Multicellular tumour spheroids capture many characteristics of human tumour microenvironments, including hypoxia, and represent an experimentally tractable in vitro model for studying interactions between radiotherapy and anticancer drugs. However, interpreting spheroid data is challenging because of limited ability to observe cell fate within spheroids dynamically. To overcome this limitation, we have developed a hybrid continuum/agent-based model (ABM) for HCT116 tumour spheroids, parameterised using experimental models (monolayers and multilayers) in which reaction and diffusion can be measured directly. In the ABM, cell fate is simulated as a function of local oxygen, glucose and drug concentrations, determined by solving diffusion equations and intracellular reactions. The model is lattice-based, with cells occupying discrete locations on a 3D grid embedded within a coarser grid that encompasses the culture medium; separate solvers are employed for each grid. The generated concentration fields account for depletion in the medium and specify concentration-time profiles within the spheroid. Cell growth and survival are determined by intracellular oxygen and glucose concentrations, the latter based on direct measurement of glucose diffusion/reaction (in multilayers) for the first time. The ABM reproduces known features of spheroids including overall growth rate, its oxygen and glucose dependence, peripheral cell proliferation, central hypoxia and necrosis. We extended the ABM to describe in detail the hypoxia-dependent interaction between ionising radiation and a hypoxia-activated prodrug (SN30000), again using experimentally determined parameters; the model accurately simulated clonogenic cell killing in spheroids, while inclusion of reversible cell cycle delay was required to account for the marked spheroid growth delay after combined radiation and SN30000. This ABM of spheroid growth and response exemplifies the utility of integrating computational and experimental tools for investigating radiation/drug interactions, and highlights the critical importance of understanding oxygen, glucose and drug concentration gradients in interpreting activity of therapeutic agents in spheroid models.
Exposure to microgravity during space flight affects almost all human physiological systems. Migration, proliferation, and differentiation of stem cells are crucial for tissues repair and regeneration. However, the effect of microgravity on the migration potentials of bone marrow mesenchymal stem cells (BMSCs) is unclear, which are important progenitor and supporting cells. Here, we utilized a clinostat to model simulated microgravity (SMG) and found that SMG obviously inhibited migration of rat BMSCs. We detected significant reorganization of F-actin filaments and increased Young's modulus of BMSCs after exposure to SMG. Moreover, Y-27632 (a specific inhibitor of ROCK) abrogated the inhibited migration capacity and polymerized F-actin filament of BMSCs under SMG. Interestingly, we found that transferring BMSCs to normal gravity also attenuated the polymerized F-actin filament and Young's modulus of BMSCs induced by SMG, but could not recover migration capacity of BMSCs inhibited by SMG. Taken together, we propose that SMG inhibits migration of BMSCs through remodeling F-actin and increasing cell stiffness.
Stem cells are characterized by their self-renewal and multi-lineage differentiation potential. Stem cell differentiation is a prerequisite for the application of stem cells in regenerative medicine and clinical therapy. In addition to chemical stimulation, mechanical cues play a significant role in regulating stem cell differentiation. The integrity of mechanical sensors is necessary for the ability of cells to respond to mechanical signals. The nucleus, the largest and stiffest cellular organelle, interacts with the cytoskeleton as a key mediator of cell mechanics. Nuclear mechanics are involved in the complicated interactions of lamins, chromatin and nucleoskeleton-related proteins. Thus, stem cell differentiation is intimately associated with nuclear mechanics due to its indispensable role in mechanotransduction and mechanical response. This paper reviews several main contributions of nuclear mechanics, highlights the hallmarks of the nuclear mechanics of stem cells, and provides insight into the relationship between nuclear mechanics and stem cell differentiation, which may guide clinical applications in the future.
Introduction: Chemotherapy is often limited by resistant subpopulations of tumor cells. Tumor hypoxia contributes to resistance of tumor cells to clinical drugs via multiple mechanisms. Hypoxia-activated prodrugs selectively target hypoxic cells, among which SN30000 is a second generation of tirapazamine analogue with superior diffusion coefficient and hypoxia selectivity. Here we hypothesize that SN30000/chemotherapy drug combinations will provide improved activity. Methods: HCT116 and SiHa spheroids were used to screen schedules of SN30000 in combination with 4 clinical drugs (cisplatin, doxorubicin, gemcitabine, paclitaxel) under 5% O2 and 20% O2, with endpoints of spheroid growth delay and clonogenic cell survival. The schedules were tested on HCT116 and SiHa monolayers under 5% O2. HCT116 monolayers and spheroids exposed to SN30000 or the clinical drugs were dissociated for clonogenic survival assay. Tumor growth delay using the same schedules of the most promising combination (SN30000 + gemcitabine) were then tested in HCT116 xenografts in NIH-III mice. Body weight loss and core body temperature of mice in response to the treatments were monitored. Results: Exposing to 25 µM SN30000 and 25 µM cisplatin/5 µM doxorubicin/10 µM gemcitabine simultaneously for 2 hr, and SN30000 3 hr before gemcitabine under 5% O2 significantly delayed HCT116 and SiHa spheroid growth, but their combined activity was compromised under 20% O2. Dosing SN30000 and cisplatin simultaneously, and SN30000 3 hr before gemcitabine significantly delayed HCT116 and SiHa monolayer growth. Clonogenic survival assay demonstrated that HCT116 monolayers were more sensitive than spheroids to doxorubicin, but not the other 3 clinical compounds, consistent with the known limited diffusion of doxorubicin into spheroids.Dosing SN30000 3 hr before gemcitabine or at the same time, but not SN30000 3 hr after gemcitabine significantly delayed tumor growth, which was consistent with the spheroid data. Body weight measurements showed that the combinations of SN30000 and gemcitabine added little additional toxicity while SN30000/doxorubicin was more toxic when administered simultaneously. SN30000 caused hypothermia in mice with recovery after 3 hr. Discussion: Screening schedules of SN30000 in combination with 4 clinical drugs in HCT116/SiHa spheroids and monolayers demonstrated schedule and oxygen dependence of the activity of SN30000 and gemcitabine combinations, which was further demonstrated in HCT116 xenografts. The mechanism of the promising interaction between gemcitabine and SN30000 is being further investigated by flow cytometry and immunohistochemical staining to explore the hypoxic fraction and cell proliferation following treatments, and by evaluating the plasma pharmacokinetics of SN30000 and gemcitabine to investigate the implications of SN30000-induced hypothermia. Citation Format: Xinjian Mao, Sarah McManaway, William Robert Wilson, Kevin Owen Hicks. Potentiation of the action of chemotherapeutic drugs by the hypoxia-activated prodrug SN30000 in multicellular tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2440.
Hypoxia-activated prodrugs (HAPs) are hypothesized to improve the therapeutic index of chemotherapy drugs that are ineffective against tumor cells in hypoxic microenvironments. SN30000 (CEN-209) is a benzotriazine di-N-oxide HAP that potentiates radiotherapy in preclinical models, but its combination with chemotherapy has not been explored. Here we apply multiple models (monolayers, multicellular spheroids and tumor xenografts) to identify promising SN30000/chemotherapy combinations (with chemotherapy drugs before, during or after SN30000 exposure). SN30000, unlike doxorubicin, cisplatin, gemcitabine or paclitaxel, was more active against cells in spheroids than monolayers by clonogenic assay. Combinations of SN30000 and chemotherapy drugs in HCT116/GFP and SiHa spheroids demonstrated hypoxia-and schedule-dependent potentiation of gemcitabine or doxorubicin in growth inhibition and clonogenic assays. Co-administration with SN30000 suppressed clearance of gemcitabine in NIH-III mice, likely due to SN30000-induced hypothermia which also modulated extravascular transport of gemcitabine in tumor tissue as assessed from its diffusion through HCT116 multicellular layer cultures. Despite these systemic effects, the same schedules that gave therapeutic synergy in spheroids (SN30000 3 h before or during gemcitabine, but not gemcitabine 3 h before SN30000) enhanced growth delay of HCT116 xenografts without increasing host toxicity. Identification of hypoxic and S-phase cells by immunohistochemistry and flow cytometry established that hypoxic cells initially spared by gemcitabine subsequently reoxygenate and re-enter the cell cycle, and that this repopulation is prevented by SN30000 only when administered with or before gemcitabine. This illustrates the value of spheroids in modeling tumor microenvironment-dependent drug interactions, and the potential of HAPs for overcoming hypoxia-mediated drug resistance.
The value of automatic organ-at-risk outlining software for radiotherapy is based on artificial intelligence technology in clinical applications. The accuracy of automatic segmentation of organs at risk (OARs) in radiotherapy for nasopharyngeal carcinoma was investigated. In the automatic segmentation model which is proposed in this paper, after CT scans and manual segmentation by physicians, CT images of 147 nasopharyngeal cancer patients and their corresponding outlined OARs structures were selected and grouped into a training set (115 cases), a validation set (12 cases), and a test set (20 cases) by complete randomization. Adaptive histogram equalization is used to preprocess the CT images. End-to-end training is utilized to improve modeling efficiency and an improved network based on 3D Unet (AUnet) is implemented to introduce organ size as prior knowledge into the convolutional kernel size design to enable the network to adaptively extract features from organs of different sizes, thus improving the performance of the model. The DSC (Dice Similarity Coefficient) coefficients and Hausdorff (HD) distances of automatic and manual segmentation are compared to verify the effectiveness of the AUnet network. The mean DSC and HD of the test set were 0.86 ± 0.02 and 4.0 ± 2.0 mm, respectively. Except for optic nerve and optic cross, there was no statistical difference between AUnet and manual segmentation results ( P > 0.05). With the introduction of the adaptive mechanism, AUnet can achieve automatic segmentation of the endangered organs of nasopharyngeal carcinoma based on CT images more accurately, which can substantially improve the efficiency and consistency of segmentation of doctors in clinical applications.
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