flow cytometry in translational cancer research: believe in rare!

Flow cytometry in colon cancer: polychromatic mosaics of TIC and new

targets for tailored therapy

Marica Gemei, PhD

• Flow cytometry in solid cancer analysis

• CD66c as CCSC markers

• Multiparametric FC in CTCs analysis

• Conclusions

• Acknowledgements

Summary

A multiparametric matter

Flow cytometry in solid cancers analysis

Colon dx

Sigmoid colon

Transverse

Colon sx

Multiparametric cytometry allows «restructuration» of solid tissues

Intact live cellsNot-hematopoietic cells

Epithelial cells

damaged cells

not epithelial cells

ematopoietic cells

80% 20%50%

20%

30%

Colon biopsies composition

h

CD45 /CD326 /SYTOX BLUE

Flow cytometry in solid cancer analysisSelection of tumor cells

Solid tissues derived samples are composed mainly by «contaminant» events

Gemei et al. Unpublished data

Flow cytometry in solid cancer analysisSelection of tumor cells

The use of CD45 together with CD326 allow a better selection of tumor cell population

Migration and Metastatization

CD9 CD49f

CD81

CSC Markers

CD133 CD24

CD29

CD326

CD47

CD90

Injury resistance and malignant behavior

CD227 CD55

CD59

CD66c

CD44

CD26

CD151

CD49b

CD324

CD166

Flow cytometry in solid cancer analysisColon cancer stem cells analysis

Flow cytometry in solid cancer analysisMultiparametric analysis = huge information

CD13

3

CD13

3

CD24

CD24

CD29

CD29

CD44

CD44

CD47

CD47

CD49

b

CD49

b

CD49

f

CD49

f

CD54

CD54

CD55

CD55

CD59

CD59

CD66

b

CD66

b

CD66

c

CD66

c

CD81

CD81

CD16

4

CD16

4

CD16

6

CD16

6

CD22

7

CD22

7

100

1000

10000

100000TUMOR VS NORMAL

CD MARKERS

MFI

CD

133

CD

24

CD

29

CD

44

CD

47

CD

49b

CD

59

CD

66b

CD

66c

CD

81

CD

164

CD

166

CD

227

Me

an

Flu

ore

scen

ce In

ten

sity (

MF

I)

102

103

104

105

CD

55

CD

54

CD

49f

Normal

Cancer


Flow cytometry in solid cancer analysisNormal colon and colon cancer surface signature

CD55 and CD66c are strongly upregulated in tumor tissue

TUMORS VS METASTASES

CD24

CD24

CD26

CD26

CD66

c

CD66

c

CD15

1

CD15

1

100

1000

10000

100000

CD MARKERS

MFI

102

104

105

103

Me

an

Flu

ore

sce

nce In

ten

sity (

MF

I)

CD24 CD26 CD66c CD151

P= 0.0099

P= 0.0180

P= 0.0119

P= 0.0395

Metastases

Primary cancer


Flow cytometry in solid cancer analysisColon primary cancers and metastases surface signature

CD66c is upregulated in metastases respect to primary tumors

CCSC marker

Migration and invasion

Cell proliferation

Complement defence ?

Flow cytometry in solid cancer analysisCD66c (CEACAM6) correlation with other markers

CD66cbright cells co-expressed other markers involved in CSC behavior




• Conclusions


Summary

CD66c as CCSC markerCD66c (CEACAM6)

CD66c is a GPI-linked glycoprotein belonging to the CEA family proteins

Normal Tissue Cancer Tissue0

20

40

60

80

100

% C

D66c+

Normal Tissue Cancer Tissue100

1000

10000

100000

CD

66c M

FI

p < 0.0001

p < 0.0001

Gemei et al. Cancer, 2012

CD66c as CCSC markerCD66c expression in normal colon and in colon cancer

CD66c is upregulated in colon cancer tissue

34

0 0 0

8

4

0 014

710

0

5

10

15

20

25

30

35

40

-/+ + ++ +++

nu

mb

er

of

sam

ple

s

CD66c expression score

normal tissue

adenoma

cancer

Normal Adenoma Carcinoma


CD66c as CCSC markerCD66c expression in normal colon and in colon pathology

CD66c is upregulated in colon cancerogenesis

T1-2 T3-4100

1000

10000

100000

CD

66c

MF

I

p= 0.0073

Tis

N0

M0

T1-2

N0

M0

T3-4

N0

M0

T any

N1-2

M0

T any

N any

M1

Metastasis to

distant organs

M0 M10

10000

20000

30000

40000

CD

66c

MF

I

p= 0.0290


CD66c as CCSC markerCD66c expression in colon cancer progression

CD66c expression parallels colon cancer progression

CD133+ Normal CD133+ Tumor0

20

40

60

80

100

% C

D66c+

p< 0.0001

Patient 23

(primary tumor)

Patient 23

(normal colon)

CD66C CD66C

normal carcinoma


CD66c as CCSC markerCD66c expression in normal and cancerous stem cells

CD66c expression parallels colon cancer progression

Adherent Conditions Sphere Conditions

0.1%0.4%

CD133 CD66c

17% 94%

CD133 CD66c

Cell Line Cell Type Origin

GEO Human AdenoCa Primary

CD66c as CCSC markerCD66c expression in CCSCs: colon spheres

CD66c is upregulated in CCSCs enriched colon spheres

92,6%

MFI 30552

4,8%

MFI 410

5%

MFI 572

100%

MFI 96578

Patient 36

(fresh tissue)

Patient 36

(sphere colture)

Primary Tumors Tumor Spheres1000

10000

100000

CD

66c M

FI

p= 0.0313

40x


CD66c as CCSC markerCD66c expression in CCSCs: colon spheres

CD66c is upregulated in CCSCs enriched colon spheres

Primary Tumors Metastasis100

1000

10000

100000

1000000

CD

66c M

FI

p= 0.0102

Primary tumor metastasis

Patient 25

(primary tumor)

Patient 25

(metastasis)

Colon carcinoma

Hepatic metastasis


CD66c as CCSC markerCD66c expression in primary tumors and hepatic metastases

CD66c expressing cells are selected in hepatic metastases

20x

20x

Xenograft

Primary tumor

H&E

H&E


CD66c as CCSC markerCD66cbright and CD66negative cells tumorigenic potential in vivo

CD66cbright cells are tumorigenic in vivo

77% 24%

SSC

CD

66

c

CD66c siRNA silencing

48h48h

Aspecific siRNA CD66c siRNA0

20

40

60

80

100

%C

D66c+

p=0.0013


CD66c as CCSC marker: analysis of its functionCD66c silencing in CACO2 cell line

WST-1 colorimetric proliferation assay

24 h 48 h 72 h 96 h 120 h 144 h

0

1

2

3

4Aspecific siRNA

CD66c siRNA

Time

Ce

ll p

rolif

era

tio

n (

OD

45

0-6

50

nm

)

p < 0,0001


CD66c as CCSC marker: analysis of its functionEffects of CD66c silencing on cell proliferation

CD66c silencing impairs CACO2 cell proliferation

YO-PRO/PI apoptosis-necrosis assay

72h 96h 120h 144h0

10

20

30

Aspecific siRNA

CD66c siRNA

Time

% A

popt

osis

72h 96h 120h 144h0

20

40

60

80

Aspecific siRNA

CD66c siRNA

Time

% N

ecro

sis

p = 0,0013

p = 0,0214


CD66c as CCSC marker: analysis of its functionEffects of CD66c silencing on cell apoptosis and necrosis

CD66c silencing enhance CACO2 cell death

Aspecific

siRNA

CD66c siRNA

Clonogenic assay in plate

Clonogenic assay in soft

agar

Aspecific siRNA CD66c siRNA0

10

20

30

40

50

n

of

co

lon

ies

p=0.0009


CD66c as CCSC marker: analysis of its functionEffects of CD66c silencing on cell clonogenic potential

CD66c silencing impairs CACO2 clonogenic potential

CD66c siRNA

treated CACO2

Aspecific siRNA

treated CACO2

55%

0.2%

H&E

H&E

Human Cells

Mouse Cells

Human Cells

Mouse Cells


CD66c as CCSC marker: analysis of its functionEffects of CD66c silencing on cell tumorigenic potential

CD66c silencing impairs CACO2 tumorigenic potential

Analysis of CD66c associated pathwaysIngenuity pathway analysis

• Multiparametric FC in solid cancer analysis



• Conclusions


Summary

Primary

tumor

Metastasis

EMT

Blood vessel

Distant

organsCTC

MET

CD26 /CD151 /CD133 /CD44 /CD66c /CD45 /CD326 /SYTO 16

CD326+ CTCs

Multiparametric FC in CTCs analysisColon cancer CTCs phenotype

CD66c along with CD26 could help in the isolation of colon cancer CTCs

0 6 12 18 24 30 36 42

100

80

60

40

20

0

months

Su

rv

iva

l p

ro

ba

bil

ity

(%

)

Number at risk

Group: 0

38 38 35 27 25 9 7 2

Group: 1

34 33 24 15 13 8 5 2

0 6 12 18 24 30 36 42

100

80

60

40

20

0

months

Su

rv

iva

l p

ro

ba

bil

ity

(%

)

Number at risk

Group: 0

46 46 43 31 29 13 10 4

Group: 1

26 25 16 11 9 4 2 0

Three-year progression-free survival rate in the 72 colorectal cancer patients who underwent potentially curative surgery according to (left) pre-and (rightr) post-operative blood levels of CD26+/CD326-cells. In blue: CD26+/CD326-cell-negative patients and in red:CD26+/CD326-cell-positive patients.

Pre-operative CSC Post-operative CSC

Gemei et al. Oncology Letters, 2014

CD326- CTCs

Multiparametric FC in CTCs analysisColon cancer CTCs detection: CD326-/CD26+ cells prognostic impact

CD26+/CD326- CTCs bearing patients have a reduced progression free survival

High pre-operative levels of CD26+/CD326-CTCs correctly predicted tumor relapse in 44.4% of the cases, while 69% of post-operative CD26+/CD326-CTC-positive patients experienced cancer recurrence, with a test accuracy of 88.8%. By contrast, post-operative CD26+/CD326-CTC-negative patients showed an increase in the three-year progression-free survival rate of 86%, along with a reduced risk of tumor relapse of >90%.

Gemei et al. Oncology Letters, 2014

Multiparametric FC in CTCs analysis

Pre operative Post operative

CD26+/CD326- CTCs are predictive of cancer recurrence

Colon cancer CTCs detection: CD326-/CD26+ cells prognostic impact




• Conclusions


Summary

Conclusion

• A precise gating strategy is necessary to analyze rare populations as CSCs and

CTCs

• Multiparametric flow cytometry allowed the analysis of solid tissues and the

identification of new CSC markers

• Multiparametric flow cytometry can identify CTCs

Acknowledgements

CEINGE-Biotecnologie Avanzate

Prof. Luigi Del Vecchio

Prof. Rosa Di Noto

Dott. Peppino Mirabelli

Dott.ssa Francesca D’Alessio

SUN – Seconda Università di Napoli

Prof. Gennaro Galizia

Dott. Anna Zamboli

Università di Napoli «Federico II»

Prof. Giancarlo Troncone

Dott. Antonino Iaccarino

flow cytometry in translational cancer research: believe in rare!

Health & Medicine