Thk cancer08 final

08回Materials & Methods

Tuesday, October 12, 2010

PCR


realtime PCR; 定量PCR

enlogationの時発光する特殊なprobeを用いて、

PCRの各cycleにおける産物の量を逐一計測することで、正確に元の量を観測できる。

Ct値


mammospheres, with a SFE of !23% (PKHPOS pre-FACS inFigure S1B; note that this number is in agreement with themaximum expected SFE, Figures S1B and S1C).

We used FACS to isolate PKHPOS cells. Based on the typicalPKH26 distribution in mammospheres and the SFE of cellsfrom mammospheres, we isolated the most epifluorescent0.2%–0.4% of the total cell population (PKHPOS cells, gated at103–104 fluorescence units), as well as a ‘‘dull’’ population (gatedat 101–102 fluorescence units, PKHNEG cells) (Figure S1A, inset).Only the PKHPOS population gave rise tomammospheres (FigureS1A, inset), with a SFE of !11% (Figure S1B; note that on thebasis of this value and a series of normalizations, we estimatethat our PKHPOS population is !90% pure, Figure S1B). Ofnote, PKHPOS cells could support at least four additional gener-ations of mammosphere growth (Figure 1C, Figure S1C).

By monitoring microscopically the growth of PKHPOS cellsembedded in methylcellulose, we established that mammo-

spheres are truly the result of the clonal expansion of singlePKHPOS cells (Figure 1D). PKHPOS, but not PKHNEG, cellsshowed features of the quiescent state, as evidenced by reten-tion of BrdU and absence of expression of the proliferationmarker Ki-67 (Figure 1E). Moreover, PKHPOS cells divided asym-metrically, as demonstrated by the unequal partitioning atmitosis of the cell fate determinant Numb (Figures 1F and 1G)(Gonczy, 2008).

Characterization of PKHPOS CellsWe analyzed PKHPOS cells, by immunofluorescence (IF), witha panel of markers (Experimental Procedures). PKHPOS cellsshared features with both epithelial (CD24+/EpCAM+) andmyoe-pithelial (CD49F+/CK5+/TP63+) cells (Figure 2A, Figure S2A);however, they did not express detectable terminal differenti-ation markers, such as Muc1, E-cadherin, and ASMA (data notshown), suggesting that lineage specification has not yet

A

B

CD24 EpCAM TP63CD49F CK5

DAPI ASMA EpCAMPKH26 Epifluor.

PK

HP

OS

cells

PK

HN

EG

cells

C

G

TP63E-cadherin

Beta casein

FD

PK

HP

OS c

ells

Bright Field DAPI

hCOT mCOT

Cells:

4/4 4/4 3/10 0/6- -

- - - -0/4 0/2

1:26(1:10-1:66)

> 1:70.000

105 104 103 102 101 100

MRUfrequency

(limits)

Outgrowths/Injections

PK

HP

OS

PK

HN

EG

< 3%

PKHPOS 10 cells

PKHNEG 10000 cells

H&E stainingBright field

PK

HP

OS

cells

PK

HN

EG

cells

PKHPOS cells

PKHNEG cells

PK

HP

OS c

ells

E

Figure 2. Immunophenotypical and Func-tional Characterization of PKHPOS andPKHNEG Cells(A) PKHPOS or PKHNEG cells were analyzedwith the

indicated antibodies by IF. Green, antibody stain-

ing; red, PKH26 epifluorescence; blue, DAPI.

Scale bars represent 10 mm. Quantification is

shown in Figure S2A. Results are typical and

representative of three independent experiments.

(B) PKHPOS or PKHNEG cells were grown on Matri-

gel and analyzed in IF with the indicated anti-

bodies. Scale bars represent 10 mm.Quantification

is shown in Figure S2B. Results are typical and


(C) PKHPOS or PKHNEG cells were grown in 3D in

Matrigel (bright field) and then stained with hema-

toxylin/eosin (H&E). Scale bars represent 100 mm

(PKHPOS) and 10 mm (PKHNEG).

(D) Serial sections (3 mm thick) of organotypic

outgrowths from PKHPOS cells were analyzed in

IHC with the indicated antibodies. Scale bars

represent 50 mm. Arrowheads point to basally

located, spindle-like shaped cells that are E-cad-

herin negative (left) and TP63 positive (right).

(E) IHC analysis of b-casein accumulation in the

lumen of 3D organotypic structures stimulated

with prolactin. The scale bar represents 50 mm.

(F) Top: The indicated amounts of PKHPOS and

PKHNEG cells were transplanted into humanized

epithelium-cleared mammary fat pads of 21-day-

old immunocompromised NOD/SCID mice. MRU,

mammary-repopulating unit. Bottom: Whole-

mount analysis of 10-week-old mammary out-

growths. Scale bars represent 500 mm. The boxed

area is magnified on the right to better visualize the

outgrowth. A graphic indication (pie) of the

percentage of fat pad filled by the outgrowth is

also shown.

(G) Genomic FISH analysis to assess the human

origin of the fat pad mammary outgrowths from

PKHPOS cells, with specific human (hCOT, red) or

mouse (mCOT, green) Cot-1 probes (Extended

Experimental Procedures). Original magnification

320.

See also Figure S2.

64 Cell 140, 62–73, January 8, 2010 ª2010 Elsevier Inc.

組織標本固定した（死んだ）組織を見る方法

1. 組織をホルマリンなどで固定（この時点で細胞は死亡2. パラフィンでブロックをつくる3. 薄くスライス4. プレパラートにのせて、各種染色をする

H&E染色：　核が青～紫　細胞質が赤～ピンク

他にもいろいろな染色がある


mammospheres, with a SFE of !23% (PKHPOS pre-FACS inFigure S1B; note that this number is in agreement with themaximum expected SFE, Figures S1B and S1C).

We used FACS to isolate PKHPOS cells. Based on the typicalPKH26 distribution in mammospheres and the SFE of cellsfrom mammospheres, we isolated the most epifluorescent0.2%–0.4% of the total cell population (PKHPOS cells, gated at103–104 fluorescence units), as well as a ‘‘dull’’ population (gatedat 101–102 fluorescence units, PKHNEG cells) (Figure S1A, inset).Only the PKHPOS population gave rise tomammospheres (FigureS1A, inset), with a SFE of !11% (Figure S1B; note that on thebasis of this value and a series of normalizations, we estimatethat our PKHPOS population is !90% pure, Figure S1B). Ofnote, PKHPOS cells could support at least four additional gener-ations of mammosphere growth (Figure 1C, Figure S1C).

By monitoring microscopically the growth of PKHPOS cellsembedded in methylcellulose, we established that mammo-

spheres are truly the result of the clonal expansion of singlePKHPOS cells (Figure 1D). PKHPOS, but not PKHNEG, cellsshowed features of the quiescent state, as evidenced by reten-tion of BrdU and absence of expression of the proliferationmarker Ki-67 (Figure 1E). Moreover, PKHPOS cells divided asym-metrically, as demonstrated by the unequal partitioning atmitosis of the cell fate determinant Numb (Figures 1F and 1G)(Gonczy, 2008).

Characterization of PKHPOS CellsWe analyzed PKHPOS cells, by immunofluorescence (IF), witha panel of markers (Experimental Procedures). PKHPOS cellsshared features with both epithelial (CD24+/EpCAM+) andmyoe-pithelial (CD49F+/CK5+/TP63+) cells (Figure 2A, Figure S2A);however, they did not express detectable terminal differenti-ation markers, such as Muc1, E-cadherin, and ASMA (data notshown), suggesting that lineage specification has not yet

A

B

CD24 EpCAM TP63CD49F CK5

DAPI ASMA EpCAMPKH26 Epifluor.

PK

HP

OS

cells

PK

HN

EG

cells

C

G

TP63E-cadherin

Beta casein

FD

PK

HP

OS c

ells

Bright Field DAPI

hCOT mCOT

Cells:

4/4 4/4 3/10 0/6- -

- - - -0/4 0/2

1:26(1:10-1:66)

> 1:70.000

105 104 103 102 101 100

MRUfrequency

(limits)

Outgrowths/Injections

PK

HP

OS

PK

HN

EG

< 3%

PKHPOS 10 cells

PKHNEG 10000 cells

H&E stainingBright field

PK

HP

OS

cells

PK

HN

EG

cells

PKHPOS cells

PKHNEG cells

PK

HP

OS c

ells

E

Figure 2. Immunophenotypical and Func-tional Characterization of PKHPOS andPKHNEG Cells(A) PKHPOS or PKHNEG cells were analyzedwith the

indicated antibodies by IF. Green, antibody stain-

ing; red, PKH26 epifluorescence; blue, DAPI.

Scale bars represent 10 mm. Quantification is

shown in Figure S2A. Results are typical and


(B) PKHPOS or PKHNEG cells were grown on Matri-

gel and analyzed in IF with the indicated anti-

bodies. Scale bars represent 10 mm.Quantification

is shown in Figure S2B. Results are typical and


(C) PKHPOS or PKHNEG cells were grown in 3D in

Matrigel (bright field) and then stained with hema-

toxylin/eosin (H&E). Scale bars represent 100 mm

(PKHPOS) and 10 mm (PKHNEG).

(D) Serial sections (3 mm thick) of organotypic

outgrowths from PKHPOS cells were analyzed in

IHC with the indicated antibodies. Scale bars

represent 50 mm. Arrowheads point to basally

located, spindle-like shaped cells that are E-cad-

herin negative (left) and TP63 positive (right).

(E) IHC analysis of b-casein accumulation in the

lumen of 3D organotypic structures stimulated

with prolactin. The scale bar represents 50 mm.

(F) Top: The indicated amounts of PKHPOS and

PKHNEG cells were transplanted into humanized

epithelium-cleared mammary fat pads of 21-day-

old immunocompromised NOD/SCID mice. MRU,

mammary-repopulating unit. Bottom: Whole-

mount analysis of 10-week-old mammary out-

growths. Scale bars represent 500 mm. The boxed

area is magnified on the right to better visualize the

outgrowth. A graphic indication (pie) of the

percentage of fat pad filled by the outgrowth is

also shown.

(G) Genomic FISH analysis to assess the human

origin of the fat pad mammary outgrowths from

PKHPOS cells, with specific human (hCOT, red) or

mouse (mCOT, green) Cot-1 probes (Extended

Experimental Procedures). Original magnification

320.

See also Figure S2.

64 Cell 140, 62–73, January 8, 2010 ª2010 Elsevier Inc.

Immunohistchemistry: 免疫組織化学組織標本にて、特定のタンパク質を抗体を用いて染める方法

茶色の部分が抗体(anti-E-cadherin)が結合している部分


fluorescence staining: 蛍光染色生きた細胞/組織/生体を染色する方法

(Stingl and Caldas, 2007).With this background inmind, we usedthe hNMSC signature to interrogate expression data sets ofbreast cancers. We discovered that the signature can stratifybreast cancers according to their biological and molecularfeatures. We further showed that this is due to the differentnumber of cancer SCs (CSCs) (or cancer-initiating cells) presentwithin different types of breast tumors. Based on our data, wepropose a model of breast tumorigenesis that might resolve theincongruence between the complexity of breast cancer pheno-types and the simplicity of the normal breast parenchyma.

RESULTS

Isolation of hNMSCsCells from normal human mammary glands were labeled withPKH26 and plated in suspension to allow mammosphere

A B

Mer

geB

right

fiel

dP

KH

26

E

F

30 h

72 h 96 h

0 hD

BrdU PKH26 Ki67 PKH26

PK

HP

OS c

ells

0

25

50

75

100

BrdU Ki67

Pos

itive

cel

ls (

%)

(n = 30/each)

63%

3% 0%

30%

10-1

100

101

1 2 3 4

0

2

4

6

R2 = 1

R2 = 0.99

Passages

Cum

ulat

ive

sphe

re n

umbe

r(5

,000

cel

ls p

late

d)

0

100

200

300

400

100

101

102

103

1 2 3 4 5

R2 = 0.99

Passages

Cum

ulat

ive

sphe

re n

umbe

r(2

,500

PK

HP

OS c

ells

pla

ted)

C

PKH26Bright field

GFP-Numb Merge

Asy

mm

etric

div

isio

ns (

%)

100

75

50

25

0

82%

6%

PK

HP

OS

(n=3

0)P

KH

NE

G

(n=1

00)

G

PK

HN

EG c

ells

PKHPOS

PKHNEG

N = 16 PKHPOS cellsAsymmetric: 13 (81%)Symmetric: 3 (19%)

N = 23 PKHPOS cellsAsymmetric: 16 (70%)Symmetric: 5 (21%)Ambiguous 2 (9%)

Figure 1. Identification and Purification ofhNMSCs(A) A mammosphere derived from PKH26-labeled

epithelial cells. The scale bar represents 100 mm.

(B and C) Propagation of mammospheres for

multiple generations starting from bulk epithelial

cells (B, open and closed circles indicate two

different patients) or FACS-sorted PKHPOS cells

(C). Top, linear scale; bottom, semilog scale.

Regression analysis trend lines and coefficients

(R2) are shown. Typical experiments, in triplicate,

are shown (additional statistical analyses are in

Figures S1B and S1C).

(D) A single PKHPOS cell, embedded in methylcel-

lulose, wasmonitored at the indicated times. Scale

bars represent 10 mm, and 100 mm in the bottom-

right panel.

(E) BrdU (left) and Ki67 (right) staining of PKHPOS

and PKHNEG cells. Quantification is shown on the

right.

(F and G) Asymmetric segregation of lentivirally

transduced GFP-Numb (F) or endogenous Numb

(G, confocal sections are shown) to one daughter

cell during cell division of a single PKHPOS cell

either embedded in methylcellulose (F) or grown

in suspension (G). Top, mock-treated; bottom,

treated with blebbistatin). Scale bars represent

10 mm. The solid white bar in (G) indicates the

cleavage plane of the two daughter cells. Quantifi-

cations are also shown.

See also Figure S1.

growth (Figure S1A available online). Asexpected, very few cells within mammo-spheres retained strong epifluorescence(Figure 1A). The sphere-forming effi-ciency (SFE) of cells from the mammarygland was 0.003%–0.01% (dependingon whether bulk mammary cells or pre-enriched mammary epithelial cells wereemployed, Figure S1B). The SFE of cellsobtained from dissociated mammo-spheres was !0.1% (F2 in Figure S1B).Normal mammospheres could be propa-

gated for at least four generations (Figure 1B), and their clono-genic ability decreased exponentially (Figure 1B, Figure S1C):at every generation, the SFE was !23% of that measured inthe preceding generation (Figure S1C). Mammospheres repro-ducibly contained !300 cells per sphere (Figure S1D).We estimated that in the normal mammary gland there is one

mammosphere-initiating cell per!7500 total cells or !2000 epi-thelial cells (Figure S1D), in good agreement with previousreports in mouse and human (Shackleton et al., 2006; Stingl etal., 2006). In addition, we calculated that approximately onemammosphere-initiating cell is present per mammosphere,and that this cell is likely found within the PKH26-positive(PKHPOS) cell fraction (Figure S1D). This was confirmed by moni-toring of the ability of single cells, obtained from dissociatedmammospheres, to form second-generation mammospheres,as a function of their PKH positivity. Only PKHPOS cells formed

Cell 140, 62–73, January 8, 2010 ª2010 Elsevier Inc. 63

蛍光色素のついた物質は、蛍光顕微鏡で観察できる。

(Stingl and Caldas, 2007).With this background inmind, we usedthe hNMSC signature to interrogate expression data sets ofbreast cancers. We discovered that the signature can stratifybreast cancers according to their biological and molecularfeatures. We further showed that this is due to the differentnumber of cancer SCs (CSCs) (or cancer-initiating cells) presentwithin different types of breast tumors. Based on our data, wepropose a model of breast tumorigenesis that might resolve theincongruence between the complexity of breast cancer pheno-types and the simplicity of the normal breast parenchyma.

RESULTS

Isolation of hNMSCsCells from normal human mammary glands were labeled withPKH26 and plated in suspension to allow mammosphere

A B

Mer

geB

right

fiel

dP

KH

26

E

F

30 h

72 h 96 h

0 hD

BrdU PKH26 Ki67 PKH26

PK

HP

OS c

ells

0

25

50

75

100

BrdU Ki67

Pos

itive

cel

ls (

%)

(n = 30/each)

63%

3% 0%

30%

10-1

100

101

1 2 3 4

0

2

4

6

R2 = 1

R2 = 0.99

Passages

Cum

ulat

ive

sphe

re n

umbe

r(5

,000

cel

ls p

late

d)

0

100

200

300

400

100

101

102

103

1 2 3 4 5

R2 = 0.99

Passages

Cum

ulat

ive

sphe

re n

umbe

r(2

,500

PK

HP

OS c

ells

pla

ted)

C

PKH26Bright field

GFP-Numb Merge

Asy

mm

etric

div

isio

ns (

%)

100

75

50

25

0

82%

6%

PK

HP

OS

(n=3

0)P

KH

NE

G

(n=1

00)

G

PK

HN

EG c

ells

PKHPOS

PKHNEG

N = 16 PKHPOS cellsAsymmetric: 13 (81%)Symmetric: 3 (19%)

N = 23 PKHPOS cellsAsymmetric: 16 (70%)Symmetric: 5 (21%)Ambiguous 2 (9%)

Figure 1. Identification and Purification ofhNMSCs(A) A mammosphere derived from PKH26-labeled

epithelial cells. The scale bar represents 100 mm.

(B and C) Propagation of mammospheres for

multiple generations starting from bulk epithelial

cells (B, open and closed circles indicate two

different patients) or FACS-sorted PKHPOS cells

(C). Top, linear scale; bottom, semilog scale.

Regression analysis trend lines and coefficients

(R2) are shown. Typical experiments, in triplicate,

are shown (additional statistical analyses are in

Figures S1B and S1C).

(D) A single PKHPOS cell, embedded in methylcel-

lulose, wasmonitored at the indicated times. Scale

bars represent 10 mm, and 100 mm in the bottom-

right panel.

(E) BrdU (left) and Ki67 (right) staining of PKHPOS

and PKHNEG cells. Quantification is shown on the

right.

(F and G) Asymmetric segregation of lentivirally

transduced GFP-Numb (F) or endogenous Numb

(G, confocal sections are shown) to one daughter

cell during cell division of a single PKHPOS cell

either embedded in methylcellulose (F) or grown

in suspension (G). Top, mock-treated; bottom,

treated with blebbistatin). Scale bars represent

10 mm. The solid white bar in (G) indicates the

cleavage plane of the two daughter cells. Quantifi-

cations are also shown.

See also Figure S1.

growth (Figure S1A available online). Asexpected, very few cells within mammo-spheres retained strong epifluorescence(Figure 1A). The sphere-forming effi-ciency (SFE) of cells from the mammarygland was 0.003%–0.01% (dependingon whether bulk mammary cells or pre-enriched mammary epithelial cells wereemployed, Figure S1B). The SFE of cellsobtained from dissociated mammo-spheres was !0.1% (F2 in Figure S1B).Normal mammospheres could be propa-

gated for at least four generations (Figure 1B), and their clono-genic ability decreased exponentially (Figure 1B, Figure S1C):at every generation, the SFE was !23% of that measured inthe preceding generation (Figure S1C). Mammospheres repro-ducibly contained !300 cells per sphere (Figure S1D).We estimated that in the normal mammary gland there is one

mammosphere-initiating cell per!7500 total cells or !2000 epi-thelial cells (Figure S1D), in good agreement with previousreports in mouse and human (Shackleton et al., 2006; Stingl etal., 2006). In addition, we calculated that approximately onemammosphere-initiating cell is present per mammosphere,and that this cell is likely found within the PKH26-positive(PKHPOS) cell fraction (Figure S1D). This was confirmed by moni-toring of the ability of single cells, obtained from dissociatedmammospheres, to form second-generation mammospheres,as a function of their PKH positivity. Only PKHPOS cells formed

Cell 140, 62–73, January 8, 2010 ª2010 Elsevier Inc. 63


蛍光色素:fluorescence pigment / fluorochrome

特定の波長の光を吸収すると、特定の波長の光を発する


Immunofluorescence: 免疫蛍光蛍光色素のついた抗体を使えば、生きた細胞で見たい物を見ることが出来る。

SERPINB5TP63CK5

A

DAPI JAG1 EpCAM Merge

JAG1 EpCAM

EpCAMDAPI CK5 Merge

CK5 EpCAM

CD49FDAPI EpCAM Merge

CD49f EpCAMTOPO2A

B

Figure S3. Related to Figure 3Additional validation of hNMSCmarkers. A. IHC analysis of mammospheres with markers of hNMSCs. Mammospheres were embedded in paraffin and analyzed

in IHC with the indicated Abs. Bar, 100 mm. B. Identification of hNMSCs in vivo using markers from the hNMSC signature (supplemental data to Figure 3E). Fresh

or paraffin-embedded (last panel) sections of normal mammary glands were analyzed in IF or IHC (last panel) with the indicated Abs. For the IF experiments,

magnifications of the boxed regions are shown on the top of each panel; bar, 10 mm. In the last panel, arrows point to rare stained cells; original magnification

x40. As discussed in the main text, in IF experiments, we used double-labeling with EpCAM, to facilitate the visualization of the epithelial layer.

The experiments of this figure and of Figure 3E confirm that some markers (CD49F, CK5, JAG1) were expressed both by myoepithelial cells and by putative

hNMSCs, while DLL1 appeared expressed exclusively by putative hNMSCs.

Cell 140, 62–73, January 8, 2010 ª2010 Elsevier Inc. S11

得られたデータを光の三原色に変換して重ね合わせると、同時に発現している場所がわ

かる

DAPI: DNAに結合する蛍光色素EpCAM: 上皮マーカーJAG1: Notch1のligandMerge: 3つの合成


FACS: fluorescene-activated cell sorting

蛍光抗体で標識した細胞を分離する機械Flow-cytometry


DNA Hybridization一本鎖DNAの短い断片(oligo)は、適切な条件下で、相補的な配列と結合して安

定になる


Fluorescent in situ hybridization蛍光 in situ hybridization

http://en.wikipedia.org/wiki/Fluorescent_in_situ_hybridization




MicroarrayHybridizationによる超並列　塩基配列検出法


階層化クラスタリング

http://en.wikipedia.org/wiki/File:Heatmap.png

3次元データを、X軸、Y軸、色で表した図をHeatmapという




階層化クラスタリング

• 最初、クラスターの数は検体の数だけある。• クラスターの中で、発現が最も似通った二つを探し、その2つを合体させ一つの新しいクラスターとする。

• クラスターがなくなるまで上の操作を繰り返す。


GSEA: Gene Set Enrichiment Analysis遺伝子集合(Gene signature)が、あるPhenotypeと相関があるかどうかの統計検定

Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles

ES (Enrichment Score): 相関が高ければ高くなるNES (Normalized Enrichment Score): ESをGene Set sizeで標準化した値FDR (False Discovery Ratio): 間違った結果が導かれる可能性


Box Plot: 箱ひげ図

http://water.epa.gov/type/rsl/monitoring/vms62.cfm




Xenograft: 異種移植免疫不全マウスにヒト癌細胞を移植し、癌化能などを観察する

• NOD/SCIDマウスは、T cell, B cell,補体が欠損し、NK cellが低下した免疫不全マウス

• たいてい、左右の足のつけ根の皮下に細胞を移植（注射）して、数週間かけて癌化の有無を見る。

http://de.wikipedia.org/wiki/Xenotransplantation




癌ではしばしばstem cellの機能を制御する経路に異常がある。今回、ヒト正常乳腺幹細胞(hNMSC)が、脂質染料であるPKH26を排出しにくい（休止状態にあるため）という性質をつかって、培養マンモスフィアから高い純度で単離することに成功した。PKH26

positive細胞はhNMSCが持つすべての性質を備えていた。PKH26-positive

(hNMSC型)細胞の網羅的転写解析によって乳癌の生物学的特徴、分子的特徴をとらえることができた。hNMSCを見分けるマーカー群を使って、正常乳腺と乳癌から幹細胞と考えられる細胞を単離した。低分化癌(G3)では高分化癌(G1)よりも単離された細胞(癌幹細胞

CSC)が少なかった。G3とG1を異種移植して比べたところ、G3はCSCを多く含むことがわかった。これらのデータは、ヒト乳癌のheterogeneousな表現形や分子的特徴は、CSCの量に応じて変わるという概念を支持する。


Cancer Stem Cell

The Molecular Basis of CancerTuesday, October 12, 2010

Thk cancer08 final

Health & Medicine

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