Karel SOUČEK Speciální metody FŽ CANCER PLASTICITY Department of Cytokinetics Institute of Biophysics AS CR Kralovopolska 135 612 65 Brno , Czech Republic Tel: + 420 541 517 166 ksoucek@ibp.cz @souceklab https://www.em.muni.cz/cache/multithumb_thumbs/logo_muni_web-790x395-2008259181.jpg * podpůrné proliferační signály * deregulace supresorů růstu/proliferace * odolnost k buněčné smrti * neomezená replikace * neoangiogeneze * invaze a metastázování * mutace a genomická nestabilita * zánět * přestavba energetického metabolismu * únik před zničením imunitním systémem • Typické znaky nádorové buňky Výsledek obrázku pro hallmarks of cancer Douglas Hanahan & Robert A. Weinberg: Hallmarks of Cancer: Next Generation, Cell, 2011 Screen Shot 2018-02-13 at 11.11.28 AM.png Why is cancer so devastating? Screen Shot 2018-02-13 at 11.11.48 AM.png cancer death portion per cancer type cancer-related death cause estimate Why is cancer so devastating? https://www.svod.cz/graph/?sessid=o61edps4el1idtk6e5bst0cft0&typ=incmor&diag=C61&pohl=m&kraj=&vek_o d=1&vek_do=18&zobrazeni=graph&incidence=1&mortalita=1&mi=0&vypocet=w&obdobi_od=1977&obdobi_do=2017& stadium=&t=&n=&m=0&pt=&pn=&pm=&t=&n=&zije=&umrti=&lecba=# https://www.svod.cz/graph/?sessid=o61edps4el1idtk6e5bst0cft0&typ=incmor&diag=C61&pohl=m&kraj=&vek_o d=1&vek_do=18&zobrazeni=graph&incidence=1&mortalita=1&mi=0&vypocet=w&obdobi_od=1977&obdobi_do=2017& stadium=&t=&n=&m=1&pt=&pn=&pm=&t=&n=&zije=&umrti=&lecba=# https://www.svod.cz/graph/?sessid=o61edps4el1idtk6e5bst0cft0&typ=incmor&diag=C50,D05&pohl=z&kraj=&v ek_od=1&vek_do=18&zobrazeni=graph&incidence=1&mortalita=1&mi=0&vypocet=w&obdobi_od=1977&obdobi_do=2 017&stadium=&t=&n=&m=1&pt=&pn=&pm=&t=&n=&zije=&umrti=&lecba=## https://www.svod.cz/graph/?sessid=o61edps4el1idtk6e5bst0cft0&typ=incmor&diag=C50,D05&pohl=z&kraj=&v ek_od=1&vek_do=18&zobrazeni=graph&incidence=1&mortalita=1&mi=0&vypocet=w&obdobi_od=1977&obdobi_do=2 017&stadium=&t=&n=&m=0&pt=&pn=&pm=&t=&n=&zije=&umrti=&lecba=# Prostate cancer Breast cancer Overview of current research What kind of cells drives metastasis and how we can target them? •Cancer is heterogeneous and not single cell disease. •Complex and dynamic, NOT static “ecosystem”. •Diversity inside tumors is clinical problem limiting the efficacy of targeted therapies and compromising treatment outcomes •90% of cancer related deaths are due to metastasis Overview of current research Does EMT & chemoresistance regulates cell surface phenotype? EMT & metastatic signature of selected cancer subpopulations •British Journal Cancer, 2018 - > Follow up(s) in docetaxel resistant PCa and TNBC What kind of cells and mechanisms drive metastasis and chemoresistance? Trop-2 associates with epithelial phenotype of breast and prostate cancer cells •Carcinogenesis, 2018 -> Follow up(s) in functional role of TSPN, Trop-2 Is there a cure for advanced cancer? Toll-like receptors in chemoresistant prostate cancer •AZV project (ICRC, IBP CAS, UPOL – Z. Culig, K. Souček, V. Študent) Synthetic lethality as a concept for treatment drug resistant cancer •Molecular Cancer Therapeutics, 2017-> Follow up – Molecular Oncology, 2020, Haspin(i) Methodology Team & Collaborators Partners Does Epithelial-to-Mesenchymal Transition (EMT) & chemoresistance regulates cell surface phenotype? epithelial surface signature mesenchymal surface signature loading.gif loading.gif loading.gif loading.gif loading.gif loading.gif Epithelial-Mesenchymal Transition (EMT) •Změna buněčného fenotypu spojená se ztrátou adheze a zvýšením motility table_14_01 EMT & nádory figure_14_18b figure_14_18a Znaky a regulátory EMT nrc2620-f1 Kornelia Polyak & Robert A. Weinberg Nature Reviews Cancer 9, 265-273 (April 2009) Experimentální přístupy BPH par ctrl Benign (BPH-1) EMT TGF-b Cancer associated fibroblast - tumor derived (CAFTD) EMT TGF-b EMT • EMT markers • EMT regulators • Cell shape and behavior Analýza migračního potenciálu Floating cell Protect ring Attached cell Matrix Electrodes Invading cell figure_14_41c Flow cytometry as a tool for understanding of cell phenotype and function single cell/well up to 384 well plate re-culture after sorting (2D, 3D) analysis: CyQuant, ATP, xCelligence, images, SEQ https://qtz-prod.s3.amazonaws.com/facilities/42459/image_full?AWSAccessKeyId=AKIAJKYN3D5X6WPTFAKQ&E xpires=1441950850&Signature=RorF7WK6E8zbpgM9V4xm%2BRD0fhc%3D Fedr, R., Pernicova, Z., Slabakova, E., Strakova, N., Bouchal, J., Grepl, M., Kozubik, A. & Soucek, K. Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells. Cytometry A 83, 472-482, (2013). Kahounova, Z., Kurfurstova, D., Bouchal, J., Kharaishvili, G., Navratil, J., Remsik, J., Simeckova, S., Student, V., Kozubik, A. & Soucek, K. The fibroblast surface markers FAP, anti-fibroblast, and FSP are expressed by cells of epithelial origin and may be altered during epithelial-to-mesenchymal transition. Cytometry A 93, 941-951, (2018). Simeckova, S., Fedr, R., Remsik, J., Kahounova, Z., Slabakova, E. & Soucek, K. Multiparameter cytometric analysis of complex cellular response. Cytometry A 93, 239-248, (2018). Drápela, S., Fedr, R., Remšík, J., Souček, K., High-throughput, parallel flow cytometry screening of hundreds of cell surface antigens using fluorescent barcoding. Methods in Molecular Biology, under review, (2021) Image result for radek fedr Co-culture - media? Radek Fedr Zuzana Kahounová Šárka Šimečková Stanislav Drápela loading.gif lineage surface manifestation loading.gif plasticity Cell phenotypes associate with distinct surface antigens in vitro Epithelial cells Mesenchymal cells EMT induced by: HMLE HMLE-EMT stem cell state MCF10A MCF10A-V12 oncogene (KRasV12) BPH-1 CAFTD03 microenvironment Image result for jan remsik High-throughput cell surface screen identified epithelial- and mesenchymal-like surface signature Remsik, J. et al. Br. J. Cancer 118, 813-819, (2018). Cell phenotypes associate with distinct surface antigens in vitro Hypothesis: The 10-molecule signature associates with plasticity of cancer cells è12-color cytometric panel for analysis of tumor heterogeneity Remsik, J. et al. Br. J. Cancer 118, 813-819, (2018). Six-molecular surface fingerprint predicts docetaxel resistance in prostate cancer patients ↑CD44 homing cell adhesion molecule ↓CD9 tetraspanin ↓EpCAM epi cell adhesion molecule ↑CD59 glycoprotein protectin ↑CD95 Fas receptor ↑SSEA-4 stage-specific embryonic antigen-4 Docetaxel-resistant cell surface profile Drápela, S., et al., Pre-existing cell subpopulations in primary prostate cancer tumors display the surface fingerprint of docetaxel-resistant cells. Under revision. Co-culture - media? Stanislav Drápela Taxane resistance = serious obstacle in the therapy of advanced prostate cancer Aim: To determine unique surface fingerprint of docetaxel-resistant (DR) cells i. “personalized“ prediction of docetaxel effectiveness prior therapy ii. identification of druggable targets for the targeting of DR cells In vivo models – docetaxel-resistant patient derived xenografts docetaxel (33 mg kg−1) van Weerden et al., Br J Cancer, 2009 Výsledek obrázku pro syringe cartoon TURP Výsledek obrázku pro syringe cartoon engrafted s.c. iii. description of the mechanism of docetaxel resistance Data from Cancer Facts & Figures, ACS, 2018 Future plans 1. IHC-based validation of selected biomarkers – e.g. SSEA-4 3. Deciphering molecular mechanism of docetaxel resistance – sorting & RNAseq Output: stratification of the patients for docetaxel therapy 2. Functional validation – CRISPR knock-out models Output: clinical relevance of selected biomarkers Output: complex genomic, transcriptomic and proteomic profile of docetaxel-resistant cells sorting RNA-seq analysis Applying transcriptomic profile of “DOC resistant“ cells to already published advanced PCa & PCa metastasis signatures. Increased amount of parameters = necessity to employ AI in data computation Radek Fedr Aim: To apply machine learning and dimension reduction algorithms in search and recognition of populations with specific or unknown fingerprint Analyses with applied artificial intelligence Output: To reveal unique populations and compare genomic, transcriptomic and proteomic profiles Expert-driven machine learning/ Dimensionality reduction maps Process implementation Microscopy Flow cytometry Sequencing Cell classification/ Clustering algorithms Original parameters translation/Sorting Local structure Global structure Jiřina Procházková Created with BioRender.com Plasticity and intratumoral heterogeneity in triple-negative breast cancer •Complex analysis of tumor and microenvironmental compartments in TNBC samples by mass cytometry •Analysis of epithelial-to-mesenchymal plasticity (EMT) in TNBC patient samples •Identification of new clinically valuable biomarkers • Barbora Kvokačková Ø Complex heterogeneity in TNBC tissues (36 markers) Tumor Stromal Immune ØEMT surface fingerprint in clinical specimens collaboration with Úspěch studentů na Brno Ph.D. Talent 2016 - Agronomická fakulta Remsik, J. et al. Plasticity and intratumoural heterogeneity of cell surface antigen expression in breast cancer Br. J. Cancer 118, 813-819, (2018). Future outlook •Description of intratumoral and stromal heterogeneity in TNBC patient cohort by mass cytometry – advanced data analysis • •Identification of genetic signatures in selected subpopulations and their association with clinical observations • • • • • • • • •Validation of identified biomarkers by IHC on retrospective cohort of TNBC patients • • • • pop1 pop2 sorting RNAseq Analysis What kind of cells and mechanisms drive metastasis and chemoresistance? loading.gif loading.gif primary tumor competent cell micrometastasis Francia et al., Nat. Rev. Cancer (2011) Metastatická kaskáda Cirkulující nádorové buňky (CNB) – klíčová úloha Proč se cirkulujícími nádorovými buňkami zabývat? Ø 90% úmrtí spojených se solidními nádory – metastáze Ø Šíření primárně krví Ø Klinicky významné Ø „Liquid biopsy“ Ø Průběh terapie Ø Prognostický znak Ø Specifické mutace à cíle terapie • • • Ø Ø Schilling, et al., Nat. Rev. Urol. (2012) Vlastnosti cirkulujících nádorových buněk Ø Překonání anoikis Ø Změna fenotypu Ø Ø1g (109 buněk) tumor – uvolnění 106 buněk/24 h Ø 1 CNB na 100 mil krevních buněk Ø Poločas života: 1 – 2 hod Ø Ø Velikost a deformovatelnost Ø Exprese povrchových znaků Ø Možnosti detekce • Ø Ø Hayes et al., Science (2010) Metody detekce nádorových cirkulujících buněk Vojtěch Dvořák, BP, MU, 2016 Detekce nádorových cirkulujících buněk Příklad: Filtrace Ø Výhody – nezávislost na povrchových znacích Ø Heterogenní populace Ø Není nutná aktivace receptorů Ø Nativní stav Ø Ø ØNevýhody Ø Možný překryv s leukocyty Ø Nutné využít dalších znaků (CD45) Ø Různá velikost CNB? Ø Ø CNB: epiteliální původ à větší velikost Ø Platformy: MetaCell, CellSieve, Celsee,… Ø Ø Ø Ø C:\Zuzka P 12-02-2015\moje data BFU\circulating tumor cells\MetaCell\Mouse 4T1 MetaCell test 30-10-2015\metacell\SAM_1878small.jpg C:\Zuzka P 12-02-2015\moje data BFU\circulating tumor cells\MetaCell\Mouse 4T1 MetaCell test 30-10-2015\SAM_1880small.jpg Øpolycarbonate membrane with 8 mm pores (CTCs over 20 mm) Øcapilary force-driven filtration Příklad: Filtrace Příklad: mikrofluidní separace Příklad: mikrofluidní separace Příklad: mikrofluidní separace Lund Izolace CTC pomocí deplece CD45+ buněk krve RosetteSep™ Immunodensity Cell Isolation and Cell Separation Label for RosetteSep™ Human CD45 Depletion Cocktail Klinické využití detekce cirkulujících nádorových buněk Ø Odhad prognózy pacienta Ø Monitoring průběhu onemocnění Ø Včasná detekce • Ø Metastázující karcinomy prsu a prostaty – hranice 5 CNB/7,5ml Ø Metastázující karcinom tlustého střeva – hranice 3 CNB/7,5 ml Ø CellSearch system Veridex – schváleno FDA Ø www.cellsearchctc.com Množství cirkulujících nádorových buněk korelují s prognózou Molekulární charakterizace CNB à cílená terapie Ø Biopsie – identifikace mutací – zacílení terapie Ø Uvolňovány i z metastáz à komplexita Ø Vývoj onemocnění à chemorezistence, identifikace nových cílů Ø Využití v budoucnu? Ø Ø Ø Krebs et al., Nat. Rev. Clin. Onc., (2014) Plasticita cirkulujících nádorových buněk Ø Tvorbu metastáz ovlivňuje řada faktorů – mj. plasticita CNB Ø Ø Epiteliálně-mezenchymální přechod Ø Podíl na vzniku CNB Ø Vyšší motilita a invazivita Ø Vznik chemorezistence Ø Detailní mechanismy stále předmětem výzkumu Ø Význam popsán u řady karcinomů (prsu, prostaty, plic, tlustého střeva, vaječníků, atd.) Ø Ø Tsai et al., Cancer Cell, (2012) - upraveno Epiteliálně-mezenchymální přechod Ø U CNB popsán epiteliální i mezenchymální fenotyp Ø M+ buňky – spojeny s progresí onemocnění Ø Dynamické změny Ø Multicentric invasive ductal carcinoma of the right breast, G2, pT3 pN1a(2/9) M0 L1 V0, ER 100%, PR 0-80%, KI67 59%, Her-2 neg., dg. 5/2011, age 32 5/2011 Progress of the disease: 7/2011 Letrozol 4/2016 Relapse in skeleton Capecitabin 6/2017 3/2018 Faslodex 9/2018 Navelbin Gemzar 6/2019 9/2019 Paclitaxel w 12/2019 Carboplatin 3/2020 Mets in CNS 5/2020 exitus Blood collection CTCs isolation Implantation Xenograft establishment CTC-derived tumor histology 11/2019 Podporujeme | MUDr. J. Navrátil, Ph.D. MUDr. P. Fabián, Ph.D. Prof. MUDr. M. Svoboda, Ph.D. Adjuvant CHT TAC Adjuvant RT & tamoxifen 11/2011 Surgery Co-culture - media? Stanislav Drápela Markéta Pícková CDX surface profiling RTG Preclinical models for isolation of circulating tumor cells Model of human melanoma Syngeneic model of breast cancer Flow cytometric detection and in vitro isolation of viable CTCs In vivo progression of A375 IV luc GFP melanoma and 4T1 breast cancer Circulating tumor cells are promissing tool for analysis of cancer heterogenity and therapy response Aims: Prepare suitable in vivo preclinical models of cancer progression to study the cancer heterogenity refflected in circulating tumor cells (CTCs) and utilize the models for translational research to support development of personalized medicine of patients with metastatic disease. CTC-derived colonies Characterization of EMT-surface markers signature in CTCs and corresponding primary tumor Markéta Pícková Is there a cure for advanced cancer? §> 500 enzymes (approx. 1.7% of human genome) • Protein kinases: promising targets for anticancer therapy §Kinases = phosphotransferases •regulation of multiple cell processes •DNA damage response, DNA repair, mitosis §Protein kinase inhibitors = hot topic in pharmacology (> 30 compounds in clinical trials) Checkpoint kinase 1 (CHK1) §implemented in DNA damage response and DNA repair §promising therapeutic target SCH900776 CHK1 IC50 = 0.005 mM phase II clinical trials (with cytarabine) MU380 CHK1 IC50 = 0.002 mM more potent than SCH900776 in vitro MU379 potential metabolite less selective than SCH900776 until 2014 unknown pharmacophore Kamil Paruch §synthetic lethality (gemcitabine, cytarabine) •novel CHK1i – MU380 Pharmacokinetics MU380 = better pharmacokinetic profile CHK1 inhibition in multiple preclinical models T. Suchánková Co-culture - media? S. Drápela Docetaxel-resistant prostate cancer (PCa) Ovarian cancer - survival Pancreatic cancer Chronic lymphocytic leukaemia (CLL) Kamil Paruch, Lumír Krejčí, Martin Trbušek §effective as monotherapy in CLL §highly efficicent in combination with antimetabolites on various preclinical models §bypasses chemoresistance in prostate cancer §in vivo robust pharmacophore Obsah obrázku zeď, interiér, osoba Popis byl vytvořen automaticky The Mitotic Protein Kinase Haspin and Its Inhibitors | IntechOpen Haspin kinase – new target for preclinical development of highly selective inhibitors Tereza Suchánková Aims i.synthesis of a small library of potent compounds ii.profiling the activity in a panel (400+) of kinases iii.testing for the activity in the cancer cell models HeLa Fucci2 reporter system suitable for single cell tracking and analysis of cell division and morphology IN Cell Analyzer 2000 による Fucci 導入細胞の細胞周期測定 HASPIN Atypical human kinase that associates with chromosome and phosphorylates threonine 3 of histone 3 during mitosis Jan Novotný Obsah obrázku osoba, zeď, brýle, interiér Popis byl vytvořen automaticky Obsah obrázku zeď, interiér, osoba Popis byl vytvořen automaticky MU1464 is highly selective haspin inhibitor with a new central pharmacophore Future plans 1. Optimization and preclinical progression of our new highly selective inhibitors of the kinase Haspin and identification of compounds suitable for early phase clinical evaluation. 3. Linking haspin biology to intratumor heterogeneity, cancer plasticity, metastasis and acquiring the resistance in response to therapy 2. Development of new tools for description of complex response to new inhibitors at cellular level i. single cell tracking - Fucci4 system ii.flow cytometric multiparametric assay for haspin inhibitor screen iii.Proximity-dependent Biotin Identification (BioID) Figure 2 SP6800 Spectral Cell Analyzer - Spectral - Sony Biotechnology Bajar et al. Nature Methods. 2016 Sony SP6800 Spectral Analyzer Tereza Suchánková Jan Novotný Obsah obrázku osoba, zeď, brýle, interiér Popis byl vytvořen automaticky Obsah obrázku zeď, interiér, osoba Popis byl vytvořen automaticky The Nobel Prize in Chemistry 2008 * "for the discovery and development of the green fluorescent protein, GFP" Nobel Prize® medal - registered trademark of the Nobel Foundation http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/ Fluorescent proteins * bioluminescence resonance energy transfer (BRET) •Aequorea victoria - jellyfish * Blue bioluminescence. Ca2+ interacts with aequorin photoprotein. * Blue light excites green fluorescent protein. •Renilla reniformis – coral * luminescence appears after degradation of coelenterazine in the presence of luciferase enzyme. * Blue light excites green fluorescent protein * • Renilla reniformis "Sea Pansy" http://www.mbayaq.org/efc/living_species/default.asp?hOri=1&inhab=440 Aequorea victoria “Crystal jelly “ http://www.whitney.ufl.edu/species/seapansy.htm Fluorescent proteins * Osamu Shimomura * 1961 discovered GFP and aequorin * http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP2.htm •Science. 1994 Feb 11;263(5148): •Green fluorescent protein as a marker for gene expression. •Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. • •Department of Biological Sciences, Columbia University, New York, NY 10027. • • A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms. mice_400x300 Fluorescent proteins nDouglas Prasher nMartin Chalfie [USEMAP] Fluorescent proteins http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP2.htm Roger Tsien * ~ 2002 – mutated FP = wide spectrum of colors •http://www.tsienlab.ucsd.edu/ http://www.tsienlab.ucsd.edu/HTML/Images/IMAGE%20-%20PLATE%20-%20Beach.jpg The image “http://www.tsienlab.ucsd.edu/HTML/Images/IMAGE%20-%20Rendered%20GFP%20-%20640.jpg” cannot be displayed, because it contains errors. Roger Y. Tsien Analysis of synchronized cells Licensing control by Cdt1 and geminin http://jcs.biologists.org/content/joces/125/10/2436/F9.large.jpg?width=800&height=600&carousel=1 J Cell Sci 2012 125: 2436-2445; doi: 10.1242/jcs.100883 Fucci (fluorescent ubiquitination-based cell cycle indicator) cells Ubiquitin E3 ligase complexes G1 - APCCdh1 substrate: Geminin, inhibitor of DNA replication inhibits Cdt1 S, G2, M- SCFSkp2 substrate: DNA replication factor Cdt1 – key licensing factor Fucci sensors - 1st generation, coral FP monomeric Kusabira orange 2 – hCdt1 (30/120) Monomeric Azami-Green – hGeminin (1/110) Fucci sensors – 2nd generation, Aequorea FP red monomeric fluorescent protein - mCherry -hCdt1 (30/120) yellowish green monomeric variant of GFP –mVenus – hGeminin (1/110) Image result Fucci http://www.amalgaam.co.jp/products/advanced/img/fucci/E3.jpg http://cfds.brain.riken.jp/Fucci.html CONTROL SCH900776 MU380 …lot of questions, but how to answer them? •How many times cells divided? •What is a length of cell cycle phases? •Is there a difference in time between first and second division? •How it is all affected by my drugs? Branches (dvisions) analysis 02_02_01_01 Median 14 hours 02_02_01_01 Methodology Team & Collaborators Partners SELECTED ALUMNI (2016 – 2020/2021) 20+ "Vojtěch Dvořák" profiles | LinkedIn Art Façade - www.cemm.at Vojtěch Dvořák MSc in CAP –> PhD student at Ce-M-M, Vienna, drug resistance Ján Remšík PhD in CAP – > postdoc at MSKCC, NYC, USA, cancer spread into cerebrospinal fluid Memorial Sloan Kettering Cancer Center | LinkedIn Top Moffitt Cancer Center doctors failed to disclose payments from China, report says Stanislav Drápela PhD in CAP – > postdoc at Moffitt, FL, USA, from 5/2021, cancer metabolism Stanislav Drápela - Ph.D. student/Researcher - Fakultní nemocnice u sv. Anny v Brně (FNUSA) - St. Anne's University Hospital Brno / FNUSA-ICRC | LinkedIn THANK YOU FOR YOUR ATTENTION Acknowledgement •Souček lab •Kamil Paruch – Medicinal Chemistry •Jiří Damborský – Protein Engineering •Lumír Krejčí – Genome Integrity •Aleš Hampl – Cells and Tissue Regeneration •Lukáš Kubala – Molecular Control of Immune response •Vladimír Rotrekl – Stem Cells and Disease Modeling •Pavel Krejčí – Cell Signaling • •Jiří Navrátil, Pavel Fabian, Marek Svoboda – Masaryk Memorial Cancer Institute •Vladimír Študent – FN Olomouc •Jan Bouchal – Palacky University • •Medical University Innsbruck •Erasmus University • •Institute of Biophysics of the Czech Academy of Science •Masarykova univerzita •FNUSA-ICRC • •Grant agencies and all patients!