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Sven Hillinger
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P2.01 - Advanced NSCLC (Not CME Accredited Session) (ID 950)
- Event: WCLC 2018
- Type: Poster Viewing in the Exhibit Hall
- Track:
- Presentations: 1
- Moderators:
- Coordinates: 9/25/2018, 16:45 - 18:00, Exhibit Hall
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P2.01-51 - Study of CD26/DPP4 Expression in a Large Series of Non-Small Cell Lung Cancer Patients (ID 13794)
16:45 - 18:00 | Presenting Author(s): Sven Hillinger
- Abstract
Background
Lung cancer is a leading cause of cancer-related death worldwide and the prognosis remains poor CD26/dipeptidyl peptidase 4 (DPP4) is a transmembrane exopeptidase expressed on various hematopoietic but also somatic cells including malignancies of breast, colon, and mesothelioma. We previously found that the activity of CD26/DPP4 in human lung adenocarcinoma is four times higher than in normal lung tissue and the inhibition of CD26/DPP4 decreased the growth of lung tumors in experimental models. These data prompted us to analyze the expression of CD26/DPP4 in samples from lung cancer patients to unravel the role of CD26/DPP4 as a potential therapeutic target to reduce lung cancer burden.
a9ded1e5ce5d75814730bb4caaf49419 Method
To identify CD26/DPP4, we performed immunohistochemistry (IHC) on multi-organ tissue micro array (TMA) using four different antibodies. We finally selected the antibody produced by Cell Signaling Technology. For the analysis of CD26/DPP4 by IHC, TMAs constructed from the samples of non-small cell lung cancer patients were used. The cohort consisted of 1110 patients (Adenocarcinoma (AC): 567; Squamous carcinoma (SC): 443). Three pathologists independently scored the staining intensity from zero to three in a blinded manner. Lung AC cell lines from tissue (H2347, H522, A549, and Hop62) and pleural malignant effusion (H1437, H460, Mai9, and Gon8) were employed to assess the expression of CD26/DPP4. ELISA was used to quantify CD26/DPP4 from cell lines.
4c3880bb027f159e801041b1021e88e8 Result
Overall survival of the patients showed no difference between AC and SC groups. IHC scores revealed AC expressing significantly higher CD26/DPP4 levels vs. normal lung or SC (p=0.035, p<0.0001, respectively). In consistency with our previous findings, early stage cancer (IA) expresses significantly higher levels of CD26 than other stages IIB (p=0.0012), IIIA (p=0.0019), and IV (p=0.02) among AC samples. From our in vitro studies, we found that early stage derived cell line (H2347: stage I) expresses significantly more CD26/DPP4 compared to others (H522: stage II, A549 and HOP62 stage IV). In contrast, metastatic AC cell lines secrete significantly more CD26/DPP4 in culture medium compared to tissue derived cell lines, while the cellular level of CD26/DPP4 was higher in tissue derived cell lines.
8eea62084ca7e541d918e823422bd82e Conclusion
AC expresses significantly more CD26/DPP4 than SC. Furthermore, the expression of CD26/DPP4 was higher at early stages of AC compared to advanced stages. Human cell line data suggest that metastatic AC secretes CD26/DPP4 more actively than primary cancer. We therefore deem CD26/DPP4 to be a target for inhibition of human AC.
6f8b794f3246b0c1e1780bb4d4d5dc53
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P2.03 - Biology (Not CME Accredited Session) (ID 952)
- Event: WCLC 2018
- Type: Poster Viewing in the Exhibit Hall
- Track:
- Presentations: 1
- Moderators:
- Coordinates: 9/25/2018, 16:45 - 18:00, Exhibit Hall
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P2.03-13 - SWATH MS Analysis of Serine Hydrolase Activity in Human Lung Adenocarcinoma for Biomarker Discovery (ID 13300)
16:45 - 18:00 | Presenting Author(s): Sven Hillinger
- Abstract
Background
Serine hydrolases (SHs), one of the largest enzyme families, have previously been shown to be implicated in the development of lung cancers. Activity-based protein profiling (ABPP) is a proteomic method that uses active site-directed chemical probes to selectively target the active form of the subsets of the enzymes in question, and then by a combination of a streptavidin-biotin enrichment step and mass spectrometry quantifies the catalytically active amount of the enzyme molecule. In this project, we monitored both forms of serine hydrolase, the “catalytically active” and “inactive”, in a distinct patient cohort of lung adenocarcinoma biopsies. For this purpose, we combined the activity-based proteomics for serine hydrolase and SWATH mass spectrometry (MS), which ensures highly reproducible protein quantification in a large panel of clinical samples.
a9ded1e5ce5d75814730bb4caaf49419 Method
Twenty four lung biopsies of long- and short surviving patients with stage IIIA adenocarcinomas and their normal tissue counterparts were available as OCT-embedded tissue. The common OCT sample clean-up by organic solvents is not compatible with the ABPP protocol since organic solvents can inactivate the molecular structure of the enzymes. The additional challenge of the ABPP protocol includes a streptavidin-biotin enrichment step of the active enzymes, which also leads to sample contamination by streptavidin peptides, and negatively influences MS spectra analysis and, consequently, biomarker discovery.
Therefore, we proceeded with optimizations of sample preparation in ABPP experiments and developed an OCT clean-up protocol compatible with “enzyme-substrate binding” of activity-based chemical probes and the targeted portion of the active enzyme. To prevent digestion on streptavidin beads and MS spectra contamination, we used the reproducible and accurate SWATH MS to indirectly measure the active enzyme form in the biopsy-extract solution.
4c3880bb027f159e801041b1021e88e8 Result
We identified over 4000 lung tissue proteins from a few milligrams of OCT-embedded biopsies, and confirmed good data quality for further SH enzyme quantification. In addition to the analysis of total proteome, 278 distinct proteins were identified on the parallel streptavidin bead samples, with chemical probes being used to deplete the active enzyme from the biopsy-extract.
Each SWATH experiment reported the percentage of “active” enzyme form through the indirectly measured ratio between the “inactive SHs” (sample depleted for active SHs) and the “total” SHs (non-depleted sample). We detected around 80 enzymes with the “active” form comparably measured in three independent experiments, which amount generally accounted for between 5–55% of the total enzyme concentration.
8eea62084ca7e541d918e823422bd82e Conclusion
Combination of ABPP and SWATH MS enables highly reproducible protein quantification in biomarker discovery.
6f8b794f3246b0c1e1780bb4d4d5dc53
