ANTICANCER RESEARCH 38 : 1279-1290 (2018) doi:10.21873/anticanres.12350 Detection of Distinct Changes in Gene- expression Profiles in Specimens of Tumors and Transition Zones of Tenascin- positive/-negative Head and Neck Squamous Cell Carcinoma VERONIKA ZIVICOVA 1,2 , PETER GAL 3,4 , ALZBETA MIFKOVA 1,2 , STEPAN NOVAK 1,2 , HERBERT KALTNER 5, MICHAL KOLAR 6, HYNEK STRNAD 6, JANA SACHOVA 6, MILUSE HRADILOVA 6, MARTIN CHOVANEC 7, HANS-JOACHIM GABIUS 5, KAREL SMETANA JR. 1,8 and ZDENEK FIK 1,2 1Institute of Anatomy, 1st Faculty of Medicine, Charles University, Prague, Czech Republic; 2Department of Otorhinolaryngology, Head and Neck Surgery, 1st Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic; 3Department of Biomedical Research, East-Slovak Institute of Cardiovascular Diseases, Inc., Kosice, Slovak Republic; 4Department of Pharmacology, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Kosice, Slovak Republic; 5Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians University, Munich, Germany; 6Laboratory of Genomics and Bioinformatics, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; 7Department of Otorhinolaryngology and Head and Neck Surgery, 3rd Faculty of Medicine, University Hospital Královské Vinohrady, Charles University Prague, Prague, Czech Republic; 8BIOCEV, Charles University, 1st Faculty of Medicine, Charles University, Vestec, Czech Republic Abstract. Background/Aim: Having previously initiated Results: Histopathological examination demonstrated that genome-wide expression profiling in head and neck squamous Ten +Fn +Gal-1 + co-expression occurs more frequently in cell carcinoma (HNSCC) for regions of the tumor, the margin samples of HNSCC (55%) than in NM (9%; p<0.01). of surgical resecate (MSR) and normal mucosa (NM), we here Contrary, the Ten –Fn +Gal-1 – (45%) and Ten –Fn –Gal-1 – proceed with respective analysis of cases after stratification (39%) status occurred with significantly (p<0.01) higher according to the expression status of tenascin (Ten). Materials frequency than in HNSCC (3% and 4%, respectively). In and Methods: Tissue specimens of each anatomical site were MSRs, different immunophenotypes were distributed rather analyzed by immunofluorescent detection of Ten, fibronectin equally (Ten +Fn +Gal-1 +=24%; Ten –Fn +Gal-1 –=36%; (Fn) and galectin-1 (Gal-1) as well as by microarrays. Ten –Fn –Gal-1 –=33%), differing to the results in tumors (p<0.05). Absence/presence of Ten was used for stratification of patients into cohorts without a difference in prognosis, to comparatively examine gene-activity signatures. Microarray This article is freely accessible online. analysis revealed i) expression of several tumor progression- + Correspondence to: Dr. Zdeněk Fík, Department of associated genes in Ten HNSCC tumors and ii) a strong up- Otorhinolaryngology, Head and Neck Surgery, First Faculty of regulation of gene expression assigned to lipid metabolism in – Medicine, Charles University and University Hospital Motol V MSRs of Ten tumors, while NM profiles remained similar. Úvalu 84, 150 06 Prague 5, Czech Republic. Tel: +420 224434301, Conclusion: The presented data reveal marked and specific Fax: +420 22443430, e-mail: [email protected]; and Dr. Peter changes in tumors and MSR specimens of HNSCC without a Gál, Department of Biomedical Research, East-Slovak Institute of separation based on prognosis. Cardiovascular Diseases Inc., Ondavská 8, 040 11 Košice, Slovak Republic. Tel: +421 552343251, Fax: +421 557891613, e-mail: [email protected] or [email protected] Faced with the enormous quantity of details on cell features, it is tempting to relate cases of differential expression to Key Words: Extracellular matrix, fibronectin, galectin, tenascin, clinically-relevant properties. Conceptually, however, it tumor, stroma. could be possible that dysregulation occurs in such RNA 1279 ANTICANCER RESEARCH 38 : 1279-1290 (2018) profiles without any association to, most importantly, Table I. TNM classification. prognosis. Thus, it is an open question to what extent these G T N S profiles are similar or different in tissue specimens of tumors classified according to a certain immunophenotypical 0 - - 24 - parameter. In our recent pilot study, we initiated a 1 14 16 15 4 comparison of tumor, margin and normal tissue specimen in 2 39 36 38 8 head and neck squamous cell carcinoma (HNSCC) (1). 3 27 17 3 21 4 - 11 - 47 Building on the previously detected relationship of presence of a member of the family of adhesion/growth-regulatory G: Grade; T: size of the primary tumor; N: degree of spread to regional galectins, a reader of the sugar code (2, 3), i.e. galectin-1 lymph nodes; M: presence of distant metastasis; S: stage. (Gal-1), with tumor invasiveness (4) and its activity to promote fibronectin (Fn) expression and fibroblast conversion to α- smooth muscle actin ( α- SMA) expressing myofibroblasts (5) as well as the significance of tenascin Table II. Anatomical sites of the primary tumors analyzed in the present (Ten) expression (6), we here examine a panel of HNSCC study. cases always with specimen of the margin of surgical resecate (MSR) and normal mucosa (NM) according to Primary site Number of patients/microarray presence of these three extracellular matrix (ECM) effectors. Oral cavity 11/4 Of fundamental importance, tumors of diverse metastatic Oropharynx 51/13 potential and progression status differ in the composition of Hypopharynx 5/1 both tumor- and stroma-derived ECM components (7, 8). In Larynx 13/8 this study, we first ask the question whether detection of the three proteins, expressed individually and/or in combination, provides prognostic information in the studied cohort of HNSCC patients. Following the immunohistochemical part that results in stratification according to the status of Ten PBS for five minutes. After extensive washing in PBS (three times expression, microarray analysis between Ten +/Ten – cases was for 10 minutes), the specimens were treated with PBS containing 0.2 performed in order to answer the question on occurrence of % (v/v) Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) and then washed in PBS. Antigen-independent binding of antibody via the Fc differences on the level of RNA presence. In addition to part was precluded by preincubation of specimens with porcine tumor tissues from specimens stratified according to Ten serum (DAKO, Glostrup, Denmark) diluted as recommended by the presence, we also ran array-based RNA profiling of MSR and supplier. A murine monoclonal antibody against Ten-C and a rabbit NM of the two patient groups differing in Ten expression. polyclonal antibody against Fib (both from Sigma-Aldrich St. Louis, MO, USA) were applied as recommended by the supplier. Home- Materials and Methods made rabbit polyclonal antibody against Gal-1 (9) that had thoroughly been tested for any cross-reactivity against other Tissue samples. The set of tissue specimen of i) HNSCC (n=80; for members of the galectin family, was used at a dilution of 1:50 (v/v). details on classification, see Table I; for details on anatomical The specificity of immunodetection was ascertained by replacing the localization of analyzed tumors, see Table II), of ii) normal oral first-step antibodies by an irrelevant antibody of the same isotype (in mucosa (NM) contralaterally to the primary tumor (NE – normal the case of monoclonal antibody) or by omitting the first-step epithelium; n=47), and of iii) margin of surgical resecate (MSR, polyclonal antibody from pocessing. DNA was visualized by 4’,6- macroscopically healthy mucosa up to 1 cm to the tumor margin; diamidino-2-phenylindole (DAPI) (Vector-Laboratories, Burlingame, n=45) was obtained from the Charles University, 1st Faculty of CA, USA). All preparations were analyzed by a fluorescence Medicine, Department of Otorhinolaryngology, Head and Neck microscope Eclipse 90i (Nikon, Tokyo, Japan) equipped with filter Surgery. All tissue samples were collected with informed consent of blocks for FITC, TRITC and DAPI and a Cool-1300Q CCD camera patients and approval of local ethical committee according to the (Vosskühler, Osnabrück, Germany). Data were processed using the Helsinki Declaration. LUCIA 5.1 computer-assisted image analysis system (Laboratory Imaging, Prague, Czech Republic). Tissue processing – immunohistochemistry. Tissue specimen of NE, HNSCC and MSR were cryoprotected by Tissue-Tek (Sakkura, Microarray analysis. Material from a subset of 26 patients was Zoeterwoude, The Netherlands) and frozen in liquid nitrogen. processed using microarray techniques. Anatomical sites of Frozen sections, 7 μm thick, were prepared by a Cryocut-E specimens used for gene profiling are given in Table I. Briefly, total microtome (Reichert-Jung, Vienna, Austria). RNA was isolated using RNeasy Micro Kit reagents (QIAGEN, Germantown, MD, USA) from cryostat sections, following the Immunofluorescent detection of Ten, Fn and Gal-1 . Frozen sections procedure optimized for animal cells. The quantity and quality of were carefully washed with phosphate-buffered saline (PBS, pH 7.2) RNA were determined using a NanoDrop ND-1000 and fixed by exposure to 2% (w/v) buffered paraformaldehyde in spectrophotometer (NanoDrop Technologies LLC, Wilmington, DE, 1280 Zivicova et al : Tumor and Transition Zone Array Profiling Figure 1. Distribution (%) of specimen according to the category of immunohistochemical negativity/positivity for the