A novel 3D co-culture platform for integrating tissue interfaces for tumor growth, migration and therapeutic sensitivity: “PP-3D-S”

Garakani, Mansoureh Mohseni; Ahangar, Pouyan; Watson, Sean; Nisol, Bernard; Wertheimer, M. R.; Rosenzweig, Derek H.; Ajji, Abdellah

doi:10.1016/j.msec.2021.112566

Cited by 10 publications

(16 citation statements)

References 72 publications

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“…3D cultures, where cells grow in three dimensions ( 40 ), better represent cell-to-cell and cell-to-extracellular environment interactions, morphology and cell division, and permit access to oxygen, nutrients, metabolites, and signaling molecules or cytokines ( 37 , 41 , 42 ). The use of 3D co-cultures is also highly relevant, since the characteristics of cells and their responses, including to drugs, can differ based on whether they are cultured in two versus three dimensions ( 42 ). Yet in both cases, while the cellular environments are more realistic, the means of assessing macrophage polarization becomes more difficult.…”

Section: Introductionmentioning

confidence: 99%

Murine macrophage-based iNos reporter reveals polarization and reprogramming in the context of breast cancer

et al. 2023

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As part of the first line of defense against pathogens, macrophages possess the ability to differentiate into divergent phenotypes with varying functions. The process by which these cells change their characteristics, commonly referred to as macrophage polarization, allows them to change into broadly pro-inflammatory (M1) or anti-inflammatory (M2) subtypes, and depends on the polarizing stimuli. Deregulation of macrophage phenotypes can result in different pathologies or affect the nature of some diseases, such as cancer and atherosclerosis. Therefore, a better understanding of macrophage phenotype conversion in relevant models is needed to elucidate its potential roles in disease. However, there are few existing probes to track macrophage changes in multicellular environments. In this study, we generated an eGFP reporter cell line based on inducible nitric oxide synthase (iNos) promoter activity in RAW264.7 cells (RAW:iNos-eGFP). iNos is associated with macrophage activation to pro-inflammatory states and decreases in immune-suppressing ones. We validated the fidelity of the reporter for iNos following cytokine-mediated polarization and confirmed that reporter and parental cells behaved similarly. RAW:iNos-eGFP cells were then used to track macrophage responses in different in vitro breast cancer models, and their re-education from anti- to pro-inflammatory phenotypes via a previously reported pyrimido(5,4-b)indole small molecule, PBI1. Using two mouse mammary carcinoma cell lines, 4T1 and EMT6, effects on macrophages were assessed via conditioned media, two-dimensional/monolayer co-culture, and three-dimensional spheroid models. While conditioned media derived from 4T1 or EMT6 cells and monolayer co-cultures of each cancer cell line with RAW:iNos-eGFP cells all resulted in decreased fluorescence, the trends and extents of effects differed. We also observed decreases in iNos-eGFP signal in the macrophages in co-culture assays with 4T1- or EMT6-based spheroids. We then showed that iNos production is enhanced in these cancer models using PBI1, tracking increased fluorescence. Collectively, this work demonstrates that this reporter-based approach provides a facile means to study macrophage responses in complex, multicomponent environments. Beyond the initial studies presented here, this platform can be used with a variety of in vitro models and extended to in vivo applications with intravital imaging.

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Section: Introductionmentioning

confidence: 99%

Murine macrophage-based iNos reporter reveals polarization and reprogramming in the context of breast cancer

et al. 2023

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“…Adhering cell populations in the 3D mat network of Figure 2 are typically increased by plasma treatments (i) or (ii) by a factor of 5 or more compared with untreated (“control”) sample surfaces. [ 19,21 ]…”

Section: Introductionmentioning

confidence: 99%

“…Our group recently reported using O 2 plasma‐modified NF mat samples as 3D scaffolds, seeded with human fibroblasts to mimic a stromal tissue layer; cell adhesion on all three greatly exceeded that on untreated controls. [ 19 ] After 7 days, cell behaviors were virtually indistinguishable among the three materials, quite surprising if one considers their very different structures and mechanical stiffness characteristics: Tensile moduli (Young's modulus as an index of stiffness) of electrospun NF mats are roughly between 50 and 300, 5–50, and 0.5–2 MPa, depending on fiber sizes, for PLA, [ 26 ] PCL, [ 27 ] and PU, [ 28 ] respectively. In other words, the choice of three polymers ranging from very soft (PU) to medium (PCL) and high stiffness (PLA) values is an asset for this aging‐related investigation because Stachewicz [ 23 ] had also identified mechanical properties to have a strong bearing in wetting studies.…”

Section: Introductionmentioning

confidence: 99%

Wetting and aging behaviors for several O₂ and NH₃ plasma treated nanofibrous and flat film polymer materials: A comparison

Watson

Pattyn

Garakani

et al. 2023

Plasma Processes & Polymers

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Aging (or "hydrophobic recovery") of plasma-modified polymer surfaces has been known and documented in the literature over several decades; to the best of our knowledge, the present study appears to be the first in which this is done for two vastly different rough surface structures: (i) electrospun nanofibrous (NF) mats, and (ii) flat films (FF), for three polymers of well-documented interest in biotechnological applications: poly(lactic acid); poly(urethane); and poly(caprolactone). Two different plasma treatments are applied: low-pressure (LP) radiofrequency (rf) glow discharges in flows of O 2 and NH 3 under mild power conditions. Measured time-dependent surface compositions (from X-ray photoelectron spectroscopy survey spectra) and water contact angles (WCA) were found to tend toward asymptotic limiting values after ca. 30 days of storage in clean air, as previously reported by these and other authors. An entirely novel aspect of this work is to examine and compare time-dependent WCA behaviors of NF and FF samples in terms of Wenzel (W)

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“…37,38 3D cultures, where cells grow in three dimensions, 40 better represent cell-to-cell and cell-to-extracellular environment interactions, morphology and cell division, and access to oxygen, nutrients, metabolites, and signaling molecules or cytokines. 37,41,42 The use of 3D co-cultures is also highly relevant, since the characteristics of cells and their responses, including to drugs, can be differ based on whether they are cultured in two versus three dimensions. 42 In both cases, however, while the cellular environments are more realistic, the means of assessing macrophage polarization becomes more difficult.…”

Section: Introductionmentioning

confidence: 99%

“…37,41,42 The use of 3D co-cultures is also highly relevant, since the characteristics of cells and their responses, including to drugs, can be differ based on whether they are cultured in two versus three dimensions. 42 In both cases, however, while the cellular environments are more realistic, the means of assessing macrophage polarization becomes more difficult. For this reason, we generated a reporter cell line to track the expression of a phenotype-associated marker in relevant disease models over time and following drug treatment.…”

Section: Introductionmentioning

confidence: 99%

Macrophage-Based iNos Reporter Reveals Polarization and Reprogramming in the Context of Breast Cancer

Mas-Rosario¹,

Medor

Jeffway

et al. 2022

Preprint

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As part of the first line of defense against pathogens, macrophages possess the ability to differentiate into divergent phenotypes with varying functions. The process by which these cells change their characteristics, commonly referred to as macrophage polarization, allows them to change into broadly pro-inflammatory (M1) or anti-inflammatory (M2) subtypes, and depends on the polarizing stimuli. Deregulation of macrophage phenotypes can result in different pathologies or affect the nature of some diseases, such as cancer and atherosclerosis. For this reason, it is necessary to better understand macrophage phenotype conversion in relevant models. However, there are few existing probes to track macrophage changes in multicellular environments. In this study, we generated an eGFP reporter cell line based on inducible nitric oxide synthase (iNos) promoter activity in RAW264.7 cells (RAW:iNos-eGFP). iNos is associated with macrophage activation to pro-inflammatory states, and decreases in immune-suppressing ones. We validated the fidelity of the reporter for iNos, including following cytokine-mediated polarization, and confirmed that reporter and parental cells behaved similarly. RAW:iNos-eGFP cells were then used to track macrophage responses in different in vitro breast cancer models, and their re-education from anti- to pro-inflammatory phenotypes via a previously reported pyrimido(5,4-b)indole small molecule, PBI1. Using two mouse mammary carcinoma cell lines, 4T1 and EMT6, effects on macrophages were assessed via conditioned media, two-dimensional/monolayer co-culture, and three-dimensional spheroid models. While conditioned media derived from 4T1 or EMT6 cells and monolayer co-cultures of each with RAW:iNos-eGFP cells all resulted in decreased fluorescence, the trends and extents of effects differed. We also observed decreases in iNos-eGFP signal in the macrophages in co-culture assays with 4T1- or EMT6-based spheroids. We then showed that we are able to enhance iNos production in the context of these cancer models using PBI1, tracking increased fluorescence. Taken together, we demonstrate that this reporter-based approach provides a facile means to study macrophage responses in complex, multicomponent environments. Beyond the initial studies presented here, this platform can be used with a variety of in vitro models and extended to in vivo applications with intravital imaging.

show abstract

A novel 3D co-culture platform for integrating tissue interfaces for tumor growth, migration and therapeutic sensitivity: “PP-3D-S”

Cited by 10 publications

References 72 publications

Murine macrophage-based iNos reporter reveals polarization and reprogramming in the context of breast cancer

Murine macrophage-based iNos reporter reveals polarization and reprogramming in the context of breast cancer

Wetting and aging behaviors for several O₂ and NH₃ plasma treated nanofibrous and flat film polymer materials: A comparison

Macrophage-Based iNos Reporter Reveals Polarization and Reprogramming in the Context of Breast Cancer

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A novel 3D co-culture platform for integrating tissue interfaces for tumor growth, migration and therapeutic sensitivity: “PP-3D-S”

Cited by 10 publications

References 72 publications

Murine macrophage-based iNos reporter reveals polarization and reprogramming in the context of breast cancer

Murine macrophage-based iNos reporter reveals polarization and reprogramming in the context of breast cancer

Wetting and aging behaviors for several O2 and NH3 plasma treated nanofibrous and flat film polymer materials: A comparison

Macrophage-Based iNos Reporter Reveals Polarization and Reprogramming in the Context of Breast Cancer

Contact Info

Product

Resources

About

Wetting and aging behaviors for several O₂ and NH₃ plasma treated nanofibrous and flat film polymer materials: A comparison