Metastasis Model of Cancer Stem Cell-Derived Tumors

Protocol Metastasis Model of Cancer Stem Cell-Derived Tumors

Hager Mansour 1, Ghmkin Hassan 2,3 , Said M. Aﬁfy 2,4 , Ting Yan 5, Akimasa Seno 1,2 and Masaharu Seno 1,2,*

1 Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan; [email protected] (H.M.); [email protected] (A.S.) 2 Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama 700-8530, Japan; [email protected] (G.H.); saidafi[email protected] (S.M.A.) 3 Department of Microbiology and Biochemistry, Faculty of Pharmacy, Damascus University, Damascus 10769, Syria 4 Division of Biochemistry, Chemistry Department, Faculty of Science, Menoufia University, Shebin El Koum-Menoufia 32511, Egypt 5 Department of Pathology, Shanxi Key Laboratory of Carcinogenesis and Translational Research on Esophageal Cancer, Shanxi Medical University, Taiyuan 030001, China; [email protected] * Correspondence: [email protected]

Received: 21 July 2020; Accepted: 17 August 2020; Published: 21 August 2020

Abstract: Metastasis includes the dissemination of cancer cells from a malignant tumor and seed in distant sites inside the body forming secondary tumors. Metastatic cells from the primary tumor can move even before the cancer is detected. Therefore, metastases are responsible for more than 90% of cancer-related deaths. Over recent decades there has been adequate evidence suggesting the existence of CSCs with self-renewing and drug-resistant potency within heterogeneous tumors. Cancer stem cells (CSCs) act as a tumor initiating cells and have roles in tumor retrieve and metastasis. Our group recently developed a unique CSC model from mouse induced pluripotent stem cells cultured in the presence of cancer cell-conditioned medium that mimics tumors microenvironment. Using this model, we demonstrated a new method for studying metastasis by intraperitoneal transplantation of tumors and investigate the metastasis ability of cells from these segments. First of all, CSCs were injected subcutaneously in nude mice. The developed malignant tumors were minimized then transplanted into the peritoneal cavity. Following this, the developed tumor in addition to lung, pancreas and liver were then excised and analyzed. Our method showed the metastatic potential of CSCs with the ability of disseminated and moving to blood circulation and seeding in distant organs such as lung and pancreas. This method could provide a good model to study the mechanisms of metastasis according to CSC theory.

Keywords: metastasis; cancer stem cells; transplantation; surgery

1. Introduction Metastasis is spreading of cancer to tissues or organs far from their original sites where they originated. Cancer metastasis enables forming secondary tumors in distant organs and is major responsible for the mortality and morbidity of cancer [1]. The metastasis includes several events beginning with dissemination of cancer cells from tumors, invading stroma, intravasation and seeding in secondary sites where they form metastasis [2]. Many genes are changing during these stages changing cell phenotypes. Genes, such as E-cadherin,

Methods Protoc. 2020, 3, 60; doi:10.3390/mps3030060 www.mdpi.com/journal/mps Methods Protoc. 2020, 3, 60 2 of 8 slug and twist, contribute to give cancer cells the dissemination and movement ability driving metastasis eventsMethods [3]. Protoc. 2020, 3, x FOR PEER REVIEW 2 of 8 On the other hand, cancer stem cells (CSCs) represent the subpopulation of cancer cells with the abilitycadherin, to dislugfferentiate and twist, into contribute other cell to phenotypes give cancer andcells initiatedthe dissemination tumorigenesis. and movement CSCs were ability proved to havedriving essential metastasis roles events in metastasis [3]. and drug resistance characters of cancer cells. When a small On the other hand, cancer stem cells (CSCs) represent the subpopulation of cancer cells with the number of CSCs are injected into immunocompromised animal model, they can form new tumors [4]. ability to differentiate into other cell phenotypes and initiated tumorigenesis. CSCs were proved to CSCs also express stemness markers, Nanog, Sox2 and Oct3/4, and CSC markers such as EpCAM, have essential roles in metastasis and drug resistance characters of cancer cells. When a small number CD133,of CSCs CD44, are and injected CD24. into CSCs immunocompromised usually enrichment animal from model, either they from can cancer form new cell tumors lines or [4]. from CSCs patient derivedalso samples.express stemness Isolation markers, of CSC Nanog, is still Sox2 considered and Oct3/4, a challenging and CSC markers and demandingsuch as EpCAM, procedure CD133, [5,6]. InducedCD44, pluripotent and CD24. stemCSCs cellsusually (iPSCs) enrichment have opened from either the from door cancer for personalized cell lines or from medicine patient and derived facilitated modelingsamples. a wide Isolation range of ofCSC diseases. is still considered Our lab hasa challenging established and novel demanding CSC models procedure by [5,6]. converting Induced iPSCs into CSCs.pluripotent Conversion stem cells of iPSCs(iPSCs) into have CSCs opened has beenthe door demonstrated for personalized by culturing medicine iPSCs and infacilitated the presence of conditionedmodeling a media wide range (CM) of from diseases. different Our cancerlab has cellestablished lines secreting novel CSC cytokines, models chemokinesby converting and iPSCs growth factorsinto that CSCs. direct Conversion the conversion of iPSCs without into CSCs genetic has been manipulation demonstrated of by iPSCs. culturing Accordingly, iPSCs in the we presence successfully of conditioned media (CM) from different cancer cell lines secreting cytokines, chemokines and established different mouse cell models using CM from lung, breast, pancreas and liver cancer cell growth factors that direct the conversion without genetic manipulation of iPSCs. Accordingly, we lines [7–11]. successfully established different mouse cell models using CM from lung, breast, pancreas and liver Thecancer selection cell lines of[7–11]. the method to investigate the metastasis is critical for the identification and candidateThe genes selection and mechanisms of the method that to may investigate regulate the metastasis metastasis and is critical for the for evaluation the identification of anti-metastatic and drugs.candidate Recent genes methods and includemechanisms detecting that may metastasis regulate aftermetastasis transplantation and for the of evaluation cancer cells of anti- or tissue eithermetastatic orthotopically drugs. Recent or ectopically methods include in addition detecting to themetastasis injection after of transplantation cancer cells inof cancer blood cells circulation or or intraperitoneallytissue either orthotopically [12,13]. However, or ectopically these in methods addition still to dothe notinjection accurately of cancer present cells thein metastasisblood eventscirculation especially or intraperitoneally regarding the disposition [12,13]. However, of cells these from methods original still tumors do not and accurately injections present cancer the cells neglectsmetastasis dissemination events especially step which regarding is main the thedisposition step in theof cells metastasis from original events. tumors Therefore, and injections developing cancer cells neglects dissemination step which is main the step in the metastasis events. Therefore, new methods is becoming important to investigate metastasis and screening new drugs. Our present developing new methods is becoming important to investigate metastasis and screening new drugs. uniqueOur models present unique that enabling models that investigation enabling investigation of tumor of progression tumor progression events events according according to CSCto CSC theory. In thistheory. manner, In this we manner, present we here present a new here method a new for method studying for studying metastasis metastasis using CSC using model CSC model developed fromdeveloped iPSCs. Our from method iPSCs. investigates Our method the investigates ability of CSCsthe ability to disseminate of CSCs to fromdisseminate bulk intraperitoneally from bulk transplantedintraperitoneally tumor andtransplanted metastasis tumor into and secondary metastasis sites. into secondary sites.

2. Experimental2. Experimental Design Design We haveWe have developed developed a protocol a protocol for for tumor tumor tissuetissue tr transplantationansplantation using using cancer cancer stem stem cell developed cell developed tumor.tumor. In our In assay, our weassay, describe we describe step by step evaluatingby step evaluating the model the of metastasismodel of metastasis by tissue transplantationby tissue of CSCtransplantation derived tumor of CSC as summarizedderived tumor in as Figure summariz1. Thised in protocol Figure will1. This be veryprotocol important will be tovery guide important to guide researchers who will follow developing metastasis from CSCs to evaluate researchers who will follow developing metastasis from CSCs to evaluate treatment strategies and treatment strategies and molecular mechanisms of metastasis development from the sight of CSC molecular mechanisms of metastasis development from the sight of CSC theory. The surgical procedure theory. The surgical procedure is performed under sterile conditions. All instruments are sterilized is performedby autoclaving under before sterile the conditions. procedure. All instruments are sterilized by autoclaving before the procedure.

FigureFigure 1. Representative 1. Representative scheme scheme of the of metastaticthe metastatic procedure procedure using using transplantation transplantation method. method. (a) Injected(a) CancerInjected stem Cancer cells (CSCs) stem cells subcutaneously (CSCs) subcutaneously and leave and it for leave 4 weeks it for to4 weeks form malignantto form malignant tumor. tumor. (b) Excised and minimized(b) Excised and tumor. minimized (c) Transplantation tumor. (c) Transplantation of the minimized of the minimize malignantd malignant tumor in tumor the in in thethe in peritoneal the cavity.peritoneal (d) CSCs cavity. disseminated (d) CSCs disseminated through blood through vessels. blood vessels. (e) Disseminated (e) Disseminated CSCs CSCs reached reached the the organs organs and colonized. and colonized. Methods Protoc. 2020, 3, 60 3 of 8

2.1. Materials Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, cat. no. 31966). • Minimum Essential Media MEM non-essential amino acid solution (100 ) (Wako, Osaka, Japan, • × catalog number: 139-15651). Fetal bovine serum (FBS, Gibco, Life Technologies, Massachusetts, MA, USA, Cat. No: 10437-028). • Penicillin/streptomycin mixed solution (Nacalai Tesque, Kyoto, Japan, Cat. No-26253-84). • L-glutamine (Nacalai Tesque, Kyoto, Japan, ((catalog number: 16948-04)). • Trypsin, 0.25% Ethylenediamine tetraacetic acid (EDTA) (Atlanta Biologicals, Flowery Branch, • GA, USA; Cat. no.: B81310). Phosphate buffered saline (PBS) (Genesee Scientific, El Cajon, CA, USA; Cat. no.: 25-508). • Hank’s balanced salt solution (HBSS). • Iodine solution (7.5% (wt/vol); Medline, cat. no. MDS093908). • Isoflurane (100% (wt/wt); IsoFlo; Abbott, cat. no. B506). • KnockOutTM Serum Replacement (Gibco, NY, USA; Cat. No.: 10828028). • Collagenase (Gibco, NY, USA; Cat. No.: 17018029/). • Mouse induced pluripotent stem cells (miPSCs) (iPS-MEF-Ng-20D-17, Lot No. 012, Riken Cell • Bank, Tokyo, Japan), in which puromycin (puro) resistant gene and green fluorescent protein (GFP) gene were cloned under the control of Nanog promoter. BALB/c-nu/nu immunodeficient mice, female, four weeks old (Charles River laboratories, • kanagawa, Japan). 70% ethanol (Sigma-Aldrich; Cat. No.: 459836-2). • CaCl (Sigma-Aldrich; Cat. No.: C5670). • 2 Leukemia inhibitory factor, 1000 U/mL (LIF, Merk Millipore). • 2.2. Equipment Sanyo MCO-19AIC(UV) CO incubator (Marshall Scientific, Hampton, WV, USA). • 2 Labculture® Class II, Type A2 Biological Safety Cabinets (E-Series). • Olympus IX81 microscope (Olympus, Tokyo, Japan). • Laser scanning confocal microscope, FV-1000, Olympus, Tokyo, Japan. • Tissue culture-treated plate, 60 mm dish (TPP Techno Plastic Products AG Schaffhausen, • Switzerland, Cat. No 93060). Tissue culture-treated plate, 100 mm dish (TPP Techno Plastic Products AG Schaffhausen, • Switzerland, Cat. No 93100). Filter max 250 mL (TPP, Switzerland, Cat. No 99255). • Falcon® Conical Centrifuge Tubes (15 mL; BD Falcon, New York, NY, USA Cat. No 352095). • Falcon® Conical Centrifuge Tubes (50 mL; BD Falcon, New York, NY, USA Cat. No 352070). • Liquid N2 storage tank. • Inverted microscope with bright field (Nikon, model no. DIAPHOT 200). • Automated cell counter (TC20; Bio-Rad Laboratories, cat. no. 1450102). • 1.5-mL microcentrifuge tubes (Eppendorf, cat. no. 0030120086). • Refrigerated centrifuge (Eppendorf, cat. no. 5810 R). • 100-mm dishes (tissue culture treated; Corning, cat. no. 353003). • Anesthesia machine (Vet Tech Solutions). • Microcentrifuge 1.5-mL tubes (Eppendorf, cat. no. 0030120086). • 5/10/25-mL plastic disposable pipette. • Methods Protoc. 2020, 3, 60 4 of 8

3. Procedure I. Cancer stem cell induction for in vivo injection.

Recently Yan et al., 2014, converted iPSCs into cancer stem cells in the presence of extracellular vesicles from Lewis lung carcinoma cell lines named miPS-LLCev cells. The converted cells showed self-renewal,Methods Protoc. 2020 diﬀerentiation, 3, x FOR PEER and REVIEW tumorigenic potential. Moreover, the primary culture cells sustain4 theof 8 expression of self-renewal and CSCs markers [14,15]. The detailed protocol of generating CSCs from iPSCsself-renewal, described differentiation by Aﬁfy et al. and 2019 tumorigenic [16]. potential. Moreover, the primary culture cells sustain the expression of self-renewal and CSCs markers [14,15]. The detailed protocol of generating CSCs II.from CSCs iPSCs preparation described by for Afify injection et al. 2019 [16].

II. CSCsRevive preparation miPS-LLCev for injection cells on gelatin coated dish. •  ChangeRevive miPS-LLCev medium every cells 2 days.on gelatin coated dish. •  WhenChange cells medium are 70% every conﬂuent, 2 days. trypsinize the cells. •  CollectWhen cells cells are from 70% culturing confluent, ﬂask trypsinize and transfer the cells. it to 15 mL tube. •  SpinCollect down cells cells from at culturing 1000 rpm flask for 5 and min. transfer it to 15 mL tube. •  AspirateSpin down supernatant cells at 1000 media. rpm for 5 min. •  WashAspirate cells supernatant with sterile media. PBS. •  AddWash PBS cells to with tube, sterile and again PBS. spin down cells at 1000 rpm for 5 min. • Add PBS to tube, and again spin down cells at 1000 rpm for 5 min. Aspirate supernatant PBS. • Aspirate supernatant PBS. Resuspend cell pellet with 1 mL sterile BSS. • Resuspend cell pellet with 1 mL sterile BSS. Count the cells. • Count the cells. Suspend 106 cells in 100 µL sterile HBSS. • Suspend 106 cells in 100 μL sterile HBSS. Keep the Eppendorf in ice. • Keep the Eppendorf in ice. CRITICALCRITICAL STEP:STEP: drawdraw cellscells intointo thethe syringesyringe withoutwithout aa needleneedle toto preventprevent cellcell shearing.shearing.

 BeforeBefore injecting,injecting, ﬂickflick oror invertinvert thethe syringesyringe toto ensureensure thethe cellscells areare inin suspension.suspension. • InjectInject cellscells slowlyslowly subcutaneously.subcutaneously. • Four weeks later, malignant tumor should be observed Four weeks later, malignant tumor should be observed • Excise and minimize the tumor tissue for intraperitoneal transplantation (Figure 2). Excise and minimize the tumor tissue for intraperitoneal transplantation (Figure2). • III. Intraperitoneal (IP) transplantation

Anesthetize mouse with 2% isoﬂurane (Figure3a). • After the mouse is anesthetized, prepare the area for transplantation with 70% alcohol • (Figure3b). Using the curved iris forceps, hold the skin and make a 15-mm vertical midline incision • through the skin using the 24-mm iris scissors (Figure3c). Insert 1 mm of the tumor tissue intraperitoneal (Figure3d). • CRITICAL STEP: try to avoid damaging any organ in the abdomen.

Close the abdominal wall and skin opening, performing continuous stitching (Figure3e,f). • FigureThe day 2. Fresh after surgically surgery, checkresected on mouse the animal malignant to make tumor sure tissue that ready the to sutures be transplanted. are still correctly • in place. III. Intraperitoneal (IP) transplantation After four weeks, euthanatize mice with 5% of isoﬂurane through inhalation to ensure rapid • lossAnesthetize of consciousness mouse with and 2% respiratory isoflurane and (Figure cardiac 3a). arrest followed by cervical dislocation to ensureAfter the the mouse death is of anesthetized, mice. prepare the area for transplantation with 70% alcohol (Figure 3b). Using the curved iris forceps, hold the skin and make a 15-mm vertical midline incision through the skin using the 24-mm iris scissors (Figure 3c). Insert 1 mm of the tumor tissue intraperitoneal (Figure 3d). CRITICAL STEP: try to avoid damaging any organ in the abdomen. Close the abdominal wall and skin opening, performing continuous stitching (Figure 3e,f). The day after surgery, check on the animal to make sure that the sutures are still correctly in place. Methods Protoc. 2020, 3, x FOR PEER REVIEW 4 of 8

self-renewal, differentiation and tumorigenic potential. Moreover, the primary culture cells sustain the expression of self-renewal and CSCs markers [14,15]. The detailed protocol of generating CSCs from iPSCs described by Afify et al. 2019 [16]. II. CSCs preparation for injection  Revive miPS-LLCev cells on gelatin coated dish.  Change medium every 2 days.  When cells are 70% confluent, trypsinize the cells.  Collect cells from culturing flask and transfer it to 15 mL tube.  Spin down cells at 1000 rpm for 5 min.  Aspirate supernatant media.  Wash cells with sterile PBS.  Add PBS to tube, and again spin down cells at 1000 rpm for 5 min.  Aspirate supernatant PBS.  Resuspend cell pellet with 1 mL sterile BSS.  Count the cells. Methods Protoc.Suspend2020, 3, 60106 cells in 100 μL sterile HBSS. 5 of 8  Keep the Eppendorf in ice. CRITICALCRITICAL STEP: draw four weekscells into are the mandatory syringe without so that a theneedle cells to willprevent have cell enough shearing. time to be disseminatedMethods from Protoc. 2020 the, 3, xoriginal FOR PEER REVIEW tumor to the other organs. Before four weeks,4 of 8 metastasis will  Before injecting, flick or invert the syringe to ensure the cells are in suspension. not be visibleself-renewal, enough. differentiation and tumorigenic potential. Moreover, the primary culture cells sustain  Injectthe cells expression slowly of self-renewalsubcutaneously. and CSCs markers [14,15]. The detailed protocol of generating CSCs  TheFour mouse fromweeks iPSCs allografts later, described malignant by were Afify excisedet al.tumor 2019 [16]. and should cut intobe observed small pieces (approximately 1 mm3). • II. CSCs preparation for injection WashExcise in and the minimize PBS for three the tumor times. tissue for intraperitoneal transplantation (Figure 2). •  Revive miPS-LLCev cells on gelatin coated dish. Transfer the pieces into a 15-mL tube with 4 mL of dissociation buﬀer. •  Change medium every 2 days.  When cells are 70% confluent, trypsinize the cells. Incubate at 37 ◦C for 40 min. •  Collect cells from culturing flask and transfer it to 15 mL tube. Add 5 mL of DMEM containing 10% FBS to terminate the digestion. • Spin down cells at 1000 rpm for 5 min. Transfer suspended Aspirate supernatant cells into media. new tubes. •  Wash cells with sterile PBS. Centrifuge atAdd 1000 PBS rpmto tube, for and 5 again min spin at down 20 C.cells at 1000 rpm for 5 min. • ◦ Resuspend theAspirate cell supernatant pellet in PBS. 5 mL PBS. •  Resuspend cell pellet with 1 mL sterile BSS. Centrifuge atCount 1000 the rpm cells. for 5 min 20 ◦C. •  Suspend 106 cells in 100 μL sterile HBSS. AspirateMethods PBS. Protoc. 2020, 3, x FOR PEER REVIEW 5 of 8 •  Keep the Eppendorf in ice. Resuspend CRITICAL theAfter cell four STEP: pelletweeks, draw euthanatize in cells 5 into mL the mice 5 syringe mL with DMEM 5%without of isoflurane a containing needle through to prevent 10%inhalation cell FBSshearing. to withoutensure LIF. • 4rapid loss of consciousness and respiratory and cardiac arrest followed by cervical Seed 5 10 cells into a 60 mm dish. • × dislocationBefore injecting, to ensure flick theor invert death theof mice. syringe to ensure the cells are in suspension. Treat the cells Inject with cellspuromycin slowly subcutaneously. for 24 h to remove the host cells. • CRITICAL Four weeks STEP: later, four malignant weeks are tumor mandatory should sobe observedthat the cells will have enough time to be Figure 2. Freshdisseminated Excise surgically and from minimize theresected original the tumor mouse tumor tissue to malignant the for othe intraperitonealr organs. tumor Before transplantation tissue four readyweeks, (Figure metastasisto be 2). transplanted. will not be visible enough.

3 The mouse allografts were excised and cut into small pieces (approximately 1 mm ). III. Intraperitoneal (IP)Wash transplantation in the PBS for three times. Transfer the pieces into a 15-mL tube with 4 mL of dissociation buffer. Anesthetize Incubate mouse at with37 °C for 2% 40 isofluranemin. (Figure 3a). After the mouse Add 5 mLis anesthetized,of DMEM containing prepare 10% FBS tothe terminate area forthe digestion.transplantation with 70% alcohol (Figure 3b). Transfer suspended cells into new tubes. Centrifuge at 1000 rpm for 5 min at 20 °C. Using the curvedResuspend iris the forceps, cell pellet inhold 5 mL thePBS. skin and make a 15-mm vertical midline incision through the Centrifuge skin using at 1000 the rpm 24-mm for 5 min iris 20 °C. scissors (Figure 3c). Aspirate PBS. Insert 1 mm Resuspend of the tumor the cell pellettissue in 5 intraperitoneal mL 5 ml DMEM containing (Figure 10% 3d).FBS without LIF. 4 FigureSeed 5 2. × Fresh 10 cells surgically into a resected60 mm dish.mouse malignant tumor tissue ready to be transplanted. CRITICALFigure 2. STEP:Fresh Treat surgicallytry the to cells avoid with resected puromycin damaging mouse for 24 any malignant h to organremove tumorthe in hostthe tissuecells. abdomen. ready to be transplanted. CloseIII. theIntraperitoneal abdominal (IP) wall transplantation and skin opening, performing continuous stitching (Figure 3e,f). Anesthetize mouse with 2% isoflurane (Figure 3a). The day after surgery, check on the(b) animal to make sure(c) that the sutures are still correctly After(a) the mouse is anesthetized, prepare the area for transplantation with 70% alcohol in place. (Figure 3b). Using the curved iris forceps, hold the skin and make a 15-mm vertical midline incision through the skin using the 24-mm iris scissors (Figure 3c). Insert 1 mm of the tumor tissue intraperitoneal (Figure 3d). CRITICAL STEP: try to avoid damaging any organ in the abdomen. Close the abdominal wall and skin opening, performing continuous stitching (Figure 3e,f). The day after surgery, check on the animal to make sure that the sutures are still correctly in place.

(d) (e) (f)

Figure 3. TransplantationFigure 3. Transplantation method method steps: steps: (a(a)) Anesthetize mouse. mouse. (b) Preparing (b) the Preparing area for the area for transplantation. (c) A 15-mm vertical midline incision through the skin. (d) Inserting the tumor tissue. transplantation.(e), (fc)) Closing A 15-mm the abdominal vertical wall and midline skin opening. incision through the skin. (d) Inserting the tumor tissue. (e), (f) Closing the abdominal wall and skin opening.

Methods Protoc. 2020, 3, x FOR PEER REVIEW 6 of 8

4. Expected Results This protocol describes a technique of disposition of CSCs from primary tumor and dissemination to another organs which is main the step in the metastasis events. Our present unique models enable investigation of tumor progression events according to CSC theory. In this manner, we present here a new method for studying metastasis using CSC model developed from iPSCs. Our method investigates the ability of CSCs to disseminate from bulk of the intraperitoneally transplanted tumorMethods through Protoc. 2020 blood, 3, 60 stream and metastasis into secondary sites. Therefore, developing new methods6 of 8 is becoming important to investigate metastasis steps clearly and screening of new drugs. In Figure 4, we showed the presence of CSCs in the primary cell culture by expressing GFP and cell morphology4. Expected was Results observed and photographed using Olympus IX81 microscope equipped with a light fluorescenceThis protocol device. describes a technique of disposition of CSCs from primary tumor and dissemination to anotherOn the organs other hand, which our is main result the has step been in the confir metastasismed by events. RT-qPCR Our presentanalysis, unique lung and models pancreatic enable metastaticinvestigation cells of were tumor confirmed progression to sustain events accordingthe expression to CSC of theory.endogenous In this stemness manner, wemarker present c-Myc, here as a muchnew methodas miPSCs. for studying On the other metastasis hand, using the expression CSC model of developed CSC-marker from CD44 iPSCs. was Our extremely method investigates elevated in boththe abilitytypes of of metastatic CSCs to disseminate cells. Furthermore, from bulk the of expression the intraperitoneally of metastatic transplanted markers vimentin tumor through and E- cadherinblood stream was significantly and metastasis different into secondary between lung sites. an Therefore,d pancreatic developing metastatic new cells, methods whereas is E-cadherin becoming expressionimportant showed to investigate significantly metastasis higher steps expression clearly and of screening more than of newdouble drugs. when In compared Figure4, we to showed miPSCs cellsthe presence(p < 0.001). of CSCs At the in the same primary time, cell vimentin culture byshowed expressing relatively GFP and higher cell morphology expression wasin pancreatic observed metastaticand photographed cells in comparison using Olympus with IX81miPSCs microscope (Figure 5). equipped with a light ﬂuorescence device.

Figure 4. Primary cell cultures of metastatic tumors in pancreatic and lung. BF, bright field; GFP, Figure 4. Primary cell cultures of metastatic tumors in pancreatic and lung. BF, bright field; GFP, detection of green fluorescence. detection of green fluorescence. On the other hand, our result has been confirmed by RT-qPCR analysis, lung and pancreatic metastatic cells were confirmed to sustain the expression of endogenous stemness marker c-Myc, as much as miPSCs. On the other hand, the expression of CSC-marker CD44 was extremely elevated in both types of metastatic cells. Furthermore, the expression of metastatic markers vimentin and E-cadherin was significantly different between lung and pancreatic metastatic cells, whereas E-cadherin expression showed significantly higher expression of more than double when compared to miPSCs cells (p < 0.001). At the same time, vimentin showed relatively higher expression in pancreatic metastatic cells in comparison with miPSCs (Figure5). Methods Protoc. 2020, 3, 60 7 of 8 Methods Protoc. 2020, 3, x FOR PEER REVIEW 7 of 8

Figure 5. (a) RT-qPCR analysis of CSC marker CD44; (b) RT-qPCR analysis of stemness marker Figure 5. (a) RT-qPCR analysis of CSC marker CD44; (b) RT-qPCR analysis of stemness marker c-Myc c-Myc (c), (d) RT-qPCR analysis of metastatic markers E-cadherin and vimentin in cells derived from (c), (d) RT-qPCR analysis of metastatic markers E-cadherin and vimentin in cells derived from intraperitoneal transplantation (lung and pancreatic metastatic cells) in comparison with miPS cells. intraperitoneal transplantation (lung and pancreatic metastatic cells) in comparison with miPS cells. MET.; Metastasis. (**, p 0.01; ***, p 0.001) MET.; Metastasis. ≤ ≤ 5. Reagents Setup 5. Reagents Setup Dissociation Buﬀer Dissociation Buffer Prepared in PBS containing: Prepared in PBS containing: 0.25% trypsin •  0.1%0.25% collagenasetrypsin •  20%0.1% KnockOutcollagenaseTM Serum Replacement (Gibco, NY, USA). • 20% KnockOutTM Serum Replacement (Gibco, NY, USA). 1 mM of CaCl2. • 1 mM of CaCl2.

Author Contributions:Contributions: H.M.H.M. and and G.H. G.H. conceived conceived and and designed designed the study;the study; H.M., H.M., S.M.A. S.M.A. and G.H. and writing—reviewG.H. writing— andreview editing; and editing; H.M., G.H., H.M., T.Y. G.H., and T.Y. A.S. and conducted A.S. conducted the experiments; the experiments; M.S. directed M.S. thedirected research the projectresearch and project wrote and the manuscriptwrote the manuscript together with together H.M. Allwith authors H.M. haveAll authors read and have agreed read to and the agreed published to the version published of the manuscript.version of the Funding:manuscript.This research was supported by the Grant-in-Aid for Scientific Research (C) No. 16K07116 and for Challenging Exploratory Research No. 26640079. Funding: This research was supported by the Grant-in-Aid for Scientific Research (C) No. 16K07116 and for ConflictsChallenging of Interest:ExploratoryAll authorsResearch declare No. 26640079 no conflicts of interest.

Conflicts of Interest: All authors declare no conflicts of interest.

References

1. Seyfried, T.N.; Huysentruyt, L.C. On the origin of cancer metastasis. Crit. Rev. Oncog. 2013, 18, 43–73. Methods Protoc. 2020, 3, 60 8 of 8

References

1. Seyfried, T.N.; Huysentruyt, L.C. On the origin of cancer metastasis. Crit. Rev. Oncog. 2013, 18, 43–73. [CrossRef][PubMed] 2. Fidler, I.J. The pathogenesis of cancer metastasis: The ‘seed and soil’ hypothesis revisited. Nat. Rev. Cancer 2003, 3, 453–458. [CrossRef][PubMed] 3. Ell, B.; Kang, Y. Transcriptional control of cancer metastasis. Trends Cell Biol. 2013, 23, 603–611. [CrossRef] [PubMed] 4. Chang, J.C. Cancer stem cells: Role in tumor growth, recurrence, metastasis, and treatment resistance. Medicine (Baltimore) 2016, 95, S20–S25. [CrossRef][PubMed] 5. Khan, M.I.; Czarnecka, A.M.; Helbrecht, I.; Bartnik, E.; Lian, F.; Szczylik, C. Current approaches in identification and isolation of human renal cell carcinoma cancer stem cells. Stem Cell Res. Ther. 2015, 6, 178. [CrossRef][PubMed] 6. Shang, Z.; Xu, Y.; Liang, W.; Liang, K.; Hu, X.; Wang, L.; Zou, Z.; Ma, Y. Isolation of cancer progenitor cells from cancer stem cells in gastric cancer. Mol. Med. Rep. 2017, 15, 3637–3643. [CrossRef][PubMed] 7. Afify, S.M.; Hassan, G.; Osman, A.; Calle, A.S.; Nawara, H.M.; Zahra, M.H.; El-Ghlban, S.; Mansour, H.; Alam, M.J.; Abu Quora, H.A.; et al. Metastasis of Cancer Stem Cells Developed in the Microenvironment of Hepatocellular Carcinoma. Bioengineering 2019, 6, 73. [CrossRef][PubMed] 8. Calle, A.S.; Nair, N.; Oo, A.K.; Prieto-Vila, M.; Koga, M.; Khayrani, A.C.; Hussein, M.; Hurley, L.; Vaidyanath, A.; Seno, A.; et al. A new PDAC mouse model originated from iPSCs-converted pancreatic cancer stem cells (CSCcm). Am. J. Cancer Res. 2016, 6, 2799–2815. [PubMed] 9. Chen, L.; Kasai, T.; Li, Y.; Sugii, Y.; Jin, G.; Okada, M.; Vaidyanath, A.; Mizutani, A.; Satoh, A.; Kudoh, T.; et al. A Model of Cancer Stem Cells Derived from Mouse Induced Pluripotent Stem Cells. PLoS ONE 2012, 7, e33544. [CrossRef][PubMed] 10. Hassan, G.; Afify, M.S.; Nair, N.; Kumon, K.; Osman, A.; Du, J.; Mansour, H.; Abu Quora, A.H.; Nawara, M.H.; Satoh, A.; et al. Hematopoietic Cells Derived from Cancer Stem Cells Generated from Mouse Induced Pluripotent Stem Cells. Cancers 2019, 12, 82. [CrossRef][PubMed] 11. Seno, A.; Kasai, T.; Ikeda, M.; Vaidyanath, A.; Masuda, J.; Mizutani, A.; Murakami, H.; Ishikawa, T.; Seno, M. Characterization of gene expression patterns among artificially developed cancer stem cells using spherical self-organizing map. Cancer Inform. 2016, 15, CIN-S39839. [CrossRef][PubMed] 12. Khanna, C.; Hunter, K. Modeling metastasis in vivo. Carcinogenesis 2005, 26, 513–523. [CrossRef][PubMed] 13. Suhail, Y.; Cain, M.P.; Vanaja, K.; Kurywchak, P.A.; Levchenko, A.; Kalluri, R.; Kshitiz. Systems Biology of Cancer Metastasis. Cell Syst. 2019, 9, 109–127. [CrossRef][PubMed] 14. Yan, T.; Mizutani, A.; Chen, L.; Takaki, M.; Hiramoto, Y.; Matsuda, S. Characterization of cancer stem-like cells derived from mouse induced pluripotent stem cells transformed by tumor-derived extracellular vesicles. J. Cancer 2014, 5, 572–584. [CrossRef][PubMed] 15. Afify, S.M.; Seno, M. Conversion of Stem Cells to Cancer Stem Cells: Undercurrent of Cancer Initiation. Cancers 2019, 11, 345. [CrossRef][PubMed] 16. Afify, S.M.; Chen, L.; Yan, T.; Sanchez Calle, A.; Nair, N.; Murakami, C.; Seno, M. Method to convert stem cells into cancer stem cells. Methods Protoc. 2019, 3, 71. [CrossRef][PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).