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Article ; Online: Protocol for computationally evaluating the loss of stoichiometry and coordinated expression of proteins.

Hinz, Stefan / Todhunter, Michael E / LaBarge, Mark A

STAR protocols

2022 Volume 3, Issue 2, Page(s) 101182

Abstract: Dysregulation of the transcriptional or translational machinery can alter the stoichiometry of multiprotein complexes and occurs in natural processes such as aging. Loss of stoichiometry has been shown to alter protein complex functions. We provide a ... ...

Abstract	Dysregulation of the transcriptional or translational machinery can alter the stoichiometry of multiprotein complexes and occurs in natural processes such as aging. Loss of stoichiometry has been shown to alter protein complex functions. We provide a protocol and associated code that use omics data to quantify these stoichiometric changes via statistical dispersion utilizing the interquartile range of expression values per grouping variable. This descriptive statistical approach enables the quantification of stoichiometry changes without additional data acquisition. For complete details on the use and execution of this protocol, please refer to Hinz et al. (2021).
MeSH term(s)	Proteins/genetics ; Proteomics/methods
Chemical Substances	Proteins
Language	English
Publishing date	2022-03-16
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2022.101182
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: High-Throughput Microenvironment Microarray (MEMA) High-Resolution Imaging.

Jokela, Tiina A / Todhunter, Michael E / LaBarge, Mark A

Methods in molecular biology (Clifton, N.J.)

2022 Volume 2394, Page(s) 47–64

Abstract: The interaction between cells and their surrounding microenvironment has a crucial role in determining cell fate. In many pathological conditions, the microenvironment drives disease progression as well as therapeutic resistance. A number of challenges ... ...

Abstract	The interaction between cells and their surrounding microenvironment has a crucial role in determining cell fate. In many pathological conditions, the microenvironment drives disease progression as well as therapeutic resistance. A number of challenges arise for researchers examining these cell-microenvironment interactions: (1) Tissue microenvironments are combinatorial and dynamic systems, and in pathological situations like cancer, microenvironments become infamously chaotic and highly heterogeneous. (2) Cells exhibit heterogeneous phenotypes, and even rare cell subpopulations can have a substantial role in tissue homeostasis and disease progression. This chapter discusses technical aspects relevant to dissecting cell-microenvironment interaction using the Microenvironment Microarray (MEMA) platform, which is a cell-based functional high-throughput screening of interactions between cells and combinatorial microenvironments at the single-cell level. MEMA provides insights into how cell phenotype and function is elicited by microenvironmental components. In this chapter, we describe automating a high-throughput and high-resolution imaging pipeline for single-cell-resolution analysis.
MeSH term(s)	Cellular Microenvironment ; High-Throughput Screening Assays ; Humans ; Microarray Analysis ; Neoplasms/pathology ; Single-Cell Analysis ; Tumor Microenvironment
Language	English
Publishing date	2022-01-30
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-1811-0_4
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Article ; Online: Sustained postconfluent culture of human mammary epithelial cells enriches for luminal and c-Kit+ subtypes.

Todhunter, Michael E / Miyano, Masaru / Carlson, Eric G / Hinz, Stefan / LaBarge, Mark A

Breast cancer research : BCR

2023 Volume 25, Issue 1, Page(s) 6

Abstract: Background: A challenge in human mammary epithelial cell (HMEC) culture is sustaining the representation of competing luminal, myoepithelial, and progenitor lineages over time. As cells replicate in culture, myoepithelial cells come to dominate the ... ...

Abstract	Background: A challenge in human mammary epithelial cell (HMEC) culture is sustaining the representation of competing luminal, myoepithelial, and progenitor lineages over time. As cells replicate in culture, myoepithelial cells come to dominate the composition of the culture with serial passaging. This drift in composition presents a challenge for studying luminal and progenitor cells, which are prospective cells of origin for most breast cancer subtypes. Methods: We demonstrate the use of postconfluent culture on HMECs. Postconfluent culture entails culturing HMECs for 2-5 weeks without passaging but maintaining frequent feedings in low-stress M87A culture medium. In contrast, standard HMEC culture entails enzymatic subculturing every 3-5 days to maintain subconfluent density. Results: When compared to standard HMEC culture, postconfluent culture yields increased proportions of luminal cells and c-Kit+ progenitor cells. Postconfluent cultures develop a distinct multilayered morphology with individual cells showing decreased physical deformability as compared to cells in standard culture. Gene expression analysis of postconfluent cells shows increased expression of lineage-specific markers and extracellular matrix components. Conclusions: Postconfluent culture is a novel, useful strategy for altering the lineage composition of HMECs, by increasing the proportional representation of luminal and progenitor cells. We speculate that postconfluent culture creates a microenvironment with cellular composition closer to the physiological state and eases the isolation of scarce cell subtypes. As such, postconfluent culture is a valuable tool for researchers using HMECs for breast cancer research.
MeSH term(s)	Humans ; Female ; Breast Neoplasms/genetics ; Breast Neoplasms/metabolism ; Breast ; Epithelial Cells/metabolism ; Tumor Microenvironment
Language	English
Publishing date	2023-01-18
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2015059-3
ISSN	1465-542X ; 1465-5411
ISSN (online)	1465-542X
ISSN	1465-5411
DOI	10.1186/s13058-022-01595-z
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Zs.A 5494: Show issues

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Article ; Online: Cell and Tissue Biology Paves a Path to Breast Cancer Prevention.

Todhunter, Michael E / LaBarge, Mark A

Trends in cancer

2017 Volume 3, Issue 5, Page(s) 313–315

Abstract: We hypothesize that breast cancer susceptibility stems from interactions between difficult-to-modify cultural and dietary habits and aging processes that are modifiable. We propose a pathway to prevention that uses human organotypic systems that ... ...

Abstract	We hypothesize that breast cancer susceptibility stems from interactions between difficult-to-modify cultural and dietary habits and aging processes that are modifiable. We propose a pathway to prevention that uses human organotypic systems that recapitulate hallmarks of aging in order to better understand and to modulate the biological consequences of aging in breast.
MeSH term(s)	Breast Neoplasms/pathology ; Breast Neoplasms/prevention & control ; Female ; Humans
Language	English
Publishing date	2017
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, Non-U.S. Gov't
ZDB-ID	2852626-0
ISSN	2405-8025 ; 2405-8033 ; 2405-8033
ISSN (online)	2405-8025 ; 2405-8033
ISSN	2405-8033
DOI	10.1016/j.trecan.2017.03.001
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Article ; Online: Volume-constrained microcontainers enable myoepithelial functional differentiation in highly parallel mammary organoid culture.

Todhunter, Michael E / Miyano, Masaru / Moolamalla, Divya S / Filippov, Aleksandr / Sayaman, Rosalyn W / LaBarge, Mark A

iScience

2021 Volume 24, Issue 4, Page(s) 102253

Abstract: A long-standing constraint on organoid culture is the need to add exogenous substances to provide hydrogel matrix, which limits the study of fully human or fully native organoids. This paper introduces an approach to culture reconstituted mammary ... ...

Abstract	A long-standing constraint on organoid culture is the need to add exogenous substances to provide hydrogel matrix, which limits the study of fully human or fully native organoids. This paper introduces an approach to culture reconstituted mammary organoids without the impediment of exogenous matrix. We enclose organoids in nanoliter-scale, topologically enclosed, fluid compartments surrounded by agar. Organoids cultured in these "microcontainers" appear to secrete enough extracellular matrix to yield a self-sufficient microenvironment without exogenous supplements. In microcontainers, mammary organoids exhibit contractility and a high-level, physiological, myoepithelial (MEP) behavior that has not been previously reported in reconstituted organoids. The presence of contractility suggests that microcontainers elicit MEP functional differentiation, an important milestone. Microcontainers yield thousands of substantially identical and individually trackable organoids within a single culture vessel, enabling longitudinal studies and statistically powerful experiments, such as the evaluation of small effect sizes. Microcontainers open new doors for researchers who rely on organoid models.
Language	English
Publishing date	2021-03-01
Publishing country	United States
Document type	Journal Article
ISSN	2589-0042
ISSN (online)	2589-0042
DOI	10.1016/j.isci.2021.102253
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Article ; Online: Tissue aging: the integration of collective and variant responses of cells to entropic forces over time.

Todhunter, Michael E / Sayaman, Rosalyn W / Miyano, Masaru / LaBarge, Mark A

Current opinion in cell biology

2018 Volume 54, Page(s) 121–129

Abstract: Aging is driven by unavoidable entropic forces, physicochemical in nature, that damage the raw materials that constitute biological systems. Single cells experience and respond to stochastic physicochemical insults that occur either to the cells ... ...

Abstract	Aging is driven by unavoidable entropic forces, physicochemical in nature, that damage the raw materials that constitute biological systems. Single cells experience and respond to stochastic physicochemical insults that occur either to the cells themselves or to their microenvironment, in a dynamic and reciprocal manner, leading to increased age-related cell-to-cell variation. We will discuss the biological mechanisms that integrate cell-to-cell variation across tissues resulting in stereotypical phenotypes of age.
MeSH term(s)	Aging/physiology ; Entropy ; Humans ; Models, Biological ; Organ Specificity ; Phenotype ; Time Factors
Language	English
Publishing date	2018-06-19
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
ZDB-ID	1026381-0
ISSN	1879-0410 ; 0955-0674
ISSN (online)	1879-0410
ISSN	0955-0674
DOI	10.1016/j.ceb.2018.05.016
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Zs.A 2963: Show issues

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Article ; Online: Volume-constrained microcontainers enable myoepithelial functional differentiation in highly parallel mammary organoid culture

Michael E. Todhunter / Masaru Miyano / Divya S. Moolamalla / Aleksandr Filippov / Rosalyn W. Sayaman / Mark A. LaBarge

iScience, Vol 24, Iss 4, Pp 102253- (2021)

2021

Abstract: Summary: A long-standing constraint on organoid culture is the need to add exogenous substances to provide hydrogel matrix, which limits the study of fully human or fully native organoids. This paper introduces an approach to culture reconstituted ... ...

Abstract	Summary: A long-standing constraint on organoid culture is the need to add exogenous substances to provide hydrogel matrix, which limits the study of fully human or fully native organoids. This paper introduces an approach to culture reconstituted mammary organoids without the impediment of exogenous matrix. We enclose organoids in nanoliter-scale, topologically enclosed, fluid compartments surrounded by agar. Organoids cultured in these “microcontainers” appear to secrete enough extracellular matrix to yield a self-sufficient microenvironment without exogenous supplements. In microcontainers, mammary organoids exhibit contractility and a high-level, physiological, myoepithelial (MEP) behavior that has not been previously reported in reconstituted organoids. The presence of contractility suggests that microcontainers elicit MEP functional differentiation, an important milestone. Microcontainers yield thousands of substantially identical and individually trackable organoids within a single culture vessel, enabling longitudinal studies and statistically powerful experiments, such as the evaluation of small effect sizes. Microcontainers open new doors for researchers who rely on organoid models.
Keywords	Cell Biology ; Stem Cells Research ; Bioengineering ; Tissue Engineering ; Science ; Q
Language	English
Publishing date	2021-04-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Evidence for accelerated aging in mammary epithelia of women carrying germline

Shalabi, Sundus F / Miyano, Masaru / Sayaman, Rosalyn W / Lopez, Jennifer C / Jokela, Tiina A / Todhunter, Michael E / Hinz, Stefan / Garbe, James C / Stampfer, Martha R / Kessenbrock, Kai / Seewaldt, Victoria E / LaBarge, Mark A

Nature aging

2021 Volume 1, Issue 9, Page(s) 838–849

Abstract: During aging in the human mammary gland, luminal epithelial cells lose lineage fidelity by expressing markers normally expressed in myoepithelial cells. We hypothesize that loss of lineage fidelity is a general manifestation of epithelia that are ... ...

Abstract	During aging in the human mammary gland, luminal epithelial cells lose lineage fidelity by expressing markers normally expressed in myoepithelial cells. We hypothesize that loss of lineage fidelity is a general manifestation of epithelia that are susceptible to cancer initiation. In the present study, we show that histologically normal breast tissue from younger women who are susceptible to breast cancer, as a result of harboring a germline mutation in
MeSH term(s)	Humans ; Female ; Mammary Glands, Human ; Aging/genetics ; Breast/pathology ; Germ-Line Mutation/genetics ; Breast Neoplasms/genetics ; BRCA1 Protein/genetics ; BRCA2 Protein/genetics
Chemical Substances	BRCA1 protein, human ; BRCA1 Protein ; BRCA2 protein, human ; BRCA2 Protein
Language	English
Publishing date	2021-09-14
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	2662-8465
ISSN (online)	2662-8465
DOI	10.1038/s43587-021-00104-9
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Article ; Online: Deep proteome profiling of human mammary epithelia at lineage and age resolution.

Hinz, Stefan / Manousopoulou, Antigoni / Miyano, Masaru / Sayaman, Rosalyn W / Aguilera, Kristina Y / Todhunter, Michael E / Lopez, Jennifer C / Sohn, Lydia L / Wang, Leo D / LaBarge, Mark A

iScience

2021 Volume 24, Issue 9, Page(s) 103026

Abstract: Age is the major risk factor in most carcinomas, yet little is known about how proteomes change with age in any human epithelium. We present comprehensive proteomes comprised of >9,000 total proteins and >15,000 phosphopeptides from normal primary human ... ...

Abstract	Age is the major risk factor in most carcinomas, yet little is known about how proteomes change with age in any human epithelium. We present comprehensive proteomes comprised of >9,000 total proteins and >15,000 phosphopeptides from normal primary human mammary epithelia at lineage resolution from ten women ranging in age from 19 to 68 years. Data were quality controlled and results were biologically validated with cell-based assays. Age-dependent protein signatures were identified using differential expression analyses and weighted protein co-expression network analyses. Upregulation of basal markers in luminal cells, including KRT14 and AXL, were a prominent consequence of aging. PEAK1 was identified as an age-dependent signaling kinase in luminal cells, which revealed a potential age-dependent vulnerability for targeted ablation. Correlation analyses between transcriptome and proteome revealed age-associated loss of proteostasis regulation. Age-dependent proteome changes in the breast epithelium identified heretofore unknown potential therapeutic targets for reducing breast cancer susceptibility.
Language	English
Publishing date	2021-08-23
Publishing country	United States
Document type	Journal Article
ISSN	2589-0042
ISSN (online)	2589-0042
DOI	10.1016/j.isci.2021.103026
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Article ; Online: Opportunities for organoids as new models of aging.

Hu, Jennifer L / Todhunter, Michael E / LaBarge, Mark A / Gartner, Zev J

The Journal of cell biology

2017 Volume 217, Issue 1, Page(s) 39–50

Abstract: The biology of aging is challenging to study, particularly in humans. As a result, model organisms are used to approximate the physiological context of aging in humans. However, the best model organisms remain expensive and time-consuming to use. More ... ...

Abstract	The biology of aging is challenging to study, particularly in humans. As a result, model organisms are used to approximate the physiological context of aging in humans. However, the best model organisms remain expensive and time-consuming to use. More importantly, they may not reflect directly on the process of aging in people. Human cell culture provides an alternative, but many functional signs of aging occur at the level of tissues rather than cells and are therefore not readily apparent in traditional cell culture models. Organoids have the potential to effectively balance between the strengths and weaknesses of traditional models of aging. They have sufficient complexity to capture relevant signs of aging at the molecular, cellular, and tissue levels, while presenting an experimentally tractable alternative to animal studies. Organoid systems have been developed to model many human tissues and diseases. Here we provide a perspective on the potential for organoids to serve as models for aging and describe how current organoid techniques could be applied to aging research.
MeSH term(s)	Aging/physiology ; Animals ; Cell Culture Techniques/methods ; Cell Proliferation/physiology ; Humans ; Models, Theoretical ; Organ Culture Techniques/methods ; Organoids/physiology ; Pluripotent Stem Cells/physiology
Language	English
Publishing date	2017-12-20
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
ZDB-ID	218154-x
ISSN	1540-8140 ; 0021-9525
ISSN (online)	1540-8140
ISSN	0021-9525
DOI	10.1083/jcb.201709054
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