Epithelial and Mesenchymal-like Pancreatic Cancer Cells Exhibit Different Stem Cell Phenotypes Associated with Different Metastatic Propensities.

Pancreatic ductal adenocarcinoma (PDAC) is mostly diagnosed at advanced or even metastasized stages, limiting the prognoses of patients. Metastasis requires high tumor cell plasticity, implying phenotypic switching in response to changing environments. Here, epithelial-mesenchymal transition (EMT), being associated with an increase in cancer stem cell (CSC) properties, and its reversion are important. Since it is poorly understood whether different CSC phenotypes exist along the EMT axis and how these impact malignancy-associated properties, we aimed to characterize CSC populations of epithelial and mesenchymal-like PDAC cells. Single-cell cloning revealed CSC (Holoclone) and non-CSC (Paraclone) clones from the PDAC cell lines Panc1 and Panc89. The Panc1 Holoclone cells showed a mesenchymal-like phenotype, dominated by a high expression of the stemness marker Nestin, while the Panc89 Holoclone cells exhibited a SOX2-dominated epithelial phenotype. The Panc89 Holoclone cells showed enhanced cell growth and a self-renewal capacity but slow cluster-like invasion. Contrarily, the Panc1 Holoclone cells showed slower cell growth and self-renewal ability but were highly invasive. Moreover, cell variants differentially responded to chemotherapy. In vivo, the Panc1 and Panc89 cell variants significantly differed regarding the number and size of metastases, as well as organ manifestation, leading to different survival outcomes. Overall, these data support the existence of different CSC phenotypes along the EMT axis in PDAC, manifesting different metastatic propensities.

Ovarian cancer cells regulate their mitochondrial content and high mitochondrial content is associated with a poor prognosis.

Most cancer patients ultimately die from the consequences of distant metastases. As metastasis formation consumes energy mitochondria play an important role during this process as they are the most important cellular organelle to synthesise the energy rich substrate ATP, which provides the necessary energy to enable distant metastasis formation. However, mitochondria are also important for the execution of apoptosis, a process which limits metastasis formation. We therefore wanted to investigate the mitochondrial content in ovarian cancer cells and link its presence to the patient's prognosis in order to analyse which of the two opposing functions of mitochondria dominates during the malignant progression of ovarian cancer. Monoclonal antibodies directed against different mitochondrial specific proteins, namely heat shock proteins 60 (HSP60), fumarase and succinic dehydrogenase, were used in immunohistochemistry in preliminary experiments to identify the antibody most suited to detect mitochondria in ovarian cancer cells in clinical tissue samples. The clearest staining pattern, which even delineated individual mitochondria, was seen with the anti-HSP60 antibody, which was used for the subsequent clinical study staining primary ovarian cancers (n = 155), borderline tumours (n = 24) and recurrent ovarian cancers (n = 26). The staining results were semi-quantitatively scored into three groups according to their mitochondrial content: low (n = 26), intermediate (n = 50) and high (n = 84). Survival analysis showed that high mitochondrial content correlated with a statistically significant overall reduced survival rate In addition to the clinical tissue samples, mitochondrial content was analysed in ovarian cancer cells grown in vitro (cell lines: OVCAR8, SKOV3, OVCAR3 and COV644) and in vivo in severe combined immunodeficiency (SCID) mice.In in vivo grown SKOV3 and OVCAR8 cells, the number of mitochondria positive cells was markedly down-regulated compared to the in vitro grown cells indicating that mitochondrial number is subject to regulatory processes. As high mitochondrial content is associated with a poor prognosis, the provision of high energy substrates by the mitochondria seems to be more important for metastasis formation than the inhibition of apoptotic cell death, which is also mediated by mitochondria. In vivo and in vitro grown human ovarian cancer cells showed that the mitochondrial content is highly adaptable to the growth condition of the cancer cells.

Spontaneous metastasis xenograft models link CD44 isoform 4 to angiogenesis, hypoxia, EMT and mitochondria-related pathways in colorectal cancer.

Hematogenous metastasis limits the survival of colorectal cancer (CRC) patients. Here, we illuminated the roles of CD44 isoforms in this process. Isoforms 3 and 4 were predominantly expressed in CRC patients. CD44 isoform 4 indicated poor outcome and correlated with epithelial-mesenchymal transition (EMT) and decreased oxidative phosphorylation (OxPhos) in patients; opposite associations were found for isoform 3. Pan-CD44 knockdown (kd) independently impaired primary tumor formation and abrogated distant metastasis in CRC xenografts. The xenograft tumors mainly expressed the clinically relevant CD44 isoforms 3 and 4. Both isoforms were enhanced in the paranecrotic, hypoxic tumor regions but were generally absent in lung metastases. Upon CD44 kd, tumor angiogenesis was increased in the paranecrotic areas, accompanied by reduced hypoxia-inducible factor-1α and CEACAM5 but increased E-cadherin expression. Mitochondrial genes and proteins were induced upon pan-CD44 kd, as were OxPhos genes. Hypoxia increased VEGF release from tumor spheres, particularly upon CD44 kd. Genes affected upon CD44 kd in xenografts specifically overlapped concordantly with genes correlating with CD44 isoform 4 (but not isoform 3) in patients, validating the clinical relevance of the used model and highlighting the metastasis-promoting role of CD44 isoform 4.

HLA-DPA1*02:01~B1*01:01 is a risk haplotype for primary sclerosing cholangitis mediating activation of NKp44+ NK cells.

OBJECTIVE: Primary sclerosing cholangitis (PSC) is characterised by bile duct strictures and progressive liver disease, eventually requiring liver transplantation. Although the pathogenesis of PSC remains incompletely understood, strong associations with HLA-class II haplotypes have been described. As specific HLA-DP molecules can bind the activating NK-cell receptor NKp44, we investigated the role of HLA-DP/NKp44-interactions in PSC. DESIGN: Liver tissue, intrahepatic and peripheral blood lymphocytes of individuals with PSC and control individuals were characterised using flow cytometry, immunohistochemical and immunofluorescence analyses. HLA-DPA1 and HLA-DPB1 imputation and association analyses were performed in 3408 individuals with PSC and 34 213 controls. NK cell activation on NKp44/HLA-DP interactions was assessed in vitro using plate-bound HLA-DP molecules and HLA-DPB wildtype versus knock-out human cholangiocyte organoids. RESULTS: NKp44+NK cells were enriched in livers, and intrahepatic bile ducts of individuals with PSC showed higher expression of HLA-DP. HLA-DP haplotype analysis revealed a highly elevated PSC risk for HLA-DPA1*02:01~B1*01:01 (OR 1.99, p=6.7×10-50). Primary NKp44+NK cells exhibited significantly higher degranulation in response to plate-bound HLA-DPA1*02:01-DPB1*01:01 compared with control HLA-DP molecules, which were inhibited by anti-NKp44-blocking. Human cholangiocyte organoids expressing HLA-DPA1*02:01-DPB1*01:01 after IFN-γ-exposure demonstrated significantly increased binding to NKp44-Fc constructs compared with unstimulated controls. Importantly, HLA-DPA1*02:01-DPB1*01:01-expressing organoids increased degranulation of NKp44+NK cells compared with HLA-DPB1-KO organoids. CONCLUSION: Our studies identify a novel PSC risk haplotype HLA-DP A1*02:01~DPB1*01:01 and provide clinical and functional data implicating NKp44+NK cells that recognise HLA-DPA1*02:01-DPB1*01:01 expressed on cholangiocytes in PSC pathogenesis.

HLA-DP on Epithelial Cells Enables Tissue Damage by NKp44+ Natural Killer Cells in Ulcerative Colitis.

BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by severe inflammation and destruction of the intestinal epithelium, and is associated with specific risk single nucleotide polymorphisms in HLA class II. Given the recently discovered interactions between subsets of HLA-DP molecules and the activating natural killer (NK) cell receptor NKp44, genetic associations of UC and HLA-DP haplotypes and their functional implications were investigated. METHODS: HLA-DP haplotype and UC risk association analyses were performed (UC: n = 13,927; control: n = 26,764). Expression levels of HLA-DP on intestinal epithelial cells (IECs) in individuals with and without UC were quantified. Human intestinal 3-dimensional (3D) organoid cocultures with human NK cells were used to determine functional consequences of interactions between HLA-DP and NKp44. RESULTS: These studies identified HLA-DPA1∗01:03-DPB1∗04:01 (HLA-DP401) as a risk haplotype and HLA-DPA1∗01:03-DPB1∗03:01 (HLA-DP301) as a protective haplotype for UC in European populations. HLA-DP expression was significantly higher on IECs of individuals with UC compared with controls. IECs in human intestinal 3D organoids derived from HLA-DP401pos individuals showed significantly stronger binding of NKp44 compared with HLA-DP301pos IECs. HLA-DP401pos IECs in organoids triggered increased degranulation and tumor necrosis factor production by NKp44+ NK cells in cocultures, resulting in enhanced epithelial cell death compared with HLA-DP301pos organoids. Blocking of HLA-DP401-NKp44 interactions (anti-NKp44) abrogated NK cell activity in cocultures. CONCLUSIONS: We identified an UC risk HLA-DP haplotype that engages NKp44 and activates NKp44+ NK cells, mediating damage to intestinal epithelial cells in an HLA-DP haplotype-dependent manner. The molecular interaction between NKp44 and HLA-DP401 in UC can be targeted by therapeutic interventions to reduce NKp44+ NK cell-mediated destruction of the intestinal epithelium in UC.

Identification of potential classes of glycoligands mediating dynamic endothelial adhesion of human tumor cells.

One critical step of metastasis formation is the extravasation of circulating tumor cells from the bloodstream. This process requires the dynamic interaction of cell adhesion molecules like E-selectin on endothelial cells with carbohydrate ligands on tumor cells. To characterize these glycans in a comprehensible approach, the rolling, tethering, and firm adhesion of nine human tumor cell lines on human umbilical vein endothelial cells was analyzed using laminar flow adhesion assays. The tumor cell lines were grouped into three subsets by their canonical E-selectin ligand status (sialyl-Lewis A and X +/+, -/+, -/-) and their adhesiveness was compared after enzymatic, pharmacologic, chemical treatment or antibody blockade of the tumor cells or endothelial cells, respectively. Tumor cells were also screened regarding their glycosyltransferase expression profile. We found that although E-selectin and terminal α2,3-sialic acid largely determined firm adhesion, adhesive events did not exclusively depend on the presence of sialyl-Lewis A and/or sialyl-Lewis X. Nevertheless, two of the three sialyl-Lewis A/X-/- tumor cells additionally or fully depended on vascular cell adhesion molecule-1 for firm adhesion. The significance of O-GalNAc- and N-glycans for adhesion varied remarkably among the tumor cells. The sialyl-Lewis A/X+/+ subset showed glycoprotein-independent adhesion, suggesting a role of glycolipids as well. All sialyl-Lewis A/X-/- tumor cells lacked FUT3 and FUT7 expression as opposed to sialyl-Lewis A/X+/+ or -/+ cell lines. In summary, the glycans on tumor cells mediating endothelial adhesion are not as much restricted to sialyl-Lewis A /X as previously assumed. The present study specifically suggests α2,3-linked sialic acid, O-GalNAc glycans, glycosphingolipids, and FUT3/FUT7 products as promising targets for future studies.

PSMA-heterogeneity in metastatic castration-resistant prostate cancer: Circulating tumor cells, metastatic tumor burden, and response to targeted radioligand therapy.

BACKGROUND: We explored the interrelation between prostate-specific membrane antigen (PSMA) expression on circulating tumor cells (CTCs) and that of solid metastatic lesions as determined by whole-body PSMA-targeted positron emission tomography (PET) to refine the prediction of response to subsequent PSMA-targeted radioligand therapy (RLT). METHODS: A prospective study was performed in 20 patients with advanced mCRPC. Of these, 16 underwent subsequent RLT with [177 Lu]Lu-PSMA-617 at a dose of 7.4 GBq every 6-8 weeks. PSMA expression on CTCs using the CellSearch system was compared to clinical and serological results, and to marker expression in targeted imaging and available histological sections of prostatectomy specimens (19% of RLT patients). Clinical outcome was obtained after two cycles of RLT. RESULTS: Marked heterogeneity of PSMA expression was observed already at first diagnosis in available histological specimens. Targeted whole-body imaging also showed heterogeneous inter- and intra-patient PSMA expression between metastases. Heterogeneity of CTC PSMA expression was partially paralleled by heterogeneity of whole-body tumor burden PSMA expression. Twenty percent of CTC samples showed no PSMA expression, despite unequivocal PSMA expression of solid metastases at PET. A high fraction of PSMA-negative CTCs emerged as the sole predictor of poor RLT response (odds ratio [OR]: 0.9379 [95% confidence interval, CI, 0.8558-0.9902]; p = 0.0160), and was prognostic for both shorter progression-free survival (OR: 1.236 [95% CI, 1.035-2.587]; p = 0.0043) and overall survival (OR: 1.056 [95% CI, 1.008-1.141]; p = 0.0182). CONCLUSION: This proof-of-principle study suggests that liquid biopsy for CTC PSMA expression is complementary to PET for individual PSMA phenotyping of mCRPC.

The chemicals between us-First results of the cluster analyses on anatomy embalming procedures in the German-speaking countries.

Hands-on courses utilizing preserved human tissues for educational training offer an important pathway to acquire basic anatomical knowledge. Owing to the reevaluation of formaldehyde limits by the European Commission, a joint approach was chosen by the German-speaking anatomies in Europe (Germany, Austria, Switzerland) to find commonalities among embalming protocols and infrastructure. A survey comprising 537 items was circulated to all anatomies in German-speaking Europe. Clusters were established for "ethanol"-, formaldehyde-based ("FA"), and "other" embalming procedures, depending on the chemicals considered the most relevant for each protocol. The logistical framework, volumes of chemicals, and infrastructure were found to be highly diverse between the groups and protocols. Formaldehyde quantities deployed per annum were three-fold higher in the "FA" (223 L/a) compared to the "ethanol" (71.0 L/a) group, but not for "other" (97.8 L/a), though the volumes injected per body were similar. "FA" was strongly related to table-borne air ventilation and total fixative volumes ≤1000 L. "Ethanol" was strongly related to total fixative volumes >1000 L, ceiling- and floor-borne air ventilation, and explosion-proof facilities. Air ventilation was found to be installed symmetrically in the mortuary and dissection facilities. Certain predictors exist for the interplay between the embalming used in a given infrastructure and technical measures. The here-established cluster analysis may serve as decision supportive tool when considering altering embalming protocols or establishing joint protocols between institutions, following a best practice approach to cater toward best-suited tissue characteristics for educational purposes, while simultaneously addressing future demands on exposure limits.

Expanded ILC2s in human infant intestines promote tissue growth.

Early life is characterized by extraordinary challenges, including rapid tissue growth and immune adaptation to foreign antigens after birth. During this developmental stage, infants have an increased risk of immune-mediated diseases. Here, we demonstrate that tissue-resident, interleukin (IL)-13- and IL-4-producing group 2 innate lymphoid cells (ILC2s) are enriched in human infant intestines compared to adult intestines. Organoid systems were employed to assess the role of infant intestinal ILC2s in intestinal development and showed that IL-13 and IL-4 increased epithelial cell proliferation and skewed cell differentiation toward secretory cells. IL-13 furthermore upregulated the production of mediators of type-2 immunity by infant intestinal epithelial cells, including vascular endothelial growth factor-A and IL-26, a chemoattractant for eosinophils. In line with these in vitro findings increased numbers of eosinophils were detected in vivo in infant intestines. Taken together, ILC2s are enriched in infant intestines and can support intestinal development while inducing an epithelial secretory response associated with type 2 immune-mediated diseases.

An Accessory Muscle Originating From the Hyoid Bone and Inserting Into the Sternocleidomastoid Muscle.

Irregular bellies and insertions into neck muscles have been described in the literature. To the best of our knowledge, a right accessory muscle originating from the hyoid bone and inserting into the sternocleidomastoid muscle has not been reported to date. Here, we report the case of a 72-year-old male patient with an irregular muscle originating from the lesser horn of the hyoid bone and inserting into sternocleidomastoid muscle fibers.

Amount and spatial arrangement of muscle fibers in the human laryngeal Musculus ventricularis.

Textbooks and atlases of human macroscopic and microscopic anatomy of the larynx generally provide, if at all, only sparse information on the laryngeal Musculus ventricularis. However, several studies indicate that this muscle takes over the function of vestibular (ventricular) fold phonation after denervation of the Musculus vocalis. In the present study, 29 laryngeal specimens were coronally dissected at different levels, i.e. the anterior (L1), middle (L2), and posterior third of the vestibular fold (L3), and they underwent histological analysis. In all specimens the vestibular folds of both hemi-larynxes contained striated muscle bundles in variable amounts, representing a ventricularis muscle. These muscle bundles obviously originated from the lateral (external) and thyroepiglottic part of the thyroarytenoid muscle and the aryepiglottic part of the oblique arytenoid muscle, as has been described by other authors. The areas of vestibular folds and their amounts of ventricularis muscle bundles were measured using image analysis software (imageJ) by manual tracing. The mean area of the vestibular folds of both hemi-larynxes was 27.9 mm2 (SD [standard deviation] ± 9.17), and the area occupied by fibers of the ventricularis muscle was 1.5 mm2 (SD ± 1.78). Statistical analysis comparing the areas of both hemi-larynxes and levels resulted in no significant differences, except for the levels 2 and 3. In level 2, significantly more muscle fibers (2.0 mm2 ; SD ± 2.21) were detectable within the vestibular fold than in level 3 (0.9 mm2 ; SD ± 1.43). Level 1 also contained more muscle fibers (1.1 mm2 ; SD ± 1.06) than level 3, however, without significance. In conclusion, the laryngeal ventricularis muscle is present in the majority of reported cases. Since the muscle is of clinical relevance, it should be included in anatomical textbooks by default.

Efficacy of zoledronic acid for the elimination of disseminated tumor cells in a clinically relevant, spontaneously metastatic prostate cancer xenograft model.

Bone metastases develop in >90 % of patients with castration-resistant prostate cancer (PCa) through complex interactions between the bone microenvironment and tumor cells. Previous androgen-deprivation therapy (ADT), which is known to cause bone loss, as well as anti-resorptive agents such as zoledronic acid (ZA), used to prevent skeletal complications, may influence these interactions and thereby the growth of disseminated tumor cells (DTC) in the bone marrow (BM). Here, a spontaneously metastatic xenograft tumor model of human PCa was further optimized to mimic the common clinical situation of ADT (castration) combined with primary tumor resection in vivo. The effects of these interventions, alone or in combination with ZA treatment, on tumor cell dissemination to the BM and other distant sites were analyzed. Metastatic burden was quantified by human-specific Alu-qPCR, bioluminescence imaging (BLI), and immunohistochemistry. Further, bone remodeling was assessed by static histomorphometry and serum parameters. Initial comparative analysis between NSG and SCID mice showed that spontaneous systemic dissemination of subcutaneous PC-3 xenograft tumors was considerably enhanced in NSG mice. Primary tumor resection and thereby prolonged observational periods resulted in a higher overall metastatic cell load at necropsy and tumor growth alone caused significant bone loss, which was further augmented by surgical castration. In addition, castrated mice showed a strong trend towards higher bone metastasis loads. Weekly treatment of mice with ZA completely prevented castration- and tumor-induced bone loss but had no effect on bone metastasis burden. Conversely, the total lung metastasis load as determined by BLI was significantly decreased upon ZA treatment. These findings provide a basis for future research on the role of ZA not only in preventing skeletal complications but also in reducing metastasis to other organs.

Tumor cell integrin ß4 and tumor stroma E-/P-selectin cooperatively regulate tumor growth in vivo.

BACKGROUND: The immunological composition of the tumor microenvironment has a decisive influence on the biological course of cancer and is therefore of profound clinical relevance. In this study, we analyzed the cooperative effects of integrin ß4 (ITGB4) on tumor cells and E-/P-selectin on endothelial cells within the tumor stroma for regulating tumor growth by shaping the local and systemic immune environment. METHODS: We used several preclinical mouse models for different solid human cancer types (xenograft and syngeneic) to explore the role of ITGB4 (shRNA-mediated knockdown in tumor cells) and E-/P-selectins (knockout in mice) for tumor growth; effects on apoptosis, proliferation and intratumoral signaling pathways were determined by histological and biochemical methods and 3D in vitro experiments; changes in the intratumoral and systemic immune cell composition were determined by flow cytometry and immunohistochemistry; chemokine levels and their attracting potential were measured by ELISA and 3D invasion assays. RESULTS: We observed a very robust synergism between ITGB4 and E-/P-selectin for the regulation of tumor growth, accompanied by an increased recruitment of CD11b+ Gr-1Hi cells with low granularity (i.e., myeloid-derived suppressor cells, MDSCs) specifically into ITGB4-depleted tumors. ITGB4-depleted tumors undergo apoptosis and actively attract MDSCs, well-known to promote tumor growth in several cancers, via increased secretion of different chemokines. MDSC trafficking into tumors crucially depends on E-/P-selectin expression. Analyses of clinical samples confirmed an inverse relationship between ITGB4 expression in tumors and number of tumor-infiltrating leukocytes. CONCLUSIONS: These findings suggest a distinct vulnerability of ITGB4Lo tumors for MDSC-directed immunotherapies.

The role of the desmosomal protein desmocollin 2 in tumour progression in triple negative breast cancer patients.

BACKGROUND: The disruption of epithelial features represents a critical step during breast cancer spread. In this context, the dysregulation of desmosomal proteins has been associated with malignant progression and metastasis formation. Curiously, both tumour suppressive and pro-metastatic roles have been attributed to desmosomal structures in different cancer entities. In the present study, we describe the pro-metastatic role of the desmosomal protein desmocollin 2 (DSC2) in breast cancer. METHODS: We analysed the prognostic role of DSC2 at mRNA and protein level using microarray data, western blot analysis and immunohistochemistry. Functional consequences of DSC2 overexpression and DSC2 knock down were investigated in the triple negative breast cancer (TNBC) cell line MDA-MB-231 and its brain-seeking subline MDA-MB-231-BR, respectively in vitro and in vivo. RESULTS: We found a significantly higher DSC2 expression in the more aggressive molecular subtypes HER2-positive and TNBC than in luminal breast cancers, as well as a significant correlation between increased DSC2 expression and a shorter disease-free-also in multivariate analysis-and overall survival. Additionally, a significant association between DSC2 expression in the primary tumour and an increased frequency of cerebral and lung metastasis could be observed. In vitro, ectopic DSC2 expression or DSC2 down-regulation in MDA-MB-231 and MDA-MB-231-BR led to a significant tumour cell aggregation increase and decrease, respectively. Furthermore, tumour cells displaying higher DSC2 levels showed increased chemoresistance in 3D structures, but not 2D monolayer structures, suggesting the importance of cell aggregation as a means for reduced drug diffusion. In an in vivo brain dissemination xenograft mouse model, reduced expression of DSC2 in the brain-seeking TNBC cells led to a decreased amount of circulating tumour cells/clusters and, in turn, to fewer and smaller brain metastatic lesions. CONCLUSION: We conclude that high DSC2 expression in primary TNBC is associated with a poorer prognosis, firstly by increasing tumour cell aggregation, secondly by reducing the diffusion and effectiveness of chemotherapeutic agents, and, lastly, by promoting the circulation and survival of tumour cell clusters, each of which facilitates distant organ colonisation.

CXCR5+PD-1++ CD4+ T cells colonize infant intestines early in life and promote B cell maturation.

Gastrointestinal infections are a major cause for serious clinical complications in infants. The induction of antibody responses by B cells is critical for protective immunity against infections and requires CXCR5+PD-1++ CD4+ T cells (TFH cells). We investigated the ontogeny of CXCR5+PD-1++ CD4+ T cells in human intestines. While CXCR5+PD-1++ CD4+ T cells were absent in fetal intestines, CXCR5+PD-1++ CD4+ T cells increased after birth and were abundant in infant intestines, resulting in significant higher numbers compared to adults. These findings were supported by scRNAseq analyses, showing increased frequencies of CD4+ T cells with a TFH gene signature in infant intestines compared to blood. Co-cultures of autologous infant intestinal CXCR5+PD-1+/-CD4+ T cells with B cells further demonstrated that infant intestinal TFH cells were able to effectively promote class switching and antibody production by B cells. Taken together, we demonstrate that functional TFH cells are numerous in infant intestines, making them a promising target for oral pediatric vaccine strategies.

The actin bundling activity of ITPKA mainly accounts for its migration-promoting effect in lung cancer cells.

Expression of Ins(1,4,5)P3-kinase-A (ITPKA), the neuronal isoform of Ins(1,4,5)P3-kinases, is up-regulated in many tumor types. In particular, in lung cancer cells this up-regulation is associated with bad prognosis and it has been shown that a high level of ITPKA increases migration and invasion of lung cancer cell lines. However, since ITPKA exhibits actin bundling and Ins(1,4,5)P3-kinase activity, it was not clear which of these activities account for ITPKA-promoted migration and invasion of cancer cells. To address this issue, we inhibited endogenous actin bundling activity of ITPKA in lung cancer H1299 cells by overexpressing the dominant negative mutant ITPKAL34P. Analysis of actin dynamics in filopodia as well as wound-healing migration revealed that ITPKAL34P inhibited both processes. Moreover, the formation of invasive protrusions into collagen I was strongly blocked in cells overexpressing ITPKAL34P. Furthermore, we found that ATP stimulation slightly but significantly (by 13%) increased migration of cells overexpressing ITPKA while under basal conditions up-regulation of ITPKA had no effect. In accordance with these results, overexpression of a catalytic inactive ITPKA mutant did not affect migration, and the Ins(1,4,5)P3-kinase-inhibitor GNF362 reversed the stimulating effect of ITPKA overexpression on migration. In summary, we demonstrate that under basal conditions the actin bundling activity controls ITPKA-facilitated migration and invasion and in presence of ATP the Ins(1,4,5)P3-kinase activity slightly enhances this effect.

Description and nomenclature of organ subdivisions in the Terminologia Anatomica and in anatomical education: Comparison with cancer classifications.

Terminology is the basis for communication among medical professionals. For anatomists, their daily work is based on the Terminologia Anatomica (TA), while pathologists cite the Tumor Node Metastasis (TNM) classification when referring to the anatomical boundaries and regions of malignant tumors. Terminologia Anatomica and clinical-based classifications, including the TMN classification of tumors, use a world-wide standardized nomenclature, which has been revised regularly to incorporate new anatomical discoveries and clinically relevant structures. In medical education, students are familiarized with medical nomenclatures in anatomy textbooks and online learning platforms. Sometime, descriptions and illustrations in anatomy teaching materials put a different focus on the importance of anatomical subdivisions and their borders than is found in cancer classifications. This discrepancy contrasts with the efforts of medical societies to define and implement clinically relevant anatomical structures, including organ subdivisions and their boundaries, in daily clinical practice. Here, we illustrate this problem using the larynx and pancreas as examples. Anatomy education should coordinate teaching content with the requirements of the clinical disciplines.

Nestin expression in osteocytes following myeloablation and during bone marrow metastasis.

Nestin is an intermediate filament protein, which was originally detected in neuroepithelial stem cells. Besides its use as a phenotypic marker of mesenchymal stem cells in the hematopoeitic stem cell niche, the functional interpretation of nestin+ cells remains elusive. We investigated the cellular expression of nestin in bone marrow trephine biopsies of MPN patients, following myeloablation at a stage of hypocellularity during early regeneration. Here, nestin is highly expressed in mature osteocytes, arteriolar endothelial and perivascular cells and small capillaries within the bone marrow space, but not in sinusoid lining cells. This is in stark contrast to nestin expression pattern in myeloproliferative neoplasms that show hypercellularity due to oncogenic driver mutations. Here, nestin is expressed exclusively in endothelial cells of arterioles, but not in osteocytes or small capillaries. Thus, the pattern of nestin expression following myeloablation inversely correlates with cellularity in the bone marrow. This nestin expression pattern is mimicking early postnatal transcriptional programming during bone marrow development. We show that nestin expression in osteocytes occurs across different species following transplant and also in bone marrow metastasis.

Description of the normal gastric mucosa in anatomy education: How many leukocytes are acceptable?

Textbooks covering normal human histology illustrate an allegedly normal gastric mucosa containing significant infiltrates of mononuclear cells in the lamina propria. This standard description seems to conflict with the pathologist's criterion for normality, which specifies only a few or a complete absence of inflammatory cells. Eventually, both anatomists and pathologists face the dilemma: how much infiltrate should their students and medical colleagues be told is acceptable for the gastric mucosa to be classified as normal? Summarizing the suggestions of experts in gastroenterology and our own experience, a simple approach could be to accept no more than five mononuclear and plasma cells per high power field as normal (400-fold magnification with a field area of 0.24 mm2 ). The divergence of views on this topic illustrates the need for anatomists and pathologists to find a consensus about the definition of "normal" tissues.

Generating Patient-Derived HCC Cell Lines Suitable for Predictive In Vitro and In Vivo Drug Screening by Orthotopic Transplantation.

Hepatocellular carcinoma (HCC) results in high mortality due to ineffective systemic therapy. Human immortalized cell lines are commonly used to study anti-tumor effects in the context of new anti-tumor therapies and tumor biology. As immortalized cell lines have limited biological relevance and heterogeneity compared to primary cells, patient-derived tumor tissues, and corresponding immune cells are the gold standards for studying the complexity of individual tumor entities. However, culturing primary HCC cells has a low success rate. Here, we aimed to establish a reproducible approach to preserve the patient-derived liver cancer cells for in vitro and in vivo studies. The underlying study aimed to establish an in vitro pre-screening platform to test treatment options' effectivity and dosage, e.g., for new substances, autologous modified immune cells, or combined therapies in HCC. We initially employed 15 surgical resection specimens from patients with different HCC entities for isolation and preservation. The isolated liver cancer cells from four HCC-diagnosed patients were used for orthotopic transplantation into the healthy liver of immunodeficient mice, allowing them to grow for six months before human liver cancer cells were isolated and cultured. As a result, we generated and characterized four new primary-like liver cancer cell lines. Compared to immortalized HCC cell lines, freshly generated liver cancer cells displayed individual morphologies and heterogeneous protein-level characteristics. We assessed their ability to proliferate, migrate, form spheroids, and react to common medications compared to immortalized HCC cell lines. All four liver cancer cell lines exhibit strong migration and colony-forming characteristics in vitro, comparable to extensively investigated immortalized HCC cell lines. Moreover, the four etiological different liver cancer cell lines displayed differences in the response to 5-FU, Sorafenib, Axitinib, and interferon-alpha treatment, ranking from non-responders to responders depending on the applicated medication. In sum, we generated individual patient-derived liver cancer cell lines suitable for predictive in vitro drug screenings and for xenograft transplantations to realize the in vivo investigation of drug candidates. We overcame the low cultivation success rate of liver cancer cells derived from patients and analyzed their potential to serve a pre-clinical model.