Establishment and characterization of breast cancer organoids from a patient with mammary Paget’s disease

Abstract

Background Mammary Paget’s disease (MPD) is an uncommon cutaneous intraepithelial malignancy with ulceration of the nipple or areola. Its pathogenesis and genomic mutation remain largely unknown and no cell lines are established from primary tumors. Methods We collected surgical tumor specimens from a 65-year-old Chinese woman diagnosed with MPD and established patient-derived breast cancer (BC) organoids from MPD using organoid culture technology. Results We successfully propagated BC organoids from a patient with MPD for more than 6 months. The organoids were cultured for long-term expansion without any change in spherical organoid morphology. Besides, the spherical organoid morphology did not change when they underwent cryopreservation after resuscitation. The H&E staining and immunohistochemistry analyses showed the similar morphological and histological features of the organoids compared with their paired original BC tissues. The organoids retained positive expression of breast cancer biomarkers: estrogen receptor, progesterone receptor, antigen Ki-67 and negative expression of human epidermal growth factor receptor 2. We also showed that MPD organoids recapitulated the unique genomic landscape including copy number alterations, mutational load, mutational signatures and cancer gene mutations by whole genome sequencing. In situ senescence-associated acid beta galactosidase assay confirmed senescence phenomenon existed in the process of organoids culture and there was no significant difference in the proportion of senescent organoids after organoid passage and resuscitation. Conclusions Our results suggested that an effective platform for ex vivo BC organoids from MPD patients could be used to explore clinicopathological and genomic characteristics of these patients.

Optimizing individualized treatment strategy based on breast cancer organoid model

“…8 Recently, we have successfully established patient-derived tumor organoids (PDTOs) from the uncommon pathological types of papillary carcinoma and mammary Paget's disease. 9,10 To study molecular pathogenesis of more Chinese population BC patients, we seek to establish a larger organoid platform. To the best of our knowledge, no Chinese population-derived BC organoids have been established in China yet.…”

Patient-Derived Organoids as a Model for Cancer Drug Discovery

“…However, it may be useful in the future to develop media containing the relevant supplements and growth factors, as this has previously been demonstrated to improve the metabolic fidelity and biological relevance for in vitro models [ 22 ]. Organoids which maintain the morphological and histological features of the original tumour have been developed to establish and culture PDOs from many common tumour types, including glioblastoma [ 23 ], breast [ 24 ], pancreas [ 25 ], prostate [ 26 , 27 ], liver [ 28 ], lung [ 4 , 29 ] and colon [ 30 ], with varying degrees of success and reproducibility.…”

“…However, methods are being developed to overcome this, with the potential of maintaining cancer-stem-cell features that may facilitate organoid development. Refined cryopreservation solutions have also led to improvements in the recovery rates of organoids derived from several tumour types, including pancreatic and breast tumours, retaining their morphological and histological features [ 24 , 32 ]. Some of the limitations of using organoids, which can deter researchers from using these models, or hinder their more widespread use, are summarized in Table 2 , along with some potential solutions.…”

Advance in Human Epithelial-Derived Organoids Research

“…Of note, a living biobank with >100 primary and metastatic BC organoid lines that recapitulated the diversity of the disease and typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor was generated . Briefly, tumor tissues were digested in BC organoid medium containing collagenase at 37 °C for 1–2 h. The obtained tumor cells were seeded into basement membrane extract (BME) with BC organoid medium supplemented with FGF7, R-spondin 3- or R-spondin1-conditioned medium, Neuregulin1, FGF10, Noggin, A83–01, SB202190, N -acetylcysteine, nicotinamide, and Y-27632. ,− The mitogen Neuregulin 1, a key factor, is a ligand of the human EGF receptor (HER) tyrosine kinases-3 and -4 and has been implicated in mammary development as well as tumorigenesis. , The addition of Neuregulin 1 allowed both the efficient generation and long-term expansion of BC organoids for >20 passages. Notably, BC organoids did not depend on Wnt3a.…”

“…Y-27632 indeed improved culture conditions but needed to be removed starting 3 days after initial seeding. , Similar to its role in stomach cancer organoids, Nutlin-3a can be added to select for cancer organoids with p53 mutations . Similar to the original tissue, BC organoid lines exhibit a series of distinct phenotypes, such as solid organoids of different sizes, cystic organoids, “grape-like” organoids, and noncompact organoids. ,,, Under the above culture conditions, BC organoids can propagate for long-term in vitro with a success rate of establishment of >80% and can be passaged every 1–4 weeks at a 1:1 to 1:6 ratio. , For passage, cystic organoids were mechanically sheared, while dense organoids were dissociated by incubation in TrypLE Express and mechanical shearing through sterile glass Pasteur pipettes …”