36 research outputs found
Calcium Electroporation Reduces Viability and Proliferation Capacity of Four Uveal Melanoma Cell Lines in 2D and 3D Cultures
Electrochemotherapy (ECT) is the combination of transient pore formation following electric
pulse application with the administration of cytotoxic drugs, which enhances the cytotoxic effect
of the applied agent due to membrane changes and permeabilization. Although EP represents an
established therapeutic option for solid malignancies, recent advances shift to the investigation of non cytotoxic agents, such as calcium, which can also induce cell death. The present study aims to evaluate
the cytotoxic effect, the morphological changes in tumor spheroids, the effect on the cell viability, and
the cell-specific growth rate following calcium electroporation (CaEP) in uveal melanoma (UM) 2D
monolayer cell cultures as well as in 3D tumor spheroid models. The experiments were conducted
in four cell lines, UM92.1, Mel270, and two primary UM cell lines, UPMD2 and UPMM3 (UPM).
The 2D and 3D UM cell cultures were electroporated with eight rectangular pulses (100 µs pulse
duration, 5 Hz repetition frequency) of a 1000 V/cm pulse strength alone or in combination with
0.11 mg/mL, 0.28 mg/mL, 0.55 mg/mL or 1.11 mg/mL calcium chloride or 1.0 µg/mL or 2.5 µg/mL
bleomycin. The application of calcium chloride alone induced an ATP reduction only in the UM92.1
2D cell cultures. Calcium alone had no significant effect on ATP levels in all four UM spheroids.
A significant decrease in the intracellular adenosine triphosphate (ATP) level was documented in
all four 2D and 3D cell cultures for both CaEP as well as ECT with bleomycin. The results suggest
a dose-dependent ATP depletion with a wide range of sensitivity among the tested UM cell lines,
control groups, and the applied settings in both 2D monolayer cell cultures and 3D tumor spheroid
models. The colony formation capacity of the cell lines after two weeks reduced significantly after
CaEP only with 0.5 mg/mL and 1.1 mg/mL, whereas the same effect could be achieved with both
applied bleomycin concentrations, 1.0 µg/mL and 2.5 µg/mL, for the ECT group. The specific growth
rate on day 7 following CaEP was significantly reduced in UM92.1 cell lines with 0.5 and 1.1 mg/mL
calcium chloride, while Mel270 showed a similar effect only after administration of 1.1 mg/mL.
UM92.1 and Mel270 spheroids exhibited lower adhesion and density after CaEP on day three in comparison to UPM spheroids showing detachment after day 7 following treatment. CaEP and
bleomycin electroporation significantly reduce cell viability at similar applied voltage settings. CaEP
may be a feasible and inexpensive therapeutic option for the local tumor control with fewer side
effects, in comparison to other chemotherapeutic agents, for the treatment of uveal melanoma. The
limited effect on normal cells and the surrounding tissue has already been investigated, but further
research is necessary to clarify the effect on the surrounding tissue and to facilitate its application in a
clinical setting for the eye
Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling
The human prolyl-4-hydroxylase domain (PHD) proteins 1-3 are known as cellular oxygen sensors, acting via the degradation of hypoxia-inducible factor (HIF) α-subunits. PHD2 and PHD3 genes are inducible by HIFs themselves, suggesting a negative feedback loop that involves PHD abundance. To identify novel regulators of the PHD2 gene, an expression array of 704 transcription factors was screened by a method that allows distinguishing between HIF-dependent and HIF-independent promoter regulation. Among others, the E-twenty six transcription factor ETS translocation variant 4 (ETV4) was found to contribute to PHD2 gene expression particularly under hypoxic conditions. Mechanistically, complex formation between ETV4 and HIF-1/2α was observed by mammalian two-hybrid and fluorescence resonance energy transfer analysis. HIF-1α domain mapping, CITED2 overexpression and factor inhibiting HIF depletion experiments provided evidence for cooperation between HIF-1α and p300/CBP in ETV4 binding. Chromatin immunoprecipitation confirmed ETV4 and HIF-1α corecruitment to the PHD2 promoter. Of 608 hypoxically induced transcripts found by genome-wide expression profiling, 7.7% required ETV4 for efficient hypoxic induction, suggesting a broad role of ETV4 in hypoxic gene regulation. Endogenous ETV4 highly correlated with PHD2, HIF-1/2α and several established markers of tissue hypoxia in 282 human breast cancer tissue samples, corroborating a functional interplay between the ETV4 and HIF pathway
Chick Chorioallantoic Membrane as a Patient-Derived Xenograft Model for Uveal Melanoma : Imaging Modalities for Growth and Vascular Evaluation
Background: Patient-derived tumor xenografts (PDXs) have emerged as valuable preclinical
in vivo models in oncology as they largely retain the polygenomic architecture of the human tumors
from which they originate. Although animal models are accompanied by cost and time constraints
and a low engraftment rate, PDXs have primarily been established in immunodeficient rodent models
for the in vivo assessment of tumor characteristics and of novel therapeutic cancer targets. The chick
chorioallantoic membrane (CAM) assay represents an attractive alternative in vivo model that has
long been used in the research of tumor biology and angiogenesis, and can overcome some of these
limitations. Methods: In this study, we reviewed different technical approaches for the establishment
and monitoring of a CAM-based uveal melanoma PDX model. Forty-six fresh tumor grafts were
acquired after enucleation from six uveal melanoma patients and were implanted onto the CAM on
ED7 with Matrigel and a ring (group 1), with Matrigel (group 2), or natively without Matrigel or a ring
(group 3). Real-time imaging techniques, such as various ultrasound modalities, optical coherence
tomography, infrared imaging, and imaging analyses with Image J for tumor growth and extension, as
well as color doppler, optical coherence angiography, and fluorescein angiography for angiogenesis,
were performed on ED18 as alternative monitoring instruments. The tumor samples were excised on
ED18 for histological assessment. Results: There were no significant differences between the three
tested experimental groups regarding the length and width of the grafts during the development
period. A statistically significant increase in volume (p = 0.0007) and weight (p = 0.0216) between
ED7 and ED18 was only documented for tumor specimens of group 2. A significant correlation of the
results for the cross-sectional area, largest basal diameter, and volume was documented between the
different imaging and measurement techniques and the excised grafts. The formation of a vascular
star around the tumor and of a vascular ring on the base of the tumor was observed for the majority
of the viable developing grafts as a sign of successful engraftment. Conclusion: The establishment of
a CAM-PDX uveal melanoma model could elucidate the biological growth patterns and the efficacy
of new therapeutic options in vivo. The methodological novelty of this study, investigating different
implanting techniques and exploiting advances in real-time imaging with multiple modalities, allows precise, quantitative assessment in the field of tumor experimentation, underlying the feasibility of
CAM as an in vivo PDX model
Electrochemotherapy with Bleomycin Enhances Radiosensitivity of Uveal Melanomas: First In Vitro Results in 3D Cultures of Primary Uveal Melanoma Cell Lines
Electrochemotherapy (ECT) is emerging as a complementary treatment modality for local
tumor control in various cancer entities. Irradiation is an established therapeutic option for oncologic
patients, which is commonly combined with chemotherapy due to its insufficient targeting ability.
The efficiency of radiotherapy for tumors can be enhanced with different radiosensitizers. ECT can
potentiate the radiosensitizing effect of chemotherapeutic agents such as bleomycin. The present
study aims to evaluate the radiosensitizing effect of concomitant ECT with bleomycin on 3D tumor
spheroids with primary and radioresistant uveal melanoma cell lines (UPMD2, UPMM3, UM92.1,
Mel270) and irradiation. The changes in the spheroid growth and the cell viability as well the
cytotoxic long-term effect of the combination treatment were evaluated with various combinations
of electroporation settings and bleomycin concentrations as well as radiotherapy doses. A broad
range of radiosensitivity was documented among the spheroids from different uveal melanoma cell
lines. The primary cell lines showed a higher radiosensitivity and required lower irradiation and
bleomycin doses. The maximal tumor control with a reduction of cell survival <10% was achieved
with a 5 Gy irradiation only in the primary uveal melanoma cell lines and in combination with all
tested ECT settings, whereas the same result could be obtained in UM92.1 spheroids only after ECT
with 20 Gy irradiation. Based on the spheroid growth and the measurement of the cross-sectional
area, the Mel270 spheroids, originating from a previously irradiated recurrent uveal melanoma,
required higher doses of bleomycin and ECT settings after irradiation with 5 Gy in order to achieve a
significant growth reduction. No significant difference could be demonstrated for the reduction of cell
viability in the combination therapy with 20 Gy and 1000 V/cm between 1 and 2.5 µg/mL bleomycin even in Mel270 spheroids, underlying the importance of a drug delivery system to potentiate the
radiosensitizing effect of agents in lower doses. ECT should be further assessed for its applicability
in clinical settings as a therapeutic radiosensitizing option for radioresistant tumors and a sufficient
local tumor control with lower chemotherapy and irradiation doses
Optimisation of the Chicken Chorioallantoic Membrane Assay in Uveal Melanoma Research
The treatment of uveal melanoma and its metastases has not evolved sufficiently over
the last decades in comparison to other tumour entities, posing a great challenge in the field of
ocular oncology. Despite improvements in the conventional treatment regime and new discoveries
about the genetic and molecular background of the primary tumour, effective treatment strategies to
either prevent tumours or treat patients with advanced or metastatic disease are still lacking. New
therapeutic options are necessary in order to achieve satisfactory local tumour control, reduce the
risk of metastasis development, and preserve the eyeball and possibly the visual function of the eye.
The development of in vivo model systems remains crucial for the identification and investigation of
potential novel treatment modalities. The aim of this study was the optimisation of the chorioallantoic
membrane (CAM) model for uveal melanoma research. We analysed the established CAM assay
and its modification after the implantation of three-dimensional spheroids. The chorioallantoic
membrane of a chick embryo was used to implant uveal melanoma-cell-line-derived spheroids in
order to study their growth rate, angiogenic potential, and metastatic capability. Using the UM 92.1,
UPMD2, UPMM3, and Mel270 cell lines, we were able to improve the viability of the embryos from
20% to >80% and to achieve up to a fourfold volume increase of the transplanted spheroid masses.
The results point to the value of an optimised chicken embryo assay as an in vivo model for testing
novel therapies for uveal melanoma by simplifying the research conditions and by contributing to a
considerable reduction in animal experiments
Gut microbiome in BALB/c and C57BL/6J mice undergoing experimental thyroid autoimmunity associate with differences in immunological responses and thyroid function
Experimental models of hyperthyroid Graves’ disease (GD) and Graves’ orbitopathy (GO) are efficiently developed by genetic immunisation by electroporation with human thyrotropin hormone receptor (hTSHR) A-subunit plasmid in female BALB/c (H-2d) mice. We investigated susceptibility in C57BL/6 J (H-2b) animals to allow studies on disease mechanisms in transgenic and immune response gene knock-out mice. Higher numbers of female C57BL/6 J were positive for pathogenic thyroid stimulating antibodies, but induced hyperthyroidism remained at a low frequency compared to BALB/c animals. Assessment of hTSHR specific T cells showed reduced proliferation in C57BL/6 J animals accompanied with anti-inflammatory IL-10, with less pro-inflammatory IFN-γ compared to BALB/c. Whilst the orbital tissue from immune BALB/c mice showed inflammation and adipogenesis, in contrast C57BL/6 J animals showed normal pathology. We characterised the gut microbiota using 16 S ribosomal RNA gene sequencing to explore its possible pathogenic role in the model. Despite being housed under identical conditions, we observed significantly different organisation of the microbiota (beta-diversity) in the two strains. Taxonomic differences were also noted, with C57BL/6 J showing an enrichment of Operational Taxonomic Units (OTUs) belonging to the Paludibacter and Allobaculum, followed by Limibacter, Anaerophaga and Ureaplasma genera. A higher number of genera significantly correlating with clinical features was observed in C57BL/6 J compared to BALB/c; for example, Limibacter OTUs correlated negatively with thyroid-stimulating antibodies in C57BL/6 J mice. Thus, our data suggest gut microbiota may play a pivotal immunomodulatory role that differentiates the thyroid function and orbital pathology outcome in these two inbred strains undergoing experimental GO
Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling
The human prolyl-4-hydroxylase domain (PHD) proteins 1–3 are known as cellular oxygen sensors, acting via the degradation of hypoxia-inducible factor (HIF) α-subunits. PHD2 and PHD3 genes are inducible by HIFs themselves, suggesting a negative feedback loop that involves PHD abundance. To identify novel regulators of the PHD2 gene, an expression array of 704 transcription factors was screened by a method that allows distinguishing between HIF-dependent and HIF-independent promoter regulation. Among others, the E-twenty six transcription factor ETS translocation variant 4 (ETV4) was found to contribute to PHD2 gene expression particularly under hypoxic conditions. Mechanistically, complex formation between ETV4 and HIF-1/2α was observed by mammalian two-hybrid and fluorescence resonance energy transfer analysis. HIF-1α domain mapping, CITED2 overexpression and factor inhibiting HIF depletion experiments provided evidence for cooperation between HIF-1α and p300/CBP in ETV4 binding. Chromatin immunoprecipitation confirmed ETV4 and HIF-1α corecruitment to the PHD2 promoter. Of 608 hypoxically induced transcripts found by genome-wide expression profiling, 7.7% required ETV4 for efficient hypoxic induction, suggesting a broad role of ETV4 in hypoxic gene regulation. Endogenous ETV4 highly correlated with PHD2, HIF-1/2α and several established markers of tissue hypoxia in 282 human breast cancer tissue samples, corroborating a functional interplay between the ETV4 and HIF pathways
Visualization of the three-dimensional organization of hypoxia-inducible factor-1α and interacting cofactors in subnuclear structures
Hepatocyte nuclear factor-4 (HNF-4α) is not involved in hypoxia induced Erythropoietin regulation in human neuroblastoma cell lines
Oxygen dependent Erythropoietin (EPO) production is regulated by hypoxia inducible factor-1 (HIF-1) which consists of the O2-labile α- and the constitutive β-subunit. In human hepatoma cells (HepG2) HNF-4α enhances hypoxia-induced EPO expression. Herein, we investigated EPO gene regulation in human neuroblastoma (NB) cell lines. Methods: EPO production in SH-SY5Y, Kelly and HepG2 cells was measured by real time PCR and EPO elisa. Intracellular distribution of HIF-1α, HIF-1β and HNF-4α was studied by Western blot and immunohistochemistry. Cells were transiently transfected with HIF-1-driven luciferase and a dominant negative (dn) HNF-4 plasmid. Results: Hypoxia induced EPO gene expression in NB cells was maximal at <0.1% O2. HIF-1α protein accumulated and translocated to the nucleus at an oxygen threshold of 7% O2 in HepG2 cells but at 3% O2 in NB. Differences were also seen in HIF-1α protein degradation upon reoxygenation. Very little HNF-4α was expressed in NB, but did not colocalize with HIF-1α under hypoxic conditions. Transfection with dnHNF-4 did not affect hypoxic induction of EPO in NB cells. Conclusions: HIF-1, but not HNF-4α is involved in EPO gene regulation in human NB cell lines.
(This abstract is as it is in the printed issue of the journal)