Center for Biomaterials, Cellular, Molecular, and Theranostics (CBCMT) DOCTORAL COMMITTEE MEETING "HPV Induced EMT in Oral Squamous Cell Carcinoma" By- Shantanu Saraf Guide - Dr. Raun

Center for Biomaterials, Cellular, Molecular, and Theranostics (CBCMT) DOCTORAL COMMITTEE MEETING “ HPV Induced EMT in Oral Squamous Cell Carcinoma ” B y - Shantanu Saraf G uide - D r . Raunak Kumar Das Scholar No - 20PHD2126 (Assistant Professor Sr. Grade 1).

Contents. Introduction Significance of the study Lacunae in the field Hypothesis Overall Objective Experimental Design Expected Outcomes Course Works.

Oral Squamous Cell carcinoma. Oral squamous cell carcinoma (OSCC) is the most common sub-site of head and neck cancer. Defined as- Malignant epithelial neoplasm exhibiting squamous differentiation as characterised by the formation of keratin and /or the presence of intercellular bridges (Pindborg et.al. 1997). Ominous characteristic of squamous carcinoma is its ability to surround nerves and to infiltrate for long distances in a perineurial fashion. OSCC is a field-defect phenomenon..

Oral Squamous cell carcinoma has a high malignant potential with great propensity for metastasis to the regional lymph nodes through vascular invasion and thereby leading to loco-regional failure..

STATISTICS. Lip, oral cavity Source: Globocan 2020 Number of new cases in 2020, both sexes, all ages Number of deaths in 2020, both sexes, all ages Breast Lung Colorectum Prostate Stomach Liver Cervix uteri Oesophagus Thyroid Bladder Non-Hodgkin lymphoma Pancreas Leukaemia Kidney Corpus uteri Lip, oral cavity Melanoma of skin Ovary Brain, central nervous system Larynx Multiple myeloma Nasopharvnx Gallbladder Oropharynx Hypopharynx Hodgkin lymphoma Testis Salivary glands Vulva Penis Kaposi sarcoma Mesothelioma Vagina 1 796144 1 414 259 1 089 103 905 677 604 127 604 100 586 202 573 278 352 495 773 474 519 431 288 417 367 377 713 324 635 313 959 308 102 184615 176404 133354 115 949 98 412 • 84 254 83 087 74458 53 583 45 240 36 068 34 270 30 870 17908 2 261 419 2 206 771 1 931 590 2 ooo ooo Lung Colorectum Liver Stomach Breast Oesophagus Pancreas Prostate Cervix uteri Leukaemia Non-Hodgkin lymphoma Brain, central nervous svstem Bradder Ovary Kidney Lip, oral cavity Multiple myeloma Larynx Corpus uteri Gallbladder Nasopharynx Melanoma of skin Oropharynx Thyroid Hypopharynx Mesothelioma Hodgkin lymphoma Salivary glands Vulva Kaposi sarcoma Penis Testis Vagina 935 173 830 180 768 793 684 996 544 076 466 003 375 304 341 831 311 594 259 793 251 329 212 536 207 252 179 368 177757 117077 97 370 84 695 80 008 57 043 48 143 43 646 38 599 26 278 23 376 22 778 17 427 15 086 13211 9 334 7 995 0 500 ooo 1 ooo ooo 1 500 ooo 500 ooo 1 ooo ooo 1 500 ooo.

5-year prevalence, both sexes Africa (3%) LAC** (4.9%) N.America* (9.2%) Asia (60.9%) Europe (20.6%) Population Asia Europe *Northern America **Latin America and the Caribbean Africa Oceania Total Number 584 403 197 515 88 196 47 348 29 134 12 652 959 248.

Age standardized (World) incidence rates, lip, oral cavity, by sex Males Females Age standardized (World) incidence and mortality rates, lip, oral cavity Melanesia South-Central Asia Central and Eastern Europe Australia and New Zealand Western Europe Northern America Northern Europe World Southern Africa Southern Europe Micronesia Caribbean Polynesia South America South-Eastern Asia Eastern Asia Eastern Africa Western Asia Middle Africa Northern Africa Western Africa Central America 30 20 3.6 6.2 3.1 2.6 3.1 2.3 5.3 2.6 5.2 2.1 4.5 — 1.4 2.6 A 1.2 2.4 1 •5 2.2 n 1.3 2.041.1 1.7111.3 1.511.1 1.011.1 10 o 10 ASR (world) per 100 000 30 Melanesia South-Central Asia Australia and New Zealand Central and Eastern Europe Western Europe Northern Europe Northern America World Southern Africa Southern Europe Caribbean Micronesia Polynesia South America South-Eastern Asia Eastern Africa Eastern Asia Western Asia Middle Africa Northern Africa Western Africa Central America 25 Incidence 9.0 6.0 5.1 4.6 4.5 4.2 4.1 3.8 3.6 2.9 2.8 2.7 2.6 2.5 1.9 1.9 1.7 1.6 1.5 1.3 1.1 Mortality 6.5 5.1 0.76 2.4 1.1 1.2 0.66 1.9 1.7 1.1 1.1 1.2 1.1 1.1 1.4 1.3 0.75 0.69 1.0 0.69 0.91 0.44 20 16.7 20 15 10 5.0 0 5.0 10 ASR (world) per 100 OOO 15 20 25.

ORAL CANCER CAPITAL- INDIA. India contributes one-third of the total burden of oral cancer cases in the world. And 57.5% of oral cancer cases occur in Asia, particularly in India. 60 - 80% of Indian patients report with advanced disease as compared to 40% in developed countries, accounting for poor 5 years survival rate (overall). Survival rate in India is only 27% for patient with advanced stage of oral cancer..

RISK FACTORS. The prominent predisposed factors reported are tobacco consumption, smokeless tobacco (khaini, mava, paan betel quid, zarda, snuff, mashiri, areca nut etc.), alcohol consumption, nutritional deficiencies, poor oral hygiene, local chronic trauma, immuno - suppression, exposure to UV radiations, and human papilloma virus (HPV) infection. Poor oral hygiene is seen in 85% of oral cancer patients. Areca nut alone is a confirmed carcinogen and causally associated with a premalignant condition. Prolonged abrasion of epithelial lining in any of these sub-sites causes potentially malignant oral disorders (PMDs) which eventually leads to OSCC..

OCCURRENCE OF OSCC. The development of OSCC is considered a complex multistep process. Normal oral mucosal keratinocytes are chronically exposed to risk factors, which can break the homeostasis and generate genetic instability . The proliferation and uncontrolled growth, along with genetic instability granted adaptive advantages over the surrounding cells, which promote local invasion and orchestrate a collaboration of the surrounding stromal cells. This microenvironment promotes loss of cell adhesion and facilitates epithelium mesenchymal transition (EMT)..

PRE - MALIGNANT DISORDERS (PMDs). The etiology of precancerous lesions of oral mucosa is not well-known. A precancerous condition is “a generalised state associated with significantly increased risk of cancer.” Potentially malignant disorders (PMDs) such as erythroplakia, leukoplakia, tobacco pouch keratosis, oral epithelial dysplasia, inflammatory oral sub-mucosa fibrosis(OSMF), and lichen planus are indicators of the preclinical phase of oral cancer. The treatment of oral malignancy primarily depends on the location and size of the tumor, and the feasibility of organ preservation in patients. Several cellular states expressing different levels of epithelial and mesenchymal markers exhibits transition of epithelial to mesenchymal in a gradual manner..

OSMF. Erythroplakia. Leukoplakia. Tobacco pouch keratosis.

Epithelial-to-Mesenchymal transitions in oscc. EMT has increasingly gained significance as an essential process, responsible for 40-50% of OSCC relapse and is considered to be important in the early change from benign to invasive carcinoma. Altered connections between the cells and the EMT-inducing transcription factors lead to over-expression of mesenchymal characteristics. Cells undergoes EMT progressively losing typical epithelial cell-to-cell junctions, such as adherent and tight junctions, and the apical-basal polarity. At the same time, cells acquire a front-rear polarity, while gaining migratory and invasive abilities..

Tight junction Ad herent junction Apical—basal polarity Stress fibers Basen•e-nt rane E—cad he rin N —cadherin E pithelial phenotype E—cadherin Cytokeratin NF-KB ß—cat e n i n F ront—rea r rity Mesenchyrnal phenotype Virnentin a-SMA EMT Snaill EMT-TFs TVVISTI ZEBI TGF—ß signalling path v•ays.

Human PapillomaVirus (hpv) in OSCC. HPV is detected in ~26% of global oral squamous cell carcinoma (OSCC) cases. The oncogenic potential of high-risk HPV genotypes is related to its ability of integrating DNA fragments (E5, E6 and E7) in the host cell, annulling the function of tumour suppressor factors such as p21, p53 and pRb. HPV (low and high risk serotypes) are 2-3 times more detected in precancerous mucosa and almost 5 times more detected in carcinoma 22.2% in benign leukoplakia; 26.2% in intraepithelial neoplasias; and 46.5% in OSCC, with a detection probability of high-risk ones 2.8 times higher than low risk. HPV related oral SSCs is more common in India than other nations. Its incidence may be noticed as high as in 74% of the cases. According to a study done by Balaram et al, it was found that the prevalence of oral SSCs cases recognized as HPV-positive was 67% in southern India, 34% in eastern India and 15% in western India. It is more frequently seen in younger patients less than 50 years of age and common sites include the base of the tongue and tonsil region. Probable connection exists with sexual practice such as oral sex, frequent sexual indulgence without use of barriers, sex with multiple partners, sex at an earlier age, etc..

Chewable Tobacco Betel nuts Paan Oral mucosa Gutka I-IPV 18 Rough mucosa Swelling, ulcers, cuts Leukopla kia/ Erythropl akia HPV 16 HPV Invasion oscc.

Significance of Study. Since India has the highest incidence of oral cancer, especially associated with HPV, this study will have high relevance for our country. Such a systematic study, addressing important aspects of HPV induced tumorigenicity and prognostication of metastatic potential, will have a great impact on understanding cancer induction and pathobiology of oral cancers common in India ..

Lacunae in the field. EMT markers may appear to have different roles in the pathogenesis of OSCC. Additionally, E-cadherin and β-catenin may serve as a marker for the occurrence of regional metastasis. Moreover, in HPV-positive OSCC cell lines, HPV infection may exploit the EMT markers and cellular pathways in the carcinogenesis process..

Hypothesis. Induction of epithelial to mesenchymal transition by high risk human papilloma virus (HPV) 16/18 (E6 and E7) in normal oral cells and oral cancer cell lines..

OVERALL OBJECTIVE •To transfect HPV E6/E7 in oral keratinocytes or in oral cancer cells •To observe changes in migratory potential of transfected cells •To observe the expression of EMT markers •To observe the nuclear changes during EMT under HPV condition.

Picture 2. Experimental Design.

Expected outcome. Migration potential of oral cells and oral cancer cells will increase on transfection with HPV. Expression of epithelial to mesenchymal transition and cellular markers will show alterations such as E-cadherin expression— Down-regulated, β-catenin expression — Up-regulated, c-myc expression — Up - regulated. Cellular Morphological changes may occur as in Cellular circularity will disrupt, Area and Factor dimension will increase, Entropy will decrease. Expression of transcription factors will up-regulate or down-regulate..

Course work completed. Research Methodology (RES 702) MODULE I—Research Philosophy:- Philosophy of research – definition and type of research – research methods in science: descriptive methods, predictive (relational) methods, explanatory method. MODULE II—Literature Sources:- Sources of scientific information – information literacy, systematic literature search, how to formulate a query, search technique, principal bibliographic databases. Medical and scientific internet search engines – building personal reference databases. Quality of papers – indices and critical appraisal. MODULE III—Descriptive Statistics:- Data type and sources – variables and types. Descriptive statistics of categorical data and continuous data – calculations and interpretation. Estimation of parameters – hypothesis testing: tests of significance, type I and type II errors, z test, t test, chi – square test, goodness – of – fit test: calculations and interpretations, correlation analysis – calculations, types and interpretations. MODULE IV—Design of Experiments Statistical concepts for designed experiments – factor types, experimental space, factor domain. Single factor experiments, multifactor experiments, analysis of variance (ANOVA) and residual analysis – fractional factorial and screening designs – design for optimisation..

MODULE V—Life Science Statistics Survivor function and hazard function – the life table analysis: the Kaplan – Mier analysis. Comparing two groups of survival data and models for survival data. Linear regression, multiple and logistic regression – analysis and interpretation. Study designs – types of clinical designs – Phase I and phase II trials – case – control studies. MODULE VI—Research Publications Research reporting – publication types – format, structure and styles. Scientific word processors – LaTeX / MSWord / LibreOffice. Scientific tables, graphs and illustrations – preparation of presentations and posters. Research Grants – national and international agencies – writing for grant applications. Patenting – types – copyright, trademark, trade secret and patents, how to apply and maintain – national and international patents and Patent Cooperation Treaty (PCT). MODULE VII—Ethics in Research and Publications Brief introduction – ethics, morals, values, philosophy, research, scientific conduct and misconduct – fabrication, falsification and plagiarism – affecting the claims of scientific findings, research design, conduct of research and research dissemination. Publication ethics – collaborating and authorship issues – Salami, imalas, duplicate publications, research management, history and essence of research philosophy – science, biology. Moral philosophy – metaethics, normative ethics and applied ethics and bio-ethics. Predatory journals – how to choose the right publication journal – COPE, WAME, DOAJ and OASPA. Open Access publications and initiatives, publication frauds and citation databases..

English for Researchers(ENG5004) MODULE I Reading for Research - Skimming, Scanning, Intensive, Extensive, Critical Reading Skills MODULE II Vocabulary for Research - Academic Words, Phrases and Clauses MODULE III Grammar for Research - Specific Grammar Use: Concord and Tense Shift, Run-on sentences, Voice, Conditionals, Prepositional Phrases, Articles, Adjectives and Adverbs. MODULE IV Study Skills for Research - Note –taking, Dictionary Skills, Reference Skills, Note-making skills and Summarizing, Quotations and Citations. MODULE V Writing Process - Prewriting Strategies, Writing Strategies, Revision Strategies: Deleting, Elaborating. MODULE VI Oral Presentation Skills - Challenges in Presentation, Verbal Ability, Audience orientation, Body Language, Etiquette MODULE VII Writing and Presentation - Research Writing and Presentation: Types of Academic Writing, Characteristics, Structure and Style. MODULE VIII Writing and Analyzing Research Papers.

RESEARCH AND PUBLICATION ETHICS(RMC0001). Module I: Philosophy and Ethics Introduction to Philosophy: Definition, nature and scope, concept, branches, Ethics: Moral Philosophy, nature of moral judgments and reactions. Module II: Scientific Conduct Ethics with respect to Science and Research, Intellectual honesty and Research integrity Scientific misconducts: Falsification, Fabrication and Plagiarism(FFP), Redundant Publications: Duplicate Publications and overlapping Publication, salami slicing, Selective Reporting misrepresentation of data. Module III: Publication Ethics Publication Ethics: Definition, introduction and importance, Best Practices/standards setting initiatives and guidelines : Committee on Publication Ethics (COPE), World Association of Medical Editors (WAME) etc., Conflicts of interest, Publication misconduct: definition, concept, problems that lead to unethical behavior and vice versa types, Violation of Publication ethics, authorship and contributorship, Identification of Publication misconduct, complaints and appeals, Predatory Publishers and Journals..

Module 4 Practice 1: OPEN Access Publishing Open access Publications and initiatives, SHERPA/ROMEO online resource and check publisher copy right & Self archiving policies 3. Software tool to identify predatory publications developed by SPPU Journal finder/ Journal suggestion tools Viz. JANE, Elsevier journal finder, Springer, Journal suggester etc., Module 5 Practice 2:Publication Misconduct Group Discussion: Subject specific ethical issues , FFP and authorship, Conflict of interest, Complaints and appeals: example fraud from India and abroad, Software tools: Use of Plagiarism software Turnitin, Urkund and other open source software tools Module 6 Practice 3: Databases and Research Metrics Data Bases: Indexing DataBase, Citation Data Bases: Web of Science, Scopus etc, Impact factor of Journal as per Journal Citation Report, SNIP,SJR,IPP, Cite Score, Metrics: h-index, g index, i10 index, almetrics.

Upcoming Course work. Guide Paper - I Cancer Biology-Focus on Oral Cancer Module I - Cancer Hallmarks Cancer Hallmarks -Sustained Proliferative Signaling; Evading Growth Suppressors; Enabling Replicative Immortality (Telomeres and Telomerases); Resisting Cell Death; Inducing Angiogenesis; Activating Invasion and Metastasis. Module II - Model Systems 2D cultures, Scaffolds (Hydrogels -PLGA, PCL, Polystyrene), tumorospheres, immunocompromised mice, inbred strains of mice; chemical-induced cancer models, K.O mice; conditional K.O. (inducible expression systems in mice) Module III - Benign and Malignant Tumors of the Oral Cavity B enign tumors of epithelial tissue origin, m alignant tumors of the epithelial tissue origin, histopathological grading of oral squamous cell carcinoma, Epithelial-Mesenchymal Transitions in oral cancer, structural and functional regulation of desmosomes, epithelial cell plasticity by dynamic transcriptional regulation of E-cadherin, regulation of catenins in oral cancer, cadherin-mediated cell-cell adhesion and the microtubule network, matrix metalloproteinases and epithelial-to-mesenchymal transition: implications for carcinoma metastasis..

Guide Paper - I Cancer Biology-Focus on Oral Cancer Module IV- Oral Mucosa Stratified squamous epithelium, and Submucosa, different layers of epithelium, structure and function of oral mucosa, cell-cell interaction in oral mucosa (tight and gap junction), anchoring junctions (adherens junction and desmosomes), cell kinetics, cytokeratin, factors controlling oral epithelium phenotype, non-keratinocytes, lamina propria, epithelial-connective tissue interface, regional variation in the structure of oral mucosa, lip, cheek, gingiva and alveolar mucosa, palate, tongue and floor of mouth, infections of oral mucosa (bacterial and viral- Epstein-Barr, HSV1&2, HPV). Module V- HPV in Oral Cancer Basic Virology-HPV nomenclature, HPV structure, Classification, functions of viral proteins (E1, E2, E4, E6, E7, L1 and L2), transmission of HPV, Pathogenesis (life cycle, interaction of HPV proteins and cell factors), HPV association with oral cancer, Diagnosis of HPV- methods of specimen collection, conventional and monolayer cytology, HPV nucleic acid detection, Diagnostic utility of p16INKa, evaluation of Dan testing and HPV mRNA liquid based cytology..

Guide Paper - II Cellular Imaging/Microscopy Module I - Fundamentals of Light and Optics Dual nature of light, vector notation and wavefronts, interactions of light and matter, refractive Index, lenses, image formation, resolution, depth of field. Module II - Types of Microscopes Light microscope- bright field microscope, microscope resolution, dark field microscope, phase-contrast microscope, differential interference contrast microscope (DIC), Fluoresces microscope, Polarized light microscopy, other methods. Module III - Illuminators, Filters, and the isolation of specific wavelengths Illuminators and their spectra, illuminator alignment and bulb replacement, filters for adjusting the intensity and wavelength of illumination, effects of light on living cells. Area detectors, CCDs, emCCDs, sCMOS, comparison, read noise, speed and other sensor characteristics..

Guide Paper - II Cellular Imaging/Microscopy Module IV - Fluorescence Microscopy Applications of Fluorescence Microscopy, Physical Basis of Fluorescence, Properties of Fluorescent Dyes, Fluorescent Dyes and Proteins in Fluorescence Microscopy, Arrangement of Filters and the Epi-illuminator in the Fluorescence Microscope, Objectives and Spatial Resolution in Fluorescence Microscopy, Causes of High Fluorescence Background, Quenching, Blinking, and Photobleaching, Examining Fluorescent Molecules in Living Cells. Module V - Confocal Laser Scanning Microscope The optical principle of confocal imaging, advantages of CLSM over wide-field fluorescence systems, criteria defining image quality and the performance of a electronic imaging system, confocal adjustments and their effects on imaging, Photobleaching, general procedure for acquiring a confocal image, performance check of a confocal system, fast (real-time) imaging in confocal microscopy, spectral analysis: a valuable enhancement for confocal imaging, optical sectioning by structured illumination..

NPTEL COURSE-CELL CULTURE TECHNOLOGIES By- Dr. Mainak Das Department of Biological Sciences & Bioengineering IIT Kanpur TYPE OF COURSE - Online COURSE DURATION - 8 weeks (26 July' 22 - 17 Sep' 22) EXAM DATE - 26 Sep 2022 COURSE OUTLINE : The course will be a short primer to understand how ‘animal cell culture technologies’ have strengthen the bio-medical research from basic research to the modern drug discovery. Animal cell culture was first performed in the very first decade of 19th century. Since then, tremendous development has taken place in this field. The lectures will help the researcher to appreciate the developments during last hundred years and will help them to independently set up cell culture laboratories. For non-biologist, it will be an informal way to demystify the intriguing routes of biomedical research where cell culture is a very ‘potent tool’. COURSE PLAN : Week 1: Introduction & biology of cultured cells Week 2: Equipments, aseptic techniques, safety protocols Week 3: Culture vessels & media development Week 4: Serum-free medium development & sterilization Week 5: Primary culture, secondary culture, cloning & selection Week 6: Cell separation, characterization, differentiation & transformation Week 7: Contamination, cryo-preservation & cyto-toxicity Week 8: Organo-typic culture & specialized cell culture techniques.

Thank you.