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R. Morales-Flores
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MINI 02 - Immunotherapy (ID 92)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:P. Forde, S.J. Antonia
- Coordinates: 9/07/2015, 10:45 - 12:15, Four Seasons Ballroom F3+F4
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MINI02.03 - Overexpression of CD47, Decrease of Apoptosis and Phagocytosis of Neutrophils in Advanced Non-Small Cell Lung Cancer Patients (ID 2265)
10:55 - 11:00 | Author(s): R. Morales-Flores
- Abstract
- Presentation
Background:
Lung cancer is the leading cause of cancer death worldwide and most of the patients are diagnosed with advanced disease. Inflammatory components play a key role in tumor progression and survival. Neutrophils are increased in blood of patients with lung cancer and they are associated with poor clinical outcomes. CD47 is a protein which control cell communication, apoptosis, adhesion and proliferation and it has been found increased in cancer and related with phagocytosis evasion mechanism.The aim of this study was to evaluate CD47 expression levels on peripheral neutrophils, also assess the phenotype, apoptosis, activation state, reactive oxygen species production of neutrophils between patients with Non-Small Cell Lung Cancer (NSCLC) and healthy subjects.
Methods:
Fifty NSCLC patients (stage IIIB and IV) naive to treatment and 25 healthy subjects were analized for: CD47 peripheral blood expression, neutrophils phenotype and activation state, evaluation of apoptosis and phagocytosis by flow cytometry. Reactive oxygen species (ROS) production by circulating neutrophils upon stimulation with PMA was assessed by flow cytometry. For the phagocytosis assay, PMNC were labeled with CMFDA and were cultured in RPMI for 24 hrs. To obtain apoptotic target cells, 24h PMNC were labeled with Annexin-V. For the evaluation of phagocytosis, the neutrophils from NSCLC patients were co-cultured with THP-1 cells. The percentage of phagocytosis was assessed by flow cytometry.
Results:
Our results showed a lower percentage of total CD47 in peripheral blood cells in NSCLC patients compared to controls [P=0.042]. Mean Fluorescence Intensity (MFI) of CD47 was higher in patients [P<0.001]. The percentage of CD66b+ cells characterized as neutrophils was higher in patients as well as their MFI of CD47 [P< 0.001]. MFI of CD66b was higher in patients [P< 0.0178]. This would be related with a more activated state. We found that a higher disease stage (IIIB vs. IV) associated with a higher MFI of CD47 [P=0.020]. Plasma pro-inflammatory cytokines, was increased in patients compare to controls IL-6 (P<0.002), IL-8 (P<0.001), IL-12p70 (P<0.008), TNF (0.010) and IFN-g (P<0.001). MFI of CD47 >1635.5 was associated with a higher median Overall survival (P= 0.007). We found a decrease of AnnexinV+/7AAD+ in neutrophils of patients [P=0.0317]. Caspases 3 and 7 were found decreased in neutrophils of patients [P= 0.049]. Oxygen species (ROS) production of neutrophils upon PMA stimulation was increased in patients [P=0.029], suggesting it might play a role in immune effector function. Phagocytosis of apoptotic neutrophils by differentiated THP-1 cells was decrease in cancer patients cells (P=0.0445). Mean fluorescence Intensity of CD47 was increased after 24 hrs in patients [P=0.0408]. This result suggests that neutrophils from patients avoid being engulfed and this may be associated with overexpression of CD47.
Conclusion:
Taken together, these findings suggest that these are altered mechanisms by which neutrophils evade anti-tumor immune response and their increased expression of CD47 is a potential therapeutic target for NSCLC.
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ORAL 41 - Immune Biology, Microenvironment and Novel Targets (ID 159)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:S.K. Padda, R. Nemenoff
- Coordinates: 9/09/2015, 18:30 - 20:00, Four Seasons Ballroom F1+F2
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ORAL41.03 - Myeloid Derived Supressor Cells and Their Clinical Relevance in Non-Small Cell Lung Cancer (ID 2946)
18:52 - 19:03 | Author(s): R. Morales-Flores
- Abstract
- Presentation
Background:
Lung cancer is the leading cause of cancer death worldwide and most of the patients are diagnosed with advanced disease. Myeloid-derived suppressor cells (MDSCs) are major contributors to tumor immune tolerance and targeting them can improve antitumor activity.
Methods:
We investigated the CD33[+]CD11b[+]CD66b[+]CD15[+]VEGFR-1[hi] MDSCs frequency in 120 non-small cell lung cancer (NSCLC) treatment-naive patients, with stage IIIB and IV of disease. We analyzed 1-year survival and its prognostic significance in relation to outcome analysis as well as its potential immunosuppression over cytotoxic CD8[+] T lymphocytes. The immunophenotyping of cell population was performed with multiparametric technique by flow cytometry.
Results:
We found a significant increase compared with controls in: Percentage of CD33[+]CD14[-]CD11b[+]CD66b[+]CD15[+ ](10.4 ± 5.01% vs. 3.1 ± 1.7% P<0.0001); Mean Fluorescence intensity (MFI) of VEGFR on MDSCs (P<0.001); plasma levels of arginase-1 (P<0.01); arginase-1 enzymatic activity (P<0.05); plasma levels of TGF-β (P<0.0001), IL-10 (P=0.0027) and IL-6 (P<0.0001). On the other hand, we found a significant decrease compared with controls in: Plasma levels of IFN-γ (P<0.0001); CD8[+] T cells (P<0.001); CD8[+]T cells IFN-γ production co-cultured with MDSCs (N=10; P<0.001) and MFI of CD3ζ chain (N=10; P<0.05). The percentage of MDSCs was negatively related to the percentage of CD8[+] T cells in the peripheral blood (N=155, R=-0.3045, P=0.0167). Finally, we found an inverse correlation between circulating MDSCs percentages and overall survival (P=0.09).
Conclusion:
Our study provides evidence of an increased pool of CD33+CD11b+CD66b+CD15+VEGFR-1hi MDSCs in the peripheral blood of NSCLC patients. The suppressive effect, of MDSCs on CD8+ T lymphocytes, suggests an important role in mediating immunosuppression in NSCLC that should enable the development of a novel biomarker and thus might represent a potential target for therapeutic intervention.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-078 - Functional Characterization of NK Cells in Non-Small Cell Lung Cancer (ID 2975)
09:30 - 09:30 | Author(s): R. Morales-Flores
- Abstract
Background:
Lung cancer is the leading cause of cancer death worldwide and most of the patients are diagnosed with advanced disease. Lung cancer is the leading cause of cancer death worldwide. Natural killer (NK) cells are important effector cells in control of infected, malignant, and tumor cells. The aim of this study was to investigate the activation state and cytotoxic potential of NK peripheral cells in patients with Non-Small Cell Lung Cancer (NSCLC).
Methods:
We investigated the relationship between NK cells apoptosis and Fas expression. NK cell apoptosis, Fas and Fas-L, NKG2D, CD69, KIR, CD244, CD122 and CD161 receptors were evaluated with multiparametric flow cytometry. We further evaluated the cytotoxic activity of NK cells and IFN-gamma expression. For this purpose, we simultaneously analyzed the loss of intracellular perforin and the surface expression of CD107a/b as well as the intracellular IFN-gamma expression with multiparametric flow cytometry.
Results:
Our results showed that Fas-positive NK cells in lung cancer patients were higher than healthy controls (P<0.001). These results also showed that up-regulation of Fas expression is related to increased apoptosis of circulating NK cells. Regarding the cytotoxic capacity, our results showed that upon PMA stimulation, the expression of surface CD107a/b and loss of intracellular perforin of NK cells from patients with NSCLC were not correlated indicating an impaired functional cytotoxic activity. Interestingly, we also found that, IFN-gamma (P<0.005) and NKG2D expression were also impaired significantly (P<0.001).
Conclusion:
The results from this study suggest a possible NK cells anergy state. Our description will help to provide a mechanistic insight into tumor immune escape via negative regulation of NK cell innate function; however, the underlying mechanisms remained to be addressed.
