Search

Supplemental Methods and Figures

Purpose:Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors significantly extend tumor response in patients with metastatic estrogen receptor–positive (ER+) breast cancer, relapse is almost inevitable. This may, in part, reflect the failure of CDK4/6 inhibitors to induce apoptotic cell death. We therefore evaluated combination therapy with ABT-199 (venetoclax), a potent and selective BCL2 inhibitor.Experimental Design:BCL2 family member expression was assessed following treatment with endocrine therapy and the CDK4/6 inhibitor palbociclib. Functional assays were used to determine the impact of adding ABT-199 to fulvestrant and palbociclib in ER+ breast cancer cell lines, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. A syngeneic ER+ mouse mammary tumor model was used to study the effect of combination therapy on the immune system.Results:Triple therapy was well tolerated and produced a superior and more durable tumor response compared with single or doublet therapy. This was associated with marked apoptosis, including of senescent cells, indicative of senolysis. Unexpectedly, ABT-199 resulted in Rb dephosphorylation and reduced G1–S cyclins, most notably at high doses, thereby intensifying the fulvestrant/palbociclib–induced cell-cycle arrest. Interestingly, a CRISPR/Cas9 screen suggested that ABT-199 could mitigate loss of Rb (and potentially other mechanisms of acquired resistance) to palbociclib. ABT-199 did not abrogate the favorable immunomodulatory effects of palbociclib in a syngeneic ER+ mammary tumor model and extended tumor response when combined with anti-PD1 therapy.Conclusions:This study illustrates the potential for targeting BCL2 in combination with CDK4/6 inhibitors and supports investigation of combination therapy in ER+ breast cancer.

Differential sgRNA enrichment from CRISPR-Cas9 genome-wide screens

Bone marrow is a preferred metastatic site for multiple solid tumours and is associated with poor prognosis and significant morbidity. Accumulating evidence indicates that cancer cells colonise specialised niches within the bone marrow to support their long-term propagation, but the precise location and mechanisms that mediate niche interactions are unknown. Using breast cancer as a model of solid tumour metastasis to the bone marrow, we applied large-scale quantitative three-dimensional imaging to characterise temporal changes in the bone marrow microenvironment during disease progression. We show that mouse mammary tumour cells preferentially home to a pre-existing metaphyseal domain enriched for type H vessels. Metastatic lesion outgrowth rapidly remodelled the local vasculature through extensive sprouting to establish a tumour-supportive microenvironment. The evolution of this tumour microenvironment reflects direct remodelling of the vascular endothelium through tumour-derived granulocyte-colony stimulating factor (G-CSF) in a hematopoietic cell-independent manner. Therapeutic targeting of the metastatic niche by blocking G-CSF receptor inhibited pathological blood vessel remodelling and reduced bone metastasis burden. These findings elucidate a mechanism of ‘host’ microenvironment hijacking by mammary tumour cells to subvert the local microvasculature to form a specialised, pro-tumorigenic niche., The visualisation of the bone metastasis process in a spatial temporal manner is lacking. Here, the authors use three-dimensional quantitative imaging and show that mouse mammary tumour cells preferentially home to endothelial subtype type H vessels within the bone marrow and remodel this vasculature by producing granulocyte-colony stimulating factor.

Background Heterogeneity within the mouse mammary epithelium and potential lineage relationships have been recently explored by single-cell RNA profiling. To further understand how cellular diversity changes during mammary ontogeny, we profiled single cells from nine different developmental stages spanning late embryogenesis, early postnatal, prepuberty, adult, mid-pregnancy, late-pregnancy, and post-involution, as well as the transcriptomes of micro-dissected terminal end buds (TEBs) and subtending ducts during puberty. Methods The single cell transcriptomes of 132,599 mammary epithelial cells from 9 different developmental stages were determined on the 10x Genomics Chromium platform, and integrative analyses were performed to compare specific time points. Results The mammary rudiment at E18.5 closely aligned with the basal lineage, while prepubertal epithelial cells exhibited lineage segregation but to a less differentiated state than their adult counterparts. Comparison of micro-dissected TEBs versus ducts showed that luminal cells within TEBs harbored intermediate expression profiles. Ductal basal cells exhibited increased chromatin accessibility of luminal genes compared to their TEB counterparts suggesting that lineage-specific chromatin is established within the subtending ducts during puberty. An integrative analysis of five stages spanning the pregnancy cycle revealed distinct stage-specific profiles and the presence of cycling basal, mixed-lineage, and 'late' alveolar intermediates in pregnancy. Moreover, a number of intermediates were uncovered along the basal-luminal progenitor cell axis, suggesting a continuum of alveolar-restricted progenitor states. Conclusions This extended single cell transcriptome atlas of mouse mammary epithelial cells provides the most complete coverage for mammary epithelial cells during morphogenesis to date. Together with chromatin accessibility analysis of TEB structures, it represents a valuable framework for understanding developmental decisions within the mouse mammary gland.

To examine global changes in breast heterogeneity across different states, we determined the single‐cell transcriptomes of > 340,000 cells encompassing normal breast, preneoplastic BRCA1 +/– tissue, the major breast cancer subtypes, and pairs of tumors and involved lymph nodes. Elucidation of the normal breast microenvironment revealed striking changes in the stroma of post‐menopausal women. Single‐cell profiling of 34 treatment‐naive primary tumors, including estrogen receptor (ER)+, HER2+, and triple‐negative breast cancers, revealed comparable diversity among cancer cells and a discrete subset of cycling cells. The transcriptomes of preneoplastic BRCA1 +/– tissue versus tumors highlighted global changes in the immune microenvironment. Within the tumor immune landscape, proliferative CD8+ T cells characterized triple‐negative and HER2+ cancers but not ER+ tumors, while all subtypes comprised cycling tumor‐associated macrophages, thus invoking potentially different immunotherapy targets. Copy number analysis of paired ER+ tumors and lymph nodes indicated seeding by genetically distinct clones or mass migration of primary tumor cells into axillary lymph nodes. This large‐scale integration of patient samples provides a high‐resolution map of cell diversity in normal and cancerous human breast., A large‐scale gene expression resource integrates diverse tissue samples and reveals unexpected heterogeneity of breast cancer subtypes.

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types.

Purpose: Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors significantly extend tumor response in patients with metastatic estrogen receptor–positive (ER+) breast cancer, relapse is almost inevitable. This may, in part, reflect the failure of CDK4/6 inhibitors to induce apoptotic cell death. We therefore evaluated combination therapy with ABT-199 (venetoclax), a potent and selective BCL2 inhibitor. Experimental Design: BCL2 family member expression was assessed following treatment with endocrine therapy and the CDK4/6 inhibitor palbociclib. Functional assays were used to determine the impact of adding ABT-199 to fulvestrant and palbociclib in ER+ breast cancer cell lines, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. A syngeneic ER+ mouse mammary tumor model was used to study the effect of combination therapy on the immune system. Results: Triple therapy was well tolerated and produced a superior and more durable tumor response compared with single or doublet therapy. This was associated with marked apoptosis, including of senescent cells, indicative of senolysis. Unexpectedly, ABT-199 resulted in Rb dephosphorylation and reduced G1–S cyclins, most notably at high doses, thereby intensifying the fulvestrant/palbociclib–induced cell-cycle arrest. Interestingly, a CRISPR/Cas9 screen suggested that ABT-199 could mitigate loss of Rb (and potentially other mechanisms of acquired resistance) to palbociclib. ABT-199 did not abrogate the favorable immunomodulatory effects of palbociclib in a syngeneic ER+ mammary tumor model and extended tumor response when combined with anti-PD1 therapy. Conclusions: This study illustrates the potential for targeting BCL2 in combination with CDK4/6 inhibitors and supports investigation of combination therapy in ER+ breast cancer.

Long-lived quiescent mammary stem cells (MaSCs) are presumed to coordinate the dramatic expansion of ductal epithelium that occurs through the different phases of postnatal development, but little is known about the molecular regulators that underpin their activation. We show that ablation of the transcription factor Foxp1 in the mammary gland profoundly impairs ductal morphogenesis, resulting in a rudimentary tree throughout life. Foxp1-deficient glands were highly enriched for quiescent Tspan8hi MaSCs, which failed to become activated even in competitive transplantation assays, thus highlighting a cell-intrinsic defect. Foxp1 deletion also resulted in aberrant expression of basal genes in luminal cells, inferring a role in cell-fate decisions. Notably, Foxp1 was uncovered as a direct repressor of Tspan8 in basal cells, and deletion of Tspan8 rescued the defects in ductal morphogenesis elicited by Foxp1 loss. Thus, a single transcriptional regulator Foxp1 can control the exit of MaSCs from dormancy to orchestrate differentiation and development.

Breast tumors are inherently heterogeneous, but the evolving cellular organization through neoplastic progression is poorly understood. Here we report a rapid, large-scale single-cell resolution 3D imaging protocol based on a one-step clearing agent that allows visualization of normal tissue architecture and entire tumors at cellular resolution. Imaging of multicolor lineage-tracing models of breast cancer targeted to either basal or luminal progenitor cells revealed profound clonal restriction during progression. Expression profiling of clones arising in Pten/Trp53-deficient tumors identified distinct molecular signatures. Strikingly, most clones harbored cells that had undergone an epithelial-to-mesenchymal transition, indicating widespread, inherent plasticity. Hence, an integrative pipeline that combines lineage tracing, 3D imaging, and clonal RNA sequencing technologies offers a comprehensive path for studying mechanisms underlying heterogeneity in whole tumors.