Principal component-based image segmentation: a new approach to outline in vitro cell colonies

The in vitro clonogenic assay is a technique to study the ability of a cell to form a colony in a culture dish. By optical imaging, dishes with stained colonies can be scanned and assessed digitally. Identification, segmentation and counting of stained colonies play a vital part in high-throughput screening and quantitative assessment of biological assays. Image processing of such pictured/scanned assays can be affected by image/scan acquisition artifacts like background noise and spatially varying illumination, and contaminants in the suspension medium. Although existing approaches tackle these issues, the segmentation quality requires further improvement, particularly on noisy and low contrast images. In this work, we present an objective and versatile machine learning procedure to amend these issues by characterizing, extracting and segmenting inquired colonies using principal component analysis, k-means clustering and a modified watershed segmentation algorithm. The intention is to automatically identify visible colonies through spatial texture assessment and accordingly discriminate them from background in preparation for successive segmentation. The proposed segmentation algorithm yielded a similar quality as manual counting by human observers. High F1 scores (>0.9) and low root-mean-square errors (around 14%) underlined good agreement with ground truth data. Moreover, it outperformed a recent state-of-the-art method. The methodology will be an important tool in future cancer research applications

Principal component-based image segmentation: a new approach to outline in vitro cell colonies

publishedVersio

Advancing Creativity Theory and Research: A Socio-cultural Manifesto

This manifesto, discussed by 20 scholars, representing diverse lines of creativity research, marks a conceptual shift within the field. Socio-cultural approaches have made substantial contributions to the concept of creativity over recent decades and today can provide a set of propositions to guide our understanding of past research and to generate new directions of inquiry and practice. These propositions are urgently needed in response to the transition from the Information Society to the Post-Information Society. Through the propositions outlined here, we aim to build common ground and invite the community of creativity researchers and practitioners to reflect up, study, and cultivate creativity as a socio-cultural phenomenon

Solid Stress and Nanoparticle Microdistribution in Xenografts: Effects of Ultrasound and Microbubble Cavitation

A drug delivery system (DDS) was characterized for two different xenografts in mice: OHS and PC3. Nanoparticles (NP) encapsulating fluorescent model drugs were used as stabilizing shells around gas microbubbles (MB), and ultrasound (US) was used to locally cavitate and collapse the MBs at the tumor site. MB cavitation is known to induce several effects which are beneficial for NP drug delivery, causing increased accumulation, extravasation and extracellular matrix (ECM) penetration by the NPs. These effects all contribute to a higher tumor specificity and ability to reach all tumor cells, allowing for a safer and more efficient method of cancer treatment. Frozen tumor sections were imaged using confocal laser scanning microscopy (CLSM), and analyzed in terms of NP microdistribution. The distribution of solid stress (SS) in the tumors was characterized using the Planar cut method and subsequent US imaging. Tile scans from frozen tumor sections were fitted to surface plots (SP) of SS distribution in the same tumor, and a connection between SS levels and NP microdistribution was established. The baseline enhanced permeability and retention (EPR) effect of both tumor models was established, and found significantly higher for OHS tissue: the NP accumulation was 64.9% higher, the NP extravasation was 50.3% higher, and the ECM penetration was 35.2% higher in OHS tissue (p=0.01, p<0.0001 and p<0.0001, respectively, t-test). The DDS with US proved to be more effective for tissue with a lower baseline EPR effect. SS distribution was shown to be highly heterogeneous between tumors, and the level of SS was significantly higher in OHS than in PC3 tumors, by 99.5% (p<0.0001, t-test). For the first time, it was shown that treatment with US induced MB collapse lowered the SS in both tumor models: by 11.1 % in OHS tumors and 8.8 % in PC3 tumors (p=0.029 and p=0.030, respectively, t-test). The correlation between SS levels and NP microdistribution was heterogeneous. PC3 tumors showed no effect of the US treatment on the correlation between SS level and NP accumulation, extravasation or ECM penetration, which were all negative for both groups. OHS tumors, on the other hand, experienced a change from positive to negative correlation between SS level and NP accumulation, and a change from no correlation to a positive correlation between SS level and NP extravasation after treatment

The Role of TGF-β3 in Radiation Response

Transforming growth factor-beta 3 (TGF-β3) is a ubiquitously expressed multifunctional cytokine involved in a range of physiological and pathological conditions, including embryogenesis, cell cycle regulation, immunoregulation, and fibrogenesis. The cytotoxic effects of ionizing radiation are employed in cancer radiotherapy, but its actions also influence cellular signaling pathways, including that of TGF-β3. Furthermore, the cell cycle regulating and anti-fibrotic effects of TGF-β3 have identified it as a potential mitigator of radiation- and chemotherapy-induced toxicity in healthy tissue. This review discusses the radiobiology of TGF-β3, its induction in tissue by ionizing radiation, and its potential radioprotective and anti-fibrotic effects

Low-Dose-Rate Radiation-Induced Secretion of TGF-&beta;3 Together with an Activator in Small Extracellular Vesicles Modifies Low-Dose Hyper-Radiosensitivity through ALK1 Binding

Hyper-radiosensitivity (HRS) is the increased sensitivity to low doses of ionizing radiation observed in most cell lines. We previously demonstrated that HRS is permanently abolished in cells irradiated at a low dose rate (LDR), in a mechanism dependent on transforming growth factor &beta;3 (TGF-&beta;3). In this study, we aimed to elucidate the activation and receptor binding of TGF-&beta;3 in this mechanism. T-47D cells were pretreated with inhibitors of potential receptors and activators of TGF-&beta;3, along with addition of small extracellular vesicles (sEVs) from LDR primed cells, before their radiosensitivity was assessed by the clonogenic assay. The protein content of sEVs from LDR primed cells was analyzed with mass spectrometry. Our results show that sEVs contain TGF-&beta;3 regardless of priming status, but only sEVs from LDR primed cells remove HRS in reporter cells. Inhibition of the matrix metalloproteinase (MMP) family prevents removal of HRS, suggesting an MMP-dependent activation of TGF-&beta;3 in the LDR primed cells. We demonstrate a functional interaction between TGF-&beta;3 and activin receptor like kinase 1 (ALK1) by showing that TGF-&beta;3 removes HRS through ALK1 binding, independent of ALK5 and TGF-&beta;RII. These results are an important contribution to a more comprehensive understanding of the mechanism behind TGF-&beta;3 mediated removal of HRS

Sonopermeation with nanoparticle-stabilized microbubbles reduces solid stress and improves nanomedicine delivery to tumors

Drug delivery to tumors is challenging due to biological barriers obstructing effective delivery. Sonopermeation with ultrasound and microbubbles has been shown to improve therapeutic effect of many classes of drugs, but the underlying mechanism is not fully understood. In this study, two subcutaneous xenograft tumor models, that differed substantially in blood vessel density and stiffness, is treated with poly(alkyl cyanoacrylate) nanoparticles and nanoparticle-stabilized microbubbles combined with ultrasound. Improved nanoparticle accumulation and extracellular matrix (ECM) penetration is found. The stiffness and solid stress in the tumors are measured and it is discovered that sonopermeation can reduce the solid stress in both models, with the highest effect in the stiffest tumor model. This suggests that sonopermeation affects not only the blood vessel wall which has been described previously, but also the ECM to reduce solid stress and increase diffusion and transport of nanomedicines.publishedVersio

Sonopermeation with nanoparticle-stabilized microbubbles reduces solid stress and improves nanomedicine delivery to tumors

Drug delivery to tumors is challenging due to biological barriers obstructing effective delivery. Sonopermeation with ultrasound and microbubbles has been shown to improve therapeutic effect of many classes of drugs, but the underlying mechanism is not fully understood. In this study, two subcutaneous xenograft tumor models, that differed substantially in blood vessel density and stiffness, is treated with poly(alkyl cyanoacrylate) nanoparticles and nanoparticle-stabilized microbubbles combined with ultrasound. Improved nanoparticle accumulation and extracellular matrix (ECM) penetration is found. The stiffness and solid stress in the tumors are measured and it is discovered that sonopermeation can reduce the solid stress in both models, with the highest effect in the stiffest tumor model. This suggests that sonopermeation affects not only the blood vessel wall which has been described previously, but also the ECM to reduce solid stress and increase diffusion and transport of nanomedicines

Sonopermeation with nanoparticle-stabilized microbubbles reduces solid stress and improves nanomedicine delivery to tumors

Drug delivery to tumors is challenging due to biological barriers obstructing effective delivery. Sonopermeation with ultrasound and microbubbles has been shown to improve therapeutic effect of many classes of drugs, but the underlying mechanism is not fully understood. In this study, two subcutaneous xenograft tumor models, that differed substantially in blood vessel density and stiffness, is treated with poly(alkyl cyanoacrylate) nanoparticles and nanoparticle-stabilized microbubbles combined with ultrasound. Improved nanoparticle accumulation and extracellular matrix (ECM) penetration is found. The stiffness and solid stress in the tumors are measured and it is discovered that sonopermeation can reduce the solid stress in both models, with the highest effect in the stiffest tumor model. This suggests that sonopermeation affects not only the blood vessel wall which has been described previously, but also the ECM to reduce solid stress and increase diffusion and transport of nanomedicines

TGF-β3 increases the severity of radiation-induced oral mucositis and salivary gland fibrosis in a mouse model

Toxicities from head and neck (H&N) radiotherapy (RT) may affect patient quality of life and can be dose-limiting. Proteins from the transforming growth factor beta (TGF-β) family are key players in the fibrotic response. While TGF-β1 is known to be pro-fibrotic, TGF-β3 has mainly been considered anti-fibrotic. Moreover, TGF-β3 has been shown to act protective against acute toxicities after radio- and chemotherapy. In the present study, we investigated the effect of TGF-β3 treatment during fractionated H&N RT in a mouse model. 30 C57BL/6J mice were assigned to three treatment groups. The RT + TGF-β3 group received local fractionated H&N RT with 66 Gy over five days, combined with TGF-β3-injections at 24-hour intervals. Animals in the RT reference group received identical RT without TGF-β3 treatment. The non-irradiated control group was sham-irradiated according to the same RT schedule. In the follow-up period, body weight and symptoms of oral mucositis and lip dermatitis were monitored. Saliva was sampled at five time points. The experiment was terminated 105 d after the first RT fraction. Submandibular and sublingual glands were preserved, sectioned, and stained with Masson’s trichrome to visualize collagen. A subset of mice in the RT + TGF-β3 group displayed increased severity of oral mucositis and increased weight loss, resulting in a significant increase in mortality. Collagen content was significantly increased in the submandibular and sublingual glands for the surviving RT + TGF-β3 mice, compared with non-irradiated controls. In the RT reference group, collagen content was significantly increased in the submandibular gland only. Both RT groups displayed lower saliva production after treatment compared to controls. TGF-β3 treatment did not impact saliva production. When repeatedly administered during fractionated RT at the current dose, TGF-β3 treatment increased acute H&N radiation toxicities and increased mortality. Furthermore, TGF-β3 treatment may increase the severity of radiation-induced salivary gland fibrosis.</p