Rapid and Non-Enzymatic <em>In Vitro</em> Retrieval of Tumour Cells from Surgical Specimens

<div><p>The study of tumourigenesis commonly involves the use of established cell lines or single cell suspensions of primary tumours. Standard methods for the generation of short-term tumour cell cultures include the disintegration of tissue based on enzymatic and mechanical stress. Here, we describe a simple and rapid method for the preparation of single cells from primary carcinomas, which is independent of enzymatic treatment and feeder cells. Tumour biopsies are processed to 1 mm³ cubes termed explants, which are cultured 1–3 days on agarose-coated well plates in specified medium. Through incisions generated in the explants, single cells are retrieved and collected from the culture supernatant and can be used for further analysis including <em>in vitro</em> and <em>in vivo</em> studies. Collected cells retain tumour-forming capacity in xenotransplantation assays, mimic the phenotype of the primary tumour, and facilitate the generation of cell lines.</p> </div

The indicated cell surface markers were measured in three independent experiments with specific antibodies using flow cytometry.

<p>Shown are mean fluorescence intensity ratios (MFI ratio) and standard deviations (SD).</p

Single cells from head and neck specimens recapitulate primary tumours.

<p>(<b>A</b>) Biopsy of a hypopharynx carcinoma was processed to explants of approx. 1 mm³ and transferred into agarose-coated 24-well plates. Shown is the primary tumour biopsy and explants in wells. (<b>B</b>) Upon incision and cultivation in specified medium, cells migrate out of the explant and settle in the well. Shown is one representative example at day one. (<b>C</b>) Upper panels: Explants were collected after one day, cryo-preserved and processed to sequential sections before staining with EpCAM-, CD133-specific or an isotype control antibody as indicated. The structure of explants begins to disintegrate and cells migrate away from the major tumour area and are released into the supernatant. Cells express EpCAM but no CD133. Lower panels: Single cells migrating out of explants were subjected to an EpCAM-, CD133 or isotype control antibody staining. The majority of cells expressed EpCAM and only rarely expressed CD133. (<b>D</b>) Matched primary carcinoma was stained for the expression of EpCAM and Ki-67. Shown are 40× (upper panel) and 200× magnifications (middle and lower panels) of the indicated stainings. (<b>E</b>) Single cells from explants of primary specimen shown in (D) were stained in cytospins with EpCAM-specific (upper panel) or an isotype control antibody (lower panel). (<b>F</b>) Cell surface expression of EpCAM on single cells from explants of primary specimen shown in (D) was analysed upon flow cytometry with specific antibodies. Left dot plot represents isotype control, while right dot plot represents EpCAM staining. Dot plot statistics according to the marker set for both graphs are given for isotype control and EpCAM staining. (<b>G</b>) Single cells (1–5×10⁶) from explants of primary specimen shown in (D) were xenotransplanted subcutaneously in immunocompromised NOD-SCID mice. After 28 days tumours were surgically removed, cryo-preserved, and stained for the expression of EpCAM and Ki-67. Shown are 100× (upper panel) and 200× magnifications (middle and lower panels) of the indicated stainings.</p

Schematic representation of the method of tumour explant formation.

<p>Specimens of primary carcinomas (>5 mm³) are cut into approximately 1 mm³ cube-like structures termed explants. One part of the primary carcinoma is cryo-preserved as a matched control for explanted cells. Incisions are generated in explants, which are then transferred into agarose-coated 96- or 24-well plates for a time period of 1–3 days. Supernatants are collected and cells harvested upon centrifugation for subsequent analysis or examination.</p

Generation of a permanent cell line from tumour explants.

<p>(<b>A</b>) Matched sample (upper left panel) and single cells (lower left panel) obtained from explants of a larynx carcinoma were stained for the expression of EpCAM. Harvested cells were passaged to establish a permanently growing cell line. PiCa cells grew in adherent manner and displayed heterogeneity in morphology (right panel). (<b>B</b>) PiCa cells were monitored for the cell surface expression of EpCAM, EGF-R, and CD44 using specific antibodies. Cells represented one determined population (FSC) and were vital (PI) as shown in the dot plot graph (upper left). (<b>C</b>) PiCa cells were transiently transfected with control or EpCAM-specific siRNA. After 24 hrs, cells were analysed for the cell surface expression of EpCAM upon flow cytometry. Isotype control staining is depicted as solid, purple curve, control siRNA as green line, and EpCAM-siRNA as dashed pink line. (<b>D</b>) PiCa cells were transiently transfected with the eGFP-C1 expression plasmid. After 24 hrs, expression of GFP was assessed upon flow cytometry. Dot plots graphs represent control transfectants (upper panel) and GFP transfectants (lower panel). Quadrants were set according to control transfectants and statistics are given below. (<b>E</b>) PiCa cells (1×10⁴, 1×10⁵, 1×10⁶) were subcutaneously injected into the left and right flanks of immunocompromised NOD-SCID mice. After 21 days, tumours were surgically removed, cryo-preserved and stained for the expression of EpCAM. Matched primary specimens were stained in parallel (left panels) to xenotransplants (right panels) and are depicted in 100× and 200× magnification. Injected cell numbers and tumour weights with mean and standard deviations are given in tabular manner.</p

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

Apoptotic OSC-19 cells after different CAP treatment times.

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

Annexin V/PI Staining of HNSCC cells.

<p>Results obtained by fluorescence microscopy show that early apoptotic cells have bound Annexin-FITC to the phosphatidylserin on the membrane surface (green cell). As apoptosis progressed, the plasma membrane integrity gets lost, and the propidium iodide is able to bind to nucleotid DNA, so that late apoptotic or necrotic cells appear in red.</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

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