Abstract P5-06-03: Generation and characterization of a novel invasive lobular breast carcinoma cell line WCRC-25
Authors: Kota K , Bossart E , Basudan A , Minteer T , Meier C , Brown D , Gurda GT , Miller L , Dabbs DJ , Lee A , Puhalla S , Jankowitz R , McAuliffe P , Lucas P , Oesterreich S 
Published: February 2018

Abstract
Background: Invasive Lobular Breast Carcinoma (ILC) is the second most common histologic subtype of breast cancer, comprising 10-15% of all cases. ILC is clinically and molecularly distinguished from the major subtype–Invasive Ductal Carcinoma (IDC)–by loss of E-cadherin (CDH1). Studies on ILC remain sparse, in part due to limited cell culture models. The Women's Cancer Research Center (WCRC) has therefore set up a combined effort of breast surgical oncologists, medical oncologists, pathologists, and cancer biologists to collect fresh tissues and establish additional ILC cell lines.
Methods: Tumor cells were grown using irradiated fibroblast conditioned media (CM) in hypoxic (5% O2) conditions. Sanger sequencing and Droplet Digital PCR (ddPCR) were utilized to test for mutation in CDH1 in tumor and circulating DNA (cfDNA), using germline DNA as control. DNA copy number status in the tumor was detected using nanoString approach. Expression of E-cadherin and a series of lineage markers was elucidated using Immunoblotting (IB) and Immunofluorescence (IF). A panel of ILC (MDA-MB-134, Sum44PE, IPH-926) and IDC (MCF-7, MDA-MB-231) cell lines was included for comparison. After establishment, WCRC-25 population doubling was compared between growth in CM or Dulbecco's Modified Eagle's Medium (DMEM), and hypoxic (5% O2) or normoxic (21% O2) conditions. Growth phenotypes were characterized in 2D, Ultra Low Attachment (ULA), and soft agar.
Results: WCRC-25 was successfully established from the pleural effusion of a 77-year old patient with metastatic ILC. The patient had stage IV (T3N3M1) ER+/PR-/HER2- ILC; was treated with bilateral mastectomy, radiation therapy, and multiple lines of chemotherapy (FOLFOX due to initial misdiagnosis; 2 cycles carboplatin/paclitaxel; anastrazole, fulvestrant, pegylated liposomal doxorubicin, gemcitabine, exemastane, and eribulin (with denosumab for bone lesions); had metastatic lesions in stomach, bone, pleura, and pericardium; and ultimately passed away from progressive pleural effusions roughly 3 years after diagnosis. A novel nonsense mutation of CDH1 was observed in exon 13 (Q705*) in cell line DNA, resulting in a premature stop codon. The same mutation was confirmed in cfDNA obtained from longitudinal blood samples from the patient. Copy number analysis revealed deletion of the second CDH1 copy in tumor, and E-cadherin protein loss was confirmed by IB and IF. WCRC-25 cells expressed epithelial cell markers CK8/18 and EpCAM, and as expected did not express stromal marker αSMA. ERα expression was very low, and not sufficient for measurable hormone response. Population doubling was significantly faster in hypoxic compared to normoxic conditions, regardless of media type, but minimal in 3D.
Conclusions: WCRC-25 is a novel ILC cell line, defined by CDH1 nonsense truncating mutation and LOH, resulting in loss of E-cadherin protein expression. Cells show favorable growth characteristics in hypoxic conditions and maintain epithelial-dominated protein expression. We are currently performing RNA seq analysis of the matched primary tumor and metastatic samples from the stomach, peritoneum/falciform ligament, pleural effusion and skin, which we will compare and contrast with that of the established WCRC-25 cell line.
Citation Format: Kota K, Bossart E, Basudan A, Minteer T, Meier C, Brown D, Gurda GT, Miller L, Dabbs DJ, Lee A, Puhalla S, Jankowitz R, McAuliffe P, Lucas P, Oesterreich S. Generation and characterization of a novel invasive lobular breast carcinoma cell line WCRC-25 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-06-03.