Initial activation of EpCAM cleavage via cell-to-cell contact

<p>Abstract</p> <p>Background</p> <p>Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is frequently over-expressed in simple epithelia, progenitors, embryonic and tissue stem cells, carcinoma and cancer-initiating cells. Besides functioning as a homophilic adhesion protein, EpCAM is an oncogenic receptor that requires regulated intramembrane proteolysis for activation of its signal transduction capacity. Upon cleavage, the extracellular domain EpEX is released as a soluble ligand while the intracellular domain EpICD translocates into the cytoplasm and eventually into the nucleus in combination with four-and-a-half LIM domains protein 2 (FHL2) and β-catenin, and drives cell proliferation.</p> <p>Methods</p> <p>EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were investigated under varying density conditions using confocal laser scanning microscopy, immunoblotting, cell counting, and conditional cell systems.</p> <p>Results</p> <p>EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were dependent on adequate cell-to-cell contact. If cell-to-cell contact was prohibited EpCAM did not provide growth advantages. If cells were allowed to undergo contact to each other, EpCAM transmitted proliferation signals based on signal transduction-related cleavage processes. Accordingly, the pre-cleaved version EpICD was not dependent on cell-to-cell contact in order to induce <it>c-myc </it>and cell proliferation, but necessitated nuclear translocation. For the case of contact-inhibited cells, although cleavage of EpCAM occurred, nuclear translocation of EpICD was reduced, as were EpCAM effects.</p> <p>Conclusion</p> <p>Activation of EpCAM's cleavage and oncogenic capacity is dependent on cellular interaction (juxtacrine) to provide for initial signals of regulated intramembrane proteolysis, which then support signalling via soluble EpEX (paracrine).</p

German S3 guideline "actinic keratosis and cutaneous squamous cell carcinoma" – long version of the update 2023

Actinic keratosis (AK) are common lesions in light-skinned individuals that can potentially progress to cutaneous squamous cell carcinoma (cSCC). Both conditions may be associated with significant morbidity and constitute a major disease burden, especially among the elderly. To establish an evidence-based framework for clinical decision making, the guideline “actinic keratosis and cutaneous squamous cell carcinoma” was updated and expanded by the topics cutanepus squamous cell carcinoma in situ (Bowen’s disease) and actinic cheilitis. This guideline was developed at the highest evidence level (S3) and is aimed at dermatologists, general practitioners, ear nose and throat specialists, surgeons, oncologists, radiologists and radiation oncologists in hospitals and office-based settings, as well as other medical specialties, policy makers and insurance funds involved in the diagnosis and treatment of patients with AK and cSCC

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

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

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

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

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

miR-323a regulates ERBB4 and is involved in depression.

International audienceMajor depressive disorder (MDD) is a complex and debilitating illness whose etiology remains unclear. Small RNA molecules, such as micro RNAs (miRNAs) have been implicated in MDD, where they display differential expression in the brain and the periphery. In this study, we quantified miRNA expression by small RNA sequencing in the anterior cingulate cortex and habenula of individuals with MDD and psychiatrically-healthy controls. Thirty-two miRNAs showed significantly correlated expression between the two regions (False Discovery Rate < 0.05), of which four, miR-204-5p, miR-320b, miR-323a-3p, and miR-331-3p, displayed upregulated expression in MDD. We assessed the expression of predicted target genes of differentially expressed miRNAs in the brain, and found that the expression of erb-b2 receptor tyrosine kinase 4 (ERBB4), a gene encoding a neuregulin receptor, was downregulated in both regions, and was influenced by miR-323a-3p in vitro. Finally, we assessed the effects of manipulating miRNA expression in the mouse ACC on anxiety- and depressive-like behaviors. Mice in which miR-323-3p was overexpressed or knocked-down displayed increased and decreased emotionality, respectively. Additionally, these mice displayed significantly downregulated and upregulated expression of Erbb4, respectively. Overall, our findings indicate the importance of brain miRNAs in the pathology of MDD, and emphasize the involvement of miR-323a-3p and ERBB4 in this phenotype. Future studies further characterizing miR-323a-3p and neuregulin signaling in depression are warranted

S3 guideline for actinic keratosis and cutaneous squamous cell carcinoma – short version, part 1: diagnosis, interventions for actinic keratoses, care structures and quality‐of‐care indicators

Actinic keratoses (AK) are common lesions in light‐skinned individuals that can potentially progress to cutaneous squamous cell carcinoma (cSCC). Both conditions may be associated with significant morbidity and constitute a major disease burden, especially among the elderly. To establish an evidence‐based framework for clinical decision making, the guideline “actinic keratosis and cutaneous squamous cell carcinoma” was developed using the highest level of methodology (S3) according to regulations issued by the Association of Scientific Medical Societies in Germany (AWMF). The guideline is aimed at dermatologists, general practitioners, ENT specialists, surgeons, oncologists, radiologists and radiation oncologists in hospitals and office‐based settings as well as other medical specialties involved in the diagnosis and treatment of patients with AK and cSCC. The guideline is also aimed at affected patients, their relatives, policy makers and insurance funds. In the first part, we will address aspects relating to diagnosis, interventions for AK, care structures and quality‐of‐care indicators.Peer Reviewe