INVAZIVITA A METASTAZOVANI Karel Souček E-mail: ksoucek@ibp.cz, tel.: 541 517 166 Typické znaky nádorové buňky o podpůrné proliferační signály o deregulace supresorů růstu/proliferace o odolnost k buněčné smrti o neomezená replikace o neoangiogeneze o invaze a metastázování ► mutace a genomická nestabilita c> zánět Sustaining proliferative signaling Evading growth suppressors Resisting cell death N on mutational epigenetic reprog ramming Avoiding immune destruction Enabling replicative immortality Tumor-promoting inflammation Polymorphic microbiomes Inducing or accessing vasculature Activating invasion & metastasis o přestavba energetického metabolismu o únik před zničením imunitním systémem ► senescence o plasticita o epigenetika o mikrobiom Douglas Hanahan & Robert A. Weinberg: Hallmarks of Cancer: Next Generation, Cell, 2011 Douglas Hanahan: Hallmarks of Cancer: New Dimension, Cancer Discovery, 2022 Proč je rakovina tak devastující? 2012 > 2030 WORLDWIDE CANCER CASES ARE PROJECTED TO INCREASE BY 50% FROM 14 million TO 21 million WORLDWIDE CANCER DEATHS ARE PROJECTED TO INCREASE BY 60% cancer-related death cause estimate other cause 10 FROM 8 million TO 13 million metastasis 90 Source: American Cancer Society: Global Cancer Facts & Figures, Second Edition cancer.gov FB: AdvocatesForBreastCancer T: a Brea stCancerABC ADVOCATES FOR BREAST CANCER Proč je rakovina tak devastující? Nádory prostaty Nádory prsu C61 - ZN předstojné žlázy - prostaty, nužl, M:G Vývoj v čase Incidence Mortalita Analyzovaná data: N(incl = 99930. N(mor)=2114B http://www.sv0d.c2 C58,D85 - Nádory prsu, ženy, M;B Vývoj u čase Analyzovaná data: N(ind=1653Sl. N[mor)=44436 t—i—i—i—r http://www.svod.cz C61 - ZN predstojné žlázy - prostaty, nuži, M:l Vývoj v čase Incidence Mortalita Analyzovaná data: N(inc] = 18698. N(mor)=14037 http://www.svod.cz C58,DB5 - Nádory prsu, ženy, H;l Vývoj v čase r K K »v K »v »v K »v K K »v K »v »v K* K* V Analyzovaná data: N(inc)=15357. N(morJ=12093 ŕ1 ^lO'ŕ1 kÍ1 i?&ŕ ■v '''.i; r',{^ 00,0 http://www.5vod.cz Transforming growth factor - ß (TGF- ß) TGF-ß rodina ~ TGF- ßs, activins, bone morphogenic proteins (BMP) TGF-ß, • pleiotropní cytokin • negativní regulátor Gene transcripti Epstein, F.H., N Engl J Med 2000; 342:1350-1358, 2000 * Hraje klíčovou úlohu během embryogenéze; o> reguluje proliferaci, diferenciaci, buněčnou smrt, motilitu, adhezi (v závislosti na buněčném typu) = ovlivňuje homeostázu; 0 reguluje expresi extracelulární matrix; - indukuje fibrilární kolagen a fibronectin; - inhibuje degradaci ECM (inhibicí MMPs a indukci TIMPs). o Fibróza - deregulace exprese ECM prostřednictvím indukce proliferace fibroblastů a jejich myofibroblastového fenotypu. ► Nádorová onemocnění - ztráta citlivosti epiteliálních buněk k inhibičnímu působeni TGF-p; - indukce angiogeneze. TGF-jtr signaling component TGF-J3 # Endoglin I Type II recepto rs * Type 1 receptors I Smad2 Smad4 • Cancers Increased ex- Colorectal (30%) Breast (16%) Colorectal (11%) Pancreatic (50%) (somatic mutations) pression leads Gastric (15%) Pancreatic Lung (7%) Colorectal (30%) to enhanced Endometrial Biliary Hepatocellular Lung (10%) Invasion and Prostate Ce rvical Breast metastasis Breast Chronic lympho- Prostate Lung cytic leukemia Ovarian Hepatic Head and neck Pancreatic Esophageal Cervical Gastric Glioma Bladder Head and neck Hepatocellular Renal cell Other diseases (germ-line mutations or polymorphisms) Fibrosis Hypertension Osteoporosis Atherosclerosis Hereditary hemorrhagic telangiectasia Atherosclerosis Familial juvenile polyposis NEJM 342. 18 (2000) 1350-1358 Role TGF-ß v carcinogenezi SMAD3 Smad, mothers against dpp homolog 3 (drosophila) TGFBR2 Transforming growth factor, beta receptor ii (7Q/80kda) 1.5 ■tt i.o c □ c 0 W 0-5 01 2,0 0,0 * QJ TJ (L) N In -0.5 E o Z -1.0 -1.5 Sub Class CI ass Box Plot - Description Prostate - normal vs. cancer -2.0 Sub Class CI ass Box Plot - Description normal, hyperplasia vs. cancer CAM"] RY'Yii >l:][ .[Nli OAI AUAS]' ONCOMINE™ Role TGF-ß v carcinogenezi Normal cells Cell enlarges and makes new proteins G1 ■J Arrest of growth Inhibition of proliferation Rapid growth Clonal expansion Carcinoma Loss of TGF-ß signaling, e.g.TßRIl mutation Increased mutation rate 1 Metastasis Increased mutation rate TGF-ß Normal -L cell ' Benign lesion Perturbation of TGF-ß signaling, e.g. altered Smad2 T Migration/invasion Reversible E MT T Slowly expanding tumor Carcinoma Metastasis TRENDS in Cell Biology TRENDS in Cell Biology Vol.11 No. 11 2001 10 Vi (0 +-» Vi (D (U E CTI C a Of > "O IĎ > o 80 70 60 50 40 30 20 s 10 -Q (Q -Q O 0.0-1.0 1.1-2.0 2.1-3.0 3.1-4.0 4.0-5.1 5.1-6.0 6.1-8.0 8.1-10.0 >10.1 diameter (cm) of primary tumor Figure 14.3 The Biology of Cancer (© Garland Science 2007) primary tumor localized intravasation transport arrest in extravasation formation invasion through micro vessels of circulation various organs colonization -formation of a macrometastasis ure 14.4 The Biology of Cancer (© Garland Science 2007) PGR signaling increases migrating potential of PGR negative, HER2-expressing cells Accrual of additional l yjme genetic changes results in proliferation of disseminated cells to form a metastasi Figure 1. A Model of Parallel Progression after Early Dissemination. Recent experiments with a mouse model of human epidermal growth factor receptor 2 (HER2)-positive breast cancer3,2 have uncovered events critical to the dissemination of cancer cells from the early lesion (Panel A) and cefis of the established tumor (Panel B). The cells in the early lesion are loosely arranged; some of them express the progesterone receptor (PGR) in addition to HER2. These PGR-positive cells secrete soluble factors, such as receptor activator of nuclear factor-K/3 ligand (RANKL) and Wnt4, that induce the migration of PGR-negative cells from the lesion. The cells in the established tumor (Panel B) are packed more tightly, typically do not express the PGR, and are less likely to metastasize. The cells that disseminate from established tumors to the bone marrow carry many of the genetic variants, such as loss of chromosome 8p, that are found in established tumors, whereas those that disseminate from early lesions do not and instead evolve in parallel with the primary tumor and other disseminated cells. N ENGLJ MED 376:25 NEJM.ORG JUNE 22, 2017 Lokální invaze a intravazace * Invaze do cév, závislá na degradaci ECM * Závislá na proteázové aktivitě o Nádorové buňky mohou produkovat své vlastní proteázy (MMP-2, -9) nebo kooptují stromální buňky a využívají jejich aktivity Figuře 14.6a The Biology af Cancer {ft Garland Science 2014) leading fibroblasts 7f "V following SSCs :n.v ■-■..í* i ■,= ľi..:.=i.-:;j tdi jud kit-.- figure Hl ineCirtagyoJrjirar[5ídldnrikkw»!■(> Tumor cells, fibroblasts Macrophages, cathepsin t v- lilív^' x* . \ ' ; -\ KS 1 T TA*---. 'V-- * ■' .As CD34+ ) V" ;......;..... S " -l.r-J 'ľrť duy. :!'Idii:s-. Ú: Immature myeloid cells lagging edge actin cortex extension of new lamellipodium control + heregulin actin staining actin staining (E) Rho ACTIVATION (C) Rat ACTIVATION (D) Cdc12 ACTIVATION Figure 14.3? The Biology of Canter |£ ia-rlantf itiente 20141 Figure 1436 The Biology of Career [© Garland Science 2014) Pohyb buněk 2X RhoA 4X Cdc42 N-WASP- ZBP1 [i-actin mRNA targeting EGF source é EGF-R + EGF Ras -—'ÍL -PLC-y ■ Tiaml Rac —»■ PAK1 -j-cortactin Src 3XPI(4,5)K \ lOx 6XArp2/3 PI(4,5)P2 complex j 5X capping protein I— 4X MENA PI3K /( 2X Rho 3X ROCK + 3X LIMK T \SSH 3X PKC^ P-cofilin PI(4,5)P2 —- DAG cofilin ^ + IP3 cofilin J cofilin (total) 2X 1 barbed ends •+-F-actin severing -i— I -G-actin filopodia lamellipodia -| chemotaxís toward EGF Figure 14.40 The Biology of Cancer (O Garland Science 2014) / hearty liver lungs arterial circulation venous circulation m 1 jut spleen kidneys brain skin muscles | breast t T ITT IT Figure R4S The Biolcijv af (m*< f ^rUnri ^rien^ 2 90% úmrtí spojených se solidními nádory -metastáze > Šíření primárně krví l> Klinicky významné > „Liquid biopsy" > Průběh terapie > Prognostický znak > Specifické mutace -> cíle terapie íCL Primární tumor Sekundární metastáza Krevní oběh CNB Z)' Vlastnosti cirkulujících nádorových buněk > Překonání anoikis i > Změna fenotypu >1g (109 buněk) tumor- . uvolnění 106 buněk/24 h > 1 CNB na 100 mil krevních buněk i > Poločas života: 1 - 2 hod | > Velikost a deformovatelnost > Exprese povrchových znaki > Možnosti detekce Filtrace Shlukování Obalování Epiteliální buňky v cirkulaci Heterogenita 1_ Potenciál (částečné) EMT 1 Analýza Detekce nádorových cirkulujících buněk Table 2. List of commercial^ available CTC enrichment technologies Method Company Enrichment Valdatbn Advantages Limitations Rets ApoStream® ApoCell Combined microfluidic and microelectronic: CTCs are enriched based on the d ifferential d ietectnc ptoperty compared with PBMCs Quantitative imiTuncfluorescence Artibooynnoerpendert capture of viable CTCs available for downstream analysis Larger leukocytes with similar dielectric property like CTCs may get captured. Similarly, CTCs of the same size as bukocytes may not be captured [95,96] CellCo Hector® GILUPI Affinity: CTCs are isolated using functionalized polymer surface stainless steel wire with anti-EpCAM antibodies 1 mmunocytochemical staining in wo technology. Steel inserts capture CTCs directly from patients without blood draws Cannot isolate EpCA^CTCs [97-99] CellSearch® Menarini-Silicon Biosystems 1 mm jno magnetic: EpCAM+ CTCs are enriched by antibody nanopartides Fluorescence imaging US FDA-approved for detection and enumeration of CTCs in metastatic breast, prostate, and colorectal cancer patients Failed to enrich EpCAWCTCs. CTCs are not viable [100) CellSieve™ Creatv Microtech Inc. Size! CTCs are enriched by filtering cells having diameter of <7 urn using mice of] Iters Multiple options can be exploited since cells are not fixed Recovery of CTC dusters and immune-cancer fusion cells along with CTCs Larger leukocytes and fi bro blast contamination [35] ClearCeJh&FX ClearbrkJge BioMedics Size: CTCs are enriched by automated microti Jdic chip Multiple options can be exploited since cells are not fixed Label-free CTCs readily available for downstream anaysis Loss of CTCs d ue to red blood cell lysis step [101) DEPArray™ Menarini Silicon Biosystans Combined microfluidic and microelectrcnic: CTCs are enriched by dietectropooretic movement and trapped in electronic cages Fluorescence imaging Individual CTCs from heterogeneoLE population of cells can be isolated for downstream anaysis allowing both EpCAM+CTC characterization Pre-enrichment and staining are required for rarecells like CTCs before loading to cartridge [102) EasySep™ Direct Human CTC Enrichment Cocktail Stemcell Technologies 1 mmu no magnetic: CTCs are enriched by depleting hematopoietic cells and platelets with a cocktail of antibodies Multiple options can be exploited since cells are not fixed Cells are not fixed and viable, which keeps multip ie options open, such as culturing, characterization, injecting to animals, etc. Beth EpCAM+CTCs are enriched Unable to achieve complete depletion of leukocytes and other eel Is [103) EasySep™ Human CD45 Depletion Wi Stemcell Technologies 1 mm jno magnetic: CTCs are enriched by depleting CD45* cells Multiple options can be exploited since cells are not fixed Isolated cells are immediatey availaPlefdr downstream applications such as flow cytometry, culture, or DMVRNA extraction Depletion of only CD45+ cells does not yield homogeneous CTCs [104) ISET® Technology Rarecells Size: leukocytes and other ceils are removed by p lessuie-cortrdled filtration system, thereby enriching CTCs ImmunoflLBrescence Fixed CTCs are processed with a specific SET buffer and can be collected for downstream processing Larger leukocytes may be captured, limiting downstream anaysis of CTCs [105) Table 2. [continued) Method Company Enrichment Validation Advantages Limitatbns Refs IsoFlux™ Fluxion Combined microfluidic and immuno magnetic: CTCs are enriched by anti-EpCAM antibody magnetic beads in microfluidic cartridges Fluorescent imaging High recovery of EpCAM-CTCs Only EpCAM+ CTCs are enriched [106] OncoOuick® Greiner Bio-One Size and density: CTCs are enriched by density gradient centrifugatbn over a porous barrier Multiple options can be exploited since cells are not fixed One-step enrichment of CTCs by simple oentrifugation Due to sparse numbers of CTCs, no visibility of distinct interphase layer that leads to loss of tumor cell on collection. Platelet contaminatbn is another challenge |107] Parsortix® PRl Angle Size and deformabilhy: CTCs are enriched by automated filtration cassette that separates leukocytes Multiple options can be exploited since cells are not fixed CTCs can be enumerated by in-cassette staining or harvested for downstream analysis Possible contamination of uncompressible large cells like fibroblasts [106] RosetteSep™ CTC Enrichment Cocktail Containing Anti-CD36/CD56 Stemcell Technologies Immuno density: j nwanted cells are crosslinked by an antibody cocktail that increases density and is pelleted by centrifugatbn Multiple options can be exploited since cells are not fixed Enriched CTCs are readily available for various downstream applications Additional centrifugatian step may result in toss of few CTCs [12,61] RosetteSep™ Human CD45 Depletion Cocktail Stemcell Technologies Immuno density: j nwanted cells are depleted by tetrameric antibody complex followed by density gradient centrifugatbn Multiple options can be exploited since cells are not fixed Enriched CTCs are readily available tar various downstream applicatbns. Depletion of only CD45+ cells does not remove other cell types [46] vTX-1 Vortex Biosciences Size: automated microti ud ic technology is adapted to enrich CTCs Multiple options can be exploited since cells are not fixed LabeJ-free CTCs readily availab le for culture or oharacterizatbn Recovery of only 60-70% CTCs [109] C> CelPress Trends in Cancer Review Preclinical models to study patient-derived circulating tumor cells and metastasis Kanve N. Suvilesh,1 '* Yariswainy Marijunath,1,2 Klaus Pantel,3 and Jussuf T. Kaifi 1 -2A* I Příklad: Filtrace > CNB: epiteliální původ -> větší velikost > Platformy: MetaCell, CellSieve, Buňky Průměr [jim] Erytrocyty 6-8 Granulocyty 12-15 Monocyty 15-25 Lymfocyty 7-10,14-20 CNB 17-52 > Výhody - nezávislost na povrchových znacích > Heterogenní populace > Není nutná aktivace receptoru > Nativní stav > Nevýhody > Možný překryv s leukocyty > Nutné využít dalších znaků (CD45) > Různá velikost CNB? Příklad: Filtrace > polycarbonate membrane with 8 |um pores (CTCs over 20 |um) > capilary force-driven filtration Experimental design En, Enriched CTCs fraction The " membrane" cell population Polycarbonate membrane (PCM) Growing media (without any concentration gradient) A Flux driven by diffusion supported by the shape of the ring The " bottom" cell population 7 (TleLaCeLL IMC slide Hb Chip Micro mixers CTC Chip HT CTC Chip Thermoplastics GEDI Chip Immunocapture VeriFAST Silicon microposts im»" «www?? bJtlar Immunomagnetic separat Nanomaterials Current Opinion in Chemical Engineering Size Based Separation Microfiiter Inertia} Effects I FMSA device Vortex technology f * • to Captured tumor cells Leukocytes Red blood cells parylene-C membrane o»o l J MO ( }T>oo ,wf 1 coo f J o of J OOO ( J ooo f OOwO/-\OV«OlOv OOO I J OOO (JO I ) Do ü f 1 OOO I r- c—' O OOO OOO I J OOO ( J OOO (10 c j-"\0^—oy>Qwo/-\0^_g 0 M OOO f) OOO M OOO ÖO(J f ) Crpö r J Oo<3 f J^P SB microfiiter Spiral devices Current Opinion in Chemical Engineering 20 p.S.i 20 p.S.i Size-based Soiling ingle File Alignment RBCs, platelets, other blood components Magnetic Separation ~630|il/min Buffer Syringe pump -600 ul/min Ruriiiiftj buffer Red blood cell (8x 10*/mlJ White blood cell (S x 107ml] CTC labe led with magnetic beads (1-100/m I; Waste 2a (WBC-hi) I -60 nl'min [J Product (CTC) -54 u.l/rnin Blood 120jiVmin Waste 1 (RBC, PLT) u CTC-iChip Stage 2 Waste 2b (WBC-low) -36 nl/min CTC-iChip running platform PROTOCOL Microfluidic, marker-free isolation of circulating tumor cells from blood samples Nezihi Murat Karabacak1,4, Philipp S Spuhler1'4, Fabio Fachin1, Eugene J Lim1, Vincent Pai1, Emre Ozkumur1, Joseph M Martel1, Nikola Kojic1, Kyle Smith1, Pin-i Chen1, Jennifer Yang1, Henry Hwang1, Bailey Morgan1, Julie Trautwein2, Thomas A Barber1, Shannon L Stott1-2, Shyamala Maheswaran2, Ravi Kapur1, Daniel A Haber2-3 & Mehmet Toner1 1 Department of Surgery and Center for Engineering in Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA. 2Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA. 3Howard Hughes Medical Institute, Chew Chase, Maryland, USA. 4These authors contributed equally to this work. Correspondence should be addressed to M.T. (mtoner@hms.harvard.edu). Published online 27 February 2014; doi: 10.1038/nprot.2014.044 Příklad: mikrofluidní separace Blood Buffer Bit*«? Ceils*'*" V Small Waste Clusters Stage i; Sorting by StSge 2: Sorting fry Stage li Oriefitetiůrt-Transvcrsc Axrt Longitudinal Axis Independent Sorting SCIENTIFIC REPpRTS open Microfluidic Isolation of Circulating Tumor Cell Clusters by Size and Asymmetry Received 20 Januar.' 2017 liiqihil TTMinliTTtlT Publishiil mine: 26 Ma> 2017 Sam H. Au'2, Jon Edd1, Amy E. Stoddard*, Keith H. K-Wong1-2, Ft. bio Fadiin1, Shyamala Maheswaran1.', Daniel A. Habere Shannon L. Stott1-1-', Ravi Kapur1 & MehnietTonerV," Add RosetteSep™ Antibody Cocktai Whole blood (D Layer over density gradient medium Incubate 20 minutes Density gradient medium ® ® Centrifuge for 20 minutes* Collect cells Plasma Enriched cells Density gradient medium Red blood cells and unwanted cells (rosetted) Highly purified cells \®/ are left untouched *Use SepMate™ to reduce centrifugation time to 10 minutes with brake on. ^RosetteSep Unique Immunodensity Cell Isolation RosetteSep™ kits offer one-step enrichment of cells directly from human whole blood. By crosslinking unwanted cells to red blood cells (RBCs) present in the sample, CTCs are enriched during standard density gradient centrifugation. RosetteSep™ is easy to use, does not require additional equipment, reduces sample handling time and maximizes convenience. RosetteSep™ can be easily combined with SepMate™, a specialized isolation tube that standardizes and minimizes variability when isolating cells using density gradient centrifugation. Learn more at www.RosetteSep.com and www.SepMate.com. Klinické využití detekce cirkulujících nádorových buněk > Odhad prognózy pacienta > Monitoring průběhu onemocnění > Včasná detekce > Metastázující karcinomy prsu a prostaty - hranice 5 CNB/7,5ml > Metastázující karcinom tlustého střeva - hranice 3 CNB/7,5 ml > CellSearch systém Veridex - schváleno FDA www.cellsearchctc.com Množství cirkulujících nádorových buněk korelují s prognózou (A) CTC at baseline ■<5 (B) CTC at baseline -<5 £5 1.00 -O Ě o a. > > i— co T c o 'co co Z' ±-CL 0.75 0.50 0.25- 0.00 Logrgnk p < 0,0001 Cox HR (95% Cl) = 2.52 (1.74-3.67) p<00001 c. I— w 0 3 6 9 12 15 18 21 24 Time Since Start of Treatment (months) Number at risk by time <5 ž5 101 88 58 47 34 20 8 8 3 69 39 23 12 9 4 1 0 0 0 3 R 9 12 15 18 21 24 f 'n a n I 1.00 0.75 0.50- -i 00 2 0.25 O 0.00 Logrank p < 0.0001 Cox HR (95% Cl) = 4.76 (2 72-3.32) p< 00001 C O "OJ OCÖ -O cd 0 3 6 9 12 15 18 21 24 27 30 33 36 39 Time Since Start of Treatment (months) Number at risk by time <■ 5 > 5 101 96 80 68 54 39 26 19 10 3 1 1 1 1 69 51 40 30 21 15 7 4 4 2 0 0 0 0 0 3 6 9 12 15 18 21 24 27 30 33 36 39 Time Since Start of Treatment (months) Time Since Start of Treatment (months) D l0O2.'pnH..234BB ORIGINAL ARTICLE Wiley ■ Circulating tumor cells and survival in abiraterone- and enzalutamide-treated patients with castration-resistant prostate cancer Bram De Laere1 Steffi Qeyen1 Peter Van Oyen2 I Christophe Ghysel2 Jozef Ampe2 Piet Ost3 Wim Demey1* I Lucien Hoekx5 Dirk Schrijvers* Barbara Brouwers7 I Willem Lybaert0 I Els Everaert3 I Piet Van Kerckhove7 Daan De Maeseneer9 I Mkhiel Strijbos4 I Alain Bols7 Karen Frarrsis5 Nick Beije10 I Inge de Kruijff10 I Valerie van Dam1 I Anja Brouwer1 I Pieter-Jan van Dam1 Gert Van den Eynden111 Annemie Rutten12 Stefan Sleijfer10 Jean Vandebroek12 Steven Van Laere1 I Luc Dirix1-" Molekulární charakterizace CNB -> cílená terapie > Biopsie - identifikace mutací - zacílení terapie > Uvolňovány i z metastáz -> komplexita > Vývoj onemocnění -> chemorezistence, identifikace nových cílů > Využití v budoucnu? Počáteční stav Progrese po počáteční terapii Progrese po další terapii Iniciační klon v převaze Převaha vyselektovaných klonů Větší komplexita? Krebs et al., Nat. Rev. Clin. One, (2014) Parsortix® Technology ► Pacientovi je odebrána tekutá biopsie (vzorek krve) - obvykle 10 ml do vakuové zkumavky EDTA. o Vzorek se pripojí k prístroji Parsortix. Není nutné žádné predbežné zpracování. Do prístroje se vloží filtrační kazeta Parsortix a systém se pripraví k použití. o Prístroj Parsortix automaticky propustí krev přes filtrační kazetu. o CTC jsou zachyceny v kazetě Parsortix díky své větší velikosti a nižší stlačitelnosti než ostatní složky krve. o První zdravotnický prostředek schválený FDA pro zachycení a sběr cirkulujících nádorových buněk (CTC) z krve pacientů s metastazujícím karcinomem prsu (MBC) pro následnou analýzu. https://angleplc.com/parsortix-technology/how-it-works/ Cirkulující nádorové buňky - prekurzor metastáz > Množství CNB nekoreluje s množstvím metastáz > Rozsev nádorových buněk může probíhat v časných stádiích (b) > K vytvoření metastáz nutná další stimulace -mikroprostředí, mutageneze > Relaps: metastáze původem z rozesetých buněk (a) > „Tumor self-seeding" > Oblast intenzivního výzkumu Primary tumour O O O Oj3 O O O O O O O O O O O A A Lung Liver metastasis metastasis OOOOOOOOOOOOOOOO Liver Lung Primary metastasis metastasis tumour Plasticita cirkulujících nádorových buněk Krevní destičky TGF-B1 eno Epiteliálně-mezenchymální přechod > U CNB popsán epiteliální i mezenchymální fenotyp > M+ buňky - spojeny s progresí onemocnění > Dynamické změny Disease progression (r) Treatment response IE E>M E=M |M>E M Month Circulating Breast Tumor Cells Exhibit Dynamic Changes in Epithelial and Mesenchymal Composition Min Yu,w* Aditya Bardia,1,3* Ben S. Wittner,1 Shannon L Statt,1-2 Malgorzata E. Smas,1 David T. Ting,1 Steven ], Isakoff,1,3 Jordan C. Cidliano,1 Marissa N. Wells,1 Ajay M. Shah/ Kyle F. Concannon,1 Maria C. Donaldson,1 Leda V. Sequist,1,3 Elena Brachtel,1'' Dennis Sgmi,1,fl Jose Baselga,1,3 Sridhar Ramaswamy,1,3 Mehmet Toner,2,5 Daniel A. Haber,1,w| Shyamala Maheswaran1,5! Yu etal., Science, (2014) enmneiHlipn CellSeanch* RNA-ieq Gene Expression Cell Death Pis. 2023 Aug: 14(8): 530. Nádorové buňky uvolněné do cirkulace > Klíčová úloha ve vzniku metastáz > Heterogenita a plasticity > Detekce > Oblast intenzivního výzkumu > Klinicky významné > Počet koreluje s prognózou > Molekulární charakterizace - personalizace medicíny Extravazace tissue (1) cancer cell parenchyma (2) platelets endothelial cells (3) capillary basement membrane Figure 14.9d The Biology of Cancer (© Garland Science 2014) Kolonizace * Velice nízká účinnost o Dormantní mikrometastázy > Metastatický relaps j\ genetically heterogeneous primary tumor dissemination of metastatic cells, subsequent removal of primary tumor I acquisition of ability to colonize micrometastases scattered throughout the body (minimal residual disease) macroscopic metastasis new, secondary shower of metastatic dissemination new colonization-competent micrometastases III I lr*d multiple macroscopic metastases, disease relapse expansion 1.0 CK+ buňky v kostní dřeni negative N = 47 0.8 re > E l/V 4- O 0.6 ~ro 0.4 -Q O 0.2 0 5 10 15 20 25 30 35 40 45 months after diagnosis Figure RIM TheEiologyoíCariíerí^GuilandStieiiíeiOU) Figure 14.11c The Biology of Cancer ( Garland Science2014) invasive edge liver metastasis carcinoma in situ invasive carcinoma EMT epithelial | mesenchymal PROGRESSION basement membrane normal stroma INTRAVASATION reactive stroma micrometastasis MET basement membrane macrometastasis TRANSPORT through circulation EXTRAVASATION normal stroma Figii re 14.18b The Biology of Cancer [£> Garland Science 2014) Znaky a regulátory E M T Invasive carcinoma Kornelia Polyak & Robert A. Weinberg Nature Reviews Cancer 9, 265-273 (April 2009) Nature Review* i Cancer Key figure Phenotypic plasticity ot disseminated tumor cells during metastatic colonization /dtc .Primary tumor I J (A) Plasticity and E MT Epitheäíl . *^~\ L-J íl citato potential Cell 1 state Hytrid Pla icily Cell death (B) Plasticity in sternness Sfem ^^Jŕŕŕer e rrtH red Safŕ-JBnawuľ | Osteogeniccells Stem-like dab MkiD- MaĽrometaiíaEei Cell state: Stem-Hta Lummleprrheliall (c) Plasticity in metabolism Meraimŕiic — IHefabortc state ľ state £ radie nts of 0=, nutrients, meta to life s J-RE A~ f».(.. Crqddbfl Outfit UK 4" Vj**y -state N62V neiťnMSOs 1#® II biAih-111 V (E) Plasticity in host-organ mimicry Haiti Hasti TV .: L^iid mefeflnfan r. ifri iniiintfilm' -------( MM DAR -i-a 'Kj Trends in Cell Biology (A) Plasticity and EMT Epithelial Hybrid Mesenchymal Metastatic potential -> A--- Plasticity —► •<— Epithelial Hybrid Mesenchymal CD51, CD61, CD106 Hybrid 1 V_jf cell state ROS Cell death (B) Plasticity in sternness Stem-like Clio HR+ DTCs a Self-renewal Differentiated FGF2 ligands A EZH2 Osteogenic cells ^ _____ _ Sternness Single CTC 0CT4, NANOG, SOX2 ( DNA methylation CTC cluster Micro- ® ESR1 [__^> Stem-like state Macrometastases OJ^O Cell state:[stem-like Luminal epith elial (A) Epithelia Stable cell-cell junctions Apical-basal polarity Canonical markers CDH1, EPCAM, GRHL2 EMT MET Mesenchymal Front-rear polarity Migratory/invasive Canonical markers VIM, CDH2, SNA! 1/2, ZEB1/2, TWIST 1 (B) Multivariate epithelial phenotypes o cz X LU Pancreatic^ acinar cell Hepatocyte <^** Epidermal epithelium' Cell junction -C3 a; :_ re EMT diversity Feature a Multidimensional space of hybrid phenotypes -t—1 Mes1 Mes2 Feature a Trends in Cancer EMP ovlivňuje interakce mezi nádorovými buňkami a imunitními buňkami a utváří imunosupresivní TME. review article open Harnessing epithelial-mesenchymal plasticity to boost cancer immunotherapy I Vuaruhmo Cu1*, Zhengkui Itaft/**1 Sid Perer tin C>ijtcl"J c TS« »uthoKs) 202J TGF-ß JJMMJMJ JiJJJJAWWJAtA» >*»JJJA»JJ*>****J JS****+*m-*J*+*JJ MMMAWMMMN f»»»»»^^ ^^wvwvwnwvw vwyw>vmww v>vm>v>-iiivvtrv>i -»vw^^r»**»»* Smad2/3 Transcription factor I Srail family SnaiH Snail2 audins Occluclir E-cadherin Desmoplakin Plakophilin Crumbs3 i Cytokeratins j Fibnonectin Vitra necti ri N-cadherin Collagen MMPs Twist, Ids ZEB 1/2 Jian Xu, Samy Lamouille, Rik Derynck Cell Research (2009) 19:156-172. ZEB family ZEB1 ZEB2 i í I Claudins ZO E-cac! herir Plakophilin CrumbsS y < Vitra necti n IVcacherin MMPs bHLH family E12/E47 Twist CS I I Claudins OcclLcin E-cad hierin Desmoplakin Plakoglobin y Fibranectin Vitronectin N-cacfherin SPARC ot5-integrin Klíčové objevy v EMT a rakovině 0 EMT creates cells with cancer stem cell characteristics Mani SA, et al., Cell. 2008 May 16;133(4):704-15. 0 Cross-regulation between ZEB1/2 and miR-200 family Gregory PA, et al., Nat Cell Biol. 2008 May;10(5):593-601. ► Cross-regulation between Twist and Slug Epithelial Mesenychmal transition Cadherin Cancer stem eel rtilKJOOf IMH/U1 1, t t Twist lsmeralda Casas, Jihoon Jim, Andres Bendesky, et |ancer Res; 71(1) January 111 j-achi Jain, Suresh K. Alaharij fontiers in Bioscience 16, 524-1832, January 1, 2011 |zhang, Y Li and M Lai icogene 29, 937-948 ||S February 2010) _1 14 Metastasis Self renewal - malur* miR-U ' matur« mlR-20 ■matur« mlR-14 Tumorigenesis Experimentální přístupy ESTABLISHMENT AND CHARACTERIZATION OF AN IMMORTALIZED BUT Malignant Transformation in a Noniumorigcnic Human Prostatic Epithelial NON-TRANSFORMED HUMAN PROSTATE EPITHELIAL CELL LINE: BPH-1 Cell Line1 S. W. HAYWARD, R. DAHIVA, C, R. CUNHA, J BARTER, N. DESHPAINDE, uro P, NARAYAN Simon W. ]lavivard,ľ Yiizhuu Wang. Mei Clio. ^ un Kit [loin. Baoliui Zliiír^. Ghi; I)l Grossfeld, Ihuiid Sudilovskv, LL]ld G?ľLlld R. Cl] ll] ILL +CAF AF TD Ol. 03 CAF TD 02. 04 BPH BPH-1 i Enlarged prostale/BPH Blande Urethra TGF-ß • EMT markers • EMT regulators • Cell shape and behavior Cancer associated fibroblast - tumor derived TGF-ß 1 M complete medium carcinoma cell macrophage (M - Bone Colonization LM2 cells • -TGFß ■ + TGFp lm2 cm34.2a 5= E0 - Bone Colonization I 60 BoM-1833 cells "g A -TGFp ♦ + TGFp I £0 Lit TGFp , - , , " *■ i LM2 BoM-1833 Days Vol 459 1B June 2009 doi:10.1038/nat nreOB021 nature LETTERS Genes that to the brain breast cancer metastasis Paula D. Bos1, Xiang H.-F. Zhang1 rCri5tina Nadal]tr WeipingShu], Roger R. Gomis't, Dor X. Nguyen1, Andy J. Minn:, Marc J. van de Vijver\ William L Gerald4, John A. Foekens^ & Joan Massague1'* Competence to seed Breast tumour Brain Latent disease Competence to colonize (years to decades) Brain metastasis (brain colonization genes) ST6GALNAC5, COX2, HBEGF, ANGPTL4? Q% . -^ I T?A Lung metastasis COX2. EREG. ANGPTL4 U Q mobilizace vápníku Nádorové buňky - adaptují tento mechanismus -osteolýza kostí vede k uvolnění řady růsových faktorů stimulujících nádorové buňky dn TGF-ßRIl dn TGF-ßRIl + ca TGF-ßRI Figuře 14.49 TheBioltmy of tanier [oGarland Science 2014] Ca?* dn TGF-ßRIl + PTHrP pump FiaweIMSh The bíoiouvjiícii.nti ľ.jil im.lítivice ÍCH] Aktivace růstových signálů Autocrine activation Autocrine and intracellular amplifi cation Pa racrine activation Pa racrine amplification Cell-cell contact and activation Stem-cell growth and survival pathways NATURE I VOL 5 2 0 | 2 1 JANUARY 2016 Seeding from primary tumour lour —v x 5> Survival Drug-sensitive cancer cell Protected niche Therapy-induced secretome Accelerated growth Stromal cell Soluble factor-• f Overt colonization Cancer therapy ncomplete elimination of tumour Drug-resistant mutant ce Relapse Drug-resistant tumou NATU R E I "\ Terapie cílená na metastázování Table 11 Preclinical and clinical history of four metastasis-directed drug development efforts Description Denosumab Monoclonal antibody to RANKl Pathway Preclinical validation RANKL activates osteoclasts and promotes bone destruction; denosumab reduced bone resorption in mice expressing human RANKL" Pivotal trials and end points Outcomes SREs* in metastatic setting; adjuvant trials used time to first bone metastasis or fracture10"" FDA approved for prevention of SREs in solid tumours; approved as adjuvant therapy in prostate cancer Bevarizumnb Monoclonal antibody to VEGF Cilengitide avß3 and avß5 integrin peptide inhibitor • Bevacizumab inhibited corneal angiogenesis and lymphangiogenesisJ" • In multiple cancerxenograft models, bevacizumab reduced primary tumourgrowth rates and, in some studies, enhanced survival- Reduced angiogenesis and vessel normalization was observed"5 • Prevention or, less frequently, abrogation of metastasis*4*247 * Recurrent ovarian cancer, * Metastatic colorectal cancer, OS""-2" * Metastatic or resistant HER2i breast cancer. PFSJ* * Metastatic renal cancer, PFSZ" ►Glioblastoma, 0St PFS:" fc Advanced lung cancer, OS3J »Adjuvant therapy in triple-negative breast cancer, DFS41 Tu m our cell Desatmiband saracatenib SRC kinase and BCR-ABL kinase inhibitor Endothelial cell Integrins ■ Stabilization of glioma growth and angiogenesis, Synergistic inhibition of glioma with I MZ*1_fr* • Synergy with therapeutics in melanoma primary tumour growth*1, synergy with radio-immunotherapy in breast cancer tumour growth2*5 • Inhibition of metastasis62 ■ Synergy with verapamil increased angiogenesisand reduced metastasis"9 ■ Inhibition of CML models"0 • Inhibition of primary tumour growth inmultiple model systems, as monotherapy or in combination'*1-"1 • Prevention of metastasis in multiple cancer model sysfce,msiS*~B,l but not osteosarcoma259 • Inhibition of prostate cancer growing in bone and bone remodelling*1-" •Phase III CENTRIC EORTC, with radiation therapy and 1MZ, for glioma, OS. Newly diagnosed glioma, same combination, recurrence65, • Phase II trials in melanoma and lung and prostate cancers, PFS*6-46 • Cytogenetic response end points for CML • Response for advanced solid tumours71-80 • OS in Phase III prostate cancer*7 * FDA approved for resistant ovarian, ceivical and colorectal cancers, glioblastoma, also advanced or metastatic lung, colorectal and renal cancers * Revoked for metastatic breast cancer ■ Negative trials for first line treatment of glioblastoma All advanced trials were negative • FDA approved for CML and resistant ALL • Discontinued in advanced lung, ovarian, colorectal and breast cancers * Negative in prostate cancer Phase III trial with docetaxel * Multiple adjuvant trials terminated ALL. acute lymphoblastic leukaemia; CML.chronic myelogenous leukaemia; DFS. disease-free survival; ECM. extracellular matrix; FAK. focal adhesion kinase; FDA, US Food and Drug Administration; OS. overall survival; PFS, progression-free survival; RANK, receptor activator of NF-kB; RANKL, RANK ligand; RTK, receptor tyrosine kinase; TMZ.temozolomide; VEGF, vaseular endothelial growth factor. *5keletal-related event (SRE) captures the deleterious effects of now lesions and progression of existing lesions to cause patient morbidity. NATURE REVIEWS I CANCER Selektivní tlak řídí proces selekce a driftingu během vývoje rakoviny Proliferation and mutation Oedifferenliation Proliferation, increased mutation rate and niche differences between colonies increased proliferation rate of the selected colony, mutation, and sub-cional heterogeneity Some colonies dominate High selective pressure Clonal selection constantly continues in response to altered heterogeneity and niche conditions -Pericyte }— Basement membrane Endothelial cell Red blood cell Trends inGenetics https://doi.org/10.1016/j.tig.2023.01.007 Paralely v klonální selekci/driftu mezi vývojem rakoviny a normální tkáně a jejich přechodem. I Cancer (wound that never heals) | I Normal tissue | ■ Inaccessibility to niche nutrients - Disadvantageous mutations ■ Epigenetic dysregvtetton ■ Spatiai constraints - Hypoxia. Several colonies initially co-arise Advantageous mutations High access to niche resources ■ inaccessibility to niche nutrients - Disadvantageous mutations ■ Epigenese deregulation ■ Spatiai constraints -Hypoxia ^Clonal selection^ ^Clonal selection^ (ĎormantN CSCs J ) _L ™ Absorbing niche resources (e.g. oxygen v , and nutrients) and space occupation > - (Active CSCs) (Active NSCs) Tissue replenishment (Dormant^ NSCs J • Niche alteration and mutation in epigenetic modifiers (Differentiated cells J J Dedifferentiation 1 (Neoplastic cel^ Inflammation increment, resisting cell death, induction of angiogenesis, cell dissociation, invasion, and migration ( Metastasis ) ( Wound healing ) Removal of differentiated cells and active stem cells Y CSC type-non-specific treatment Tissue damage Removal of negative selective pressures (e.g. physica constraints and hypoxia) and enhancement of niche resources around dormant (cancer) stem cells Trends in Genetics https://doi.org/10.1016/j.tig.2023.01.007 Shrnutí o 90% úmrtí na nádorová onemocnění souvisí s metastázováním o Invazivní kaskáda zahrnuje: lokální invazi, intravazaci, transport, extravazaci, formovaní mikrometastáz a kolonizaci o Nízká efektivita celé kaskády, nejméně efektivní je kolonizace C> EMT, řízena pleiotropními TF v různých fázích embryogenéze, adaptována během tumorigeneze ► Motilita je řízena malými GTPasami, Rho rodina c> Proteázy (MMP) umožňují invazi nádorových buněk, degradace ECM o Tkáňový tropismus nádorových buněk lze v některých případech vysvětlit organizací oběhového systému, často prozatím neobjasněn