Development of a Folding Paper System To Enable the Analysis of Gene Profile of Short- and Long-Distance Cancer Cell Migration.

In cancer metastasis, where mortality rates remain high despite advancements in medical treatments, understanding the molecular pathways and cellular dynamics underlying tumor spread is critical for devising more effective therapeutic strategies. Here, a folding paper system was proposed and developed to mimic native tumor microenvironment. This system, composed of 7 stacked layers of paper enclosed in a holder, allows for the culture of cancer cells under conditions mimicking those found in solid tumors, including limited oxygen and nutrients. Because of the migratory capabilities of cancer cells, the cells in the center layer could migrated to outer layers of the paper stack, enabling the differentiation of cells based on their migratory potential. Subsequent gene expression analysis, conducted through RT-PCR and RNA sequencing, revealed significant correlations between cancer cell migration distance and the expression of genes associated with hypoxia, metabolism, ATP production, and cellular process. Moreover, our study identified cells with aggressive phenotypic traits from the outer layers of the paper stack, highlighting the potential of this system for enabling the study of aggressive cancer cell characteristics. Validation of the folding paper system against clinical carcinoma tissue demonstrated its ability to faithfully mimic the native tumor microenvironment. Overall, our findings underscore the utility of the folding paper system as a valuable tool for investigating and identifying critical molecular pathways involved in cancer metastasis.

Investigation of Stem Cell-Like Characteristics and Immune Cell Interaction of Tumor Cells Survived from Continuous Shear Flow Environment.

When tumor cells are released from a primary tumor into the bloodstream or lymphatic circulation system, they are exposed to a continuous shear flow environment. This environment exerts physical stresses on the tumor cells, which can activate apoptotic pathways. However, certain tumor cells have the ability to adapt to these mechanical stresses, enhancing their likelihood of survival and promoting metastasis. In this study, these tumor cells survived from shear flow environment are examined and revealed to closely link to stem cell-like characteristics. Higher gene expression levels of self-renewal and differentiation markers and enhanced abilities of migration, spheroid formation, and colony formation are shown. Moreover, the interaction between immune cells and the surviving cells is investigated. The results show that the surviving cells possess immune escape capabilities, implying their ability to evade immune surveillance. Additionally, these surviving cells display characteristics reminiscent of stem cells. This study holds great importance in advancing the understanding of tumor biology. By comprehending the behavior and properties of these surviving cells, new therapeutic strategies can be developed to specifically target circulating tumor cells (CTCs) and enhance cancer treatment outcomes.

Cell marathon: long-distance cell migration and metastasis-associated gene analysis using a folding paper system.

A high mortality rate was found in cancer patients with distant metastasis. Development of targeted drugs for effectively inhibiting cancer metastasis is the key for increasing therapeutic success. In the current study, a folding paper system was developed to mimic a tumor-vascular interface for the study of long-distance cell migration. Correlation between the cell migration distance and metastasis-associated gene was successfully analyzed by disassembling the stacked paper construct. The result revealed that the migration distance and number of migrated cells were highly correlated to cell characteristics. Moreover, immunocytochemistry was directly conducted on the paper layer to study the signaling pathway. Kelch-like and protein tyrosine phosphatase families were examined and the PTPN13 gene was shown to regulate long-distance cell migration. By analyzing the phosphorylated mTOR, the PTPN13 gene was further confirmed to be a tumor suppressor gene that inhibits long-distance cell migration. The folding paper system provides an alternative approach for long-distance cell migration. Metastasis-associated gene expression can be analyzed to potentially develop targeted drugs for cancer metastasis inhibition.

Highly sensitive and selective electrochemical detection of lipocalin 2 by NiO nanoparticles/perovskite CeCuOx based immunosensor to diagnose renal failure.

The early detection of lipocalin 2 (LCN 2), a biomarker to diagnose acute kidney injury (AKI) and its consequences leading to renal failure, is highly challenging due to the lack of proper investigating tools. To overcome this issue, we developed nickel oxide nanoparticles modified cerium copper oxide (NiO Nps/CeCuOx) thin film-based immunosensor to determine the presence of LCN 2 in the analyte. The sol-gel deposited CeCuOx (on a silicon (Si) substrate) was post-annealed at different temperatures (700 °C, 800 °C, and 900 °C), where 800 °C showed the optimum electrochemical performance. NiO nanoparticles, prepared by co-precipitation method, were coated on the pre-silanized CeCuOx to obtain the NiO Nps/CeCuOx/Si electrode. The fast electron transfer, excellent redox property, and high surface-to-volume ratio of NiO Nps/CeCuOx based sensing electrode facilitate the successful immobilization of anti-LCN2 by carbodiimide coupling. The structural, compositional, and stoichiometric properties of the electrode were evaluated by X-ray diffraction, field emission scanning electron microscopy, X-ray photoelectron spectroscopy, high-resolution transmission electron microscopy, and ultraviolet-visible spectroscopy. The immunosensor achieved a significantly low limit of detection (LOD) of 4.23 ng ml-1 for an acceptable linear range of 25-400 ng ml-1 in voltammetric measurement. The device showed high selectivity compared to other related biomolecules. The excellent recovery rates in human serum spiked with LCN2 confirmed the feasibility of the biosensor to use in the actual clinical samples. Therefore, the NiO Nps/CeCuOx/Si based LCN2 immunosensor paves a new route to diagnose kidney-related end-stage diseases.

Analysis of the Internal Hypoxic Environment in Solid Tumor Tissue Using a Folding Paper System.

Hypoxia is a nonphysiological oxygen tension which is common in most malignant tumors. Hypoxia stimulates complicated cell signaling networks in cancer cells, e.g., the HIF, PI3K, MAPK, and NFκB pathways. Then, cells release a number of cytokines such as VEGFA to promote the growth of peripheral blood vessels and lead to metastasis. In the current work, understanding of the internal hypoxic environment in solid tumor tissue was attempted by developing a folding paper system. A paper-based solid tumor was constructed by folding a filter paper cultured with cancer cells. The cellular response in each layer could be analyzed by disassembling the folded paper after the culture course. The result showed that an internal hypoxic environment was successfully reproduced in the paper-based solid tumor. The cells in the inner layer expressed high levels of HIF1-α and VEGFA. Hence, proliferation and migration of endothelial cells were shown to be induced by the cells located in the internal hypoxic environment. Moreover, the paper-based solid tumor was transplanted into nude mice for the study of hypoxic response and angiogenesis. The crosstalk between internal and external parts of solid tumor tissue could be analyzed by sectioning each layer of the paper-based solid tumor. This approach provides a favorable analytical method for the discovery of the interaction between cancer cells, hypoxia, and peripheral angiogenesis.

Characterization of stem cell-like property in cancer cells based on single-cell impedance measurement in a microfluidic platform.

Investigation of stem cell-like property in cancer cells is important for the development of new therapeutic drugs targeting at malignant tumors. Currently, the standard approach for identifying cancer stem cell-like cells relies on the recognition of stem cell surface markers. However, the reliability remains controversial among biologists. In the current work, a dielectrophoretic and impedimetric hybrid microfluidic platform was developed for capturing single cells and characterizing their stem cell-like property. Single cells were captured in 20 µm trapping wells by dielectrophoretic force and their impedance spectra were measured by an impedance analyzer. The result showed that different cancer cell lines could be differentiated by impedance magnitude ranging between 2 and 20 kHz. Moreover, cancer cells and cancer stem cell-like cells could be categorized by a 2-dimensional graph of the impedance magnitudes at 2 and 20 kHz. The stem cell-like property in cancer cells was verified by stem cell surface markers and single-cell derived colony assay. Comparing with bio-chemical approach, i.e., surface markers, bio-physical approach, i.e., cell impedance, is a label-free technique to identify cancer stem cell-like cells.

Analysis of a Cancer Stem Cell-Derived Single Colony Raised in a Microwell Array.

Cancer stem cells (CSCs) were reported to play important roles in cancer initialization, progression, and metastasis. In order to study the variation between CSCs and non-CSCs, single-cell analysis is conducted but technically complicated. In the current work, a microwell array made by an agarose hydrogel was developed for the study of a CSC-derived single colony. This approach is simple, convenient, and compatible with the setting and skill set of existing biological laboratories. Single cells, double cells, and multiple cells were distributed in the microwells. Isolation of CSCs could be achieved after a 5 day starvation culture course. Then, a CSC-derived single colony was formed by culturing the CSCs in a nutritious culture medium for another 5 days. The results revealed that a single CSC presented a high colony formation rate. Multiple cells containing CSCs and non-CSCs could raise a larger single colony than the multiple cells with CSCs only. Although CSCs possess an aggressive characteristic, development of a solid tumor requires the proactive involvement of non-CSCs. This work showed a practical demonstration of using a microwell array for the investigation of a CSC-derived single colony.

Impedimetric quantification of migration speed of cancer cells migrating along a Matrigel-filled microchannel.

Cancer metastasis, that cancer cells migrate from primary to distance site, is the major cause of death for cancer patients. Investigation of the correlation between cell migration and extracellular stimulation is critical to develop effective therapy for suppressing cancer metastasis. However, the existing cell migration assays remain limitations to faithfully investigate cell migration capability. In this work, a microfluidic device embedded with impedance measurement system was developed for the quantification of cancer cell migration process. Cancer cells were guided and migrated along a Matrigel-filled microchannel mimicking the basement membrane. The microchannel was embedded with 5 pairs of opposite electrodes. Cell migration process was monitored by impedance measurement and migration speed was calculated from the traveling distance divided by the time taken. Impedimetric quantification of cell migration under extracellular stimulation of interleukin-6 was demonstrated. The result showed a higher measurement sensitivity compared to the conventional Transwell assay. The current microfluidic device provides a reliable and quantitative assessment of cellular response under tested conditions. It is potentially beneficial to the study of suppressing cancer metastasis.

A Review on Microdevices for Isolating Circulating Tumor Cells.

Cancer metastasis is the primary cause of high mortality of cancer patients. Enumeration of circulating tumor cells (CTCs) in the bloodstream is a very important indicator to estimate the therapeutic outcome in various metastatic cancers. The aim of this article is to review recent developments on the CTC isolation technologies in microdevices. Based on the categories of biochemical and biophysical isolation approaches, a literature review and in-depth discussion will be included to provide an overview of this challenging topic. The current excellent developments suggest promising CTC isolation methods in order to establish a precise indicator of the therapeutic outcome of cancer patients.

Association of subcutaneous and visceral adipose tissue with overall survival in Taiwanese patients with bone metastases - results from a retrospective analysis of consecutively collected data.

BACKGROUND: Growing evidence indicates that measures of body composition may be related to clinical outcomes in patients with malignancies. The aim of this study was to investigate whether measures of regional adiposity-including subcutaneous adipose tissue index (SATI) and visceral adipose tissue index (VATI)-can be associated with overall survival (OS) in Taiwanese patients with bone metastases. METHODS: This is a retrospective analysis of prospectively collected data. We examined 1280 patients with bone metastases who had undergone radiotherapy (RT) between March 2005 and August 2013. Body composition (SATI, VATI, and muscle index) was assessed by computed tomography at the third lumbar vertebra and normalized for patient height. Patients were divided into low- and high-adiposity groups (for both SATI and VATI) according to sex-specific median values. RESULTS: Both SATI (hazard ratio [HR]: 0.696; P<0.001) and VATI (HR: 0.87; P = 0.037)-but not muscle index-were independently associated with a more favorable OS, with the former showing a stronger relationship. The most favorable OS was observed in women with high SATI (11.21 months; 95% confidence interval: 9.434-12.988; P<0.001). CONCLUSIONS: High SATI and VATI are associated with a more favorable OS in Taiwanese patients with bone metastases referred for RT. The question as to whether clinical measures aimed at improving adiposity may improve OS in this clinical population deserves further scrutiny.

Visualization and Quantification of 3D Tumor Cell Migration under Extracellular Stimulation.

To investigate tumor cell migration capability, the scratch/wound healing assay and the Transwell assay are the most commonly used assays in the current biomedical research laboratory. However, both assays have their limitations and may mislead the interpretation of the results. In the current study, visualization and quantification of tumor cell migration process was realized in a three-dimensional (3D) environment. The tumor cells horizontally migrated along a Matrigel-filled microchannel under extracellular stimulation. The cell migration process was visualized under a microscope, and the migration speed could be calculated based on the traveling distance of the cells and the time required. Here, three demonstrations were conducted, respectively, including cells attracted by nutrient gradient, stimulated by cytokine, and coculturing with fibroblasts. The results revealed that the cell migration capability could be visually and quantitatively correlated to the extracellular stimulation. The current protocol is compatible to the existing laboratory setup and provides a persuasive result for the study of the 3D cell migration process. Understanding of the molecular and intercellular mechanism of cancer metastasis can potentially develop effective therapeutic strategy.

Investigation of uniform sized multicellular spheroids raised by microwell arrays after the combined treatment of electric field and anti-cancer drug.

Nowadays, cancer disease is continuously identified as the leading cause of mortality worldwide. Cancer chemotherapeutic agents have been continuously developing to achieve high curative effectiveness and low side effects. However, solid tumors present the properties of low drug penetration and resistance of quiescent cells. Radiation therapy is concurrently given in some cases; but it induces different levels of adverse effects. In the current work, uniform sized multicellular spheroids were raised by microwell arrays to mimic the architecture of solid tumors. Investigation of the response of the spheroids was conducted after the treatment of alternating electric field. The result showed that the electric field could induce early apoptosis by disturbing cell membrane. Moreover, combined treatment of electric field and anti-cancer drug was applied to the spheroids. The electric field synergistically enhanced the treatment efficacy because the anti-cancer drug could permeate through the disrupted cell membrane. Significant improvement of late apoptosis was shown by the combined treatment. Because the electric field treatment induces limited side effect to the patient, lower dosage of anti-cancer drug may be applied to the patients for achieving curative effectiveness.

Paper/polymer composited microfluidic platform for screening cell viability and protein expression under a chemical gradient environment.

Culturing cells in three-dimensional (3D) environment can obtain a better clinical prediction for evaluating chemotherapy protocols and become a standard culture practice in cancer research. However, it involves tedious and time consuming operation. In the current work, a paper/polymer composited microfluidic platform was developed for screening cell viability and protein expression under chemical gradient environment. Cells were cultured in a paper sheet and expressed cell properties in 3D environment. The paper sheet was encapsulated in the microfluidic platform generating chemical gradient. After the culture course, investigations of cell viability and protein expression were respectively achieved by directly adding reagent and conducting on-paper immunoassay. Activation of respective signaling pathway could be identified and responded to different stimulations including nutrient gradient, IL-6 cytokine gradient, and anti-cancer drug gradient. On-paper analysis of protein expression could be completed within 1.5 h. The present technique integrates tedious operations on a single paper substrate. It provides a first-tier screening tool for cellular response under chemical gradient.

Real-time detection of antibiotics cytotoxicity in rabbit periosteal cells using microfluidic devices with comparison to conventional culture assays.

BACKGROUND: Local antibiotic application has been widely used in orthopedic surgery. The dose-related toxicity of antibiotics towards periosteal tissues and resulting effects on osteogenic expression are yet to be studied. METHODS: Periosteal cells harvested from the medial tibia of New Zealand White rabbits were used. A seeding density of 5 × 103 cells/cm2 was determined to be optimal for testing in the pilot study; the cells were cultured in xCELLigence 96-well plates. Microfluidic impedance analyzers were used to monitor cellular proliferation in microfluidic culture systems with exposure to three different concentrations (10 µg/mL, 100 µg/mL, and 1000 µg/mL) of cefazolin, ciprofloxacin, and vancomycin, respectively. The correlation of cell index at day 7 with optical density values from WST-1 assays using conventional cultures was evaluated by calculating the Pearson's coefficient. RNA analysis was performed to investigate the expression of osteogenic markers in the cultured cells, including core-binding factor alpha 1 (Cbfa1), osteopontin (OPN), and osteopontin promoter (OPNp), relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous control. RESULTS: A significant dose-related inhibition of cell index was found for all the 3 antibiotics, whereas the WST-1 assays showed a significant dose-related inhibition of cellular proliferation only at a high dose of cefazolin (1000 µg/mL) and medium-to-high dose of ciprofloxacin (100 µg/mL and 1000 µg/mL). Pearson's coefficient analysis indicated a high correlation between the cell index and optical density values of WST-1 assays only for medium and high doses of ciprofloxacin (100 µg/mL and 1000 µg/mL); a moderate correlation was seen for cefazolin, and a low dose of ciprofloxacin (10 µg/mL). RNA analysis confirmed significant dose-related inhibition of cfba1, OPN, and OPNp expression by all three antibiotics. CONCLUSION: With optimal seeding amounts, rabbit periosteal cells can be dynamically monitored in the xCELLigence microfluidic system. Dose-related inhibition of cellular proliferation and osteogenic expression was found after exposure to cefazolin and ciprofloxacin. By providing real-time detection and exhibiting comparable correlation, microfluidic impedance-based analyzer is a feasible alternative to the conventional WST-1 assays.

Prognostic significance of pretreatment neutrophil-to-lymphocyte ratio in older patients with metastatic cancer.

BACKGROUND: Treatment options for older patients with malignancies remain suboptimal. An accurate prognostic stratification could inform treatment decisions, which can potentially improve patient outcomes. Here, we sought to investigate whether the neutrophil-to-lymphocyte ratio (NLR) may have prognostic significance in patients with metastatic malignant tumors, with a special focus on older individuals. METHODS: We retrospectively reviewed the clinical records of 3981 patients with histology-proven metastatic cancer who underwent radiotherapy between 2000 and 2013. The pretreatment NLR was determined within 7 days before treatment initiation. Patients aged ≥65 years were considered as older. We analyzed the prognostic significance of NLR for overall survival (OS) across all age groups. RESULTS: Compared with their younger counterparts, older patients showed a higher NLR (P < 0.001) and a lower OS (P < 0.001). Multivariate analysis revealed that a pretreatment NLR below the median was an independent favorable predictor of OS in both older (hazard ratio [HR]: 0.669, 95.0% CI: 0.605-0.740; P < 0.001) and younger patients (HR: 0.704; 95.0% CI: 0.648-0.765; P < 0.001). Regardless of age, patients who underwent systemic therapy showed more favorable OS, especially when NLR was low. In the older subgroup, the OS of patients with a low pretreatment NLR who did not undergo systemic therapy and of those with high pretreatment NLR who underwent systemic therapy was similar. CONCLUSION: A low pretreatment NLR predicts a more favorable OS in older patients with metastatic cancer. The most favorable OS was observed in patients with a low pretreatment NLR who received systemic therapy.

The influence of laser intensity activated plasmonic gold nanoparticle-generated photothermal effects on cellular morphology and viability: a real-time, long-term tracking and monitoring system.

In this study, a microfluidic apparatus embedded with microstructures was designed and aligned with a laser and dark-field microscope for real-time, long-term observation of photothermal effects on cells. Gold nanorods (AuNRs, 10 ppm) were incubated with MG-63 human osteosarcoma cells for 3 h. Then, the cells were exposed to a continuous-wave laser at a wavelength of 830 nm for 10, 20, and 30 min at 5, 9, 14, 24, and 32 W cm-2. Subsequent changes in morphology were observed. Under different conditions, cell membrane blebbing occurred at different times, indicating that actin filaments were destroyed in large quantities and apoptosis was induced. In suitable conditions, we first induced slight cell injury by causing cytoskeletal fractures with a high-energy laser; then, the cells were irradiated with a low-energy laser at 0.3 W cm-2. We found that among cells treated with the high-energy laser, cells treated additionally with a low-energy laser showed extended viability compared with cells that did not receive the additional treatment.

Apoptosis and cell cycle arrest of hepatocellular carcinoma spheroids treated by an alternating electric field.

Most of the current cancer therapies may induce serious side effects and affect patient quality of life. Recently, a novel treatment using an alternating low-intensity and intermediate-frequency electric field was proposed and found to be a noninvasive and minimally toxic approach. However, additional fundamental studies and scientific evidence are required to further support the development of this treatment into a standard cancer therapy. In the current work, an in-house fabricated culture plate was developed to study the responses of hepatocellular carcinoma spheroids to treatment with an alternating electric field. From the results of the viability study, the electric field was confirmed to influence the dividing cells in the spheroids. Fluorescent staining of live and dead cells revealed that a fraction of the cells were damaged in the field-treated spheroids. Moreover, flow cytometry analyses were conducted and showed that a fraction of the cells in the spheroids underwent apoptosis and cell cycle arrest. Additionally, the apoptosis pathway (Bax/caspase) and cell cycle arrest pathway (p53/p21) were found to be activated after exposure to the electric field. In summary, the results further elucidated the cellular and molecular mechanism inducing apoptosis and cell cycle arrest in the field-treated hepatocellular carcinoma spheroids. This study provides more evidence to support the efficacy of electric-field-based cancer therapy.

Non-Invasive Quantification of the Growth of Cancer Cell Colonies by a Portable Optical Coherence Tomography.

Investigation of tumor development is essential in cancer research. In the laboratory, living cell culture is a standard bio-technology for studying cellular response under tested conditions to predict in vivo cellular response. In particular, the colony formation assay has become a standard experiment for characterizing the tumor development in vitro. However, quantification of the growth of cell colonies under a microscope is difficult because they are suspended in a three-dimensional environment. Thus, optical coherence tomography (OCT) imaging was develop in this study to monitor the growth of cell colonies. Cancer cell line of Huh 7 was used and the cells were applied on a layer of agarose hydrogel, i.e., a non-adherent surface. Then, cell colonies were gradually formed on the surface. The OCT technique was used to scan the cell colonies every day to obtain quantitative data for describing their growth. The results revealed the average volume increased with time due to the formation of cell colonies day-by-day. Additionally, the distribution of cell colony volume was analyzed to show the detailed information of the growth of the cell colonies. In summary, the OCT provides a non-invasive quantification technique for monitoring the growth of the cell colonies. From the OCT images, objective and precise information is obtained for higher prediction of the in vivo tumor development.

Proliferation arrest, selectivity, and chemosensitivity enhancement of cancer cells treated by a low-intensity alternating electric field.

Elimination of serious side effects is a desired feature of cancer therapy. Alternating electric field treatment is one approach to the non-invasive treatment of cancer. The efficacy and safety of this novel therapy are confirmed for the treatment of glioblastoma multiforme. In the current study, we co-cultured cancer cells and normal cells to investigate the selectivity and chemosensitivity enhancement of an electric field treatment. Cancer cells (cell line: HeLa and Huh7) and fibroblasts (cell line: HEL299) were cultured in an in-house-developed cell culture device embedded with stimulating electrodes. A low-intensity alternating electric field was applied to the culture. The field significantly induced proliferation arrest of the cancer cells, while had limited influence on the fibroblasts. Moreover, in combination with the anti-cancer drug, damage to the cancer cells was enhanced by the electric field. Thus, a lower dosage of the drug could be applied to achieve the same treatment effectiveness. This study provides evidence that low-intensity electric field treatment selectively induced proliferation arrest and enhanced the chemosensitivity of the cancer cells. This electro-chemotherapy could be developed and applied as a regional cancer therapy with minimal side effects.

Dissociated effect and Chemosensitive enhancement of tumor spheroids influenced by an electric field in a microdevice.

The use of electric field for cancer therapy has been proposed for a novel non-invasive cancer therapeutic approach that provides better quality of life for patients. However, argument of the efficacy hampers the therapeutic development for various cancer diseases. More scientific evidences are necessary to be addressed by basic research. The current in vitro cell culture study reports the responses of tumor spheroids after the application of an alternating electric field. Human hepatocarchinoma cells suspended in soft hydrogel were cultured in a cell culture device embedded with stimulating electrodes. Tumor spheroids gradually formed and alternating electric field was then applied during the culture course. Investigation of cell viability and cell cycle were conducted to optimize the treatment conditions. The results showed that the electric potential of 1.0 Vpp and frequency of 130 kHz was the minimal effective conditions for inhibiting tumor spheroids. Importantly, dissociation of tumor spheroids was observed after the treatment. The effectiveness of chemotherapeutic agents was shown to be enhanced while the electric filed was simultaneously applied to the tumor spheroids. These results provided solid foundation for developing the effective therapeutic strategies.