Abstract P1-05-09: Analysis of immunophenotyping of patient-derived primary breast cancer cells according to the medium composition and culture method
Authors: RYU Seungyeon , Kim Hoe Suk , Yoon So-Hyun , Lee Sangeun , Song Junhyuk , Baek Moonjou , Lee Han-Byoel , Jon Sangyong , Han Wonshik 
Published: March 2022

Abstract
Purpose: Immunophenotyping is a useful tool for diagnostic and therapeutic assessment of heterogenous breast cancer cells that depend on external factors and growth conditions. The objective of this study was to characterize the phenotype of patient-derived breast cancer cells (PDBCCs) grown in different culture medium and under different conditions including 2D monolayer and non-adherent 3D spheroid culture by using flow cytometry. Methods: PDBCCs and normal breast cells were harvested from fresh surgical specimens (N=11) of breast tumor and normal tissues. PDBCCs were grown in various culture media under monolayer, spheroid and organoid conditions. Flow cytometry, qRT-PCR, ALDEFLUOR assay, β-galactosidase staining and RNA-seq were performed. Results: PDBCC growth rates were different for each patient case. As compared with full media, A83-01 removal increased EpCAM+CD24+ cells (1.8-fold, p&lt;0.029) and decreased EpCAM-CD44high/+CD24- (0.45-fold, p=0.029). EGF removal increased EpCAM+CD49f− cells (14.1-fold, p=0.029). Removal of both EGF and A83-01 induced senescent cells with high β-galactosidase activity. EpCAM-CD44high/+CD24- cells, ALDHhigh cells, and EpCAMlow/-CD49f+ cells were enriched by 3D spheroids relative to 2D monolayer. 562 genes with 2-fold change and, p-value&lt;0.05 were differentially expressed in 3D spheroids compared to 2D monolayers. Based on public database, many genes associated with poor prognosis in 3D spheroids were identified (Table 1). Table 1. Clinically poor prognosis-associated DEGs were selected from tumor spheroids based on recurrence-free survival analysis using Kaplan-Meier Plotter on database that includes estrogen receptor positive patients. B, coefficient of regression; HR, hazard ratio; CI, confidence interval; ER, estrogen receptor, HER2, human epidermal receptor 2; RFS, recurrent free survival; FC, fold change. Conclusion: PDBCC phenotypes can be modulated by external factors and culture methods. Cancer stem-like properties of PDBCCs can be enriched by 3D spheroids. Future studies to explore the specific molecular mechanisms underlying these observations may provide new molecular targets for the development of therapies for heterogenous breast cancer
Clinically poor prognosis-associated DEGs based on recurrence-free survival analysisGenes symbolLog2(FC) P&lt;0.05Prognosis95% CI for Exp(B)HRlowerupperP-valueTop 30 of DEGsUpregulated genesMMP110.90Poor1.611.381.877.5e-10BMP68.85Poor1.21.021.040.024MYCN7.20Poor1.231.041.460.015COL22A17.12Poor1.571.172.110.0026CYP1A1(CP11)7.01Poor1.271.051.530.012PGBD56.78Poor1.221.051.420.011S100P6.70Poor1.491.281.733.3e-07CALML56.24Poor1.271.0810480.0034PPP2R2C6.19Poor1.491.131.970.005PMEPA16.07Poor1.181.011.40.043Downregulated geneFOSB-6.36Poor0.630.520.762e-06LINC00844-6.01Poor0.670.480.930.017DLK2-5.46Poor0.820.710.960.011IL33-5.31Poor0.650.550.773.1e-7SCN2B-5.09Poor0.80.670.950.0099CLDN19-4.80Poor0.660.490.890.0065FOS-4.67Poor0.670.570.798.5e-7EGR1-4.63Poor0.680.580.822.3e-0.5JAM2-4.62Poor0.80.680.950.0092MYH11-4.38Poor0.760.660.890.00045ALPL-4.35Poor0.820.710.960.015
Citation Format: Seungyeon RYU, Hoe Suk Kim, So-Hyun Yoon, Sangeun Lee, Junhyuk Song, Moonjou Baek, Han-Byoel Lee, Sangyong Jon, Wonshik Han. Analysis of immunophenotyping of patient-derived primary breast cancer cells according to the medium composition and culture method [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-05-09.