Selective adhesion inhibition and hyaluronan envelope reduction of dermal tumor cells by cold plasma-activated medium

The sensitivity to cold plasma is specific to tumor cells while leaving normal tissue cells unaffected. This is the desired challenge in cancer therapy. Therefore, the focus of this work was a comparative study concerning the plasma sensitivity of dermal tumor cells (A-431) versus non-tumorigenic dermal cells (HaCaT) regarding their adhesion capacity. We found a selective inhibiting effect of plasma-activated medium on the adhesion of tumor cells while hardly affecting normal cells. We attributed this to a lower basal gene expression for the adhesion-relevant components CD44, hyaluronan synthase 2 (HAS2), HAS3, and the hyaluronidases in A431. Noteworthy, after plasma exposure, we revealed a significantly higher expression and synthesis of the hyaluronan envelope, the HAS3 gene, and the transmembrane adhesion receptors in non-tumorigenic HaCaTs.</p

Tumor-Preferential Induction of Immune Responses and Epidermal Cell Death in Actinic Keratoses by Ingenol Mebutate

<div><p>The rapid and strong clinical efficacy of the first-in-class, ingenol mebutate, against actinic keratosis (AK) has resulted in its recent approval. We conducted the first comprehensive analysis of the cellular and molecular mode of action of topical ingenol mebutate 0.05% gel in both AK and uninvolved skin of 26 patients in a phase I, single-center, open-label, within-patient comparison. As early as 1 day after application, ingenol mebutate induced profound epidermal cell death, along with a strong infiltrate of CD4⁺ and CD8⁺ T-cells, neutrophils, and macrophages. Endothelial ICAM-1 activation became evident after 2 days. The reaction pattern was significantly more pronounced in AK compared with uninvolved skin, suggesting a tumor-preferential mode of action. Extensive molecular analyses and transcriptomic profiling of mRNAs and microRNAs demonstrated alterations in gene clusters functionally associated with epidermal development, inflammation, innate immunity, and response to wounding. Ingenol mebutate reveals a unique mode of action linking directly to anti-tumoral effects.</p><p><b><i>Trial Registration</i>:</b> ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01387711" target="_blank">NCT01387711</a></p></div

Cold Atmospheric Plasma: A Promising Complementary Therapy for Squamous Head and Neck Cancer

<div><p>Head and neck squamous cell cancer (HNSCC) is the 7^th most common cancer worldwide. Despite the development of new therapeutic agents such as monoclonal antibodies, prognosis did not change for the last decades. Cold atmospheric plasma (CAP) presents the most promising new technology in cancer treatment. In this study the efficacy of a surface micro discharging (SMD) plasma device against two head and neck cancer cell lines was proved. Effects on the cell viability, DNA fragmentation and apoptosis induction were evaluated with the MTT assay, alkaline microgel electrophoresis (comet assay) and Annexin-V/PI staining. MTT assay revealed that the CAP treatment markedly decreases the cell viability for all tested treatment times (30, 60, 90, 120 and 180 s). IC 50 was reached within maximal 120 seconds of CAP treatment. Comet assay analysis showed a dose dependent high DNA fragmentation being one of the key players in anti-cancer activity of CAP. Annexin-V/PI staining revealed induction of apoptosis in CAP treated HNSCC cell lines but no significant dose dependency was seen. Thus, we confirmed that SMD Plasma technology is definitely a promising new approach on cancer treatment.</p></div

Inflammatory skin reactions induced by ingenol mebutate 0.05% gel are more pronounced in actinic keratosis (AK)-lesioned areas compared with uninvolved skin.

<p><b>(A)</b> CONSORT study diagram. This was a phase I, single-center, open-label, within-patient comparison trial to explore the biological effects of ingenol mebutate gel applied once daily for 2 consecutive days in patients with actinic keratosis (AK) in a 25-cm² area on the extremities and a 25-cm² area of uninvolved-skin (US) on the inner upper arm. <b>(B)</b> Typical skin reactions during the course of the trial in AK treatment areas of three representative patients at the dorsum of the hand (upper panel) (<i>n</i> = 26) and uninvolved skin of the inner upper arm (lower panel) (<i>n</i> = 26) at day 0 (baseline), day 1 (after one treatment application), and day 2 (after two treatment applications) as well as during follow-up at days 8 and 29. <b>(B)</b> The composite local skin response (LSR) score is the sum of six individual LSR scores including erythema, flaking/scaling, crusting, swelling, vesiculation/pustulation, and erosion/ulceration, which range from 0 to 4, with higher numbers indicating more severe reactions. This was calculated at each study visit for each patient, with a theoretical maximum composite score of 24. Patients were assessed on days 0, 1, 2, 8, and 29. No unexpected signs of local or systemic toxicity were noted. Illustrated are averages of the LSR; error bars indicate standard error of the mean.</p

Ingenol mebutate 0.05% gel treatment causes rapid infiltration of T-cells, macrophages, and neutrophilic granulocytes.

<p>Biopsy specimens from all patients (<i>n</i> = 26) were subjected to histopathological evaluation. Skin tissues from the two treatment areas and all time points were assessed, and representative images are depicted. The panels depict hematoxylin & eosin staining as well as immunohistochemical staining for CD4⁺ T-lymphocytes, CD8⁺ T-lymphocytes, CD68⁺ macrophages/histiocytes, and myeloperoxidase (MPO⁺) neutrophils as indicated. The scale bar represents 100 μm.</p

Topical treatment with ingenol mebutate 0.05% gel affects expression of gene clusters relevant for inflammatory and wound healing responses, and induces a characteristic microRNA (miRNA) pattern.

<p>The profiles illustrated are heat-maps and 2-way hierarchical clusterings of deregulated genes of <b>(A)</b> the 95 most variable mRNAs, and <b>(B)</b> the 64 most variable miRNAs across samples (<i>n</i> = 24) for the first six patients in the study. Differentially expressed genes and miRNAs were identified by pair-wise comparison of the following groups: actinic keratosis day 0 (AK0) versus uninvolved skin day 0 (US0), AK2 versus AK0, US2 versus US0, and AK2 versus US2. The expression analysis included variance filtering (VAR >0.2), statistical significance test by analysis of variance (<i>P</i> < 0.01), expression cut-off (>2-fold), and the false-discovery-rate was controlled by the Benjamini-Hochberg procedure (q < 0.05). Microarray data can be found in the GEO repository (GSE63107). Arrows indicate mRNA genes and miRNAs that were selected for validation by quantitative polymerase chain reaction. The colors above the heat map indicate: US0 (dark blue, <i>n</i> = 6), US2 (light blue, <i>n</i> = 6), AK0 (green, <i>n</i> = 6), and AK2 (red, <i>n</i> = 6) samples, respectively. The red and green shades on the heatmap represent up- and down-regulated genes, respectively.</p

Standard box-plot of apoptotic cells for FaDu cell line after different CAP treatment times.

<p>Standard box-plot of apoptotic cells for FaDu cell line after different CAP treatment times.</p

Apoptotic OSC-19 cells after different CAP treatment times.

<p>Apoptotic OSC-19 cells after different CAP treatment times.</p

Topical ingenol mebutate 0.05% gel activates cutaneous blood vessels, attracts B-cells, and induces epidermal cell death.

<p>Automated histomorphometric analyses on all samples from all patients (<i>n</i> = 26) were performed to quantitate expression of CD1a⁺ dendritic cells <b>(A)</b>, CD20⁺ B-cells <b>(B)</b>, and CD54 (ICAM-1) expressing cutaneous blood vessels and inflammatory cells within the dermis <b>(C)</b>. Expression of both CD20 and CD54 increased rapidly and strongly upon treatment with ingenol mebutate, and actinic keratosis (AK) lesions showed generally higher expression levels compared with uninvolved skin. <b>(D)</b> Dyskeratotic (dead) keratinocytes within the epidermis of all samples (<i>n</i> = 26 patients; five biopsy specimens each) were assessed using a scoring system ranging from 0 (absent, not depicted), 1 (few dyskeratotic cells), 2 (several dead cells) to 3 (numerous necrotic cells). No necrosis was evident in the dermis. TdT-mediated dUTP-biotin nick end labeling (TUNEL)⁺ apoptotic cells <b>(E)</b> and cleaved caspase 3 (CC3)⁺ nuclei <b>(F)</b> were determined in biopsies from five patients. A full statistical overview is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160096#pone.0160096.s007" target="_blank">S1 Table</a>.</p

Comet assay images after DNA-staining with ethidium bromide.

<p><b>A</b> Undamaged OSC-19 cell with intact DNA and no migration. DNA fragmentation leads to a faster and further migration into the electric field, which results in a figure shaped like a comet with undamaged DNA in the head and damaged DNA in the tail (<b>B+C</b>). The brighter and longer the tail, the higher the level of DNA fragmentation. <b>B</b> OSC-19 cell with a moderate CAP induced DNA-damage. <b>C</b> Representative image of high DNA-fragmentation.</p