Abstract 3280
Background
Colorectal cancer (CRC) is one of the leading cancer with the 55% survival rate. The analysis of tumor microenviroment in fresh surgical samples by flow cytometry focuses on the presence of immune cells. However, no direct access to fresh tumor sample is always possible. Moreover, transport of interesting fresh tumor tissue specimens to distant labs is sometimes neccesary. Therefore, we analyzed both fresh and frozen cells isolated from tumor tissue, stored in hibernation buffer for 1 to 3 days and compared the percentage of EpCAM+, CD45+ and CD3+ cells. We analyzed 26 surgical samples of primary CRC and metastatic samples.
Methods
Surgical samples were obtained from patients with CRC and processed for an isolation of a single cell suspension (SCS) prepared using standard protocol with collagenase. After washing and blocking with mouse serum, cells were stained with fluorescently labeled antibodies (EpCAM-PE, CD45-FITC, CD3-APC). The cells were phenotyped using the FACS ARIAII instrument. Samples were obtained from freshly prepared (n = 19) and frozen cells, stored in DMSO supplemented medium (n = 8). 1 sample was phenotyped both from freshly prepared and frozen cells.
Results
From freshly prepared SCS, the average amount of EpCAM+ and CD45+ cells was 78.7% and 14.7%, respectively. In 12 out of 19 cases we also used anti CD3 antibodies. 2% of infiltrated cells were found to be positive for CD3. When using frozen SCS, we detected a higher positivity for EpCAM (92.85%), CD45 (62%) and CD3 (6%) compared to freshly prepared SCS.
Conclusions
We phenotyped human CRC samples for EpCAM, CD45 and some samples for CD3 marker. We found that for the FACS analysis, the stability of tumor tissue samples seems to be acceptable for isolation of SCS from CRC if stored in hibernation buffer, at 4 °C for 1 to 3 days. When isolated SCS from frozen stocks were used, we detected higher percentages of positive cells for EpCAM, CD45, CD3 markers. Therefore, we do not recommend to compare freshly isolated cells with previously frozen cells in FACS experiments.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
The authors.
Funding
grants APVV (no.16–0066), VEGA (no. 1/0380/18).
Disclosure
All authors have declared no conflicts of interest.
Resources from the same session
1707 - Clinical utility of precision immunoprofiling and monitoring of the tumor microenvironment using flow cytometry and CyTOF in patients with advanced NSCLC treated with atezolizumab: results from a phase II study for biomarker analysis (EPOC1702)
Presenter: Keisuke Kirita
Session: Poster Display session 3
Resources:
Abstract
3594 - Tumor mutation burden (TMB), PD-L1, IFN-γ signaling identify subgroups of patients (pts) who benefit from durvalumab (D, anti-PDL1) or D and tremelimumab (T, anti-CTLA4) treatment in urothelial bladder cancer (UC)
Presenter: Christophe Massard
Session: Poster Display session 3
Resources:
Abstract
744 - The decrease of TMB, TNB and HLA expression are the Mechanism of Drug Resistance of NSCLC to immunosuppressive PD-1/PD-l1.
Presenter: Sheng Yu
Session: Poster Display session 3
Resources:
Abstract
2350 - Eosinophilia during treatment of immune checkpoint inhibitors (ICIs) predicts succeeding onset of immune-related adverse events (irAEs)
Presenter: Rika Kizawa
Session: Poster Display session 3
Resources:
Abstract
5930 - A transcriptomic immunologic signature predicts favorable outcome in neoadjuvant chemotherapy treated triple negative breast tumors.
Presenter: Javier Pérez-peña
Session: Poster Display session 3
Resources:
Abstract
6127 - Alterations of TMB and TCR repertoires during Chemotherapy in East Asian lung cancer patients without TKI-related driver gene mutations
Presenter: Lele Song
Session: Poster Display session 3
Resources:
Abstract
1310 - Association of SCFA in gut microbiome and clinical response in solid cancer patients treated with andi-PD-1 antibody.
Presenter: Motoo Nomura
Session: Poster Display session 3
Resources:
Abstract
2286 - Extracellular matrix and tissue derived metabolites in a liquid biopsy identifies endotypes of metastatic melanoma patients with differential response to immune checkpoint inhibitor treatment
Presenter: Nicholas Willumsen
Session: Poster Display session 3
Resources:
Abstract
4107 - Pathologic scoring of pre-treatment H&E biopsies predicts overall survival in patients with metastatic clear cell renal cell carcinoma receiving nivolumab monotherapy
Presenter: Julie Stein
Session: Poster Display session 3
Resources:
Abstract
1291 - PD-L1 expression in uncommon EGFR-mutant non-small cell lung cancer and its response to immunotherapy
Presenter: Yun Fan
Session: Poster Display session 3
Resources:
Abstract