Background: To adequately recapitulate the influence of the mammary immune microenvironment in early ER+ breast cancer, ER+ syngeneic mouse models are used. At present, there is only one validated ER+ cell line, the SSM3 model developed from Stat1 deficient mice. There are numerous syngeneic lines which have inconsistent receptor positivity between studies. We aimed to characterise the syngeneic lines indicated to be ER+ using pathology, IHC for ERa, FACS for luminal and basal cells as well as molecular assessment. Additionally, as there are no lines recapitulating PIK3CA/PTEN mutant ER+ tumours, we have begun to develop a new ER+ syngeneic line on this mutant background.
Methods: We collated primary tumours from in-vivo studies of a range of different syngeneic tumours lines (SSM3, J110, E0771, 4T1, D2A1 and D2OR) and made formalin fixed paraffin embedded tissue sections. ERα expression (ERa (6F11), Abcam) was detected using a standard immunohistochemical staining protocol and analysed using ImageJ. A positive sample was considered as >1% nuclear ERα staining. For the syngeneic PIK3CA/PTEN tumour cell line development, tumours were harvested from 6-8-month-old K8-PIK3CAHR1047R/-/PTENGE/- mutant mice, processed to single cells and FACS sorted for ER+ luminal mature (CD49b-/Sca-1+) and ER+ luminal progenitor (CD49b+Sca-1+) cells. Freshly sorted cells were then bulk seeded into various in vitro conditions or single cell cloned into 96 well plates.
Results: SSM3 tumours were highly positive (>80%) for ERα as were 67NR, but to a lesser degree. D1A1 and 4T1 tumours had scattered areas of positivity (<1%). J110, D2OR and E0771 tissues were negative for ERα, however, some regions of positivity were ambiguous due to immune infiltrates and require further characterisation.
Five tumours were harvested from a K8-PIK3CA/PTEN mouse. Of the various in vitro growth conditions, ER+ luminal mature and ER+ luminal progenitor cells show increased proliferative activity in mammary organoid media either on an irradiated feeder layer of 3T3 cells or as a monolayer.
Conclusions/future directions: SSM3 and 67NR may be considered bonafide ER+ syngeneic models, however further characterisation of these tumours are required (i.e molecular/estrogen sensitivity assays). The PIK3CA/PTEN syngeneic model is a novel ER+ cell line that will also undergo similar characterisation.