Intratumoral injection of melatonin enhances tumor regression in cell line-derived and patient-derived xenografts of head and neck cancer by increasing mitochondrial oxidative stress.

Head and neck squamous cell carcinoma present a high mortality rate. Melatonin has been shown to have oncostatic effects in different types of cancers. However, inconsistent results have been reported for in vivo applications. Consequently, an alternative administration route is needed to improve bioavailability and establish the optimal dosage of melatonin for cancer treatment. On the other hand, the use of patient-derived tumor models has transformed the field of drug research because they reflect the heterogeneity of patient tumor tissues. In the present study, we explore mechanisms for increasing melatonin bioavailability in tumors and investigate its potential as an adjuvant to improve the therapeutic efficacy of cisplatin in the setting of both xenotransplanted cell lines and primary human HNSCC. We analyzed the effect of two different formulations of melatonin administered subcutaneously or intratumorally in Cal-27 and SCC-9 xenografts and in patient-derived xenografts. Melatonin effects on tumor mitochondrial metabolism was also evaluated as well as melatonin actions on tumor cell migration. In contrast to the results obtained with the subcutaneous melatonin, intratumoral injection of melatonin drastically inhibited tumor progression in HNSCC-derived xenografts, as well as in patient-derived xenografts. Interestingly, intratumoral injection of melatonin potentiated CDDP effects, decreasing Cal-27 tumor growth. We demonstrated that melatonin increases ROS production and apoptosis in tumors, targeting mitochondria. Melatonin also reduces migration capacities and metastasis markers. These results illustrate the great clinical potential of intratumoral melatonin treatment and encourage a future clinical trial in cancer patients to establish a proper clinical melatonin treatment.

Recurrent Respiratory Papillomatosis (RRP)-Meta-analyses on the use of the HPV vaccine as adjuvant therapy.

Recurrent Respiratory Papillomatosis(RRP) is a rare disease with severe morbidity. Treatment is surgical. Prevailing viewpoint is that prophylactic HPV vaccines do not have therapeutic benefit due to their modus operandi. Studies on HPV vaccination alongside surgery were meta-analysed to test effect on burden of disease. Databases were accessed Nov and Dec 2021 [PubMed, Cochrane, Embase and Web of Science]. Main outcome measured was: Mean paired differences in the number of surgeries or recurrences per month. Analyses was performed using: Random effect maximal likelihood estimation model using the Stata module Mataan(StataCorp. 2019. Stata Statistical Software: Release 16. College Station, TX:StataCorp LLC.) Our results found n = 38 patients, suitable for syntheses with one previous meta-analyses (4 published, 2 unpublished studies) n = 63, total of n = 101 patients. Analyses rendered an overall reduction of 0.123 recurrences or surgeries per month (95% confidence interval [0.064, 0.183]). Our meta-analyses concludes that HPV vaccine is a beneficial adjunct therapy alongside surgery.

Primary head and neck cancer cell cultures are susceptible to proliferation of Epstein-Barr virus infected lymphocytes.

BACKGROUND: New concepts for a more effective anti-cancer therapy are urgently needed. Experimental flaws represent a major counter player of this development and lead to inaccurate and unreproducible data as well as unsuccessful translation of research approaches into clinics. In a previous study we have created epithelial cell cultures from head and neck squamous cell carcinoma (HNSCC) tissue. METHODS: We characterize primary cell populations isolated from human papillomavirus positive HNSCC tissue for their marker expression by RT-qPCR, flow cytometry, and immunofluorescence staining. Their sensitivity to MDM2-inhibition was measured using cell viability assays. RESULTS: Primary HNSCC cell cultures showed the delayed formation of spheroids at higher passages. These spheroids mimicked the morphology and growth characteristics of other established HNSCC spheroid models. However, expression of epithelial and mesenchymal markers could not be detected in these cells despite the presence of the HNSCC stem cell marker aldehyde dehydrogenase 1 family member A1. Instead, strong expression of B- and T-lymphocytes markers was observed. Flow cytometry analysis revealed a heterogeneous mixture of CD3 + /CD25 + T-lymphocytes and CD19 + B-lymphocytes at a ratio of 4:1 at passage 5 and transformed lymphocytes at late passages (≥ passage 12) with CD45 + CD19 + CD20 + , of which around 10 to 20% were CD3 + CD25 + CD56 + . Interestingly, the whole population was FOXP3-positive indicative of regulatory B-cells (Bregs). Expression of transcripts specific for the Epstein-Barr-virus (EBV) was detected to increase in these spheroid cells along late passages, and this population was vulnerable to MDM2 inhibition. HPV + HNSCC cells but not EBV + lymphocytes were detected to engraft into immunodeficient mice. CONCLUSIONS: In this study we present a primary cell culture of EBV-infected tumor-infiltrating B-lymphocytes, which could be used to study the role of these cells in tumor biology in future research projects. Moreover, by describing the detailed characteristics of these cells, we aim to caution other researchers in the HNSCC field to test for EBV-infected lymphocyte contaminations in primary cell cultures ahead of further experiments. Especially researchers who are interested in TIL-based adopted immunotherapy should exclude these cells in their primary tumor models, e.g. by MDM2-inhibitor treatment. BI-12-derived xenograft tumors represent a suitable model for in vivo targeting studies.

p53 Pathway Inactivation Drives SMARCB1-deficient p53-wildtype Epithelioid Sarcoma Onset Indicating Therapeutic Vulnerability Through MDM2 Inhibition.

Loss of the gene SMARCB1 drives the development of malignant rhabdoid tumors, epithelioid sarcomas, and other malignancies. The SMARCB1 protein is a core component of the SWI/SNF (SWItch/Sucrose Non-Fermentable) family of chromatin remodeling complexes, which are important regulators of gene expression and cell differentiation. Here, we use CRISPR-Cas9 to create germline smarcb1 loss of function in zebrafish. We demonstrate that the combination of smarcb1 deficiency with mutant p53 results in the development of epithelioid sarcomas, angiosarcomas, and carcinomas of the thyroid and colon. Although human epithelioid sarcomas do not frequently harbor p53 mutations, smarcb1-deficient tumors in zebrafish were only observed following disruption of p53, indicating that p53 signaling in human tumors might be attenuated through alternative mechanisms, such as MDM2-mediated proteasomal degradation of p53. To leverage this possibility for the treatment of human epithelioid sarcoma, we tested small molecule-mediated disruption of the p53-MDM2 interaction, which stabilized p53 protein leading to p53-pathway reactivation, cell-cycle arrest, and increased apoptosis. Moreover, we found that MDM2 inhibition and the topoisomerase II inhibitor doxorubicin synergize in targeting epithelioid sarcoma cell viability. This could be especially relevant for patients with epithelioid sarcoma because doxorubicin represents the current gold standard for their clinical treatment. Our results therefore warrant reactivating p53 protein in SMARCB1-deficient, p53-wildtype epithelioid sarcomas using combined doxorubicin and MDM2 inhibitor therapy.

Viral and Clinical Oncology of Head and Neck Cancers.

PURPOSE OF REVIEW: This study assesses the current state of knowledge of head and neck squamous cell carcinomas (HNSCC), which are malignancies arising from the orifices and adjacent mucosae of the aerodigestive tracts. These contiguous anatomical areas are unique in that 2 important human oncoviruses, Epstein-Barr virus (EBV) and human papillomavirus (HPV), are causally associated with nasopharyngeal and oropharyngeal cancers, respectively. Mortality rates have remained high over the last 4 decades, and insufficient attention paid to the unique viral and clinical oncology of the different subgroups of HNSCC. RECENT FINDINGS: We have compared and contrasted the 2 double-stranded DNA viruses and the relevant molecular oncogenesis of their respective cancers against other head and neck cancers. Tobacco and alcohol ingestion are also reviewed, as regard the genetic progression/mutation accumulation model of carcinogenesis. The importance of stringent stratification when searching for cancer mutations and biomarkers is discussed. Evidence is presented for a dysplastic/pre-invasive cancerous phase for HPV+ oropharyngeal cancers, and analogous with other HPV+ cancers. This raises the possibility of strategies for cancer screening as early diagnosis will undoubtedly save lives. Staging and prognostication have changed to take into account the distinct biological and prognostic pathways for viral+ and viral- cancers. Diagnosis of pre-cancers and early stage cancers will reduce mortality rates. Multi-modal treatment options for HNSCC are reviewed, especially recent developments with immunotherapies and precision medicine strategies. Knowledge integration of the viral and molecular oncogenic pathways with sound planning, hypothesis generation, and clinical trials will continue to provide therapeutic options in the future.

Retinoic acid rewires the adrenergic core regulatory circuitry of childhood neuroblastoma.

Neuroblastoma cell identity depends on a core regulatory circuit (CRC) of transcription factors that collaborate with MYCN to drive the oncogenic gene expression program. For neuroblastomas dependent on the adrenergic CRC, treatment with retinoids can inhibit cell growth and induce differentiation. Here, we show that when MYCN-amplified neuroblastoma cells are treated with retinoic acid, histone H3K27 acetylation and methylation become redistributed to decommission super-enhancers driving the expression of PHOX2B and GATA3, together with the activation of new super-enhancers that drive high levels of MEIS1 and SOX4 expression. These findings indicate that treatment with retinoids can reprogram the enhancer landscape, resulting in down-regulation of MYCN expression, while establishing a new retino-sympathetic CRC that causes proliferative arrest and sympathetic differentiation. Thus, we provide mechanisms that account for the beneficial effects of retinoids in high-risk neuroblastoma and explain the rapid down-regulation of expression of MYCN despite massive levels of amplification of this gene.

Sensorineural Hearing Loss After Balloon Eustachian Tube Dilatation.

Objective: Eustachian tube function is of central importance for the ventilation of the middle ear. A dysfunction can be associated with chronic otitis media, and cholesteatoma. Balloon Eustachian tube dilatation (BET) is a treatment option used to solve eustachian tube dysfunction. Although BET is widely performed, little is known about the occurrence rate of the complications associated with BET. The aim of the present study was to observe the rate of sensorineural hearing loss (SNHL) after BET. Methods: We retrospectively evaluated in a chart review 1,547 patients and 2,614 procedures of BET performed in a single center between 2015 and 2019 using the Spiggle and Theis, Overath, Germany eustachian tube dilatation system. Results: We observed seven cases of SNHL after BET. In two cases, the SNHL persisted, and in five cases, the SNHL was transient. In two cases of SNHL, a simultaneous tympanoplasty was performed. The overall rate of SNHL per procedure is 0.3%. The rate of permanent SNHL is 0.08%. Conclusion: BET has a low rate of SNHL. Rapid middle ear pressure changes are assumed to cause BET-related hearing loss.

Disulfiram Acts as a Potent Radio-Chemo Sensitizer in Head and Neck Squamous Cell Carcinoma Cell Lines and Transplanted Xenografts.

The poor prognosis of locally advanced and metastatic head and neck squamous cell carcinoma (HNSCC) is primarily mediated by the functional properties of cancer stem cells (CSCs) and resistance to chemoradiotherapy. We investigated whether the aldehyde dehydrogenase (ALDH) inhibitor disulfiram (DSF) can enhance the sensitivity of therapy. Cell viability was assessed by the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) and apoptosis assays, and the cell cycle and reactive oxygen species (ROS) levels were evaluated by fluorescence-activated cell sorting (FACS). The radio-sensitizing effect was measured by a colony formation assay. The synergistic effects were calculated by combination index (CI) analyses. The DSF and DSF/Cu2+ inhibited the cell proliferation (inhibitory concentration 50 (IC50) of DSF and DSF/Cu2+ were 13.96 µM and 0.24 µM). DSF and cisplatin displayed a synergistic effect (CI values were < 1). DSF or DSF/Cu2+ abolished the cisplatin-induced G2/M arrest (from 52.9% to 40.7% and 41.1%), and combining irradiation (IR) with DSF or DSF/Cu2+ reduced the colony formation and attenuated the G2/M arrest (from 53.6% to 40.2% and 41.9%). The combination of cisplatin, DSF or DSF/Cu2+, and IR enhanced the radio-chemo sensitivity by inducing apoptosis (42.04% and 32.21%) and ROS activity (46.3% and 37.4%). DSF and DSF/Cu2+ enhanced the sensitivity of HNSCC to cisplatin and IR. Confirming the initial data from patient-derived tumor xenograft (PDX) supported a strong rationale to repurpose DSF as a radio-chemosensitizer and to assess its therapeutic potential in a clinical setting.

Chronic inflammation of middle ear cholesteatoma promotes its recurrence via a paracrine mechanism.

BACKGROUND: Cholesteatoma disease is an expanding lesion in the middle ear. Hearing loss and facial paralysis alongside with other intracranial complications are found. No pharmaceutical treatment is available today and recurrence after surgical extraction occurs. We investigated possible TLR4-based mechanisms promoting recurrence and explore possible treatments strategies. METHODS: We isolated fibroblasts and epidermal stem cells from cholesteatoma tissue and healthy auditory canal skin. Subsequently, their expression under standard culture conditions and after stimulation with LPS was investigated by RT-qPCR. Cell metabolism and proliferation were analysed upon LPS treatment, with and without TLR4 antagonist. An indirect co-culture of fibroblasts and epidermal stem cells isolated from cholesteatoma tissue was utilized to monitor epidermal differentiation upon LPS treatment by RT-qPCR and immunocytochemistry. RESULTS: Under standard culture conditions, we detected a tissue-independent higher expression of IL-1ß and IL-8 in stem cells, an upregulation of KGF and IGF-2 in both cell types derived from cholesteatoma and higher expression of TLR4 in stem cells derived from cholesteatoma tissue. Upon LPS challenge, we could detect a significantly higher expression of IL-1α, IL-1ß, IL-6 and IL-8 in stem cells and of TNF-a, GM-CSF and CXCL-5 in stem cells and fibroblasts derived from cholesteatoma. The expression of the growth factors KGF, EGF, EREG, IGF-2 and HGF was significantly higher in fibroblasts, particularly when derived from cholesteatoma. Upon treatment with LPS the metabolism was elevated in stem cells and fibroblasts, proliferation was only enhanced in fibroblasts derived from cholesteatoma. This could be reversed by the treatment with a TLR4 antagonist. The cholesteatoma fibroblasts could be triggered by LPS to promote the epidermal differentiation of the stem cells, while no LPS treatment or LPS treatment without the presence of fibroblasts did not result in such a differentiation. CONCLUSION: We propose that cholesteatoma recurrence is based on TLR4 signalling imprinted in the cholesteatoma cells. It induces excessive inflammation of stem cells and fibroblasts, proliferation of perimatrix fibroblasts and the generation of epidermal cells from stem cells thru paracrine signalling by fibroblasts. Treatment of the operation site with a TLR4 antagonist might reduce the chance of cholesteatoma recurrence. Video Abstract.

Insights into Nanomedicine for Immunotherapeutics in Squamous Cell Carcinoma of the head and neck.

Immunotherapies such as immune checkpoint blockade benefit only a portion of patients with head and neck squamous cell carcinoma. The multidisciplinary field of nanomedicine is emerging as a promising strategy to achieve maximal anti-tumor effect in cancer immunotherapy and to turn non-responders into responders. Various methods have been developed to deliver therapeutic agents that can overcome bio-barriers, improve therapeutic delivery into the tumor and lymphoid tissues and reduce adverse effects in normal tissues. Additional modification strategies also have been employed to improve targeting and boost cytotoxic T cell-based immune responses. Here, we review the state-of-the-art use of nanotechnologies in the laboratory, in advanced preclinical phases as well as those running through clinical trials assessing their advantages and challenges.

suz12 inactivation in p53- and nf1-deficient zebrafish accelerates the onset of malignant peripheral nerve sheath tumors and expands the spectrum of tumor types.

Polycomb repressive complex 2 (PRC2) is an epigenetic regulator of gene expression that possesses histone methyltransferase activity. PRC2 trimethylates lysine 27 of histone H3 proteins (H3K27me3) as a chromatin modification associated with repressed transcription of genes frequently involved in cell proliferation or self-renewal. Loss-of-function mutations in the PRC2 core subunit SUZ12 have been identified in a variety of tumors, including malignant peripheral nerve sheath tumors (MPNSTs). To determine the consequences of SUZ12 loss in the pathogenesis of MPNST and other cancers, we used CRISPR-Cas9 to disrupt the open reading frame of each of two orthologous suz12 genes in zebrafish: suz12a and suz12b We generated these knockout alleles in the germline of our previously described p53 (also known as tp53)- and nf1-deficient zebrafish model of MPNSTs. Loss of suz12 significantly accelerated the onset and increased the penetrance of MPNSTs compared to that in control zebrafish. Moreover, in suz12-deficient zebrafish, we detected additional types of tumors besides MPNSTs, including leukemia with histological characteristics of lymphoid malignancies, soft tissue sarcoma and pancreatic adenocarcinoma, which were not detected in p53/nf1-deficient control fish, and are also contained in the human spectrum of SUZ12-deficient malignancies identified in the AACR Genie database. The suz12-knockout tumors displayed reduced or abolished H3K27me3 epigenetic marks and upregulation of gene sets reported to be targeted by PRC2. Thus, these zebrafish lines with inactivation of suz12 in combination with loss of p53/nf1 provide a model of human MPNSTs and multiple other tumor types, which will be useful for mechanistic studies of molecular pathogenesis and targeted therapy with small molecule inhibitors.

The Potential of Tumor Debulking to Support Molecular Targeted Therapies.

Tumors may consist of billions of cells, which in malignant cases disseminate and form distant metastases. The large number of tumor cells formed by the high number of cell divisions during tumor progression creates a heterogeneous set of genetically diverse tumor cell clones. For cancer therapy this poses unique challenges, as distinct clones have to be targeted in different tissue locations. Recent research has led to the development of specific inhibitors of defined targets in cellular signaling cascades which promise more effective and more tumor-specific therapy approaches. Many of these molecular targeted therapy (MTT) compounds have already been translated into clinics or are currently being tested in clinical studies. However, the outgrowth of tumor cell clones resistant to such inhibitors is a drawback that affects specific inhibitors in a similar way as classical cytotoxic chemotherapeutics, because additionally acquired genetic alterations can enable tumor cells to circumvent the particular regulators of cellular signaling being targeted. Thus, it might be desirable to reduce genetic heterogeneity prior to molecular targeting, which could reduce the statistical chance of tumor relapse initiated by resistant clones. One way to achieve this is employing unspecific methods to remove as much tumor material as possible before MTT, e.g., by tumor debulking (TD). Currently, this is successfully applied in the clinical treatment of ovarian cancer. We believe that TD followed by treatment with a combination of molecular targeted drugs, optimally guided by biomarkers, might advance survival of patients suffering from various cancer types.

Stem Cell-Induced Inflammation in Cholesteatoma is Inhibited by the TLR4 Antagonist LPS-RS.

Cholesteatoma is a severe non-cancerous lesion of the middle ear characterized by massive inflammation, tissue destruction, and an abnormal growth of keratinized squamous epithelium. We recently demonstrated the presence of pathogenic stem cells within cholesteatoma tissue, unfortunately their potential roles in regulating disease-specific chronic inflammation remain poorly understood. In the presented study, we utilized our established human in vitro cholesteatoma stem cell model for treatments with lipopolysaccharides (LPS), tumor necrosis factor α (TNFα), and the TLR4-antagonist LPS from R.sphaeroides(LPS-RS) followed by qPCR, western blot, and immunocytochemistry. Middle ear cholesteatoma stem cells (ME-CSCs) showed a significantly increased expression of TLR4 accompanied by a significantly enhanced LPS-dependent pro-inflammatory gene expression pattern of TNFα, IL-1α, IL-1ß, IL-6, and IL-8 compared to non-pathogenic control cells. LPS-dependent pro-inflammatory gene expression in ME-CSCs was driven by an enhanced activity of NF-B p65 leading to a TNFα-mediated feed-forward-loop of pro-inflammatory NF-B target gene expression. Functional inactivation of TLR4 via the TLR4-antagonist LPS-RS blocked chronic inflammation in ME-CSCs, resulting in a nearly complete loss of IL-1ß, IL-6, and TNFα expression. In summary, we determined that ME-CSCs mediate the inflammatory environment of cholesteatoma via TLR4-mediated NF-B-signaling, suggesting a distinct role of ME-CSCs as drivers of cholesteatoma progression and TLR4 on ME-CSCs as a therapeutic target.

The Therapeutic Effect of 1,8-Cineol on Pathogenic Bacteria Species Present in Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is marked by an inflamed mucosa of sinuses and is accompanied by a significantly reduced quality of live. Since no guidelines for the treatment of CRS are available, long lasting clinical histories with health care costs adding up to dozens of billion $ annually are caused by CRS. The progression of CRS is often induced by bacterial infections and/or a shift in microbiome as well as biofilm formation. The exact microbiome alterations are still unclear and the impenetrable biofilm renders the treatment with common antibiotics ineffective. This study focuses on characterizing the microbiome changes in CRS and investigating the inhibition of biofilm growth by 1,8-Cineol, a small, non-polar and hence biofilm penetrating molecule with known antimicrobial potential. We performed MALDI-TOF MS based characterization of the microbiomes of healthy individuals and CRS patients (n = 50). The microbiome in our test group was shifted to pathogens (Staphylococcus aureus, Escherichia coli, and Moraxella catarrhalis). In contrast to published studies, solely based on cell culture techniques, we could not verify the abundance of Pseudomonas aeruginosa in CRS. The inhibition of bacterial proliferation and biofilm growth by 1,8-Cineol was measured for these three pathogens. Interestingly, S. aureus, the most prominent germ in CRS, showed a biofilm inhibition not simply correlated to its inhibition of proliferation. RT-qPCR confirmed that this was due to the downregulations of major key players in biofilm generation (agrA, SarA and σB) by 1,8-Cineol. Furthermore we verified this high biofilm inhibition potential in a model host system consisting out of S. aureus biofilm grown on mature respiratory epithelium. A second host model, comprising organotypic slices, was utilized to investigate the reaction of the innate immune system present in the nasal mucosa upon biofilm formation and treatment with 1,8-Cineol. Interestingly Staphylococcus epidermidis, the cause of very common catheter infections, possesses a biofilm generation pathway very similar to S. aureus and might be treatable in a similar fashion. The two presented in vitro model systems might be transferred to combinations of every biofilm forming bacterial with most kind of epithelium and mucosa.

Mechanisms underlying synergy between DNA topoisomerase I-targeted drugs and mTOR kinase inhibitors in NF1-associated malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas that frequently arise in patients with neurofibromatosis type 1 (NF1). Most of these tumors are unresectable at diagnosis and minimally responsive to conventional treatment, lending urgency to the identification of new pathway dependencies and drugs with potent antitumor activities. We therefore examined a series of candidate agents for their ability to induce apoptosis in MPNST cells arising in nf1/tp53-deficient zebrafish. In this study, we found that DNA topoisomerase I-targeted drugs and mTOR kinase inhibitors were the most effective single agents in eliminating MPNST cells without prohibitive toxicity. In addition, three members of these classes of drugs, either AZD2014 or INK128 in combination with irinotecan, acted synergistically to induce apoptosis both in vitro and in vivo. In mechanistic studies, irinotecan not only induces apoptosis by eliciting a DNA damage response, but also acts synergistically with AZD2014 to potentiate the hypophosphorylation of 4E-BP1, a downstream target of mTORC1. Profound hypophosphorylation of 4E-BP1 induced by this drug combination causes an arrest of protein synthesis, which potently induces tumor cell apoptosis. Our findings provide a compelling rationale for further in vivo evaluation of the combination of DNA topoisomerase I-targeted drugs and mTOR kinase inhibitors against these aggressive nerve sheath tumors.

An Effective Primary Head and Neck Squamous Cell Carcinoma In Vitro Model.

Head and neck squamous cell carcinoma is a highly malignant disease and research is needed to find new therapeutic approaches. Faithful experimental models are required for this purpose. Here, we describe the specific cell culture conditions enabling the efficient establishment of primary cell culture models. Whereas a classical 10% serum-containing medium resulted in the growth of fibroblast-like cells that outcompeted epithelial cells, we found that the use of specific culture conditions enabled the growth of epithelial tumor cells from HPV+ and HPV- head and neck cancer tissue applicable for research. EpCAM and high Thy-1 positivity on the cell surface were mutually exclusive and distinguished epithelial and fibroblast-like subpopulations in all primary cultures examined and thus can be used to monitor stromal contamination and epithelial cell content. Interestingly, cells of an individual patient developed tumor spheroids in suspension without the use of ultra-low attachment plates, whereas all other samples exclusively formed adherent cell layers. Spheroid cells were highly positive for ALDH1A1 and hence displayed a phenotype reminiscent of tumor stem cells. Altogether, we present a system to establish valuable primary cell culture models from head and neck cancer tissue at high efficiency that might be applicable in other tumor entities as well.

Loss of atrx cooperates with p53-deficiency to promote the development of sarcomas and other malignancies.

The SWI/SNF-family chromatin remodeling protein ATRX is a tumor suppressor in sarcomas, gliomas and other malignancies. Its loss of function facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells, while it also affects Polycomb repressive complex 2 (PRC2) silencing of its target genes. To further define the role of inactivating ATRX mutations in carcinogenesis, we knocked out atrx in our previously reported p53/nf1-deficient zebrafish line that develops malignant peripheral nerve sheath tumors and gliomas. Complete inactivation of atrx using CRISPR/Cas9 was lethal in developing fish and resulted in an alpha-thalassemia-like phenotype including reduced alpha-globin expression. In p53/nf1-deficient zebrafish neither peripheral nerve sheath tumors nor gliomas showed accelerated onset in atrx+/- fish, but these fish developed various tumors that were not observed in their atrx+/+ siblings, including epithelioid sarcoma, angiosarcoma, undifferentiated pleomorphic sarcoma and rare types of carcinoma. These cancer types are included in the AACR Genie database of human tumors associated with mutant ATRX, indicating that our zebrafish model reliably mimics a role for ATRX-loss in the early pathogenesis of these human cancer types. RNA-seq of p53/nf1- and p53/nf1/atrx-deficient tumors revealed that down-regulation of telomerase accompanied ALT-mediated lengthening of the telomeres in atrx-mutant samples. Moreover, inactivating mutations in atrx disturbed PRC2-target gene silencing, indicating a connection between ATRX loss and PRC2 dysfunction in cancer development.

Systematic Generation of Patient-Derived Tumor Models in Pancreatic Cancer.

In highly aggressive malignancies like pancreatic cancer (PC), patient-derived tumor models can serve as disease-relevant models to understand disease-related biology as well as to guide clinical decision-making. In this study, we describe a two-step protocol allowing systematic establishment of patient-derived primary cultures from PC patient tumors. Initial xenotransplantation of surgically resected patient tumors (n = 134) into immunodeficient mice allows for efficient in vivo expansion of vital tumor cells and successful tumor expansion in 38% of patient tumors (51/134). Expansion xenografts closely recapitulate the histoarchitecture of their matching patients' primary tumors. Digestion of xenograft tumors and subsequent in vitro cultivation resulted in the successful generation of semi-adherent PC cultures of pure epithelial cell origin in 43.1% of the cases. The established primary cultures include diverse pathological types of PC: Pancreatic ductal adenocarcinoma (86.3%, 19/22), adenosquamous carcinoma (9.1%, 2/22) and ductal adenocarcinoma with oncocytic IPMN (4.5%, 1/22). We here provide a protocol to establish quality-controlled PC patient-derived primary cell cultures from heterogeneous PC patient tumors. In vitro preclinical models provide the basis for the identification and preclinical assessment of novel therapeutic opportunities targeting pancreatic cancer.

Specific Targeting of Oncogenes Using CRISPR Technology.

In recent decades, tools of molecular biology have enabled researchers to genetically modify model organisms, including human cells. RNAi, zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats and CRISPR-associated protein 9), retro- or lentiviral gene transfer, and many other methods can be utilized to remove genes, add genes, or change their expression. Within the same timeframe, survival rates for many highly malignant tumor diseases have not improved substantially. If modern medicine could apply even a subset of research methods in clinical management, which are already well established and controllable in basic research laboratories, this could strongly impact patients' prognosis. CRISPR-Cas9 is a method to precisely target and manipulate genomic loci and recent studies have attempted to use this method as a genetic treatment for Duchenne muscular dystrophy, blood disorders, autosomal-dominant hearing loss, and cancer. Some of these approaches target mutant genomic sequences specifically and try to avoid affecting the respective normal loci. Considering obvious genetic risks opposing the objected benefits, data are needed to show whether CRISPR technology is suitable as a future cancer therapy approach or not. Here, we develop strategies for the specific targeting of viral cancer drivers and oncogenes activated by mutation, using the latest CRISPR technology. Cancer Res; 78(19); 5506-12. ©2018 AACR.

Succession of transiently active tumor-initiating cell clones in human pancreatic cancer xenografts.

Although tumor-initiating cell (TIC) self-renewal has been postulated to be essential in progression and metastasis formation of human pancreatic adenocarcinoma (PDAC), clonal dynamics of TICs within PDAC tumors are yet unknown. Here, we show that long-term progression of PDAC in serial xenotransplantation is driven by a succession of transiently active TICs producing tumor cells in temporally restricted bursts. Clonal tracking of individual, genetically marked TICs revealed that individual tumors are generated by distinct sets of TICs with very little overlap between subsequent xenograft generations. An unexpected functional and phenotypic plasticity of pancreatic TICs in vivo underlies the recruitment of inactive TIC clones in serial xenografts. The observed clonal succession of TIC activity in serial xenotransplantation is in stark contrast to the continuous activity of limited numbers of self-renewing TICs within a fixed cellular hierarchy observed in other epithelial cancers and emphasizes the need to target TIC activation, rather than a fixed TIC population, in PDAC.