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.

Bionanotechnology and bioMEMS (BNM): state-of-the-art applications, opportunities, and challenges.

The development of micro- and nanotechnology for biomedical applications has defined the cutting edge of medical technology for over three decades, as advancements in fabrication technology developed originally in the semiconductor industry have been applied to solving ever-more complex problems in medicine and biology. These technologies are ideally suited to interfacing with life sciences, since they are on the scale lengths as cells (microns) and biomacromolecules (nanometers). In this paper, we review the state of the art in bionanotechnology and bioMEMS (collectively BNM), including developments and challenges in the areas of BNM, such as microfluidic organ-on-chip devices, oral drug delivery, emerging technologies for managing infectious diseases, 3D printed microfluidic devices, AC electrokinetics, flexible MEMS devices, implantable microdevices, paper-based microfluidic platforms for cellular analysis, and wearable sensors for point-of-care testing.

Analysis of Cellular Crosstalk and Molecular Signal between Periosteum-Derived Precursor Cells and Peripheral Cells During Bone Healing Process Using a Paper-Based Osteogenesis-On-A-Chip Platform.

Periosteum-derived progenitor cells (PDPCs) are highly promising cell sources that are indispensable in the bone healing process. Adipose-derived stem cells (ADSCs) are physiologically close to periosteum tissue and release multiple growth factors to promote the bone healing process. Co-culturing PDPCs and ADSCs can construct periosteum-bone tissue microenvironments for the study of cellular crosstalk and molecular signal in the bone healing process. In the current work, a paper-based osteogenesis-on-a-chip platform was successfully developed to provide an in vitro three-dimensional coculture model. The platform was a paper substrate sandwiched between PDPC-hydrogel and ADSC-hydrogel suspensions. Cell secretion could be transferred through the paper substrate from one side to another side. Growth factors including BMP2, TGF-ß, POSTN, Wnt proteins, PDGFA, and VEGFA were directly analyzed by a paper-based immunoassay. Cellular crosstalk was studied by protein expression on the paper substrate. Moreover, osteogenesis of PDPCs was investigated by examining the mRNA expressions of PDPCs after culture. Neutralizing and competitive assays were conducted to understand the correlation between growth factors secreted from ADSCs and the osteogenesis of PDPCs. In vitro periosteum-bone tissue microenvironment was established by the paper-based osteogenesis-on-a-chip platform. The proposed approach provides a promising assay of cellular crosstalk and molecular signal in 3D coculture microenvironment that may potentially lead to the development of effective bone regeneration therapy.

A Review on Microfluidics-Based Impedance Biosensors.

Electrical impedance biosensors are powerful and continuously being developed for various biological sensing applications. In this line, the sensitivity of impedance biosensors embedded with microfluidic technologies, such as sheath flow focusing, dielectrophoretic focusing, and interdigitated electrode arrays, can still be greatly improved. In particular, reagent consumption reduction and analysis time-shortening features can highly increase the analytical capabilities of such biosensors. Moreover, the reliability and efficiency of analyses are benefited by microfluidics-enabled automation. Through the use of mature microfluidic technology, complicated biological processes can be shrunk and integrated into a single microfluidic system (e.g., lab-on-a-chip or micro-total analysis systems). By incorporating electrical impedance biosensors, hand-held and bench-top microfluidic systems can be easily developed and operated by personnel without professional training. Furthermore, the impedance spectrum provides broad information regarding cell size, membrane capacitance, cytoplasmic conductivity, and cytoplasmic permittivity without the need for fluorescent labeling, magnetic modifications, or other cellular treatments. In this review article, a comprehensive summary of microfluidics-based impedance biosensors is presented. The structure of this article is based on the different substrate material categorizations. Moreover, the development trend of microfluidics-based impedance biosensors is discussed, along with difficulties and challenges that may be encountered in the future.

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.

Excellent physicochemical and sensing characteristics of a Re x O y based pH sensor at low post-deposition annealing temperature.

pH monitoring in clinical assessment is pivotal as pH imbalance significantly influences the physiological and extracellular functions of the human body. Metal oxide based pH sensors, a promising alternative to bulky pH electrodes, mostly require complex fabrication, high-temperature post-deposition treatment, and high expenses that inhibit their practical applicability. So, there is still room to develop a straightforward and cost-effective metal oxide based pH sensor comprising high sensitivity and reliability. In this report, a novel solution-processed and low-temperature annealed (220 °C) mixed-valence (vii/vi) oxide of rhenium (Re x O y ) was applied in an electrolyte-insulator-semiconductor (EIS) structure. The annealing effect on morphological, structural, and compositional properties was scrutinized by physical and chemical characterizations. The post-annealed Re x O y exhibited a high pH sensitivity (57.3 mV pH-1, R 2 = 0.99), a lower hysteresis (4.7 mV), and a reduced drift rate (1.7 mV h-1) compared to the as-prepared sample for an analytically acceptable pH range (2-12) along with good stability and reproducibility. The magnified sensing performance originated due to the valence state of Re from Re6+ to Re7+ attributed to each electron transfer for a single H+ ion. The device showed high selectivity for H+ ions, which was confirmed by the interference study with other relevant ions. The feasibility of the sensor was verified by measuring the device in real samples. Hence, the ease-of-fabrication and notable sensing performance of the proposed sensor endorsed its implementation for diagnosing pH-related diseases.

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.

Leukocyte-Rich Platelet-Rich Plasma Has Better Stimulating Effects on Tenocyte Proliferation Compared With Leukocyte-Poor Platelet-Rich Plasma.

Background: Rotator cuff (RC) tendinopathy is one of the most common causes of shoulder pain. Platelet-rich plasma (PRP) has been frequently used in clinical scenarios, but its efficacy remains inconsistent. Purpose: To investigate the different responses of human tenocytes from torn RCs to leukocyte-rich PRP (LR-PRP) and leukocyte-poor PRP (LP-PRP) in a 2-chamber coculture device. Study Design: Controlled laboratory study. Methods: PRP was prepared using different platelet and leukocyte concentrations according to 5 groups: (1) LR-PRP with 5000 platelets/µL, (2) LR-PRP with 10,000 platelets/µL, (3) LP-PRP with 5000 platelets/µL, (4) LP-PRP with 10,000 platelets/µL, and (5) control with only culture medium supplementation and without PRP stimulation. Platelet-derived growth factor-AB (PDGF-AB) and transforming growth factor-ß1 (TGF-ß1) were measured in LR-PRP and LP-PRP via enzyme-linked immunosorbent assay. Microscopy, water-soluble tetrazolium salt assay, and quantitative real-time polymerase chain reaction were used to investigate the morphology, proliferation, and gene expression of RC tenocytes exposed to different PRP formulations. Data were collected from at least 3 independent measurements. The results were analyzed via 1-way analysis of variance, followed by the post hoc Bonferroni test. Results: The ratio of leukocytes to 5000 platelets/µL was 29.5 times higher in LR-PRP than in LP-PRP (P < .05). In the 5000 platelets/µL groups, the levels of TGF-ß1 and PDGF-AB were both significantly higher in LR-PRP versus LP-PRP (TGF-ß1: 367.0 ± 16.5 vs 308.6 ± 30.3 pg/mL, respectively [P = .043]; PDGF-AB: 172.1 ± 1.8 vs 94.1 ± 4.2 pg/mL, respectively [P < .001]). Compared with the control group, RC tenocyte proliferation was 1.42 ± 0.01 and 1.41 ± 0.03 times higher in the LR-PRP groups with 5000 platelets/µL and 10,000 platelets/µL, respectively (P < .05). The expression of tenocyte-related genes was higher in tenocytes cultured in LR-PRP. Conclusion: Both the LR-PRP groups with 5000 platelets/µL and 10,000 platelets/µL induced more growth factor release and increased RC tenocyte proliferation than did the LP-PRP groups. Clinical Relevance: In RC repair, LR-PRP may be better than LP-PRP for increasing the proliferation of tenocytes.

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.

Experimental Characterization of an Embossed Capacitive Micromachined Ultrasonic Transducer Cell.

Capacitive Micromachined Ultrasonic Transducer (CMUT) is a promising ultrasonic transducer in medical diagnosis and therapeutic applications that demand a high output pressure. The concept of a CMUT with an annular embossed pattern on a membrane working in collapse mode is proposed to further improve the output pressure. To evaluate the performance of an embossed CMUT cell, both the embossed and uniform membrane CMUT cells were fabricated in the same die with a customized six-mask sacrificial release process. An annular nickel pattern with the dimension of 3 µm × 2 µm (width × height) was formed on a full top electrode CMUT to realize an embossed CMUT cell. Experimental characterization was carried out with optical, electrical, and acoustic instruments on the embossed and uniform CMUT cells. The embossed CMUT cell achieved 27.1% improvement of output pressure in comparison to the uniform CMUT cell biased at 170 V voltage. The fractional bandwidths of the embossed and uniform CMUT cells were 52.5% and 41.8%, respectively. It substantiated that the embossed pattern should be placed at the vibrating center of the membrane for achieving a higher output pressure. The experimental characterization indicated that the embossed CMUT cell has better operational performance than the uniform CMUT cell in collapse region.

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.