Single-cell analysis of breast cancer metastasis reveals epithelial-mesenchymal plasticity signatures associated with poor outcomes
Juliane Winkler, Weilun Tan, Catherine M.M. Diadhiou, Christopher S. McGinnis, Aamna Abbasi, Saad Hasnain, Sophia Durney, Elena Atamaniuc, Daphne Superville, Leena Awni, Joyce V. Lee, Johanna H. Hinrichs, Patrick S. Wagner, Namrata Singh, Marco Y. Hein, Michael Borja, Angela M. Detweiler, Su-Yang Liu, Ankitha Nanjaraj, Vaishnavi Sitarama, Hope S. Rugo, Norma Neff, Zev J. Gartner, Angela Oliveira Pisco, Andrei Goga, Spyros Darmanis, Zena Werb
Juliane Winkler, Weilun Tan, Catherine M.M. Diadhiou, Christopher S. McGinnis, Aamna Abbasi, Saad Hasnain, Sophia Durney, Elena Atamaniuc, Daphne Superville, Leena Awni, Joyce V. Lee, Johanna H. Hinrichs, Patrick S. Wagner, Namrata Singh, Marco Y. Hein, Michael Borja, Angela M. Detweiler, Su-Yang Liu, Ankitha Nanjaraj, Vaishnavi Sitarama, Hope S. Rugo, Norma Neff, Zev J. Gartner, Angela Oliveira Pisco, Andrei Goga, Spyros Darmanis, Zena Werb
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Research Article Oncology

Single-cell analysis of breast cancer metastasis reveals epithelial-mesenchymal plasticity signatures associated with poor outcomes

Abstract

Metastasis is the leading cause of cancer-related deaths. It is unclear how intratumor heterogeneity (ITH) contributes to metastasis and how metastatic cells adapt to distant tissue environments. The study of these adaptations is challenged by the limited access to patient material and a lack of experimental models that appropriately recapitulate ITH. To investigate metastatic cell adaptations and the contribution of ITH to metastasis, we analyzed single-cell transcriptomes of matched primary tumors and metastases from patient-derived xenograft models of breast cancer. We found profound transcriptional differences between the primary tumor and metastatic cells. Primary tumors upregulated several metabolic genes, whereas motility pathway genes were upregulated in micrometastases, and stress response signaling was upregulated during progression. Additionally, we identified primary tumor gene signatures that were associated with increased metastatic potential and correlated with patient outcomes. Immune-regulatory control pathways were enriched in poorly metastatic primary tumors, whereas genes involved in epithelial-mesenchymal transition were upregulated in highly metastatic tumors. We found that ITH was dominated by epithelial-mesenchymal plasticity (EMP), which presented as a dynamic continuum with intermediate EMP cell states characterized by specific genes such as CRYAB and S100A2. Elevated expression of an intermediate EMP signature correlated with worse patient outcomes. Our findings identified inhibition of the intermediate EMP cell state as a potential therapeutic target to block metastasis.

Authors

Juliane Winkler, Weilun Tan, Catherine M.M. Diadhiou, Christopher S. McGinnis, Aamna Abbasi, Saad Hasnain, Sophia Durney, Elena Atamaniuc, Daphne Superville, Leena Awni, Joyce V. Lee, Johanna H. Hinrichs, Patrick S. Wagner, Namrata Singh, Marco Y. Hein, Michael Borja, Angela M. Detweiler, Su-Yang Liu, Ankitha Nanjaraj, Vaishnavi Sitarama, Hope S. Rugo, Norma Neff, Zev J. Gartner, Angela Oliveira Pisco, Andrei Goga, Spyros Darmanis, Zena Werb

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Figure 1

BC PDX models show varying metastatic potential.

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BC PDX models show varying metastatic potential. (A) Experimental overvi...
(A) Experimental overview: Lung metastases and primary tumor tissues were isolated from orthotopically transplanted BC PDX models and dissociated. The resulting single-cell suspensions were FACS enriched for human cells using a human specific antibody (hCD298) and sorted into 384-well plates (1 cell per well), and scRNA-Seq was performed using Smart-Seq2. Data analysis investigated tumor heterogeneity and differences between primary tumor and metastatic cells. (B) Bar chart shows the median number of metastatic foci per mm2 lung tissue area per model (upper panel), determined by histology. Metastatic foci were classified as micrometastasis (< 10 cells), intermediate (10–100 cells), and macrometastasis (>100 cells). Box plot shows the fraction of metastatic tissue per total lung tissue area, determined by histology (lower panel). The x-axis shows the model, BC subtype, and metastatic potential. (C) Representative H&E-stained images of metastatic lung tissue for low, moderate, and high metastatic potential models. Scale bars: 100 μm. (D) Bubble plot showing the expression of receptors in primary tumor (PT) and metastatic cells (Met) per model. (E) Representative images showing immunohistochemical staining for ER, PR, and HER2 in primary tumors and metastatic lungs of ER+ tumor models. Arrowheads indicate metastasis. When possible, the same metastasis is shown in consecutive sections. Scale bars: 100 μm.