Asian Journal of Bio-Medical Research
Original Article
Ex-vivo Activation of Dendritic Cells Isolated from Breast Cancer Patients Motawa E El-Houseini1, Mona S Abdel-Lateif1, Eman Z Kandeel2, Ahmed H El- Habashy4, Aml R El-Shehaby5, Waleed S Mohamed3 Unit of Biochemistry and Molecular Biology, Department of Cancer Biology, National Cancer Institute, Cairo University, 2Unit of Immunology, Department of Clinical Pathology, National Cancer Institute, Cairo University, 3Unit of Virology and Immunology, Department of Cancer Biology, National Cancer Institute, Cairo University, 4Unit of Cytology, Department of Pathology, National Cancer Institute, Cairo University, 5 Department of Medical Biochemistry, Faculty of Medicine, Cairo University 1
Address for Correspondence: Waleed Seif El-Din Mohamed, Tel: +202-25203553, Fax: +202 -23644720, E-mail:
[email protected]
Submission : 11-06-2015 Revision : 26-06-2015 Publication : 10-07-2015 URN: pmi:jr:0019ajbr.v1i1.7445 How to Cite this Article: EL-DIN MOHAMED, Waleed Seif. Ex-vivo activation of Dendritic Cells isolated from Breast cancer Patients. Asian Journal of Bio-Medical Research, [S.l.], v. 1, n. 1, june. 2015. Available at: .
ABSTRACT Background: One of the new approaches for breast cancer treatment is dendritic cellsbased vaccine which requires generation of large quantities of active dendritic cells (DCs). Objective: We aimed to optimize conditions in vitro to generate DCs capable of inducing an effective anti-cancer cell-mediated immune response. Methods: DCs were generated from monocytes culture isolated from peripheral blood and axillary lymph nodes of 35 breast cancer cases and 35 matched age and sex controls. Flow cytometry was done for analysis of DCs phenotypes. A mixed lymphocyte dendritic cell reaction (MLDCR) was performed, and INF-γ and IL12 were measured by ELISA. Results: DCs cultured with GM-CSF, IL4, TNF α and IFN α were optimally differentiated into activated DCs as demonstrated by increased cell density and viability (P-value 0.05
I
20
II
28
III
7
IV
‑
Tumortype
>0.05
Ductal
51
Lobular
4
LN status
>0.05
Positive
35
Negative
20
Distant metastasis
>0.05
Yes
‑
No
55
*p‑value is >0.05 not significant
AJBR • Vol 1 • Issue 1 • 2015
El-Houseini, et al.: Dendritic cells activation of breast cancer patients
Immunophenotyping of activated D.Cs Patients were classified into 5 groups according to the method and site of cells isolation (either from peripheral blood or from axillary lymph nodes). There was no significant deference between the expressions of CD83 & CD86 in MDDCs isolated either from P.B (P value= 0.057 & 0.072) or axillary L.N (P value 0.05) for IL12 level in the different groups. Also, there was a highly significant difference of IL12 level
a
b Figure 1: Phenotype characterization of activated dendritic cells drawn from peripheral blood sample of breast cancer patients. (A) Histograms of the PE/ FITC isotype control of the sample to set the suitable regions. The left one quadrant gate of the monoclonal antibodies, the next single histograms of isotype PE and FITC respectively. (B) Histograms of the stained sample CD83 PE and CD86 FITC The left one quadrant gate to show the co expression percentage illustrated results using the geometric mean of each monoclonal antibodies. Data were analyzed using WinList multiparameter analysis software (Verity Software House, Topsham, ME)
AJBR • Vol 1 • Issue 1 • 2015
among the benign breast lesions group after activation and the other groups (P value< 0.001). Moreover, there was a significant increase in IL12 level in the culture media for the 4 groups after activation with TNFα and INFα (P- value < 0.001), as shown in Table 3.
Detection of IFNγ level Table 3 showed the IFNγ level (pg/ml) secreted by D.C (IFNγ D) after activation, and also by T-cell (IFNγ T) before and after MLDCR. There was significant (P value < 0.001) increase in IFNγ mean level in the culture media of D.Cs before MLDCR for G4 as compared to other groups. The IFNγ level after MLDCR in the culture media of G4 was significally higher than that of G3(P value < 0.038), and G1(P value 0.014). In addition, there was significant increase in IFNγ level in the culture media for the four groups after activation with D.Cs in MLDCR (P value < 0.001).
Discussion Previous studies identified dendritic cells as the key player involved in controlling the immune response. So far, DC-based vaccine approaches have been most extensively pursued because DCs are considered to be the most potent professional antigen presenting cells (APCs).2 In the current study, DCs were optimally differentiated into activated DCs as demonstrated by immunophenotypic profile, MLDCR and IL12cytokine secretion when they were cultured with cytokine conditioned medium (CCM), containing GM-CSF, IL4, TNF α and IFN α. This finding was in accordance with Satthaporn et al.4 who stated that IFN-α was critical for optimal phenotypic maturity of monocyte-derived DC from patients with breast cancer. Also, Hou et al.10 used DCs co-cultured with GM-CSF, IL4 and TNF-α as an inflammatory cytokine alone, and they found that the DCs phenotype of healthy donors were CD83 (60 ± 16%) and CD86 (93 ± 5%) and of cancer patients were CD83 (43 ±18%) and CD86 (93 ± 5%). However, in the current study, phenotype of DCs co-cultured with TNF α and IFN α was in healthy donors; CD83 (87.0 ± 9.6%) and CD86 (74.2 ± 12.9%) and in breast cancer patients; CD83 (75.7 ± 10.9%) and CD86 (85.5 ±11.5%). Thus, IFN α increased maturity of DCs by increasing the expression of CD83, but in CD86 there was a little increase as this molecule was also expressed at high (maximal) levels by 3 CCM (GM-CSF, IL4, TNF α) treated-DCs. Our results were comparable to those findings reported by Satthaporn et al.4 Meanwhile, Svane et al.11 reported that IFN-α when used as a single maturation reagent for monocyte-derived DC generated in GM-CSF + IL-4 leads to the production of DC with a semimature phenotype (low CD83) and an increased capacity for allogeneic T-cell stimulation. However, this maturation regimen did not lead to effective secretion of IL12. So, applying of TNF-α and IFN-α as a maturation reagents for MDDCs were effective and possible in clinical use. Our results showed that CD86 (Co-stimulatory molecules) and CD83 (marker of terminally mature DCs) expressions in MDDCs isolated from peripheral blood and L.Ns of control and malignant cases were high. This finding was in agreement with that reported by Satthaporn et al.4 for CD86 expression, while CD83 was expressed by only a minority of DCs prepared using all four cytokines 4 CCM (GM-CSF, IL4, TNF-α and IFN-α). This difference might be due to the addition of IFN-α at the initiation of DC development along with GM-CSF, IL-4, and TNF-α during DC maturation process (5–7 day culture period). However, in our study IFN-α was added later (for
5
El-Houseini, et al.: Dendritic cells activation of breast cancer patients
Table 2: D.Cs in relation to CD83 and CD86 expression after activation with TNFα and INFα Parameter
Mean±SD
P value
G1
G2
G3
G4
G5
CD83
87.0±9.6A
75.7±10.9AB
40.5±12.4C
39.5±13.6C
63.9±17.4B
