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SARCOSPHERE METHOD FOR ISOLATION AND CULTURE

OSTEOSARCOMA CANCER STEM CELLS

DEDED YUDHA PRANATHA

INTRODUCTION

OSTEOSARCOMA PROBLEMS : METASTASIS, RECCURENCE AND RESISTANT FROM CHEMOTHERAPY

RECENT STUDY CANCER STEM CELLS SELF RENEWAL, DIFFERENTIATION, PROLIFERATION

OSTEOSARCOMA CSCs RESEARCH HAS NOT BEEN REPORTED IN INDONESIA

OUR STUDY : ISOLATION, CULTURE AND CHARACTERIZATION OF HUMAN OSTEOSARCOMA CELLS

Gibbs CP, et al. Stem-like cells in bone sarcomas: Implications for Tumorigenesis. Neoplasia. 2005;7:11. Wilson H, Huelsmeyer M, Chun R, Young KM, Friedrichs K, Argyle DJ. Isolation and characterisation of cancer stem cells from canine osteosarcoma. Veterinary journal. 2008 Jan;175(1):69-75.

Research Question

1. ARE OSTEOSARCOMA CSCs CAN BE ISOLATED AND CULTURED BY INVITRO MEANS ?

2. HOW ARE THE MORPHOLOGY AND CHARACTERISTIC OF OSTEOSARCOMA CSCs ?

Research Hypotheses

1. CSCs CAN BE ISOLATED AND CULTURED FROM OSTEOSARCOMA CELLS

2. OSTEOSARCOMA CSCs MORPHOLOGY AND CHARACTERISTIC EXAMINATION CAN BE PERFORMED BY USING ALIZARIN RED S STAINING, RT-PCR METHOD, AND IFA

Objectives Research Benefits

1. THEORETICAL BENEFITS– TO PROVE THAT CSCs CAN BE

ISOLATED, CULTURED, AND CHARACTERIZED BY IN VITRO MEANS FROM HUMAN OSTESOSARCOMA CELLS

2. METHODOLOGICAL BENEFITS– PROVIDE DONATION IN

PROBLEM SOLVING OF OSTEOSARCOMA CSCs STUDY

– HAVING AN INTELLECTUAL PROPERTY

3. APPLICATIVE BENEFITS– MAKE A STUDY GUIDE FOR

RESEARCH DEVELOPMENT OF HUMAN OSTEOSARCOMA IN CMH AND UI

1. GENERAL OBJECTIVESa. TO KNOW WHETHER

OSTEOSARCOMA STEM CELLS CAN BE ISOLATED, CULTURED, AND CHARACTERIZED BY IN VITRO MEANS

b. TO KNOW THE MORPHOLOGY AND CHARACTERISTIC OF OSTEOSARCOMA CSCs

2. SPECIFIC OBJECTIVES– TO MAKE THIS STUDY AS AN

INTRODUCTION OF OSTEOSARCOMA CSCs STUDY IN CMH AND UI

CONCEPTUAL FRAMEWORK

SARCOSPHERE-FORMING ASSAY

ISOLATION AND CULTURE OF CANCER STEM CELLS

CHARACTERIZATION OF CANCER STEM CELL

MORPHOLOGY RECULTURIZATION OF ADHEREN CULTURE

RT-PCR

NANOG CD 133

OCT3/4STAT3

IMMUNOFLUORESCENCE

PHOSPHATASE ALKALINE

CD133

OSTEOCALCIN

ALIZARIN RED S STAINING

OSTEOSARCOMA CELLS

MATERIALS AND METHODS

Inclusion :age 10-25, CPC, conventionalExclusion :Damage of tissue tumor or specimen failed to grow

• Number of samples are

assigned from an osteosarcoma

cell of predecessor

research

• 2015, CMH and PSSP Bogor

• Descriptive

Design Time and Location

SampleCharacteristics of Inclusion and Exclusion

RESEARCH PROTOCOL

CHARACTERIZED BY ALIZARIN RED S STAINING, RT-PCR, AND IFA

CSC MORPHOLOGY

SARCOSPHERES FORMING ASSAY

ISOLATION AND CULTURALIZATION OF OSTEOSARCOMA CELL INSIDE OBM + OGM SINGLEQUOTS MEDIUM, DMEM, THEN CELLS ARE SUPPLEMENTED WITH 10% FBS

AND PS 1% IN 370C, CO2 5%, 95% AIR

CANCER CELL IS OBTAINED BY OPEN BIOPSY AND SURGERY

OSTEOSARCOMA

Gibbs CP, et al. Stem-like cells in bone sarcomas: Implications for Tumorigenesis. Neoplasia. 2005;7:11. Wilson H, Huelsmeyer M, Chun R, Young KM, Friedrichs K, Argyle DJ. Isolation and characterisation of cancer stem cells from canine osteosarcoma. Veterinary journal. 2008 Jan;175(1):69-75.

RESULTSSTAGE I :

ISOLATION AND CULTURALIZATION OF OSTEOSARCOMA CELLS

STAGE II SARCOSPHERE FORMING ASSAYS

RESULTSSTAGE III CHARACTERIZATION

ALIZARIN RED S STAINING

DAY 3 DAY 7

RESULTSSTAGE III CHARACTERIZATION

RT-PCR

NANOG, OCT3/4, STAT3 CD133

RESULTS

STAGE III CHARACTERIZATION IFA

ALKALINE PHOSPATASE CD 133 OSTEOCALSIN NEGATIVE

CONTROL

DISCUSSION

GIBBS, ET AL (2005) : 3 human primary

osteosarcoma cells and 1 human osteosarcoma

cell line

WILSON H, ET AL (2007) : 1 human osteosarcoma

cell line and 3 osteosarcoma canine

lines

WANG L, PARK P, LIN CY (2009):

4 human osteosarcoma cell lines

NEVES (2011) : 1 human osteosarcoma

cell line

ISOLATION AND CULTURE OSTEOSARCOMA CANCER STEM CELLS

ISOLATION AND CULTURE OF OSTEOSARCOMA STEM CELL

THOSE FINDINGS WERE APPROPRIATE WITH OUR STUDY. ABILITY TO PROLIFERATE AND SELF RENEW WAS SHOWN BY THE ABILITY TO FORM THE SPHERE ON ANOIKIS CONDITION AND WHEN THE SPHERE WAS REGROWN BACK AT THE TISSUE CULTURE PLATE, THE CELLS COULD RE ATTACH TO THE PLATE SURFACE

PROVED THAT OUR STUDIED OSTEOSARCOMA CELLS CONTAINED A SMALL NUMBER OF PRIMITIVE CELLS THAT WERE PROBABLY CANCER STEM CELLS

OSTEOSARCOMA STEM CELL CHARACTERIZATION

DISCUSSION

FIRST TECHNIQUE : ALIZARIN RED S STAINING

SECOND TECHNIQUE : RT-PCR

THIRD TECHNIQUE : IFA


DISCUSSION

OUR STUDY SHOWED POSITIVE RESULTS, SARCOSPHERE SHOWED BRIGHT RED-ORANGE COLOR, PROVING A COMPLEX BOND BETWEEN ALIZARIN RED S AND

CALSIUM

FIRST TECHNIQUE : ALIZARIN RED S STAINING

LUK F,YU Y, DONG HT, WALSH WR, YANG JL (2011):

Alizarin red S staining was used to stain Osteosarcoma cell line : Sao2-U-2 OS

Cells


DISCUSSION


GIBBS, ET AL (2005) : Express actived STAT3 and

marker genes of pluripotent marker, Oct ¾ and Nanog

WILSON H, ET AL (2007) : Expression of genes

characteristic of Oct4, NANOG AND STAT3 and Stro-

1

WANG L, PARK P, LIN CY (2009):

Demonstrated Oct ¾ and Nanog were expresed in all 4

cell lines of human Osteosarcoma

LOU N,WANG Y, SUN D, ZHAO J, WANG Y, GAO Z (2010) :

RT-PCR : Oct ¾, Nestin, Nanog were highly expressed

MG-63-M cells

OUR FINDING SUPPORTED THOSE RESULTS: RT-PCR SHOWED EXPRESSIONS OF OCT ¾, NANOG, STAT3 IN ALL OF THE CELL LINEAGE


DISCUSSION


TIRINO, ET AL (2008) :Identified CD133+ cells

within osteosarcoma cell lines

TIRINO, ET AL (2011) :Show existence of CSCs in

human primary bone sarcomas and highlight

CD133 as a pivotal marker for identification

DI FIORE, ET AL (2013):Demonstrated highly expresed CD 133 on

3AB-OS Cell Line


DISCUSSION

THE POSIBILITY OF CD133 WAS NOT DETECTED BECAUSE OF THE NUMBER OF OSTEOSARCOMA CELLS WERE VERY SMALL IN SARCOSPHERE. THIS WAS ACTUALLY CAN PREVENTABLE BY DOING

FACS ANALYSIS PRIOR SARCOSPHERE TO ENSURE THAT CELLS DERIVED FROM CELL CANCER CELLS WAS OSTEOSARCOMA CELLS OR BY USING qPCR METHOD

OUR STUDY DEMONSTRATED DIFFRENTIATED RESULTS, CD 133 DID NOT EXPRESS ON RT-PCR AND WEAK POSITIVE ON IMUNNOFLUORECENCE ANALYSIS.

LOU N,WANG Y, SUN D, ZHAO J, WANG Y, GAO Z

(2010) :Demonstrated highly

expresed CD 133 MG-63-M cells

HE, ET AL (2012) :Demonstrated clinical

relevance expressed CD 133 in Osteosarcoma Patients

with Pulmonary Metastasis



DISCUSSION

THIS PROVED THAT ON THE SARCOSPHERE THAT WE STUDIED OCCURRED IN VITRO OSTEOBLAST’S ACTIVITIES. POSITIVE ALKALINE PHOSPHATASE SHOWED PLURIPOTENCY THAT

WAS ALSO PRESENT AT THE SARCOSPHERETHAT WE ANALYZED

OUR RESULTS OF IMMUNOFLUOROSENCE ANALYSIS SHOWED ALKALINE PHOSPHATASE MARKERS AND OSTEOCALCIN GAVE POSITIVE PROTEIN EXPRESSION RESULTS

SINGH U, ET AL (2012) :Demonstrated :

flurogenic AP Stain that enables characterization

of ESC and iPSC

O CONNOR, ET AL (2008) :

Alkaline Phosphatase (AP+) optimized for

propagating undifferentiated hESCs.

PALMQVIST, ET AL (2005):

Found : Cells with ability to develop in vivo produce alkaline phosphatase cell colonies at high

frequency

THIRD TECHNIQUE : IFA

IN OUR STUDY, OSTEOSARCOMA CSCs CHARACTERIZATION CAN BE PROVEN BY:

DISCUSSION

• POSITIVE EXPRESSION OF OSTEOBLAST COMPLEX WITH CALCIUM ON ALIZARIN RED STAINING

• POSITIVE EXPRESSION FROM PLURIPOTENCY MARKER ON RT-PCR.

• POSITIVE PROTEIN EXPRESSION AS A MARKER FOR OSTEOBLAST AND SURFACE MARKER ON IMMUNOPLUOROSENCE ANALYSIS

ISOLATION AND CULTURE OF OSTEOSARCOMA CANCER STEM CELLS

OSTEOSARCOMA CSCs COULD BE ISOLATED AND CULTURED IN VITRO FROM OSTEOSARCOMA CELLS BY METHODS OF SPHERE FORMING ASSAY

MORPHOLOGICALLY , OSTEOSARCOMA STEM CELLS ON THE SARCOSPHERE WAS ROUND SHAPED, THREE DIMENSION AND DID NOT ADHERE TO THE SUBSTRATE WHEN REPLANTED INTO THE TISSUE

CULTURE PLATE, SARCOSPHERE WAS REATTECHED TO THE SUBSTRATE, FORMING SPINDLE-LIKE FIBROBLASTS

OSTEOSARCOMA CANCER STEM CELLS CHARACTERIZATION

ALIZARIN RED S STAINING SHOWED POSITIVE EXPRESSION ON OSTEOSARCOMA CSCs

OSTEOSARCOMA CSCs SHOWED GENE EXPRESSION, PROVED BY RT-PCR WITH PROTEIN EXPRESSION CONFIRMED BY IFA

CONCLUSION

ISOLATION AND CELL CULTURE OF OSTEOSARCOMA CANCER STEM CELLS

FACS ANALYSIS IS NEEDED ON SARCOSPHERE STAGE WITH CD133 GENE MARKER FOR PURIFICATION OF FORMED SACOSPHERE SO THAT CELLS THAT WERE OBTAINED

CAN BE PROVEN AS CD133 POSITIVE

CULTURE OF OSTEOSARCOMA FROM OTHER PATIENT TO STANDARIZED THE CULTURE PROCEDURE OF CSCs OSTEOSARCOMA

CHARACTERIZATION OF OSTEOSARCOMA CANCER STEM CELLS

qPCR METHOD IS NEEDED FOR CHARACTERIZATION OF OSTEOSARCOMA CSCs

RECOMMENDATION

THANK YOU

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