源自循环肿瘤细胞的三阴性乳腺癌类器官,推进转移性癌症研究与耐药性研究

Triple-negative breast cancer (TNBC) remains challenging to treat due to a lack of effective targeted therapies, and a higher rate of metastasis compared to other breast cancer subtypes. Circulating tumor cells (CTCs) are a subset of cancer cells that shed from the primary tumor, survive in the circulatory system, and then form new metastatic tumors in new environments (s). We believe that a clinically relevant model of metastatic TNBC could be built from cultivation of functional CTCs in vitro, as we have done so for several solid tumors. To better represent the physiology of metastatic tumors, we aimed to culture these cells into 3D tumor organoids to mimic the establishment of early metastasis from CTCs. These CTC-derived tumor organoids would be a valuable platform for identifying novel drugs or drug combinations capable of effectively treating metastatic TNBC. We successfully established a TNBC CTC organoid line from the blood of a treatment-naïve stage 4 TNBC patient. From whole blood, we isolated CTC-containing PBMC, lysing residual red blood cells and seeding them into a proprietary media and plate system. To evaluate whether PBMC depletion would improve the cultivation of CTC organoids, we tested the use of anti-CD45 magnetic beads to deplete immune cells from the PBMC fraction. The cultures were closely monitored and replenished with fresh media every 2-3 days, and passaged to fresh plates every 2-3 weeks, enabling the steady removal of dead cells. This CTC organoid line of TNBC has been continuously passaged for four months now, consistently forms organoids of various sizes and as large as 200 microns, and slightly increases its growth rate over time. Morphology shows loose and irregular aggregates, similar to other breast cancer organoids, but unlike the spherical and densely packed organoids observed from CTC organoids from colon, renal, and other solid tumors. Due to the looser structure of these organoids, we found that cell health was best maintained during passaging by dissociating mechanically using a P1000 pipette, as opposed to enzymatic dissociation. This organoid line expresses EpCAM and cytokeratin, but not CD45, confirming an epithelial origin. Following our previous success in generating drug-resistant colonies from treating CTC organoid lines from colon and renal cancers with standard of care chemotherapy drugs, we are performing in vitro drug resistance colony selection on this TNBC CTC organoid line and applying RNA-Seq on these drug-resistant clones. Gene expression data are analyzed against public domain data from real patients and their treatment outcomes using an in-house AI algorithm to identify mechanisms of drug resistance. We intend to culture TNBC CTCs from new patient samples to increase our bank size, to improve our representation of the diversity of TNBC and validate the identified drug resistance mechanisms