signaling, addressing both safety and efficacy. Hence, developing a treatment to potentially recondition numerous pathological processes mediated by elevated TGFß signaling, we have chosen to create our data in human lung cell lines and human neuronal stem cell lines, each representative for prospective drug developments in pulmonary fibrosis and neurodegeneration. We show that TGFBR2 mRNA as a single gene target for NVP-13 responds well, and that it bears great potential to be safe and efficient in TGFß signaling related disorders.

Effects of continuous high-dose G-CSF administration on hematopoietic stem cell mobilization and telomere length in patients with amyotrophic lateral sclerosis - a pilot study.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of complex and still poorly understood etiology. Loss of upper and lower motoneurons results in death within few years after diagnosis. Recent studies have proposed neuroprotective and disease-slowing effects of granulocyte-colony stimulating factor (G-CSF) treatment in ALS mouse models as well as humans. In this study, six ALS patients were monitored up to 3.5 years during continuous high-dose G-CSF administration. Repetitive analyses were performed including blood count parameters, CD34+ hematopoietic stem and progenitor cell (HSPC) and colony forming cell (CFC) counts, serum cytokine levels and leukocyte telomere length. We demonstrate that continuous G-CSF therapy was well tolerated and safe resulting in only mild adverse events during the observation period. However, no mobilization of CD34+ HSPC was detected as compared to baseline values. CFC mobilization was equally low and even a decrease of myeloid precursors was observed in some patients. Assessment of telomere length within ALS patients' leukocytes revealed that G-CSF did not significantly shorten telomeres, while those of ALS patients were shorter compared to age-matched healthy controls, irrespective of G-CSF treatment. During G-CSF stimulation, TNF-alpha, CRP, IL-16, sVCAM-1, sICAM-1, Tie-2 and VEGF were significantly increased in serum whereas MCP-1 levels decreased. In conclusion, our data show that continuous G-CSF treatment fails to increase circulating CD34+ HSPC in ALS patients. Cytokine profiles revealed G-CSF-mediated immunomodulatory and proteolytic effects. Interestingly, despite intense G-CSF stimulation, telomere length was not significantly shortened.

Biomarker Supervised G-CSF (Filgrastim) Response in ALS Patients.

Objective: To evaluate safety, tolerability and feasibility of long-term treatment with Granulocyte-colony stimulating factor (G-CSF), a well-known hematopoietic stem cell factor, guided by assessment of mobilized bone marrow derived stem cells and cytokines in the serum of patients with amyotrophic lateral sclerosis (ALS) treated on a named patient basis. Methods: 36 ALS patients were treated with subcutaneous injections of G-CSF on a named patient basis and in an outpatient setting. Drug was dosed by individual application schemes (mean 464 Mio IU/month, range 90-2160 Mio IU/month) over a median of 13.7 months (range from 2.7 to 73.8 months). Safety, tolerability, survival and change in ALSFRS-R were observed. Hematopoietic stem cells were monitored by flow cytometry analysis of circulating CD34+ and CD34+CD38- cells, and peripheral cytokines were assessed by electrochemoluminescence throughout the intervention period. Analysis of immunological and hematological markers was conducted. Results: Long term and individually adapted treatment with G-CSF was well tolerated and safe. G-CSF led to a significant mobilization of hematopoietic stem cells into the peripheral blood. Higher mobilization capacity was associated with prolonged survival. Initial levels of serum cytokines, such as MDC, TNF-beta, IL-7, IL-16, and Tie-2 were significantly associated with survival. Continued application of G-CSF led to persistent alterations in serum cytokines and ongoing measurements revealed the multifaceted effects of G-CSF. Conclusions: G-CSF treatment is feasible and safe for ALS patients. It may exert its beneficial effects through neuroprotective and -regenerative activities, mobilization of hematopoietic stem cells and regulation of pro- and anti-inflammatory cytokines as well as angiogenic factors. These cytokines may serve as prognostic markers when measured at the time of diagnosis. Hematopoietic stem cell numbers and cytokine levels are altered by ongoing G-CSF application and may potentially serve as treatment biomarkers for early monitoring of G-CSF treatment efficacy in ALS in future clinical trials.

Metformin inhibits proliferation and migration of glioblastoma cells independently of TGF-ß2.

To this day, glioblastoma (GBM) remains an incurable brain tumor. Previous research has shown that metformin, an oral anti-diabetic drug, may decrease GBM cell proliferation and migration especially in brain tumor initiating cells (BTICs). As transforming growth factor ß 2 (TGF-ß2) has been reported to promote high-grade glioma and is inhibited by metformin in other tumors, we explored whether metformin directly interferes with TGF-ß2-signaling. Functional investigation of proliferation and migration of primary BTICs after treatment with metformin+/-TGF-ß2 revealed that metformin doses as low as 0.01 mM metformin thrice a day were able to inhibit proliferation of susceptible cell lines, whereas migration was impacted only at higher doses. Known cellular mechanisms of metformin, such as increased lactate secretion, reduced oxygen consumption and activated AMPK-signaling, could be confirmed. However, TGF-ß2 and metformin did not act as functional antagonists, but both rather inhibited proliferation and/or migration, if significant effects were present. We did not observe a relevant influence of metformin on TGF-ß2 mRNA expression (qRT-PCR), TGF-ß2 protein expression (ELISA) or SMAD-signaling (Western blot). Therefore, it seems that metformin does not exert its inhibitory effects on GBM BTIC proliferation and migration by altering TGF-ß2-signaling. Nonetheless, as low doses of metformin are able to reduce proliferation of certain GBM cells, further exploration of predictors of BTICs' susceptibility to metformin appears justified.