Characterisation of acini formed in Matrigel 3D cultures

The mechano-electrical transduction (MET) channel of the inner ear receptor cells, termed hair cells, is a protein complex that enables our senses of hearing and balance. Hair cell MET requires an elaborate interplay of multiple proteins that form the MET channel. One of the MET complex components is the transmembrane protein LHFPL5, which is required for hair cell MET and hearing. LHFPL5 is thought to form a multi-protein complex with other MET channel proteins, such as PCDH15, TMIE, and TMC1. Despite localizing to the plasma membrane of stereocilia, the mechanosensing organelles of hair cells, LHFPL5 requires its binding partner within the MET complex, PCDH15, to localize to the stereocilia tips in hair cells and to the plasma membrane in heterologous cells. Using the Aquaporin 3-tGFP reporter (AGR) for plasma membrane localization, we found that a region within extracellular loop 1, which interacts with PCDH15, precludes the trafficking of AGR reporter to the plasma membrane in heterologous cell lines. Our results suggest that the presence of protein partners may mask endoplasmic reticulum retention regions or enable the proper folding and trafficking of the MET complex components, to facilitate expression of the MET complex at the stereocilia membrane.

Supplementary figure legends, tables and macro

BPEC culture characterisation

Video containing time-lapse images acquired during the first 9 hours after seeding.

Speed of movement of BPECs in 3D culture

Multiple myeloma (MM) remains a lethal hematologic cancer characterized by the expansion of transformed plasma cells within the permissive bone marrow (BM) milieu. The emergence of relapsed and/or refractory MM (RRMM) is provoked through clonal evolution of malignant plasma cells that harbor genomic, metabolic and proteomic perturbations. For most patients, relapsed disease remains a major cause of overall mortality. Transforming growth factors (TGFs) have pleiotropic effects that regulate myelomagenesis as well as the emergence of drug resistance. Moreover, TGF-β modulates numerous cell types present with the tumor microenvironment, including many immune cell types. While numerous agents have been FDA-approved over the past 2 decades and significantly expanded the treatment options available for MM patients, the molecular mechanisms responsible for drug resistance remain elusive. Multiple myeloma is uniformly preceded by a premalignant state, monoclonal gammopathy of unknown significance, and both conditions are associated with progressive deregulation in host immunity characterized by reduced T cell, natural killer (NK) cell and antigen-presenting dendritic cell (DC) activity. TGF-β promotes myelomagenesis as well as intrinsic drug resistance by repressing anti-myeloma immunity to promote tolerance, drug resistance and disease progression. Hence, repression of TGF-β signaling is a prerequisite to enhance the efficacy of current and future immunotherapeutics. Novel strategies that incorporate T cells that have been modified to express chimeric antigen receptor (CARs), T cell receptors (TCRs) and bispecific T cell engagers (BiTEs) offer promise to block TGF-β signaling, overcome chemoresistance and enhance anti-myeloma immunity. Here, we describe the effects of TGF-β signaling on immune cell effectors in the bone marrow and emerging strategies to overcome TGF-β-mediated myeloma growth, drug resistance and survival.

Study of human monocytic Myeloid-Derived Suppressor cells Mo-MDSC (CD14(+) HLA-DRneg/low) has been hampered by the lack of positive cell-surface markers. In order to identify positive markers for Mo-MDSC, we performed microarray analysis comparing Mo-MDSC cells from healthy subjects versus CD14(+) HLA-DRhigh monocytes. We have identified the surface ectoenzyme Vanin-2(VNN2) protein as a novel biomarker highly-enriched in healthy subjects Mo-MDSC. Indeed, healthy subjects Mo-MDSC cells expressed 68 % VNN2, whereas only 9% VNN2 expression was observed on CD14(+) HLA-DRhigh cells (n = 4 p < 0.01). The top 10 percent positive VNN2 monocytes expressed CD33 and CD11b while being negative for HLA-DR, CD3, CD15, CD19 and CD56, consistent with a Mo-MDSC phenotype. CD14(+)VNN2(high) monocytes were able to inhibit CD8 T cell proliferation comparably to traditional Mo-MDSC at 51 % and 48 % respectively. However, VNN2 expression on CD14(+) monocytes from glioma patients was inversely correlated to their grade. CD14(+)VNN2(high) monocytes thus appear to mark a monocytic population similar to Mo-MDSC only in healthy subjects, which may be useful for tumor diagnoses.

Abstract INTRODUCTION Glioblastoma multiforme (GBM) is the most common primary central nervous system malignancy associated with a 12-15 month survival after surgery and radio-chemotherapy. Utilizing adoptive cellular immunotherapy using natural killer (NK) cells has developed over the past two decades for a variety of hematologic malignancies. This approach in solid malignancies is limited by questions of cell dose versus tumor burden, insufficient tumor infiltration, and a tumor microenvironment that suppresses NK cell function. METHODS We isolated NK cells from healthy volunteers and activated them using IL-2, -15, -12, -18, then perform cytotoxic assays in the presence of glioma stem cells. We also tested the efficacy of the NK cells with intracranial delivery in a pre-clinical murine model of glioma. We tested various concentrations of IL-2 and IL-15 on the cytokine culture platform. RESULTS In this study, we demonstrate human NK cells, activated using a cytokine cocktail of interleukins-2, -15, -12, and -18, exert strong cytotoxic events against glioma cell lines. To further examine the efficacy of activated NK cells in vitro, we utilized intracranially xenografted glioma lines and demonstrated a survival benefit with tumor bed injections of these cytokine-activated NK cells (p=0.0089). We were able to confirm that NK cells cultured with low doses (200u IL2; 50ng/ml IL15) of both cytokines are just as effective as higher doses. This is important, as in vivoexhaustion of NK cells stimulated with high doses of either cytokine has been well validated. We also found that low-dose irradiation (4Gy) of glioma cells prior to co-culture with cytokine-activated NK cells promoted increased targeted glioma cell killing within 4 hours(32% cell killing). CONCLUSIONS These findings suggest that in a clinical study, injection of cytokine-activated NK cells into the glioblastoma tumor bed could be used as adjuvant treatment following either stereotactic radiation or surgical resection.

Chromosomal instability, the most frequent form of plasticity in cancer cells, often proceeds through the formation of chromosome bridges. Despite the importance of these bridges in tumor initiation and progression, debate remains over how and when they are resolved. In this study, we investigated the behavior and properties of chromosome bridges to gain insight into the potential mechanisms underlying bridge-induced genome instability. We report that bridges may break during mitosis or may remain unbroken until the next interphase. During mitosis, we frequently observed discontinuities in the bridging chromatin, and our results strongly suggest that a substantial fraction of chromosome bridges are broken during this stage of the cell cycle. This notion is supported by the observation that the chromatin flanking mitotic bridge discontinuities is often decorated with the phosphorylated form of the histone H2AX, a marker of DNA breaks, and by MDC1, an early mediator of the cell response to DNA breaks. Also, free 3 ' OH DNA ends were detected in more than half of the bridges during the final stages of cell division. However, even if detected, the DNA ends of broken bridges are not repaired in mitosis. To investigate whether mitotic bridge breakage depends on mechanical stress, we used experimental models in which chromosome bridges with defined geometry are formed. Although there was no association between spindle pole separation or the distance among non-bridge kinetochores and bridge breakage, we found a direct correlation between the distance between bridge kinetochores and bridge breakage. Altogether, we conclude that the discontinuities observed in bridges during mitosis frequently reflect a real breakage of the chromatin and that the mechanisms responsible for chromosome bridge breakage during mitosis may depend on the separation between the bridge kinetochores. Considering that previous studies identified mechanical stress or biochemical digestion as possible causes of bridge breakage in interphase cells, a multifactorial model emerges for the breakage of chromosome bridges that, according to our results, can occur at different stages of the cell cycle and can obey different mechanisms.

The inability to over-express Aquaporin 6 (AQP6) in the plasma membrane of heterologous cells has hampered efforts to further characterize the function of this aquaglyceroporin membrane protein at atomic detail using crystallographic approaches. Using an Aquaporin 3-tGFP Reporter (AGR) system we have identified a region within loop C of AQP6 that is responsible for severely hampering plasma membrane expression. Serine substitution corroborated that amino acids present within AQP6 194–213 of AQP6 loop C contribute to intracellular endoplasmic reticulum (ER) retention. This intracellular retention signal may preclude proper plasma membrane trafficking and severely curtail expression of AQP6 in heterologous expression systems.

BACKGROUND:Brain metastases (BM) are the most common type of brain tumor malignancy in the US. They are also the most common indication for stereotactic radiosurgery (SRS). However, the incidence of both local recurrence and radiation necrosis (RN) is increasing as treatments improve. MRI imagery often fails to differentiate BM from RN; thus, patients must often undergo surgical biopsy or resection to obtain a definitive diagnosis. OBJECTIVE:To hypothesize that a marker of immunosuppression might serve as a surrogate marker to differentiate patients with active vs inactive cancer-including RN. METHODS:We thus purified and quantified Monocytic Myeloid-Derived Suppressor Cells (Mo-MDSC) by flow cytometry in patients proven by biopsy to represent BM or RN. RESULTS:We report the utility of the previously reported HLA-Dr-Vnn2 Index or DVI to discriminate recurrent BM from RN using peripheral blood. The presence of CD14+ HLA-DRneg/low Mo-MDSC is significantly increased in the peripheral blood of patients with brain metastasis recurrence compared to RN (Average 61.5% vs 7%, n = 10 and n = 12, respectively, P < .0001). In contrast, expression of VNN2 on circulating CD14+ monocytes is decreased in BM patients compared to patients with RN (5.5% vs 26.5%, n = 10 and n = 12, respectively, P = .0008). In patients with biopsy confirmed recurrence of brain metastasis, the average DVI was 11.65, whereas the average DVI for RN patients was consistently <1 (Avg. of 0.17). CONCLUSION:These results suggest that DVI could be a useful diagnostic tool to differentiate recurrent BM from RN using a minimally invasive blood sample.

Abstract Radiation is used in multiple procedures as a therapeutic and diagnostic tool. However, ionizing radiation can induce mutations in the DNA of irradiated cells, which can promote tumorigenesis. As malignant transformation is a process that takes many years, there are intermediate stages of cells that have initiated the process but have not yet evolved into cancer. The study here aimed to investigate the effect of ionizing radiation on normal and partially transformed human mammary epithelial cells. Breast primary epithelial cells were derived from normal breast tissue from two different donors and modified by transduction with the SV40 small and large T antigen and hTERT genes to obtain partially transformed cells and also with HRAS to completely and experimentally transform them. After exposure to different doses of ionizing radiation, oncogenic features were analyzed by means of an anchorage-independent growth assay and 3D cell culture. The addition of radiation exposure resulted in an increase in the number and size of colonies formed in each of the conditions analyzed and in the reduction of the capacity of partially transformed cells to form properly polarized 3D structures. Moreover, partially transformed cells require lower doses of radiation than healthy cells to enhance anchorage-independent growth capacity. Although cells from different donors have a different degree of sensitivity in the response to radiation, a higher sensitivity to the radiation-induced cell transformation process was observed in those cells that had already initiated the oncogenic process, which require higher doses of radiation to complete the transformation process. Implications: Individuals carrying accumulation of genetic alterations may have an increased susceptibility to radiation-induced neoplastic transformation.

Mechanotransduction by hair cell stereocilia lies at the heart of sound detection in vertebrates. Considerable effort has been put forth to identify proteins that comprise the hair cell mechanotransduction apparatus. TMC1, a member of the transmembrane channel-like (TMC) family, was identified as a core protein of the mechanotransduction complex in hair cells. However, the inability of TMC1 to traffic through the endoplasmic reticulum in heterologous cellular systems has hindered efforts to characterize its function and fully identify its role in mechanotransduction. We developed a novel approach that allowed for the detection of uncharacterized protein regions, which preclude trafficking to the plasma membrane (PM) in heterologous cells. Tagging N-terminal fragments of TMC1 with Aquaporin 3 (AQP3) and GFP fusion reporter, which intrinsically label PM in HEK293 cells, indicated that residues at the edges of amino acid sequence 138–168 invoke intracellular localization and/or degradation. This signal is able to preclude surface localization of PM protein AQP3 in HEK293 cells. Substitutions of the residues by alanine or serine corroborated that the information determining the intracellular retention is present within amino acid sequence 138–168 of TMC1 N-terminus. This novel signal may preclude the proper trafficking of TMC1 to the PM in heterologous cells.

The well-characterized cell line Chinese hamster ovary (CHO) has been used to produce numerous biopharmaceuticals and is an important tool for basic research. However, introducing foreign DNA into specially modified CHO cells such as DG44 and Lec 3.2.8.1 can sometimes be an arduous process. Here we show that the Flp-intm plasmid can be modified to produce a fluorescent tracer protein tag (mCherrytm) as a fusion reporter, to allow for the rapid selection of single-cell sorted, isogenic Flp-intm-ready DG44 and Lec 3.2.8.1 cell lines. These two cell lines are stable and viable and may be useful for applications such as antibody production and crystallographic studies. Here we provide key details on how the modified pFRT/CherryZeo plasmid may be used to incorporate Flp-intm technology into virtually any desired target cell line in a fast, safe and reliable manner.

Glioblastoma (GBM) is the most aggressive and lethal type of brain cancer with a median survival of less than two years even following aggressive treatment (Stupp et al., N Engl J Med 352:987–996, 2005). Among the many challenges in treating patients with this devastating disease is the ability to differentiate Magnetic Resonance Imaging (MRI) images that appear following radiation therapy, often termed “radiation necrosis” from true GBM recurrence. Radiation necrosis (RN) and GBM are very difficult to distinguish and currently only a brain biopsy can conclusively differentiate these pathologies. In the present study, we introduce a differential diagnostic approach using a newly identified Myeloid-Derived Suppressor Cell (MDSC) biomarker, vascular non-inflammatory molecule 2 (VNN2+), in combination with expression of traditional HLA-DR on peripheral blood CD14+ monocytes isolated from GBM and/or RN patients. We performed proof-of-principle experiments confirming the sensitivity and specificity of this approach based upon the combined expression levels of HLA-DR and VNN2 among CD14+ Mo-MDSC, which we called the DR-Vanin Index or DVI. The DVI was able to distinguish GBM from RN patients with a high degree of certainty (n = 18 and n = 6 respectively; p = 0.0004). This novel, quick and inexpensive blood-based liquid biopsy could potentially replace invasive brain biopsies in differentiating GBM from RN patients using a minimally-invasive technique.

We previously observed that aquaporin-3 and aquaporin-10 are upregulated in the epidermis of hand dermatitis patients (Med. Hypotheses, 84, 2015, 498). To address the functional relevance of this upregulation, we overexpressed AQP3/AQP10 in mice using the human K1 promoter. Combining imiquimod with detergent-containing water challenge, a common trigger in hand and other dermatitis, resulted in an increase in acanthosis in mice overexpressing AQP3 or AQP3 and AQP10. Aquaporin overexpression also drove a trend towards greater weight loss in these animals. These data support a role for cutaneous aquaporins in the pathogenesis of dermatitis and as a potential target in their treatment.

Brain metastasis (BM) are the most common type of brain tumor with an estimated incidence of 200,000–400,000/yr. in the US alone. BM are also the most common indication for stereotactic radiosurgery (SRS). However, while SRS is highly effective in achieving local control, the incidence of both local recurrence and radiation necrosis (RN) is increasing as treatment for primary systemic cancers improves. Among the challenges at the time of apparent imaging recurrence is differentiating “true” recurrence of BM from RN. Since conventional imaging is not always reliable for this, many patients must undergo surgical biopsy or resection due to this uncertainty. Here, we demonstrate the utility of the novel biomarker Vanin-2 (VNN2) combined with expression of MHC Class II molecule HLA-DR on CD14+ cells in order to differentiate recurrent BM from RN. Our preliminary data indicate that presence of Mo-MDSC is significantly increased in BM compared to RN (49% vs 4%, p=0.005). In contrast, VNN2 expression on CD14+ in BM is decreased compared to RN (4.3% vs 36%, p=0.02). In order to further decrease overlap between outliers in each group of patients, we define the ratio of HLA-DR expression to VNN2 expression on CD14+ cells from as the DR-VNN Index or DVI. While a DVI >1 is considered BM (average= 15.5), a DVI<1 is considered RN (Average 0.07). This increases the receiver operator curve (ROC) and thus the reliability of the biomarker. These results suggest that the novel biomarker VNN2 in conjunction with HLA-DRneg/low on CD14+cells, could be useful to differentiate BM from RN using a minimally invasive blood sample. Thus, the DVI has the potential to serve as an inexpensive and non-invasive biomarker in helping to manage clinical care. Further studies are indicated.

Background/PurposePsoriasis continues to be a debilitating skin disease affecting 1-3% of the United States population. Although the effectiveness of several current biologic therapies have described this pathology as a IL-23, TNF-a and Th17-mediated disease, less invasive approaches are still in use and in need of refinement. One of these is the usage of narrow band-UVB (NB-UVB) therapy to deplete specifically intra-epidermal CD3+, CD4+ and CD8+ cells to clear psoriatic plaques.Aims/ObjectivesIn order to improve NB-UVB therapy, we sought to determine whether skin pre-treatment with the TLR7 agonist imiquimod (IMQ) would help increase the efficiency of the former at resolving psoriatic plaques.Materials and MethodsEucerin((R)) Original Moisturizing Lotion (topical vehicle) or Aldara((R)) (imiquimod 5% topical cream) were applied for 5 days once daily to a maximum contiguous area of 25 cm(2) (5 cm x 5 cm area). Patients were provided with sachets containing 12.5 mg of imiquimod each and were instructed to apply imiquimod (I) to two psoriasis plaques (5 sachets of imiquimod allotted to each plaque). A PHAROS excimer Laser EX-308 (Ra Medical Systems, Inc. Carlsbad, CA, USA) with an output of monochromatic 308-nm light and pulse width of 20-50 ns was used for all patients. Punch biopsies of psoriatic lesions (6 mm) were taken at 4 and 48 h after final application of topical treatment with or without excimer laser treatment. Real-time quantitative RT-PCR was performed according to manufacturer's instructions and Inmunohistochemistry was used as described before.ResultsOur results suggests that although IMQ seemed to activate the type I interferon pathway as previously described, its concomitant usage with NB-UVB for clearing psoriatic skin was ineffective. Although upregulation of genes MxA, GRAMD1A and DMXL2 suggested that IMQ treatment did induce skin changes in psoriasis patients, more optimal dosing of IMQ and NB-UVB might be necessary to achieve desired treatment responses.ConclusionThe observation that psoriasis involvement was not aggravated by usage of topical IMQ was encouraging. Additional observational studies might be necessary to further tailor the combination of IMQ with NB-UVB therapy to reliably improve the psoriatic pathology.

Introduction: Crusted or Norwegian scabies is an infectious skin dermatopathology usually associated with an underlying immunodeficiency condition. It is caused when the mite Sarcoptes scabiei infects the skin, and the immune system is unable to control its spread, leading to a massive hyperinfestation with a simultaneous inflammatory and hyperkeratotic reaction. This is the first report of a novel 1p36 duplication associated with a recurrent infection of crusted scabies.Case report: We describe a 34-year-old patient with a cutaneous immunodeficiency characterized by recurrent crusted scabies infestation, diffuse tinea, and recurrent staphylococcal cellulitis, who we suspected had an undiagnosed syndrome. The patient also suffered from mental retardation, renal failure, and premature senescence. A cytogenetic fluorescence in situ hybridization analysis revealed a 9.34 Mb duplication within the short (p)arm of chromosome 1, precisely from 1p36.11 to 1p36.21, with an adjacent 193 kb copy gain entirely within 1p36.11. In addition, chromosome 4 had a 906 kb gain in 4p16.1 and chromosome 9 had a 81 kb copy gain in 9p24.3. Over 100 genes localized within these duplicated regions. Gene expression array revealed 82 genes whose expression changed >1.5-fold compared to a healthy age-matched skin control, but among them only the lipolytic enzyme arylacetamide deacetylase-like 3 was found within the duplicated 1p36 region of chromosome 1.Discussion: Although genetic duplications in the 1p36 region have been previously described, our report describes a novel duplicative variant within the 1p36 region. The patient did not have a past history of immunosuppression but was afflicted by a recurrent case of crusted scabies, raising the possibility that the recurrent infection was associated with the 1p36 genetic duplication.Conclusion: To our knowledge, the specific duplicated sequence between 1p36.11 and p36.21 found in our patient has never been previously reported. We reviewed and compared the clinical, genotyping, and gene microarray results of our patient in order to characterize this novel 1p36 duplication syndrome, which might have contributed to the recurrent scabies infection in this patient.