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
2115 - Preclinical in vivo screening to predict responder patients depend on EGFR status
Presenter: Yejin Kim
Session: Poster Display session 3
Resources:
Abstract
3349 - Interplay between miR-17-5p and MALAT-1 Shapes The Cytokine Storm in Triple Negative Breast Cancer (TNBC) Tumor Microenvironment
Presenter: Raghda Soliman
Session: Poster Display session 3
Resources:
Abstract
4014 - Clinical verification on the relationship between lipid metabolism and the immune microenvironment of breast cancer
Presenter: Wataru Goto
Session: Poster Display session 3
Resources:
Abstract
4158 - The clinical and transcriptional signatures of human CD204 reveal an applicable marker for tumor associated macrophage in breast cancer
Presenter: Yunjie He
Session: Poster Display session 3
Resources:
Abstract
5392 - Activated effector T cells co-expressing multiple inhibitory receptors (IRs) are enriched in the tumor immune microenvironment in high grade serous ovarian cancer (HGSOC)
Presenter: Alice Bergamini
Session: Poster Display session 3
Resources:
Abstract
2617 - Oncolytic reovirus as a new anti-tumor strategy in castration resistant prostate cancer
Presenter: Yunlim Kim
Session: Poster Display session 3
Resources:
Abstract
2995 - Dysregulation of helper T lymphocytes in esophageal squamous cell carcinoma (ESCC) patients is highly associated with aberrant production of miR-21
Presenter: Ali Memarian
Session: Poster Display session 3
Resources:
Abstract
3597 - Myeloid derived suppressor cells but not regulatory T cells are associated with adaptive immunity and clinical outcomes in anal squamous cell carcinoma
Presenter: Christophe Borg
Session: Poster Display session 3
Resources:
Abstract
3430 - Evaluation of immune responses among responders (R) and non-responders (non-R) in a humanized mouse model with colorectal cancer (CRC) xenografts treated with combination immunotherapy
Presenter: Juan Marín Jiménez
Session: Poster Display session 3
Resources:
Abstract
1995 - ¬¬Advanced melanoma patients with high CD16+ macrophages have better response and survival to anti-PD-1 based immunotherapy
Presenter: Hansol Lee
Session: Poster Display session 3
Resources:
Abstract