Analysis of Tumor Vasculature developed
from Cancer Stem Cells.

September 2016

Marta Prieto-Vila

Graduate School of Natural Science and Technology(Doctor Course)

OKAYAMA UNIVERSITY

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Abstract
To grow beyond a size of approximately 1-2 mm3, tumor cells activate many
processes to develop blood vasculature. Growing evidences indicate that the formationof the tumor vascular network is very complex, and is not restricted to angiogenesis.
Cancer cell-derived tumor vasculatures have been recently described. Among them,endothelial differentiation of tumor cells have been directly related to cancer stem cells,
which are cells within a tumor that possess the capacity to self-renew, and to exhibit
multipotential heterogeneous lineages of cancer cells. Vasculogenic mimicry has been
described to be formed by cancer cells expressing stemness markers. Thus, Cancer StemCells have been proposed to contribute to vasculogenic mimicry, though its relation is
yet to be clarified. Here, we analyzed the tumor vasculature by using a model of mousecancer stem cells, miPS-LLCcm cells, which we have previously established frommouse induced pluripotent stem cells and we introduced the DsRed gene in miPSLLCcm to trace them in vivo. Various features of vasculature were evaluated in ovo, in
vitro, and in vivo. The tumors formed in allograft nude mice exhibited angiogenesis inchick chorioallantoic membrane assay. In those tumors, along with penetrated hostendothelial vessels, we detected endothelial differentiation from cancer stem cells andformation of vasculogenic mimicry. The angiogenic factors such as VEGF-A and FGF2
were expressed predominantly in the cancer stem cells subpopulation of miPS-LLCcm
cells. Our results suggested that cancer stem cells play key roles in not only therecruitment of host endothelial vessels into tumor, but also in maturation of endothelial
linage of cancer stem cell’s progenies. Furthermore, the undifferentiated subpopulationof the miPS-LLCcm participates directly in the vasculogenic mimicry formation.
Collectively, we show that miPS-LLCcm cells have advantages to further study tumor
vasculature and to develop novel targeting strategies in the future.

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Contents
page

Abstract

1

Chapter 1. General introduction

5

Chapter 2. DsRed-LLCcm is an improved model from miPS-LLCcm cells.

15

Introduction

17

Materials and methods

18

Results

19

Discussion

22

Chapter 3. miPS-LLCcm and DsRed-LLCcm cells release factors that 25
induce angiogenic response
Introduction

27

Materials and methods

27

Results

30

Discussion

37

Chapter 4. miPS-LLCcm cells differentiate into endothelial cells in vitro 41
and in vivo.
Introduction

43

Materials and methods

43

Results

45

Discussion

51

Chapter 5. DsRed-LLCcm cells participate in the formation of Vasculature 57
Mimicry.
Introduction

59

Materials and methods

59

Results

60

Discussion

62
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Chapter 6. General conclusions and futures challenges

65

List of publications

69

Acknowledgements

71

4

CHAPTER 1
ooo ooo

General Introduction

5

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1.1 Cancer Stem Cell concept and concern.
Cancer stem cells (CSCs) are cells within a tumor that possess the capacity to selfrenew and to exhibit multipotential heterogeneous lineages of cancer cells that comprisethe tumor [1].
The CSC hypothesis was firstly postulated more than 150 years ago [2], and cancer
cells were thought to arise from germ cells, however this hypothesis was forgotten andnot reconsidered and intense researched until in 1997 when John Dick described cancer
stem cells in leukemia. [3]
CSC are thought to be responsible for various cancer pathologies, such as metastasisor recurrence due to CSC exhibiting higher drug resistance compared to other tumor
cells. And although the primary tumors may be eliminated from the patient with thetreatment, the tumor reappears after a period of time, arising from the small
subpopulation of CSC that survived, either in the primary location or a new position
after metastasis (Figure 1).

Figure 1. Scheme of tumor relapse due to Cancer stem cells. After treatment mostof tumor cells die, however a small number of cells that are more resistant to thetreatment, called cancer stem cells. Those, while self-renewing to maintain the CSC
subpopulation (up), are also able to differentiate again (right), generating a tumor on
the same place. CSC can also migrate to distant places with blood stream and generate a
new tumor there (down).

Normal stem cells and CSC shares several properties, including the capacity of selfrenew, differentiation capacity, the active telomerase expression, the activation of7

antiapoptotic pathways, the increase of membrane transporter activity and he ability to
migrate [2, 4]. However, the origin of cancer stem cells is yet to be clarified. Fialow etal. showed that a single progenitor cell that is responsible for the repeatable clonal
formation and cancer creation, was provided in the study of chronic mylogenous
leukemia and acute leukemia [5], evidencing that normal tissue stem cells are the targetof mutational accumulation resulting in CSC, which afterwards differentiate toheterogeneous tumor cell population. On the other hand recent studies showed that CSC
can arise from progenitor cells which acquired self-renewal capacity and generate moredifferentiate progenies [6, 7].
During the last 20 years, CSCs have been described and isolated from various
cancers [8-12] allowing the CSC hypothesis to be increasingly accepted.

1.2 Tumor niche.
Normal stem cells in the body are responsible for the organic generation, due to theircapacity to self-renew as well as differentiate into various lineages that allows for thepreservation of homeostatic pool, necessary for maintenance of a tissue for the life of anorganism. Without stem cells the tissue organ would eventually degenerate [13]. A
refined balance between these two opposite processes is significant for the proper
maintenance and repair [14], therefore the expansion of stem cells pool is restricted toprevent uncontrolled growth [15]. This control is done by the stem cell niche. Niches
are specialized microenvironments that regulate adult stem cells fate by providing cuesin the form of both cell-cell contacts and secreted factors. They are comprised offibroblastic cells, immune cells, endothelial and perivascular cells, extracellular matrix(ECM) components and networks of cytokines and growth factors [16, 17].
CSCs, as well as normal stem cells, due to their similarities, are believed to reside inniches [17]. Cells within the CSC niche produce factors that stimulate CSC self-renewal,
induce angiogenesis and recruit immune and other stromal cells that, at the same time,those will secrete additional factors to promote tumor cell invasion and metastasis [18,19].
Among all the elements in tumor niche, tumor vasculature is an essential factor since8vessels are in charge of the supply of nutrients and oxygen to sustain tumor tissues.

1.3 CSCs in tumor vasculature
Considering the tumor vasculature is one element from CSCs niche, it is to assume
that CSC would participate either directly or indirectly in tumor vasculature.
Several groups reported that the CSCs reside adjacent to the tumor blood vessels, whichtermed the vascular CSC niche [20]. For example, endothelial cells (EC) were proved toreside adjacent to nestin positive cells in glioblastoma, promoted the growth of tumor
cell population and self-renewal of the CSCs inside [20, 21] andsustained theovercoming of CSCs from radiotherapy [22], suggesting that the EC cells play a role in
CSC niche. Its seems to be regulated by several pathways in the microenviroment that
the EC encourage the maintenance of CSC, such as Notch signaling pathway [21] .
Although, the source and the exact mechanism of this process is remaining unknown
and it should be the critical factor for the controlling of CSC maintenance.
One of the most significant findings regarding tumor vasculogenesis and CSCs is the
direct differentiation of glioblastoma CD133+ CSCs into vascular endothelial cells
expressing CD31 when these cells were injected into nude mice. Moreover, when CSC
were killed, tumor growth was greatly suppressed, indicating that EC directly contributeto blood vessel formation in glioblastoma [23 24]. Additionally, breast and renal CSCs
differentiated after 14 days of incubation under hypoxia and with VEGF containing
complete medium in vitro [25].
In addition to the direct differentiation into endothelial cells, the high degree ofplasticity of CSCs and the fact that cells lining the channels express stemness-related
genes [26, 27] indicate that CSCs might be involved in Vasculogenic mimicry (VM).
VM are vascular channels that supply blood to the tumor is lined not by EC, but tumor
cells itself, and is rich in ECM. [28, 29] Despite the undifferentiated state of cells lining
the VM channels, currently there is no clear evidence that demonstrates the direct
relation of CSCs and VM

The processes of tumor vasculogenesis should therefore be investigated with the9consideration of the properties of CSCs. To understand the entire process of tumor
vasculogenesis, our laboratory developed a model of CSCs, which formed highly
angiogenic tumors in nude mice, and exhibited the capacity of differentiation into
endothelial cells in vitro [30]. However, the origin of the cells in the vascular structuresin the tumor formed by miPS-LLCcm has not been assessed directly. In this study, I
evaluated angiogenesis, CSC’s differentiation into ECs, and VM, along with their
possible correlations during the vasculature development in the tumors of miPS-LLCcm
cells.

1.4 References[1] Clarke MF, Dick JE, Dirs PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J,
Weissman IL, Wahl GM. Cancer stem cells-prespectives on current status andfuture directions:ACR Workshop on cancer stem cells. Cancer Res. 2006; 66:9339934[2] Wisha MS, Liu S, Dontu G. Cancer stem cells: an old idea –a paradigm shift. Cancer
Res. 2006; 66: 1883-1890; discussion 1895-1886.

[3] Human acute myeloid leukemia is organized as a hierarchy that originates from aprimitive hematopoietic cell. Bonnet D, Dick JE. Nature Med. 1997; (3): 730 – 737.

[4] The biology of cancer stem cells. Lobo NA, Shimono Y, Qian D, Clarke MF. Annu
Rev Cell Dev Biol. 2007; 23: 675-699.

[5] Fialow PJ. Stem cell origin of human myeloid blood cell neoplasm. Verh Dstch Gen
Pahol. 1990; 74:43-47.

[6] Lavau C, Szilvassy SJ, Snay R, Cleary ML. Immoralization and leukemictransformation of myelomonocytic precursor by retrovirally transduced. HRX-ENL.
Embo J. 1997; 16:4226-4237.
10[7] Kristov AV, Twomey D, Feng Z, Stubbs MC, Wang Y, faber J et al. Transformationfrom commited progenitor to leukemia stem cells initiated by MLL-AF9. Nature.
2006; 442: 818-822.

[8] Miyoshi N, Ishii H, Sekimoto M, Haraguchi N, Doki Y, Mori M. Properties andidentification of cancer stem cells: a changing insight into intractable cancer. Surg
Today. 2010; 40:608-613.

[9] Suva ML, Riggi N, Stehle JC, Baumer K, Tercier S, Joseph JM et al. Identificationof cancer stem cells in Edwings Sarcoma. Cancer Res. 2009; 69:1776-1781.

[10] Bussolati B, Bruno S, Grange C, Ferrando U, Camussi G. Idenification of a tumorinitiating stem cells population in human renal carcinomas. FASEB J. 2008;
22:3698-3705.

[11] Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S, et al. Isolation andcharacterization of tumorigenic, stem-like neral precursors from humanglioblastoma. Cancer Res. 2004; 64: 7011-7021.

[12] Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V et al. Identification of
pancreatic cancer stem cells. Cancer res. 2007; 67: 1030-1037.

[13] Clains J. The origin of human cancers. Nature. 1981; 289:353-357.

[14] Sneddon JB, Werb Z. Location, location, location: the cancer stem cell niche. Cell
stem cell. 2007; 1:607-611.

[15] Morrison Sj, Quian D, Jerebek L, Thiel BA, Park I-K, et al. A genetic determinantthat specifically regulates the frequency of hematopietic stem cells. J Immunol.
2002; 168:635-642.

[16] Hanahan D, Coussens LM. Accessories to the crime: functions of cells recruited to11the tumor microenviroment. Cancer cell. 2012; 21:309-322.

[17] Plaks V, Kong N, Werb Z. The cancer stem cell niche: How essential is he niche inregulating stemness of tumor cells?. Cell Stem cell. 2015; 16: 225-238.

[18] Oskarsson T, Batlle E, Massagué J. Mestastatic stem cells: sources. Niches andvital pathways. Cell stem cell. 2015; 14: 306-321.

[19] Kreso A, Dick J. Evolution of cancer stem cell model. Cell stem cell. 2014; 4:275291.
[20] Cabarcas SM, Mathews LA, Farrar WL. The cancer stem cell niche –there goes theneighborhood?. Int J Cancer. 2001; 129:2315-2327.

[21 ] Zhu TS, Costello MA, Talsma CE, Flack CG, Crowley JG, Hamm LL, et al.
Endothelial cells create a stem cell niche in glioblastoma by providing NOTCH
ligands that nurture self-renewal of cancer stem-like cells. Cancer Res.
2011;71:6011-6072.

[22] Hambardumayn D, Becher OJ, Rosenblum MK, Pandolfi PP, Manova-Todorova K,
Holland EC. PI3K pathway regulates survival of cancer stem cells residing in theperivascular miche following radiation in medulloblastoma in vivo. Genes Des.
2008; 22:436-448.

[23] Ricci-Vitiani L, Pallini R, Biffoni M, Todaro M, Invernici G, Cenci T, Maira G,
Parati EA, Stassi G, Larocca LM, De Maria R. Tumour vascularization via
endothelial differentiation of glioblastoma stem-like cells. Nature. 2011; 468:824–
828[24] Wang R, Chadalavada K, Wilshire J, Kowalik U, Hovinga KE, Geber A, Fligelman
B, Leversha M, Brennan C, Tabar V. Glioblastoma stem-like cells give rise totumour endothelium. Nature. 2010; 468:829–833[25] Brossa A, Grange C, Mancuso L, Annaratone L, Maria Satolli MA, Mazzone M,
Camussi G, Bussolati B. Sunitinib but not VEGF blockade inhibits cancer stem cell
12

endothelial differentiation. Oncotarget. 2015;6(13):11295-11309.
[26] Kaur S, Bajwa P. A ‘tête-à tête’ between cancer stem cells and endothelial
progenitor cells in tumor angiogenesis. Clin Transl Oncol. 2014;16(3):115-121.

[27] Yue W-Y. Does Vasculogenic Mimicry Exist in Astrocytoma? J Histochem
Cytochem. 2005;53(8):997-1002. doi:10.1369/jhc.4A6521.2005.

[28] Chen YS, Chen ZP. Vasculogenic mimicry: A novel target for glioma therapy.
Chin J Cancer. 2014;33(2):74-79.
[29] Maniotis AJ, Folberg R, Hess A, Sefto EA, Gardner LMG, Pe’er J, Trent JM,
Meltzer PS, Hendrix MJC. Vascular channel formation by human melanoma cells
in vivo and in vitro: vasculogenic mimicry. Am J Pathol. 1999;155(3):739-752.

[30] Chen L, Kasai T, Li Y, et al. A model of cancer stem cells derived from mouse
induced pluripotent stem cells. PLoS One. 2012;7(4):e33544.

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CHAPTER 2
ooo ooo

DsRed-LLCcm is an improved model frommiPS-LLCcm cells.

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2.1 Introduction
We have previously established models of CSCs, that were spontaneously converted
from mouse induced pluripotent stem cells (miPS) cultured in the presence ofconditioned medium (CM) from various cancer cell lines. We reported previously thatmiPS-LLCcm, a representative miPS-CSC converted with CM of Lewis lung carcinoma(LLC) cells or LLC-derived microvesicles/exosomes (Figure 1) [1, 2]. We haveproposed that these models are very promising to study cancer’s nature from the aspectof CSCs properties, such as the relation between CSCs and differentiated cancer cells [1,3]. miPS-LLCcm cells generated malignant, highly vascularized tumors when they weretransplanted in nude mice. In addition, we could reveal the differentiation capacity ofmiPS-LLCcm cells into endothelial cells in vitro [2, 3].

Figure 1. Scheme of miPS-LLCcm generation. miPS cells were cultured without
feeder cells and one day without LiF, before the conversion. During the conversion, theCM was daily changed up to one month. After that the resulting cells were injected into
nude mouse, where they generated malignant tumor.

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In the previous study, however, it remained unclear whether the cells forming thevasculature in the tumor are derived from miPS-LLCcm or not. To confirm whether thecells forming the vasculature in the tumor originated from miPS-LLCcm cells or hostmouse cells, a previous student, Yan Ting, established DsRed-LLCcm cells introducing
pEF-DsRed into miPS-LLCcm cells. Since miPS-LLCcm cells were derived from
Nanog-GFP-iPSCs [4], the undifferentiated state could be monitored by the expressionof GFP together with that of DsRed. On the other hand, the cells differentiated frommiPS-LLCcm cells would display only DsRed fluorescence .
Once DsRed-LLCcm cells were established, their characteristic had to been analyzed
to confirm whether DsRed-LLCcm cells maintained miPS-LLCcm properties.

2. 2 Materials and methods
2.2.1 Construction of DsRed expression vector.
The DsRed2 gene was amplified from pCI-EGFP/DsRed2-puro by PCR with aprimer pair; EcoRI-DsRed (5’-CCGGAATTCATGGCCTCTCC-3’) and SalI-DsRed(5’-TCCGGTCGACCTACAGGAACAG-3’) to add EcoRI and SalI sites to the 5’ and 3’
end of Ds-Red gene, respectively. Then, the PCR product was cloned downstream of
EF1 constitutive promoter in pEF-EX-HA vector, which was constructed by adding an
HA tag to pEF-BOS-EX [5] with the similar construction procedure of Flag tag
described in [6], to create pEF-DsRed. For pEF-neo plasmid construction, the G418
resistant gene was amplified from pST-Neo by PCR with a primer pair EcoRI-neo (5’GCCGGAATTCATGATTGAACAAGATGGA-3’)

and

(5’-

SalI-neo

TGTAGTCGACTCAGAAGAACTCGTCAAG-3’) to add EcoRI and SalI sites to the 5’
and 3’ sites of the gene. Amplified gene was cloned into pEF-EX-HA.

2.2.2 Cell culture and transfection.
miPS-LLCcm and DsRed-LLCcm cells were cultured in DMEM containing 15%
FBS, 0.1 mM MEM Non-Essential amino acids, (100X NEAA, Gibco, NY), 2 mM LGlutamine(NacalaiTesque,Japan),

0.1mM

2-mercaptoethanol,

50

U/ml

penicillin/streptomycin and 1000 U/mL and without LIF. All the cells were maintained18

at 37 ºC in the atmosphere of 5% CO2. Medium was changed every two days.
For transfection, 1x107 miPS-LLCcm cells, which had been maintained insuspension culture for 8 days, in 600µL electroporation buffer for ES cells (Millipore,MA) were mixed with 1µg of linearized pEF-neo DNA and 10 µg of linearized pEFDsRed DNA. Then, the cell suspension was transferred into a 0.4cm gap-cuvette andelectroporated by using Gene Pulser II (Bio Rad Labs, VA). The cells were then plated
into gelatin-coated dishes without antibiotics for 2 days. Then the DsRed positivetransfectants (termed DsRed-LLCcm) were established by culturing for 1 to 2 weeks inthe presence of G418 at a concentration of 0.3 mg/mL.

2.2.3 Sphere formation assay.
4x104 cells (1x104 cells/ml) were seeded on 6cm ultra low attachment dishes(Corning incorporated, NY) with the miPS-LLCcm media without FBS, but suppliedwith Insulin-Transferrin-Selenium-X (ITS-X, Life Technologies, CA). Cells were
cultured at 37 ºC under 5% CO2 for 4 days, and the spheroids with diameters above 100
μm were judged as self-renewing spheroids.

2.2.4 In vitro Tube formation assay
5x104 cells were seeded on growth factors reduced Matrigel (Corning, NY) -coated96well plate and cultured in 50uL of EBM2 media (EBM-2 Single Quots Kit, Lonza,
Switzerland) for 24hr in the presence of angiogenic factors (FBS, hydrocortisone,hFGF-B, VEGF, R3-IGF-1, hEGF, ascorbic acid, GA-1000 and heparin). The
experiments were performed in triplicate. Images of formed tubes were captured by
Olympus CKX41 microscope, or Keyence BZ-X700.

2.3 Results
2.3.1 DsRed-LLCcm are equivalent to miPS-LLCcm cells.
The newly established DsRed-LLCcm cells are an improved model of CSC derived
from miPS-LLCcm. Since miPS-LLCcm cells were derived from Nanog-GFP-iPSCs[4], the undifferentiated state could be monitored by the expression of GFP. DsRed19

LLCcm introduced the DsRed2 gene expressed constitutively, that allowed to trace thecells even when they differentiate (Figure 2.1).
miPS-LLCcm have been described to generate malignant, highly vascularized tumors
when they were transplanted in nude mice and, in vitro to present self-renewal anddifferentiation capacity of miPS-LLCcm cells into endothelial [2,3]. First I evaluatedwhether DsRed-LLCcm maintained miPS-LLCcm properties.
Self-renewal was assessed by sphere formation assay [7]. DsRed-LLCcm cells wereable to form spheres under non-adherent conditions as miPS-LLCcm did (Figure2.2).
All the spheres derived from DsRed-LLCcm were GFP and DsRed positive. Supporting
their stemness state, the expression levels of endogenous Oct3/4, Sox2, Klf4 and c-Myc
were comparable to those in miPS-LLCcm cells (Figure 2.3). DsRed-LLCcm cells werealso able to generate tube-like structures when the cells were seeded on Matrigel (Figure2.4). Similar to miPS-LLCcm cells, this tube formation was not dependent on the
supplement of endothelial cell growth factor such as VEGF or FGF (Chapter 3, Figure2.5). The entire tubular structures possessed DsRed fluorescence. In contrast, GFP
positive cells were found to be concentrated on the nodules of the tubes, where two or
more tubular structures are connected (Figure 2.4). As described below in Chapter 3,
and as miPS-LLCcm cells do, DsRed-LLCcm cells generated malignant and highly
vascularized tumors (Chapter 3, Figure 1).
Taken all together, I could confirm that DsRed-LLCcm cells remained thecharacteristics of miPS-LLCcm cells.

20

Figure 2.1 Morphology of miPS-LLCcm and DsRed-LLCcm cells. Under thecontrol of EF1 promoter, DsRed was expressed constitutively, while GFP was only
expressed in undifferentiated cells. Scale bar: 50µm.

Figure 2.2 Sphere formation of DsRed-LLCcm cells. All the spheres are GFP+ and21

DsRed+, supporting ability to self renewal of undifferentiated cells. Scale bar: 100µm.

Figure 2.3 Expression levels of Sox2, Oct3/4, Klf4, and C-myc in DsRed-LLCcm
cells analyzed by RT-qPCR. The expressions of those genes in DsRed-LLCcm cells
were equivalent to those in miPS-LLCcm cells (Sox2 p=0,71; Oct3/4 p=0,45; Klf4 =
0,20; c-myc p= 0,73). Expression values are normalized to GAPDH

Figure 2.4 In vitro tube formation of DsRed-LLCcm cells. While all the cells
expressed DsRed, the GFP+ cells were found mostly in the nodules. Scale bar: 200µm.

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2.4 Discussion
The growing evidences indicate that formation of the tumor vascular network is acomplex event in the cancerous niche [8]. Recently two models of tumor vasculogenesis,
CSC differentiation into EC and vasculogenic mimicry, emphasize the contribution of
CSCs towards tumor vascularization [9,10]. To analyze the relationship of the twomodels, we adopted our miPS-LLCcm cell, which is a model of CSC that exhibits selfrenewal [7], differentiation and ability to generate a malignant tumor in nude mice [1,3].
First of all, we introduced DsRed2 gene into miPS-LLCcm cells, establishing DsRedLLCcm cells, which besides from the GFP fluorescence under the expression of Nanog,
constitutively display red fluorescence. The introduction of DsRed2 gene represents an
improvement of miPS-LLCcm model since it allows us to trace miPS-LLCcm
throughout the cell differentiation and localization both in vitro and in vivo. Studies inself-renewal and differentiation capacity along with tumorigenicity, indicated that theinsertion of DsRed2 gene did not affect miPS-LLCcm properties.

2.5 References[1] Chen L, Kasai T, Li Y, et al. A model of cancer stem cells derived from mouse
induced pluripotent stem cells. PLoS One. 2012;7(4):e33544.

[2] Yan T, Mizutani A, Chen L, Sugii Y, Jin G, Okada M, Vaidyanath A, Mizutani A,
Satoh A, Kudoh T, Hendrix MJC, Salomon DS, Fu L, Seno M. Characterization ofcancer stem-like cells derived from mouse induced pluripotent stem cells
transformed by tumor-derived extracellular vesicles. J Cancer. 2014;5(7):572-584.

[3] Matsuda S, Yan T, Mizutani A, Sota T, Hiramoto Y, Prieto-Vila M, Chen L, SatohA, Kudoh T, Kasai T, Murakami H, Fu L, Salomon DS, Seno M. Cancer stem cells
maintain a hierarchy of differentiation by creating their niche. Int J Cancer.
2014;135(1):27-36.

[4] Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced
23

pluripotent stem cells. Nature. 2007; 448:313-317[5] Murai K, Murakami H, Nagata S. Myeloid-specific transciptional activation bymurine myeloid zinc-finger protein 2. Porc. Natl. Acad. Sci. 1998; 95:3461-3466[6] Omura T, Sakai H, Murakami H. Acceleration of granulocyte colony-stimulating
factor-induced neutrophilic nuclear lobulation by overexpression of Lyn tirosine
kinase. Eur. J. Biochem. 2002; 269:381-389.

[7] Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating
evidence and unresolved questions. Nat. Rev. 2008; 8:755-768[8] Butler J, Kobayashi H, Rafii S.Mechanisms of angiogenesis and arteriogenesis. Nat
Med. 2000;6(4):389-395.

[9] Ricci-Vitiani L, Pallini R, Biffoni M, Todaro M, Invernici G, Cenci T, Maira G,
Parati EA, Stassi G, Larocca LM, De Maria R. Tumour vascularization via
endothelial differentiation of glioblastoma stem-like cells. Nature. 2011; 468:824–
828[10] Wang R, Chadalavada K, Wilshire J, Kowalik U, Hovinga KE, Geber A, Fligelman
B, Leversha M, Brennan C, Tabar V. Glioblastoma stem-like cells give rise totumour endothelium. Nature. 2010; 468:829–833.

24

CHAPTER 3
ooo ooomiPS-LLCcm and DsRed-LLCcm cells releasefactors that induce angiogenic response

25

26

3.1 Introduction
Tumors can only grow to a size of approximately 1-2mm3 before reaching their
metabolic demand [1]. To grow beyond this size, tumor cells undergo the process
known as angiogenesis [1]. Angiogenesis, firstly described by Folkman in 1971 [2], is a
progressive, multistep physiological process by which new blood vessels are developed
from preexisting vessels [3]. These vessels are in charge of the supply of nutrients andoxygen to sustain tumor tissues.
Tumor angiogenesis starts with the activation of endothelial cells (EC) by
inflammatory cytokines and angiogenic factors released by tumor cells, such as vascularendothelial growth factor (VEGF), fibroblast growth factor (FGF) and platelet derived
growth factor (PDGF), which stimulate a cascade of endothelial changes and promote
them to secrete several proteases and plasminogen activators, resulting in the
degradation of the vessel basement membrane, allowing EC to invade the surrounding
membrane [4, 5]. Then, cells proliferate and eventually migrate to form new vessels.
Finally, the endothelial cells deposit a new basement membrane and secret growth
factors that attract support cells such as pericytes, ensuring the stability of the newvessels [6].
Many studies showed the importance of CSC in angiogenesis, secreting angiogenic
factors such as VEGF [7-10]. In previous studies we observed that miPS-LLCcm
exhibited highly vascularized tumors [11]. However, the origin of these vessels still
remains unknown. Taking in consideration all the studies, I hypothesized that miPSLLCcm could actively trigger angiogenesis.

3.2 Materials and methods
3.2.1 Cell culture.
Mouse Lewis Lung Carcinoma (LLC) cells were maintained in Dulbecco’s Modified
Eagle’s Medium-high glucose (DMEM, Sigma, MO) containing 10% FBS (Gibco,NY)
and 100U/mL penicillin/streptomycin (Wako, Japan).
miPS cells were cultured in DMEM containing 15% FBS, 0.1 mM MEM Non27

Essential amino acids, (100X NEAA, Gibco, NY), 2 mM L-Glutamine (Nacalai Tesque,
Japan), 0.1 mM 2-mercaptoethanol, 50 U/ml penicillin/streptomycin and 1000 U/mL
Leukemia inhibitory factor (LIF, Millipore, MA).
miPS-LLCcm cells and DsRed-LLCcm cells were cultured in miPS media without
LIF.
All the cells were maintained at 37 ºC in the atmosphere of 5% CO2 and medium waschanged every two days.

3.2.2 Animal experiments.
Nude mice (Balb/c-nu/nu, female, 4 weeks old) were purchased from Charlesriver,Japan. For transplantation studies, 1x106 cells were suspended in 200µL in the miPSmedia and injected subcutaneously in both flanks of mice. Tumor volumes were
measured every 3-4 days and calculated as following formula (0.5 x longer diameter x
shorter diameter2). After 5 weeks, tumors were extracted and separated in equal parts
that were used for the histological analysis, immunofluorescent analysis, and CAM
assay experiment as described below.
The plan of animal experiments was reviewed and approved by the ethics committee
for animal experiments of Okayama University under the ID OKU-2013252 and OKU2016078.

3.2.3 Chick embryo chorioallantoic membrane (CAM) Assay.
Fertilized eggs were obtained from Yamagishi (Mie, Japan), and sterilized withethanol 70% to a posteriori be incubated at 37 ºC in 60 % of humidity in a incubator P008(B) (Showa Furani, Japan). After acclimatization for 2 days in the incubator, 3 mLof egg white was extracted using an 18G hypodermic needle and 5 mL syringe
generating an air sac directly over the chick embryo chorioallantoic membrane (CAM).
On the day 8, an approximately 1 cm2 window was opened in the shell of an egg and the
5 mm3 portions of the tumor extracted from the grafted mice was collocated over the
CAM with sterilized plastic ring. For the control, only the plastic ring was located on
CAM. The window was sealed with transparent tape, then, the eggs were incubated.
Images of vasculature were taken on day 12 after injecting 2 mL of 20% Intralipos(Otsuka Pharmaceutical, Japan) under the membrane to increase the contrast.

28

3.2.4 Histological analysis and Immunohistochemistry.
Extracted tumors were enveloped with paraffin and sectioned at 5 µm of thickness.
After deparaffinization, sections were stained with hematoxylin-eosin (Hemaoxylin
solution, Sigma-Aldrich, MO; 0,5% Eosin Y, Sigma Aldrich, MO) for histologicalanalysis. For immunohistochemistry, antigen retrieval was carried out by boiling in
10mM citrate sodium (pH6) with 0.05 % Tween20 for 15min. After cooling down thesamples, the endogenous peroxidase was blocked with 3 % H2O2 for 5 min. Ellite antirabbit ABC staining Vectastain kit (Vector, MI, ) and 3, 30-diaminobenzidine
tetrahydrochloride (DAB, Vector, MI) were used for detection of Ki67 and DsRed withrabbit monoclonal Ki67 (1:200, #ab66155, Abcam, UK) and rabbit polyclonal antiDsRed (1:100, #ab62341, Abcam, UK), respectively. Counter staining was carried out
using hematoxylin.

3.2.5 Immunofluorescence analysis of tumor tissue.
The freshly extracted tumor tissue samples were embedded with Tissue-Tek OCT
compounds (Sakura Finetek, CA), frozen and sectioned into the 10µm thickness.
Cryosections were fixed with Paraformaldehyde phosphate buffer solution 4% (PFA,
Nacalai Tesque, Japan) for 20 min at room temperature, followed by permeabilization
with 0.05 % Tween20 in PBS (PBS-T). After blocking with PBS-T containing 5% BSA,
cryosections were incubated with primary antibodies rat monoclonal anti-PECAM1(1:50, sc-101454, Santa Cruz) and rabbit polyclonal anti-DsRed (1:100, ab62341,
Abcam) for overnight at 4ºC. Following washes with PBS-T, sections were incubatedwith secondary antibodies Texas Red-X conjugated goat anti-rat IgG (1:500, #6392,
Invitrogen, CA) and Alexa Fluor 488 donkey anti rabbit IgG (1:500, #A21206, Life
Technologies, CA). Nuclei were counterstained with 4`,6-diamidino-3-phenylidole,
dihydrochloride (DAPI, Vector, MI).
Images were acquired using an Olympus IX81 microscope equipped with a lightfluorescence device (Olympus, Japan).

3.2.6 RNA extraction, cDNA synthesis and quantitative real time PCR.
Total RNA from cells was isolated using RNeasy Mini Kit (QIAGEN, Germany),
then treated with DNase I (Takara, Japan). 1µg of RNA was reverse transcribed using
SuperScript III First strand kit (Invitrogen, CA).
Quantitative real-time PCR was performed with Cycler 480 SYBR Green I Master29

mix (Roche,Switzerland) according to manufacturer’s instructions. Primers used for thereal time qPCR were as following (forward and reverse). VEGF-A 5’ATGAACTTTCTGCTCTCTTGGGTGC-3’

and

5’-

CATGGGACTTCTGCTCTCCTTCTG-3’;

FGF2

5’-

CCTTCCCACCAGGCCACTTCAA-3’and 5’-GGTCCCGTTTTGGATCCGAGTTT-3’.
Gene expression level was normalized with that of GAPDH mRNA.

3.2.7 In vitro Tube formation assay
5x104 cells were seeded on growth factors reduced Matrigel (Corning, NY) -coated96well plate and cultured in 50uL of EBM2 media (EBM-2 Single Quots Kit, Lonza,
Switzerland) for 24hr in the presence or absence ofangiogenic factors (FBS,hydrocortisone, hFGF-B, VEGF, R3-IGF-1, hEGF, ascorbic acid, GA-1000 and heparin).
The experiments were performed in triplicate. Images of formed tubes were captured by
Olympus CKX41 microscope, or Keyence BZ-X700.

3.2.8 Statistical analysis.
All quantitative data is expressed as mean ± SD and statistically analyzed byStudent’s t-test. A p-value lower than 0.05 was considered as statistically significant.

3.3 Results
3.3.1 DsRed tumorigenicity.
Firstly the tumorigenicity and angiogenic activity of DsRed-LLCcm cells wasevaluated and by transplanting 106 DsRed-LLCcm cells subcutaneously into nude mice.
miPS-LLCcm cells, miPS cells, and LLC cells were also transplanted for comparison.
The tumor derived from LLC cells had the highest growth rate (Figure 1.1). However,on the third week the tumor showed significant necrotic features (Figure 1.2). On theother hand, tumors derived by DsRed-LLCcm and miPS-LLCcm cells, which exhibit
similar growth, kept growing without any apparent signatures of necrosis, and hostvessels were found to be directed towards the tumor (Figure 1.3).

30

When LLC tumor and DsRed-LLCcm tumor were histologically analyzed, I foundthat LLC tumor showed relatively homogenous structure and few and small bloodvessels (Figure 1.4 left). On the other hand DsRed-LLCcm tumors constructedunorganized, heterogeneous structures with higher amount of blood vessels (Figure 1.4,
right). The vessel structures containing blood cells in the three replicates of tumor of
DsRed-LLCcm were significantly abundant when compared to those found in the tumorof LLC cells (Figure 1.5). These could help to explain the observation of necrosis in the
LLC derived tumor, but not in the DsRed-LLCcm derived tumor which continuously
grew without visible necrosis.
Further analysis of DsRed-LLCcm tumors showed that most of the cells forming thetumor expressed DsRed (Figure 1.6, right), and were stained with Ki67 antibody
revealeing that the cells in the DsRed-LLCcm tumors were highly proliferative (Figure
1.6, left), supporting the rapid tumor growth as it has been previously shown in miPSLLCcm [11].

10000
9000
Volumme (mm3)

8000
7000
6000
DsRed-LLCcm

5000
4000

LLC

3000

miPS

2000

miPS-LLCcm

1000
0
14

16

18

20

22

24

26

28

30

32

34

Days after injection

Figure 1.1 Growth rates of tumors. LLC (n=2), DsRed (n= 4), miPS (n=3), miPSLLCcm (n=1). Mice tumors were measured every 3-4 days and the volume wascalculated. After 2 weeks, all the cells except for miPS cells, exhibited a tumor thatgrew rapidly.

31

Figure 1.2 Images of tumors. miPS was used as a negative control. LLC cells, even
they grew very fast, soon showed necrosis region that stop tumor growth. CSC-modelcells (DsRed-LLCcm and miPS-LLCcm) generated big tumors that remained nonnecrotic.

Figure 1.3 Host blood vessels towards the tumor. Image of tumor extraction in which
host-blood vessels we found surrounding the tumor of DsRed-LLCcm.

Figure 1.4 Hematoxylin-Eosin staining images of LLC and DsRed-LLCcm derived
32

tumor. While LLC derived tumor showed a homogeneous structure, the tumor formedby DsRed-LLCcm cells was heterogeneous and highly angiogenic. Scale bar: 100µm.

Figure 1.5 Blood vessel densities of LLC and DsRed-LLCcm derived tumor.
Random camps (n=15) were pictured and all the channels that contained red bloodvessels were counted showing that all the replicates of DsRed-LLCcm tumors contained
significantly more blood vessels rather than LLC tumor.*:P<0.05.

Figure 1.6 Immunohistochemistry analysis of DsRed and Ki67 of tumor derived
from DsRed-LLCcm cells. Most of the cells were stained against DsRed and Ki67,
showing that the cells within the tumor were proliferating. Scale bar: 50µm.

3.3.2 The undifferentiated subpopulation of DsRed-LLCcm secrete proangiogenic
factors
Since we had observed host blood vessels surrounding the tumors derived frommiPS-LLCcm and DsRed-LLCcm (Figure 1.3) and high amount of vessels within thosetumors (Figure 1.4-5), I further confirmed the recruitment of preexisting blood vessels
33

to the tumors by using CAM assay [12]. In this study, unlike the general protocol that
injects cells in a sponge, [13] I implanted a whole tumor portion of 5-mm-cube on CAM
that had been freshly extracted from grafted mouse (Figure 2.1).
In CAM assay both tumors of DsRed-LLCcm and LLC promoted chick bloodvessels formation when compared with the negative control (Figure 2.2). In addition, asignificantly higher number of blood vessels inside the plastic ring and towards thetumor of DsRed-LLCcm cells rather than of LLC cell-derived tumor was observed(8.8±3.3 and 5.6±1.9, respectively), indicating a higher ability of recruitment of bloodvessels in DsRed-LLCcm tumor (Figure 2.3).
The expression of VEGF-A and FGF2, angiogenic factors that have been describedin CAM assay [14, 15] were analyzed. In accordance with CAM assay results, VEGF-Aand FGF2 expression was found to be higher DsRed-LLCcm cells than that in LLC
cells (Figure 2.4). Interestingly, the expression level the factors were dramaticallyhigher when the undifferentiated subpopulation of DsRed-LLC cells was concentrated,suggesting that the expression of VEGF-A and FGF2 could be secreted from CSCs
predominantly. The expression of angiogenic factors in CSCs could explain the resultsof the in vitro tube formation assay that were independent of exogenous angiogenic
factors (Figure 2.5) [16].

Figure 2.1 Experimental scheme of CAM assay. On day 6, a 5-mm-cube piece offreshly isolated tumor was added on the CAM, and on day 10, the pictures were taken
and results analyzed.

34

Figure 2.2 Typical results of CAM assay. Both DsRed-LLCcm and LLC tumors
promoted blood vessels formations; however, when compared, DsRed-LLCcm tumor
was significantly more angiogenic than LLC tumor.

Figure 2.3 Quantitative evaluation of blood vessel number in CAM assay. Both
DsRed-LLCcm and LLC tumors, promoted blood vessels formations in comparison to
the negative control. DsRed-LLCcm tumor was significantly more proangiogenic than
LLC tumor *: p<0.05.

35Figure 2.4 Angiogenic factors expression. Expression of VEGF-A and FGF2 in
DsRed-LLCcm and DsRed-LLCcm selected with puromycin in comparison to LLC
cells were quantified by RT-qPCR. Expression values are normalized to GAPDH.
*:p<0.05. In both factors, the expression was significantly higher in DsRed-LLCcm
cells rather than LLC cells and even higher when the CSC-subpopulation was enriched.

Figure 2.5 Tube formation of miPS cells, LLC cells, miPS-LLCcm cells and DsRedLLCcm. While miPS and LLC cells were not able to form tube formation, miPSLLCcm and DsRed-LLCcm cells could form tubular structures independent ofexogenous angiogenic factors, indicating that the cells are able to secrete these factor bythemselves.

36

2.3.3 DsRed-LLCcm cells capacity of recruit host-derived endothelial cells
Next, I confirmed the existence of penetrated host blood vessels in the tumor
generated by DsRed-LLCcm cells. The tumor was stained with anti-CD31 antibody andanti-DsRed antibody. CD31+/DsRed- cells with channel-like structure were foundfrequently, showing that these blood vessels were originated from host animal (Figure3.1). Thus, the results suggest that CSCs should contribute to angiogenesis directly bysecreting angiogenic factors.

Figure3.1

Host-derived

endothelial

cells

withinDsRed-LLCcm

tumor.

Immunofluorescence analysis using CD31 (red)- and DsRed (green)-antibodies intumors derived from DsRed-LLCcm cells. The tubular structure is CD31+ but DsRedindicating that these endothelial cells have an origin in the host animal. Scale bar: 25µm.

3.4 Discussion
The histological analysis of the tumor formed by DsRed-LLCcm cells, in comparison
to the necrotic tumor formed by LLC cells, showed that their tumors were highly
vascularized (Figure 1.4). This, along with the big vessels from the host found towards
the tumor, prompted me to hypothesis that DsRed-LLCcm cells present high angiogenic
activity (Figure 1.3).
The angiogenic activity of DsRed-LLCcm, which was further confirmed by CAM
assay (Figure 2.2-3) and by the presence of host CD31+/DsRed- endothelial vessels
derived from the host in the tumor (Figure 3.1), might be corroborated with the fact thatmiPS-LLCcm cells were able to do tube formation without exogenous angiogenic
factors addition (Figure 2.5) and by the high expression of angiogenic factors in CSCs
(Figure 2.4). It has been shown that Glioma CSCs, defined as the subpopulation
expressing the CSC marker CD133, expressed 10 fold more VEGF than the CD13337

subpopulation in both hypoxia and normoxia, inducing the angiogenesis response [7].
This ability of CSC to secrete higher amount of VEGF has been described in breast
cancer and malignant gliomas [7-10]. Although it has not been considered directly
related with the secretion from CSCs, FGF2 stimulates survival, proliferation, migrationand differentiation of EC. Both VEGF and FGF2 have been described to induce
angiogenesis in CAM assay [14,15]. In accordance with these observations, VEGF-Aand FGF2 were expressed 6 and 3 folds higher, respectively, in the undifferentiated
GFP+ population of DsRed-LLCcm cells compared to the bulk culture of DsRedLLCcm cells (Figure 2.4). Thus the CSC subpopulation in DsRed-LLCcm cells presents
high angiogenic activity in ovo and in vivo inducing the differentiation and proliferationof ECs by the secretation of angiogenic factors such as VEGF-A or FGF2.

2.5 References[1] Kaur S, Bajwa P. A ‘tête-à tête’ between cancer stem cells and endothelial
progenitor cells in tumor angiogenesis. Clin Transl Oncol. 2014;16(3):115-121.

[2] Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch
during tumorigenesis. Cell. 1996;86(3):353-364.

[3] Butler J, Kobayashi H, Rafii S.Mechanisms of angiogenesis and arteriogenesis. Nat
Med. 2000;6(4):389-395.

[4] Buttler JM, Kobayashi H, Rafii S. Instructive role of the vascular niche in promoting
tumour growth and tissue repair by angiocrine factors. Nat Rev Cancer.
2010;10(2):138–146[5] Otrocka ZK, Mahfouzb RAR, Makarema JA, Shamseddine AI. Understanding thebiology of angiogenesis: Review of the most important molecular mechanisms.
Blood Cells Mol Dis. 2007; 39(2):212-220.

38

[6] Cross MJ, Claesson-Welsh L. FGF and VEGF function in angiogenesis: Signalling
pathways, biological responses and therapeutic inhibition. Trends Pharmacol Sci.
2001;22(4):201-207.

[7] Bao S, Wu Q, Sathornsumetee S, Hao Y, Li Z, Hjelmeland AB, Shi Q, McLendon
RE, Bigner DD, Rich JN. Stem cell-like glioma cells promote tumor angiogenesis
through vascular endothelial growth factor. Cancer Res. 2006;66(16):7843-7848.

[8] Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, Pilotti S,
Pierotti MA, Daidone MG. Isolation and In vitro Propagation of Tumorigenic
Breast Cancer Cells with Stem / Progenitor Cell Properties. 2005;(13):5506-5512.

[9] Pellegatta S, Poliani PL, Corno D, Menghi F, Ghielmetti F, Suarez-Merino B,
Caldera V, Nava S, Ravanini M, Facchetti F, Bruzzone MG, Finocchiaro G.
Neurospheres enriched in cancer stem-like cells are highly effective in eliciting adendritic cell-mediated immune response against malignant gliomas. Cancer Res.
2006;66(21):10247-10252.

[10] Lever E, Sheer D. The role of nuclear organization in cancer. J Pathol.
2010;220(September):114-125.

[11] Chen L, Kasai T, Li Y, et al. A model of cancer stem cells derived from mouse
induced pluripotent stem cells. PLoS One. 2012;7(4):e33544.

[12] Blacher S, Devy L, Hlushchuk R, Blacher S, Devy L, Hlushchuk R, Larger E,
Lamande N, Burri P, Corvol P, Djonov V, Foidart JM, Noel A. Quantification ofangiogenesis in the chicken chorioallantoic membrane (CAM). Image Anal Stereol.
2005;24(c):169-180.

[13] Ribatti D, Nico B, Vacca A, Presta M. The gelatin sponge–chorioallantoic
membrane assay. Nat protocols. 2006; 1:85- 91.

[14] Eliceiri BP, Klemke R, Strömblad S, Cheresh D a. Integrin avβ3 requirement forsustained mitogen-activated protein kinase activity during angiogenesis. J Cell Biol.
39

1998;140(5):1255-1263.

[15] Eliceiri BP, Paul R, Schwartzberg PL, Hood JD, Leng J, Cheresh D a. Selective
requirement for Src kinases during VEGF-induced angiogenesis and vascularpermeability. Mol Cell. 1999;4(6):915-924.

[16] Matsuda S, Yan T, Mizutani A, Sota T, Hiramoto Y, Prieto-Vila M, Chen L, SatohA, Kudoh T, Kasai T, Murakami H, Fu L, Salomon DS, Seno M. Cancer stem cells
maintain a hierarchy of differentiation by creating their niche. Int J Cancer.
2014;135(1):27-36.

40

CHAPTER 4.
ooo ooomiPS-LLCcm cells differentiate into endothelial
cells in vitro and in vivo.

41

42

4.1 Introduction
Tumor vasculature is mainly developed through angiogenesis sprouting frompreexisting vessels and vasculogenesis of tumor cells differentiating into ECs. Bothangiogenesis and vasculogenesis are initiated and promoted by angiogenic factors suchas VEGF, that even though it has been widely described to be produced by stromal or
inflammatory cells [1, 2], in our miPS-LLCcm model is mostly secreted by our CSC
subpopulation. As well as CSCs have been described to participate in angiogenesis [3-6]recent studies reported them in other kinds of tumor vasculature.
One of the most significant findings regarding tumor vasculogenesis and CSC is the
direct differentiation of those into endothelial cells to fuel the fast tumor growth. For
example, glioblastoma stem cells, expressing the stemness marker CD133+ were foundto differentiate into vascular endothelial cells expressing CD31 when these cells were
injected into nude mice [7, 8]. Furthermore, environmental changes, such us hypoxia or
glucose deprivation, induced CSCs to acquire EC markers including CD31, CD34, andvWF [9]. Additionally, breast and renal CSCs differentiated after 14 days of incubation
under hypoxia and with VEGF containing complete medium in vitro [10].
In previous studies our laboratory showed that miPS-LLCcm cells were able to form
tube formation when cells were seeded on matrigel, and contained CD31positive cells.
Thus here, I assessed further this differentiation capacity in vitro and in vivo.

4.2 Materials and methods
4.2.1 Cell culture.
miPS-LLCcm and DsRed-LLCcm cells were cultured in DMEM containing 15%
FBS, 0.1 mM MEM Non-Essential amino acids, (100X NEAA, Gibco, NY), 2 mM LGlutamine(NacalaiTesque,Japan),

0.1mM

2-mercaptoethanol,

50

U/ml

penicillin/streptomycin and 1000 U/mL and without LIF. All the cells were maintained
at 37 ºC in the atmosphere of 5% CO2. Medium was changed every two days.
For selection of GFP-positive undifferentiated cells of miPS-LLCcm and DsRedLLCcm, cells were cultured in the miPS-LLCcm medium containing 1 mg/mL
43

puromycin for 7 days, changing the medium daily [11, 12].

4.2.2 In vitro Tube formation assay.
5x104 cells were seeded on growth factors reduced Matrigel (Corning, NY) -coated96well plate and cultured in 50uL of EBM2 media (EBM-2 Single Quots Kit, Lonza,
Switzerland) for 24hr in the presence or absence ofangiogenic factors (FBS,hydrocortisone, hFGF-B, VEGF, R3-IGF-1, hEGF, ascorbic acid, GA-1000 and heparin).
The experiments were performed in triplicate. Images of formed tubes were captured by
Olympus CKX41 microscope, or Keyence BZ-X700.

4.2.3 Sphere formation assay.
4x104 cells (1x104 cells/ml) were seeded on 6cm ultra low attachment dishes(Corning incorporated, NY) in miPS-LLCcm media without FBS, but supplied with
Insulin-Transferrin-Selenium-X (ITS-X, Life Technologies, CA). Cells were cultured at
37 ºC under 5% CO2 for 4 days, and the spheroids with diameters above 100 μm were
judged as self-renewing spheroids.

4.2.4 Animal experiments.
Nude mice (Balb/c-nu/nu, female, 4 weeks old) were purchased from Charlesriver,Japan. For transplantation studies, 1x106 cells were suspended in 200µL in the miPSmedia and injected subcutaneously in both flanks of mice. Tumor volumes were
measured every 3-4 days and calculated as following formula 0.5x (longer diameter x
shorter diameter2). After 5 weeks, tumors were extracted and frozen for
immunofluorescent analysis, experiment as described below.
The plan of animal experiments was reviewed and approved by the ethics committee
for animal experiments of Okayama University under the ID OKU-2013252 and OKU2016078.

4.2.5 Immunofluorescence analysis.
For staining of in vitro formed tubes, 1.5x105 cells were seeded on 8-well chamberslides (Corning, NY) coated with matrigel and for the staining of in vitro culture, cells
were seeded on gelatin-coated cover glasses. After incubation, cells were fixed,
permeabilized, and stained with rabbit polyclonal anti-CD31 antibody (1:100, #ab28364,
Abcam, UK) and Alexa Fluor 555 conjugated anti-rabbit IgG (1:500, #A21428, Life44

technologies, CA). Nuclei were counterstained with DAPI (Vector, MI).
For tissue samples, freshly extracted tumors were embedded with Tissue-Tek OCT
compounds (Sakura Finetek, CA), frozen and sectioned into the 10µm thickness.
Cryosections were fixed with Paraformaldehyde phosphate buffer solution 4% (PFA,
Nacalai Tesque, Japan) for 20 min at room temperature, followed by permeabilization
with 0.05 % Tween20 in PBS (PBS-T). After blocking with PBS-T containing 5% BSA,
cryosections were incubated with primary antibodies (rat monoclonal anti-PECAM1(1:50, sc-101454, Santa Cruz) and rabbit polyclonal anti-DsRed (1:100, ab62341,
Abcam) for overnight at 4ºC. Following washes with PBS-T, sections were incubatedwith secondary antibodies Texas Red-X conjugated goat anti-rat IgG (1:500, #6392,
Invitrogen, CA) and Alexa Fluor 488 donkey anti rabbit IgG (1:500, #A21206, Life
Technologies, CA). Nuclei were counterstained with DAPI (Vector, MI).
Images were acquired using an Olympus IX81 microscope equipped with a lightfluorescence device (Olympus, Japan).

4.3 Results
4.3.1 The differentiated subpopulation of miPS-LLCcm is required for tube
formation.
As reported in previous studies, miPS-LLCcm cells were able to differentiate into
endothelial cells, and were able to form tube-like structures composed by CD31+/GFPendothelial differentiated cells, CD31-/GFP+ undifferentiated cells and CD31+/GFP+
bipotential cells on Matrigel (Figure 1.1) [11, 13]. The formation of the tubular structure
required differentiated population, since GFP+ cells concentrated by puromycin in
adherent culture, which contains CD31-/GFP+ and CD31+/GFP+ cells (Figure 1.2) wereable to form the structure when seeded on Matrigel (Figure 1.3). GFP+ self-renewing
spheres cultured in non-adherent dishes were not able to do tube formation neither(Figure 1.4) [11]. In contrast, when spheres were seeded into adherent culture for twodays to give rise to GFP- differentiated cells; an seeded a posteriori on Matrigel, thesecells were able to generate tube structures (Figure 1.5).

45

Figure 1.1 Staining of CD31 and GFP in tube formation of miPS-LLCcm cells.
Staining of CD31 (red) and GFP (green) showed 4 kinds of cells: CD31+/GFPendothelial

differentiatedcells,

CD31-/GFP-

differentiatedcells, CD31-/GFP+

undifferentiated cells and CD31+/GFP+ bipotential cells. Original magnification x20.

Figure 1.2 Analysis of cells present in puromycin-selected miPS-LLCcm culture.
The

Immunofluorescencestaining

showsapopulation

mostlyCD31-/GFP+

undifferentiated with some bipotential cells (CD31+/GFP+). Original magnification x60.

Figure 1.3 Incomplete tube formation of puromycin-selected miPS-LLCcm cells.
Puromycin selected cells were seeded on Matrigel and stained with CD31 antibody(red). Cells were no able to differentiate and to form tubular structures. Original
magnification x20.

46

Figure 1.4 Tube formation ability of GFP+ spheroids. Sphere forming selfrenewing miPS-LLCcm cells were not able to do tube formation. When the cells wereallowed to differentiated under adherent conditions, the cells could do tube formation.
Scale bar: Day 0: 200µm; Day 2: 50µm; Tube formation Original magnification x2.

4.3.2 miPS-LLCcm are able to differentiate into endothelial cells in vitro
The bulk culture of miPS-LLCcm cells consists of 2 distinct kinds of morphologies:
cells forming GFP+ embryonic stem (ES)-like colonies and GFP- differentiated cells(Figure 2.1) [14].
When culture of miPS-LLCcm cells was stained with anti-CD31 antibody, CD31+
cells were detected (Figure 2.2). As found in the tube formation, four kind of cells were
found: CD31-/GFP- differentiated cells, CD31+/GFP- differentiated endothelial cells,
CD31-/GFP+ undifferentiated cells, and CD31+/GFP+ bipotential cells. Outside thecolonies, most the cells were CD31-/GFP- differentiated cells (Figure 2.3), while all thekinds of cells inside forming ES-like colonies could be find. CD31+/GFP- differentiatedcells were predominantly located at the bottom of the ES-like colonies (Figure 2.2).
47

This observation implied that miPS-LLCcm formed a structure that resembles anendothelial niche in vitro (Figure 2.4).
Even though I could find some CD31+ cells in the bulk culture, it was not until thecells were seeded on Matrigel that most of them differentiated (Figure 2.5). It is highly
possible that some of the CD31-/GFP- cells acted as endothelial precursor cells, such as
VEGFR2+ cells, mature into endothelial cells as CD31+/GFP- cells during the culture on
Matrigel.

Figure 2.1 miPS-LLCcm bulk culture. miPS-LLCcm bulk culture present two kind of
subpopulations: GFP+ embryonic stem (ES)-like colonies and GFP- differentiated cells.
Scale bar: 100 µm.

Figure 2.2 Kinds of cells in the colony within the bulk culture. Immunofluorescenceanalysis of CD31 (red) and GFP (green) in bulk culture of miPS-LLCcm cells. In thelower section, a 3D image of the colony cut in three different height sections, showing
the predisposition of differentiated cells in the bottom and undifferentiated cells in the
top of the colony. Original magnification x100
48

Figure 2.3 Kinds of cells outside the colony in the bulk culture. Immunofluorescenceanalysis of CD31 (red) and GFP (green) in bulk culture of miPS-LLCcm cells showsthat most of the cells are CD31-/GFP- differentiated cells with few CD31+/GFPendothelial cells. Original magnification x100.

Figure 2.4 Scheme of the position of the cells in the bulk culture, resembling aniche in vitro. Outside the colony were differentiated cells (CD31-/GFP-). CD31+/GFPendothelial cells are concentrated at the bottom of the colony, while undifferentiatedcells are situated at the edges and top of the colony. Inside the colony bipotential
CD31+/GFP+ can be found.

49

Figure 2.5 Time course analysis of in vitro tube formation. Cells were stainedwith CD31 (red) antibody at indicated time. Even though the tubular structures could be
identified after 6hr, it was not until 24hr that CD31+ cells were mostly found. Original
magnification x20.

4.3.3 DsRed-LLCcm cells differentiate into endothelial cells in vivo
The observation of endothelial cell differentiation of miPS-LLCcm in vitro promptedme to investigate whether CSC originated blood vessels in vivo. I looked for tubular
structures that were CD31+/DsRed+ double positive in the tumor derived from DsRedLLCcm cells by immunofluorescence staining. I could find not only these structures(Figure 3.1), but also mosaic vasculatures, in which blood vessels were mostly formedby CD31+/DsRed- cells, which should be derived from host, and CD31+/DsRed+ cells,
which should be derived from DsRed-LLCcm cells (Figure 3.2). The physicalinteraction between host-derived ECs and tumor-derived ECs, explains the functionalityof CSC-derived blood vessel.

50

Figure 3.1 CSC differentiate into EC in vivo. Tumors derived from DsRed-LLCcm
cells were stained with CD31 (red) and DsRed (green) antibodies. A typicalCD31+endothelial vessel comprised of DsRed+ cells indicate the differentiation of CSC
into EC in vivo. Scale bar: 25µm.

Figure 3.2 Mosaic vessels in vivo. Tumors derived from DsRed-LLCcm cells were
stained with CD31 (red) and DsRed (green) antibodies. A blood vessel comprised of
CD31+/DsRed- cells and CD31+/DsRed+ explains the functionality of CSC-derived
vessels. Scale bar: 58µm.

4.4 Discussion
The angiogenic factors secreted predominantly by the CSC subpopulation, as
described in chapter 3, should contribute not only in the recruitment of host-EC but also
into the differentiation of CSCs into ECs. CSC differentiation into EC was first reported
in glioblastoma, in which some EC were found contained glioma-specific chromosomal
aberration, indicating that the endothelial cells were derived from tumor cells and notfrom the host [15]. Brossa et al. described the differentiation of breast-CSC into
endothelial cells but this required of addition of external VEGF or hypoxia stimulation[10]. We have also reported that our miPS-LLCcm differentiated into endothelial cells
in vitro (Figure 1.1) [11]. Contrasting to breast-CSCs, EC differentiation from miPSLLCcm cells occurred spontaneously on gelatin-coated dish exhibiting CD31 positive
cells, which were readily detectable in the bulk culture (Figure 2.2), resembling an invitro niche (Figure 2.4). This finding is consistent with the reports describing that ECs
secrete factors that will promote survival and self-renewal of CSCs [11, 16, 17].
51

When miPS-LLCcm cells were seeded on matrigel, endothelial CD31+ cells appeared,
however we could not determine which was the origin of CD31+/GFP- ECs in vitro.
Studies in glioblastoma have shown a bipotential CD31+/CD133+ subpopulation thatgave rise to EC via differentiation [7]. These bipotential cells in glioblastoma seems tobe equivalents to our CD31+/GFP+ cells found in the bulk (Figure 2.2, 2.4) since both
cells expressed stem cell marker and endothelial marker. However, as shown in ourprevious study [11], concentrated GFP+ miPS-LLCcm cells, which contain GFP+/CD31and GFP+/CD31+ cells (Figure 1.2), alone were not able to generate tube-like structures
on Matrigel (Figure 1.3). Similarly, self-renewing spheroids of miPS-LLCcm did notform tube-like structures directly (Figure 1.4, left). On the other hand, these cells gained
the ability to form tube-like structures when the spheroids are allowed to differentiate
into adhesive GFP- cells, which contain GFP-/CD31- and GFP-/CD31+ cells (Figure1.4,
right). This suggests that the dominant cells responsible to form tubes were GFP- cells,
which were also found to be mostly CD31- before tube formation and resided outside of
ES-like colonies in bulk culture of miPS-LLCcm cells (Figure 2.3-4). We havepreviously reported the presence of VEGFR2+ cells, which are considered to be EC
precursors, in the GFP- subpopulation [11]. Taking our previous results together, it islikely that ECs were maturated from CD31-/GFP- with VEGFR2 expressing cells as
precursors of ECs derived from ES cells were maturated in VEGF dependent manner[18]. Considering the significant expression of VEGF-A and FGF2 from GFP+ cells(Chapter 3, Figure 2.4), these angiogenic factors should be responsible for thematuration of miPS-LLCcm progenies as ECs in paracrine manner.
The CD31+/GFP+ bipotential cells in miPS-LLCcm cells might be corresponding to
the stem cell/progenitor-like cells, which were considered to differentiate into
endothelial cells, present in vascular structures [19]. Recently, stem cell/progenitors
excluding Hoechst with CD31 and other endothelial markers were isolated from mousenormal lung [20]. These cells were found to be involved in the tumor angiogenesis by
supplying ECs. Hoechst excluding side population has been recognized as stem cellenriched population in both normal and cancer tissues [21]. Collectively, CD31+/GFP+
bipotential cells in miPS-LLCcm cells could be one of the origins of CSC-derived ECs
in vivo since the differentiation of DsRed-LLCcm into ECs was confirmed in vivo as
CD31+/DsRed+ vessels were found (Figure 3.1-2).
However, further investigation is required to identify the exact pathway of CSC
differentiation into ECs since the differentiation process is not clear. Also the CSCs
52

properties, such as the ability to form malignant tumors, of CD31+/GFP+ bipotential
cells should be assessed.

4.5 References[1] Shweiki D, Itin A, Soffer D et al. Vascular endothelial growth factor induced byhypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992; 359:843–845.

[2] Shing Y, Folkman J, Haudenschild C et al. Angiogenesis is stimulated by a tumorderived endothelial cell growth factor. J Cell Biochem 1985; 29:275–287.

[3] Bao S, Wu Q, Sathornsumetee S, Hao Y, Li Z, Hjelmeland AB, Shi Q, McLendon
RE, Bigner DD, Rich JN. Stem cell-like glioma cells promote tumor angiogenesis
through vascular endothelial growth factor. Cancer Res. 2006; 66(16):7843-7848.

[4] Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, Pilotti S,
Pierotti MA, Daidone MG. Isolation and In vitro Propagation of Tumorigenic
Breast Cancer Cells with Stem / Progenitor Cell Properties. 2005; 13:5506-5512.

[5] Pellegatta S, Poliani PL, Corno D, Menghi F, Ghielmetti F, Suarez-Merino B,
Caldera V, Nava S, Ravanini M, Facchetti F, Bruzzone MG, Finocchiaro G.
Neurospheres enriched in cancer stem-like cells are highly effective in eliciting adendritic cell-mediated immune response against malignant gliomas. Cancer Res.
2006; 66(21):10247-10252.

[6] Lever E, Sheer D. The role of nuclear organization in cancer. J Pathol. 2010;
220(9):114-125.

[7] Ricci-Vitiani L, Pallini R, Biffoni M, Todaro M, Invernici G, Cenci T, Maira G,
Parati EA, Stassi G, Larocca LM, De Maria R. Tumour vascularization via
endothelial differentiation of glioblastoma stem-like cells. Nature. 2011; 468:824–
828

53

[8] Wang R, Chadalavada K, Wilshire J, Kowalik U, Hovinga KE, Geber A, Fligelman
B, Leversha M, Brennan C, Tabar V. Glioblastoma stem-like cells give rise totumour endothelium. Nature. 2010; 468:829–833[9] Zhao Y, Dong J, Huang Q et al. Endothelial cell transdifferentiation of humanglioma stem progenitorcells in vitro. Brain Res Bull 2010; 82:308–312.

[10] Brossa A, Grange C, Mancuso L, Annaratone L, Maria Satolli MA, Mazzone M,
Camussi G, Bussolati B. Sunitinib but not VEGF blockade inhibits cancer stem cell
endothelial differentiation. Oncotarget. 2015; 6(13):11295-11309.

[11] Matsuda S, Yan T, Mizutani A, Sota T, Hiramoto Y, Prieto-Vila M, Chen L, SatohA, Kudoh T, Kasai T, Murakami H, Fu L, Salomon DS, Seno M. Cancer stem cells
maintain a hierarchy of differentiation by creating their niche. Int J Cancer.
2014;135(1):27-36.

[12] Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced
pluripotent stem cells. Nature. 2007; 448:313-317[13] Yan T, Mizutani A, Chen L, Sugii Y, Jin G, Okada M, Vaidyanath A, Mizutani A,
Satoh A, Kudoh T, Hendrix MJC, Salomon DS, Fu L, Seno M. Characterization ofcancer stem-like cells derived from mouse induced pluripotent stem cells
transformed by tumor-derived extracellular vesicles. J Cancer. 2014; 5(7):572-584.

[14] Chen L, Kasai T, Li Y, et al. A model of cancer stem cells derived from mouse
induced pluripotent stem cells. PLoS One. 2012; 7(4):e33544.

[15] Wang R, Chadalavada K, Wilshire J, Kowalik U, Hovinga KE, Geber A, Fligelman
B, Leversha M, Brennan C, Tabar V. Glioblastoma stem-like cells give rise totumour endothelium. Nature. 2010; 468:829–833[16] Yao X hong, Ping Y fang, Bian X wu. Contribution of cancer stem cells to tumor
vasculogenic mimicry. Protein Cell. 2011; 2(4):266-272.

54

[17] Krishnamurthy S, Dong Z, Vodopyanov D, Imai A, Helman JI, Prince ME, Wicha
MS, Nör JE. Endothelial cell-initiated signaling promotes the survival and selfrenewal of cancer stem cells. Cancer Res. 2010; 70(23): 9969–9978.

[18] Yamashita J, Itoh H, irashima M, Ogawa M, Nishikawa S, Yurugi T, Naito M,
Nakao K, Nishikawa S. Flk-1positive cells derived from embryonic stem cells serve
as vascular progenitors. Nature. 2000; 408(6808):92-96.

[19] Alvarez DF, Huang L, King J a, ElZarrad MK, Yoder MC, Stevens T. Lung
microvascular endothelium is enriched with progenitor cells that exhibit
vasculogenic capacity. Am J Physiol Lung Cell Mol Physiol. 2008; 294(3):L419L430.

[20] Naito H, Wakabayashi T, Kidoya H, Muramatsu F, Takara K, Eino D, Yamane K,
Iba T, Takakura N. Endothelial Side Population Cells Contribute to Tumor
Angiogenesis

andAntiangiogenic

Drug

Resistance.

Cancer

Res.

2016;

76(13):3200-3210.

[21] Golebiewska A, Brons NHC, Bjerkvig R, Niclou SP. Critical appraisal of the side
population assay in stem cell and cancer stem cell research. Cell Stem Cell. 2011;8(2):136-147.

55

56

CHAPTER 5.
ooo ooo

DsRed-LLCcm cells participate in the formationof Vasculature Mimicry.

57

58

5.1 Introduction
Recent therapies have being focusing in tumor vasculature. With targeting EC
specific molecules, many therapeutic strategies have been developed to interfere withthe formation of tumor blood vessels. For example, bevacizumab, an antibody against
VEGF receptors, has been tested in many types of solid tumor [1, 2]. Unfortunately,
despite the efforts, most of these treatments failed to reveal the expected results[3].Accumulating knowledge regarding the complexity of tumor vasculogenesis might
explain, at least in part, the failure of the classical anti-angiogenic drugs targeting ECs.
Vasculogenic mimicry (VM), a concept which was first described in 1999 by
Maniotis et al., is a process for tumor vasculature generation found in malignant andpoorly differentiated tumors, in which tumor cells themselves generate organizednetworks of vessels. The VMs channels are rich in extracellular matrix (ECM), but are
typically without CD31 positive endothelial cells [4, 5]; therefore, VM should not rely
on typical essential angiogenic markers [6].
EphA2-VE cadherin connection, Tie1, Slpi and Serpine2 have been described asimportant factors for the structure of VM [7-10]. VM has been found in many kinds oftumors such as glioblastoma [4,11], hepatoma [6], melanoma [5] and colon cancer [12].
It has been described [5] that cells forming VM forming cells express stemness markers,
and it has proposed that CSC could be responsible for VM. However there are not clear
evidences.

5.2 Materials and methods
5.2.1 Animal experiments.
Nude mice (Balb/c-nu/nu, female, 4 weeks old) were purchased from Charlesriver,Japan. For transplantation studies, 1x106 cells were suspended in 200µL in the miPSmedia and injected subcutaneously in both flanks of mice. Tumor volumes were
measured every 3-4 days and calculated as following formula (0.5 x longer diameter x
shorter diameter2). After 5 weeks, tumors were fixed for the histological analysis.
The plan of animal experiments was reviewed and approved by the ethics committee
for animal experiments of Okayama University under the ID OKU-2013252 and OKU2016078.
595.2.2 Immunohistochemistry analysis.
For CD31-PAS double staining, extracted tumors were enveloped with paraffin andsectioned at 5 µm of thickness. After deparaffinization, antigen retrieval was carried out
by boiling in 10mM citrate sodium (pH6) with 0.05 % Tween20 for 15min. Aftercooling down the samples, the endogenous peroxidase was blocked with 3 % H2O2 for 5
min. Then, Ellite anti-rabbit ABC Vectastain kit was used following manufactures
protocol with rabbit polyclonal anti CD31 (1:100, #ab28364, Abcam) incubation of hr atroom temperature. Immunoreactivity was detected by using DAB. Afterwards, tissues
were stained with PAS solution by following manufactures protocol, omitting
hematoxylin counterstaining to reduce visual noise. These sections were viewed underlight microscopy (FSX100, Olympus, Japan).
For GFP staining, rabbit monoclonal anti-GFP antibody (1:400, #2956, Cell
Signaling, MA) and Ellite anti-rabbit ABC staining Vectastain kit (Vector, MI) wereused folloing manufactures protocol.

DAB (Vector, MI) were used for detection.

Counter staining was carried out using hematoxylin. Then, the following section was
stained with PAS solution as described previously.

5.3 Results
5.3.1 DsRed-LLCcm cells are able to generate Vasculogenic mimicry
While blood vessels composed of ECs were observed in the tumor of miPS-LLCcm
cells and DsRed-LLCcm cells, I also looked into the presence of VM in the tumors.
WM are blood channels rich in ECM which are lined by tumor cells instead of vascularendothelial cells. These VM-forming cells have been described as very malignant andpoorly differentiated [4].
I stained paraffin-embedded sections of the tumors with anti-CD31 antibody and PAS,
to investigate ECM rich structures to find VM, which is defined as CD31-/PAS+ patterns.
As the result, I found abundant PAS+ patterns and tubular blood vessels-like outlines. In
this context, approximately 7.6% of all the channels were judged VM while most partsof the structure were not VM as CD31+. Blood cells were frequently observed in the60

cavities surrounding PAS+ structures (Figure 1.1). This indicates that VM wasdeveloped in the tumor of DsRed-LLCcm cells. In contrast, tumor derived from LLC
cells exhibited little PAS+ staining (data not shown).

Figure 1.1 Vasculogenic mimicry in DsRed-LLCcm tumor. Tumor sections were
stained with CD31 antibody (brown) and PAS (pink). EC lined vessels (CD31+/PAS-)and tumor cell lined VMs (CD31-/PAS+) were observed. Arrows show VM. Scale bar:
50µm (left), 25µm (right).

5.3.2 Vasculogenic mimcry is formed by the undifferentiated subpopulation of
DsRed-LLCcm cells
GFP+ cells lining vasculatures were also found (Figure 2.1, up). Since GFP was used
as a reporter of Nanog expression [13, 14], this indicates that the channel was structured
by undifferentiated cells. Moreover, these vessels were also PAS+ (Figure 2.1, down).
Both undifferentiated cells and PAS+ channels should illustrate VM, and suggests thatundifferentiated subpopulation of the CSC model, DsRed-LLCcm cells, participate in
the VM formation.

61

Figure 2.1 The undifferentiated subpopulation of DsRed-LLCcm cells form VM.
Immunohistochemistry of GFP (brown) and PAS (Pink) staining in serial sections oftumor derived from DsRed-LLCcm cells showing GFP+/PAS+ channels. Scale bar:
50µm.

5.4 Discussion
Despite its clinical importance [5], the biological features of the tumor cells that form
VM, as well as the cellular and molecular events underlying its formation remain
largely unknown. Maniotis et al. suggested that melanoma cells lining VM could havebeen reverted to an embryonic-like phenotype [9] since they were found to express
multiple stemness markers. We observed that 7.6% of all the tumor vasculature in
DsRed-LLCcm tumor could be defined as VM, which were ECM-rich vessels lackingof ECs (Figure 1.1). Furthermore, we detected the presence of GFP+ undifferentiatedcells, reflecting the stemness gene Nanog expression, in PAS+ channels (Figure 2.1).
62

These channels were frequently found in the region where most of stromal cells were
GFP+. Although we cannot state that the CSC ability remains in all of the GFP+ cells
including those forming the PAS+ channel, GFP+ cells are indeed the ones with theability of self-renewal (Chapter 2, Figure 2.2 ) and differentiation (Chapter 2, Figure
2.4 ) giving new insides in the direct relation between CSC and VM.

5.5 References[1] Cross MJ, Claesson-Welsh L. FGF and VEGF function in angiogenesis: Signalling
pathways, biological responses and therapeutic inhibition. Trends Pharmacol Sci.
2001;22(4):201-207.

[2] Folkman J. Angiogenesis: an organizing principle for drug discovery?. Nat. Rev.
Drug Discov. 2007; 6: 273-286[3] Bergers G, Hanahan D. Mode of resistance to anti-angiogenic therapy. Nat Rev
Cancer. 2008;8(8):592-603.

[4] Chen YS, Chen ZP. Vasculogenic mimicry: A novel target for glioma therapy. Chin
J Cancer. 2014;33(2):74-79.
[5] Maniotis AJ, Folberg R, Hess A, Sefto EA, Gardner LMG, Pe’er J, Trent JM,
Meltzer PS, Hendrix MJC. Vascular channel formation by human melanoma cells
in vivo and in vitro: vasculogenic mimicry. Am J Pathol. 1999;155(3):739-752.

[6] Lirdprapamongkol K, Chiablaem K, Sila-Asna M, Surarit R, Bunyaratvej A, Svasti J.
Exploring stemness gene expression and vasculogenic mimicry capacity in welland poorly-differentiated hepatocellular carcinoma cell lines. Biochem Biophys Res
Commun. 2012;422(3):429-435.

[7] Alameddine RS, Hamieh L, Shamseddine A. From sprouting angiogenesis to63erythrocytes generation by cancer stem cells: Evolving concepts in tumor
microcirculation. Biomed Res Int. 2014;2014.

[8] Wagenblast E, Soto M, Gutierrez-Angel S, Hartl CA, Gable AL, Maceli AR, Erard
N, Williams AM, Kim SY, Dickopf S, Harrell JC, Smith AD, Perou CM,
Wilkinson JE, Hannon GJ, Knott SRV. A model of breast cancer heterogeneity
reveals vascular mimicry as a driver of metastasis. Nat 2014;67(3):223-230.

[9] Hendrix MJ, Seftor E a, Meltzer PS, et al. Expression and functional significance of
VE-cadherin in aggressive human melanoma cells: role in vasculogenic mimicry.
Proc Natl Acad Sci U S A. 2001;98(14):8018-8023.

[10] Hess AR, Seftor EA, Gruman LM, Kinch MS, Seftor REB, Hendrix MJC. VEcadherin regulates EphA2 in aggressive melanoma cells through a novel signaling
pathway: Implications for vasculogenic mimicry. Cancer Biol Ther. 2006;5(2):228233.

[11] Yao X hong, Ping Y fang, Bian X wu. Contribution of cancer stem cells to tumor
vasculogenic mimicry. Protein Cell. 2011;2(4):266-272.

[12] Chang YS, di Tomaso E, McDonald DM, Jones R, Jain RK, Munn LL. Mosaic
blood vessels in tumors: frequency of cancer cells in contact with flowing blood.
Proc Natl Acad Sci U S A. 2000;97(26):14608-14613.

[13] Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced
pluripotent stem cells. Nature. 2007; 448:313-317[14] Takahashi K, Yamanaka S. Induction of Pluripotent Stem Cells from Mouse
Embryonic

and

Adult

Fibroblast

Culturesby

DefinedFactors.

Cell.

2006;126(4):663-676.

64

CHAPTER 6
ooo ooo

General conclusions and futures challenges

65

66

6.1 Conclusion and Important findings
In this research I found that miPS-LLCcm and DsRed-LLCcm cells simultaneously
exhibited different three types of tumor vasculature in a tumor tissue as angiogenesis,
endothelial differentiation from CSCs and formation of VM in vivo. As far as I know,
this is the first model of CSC cells that exhibit these three kinds of vasculature together.
It is highly possible that miPS-LLCcm also present other kinds of vasculature. Tumor
vasculature is essential for tumor growth; however current therapeutic strategies
targeting only angiogenesis are leading to insufficient outcomes. Taking these underconsideration, miPS-LLCcm could be an appropriate model to understand entire tumor
vascularization and to develop novel drugs and therapeutic strategies.

The CSC subpopulation of miPS-LLCcm cells predominantly donates angiogenic
factors that not only attract host endothelial vessels into tumor, but also to promotematuration of endothelial linage of CSC’s, probably VEGFR2 expressing GFP -/CD31progenies. Moreover, GFP expressing cells, and CD31- cells were found lining VM(Figure 1). Thus, it seems that all the vasculatures types are interconnected.

Figure 1. Scheme of the cells found in bulk culture and its relation with thedifferent kinds of vasculature.
67

We have shown that CSCs can differentiate into ECs which are able form vessels aswell as contribute to VM formation in vivo. At the same time, early stages of in vitro
tube formation showed that these were formed mostly by CD31- cells (Figure 2.5).
Taking these observations into consideration, I hypothesized that VM could be anintermediate stage of CSC differentiation. Zhang et al. discussed the presence of threekinds of vessels in melanoma, which were VM, mosaic vessels and endothelium
patterned vessels. VM was described as the dominant in the early stage of tumor growth,following EC differentiation and proliferation and mosaic vessels would appear as atransitional pattern [16, 43]. Other authors proposed that VM might be CSCs thatremain incomplete in differentiation towards the endothelial lineage [19] theseresembles the cells that remained GFP expression within the tubular structures (Figure
2.5).

6.2 References[1] Yao X hong, Ping Y fang, Bian X wu. Contribution of cancer stem cells to tumor
vasculogenic mimicry. Protein Cell. 2011;2(4):266-272.

[2] Golebiewska A, Brons NHC, Bjerkvig R, Niclou SP. Critical appraisal of the side
population assay in stem cell and cancer stem cell research. Cell Stem Cell.
2011;8(2):136-147.

[3] Ricci-Vitiani L, Pallini R, Biffoni M, Todaro M, Invernici G, Cenci T, Maira G,
Parati EA, Stassi G, Larocca LM, De Maria R. Tumour vascularization via
endothelial differentiation of glioblastoma stem-like cells. Nature. 2011; 468:824–
828

68

List of publications
Papers.
1. iPSC-derived Cancer Stem Cells Provide a Model of Tumor Vasculature.
Marta Prieto-Vila, Ting Yan, Anna Sanchez Calle, Neha Nair, Laura Hurley,Tomonari Kasai, Hiroki Kakuta, Junko Masuda, Hiroshi Murakami, Akifumi
Mizutani Masaharu Seno. American Jounal of Cancer Research. Accepted.

2. Cancer stem cells maintain a hierarchy of differentiation by creating their niche.
Shuichi Matsuda, Ting Yan, Akifumi Mizutani, Tatsuyuki Sota, Yuki Hiramoto,Marta Prieto-Vila, Ling Chen, Ayano Satoh, Takayuki Kudoh, Tomonari Kasai,
Hiroshi Murakami, Li Fu, David S. Salomon and Masaharu Seno. International
Journal of Cancer. Volume 135, 27–36, (2014)

Oral presentations.
1. Studies of the mechanism inducing Cancer Stem Cells from normal Stem Cells
Marta Prieto-Vila.
平成２６高校生・大学院生による研究紹介と交流の会(Okayama, 2014.07)
2. Acquisition of immortalization prior to malignancy during the miPS-CSC
generation.
Marta Prieto-Vila, Tsukasa Shigehiro, Anna Sanchez Calle, Tomonari Kasai,
Hiroshi Murakami, Akifumi Mizutani, Masaharu Seno.
The 74th Japanese Cancer Association meeting (Nagoya 2015.10).

3. iPSC-derived Cancer Stem Cells Provide a Model of Tumor Vasculature
Marta Prieto-Vila, Anna Sanchez Calle, Neha Nair, Akifumi Mizutani, Masaharu
Seno.
5th Mouse Resource Workshop 2016 (Tokyo, 2016.07).

4. The significance of c-kit proto-oncogene in CSC derived PDAC
Anna Sanchez Calle, Kenta Hoshikawa, Neha Nair, Marta Prieto-Vila, Arun
Vaidyanath, Tomonari Kasai, Masaharu Seno.

69

平 成 ２ ７ 高 校 生 ・ 大 学 院 生 に よ る 研 究 紹 介 と 交 流 の 会 (Okayama, Japan
2015.07)
5. Generation of Mammary ductal carcinoma associated carcinoma fibroblast fromcancer stem cells.
Neha Nair, Tomonari Kasai, Kenta Hoshikawa, Anna Sanchez Calle, Marta
Prieto-Vila, Arun Vaidyanath, Masaharu Seno.
平成２７高校生・大学院生による研究紹介と交流の会(Okayama, Japan 2015.07)
6. The significance of c-kit proto-oncogene in CSC-derived PDAC model
Anna Sanchez Calle, Kenta Hoshikawa, Neha Nair, Marta Prieto-Vila, Arun
Vaidyanath, Tomonari Kasai, Masaharu Seno.
AACR annual meeting, (New Orleans, U.S.A, 2016.04).

7. The significance of c-kit proto-oncogene in CSC-derived PDAC model
Anna Sanchez Calle, Kenta Hoshikawa, Neha Nair, Marta Prieto-Vila, Arun
Vaidyanath, Tomonari Kasai, Masaharu Seno.
CIRA/ISSCR 2016 International symposium (Kyoto, Japan 2016.03).

8. Intracellular localization of Nanog in cells within caner stem cell niche.
Kazuki Kumon, T Motoyama, Junko Matsuda, Marta Prieto-Vila, Kazuki
Aizawa ,Ryouma Onoue, Tomonari Kasai, Hiroshi Murakami, Akifumi Mizutani,
Masaharu Seno.
Molecular Biology Society of Japan (Kobe, 2015.12)

70

Acknowledgements
I would like to thank all the people who made this thesis possible, specially to mysupervisor, Professor Masaharu Seno in the Laboratory of Nanobiotechnology, Graduate
school of Natural Science and Technology, Okayama University, who gave me theopportunity to do my doctoral course under his guidance and for all his help along thesethree years, without him, I could not complete my study of doctor course and thisdissertation.

I would like to express my sincere appreciation to Assistant Professor Akifumi Mizutani,for his valuables advices and who helped and encouraged me daily, reviewing andcorrection of the manuscript.

I additional extend my great thanks to Dr. Tomonari Kasai, who helped me, not only
giving me instructions of CAM assay, but many helpful suggestions and humorouswords.

I would like to thank Associate Professor Hiroshi Murakami, Assistant Professor Junko
Masuda, Professor Hiroki Kakuta, Research Assistant Professor Arun Vaidyanath,Research Assistant Professor Akimasa Seno, Ms Mami Asakura and Ms Kaoru Furuse
for their kindly help. Without their cooperation it would had been very difficult for mylab time.

Special thanks should go to Anna Sanchez Calle, Neha Nair, Ting Yan, Kenta
Hoshikawa and all the students from the laboratory for their valuable suggestion,encouragement and friendliness giving me a wonderful memory of my doctoral course.
Finally, I want to thank my parents, my brother and my boyfriend who supported me inevery possible way and always encouraged me.

I gratefully acknowledge the funding received towards my PhD from MEXT (Ministryof Education, Culture, Sports, Science and Technology).
Marta Prieto
Okayama University
September 2016
71