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Minimally invasive robotic surgery offer benefits such as reduced physical trauma, faster recovery and lesser pain for the patient. For these procedures, visual and haptic feedback to the surgeon is crucial when operating surgical tools without line-of-sight with a robot. External force sensors are biased by friction at the tool shaft and thereby cannot estimate forces between tool tip and tissue. As an alternative, vision-based force estimation was proposed. Here, interaction forces are directly learned from deformation observed by an external imaging system. Recently, an approach based on optical coherence tomography and deep learning has shown promising results. However, most experiments are performed on ex-vivo tissue. In this work, we demonstrate that models trained on dead tissue do not perform well in in vivo data. We performed multiple experiments on a human tumor xenograft mouse model, both on in vivo, perfused tissue and dead tissue. We compared two deep learning models in different training scenarios. Training on perfused, in vivo data improved model performance by 24% for in vivo force estimation.

First described as a neuronal cell adhesion molecule, L1CAM was later identified to be present at increased levels in primary tumors and metastases of various types of cancer. Here, we describe the multifaceted roles of L1CAM that are involved in diverse fundamental steps during tumor initiation and progression, as well as in chemoresistance. Recently, Ganesh et al reported that L1CAM identifies metastasis-initiating cells in colorectal carcinoma exhibiting stem-like cell features, increased tumorigenic potential and enhanced chemoresistance. In this review, we highlight recent advances in L1CAM research with particular emphasis on its role in de-differentiation processes and cancer cell stemness supporting the view that L1CAM is a powerful prognostic factor and a suitable target for improved therapy of metastatic and drug-resistant tumors.

The majority of cancer-related deaths are due to hematogenous metastases, and the bone marrow (BM) represents one of the most frequent metastatic sites. To study BM metastasis formation in vivo, the most efficient approach is based on intracardiac injection of human tumor cells into immunodeficient mice. However, such a procedure circumvents the early steps of the metastatic cascade. Here we describe the development of xenograft mouse models (balb/c rag2-/- and severe combined immunodeficient (SCID)), in which BM metastases are spontaneously derived from subcutaneous (s.c.) primary tumors (PTs). As verified by histology, the described methodology including ex vivo bioluminescence imaging (BLI) even enabled the detection of micrometastases in the BM. Furthermore, we established sublines from xenograft primary tumors (PTs) and corresponding BM (BM) metastases using LAN-1 neuroblastoma xenografts as a first example. In vitro &ldquo, metastasis&rdquo, assays (viability, proliferation, transmigration, invasion, colony formation) partially indicated pro-metastatic features of the LAN-1-BM compared to the LAN-1-PT subline. Unexpectedly, after s.c. re-injection into mice, LAN-1-BM xenografts developed spontaneous BM metastases less frequently than LAN-1-PT xenografts. This study provides a novel methodologic approach for modelling the spontaneous metastatic cascade of human BM metastasis formation in mice. Moreover, our data indicate that putative bone-metastatic features get rapidly lost upon routine cell culture.

A major limitation in the pharmacological treatment of clinically detectable primary cancers and their metastases is their limited accessibility to anti-cancer drugs (cytostatics, inhibitory antibodies, small-molecule inhibitors) critically impairing therapeutic efficacies. Investigations on the tissue distribution of such drugs are rare and have only been based on fresh frozen material or methanol-fixed cell culture cells so far. In this paper, we expand the detection of cisplatin-induced DNA adducts and anthracyclines as well as therapeutic antibodies to routinely prepared formalin-fixed, paraffin-embedded sections (FFPE). Using pre-treated cell lines prepared as FFPE samples comparable to tissues from routine analysis, we demonstrate that our method allows for the detection of chemotherapeutics (anthracyclines by autofluorescence, cisplatin by immune detection of DNA adducts) as well as therapeutic antibodies. This methodology thus allows for analyzing archival FFPE tissues, as demonstrated here for the detection of cisplatin, doxorubicin and trastuzumab in FFPE sections of tumor xenografts from drug-treated mice. Analyzing human tumor samples, this will lead to new insights into the tissue penetration of drugs.

Leukocytes travel within the circulation and enter connective tissues by interactions with endothelium of postcapillary venules mediated by cell adhesion molecules, summarized as the leukocyte adhesion cascade. In the severe combined immunodeficient (SCID) mouse model, rheumatoid arthritis (RA) synovial fibroblasts (SF) migrated to distant cartilage through the vasculature. Therefore, RASF adhesion toward endothelial cells (EC) and E- and P-selectins were analyzed. Cell-to-cell binding assays between SF and EC were performed. Interactions of SF with tumor necrosis factor α (TNFα)-activated EC or selectins were analyzed in flow adhesion assays. Immunohistochemistry for E-selectin ligand CD15s was performed. CD15s induction in RASF by human serum or media was evaluated. Wild-type and E-/-/ P-/- Selectin-SCID mice were used for inverse-wrap surgery. After laser-mediated microdissection, real-time PCR for E-/P-selectin/vascular cell adhesion molecule 1 was performed. Adhesion between SF/EC under static conditions was highest in Roswell Park Memorial Institute-cultured RASF to TNFαα-activated human umbilical vein endothelial cells (2.25-fold) and RASF adhesion was higher toward venous than arterial EC (Dulbecco's modified eagle medium P = 0.0419, RPMI P = 0.0119). In flow chamber assays, RASF adhesion to E-selectin was higher than to P-selectin (e.g. 0.9 dyn cm-2 P = 0.0001). Osteoarthritis synovial fibroblasts showed lower rolling/adhesion properties (e.g. 0.5 dyn cm-2 , P = 0.0010). RASF adhesion to TNFαα-activated EC was increased (e.g. 0.9 dyn cm-2 , P = 0.0061). CD15s induction in RASF was strongest in RA serum. Vimentin/CD15s double-positive cells were detectable. In E-/P-selectin-deficient mice, contralateral invasion was reduced (P = 0.023). E- and P-selectin, and vascular cell adhesion molecule 1 expression in EC of implants was confirmed. Our data indicate that the milieu within vessels induces CD15s which enables RASF to interact with E-selectin/EC under flow. Therefore, RASF may migrate to distant sites and leave the vasculature similarly to leukocytes.

Introduction: Elevated tumor interstitial fluid pressure (TIFP) is a major barrier to successful cancer treatment. It is mainly caused by an inadequate tumor perfusion resulting from missing lymphatic vessels, a leaky and immature tumor vasculature, an overly stimulated angiogenesis, and interstitial fibrosis. TIFP hampers convection-driven fluid transport within tumors and causes inefficient penetration and distribution of anti-neoplastic drugs.Areas covered: This review covers mechanisms and regulation of TIFP and gives an overview of strategies to overcome this barrier.Expert opinion: Increased TIFP occurs in vivo and hence may be overlooked in molecular biology-driven cancer research. Its pervasiveness means that it can reduce the effectiveness of anti-neoplastic drugs. This is often misinterpreted as drug resistance; it precedes all drug resistance mechanisms operating at the cell level. The key challenge in the field of cancer therapeutics is to find ways to lower TIFP. Several drug therapies have been tried but none of them have had an impact in the clinic. This is an overarching problem in the field of cancer research.

Understanding the mechanisms regulating human mammary epithelium requires knowledge of the cellular constituents of this tissue. Different and partially contradictory definitions and concepts describing the cellular hierarchy of mammary epithelium have been proposed, including our studies of keratins K5 and/or K14 as markers of progenitor cells. Furthermore, we and others have suggested that the p53 homolog p63 is a marker of human breast epithelial stem cells. In this investigation, we expand our previous studies by testing whether immunohistochemical staining with monospecific anti-keratin antibodies in combination with an antibody against the stem cell marker p63 might help refine the different morphologic phenotypes in normal breast epithelium. We used in situ multilabel staining for p63, different keratins, the myoepithelial marker smooth muscle actin (SMA), the estrogen receptor (ER), and Ki67 to dissect and quantify the cellular components of 16 normal pre- and postmenopausal human breast epithelial tissue samples at the single-cell level. Importantly, we confirm the existence of K5+ only cells and suggest that they, in contrast to the current view, are key luminal precursor cells from which K8/18+ progeny cells evolve. These cells are further modified by the expression of ER and Ki67. We have also identified a population of p63+K5+ cells that are only found in nipple ducts. Based on our findings, we propose a new concept of the cellular hierarchy of human breast epithelium, including K5 luminal lineage progenitors throughout the ductal-lobular axis and p63+K5+ progenitors confined to the nipple ducts.

Purpose To investigated the influence of radiation therapy (RT), surgery (OP), radio-chemotherapy (RChT), or chemotherapy (ChT) on small cell lung cancer metastases in 2 xenograft models. Methods and Materials A total of 1 × 106 human small cell lung cancer cells (OH1, H69) were subcutaneously injected into severe combined immunodeficiency mice to form a local primary tumor node at the lower trunk. Radiation therapy, OP, RChT, or ChT were started after development of palpable tumors. Chemotherapy was given as a single intraperitoneal injection of cisplatin. Radiation therapy was 5 × 10 Gy on the local tumor node. Two additional groups were implemented to assess primary tumors and distant metastases in untreated mice at the beginning (control group A) and at the end of the experiment (control group B). Proapoptotic, antiproliferative, antiangiogenic, and hypoxic effects were assessed by Feulgen, Ki67, S1P1 receptor, and hypoxia-inducible factor 1α staining, respectively. Quantitative Alu–polymerase chain reaction was used to determine circulating tumor cells in the blood, and disseminated tumor cells in the lungs, bone marrow, liver, and brain. Results In both xenograft models, RT and RChT abrogated local tumor growth, indicated by increased apoptosis, decreased cell proliferation, and reduced microvessel density (equally affecting vessels of all diameters). Regarding metastases, RT and RChT not only counteracted the time-dependent increase of dissemination but also decreased the metastatic load pre-existing at therapy induction in the blood, lungs, and liver. Only in the case of relapse-free surgery could similar effects be achieved by OP. Conclusions Our models provide evidence that RT and RChT ablate the primary tumor and inhibit metastasis development over time. Upon local recurrence, RT showed beneficial effects compared with OP with regard to suppression of circulating tumor cells and disseminated tumor cells.

BMC cancer 21(1), 1024 (2021). doi:10.1186/s12885-021-08754-z, Published by BioMed Central, London

Purpose Ovarian cancer (OvCa) progression mainly takes place by intraperitoneal spread. Adhesion of tumor cells to the mesothelial cells which form the inner surface of the peritoneum is a crucial step in this process. Cancer cells use in principle different molecules of the leukocyte adhesion cascade to facilitate adhesion. This cascade is initiated by selectin-ligand interactions followed by integrin - extracellular matrix protein interactions. Here we address the question whether all tumor cells predominantly employ selectin-dependent leukocyte-like adhesion cascade (SDAC) or whether they use integrin mediated adhesion for OvCa progression as well. Methods A comparative transcriptomic analysis of the human OvCa cell lines OVCAR8 and SKOV3 was performed. Intraperitoneal xenograft model of OVCAR8 cells was used to determine whether there is a correlation between SDAC gene expression and the metastatic potential of the control cells and the cells overexpressing c-Fos. Transcriptomic analysis of OVCAR8 and SKOV3 samples was performed using microarrays. Results One-third of the protein-coding genes involved in SDAC exhibited lower expression levels in OVCAR8 than in SKOV3 cells. In contrast to SKOV3 cells, c-Fos overexpression in OVCAR8 cells did not significantly influence the expression of SDAC genes. Intraperitoneal xenograft model of OVCAR8 cells unexpectedly demonstrated that the aggressiveness of OVCAR8 tumors was not depended on the c-Fos expression level and was comparable to that of SKOV3 control tumors. Gene expression analysis of tumors suggests that SKOV3-derived tumor progression was mainly depended on SDAC. Progression of OVCAR8 tumors relied on other cell adhesion molecules that do not interact with selectins. Conclusions High expression of c-Fos in ovarian cancer cells is not always associated with reduced metastatic potential. Low expression level of SDAC genes may not ensure low OvCa metastatic potential hence alternative adhesion mechanisms involving laminin-integrin interactions exist as well.

Changes in cell adhesion molecule (CAM) expression and miRNAs regulating them are known to be involved in malignant progression in colon cancer. We investigated expression profiles of CAM genes and non-coding RNAs in CaCo2 colon cancer cells in static culture and under dynamic flow conditions perfused in microfluidic chip emulating physiological microenvironment. We incubated monolayers of CaCo2 cells in Transwell® units either under static conditions or under flow in a microfluidic chip. We identified 7 up-regulated CAM genes (CD44, CDH7, CEACAM5, CEACAM6, CYR61, L1CAM and VCAN), 7 down-regulated genes (COL12A1, FGA, FGB, FGG, GJA1, ITGA5 and LAMA1) and 69 miRNAs targeting them under the influence of microcirculation. The revealed network comprised CAM genes known to interact with each other and 13 miRNAs simultaneously regulating more than one of them. The discovered regulatory network comprising CAM genes and miRNAs is likely involved in normal functioning of intestine epithelium as well as in cancer progression.

Merkel cell carcinoma (MCC) is an aggressive skin cancer entity that frequently leads to rapid death due to its high propensity to metastasize. The etiology of most MCC cases is linked to Merkel cell polyomavirus (MCPyV), a virus which is monoclonally integrated in up to 95% of tumors. While there are presently no animal models to study the role of authentic MCPyV infection on transformation, tumorigenesis or metastasis formation, xenograft mouse models employing engrafted MCC-derived cell lines (MCCL) represent a promising approach to study certain aspects of MCC pathogenesis. Here, the two MCPyV-positive MCC cell lines WaGa and MKL-1 were subcutaneously engrafted in scid mice. Engraftment of both MCC cell lines resulted in the appearance of circulating tumor cells and metastasis formation, with WaGa-engrafted mice showing a significantly shorter survival time as well as increased numbers of spontaneous lung metastases compared to MKL-1 mice. Interestingly, explanted tumors compared to parental cell lines exhibit an upregulation of MCPyV sT-Antigen expression in all tumors, with WaGa tumors showing significantly higher sT-Antigen expression than MKL-1 tumors. RNA-Seq analysis of explanted tumors and parental cell lines furthermore revealed that in the more aggressive WaGa tumors, genes involved in inflammatory response, growth factor activity and Wnt signalling pathway are significantly upregulated, suggesting that sT-Antigen is the driver of the observed differences in metastasis formation.

We contend that knowledge about the cellular composition of normal breast epithelium is a prerequisite for understanding proliferative breast disease. Against this background, we used multicolor immunofluorescence to study normal breast epithelium and two types of intraepithelial proliferative breast lesion for expression of the p63, basal keratin K5, glandular keratin K8/18, SMA, ER-alpha, and Ki67. We studied eight normal breast epithelium samples, 12 cases of usual ductal hyperplasia, and 33 cases of low-grade intraepithelial neoplasia (9 flat epithelial atypia, 14 low-grade ductal carcinoma in situ and 10 cases of lobular neoplasia). Usual ductal hyperplasia showed striking similarity to normal luminal breast epithelium including p63+ and/or K5+ luminal progenitor cells and the full spectrum of luminal progeny cells. In normal breast epithelium and usual ductal hyperplasia, expression of ER-alpha was associated with lack of expression of the proliferation antigen Ki67. In contrast, we found in both types of low-grade intraepithelial neoplasia robust expression of keratin K8/18 and a positive association between ER-alpha and Ki67 expression. However, these lesions were consistently negative for p63 and/or K5. Our observational study supports the view that usual ductal hyperplasia and low-grade intraepithelial neoplasia are different entities rather than part of a spectrum of the same disease. We propose a new operational model of cell differentiation that may serve to better understand correlations between normal breast epithelium and proliferative breast diseases. From our data we conclude that p63+ and/or K5+ progenitor cells contribute to maintenance of normal epithelium and usual ductal hyperplasia, but not to low-grade intraepithelial neoplasia of the breast.

Aims Radiotherapy of small laboratory animals (SLA) is often not as precisely applied as in humans. Here we describe the use of a dedicated SLA magnetic resonance imaging (MRI) scanner for precise tumor volumetry, radiotherapy treatment planning, and diagnostic imaging in order to make the experiments more accurate. Methods and materials Different human cancer cells were injected at the lower trunk of pfp/rag2 and SCID mice to allow for local tumor growth. Data from cross sectional MRI scans were transferred to a clinical treatment planning system (TPS) for humans. Manual palpation of the tumor size was compared with calculated tumor size of the TPS and with tumor weight at necropsy. As a feasibility study MRI based treatment plans were calculated for a clinical 6 MV linear accelerator using a micro multileaf collimator (μMLC). In addition, diagnostic MRI scans were used to investigate animals which did clinical poorly during the study. Results MRI is superior in precise tumor volume definition whereas manual palpation underestimates their size. Cross sectional MRI allow for treatment planning so that conformal irradiation of mice with a clinical linear accelerator using a μMLC is in principle feasible. Several internal pathologies were detected during the experiment using the dedicated scanner. Conclusion MRI is a key technology for precise radiotherapy of SLA. The scanning protocols provided are suited for tumor volumetry, treatment planning, and diagnostic imaging.

The formation of distant metastases often determines the fate of patients with head and neck squamous cell carcinoma (HNSCC). The expression of cell adhesion molecules (CAMs) and their ligands of the leukocyte adhesion cascade has been associated with metastatic competence in several malignant entities. In this study, human HNSCC cell lines were analyzed in vitro and in a spontaneous metastatic xenograft model. Immunohistochemical analyses of several CAMs were performed on xenograft tumors and tissue microarrays (TMA) from 453 patients with head and neck squamous cell carcinomas with full histo-pathological and clinical follow-up data. UTSCC 24A and 24B cells bind to E-selectin in vitro, show E-selectin dependent binding to human umbilical vein endothelial cells (HUVECs), and express sLeX. All HNSCC cells engrafted into severe combined immunodeficient (SCID) mice, and UTSCC 24A cells formed sporadically spontaneous lung metastases. The expression of CAMs varied between the cell lines, but a correlation between tumor growth and metastatic potential did not exist. None of the CAMS or their ligands could be identified to be of prognostic relevance in the TMA study. The in vitro results indicate that E-selectin and sLeX are involved in the adhesion of HNSCC cells to endothelium. However, specific prognostic markers chosen from the leukocyte adhesion cascade for HNSCC were not identified.

Stable integration of HIV proviral DNA into host cell chromosomes, a hallmark and essential feature of the retroviral life cycle, establishes the infection permanently. Current antiretroviral combination drug therapy cannot cure HIV infection. However, expressing an engineered HIV-1 long terminal repeat (LTR) site-specific recombinase (Tre), shown to excise integrated proviral DNA in vitro, may provide a novel and highly promising antiviral strategy. We report here the conditional expression of Tre-recombinase from an advanced lentiviral self-inactivation (SIN) vector in HIV-infected cells. We demonstrate faithful transgene expression, resulting in accurate provirus excision in the absence of cytopathic effects. Moreover, pronounced Tre-mediated antiviral effects are demonstrated in vivo, particularly in humanized Rag2−/−γc−/− mice engrafted with either Tre-transduced primary CD4+ T cells, or Tre-transduced CD34+ hematopoietic stem and progenitor cells (HSC). Taken together, our data support the use of Tre-recombinase in novel therapy strategies aiming to provide a cure for HIV., Author Summary Current antiretroviral combination therapy can efficiently suppress virus replication, but cannot eliminate HIV. Therefore, no cure for HIV exists. A main hurdle for virus eradication is seen in the existence of resting cells that contain integrated replication-competent, but temporarily silenced, HIV genomes. Therefore, the most direct approach to eliminating virus reservoirs is to remove HIV genomes from infected cells. As previous studies suggested, this may be achievable by Tre-recombinase, an engineered enzyme that can excise integrated HIV from host cell chromosomes. The present work analyzes the expression of Tre-recombinase in human cells and demonstrates highly accurate Tre activity in complete absence of Tre-related cytopathic effects. Furthermore, in vivo analysis of Tre-recombinase demonstrates highly significant antiviral effects of Tre in HIV-infected humanized mice. The presented data suggest that Tre-recombinase might become a valuable component of a future therapy that aims at virus eradication.

The development of a myeloid neoplasm is a step-wise process that originates from leukemic stem cells (LSC) and includes pre-leukemic stages, overt leukemia and a drug-resistant terminal phase. Organ-invasion may occur in any stage, but is usually associated with advanced disease and a poor prognosis. Sometimes, extra-medullary organ invasion shows a metastasis-like or even sarcoma-like destructive growth of neoplastic cells in local tissue sites. Examples are myeloid sarcoma, mast cell sarcoma and localized blast phase of chronic myeloid leukemia. So far, little is known about mechanisms underlying re-distribution and extramedullary dissemination of LSC in myeloid neoplasms. In this article, we discuss mechanisms through which LSC can mobilize out of the bone marrow niche, can transmigrate from the blood stream into extramedullary organs, can invade local tissue sites and can potentially create or support the formation of local stem cell niches. In addition, we discuss strategies to interfere with LSC expansion and organ invasion by targeted drug therapies.

The impedance profile of the human PC-3 and DU 145 prostate cancer cells were recorded and compared using Electrical Impedance Spectroscopy. Cells were measured in a special chamber using a four terminal setup to avoid parasitic effects of electrode polarization in low frequencies. Our results show that the two cancer cell lines are readily distinguishable by their impedance spectrum. As PC-3 cells have been shown to be spontaneously metastatic in previous xenograft experiments while DU 145 cells were non-metastatic, Electrical Impedance Spectroscopy has the potential to be developed into a simple diagnostic tool to distinguish metastatic from non-metastatic cells.

Formation of distant metastases is by far the most common cause of cancer-related deaths. The process of metastasis formation is complex, and within this complex process the formation of migratory cells, the so called epithelial mesenchymal transition (EMT), which enables cancer cells to break loose from the primary tumor mass and to enter the bloodstream, is of particular importance. To break loose from the primary cancer, cancer cells have to down-regulate the cell-to-cell adhesion molecuIes (CAMs) which keep them attached to neighboring cancer cells. In contrast to this downregulation of CAMS in the primary tumor, cancer cells up-regulate other types of CAMs, that enable them to attach to the endothelium in the organ of the future metastasis. During EMT, the expression of cell-to-cell and cell-to-matrix adhesion molecules and their down- and upregulation is therefore critical for metastasis formation. Tumor cells mimic leukocytes to enable transmigration of the endothelial barrier at the metastatic site. The attachment of leukocytes/cancer cells to the endothelium are mediated by several CAMs different from those at the site of the primary tumor. These CAMs and their ligands are organized in a sequential row, the leukocyte adhesion cascade. In this adhesion process, integrins and their ligands are centrally involved in the molecular interactions governing the transmigration. This review discusses the integrin expression patterns found on primary tumor cells and studies whether their expression correlates with tumor progression, metastatic capacity and prognosis. Simultaneously, further possible, but so far unclearly characterized, alternative adhesion molecules and/or ligands, will be considered and emerging therapeutic possibilities reviewed.

Here we describe the establishment and characterization of an AR+, PSMA+, ERG+, PTEN−/−, CHD1+/− patient-derived xenograft (PDX) model termed ‘C5’, which has been developed from a 60 years old patient suffering from castration-resistant prostate cancer (CRPC). The patient underwent radical prostatectomy, showed early tumor marker PSA recurrence and, one year after surgery, abiraterone resistance. Subcutaneous C5 tumors can be serially transplanted between mice and grow within ~90 days to 1.5–2 cm³ tumors in SCID Balb/c mice (take rate 100%), NOD-scid IL2Rgnull (NSG) mice (100%) and C57BL/6 pfp−/−/rag2−/− mice (66%). In contrast, no tumor growth is observed in female mice. C5 tumors can be cryopreserved and show the same growth characteristics in vivo afterwards. C5 tumor cells do not grow stably in vitro, neither under two- nor three-dimensional cell culture conditions. Upon serial transplantation, some C5 tumors spontaneously disseminated to distant sites with an observable trend towards higher metastatic cell loads in scid compared to NSG mice. Lung metastases could be verified by histology by means of anti-PSMA immunohistochemistry, exclusively demonstrating single disseminated tumor cells (DTCs) and micro-metastases. Upon surgical resection of the primary tumors, such pulmonary foci rarely grew out to multi-cellular metastatic colonies despite doubled overall survival span. In the brain and bone marrow, the metastatic cell load present at surgery even disappeared during the post-surgical period. We provide shallow whole genome sequencing and whole exome sequencing data of C5 tumors demonstrating the copy number aberration/ mutation status of this PCa model and proving genomic stability over several passages. Moreover, we analyzed genomic and transcriptomic alterations during metastatic progression achieved by serial transplantation. This study describes a novel PCa PDX model that enables future research on several aspects of metastatic PCa, particularly for the AR+ , ERG+ , PTEN−/− PCa subtype.

104 Background: The Warburg effect describes the ability of cancer cells to consume larger amounts of glucose in comparison to normal tissue, due to the overexpression of insulin-independent glucose transporters (e.g. GLUT1). This effect can be used to enhance the selectivity and reduce side effects of cytotoxic anticancer molecules by its conjugation to sugar residues, thus, generating cytotoxic agents showing higher selectivity to cancer cells. In continuation of our research on anticancer natural 1,4-naphthoquinones we have investigated a large series of novel semi-synthetic molecules containing 1,4-naphthoquinones element conjugated with glucose molecule via -S-CH2- bond. Methods: We performed screening examinations for 35 novel synthetic molecules in human prostate cancer in vitro. The selected most active compounds were tested in several human prostate cancer cell lines harboring different levels of drug resistance, as well as in non-malignant cells to specify their selectivity. Compounds with the highest cytotoxicity and selectivity were further investigated. The mode of action was assessed including effects on apoptosis induction, oxidative stress, mitochondria, AR-signaling as well as glucose uptake and ER stress were assessed. In vivo dose finding and efficacy analyses were performed. Results: We identified two promising derivatives, showing IC50s at low micro- and nanomolar concentrations. Glucose depletion from the culture media led to increased cytotoxicity and cotreatment with a GLUT1-inhibitor showed an antagonistic effect, suggesting a concurrent uptake and therefore a Warburg effect targeting. The selected compounds exhibited most pronounced cytotoxic activity in DU145 cells as well as 22Rv1 cells. Non-malignant cells were generally less affected. The mode of action involves a loss of mitochondrial membrane potential, a release of cytochrome c and AIF into the cytosol and an upregulation of caspase-9, caspase-3 and cleaved PARP, as well as downregulation of Bcl-2 and Survivin, indicating that mitochondria are a major target, leading to the activation of the intrinsic apoptotic pathway. Early events in treated cells are ROS production and calcium release into the cytosol, a marker of ER-stress. Furthermore, downregulation of the AR and its signaling was observed on mRNA- and protein-level. In vivo experiments revealed antitumor activity in a 22Rv1-xenograft mouse model without severe side effects. Conclusions: In conclusion, we were able to identify two glucose-conjugated 1,4-naphthoquinones exhibiting potent in vitro and in vivoactivity and selectivity in human prostate cancer cells due to the Warburg effect targeting. Cytotoxic activity was exerted via initial ROS production and ER stress leading to mitochondrial damage and the induction of the intrinsic apoptotic pathway.

// Michael Bruns 1 , Jara Wanger 1,4 , Udo Schumacher 2 and Wolfgang Deppert 1,3 1 Heinrich-Pette-Institute, Leibniz-Institute for Experimental Virology, Hamburg, Germany 2 Institute for Anatomy and Experimental Morphology, University Medical Center Hamburg-Eppendorf (UKE), University of Hamburg, Hamburg, Germany 3 Institute for Tumor Biology, University Medical Center Hamburg-Eppendorf (UKE), University of Hamburg, Hamburg, Germany 4 Woldsenweg, Hamburg, Germany Correspondence to: Wolfgang Deppert, email: // Keywords : transgenic breast cancer mouse model, SV40 T-antigen, LCMV NP T-cell epitope, CTL response, differential response to immune checkpoint blockade therapy Received : July 01, 2016 Accepted : August 21, 2016 Published : August 25, 2016 Abstract Using the SV40 transgenic WAP-T/WAP-T NP mouse models for mammary carcinomas, we compared the response to immune checkpoint blockade therapy in tumor mice expressing either SV40 T-antigen containing the LCMV NP-epitope (T-Ag NP in WAP-T NP mice), or the unmodified T-antigen (T-Ag in WAP-T mice). Specifically, we asked, whether the presence of the highly immunogenic NP-epitope in T-Ag NP influences this response in comparison to the weakly immunogenic T-cell epitopes of T-Ag in WAP-T tumor mice. Treatment of WAP-T NP tumor mice with either anti-PD1 or anti-PD-L1 antibodies led to tumor regression, with anti-PD-L1 treatment being more effective. However, tumors had fully re-appeared after 21 days, indicating that CTL exhaustion had been rapidly re-established. Surprisingly, the same treatment applied to WAP-T tumor mice resulted in a significantly prolonged period of tumor regression. We provide evidence that in contrast to the weak antigenic stimuli exerted by T-cell epitopes of T-Ag, the strong antigenic stimulus of the NP-epitope in T-Ag NP has a dual effect: (i) a rapid generation of active NP-specific CTLs, accompanied (ii) by accelerated CTL exhaustion. Our data support the hypothesis that the immunogenicity of tumor antigen T-cell epitopes strongly influences the success of immune checkpoint blockade therapy.

The human-on-chip technology provides an efficient basis for preclinical studies and has potentially a greater predictive power for human drug response than classical 2D cell culture. Here we report the expression profile of druggable genes in the human colon cancer cells CaCo2 in static culture and within a microfluidic chip. We identified gene expression pattern under flow to be closer to the one of CaCo2 primary xenograft tumours as compared to those cells grown without circulation. The obtained results indicate that a microenvironment connected to a circulation within a chip brings the cells closer to in vivo situation. Hence the human-on-chip technology is a more powerful tool for drug development than conventional 2D cell culture.

To compare multidetector computed tomography (MDCT) and cone-beam computed tomography (CBCT) regarding radiation, resolution, image noise, and image quality. CBCT and 256-MDCT were compared based on three scan protocols: Standard-dose (≈24 mGy), reduced-dose (≈9 mGy), and low-dose (≈4 mGy). MDCT images were acquired in standard- and high-resolution mode (HR-MDCT) and reconstructed using filtered back projection (FBP) and iterative reconstruction (IR). Spatial resolution in linepairs (lp) and objective image noise (OIN) were assessed using dedicated phantoms. Image quality was assessed in scans of 25 cadaver heads using a Likert scale. OIN was markedly higher in FBP-MDCT when compared to CBCT. IR lowered the OIN to comparable values in standard-mode MDCT only. CBCT provided a resolution of 13 lp/cm at standard-dose and 11 lp/cm at reduced-dose vs. 11 lp/cm and 10 lp/cm in HR-MDCT. Resolution of 10 lp/cm was observed for both devices using low-dose settings. Quality scores of MDCT and CBCT did not differ at standard-dose (CBCT, 3.4; MDCT, 3.3-3.5; p > 0.05). Using reduced- and low-dose protocols, CBCT was superior (reduced-dose, 3.2 vs. 2.8; low dose, 3.0 vs. 2.3; p

Molecule L1CAM is specific for nerve cells and tumors of various localizations. The expression of L1CAM is significantly higher in melanoma in comparison with benign nevi and correlates with the progress of melanoma and transition from radial to vertical growth. Monoclonal antibodies to L1CAM effectively and specifically attenuate melanoma growth, though stimulates the epithelial-mesenchymal transition. shRNA-mediated knock-down of L1CAM showed the involvement of L1CAM in regulation of activity of the canonical Wnt pathway and expression of genes of class I melanoma-associated antigens (MAGE).

Despite recent advances in the treatment of metastatic castration-resistant prostate cancer (CRPC), outcome of patients remains poor due to the development of drug resistance. Thus, new drugs are urgently needed. We investigated efficacy, toxicity and mechanism of action of marine triterpene glycoside frondoside A (FrA) using CRPC cell lines in vitro and in vivo. FrA revealed high efficacy in human prostate cancer cells, while non-malignant cells were less sensitive. Remarkably, proliferation and colony formation of cells resistant to enzalutamide and abiraterone (due to the androgen receptor splice variant AR-V7) were also significantly inhibited by FrA. The marine compound caused cell type specific cell cycle arrest and induction of caspase-dependent or -independent apoptosis. Up-regulation or induction of several pro-apoptotic proteins (Bax, Bad, PTEN), cleavage of PARP and caspase-3 and down-regulation of anti-apoptotic proteins (survivin and Bcl-2) were detected in treated cells. Global proteome analysis revealed regulation of proteins involved in formation of metastases, tumor cell invasion, and apoptosis, like keratin 81, CrkII, IL-1β and cathepsin B. Inhibition of pro-survival autophagy was observed following FrA exposure. In vivo, FrA inhibited tumor growth of PC-3 and DU145 cells with a notable reduction of lung metastasis, as well as circulating tumor cells in the peripheral blood. Increased lymphocyte counts of treated animals might indicate an immune modulating effect of FrA. In conclusion, our results suggest that FrA is a promising new drug for the treatment of mCRPC. Induction of apoptosis, inhibition of pro-survival autophagy, and immune modulatory effects are suspected modes of actions.

The rhizome of Coptis chinensis is commonly used in traditional Chinese medicine alone or in combination with other herbs to treat diseases characterized by causing oxidative stress including inflammatory diseases, diabetes mellitus and neurodegenerative diseases. In particular, there is emerging evidence that Coptis chinensis is effective in the treatment of neurodegenerative diseases associated with oxidative stress. Hence, the aim of this study was to investigate the neuroprotective effect of Coptis chinensis in vitro and in vivo using MPP[Formula: see text] and MPTP models of Parkinson’s disease. MPP[Formula: see text] treated human SH-SY5Y neuroblastoma cells were used as a cell model of Parkinson’s disease. A 24[Formula: see text]h pre-treatment of the cells with the watery extract of Coptis chinensis significantly increased cell viability, as well as the intracellular ATP concentration and attenuated apoptosis compared to the MPP[Formula: see text] control. Further experiments with the main alkaloids of Coptidis chinensis, berberine, coptisine, jaterorrhizine and palmatine revealed that berberine and coptisine were the main active compounds responsible for the observed neuroprotective effect. However, the full extract of Coptis chinensis was more effective than the tested single alkaloids. In the MPTP-induced animal model of Parkinson’s disease, Coptis chinensis dose-dependently improved motor functions and increased tyrosine hydroxylase-positive neurons in the substantia nigra compared to the MPTP control. Based on the results of this work, Coptis chinensis and its main alkaloids could be considered potential candidates for the development of new treatment options for Parkinson’s disease.

Computer simulations of the spread of malignant tumor cells in an entire organism provide important insights into the mechanisms of metastatic progression. Key elements for the usefulness of these models are the adequate selection of appropriate mathematical models describing the tumor growth and its parametrization as well as a proper choice of the fractal dimension of the blood vessels in the primary tumor. In addition, survival in the bloodstream and evasion into the connective spaces of the target organ of the future metastasis have to be modeled. Determination of these from experimental models is complicated by systematic and unsystematic experimental errors which are difficult to assess. In this chapter, we demonstrate how to select the best-suited mathematical function to describe tumor growth for experimental xenograft mouse tumor models and how to parametrize them. Common pitfalls and problems are described as well as methods to avoid them.

Computer simulations of the spread of malignant tumor cells in an entire organism provide important insights into the mechanisms of metastatic progression. Key elements for the usefulness of these models are the adequate selection of appropriate mathematical models describing the tumor growth and its parametrization as well as a proper choice of the fractal dimension of the blood vessels in the primary tumor. In addition, survival in the bloodstream and evasion into the connective spaces of the target organ of the future metastasis have to be modeled. Determination of these from experimental models is complicated by systematic and unsystematic experimental errors which are difficult to assess. In this chapter, we demonstrate how to select the best-suited mathematical function to describe tumor growth for experimental xenograft mouse tumor models and how to parametrize them. Common pitfalls and problems are described as well as methods to avoid them.

We investigated the functional role of CEACAM1 in a spontaneous metastasis xenograft model of human melanoma in scid mice using BRAF wildtype MeWo cells with and without RNAi mediated knockdown of CEACAM1. Tumors from the xenograft model were subjected to whole genome expression analysis and metastasis was quantified histologically. Results and identified markers were verified using tissue samples of over 100 melanoma patients. Knockdown of CEACAM1 prolonged the animals’ survival by significantly reducing subcutaneous growth of MeWo tumors and spontaneous lung metastasis. Microarray analysis revealed a strong influence of CEACAM1 knockdown on the network of EMT associated genes in the xenograft tumors (e.g. downregulation of BRAF, FOSL1, NRAS and TWIST). IGFBP7 and Latexin (highest up- and downregulated expression in microarray analysis) were found to be associated with longer and shorter survival, respectively, of melanoma patients. High FOSL1 and altered TWIST1 expression were found to be correlated with shortened survival in the cohort of melanoma patients. After a stepwise selection procedure combining above markers, multivariate analysis revealed IGFBP7, Latexin and altered TWIST to be prognostic markers for death. CEACAM1 could be a target for melanoma therapy as an alternative to (or in combination with) immune checkpoint and BRAF inhibitors.

Ovarian cancer has a high mortality rate and up to now no reliable molecular prognostic biomarkers have been established. During malignant progression, the cytoskeleton is strongly altered. Hence we analyzed if expression of certain cytoskeleton-associated proteins is correlated with clinical outcome of ovarian cancer patients. First, in silico analysis was performed using the cancer genome atlas (TCGA), the human expression atlas and Pubmed. Selected candidates were validated on 270 ovarian cancer patients by qRT-PCR and/or by western blotting. In silico analysis revealed that mRNAs of 214 cytoskeleton-associated proteins are detectable in ovarian cancer tissue. Among these, we selected 17 proteins that participate in cancer disease progression and cytoskeleton modulation: KIF14, KIF20A, KIF18A, ASPM, CEP55, DLGAP5, MAP9, EB1, KATNA1, DIAPH1, ANLN, SCIN, CCDC88A, FSCN1, GSN, VASP and CDC42. The first ten candidates interact with microtubules (MTs) and the others bind to actin filaments. Validation on clinical samples of ovarian cancer patients revealed that the expression levels of DIAPH1, EB1, KATNA1, KIF14 and KIF18A significantly correlated with clinical and histological parameters of ovarian cancer. High DIAPH1, EB1, KATNA1 and KIF14 protein levels were associated with increased overall survival (OAS) of ovarian cancer patients, while high DIAPH1 and EB1 protein levels were also associated with low differentiation of respective tumors (G2/3). Moreover, DIAPH1 was the only protein, whose expression significantly correlated with increased recurrence-free interval (RFI). Mainly the expression levels of the MT-associated proteins analyzed in this study, correlated with prolonged survival of ovarian cancer patients. From > 200 genes initially considered, 17 cytoskeletal proteins are involved in cancer progression according to the literature. Among these, four proteins significantly correlated with improved survival of ovarian cancer patients.

Objective Using a contact-free laser technique for stapedotomy reduces the risk of mechanical damage of the stapes footplate. However, the risk of inner ear dysfunction due to thermal, acoustic, or direct damage has still not been solved. The objective of this study was to describe the first experiences in footplate perforation in cadaver tissue performed by the novel Picosecond-Infrared-Laser (PIRL), allowing a tissue preserving ablation. Patients and intervention Three human cadaver stapes were perforated using a fiber-coupled PIRL. The results were compared with footplate perforations performed with clinically applied Er:YAG laser. Therefore, two different laser energies for the Er:YAG laser (30 and 60 mJ) were used for footplate perforation of three human cadaver stapes each. Main outcome measure Comparisons were made using histology and environmental scanning electron microscopy (ESEM) analysis. Results The perforations performed by the PIRL (total energy: 640-1070 mJ) revealed a precise cutting edge with an intact trabecular bone structure and no considerable signs of coagulation. Using the Er:YAG-Laser with a pulse energy of 30 mJ (total energy: 450-600 mJ), a perforation only in the center of the ablation zone was possible, whereas with a pulse energy of 60 mJ (total energy: of 195-260 mJ) the whole ablation zone was perforated. For both energies, the cutting edge appeared irregular with trabecular structure of the bone only be conjecturable and signs of superficial carbonization. Conclusion The microscopic results following stapes footplate perforation suggest a superiority of the PIRL in comparison to the Er:YAG laser regarding the precision and tissue preserving ablation.

The Hermes receptor CD44 is a multifunctional adhesion molecule that plays an essential role in the homing and invasion of neoplastic stem cells in various myeloid malignancies. Although mast cells (MCs) reportedly express CD44, little is known about the regulation and function of this receptor in neoplastic cells in systemic mastocytosis (SM). We found that clonal CD34+/CD38- stem cells, CD34+/CD38+ progenitor cells, and CD117++/CD34- MCs invariably express CD44 in patients with indolent SM (ISM), SM with an associated hematologic neoplasm, aggressive SM, and MC leukemia (MCL). In addition, all human MCL-like cell lines examined (HMC-1, ROSA, and MCPV-1) displayed cytoplasmic and cell-surface CD44. We also found that expression of CD44 in neoplastic MCs depends on RAS-MEK and STAT5 signaling and increases with the aggressiveness of SM. Correspondingly, higher levels of soluble CD44 were measured in the sera of patients with advanced SM compared with ISM or cutaneous mastocytosis and were found to correlate with overall and progression-free survival. To investigate the functional role of CD44, a xenotransplantation model was employed using severe combined immunodeficient (SCID) mice, HMC-1.2 cells, and a short hairpin RNA (shRNA) against CD44. In this model, the shRNA-mediated knockdown of CD44 resulted in reduced MC expansion and tumor formation and prolonged survival in SCID mice compared with HMC-1.2 cells transduced with control shRNA. Together, our data show that CD44 is a RAS-MEK/STAT5-driven MC invasion receptor that correlates with the aggressiveness of SM. Whether CD44 can serve as therapeutic target in advanced SM remains to be determined in forthcoming studies.

Metastasis formation is the major cause for cancer-related deaths and the underlying mechanisms remain poorly understood. In this study we describe spontaneous metastasis xenograft mouse models of human neuroblastoma used for unbiased identification of metastasis-related proteins by applying an infrared laser (IR) for sampling primary tumor and metastatic tissues, followed by mass spectrometric proteome analysis. IR aerosol samples were obtained from ovarian and liver metastases, which were indicated by bioluminescence imaging (BLI), and matched subcutaneous primary tumors. Corresponding histology proved the human origin of metastatic lesions. Ovarian metastases were commonly larger than liver metastases indicating differential outgrowth capacities. Among ~1,900 proteins identified at each of the three sites, 55 proteins were differentially regulated in ovarian metastases while 312 proteins were regulated in liver metastases. There was an overlap of 21 and 7 proteins up- and down-regulated at both metastatic sites, respectively, most of which were so far not related to metastasis such as LYPLA2, EIF4B, DPY30, LGALS7, PRPH, and NEFM. Moreover, we established in vitro sublines from primary tumor and metastases and demonstrate differences in cellular protrusions, migratory/invasive potential and glycosylation. Summarized, this work identified several novel putative drivers of metastasis formation that are tempting candidates for future functional studies.

Finding additional functional targets for combination therapy could improve the outcome for melanoma patients. In a spontaneous metastasis xenograft model of human melanoma a shRNA mediated knockdown of L1CAM more than sevenfold reduced the number of lung metastases after the induction of subcutaneous tumors for two human melanoma cell lines (MeWo, MV3). Whole genome expression arrays of the initially L1CAM high MeWo subcutaneous tumors revealed unchanged or downregulated genes involved in epithelial to mesenchymal transition (EMT) except an upregulation of Jagged 1, indicating a compensatory change in Notch signaling especially as Jagged 1 expression showed an increase in MeWo L1CAM metastases and Jagged 1 was expressed in metastases of the initially L1CAM low MV3 cells as well. Expression of 17 genes showed concordant regulation for L1CAM knockdown tumors of both cell lines. The changes in gene expression indicated changes in the EMT network of the melanoma cells and an increase in p53/p21 and p38 activity contributing to the reduced metastatic potential of the L1CAM knockdowns. Taken together, these data make L1CAM a highly interesting therapeutic target to prevent further metastatic spread in melanoma patients.