A dose-response study of a novel method of selective tissue modification of cellular structures in the skin with nanosecond pulsed electric fields.

BACKGROUND AND OBJECTIVES: This study describes the effects of nanosecond pulsed electric fields (nsPEF) on the epidermis and dermis of normal skin scheduled for excision in a subsequent abdominoplasty. NsPEF therapy applies nanosecond pulses of electrical energy to induce regulated cell death (RCD) in cellular structures, with negligible thermal effects. Prior pre-clinical studies using nsPEF technology have demonstrated the ability to stimulate a lasting immune response in animal tumor models, including melanoma. This first-in-human-use of nsPEF treatment in a controlled study to evaluate the dose-response effects on normal skin and subcutaneous structures is intended to establish a safe dose range of energies prior to use in clinical applications using nsPEF for non-thermal tissue modification. STUDY DESIGN/MATERIALS AND METHODS: Seven subjects with healthy tissue planned for abdominoplasty excision were enrolled. Five subjects were evaluated in a longitudinal, 60-day study of effects with doses of six nsPEF energy levels. A total of 30 squares of spot sizes 25mm2 or less within the planned excision area were treated and then evaluated at 1 day, 5 days, 15 days, 30 days, and 60 days prior to surgery. Photographs were taken over time of each treated area and assessed by three independent and blinded dermatologists for erythema, flaking and crusting using a 5-point scale (0 = low, 4 = high). Punch biopsies of surgically removed tissue were processed and evaluated for tissue changes using hematoxylin and eosin, trichome, caspase-3, microphthalmia transcription factor, and elastin stains and evaluated by a dermatopathologist. The skin of two subjects received additional treatments at 2 and 4 hours post-nsPEF and was evaluated in a similar manner. RESULTS: Most energy settings exhibited delayed epidermal loss followed by re-epithelization by day 15 and a normal course of healing. Histologic analysis identified the appearance of activated caspase-3 at two and four hours after nsPEF treatment, but not at later time points. At the 1-day time point, a nucleolysis effect was observed in epidermal cells, as evidenced by the lack of nuclear staining while the epidermal plasma membranes were still intact. Cellular structures within the treatment zone such as melanocytes, sebaceous glands, and hair follicles were damaged while acellular structures such as elastic fibers and collagen were largely unaffected except for TL6 which showed signs of dermal damage. Melanocytes reappeared at levels comparable with untreated controls within 1 month of nsPEF treatment. CONCLUSIONS: The selective effect of nsPEF treatment on cellular structures in the epidermal and dermal layers suggests that this non-thermal mechanism for targeting cellular structures does not affect the integrity of dermal tissue within a range of energy levels. The specificity of effects and a favorable healing response makes nsPEF ideal for treating cellular targets in the epidermal or dermal layers of the skin, including treatment of benign and malignant lesions. NsPEF skin treatments provide a promising, non-thermal method for treating skin conditions and removing epidermal lesions. © 2019 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

SM22 is required for the maintenance of actin-rich structures generated during bacterial infections.

The host actin cytoskeleton is utilized by an assortment of pathogenic bacteria to colonize and cause disease in their hosts. Two prominently studied actin-hijacking bacteria are enteropathogenic Escherichia coli (EPEC) and Listeria monocytogenes. EPEC form actin-rich pedestals atop its host cells to move across the intestinal epithelia, while Listeria monocytogenes generate branched actin networks arranged as actin clouds around the bacteria and as comet tails for propulsion within and amongst their host cells. Previous mass spectrometry analysis revealed that a member of the calponin family of actin-bundling proteins, transgelin/SM22 was enriched in EPEC pedestals. To validate that finding and examine the role of SM22 during infections, we initially immunolocalized SM22 in EPEC and L. monocytogenes infected cells, used siRNA to deplete SM22 and EGFP-SM22 to overexpress SM22, then quantified the alterations to the bacterially generated actin structures. SM22 concentrated at all bacterially-generated actin structures. Depletion of SM22 resulted in fewer pedestals and comet tails and caused comet tails to shorten. The decrease in comet tail abundance caused a proportional increase in actin clouds whereas overexpression of SM22 reversed the actin cloud to comet tail proportions and increased comet tail length, while not influencing EPEC pedestal abundance. Thus, we demonstrate that SM22 plays a role in regulating the transitions and morphological appearance of bacterially generated actin-rich structures during infections.

Systems microscopy approaches to understand cancer cell migration and metastasis.

Cell migration is essential in a number of processes, including wound healing, angiogenesis and cancer metastasis. Especially, invasion of cancer cells in the surrounding tissue is a crucial step that requires increased cell motility. Cell migration is a well-orchestrated process that involves the continuous formation and disassembly of matrix adhesions. Those structural anchor points interact with the extra-cellular matrix and also participate in adhesion-dependent signalling. Although these processes are essential for cancer metastasis, little is known about the molecular mechanisms that regulate adhesion dynamics during tumour cell migration. In this review, we provide an overview of recent advanced imaging strategies together with quantitative image analysis that can be implemented to understand the dynamics of matrix adhesions and its molecular components in relation to tumour cell migration. This dynamic cell imaging together with multiparametric image analysis will help in understanding the molecular mechanisms that define cancer cell migration.

Effects of surgical excision and radiation on medulloblastoma cell invasiveness.

OBJECTIVES: Surgical resection and adjuvant radiation are mainstays of medulloblastoma (MB) patient management. We utilized a novel 3-dimensional assay to identify how (a) radiation, (b) excision of the primary tumour aggregate, and (c) both treatments combined influence MB cell invasiveness. METHODS: Five MB cell lines (UW228-1, 2 and 3; Daoy, and Madsen) were implanted onto a 3-dimensional, type I collagen gel assay to assess tumour invasion distance over five days, in response to (1) needle-assisted excision of the central cell aggregate; (2) pre-exposure to single-dose and fractionated dose irradiation in doses from 6-25 and 8-24 Gy, respectively; and (3) excision plus either single-dose or fractionated radiation. RESULTS: Within hours, individual MB cells detached from the surface of the cell aggregates and invaded the collagen matrix, to distances up to 1200 microm and at rates up to 300 microm daily. The UW228-1 cell line was less invasive than the other cell lines and was dropped from further analysis. In the four remaining lines, a dose-dependent decline in tumour invasiveness was identified, both for single-dose and fractionated radiation, which achieved statistically decreased invasion distances at higher doses, especially of fractionated irradiation. Excision of the central tumour aggregate tended towards exerting a late effect on cell invasion, but exerted no significant influence on the radio-sensitivity of residual cells. CONCLUSIONS: Both single-dose and fractionated dose irradiation appear to inhibit MB cell invasiveness in a dose-dependent manner, whereas excision of the central cell aggregate exerts no effect on residual invading cells.

Understanding molecular mechanisms of disease through spatial proteomics.

Mammalian cells are organized into different compartments that separate and facilitate physiological processes by providing specialized local environments and allowing different, otherwise incompatible biological processes to be carried out simultaneously. Proteins are targeted to these subcellular locations where they fulfill specialized, compartment-specific functions. Spatial proteomics aims to localize and quantify proteins within subcellular structures.

Acinic cell carcinoma of salivary gland with massive deposits of globular amyloid.

We present the case of a 23-year-old woman with a parotid gland tumor, the fine-needle aspiration biopsy smears of which showed epithelial cells with wide cytoplasm, isolated or arranged in micropapillary groups together with psammoma bodies. The surgical specimen contained a 5-cm tumor with the histologic features of an acinic cell carcinoma (ACC) with papillary areas. Notably, the cells of the tumor seemed to follow a sequence from large cells with rounded nuclei with open chromatin and prominent nucleoli to vacuolated cells with granular material, and finally to cells undergoing apoptosis. This finding was followed by the appearance of concentrically laminated, round to polygonal, Congo red-positive, birefringent bodies that in areas accumulated and formed extensive areas with massive deposits. The picture suggested that those amyloid bodies (psammoma bodies) resulted from the accumulation of residual masses of apoptotic cells. Huge globular amyloid deposits, the suggested name for this material irrespective of the type of amyloid, have not been previously reported in ACC of salivary gland.

Nanoscale dose deposition in cell structures under X-ray irradiation treatment assisted with nanoparticles: An analytical approach to the relative biological effectiveness.

In this study, an analytical model for the assessment of the modification of cell culture survival under ionizing radiation assisted with nanoparticles (NPs) is presented. The model starts from the radial dose deposition around a single NP, which is used to describe the dose deposition in a cell structure with embedded NPs and, in turn, to evaluate the number of lesions formed by ionizing radiation. The model is applied to the calculation of relative biological effectiveness values for cells exposed to 0.5mg/g of uniformly dispersed NPs with a radius of 10nm made of Fe, I, Gd, Hf, Pt and Au and irradiated with X-rays of energies 20keV higher than the element K-shell binding energy.

Histological examination of primary posterior capsule plaques.

PURPOSE: To understand the source and evolution of the cellular elements of posterior capsule plaques. SETTING: Department of Ophthalmology, University of Sciences, Faculty of Medicine, Pécs, Hungary. METHODS: In 24 eyes of 24 patients, the primary fibrotic plaques were carefully peeled off the posterior capsule with forceps and sent for histological examination. The samples were stained with hematoxylin-eosin and with picrosirius to demonstrate collagen content. RESULTS: Most plaques were found in elderly patients with mature cataracts. However, one was found in a patient with congenital cataract. The histological examination revealed different stages of plaques, permitting discrimination of early (cellular), transitional, and late (fibrotic) stages. The plaques in the early stage contained cuboidal cells with nuclei; collagen deposition led to the development of the transitional stage; and further collagen deposition led to the fibrotic stage. There was no significant correlation between the maturity of the cataract and the plaque stage, but other factors such as previous eye surgery (vitrectomy, glaucoma surgery), long-standing diabetes mellitus (DM), and a high degree of myopia appeared to contribute to the development of plaques. CONCLUSIONS: Primary posterior capsule plaques were found primarily in elderly patients with mature cataracts but were also present post vitrectomy in those who had DM from a young age and had early stages of cataract. In the early plaque stage, cells with nuclei were found. It follows that plaques are capable of proliferation at this stage, aggravating visual disturbance. Therefore, removing the plaques by peeling from the posterior capsule or by posterior capsulorhexis during surgery is suggested.

Cervical cytology of recurrent psammoma bodies with atypical cells in a postmenopausal woman: a case report.

BACKGROUND: Psammoma bodies (PBs) are an unusual finding in cervical cytology preparations. They have been identified in association with a wide range of benign and malignant conditions within the female genital tract. Portents of a significant underlying pathology include their occurrence in postmenopausal patients, the presence of unexplained vaginal bleeding and their occurrence in association with atypical cells. CASE: PBs associated with atypical cells were detected in repeated cervical cytology smears of an asymptomatic, 55-year-old postmenopausal woman over a 4-year period. She was extensively investigated, and, in the absence of a bleeding) have definitive cause, she underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy. Histologic examination of the specimen demonstrated the presence of bilateral benign ovarian serous cystadenofibromas with large numbers of PBs. Focally the PBs were closely apposed to the serosal surface and invested in a blanket of mesothelial cells. CONCLUSION: Caution is required when assessing the significance of PBs associated with atypical cells in a cervical cytology specimen. Our case demonstrates the presence of ovarian mesothelial cells mimicking atypical glandular cells.

High-resolution immunophenotyping of subcellular compartments in tissue microarrays by enzyme metallography.

Ultrasensitive bright field in situ hybridization assays using enzyme metallography (EnzMet) have been developed and validated, but little is known regarding the applicability of EnzMet for immunophenotypic detection of protein via IHC. Superior resolution via discrete metallographic deposits offers the potential for enhancing high-resolution immunophenotyping. Using high-complexity tissue microarrays (TMAs), 88 common solid tumors were evaluated by automated EnzMet (Nanoprobes and Ventana). Targets were chosen to assess the ability of EnzMet to specifically localize encoded antigens in the nucleus (estrogen receptor), cytoplasm (cytokeratins), and cytoplasmic membrane (HER2) in TMAs. Results were compared with conventional IHC diaminobenzidine (DAB) immunostaining. There was full concordance between the EnzMet and conventional IHC results. Furthermore, the EnzMet reaction products did not appreciably diffuse, were dense and sharply defined, and provided excellent high-resolution differentiation of cellular compartments in paraffin sections for the nuclear, cytoplasmic, and cell membrane-localized antigens evaluated. The higher density of elemental silver deposited during enzyme metallography permitted evaluation of core immunophenotypes at a relatively low magnification, allowing more tissue to be screened in an efficient manner. This preliminary study shows the utility of using enzyme metallography for high-resolution immunophenotyping in TMAs.

Effect of dose-rate and irradiation geometry on the biological response of normal cells and cancer cells under radiotherapeutic conditions.

Biological efficacy of radiation depends on its energy, dose, dose rate, and on the type of cell irradiated. Changes in the radiation-energy spectrum due to passage through absorbing and scattering media affect the variability of biological responses of the cells. We investigated the impact of photon-radiation dose rate on the biological response of both normal and cancer cells in culture exposed to radiation in various positions (relative to the axis of the radiation beam) and depth of the absorbing medium (water). Human cancer cells (A549 and HCT116) as well as normal human cells (BEAS-2B) were placed in a water phantom at different medium depths (3 cm, 15 cm) and exposed to 6-MV photon radiation delivered at a beam rate of either 100 or 600 MU/min (Monitor Units per minute). The applied dose was 5 Gy. Cells were exposed in the axis and four cm outside the radiation field. Radiation-induced genetic changes were estimated as frequency of micro-nucleated and apopototic-like cells, by use of a cytokinesis-block micronucleus test. A smaller dose rate induced more severe cytogenetic damage (formation of micro-nucleated and apoptotic cells) than a higher dose rate, both in normal and in cancer cells. More micro-nucleated and apoptotic cells were formed at larger depth than at smaller depth. This holds true for both the normal and the two types of cancer cell investigated. The extent of cytogenetic damage arising in cells placed outside the irradiation field is independent of positioning depth and dose rate. Exposure of cells to smaller dose rates and larger depths in water medium resulted in a better ratio of cytogenetic damage to cancer cells irradiated in the beam axis vs damage to normal cells exposed outside the radiation field.

Breast metaplastic carcinoma with unusual small cell component.

Small round cell mesenchymal component in breast metaplastic carcinoma is very rare and could be confused with other small round cell neoplasms of the breast. Synovial sarcoma exceptionally involves the breast and rarely may show a poorly differentiated small round cell component. These unusual small round cell components in biphasic metaplastic carcinoma and synovial sarcoma as well as collision tumor of ductal carcinoma and Ewing sarcoma might have overlapping clinical, histologic and immunohistochemical features which make distinction between these neoplasms difficult for the pathologists. Separation between these neoplasms is important for the treating oncologists because each tumor type has different prognostic implications and treatment modalities. Certain immunohistochemical markers might help, but cytogenetics study is the final confirmatory test. We report a unique example of a biphasic breast neoplasm in a 43-year-old woman that showed a combination of a minor central component of adenocarcinoma distinct from the bulky surrounding component of small round cell tumor. The histologic and immunohistochemical features were overlapping between metaplastic carcinoma, poorly differentiated synovial sarcoma and a composite collision tumor of invasive ductal carcinoma and Ewing sarcoma.

High-resolution optical coherence tomography of subpigment epithelial structures in patients with pigment epithelium detachment secondary to age-related macular degeneration.

BACKGROUND: The pathophysiology of pigment epithelial detachment (PED) secondary to age-related macular degeneration (AMD) is as yet incompletely understood and treatment remains challenging. Spectral domain optical coherence tomography (SD-OCT) allows for improved morphological characterisation of the space underneath the retinal pigment epithelium (RPE). OBJECTIVE: To investigate eyes with PED for structures underneath the detached RPE cell layer. METHODS: In a retrospective observational case study, SD-OCT scans of AMD-related PEDs were assessed for the presence of distinctive morphological features in the space between the detached RPE and inner Bruch's membrane. RESULTS: Structures present in the space between the detached RPE and Bruch's membrane were found in 14 of 90 eyes with AMD-related PED. Each of these eyes shows hyper-reflective material underneath the PED, presenting as highly reflective, multilayered, laminar structures, usually orientated parallel to Bruch's membrane. CONCLUSIONS: The findings indicate that SD-OCT may be useful for a more refined phenotypic stratification of AMD-associated PED. Further studies are warranted to explore the correlates on other imaging modalities, to investigate the composition of this material and to assess the potential prognostic relevance of this new finding.

Noninvasive identification of subcellular organization and nuclear morphology features associated with leukemic cells using light-scattering spectroscopy.

Leukemia is the most common and deadly cancer among children and one of the most prevalent cancers among adults. Improvements in its diagnosis and monitoring of leukemic patients could have a significant impact in their long-term treatment. We demonstrate that light-scattering spectroscopy (LSS)-based approaches could serve as a tool to achieve this goal. Specifically, we characterize the light scattering properties of leukemic (NALM-6) cells and compare them to those of normal lymphocytes and granulocytes in the 440-710 nm range, over ±4 deg about the exact backscattering direction. We find that the LSS spectra are well described by an inverse power-law wavelength dependence, with a power exponent insensitive to the scattering angle but significantly higher for leukemic cells than for normal leukocytes. This is consistent with differences in the subcellular morphology of these cells, detected in differential interference contrast images. Furthermore, the residual light-scattering signal, extracted after subtracting the inverse power-law fit from the data, can be analyzed assuming a Gaussian distribution of spherical scatterers using Mie theory. This analysis yields scatterer sizes that are consistent with the diameters of cell nuclei and allows the detection of the larger nuclei of NALM-6 cells compared to those of lymphocytes and granulocytes.

Tumor cells disseminate early, but immunosurveillance limits metastatic outgrowth, in a mouse model of melanoma.

Although metastasis is the leading cause of cancer-related death, it is not clear why some patients with localized cancer develop metastatic disease after complete resection of their primary tumor. Such relapses have been attributed to tumor cells that disseminate early and remain dormant for prolonged periods of time; however, little is known about the control of these disseminated tumor cells. Here, we have used a spontaneous mouse model of melanoma to investigate tumor cell dissemination and immune control of metastatic outgrowth. Tumor cells were found to disseminate throughout the body early in development of the primary tumor, even before it became clinically detectable. The disseminated tumor cells remained dormant for varying periods of time depending on the tissue, resulting in staggered metastatic outgrowth. Dormancy in the lung was associated with reduced proliferation of the disseminated tumor cells relative to the primary tumor. This was mediated, at least in part, by cytostatic CD8+ T cells, since depletion of these cells resulted in faster outgrowth of visceral metastases. Our findings predict that immune responses favoring dormancy of disseminated tumor cells, which we propose to be the seed of subsequent macroscopic metastases, are essential for prolonging the survival of early stage cancer patients and suggest that therapeutic strategies designed to reinforce such immune responses may produce marked benefits in these patients.

Targeting hematogenous spread of circulating tumor cells by a chemotactic drug-eluting IVC filter to prevent pulmonary and systemic metastasis.

The formation of distant metastases is the main cause of morbidity and mortality in patients with cancer. Hematogenous spread through venous drainage of tumors is the main route of metastatic spread. We propose that the use of a drug-eluting chemotactic Inferior vena cava (IVC) filter coated with specific antibodies to surface markers of circulating tumor cells will attract these malignant cells and lyse them thus theoretically decreasing formation of distant systemic metastases. This could have a major impact on cancer related morbidity and mortality caused by metastatic disease.

Correlation of [18F]-2-fluoro-deoxy-D-glucose positron emission tomography standard uptake values with the cellular composition of stage I nonsmall cell lung cancer.

BACKGROUND: The aim of the current study was to determine whether the [18F]-2-fluoro-deoxy-D-glucose (FDG) positron emission tomography (PET) standardized uptake value (SUV) in patients with a new diagnosis of stage I lung cancer correlates with a specific cellular component in the primary tumor. METHODS: This study was Health Insurance Portability and Accountability Act compliant and approved by our Institutional Review Board with a waiver of informed consent. Forty patients with stage I nonsmall cell lung cancer (NSCLC) who underwent FDG-PET imaging at the time of diagnosis followed by surgical resection were retrospectively identified. Histologic sections of the primary tumor were reviewed by a pathologist without any clinical data and scored according to the percentage of each cellular component (tumor cells, normal stroma, inflammatory cells, necrosis, fibrosis, and other). Each component was compared with maximal (SUV(max)) and mean (SUV(mean)) SUVs using Pearson correlation coefficient analysis. RESULTS: The mean SUV(max) and SUV(mean) values for 40 stage I NSCLC tumors were 8.8 and 5.4, respectively. The mean histologic composition of tumor specimens in order of frequency was as follows: tumor cells (38.9%), fibrosis (30.8%), inflammatory cells (14.8%), normal stroma (5.2%), necrosis (5.8%), and other components (4.5%); however, there was considerable variation noted among individual tumors. There was no statistically significant correlation between SUV(max) (r = .19; P = .24) or SUV(mean) (r = .017; P = .29) and the proportion of tumor cells in the tumor mass or any other cellular components. CONCLUSIONS: The cellular composition of stage I NSCLC appears to be highly variable, with no correlation noted between a specific tumor cellular component and FDG activity.

A hierarchy of self-renewing tumor-initiating cell types in glioblastoma.

The neural stem cell marker CD133 is reported to identify cells within glioblastoma (GBM) that can initiate neurosphere growth and tumor formation; however, instances of CD133(-) cells exhibiting similar properties have also been reported. Here, we show that some PTEN-deficient GBM tumors produce a series of CD133(+) and CD133(-) self-renewing tumor-initiating cell types and provide evidence that these cell types constitute a lineage hierarchy. Our results show that the capacities for self-renewal and tumor initiation in GBM need not be restricted to a uniform population of stemlike cells, but can be shared by a lineage of self-renewing cell types expressing a range of markers of forebrain lineage.

Shank-interacting protein-like 1 promotes tumorigenesis via PTEN inhibition in human tumor cells.

Inactivation of phosphatase and tensin homolog (PTEN) is a critical step during tumorigenesis, and PTEN inactivation by genetic and epigenetic means has been well studied. There is also evidence suggesting that PTEN negative regulators (PTEN-NRs) have a role in PTEN inactivation during tumorigenesis, but their identity has remained elusive. Here we have identified shank-interacting protein-like 1 (SIPL1) as a PTEN-NR in human tumor cell lines and human primary cervical cancer cells. Ectopic SIPL1 expression protected human U87 glioma cells from PTEN-mediated growth inhibition and promoted the formation of HeLa cell-derived xenograft tumors in immunocompromised mice. Conversely, siRNA-mediated knockdown of SIPL1 expression inhibited the growth of both HeLa cells and DU145 human prostate carcinoma cells in vitro and in vivo in a xenograft tumor model. These inhibitions were reversed by concomitant knockdown of PTEN, demonstrating that SIPL1 affects tumorigenesis via inhibition of PTEN function. Mechanistically, SIPL1 was found to interact with PTEN through its ubiquitin-like domain (UBL), inhibiting the phosphatidylinositol 3,4,5-trisphosphate (PIP3) phosphatase activity of PTEN. Furthermore, SIPL1 expression correlated with loss of PTEN function in PTEN-positive human primary cervical cancer tissue. Taken together, these observations indicate that SIPL1 is a PTEN-NR and that it facilitates tumorigenesis, at least in part, through its PTEN inhibitory function.