Anti-tumor withanolides as signal transducers and activators of transcription 3 (STAT3)-inhibition from Withania obtusifolia.

The Solanaceae family and the Withania genus specifically are rich sources of medicinal plants. Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS/MS) revealed a predominance of withanolides from an organic extract of Withania obtusifolia. A constructed molecular network uncovered the presence of potentially novel withanolides. A series of withanolides were then isolated and structurally characterized from the extract including two new withanolides (withafolia A and withafolia B) and seven previously reported metabolites. Of the isolated compounds, cytotoxicity of withanolide J, physaperuvin G, and a commercial STAT3 inhibitor (S3I-201) were assessed against a human leukemia HL-60 cell line resulting in IC50 values of 26, 29, and 120 µM, respectively. In silico molecular docking simulations indicate that withanolide J and physaperuvin G can bind as an inhibitor in the active site of STAT3 with docking scores comparable to the selective STAT3 inhibitor, S3I-201.

Cancer Cell Targeting Via Selective Transferrin Receptor Labeling Using Protein-Derived Carbon Dots.

Carbon dot (CD) nanoparticles offer tremendous advantages as fluorescent probes in bioimaging and biosensing; however, they lack specific affinity for biomolecules, limiting their practical applications in selective targeting. Nanoparticles with intrinsic affinity for a target have applications in imaging, cytometry, therapeutics, etc. Toward that end, we report the transferrin receptor (CD71) targeting CDs, synthesized for the first time. The formation of these particles is truly groundbreaking, as direct tuning of nanoparticle affinity was achieved by simple and careful precursor selection of a protein, which has the targeting characteristic of interest. We hypothesized that the retention of the original protein's peptides on the nanoparticle surface provides the CDs with some of the function of the precursor protein, enabling selective binding to the protein's receptor. This was confirmed with FTIR (Fourier transform infrared) data and subsequent affinity-based cell assays. These transferrin (Tf)-derived CDs have been shown to possess an affinity for CD71, a cancer biomarker that is ubiquitously expressed in nearly every cancer cell line due to its central role mediating the uptake of cellular iron. The CDs were tested using the human leukemia cell line HL60 and demonstrated the selective targeting of CD71 and specific triggering of transferrin-mediated endocytosis via clathrin-coated pits. The particle characterization results reflect a carbon-based nanoparticle with bright violet fluorescence and 7.9% quantum yield in aqueous solution. These unpresented CDs proved to retain the functional properties of the precursor protein. Indicating that this process can be repeated for other disease biomarkers for applications ranging from biosensing and diagnostic bioimaging to targeted therapeutics.

Isolation of Cancer Cells from Liquid Biopsies Using 3D-Printed Affinity Devices.

Liquid biopsies are examination procedures for deciding the grouping of malignant growth cells tracked down in samples of blood and other body fluids. Liquid biopsies are likewise significantly less intrusive than tissue biopsies as they just require small amount of blood or body fluids from the patient. With the utilization of microfluidics, cancer cells can be isolated from the fluid biopsy and achieve early diagnosis. 3D printing is turning out to be progressively well known for microfluidic devices creation. 3D printing has shown multiple advantages compared to traditional microfluidic devices production, including effortless large-scale manufacturing of precise copies, the fuse of new materials, and execution of additional complicated or drawn-out plans that are hard to execute in conventional microfluidic devices. Combining 3D printing with microfluidics makes for a relatively inexpensive analysis of liquid biopsies with a chip that can be more advantageous to use over traditional microfluidic chips. In this chapter, a method for affinity-based separation of cancer cells in a liquid biopsy using a 3D microfluidic chip will be discussed, along with the rationale behind the method.

Novel synthesis of fibronectin derived photoluminescent carbon dots for bioimaging applications.

Fibronectin (FN) derived from human plasma has been used for the first time as the carbon precursor in the top-down, microwave-assisted hydrothermal synthesis of nitrogen doped carbon dots (CDs). FN is a large glycoprotein primarily known for its roles in cell adhesion and cell growth. Due to these properties FN can be over expressed in the extracellular matrix (ECM) of some cancers allowing FN to be used as an indicator for the detection of cancerous cells over non-cancerous cells. These FN derived CDs display violet photoluminescence with UV excitation and appear to possess similar functional groups on their surface to their carbon precursor (-COOH and -NH2). This is believed to be due to the self-passivation of the CDs' nitrogen-containing surface functional groups during the heating process. These CDs were then used to stain MCF-7 and MDA-231 breast cancer cells and were observed to interact primarily with the cell membrane rather than intercalating into the cell like the many other types of CDs. This led to the hypothesis that the CDs are selectively binding to the FN overexpressed within the cancer cells' ECM via amide linkages. This is in agreement with the EDX and FTIR spectra of the FN CDs which indicate the presence of -COOH and nitrogen containing surface groups like -NH3. The inherent selectivity of the CDs combined with their ability to photoluminesce enables their use as a fluorophore for bioimaging applications.

High-recovery sorting of cancer cells from whole blood via periodic-focusing inertial microchip.

Inertial microfluidic devices continue to show promise for label-free separation of cells from liquid biopsies and other biological samples. Serpentine-channel microfluidic devices capitalizing on inertial forces such as Dean flow have been demonstrated for cell separation, but are limited in performance due to the magnitude of the inertial lift and drag force gradients across the separation channel. We have developed a new flow design that uses periodic channel contractions to enhance the magnitude of the force gradient. Separation recover was 97% with the final sorter output consisting of 78% target cells. Separation efficiency was 87% for whole blood, which could be increased to 97% if the sample was diluted prior to sorting. The enrichment of cancer cells was over 1000-fold, and sorted cancer cells maintained a viability of 93.8% for 96 hours after sorting. In the analysis of blood plasma, breast cancer cells from a clinical patient were enriched 20×. The incorporation of periodic channel contractions in a Dean flow circuit resulted in an increase in Dean flow gradient according to simulation, resulting in sorting of small-diameter cancer cells in blood samples.

Microfluidic Chips for Sepsis Diagnosis.

This chapter discusses two microfluidic-based approaches for early sepsis detection that achieve a higher accuracy than traditional blood culture analysis. Patient blood samples were included in this work to validate the performance of our chips in diagnosing sepsis. The single-parameter chip demonstrated the increased accuracy if using CD64 as a biomarker for sepsis detection compared with C-reactive protein (CRP) and procalcitonin (PCT) when applied alone. In addition, a multiparameter chip measuring a combined panel of CD25, CD64, and CD69, and achieved a high accuracy with an Area Under the Receiver Operating Characteristic Curve (AUROC) of 0.978. The combined panel was also able to detect culture-negative patients and provided a faster diagnosis. Besides, microfluidics has advantages of less time consuming, easier to manufacture, less sample loading, less complex, and portable. Therefore, our approach is of great potential to become a bedside sepsis detection method.

A comparison of transferrin-receptor and epithelial cellular adhesion molecule targeting for microfluidic separation of cancer cells.

Microfluidic, flow cytometry, and immunomagnetic methods for cancer cell isolation have heavily relied on the Epithelial Cellular Adhesion Molecule (EpCAM) for affinity separation. While EpCAM has been used extensively for circulating tumor cell isolation, it cannot be used to isolate non-epithelial cells. The human transferrin receptor (CD71) can also be used for cancer cell isolation and has the advantage that as an affinity target it can separate virtually any cancer cell type, regardless of disease origin. However, direct comparison of the capture ability of EpCAM and CD71 has not been reported previously. In this work, cell capture with both EpCAM and CD71 were studied using a novel higher-throughput herringbone cell separation microfluidic device. Five separation chip models were designed and the one with the highest capture efficiency (average 90 ± 10%) was chosen to compare antigen targets for cell capture. Multiple cancer cell lines including CCRF-CEM, PC-3 and MDA-MB-231 were tested for cell capture performance using both ligands (anti-CD71 and anti-EpCAM) in the optimized chip design. PC-3 and MDA-MB-231 cells were spiked into blood at concentrations ranging from 0.5%-10%. PC-3 cells were separated by anti-CD71 and anti-EpCAM with 32-37% and 31-50% capture purity respectively, while MDA-MB-231 were separated with 35-53% and 33-56% capture purity using anti-CD71 and anti-EpCAM for all concentrations. The enrichment factor for the lowest concentrations of cells in blood ranged from 66-74X. The resulting enrichment of cancer cells shows that anti-CD71 was found to be statistically similar to anti-EpCAM for epithelial cancer cells, while anti-CD71 can be further used for non-epithelial cells, where anti-EpCAM cannot be used.

Evaluating the Timeliness and Specificity of CD69, CD64, and CD25 as Biomarkers of Sepsis in Mice.

ABSTRACT: Sepsis occurs when an infection induces a dysregulated immune response, and is most commonly bacterial in origin. This condition requires rapid treatment for successful patient outcomes. However, the current method to confirm infection (blood culture) requires up to 48 h for a positive result and many true cases remain culture-negative. Therefore, new diagnostic tests are urgently needed. Recent clinical studies suggest that CD69, CD64, and CD25 may serve as useful biomarkers of sepsis. In this study, we evaluated the cecal ligation and puncture and cecal slurry mouse models as tools to study these biomarkers in young and aged mice, and elucidate the timeliness and specificity of sepsis diagnosis. Fluorescence-activated cell sorting analysis revealed that all three biomarkers were elevated on blood leukocytes during sepsis. CD69 was specifically upregulated during sepsis, while CD64 and CD25 were also transiently upregulated in response to sham surgery. The optimal biomarker, or combination of biomarkers, depended on the timing of detection, mouse age, and presence of surgery. CD69 demonstrated an excellent capacity to distinguish sepsis, and in some scenarios the diagnostic performance was enhanced by combining CD69 with CD64. We also analyzed biomarker expression levels on specific cell populations (lymphocytes, monocytes, and neutrophils) and determined the cell types that upregulate each biomarker. Elevations in blood biomarkers were also detected via microfluidic analyses; in this case CD64 distinguished septic mice from naive controls. Our results suggest that CD69 and CD64 are valuable biomarkers to rapidly detect sepsis, and that mouse models are useful to study and validate sepsis biomarkers.

Tandem microfluidic chip isolation of prostate and breast cancer cells from simulated liquid biopsies using CD71 as an affinity ligand.

The use of blood as a liquid biopsy provides a minimally invasive and less traumatic approach for initial cancer screens as well as patient monitoring. However, current clinical protocols require a priori knowledge of cancer type for liquid biopsy analyses. Previously, we proposed the use of the human transferrin 1 receptor protein (CD71) as a universal capture target for cancer cells analyses. In this study we have attempted to identify the lowest limit of detection for circulating tumor cells of prostate (PC-3) and breast cancers (MDA-MB-231) using CD71. We used a novel high-throughput herringbone chip design which could extract PC-3 cells at 34 ± 5% purity and MDA-MB-231 cells at 43 ± 35% purity when spiked to lysed blood at 0.1%. MDA-MB-231 cell spiked samples showed higher standard deviation, but the system captured 55 ± 16 cells, which is a sufficient number of cells for subsequent analyses. Further, this herringbone chip design has been shown to be compatible with an erythrocyte lysis chip we have described in previous studies. This circuit was capable of capturing 510 ± 120 cells with a purity of 82 ± 14% using <7 µL of a whole blood sample spiked with 10% MDA-MB-231 cells. Using an erythrocyte lysis circuit eliminates the need for human intervention for target cell enrichment, thereby reducing cell loss and sample contamination. We have shown that, when used with the high-throughput herringbone chip CD71 has the capacity to sensitively detect rare target cells for routine low-cost cancer screens.

Nanoparticle modification of microfluidic cell separation for cancer cell detection and isolation.

Cancer is a major health problem in the United States with extremely high mortality. The detection and isolation of cancer cells are becoming increasingly important for cancer diagnosis. We describe a microfluidic device modified with silica nanoparticles to enhance the isolation of cancer cells using affinity separation. The isolation of seven different cancer cell lines spiked into liquid biopsies was demonstrated and compared with unmodified separation devices. Cancer cells were isolated using CD71 which has already been demonstrated to be a widespread "net" for capturing cancer cells of any phenotype as the affinity target. The capture efficiency of our nanoparticle (NP)-modified HB chip showed significant differences compared with the normal HB chip, exhibiting an average increase of 16%. The cell enrichment increased by a factor of 2 over unmodified chips. Patient-derived ALL cells, COG-LL-332, were spiked into blood with concentrations ranging from 1% to 20% of total leukocytes, and isolated with the purity of 41%-65%. The results of this study demonstrated that the increase of cell-chip interactions after nanoparticle modification improved capture efficiency and capture purity, and can be applied to a wide range of cell separations.

Isolation of proliferating cells from whole blood using Human Transferrin Receptor in a two-stage separation system.

Blood is a routinely tested biological fluid for diagnosis and monitoring of diseases as many diseases would trigger a change in white blood cell count. Thus, several methods have been established to isolate or enrich white blood cells from patient blood samples for such analyses. One method of preparing an enriched white blood cell sample is through the selective lysis of red blood cells by hypotonic shock and restoration of osmolarity to maintain viability of target white blood cells. An inherent problem with this approach is the loss of target cells during sample handling. We report a two-stage separation system that can perform lysis and restoration of osmolarity of blood on-chip and direct the resultant sample to the second step of the analysis. Hence, there is no loss of sample. The post-lysis makeup features a protein-rich buffer to help stabilize cells. As proof of concept, we spiked HL-60 cells into a whole blood and a pre-lysed blood sample and compared capture metrics of each method using a downstream affinity separation. The capture efficiency of the whole blood sample ranged between 40 and 80% using <7 µL of sample compared to 10-52% from 60 µL of pre-lysed blood required for similar analysis. In addition, both pre-lysed and whole blood samples showed no significant difference in purity and viability. This two-stage separation system has demonstrated the capacity to replace centrifugation and wash steps required for the preparation of lysed blood, for white blood cell analyses.

The effect of protein expression on cancer cell capture using the Human Transferrin Receptor (CD71) as an affinity ligand.

Cancer cell detection in liquid biopsies has been a widely studied application in many microfluidic devices. The use of a common antibody, such as the epithelial cell adhesion molecule (Anti-EpCAM) or other specific antibodies, has facilitated the detection and study of many cancers. However, the use of such antibodies requires a priori knowledge of the cancer source, and many cancer subtypes are missed in screening applications. There remains a need to study a wider range of cancers that maintain the streamlined antibody approach in cell affinity separations. The Human transferrin receptor (CD71) has recently been demonstrated as a cancer cell affinity target in blood samples. CD71 expression in blood cells is low, whereas proliferating cancer cells have a higher expression of the surface protein. CD71 expression is variable with cell cycle, which can impact cell separations. In this work, we investigated the effects of cell cycle and CD71 expression on cell capture metrics. Six cancer cell lines were isolated from blood via CD71 affinity capture, with a capture efficiency and purity that varied with CD71 expression. Despite variation in CD71 expression, the affinity was sufficient to isolate cancer cells spiked into blood; under optimal conditions, CD71-based capture resulted in capture purity >80%. We conclude that CD71 affinity separations show potential as a biomarker for cancer studies without sacrificing sensitivity and selectivity, and that cancer cells can be isolated from liquid biopsies over a range of expression of the target protein.

Affinity separation and subsequent terminal differentiation of acute myeloid leukemia cells using the human transferrin receptor (CD71) as a capture target.

The microfluidic detection of myeloblasts in blood via the human transferrin receptor (CD71) can serve as a diagnostic marker for acute myeloid leukemia (AML). Furthermore, CD71 expression is present in all proliferating cells and can capture target cells without prior knowledge of AML subtype. The use of anti-CD71 as the affinity ligand for AML detection in this work yields a capture efficiency and purity during peak CD71 expression of 92% and 62%, respectively. Additionally, target cells were isolated from lysed, preserved blood samples with a capture purity of 32% at a concentration of 10% myeloblasts in blood, half of the current diagnosis threshold determined by the World Health Organization. Cells isolated using this capture ligand were then subjected to post-separation differentiation therapy. HL60 cells were differentiated into non-proliferating, neutrophil-like cells. After 48 hours of incubation with 1.5% DMSO, there was a decrease in the CD71 antigen expression in differentiated cells. This separation approach can be used to screen blood samples for AML cells, and the effluent of the separation is available for post-separation analyses.

Enhanced capture and release of circulating tumor cells using hollow glass microspheres with a nanostructured surface.

Self-floating hollow glass microspheres (HGMS) modified with tumor-specific antibodies have been developed for the capture of circulating tumor cells (CTCs), and have demonstrated effective cell isolation and good viability of isolated cancer cells. However, the capture efficiency decreases dramatically if the spiked cell concentration is low, possibly due to insufficient interactions between cancer cells and the HGMS surface. In order to apply HGMS-based CTC isolation to clinically relevant samples, it is desirable to create nanostructures on the surface of HGMS to enhance cell-surface interactions. Nevertheless, current microfabrication methods cannot generate nanostructured-surfaces on microspheres. The authors have developed a new HGMS with a controlled nanotopographical surface structure (NSHGMS), and demonstrated isolation and recovery of rare cancer cells. NSHGMS are achieved by applying layer-by-layer (LbL) assembly of negatively charged SiO2 nanoparticles and positively charged poly-l-arginine molecules, then sheathing the surface with an enzymatically degradable LbL film made from biotinylated alginate and poly-l-arginine, and capping with anti-EpCAM antibodies and anti-fouling PEG molecules. Compared to smooth-surfaced HGMS, NSHGMS showed shorter isolation time (20 min), enhanced capture efficiency (93.6 ± 4.9%) and lower detection limit (30 cells per mL) for commonly used cancer cell lines (MCF7, SK-BR-3, PC-3, A549 and CCRF-CEM). This NSHGMS-based CTC isolation method does not require specialized lab equipment or an external power source, and thus, can be used for the separation of targeted cells from blood or other body fluids in a resource-limited environment.

Microfluidic Separation of Lymphoblasts for the Isolation of Acute Lymphoblastic Leukemia Using the Human Transferrin Receptor as a Capture Target.

Acute lymphocytic leukemia (ALL) is the most prevalent pediatric cancer, and the peripheral blood lymphoblast percentage is an important index for ALL diagnosis and prognosis. We describe a microfluidic device that isolates and enumerates peripheral blood lymphoblasts using affinity separations. The innovative use of a nonspecific ligand allows a widespread "net" for cancer cells, without a priori knowledge of the cancer type. Using lymphoblasts spiked into blood, we simulated leukemia cases with lymphoblast concentrations ranging from 1 to 30% of total leukocytes. Lymphoblasts were isolated using monoclonal antibodies for the Human Transferring Receptor (CD71). Anti-CD71 antibodies were found to be more effective for capturing lymphoblasts than commonly used, ALL-specific antibodies for CD7 and CD10. CCRF-CEM lymphoblasts were isolated in the chip with 82-97% purity, with lower concentrations tested (7%) still showing >80% purity for cell capture. Patient-derived ALL cell lines COG-LL-332 and COG-LL-317 were isolated in the chip with 80%-97% and 57% -92% of purity, respectively, with the initial spike concentrations as low as 1%. The ability to capture ALL lymphoblasts present in blood at low concentrations provides a novel approach for characterization of ALL cells, including patients with low leukemic burdens during and after therapy.

Synthesis and Antineoplastic Evaluation of Mitochondrial Complex†II (Succinate Dehydrogenase) Inhibitors Derived from Atpenin†A5.

Mitochondrial complex II (CII) is an emerging target for numerous human diseases. Sixteen analogues of the CII inhibitor natural product atpenin A5 were prepared to evaluate the structure-activity relationship of the C5 pyridine side chain. The side chain ketone moiety was determined to be pharmacophoric, engendering a bioactive conformation. One analogue, 1-(2,4-dihydroxy-5,6-dimethoxypyridin-3-yl)hexan-1-one (16 c), was found to have a CII IC50 value of 64 nm, to retain selectivity for CII over mitochondrial complex I (>156-fold), and to possess a ligand-lipophilicity efficiency (LLE) of 5.62, desirable metrics for a lead compound. This derivative and other highly potent CII inhibitors show potent and selective anti-proliferative activity in multiple human prostate cancer cell lines under both normoxia and hypoxia, acting to inhibit mitochondrial electron transport.

Observation of reversible, rapid changes in drug susceptibility of hypoxic tumor cells in a microfluidic device.

Hypoxia is a major stimulus for increased drug resistance and for survival of tumor cells. Work from our group and others has shown that hypoxia increases resistance to anti-cancer compounds, radiation, and other damage-pathway cytotoxic agents. In this work we utilize a microfluidic culture system capable of rapid switching of local oxygen concentrations to determine changes in drug resistance in prostate cancer cells. We observed rapid adaptation to hypoxia, with drug resistance to 2 µM staurosporine established within 30 min of hypoxia. Annexin-V/Sytox Green apoptosis assays over 9 h showed 78.0% viability, compared to 84.5% viability in control cells (normoxic cells with no staurosporine). Normoxic cells exposed to the same staurosporine concentration had a viability of 48.6% after 9 h. Hypoxia adaptation was rapid and reversible, with Hypoxic cells treated with 20% oxygen for 30 min responding to staurosporine with 51.6% viability after drug treatment for 9 h. Induction of apoptosis through the receptor-mediated pathway, which bypasses anti-apoptosis mechanisms induced by hypoxia, resulted in 39.4 ± 7% cell viability. The rapid reversibility indicates co-treatment of oxygen with anti-cancer compounds may be a potential therapeutic target.

A review of chemical gradient systems for cell analysis.

Microfluidic spatial and temporal gradient generators have played an important role in many biological assays such as in the analysis of wound healing, inflammation, and cancer metastasis. Chemical gradient systems can also be applied to other fields such as drug design, chemical synthesis, chemotaxis, etc. Microfluidic systems are particularly amenable to gradient formation, as the length scales used in chips enable fluid processes that cannot be conducted in bulk scale. In this review we discuss new microfluidic devices for gradient generation and applications of those systems in cell analysis.

Microfluidic cell surface antigen expression analysis using a single antibody type.

Antigen expression plays a significant role in clinical studies, pathology, biology and chemistry. The type and degree of antigen expression can provide information for disease diagnosis/monitoring and is used for phenotype analysis of cells. In this work, an affinity capture method was developed to capture cells based on antigen expression differences in a single microfluidic chip. Microfluidic chips with two affinity regions-at different antibody concentrations-captured two cell types based on differences in the expression of a single antigen. Using herringbone-modified capture channels, a separation purity of 95% and a capture efficiency of 15% were achieved under continuous-flow conditions. We observed that the capture ratio of Ramos B lymphocytes and HuT 78 T lymphocytes matched the expression ratio of CD71 for the two cell lines (R(2) = 0.94). To further validate our analytical method, Ramos B lymphocytes were spiked into blood samples to demonstrate performance with a complex sample. Expression ratios matched conventional flow cytometry measurements over a 40-fold difference, and the sample enrichment was 9.5×. This method has proven to be a robust system to measure the differences in antigen expression, and can be used to distinguish cells without having a unique surface antigen if the expression level is sufficiently high in one cell type.

Microfluidics and cancer analysis: cell separation, cell/tissue culture, cell mechanics, and integrated analysis systems.

Among the growing number of tools available for cancer studies, microfluidic systems have emerged as a promising analytical tool to elucidate cancer cell and tumor function. Microfluidic methods to culture cells have created approaches to provide a range of environments from single-cell analysis to complex three-dimensional devices. In this review we discuss recent advances in tumor cell culture, cancer cell analysis, and advanced studies enabled by microfluidic systems.