Michael T. Lewis

BACKGROUND: Tumorigenic breast cancer cells that express high levels of CD44 and low or undetectable levels of CD24 (CD44(>)/CD24(>/low)) may be resistant to chemotherapy and therefore responsible for cancer relapse. These tumorigenic cancer cells can be isolated from breast cancer biopsies and propagated as mammospheres in vitro. In this study, we aimed to test directly in human breast cancers the effect of conventional chemotherapy or lapatinib (an epidermal growth factor receptor [EGFR]/HER2 pathway inhibitor) on this tumorigenic CD44(>) and CD24(>/low) cell population. METHODS: Paired breast cancer core biopsies were obtained from patients with primary breast cancer before and after 12 weeks of treatment with neoadjuvant chemotherapy (n = 31) or, for patients with HER2-positive tumors, before and after 6 weeks of treatment with the EGFR/HER2 inhibitor lapatinib (n = 21). Single-cell suspensions established from these biopsies were stained with antibodies against CD24, CD44, and lineage markers and analyzed by flow cytometry. The potential of cells from biopsy samples taken before and after treatment to form mammospheres in culture was compared. All statistical tests were two-sided. RESULTS: Chemotherapy treatment increased the percentage of CD44(>)/CD24(>/low) cells (mean at baseline vs 12 weeks, 4.7%, 95% confidence interval [CI] = 3.5% to 5.9%, vs 13.6%, 95% CI = 10.9% to 16.3%; P < .001) and increased mammosphere formation efficiency (MSFE) (mean at baseline vs 12 weeks, 13.3%, 95% CI = 6.0% to 20.6%, vs 53.2%, 95% CI = 42.4% to 64.0%; P < .001). Conversely, lapatinib treatment of patients with HER2-positive tumors led to a non-statistically significant decrease in the percentage of CD44(>)/CD24(>/low) cells (mean at baseline vs 6 weeks, 10.0%, 95% CI = 7.2% to 12.8%, vs 7.5%, 95% CI = 4.1% to 10.9%) and a statistically non-significant decrease in MSFE (mean at baseline vs 6 weeks, 16.1%, 95% CI = 8.7% to 23.5%, vs 10.8%, 95% CI = 4.0% to 17.6%). CONCLUSION: These studies provide clinical evidence for a subpopulation of chemotherapy-resistant breast cancer-initiating cells. Lapatinib did not lead to an increase in these tumorigenic cells, and, in combination with conventional therapy, specific pathway inhibitors may provide a therapeutic strategy for eliminating these cells to decrease recurrence and improve long-term survival.

Some breast cancers have been shown to contain a small fraction of cells characterized by CD44(+)/CD24(-/low) cell-surface antigen profile that have high tumor-initiating potential. In addition, breast cancer cells propagated in vitro as mammospheres (MSs) have also been shown to be enriched for cells capable of self-renewal. In this study, we have defined a gene expression signature common to both CD44(+)/CD24(-/low) and MS-forming cells. To examine its clinical significance, we determined whether tumor cells surviving after conventional treatments were enriched for cells bearing this CD44(+)/CD24(-/low)-MS signature. The CD44(+)/CD24(-/low)-MS signature was found mainly in human breast tumors of the recently identified "claudin-low" molecular subtype, which is characterized by expression of many epithelial-mesenchymal-transition (EMT)-associated genes. Both CD44(+)/CD24(-/low)-MS and claudin-low signatures were more pronounced in tumor tissue remaining after either endocrine therapy (letrozole) or chemotherapy (docetaxel), consistent with the selective survival of tumor-initiating cells posttreatment. We confirmed an increased expression of mesenchymal markers, including vimentin (VIM) in cytokeratin-positive epithelial cells metalloproteinase 2 (MMP2), in two separate sets of postletrozole vs. pretreatment specimens. Taken together, these data provide supporting evidence that the residual breast tumor cell populations surviving after conventional treatment may be enriched for subpopulations of cells with both tumor-initiating and mesenchymal features. Targeting proteins involved in EMT may provide a therapeutic strategy for eliminating surviving cells to prevent recurrence and improve long-term survival in breast cancer patients.

Breast cancer research is hampered by difficulties in obtaining and studying primary human breast tissue, and by the lack of in vivo preclinical models that reflect patient tumor biology accurately. To overcome these limitations, we propagated a cohort of human breast tumors grown in the epithelium-free mammary fat pad of severe combined immunodeficient (SCID)/Beige and nonobese diabetic (NOD)/SCID/IL-2γ-receptor null (NSG) mice under a series of transplant conditions. Both models yielded stably transplantable xenografts at comparably high rates (∼21% and ∼19%, respectively). Of the conditions tested, xenograft take rate was highest in the presence of a low-dose estradiol pellet. Overall, 32 stably transplantable xenograft lines were established, representing 25 unique patients. Most tumors yielding xenografts were "triple-negative" [estrogen receptor (ER)-progesterone receptor (PR)-HER2+; n = 19]. However, we established lines from 3 ER-PR-HER2+ tumors, one ER+PR-HER2-, one ER+PR+HER2-, and one "triple-positive" (ER+PR+HER2+) tumor. Serially passaged xenografts show biologic consistency with the tumor of origin, are phenotypically stable across multiple transplant generations at the histologic, transcriptomic, proteomic, and genomic levels, and show comparable treatment responses as those observed clinically. Xenografts representing 12 patients, including 2 ER+ lines, showed metastasis to the mouse lung. These models thus serve as a renewable, quality-controlled tissue resource for preclinical studies investigating treatment response and metastasis.

Models that recapitulate the complexity of human tumors are urgently needed to develop more effective cancer therapies. We report a bank of human patient-derived xenografts (PDXs) and matched organoid cultures from tumors that represent the greatest unmet need: endocrine-resistant, treatment-refractory and metastatic breast cancers. We leverage matched PDXs and PDX-derived organoids (PDxO) for drug screening that is feasible and cost-effective with in vivo validation. Moreover, we demonstrate the feasibility of using these models for precision oncology in real time with clinical care in a case of triple-negative breast cancer (TNBC) with early metastatic recurrence. Our results uncovered a Food and Drug Administration (FDA)-approved drug with high efficacy against the models. Treatment with this therapy resulted in a complete response for the individual and a progression-free survival (PFS) period more than three times longer than their previous therapies. This work provides valuable methods and resources for functional precision medicine and drug development for human breast cancer.