Browsing by Subject "CANCER-CELLS"

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  • Zhou, Kecheng; Dichlberger, Andrea; Martinez-Seara, Hector; Nyholm, Thomas K. M.; Li, Shiqian; Kim, Young Ah; Vattulainen, Ilpo; Ikonen, Elina; Blom, Tomas (2018)
    Membrane proteins are functionally regulated by the composition of the surrounding lipid bilayer. The late endosomal compartment is a central site for the generation of ceramide, a bioactive sphingolipid, which regulates responses to cell stress. The molecular interactions between ceramide and late endosomal transmembrane proteins are unknown. Here, we uncover in atomistic detail the ceramide interaction of Lysosome Associated Protein Transmembrane 4B (LAPTM4B), implicated in ceramide-dependent cell death and autophagy, and its functional relevance in lysosomal nutrient signaling. The ceramide-mediated regulation of LAPTM4B depends on a sphingolipid interaction motif and an adjacent aspartate residue in the protein's third transmembrane (TM3) helix. The interaction motif provides the preferred contact points for ceramide while the neighboring membrane-embedded acidic residue confers flexibility that is subject to ceramide-induced conformational changes, reducing TM3 bending. This facilitates the interaction between LAPTM4B and the amino acid transporter heavy chain 4F2hc, thereby controlling mTORC signaling. These findings provide mechanistic insights into how transmembrane proteins sense and respond to ceramide.
  • Clark, Christine; Palta, Priit; Joyce, Christopher J.; Scott, Carol; Grundberg, Elin; Deloukas, Panos; Palotie, Aarno; Coffey, Alison J. (2012)
  • Salo, Tuula; Sutinen, Meeri; Apu, Ehsanul Hoque; Sundquist, Elias; Cervigne, Nilva K.; de Oliveira, Carine Ervolino; Akram, Saad Ullah; Ohlmeier, Steffen; Suomi, Fumi; Eklund, Lauri; Juusela, Pirjo; Astrom, Pirjo; Bitu, Carolina Cavalcante; Santala, Markku; Savolainen, Kalle; Korvala, Johanna; Paes Leme, Adriana Franco; Coletta, Ricardo D. (2015)
    Background: The composition of the matrix molecules is important in in vitro cell culture experiments of e.g. human cancer invasion and vessel formation. Currently, the mouse Engelbreth-Holm-Swarm (EHS) sarcoma -derived products, such as Matrigel (R), are the most commonly used tumor microenvironment (TME) mimicking matrices for experimental studies. However, since Matrigel (R) is non-human in origin, its molecular composition does not accurately simulate human TME. We have previously described a solid 3D organotypic myoma disc invasion assay, which is derived from human uterus benign leiomyoma tumor. Here, we describe the preparation and analyses of a processed, gelatinous leiomyoma matrix, named Myogel. Methods: A total protein extract, Myogel, was formulated from myoma. The protein contents of Myogel were characterized and its composition and properties compared with a commercial mouse Matrigel (R). Myogel was tested and compared to Matrigel (R) in human cell adhesion, migration, invasion, colony formation, spheroid culture and vessel formation experiments, as well as in a 3D hanging drop video image analysis. Results: We demonstrated that only 34 % of Myogel's molecular content was similar to Matrigel (R). All test results showed that Myogel was comparable with Matrigel (R), and when mixed with low-melting agarose (Myogel-LMA) it was superior to Matrigel (R) in in vitro Transwell (R) invasion and capillary formation assays. Conclusions: In conclusion, we have developed a novel Myogel TME matrix, which is recommended for in vitro human cell culture experiments since it closely mimics the human tumor microenvironment of solid cancers.
  • Kohonen, Pekka; Parkkinen, Juuso A.; Willighagen, Egon L.; Ceder, Rebecca; Wennerberg, Krister; Kaski, Samuel; Grafstrom, Roland C. (2017)
    Predicting unanticipated harmful effects of chemicals and drug molecules is a difficult and costly task. Here we utilize a 'big data compacting and data fusion'-concept to capture diverse adverse outcomes on cellular and organismal levels. The approach generates from transcriptomics data set a 'predictive toxicogenomics space' (PTGS) tool composed of 1,331 genes distributed over 14 overlapping cytotoxicity-related gene space components. Involving similar to 2.5 x 10(8) data points and 1,300 compounds to construct and validate the PTGS, the tool serves to: explain dose-dependent cytotoxicity effects, provide a virtual cytotoxicity probability estimate intrinsic to omics data, predict chemically-induced pathological states in liver resulting from repeated dosing of rats, and furthermore, predict human drug-induced liver injury (DILI) from hepatocyte experiments. Analysing 68 DILI-annotated drugs, the PTGS tool outperforms and complements existing tests, leading to a hereto-unseen level of DILI prediction accuracy.
  • Tervaniemi, Mari H.; Siitonen, H. Annika; Soderhall, Cilla; Minhas, Gurinder; Vuola, Jyrki; Tiala, Erica Inkeri; Sormunen, Raija; Samuelsson, Lena; Suomela, Sari; Kere, Juha; Elomaa, Outi (2012)
  • Saare, Merli; Rekker, Kadri; Laisk-Podar, Triin; Rahmioglu, Nilufer; Zondervan, Krina; Salumets, Andres; Götte, Martin; Peters, Maire (2017)
    In order to uncover miRNA changes in endometriosis pathogenesis, both endometriotic lesions and endometrial biopsies, as well as stromal and epithelial cells isolated from these tissues have been investigated and a large number of dysregulated miRNAs have been reported. However, the concordance between the result of different studies has remained small. One potential explanation for limited overlap between the proposed disease-related miRNAs could be the heterogeneity in tissue composition, as some studies have compared highly heterogeneous whole-lesion biopsies with endometrial tissue, some have compared the endometrium from patients and controls, and some have used pure cell fractions isolated from lesions and endometrium. This review focuses on the results of published miRNA studies in endometriosis to reveal the potential impact of tissue heterogeneity on the discovery of disease-specific miRNA alterations in endometriosis. Additionally, functional studies that explore the roles of endometriosis-involved miRNAs are discussed.
  • Lavoginal, Darja; Samuel, Kulli; Lavrits, Arina; Meltsovl, Alvin; Soritsa, Deniss; Kadastik, Ulle; Peters, Maire; Rinken, Ago; Salumets, Andres (2019)
    Research question: Endometriosis is a common gynaecological disease defined by the presence of endometrium-like tissue outside the uterus. This complex disease, often accompanied by severe pain and infertility, causes a significant medical and socioeconomic burden; hence, novel strategies are being sought for the treatment of endometriosis. Here, we set out to explore the cytotoxic effects of a panel of compounds to find toxins with different efficiency in eutopic versus ectopic cells, thus highlighting alterations in the corresponding molecular pathways. Design: The effect on cellular viability of 14 compounds was established in a cohort of paired eutopic and ectopic endometrial stromal cell samples from 11 patients. The biological targets covered by the panel included pro-survival enzymes, cytoskeleton proteins, the proteasome and the cell repair machinery. Results: Protein kinase inhibitors GSK690693, ARC-775 and sorafenib, proteasome inhibitor bortezomib, and microtubuledepolymerizing toxin monomethyl auristatin E were more effective in eutopic cells. In contrast, 10 mu mol/l of the anthracycline toxin doxorubicin caused cellular death in ectopic cells more effectively than in eutopic cells. The large-scale sequencing of mRNA isolated from doxorubicin-treated and control cells indicated different survival strategies in eutopic versus ectopic endometrium. Conclusions: Overall, the results confirm evidence of large-scale metabolic reprogramming in endometriotic cells, which underlies the observed differences in sensitivity towards toxins. The enhanced efficiency of doxorubicin interfering with redox equilibria and/or DNA repair mechanisms pinpoints key players that can be potentially used to selectively target ectopic lesions in endometriosis.
  • Galluzzi, Lorenzo; Vitale, Ilio; Warren, Sarah; Adjemian, Sandy; Agostinis, Patrizia; Martinez, Aitziber Buqué; Chan, Timothy A; Coukos, George; Demaria, Sandra; Deutsch, Eric; Draganov, Dobrin; Edelson, Richard L; Formenti, Silvia C; Fucikova, Jitka; Gabriele, Lucia; Gaipl, Udo S; Gameiro, Sofia R; Garg, Abhishek D; Golden, Encouse; Han, Jian; Harrington, Kevin J; Hemminki, Akseli; Hodge, James W; Hossain, Dewan Md Sakib; Illidge, Tim; Karin, Michael; Kaufman, Howard L; Kepp, Oliver; Kroemer, Guido; Lasarte, Juan Jose; Loi, Sherene; Lotze, Michael T; Manic, Gwenola; Merghoub, Taha; Melcher, Alan A; Mossman, Karen L; Prosper, Felipe; Rekdal, Øystein; Rescigno, Maria; Riganti, Chiara; Sistigu, Antonella; Smyth, Mark J; Spisek, Radek; Stagg, John; Strauss, Bryan E; Tang, Daolin; Tatsuno, Kazuki; van Gool, Stefaan W; Vandenabeele, Peter; Yamazaki, Takahiro; Zamarin, Dmitriy; Zitvogel, Laurence; Cesano, Alessandra; Marincola, Francesco M (2020)
    Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation.
  • Al-Samadi, Ahmed; Awad, Shady Adnan; Tuomainen, Katja; Zhao, Yue; Salem, Abdelhakim; Parikka, Mataleena; Salo, Tuula (2017)
    The crosstalk between immune cells, cancer cells, and extracellular vesicles (EVs) secreted by cancer cells remains poorly understood. We created three-dimensional (3D) cell culture models using human leiomyoma discs and Myogel to study the effects of immune cells on highly (HSC-3) and less (SCC-25) invasive oral tongue squamous cell carcinoma (OTSCC) cell lines. Additionally, we studied the effects of EVs isolated from these cell lines on the cytotoxicity of CD8(+) T and NK cells isolated from three healthy donors. Our analysis included the effects of these EVs on innate immunity in zebrafish larvae. Activated immune cells significantly decreased the proliferation of both OTSCC cell lines and associated with a diminished invasion area of HSC-3 cells. In general, EVs from SCC-25 increased the cytotoxic activity of CD8(+) T and NK cells more than those from HSC-3 cells. However, this effect varied depending on the source and the immune and cancer cell subgroups. In zebrafish, the amount of IL-13 mRNA was decreased by SCC-25 EVs. This study describes promising in vitro and in vivo models to investigate interactions between immune cells, cancer cells, and EVs.
  • Tykesson, Emil; Hassinen, Antti; Zielinska, Katarzyna; Thelin, Martin A.; Frati, Giacomo; Ellervik, Ulf; Westergren-Thorsson, Gunilla; Malmström, Anders; Kellokumpu, Sakari; Maccarana, Marco (2018)
    During the biosynthesis of chondroitin/dermatan sulfate (CS/DS), a variable fraction of glucuronic acid is converted to iduronic acid through the activities of two epimerases, dermatan sulfate epimerases 1 (DS-epi1) and 2 (DS-epi2). Previous in vitro studies indicated that without association with other enzymes, DS-epi1 activity produces structures that have only a few adjacent iduronic acid units. In vivo, concomitant with epimerization, dermatan 4-O-sulfotransferase 1 (D4ST1) sulfates the GalNAc adjacent to iduronic acid. This sulfation facilitates DS-epi1 activity and enables the formation of long blocks of sulfated iduronic acid-containing domains, which can be major components of CS/DS. In this report, we used recombinant enzymes to confirm the concerted action of DS-epi1 and D4ST1. Confocal microscopy revealed that these two enzymes colocalize to the Golgi, and FRET experiments indicated that they physically interact. Furthermore, FRET, immunoprecipitation, and cross-linking experiments also revealed that DS-epi1, DS-epi2, and D4ST1 form homomers and are all part of a hetero-oligomeric complex where D4ST1 directly interacts with DS-epi1, but not with DS-epi2. The cooperation of DS-epi1 with D4ST1 may therefore explain the processive mode of the formation of iduronic acid blocks. In conclusion, the iduronic acid-forming enzymes operate in complexes, similar to other enzymes active in glycosaminoglycan biosynthesis. This knowledge shed light on regulatory mechanisms controlling the biosynthesis of the structurally diverse CS/DS molecule.
  • Kylmä, Anna Kaisa; Tolvanen, Tuomas Aleksi; Carpén, Timo; Haglund, Caj; Mäkitie, Antti; Mattila, Petri S.; Grenman, Reidar; Jouhi, Lauri; Sorsa, Timo; Lehtonen, Sanna; Hagström, Jaana (2020)
    In oropharyngeal squamous cell carcinoma (OPSCC), the expression pattern of toll-like receptors (TLRs), in comparison between human papillomavirus (HPV)-positive and -negative tumors differs. TLRs control innate immune responses by activating, among others, the nuclear factor-κΒ (NF-κΒ) signaling pathway. Elevated NF-κΒ activity is detectable in several cancers and regulates cancer development and progression. We studied TLR5 expression in 143 unselected consecutive OPSCC tumors, and its relation to HPV-DNA and p16 status, clinicopathological parameters, and patient outcome, and studied TLR5 stimulation and consecutive NF-κB cascade activation in vitro in two human OPSCC cell lines and immortalized human keratinocytes (HaCat). Clinicopathological data came from hospital registries, and TLR5 immunoexpression was evaluated by immunohistochemistry. Flagellin served to stimulate TLR5 in cultured cells, followed by analysis of the activity of the NF-κB signaling cascade with In-Cell Western for IκΒ and p-IκΒ. High TLR5 expression was associated with poor disease-specific survival in HPV-positive OPSCC, which typically shows low TLR5 immunoexpression. High TLR5 immunoexpression was more common in HPV-negative OPSCC, known for its less-favorable prognosis. In vitro, we detected NF-κΒ cascade activation in the HPV-positive OPSCC cell line and in HaCat cells, but not in the HPV-negative OPSCC cell line. Our results suggest that elevated TLR5 immunoexpression may be related to reduced NF-κΒ activity in HPV-negative OPSCC. The possible prognosis-worsening mechanisms among these high-risk OPSCC patients however, require further evaluation.
  • Aho, Joonas; Helenius, Mikko; Vattulainen-Collanus, Sanna; Alastalo, Tero-Pekka; Koskenvuo, Juha (2016)
    Cell damage can lead to rapid release of ATP to extracellular space resulting in dramatic change in local ATP concentration. Evolutionary, this has been considered as a danger signal leading to adaptive responses in adjacent cells. Our aim was to demonstrate that elevated extracellular ATP or inhibition of ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39) activity could be used to increase tolerance against DNA-damaging conditions. Human endothelial cells, with increased extracellular ATP concentration in cell proximity, were more resistant to irradiation or chemically induced DNA damage evaluated with the DNA damage markers gamma H2AX and phosphorylated p53. In our rat models of DNA damage, inhibiting CD39-driven ATP hydrolysis with POM-1 protected the heart and lung tissues against chemically induced DNA damage. Interestingly, the phenomenon could not be replicated in cancer cells. Our results show that transient increase in extracellular ATP can promote resistance to DNA damage.
  • Rodrigues, Priscila Campioni; Sawazaki-Calone, Iris; de Oliveira, Carine Ervolino; Soares Macedo, Carolina Carneiro; Dourado, Mauricio Rocha; Cervigne, Nilva K.; Miguel, Marcia Costa; do Carmo, Andreia Ferreira; Lambert, Daniel W.; Graner, Edgard; da Silva, Sabrina Daniela; Alaoui-Jamali, Moulay A.; Paes Leme, Adriana Franco; Salo, Tuula A.; Coletta, Ricardo D. (2017)
    Oral squamous cell carcinoma (OSCC) prognosis is related to clinical stage and histological grade. However, this stratification needs to be refined. We conducted a comparative proteome study in microdissected samples from normal oral mucosa and OSCC to identify biomarkers for malignancy. Fascin and plectin were identified as differently expressed and both are implicated in several malignancies, but the clinical impacts of aberrant fascin and plectin expression in OSCCs remains largely unknown. Immunohistochemistry and real-time quantitative PCR were carried out in ex vivo OSCC samples and cell lines. A loss-of-function strategy using shRNA targeting fascin was employed to investigate in vitro and in vivo the fascin role on oral tumorigenesis. Transfections of microRNA mimics were performed to determine whether the fascin overexpression is regulated by miR-138 and miR-145. We found that fascin and plectin are frequently upregulated in OSCC samples and cell lines, but only fascin overexpression is an independent unfavorable prognostic indicator of disease-specific survival. In combination with advanced T stage, high fascin level is also an independent factor of disease-free survival. Knockdown of fascin in OSCC cells promoted cell adhesion and inhibited migration, invasion and EMT, and forced expression of miR-138 in OSCC cells significantly decreased the expression of fascin. In addition, fascin downregulation leads to reduced filopodia formation and decrease on paxillin expression. The subcutaneous xenograft model showed that tumors formed in the presence of low levels of fascin were significantly smaller compared to those formed with high fascin levels. Collectively, our findings suggest that fascin expression correlates with disease progression and may serve as a prognostic marker and therapeutic target for patients with OSCC.
  • Vilen, Suvi-Tuuli; Salo, Tuula; Sorsa, Timo; Nyberg, Pia (2013)
  • Katayama, Shintaro; Skoog, Tiina; Jouhilahti, Eeva-Mari; Siitonen, H. Annika; Nuutila, Kristo; Tervaniemi, Mari H.; Vuola, Jyrki; Johnsson, Anna; Lonnerberg, Peter; Linnarsson, Sten; Elomaa, Outi; Kankuri, Esko; Kere, Juha (2015)
    Background: Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact skin has been questioned. Moreover, when analyzing KC transcriptomes, fluctuation of polyA+ RNA content during the KCs' lifecycle has been omitted. Results: We performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types. Conclusions: The epidermal keratinocytes derived from tissue and cell culture samples showed highly different polyA+ RNA contents. The use of SAMstrt and synthetic RNA based normalization allowed the comparison between tissue and cell culture samples and thus proved to be valuable tools for RNA-seq analysis with translational approach. Transciptomics revealed clear difference both between tissue and cell culture samples and between primary KCs and immortalized HaCaT cells.
  • Ravikumar, Balaguru; Aittokallio, Tero (2018)
    Introduction: Polypharmacology has emerged as an essential paradigm for modern drug discovery process. Multiple lines of evidence suggest that agents capable of modulating multiple targets in a selective manner may offer also improved balance between therapeutic efficacy and safety compared to single-targeted agents. Areas covered: Herein, the authors review the recent progress made in experimental and computational strategies for addressing the critical challenges with rational discovery of selective multi-targeted agents within the context of polypharmacological modelling. Specific focus is placed on multi-targeted mono-therapies, although examples of combinatorial polytherapies are also covered as an important part of the polypharmacology paradigm. The authors focus mainly on anti-cancer treatment applications, where polypharmacology is playing a key role in determining the efficacy-toxicity trade-off of multi-targeting strategies. Expert opinion: Even though it is widely appreciated that complex polypharmacological interactions can contribute both to therapeutic and adverse side-effects, systematic approaches for improving this balance by means of integrated experimental-computational strategies are still lacking. Future developments will be needed for comprehensive collection and harmonization of systems-wide target selectivity data, enabling better utilization and control for multi-targeted activities in the drug development process. Additional areas of future developments include model-based strategies for drug combination screening and improved pre-clinical validation options with animal models.
  • Dickinson, Amy; Saraswat, Mayank; Joenväärä, Sakari; Agarwal, Rahul; Jyllikoski, Daniel; Wilkman, Tommy; Mäkitie, Antti; Silen, Suvi (2020)
    Lipid metabolic reprogramming is one hallmark of cancer. Lipid metabolism is regulated by numerous enzymes, many of which are targeted by several drugs on the market. We aimed to characterize the lipid alterations in oral squamous cell carcinoma (OSCC) as a basis for understanding its lipid metabolism, thus identifying potential therapeutic targets. We compared lipid species, classes, and glycerophospholipid (GPL) fatty acid species between paired tumor tissue and healthy oral tongue mucosa samples from 10 OSCC patients using a QExactive mass spectrometer. After filtering the 1370 lipid species identified, we analyzed 349 species: 71 were significantly increased in OSCC. The GPL metabolism pathway was most represented by the lipids differing in OSCC (P = .005). Cholesterol and the GPLs phosphatidylcholines, phosphatidylethanolamines, and phosphatidylinositols were most significantly increased in OSCC tissue (FC 1.8, 2.0, 2.1, and 2.3 and, P = .003, P = .005, P = .002, P = .007). In conclusion, we have demonstrated a shift in the lipid metabolism in these OSCC samples by characterizing the detailed landscape. Predominantly, cholesterol and GPL metabolism were altered, suggesting that interactions with sterol regulatory binding proteins may be involved. The FA composition changes of the GPLs suggest increased de novo lipogenesis.
  • Eloranta, Katja; Nousiainen, Ruth; Cairo, Stefano; Pakarinen, Mikko P.; Wilson, David B.; Pihlajoki, Marjut; Heikinheimo, Markku (2021)
    The neuropilins NRP1 and NRP2 are multifunctional glycoproteins that have been implicated in several cancer-related processes including cell survival, migration, and invasion in various tumor types. Here, we examine the role of neuropilins in hepatoblastoma (HB), the most common pediatric liver malignancy. Using a combination of immunohistochemistry, RNA analysis and western blotting, we observed high level expression of NRP1 and NRP2 in 19 of 20 HB specimens and in a majority of human HB cell lines (HUH6 and five cell lines established from patient-derived xenografts) studied but not in normal hepatocytes. Silencing of NRP2 expression in HUH6 and HB-282 HB cells resulted in decreased cell viability, impaired cytoskeleton remodeling, and reduced cell motility, suggesting that NRP2 contributes to the malignant phenotype. We propose that neuropilins warrant further investigation as biomarkers of HB and potential therapeutic targets.
  • Santos, João Manuel; Heiniö, Camilla; Cervera-Carrascon, Victor; Quixabeira, Dafne C A; Siurala, Mikko; Havunen, Riikka; Butzow, Ralf; Zafar, Sadia; de Gruijl, Tanja; Lassus, Heini; Kanerva, Anna; Hemminki, Akseli (2020)
    BACKGROUND: Ovarian cancers often contain significant numbers of tumor-infiltrating lymphocytes (TILs) that can be readily harnessed for adoptive T-cell therapy (ACT). However, the immunosuppressive ovarian tumor microenvironment and lack of tumor reactivity in TILs can limit the effectiveness of the therapy. We hypothesized that by using an oncolytic adenovirus (Ad5/3-E2F-D24-hTNFa-IRES-hIL2; TILT-123) to deliver tumor necrosis factor alpha (TNFa) and interleukin-2 (IL-2), we could counteract immunosuppression, and enhance antitumor TIL responses in ovarian cancer (OVCA). METHODS: We established ex vivo tumor cultures freshly derived from patients with advanced OVCA and evaluated the effects of Ad5/3-E2F-D24-hTNFa-IRES-hIL2 or Ad5/3-E2F-D24 (the control virus without TNFa and IL-2) on TILs, cytokine response and tumor viability. Tumor reactivity was assessed by determining interferon gamma (IFNg) response of clinically relevant TILs towards autologous T-cell-depleted ex vivo tumor cultures pretreated with or without the aforementioned oncolytic adenoviruses. RESULTS: Treatment of ex vivo tumor cultures with Ad5/3-E2F-D24-hTNFa-IRES-hIL2 caused a substantial rise in proinflammatory signals: increased secretion of IFNg, CXCL10, TNFa and IL-2, and concomitant activation of CD4+ and CD8+ TILs. Potent tumor reactivity was seen, as clinically relevant TIL secreted high levels of IFNg in response to autologous T-cell-depleted ovarian ex vivo tumor cultures treated with Ad5/3-E2F-D24-hTNFa-IRES-hIL2. This phenomenon was independent of PD-L1 expression in tumor cells, a factor that determined the variability of IFNg responses seen in different patient samples. CONCLUSIONS: Overall, oncolytic adenovirus Ad5/3-E2F-D24-hTNFa-IRES-hIL2 was able to rewire the ovarian tumor microenvironment to accommodate heightened antitumor TIL reactivity. Such effects may improve the clinical effectiveness of ACT with TILs in patients with advanced OVCA.
  • Acheva, Anna; Schettino, Giuseppe; Prise, Kevin M. (2017)
    Nearly 85% of radiotherapy patients develop acute radiation dermatitis, which is an inflammatory reaction of the skin at the treatment field and in the surrounding area. The aims of this study were to unravel the mechanisms of radiation-induced inflammatory responses after localized irradiation in a human 3D organotypic skin culture model. This could provide possible inflammatory targets for reduction of skin side effects. 3D organotypic skin cultures were set up and locally irradiated with 225 kVp X-rays, using a combination of full exposure and partial shielding (50%) of the cultures. The secretion of pro-inflammatory cytokines, the phenotype, and the differentiation markers expression of the cultures were assessed up to 10 days postirradiation. The pro-inflammatory transcription factor nuclear factor kappa B (NF-kappa B) and cyclooxygenase-2 (COX-2) pathways have been studied. The results showed fast activation of NF-kappa B, most likely triggered by DNA damage in the irradiated cells, followed by upregulation of p38 MAPK and COX-2 in the irradiated and surrounding, non-irradiated, areas of the 3D cultures. The application of the COX-2 inhibitor sc-236 was effective at reducing the COX-2 mRNA levels 4 h postirradiation. The same inhibitor also suppressed the PGE2 secretion significantly 72 h after the treatment. The expression of a pro-inflammatory phenotype and abnormal differentiation markers of the cultures were also reduced. However, the use of an NF-kappa B inhibitor (Bay 11-7085) did not have the predicted positive effect on the cultures phenotype postirradiation. Radiation-induced pro-inflammatory responses have been observed in the 3D skin model. The activated signaling pathways involved NF-kappa B transcription factor and its downstream target COX-2. Further experiments aiming to suppress the inflammatory response via specific inhibitors showed that COX-2 is a suitable target for reduction of the normal skin inflammatory responses at radiotherapy, while NF-kappa B inhibition had detrimental effects on the 3D skin model development.