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(2) Doctoral Thesis. Michał Sarna. Melanin granules modify nanomechanical properties of melanoma cells and inhibit invasive abilities of the cells Supervisor: prof. dr hab. Květoslava Burda. Cracow, 2015.

(3) Declaration of the author of this dissertation: Aware of legal responsibility for making untrue statements I hereby declare that I have written this dissertation myself and all the contents of the dissertation have been obtained by legal means.. Date, signature. Declaration of the thesis Supervisor: This dissertation is ready to be reviewed.. Date, signature.

(4) Preface The following dissertation is based on novel ideas and original concepts, which were developed and experimentally tested by the author during his PhD studies in the Group of Molecular Biophysics and Bioenergetics, Department of Medical Physics and Biophysics, Faculty of Physics and Applied Computer Science, AGH University of Science and Technology. Results discussed in this PhD Thesis were published as original scientific papers. Two of the papers appeared in international scientific journals from the ‘Philadelphia List’ while one was published as a conference proceeding. Selected results were presented during several scientific conferences and were recently discussed at an international forum of leading specialists in the field of melanoma and melanin pigmentation research during the 22nd International Pigment Cell Conference held in Singapore in September, 2014..

(5) I would like to express my sincere gratitude to: Professor Květoslava Burda for allowing me to conduct this PhD study under her supervision, for the freedom that she gave me during the work, and for all her help and kindness; Members of the Group of Molecular Biophysics and Bioenergetics for creating a friendly environment during my work; Professor Krystyna Urbańska and Members of the Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University for facilitating EPR measurements and for collaboration; Professor Zbigniew Madeja and Members of the Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University for valuable scientific collaboration; Mrs. Barbara Czuba-Pełech for helpful assistance with cell culture; Mr. Paweł Hermanowicz for fruitful collaboration..

(6) Contents Scientific achievements......................................8 Summary in Polish............................................15 Summary in English..........................................17 1 Background Information...............................19 1.1 Melanoma & m elanin synthesi s.... ..... ..... ..... 19 1 . 1.1 Melan in. pigm en tat ion. and. m eta stat ic. b eh a v io r o f m elan o m a cells. . . . . . . . . . . . . . . . . . . . . 2 0 1 . 1.2 Ph ysicoch em ica l. p ro pert ies. of. m ela n in. p igment .. . .. .. .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. .. . ... . .. .. .. .. .. 2 0. 1.2 Metastasis & elasticity of cancer cells.........21 1 . 2 . 1 Met a st a sis o f ca n cer cel ls. . . . . . . . . . . . . . . . . . . . . . . . . 2 1 1 . 2 . 2 Defo r m a t io n o f ca n cer ce ll b o dy du r in g t r a n sm ig r a t io n t h r o u g h differ en t . . . . . . . . . . . . 2 2 1 . 2 . 3 Ela st i cit y a s a u n i q u e m a r k er o f ca n c er t r a n sfo r m a t io n ,. p r o g r e ss io n. and. inv a s io n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2 4. 1.3 Atomic. fo rce. mi croscopy. &. el ast icit y. analy sis..... ..... .. ....... ..... ..... ..... .... ....... ..... ..... ..25 1 . 3. 1 W o r kin g p r in cip le o f A F M. . . . . . . . . . . . . . . . . . . . . . . . 2 5 1 . 3. 2 I n denta t io n exper im ent .. .. .. .. . .. .. .. . .. .. .. .. .. . .2 7 1 . 3. 3 Her t z m o del o f e la st ic de fo r m a t io n s. . . . . . . 2 8. 2 Objectives of the work.................................29 3 Experimental section.....................................31 3.1 Cells...............................................................31 3.2 Optical microscopy.......................................32 3. 3 Electron paramagnetic resonance...............33 3. 4 Atomic for ce m icro scopy. ..... .... ....... ..... ..... ..33. 6.

(7) 3.5 Force spectroscopy data analysis....... ..... ....33 3. 6 Cell biology methods............ .... ....... ..... ..... ...34. 4 Results and Discussion.................................35 4.1 Melanin. granules. modify. nanomechanical. properties of pigmented melanoma cells....35 4.2 Presence of melanin granules in melanoma cells is likely to make difficult nanodiagnosis of mel anoma.. .. ....... ..... ..... ..... .... ....... ..... ..... ..35 4 .3 Melanin granules inhibit invasive abilities of me l ano m a cel l s in v itr o. . .. .. .. .. . .. . .. .. . .. . .. .. 36 4.4 Melanin pigme nt i s a mechani cal modu lator of bot h e lasti c propert ies and metast ati c cap abil itie s o f melanoma cel ls....... ..... ..... ...36 4.5 Quantitative analysis of melanin content in melanoma metastatic. cells. could. potential. of. determine melanoma. the cells. leading to improved melanoma diagnosis.. . .3 7. 5 Concluding remarks......................................39 5.1 Key findings of the work. . . . . . . . . . .. . . . . . . . . . . . . . . . . . 3 9 5.2 Postulates formulated based on the observed effect...... ..... ..... ....... ..... ..... ..... .... ....... ..... ..... ..39 5.3 Issues to be addressed in future studies....40. 6 References ............. ..... ..... ..... .... ....... ..... ..... ..... .. 41 Appendix ... .. . .. . .. .. . .. . .. .. . .. . .. .. . .. .. .. .. . .. . .. .. . .. . .. .. . .. .. .. . 48. 7.

(8) Scientific achievements. Scientific achievements Publications Papers being part of this PhD Thesis: 1. Sarna M, Zadlo A, Hermanowicz P, Madeja Z, Burda K, Sarna T. Cell elasticity is an important indicator of the metastatic phenotype of melanoma cells. Experimental Dermatology, 2014; 23(11): 813-818.. 2 Sarna M, Zadlo A, Pilat A, Olchawa M, Gkogkolou P, Burda K, Böhm M, Sarna T. Nanomechanical analysis of pigmented human melanoma cells. Pigment Cell and Melanoma Research, 2013; 26(5): 727-730. 3 Sarna M, Zadlo A, Koczurkiewicz P, Burda K, Sarna T. Melanin modifies nanomechanical properties of pigmented melanoma cells. In: Melanocyte and its Environment. Medimond; Bologna, Italy, 2012; 2327.. Remaining papers: 4 Sarna M, Wojcik K A, Hermanowicz P, Wnuk D, Burda K, Sanak M, Czyż J, Michalik M. Undifferentiated bronchial fibroblasts derived from asthmatic patients display higher elastic modulus than their nonasthmatic counterparts. PLoS ONE, 2015; in press. 5 Wytrwal M, Leduc C, Sarna M, Goncalves C, Kepczynski M, Midoux P, Nowakowska M, Pichon C. Gene delivery efficiency and intracellular trafficking of novel poly(allylamine) derivatives. International Journal of Pharmacology, 2014; 478(1): 372-382. 6 Hermanowicz P, Sarna M, Burda K, Gabrys H. AtomicJ: an open source software for analysis of force curves. Review of Scientific Instruments, 2014; 85(6): 063703.. 8.

(9) Scientific achievements. 7 Ryszawy D, Sarna M, Rak M, Szpak K, Kędracka-Krok S, Michalik M, Siedlar M, Zuba-Surma E, Burda K, Korohoda W, Madeja Z, Czyż J. Functional links between Snail-1 and Cx43 account for the recruitment of Cx43-positive cells into the invasive front of prostate cancer. Carcinogenesis, 2014; 35(9): 1920-1930. 8 Koczurkiewicz P, Podolak I, Skrzeczyńska-Moncznik J, Sarna M, Wójcik K A, Ryszawy D, Galanty A, Lasota S, Madeja Z, Czyż J, Michalik. M.. Triterpene. saponosides. from. Lysimachia. ciliata. differentially attenuate invasive potential of prostate cancer cells. Chemico-Biological Interactions, 2013; 206(1): 6-17. 9 Wytrwal M, Sarna M, Bednar J, Kozik B, Nowakowska M, Kepczynski M. Formation of micelles by hydrophobically modified poly(allylamine hydrochloride). Polish Journal of Applied Chemistry, 2011; 55: 11-17. 10 Sarna M, Wybieralska E, Miekus K, Drukala J, Madeja Z. Topographical control of prostate cancer cell migration. Molecular Medicine Reports, 2009; 2(5): 865-871.. Oral presentations: 1. Sarna M. Atomic force microscopy – fine biophysical tool in life science research. Invited guest of the Centre de Biophysique Moleculaire. Orleans, France. April 18, 2013.. 2 Sarna M. Nanomechanical analysis of melanoma cells. 40th Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology. Zakopane, Poland. February 16-17, 2013. 3 Sarna M. Nanomechanical analysis of human prostate cancer cells. International conference of nanotechnology – Nano Measure. Cracow, Poland. June 3-4, 2010.. 9.

(10) Scientific achievements. Conference communications: 1. Mostert A B, Hanson G R, Gentle I R, Powell B J, Meredith P, Zadlo A, Sarna M, Sarna T. The Effect of Hydration on Photophysical and Photochemical Reactivity of Melanin. IInd International Scientific Conference OXYGENALIA. Poznan, Poland. November 7-8, 2014.. 2 Sarna T, Burke J M, Olchawa M, Pilat A, Sarna M, Szewczyk G, Zareba M. Photodynamic Properties of Lipofuscin Granules from Retinal Pigment Epithelium. NannBioPhoton Workshop – Light and Nanostructures for Environment and Health. Jagiellonian University, Faculty of Chemistry. Krakow, Poland. October 29-30, 2014. 3 Sarna M, Zadlo A, Burda K, Sarna T. Cell elasticity is an important indicator of the invasive abilities of pigmented melanoma cells. 22nd International Pigment Cell Conference. Singapore. September 4-7, 2014 (Abstract published in Pigment Cell and Melanoma Research, 2014; 27(5): 952, 2014). 4 Zareba M, Burke J M, Skumatz C M, Sarna M, Pilat A K, Sarna T. The modulatory effect of melanin on susceptibility of retinal pigment epithelial cells to photic stress. 22nd International Pigment Cell Conference. Singapore. September 4-7, 2014 (Abstract published in Pigment Cell and Melanoma Research, 2014; 27(5): 893, 2014. 5 Zadlo A, Pilat A, Sarna M, Broniec A, Sarna T. The influence of selected transition metal ions on the ability of melanin to decompose hydrogen peroxide. 22nd International Pigment Cell Conference. Singapore. September 4-7, 2014 (Abstract published in Pigment Cell and Melanoma Research, 2014; 27(5): 929, 2014). 6 Olchawa M, Pilat A, Sarna M, Szewczyk G, Zareba M, Burke J, Sarna T. Oxidative stress and age-related macular degeneration; sub-lethal photodynamic damage to retinal pigment epithelial cells impairs their vital functions. First Adriatic Symposium on Biophysical Approaches in Biomedical Sciences. Split, Croatia. August 24-29, 2014.. 10.

(11) Scientific achievements. 7 Wytrwal M, Stepniewski J, Sarna M, Jozkowicz A, Dulak J, Kepczynski M, Nowakowska M. The novel PAH derivatives and their transfection effiviency in vitro. Silesian Meetings on Polymer Materials POLYMAT60. Zabrze, Poland. June 30-July 01, 2014. 8 Sarna T, Sarna M, Pilat A, Olchawa M. Atomic force microscopy (AFM) and fluorescence imaging of ARPE-19 cells subjected to sublethal oxidative stress. Association for Research in Vision and Ophthalmology. Orlando, Florida, USA. May 4-8, 2014 (Abstract published in Investigative Ophthalmology and Visual Science, 2014; 55: 381, 2014). 9 Chmielowiec E, Orzechowska A, Sarna M, Ząbczyk M, Undas A, Burda K. Influence of fibrinogen on the stability of erythrocytes. Innovative technologies in biomedicine: the first international conference, Krakow, Poland. October 15-16, 2013. 10 Gkogkolou P, Sarna M, Luger T A, Böhm M. KdPT: Novel protective role against impaired wound healing in diabetes? 10th Meeting of the German Endocrine Brain Immune Network (GEBIN). Regensburg, Germany. March 21-23, 2013 (Abstract published in Brain, Behavior, and Immunity, 2013; 29(15) Suppl: S3, 2013). 11 Koczurkiewicz P, Sarna M, Podolak I, Wójcik K A, Czyż J, Michalik M. Effects of triterpene saponosides on human prostate cancer and normal cell in vitro. 40th Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology. Zakopane, Poland. February 16-17, 2013. 12 Ryszawy D, Sarna M, Szpak K, Rak M, Michalik M, Siedlar M, ZubaSurma E, Korochoda W, Madeja Z, Czyż J. Cx43 participates in the pre-selection of metastatic progenitors during prostate cancer metastatic cascade in vivo. 40th Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology. Zakopane, Poland. February 16-17, 2013.. 11.

(12) Scientific achievements. 13 Sarna M, Pilat A, Olchawa M, Zadlo A, Gkogkolou P, Burda K, Bohm M, Sarna T. Melanin modifies nanomechanical properties of melanoma cells. 17th Meeting of the European Society for Pigment Cell Research. Geneva, Switzerland. September 11-13, 2012. 14 Hermanowicz P, Sarna M, Burda K, Gabrys H. JRobust: an open source application for analysis of AFM force curves. 7th Plant Biomechanics International Conference. Clermont-Ferrand, France. August 20-24, 2012. 15 Wytrwal M, Sarna M, Bednar J, Kozik B, Kepczynski M, Nowakowska M. Interactions of a hydrophobically modified poly(allylamine hydrochloride). with. zwitterionic. lipid. membranes.. The. 9th. International Symposium Polyelectrolites. Lausanne, Switzerland. July 9-12, 2012. 16 Sarna M, Wojcik K A, Hermanowicz P, Burda K, Michalik M. Nanomechanical analysis of bronchial fibroblasts from asthmatic patients - role of cell stiffness in fibroblasts-to-myofibroblasts transition. 35th Annual Meeting of the German Society for Cell Biology. Dresden, Germany. March 21-24, 2012. 17 Wojcik K A, Sarna M, Czyz J, Sanak M, Michalik M. TGF-β-induced fibroblast to myofibroblast transition is enhanced in bronchial fibroblasts derived from asthmatic patients. 35th Annual Meeting of the German Society for Cell Biology. Dresden, Germany. March 21-24, 2012. 18 Hermanowicz P, Sarna M, Burda K, Gabrys H. Light induces changes in the mechanical properties of chloroplasts. 39th Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology. Zakopane, Poland. February 4-8, 2012. 19 Sarna M, Olchawa M, Pilat A, Szewczyk G, Burda K, Sarna T. Atomic force microscopy analysis of retinal pigment epithelium cells subjected to photodynamic stress. 21st International Pigment Cell Conference.. 12.

(13) Scientific achievements. Bordeaux, France. September 8-12, 2011 (Abstract published in Pigment Cell and Melanoma Research, 2011; 24(4): 818, 2011). 20 Sarna M, Wojcik K A, Burda K, Michalik M. Atomic force microscopy analysis of human bronchial fibroblasts from asthmatic patients. 2nd Polish Congress of Biochemistry and Cell Biology. Cracow, Poland. September 5-9, 2011. 21 Sarna M, Koczurkiewicz P, Michalik M, Podolak I, Burda K. The influence of triterpenoid sapponins on nanomechanical properties of human prostate cancer cells. 2nd Polish Congress of Biochemistry and Cell Biology. Cracow, Poland. September 5-9, 2011. 22 Wlodarczyk A, Sarna M, Zabczyk M, Michalik M, Burda K, Undas A. The influence of dust nanoparticles on the nanomechanical properties of human endothelial cells. 2nd Polish Congress of Biochemistry and Cell Biology. Cracow, Poland. September 5-9, 2011. 23 Wytrwal M, Sarna M, Bednar J, Kozik B, Kepczynski M, Nowakowska M. Hydrophobic modifications of poly(allylamine hydrochloride) and its characteristic. International Conference – Polymers on the Odra River. Opole, Poland. June 6-7, 2011. 24 Sarna M, Ryszawy D, Hermanowicz P, Wojcik K A, Madeja Z, Burda K. Elastic analysis of rat prostate cancer cells. 38th Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology Zakopane, Poland. February 13-17, 2011. 25 Sarna M, Ryszawy D, Hermanowicz P, Wojcik K A, Madeja Z, Burda K. Elastic analysis of Dunning R3327 rat prostate cancer cells. FEBS Advanced Lecture Course: Analysis and Engineering of Biomolecular Systems. Spetses, Greece. September 11-17, 2010. 26 Wytrwał M, Kępczyński M, Sarna M, Jachimska B, Nowakowska M. Stabilization of liposomes covered with modified poly(allylamine) hydrochloride. III International Conference: YES (“Toxic substances in the environment”), Cracow, Poland. September 6-10, 2010.. 13.

(14) Scientific achievements. 27 Szpak K, Wybieralska E, Niedzialkowska E, Sarna M, Bechyne I, Michalik M, Burda K, Madeja Z, Czyz J. Cx43 alleviates the transmigration of human prostate Du145 cells trough artificial micropores. 37th Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology. Zakopane, Poland. February 13-17, 2010. 28 Rabbi M, Sarna M, Manson L, Jiang Y, Marszalek P. High force elastic profiles of single and double stranded polynucleotides probed with AFM force spectroscopy. 54th Annual Meeting of the Biophysical Society. San Francisco, California, USA. February 23, 2010 (Abstract published in Biophysical Journal, 2010; 3: 594a). 29 Sarna M, Wybieralska E, Drukala J, Madeja Z. Topographical control of cancer cell migration. 90th anniversary of the AGH University of Science and Technology. Cracow, Poland. May 28-30, 2009. 30 Sarna M, Drukala J, Wybieralska E, Madeja Z. Topographical control of tumor cell migration. 1st Polish Congress of Biochemistry and Cell Biology. Olsztyn, Poland. September 7-11, 2008.. 14.

(15) Summary in Polish. Streszczenie Czerniak jest jednym z najbardziej złośliwych nowotworów, który charakteryzuje się wysokim stopniem śmiertelności jeżeli zostanie wykryty na późnym etapie rozwoju. Warto zaznaczyć, że wczesna oraz prawidłowa diagnoza gwarantuje niemalże całkowite wyleczenie. Obecnie diagnoza czerniaka polega w głównej mierze na analizie histopatologicznej. Pomimo, iż takie badanie może ustalić, czy komórki nowotworowe zaczęły naciekać sąsiadujące tkanki, pozostaje co najwyżej oceną jakościową gdyż nie pozwala przewidzieć ile komórek nowotworowych może dać przerzut. W konsekewncji nie jest możliwe precyzyjne rokowanie co do dalszego rozwoju choroby. Od. lat. prowadzone. rezeprezentujących. różne. są. badania. dziedziny. nauki. przy od. udziale biologii. specjalistów komórki. po. nanotechnologię w celu opracowania nowych, niezawodnych markerów zdolności komórek rakowych do rozprzestrzeniania się i przerzutowania. W ostatnim czasie właściwości nanomechaniczne komórek rakowych, szczególnie ich elastyczność, wzbudzają duże oczekiwania w tym zakresie. Jest to związane z dużą specyfiką pomiarów elastyczności komórek rakowych oraz unikatowym charakterem tego markera komórkowego. Przeprowadzone w ostatnim czasie badania pokazały, że komórki rakowe, mające niską wartość modułu Younga (wielkości opisującej elastyczność) wykazują jednocześnie zwiększoną inwazyjność. W celu opracowania nowej metody diagnozy czerniaka niezbędne jest dokładne poznanie procesu przerzutowania tego złośliwego nowotworu. Warto zaznaczyć, że pomimo znacznego wysiłku badaczy, proces ten pozostaje w pełni nie wyjaśniony. Co ważne w rozważaniach tych jak dotąd nie brano poważnie pod uwagę jednej z najbardziej charakterystycznych cech tego nowotworu, jaką jest zdolność komórek czerniaka do syntezy barwnika melaninowego. Ponadto nie wiadomo, jaki jest związek pomiędzy obecnością melaniny w komórkach czerniaka a ich fenotypem metastatycznym. Należy zaznaczyć, że do tej pory nie rozważano efektu mechanicznego ziaren melaniny na właściwości elastyczne komórek czerniaka a w konsekwencji na zdolności inwazyjne tych komórek.. 15.

(16) Summary in Polish. Głównym celem niniejszej pracy doktorskiej było zbadanie wpływu ziaren melaniny na właściwości nanomechaniczne komórek czerniaka oraz zdolności inwazyjne tych komórek w warunkach in vitro. Wykorzystując unikatowe narzędzie nanotechnologii jakimm jest mikroskop sił atomowych wykazano, że obecność twardych, niedeformowalnych ziaren melaniny w komórkach czerniaka istotnie modyfikuje właściwości nanomechaniczne tych komórek, powodując wzrost modułu elastyczności. Ponadto pokazano, że obecność ziaren melaniny obniża zdolności inwazyjne komórek czerniaka in vitro, poprzez zmniejszenie podatności do deformacji ciała komórkowego, co warunkuje przenikanie komórek przez bariery mechaniczne podczas inwazji. Należy podkreślić, że obserwowany w tej pracy efekt zależy od liczby ziaren wewnątrz komórek. Szczegółowa analiza kluczowych parametrów biologicznych komórek czerniaka wykazała, że wpływ melaniny na inwazyjność czerniaka na poziomie komórkowym ma czysto mechaniczny charakter. Wyniki uzyskane w niniejszej pracy po raz pierwszy dostaraczają dowodów na to, że obecność pigmentu w komórkach czerniaka może zmniejszać zdolności inwazyjne tych komórek. Wyniki te mają kluczowe znaczenie dla zrozumienia procesu przerzutowania komórek czerniaka jak również mogą przyczynić się do opracowania nowej metody diagnozy tego złośliwego nowotworu. Stwierdzona w tej pracy zależność pomiędzy ilością ziaren melaniny a zdolnościami inwazyjnymi komórek czerniaka wskazuje na możliwość oceny stopnia ryzyka wystąpienia przerzutu w oparciu o pomiar zawartości melaniny w komórkach pozyskanych od pacjenta ze zdiagnozowanym nowotworem. Komórki zawierające więcej ziaren melaniny powinny mieć mniejsze prawdopodobieństwo dania przerzutu. W połączeniu z istniejącymi metodami, taka nowa metoda mogłaby przyczynić się do lepszej diagnozy tego złośliwego nowotworu skóry.. 16.

(17) Summary in English. Summary Melanoma is one of the deadliest types of cancer with very high mortality rates when diagnosed in the late stage of its development. Importantly, if diagnosed early, the treatment of melanoma can be very successful. The most common and generally accepted method of melanoma diagnosis today is histopathology analysis. Although such analysis can determine if cancer cells have already begun to invade the surrounding tissues, it is not quantitative and cannot provide any information on how many cells are capable of undergoing metastasis. Therefore, such analysis cannot predict with any levels of certainty the outcomes of the disease. Over the years extensive research efforts were made to identify new and reliable markers of the ability of cancer cells to spread and metastasize, involving researchers representing different fields of science ranging from cell biology to nanotechnology.. Recently,. nanomechanical. properties. of. cancer. cells,. particularly their elasticity has shown considerable promise in this regard. This is justified by high specificity of elasticity measurements and the unique character of such a cellular marker. Different studies have demonstrated that cancer cells with low values of the Young’s modulus (the measure of cell elasticity) also exhibit increased invasive abilities. In order to develop an improved method of melanoma diagnosis it is necessary to fully comprehend the process of metastasis of malignant melanoma, which in spite of significant research efforts is not completely understood. Importantly, one of the most characteristic features of melanoma i.e. the ability of melanoma cells to synthesize melanin pigment was typically omitted. Moreover, the relationship between melanin pigmentation and metastatic phenotype of melanoma cells remains unclear. It is important to stress that until now, the mechanical effect of melanin granules on elastic properties of melanoma cells and their invasive abilities has not been taken into consideration. The primary objective of this PhD Thesis was to examine the effect of melanin granules on nanomechanical properties of melanoma cells and the. 17.

(18) Summary in English. impact of the pigment granules on the cells invasive abilities in vitro. The obtained results demonstrate that melanin granules dramatically modify nanomechanical properties of melanoma cells by increasing the cells elastic modulus. Moreover, the presence of melanin granules inhibits the invasive abilities of melanoma cells in vitro by reducing the cell capability to undergo extensive deformation when transmigrating through a mechanical barrier. It is important to stress that the observed effect depends on the number of melanin granules inside the cells. Furthermore, analysis of the cell vital functions indicates that the inhibitory effect of melanin granules is exclusively mechanical in nature. Results of this work provide the first experimental evidence that melanin granules can have an inhibitory effect on the invasive abilities of melanoma cells. This observation is of key importance considering that the current concepts of melanoma metastasis ignore the mechanical effect of melanin granules on melanoma invasiveness. The revealed correlation between the number of melanin granules in melanoma cells and their metastatic abilities can have significant impact on future melanoma diagnosis. Quantitative analysis of melanin inside melanoma cells, obtained from patients with diagnosed melanoma may facilitate simple and accurate determination of the cell metastatic phenotype. Thus, cells containing more melanin granules would likely indicate lower invasive potential. Together with existing diagnostic methods, this could lead to a more complete melanoma diagnosis.. 18.

(19) Background information. 1. Background information. This chapter provides concise background information about the undertaken study emphasizing its key aspects, such as the presence of melanin granules in melanoma cells as a unique feature of this cancer, elasticity of cancer cells as an important mechanical property of the cells during the process of metastasis, and the principles of atomic force microscopy and spectroscopy as a method for analyzing complex nanomechanical properties of living cells. While the chapter introduces the reader to the most important biophysical and methodological issues addressed by this PhD study, it is organized in a way to avoid overlap with the introductory paragraphs that highlight major results described in each paper of the PhD Thesis.. 1.1. Melanoma & melanin synthesis. Melanoma. is. a. malignant. tumor. originating. from. transformed. melanocytes – specialized cells that normally produce melanin (Lin and Fisher, 2007). Unlike any other cancer cells, melanoma cells can synthesize melanin in large amounts (Riley, 2003). However, while in normal melanocytes melanogenesis is regulated by different factors (Slominski et al., 2012); melanin synthesis in melanoma cells is highly deregulated (Slominski et al., 1998). This, together with the fact that melanoma cells do not excrete the pigment, can result in heavy pigmentation of the cells due to the accumulation of large number of melanin granules (Lazova nad Pawelek, 2009). Although in vivo, melanoma cells can synthesize melanin in large amounts; their pigmentation under in vitro conditions is rather rare (Imokawa and Mishima, 1982). This makes it very difficult to study the effects of melanin pigmentation in melanoma cells. Melanoma is one of the most invasive types of cancer with very poor prognosis when diagnosed in the metastatic stage (Gaggioli and Sahai, 2007). Importantly, if diagnosed early the treatment of melanoma can be very. 19.

(20) Background information. successful. Therefore, a reliable method for early diagnosis of melanoma and determination of the melanoma cell metastatic potential is of key importance.. 1.1.1 Melanin. pigmentation. and. metastatic. behavior. of. melanoma cells The main biological function of melanin pigment in normal cells is generally well understood and is usually related to photoprotection and mimicry (d’Ishia et al., 2013). On the other hand, the role of melanin in melanoma remains unclear. Moreover, the relationship between melanin pigmentation and metastatic behavior of melanoma cells is controversial. It has been postulated that melanin synthesis in melanoma cells can inhibit the host immune system, making the cancer cells less vulnerable to the host’s defense mechanisms (Slominski et al., 2009). Under these circumstances, pigmented melanoma cells can be viewed as the more aggressive cell subset of a heterogeneously pigmented tumor. On the other hand pigmentation of melanoma cells is also viewed as an indicator of the cells differentiation state. Following this reasoning pigmented melanoma cells are considered to be more differentiated and therefore less aggressive (Carreira et al., 2006). Importantly, none of the explanations take into consideration biophysical aspects of the phenomenon such as the mechanical effect of melanin granules on elastic properties and invasive abilities of pigmented melanoma cells.. 1.1.2 Physicochemical properties of melanin pigment Melanin is a biological pigment found in a variety of normal pigmented tissues including the skin, hair, eyes, brain, and inner ear (Hearing, 2005). Melanin is commonly divided into two main types: the brown-black eumelanin, and the yellow-reddish pheomelanin (Samokhvalov et al., 2005). Both types of. 20.

(21) Background information. melanin can be found in human melanoma cells (Hu et al., 2009; Matthews et al., 2011). Melanin in pigmented cells including melanoma cells is present in the form of distinct granules called melanosomes (Dell’Angelica, 2003). These organelles display a range of different shapes and sizes (Liu et al., 2005), and have unusual mechanical properties. Elasticity analysis of the pigment granules revealed that melanosomes were very stiff and hard to deform (Guo et al., 2008). Over the years melanin has been of interest to researchers from different fields due to unique physicochemical properties of melanin pigment, including its paramagnetism (Meredith and Sarna, 2006; d’Ischia et al., 2009; Mostert et al., 2012).. 1.2 Metastasis & elasticity of cancer cells The ability of cancer cells to spread all over the host body and colonize different organs is one of the main causes of death of patients with diagnosed cancer (Nguyen et al., 2009). Among cellular properties that determine the efficiency of cancer cells to spread, elasticity of the cells is viewed as a key parameter (Makale, 2007).. 1.2.1 Metastasis of cancer cells Metastasis of cancer cells is a complex and multistep process that consists of detachment of cancer cells from the primary tumor, active migration, invasion of cancer cells into the blood vessels, extravasation, and subsequent growth in a distant tissue (Chambers, 1999). In order to metastasize, cancer cells have to acquire many specific features, which determine the cells’ metastatic behavior (Gupta and Massague, 2006). Figure 1 shows a schematic description of the process of metastasis.. 21.

(22) Background information. Figure 1. Schematic description of the process of metastasis. During the first stage of metastasis, cancer cells invade the basement membrane surrounding the tumor from normal tissues (1). Subsequently, the cells pass through the extracellular matrix deposited by normal cells (2) and enter the blood vessels (3). After circulating in the bloodstream, cancer cells arrest (4), extravagate and reach a metastatic site (modified from: Robbins and Kumar, 2010).. 1.2.2 Deformation of cancer cell body during transmigration through a mechanical barrier One of the most critical steps in the process of metastasis is the invasion of host tissues (Friedl and Wolf, 2003). During this stage of metastasis, cancer cells encounter multiple barriers inside the tissues they invade (Sherwood, 2006; Deeken and Loscher, 2007; Reymond et al., 2013), with the most frequent being. 22.

(23) Background information. the basement membranes (Kelley et al., 2014). In order to pass through these barriers, cancer cells must be able to undergo extensive deformation of their cellular body (Ochalek et al., 1988; Guck et al., 2005). This is because normal tissues are built up of tightly joined constituents such as: cells, extracellular matrix and microvessels, which form such mechanical barriers. Invasion of cancer cells into the tissues begins with the formation of invadopodia – thin cellular structures that enable penetration of the cancer cell into adjacent normal cells (Schoumacher et al., 2010). To facilitate this process, cancer cells express metaloproteinases, which degrade extracellular matrix. As a result, cancer cells form an opening in the tissue, through which the cells then transmigrate. Figure 2 shows key stages of transmigration of a cancer cell through a mechanical barrier such as the basement membrane.. Figure 2. Key stages of transmigration of a cancer cell through a basement membrane. At first stage non-invasive tumor cell do not degrade the surrounding tissue (1). Once a cancer cell becomes invasive, invadopodia are formed and the cell penetrates the basement membrane (2). Next, the cell creates an opening in the basement membrane by expressing metalloproteinases, which degrade the extracellular matrix. Transmigration is then possible by squeezing the cellular body into a narrow opening (modified from: Schoumacher et al., 2010).. 23.

(24) Background information. As evident from Figure 2, once a cancer cell breaches the basement membrane, it then must drag its entire body through a narrow opening. It is during this stage of invasion that cancer cells must significantly deform their cellular body in order to transmigrate through a tissue. It is important to stress that cell central body is the thickest part of a cell, which must undergo extensive deformation, compared to outer regions of the cell such as thin lamellipodia (Manimaran et al., 2006; Fu et al., 2012; Krause et al., 2013). This is because this region of the cell contains nucleus with densely packed chromatin, which makes it the most critical area of a cancer cell during transmigration trough different mechanical barriers in vitro and in vivo.. 1.2.3 Elasticity as a unique marker of cancer transformation, progression and invasion Elasticity is an important mechanical property of cancer cells, which is acquired by the cells during their transformation and is due to significant remodeling of the cell cytoskeleton (Bhadriraju and Hansen, 2002; Ketene et al., 2012). As a result of these changes, cancer cells become very soft and highly deformable. This mechanical property of cancer cells is believed to be one of the main reasons for the cell ability to pass through mechanical barriers much more efficiently. Different in vitro biomechanical assays demonstrated that cancer cells with higher elastic properties exhibited increased transmigration abilities, compared to cancer cells with lower elasticity (Watanabe et al., 2012; Koczurkiewicz et al., 2013; Zhou et al., 2013; Ryszawy et al., 2014). Based on such observations, elasticity is viewed as an important indicator of the metastatic phenotype of cancer cells and is even considered as a potential diagnostic marker (Cross et al., 2007). Indeed, correlation between elasticity of cancer cells isolated from patients and their clinical conditions has been reported (Cross et al., 2010; Plodinec et al., 2012). The relationship between elasticity of cancer cells and their metastatic behavior can simplistically be depicted as shown below:. 24.

(25) Background information. Elasticity. Metastasis. (1). It is believed that most if not all cancer cells fulfill this relationship.. 1.3 Atomic force microscopy & elasticity analysis Atomic force microscope (AFM) (Binnig et al., 1986) is by far the most versatile member of a family of microscopes known as scanning probe microscopes (SPMs) (Bhushan, 2010). These instruments can generate high resolution images of different samples by measuring some form of interaction between the probe and the sample. One of the most spectacular features of the AFM is the ability to examine mechanical properties of samples, in particular their elasticity. Over the years AFM has become a method of choice for investigating mechanical properties of different biological materials, such as: human platelets (Radmacher et al., 1996), fibroblasts (Rotsch et al., 1999), red blood cells (Bremmell et al., 2006), bone marrow (Tao et al., 1992) and bacteria (Fritz et al., 1994). The applicability of AFM to analyze elasticity of cells has been mostly demonstrated in case of cancer cells (e.g. Cross et al., 2007; Cross et al., 2010; Plodinec et al., 2012).. 1.3.1 Working principle of AFM Unlike optical microscopes, which ‘look’ at a sample from a distance, AFM operates in the near field and virtually ‘touches’ the sample. AFM ‘senses’ the surface of a sample by using a sharpened tip mounted on the end of a flexible cantilever. Changes in the surface topography of a sample cause the cantilever to bend. Measurement of this bending is made with a photodiode, by detecting laser light reflected from the cantilever. The registered bending is then ‘translated’ into an operating signal, which runs the system using a feedback loop. 25.

(26) Background information. mechanism. This simple yet elegant method of surface analysis can deliver high resolution images without the need of any sample preparation. Figure 3 shows a schematic layout of the working principal of AFM.. Figure 3. Schematic layout of the working principal of AFM. AFM scans the surface of a sample using a sharpened tip mounted on the end of a flexible cantilever. Once the tip encounters a change in the topography the cantilever bends due to atomic interactions between the tip and the sample. This bending is registered using an optical method, in which laser and photodiode detector are employed.. Positioning of the probe relative to the sample is made using a piezoelectric scanner, which moves the sample (in a sample scanning system) or the probe (in a probe scanning system) in all three axes. Piezoelectric crystals are used for their great precision and excellent repeatability. The entire system is controlled by a computer and dedicated software. AFM can be coupled with other imaging techniques such as optical microscopy to increase the measuring capabilities of the system (Madl et al., 2006).. 26.

(27) Background information. 1.3.2 Indentation experiment AFM is frequently used to study mechanical properties of different samples, including living cells. Mechanical analysis with AFM is performed in the so-called force spectroscopy mode (Dufrene, 2003). The simplest way to investigate elasticity of a sample with AFM is to perform an indentation experiment (Sokolov et al., 2013). The principle of such experiment consists of determining the relationship between the force exerted on a sample and the depth of indentation. During indentation experiment the probe is brought into close proximity with the sample, so that the tip starts to deform the sample. Figure 4 shows a schematic description of an indentation experiment. The deflection of the cantilever, a measure of the force exerted on a sample by the tip, is recorded as a function of the distance covered by the cantilever. Based on the Hooke’s law, deflection can be converted into force, transforming the deflectiondistance curve into the force-indentation curve. Using a proper contact mechanics model, the value of the Young’s modulus (the quantitative measure of elasticity) can be determined.. Figure 4. Schematic description of an indentation experiment. At the point of initial contact (position A), the cantilever (light grey) experiences relatively small adhesion forces. When these forces are negligible, the cantilever is virtually un-deflected and the sample remains un-deformed. When the cantilever (dark gray) is pushed into the sample and starts to bend (position B), the sample begins to deform. The sum of the depth of the indentation δ and the corresponding deflection d of the cantilever is equal to the distance ∆z covered by the cantilever. ∆z is the distance traveled by the cantilever after the initial contact and represents the measured value (modified from: Hermanowicz, 2010).. 27.

(28) Background information. 1.3.3 Hertz model of elastic deformations The relationship between the applied force F and the depth of indentation δ depends on the tip geometry and inherent properties of the sample. In order to express it in a simple fashion, a number of assumptions have to be made. In most cases, the sample is assumed to be thick, homogenous, its deformation is considered to be purely elastic (i.e. viscoelastic and plastic effects are omitted) and adhesive forces deemed negligible. Such samples are often said to exhibit a Hertzian behavior (Lin et al., 2007a). Under these assumptions, for basic tip profiles, the force-indentation relation may be expressed as a polynomial: F = λδβ,. [1]. where λ and β are profile specific parameters. For instance, when a sample is deformed by a paraboloidal or a sharp conical tip, force-indentation relation is given by the following equations:. =. √ (

(29) ). =. / (for a paraboloid);. π (

(30) ). (for a cone),. [2]. [3]. where R is the paraboloid’s end radius, is the cone’s half-opening angle, E and ν are sample specific parameters – the Young’s modulus and the Poisson ratio, respectively. It should be emphasized, that polynomial relation holds for a sharp cone and sharp pyramid, but not for a pyramid or cone with a rounded apex that is shapes of common AFM tips. Although the relations for rounded tips have been derived (Costa and Yin 1999; Na et al., 2004; Rico et al., 2005), they are too cumbersome for routine use. As a result, the common practice is to apply the polynomial equation for the sharp tip.. 28.

(31) Objectives of the work. 2. Objectives of the work. The primary objective of this PhD study was to examine the impact of melanin granules on nanomechanical properties of melanoma cells and to determine whether the pigment granules affect the invasive abilities of melanoma cells in vitro. In order to achieve the main objective, the following specific goals were addressed: i.. To compare the elasticity of pigmented melanoma cells with the elasticity of normal melanocytes. Melanoma cells can contain large amounts of melanin. Although elastic properties of individual pigment granules were recently determined, their impact on nanomechanical properties of melanoma cells, has not been examined. Furthermore, it is believed that most if not all cancer cells are significantly softer than their normal counterparts. Importantly, the difference in elastic properties between cancer and normal cells is the basis of the nanodiagnosis of cancer. Therefore, such comparative analysis was necessary considering that until now no studies of the impact of melanin on nanomechanical properties of pigmented cells were reported.. ii. To investigate whether melanin granules have any impact on the invasive abilities of melanoma cells in vitro. It is important to stress that current concepts of melanoma metastasis do not take into consideration the effect of melanin granules on nanomechanical properties of melanoma cells. Considering the importance of elastic properties of cancer cells and their metastatic behavior, the mechanical effect of melanin granules on the invasive abilities of melanoma cells was to be elucidated.. 29.

(32) Objectives of the work. iii. To. determine if. the invasive abilities of pigmented. melanoma cells in vitro depend on the number of pigment granules inside the cells. This study was based on a working hypothesis that the expected effect of melanin granules on the ability of melanoma cells to transmigrate through a mechanical barrier is mainly mechanical in nature. To confirm this hypothesis, the effect was tested as a function of the number of intracellular melanin granules. In parallel, the impact of varying content of melanin granules on the cell key functions was examined.. 30.

(33) Experimental section. 3. Experimental section. This chapter provides brief information about the materials and methods used in this work. Detailed description of the actual materials and specific methods employed in each study can be found in appropriate experimental sections of the papers included in the Appendix of this PhD Thesis.. 3.1. Cells. Different melanoma cells were used to study the effects of melanin pigment on nanomechanical properties of these cancer cells. For comparison, normal human melanocytes were used. All cell cultures described in this work were performed in the Group of Molecular Biophysics and Bioenergetics, Department of Medical Physics and Biophysics, Faculty of Physics and Applied Computer Science, AGH University of Science and Technology, Cracow, Poland. The following cells were studied: •. Normal human melanocytes (NHMs) kindly provided by Professor Markus Böhm from the Department of Dermatology, Laboratory. for. Neuroendocrinology. of. the. Skin. and. Interdisciplinary Endocrinology, University of Münster, Münster, Germany. •. Human melanoma cells (SKMEL-188) kindly provided by Professor Andrzej Słomiński from the Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, USA.. •. Murine melanoma cells (B16F10) made available as a result of collaboration with the Department of Biophysics, Faculty of. 31.

(34) Experimental section. Biochemistry,. Biophysics. and. Biotechnology,. Jagiellonian. University, Cracow, Poland.. 3.2 Optical microscopy An array of different optical microscopes was used to examine the cells vital functions. These included: •. Olympus IX71 microscope was employed for routine inspection of the cells during cell culture. This microscope was available at the Department of Medical Physics and Biophysics, Faculty of Physics and Applied Computer Science, AGH University of Science and Technology, Cracow, Poland.. •. Leica DM IRE2 microscope was used to examine the cytoskeleton of B16F10 cells. This microscope was made available through collaboration with the Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, Poland.. •. Leica DMI6000b microscope was used to analyze migration abilities of SKMEL-188 cells. This microscope was made available through collaboration with the Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, Poland.. •. Nikon A1 confocal microscope was used to examine the cytoskeleton of SKMEL-188 cells. These measurements were kindly facilitated by Dr. Mariusz Kępczyński from the Department of. 32.

(35) Experimental section. Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian University, Cracow, Poland.. 3.3 Electron paramagnetic resonance Different techniques are commonly used for the detection of melanin pigment in biological samples. However, most of the methods are not specific and can lead to erroneous conclusions (Sarna and Swartz, 2006). The presence of persistent free radical centers in the melanin pigment enables its specific detection and characterization by electron paramagnetic resonance (EPR) spectroscopy (Sealy et al., 1982). This nondestructive method can be used to analyze melanin in different biological materials, including cells. EPR analysis was carried in the Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, Poland.. 3.4 Atomic force microscopy AFM measurements were conducted employing Agilent 5500 atomic force microscope and were performed in the Laboratory of Molecular Biophysics and Bioenergetics, Department of Medical Physics and Biophysics, Faculty of Physics and Applied Computer Science, AGH University of Science and Technology, Cracow, Poland.. 3.5 Force spectroscopy data analysis Reliable determination of the Young’s modulus from force curves can be problematic. The key difficulty is associated with limited applicability of simple contact mechanics models to complex materials. The most commonly employed. 33.

(36) Experimental section. Hertz model of elastic deformations requires the sample to be thick, linearly elastic and homogenous. Real samples often exhibit non-Hertzian behavior, which makes the determination of the contact point and extraction of the Young’s modulus values from the force-indentation data ambiguous. Most of the automatic approaches to contact point identification developed so far exhibit compromised performance in the presence of high levels of noise or nonHertzian behavior (Lin et al. 2007b). To overcome these obstacles, dedicated software for the analysis of force curves was developed in the course of the study (Hermanowicz et al., 2014).. 3.6 Cell biology methods To examine selected cell vital functions, the following cell biology methods were employed: migration analysis, proliferation assay, cytoskeleton analysis, metalloproteinase expression by zymography assay and Transwell invasion test. Detailed description of the methods used in this work can be found in the materials and methods section of each paper included in the Appendix of this PhD Thesis.. 34.

(37) Results and Discussion. 4. Results and Discussion. Results discussed in this PhD Thesis where published as original scientific papers. Below a short description of key findings made in this work is given.. 4.1 Melanin granules modify nanomechanical properties of pigmented melanoma cells It should be emphasized that until this PhD study was undertaken, the effect of melanin granules on nanomechanical properties of pigmented cells, including melanoma cells, was not examined. This important issue has been addressed in carefully designed experiments, in which cell elasticity of two pigmented melanoma cell lines – murine melanoma B16F10 and human melanoma SKMEL-188 – was measured. The study revealed that melanin granules dramatically modified nanomechanical properties of the cells. The values of the Young’s modulus were significantly higher in pigmented cells when compared to non-pigmented cells. Furthermore, the Young’s modulus of pigmented cells depended on the number melanin granules inside the cells.. 4.2 Presence of melanin granules in melanoma cells is likely to make difficult nanodiagnosis of melanoma Cancer cells are generally considered to be much softer than their normal counterparts. This difference between normal and cancer cells has been utilized in so-called nanodiagnosis of cancer (Choi et al., 2006). Such diagnostic approach is based on nanomechanical analysis of living cells isolated from patient with suspected cancer typically employing atomic force microscopy. However, as demonstrated in this PhD study, pigmented melanoma cells were. 35.

(38) Results and Discussion. actually much stiffer than normal melanocytes. Comparative elasticity analysis of melanoma cells and normal melanocytes revealed that it would be very hard, if possible at all, to distinguish melanoma cells from melanocytes based solely on nanomechancial measurements. Furthermore, considering that the amount of melanin in melanoma cells often varies and can be higher than in normal melanocytes, nanodiagnosis of melanoma is expected to be very difficult. Such analysis could even lead to erroneous conclusions.. 4.3 Melanin. granules. inhibit. invasive. abilities. of. melanoma cells in vitro Although the relationship between metastatic phenotype of melanoma cells and melanin presence in the cells has been previously considered, the exact impact of melanin granules on the invasive abilities of melanoma cells was not examined. Furthermore, the mechanical effect of melanin granules on transmigration capabilities of pigmented melanoma cells was not addressed. Results of this PhD study have clearly shown that melanin granules inhibit the invasive abilities of melanoma cells in vitro. Furthermore, the observed inhibitory effect depended on the number of the pigment granules inside the cells. Detailed analysis of key cellular parameters including tumorigenic properties of melanoma cells indicated that the observed effect was exclusively mechanical in nature.. 4.4 Melanin pigment is a mechanical modulator of both elastic properties and metastatic capabilities of pigmented melanoma cells The inhibitory effect of melanin on the invasive abilities of pigmented melanoma cells is a novel observation, which has not been reported yet by other. 36.

(39) Results and Discussion. researchers. It may give new insight into the role of melanin in metastatic behavior of pigmented melanoma melanoma cells. Consequently, the relationship (1) in the case of pigmented melanoma cells should be modified to account for the presence of melanin inside the cells that act as a unique nanomechanical modulator of both the elastic properties and metastatic capabilities capabilities of the cells, as follows: Melanin (2) Elasticity. Metastasis. 4.5 Quantitative analysis of melanin content in melanoma cells could determine the metastatic potential of melanoma cells leading to improved melanoma diagnosis The observed correlation in this PhD study between the number of melanin granules in melanoma cells and their invasive abilities may have a significant impact on future melanoma diagnosis. diagnosis. As a highly aggressive tumor, melanoma once diagnosed, is immediately excised and and sent to histopathology. Although such analysis can determine whether cancer cells have already begun to invade the surrounding tissues, it is not quantitative and cannot provide any information on how many cells are capable of undergoing metastasis. Therefore it cannot predict with any level of certainty the outcome of the disease. Quantitative uantitative analysis of the melanin content in melanoma cells, obtained from patients with diagnosed melanoma could facilitate simple and accurate determination of the cell metastatic me potential.. Cells, containing more pigment granules, would likely indicate lower invasive potential. This, together with existing diagnostic methods, could lead to a more reliable melanoma diagnosis. Such a diagnosis could better assess the risk of developing metastatic tumors,. 37.

(40) Results and Discussion. helping to employ optimized treatment. Of course, many additional experiments are necessary before such approach can be tested in a pre-clinical setup.. 38.

(41) Concluding remarks. 5. Concluding remarks. This PhD dissertation focused on the mechanical effect of melanin granules on the invasive abilities of melanoma cells in vitro.. 5.1. Key findings of the work. The most important results of this PhD study can be summarized as following: i.. Melanin granules dramatically modify nanomechanical properties of pigmented melanoma cells.. ii.. Melanin granules inhibit invasive abilities of melanoma cells in vitro.. iii.. The observed inhibitory effect depends on the number of melanin granules inside the cells and is mechanical in nature.. 5.2 Postulates formulated based on the observed effect The observed inhibitory effect of melanin granules on the invasive abilities of melanoma cells in vitro justifies formulating the following postulates: i.. Nanodiagnosis of melanoma based entirely on the nanomechanical analysis of the cells could be unreliable.. ii.. Quantitative analysis of the melanin content in melanoma cells isolated from patients with diagnosed melanoma could lead to the. 39.

(42) Concluding remarks. development of a novel, relatively simple method for determination of the metastatic phenotype of these cancer cells. iii.. Pigmentation of melanoma cells should thoroughly be analyzed when studying melanoma cells in vitro and in vivo.. 5.3 Issues to be addressed in future studies Although results of this PhD study validate specific conclusions that have been reached, the work raises the following important issues that should be addressed in future studies: i.. Demonstration of the inhibitory effect of melanin granules on melanoma cells invasive abilities under in vivo conditions employing an appropriate animal model.. ii.. Examination if the inhibitory effect of melanin granules depends on the type of melanin pigment.. 40.

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