High-Dimensional Phenotyping Identifies Age-Emergent Cells in Human Mammary Epithelia

Aging is associated with tissue-level changes in cellular composition that are correlated with increased susceptibility to disease. Aging human mammary tissue shows skewed progenitor cell potency, resulting in diminished tumor-suppressive cell types and the accumulation of defective epithelial progenitors. Quantitative characterization of these age-emergent human cell subpopulations is lacking, impeding our understanding of the relationship between age and cancer susceptibility. We conducted single-cell resolution proteomic phenotyping of healthy breast epithelia from 57 women, aged 16–91 years, using mass cytometry. Remarkable heterogeneity was quantified within the two mammary epithelial lineages. Population partitioning identified a subset of aberrant basal-like luminal cells that accumulate with age and originate from age-altered progenitors. Quantification of age-emergent phenotypes enabled robust classification of breast tissues by age in healthy women. This high-resolution mapping highlighted specific epithelial subpopulations that change with age in a manner consistent with increased susceptibility to breast cancer

High-Dimensional Phenotyping Identifies Age-Emergent Cells in Human Mammary Epithelia.

Aging is associated with tissue-level changes in cellular composition that are correlated with increased susceptibility to disease. Aging human mammary tissue shows skewed progenitor cell potency, resulting in diminished tumor-suppressive cell types and the accumulation of defective epithelial progenitors. Quantitative characterization of these age-emergent human cell subpopulations is lacking, impeding our understanding of the relationship between age and cancer susceptibility. We conducted single-cell resolution proteomic phenotyping of healthy breast epithelia from 57 women, aged 16-91 years, using mass cytometry. Remarkable heterogeneity was quantified within the two mammary epithelial lineages. Population partitioning identified a subset of aberrant basal-like luminal cells that accumulate with age and originate from age-altered progenitors. Quantification of age-emergent phenotypes enabled robust classification of breast tissues by age in healthy women. This high-resolution mapping highlighted specific epithelial subpopulations that change with age in a manner consistent with increased susceptibility to breast cancer

Microenvironment-Induced Non-sporadic Expression of the AXL and cKIT Receptors Are Related to Epithelial Plasticity and Drug Resistance

The existence of rare cancer cells that sporadically acquire drug-tolerance through epigenetic mechanisms is proposed as one mechanism that drives cancer therapy failure. Here we provide evidence that specific microenvironments impose non-sporadic expression of proteins related to epithelial plasticity and drug resistance. Microarrays of robotically printed combinatorial microenvironments of known composition were used to make cell-based functional associations between microenvironments, which were design-inspired by normal and tumor-burdened breast tissues, and cell phenotypes. We hypothesized that specific combinations of microenvironment constituents non-sporadically impose the induction of the AXL and cKIT receptor tyrosine kinase proteins, which are known to be involved in epithelial plasticity and drug-tolerance, in an isogenic human mammary epithelial cell (HMEC) malignant progression series. Dimension reduction analysis reveals type I collagen as a dominant feature, inducing expression of both markers in pre-stasis finite lifespan HMECs, and transformed non-malignant and malignant immortal cell lines. Basement membrane-associated matrix proteins, laminin-111 and type IV collagen, suppress AXL and cKIT expression in pre-stasis and non-malignant cells. However, AXL and cKIT are not suppressed by laminin-111 in malignant cells. General linear models identified key factors, osteopontin, IL-8, and type VIα3 collagen, which significantly upregulated AXL and cKIT, as well as a plasticity-related gene expression program that is often observed in stem cells and in epithelial-to-mesenchymal-transition. These factors are co-located with AXL-expressing cells in situ in normal and breast cancer tissues, and associated with resistance to paclitaxel. A greater diversity of microenvironments induced AXL and cKIT expression consistent with plasticity and drug-tolerant phenotypes in tumorigenic cells compared to normal or immortal cells, suggesting a reduced perception of microenvironment specificity in malignant cells. Microenvironment-imposed reprogramming could explain why resistant cells are seemingly persistent and rapidly adaptable to multiple classes of drugs. These results support the notion that specific microenvironments drive drug-tolerant cellular phenotypes and suggest a novel interventional avenue for preventing acquired therapy resistance

Microenvironment-Induced Non-sporadic Expression of the AXL and cKIT Receptors Are Related to Epithelial Plasticity and Drug Resistance

The existence of rare cancer cells that sporadically acquire drug-tolerance through epigenetic mechanisms is proposed as one mechanism that drives cancer therapy failure. Here we provide evidence that specific microenvironments impose non-sporadic expression of proteins related to epithelial plasticity and drug resistance. Microarrays of robotically printed combinatorial microenvironments of known composition were used to make cell-based functional associations between microenvironments, which were design-inspired by normal and tumor-burdened breast tissues, and cell phenotypes. We hypothesized that specific combinations of microenvironment constituents non-sporadically impose the induction of the AXL and cKIT receptor tyrosine kinase proteins, which are known to be involved in epithelial plasticity and drug-tolerance, in an isogenic human mammary epithelial cell (HMEC) malignant progression series. Dimension reduction analysis reveals type I collagen as a dominant feature, inducing expression of both markers in pre-stasis finite lifespan HMECs, and transformed non-malignant and malignant immortal cell lines. Basement membrane-associated matrix proteins, laminin-111 and type IV collagen, suppress AXL and cKIT expression in pre-stasis and non-malignant cells. However, AXL and cKIT are not suppressed by laminin-111 in malignant cells. General linear models identified key factors, osteopontin, IL-8, and type VIα3 collagen, which significantly upregulated AXL and cKIT, as well as a plasticity-related gene expression program that is often observed in stem cells and in epithelial-to-mesenchymal-transition. These factors are co-located with AXL-expressing cells in situ in normal and breast cancer tissues, and associated with resistance to paclitaxel. A greater diversity of microenvironments induced AXL and cKIT expression consistent with plasticity and drug-tolerant phenotypes in tumorigenic cells compared to normal or immortal cells, suggesting a reduced perception of microenvironment specificity in malignant cells. Microenvironment-imposed reprogramming could explain why resistant cells are seemingly persistent and rapidly adaptable to multiple classes of drugs. These results support the notion that specific microenvironments drive drug-tolerant cellular phenotypes and suggest a novel interventional avenue for preventing acquired therapy resistance

DataSheet1_Microenvironment-Induced Non-sporadic Expression of the AXL and cKIT Receptors Are Related to Epithelial Plasticity and Drug Resistance.zip

<p>The existence of rare cancer cells that sporadically acquire drug-tolerance through epigenetic mechanisms is proposed as one mechanism that drives cancer therapy failure. Here we provide evidence that specific microenvironments impose non-sporadic expression of proteins related to epithelial plasticity and drug resistance. Microarrays of robotically printed combinatorial microenvironments of known composition were used to make cell-based functional associations between microenvironments, which were design-inspired by normal and tumor-burdened breast tissues, and cell phenotypes. We hypothesized that specific combinations of microenvironment constituents non-sporadically impose the induction of the AXL and cKIT receptor tyrosine kinase proteins, which are known to be involved in epithelial plasticity and drug-tolerance, in an isogenic human mammary epithelial cell (HMEC) malignant progression series. Dimension reduction analysis reveals type I collagen as a dominant feature, inducing expression of both markers in pre-stasis finite lifespan HMECs, and transformed non-malignant and malignant immortal cell lines. Basement membrane-associated matrix proteins, laminin-111 and type IV collagen, suppress AXL and cKIT expression in pre-stasis and non-malignant cells. However, AXL and cKIT are not suppressed by laminin-111 in malignant cells. General linear models identified key factors, osteopontin, IL-8, and type VIα3 collagen, which significantly upregulated AXL and cKIT, as well as a plasticity-related gene expression program that is often observed in stem cells and in epithelial-to-mesenchymal-transition. These factors are co-located with AXL-expressing cells in situ in normal and breast cancer tissues, and associated with resistance to paclitaxel. A greater diversity of microenvironments induced AXL and cKIT expression consistent with plasticity and drug-tolerant phenotypes in tumorigenic cells compared to normal or immortal cells, suggesting a reduced perception of microenvironment specificity in malignant cells. Microenvironment-imposed reprogramming could explain why resistant cells are seemingly persistent and rapidly adaptable to multiple classes of drugs. These results support the notion that specific microenvironments drive drug-tolerant cellular phenotypes and suggest a novel interventional avenue for preventing acquired therapy resistance.</p

Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids

We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling. For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020).Funding agencies: he National Cancer Institute, United States CA224175 (D.L.), CA210240 (D.L.), CA232093(D.L.), CA163117 and CA207983 (D.L.), CA163120 (D.L.), CA169416 (D.L.), CA169538 (D.L.),CA218513 (D.L., H.Z.) and AI144301 (D.L., V.P.); the United States Department of Defense, UnitedStates W81XWH-13-1-0425 (D.L.), W81XWH-13-1-0427, W81XWH-13-1-0249 (D.L.), and W81XWH-14-1-0199 (D.L.); National Institutes of Health, United States/WCM CTSC, United States (NIH/NCATS (UL1TR00457) (H.Z.); NIH/NCATS (UL1TR002384) (D.L. and H.Z.); the Hartwell Foundation,United States (D.L.); the Thompson Family Foundation (D.L., D.K.); the STARR Consortium I9-A9-056(D.L., H.Z.) and I8-A8-123 (D.L.); the Pediatric Oncology Experimental Therapeutics Investigator’sConsortium (D.L., T.M.T.); Alex’s Lemonade Stand Foundation, United Sates (D.L.); the Breast Can-cer Research Foundation, United States (D.L.); the Feldstein Medical Foundation (D.L.); the TortolaniFoundation (D.L.); the Clinical &amp; Translational Science Center (D.L., H.Z.); the Mary Kay Ash Chari-table Foundation (D.L., I.M.); the Malcolm Hewitt Weiner Foundation (D.L.); the Manning Founda-tion (DL., A.H.); the Daniel P. and Nancy C. Paduano Family Foundation (D.L); the James PaduanoFoundation (D.L.); the Sohn Foundation (D.L.); the AHEPA Vth District Cancer Research Foundation(D.L., L.B., S.L.); the Daedalus Fund Selma and Lawrence Ruben Science to Industry Bridge Award(D.L.); the Children’s Cancer and Blood Foundation, United States (D.L.); Susan G. Komen Postdoc-toral Fellowship PDF15331556, JST PRESTO, Japan 30021, and JSPS KAKENHI, Japan JP19K23743(A.H.); the National Research Foundation of Korea, South Korea (NRF) grant funded by the Korea government (MSIT) (2019R1C1C1006709, 2018R1A5A2025079, and 2020M3F7A1094093); a grantof the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Insti-tute (KHIDI), funded by the Ministry of Health &amp; Welfare, South Korea (KHIDIHI19C1015010020);"The Alchemist Project" through the Ministry of Trade, Industry and Energy (MOTIE, Korea)(20012443), and Severance Hospital Research fund for Clinical excellence (SHRC) (C-2020-0032and C-2020-0025) (H.S.K.); The Swedish Cancer Society Pancreatic Cancer Fellowship, the Lions In-ternational Postdoctoral fellowship and the Sweden-America stipend (L.B.); the Sa ̃ o Paulo ResearchFoundation (FAPESP) Sa ̃ o Paulo, Brazil (2017/07117-7) (G.C.T); the Swiss National Science Founda-tion (SFNS), Switzerland Postdoc Mobility grant (P2SKP3_174785 and P400PB_186791) (F.A.V.); andthe DoD PRCRP Horizon Award, United States (W81XWH-19-PRCRP-HA) (S.L).</p

AXL is a driver of stemness in normal mammary gland and breast cancer

The receptor tyrosine kinase AXL is associated with epithelial plasticity in several solid tumors including breast cancer and AXL-targeting agents are currently in clinical trials. We hypothesized that AXL is a driver of stemness traits in cancer by co-option of a regulatory function normally reserved for stem cells. AXL-expressing cells in human mammary epithelial ducts co-expressed markers associated with multipotency, and AXL inhibition abolished colony formation and self-maintenance activities while promoting terminal differentiation in vitro. Axl-null mice did not exhibit a strong developmental phenotype, but enrichment of Axl+ cells was required for mouse mammary gland reconstitution upon transplantation, and Axl-null mice had reduced incidence of Wnt1-driven mammary tumors. An AXL-dependent gene signature is a feature of transcriptomes in basal breast cancers and reduced patient survival irrespective of subtype. Our interpretation is that AXL regulates access to epithelial plasticity programs in MaSCs and, when co-opted, maintains acquired stemness in breast cancer cells

AXL Is a Driver of Stemness in Normal Mammary Gland and Breast Cancer

The receptor tyrosine kinase AXL is associated with epithelial plasticity in several solid tumors including breast cancer and AXL-targeting agents are currently in clinical trials. We hypothesized that AXL is a driver of stemness traits in cancer by co-option of a regulatory function normally reserved for stem cells. AXL-expressing cells in human mammary epithelial ducts co-expressed markers associated with multipotency, and AXL inhibition abolished colony formation and self-maintenance activities while promoting terminal differentiation in&nbsp;vitro. Axl-null mice did not exhibit a strong developmental phenotype, but enrichment of Axl + cells was required for mouse mammary gland reconstitution upon transplantation, and Axl-null mice had reduced incidence of Wnt1-driven mammary tumors. An AXL-dependent gene signature is a feature of transcriptomes in basal breast cancers and reduced patient survival irrespective of subtype. Our interpretation is that AXL regulates access to epithelial plasticity programs in MaSCs and, when co-opted, maintains acquired stemness in breast cancer cells