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
5368 - Durvalumab and Paclitaxel Combination for treatment of metastatic triple negative breast cancer is safe with very promising efficacy
Presenter: Hazem Ghebeh
Session: Poster Display session 3
Resources:
Abstract
1520 - A prospective cohort study on the pharmacokinetics of nivolumab in metastatic non-small cell lung cancer, melanoma, and renal cell cancer patients
Presenter: Daan Hurkmans
Session: Poster Display session 3
Resources:
Abstract
1603 - Safety and clinical activity of subcutaneously (SC) administered anti-PD-1 antibody PF-06801591 in phase I dose-expansion cohorts of locally advanced or metastatic non-small-cell lung cancer (NSCLC) and urothelial carcinoma (UC)
Presenter: Byoung Cho
Session: Poster Display session 3
Resources:
Abstract
3922 - Development of the Functional Assessment of Cancer Therapy-Immune Checkpoint Modulator (FACT-ICM): A scale to measure quality of life in cancer patients treated with ICMs
Presenter: Aaron Hansen
Session: Poster Display session 3
Resources:
Abstract
2408 - Immune checkpoint inhibitors (ICIs) as “chemotherapy (Ctx) sensitization” strategy in advanced solid tumors
Presenter: Francisco Javier Ros Montana
Session: Poster Display session 3
Resources:
Abstract
3612 - Validation of progression-free survival (PFS) as surrogate endpoint in randomised trials of immune checkpoint inhibitors in advanced solid cancers
Presenter: Peey Sei Kok
Session: Poster Display session 3
Resources:
Abstract
3827 - Pharmacokinetic (PK) analysis of weight-based and fixed dose cemiplimab in patients (pts) with advanced malignancies
Presenter: Michael Migden
Session: Poster Display session 3
Resources:
Abstract
2120 - A burst of highly differentiated CD4 TL identifies a subset of fast progressors, and correlates with hyperprogressive disease in NSCLC patients treated with ICI
Presenter: Hugo Arasanz
Session: Poster Display session 3
Resources:
Abstract
4254 - Nivolumab treatment in advanced non-small cell lung cancer (aNSCLC): a French nationwide retrospective cohort (UNIVOC Study)
Presenter: Christos Chouaid
Session: Poster Display session 3
Resources:
Abstract
1084 - Dissociated responses in patients with metastatic solid tumors treated with immunotherapy
Presenter: Pauline Vaflard
Session: Poster Display session 3
Resources:
Abstract