New generation breast cancer cell lines developed from patient-derived xenografts

Breast cancer comprises a complex and diverse group of neoplasms that are stratified into various subtypes to guide treatment [1]. The three-marker diagnostic system of estrogen receptor alpha (ER), progesterone receptor (PR), and HER2 remains the cornerstone of clinical decision-making with prognostic and predictive value. In addition, the WHO classifies invasive carcinomas into a dozen more common histological subtypes, plus several rare subtypes, based on architectural and morphological features [2]. Among these, invasive ductal carcinomas (IDC) comprise the vast majority of cases (> 70%). However, it is increasingly recognized that other histological subtypes such as invasive lobular carcinomas (ILC) have unique morphologic and clinical attributes [3, 4]. Molecular classifications that emerged in the 2000s use gene expression analysis to define subtypes with different recurrence and survival patterns, for example, the PAM50 classifier [5], or a specific triple-negative breast cancer (TNBC) classifier [6]. Breast cancers can also be characterized by gene mutation status such as germline BRCA1/2 and somatic PIK3CA mutations [7‐9], which can indicate eligibility for targeted inhibitors to PARP or PIK3CA, respectively, or by progressive mutation in the gene for ER (ESR1) which indicates loss of sensitivity to some endocrine therapies [10]. Existing human breast cancer cell lines, patient-derived xenografts (PDX), patient-derived organoids (PDO), and murine models with intact immune systems capture many of these unique histopathological, molecular, and genetic subtypes. Here we describe generation of six PDX-derived breast cancer cell lines including three ER+ lines, one with ESR1 mutation, that add to the collection of research models available to investigators.

In vitro propagating cell lines are the most long-standing and heavily used models for conducting basic breast cancer research. The first breast cancer cell line was generated from a primary breast IDC by Lasfargues et al. over 60 years ago named BT-20, a basal-like TNBC [11]. In the 1970s, the first ER+ lines were developed including two from late stage pleural effusions (MCF7 [12] and T47D [13]), and two from primary IDCs (BT-474, also HER2 amplified, and BT-483 [14]). Larger collections were described over a 20-year period including 19 from the MD Anderson (MDA) [15], 18 from the Hamon Cancer Center (HCC, UT Southwestern) [16], and 12 from the University of Michigan (SUM) series [17]. There are numerous other descriptions of generating single or several cell lines within focused research articles. Collectively, these endeavors have produced approximately 51 human breast cancer cell lines available for research [18]. More recently, breast cancer cell lines have been developed from circulating tumor cells [19, 20]. A survey of PubMed shows > 40,000 original research articles utilizing MCF7 cells and > 14,000 for MDA-MB-231 cells. These well-characterized cell lines are commonly manipulated to over or under express specific genes, test specific drugs, and perform genome-wide functional genetic and drug screens. This research has led to the discovery of a multitude of (mostly) cell intrinsic behaviors. Despite their historic importance, the evolving emphasis on personalized therapies necessitates continued generation of newer models that capture tumor diversity found in the population.

We previously reported the generation of a collection of PDX with an emphasis on ER+ models [21]. These provide a continuously propagating in vivo resource for testing therapeutics in a heterogeneous context, and there are > 500 such models developed by groups worldwide [22]. PDO offer an ex vivo approach to screen different patients’ tumors for drug sensitivity and can potentially include other cell types contained in the microenvironment [23]. However, PDX and PDO are time-consuming and molecular biology approaches including shRNA knockdown or CRISPR/Cas9 gene editing remain difficult in such models. We sought to generate cell lines from PDX that harbor underrepresented phenotypes and genotypes that allow basic molecular manipulations and mechanistic studies and that can be used in parallel with in vivo studies. We have created six such cell lines that capture several important clinical features and subtypes and present here their initial characterization.

The innate intertumoral heterogeneity among breast cancers and an increasing emphasis on individualizing therapies necessitate that we continue to generate research models to meet this challenge. Advances in measuring CTCs and circulating tumor DNA further facilitate real-time monitoring of disease progression and personalized care. Our group and others have derived collections of breast cancer PDX that can be utilized for pre-clinical drug testing [22]. However, some fundamental research questions still require novel human disease models that can be more feasibly engineered. Here we describe the generation of six PDX-derived passageable breast cancer cell lines that are amenable to manipulations such as viral transduction. These complement existing models, with well-annotated oncogenic driver mutations and expression profiles, to provide depth in conducting basic and translational research on breast cancer.

One of our primary goals was to increase the number of workable ER+ breast cancer cell lines, which are relatively underrepresented compared to their clinical predominance. The primary “workhorse” ER+ breast cancer models include IDC subtypes (MCF7, T47D, ZR75-1, and the ER+HER2+/amplified BT474) with several ER+ ILC cell lines seeing increased use (MDA-MB-134, MDA-MB-330, SUM44, and BCK4) [25, 30]. Several additional cell lines are reported to have ERα mRNA transcripts [18]; however, ER protein expression has not always been documented. PR is expressed only in UCD4 and UCD65; UCD65 cells have some constitutive expression of PR in the absence of estrogen likely due to their naturally high ER level. AR is present in all three cell lines to some degree but is highest in UCD4 cells where it is stabilized with DHT. A drawback of these ER+ cell lines is their relatively long doubling times compared to long-term cultured ER+ cell lines. UCD65 has the longest doubling time, which is typical of the slower growing luminal A subtype breast cancers. A slow proliferation rate is also typical of newly developed breast cancer cell lines [17] and may more accurately reflect growth rates in ER+ patients.

It is now recognized that up to 30% of advanced breast cancer patients contain somatic genetic anomalies in the ER gene (ESR1), prospectively driven by long-term estrogen deprivation with aromatase inhibitors (AIs) [31]. Existing breast cancer cell lines do not harbor ESR1 mutations naturally (cbioportal.​org), even though some were derived from metastatic patients, prospectively because these patients were either untreated or treated prior to standard use of AIs in the 2000s. To functionally study the mutant ERs, laboratories have used exogenous expression or generated CRISPR knock-in models of the D538G and Y537S ESR1 mutations, or forced mutations by long-term endocrine treatment of ER+ breast cancer cells [31‐34]. Some PDX models contain ESR1 mutations, notably in the WHIM collection [35]. To our knowledge, a cell line has not been derived from a specimen with a natural ESR1 mutation, without the potential unintended off-target effects of CRISPR-derived clonal cell lines, and that likely contain other anomalies that co-occur with ESR1 mutation. The UCD4 cell line is homozygous for the ESR1 D538G mutation and is Fulv but minimally Tam responsive (Fig. 3). UCD65 cells are the first cell line, to our knowledge, that is luminal A and expresses high levels of ER comparatively to the other cell lines. UCD12 (formerly PT12 cells) are ER+ have been used to study mucin secretion dynamics, anti-AR therapies, and endocrine resistance in diet-induced obesity models of breast cancer [28, 36, 37]. UCD4 and UCD12 contain BRCA2 mutations that are likely somatic although the germline status of the patients is unknown. All three cell lines contain amplification of the FGF-FGFR signaling axis including FGFR1 (UCD12 and UCD65), FGFR3 (UCD4), and FGF3 and FGF19 in UCD65. This underscores the significance of FGF signaling as driving ER+ breast cancers, and here we note these anomalies in early (primary tumor, UCD12), intermediate (lymph node, UCD65), and advanced (pleural effusion, UCD4) disease settings. Thus, the ER+ cell lines provide additional platforms for testing FGFR inhibitors [38].

The three ER−PR− cell lines (by our limits of detection) were derived from PDX which each contained rare ER+ cells (Supplemental Figure 1). However, the patient tumors were defined as ER−PR+ (UCD46), ER−PR− (UCD115), and ER+ at original diagnosis but untested in the recurrent pleural effusion (UCD178). Total loss of ER upon tumor progression occurs in only 10–20% of patients [39], and we speculate this could be the case for UCD178. We have observed that most TNBC specimens contain rare ER+ cells as PDX, prospectively due to estrogen used at initial implantation or a change in microenvironment. We speculate that the predominant ER− populations were likely selected for in culture. All three ER− cell lines also have alterations associated with impaired P53 function, either mutation in the TP53 gene (UCD115, UCD178) or amplification of its negative regulator MDM2 (UCD46). Basal-like UCD46 cells additionally have amplification of cyclin E (CCND2), PIK3CA, and CREBBP. UCD115 is peculiar as its parent PDX is epithelial-like with CK5+ and CK18+ cells, whereas the cells that grew in culture may have undergone a partial epithelial-mesenchymal transition (EMT), evidenced by loss of CKs, gain of vimentin, and increase in transcripts for EMT transcription factors (Fig. 1, Supplemental Figure 5, Supplementary Table 1). The UCD178 line was derived from a patient with ILC at the time of recurrence in the lungs (pleural effusion). Although this cell line aligned with the luminal B molecular subtype, it has low ER transcript expression and lack of ER protein. RNA-seq data for UCD178 shows expression of both E-cadherin and N-cadherin transcripts, which we confirmed by ICC (not shown). We presently describe the UCD178 cell line as mixed ductal/lobular, although further characterization is needed to define their unusual histological type. UCD178 also contains a SEC16A-NOTCH1 fusion protein occasionally found in breast cancers [40]. Thus, the three ER− cell lines share some common features of TNBC cell lines (i.e., mutation in the P53 signaling axis, MYC amplification) and harbor some less common mutations (i.e., SEC16A-NOTCH fusion) and histological features (i.e., ILC/IDC subtype).

In this manuscript, we describe six PDX-derived breast cancer cell lines that can be used in parallel with existing cell lines to provide depth and rigor to experimental approaches. These include three new ER+ cell lines, including two that are ER+PR+ and one with a mixed ductal/lobular phenotype. While each of these cell lines grows comparatively slower than long-established ER+ and ER− cell lines, this may be more reflective of their true proliferative rates, and they remain amenable to serial passaging and manipulations such as viral transduction. The expanding repertoire of cells that represent the individualistic nature of breast cancer will bring us closer to realizing prevention and cure for a wider group of patients.