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D.C. Chan
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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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ORAL41.02 - Novel Mechanism of Immune-Tolerance and Cancer Metastasis Due to Aberrant Expression of Natural Killer Immunoglobulin-Like Receptors (KIRs) (ID 2199)
18:30 - 20:00 | Author(s): D.C. Chan
- Abstract
- Presentation
Background:
Natural Killer (NK) cells are a major defense to eliminate cancer cells. Cancer cells and metastases may have aberrantly expressed KIRs to prevent killing by NK cells. In addition, platelets may inhibit NK killing of cancer cells. Metastatic cancer cells spread through blood vessels where they constantly interact with platelets by forming tumor microemboli and thereby protected from otherwise rapid elimination from host immune defense cells such as NK cells. Here, using an in vivo model of cancer metastasis in athymic nude mice by directly injecting cancer cells into the blood stream, we study the ability of platelets and KIRs in helping cancer cells to escape from immune surveillance and promote metastasis.
Methods:
GFP-luciferase tagged human lung adenocarcinoma cell line, H2122-GL, was further transfected with KIR2DL1 (LL454) plasmids. Stable transformants were enriched by cell sorting. In vivo experimental metastasis were performed in both athymic nude mice and in Nbeal2 knockout and wild type C57 black mice, by tail vein injections of H2122 parental and KIR expressing cells, with and without pre-infusion of human platelets. Levels of tumor cells detected in the lung and other sites were closely monitored by bioluminescence imaging at various time intervals, using an IVIS200 imager.
Results:
24 hours after tail vein injection of a million parental H2122-GL, as low as 0.4 million photons were detectable in the lungs of nude mice (n=5), while those mice injected with a same number of H2122-GL-KIR2DL1 cells, they produced 1.85 million photons in the lungs, showing a 4.6 fold increase in accumulation of KIR-expressed cancer cells than those parental cells in the lung. When the nude mice were pre-infused with iv injection of human platelets followed by tail vein injection of parental or KIR-expressed H2122 cells, enhancement up to 7 fold of lung metastases of KIR expressed H2122 were detected relative to the parental cells as early as 24 hours. 5 weeks post injection, an enhancement up to 190 fold in bioluminescence intensity was found with KIR expressed cells relative to the parental cells. Interestingly, the enhancement of lung metastases was abrogated when similar experiments were repeated in the NBeal2 knockout mice, whose platelets were nonfunctional due to defective alpha-granules and deficiency in their cargo, including von Willebrand factor, thrombospondin-1, and platelet factor 4. One hour after tail vein injection, both parental and KIR expressed H2122 cells produced same but low number of lung metastases, indicating that the defective platelets in the ko mice had failed to promote lung metastases. In the wild type mice, significantly more KIR expressed H2122 cells were detected in the lung relative to parental cells. However, as expected, these early lung metastases were rejected later by the host intact immune cells.
Conclusion:
Our studies demonstrated that metastatic cancer cells acquire immune-resistance by aberrantly express Natural Killer-Cell Immunoglobulin-like Receptors (KIRs) on their surface and that KIR-expressing cancer cells interact more strongly with platelets leading to significantly increase in NK tolerance and enhancing cancer metastases in pre-clinical models.
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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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P2.04-007 - In Vitro and in Vivo Efficacy of AZD9291 Is Enhanced by Combination with AZD4547 in EGFR Mutant Lung Cancer Cells (ID 2456)
09:30 - 17:00 | Author(s): D.C. Chan
- Abstract
Background:
EGFR-specific tyrosine kinase inhibitors (TKIs) provide marked clinical responses in patients bearing EGFR mutated lung tumors, although acquired resistance limits the durability of the response. In light of the frequent emergence of erlotinib and gefitinib-resistant EGFR T790M mutations upon tumor progression, 3[rd] generation EGFR-specific TKIs have been developed that specifically inhibit gain-of-function EGFR mutants irrespective of T790M status. Recently, we reported a distinct mechanism of acquired resistance whereby specific EGFR mutant lung cancer cell lines including H1650 and HCC4006 cells, but not PC9 cells, undergo an epithelial-mesenchymal transition (EMT) upon chronic in vitro treatment with gefitinib. As a result, the adapted cells acquire vulnerability to FGFR inhibitors by virtue of EMT-mediated FGF2 and FGFR1 induction. Herein, we have tested the hypothesis that combination of the FGFR inhibitor, AZD4547, with the 3[rd] generation EGFR TKI, AZD9291 will yield superior anti-tumor activity relative to AZD9291 alone.
Methods:
Lung cancer cell lines bearing gain-of-function EGFR mutations (HCC4006, H1650 and PC9) were submitted to in vitro clonogenic growth assays in the presence of AZD9291 and/or AZD4547 over concentration ranges for 1 to 300 nM for each drug. For in vivo measurement of the activity of these drugs, flank xenografts were established in Nu/Nu mice with the 3 lung cancer cell lines and treated by daily oral gavage (5 days on, 2 days off) with diluent, AZD9291 (5 mg/kg), AZD4547 (12.5 mg/kg) and the combination of the two drugs at these doses. Tumor size was measured with calipers and volume was calculated using the formula, Volume=3.14(short diameter)[2](long diameter)/6.
Results:
HCC4006, H1650 and PC9 cells were highly sensitive to ZD9291 in vitro with IC~50~ values of 1.6, 7.4 and 3.3 nM, respectively. In a 2 week clonogenic growth assay, AZD9291 reduced growth of all cell lines by >95%, although viable drug resistant persisters clearly remained. While none of these cell lines exhibited significant growth inhibition in response to AZD4547 alone, combination of AZD9291 and AZD4547 further reduced clonogenic growth of HCC4006 and H1650 cells, but not PC9 cells. In flank xenograft studies, AZD9291 monotherapy induced marked tumor shrinkage (H1650, ~80% at day 10; HCC4006, ~90% at day 30; PC9, 89% at day 25), although regrowth of the tumors occurred with all three xenografts. AZD4547 yielded little or no growth inhibition as a monotherapy, but significantly enhanced the degree of tumor shrinkage and delayed the time to tumor progression in H1650 and HCC4006 tumors, but not PC9 tumors.
Conclusion:
Combination of the FGFR inhibitor AZD4547 with AZD9291 affords greater growth suppression relative to AZD9291 alone in HCC4006 and H1650 cells that undergo EMT and induction of an FGF2-FGFR1 pathway. Predictably, this combination was not more effective compared to AZD9291 alone in PC9 cells that fail to undergo EMT in response to EGFR TKI treatment. The studies support the efficacy of combined AZD9291 and AZD4547 treatment of a subset of lung tumors driven by mutated EGFR, although the features of these particular lung tumors that predict this response is unknown at this time.
