Effects of ionizing radiation in combination with Erufosine on T98G glioblastoma xenograft tumours: a study in NMRI nu/nu mice

BACKGROUND: Erufosine is a promising anticancer drug that increases the efficacy of radiotherapy in glioblastoma cell lines in vitro. Moreover, treatment of nude mice with repeated intraperitoneal or subcutaneous injections of Erufosine is well tolerated and yields drug concentrations in the brain tissue that are higher than the concentrations required for cytotoxic drug effects on glioblastoma cell lines in vitro. METHODS: In the present study we aimed to evaluate the effects of a combined treatment with radiotherapy and Erufosine on growth and local control of T98G subcutaneous glioblastoma xenograft-tumours in NMRI nu/nu mice. RESULTS: We show that repeated intraperitoneal injections of Erufosine resulted in a significant drug accumulation in T98G xenograft tumours on NMRI nu/nu mice. Moreover, short-term treatment with 5 intraperitoneal Erufosine injections caused a transient decrease in the growth of T98G tumours without radiotherapy. Furthermore, an increased radiation-induced growth delay of T98G xenograft tumours was observed when fractionated irradiation was combined with short-term Erufosine-treatment. However, no beneficial drug effects on fractionated radiotherapy in terms of local tumour control were observed. CONCLUSIONS: We conclude that short-term treatment with Erufosine is not sufficient to significantly improve local control in combination with radiotherapy in T98G glioblastoma xenograft tumours. Further studies are needed to evaluate efficacy of extended drug treatment schedules

Targeting the Immunomodulatory CD73/Adenosine System to Improve the Therapeutic Gain of Radiotherapy

Extracellular adenosine is a potent endogenous immunosuppressive mediator critical to the maintenance of homeostasis in various normal tissues including the lung. Adenosine is either released from stressed or injured cells or generated from extracellular adenine nucleotides by the concerted action of the ectoenzymes ectoapyrase (CD39) and 5′ ectonucleotidase (CD73) that catabolize ATP to adenosine. An acute CD73-dependent increase of adenosine in normal tissues mostly exerts tissue protective functions whereas chronically increased adenosine-levels in tissues exposed to DNA damaging chemotherapy or radiotherapy promote pathologic remodeling processes and fibrosis for example in the skin and the lung. Importantly, cancer cells also express CD73 and high CD73 expression in the tumor tissue has been linked to poor overall survival and recurrence free survival in patients suffering from breast and ovarian cancer. CD73 and adenosine support growth-promoting neovascularization, metastasis, and survival in cancer cells. In addition, adenosine can promote tumor intrinsic or therapy-induced immune escape by various mechanisms that dampen the immune system. Consequently, modulating CD73 or cancer-derived adenosine in the tumor microenvironment emerges as an attractive novel therapeutic strategy to limit tumor progression, improve antitumor immune responses, avoid therapy-induced immune deviation, and potentially limit normal tissue toxicity. However, the role of CD73/adenosine signaling in the tumor and normal tissue responses to radiotherapy and its use as therapeutic target to improve the outcome of radiotherapy approaches is less understood. The present review will highlight the dual role of CD73 and adenosine in tumor and tissue responses to radiotherapy with a special focus to the lung. It will also discuss the potential benefits and risks of pharmacologic modulation of the CD73/adenosine system to increase the therapeutic gain of radiotherapy or combined radioimmunotherapy in cancer treatment

Nestin(+) Tissue-Resident Multipotent Stem Cells Contribute to Tumor Progression by Differentiating into Pericytes and Smooth Muscle Cells Resulting in Blood Vessel Remodeling

Tumor vessels with resistance to anti-angiogenic therapy are characterized by the normalization of the vascular structures through integration of mature pericytes and smooth muscle cells (SMC) into the vessel wall, a process termed vessel stabilization. Unfortunately, stabilization-associated vascular remodeling can result in reduced sensitivity to subsequent anti-angiogenic therapy. We show here that blockade of VEGF by bevacizumab induces stabilization of angiogenic tumor blood vessels in human tumor specimen by recruiting Nestin-positive cells, whereas mature vessels down-regulated Nestin-expression. Using xenograft tumors growing on bone-marrow (BM) chimera of C57Bl/6 wildtype and Nestin-GFP transgenic mice, we show for first time that Nestin(+) cells inducing the maturation of tumor vessels do not originate from the BM but presumably reside within the adventitia of adult blood vessels. Complementary ex vivo experiments using explants of murine aortas revealed that Nestin(+) multipotent stem cells (MPSCs) are mobilized from their niche and differentiated into pericytes and SMC through the influence of tumor-cell-secreted factors. We conclude that tissue-resident Nestin(+) cells are more relevant than BM-derived cells for vessel stabilization and therefore have to be considered in future strategies for anti-angiogenic therapy. The identification of proteins mediating recruitment or differentiation of local Nestin(+) cells with potential stem cell character to angiogenic blood vessels may allow the definition of new therapeutic targets to reduce tumor resistance against anti-angiogenic drugs

The Akt-inhibitor Erufosine induces apoptotic cell death in prostate cancer cells and increases the short term effects of ionizing radiation

<p>Abstract</p> <p>Background and Purpose</p> <p>The phosphatidylinositol-3-kinase (PI3K)/Akt pathway is frequently deregulated in prostate cancer and associated with neoplastic transformation, malignant progression, and enhanced resistance to classical chemotherapy and radiotherapy. Thus, it is a promising target for therapeutic intervention. In the present study, the cytotoxic action of the Akt inhibitor Erufosine (ErPC3) was analyzed in prostate cancer cells and compared to the cytotoxicity of the PI3K inhibitor LY294002. Moreover, the efficacy of combined treatment with Akt inhibitors and ionizing radiation in prostate cancer cells was examined.</p> <p>Materials and methods</p> <p>Prostate cancer cell lines PC3, DU145, and LNCaP were treated with ErPC3 (1-100 µM), LY294002 (25-100 µM), irradiated (0-10 Gy), or subjected to combined treatments. Cell viability was determined by the WST-1 assay. Apoptosis induction was analyzed by flow cytometry after staining with propidium iodide in a hypotonic citrate buffer, and by Western blotting using antibodies against caspase-3 and its substrate PARP. Akt activity and regulation of the expression of Bcl-2 family members and key downstream effectors involved in apoptosis regulation were examined by Western blot analysis.</p> <p>Results</p> <p>The Akt inhibitor ErPC3 exerted anti-neoplastic effects in prostate cancer cells, however with different potency. The anti-neoplastic action of ErPC3 was associated with reduced phosphoserine 473-Akt levels and induction of apoptosis. PC3 and LNCaP prostate cancer cells were also sensitive to treatment with the PI3K inhibitor LY294002. However, the ErPC3-sensitive PC3-cells were less susceptible to LY294002 than the ErPC3-refractory LNCaP cells. Although both cell lines were largely resistant to radiation-induced apoptosis, both cell lines showed higher levels of apoptotic cell death when ErPC3 was combined with radiotherapy.</p> <p>Conclusions</p> <p>Our data suggest that constitutive Akt activation and survival are controlled by different different molecular mechanisms in the two prostate cancer cell lines - one which is sensitive to the Akt-inhibitor ErPC3 and one which is more sensitive to the PI3K-inhibitor LY294002. Our findings underline the importance for the definition of predictive biomarkers that allow the selection patients that may benefit from the treatment with a specific signal transduction modifier.</p

The membrane targeted apoptosis modulators erucylphosphocholine and erucylphosphohomocholine increase the radiation response of human glioblastoma cell lines in vitro

Background: Alkylphosphocholines constitute a novel class of antineoplastic synthetic phospholipid derivatives that induce apoptosis of human tumor cell lines by targeting cellular membranes. We could recently show that the first intravenously applicable alkylphosphocholine erucylphosphocholine (ErPC) is a potent inducer of apoptosis in highly resistant human astrocytoma/glioblastoma cell lines in vitro. ErPC was shown to cross the blood brain barrier upon repeated intravenous injections in rats and thus constitutes a promising candidate for glioblastoma therapy. Aim of the present study was to analyze putative beneficial effects of ErPC and its clinically more advanced derivative erucylphosphohomocholine (erucyl-N, N, N-trimethylpropanolaminphosphate, ErPC3, Erufosine (TM) on radiation-induced apoptosis and eradication of clonogenic tumor cells in human astrocytoma/glioblastoma cell lines in vitro. Results: While all cell lines showed high intrinsic resistance against radiation-induced apoptosis as determined by fluorescence microscopy, treatment with ErPC and ErPC3 strongly increased sensitivity of the cells to radiation-induced cell death (apoptosis and necrosis). T98G cells were most responsive to the combined treatment revealing highly synergistic effects while A172 showed mostly additive to synergistic effects, and U87MG cells sub-additive, additive or synergistic effects, depending on the respective radiation-dose, drug-concentration and treatment time. Combined treatment enhanced therapy-induced damage of the mitochondria and caspase-activation. Importantly, combined treatment also increased radiation-induced eradication of clonogenic T98G cells as determined by standard colony formation assays. Conclusion: Our observations make the combined treatment with ionizing radiation and the membrane targeted apoptosis modulators ErPC and ErPC3 a promising approach for the treatment of patients suffering from malignant glioma. The use of this innovative treatment concept in an in vivo xenograft setting is under current investigation

Pharmacokinetics and biodistribution of Erufosine in nude mice - implications for combination with radiotherapy

<p>Abstract</p> <p>Background</p> <p>Alkylphosphocholines represent promising antineoplastic drugs that induce cell death in tumor cells by primary interaction with the cell membrane. Recently we could show that a combination of radiotherapy with Erufosine, a paradigmatic intravenously applicable alkylphosphocholine, <it>in vitro </it>leads to a clear increase of irradiation-induced cell death. In view of a possible combination of Erufosine and radiotherapy <it>in vivo </it>we determined the pharmacokinetics and bioavailability as well as the tolerability of Erufosine in nude mice.</p> <p>Methods</p> <p>NMRI (nu/nu) nude mice were treated by intraperitoneal or subcutaneous injections of 5 to 40 mg/kg body weight Erufosine every 48 h for one to three weeks. Erufosine-concentrations were measured in brain, lungs, liver, small intestine, colon, spleen, kidney, stomach, adipoid tissue, and muscle by tandem-mass spectroscopy. Weight course, blood cell count and clinical chemistry were analyzed to evaluate general toxicity.</p> <p>Results</p> <p>Intraperitoneal injections were generally well tolerated in all dose groups but led to a transient loss of the bodyweight (<10%) in a dose dependent manner. Subcutaneous injections of high-dose Erufosine caused local reactions at the injection site. Therefore, this regimen at 40 mg/kg body weight Erufosine was stopped after 14 days. No gross changes were observed in organ weight, clinical chemistry and white blood cell count in treated compared to untreated controls except for a moderate increase in lactate dehydrogenase and aspartate-aminotransferase after intensive treatment. Repeated Erufosine injections resulted in drug-accumulation in different organs with maximum concentrations of about 1000 nmol/g in spleen, kidney and lungs.</p> <p>Conclusion</p> <p>Erufosine was well tolerated and organ-concentrations surpassed the cytotoxic drug concentrations <it>in vitro</it>. Our investigations establish the basis for a future efficacy testing of Erufosine in xenograft tumor models in nude mice alone and in combination with chemo- or radiotherapy.</p

CD47 restricts antiviral function of alveolar macrophages during influenza virus infection

CD47 is an ubiquitously expressed surface molecule with significant impact on immune responses. However, its role for antiviral immunity is not fully understood. Here, we revealed that the expression of CD47 on immune cells seemed to disturb the antiviral immune response as CD47-deficient mice (CD47−/−) showed an augmented clearance of influenza A virus (IAV). Specifically, we have shown that enhanced viral clearance is mediated by alveolar macrophages (aMФ). Although aMФ displayed upregulation of CD47 expression during IAV infection in wildtype mice, depletion of aMФ in CD47−/− mice during IAV infection reversed the augmented viral clearance. We have also demonstrated that CD47 restricts hemoglobin (HB) expression in aMФ after IAV and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, with HB showing antiviral properties by enhancing the IFN-β response. Our study showed a negative role for CD47 during antiviral immune responses in the lung by confining HB expression in aMФ