Model systems that recapitulate the complexity of human tumors and the reality of variable treatment responses are urgently needed to better understand cancer biology and to develop more effective cancer therapies. Here we report development and characterization of a large bank of patient-derived xenografts (PDX) and matched organoid cultures from tumors that represent some of the greatest unmet needs in breast cancer research and treatment. These include endocrine-resistant, treatment-refractory, and metastatic breast cancers and, in some cases, multiple tumor collections from the same patients. The models can be grown long-term with high fidelity to the original tumors. We show that development of matched PDX and PDX-derived organoid (PDxO) models facilitates high-throughput drug screening that is feasible and cost-effective, while also allowing in vivo validation of results. Our data reveal consistency between drug screening results in organoids and drug responses in breast cancer PDX. Moreover, we demonstrate the feasibility of using these patient-derived models for precision oncology in real time with patient care, using a case of a triple negative breast cancer with early metastatic recurrence as an example. Our results uncovered an FDA-approved drug with high efficacy against the models. Treatment with the PDxO-directed therapy resulted in a complete response for the patient and a progression-free survival period more than three times longer than her previous therapies. This work provides valuable new methods and resources for functional precision medicine and drug development for human breast cancer.
Studies of reticulocyte maturation have been limited by the inability to obtain pure populations of age-synchronized reticulocytes and by the absence of well-defined methods for the maturation of reticulocytes in vitro. Many of these problems were overcome using temporary suppression of erythropoiesis with thiamphenicol and phlebotomy resulting in a highly reproducible reticulocyte response, Percoll density gradient separation of cells yielding essentially pure populations of age- synchronized reticulocytes, and liquid culture techniques where cell lysis is minimal. The system allows reproducible study of well-defined cohorts of reticulocytes as they mature into erythrocytes. During in vitro maturation we serially monitored changes in reticulocyte count, glucose consumption, 125I-transferrin binding, fluorescein (FITC)- labeled transferrin binding, the activities of four erythrocyte enzymes (glucose-6-phosphate dehydrogenase, pyruvate kinase, phosphofructokinase, and lactate dehydrogenase) and the appearance of cells on scanning electron microscopy. These variables changed at different rates suggesting that multiple mechanisms underlie these maturational events. Transferrin binding and reticulocyte count decreased most rapidly and reached values near zero after three to four days in culture. The four enzyme activities decreased much more slowly, and only two reached pretreatment values after seven days in culture. In contrast to the findings of others, scanning electron microscopy suggested that cells do not assume the normal biconcave shape in this system. The methods described make it feasible to study the process of reticulocyte maturation in vitro. The data presented represent a first step in the study of the coordination and interrelationships of various maturational processes.
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