ReN VM spheroids in matrix: A neural progenitor three-dimensional in vitro model reveals DYRK1A inhibitors as potential regulators of radio-sensitivity.

INTRODUCTION: Pre-clinical testing of small molecules for therapeutic development across many pathologies relies on the use of in-vitro and in-vivo models. When designed and implemented well, these models serve to predict the clinical outcome as well as the toxicity of the evaluated therapies. The two-dimensional (2D) reductionist approach where cells are incubated in a mono-layer on hard plastic microtiter plates is relatively inexpensive but not physiologically relevant. In contrast, well developed and applied three dimensional (3D) in vitro models could be employed to bridge the gap between 2D in vitro primary screening and expensive in vivo rodent models by incorporating key features of the tissue microenvironment to explore differentiation, cortical development, cancers and various neuronal dysfunctions. These features include an extracellular matrix, co-culture, tension and perfusion and could replace several hundred rodents in the drug screening validation cascade. METHODS: Human neural progenitor cells from middle brain (ReN VM, Merck Millipore, UK) were expanded as instructed by the supplier (Merck Millipore, UK), and then seeded in 96-well low-attachment plates (Corning, UK) to form multicellular spheroids followed by adding a Matrigel layer to mimic extracellular matrix around neural stem cell niche. ReN VM cells were then differentiated via EGF and bFGF deprivation for 7 days and were imaged at day 7. Radiotherapy was mimicked via gamma-radiation at 2Gy in the absence and presence of selected DYRK1A inhibitors Harmine, INDY and Leucettine 41 (L41). Cell viability was measured by AlamarBlue assay. Immunofluorescence staining was used to assess cell pluripotency marker SOX2 and differentiation marker GFAP. RESULTS: After 7 days of differentiation, neuron early differentiation marker (GFAP, red) started to be expressed among the cells expressing neural stem cell marker SOX2 (green). Radiation treatment caused significant morphology change including the reduced viability of the spheroids. These spheroids also revealed sensitizing potential of DYRK1A inhibitors tested in this study, including Harmine, INDY and L41. DISCUSSION & CONCLUSIONS: Combined with the benefit of greatly reducing the issues associated with in vivo rodent models, including reducing numbers of animals used in a drug screening cascade, cost, ethics, and potential animal welfare burden, we feel the well-developed and applied 3D neural spheroid model presented in this study will provide a crucial tool to evaluate combinatorial therapies, optimal drug concentrations and treatment dosages.

3D tumour spheroids for the prediction of the effects of radiation and hyperthermia treatments.

For multimodality therapies such as the combination of hyperthermia and radiation, quantification of biological effects is key for dose prescription and response prediction. Tumour spheroids have a microenvironment that more closely resembles that of tumours in vivo and may thus be a superior in vitro cancer model than monolayer cultures. Here, the response of tumour spheroids formed from two established human cancer cell lines (HCT116 and CAL27) to single and combination treatments of radiation (0-20 Gy), and hyperthermia at 47 °C (0-780 CEM43) has been evaluated. Response was analysed in terms of spheroid growth, cell viability and the distribution of live/dead cells. Time-lapse imaging was used to evaluate mechanisms of cell death and cell detachment. It was found that sensitivity to heat in spheroids was significantly less than that seen in monolayer cultures. Spheroids showed different patterns of shrinkage and regrowth when exposed to heat or radiation: heated spheroids shed dead cells within four days of heating and displayed faster growth post-exposure than samples that received radiation or no treatment. Irradiated spheroids maintained a dense structure and exhibited a longer growth delay than spheroids receiving hyperthermia or combination treatment at (thermal) doses that yielded equivalent levels of clonogenic cell survival. We suggest that, unlike radiation, which kills dividing cells, hyperthermia-induced cell death affects cells independent of their proliferation status. This induces microenvironmental changes that promote spheroid growth. In conclusion, 3D tumour spheroid growth studies reveal differences in response to heat and/or radiation that were not apparent in 2D clonogenic assays but that may significantly influence treatment efficacy.

Preclinical evaluation of binimetinib (MEK162) delivered via polymeric nanocarriers in combination with radiation and temozolomide in glioma.

BACKGROUND AND PURPOSE: Glioblastoma multiforme (GBM) is the most aggressive subtype of malignant gliomas, with an average survival rate of 15 months after diagnosis. More than 90% of all GBMs have activating mutations in the MAPK/ERK pathway. Recently, we showed the allosteric MEK1/2 inhibitor binimetinib (MEK162) to inhibit cell proliferation and to enhance the effect of radiation in preclinical human GBM models. Because the free drug cannot pass the blood-brain barrier (BBB), we investigated the use of nanocarriers for transport of the drug through the BBB and its efficacy when combined with radiotherapy and temozolomide (TMZ) in glioma spheroids. METHODS: In vitro studies were performed using multicellular U87 human GBM spheroids. Polymeric nanocarriers (polymersomes) were loaded with MEK162. The interaction between nanocarrier delivered MEK162, irradiation and TMZ was studied on the kinetics of spheroid growth and on protein expression in the MAPK/ERK pathway. BBB passaging was evaluated in a transwell system with human cerebral microvascular endothelial (hCMEC/D3) cells. RESULTS: MEK162 loaded polymersomes inhibited spheroid growth. A synergistic effect was found in combination with fractionated irradiation and an additive effect with TMZ on spheroid volume reduction. Fluorescent labeled polymersomes were taken up by human cerebral microvascular endothelial cells and passed the BBB in vitro. CONCLUSION: MEK162 loaded polymersomes are taken up by multicellular spheroids. The nanocarrier delivered drug reduced spheroid growth and inhibited its molecular target. MEK162 delivered via polymersomes showed interaction with irradiation and TMZ. The polymersomes crossed the in vitro BBB model and therewith offer exciting challenges ahead for delivery of therapeutics agents to brain tumours.

Thermal monitoring during photothermia: hybrid probes for simultaneous plasmonic heating and near-infrared optical nanothermometry.

The control of temperature during photothermal therapy is key to preventing unwanted damage in surrounding tissue or post-treatment inflammatory responses. Lack of accurate thermal control is indeed one of the main limitations that hyperthermia techniques present to allow their translation into therapeutic applications. We developed a nanoprobe that allows controlled local heating, combined with in situ nanothermometry. The design of the probe follows a practical rationale that aims at simplifying experimental requirements and exploits exclusively optical wavelengths matching the first and second biological windows in the near-infrared. Methods: Hybrid nanostructures were chemically synthesized, and combine gold nanostars (photothermal agents) with CaF2:Nd3+,Y3+ nanoparticles (luminescent nanothermometers). Both components were simultaneously excited in the near-infrared range, at 808 nm. Following the goal of simplifying the thermal monitoring technique, the luminescent signal was recorded with a portable near-infrared detector. The performance of the probes was tested in 3D tumor spheroids from a human glioblastoma (U87MG) cell line. The location of the beads within the spheroids was determined measuring Nd3+ emission in a commercial Lightsheet microscope, modified in-house to be able to select the required near-infrared wavelengths. The temperature achieved inside the tumor spheroids was deduced from the luminescence of Nd3+, following a protocol that we developed to provide reliable thermal readings. Results: The choice of materials was shown to work as an optically excited hybrid probe. Depending on the illumination parameters, temperature can be controlled in a range between 37 ºC and 100 ºC. The near-infrared emission of nanothermometers also allows microscopic tracking of the hybrid nanostructures, confirming that the probes can penetrate deeper into the spheroid mass. We observed that, application of optical thermometry in biological environments requires often neglected considerations, since the optical signal changes along the optical path. Accordingly, we developed data analysis protocols that guarantee reliable thermal readings. Conclusions: The prepared hybrid probes are internalized in 3D tumor spheroids and can be used to induce cell death through photothermal effects, while simultaneously measuring the local temperature in situ. We show that luminescent thermometry in biomedical applications requires the development of protocols that guarantee accurate readings. Regarding photothermal treatments, we observe a sharp thermal threshold at around 55 ºC (for 10 min treatments) that separates high survival ratio from complete cell death.

A cellular automaton model for spheroid response to radiation and hyperthermia treatments.

Thermo-radiosensitisation is a promising approach for treatment of radio-resistant tumours such as those containing hypoxic subregions. Response prediction and treatment planning should account for tumour response heterogeneity, e.g. due to microenvironmental factors, and quantification of the biological effects induced. 3D tumour spheroids provide a physiological in vitro model of tumour response and a systems oncology framework for simulating spheroid response to radiation and hyperthermia is presented. Using a cellular automaton model, 3D oxygen diffusion, delivery of radiation and/or hyperthermia were simulated for many ([Formula: see text]) individual cells forming a spheroid. The iterative oxygen diffusion model was compared to an analytical oxygenation model and simulations were calibrated and validated against experimental data for irradiated (0-10 Gy) and/or heated (0-240 CEM43) HCT116 spheroids. Despite comparable clonogenic survival, spheroid growth differed significantly following radiation or hyperthermia. This dynamic response was described well by the simulation ([Formula: see text] > 0.85). Heat-induced cell death was implemented as a fast, proliferation-independent process, allowing reoxygenation and repopulation, whereas radiation was modelled as proliferation-dependent mitotic catastrophe. This framework stands out both through its experimental validation and its novel ability to predict spheroid response to multimodality treatment. It provides a good description of response where biological dose-weighting based on clonogenic survival alone was insufficient.

Evaluation of combined effect of hyperthermia and ionizing radiation on cytotoxic damages induced by IUdR-loaded PCL-PEG-coated magnetic nanoparticles in spheroid culture of U87MG glioblastoma cell line.

PURPOSE: Glioblastoma multiform (GBM) is the most prevalent and aggressive type of primary brain tumor. None of the current conventional treatment methods has improved treatment considerably. Therefore, in this study the effect of magnetic nanoparticles coated with poly (caprolactone)-poly (ethylene glycol) (PCL-PEG) as a 5-iodo 2'deoxyuridine (IUdR) carrier in the presence of hyperthermia and 6 MV (megavoltage) X-ray radiation, were investigated in a spheroid model of U87MG glioblastoma cell line using colony formation assay. MATERIALS AND METHODS: First, the human glioblastoma cell line U87MG was cultured as a spheroid using the liquid overlay technique. Spheroids on day 10 with 100 mm diameters were treated with 1 µM IUdR or nanoparticles as IUdR carriers for one volume doubling time (VDT) of spheroids (67 h) and hyperthermia at 43 °C for 1 h, and then irradiated with 2 Gy of 6 MV X-ray in different groups. Finally, the effect of treatment on colony-forming ability was obtained by colony formation and alkaline assay. RESULTS: Our results revealed that hyperthermia in combination with radiation could significantly reduce the colony number of glioblastoma spheroid cells treated with IUdR or nanoparticles as IUdR carriers. However, the extent of reduction in colony number following treatment with IUdR-loaded nanoparticles in combination with hyperthermia and then X-ray radiation was significantly more than free IUdR. CONCLUSION: According to this study, PCL-PEG-coated magnetic nanoparticles are effective delivery vehicles for IUdR into cells. Moreover, they can act as a radiosensitizer and thermosensitizer in the treatment of the glioblastoma cell line.

Tumor-on-a-chip platforms for assessing nanoparticle-based cancer therapy.

Cancer has become the most prevalent cause of deaths, placing a huge economic and healthcare burden worldwide. Nanoparticles (NPs), as a key component of nanomedicine, provide alternative options for promoting the efficacy of cancer therapy. Current conventional cancer models have limitations in predicting the effects of various cancer treatments. To overcome these limitations, biomimetic and novel 'tumor-on-a-chip' platforms have emerged with other innovative biomedical engineering methods that enable the evaluation of NP-based cancer therapy. In this review, we first describe cancer models for evaluation of NP-based cancer therapy techniques, and then present the latest advances in 'tumor-on-a-chip' platforms that can potentially facilitate clinical translation of NP-based cancer therapies.

Evaluation of the effect of hyperthermia and electron radiation on prostate cancer stem cells.

The aim of this study was to investigate the effect of hyperthermia, 6 MeV electron radiation and combination of these treatments on cancer cell line DU145 in both monolayer culture and spheroids enriched for prostate cancer stem cells (CSCs). Flowcytometric analysis of the expression of molecular markers CD133+/CD44+ was carried out to determine the prostate CSCs in cell line DU145 grown as spheroids in serum-free medium. Following monolayer and spheroid culture, DU145 cells were treated with different doses of hyperthermia, electron beam and combination of them. The survival and self-renewing of the cells were evaluated by colony formation assay (CFA) and spheroid formation assay (SFA). Flowcytometry results indicated that the percentage of CD133+/CD44+ cells in spheroid culture was 13.9-fold higher than in the monolayer culture. The SFA showed significant difference between monolayer and spheroid culture for radiation treatment (6 Gy) and hyperthermia (60 and 90 min). The CFA showed significantly enhanced radiosensitivity in DU145 cells grown as monolayer as compared to spheroids, but no effect of hyperthermia. In contrast, for the combination of radiation and hyperthermia the results of CFA and SFA showed a reduced survival fraction in both cultures, with larger effects in monolayer than in spheroid culture. Thus, hyperthermia may be a promising approach in prostate cancer treatment that enhances the cytotoxic effect of electron radiation. Furthermore, determination and characterization of radioresistance and thermoresistance of CSCs in the prostate tumor is the key to develop more efficient therapeutic strategies.

Adipose-derived stem cell spheroid treated with low-level light irradiation accelerates spontaneous angiogenesis in mouse model of hindlimb ischemia.

BACKGROUND AIMS: We investigated whether low-level light irradiation (LLLI) before adipose-derived stromal cells (ASCs) spheroid transplantation improved hind-limb functional recovery by stimulation of angiogenesis. METHODS: The spheroid, composed of ASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor and basic fibroblast growth factor. From immunochemical staining analysis, the spheroid of ASCs included CD31+, KDR+ and CD34+, whereas monolayer-cultured ASCs were negative for these markers. To evaluate the therapeutic effect of the ASC spheroid treated with LLLI in vivo, phosphate-buffered saline, monolayer ASCs, LLLI-monolayer ASCs, spheroid ASCs and LLLI-spheroid ASCs were transplanted into a hind-limb ischemia model. RESULTS: The LLLI-spheroid ASCs transplanted into the hind-limb ischemia differentiated into endothelial cells and remained differentiated. Transplantation of LLLI-spheroid ASCs into the hind-limb ischemia significantly elevated the density of vascular formations through angiogenic factors released by the ASCs and enhanced tissue regeneration at the lesion site. Consistent with these results, the transplantation of LLLI-spheroid ASCs significantly improved functional recovery compared with ASC or spheroid ASC transplantation and PBS treatment. CONCLUSIONS: These findings suggest that transplantation of ASC spheroid treated with LLLI may be an effective stem cell therapy for the treatment of hind-limb ischemia and peripheral vascular disease.

Human adipose-derived stem cell spheroid treated with photobiomodulation irradiation accelerates tissue regeneration in mouse model of skin flap ischemia.

Skin flap grafting is a form of transplantation widely used in plastic surgery. However, ischemia/reperfusion injury is the main factor which reduces the survival rate of flaps following grafting. We investigated whether photobiomodulation (PBM) precondition prior to human adipose-derived stromal cell (hASC) spheroid (PBM-spheroid) transplantation improved skin tissue functional recovery by the stimulation of angiogenesis and tissue regeneration in skin flap of mice. The LED had an emission wavelength peaked at 660 ± 20 nm (6 J/cm2, 10 mW/cm2). The expression of angiogenic growth factors in PBM-spheroid hASCs was much greater than that of not-PBM-treated spheroid or monolayer-cultured hASCs. From immunochemical staining analysis, the hASCs of PBM-spheroid were CD31+, KDR+, and CD34+, whereas monolayer-cultured hASCs were negative for these markers. To evaluate the therapeutic effect of hASC PBM-spheroid in vivo, PBS, monolayer-cultured hASCs, and not-PBM-spheroid were transplanted into a skin flap model. The animals were observed for 14 days. The PBM-spheroid hASCs transplanted into the skin flap ischemia differentiated into endothelial cells and remained differentiated. Transplantation of PBM-spheroid hASCs into the skin flap ischemia significantly elevated the density of vascular formations through angiogenic factors released by the skin flap ischemia and enhanced tissue regeneration at the lesion site. Consistent with these results, the transplantation of PBM-spheroid hASCs significantly improved functional recovery compared with PBS, monolayer-cultured hASCs, and not-PBM-spheroid treatment. These findings suggest that transplantation of PBM-spheroid hASCs may be an effective stem cell therapy for the treatment of skin flap ischemia.

Enhancement of Ischemic Wound Healing by Spheroid Grafting of Human Adipose-Derived Stem Cells Treated with Low-Level Light Irradiation.

We investigated whether low-level light irradiation prior to transplantation of adipose-derived stromal cell (ASC) spheroids in an animal skin wound model stimulated angiogenesis and tissue regeneration to improve functional recovery of skin tissue. The spheroid, composed of hASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). Immunochemical staining analysis revealed that the spheroid of the hASCs was CD31+, KDR+, and CD34+. On the other hand, monolayer-cultured hASCs were negative for these markers. PBS, human adipose tissue-derived stromal cells, and the ASC spheroid were transplanted into a wound bed in athymic mice to evaluate the therapeutic effects of the ASC spheroid in vivo. The ASC spheroid transplanted into the wound bed differentiated into endothelial cells and remained differentiated. The density of vascular formations increased as a result of the angiogenic factors released by the wound bed and enhanced tissue regeneration at the lesion site. These results indicate that the transplantation of the ASC spheroid significantly improved functional recovery relative to both ASC transplantation and PBS treatment. These findings suggest that transplantation of an ASC spheroid treated with low-level light may be an effective form of stem cell therapy for treatment of a wound bed.

Multicell tumor spheroids in photodynamic therapy.

BACKGROUND AND OBJECTIVES: Multicell spheroids (MCSs) represent a simple in vitro system ideally suited for studying the effects of a wide variety of investigational treatments including photodynamic therapy (PDT). STUDY DESIGN/MATERIALS AND METHODS: In the first section of this review study, an overview of the current literature on MCS in PDT will be presented. Knowledge of basic PDT parameters has been gained from numerous MCS studies, in particular, the mechanisms of sensitizer photobleaching have been elucidated. MCSs have also proven useful for the study of complex PDT treatment regimens including multiple treatments and combined therapies involving PDT and ionizing radiation or hyperthermia. The purpose of the second part of this review is to present results from recent studies in our laboratory aimed at developing MCS models suitable for investigating tumor cell invasion and angiogenesis-processes characteristic of high-grade gliomas. RESULTS AND CONCLUSION: To that end, progress has recently been made to develop a more accurate in vivo brain tumor model consisting of biopsy-derived human tumor spheroids implanted into the brains of immunodeficient rats. Finally, recent work suggests that computer simulations may prove useful to describe the growth of MCS and predict the effects of investigational therapies including PDT. Such in silico models have made a number of counterintuitive predictions that have been verified in vitro and, as such, could guide the development of improved therapeutics.