CD140+/CD44+/ CD24- population. âCharacterization of the different cell population in primary culture of breast tumorâ. Birlipta Pattanayak, Guadalupe Herrera, ...
“Characterization of the different cell population in primary culture of breast tumor” Birlipta Pattanayak, Guadalupe Herrera, Iris Garrido-Cano, Anna Adam-Artigues, Eduardo Tormo, Begoña Pineda, Paula Cabello, Ana Lluch & Pilar Eroles INCLIVA Biomedical Research Institute, 46010 Valencia, Spain; Oncology and Haematology Department, Hospital Clinico Universitario, 46010 Valencia, Spain ; CIBERONC
RESULTS
INTRODUCTION From decades immortal cells have been used as a model system to study the mechanism of cancer, drug resistance and explore the potential efficacy of the anticancer drugs. However, the numerous study has been suggested that considering the immortal cancer cell has a backdrop to represent the drug resistance and heterogeneity of the tumor in the patients. So, Primary tumor cultures currently begin to constitute the golden standard to study the primary cells, which has to apply to explore the mechanism of resistance and personalized therapy including in breast tumor.
Initially 10 breast tumors with different subtypes were obtained from patients, with their informed consent. They were freshly taken biopsy samples. The patient biopsies underwent an enzymatic digestion with (3 mg /ml collagenase and 5 unit/ml dispase). After enzymatic digestion cells were grown in normal complete media with different growth factors and ROCK inhibitor. All cell lines were maintained till their passage (p15-p20). The cells were characterized with different antibodies by flow cytometry, different gene expression were assessed by PCR.
ACKNOWLEDGMENT
95,3%
(A) A
C
B
(B)
Fig 3:Double positive population were analyzed by three different color antibodies using flow cytometer specific for Stem cell and CD140+/CD44+/ CD24- population.
D
Fig 1: Morphology of the primary cells of the different breast tumor subtypes. (A) Phase-contrast photography of a tissue-culture specimen of the ductal carcinoma of the Luminal A subtype.(B) Phasecontrast photography of a tissue-culture specimen of the ductal carcinoma of the HER2+ subtype.(C) Phase-contrast photography of a tissue-culture specimen of the ductal carcinoma of the Luminal B subtype. (D) Phase-contrast photography of a tissue-culture specimen of the ductal carcinoma of the Triple Negative subtype.
R E L A T IV E E X P R E S S IO N
DESIGN
98,3%
20
M C F 10 A D T 11 ***
15
10 ***
5
***
0 5 T
(A) (B)
P
B
Markers
Population (%)
CD 44
99.8 %
CD 140 a
59.4 %
CD 36
1.2 %
CD 24
2%
CD 326
19%
Fig 2: Characterization of the primary cells by flow cytometer. (A) Characterization of primary tumor (from patient with triple negative subtype) cells using different population markers such as for stem cell population cd24- and cd 44+, for epithelial cells cd 326 , for adipose-derived stem cell (ADSC) cd 36 and for cancer associated fibroblast (CAF) cd 140a by flow cytometer. (B) The table depicted the population of the expression markers in primary cells culture in percentage as compared to unstained population.
3
E
T
A
-C
A
T F
N
1
1 Z
E
B S
IR
T
2
1 Z
E
B B
M
I1
V
IM P
T
E
N S
A
L
L
4 C
D
H
1
Fig 4: Overexpression of metastatic genes in primary cell line. qRTPCR analysis revealed gene expression related to epithelial to mesenchymal transition (EMT) and proliferation are highly expressed in Primary tumour cell line with p-3.Data represent the mean ± s.d: ***p
