Short-term Cell Cultures Research Articles

Identification, expansion and characterization of cancer cells with stem cell properties from head and neck squamous cell carcinomas

Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. Recent data indicate the presence of cancer stem cells (CSC) in many solid tumors, including HNSCC. Here, we assessed the stem cell (SC) characteristics, including cell surface markers, radioresistance, chromosomal instability, and in vivo tumorigenic capacity of CSC isolated from HNSCC patient specimens. We show that spheroid enrichment of CSC from early and short-term HNSCC cell cultures was associated with increased expression of CD44, CD133, SOX2 and BMI1 compared with normal oral epithelial cells. On immunophenotyping, five of 12 SC/CSC markers were homogenously expressed in all tumor cultures, while one of 12 was negative, four of 12 showed variable expression, and two of the 12 were expressed heterogeneously. We showed that irradiated CSCs survived and retained their self-renewal capacity across different ionizing radiation (IR) regimens. Fluorescence in situ hybridization (FISH) analyses of parental and clonally-derived tumor cells revealed different chromosome copy numbers from cell to cell, suggesting the presence of chromosomal instability in HNSCC CSC. Further, our in vitro and in vivo mouse engraftment studies suggest that CD44+/CD66− is a promising, consistent biomarker combination for HNSCC CSC. Overall, our findings add further evidence to the proposed role of HNSCC CSCs in therapeutic resistance.

A Biobank of Breast Cancer Explants with Preserved Intra-tumor Heterogeneity to Screen Anticancer Compounds

SummaryThe inter- and intra-tumor heterogeneity of breast cancer needs to be adequately captured in pre-clinical models. We have created a large collection of breast cancer patient-derived tumor xenografts (PDTXs), in which the morphological and molecular characteristics of the originating tumor are preserved through passaging in the mouse. An integrated platform combining in vivo maintenance of these PDTXs along with short-term cultures of PDTX-derived tumor cells (PDTCs) was optimized. Remarkably, the intra-tumor genomic clonal architecture present in the originating breast cancers was mostly preserved upon serial passaging in xenografts and in short-term cultured PDTCs. We assessed drug responses in PDTCs on a high-throughput platform and validated several ex vivo responses in vivo. The biobank represents a powerful resource for pre-clinical breast cancer pharmacogenomic studies (http://caldaslab.cruk.cam.ac.uk/bcape), including identification of biomarkers of response or resistance.

Multifunctional T Cell Response to DosR and Rpf Antigens Is Associated with Protection in Long-Term Mycobacterium tuberculosis-Infected Individuals in Colombia.

Multifunctional T cells have been shown to be protective in chronic viral infections. In mycobacterial infections, however, evidence for a protective role of multifunctional T cells remains inconclusive. Short-term cultures of peripheral blood mononuclear cells stimulated with the Mycobacterium tuberculosis RD1 antigens 6-kDa early secretory antigenic target (ESAT6) and 10-kDa culture filtrate antigen (CFP10), which are induced in the early infection phase, have been mainly used to assess T cell multifunctionality, although long-term culture assays have been proposed to be more sensitive than short-term assays for assessment of memory T cells, which are essential for long-term immunity. Here we used a long-term culture assay system to study the T cell immune responses to the M. tuberculosis latency-associated DosR antigens and reactivation-associated Rpf antigens, compared to ESAT6 and CFP10, in patients with pulmonary tuberculosis (PTB) and household contacts of PTB patients with long-term latent tuberculosis infection (ltLTBI), in a community in which M. tuberculosis is endemic. Our results showed that the DosR antigens Rv1737c (narK2) and Rv2029c (pfkB) and the Rv2389c (rpfD) antigen of M. tuberculosis induced higher frequencies of CD4+ or CD8+ mono- or bifunctional (but not multifunctional) T cells producing interferon gamma (IFN-γ) and/or tumor necrosis alpha (TNF-α) in ltLTBI, compared to PTB. Moreover, the frequencies of CD4+ and/or CD8+ T cells with a CD45RO+ CD27+ phenotype were higher in ltLTBI than in PTB. Thus, the immune responses to selected DosR and Rpf antigens may be associated with long-term latency, correlating with protection from M. tuberculosis reactivation in ltLTBI. Further study of the functional and memory phenotypes may contribute to further discrimination between the different states of M. tuberculosis infections.

Plasma Surface Modification of Polyhedral Oligomeric Silsequioxane-Poly(carbonate-urea) Urethane with Allylamine Enhances the Response and Osteogenic Differentiation of Adipose-Derived Stem Cells.

This study present amino functionalization of biocompatible polymer polyhedral oligomeric silsequioxane-poly(carbonate-urea) urethane (POSS-PCU) using plasma polymerization process to induce osteogenic differentiation of adipose derived stem cells (ADSCs). Optimization of plasma polymerization process was carried out keeping cell culture application in mind. Thus, samples were rigorously tested for retention of amino groups under both dry and wet conditions. Physio-chemical characterization was carried out using ninhydrin test, X-ray photon spectroscopy, scanning electron microscopy, and static water contact analysis. Results from physio chemical characterization shows that functionalization of the amino group is not stable under wet conditions and optimization of plasma process is required for stable bonding of amino groups to the POSS-PCU polymer. Optimized samples were later tested in vitro in short and long-term culture to study differentiation of ADSCs on amino modified samples. Short-term cell culture shows that initial cell attachment was significantly (p < 0.001) improved on amine modified samples (NH2-POSS-PCU) compared to unmodified POSS-PCU. NH2-POSS-PCU samples also facilitates osteogenic differentiation of ADSCs as confirmed by immunological staining of cells for extracellular markers such as collagen Type I and osteopontin. Quantification of total collagen and ALP activity also shows significant (p < 0.001) increase on NH2-POSS-PCU samples compared to unmodified POSS-PCU. A pilot study also confirms that these optimized amino modified POSS-PCU samples can further be functionalized using bone inducing peptide such as KRSR using conventional wet chemistry. This further provides an opportunity for biofunctionalization of the polymer for various tissue specific applications.

Long-term vemurafenib treatment drives inhibitor resistance through a spontaneous KRAS G12D mutation in a BRAF V600E papillary thyroid carcinoma model.

The BRAF V600E mutation is commonly observed in papillary thyroid cancer (PTC) and predominantly activates the MAPK pathway. Presence of BRAF V600E predicts increasing risk of recurrence and higher mortality rate, and treatment options for such patients are limited. Vemurafenib, a BRAF V600E inhibitor, is initially effective, but cells inevitably develop alternative mechanisms of pathway activation. Mechanisms of primary resistance have been described in short-term cultures of PTC cells; however, mechanisms of acquired resistance have not. In the present study, we investigated possible adaptive mechanisms of BRAF V600E inhibitor resistance in KTC1 thyroid cancer cells following long-term vemurafenib exposure. We found that a subpopulation of KTC1 cells acquired resistance to vemurafenib following 5 months of treatment with the inhibitor. Resistance coincided with the spontaneous acquisition of a KRAS G12D activating mutation. Increases in activated AKT, ERK1/2, and EGFR were observed in these cells. In addition, the resistant cells were less sensitive to combinations of vemurafenib and MEK1 inhibitor or AKT inhibitor. These results support the KRAS G12D mutation as a genetic mechanism of spontaneously acquired secondary BRAF inhibitor resistance in BRAF V600E thyroid cancer cells.

A novel pharmacodynamic approach to assess and predict tumor response to the epidermal growth factor receptor inhibitor gefitinib in patients with esophageal cancer.

This study aimed to describe a short-term ex vivo assay to predict response to epidermal growth factor receptor (EGFR) targeted therapy (gefitinib) in adenocarcinoma patients. Four patients with locally advanced esophageal adenocarcinoma were treated with gefitinib (250 mg/day) for 14 days and pharmacokinetic (PK) studies were conducted to monitor plasma drug concentrations. Tumor cells were sampled by endoscopic biopsy prior to (baseline, day 0) and at the completion of (day 14) treatment. Cells obtained at baseline were exposed to gefitinib in short-term cell culture conditions (ex vivo assay). Western blot analyses with phospho-specific antibodies were performed to evaluate activation and biochemical response to therapy of EGFR and its downstream signaling components ERK and AKT ex vivo and in vivo. The in vivo profiles were correlated with the gefitinib-mediated alteration in proliferating cell nuclear antigen (PCNA) expression, a marker of cell proliferation. The correlation between EGFR expression and ERK activity was also investigated by immunohistochemical analysis in pretreatment biopsies. Mutational status of the genes encoding EGFR, K-RAS, and PI3KCA (the phosphoinositide 3-kinase catalytic subunit p110) as well as expression levels of PTEN protein were tested in order to investigate potential confounders of the gefitinib effect. All patients completed the gefitinib therapy. PK studies demonstrated constant gefitinib concentrations during the treatment, confirming persistent exposure of target tissue to the drug at sufficient levels to achieve EGFR blockade. Ex vivo culture with gefitinib resulted in distinct response patterns representing various states of activity of the ERK and AKT pathways. The results of the ex vivo studies correctly predicted the pharmacodynamic (PD) effects of the agents in tumor tissue in vivo. PCNA expression correlated with ERK pathway inhibition, but not with gefitinib-mediated inhibition of EGFR activity alone. Immunohistochemical analysis performed on pretreatment biopsies correlated with Western blot analysis of EGFR and phospho-ERK expression. No mutations were identified in exons 18-21 of EGFR, exons 2 and 3 of K-RAS or exons 9 and 22 of PI3KCA. Levels of PTEN were comparable across tumors. The novel pharmacodynamic approach described in this proof of principle study may be useful to refine the patient selection to maximize the potential benefits of drugs and design individualized rational therapies for cancer patients.

Modeling Breast Cancer Intertumor and Intratumor Heterogeneity Using Xenografts.

Breast cancer is a heterogeneous disease that can be stratified in at least 10 different subtypes. We present here a platform for derivation of preclinical models based on patient-derived tumor xenografts (PDTXs) that represent these subgroups. These models preserve the transcriptome, methylome, copy-number, and mutational landscape features of the tumor of origin through different passaging. Furthermore, the intratumoral composition of these models is formed by communities of clones very similar to the ones present in the originating tumor. Finally, we show that short-term cultures of cells from these models (PDTX-derived tumor cells, PDTCs) also preserve the molecular features of the tumor and can be used for high-throughput drug testing of single compounds or combinations, with high reproducibility and clinical predictive power.

PRODUCTION OF PROINFLAMMATORY CYTOKINES AND ALPHA-2-MACROGLOBULIN BY PERIPHERAL BLOOD CELLS IN THE PATIENTS WITH COLORECTAL CANCER

Colorectal cancer (CRC) is the third most common cancer worldwide, being quite complicated, with respect to diagnostics and postoperative prognosis. Proinflammatory cytokines are shown to be involved into CRC pathogenesis. However, the changes in alpha-2-macroglobulin (α2-MG), a known regulator of cytokine production, still remain unclear. The aim of this work was to compare contents and production of a2-MG and several pro-inflammatory cytokines in blood serum and supernates from short-term blood cell cultures. The samples were taken from the patients with CRC at initial terms and after surgical removal of the tumor. Studies of cytokines and a2-MG concentrations in serum and supernates of 24-h blood cell cultures from the patients with verified CRC (stages T2-3N0-1M0 and T4N0-1M0) have shown some sufficient differences from healthy volunteers (control group). Pre-surgery IL-6 and TNFα contents in blood of CRC patients was significantly increased agains healthy controls (respectively, 29.9±5.4 and 3.4±1.5 pg/mL) versus control group (1.0±0.3 and 0 pg/mL, respectively). Following surgical treatment, the cytokine levels were decreased by 40- 60% after the operation, however, without significant differences from initial values. The supernates of blood cultures stimulated with polyclonal mitogens exhibited significant reduction of IFNγ levels prior to surgery (273±123 pg/ml versus 804±154 pg/mL), and elevated IL-6 levels (14412±2570 pg/mL versus 1970±457 pg/mL). The mean α2-MG concentrations before CRC surgery comprised 1.96±0.11 g/L for blood serum, 0.0304±0.0047 g/L, for non-stimulated blood cell cultures, and 0.0300±0.0052 g/L in mitogen-induced cultures. These parameters did not significantly differ from control values (2.21±0.17 g/L, 0.0328±0.0018 g/L, and 0.0314±0.0019 g/L, respectively). Similar results have been yielded with the samples obtained after surgical treatment of the CRC patients. The obtained data indicate that surgical treatment of CRC does not lead to normalization of the serum levels and secretion of pro-inflammatory IL-6 and TNFα cytokines by the patients’ blood cells. Marginal changes in a2-MG levels in colorectal cancer seem to be not of such importance, as functional state of the molecules which may abnormally respond to changing cytokine contents. The revealed post-surgical suppression of mitogen-induced IFNγ secretion makes it necessary to suggest administration of IFNγ and IL-2 after surgical treatment of CRC.

Primary cross-resistance to BRAFV600E-, MEK1/2- and PI3K/mTOR-specific inhibitors in BRAF-mutant melanoma cells counteracted by dual pathway blockade.

Intrinsic cross-resistance to inhibition of different signaling pathways may hamper development of combinatorial treatments in melanoma, but the relative frequency of this phenotype and the strategies to overcome this hurdle remain poorly understood. Among 49 BRAF-mutant melanoma cell lines from patients not previously treated with target therapy, 21 (42.9%) showed strong primary resistance (IC50 > 1 μM) to a BRAFV600E inhibitor. Most of the BRAF-inhibitor-resistant cell lines showed also strong or intermediate cross-resistance to MEK1/2- and to PI3K/mTOR-specific inhibitors. Primary cross-resistance was confirmed in an independent set of 23 BRAF-mutant short-term melanoma cell cultures. MEK1/2 and PI3K/mTOR co-targeting was the most effective approach, compared to BRAF and PI3K/mTOR dual blockade, to counteract primary resistance to BRAF inhibition and the cross-resistant phenotype. This was shown by extensive drug interaction analysis, tumor growth inhibition assays in-vivo, p-ERK and p-AKT inhibition, promotion of melanoma apoptosis, apoptosis-related protein modulation, activation of effector caspases and selective modulation of genes involved in melanoma drug resistance and belonging to the ERK/MAPK and PI3K/AKT canonical pathways. Compared to co-targeting of mutant BRAF and PI3K/mTOR, the association of a MEK1/2 and a PI3K/mTOR inhibitor was more effective in the activation of Bax and of caspase-3 and in the induction of caspase-dependent melanoma apoptosis. Furthermore Bax silencing reduced the latter effects. These results suggest that intrinsic resistance to BRAF inhibition is frequently associated with primary cross-resistance to MEK and PI3K/mTOR blockade in BRAF-mutant melanoma and provide pre-clinical evidence for a combinatorial approach to counteract this phenotype.

Metastasis-associated MCL1 and P16 copy number alterations dictate resistance to vemurafenib in a BRAFV600E patient-derived papillary thyroid carcinoma preclinical model.

BRAF(V600E) mutation exerts an essential oncogenic function in many tumors, including papillary thyroid carcinoma (PTC). Although BRAF(V600E) inhibitors are available, lack of response has been frequently observed. To study the mechanism underlying intrinsic resistance to the mutant BRAF(V600E) selective inhibitor vemurafenib, we established short-term primary cell cultures of human metastatic/recurrent BRAF(V600E)-PTC, intrathyroidal BRAF(V600E)-PTC, and normal thyroid (NT). We also generated an early intervention model of human BRAF(V600E)-PTC orthotopic mouse. We find that metastatic BRAF(V600E)-PTC cells elicit paracrine-signaling which trigger migration of pericytes, blood endothelial cells and lymphatic endothelial cells as compared to BRAF(WT)-PTC cells, and show a higher rate of invasion. We further show that vemurafenib therapy significantly suppresses these aberrant functions in non-metastatic BRAF(V600E)-PTC cells but lesser in metastatic BRAF(V600E)-PTC cells as compared to vehicle treatment. These results concur with similar folds of down-regulation of tumor microenvironment-associated pro-metastatic molecules, with no effects in BRAF(WT)-PTC and NT cells. Our early intervention preclinical trial shows that vemurafenib delays tumor growth in the orthotopic BRAF(WT/V600E)-PTC mice. Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss of CDKN2A (P16, chromosome 9p) in metastatic BRAF(V600E)-PTC cells which are associated with resistance to vemurafenib treatment. Critically, we demonstrate that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic BRAF(V600E)-PTC cell death and ameliorates response to vemurafenib treatment as compared to single agent treatment. In conclusion, short-term PTC and NT cultures offer a predictive model for evaluating therapeutic response in patients with PTC. Our PTC pre-clinical model suggests that combined targeted therapy might be an important therapeutic strategy for metastatic and refractory BRAF(V600E)-positive PTC.

PO71METFORMIN, PHENFORMIN AND SORAFENIB AS POTENTIAL THERAPIES FOR HIGH GRADE BRAIN TUMOURS: INVESTIGATING SUPPRESSION OF TUMOUR CELL GROWTH

INTRODUCTION: Current treatments for glioblastoma multiforme (GBM) do not yield large benefits to patients and hence the repurposing of existing medications is a potentially valuable treatment option. Metformin and phenformin have been used for >60 years for the treatment of type 2 diabetes and recent research indicates that metformin may be associated with reduced risk of developing cancer. Sorafenib is a multikinase inhibitor used in the treatment of kidney, liver and thyroid cancer. A synergistic effect of metformin and sorafenib has been identified in some tumour types. METHOD: The IC50 values for metformin and phenformin were evaluated using sulforhodamine B colorimetric assay for cytotoxicity in 13 GBM-derived short-term cell cultures and the established GBM cell line U251MG. The p53 mutation status had been determined in all cultures. In 4 cultures, the cytotoxic effects of sorafenib alone and in combination with metformin were determined using non-linear regression to fit a 4-parameter hill equation. Synergy was determined by calculating combination index (Chou-Talalay) method. RESULTS: For metformin the IC50 values ranged from 4.97(±1.36)mM to 72.23(± 3.16)mM compared to phenformin with an IC50 range of 0.41(±0.299951)mM to 7.32(±0.82)mM. For both drugs, response was associated with p53 mutation status (p = 0.0152, p = 0.0140). The IC50 range for Sorafenib was 5.5(±0.28)uM to 9.4(±0.69)uM. Combining metformin and sorafenib resulted in synergy in 3 cases and an additive effect in 1 culture. CONCLUSION: Metformin and phenformin have potential as treatment for GBM. The synergistic interaction between metformin and sorafenib indicates that these drugs in combination have a potential to be used for GBM therapy.

Hydrogel arrays formed via differential wettability patterning enable combinatorial screening of stem cell behavior

Here, we have developed a novel method for forming hydrogel arrays using surfaces patterned with differential wettability. Our method for benchtop array formation is suitable for enhanced-throughput, combinatorial screening of biochemical and biophysical cues from chemically defined cell culture substrates. We demonstrated the ability to generate these arrays without the need for liquid handling systems and screened the combinatorial effects of substrate stiffness and immobilized cell adhesion peptide concentration on human mesenchymal stem cell (hMSC) behavior during short-term 2-dimensional cell culture. Regardless of substrate stiffness, hMSC initial cell attachment, spreading, and proliferation were linearly correlated with immobilized CRGDS peptide concentration. Increasing substrate stiffness also resulted in increased hMSC initial cell attachment, spreading, and proliferation; however, examination of the combinatorial effects of CRGDS peptide concentration and substrate stiffness revealed potential interplay between these distinct substrate signals. Maximal hMSC proliferation seen on substrates with either high stiffness or high CRGDS peptide concentration suggests that some baseline level of cytoskeletal tension was required for hMSC proliferation on hydrogel substrates and that multiple substrate signals could be engineered to work in synergy to promote mechanosensing and regulate cell behavior. Statement of significanceOur novel array formation method using surfaces patterned with differential wettability offers the advantages of benchtop array formation for 2-dimensional cell cultures and enhanced-throughput screening without the need for liquid handling systems. Hydrogel arrays formed via our method are suitable for screening the influence of chemical (e.g. cell adhesive ligands) and physical (stiffness, size, shape, and thickness) substrate properties on stem cell behavior. The arrays are also fully compatible with commercially available micro-array add-on systems, which allows for simultaneous control of the insoluble and soluble cell culture environment. This study used hydrogel arrays to demonstrate that synergy between cell adhesion and mechanosensing can be used to regulate hMSC behavior.

ID: 239: A functional FAS polymorphism links CD4 T stem cell memory levels and IFN-induced apoptosis in both healthy controls and Adult T-cell Leukemia patients

Introduction: Adult T-cell leukemia (ATL) is an aggressive, chemotherapy-resistant CD4+ CD25+ leukemia caused by HTLV-1 infection, which usually develops in a minority of patients several decades after infection. Recently, CD4 T stem cell memory (Tscm) cells have been identified as the hierarchical cellular apex of ATL. IFN- α , in combination with AZT, is currently the major treatment option for ATL, but therapeutic success is limited and no bona fide biomarkers for therapeutic failure are currently available. Results: In a prospective study with a five-year clinical follow-up, we found significant ex vivo antiproliferative, antiviral and immunomodulatory effects of IFN- α treatment in short-term culture of primary mononuclear cells from ATL patients (n = 28). We detected a genotype/phenotype interaction for the −670 FAS A/G promoter polymorphism, apoptosis and proliferation in ATL patients. Surprisingly, IFN-induced apoptosis occurred only in carriers of the functional A allele (corresponding to a functional STAT1 binding site), which we experimentally validated in an independent cohort of healthy controls (n = 20). This Fashigh proliferating and chemotherapy-resistant leukemic phenotype is in agreement with the recently discovered CD4 Tscm hierarchical apex of ATL. The same FAS −670 polymorphism also determined CD4 Tscm levels in a genome-wide twin study (p = 7 × 10−11, n = 669), confirming our hypothesis. Finally, we provide in vivo evidence that CD4+ Fashigh leukemic cells can be eliminated by IFN- α +AZT combination therapy, but not AZT monotherapy. Conclusion: A genetically determined IFN/STAT1/FAS axis underscores the CD4 Tscm ATL model and helps explain the observed in vivo IFN + AZT responsiveness and chemotherapy resistance in ATL patients.

Abstract 2422: Pan-pim kinases inhibitor, AZD1208 suppresses tumor growth and synergistically interacts with an AKT inhibitor in gastric cancer cells

Abstract Introduction: Pim kinases are highly conserved serine/threonine kinases that control proliferation and survival pathways in cancer cells. Different expression patterns of each isoforms (Pim-1, Pim-2, and Pim-3) have been observed in various types of human cancer including gastric cancer. AZD1208 is a potent and selective inhibitor that affects all three isoforms of Pim. We investigated the effect of AZD1208 as a single agent or in combination with an Akt inhibitor on the growth and cell death in gastric cancer cells. Materials and Methods: The antitumor activity of AZD1208 with/without an Akt inhibitor was evaluated in a large panel of gastric cancer cell lines through growth inhibition assay. The underlying mechanism was also examined by western blotting, immunofluorescence assay (IFA), and cell cycle analysis. Results: Direct substrates of Pim kinases were downregulated by the treatment with AZD1208, but we could not find any reduction of cell viability in short-term cell cultures. In contrast, cell viability decreased significantly in long-term cultures. Light chain 3B (LC3B), a marker for autophagy, increased in sensitive cells in dose dependent manner with AZD1208 treatment, which suggest that the growth inhibition effect of AZD1208 was achieved through autophagy, but not through apoptosis. Moreover, we found that ATM and Chk2, two critical components of the DNA repair pathway, were activated in an AZD1208-resistant cell line. We also confirmed that the combination of an Akt inhibitor with AZD1208 produced a highly synergistic effect in gastric cancer cell lines. Conclusion: AZD1208 induced cell death through autophagy in gastric cancer cells. Additionally, the combination of AZD1208 with an Akt inhibitor shows synergistic antitumor effects. Citation Format: Miso Lee, Kyung-Hun Lee, Ahrum Min, Jungeun Kim, Seock-Ah Im, Seon-Gyeong Kim, Hyemin Jang, Yae-Won Yang, Tae-Yong Kim, Sae-Won Han, Do-Youn Oh, Tae-You Kim, Yung-Jue Bang. Pan-pim kinases inhibitor, AZD1208 suppresses tumor growth and synergistically interacts with an AKT inhibitor in gastric cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2422. doi:10.1158/1538-7445.AM2015-2422

Abstract 4845: Microarray analysis of melanoma autologous tumor cell lines used as the source of tumor associated antigens in patient-specific dendritic cell immunotherapy phase II trial in patients with metastatic melanoma

Abstract Melanoma cells that are proliferating and self-renewing in short-term cell culture, have the phenotypic and functional characteristics of tumor stem cells, and they express unique patient-specific neoantigens, as well as numerous common melanoma associated antigens. Patient-specific therapeutic vaccines, produced by Incubating autologous dendritic cells with autologous tumor cells from such cell lines (DC-TC), have yielded promising results in phase II trials in metastatic melanoma. In this study we examined the expression of genes on the tumor cell lines which had served as the sources of tumor associated antigens loaded onto autologous dendritic cells as part of a phase II trial in which there was an observed 5-year survival rate of 50%.[Dillman 2009] There was sufficient melanoma tumor cells to analyze for 50 of the 54 patients enrolled in the phase II trial. Microarray analysis was performed by Response Genetics, Inc. (Los Angeles) for 54,000 genes. The Excel data file was entered into an Affymetrix data processing algorithm using the R programming language. Gene ranking was performed using prediction analysis of microarrays.[Tibshirani 2002] Correlation with overall survival was done for 26 patients who survived 5 years or longer, and 24 patients who survived less than 5 years. Hierarchical clustering for over and under expression yielded 9 genes, VEPH1, ZNF280B, FGF13, ST6GALNAC3, QPCT, ZNF280B, BST, PBRM1 and C21orf91 which identified 11/11 patients who had a survival greater than 5 years, and 17/18 patients who died in less than 2 years. Using the top six genes, overall survival greater than 5 years was correctly predicted for 80.3% of patients, and survival less than 5 years was correctly predicted for 74.2% of patients. Gene array data obtained from self-renewing, proliferating, autologous tumor cells was prognostic for survival. It is unclear whether these genes are predictive of an effective immune response after repeated injections of a therapeutic dendritic cell-tumor cell vaccine. Citation Format: Andrew N. Cornforth, Gary Fogel, Denysha Carbonell, Robert O. Dillman. Microarray analysis of melanoma autologous tumor cell lines used as the source of tumor associated antigens in patient-specific dendritic cell immunotherapy phase II trial in patients with metastatic melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4845. doi:10.1158/1538-7445.AM2015-4845

ANTIOXIDANT EFFICIENCY OF RESVERATROL ON OXIDATIVE STRESS-INDUCED DAMAGE IN BOVINE SPERMATOZOA

Substantial reports suggest that biologically active compounds isolated from natural sources may offer protection to semen against the incidence of free radical producers, improvement of semen quality, prevention of cryodamage and DNA fragmentation, as well as a general promotion of spermatozoa metabolism. This study was therefore designed to assess the impact of resveratrol (RES), a natural polyphenol and phytoalexin with potential antiviral, anti-inflammatory and antioxidant properties on bovine spermatozoa subjected to oxidative stress induced by ferrous ascorbate during a short-term cell culture (6h). Fresh bovine semen samples were centrifuged, the seminal plasma was removed, the sperm pellet was washed out and the resulting cells were diluted in 2.90% sodium citrate, divided into equal fractions and subjected to treatment based on a specific concentration of RES (50, 25, 10, 5, 0 μmol/L) in the presence of an oxidative stress inducer, i.e. ferrous ascorbate (FeAA) containing 150 μmol/L FeSO4 and 750 μmol/L ascorbic acid. After 6h, the spermatozoa motility (using the Computer-aided sperm analysis/CASA system) and superoxide production (via the NBT test) were assessed in each sample. Furthermore, the sperm cells were collected, lysed and examined for the activity of superoxide dismutase and catalase, as well as glutathione and malondialdehyde concentrations. The addition of RES into the culture medium was able to significantly (P

ZGDHu-1 and fludarabine have a synergistic effect on apoptosis of chronic lymphocytic leukemia cells.

Previously, it was demonstrated that the novel proteasome inhibitor N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu‑1) possesses activity against chronic lymphocytic leukemia (CLL). In the present study, we attempted to assess whether this drug has a synergistic effect with fludarabine on the apoptosis of CLL cells. AnnexinV/PI staining, mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) levels were examined by flow cytometry in short-term cell culture of blood cells from untreated newly diagnosed patients exvivo. Expression of active caspase-3 and the Bcl-2/Bax ratio for determination of apoptosis were also investigated by flow cytometry and western blot analysis. Our results revealed that the ZGDHu-1 may induce the apoptosis of CLL cells through the mitochondrial pathway and its pro-apoptotic effect is CLL-specific, not affecting normal lymphocytes. Most importantly, a combination of ZGDHu-1 and a non-cytotoxic dose of fludarabine had a synergistic apoptotic effect. To some extent, caspase-3 activation may be involved in the mechanism of the ZGDHu-1 synergistic cytotoxic effect with fludarabine, as well as the cleavage of PARP, consequently leading to apoptosis. Notably, the rate of apoptosis caused by ZGDHu-1 alone or in combination with fludarabine was independent of prognostic markers of CLL disease such as ZAP-70 and CD38 expression or clinical Rai classification stage. In conclusion, ZGDHu-1 exhibited a significant synergistic effect with fludarabine to induce the apoptosis of CLL cells, which implies a possible clinical application.

Relaxin affects cell organization and early and late stages of spermatogenesis in a coculture of rat testicular cells.

Relaxin and its receptor RXFP1 are co-expressed in Sertoli cells, and relaxin can stimulate proliferation of Sertoli cells. In this study, we investigated a role of relaxin in spermatogenesis, using a short-term culture of testicular cells of the rat that allowed differentiation of spermatogonia to spermatids. Sertoli, germ, and peritubular myoid cells were the predominant cell types in the culture. Sertoli and germ cells expressed RXFP1. Cultures were incubated without (control) or with 0.5% fetal bovine serum (FBS) or 100ng/mL H2 relaxin (RLN) for 2days. Cell organization, number, and differentiation were analyzed after 2 (D2), 5 (D5) or 8 (D8) days of culturing. Although the proportion of germ cells decayed from D2 to D5, the relative contribution of HC, 1C, 2C, and 4C germ cell populations remained constant in the control group during the whole culture. RLN did not affect the proportion of germ cell populations compared with control, but increased gene and/or protein expression of the undifferentiated and differentiated spermatogonia markers PLZF and c-KIT, and of the post-meiotic marker Odf2 in D5. RLN favored organization of cells in tubule-like structures, the arrangement of myoid cells around the tubules, arrangement of c-KIT-positive spermatogonia at the basal region of the tubules, and expression of the cell junction protein β-catenin close to the plasma membrane region. Knockdown of relaxin with small interfering RNA (siRNA) reduced expression of β-catenin at the cell junctions, and shifted its expression to the nucleus. We propose that relaxin may affect spermatogenesis by modulating spermatogonial self renewal and favoring cell contact.

Atraumatic Pulsatile Leukocyte Circulation for Long-Term In Vitro Dynamic Culture and Adhesion Assays.

Low flow rate pumping of cell suspensions finds current applications in bioreactors for short-term dynamic cell culture and adhesion assays. The aim of this study was to develop an atraumatic pump and hemodynamically adapted test circuit to allow operating periods of at least several hours. A computer-controlled mini-pump (MP) was constructed based on non-occlusive local compression of an elastic tube with commercial bi-leaflet valves directing the pulsatile flow into a compliant circuit. Cell damage and activation in the system were tested with whole blood in comparison with a set with a conventional peristaltic pump (PP). Activation of circulating THP-1 monocytes was tested by measuring the expression of CD54 (ICAM-1). Additionally, monocyte-endothelial interactions were monitored using a parallel-plate flow chamber with an artificial stenosis. The system required a priming volume of only 20 mL, delivering a peak pulsatile flow of up to 35 mL/min. After 8 h, blood hemolysis was significantly lower for MP with 11 ± 3 mg/dL compared with PP with 100 ± 16 mg/dL. CD142 (tissue factor) expression on blood monocytes was 50% lower for MP. With MP, THP-1 cells could be pumped for extended periods (17 h), with no enhanced expression of CD54 permitting the long-term co-culture of THP-1 with endothelial cells and the analysis of flow pattern effects on cell adhesion. A low-damage assay setup was developed, which allows the pulsatile flow of THP-1 cells and investigation of their interaction with other cells or surfaces for extended periods of time.

Sensitivity of endometrial cancer cells from primary human tumor samples to new potential anticancer peptide lactaptin.

Endometrial carcinoma is the most common gynecologic malignancy which is associated with a poor prognosis when diagnosed at an advanced stage; therefore, the discovery of efficacious new drugs is required to reinforce conventional chemotherapy. Short-term cultures of primary cells from endometrial tumors could be used for testing new anticancer therapeutics as well as for the development of personalized cancer therapy strategy. Here, the antitumor effect of a recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, was examined against primary human endometrial cancer cells. Primary cell cultures of malignant and normal human endometrium were performed by enzymatic digestion of endometrial tissue from biopsy material. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the messenger ribonucleic acid (mRNA) state of estrogen (ERs) and progesterone (PRs) hormone receptors and aromatase (Cyp 19) in cell cultures. Dynamic monitoring of cell adhesion and proliferation was made using the iCELLigence system (ASEA Biosciences). The sensitivity of cell cultures to conventional anticancer drugs and the lactaptin analog was estimated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, flow cytometry, and the iCELLligence system. Established short-term primary cultures of endometrial cancer cells were ERα/ERβ/PR-positive and sensitive for RL2. The IC 50 values of doxorubicin and cisplatin were determined for all of the primary cultures designed. KE normal cells displaying low Cyp19 mRNA levels and high ERβ and PR mRNA levels were more resistant to RL2 treatment as well as to cisplatin and doxorubicin. Our results indicate that the recombinant analog of lactaptin, RL2, exerts cytotoxic effects against primary hormone-dependent endometrial tumor cells in vitro with features of apoptosis.