DIAGNOSTIC APPLICATION OF MULTIPHOTON MICROSCOPY IN EPITHELIAL TISSUES

Epithelial cancer comprises more than 85%of human cancers.The detection and treatment at the early stage has been demonstrated to apparently improve patient survival.In this review,we summarize our recent research works on the diagnostic application of epithelial tissue based on multiphoton microscopy(MPM),including identification of the layered structures of esophagus,oral cavity,skin and bronchus tissues,establishment of the diagnostic features for distinguishing gastric normal tissue from cancerous tissue,linking collagen alteration and ectocervical epithelial tumor progression for evaluating epithelial tumor progression,and differentiating normal,inflammatory,and dysplastic ectocervical epithelial tissues.These results provide the groundwork for developing MPM into clinical multiphoton endoscopy.

Proteomic Comparison of Two-Dimensional Gel Electrophoresis Profiles from Human Lung Squamous Carcinoma and Normal Bronchial Epithelial Tissues

Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorp-tion/ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-mg protein-load. The average deviation of spot position was 0.733±0.101 mm in IEF direction, and 0.925±0.207 mm in SDS-PAGE direction. For tumor tissue, a total of 1241±88 spots were detected, 987±65 spots were matched with an average matching rate of 79.5%. For control, a total of 1190±72 spots were detected, and 875±48 spots were matched with an average matching rate of 73.5%. A total of 864±34 spots were matched between tumors and controls. Forty-three differential proteins were characterized: some proteins were related to oncogenes, and others involved in the regulation of cell cycle and signal transduc-tion. It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis. These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.

Mathematical modeling of evolution of cell networks in epithelial tissues

Epithelial cell networks imply a packing geometry characterized by various cell shapes and distributions in terms of number of cell neighbors and areas.Despite such simple characteristics describing cell sheets,the formation of bubble-like cells during the morphogenesis of epithelial tissues remains poorly understood.This study proposes a topological mathematical model of morphogenesis in a squamous epithelial.We introduce a new potential that takes into account not only the elasticity of cell perimeter and area but also the elasticity of their internal angles.Additionally,we incorporate an integral equation for chemical signaling,allowing us to consider chemo-mechanical cell interactions.In addition to the listed factors,the model takes into account essential processes in real epithelial,such as cell proliferation and intercalation.The presented mathematical model has yielded novel insights into the packing of epithelial sheets.It has been found that there are two main states:one consists of cells of the same size,and the other consists of“bubble”cells.An example is provided of the possibility of accounting for chemo-mechanical interactions in a multicellular environment.The introduction of a parameter determining the flexibility of cell shapes enables the modeling of more complex cell behaviors,such as considering change of cell phenotype.The developed mathematical model of morphogenesis of squamous epithelium allows progress in understanding the processes of formation of cell networks.The results obtained from mathematical modeling are of significant importance for understanding the mechanisms of morphogenesis and development of epithelial tissues.Additionally,the obtained results can be applied in developing methods to influence morphogenetic processes in medical applications.

The role of extracellular calcium in the effect of a snake venom Lys49-phospholipase A_(2) on water transport across epithelial membranes

ACLMT is a Lys49-phospholipase A2 myotoxin isolated from the venom of the Agkistrodon contortrix laticinctus snake. This study investigated the mechanisms involved in effect of ACLMT on membrane water permeability by examining the role of extracellular calcium and strontium in this effect. Water flow across the membrane was gravimetrically measured in bladder sac preparations. The decrease in extracellular calcium promoted a higher response of epithelium to ACLMT, suggesting that the extracellular calcium protects the membrane from the action of the toxin. No alteration in the effect of the toxin on water transport was observed when calcium was replaced by strontium, indicating that this effect is independent of its enzymatic activity. These findings may bring an important contribution towards the comprehension of the mechanisms involved in the effect of Lys49-phospholipase A2 myotoxins on water permeability of epithelial membranes, with implications for the understanding of renal toxicity.

Comparative Proteome Analysis of Human Lung Squamous Carcinoma Tissue

Objective： To establish the two-dimensional electrophoresis profiles with high resolution and reproducibility from human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue, and to identify differential expression tumor-associated proteins by using proteome analysis. Methods： Comparative proteome analysis with 20 human lung squamous carcinoma tissues and the paired normal bronchial epithelial tissues adjacent to tumors was carried out. The total proteins of human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue were separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis （2-DE） and silver staining. The differential expression proteins were analyzed and then identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS）. Results： （1） Well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained. For tumor tissue, average spots of 3 gels were 1567±46, and 1436±54 spots were matched with an average matching rate of 91.6%. For control, average spots of 3 gels were 1349±58, and 1228±35 spots were matched with an average matching rate of 91.03%. The average position deviation of matched spots was 0.924±0.128 mm in IEF direction, and 1.022±0.205 mm in SDS-PAGE direction; （2） A total of 1178±56 spots were matched between the eleetrophoretie maps of 20 human lung squamous carcinoma tissues and paired normal tumor-adjacent bronchial epithelial tissues. Seventy-six differentially expressed proteins were screened; （3） Sixty-eight differential proteins were identified by PMF, some proteins were the products of oneogenes, and others involved in the regulation of cell cycle and signal transduetion; （4） In order to validate the reliability of the identified results, the expression of 3 proteins mdm2, c-jun and EGFR, which was correlated with lung squamous carcinoma, was detected by immunohistoehemieal staining and Western blot analysis. The results revealed that mdm2, c-jun and EGFR were up-regulated in lung squamous carcinomas, whereas they were down-regulated in adjacent normal bronchial epithelial tissues, normal lung tissues and inflammatory pseudotumor, which was consistent with our proteome analysis results. Conclusion： The well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were established and 68 differential proteins were characterized by applying comparative proteome analysis successfully. These results will provide scientific foundation for screening the molecular biomarker used to diagnose and treat lung squamous carcinoma, as well as to improve the patient＇s prognosis and provide new clue for the research of lung squamous carcinogenic mechanism.

Kindler syndrome protein Kindlin-1 is mainly expressed in adult tissues originating from ectoderm/endoderm

Mutations of integrin-interacting protein Kindlin-1 cause Kindler syndrome and deregulation of Kindlin-1 is implicated in human cancers. The Kindlin-1-related diseases are confined in limited tissue types. However, Kindlin-1 tissue distribution and the dogma that governs Kindlin-1 expression in normal human body are elusive. This study examined Kindlin-1 expression in normal human adult organs, human and mouse embryonic organs by immunohistochemical analyses. We identified a general principle that the level of Kindlin-1 expression in tissues is tightly correlated with the corresponding germ layers from which these tissues originate. We compared the expression of Kindlin-1 with Kindlin-2 and found that Kindlin-1 is highly expressed in epithelial tissues derived from ectoderm and endoderm, whereas Kindlin-2 is mainly expressed in mesoderm-derived tissues. Likewise, Kindlin-1 was also found highly expressed in endoderm/ectoderm-derived tissues in human and mouse embryos. Our findings indicate that Kindlin-1 may play an importance role in the development of endoderm/ectoderm related tissues.

Numerical study of dynamic zigzag patterns in migrating epithelial tissue

Collective cell migration plays a crucial role in embryonic development, metastasis, and wound healing. Nevertheless, to the best of our knowledge, how the coordination between the cell motility and deformations affects the collective motion of epithelial cells is not fully understood. In this work, we propose a modified self-propelled Voronoi model for epithelial cell migration incorporating the coupling between the self-propulsion of cells and the polarization of the cell elongation. At a high coupling strength,we observe the emergence of backward traveling band structures formed by highly aligned cells, which can be regulated by cell elongations or shape anisotropy. Increasing the cell shape anisotropy, we find that large bands split into multiple small microbands. The latter essentially forms a dynamic zigzag pattern, in which the angle between the polarization direction of the bands and the migration direction switches alternatively between π/4 and-π/4 because the cells are forced to move preferentially in the anterior direction. We also analyzed the disclinations in the cell monolayer, force distribution near the domain boundaries and the shape alignment of the epithelial monolayer during the formation of this dynamic pattern. The present findings may further our understanding of stripe pattern formations in living systems and inspire potential designs for cell sorting.

Linking tyrosine kinase inhibitor-mediated inflammation with normal epithelial cell homeostasis and tumor therapeutic responses

Receptor tyrosine kinases(RTKs)bearing oncogenic mutations in EGFR,ALK and ROS1 occur in a significant subset of lung adenocarcinomas.Tyrosine kinase inhibitors(TKIs)targeting tumor cells dependent on these oncogenic RTKs yield tumor shrinkage,but also a variety of adverse events.Skin toxicities,hematological deficiencies,nausea,vomiting,diarrhea,and headache are among the most common,with more acute and often fatal side effects such as liver failure and interstitial lung disease occurring less frequently.In normal epithelia,RTKs regulate tissue homeostasis.For example,EGFR maintains keratinocyte homeostasis while MET regulates processes associated with tissue remodeling.Previous studies suggest that the acneiform rash occurring in response to EGFR inhibition is a part of an inflammatory response driven by pronounced cytokine and chemokine release and recruitment of distinct immune cell populations.Mechanistically,blockade of EGFR causes a Type I interferon response within keratinocytes and in carcinoma cells driven by this RTK.This innate immune response within the tumor microenvironment(TME)involves increased antigen presentation and effector T cell recruitment that may participate in therapy response.This TKI-mediated release of inflammatory suppression represents a novel tumor cell vulnerability that may be exploited by combining TKIs with immune-oncology agents that rely on T-cell inflammation for efficacy.However,early clinical data indicate that combination therapies enhance the frequency and magnitude of the more acute adverse events,especially pneumonitis,hepatitis,and pulmonary fibrosis.Further preclinical studies to understand TKI mediated inflammation and crosstalk between normal epithelial cells,cancer cells,and the TME are necessary to improve treatment regimens for patients with RTK-driven carcinomas.