Dual modes of motility at the leading edge of migrating epithelial cell sheets

Purse-string healing is driven by contraction of actin/myosin cables that span cells at wound edges, and it is the predominant mode of closing small round wounds in embryonic and some adult epithelia. Wounds can also heal by cell crawling, and my colleagues and I have shown previously that the presence of unconstrained, straight edges in sheets of epithelial cells is a sufficient signal to induce healing by crawling. Here, it is reported that the presence of highly concave edges, which are free or physically constrained by an inert material (agarose), is sufficient to induce formation of purse strings. It was determined that neither of the two types of healing required cell damage or other potential stimuli by using the particularly gentle procedure of introducing gaps by digesting agarose blocks imbedded in the cell sheets. Movement by crawling depends on signaling by the EGF receptor (EGFR); however, this was not required for purse-string contraction. A migrating epithelial cell sheet usually produces finger-like projections of crawling cells. The cells between fingers contain continuous actin cables, which were also determined to contain myosin IIA and exhibit additional characteristics of purse strings. When crawling was blocked by inhibition of EGFR signaling, the concave regions continued to move, suggesting that both mechanisms contribute to propel the sheets forward. Wounding epithelial cell sheets causes activation of the EGFR, which triggers movement by crawling. The EGFR was found to be activated only at straight and convex edges, which explains how both types of movement can coexist at leading epithelial edges.     

Human phosphatase CDC14A is recruited to the cell leading edge to regulate cell migration and adhesion

Cell adhesion and migration are highly dynamic biological processes that play important roles in organ development and cancer metastasis. Their tight regulation by small GTPases and protein phosphorylation make interrogation of these key processes of great importance. We now show that the conserved dual-specificity phosphatase human cell-division cycle 14A (hCDC14A) associates with the actin cytoskeleton of human cells. To understand hCDC14A function at this location, we manipulated native loci to ablate hCDC14A phosphatase activity (hCDC14A^PD) in untransformed hTERT-RPE1 and colorectal cancer (HCT116) cell lines and expressed the phosphatase in HeLa FRT T-Rex cells. Ectopic expression of hCDC14A induced stress fiber formation, whereas stress fibers were diminished in hCDC14A^PD cells. hCDC14A^PD cells displayed faster cell migration and less adhesion than wild-type controls. hCDC14A colocalized with the hCDC14A substrate kidney- and brain-expressed protein (KIBRA) at the cell leading edge and overexpression of KIBRA was able to reverse the phenotypes of hCDC14A^PD cells. Finally, we show that ablation of hCDC14A activity increased the aggressive nature of cells in an in vitro tumor formation assay. Consistently, hCDC14A is down-regulated in many tumor tissues and reduced hCDC14A expression is correlated with poorer survival of patients with cancer, to suggest that hCDC14A may directly contribute to the metastatic potential of tumors. Thus, we have uncovered an unanticipated role for hCDC14A in cell migration and adhesion that is clearly distinct from the mitotic and cytokinesis functions of Cdc14/Flp1 in budding and fission yeast.Cell migration and adhesion play key roles in embryonic development, tissue remodeling and cancer metastasis (1). Many oncoproteins, such as Yes-associated protein 1 (YAP), STAT3, and K-RAS, regulate cancer metastasis by enhancing cell migration and invasion (2–4). The dynamic behavior of the actin cytoskeleton drives migration and invasion and is regulated by a combined impact of Rho GTPases, membrane phospholipids, and protein phosphorylation (5). The switch of phosphorylation at the cell leading edge is crucial for rapid turnover of actin filaments. For example, focal adhesion kinase (FAK) can be activated by integrins and various growth factors. Once activated, FAK regulates actin polymerization, membrane protrusion and cell migration by promoting the phosphorylation of the actin cytoskeleton remodelers p130cas, GRB2/7, and WASP (5, 6). The tyrosine phosphatase SHP2 increases cell mobility through activation of the SRC kinase family to promote tumor metastasis (7). Conversely, the lipid phosphatase PTEN inhibits tumor invasion by suppressing the activation of RAC GTPases (8). There is also extensive evidence for control of cancer cell migration and invasion through the phosphatase PP2A upon Wnt/beta-catenin signaling, metal matrix proteases, and ERK kinase (9–11).At the G2/M transition, cyclin-dependent kinase 1 (CDK1) is activated to trigger mitotic entry and its kinase activity remains high until metaphase to maintain the cell in a mitotic state (12). With mitotic exit, the proteins that were phosphorylated by CDK1 are dephosphorylated, so that cells can return to the nonmitotic, interphase status (13). In Saccharomyces cerevisiae, cell-division cycle 14 (Cdc14) is the major phosphatase that counteracts Cdk1 activity after the metaphase–anaphase transition (14). The function of Cdc14 in budding yeast has been well studied and two regulatory networks have been identified that promote the release of Cdc14 from the RENT complex in the nucleolus: “Cdc Fourteen Early Anaphase Release” (FEAR) and “Mitotic Exit Network” (MEN) (15–17).Despite the conservation of the N-terminal catalytic phosphatase domain and the ability of human CDC14B to complement the essential functions of budding yeast CDC14 (18), Cdc14 phosphatases play divergent roles in different organisms. Schizosaccharomyces pombe Cdc14/Flp1 primarily participates in the regulation of the phosphatase Cdc25 and cytokinesis (19). Vertebrate CDC14s have been linked to diverse functions ranging from centrosome maturation and separation, DNA damage checkpoint control, DNA repair, and cytokinesis control (20–24). These studies have unraveled novel functions of mammalian CDC14 phosphatases; however, they reveal striking inadequacies in our understanding of this important phosphatase family.Here, we have addressed the function of hCDC14A (human cell-division cycle 14A) using human genetically engineered hCDC14A phosphatase dead cell lines (PD). Mobility and spreading were both enhanced by ablation of hCDC14A, whereas cell–cell adhesion was reduced. Moreover, ectopic hCDC14A expression inhibited migration and the actin cytoskeleton was remodeled when hCDC14A activity was impaired. Consistent with these actin-modulating functions, a pool of hCDC14A associated with F-actin filaments at the leading edge where it colocalized with the Hippo pathway component kidney- and brain-expressed protein (KIBRA). KIBRA overproduction rescued the migration and adhesion defects in the hCDC14A^PD cells. Our study therefore reveals a previously unidentified function of hCDC14A. As hCDC14A expression is down-regulated in a variety of cancers, including colorectal, and this down-regulation is associated with poor prognosis, our results suggest that hCDC14A regulates tumor metastasis and is therefore of considerable clinical relevance.     

Evolutionary dynamics at the tumor edge reveal metabolic imaging biomarkers

Human cancers are biologically and morphologically heterogeneous. A variety of clonal populations emerge within these neoplasms and their interaction leads to complex spatiotemporal dynamics during tumor growth. We studied the reshaping of metabolic activity in human cancers by means of continuous and discrete mathematical models and matched the results to positron emission tomography (PET) imaging data. Our models revealed that the location of increasingly active proliferative cellular spots progressively drifted from the center of the tumor to the periphery, as a result of the competition between gradually more aggressive phenotypes. This computational finding led to the development of a metric, normalized distance from ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) hotspot to centroid (NHOC), based on the separation from the location of the activity (proliferation) hotspot to the tumor centroid. The NHOC metric can be computed for patients using ¹⁸F-FDG PET–computed tomography (PET/CT) images where the voxel of maximum uptake (standardized uptake value [SUV]_max) is taken as the activity hotspot. Two datasets of ¹⁸F-FDG PET/CT images were collected, one from 61 breast cancer patients and another from 161 non–small-cell lung cancer patients. In both cohorts, survival analyses were carried out for the NHOC and for other classical PET/CT-based biomarkers, finding that the former had a high prognostic value, outperforming the latter. In summary, our work offers additional insights into the evolutionary mechanisms behind tumor progression, provides a different PET/CT-based biomarker, and reveals that an activity hotspot closer to the tumor periphery is associated to a worst patient outcome.

Human cancers are genetically and morphologically heterogeneous (1, 2). This is generally attributed to the evolutionary dynamics of different clonal cell populations coexisting in the tumor ecosystem and undergoing stochastic branching processes over time (3–5). Successively acquired driver mutations, somatic alterations, and nongenetic modifications may confer increased fitness on certain cancer cell phenotypes, which subsequently outcompete those that do not experience such selection benefits within their microenvironment (4, 6, 7). Cells with specific advantageous traits may not show uniform spatial distribution across the tumor, particularly in large tumors. In fact, trade-offs exist that preclude the occurrence of optimal phenotypes, as exemplified by the hallmarks of cancer (8), and thus only local selection is expected to take place. This produces the spatial phenotypic diversity found in primary tumors and distant metastases (9).Sustained metabolic reorganization during tumor progression, due to bioenergetically very demanding processes such as rapid proliferation, is a major hallmark of cancer (8, 10). This gives rise to a global metabolic plasticity and fitness optimization that confers evolutionary advantages under specific selective pressures, such as hypoxia (11). Positron emission tomography (PET) has been proposed as a way to assess macroscopic tumor heterogeneity in human patients (12). The technique is used in clinical practice with the radiotracer ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) (13), which is an analog of glucose and thus a marker of glycolysis (14). The altered tumor metabolism leads to an up-regulation of glycolysis and an increase in glucose consumption (15). This happens even in the presence of oxygen and is referred to as the Warburg effect. Even though this process is energetically inefficient (16), cancer cells may find it beneficial to satisfy the biomass demands required by their high proliferation rates (17). This is confirmed by studies that relate the uptake of ¹⁸F-FDG in PET images to proliferation markers (18, 19). Therefore, the spatial map of glucose consumption provided by ¹⁸F-FDG PET images, as measured at each voxel by the standardized uptake value (SUV), is of great utility in portraying the spatial distribution of proliferation within the tumor.The degree and impact of intertumor diversity and intratumor heterogeneity in patients has driven the need for quantitative frameworks to account for this variability (20). We considered how the metabolic activity might be distributed inside the tumor and how that information could be related to ¹⁸F-FDG PET images. Specifically, we looked at whether the location of prominent proliferation hotspots, as measured by the voxel of maximum radiotracer uptake (SUV_max), could convey information about patient prognosis. We did this by analyzing how these spots changed over time and space within the tumor in silico using two mathematical models of different levels of complexity. The implications of these results, embodied in the definition of a prognostic biomarker named NHOC, were tested on datasets of breast and lung cancer patients.     

Coherent angular motion in the establishment of multicellular architecture of glandular tissues

Glandular tissues form ducts (tubes) and acini (spheres) in multicellular organisms. This process is best demonstrated in the organization of the ductal tree of the mammary gland and in 3D models of morphogenesis in culture. Here, we asked a fundamental question: How do single adult epithelial cells generate polarized acini when placed in a surrogate basement membrane 3D gel? Using human breast epithelial cells from either reduction mammoplasty or nonmalignant breast cell lines, we observed a unique cellular movement where single cells undergo multiple rotations and then maintain it cohesively as they divide to assemble into acini. This coherent angular motion (CAMo) was observed in both primary cells and breast cell lines. If CAMo was disrupted, the final geometry was not a sphere. The malignant counterparts of the human breast cell lines in 3D were randomly motile, did not display CAMo, and did not form spheres. Upon “phenotypic reversion” of malignant cells, both CAMo and spherical architecture were restored. We show that cell-cell adhesion and tissue polarity are essential for the formation of acini and link the functional relevance of CAMo to the establishment of spherical architecture rather than to multicellular aggregation or growth. We propose that CAMo is an integral step in the formation of the tissue architecture and that its disruption is involved in malignant transformation.     

Integrin adhesion drives the emergent polarization of active cytoskeletal stresses to pattern cell delamination

Tissue patterning relies on cellular reorganization through the interplay between signaling pathways and mechanical stresses. Their integration and spatiotemporal coordination remain poorly understood. Here we investigate the mechanisms driving the dynamics of cell delamination, diversely deployed to extrude dead cells or specify distinct cell fates. We show that a local mechanical stimulus (subcellular laser perturbation) releases cellular prestress and triggers cell delamination in the amnioserosa during Drosophila dorsal closure, which, like spontaneous delamination, results in the rearrangement of nearest neighbors around the delaminating cell into a rosette. We demonstrate that a sequence of "emergent cytoskeletal polarities" in the nearest neighbors (directed myosin flows, lamellipodial growth, polarized actomyosin collars, microtubule asters), triggered by the mechanical stimulus and dependent on integrin adhesion, generate active stresses that drive delamination. We interpret these patterns in the language of active gels as asters formed by active force dipoles involving surface and body stresses generated by each cell and liken delamination to mechanical yielding that ensues when these stresses exceed a threshold. We suggest that differential contributions of adhesion, cytoskeletal, and external stresses must underlie differences in spatial pattern.     

DAB2IP coordinates both PI3K-Akt and ASK1 pathways for cell survival and apoptosis

In metastatic prostate cancer (PCa) cells, imbalance between cell survival and death signals such as constitutive activation of phosphatidylinositol 3-kinase (PI3K)-Akt and inactivation of apoptosis-stimulated kinase (ASK1)-JNK pathways is often detected. Here, we show that DAB2IP protein, often down-regulated in PCa, is a potent growth inhibitor by inducing G₀/G₁ cell cycle arrest and is proapoptotic in response to stress. Gain of function study showed that DAB2IP can suppress the PI3K-Akt pathway and enhance ASK1 activation leading to cell apoptosis, whereas loss of DAB2IP expression resulted in PI3K-Akt activation and ASK1-JNK inactivation leading to accelerated PCa growth in vivo. Moreover, glandular epithelia from DAB2IP^−/− animal exhibited hyperplasia and apoptotic defect. Structural functional analyses of DAB2IP protein indicate that both proline-rich (PR) and PERIOD-like (PER) domains, in addition to the critical role of C2 domain in ASK1 activity, are important for modulating PI3K-Akt activity. Thus, DAB2IP is a scaffold protein capable of bridging both survival and death signal molecules, which implies its role in maintaining cell homeostasis.     

A progenitor cell origin of myeloid malignancies

All cancers rely on cells that have properties of long-term self-renewal or “stemness” to maintain and propagate the tumor, but the cell of origin of most cancers is still unknown. Here, we design a stochastic mathematical model of hematopoietic stem and progenitor cells to study the evolutionary dynamics of cancer initiation. We consider different evolutionary pathways leading to cancer-initiating cells in JAK2V617F-positive myeloproliferative neoplasms (MPN): (i) the JAK2V617F mutation may arise in a stem cell; (ii) a progenitor cell may first acquire a mutation conferring self-renewal, followed by acquisition of the JAK2V617F mutation; (iii) the JAK2V617F mutation may first emerge in a progenitor cell, followed by a mutation conferring self-renewal; and (iv) a mutation conferring self-renewal to progenitors may arise in the stem cell population without causing a change in the stem cell''s phenotype, followed by the JAK2V617F mutation emerging in a progenitor cell. We find mathematical evidence that a progenitor is the most likely cell of origin of JAK2V617F-mutant MPN. These results may also have relevance to other tumor types arising in tissues that are organized as a differentiation hierarchy.     

Circulating tumor cells counts are associated with CD8+ T cell levels in programmed death-ligand 1-negative non-small cell lung cancer patients after radiotherapy: A retrospective study

This study aimed to explore the dynamics of circulating tumor cells (CTCs) and CD8+ T cells in stage II–III non-small cell lung cancer patients with CTCs in different programmed death-ligand 1 (PD-L1) status treated with radiotherapy and evaluate the correlation between CTCs and CD8+ T cells.This study was a retrospective study which reviewed 69 stage II–III non-small cell lung cancer patients underwent postoperative radiotherapy and peripheral blood tests of CTCs and T lymphocyte were available before radiation, 1 week after radiation and 1 month after radiation.In this study, 25 patients had PD-L1 positive CTCs and 44 patients had PD-L1 negative CTCs. The CTCs count was significantly decreased compared with baseline in patients with different PD-L1 status CTCs at 1 week and 1 month after radiotherapy. The proportion of CD8+ T cells was significantly increased at 1 month after radiotherapy compared with baseline in the total population (mean change, 7.24 ± 2.12; P < .05) and patients with PD-L1 negative CTCs (mean change, 7.17 ± 2.65; P < .05). One month after radiotherapy, the proportion of CD8+ T cells was negatively correlated with the CTCs count in the total population (r = −0.255, P = .034) and PD-L1 negative patients (r = –0.330, P = .029). In patients with PD-L1 negative CTCs, the CTCs count 1 week after radiotherapy (hazard ratio, 0.150 [95% confidence intervals., 0.027–0.840], P = .031) and the proportion of CD8+ T cells 1 month after radiotherapy (hazard ratio, 7.961 [95% confidence intervals, 1.028–61.68], P = .047) were independent prognostic factors for disease recurrence.After radiotherapy, only PD-L1-negative patients had a significant increase in the CD8+ T cell levels, while it was negatively correlated with CTCs count and was an independent prognostic factors of disease recurrence.     

Emerging modes of collective cell migration induced by geometrical constraints

The role of geometrical confinement on collective cell migration has been recognized but has not been elucidated yet. Here, we show that the geometrical properties of the environment regulate the formation of collective cell migration patterns through cell-cell interactions. Using microfabrication techniques to allow epithelial cell sheets to migrate into strips whose width was varied from one up to several cell diameters, we identified the modes of collective migration in response to geometrical constraints. We observed that a decrease in the width of the strips is accompanied by an overall increase in the speed of the migrating cell sheet. Moreover, large-scale vortices over tens of cell lengths appeared in the wide strips whereas a contraction-elongation type of motion is observed in the narrow strips. Velocity fields and traction force signatures within the cellular population revealed migration modes with alternative pulling and/or pushing mechanisms that depend on extrinsic constraints. Force transmission through intercellular contacts plays a key role in this process because the disruption of cell-cell junctions abolishes directed collective migration and passive cell-cell adhesions tend to move the cells uniformly together independent of the geometry. Altogether, these findings not only demonstrate the existence of patterns of collective cell migration depending on external constraints but also provide a mechanical explanation for how large-scale interactions through cell-cell junctions can feed back to regulate the organization of migrating tissues.     

A new video image analysis system to study red blood cell dynamics and oxygenation in capillary networks

OBJECTIVE: The authors present a Measurement and Analysis System for Capillary Oxygen Transport (MASCOT) to study red blood cell (RBC) dynamics and oxygenation in capillary networks. The system enables analysis of capillaries to study geometry and morphology and provides values for capillary parameters such as diameter and segment length. It also serves as an analysis tool for capillary RBC flow characteristics, including RBC velocity, lineal density, and supply rate. Furthermore, the system provides a means of determining the oxygen saturation of hemoglobin contained within RBCs, by analysis of synchronized videotapes containing images at two wavelengths, enabling the quantification of the oxygen content of individual RBCs. METHODS: Video recordings of RBC flow at two wavelengths, 420 nm (isosbestic) and 436 nm (oxygen sensitive), are made using a dual camera video microscopy system. The 420-nm recording is used to generate images based on the variance of light intensity fluctuations that help to identify capillaries in a given field of view that are in sharp focus and exhibit flow of individual RBCs separated by plasma gaps. A region of interest enclosing the desired capillary is defined and a fixed number of successive video frames at the two wavelengths are captured. Next a difference image is created, which delineates the RBC column, whose width is used to estimate the internal diameter of the capillary. The 420-nm images are also used to identify the location and centroid of each RBC within the capillary. A space-time image is generated to compute the average RBC velocity. Lineal density is calculated as the number of RBCs per unit length of a capillary segment. The mean optical density (OD) of each RBC is calculated at both wavelengths, and the average SO(2) for each cell is determined from OD(436)/OD(420). RESULTS AND CONCLUSIONS: MASCOT is a robust and flexible system that requires simple hardware, including a SGI workstation fitted with an audio-visual module, a VCR, and an oscilloscope. Since the new system provides information on an individual cell basis from entire capillary segments, the authors believe that results obtained using MASCOT will be more accurate than those obtained from previous systems. Due to its flexibility and ease of extension to other applications, MASCOT has the potential to be applied widely as an analysis tool for capillary oxygen transport measurements.     

Genes associated with telomerase activity levels in esophageal carcinoma cell lines

Telomerase activity levels have been shown to correlate with tumor progression in several malignancies. However, the genetic regulation of telomerase activity levels is not fully understood. The aim of the present study has been to identify a gene expression profile, predicting correlation with the telomerase-activity test. Ten human esophageal carcinoma cell lines were investigated using the telomerase activity assay (TRAPeze) Telomerase Detection Kit), followed by further characterization using the GeneChip Human Genome U133A 2.0 Array (Affymetrics Inc., USA), including 14 500 human genes. Telomerase activity levels were detected in all cell lines with a broad range of activity levels. Using a high correlation coefficient, r > 0.90, the following genes were found to be positively correlated with telomerase activity levels: N-myristoyltransferase 2; ribosomal protein L3; retinoblastoma-like 2 (pRb2/p130); and cyclin G2. Only one gene was negatively correlated with telomerase activity levels, zinc finger protein 207. In conclusion, the present microarray data provide primary validation data indicating possible candidates for prognostic and prediction factors in esophageal cancer in relation to telomerase activity.     

Imaging red blood cell dynamics by quantitative phase microscopy

Red blood cells (RBCs) play a crucial role in health and disease, and structural and mechanical abnormalities of these cells have been associated with important disorders such as Sickle cell disease and hereditary cytoskeletal abnormalities. Although several experimental methods exist for analysis of RBC mechanical properties, optical methods stand out as they enable collecting mechanical and dynamic data from live cells without physical contact and without the need for exogenous contrast agents. In this report, we present quantitative phase microscopy techniques that enable imaging RBC membrane fluctuations with nanometer sensitivity at arbitrary time scales from milliseconds to hours. We further provide a theoretical framework for extraction of membrane mechanical and dynamical properties using time series of quantitative phase images. Finally, we present an experimental approach to extend quantitative phase imaging to 3-dimensional space using tomographic methods. By providing non-invasive methods for imaging mechanics of live cells, these novel techniques provide an opportunity for high-throughput analysis and study of RBC mechanical properties in health and disease.     

Effects of cytotoxic T lymphocytes on hepatoma cell line SMMC-7721 induced by different subsets of dendritic cells in vitro

BACKGROUND: Dendritic cells (DCs) loaded with complex antigen are always used to induce cytotoxic T lymphocytes (CTLs) which have a specific anti-tumor activity. However, CTLs can assault autologous cells induced by DCs loaded with autologous antigen. This study aimed to explore how to weaken the autoimmune reaction induced by DC vaccine by combining mature DC (mDC) activating immunity and immature DC (imDC) leading to immune tolerance to make hepatocellular carcinoma (HCC) vaccine in vitro. METHODS: DC progenitors derived from human peripheral blood were assigned to two groups. One was cultured to mDC and pulsed with frozen-thawed antigen (FTA) of human HCC cell line SMMC-7721 cells (mDC group), and the other was cultured to imDC and pulsed with FTA of human liver cell line L-02 cells (imDC group). The morphology of DCs was monitored and cells phenotypes including HLA-DR, CD80, CD1α, CD83 were assayed by flowcytometry (FCM). The concentrations of interleukin-12 (IL-12) in the supernatant were assayed by ELISA. Methyl thiazolyl tetrazolium (MTT) was used to evaluate T cell proliferation induced by mDC and imDC and the killing rate of CTL induced by mDC and imDC respectively/together on SMMC-7721 and L-02 cells. RESULTS: Compared with the imDC group, the mDC group was characterized by the following: increased secretion of IL-12 (P<0.05); higher expression of HLA-DR, CDla, CD80, CD83; and stronger activity in stimulating proliferation of isogenic T cells (P<0.05). CTL induced by the mDC group had a significant killing response to SMMC-7721 as well as a higher killing rate for L-02 (P>0.05). CTL induced by mDC and imDC together had a higher killing response to SMMC-7721, but a lower killing rate for L-02(P<0.01). CONCLUSIONS: CTL induced by mDC and imDC together has a higher antigen-specific killing response in vitro than that induced by mDC alone. This may be of greater clinical value.     

Melatonin and verteporfin synergistically suppress the growth and stemness of head and neck squamous cell carcinoma through the regulation of mitochondrial dynamics

The prevalence of head and neck squamous cell carcinoma (HNSCC) has continued to rise for decades. However, drug resistance to chemotherapeutics and relapse, mediated by cancer stem cells (CSCs), remains a significant impediment in clinical oncology to achieve successful treatment. Therefore, we focused on analyzing CSCs in HNSCC and demonstrated the effect of melatonin (Mel) and verteporfin (VP) on SCC-25 cells. HNSCC CSCs were enriched in the reactive oxygen species-low state and in sphere-forming cultures. Combination treatment with Mel and VP decreased HNSCC viability and increased apoptosis without causing significant damage to normal cells. Sphere-forming ability and stem cell population were reduced by co-treatment with Mel and VP, while mitochondrial ROS level was increased by the treatment. Furthermore, the expression of mitophagy markers, parkin and PINK1, was significantly decreased in the co-treated cells. Mel and VP induced mitochondrial depolarization and inhibited mitochondrial function. Parkin/TOM20 was localized near the nucleus and formed clusters of mitochondria in the cells after treatment. Moreover, Mel and VP downregulated the expression of markers involved in epithelial-mesenchymal transition and metastasis. The migration capacity of cells was significantly decreased by co-treatment with Mel and VP, accompanied by the down-regulation of MMP-2 and MMP-9 expression. Taken together, these results indicate that co-treatment with Mel and VP induces mitochondrial dysfunction, resulting in the apoptosis of CSCs. Mel and VP could thus be further investigated as potential therapies for HNSCC through their action on CSCs.     

Cell culture detection of microvascular cell death in clinical specimens of human neoplasms and peripheral blood

Background. Angiogenesis studies are limited by the clinical relevance of laboratory model systems. We developed a new method for measuring dead microvascular (MV) cells in clinical tissue, fluid and blood specimens, and applied this system to make several potentially novel observations relating to cancer pharmacology. Methods. Dead MV cells tend to have a hyperchromatic, refractile quality, further enhanced during the process of staining with Fast Green and counterstaining with either haematoxylin–eosin or Wright–Giemsa. We used this system to quantify the relative degree of direct antitumour versus anti‐MV effects of cisplatin, erlotinib, imatinib, sorafenib, sunitinib, gefitinib and bevacizumab. Results. Bevacizumab had striking anti‐MV effects and minimal antitumour effects; cisplatin had striking antitumour effects and minimal anti‐MV effects. The `nib' drugs had mixed antitumour and anti‐MV effects. Anti‐MV effects of erlotinib and gefitinib were equal to those of sunitinib and sorafenib. There was no detectable VEGF in culture medium without cells; tumour cells secreted copious VEGF, reduced to undetectable levels by bevacizumab, greatly reduced by cytotoxic levels of cisplatin + anguidine, and variably reduced by DMSO and/or ethanol. We observed anti‐MV additivity between bevacizumab and other drugs on an individual patient basis. Peripheral blood specimens had numerous MV cells which were strikingly visualized for quantification with public domain image analysis software using bevacizumab essentially as an imaging reagent. Conclusions. This system could be adapted for simple, inexpensive and sensitive/specific detection of tissue and circulating MV cells in a variety of neoplastic and non‐neoplastic conditions, and for drug development and individualized cancer treatment.     

血清基质金属蛋白酶-9等对非小细胞肺癌诊断的临床意义

目的综合评价血清基质金属蛋白酶-9(MMP-9)与其它肿瘤标记物-癌胚抗原(CEA)、神经元特异性烯醇化酶(NSE)、糖类抗原125(CA125)和糖类抗原19-9(CA19-9)等对非小细胞肺癌诊断及预测疾病状态的临床意义。方法利用ELISA方法和多肿瘤标记物蛋白芯片检测系统分别测定73例非小细胞肺癌患者、21例良性肺疾病患者以及16例健康人体血清中MMP-9、CEA、NSE、CA125和CA19-9的水平,比较5项指标对非小细胞肺癌诊断的灵敏度和特异度。结果非小细胞肺癌患者血清中MMP-9、CEA、NSE、CA125和CA19-9的水平均显著高于良性肺疾病组和健康组(P<0.01),其中血清MMP-9检测对诊断非小细胞肺癌的灵敏度较高。另外,MMP-9的水平随肿瘤体积增大、淋巴结转移以及临床分期进展而显著升高(P<0.01)。结论血清MMP-9与CEA、NSE、CA125和CA19-9对非小细胞肺癌诊断都具有一定的临床价值,血清MMP-9还是一项预测非小细胞肺癌侵袭、转移等疾病状态的良好指标。