A cautionary note: Toxicity of polyethylene glycol 200 injected intraperitoneally into mice

The parenteral administration of hydrophobic substances in vivo requires the use of organic solvents to ensure sufficient solubility and avoid precipitation. Dimethyl sulfoxide is commonly used for this purpose. Based on the common assumption that polyethylene glycol (PEG) is non-toxic, our local regulatory authorities recently recommended the use of PEG instead. However, mice injected intraperitoneally (i.p.) with PEG 200 at a dose of 8 mL/kg (i.e. 9 g/kg) did not tolerate PEG 200 well, and half of the animals had to be euthanized. Our results demonstrate that although PEG 200 is generally considered to be harmless, it can be toxic when injected i.p. and is painful for the recipient mice. Nevertheless, it can be used as a solvent for repeated i.p. injections in mice at a dose of 2 mL/kg (i.e. 2.25 g/kg) without obvious signs of systemic toxicity

LiCl induces TNF-α and FasL production, thereby stimulating apoptosis in cancer cells

<p>Abstract</p> <p>Background</p> <p>The incidence of cancer in patients with neurological diseases, who have been treated with LiCl, is below average. LiCl is a well-established inhibitor of Glycogen synthase kinase-3, a kinase that controls several cellular processes, among which is the degradation of the tumour suppressor protein p53. We therefore wondered whether LiCl induces p53-dependent cell death in cancer cell lines and experimental tumours.</p> <p>Results</p> <p>Here we show that LiCl induces apoptosis of tumour cells both <it>in vitro </it>and <it>in vivo</it>. Cell death was accompanied by cleavage of PARP and Caspases-3, -8 and -10. LiCl-induced cell death was not dependent on p53, but was augmented by its presence. Treatment of tumour cells with LiCl strongly increased TNF-α and FasL expression. Inhibition of TNF-α induction using siRNA or inhibition of FasL binding to its receptor by the Nok-1 antibody potently reduced LiCl-dependent cleavage of Caspase-3 and increased cell survival. Treatment of xenografted rats with LiCl strongly reduced tumour growth.</p> <p>Conclusions</p> <p>Induction of cell death by LiCl supports the notion that GSK-3 may represent a promising target for cancer therapy. LiCl-induced cell death is largely independent of p53 and mediated by the release of TNF-α and FasL.</p> <p>Key words: LiCl, TNF-α, FasL, apoptosis, GSK-3, FasL</p

Increased Circulating Osteopontin Levels Promote Primary Tumour Growth, but Do Not Induce Metastasis in Melanoma

Osteopontin (OPN) is a phosphoprotein with diverse functions in various physiological and pathological processes. OPN expression is increased in multiple cancers, and OPN within tumour tissue has been shown to promote key stages of cancer development. OPN levels are also elevated in the circulation of cancer patients, which in some cases has been correlated with enhanced metastatic propensity and poor prognosis. However, the precise impact of circulating OPN (cOPN) on tumour growth and progression remains insufficiently understood. To examine the role of cOPN, we used a melanoma model, in which we stably increased the levels of cOPN through adeno-associated virus-mediated transduction. We found that increased cOPN promoted the growth of primary tumours, but did not significantly alter the spontaneous metastasis of melanoma cells to the lymph nodes or lungs, despite an increase in the expression of multiple factors linked to tumour progression. To assess whether cOPN has a role at later stages of metastasis formation, we employed an experimental metastasis model, but again could not detect any increase in pulmonary metastasis in animals with elevated levels of cOPN. These results demonstrate that increased levels of OPN in the circulation play distinct roles during different stages of melanoma progression

Ketogenic diet does not promote triple-negative and luminal mammary tumor growth and metastasis in experimental mice

Vol.:(0123456789)1 3Clinical & Experimental Metastasis https://doi.org/10.1007/s10585-023-10249-z RESEARCH PAPER Ketogenic diet does not promote triple-negative and luminal mammary tumor growth and metastasis in experimental mice Meret Grube1 · Arno Dimmler2 · Anja Schmaus1 · Rafael Saup1 · Tabea Wagner 1 · Boyan K. Garvalov 1 · Jonathan P. Sleeman1,3 · Wilko Thiele 1 Received: 17 October 2023 / Accepted: 21 November 2023 © The Author(s) 2023 Abstract Ketogenic diets (KDs) can improve the well-being and quality of life of breast cancer patients. However, data on the effects of KDs on mammary tumors are inconclusive, and the influence of KDs on metastasis in general remains to be investigated. We therefore assessed the impact of a KD on growth and metastasis of triple negative murine 4T1 mammary tumors, and on the progression of luminal breast tumors in an autochthonous MMTV-PyMT mouse model. We found that KD did not influence the metastasis of 4T1 and MMTV-PyMT mammary tumors, but impaired 4T1 tumor cell proliferation in vivo, and also temporarily reduced 4T1 primary tumor growth. Notably, the ketogenic ratio (the mass of dietary fat in relation to the mass of dietary carbohydrates and protein) that is needed to induce robust ketosis was twice as high in mice as compared to humans. Surprisingly, only female but not male mice responded to KD with a sustained increase in blood β-hydroxybutyrate levels. Together, our data show that ketosis does not foster primary tumor growth and metastasis, suggesting that KDs can be safely applied in the context of luminal breast cancer, and may even be advantageous for patients with triple negative tumors. Furthermore, our data indicate that when performing experiments with KDs in mice, the ketogenic ratio needed to induce ketosis must be verified, and the sex of the mice should also be taken into account

Hyaluronic acid-CD44 interactions promote BMP4/7-dependent Id1/3 expression in melanoma cells

Abstract BMP4/7-dependent expression of inhibitor of differentiation/DNA binding (Id) proteins 1 and 3 has been implicated in tumor progression and poor prognosis of malignant melanoma patients. Hyaluronic acid (HA), a pericellular matrix component, supports BMP7 signalling in murine chondrocytes through its receptor CD44. However, its role in regulating BMP signalling in melanoma is not clear. In this study we found that depletion of endogenously-produced HA by hyaluronidase treatment or by inhibition of HA synthesis by 4-methylumbelliferone (4-MU) resulted in reduced BMP4/7-dependent Id1/3 protein expression in mouse melanoma B16-F10 and Ret cells. Conversely, exogenous HA treatment increased BMP4/7-dependent Id1/3 protein expression. Knockdown of CD44 reduced BMP4/7-dependent Id1/3 protein expression, and attenuated the ability of exogenous HA to stimulate Id1 and Id3 expression in response to BMP. Co-IP experiments demonstrated that CD44 can physically associate with the BMP type II receptor (BMPR) ACVR2B. Importantly, we found that coordinate expression of Id1 or Id3 with HA synthases HAS2, HAS3, and CD44 is associated with reduced overall survival of cutaneous melanoma patients. Our results suggest that HA-CD44 interactions with BMPR promote BMP4/7-dependent Id1/3 protein expression in melanoma, contributing to reduced survival in melanoma patients

VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis

Introduction VEGFR-3 is a member of the VEGFR receptor tyrosine kinase family. It is expressed on lymphatic endothelial cells (LECs) and plays a central role in the regulation of lymphangiogenesis. 1 On binding to its ligands, VEGF-C and VEGF-D, VEGFR-3 is activated and orchestrates the outgrowth of lymphatic vessels. During murine hematopoiesis, Sca-1 ϩ hematopoietic stem cells give rise to the precursors of all hematopoietic lineages. 10 Megakaryocytes develop from CD34 ϩ progenitors. Methods Cell culture HEL cells were obtained from DSMZ and cultivated in RPMI (Gibco-BRL) containing 10% FCS and 1% penicillin-streptomycin. Differentiation was induced with 10nM tetradecanoyl phorbol acetate (TPA; Sigma-Aldrich). Primary human microvascular LECs (Cambrex) from the dermis (HMVECdLyNeo) were cultivated in EGM-2MV (Lonza) and 5% FCS supplemented with growth factors provided by the manufacturer. Bovine lymphatic endothelial cells were cultivated in DMEM (Gibco-BRL) containing 20% FCS and 1% penicillin-streptomycin on gelatin-coated plastic. HEK-293 cells were cultivated in DMEM supplemented with 10% FCS and 1% penicillin-streptomycin. Western blot analysis Cell lysates were analyzed using standard Western blotting techniques. The membranes were probed with Abs specific for VEGFR-3 (R&amp;D Systems), CD31 (Santa Cruz Biotechnology), CD34 (Abcam), CD42a (Santa Cruz Biotechnology), CD61 (R&amp;D Systems), CD144 (Santa Cruz Biotechnology), or GpA (International Blood Group Reference Laboratory). Probing with hypoxanthine phosphoribosyltransferase (HPRT) Abs (Santa Cruz Biotechnology) served as a loading control. PCR analysis RNA was prepared using peqGOLD RNAPure (PeqLab). Synthesis of cDNA using Superscript II (Invitrogen) was performed according to the manufacturer&apos;s recommendations. For PCR, cDNAs were amplified as follows: 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 90 seconds (VEGFR-2, Prox1, LYVE-1, Podoplanin, HPRT, Fli-1, Fog-2, Gata-2, and Elf-1) or 94°C for 30 seconds, 54°C for 30 seconds, and 72°C for 90 seconds (VEGFR-3). Details of the primers used are in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Tubule formation on collagen gels Collagen type 1 was prepared from rat tails. Tendons were isolated, dissolved in acetic acid, then filtered, lyophilized, and redissolved in 0.1% acetic acid at 4 mg/mL. Cells were seeded on collagen gels (2 mg/mL) and cultured in the presence of 30 ng/mL of VEGF 165 (Promokine) for 8 days. Tubule formation was analyzed as described previously. 22 Immunohistochemistry For the immunohistochemical analysis of VEGFR-3 expression in the BM, cryosections of decalcified murine femurs embedded in tissue-freezing medium (Leica) were fixed in acetone and stained with VEGFR-3 Abs (eBiosciences). The stained sections were then analysed at room temperature using an Axioskop (Zeiss) equipped with a PlanNeoflur 20ϫ/0.50 and an Axiocam (Zeiss) and Axiovision software (Ziess). MACS BM cells isolated from femurs and tibias of C57BL/6 mice were treated with Fc-block (BD Biosciences) and then incubated with Abs against VEGFR-3 (R&amp;D Systems), Sca-1, CD41, or CD38 (BD Biosciences), followed by specific secondary MACS Abs (Miltenyi-Biotec) according to the manufacturerЈs recommendations. Cell populations were then either enriched or depleted for the labeled epitope using LS or LD columns (Miltenyi-Biotec), respectively. The purity of the sorted populations was controlled by flow cytometry. CD42 FACS BM was isolated from femurs and tibias of C57BL/6 mice and stained with Abs specific for VEGFR-3 (R&amp;D Systems) and/or CD42a (Emfret) and analyzed by FACS. Lethal irradiation and BM transplantation C57BL/6 mice were irradiated with lethal doses (9 Gy) from a ␥ source. After 24 hours, the mice were all transplanted in parallel by IV injection with either complete BM, BM depleted of VEGFR-3 ϩ cells, or BM mock depleted with an appropriate isotype control using MACS. EDTA blood samples were taken from all animals on days 0, Isolation and culture of primary murine BM cells BM was isolated from femurs and tibias of C57BL6 mice. After lysis of RBCs with ammonium-chloride-potassium buffer, the cells were transferred to IMDM (Gibco-BRL) supplemented with 1% penicillin/streptomycin, 10% HEK-293 cell-conditioned DMEM, Nutridoma SP (Roche), L-glutamine, and 100 pg/mL of recombinant murine TPO (RDI Diagnostics). Depending on the experiment, the cells were cultured with either 100 g/mL of mF4-31C1 VEGFR-3-blocking Abs (kindly provided by ImClone Systems), 100 g/mL of rat IgG isotype control, or 400 ng/mL of VEGF-C-Cys, a mutant form of VEGF-C that activates VEGFR-3 but not VEGFR-2. Long-term injections C57BL/6 mice were injected daily with 25 g of VEGF-C-Cys for 3 weeks. Blood was taken on days 0, 3, 7, 10, 14, 17, and 21. In the blocking Ab experiments, mice were injected with 600 g/animal/injection of mF4-31C1 VEGFR-3-blocking Ab, isotype control Ig, or PBS on a MondayWednesday-Friday schedule for 6 weeks. Blood was taken on days 0, Recovery kinetics after sublethal irradiation Experimental C57BL/6 mice were sublethally irradiated (4.5 Gy) in a ␥ source. They were then either injected daily with VEGF-C-Cys (25 g/animal/injection) or PBS or were intraperitoneally injected with 600 g/animal/injection of mF4-31C1 VEGFR-3-blocking Abs, isotype control Ig, or PBS every other day. Blood was taken on days 0, 7, 11, 14, 18, and 21 after irradiation and analyzed. In each experiment, all animals were treated at the same time and on the same day and all animals were bled at each time point. BM was isolated from femurs and tibias 20 days after irradiation, and the number and ploidy of CD41 ϩ cells in the BM was assessed. Significance was tested using 2-tailed unpaired t tests assuming equal variance. TPO administration C57BL/6 mice were administered with 5 g of recombinant murine TPO (RDI), followed by daily injections of either 25 g of VEGF-C-Cys or PBS. One group received only PBS throughout. Blood was taken and analyzed 0, 3, 5, 7, and 10 days after TPO administration. All animals were treated at the same time and on the same day and all animals were bled at each time point. After 10 days, the animals were killed and the number and ploidy of CD41 ϩ 1900 THIELE et al BLOOD, 30 AUGUST 2012 ⅐ VOLUME 120, NUMBER 9 For personal use only. on October 6, 2016. by guest www.bloodjournal.org From cells in the BM was assessed. Significance was tested using 2-tailed unpaired t tests assuming equal variance. 5-FU treatment C57BL/6 mice were intraperitoneally injected with a single dose of 5-FU (Sigma-Aldrich) at 150 mg/kg. Control mice remained untreated. The 5-FU-treated mice then received daily injections of either 25 g of VEGF-C-Cys or PBS throughout the experiment. Blood was taken and analyzed 0, All animal experiments were approved by the local regulatory authorities and were performed according to German legal requirements. Results Expression of VEGFR-3 and other lymphatic endothelial markers is up-regulated on phorbol diester-induced megakaryocytic differentiation of HEL cells VEGFR-3 is widely used as a marker for lymphatic endothelium. Originally, however, the receptor was cloned from the HEL cell line. 7 This cell line can be induced to differentiate into the erythrocyte lineage by EPO treatment 23 and into the megakaryocyte lineage in response to TPA. Consistent with the notion that HEL cells differentiate into the megakaryocyte lineage on TPA treatment, we detected strong up-regulation of several markers and transcription factors associated with megakaryocytic differentiation A survey of the literature revealed that virtually all markers described to date as being expressed on megakaryocytes can also be expressed on endothelial cells (supplemental These observations raised the question of whether HEL cells really undergo megakaryocytic differentiation after TPA treatment or if they adopt an endothelial phenotype with LEC characteristics. To address this point, we investigated whether TPA-treated HEL cells are capable of forming capillaries, reasoning that if the cells differentiated into endothelial cells, this should be the case. However, in contrast to control bovine LECs, TPA-treated HEL cells could not be induced to form capillaries VEGFR-3 IN MEGAKARYOPOIESIS 1901 BLOOD, 30 AUGUST 2012 ⅐ VOLUME 120, NUMBER 9 For personal use only. on October 6, 2016. by guest www.bloodjournal.org From VEGFR-3 is expressed on megakaryocytic progenitors through to the promegakaryoblast stage in the BM The up-regulation of VEGFR-3 during HEL cell megakaryocytic differentiation suggested to us that VEGFR-3 may play a role in megakaryopoiesis. Because of the limited megakaryocytic differentiation capacity of HEL cells and their cancerous nature, we explored this possibility further using murine BM. First we characterized VEGFR-3 expression in the BM. FACS staining revealed that approximately 2% of murine BM cells were VEGFR-3 ϩ ( To define further the stages of megakaryopoiesis during which VEGFR-3 is expressed, costainings with the stem cell marker Sca-1 and with CD38, CD41, and VEGFR-3 were performed. Expression of Sca-1 is lost during myeloid differentiation. 25 CD38 expression, in turn, is increased early in megakaryopoiesis from the BFU-MK stage on. These observations suggested to us that VEGFR-3 might be expressed on hematopoietic stem cells through to the promegakaryoblast stage. However, Sca1 is not just expressed on hematopoietic stem cells, but also on the immediate progenitors arising from the stem cells. These data are consistent with the notion that VEGFR-3 is not expressed on hematopoietic stem cells, but rather on megakaryocyte precursors through to the premegakaryoblast stage, and that VEGFR-3 expression is lost as megakaryocytes further mature. This notion is further substantiated by the observation that VEGFR-3 ϩ BM cells coexpressed CD42, a marker for megakaryocytes that is not expressed on hematopoietic precursor cells (supplemental Manipulation of VEGFR-3 influences megakaryopoiesis in vitro To examine the role that VEGFR-3 plays during megakaryopoiesis, we cultivated primary murine BM cells with physiologic concentrations of TPO to maintain the megakaryocyte precursors. The cells were grown for 3 days in the presence or absence of VEGF-C-Cys, a mutant form of VEGF-C that specifically activates VEGFR-3 but not VEGFR-2, 20 because VEGFR-2 is also present on megakaryocytic cells. Our data suggest that the specific activation of VEGFR-3 during megakaryopoiesis impairs the transition to polyploid stages, whereas blocking the receptor promotes differentiation and endoreplication. For personal use only. on October 6, 2016. by guest www.bloodjournal.org From Neither activation nor blocking of VEGFR-3 influences steady-state megakaryopoiesis or thrombopoiesis in vivo To study the potential effects of VEGFR-3 manipulation on megakaryopoiesis and thrombopoiesis in vivo, we first injected VEGF-C-Cys to activate VEGFR-3, or PBS as a control, into mice on a daily basis for 3 weeks. Thrombocyte concentrations in the blood were monitored regularly. After 3 weeks of treatment, the mice were killed. BM cells were isolated and stained for CD41 and DNA content to evaluate the number and ploidy of the CD41 ϩ population. We observed a significant decrease in apoptotic CD41 ϩ BM cells in the VEGF-C-Cys-treated group (P Ͻ .01), a trend toward reduced polyploidy, and an increase in 2n CD41 ϩ cells, which were consistent with our in vitro observations. VEGF-C-Cys had no effect on platelet counts or the number of CD41 ϩ cells in the BM (supplemental To determine the effect of inhibiting VEGFR-3 activation on megakaryopoiesis and thrombopoiesis in vivo, mice were injected daily with VEGFR-3-blocking Abs or an appropriate isotype control for 6 weeks. Platelet counts were monitored regularly and the numbers and ploidy distribution of CD41 ϩ BM cells were analyzed at the end of the experiment. Under these conditions, no effects on the measured parameters were observed (supplemental Activation of VEGFR-3 increases platelet counts in TPO-stimulated animals, modulates 5-FU-induced thrombocytopenia and thrombocytosis, and influences ploidy distribution and numbers of CD41 ؉ BM cells after sublethal irradiation Thrombocyte homeostasis is tightly controlled in mammals, and alternative mechanisms exist that can compensate for perturbation . FACS analysis showed that 1.85% Ϯ 0.31% SEM (n ϭ 9) of the murine BM cells expressed VEGFR-3. Dot plots of 1 representative experiment are depicted. Density plots were used to define a region in which 95% (the 2 outer contours) of the negative control events were excluded. The region was then applied to a plot displaying the stained sample. The number of positive events in both the negative control and the actual sample was then assessed. The percentage of true positive cells was calculated by subtraction of the number of events in the negative control within the defined region from the number of events found in the same region for the actual sample. Identical numbers of events were acquired. (B) VEGFR-3 is expressed on isolated mononuclear cells in the murine BM. Sections of murine femurs were stained with VEGFR-3-specific Abs (left panel, VEGFR-3; right panel, control). MK indicates megakaryocyte. Scale bars indicate 100 m. (C) Ploidy of VEGFR-3 ϩ cells in the murine BM. VEGFR-3 ϩ BM cells were enriched by MACS and then analyzed in FACS. As a control, cells were treated with an appropriate isotype control. Clumping cells mimicking polyploidy were excluded from the analysis by appropriate gating strategies. The resulting histogram plot shows the DNA content of VEGFR-3 ϩ cells. Dot plots of the DNA content of the cells were used for the quantification of VEGFR-3 ϩ and isotype-treated cells within different ploidy classes or cell cycle stages, respectively (a detailed scheme of the gating strategy is provided in supplementa