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Abstract
Background - Relevant surrogates that maintain the pathological and physiological properties of patient tumors are
essential for guiding triple-negative breast cancer (TNBC) therapy. The goals are to generate patient-derived orga‑
noids (PDOs), xenografts (PDXs), and PDX-derived organoids (PDXOs), evaluate the therapeutic potential of the WEE1
inhibitor AZD1775, and compare their responses to 18 anti-cancer drugs in PDOs and PDXOs.
Methods - PDOs were produced from surgical specimens of patients with TNBC. PDXs were generated by transplant‑
ing PDOs into the mammary fat pads of NOD.Cg-Prkdcscid Il2rgtm1wjl/SzJ mice. PDXOs were derived from fresh
tumor specimens of PDXs. For drug efcacy, half-maximal inhibitory concentration (IC50) values for 18 anti-cancer
drugs on PDOs and PDXOs were calculated using the CellTiter-Glo® 3D cell viability assay in a high-throughput drug
screening system. The relationship between WEE1 expression and survival in TNBC-basal-like (BL) patients was ana‑
lyzed using the Kaplan–Meier Plotter database. Mice were treated with AZD1775 via oral gavage (30 mg/kg). Bio‑
logical mechanisms underlying the anti-cancer drug responses were evaluated by calcein-AM staining, caspase 3/7
staining, Western blot, fow cytometry, and immunohistochemistry.
Results - PDOs were established through subcultures of 2-7 passages. TNBC-BL PDXs expressing CK5, vimen‑
tin, and EGFR were generated and expanded over 3-4 generations of transplantation. PDXOs were produced
through subcultures of 4-5 passages. PDOs, PDXs, and PDXOs retained the immunohistological characteristics
of the relevant patients with TNBC. WEE1 was associated with poor survival outcomes in TNBC-BL patients. The high‑
est cytotoxicity and tumor growth suppression to AZD1775 therapy were observed in PDXOs and PDXs with high
WEE1 expression. AZD1775 inhibited WEE1 and CDK1 phosphorylation, increased γH2AX phosphorylation, induced
G2/M arrest, and activated caspase 3/7 in PDXOs and PDXs, all associated with DNA damage, mitotic catastrophe,
and apoptosis. Anti-cancer drug responses were highly concordant between matched PDOs and PDXOs. The
responses of PDOs and PDXOs to anti-cancer drugs were comparable to those of patients receiving neoadjuvant
or adjuvant chemotherapy, according to clinical records.
Conclusion - PDOs, PDXOs, and PDXs, which maintained the immunological properties of TNBC patient, provide a sci‑
entifc rationale for future WEE1-targeted clinical trials in TNBC. PDOs and PDXOs represent cost- and time-efective
surrogates for predicting prioritized personalized therapy.
Keywords Patient-derived organoid (PDO), Patient-derived xenograft (PDX), PDX-derived organoid (PDXO), Highthroughput screening (HTS), WEE1, AZD1775