Immunohistochemical Assessment of Cancer Stem Cells in

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characteristics associated with normal stem cells, specifically the ability to give ... I would like to express my sincere gratitude to my supervisor ... words to express my gratitude and never be able to ... To my dearest brothers who without their.
Immunohistochemical Assessment of Cancer Stem Cells in Mucoepidermoid Carcinoma Omnia Ibrahim Afifi*, Samia Mostafa El-Azab** and Rehab Fawzi Kasem*** Abstract Background: Cancer stem cells (CSCs) are cancer cells that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Therefore, development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients. CD133 was reported as a putative marker for identification of CSC in different tumors. However, no studies investigated the presence of CSCs in mucoepidermoid carcinoma (MEC). Materials and Methods: The presence of CSCs in paraffin embedded tissue sections of twelve MEC cases (6 low grade and 6 high grade) was investigated by the expression of CD133 immunohistochemically. Identification and counting of CD133 positive colonies of cells was done using a light microscope. Welixon test was used to detect the statistical significance and to compare CD133 positivity between low grade and high grade MEC. Results: Low grade MEC showed moderate and many CD133 + colonies of cells. Whereas high grade cases showed negative, few and moderate CD133+ cells. Conclusion: The current detection of CD133+ colonies in MEC suggests that MEC follows CSCs model, the CSCs count decrease with increasing the grade of the carcinoma. Therefore, these cells may play a role in tumor initiation. Additionally CD133 is a potential cancer stem cell marker in MEC. Key Words: Cancer Stem Cells, Mucoepidermoid Carcinoma, Salivary gland tumors, CD133

‫ﺗﻘﯿﯿﻢ اﻟﻤﻨﺎﻋﺔ اﻟﻨﺴﯿﺠﯿﺔ اﻟﻜﯿﻤﯿﺎﺋﯿﺔ ﻟﻠﺨﻼﯾﺎ اﻟﺠﺬﻋﯿﺔ‬ ‫اﻟﺴﺮطﺎﻧﯿﺔ ﻓﻰ ﺳﺮطﺎن اﻟﺨﻼﯾﺎ اﻟﻤﺨﺎطﯿﺔ اﻟﺒﺸﺮاﻧﯿﺔ‬

‫رﺳﺎﻟﺔ ﻣﻘﺪﻣﺔ إﻟﻰ ﻛﻠﯿﺔ طﺐ اﻟﻔﻢ واﻷﺳﻨﺎن ﺟﺎﻣﻌﺔ اﻟﻘﺎھﺮة ﻛﺠﺰء ﺗﻜﻤﯿﻠﻲ‬ ‫ﻟﻤﺘﻄﻠﺒﺎت اﻟﺤﺼﻮل ﻋﻠﻰ درﺟﺔ اﻟﻤﺎﺟﯿﺴﺘﯿﺮ ﻓﻲ اﻟﻌﻠﻮم اﻷﺳﺎﺳﯿﺔ ﻟﻄﺐ‬ ‫اﻷﺳﻨﺎن‬ ‫)ﺑﺎﺛﻮﻟﻮﺟﯿﺎ اﻟﻔﻢ(‬

‫ﻣﻘﺪﻣﺔ ﻣﻦ‬

‫أﻣﻨﯿﺔ إﺑﺮاھﯿﻢ ﻣﺤﻤﺪ ﻋﻔﯿﻔﻲ‬ ‫ﻛﻠﯿﺔ طﺐ اﻟﻔﻢ واﻷﺳﻨﺎن‬ ‫ﺟﺎﻣﻌﺔ ‪ ٦‬أﻛﺘﻮﺑﺮ‬

‫ﻗﺴﻢ ﺑﺎﺛﻮﻟﻮﺟﯿﺎ اﻟﻔﻢ‬ ‫ﻛﻠﯿﺔ طﺐ اﻟﻔﻢ واﻷﺳﻨﺎن ﺟﺎﻣﻌﺔ اﻟﻘﺎھﺮة‬ ‫‪٢٠١٢‬‬

Immunohistochemical Assessment of Cancer Stem Cells in Mucoepidermoid Carcinoma

Thesis Submitted to the Faculty of Oral and Dental Medicine, Cairo University in Partial Fulfillment of the Requirements for the Degree of Master in Basic Dental Science (Oral Pathology)

By

Omnia Ibrahim Mohammed Afifi B.D.S Faculty of Oral and Dental Medicine, October 6 University.

Department of Oral Pathology Faculty of Oral and Dental Medicine, Cairo University.

2012

Supervisors

Prof. Dr. Samia Mostafa El-Azab Professor of Oral Pathology Faculty of Oral and Dental Medicine, Cairo University

Dr. Rehab Fawzi Kasem Lecturer of Oral Pathology Faculty of Oral and Dental Medicine, Cairo University

‫ﺗﺤﺖ إﺷﺮاف‬ ‫اﻷﺳﺘﺎذ اﻟﺪﻛﺘﻮرة ‪ /‬ﺳﺎﻣﯿﺔ ﻣﺼﻄﻔﻰ اﻟﻌﺰب‬ ‫أﺳﺘﺎذ ﺑﺎﺛﻮﻟﻮﺟﯿﺎ اﻟﻔﻢ‬ ‫ﻛﻠﯿﺔ طﺐ اﻟﻔﻢ واﻷﺳﻨﺎن‬ ‫ﺟﺎﻣﻌﺔ اﻟﻘﺎھﺮة‬

‫اﻟﺪﻛﺘﻮرة ‪ /‬رﺣﺎب ﻓﻮزي ﻗﺎﺳﻢ‬ ‫ﻣﺪرس ﺑﺎﺛﻮﻟﻮﺟﯿﺎ اﻟﻔﻢ‬ ‫ﻛﻠﯿﺔ طﺐ اﻟﻔﻢ واﻷﺳﻨﺎن‬ ‫ﺟﺎﻣﻌﺔ اﻟﻘﺎھﺮة‬

First of all, thanks to Allah for guiding and helping me during this work. There is no word capable of expressing my gratitude and appreciation to Prof. Dr. Samia Mostafa Al-Azab, Professor of Oral Pathology, Faculty of Oral and Dental Medicine, Cairo University, for resolving the most difficult obstacles I met throughout this work. I shall never forget her wise guidance, continuous encouragement, close supervision and most valuable advices. I would like to express my sincere gratitude to my supervisor Dr. Rehab Fawzi Kasem, Lecturer of Oral Pathology, Faculty of Oral and Dental Medicine, Cairo University, for her valuable instructions,

endless

patience

that

made

her

meticulously revise this work word by word and assistance in various processes of putting this study in its final form.

Omnia Ibrahim 2012

To my lovely parents who are the most beautiful thing in my life and whom I cannot find adequate words to express my gratitude and never be able to repay them for their patience, kindness and their great support toward me.

To my dearest brothers who without their support and continuous help, none of this work would have been accomplished.

I.

Contents

Chapters

Page

Introduction and Review of literature

1

Stem Cells.

1

Types of Stem Cells.

3

Stem Cell Niche.

13

Identification of Stem Cells.

14

Cancer Stem Cells.

16

Cancer Stem Cells Hypothesis.

17

Origin of Cancer Stem Cells.

21

Identification of Cancer Stem Cells.

25

Expression of Stem Cell Markers in Different Human Tumors.

27

CD133 as a CSCs Marker.

30

Expression of CD133 as a Stem Cell Marker in Different Human Tumors. Expression of Other Stem Cell Markers in Different Salivary Gland Tumors.

32

Aim of the Study

35

39

I. Contents (continue) Chapters

Page

Materials and Methods

40

Results

45

Discussion

63

Summary 69

Conclusion

71

References

72

II.

List of Tables

Numbers

Contents

Page

CSC Markers and their

26

of tables

(1)

sources.

(2)

Statistical

difference

in

CD133+ colonies of cells

48

between low and high grade MEC.

(3)

Individual scoring data for the samples of low grade MEC

55

that represent CD133+ cells.

(4)

Individual scoring data for the samples of high grade MEC that represent CD133+ cells.

49

III.

Numbers

List of Graphs

Contents

Page

Scores of CD133+ colonies of cells in

50

of graphs

(1)

low and high grade MEC.

IV.

List of Figures

Numbe rs

Figure caption

of

Pa ge

figures 1

Stem Cell replication and differentiation.

1

2

Stem cells properities.

1

3

Stem Cells Potency.

3

4

Characteristics of ES cells.

4

5

HSCs.

6

6

UCB Stem Cells.

7

7

MSC’s are capable of differentiating into mesenchymal tissues.

8

8

DSCs.

9

9

NSCs

10

10

iPSC.

12

11

Tumor cells are heterogenous and only CSCs can proliferate extensively and form new tumors.

19

IV. List of Figures (continue) Numbe rs

Pa

Figure caption

of

ge

figures 12

Stem cell specific and conventional cancer therapies.

20

13

Origin of CSCs

24

14

A photograph illustrating the structure model of CD133.

31 50

15 Photomicrograph

of

low

grade

MEC

(H&E×100). 50

16 Photomicrograph

of

low

grade

MEC

(H&E×100).

51

17 Photomicrograph (H&E×100).

of

low

grade

MEC

51

18 Another field of previous photomicrograph of low grade (H&E×200).

52

19 Photomicrograph

of

low

grade

MEC

(H&E×200). 52

20 Photomicrograph of low grade MEC (H&E×200).

53

21 Another case of low grade MEC (H&E×100).

IV. List of Figures (continue) Numbe rs

Pa

Figure caption

of

ge

figures 53

22 Higher

magnification

photomicrograph

of

of low

grade

previous MEC

(H&E×200). 54

23 Another photomicrograph of low grade MEC (H&E×400).

56

24 Photomicrograph

of

high

grade

MEC

(H&E×100). 56

25 Photomicrograph

of

high

grade

MEC

(H&E×200). 57

26 Photomicrograph of another case of high grade MEC (H&E×200).

57

27 Photomicrograph

of

high

grade

MEC

(H&E×400). 58

28 Another case of high grade MEC (H&E×400).

58

29 Another field of the previous case (H&E×400).

59

30 Photomicrograph of low grade MEC (antiCD133×200).

59

31 Photomicrograph of another case of low grade MEC (anti-CD133×200).

IV. List of Figures (continue) Numbe rs

Figure caption

of

Pa ge

figures 61

32 Photomicrograph of low grade MEC (antiCD133×400).

61

33 Photomicrograph of high grade MEC (antiCD133×200).

62

34 Photomicrograph of high grade MEC (antiCD133×200).

62

35 Photomicrograph of high grade MEC (antiCD133×200).

V. List of Abbreviations Abbreviations

Words

ABCG2

ATP-binding cassette superfamily G member 2

Abs

Antibodies

ACC

Adenoid cystic carcinoma

AECs

Amniotic stem cells

ALDH

Aldehyde dehydrogenase

AML

Acute Myeloid Leukemia

ASCs

Adult stem cells

ATP

Adenosine triphosphate

B-SA

Biotin Streptavidin Amplified Detection System

BM

Bone Marrow

Bmi-1

Polycomb ring finger-1

Ca

Calcium

CD

Cluster of differentiation (cluster of designation)

CK5/14

Cytokeratin 5/14

CML

Chronic Myeloid Leukemia

CNS

Central Nervous System

CRC

Colorectal cancer

CSCs

Cancer Stem Cells

DNA

DeoxyriboNucleic Acid

DSCs

Dental stem cells

ESA

Epithelial specific antigen

ES cell line

Embryonic Stem Cell Lines

ESCs

Epidermal Stem Cells

FACS

Fluorescence Activated Cell Sorting

FSCs

Fetal Stem Cells

GIT

Gastro-intestinal tract

G phase

Growth phase

H&E

Hematoxylin and Eosin

HIF-1α

Hypoxia-inducible factors-1α

HMGA2

High-mobility group AT-hook 2 gene

HNSCC

Head and Neck Squamous Cell Carcinoma

HSCs

Hematopoietic Stem Cells

IHC

Immunohistochemistry

kDa

kilodalton

LGR5

Leucine-rich repeat-containing G-protien coupled Receptor 5

MB

Medulloblastoma

MEC

Mucoepidermoid Carcinomas

M phase

Mitosis phase

V. List of Abbreviations (continue) Abbreviations

Words

MSCs

Mesenchymal Stem Cells

NCAM

Neural Cell Adhesion Molecule

NCSCs

Neural Crest Stem Cells

NGF

Nerve growth factor

NK-cells

Natural Killer Cells

NOD/SCID

Non-obese diabetic /Severe Combined Immunodeficiency

NSCs

Neural Stem Cells

Oct-4

Octamer binding protien-4

OSCCs

Oral Squamous Cell Carcinoma

PBS

Phosphate buffered saline

PCR

Quantitative Reverse-Transcriptase

pH

Potential Hydrogen (a measure of acidity and alkalinity)

PSA-NCAM

Polysialic acid-neural cell adhesion molecule

P53

Protein 53

p75 NTR

p75 Neurotrophin R

Q RT-PCR

Quantitative Real-Time Polymerase Chain Reaction

Sca-1

Stem cell antigen-1