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
5760 - Landscape of PD-L1 expression status in Chinese solid tumor patients.
Presenter: Yi Zhong
Session: Poster Display session 3
Resources:
Abstract
3733 - Anti-cancer and immunomodulatory effects of cobimetinib in triple negative breast cancer
Presenter: Chun-Yu Liu
Session: Poster Display session 3
Resources:
Abstract
4426 - Differential expression of immunoregulatory molecules and highly-associated cancer genes may provide novel insights into strategic trial design for therapeutics
Presenter: Jacob Adashek
Session: Poster Display session 3
Resources:
Abstract
2752 - Insights into the Tumor Immune Microenvironment using Tissue Phenomics to Drive Cancer Immunotherapy
Presenter: Martin Groher
Session: Poster Display session 3
Resources:
Abstract
5713 - Immune competent somatic mosaic model of colorectal cancer
Presenter: Stefania Napolitano
Session: Poster Display session 3
Resources:
Abstract
1898 - Genomic correlates of response to anti-PDL1 Atezolizumab in non-small-cell lung cancer OAK and POPLAR trials
Presenter: Hari Singhal
Session: Poster Display session 3
Resources:
Abstract
3246 - Erdafitinib (erda) versus available therapies in advanced urothelial cancer: A matching adjusted indirect comparison
Presenter: Yohann Loriot
Session: Poster Display session 3
Resources:
Abstract
3311 - High level of activity of Nivolumab anti-PD-1 immunotherapy and favorable outcome in metastatic/refractory MSI-H non-colorectal cancer: Results of the MSI cohort from the French AcSé program
Presenter: Christophe Tournigand
Session: Poster Display session 3
Resources:
Abstract
2314 - TP53 and ATM Co-mutation Predicts Response to Immune Checkpoint Inhibitors in Non-Small Cell Lung Cancer
Presenter: Yu Chen
Session: Poster Display session 3
Resources:
Abstract
4692 - Immune cell biomarkers on neo-adjuvant chemo-immunotherapy treatment for resectable stage IIIA NSCLC patients
Presenter: Raquel Laza-Briviesca
Session: Poster Display session 3
Resources:
Abstract