Dual-Wave Acoustofluidic Centrifuge for Ultrafast Concentration of Nanoparticles and Extracellular Vesicles.

Extracellular vesicles (EVs) are secreted nanostructures that play various roles in critical cancer processes. They operate as an intercellular communication system, transferring complex sets of biomolecules from cell to cell. The concentration of EVs is difficult to decipher, and there is an unmet technological need for improved (faster, simpler, and gentler) approaches to isolate EVs from complex matrices. Herein, an acoustofluidic concentration of extracellular vesicles (ACEV) is presented, based on a thin-film printed circuit board with interdigital electrodes mounted on a piezoelectric substrate. An angle of 120° is identified between the electrodes and the reference flat of the piezoelectric substrate for simultaneous generation of Rayleigh and shear horizontal waves. The dual waves create a complex acoustic field in a droplet, resulting in effective concentration of nanoparticles and EVs. The ACEV is able to concentrate 20 nm nanospheres within 105 s and four EV dilutions derived from the human prostate cancer (Du145) cell line in approximately 30 s. Cryo-electron microscopy confirmed the preservation of EV integrity. The ACEV device holds great potential to revolutionize investigations of EVs. Its faster, simpler, and gentler approach to EV isolation and concentration can save time and effort in phenotypic and functional studies of EVs.

Quantitative Imaging of B1 Cyclin Expression Across the Cell Cycle Using Green Fluorescent Protein Tagging and Epifluorescence.

In this article, we report the number of cyclin B1 proteins tagged with enhanced green fluorescent protein (eGFP) in fixed U-2 OS cells across the cell cycle. We use a quantitative analysis of epifluorescence to determine the number of eGFP molecules in a nondestructive way, and integrated over the cell we find 104 to 105 molecules. Based on the measured number of eGFP tagged cyclin B1 proteins, knowledge of cyclin B1 dynamics through the cell cycle, and the cell morphology, we identify the stages of cells in the cell cycle. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Label-Free Volumetric Quantitative Imaging of the Human Somatic Cell Division by Hyperspectral Coherent Anti-Stokes Raman Scattering.

Quantifying the chemical composition of unstained intact tissue and cellular samples with high spatio-temporal resolution in three dimensions would provide a step change in cell and tissue analytics critical to progress the field of cell biology. Label-free optical microscopy offers the required resolution and noninvasiveness, yet quantitative imaging with chemical specificity is a challenging endeavor. In this work, we show that hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy can be used to provide quantitative volumetric imaging of human osteosarcoma cells at various stages through cell division, a fundamental component of the cell cycle progress resulting in the segregation of cellular content to produce two progeny. We have developed and applied a quantitative data analysis method to produce volumetric three-dimensional images of the chemical composition of the dividing cell in terms of water, proteins, DNAP (a mixture of proteins and DNA, similar to chromatin), and lipids. We then used these images to determine the dry masses of the corresponding organic components. The attribution of proteins and DNAP components was validated using specific well-characterized fluorescent probes, by comparison with correlative two-photon fluorescence microscopy of DNA and mitochondria. Furthermore, we map the same chemical components under perturbed conditions, employing a drug that interferes directly with cell division (Taxol), showing its influence on cell organization and the masses of proteins, DNAP, and lipids.

Nuclear cytometry and chromatin organization.

The nuclear-targeting chemical probe, for the detection and quantification of DNA within cells, has been a mainstay of cytometry-from the colorimetric Feulgen stain to smart fluorescent agents with tuned functionality. The level of nuclear structure and function at which the probe aims to readout, or indeed at which a DNA-targeted drug acts, is shadowed by a wide range of detection modalities and analytical methods. These methods are invariably limited in terms of the resolution attainable versus the volume occupied by targeted chromatin structures. The scalar challenge arises from the need to understand the extent and different levels of compaction of genomic DNA and how such structures can be re-modeled, reported, or even perturbed by both probes and drugs. Nuclear cytometry can report on the complex levels of chromatin order, disorder, disassembly, and even active disruption by probes and drugs. Nuclear probes can report defining features of clinical and therapeutic interest as in NETosis and other cell death processes. New cytometric approaches continue to bridge the scalar challenges of analyzing chromatin organization. Advances in super-resolution microscopy address the resolution and depth of analysis issues in cellular systems. Typical of recent insights into chromatin organization enabled by exploiting a DNA interacting probe is ChromEM tomography (ChromEMT). ChromEMT uses the unique properties of the anthraquinone-based cytometric dye DRAQ5™ to reveal that local and global 3D chromatin structures effect differences in compaction. The focus of this review is nuclear and chromatin cytometry, with linked reference to DNA targeting probes and drugs as exemplified by the anthracenediones.

Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis.

Objective: Macrophage inflammatory protein 1-alpha (CCL3) is a chemokine that regulates macrophage trafficking to the inflamed joint. The agonistic effect of CCL3 on osteolytic lesions in patients with multiple myeloma is recognized; however, its role in skeletal damage during inflammatory arthritis has not been established. The aim of the study was to explore the role of osteoclast-associated CCL3 upon bone resorption, and to test its pharmacological blockade for protecting against bone pathology during inflammatory arthritis. Methods: CCL3 production was studied during osteoclast differentiation from osteoclast precursor cells: human CD14-positive mononuclear cells. Mice with CIA were treated with an anti-CCL3 antibody. The effect of CCL3 blockade through mAb was studied through osteoclast number, cytokine production and bone resorption on ivory disks, and in vivo through CIA progression (clinical score, paw diameter, synovial inflammation and bone damage). Results: Over time, CCL3 increased in parallel with the number of osteoclasts in culture. Anti-CCL3 treatment achieved a concentration-dependent inhibition of osteoclast fusion and reduced pit formation on ivory disks (P ⩽ 0.05). In CIA, anti-CCL3 treatment reduced joint damage and significantly decreased multinucleated tartrate-resistant acid phosphatase-positive osteoclasts and erosions in the wrists (P < 0.05) and elbows (P < 0.05), while also reducing joint erosions in the hind (P < 0.01) and fore paws (P < 0.01) as confirmed by X-ray. Conclusion: Inhibition of osteoclast-associated CCL3 reduced osteoclast formation and function whilst attenuating arthritis-associated bone loss and controlling development of erosion in murine joints, thus uncoupling bone damage from inflammation. Our findings may help future innovations for the diagnosis and treatment of inflammatory arthritis.

Probing cytochrome P450 bioactivation and fluorescent properties with morpholinyl-tethered anthraquinones.

Structural features from the anticancer prodrug nemorubicin (MMDX) and the DNA-binding molecule DRAQ5™ were used to prepare anthraquinone-based compounds, which were assessed for their potential to interrogate cytochrome P450 (CYP) functional activity and localisation. 1,4-disubstituted anthraquinone 8 was shown to be 5-fold more potent in EJ138 bladder cancer cells after CYP1A2 bioactivation. In contrast, 1,5-bis((2-morpholinoethyl)amino) substituted anthraquinone 10 was not CYP-bioactivated but was shown to be fluorescent and subsequently photo-activated by a light pulse (at a bandwidth 532-587 nm), resulting in punctuated foci accumulation in the cytoplasm. It also showed low toxicity in human osteosarcoma cells. These combined properties provide an interesting prospective approach for opto-tagging single or a sub-population of cells and seeking their location without the need for continuous monitoring.

The unidirectional hypoxia-activated prodrug OCT1002 inhibits growth and vascular development in castrate-resistant prostate tumors.

BACKGROUND: OCT1002 is a unidirectional hypoxia-activated prodrug (uHAP) OCT1002 that can target hypoxic tumor cells. Hypoxia is a common feature in prostate tumors and is known to drive disease progression and metastasis. It is, therefore, a rational therapeutic strategy to directly target hypoxic tumor cells in an attempt to improve treatment for this disease. Here we tested OCT1002 alone and in combination with standard-of-care agents in hypoxic models of castrate-resistant prostate cancer (CRPC). METHODS: The effect of OCT1002 on tumor growth and vasculature was measured using murine PC3 xenograft and dorsal skin fold (DSF) window chamber models. The effects of abiraterone, docetaxel, and cabazitaxel, both singly and in combination with OCT1002, were also compared. RESULTS: The hypoxia-targeting ability of OCT1002 effectively controls PC3 tumor growth. The effect was evident for at least 42 days after exposure to a single dose (30 mg/kg) and was comparable to, or better than, drugs currently used in the clinic. In DSF experiments OCT1002 caused vascular collapse in the PC3 tumors and inhibited the revascularization seen in controls. In this model OCT1002 also enhanced the anti-tumor effects of abiraterone, cabazitaxel, and docetaxel; an effect which was accompanied by a more prolonged reduction in tumor vasculature density. CONCLUSIONS: These studies provide the first evidence that OCT1002 can be an effective agent in treating hypoxic, castrate-resistant prostate tumors, either singly or in combination with established chemotherapeutics for prostate cancer.

ProtocolNavigator: emulation-based software for the design, documentation and reproduction biological experiments.

MOTIVATION: Experimental reproducibility is fundamental to the progress of science. Irreproducible research decreases the efficiency of basic biological research and drug discovery and impedes experimental data reuse. A major contributing factor to irreproducibility is difficulty in interpreting complex experimental methodologies and designs from written text and in assessing variations among different experiments. Current bioinformatics initiatives either are focused on computational research reproducibility (i.e. data analysis) or laboratory information management systems. Here, we present a software tool, ProtocolNavigator, which addresses the largely overlooked challenges of interpretation and assessment. It provides a biologist-friendly open-source emulation-based tool for designing, documenting and reproducing biological experiments. AVAILABILITY AND IMPLEMENTATION: ProtocolNavigator was implemented in Python 2.7, using the wx module to build the graphical user interface. It is a platform-independent software and freely available from http://protocolnavigator.org/index.html under the GPL v2 license.

A 3D ex vivo mandible slice system for longitudinal culturing of transplanted dental pulp progenitor cells.

Harnessing mesenchymal stem cells for tissue repair underpins regenerative medicine. However, how the 3D tissue matrix maintains such cells in a quiescent state whilst at the same time primed to respond to tissue damage remains relatively unknown. Developing more physiologically relevant 3D models would allow us to better understand the matrix drivers and influence on cell-lineage differentiation in situ. In this study, we have developed an ex vivo organotypic rat mandible slice model; a technically defined platform for the culture and characterization of dental pulp progenitor cells expressing GFP driven by the ß-actin promoter (cGFP DPPCs). Using confocal microscopy we have characterized how the native environment influences the progenitor cells transplanted into the dental pulp. Injected cGFP-DPPCs were highly viable and furthermore differentially proliferated in unique regions of the mandible slice; in the dentine region, cGFP-DPPCs showed a columnar morphology indicative of expansion and lineage differentiation. Hence, we demonstrated the systematic capacity for establishing a dental pulp cell-micro-community, phenotypically modified in the tooth (the "biology"); and at the same time addressed technical challenges enabling the mandible slice to be accessible on platforms for high-content imaging (the biology in a "multiplex" format).

Harmonizing the Generation and Pre-publication Stewardship of FAIR Image data.

Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured image data must be shared among labs at a global scale to be compared, pooled, and reanalyzed, thus unleashing untold potential beyond the original purpose for which the data was generated. There are two broad sets of requirements to enable image data sharing in the life sciences. One set of requirements is articulated in the companion White Paper entitled "Enabling Global Image Data Sharing in the Life Sciences," which is published in parallel and addresses the need to build the cyberinfrastructure for sharing the digital array data (arXiv:2401.13023 [q-bio.OT], https://doi.org/10.48550/arXiv.2401.13023). In this White Paper, we detail a broad set of requirements, which involves collecting, managing, presenting, and propagating contextual information essential to assess the quality, understand the content, interpret the scientific implications, and reuse image data in the context of the experimental details. We start by providing an overview of the main lessons learned to date through international community activities, which have recently made considerable progress toward generating community standard practices for imaging Quality Control (QC) and metadata. We then provide a clear set of recommendations for amplifying this work. The driving goal is to address remaining challenges, and democratize access to common practices and tools for a spectrum of biomedical researchers, regardless of their expertise, access to resources, and geographical location.

Kinetic analysis of intracellular Hoechst 33342--DNA interactions by flow cytometry: misinterpretation of side population status?

We outline a simple approach involving instrument setup and calibration for the analysis of Hoechst dye 33342-loading in human cell lines for exploring heterogeneity in dye efflux efficiency and the status of side population (SP) A549 lung cancer cells. Dual excitation 488 nm/multiline UV (351-364 nm) flow cytometry was used to confirm ABCG2-specific inhibition of dye efflux using Fumitremorgin C. Transporter gene expression, assayed by qRT-PCR, confirmed higher expression of ABCG2 versus ABCB1, reiterated in a cloned subline. Coexpression of aldehyde dehydrogenase genes ranked as aldehyde dehydrogenase class 1A1 (ALDH1A1) > ALDH3A1 > ALDH3, relative expression of all genes was again reiterated in a cloned subline. Permeabilized cells were used to create red:violet (660:405 nm Em wavelengths) ratiometric references for mapping temporal changes in Hoechst 33342-DNA fluorescence in live cells. A live cell "kinetic SP gate" tracked progressive dye loading of the whole population and coapplication of the far red (>695 nm wavelength) fluorescing dye DRAQ7 enabled viable cell gating. Kinetic gating revealed a continuum for dye accumulation suggesting that SP enumeration is critically dependent upon the nonlinear relationship of the spectral shift with progressive dye-DNA binding and thus requires accurate definition. To this end, permeabilized cell reference samples permit reproducible instrument setup, guide gate boundaries for SP and compromised cells, and offer a simple means of comparing SP enumeration across laboratory sites/platforms. Our approach reports the dynamic range for the spectral shift, revealing noninformative staining conditions and explaining a source of variability for SP enumeration. We suggest that live cell kinetic sorting of all cells with the same dye:DNA load but with differences in efflux capacity can be used to explore drug resistance capability without prejudice. The SP phenotype should be regarded as a kinetic parameter and not a fixed characteristic--critical for functional assay design and the interpretation of heterogeneity.

Real-time cell viability assays using a new anthracycline derivative DRAQ7®.

The exclusion of charged fluorescent dyes by intact cells has become a well-established assay for determining viability of cells. In search for a noninvasive fluorescent probe capable of long-term monitoring of cell death in real-time, we evaluated a new anthracycline derivative DRAQ7. The novel probe does not penetrate the plasma membrane of living cells but when the membrane integrity is compromised, it enters and binds readily to nuclear DNA to report cell death. It proved to be nontoxic to a panel of cancer cell lines grown continuously for up to 72 h and did not induce any detectable DNA damage signaling when analyzed using laser scanning microscopy and flow cytometry. The DRAQ7 provided a sensitive, real-time readout of cell death induced by a variety of stressors such as hypoxia, starvation, and drug-induced cytotoxicity. The overall responses to anticancer agents and resulting pharmacological dose-response profiles were not affected by the growth of tumor cells in the presence DRAQ7. Moreover, we for the first time introduced a near real-time microflow cytometric assay based on combination of DRAQ7 and mitochondrial inner membrane potential (ΔΨ(m) ) sensitive probe TMRM. We provide evidence that this low-dosage, real-time labeling procedure provides multiparameter and kinetic fingerprint of anticancer drug action.

NCAM polysialylation during adherence transitions: live cell monitoring using an antibody-mimetic EGFP-endosialidase and the viability dye DRAQ7.

Polysialylation of neural cell adhesion molecule (NCAM) in small-cell lung cancer (SCLC) is thought to regulate NCAM-mediated cell-surface interactions, imparting antiadhesive properties to cells. However, SCLC cells in culture demonstrate anchorage-independent growth and spontaneously generate adherent forms. Here, the ability of polySia-NCAM to influence cell proliferation and adherence is unclear. We analyzed live SCLC cell polySia-NCAM expression by flow cytometry, using the novel combination of a polySia antibody-mimetic eGFP-tagged endosialidase and the viability dye DRAQ7. Enrichment for adherence (<30 population doublings) in SCLC cell lines resolved populations with increased (SHP-77 and COR-L279) or negligible (NCI-H69) polysialylation compared with nonadherent parent populations. Adherent forms retained NCAM expression as confirmed by immunofluorescence and immunoblotting. Initial transition to adherence and loss of polysialylation in NCI-H69 was linked to a reduced proliferation rate with no increase in cell death. This reduced proliferation rate was reiterated in vivo as determined by the growth of noninvasive subcutaneous xenografts in mice. Continued selection for enhanced substrate adherence in NCI-H69 (>150 population doublings) resolved cells with stable re-expression of polySia and increased growth rates both in vitro and in vivo. Endoneuraminidase removal of polySia from re-expressing cells showed that rapid adherence to extracellular matrix components was functionally independent of polySia. PolySia expression was not altered when isolated adherent forms underwent enforced cell-cell contact in three-dimensional culture. Coculture of polySia expression variants modulated overall polySia expression profiles indicating an influence of SCLC microcommunity composition independent of substrate adherence potential. We conclude that an obligatory linkage between substrate adherence potential and polySia expression is rejected for SCLC cells. We suggest that a degree of homeostasis operates to regulate polysialylation within heterogeneous cell populations. The findings suggest a new model for SCLC progression while the application of live cell profiling of polysialylation could be used to assess polySia-NCAM-targeted therapies.

Micro-community cytometry: sensing changes in cell health and glycoconjugate expression by imaging and flow cytometry.

Discerning the extent of biologically relevant heterogeneity presents unique challenges to both microscopy and flow cytometry. Micro-environmental influences and stochastic changes in cellular behaviour can act to mask the origins of both progression and therapeutic resistance in tumour cell systems. In part the dimensionality of different and frequently metastable states can be assessed by multi-parameter flow cytometry with unparalleled statistical robustness. Complementary application of imaging can provide valuable insights into the complex temporal changes that can occur in cell micro-communities either spontaneously or in response to selection pressure. With an extensive range of methodologies for the labelling of cells there are multiple options for tracking cells, defining fate and the re-construction of provenance and behavioural history. The challenge is highlighted by attempts to identify the critical glycosylation events modifying the function of cell surface proteins. Central to a cytometric approach is the availability of methods that reveal cell health and are compatible with the detection of cell surface changes within dynamic micro-communities. The review briefly addresses the options for sensing cell health and the co-application of an antibody mimetic for detection of cell surface glycoconjugate expression accessible for both imaging and flow cytometry.

Quantifying the microstructural and biomechanical changes in the porcine ventricles during growth and remodelling.

Cardiac tissue growth and remodelling (G & R) occur in response to the changing physiological demands of the heart after birth. The early shift to pulmonary circulation produces an immediate increase in ventricular workload, causing microstructural and biomechanical changes that serve to maintain overall physiological homoeostasis. Such cardiac G & R continues throughout life. Quantifying the tissue's mechanical and microstructural changes because of G & R is of increasing interest, dovetailing with the emerging fields of personalised and precision solutions. This study aimed to determine equibiaxial, and non-equibiaxial extension, stress-relaxation, and the underlying microstructure of the passive porcine ventricles tissue at four time points spanning from neonatal to adulthood. The three-dimensional microstructure was investigated via two-photon excited fluorescence and second-harmonic generation microscopy on optically cleared tissues, describing the 3D orientation, rotation and dispersion of the cardiomyocytes and collagen fibrils. The results revealed that during biomechanical testing, myocardial ventricular tissue possessed non-linear, anisotropic, and viscoelastic behaviour. An increase in stiffness and viscoelasticity was noted for the left and right ventricular free walls from neonatal to adulthood. Microstructural analyses revealed concomitant increases in cardiomyocyte rotation and dispersion. This study provides baseline data, describing the biomechanical and microstructural changes in the left and right ventricular myocardial tissue during G & R, which should prove valuable to researchers in developing age-specific, constitutive models for more accurate computational simulations. STATEMENT OF SIGNIFICANCE: There is a dearth of experimental data describing the growth and remodelling of left and right ventricular tissue. The published literature is fragmented, with data reported via different experimental techniques using tissues harvested from a variety of animals, with different gender and ages. This prevents developing a continuum of data spanning birth to death, so limiting the potential that can be leveraged to aid computational modelling and simulations. In this study, equibiaxial, non-equibiaxial, and stress-relaxation data are presented, describing directional-dependent material responses. The biomechanical data is consolidated with equivalent microstructural data, an important element for the development of future material models. Combined, these data describe microstructural and biomechanical changes in the ventricles, spanning G &R from neonatal to adulthood.

Interoperability of time series cytometric data: a cross platform approach for modeling tumor heterogeneity.

The cell cycle, with its highly conserved features, is a fundamental driver for the temporal control of cell proliferation-while abnormal control and modulation of the cell cycle are characteristic of tumor cells. The principal aim in cancer biology is to seek an understanding of the origin and nature of innate and acquired heterogeneity at the cellular level, driven principally by temporal and functional asynchrony. A major bottleneck when mathematically modeling these biological systems is the lack of interlinked structured experimental data. This often results in the in silico models failing to translate the specific hypothesis into parameterized terms that enable robust validation and hence would produce suitable prediction tools rather than just simulation tools. The focus has been on linking data originating from different cytometric platforms and cell-based event analysis to inform and constrain the input parameters of a compartmental cell cycle model, hence partly measuring and deconvolving cell cycle heterogeneity within a tumor population. Our work has addressed the concept that the interoperability of cytometric data, derived from different cytometry platforms, can complement as well as enhance cellular parameters space, thus providing a more broader and in-depth view of the cellular systems. The initial aim was to enable the cell cycle model to deliver an improved integrated simulation of the well-defined and constrained biological system. From a modeling perspective, such a cross platform approach has provided a paradigm shift from conventional cross-validation approaches, and from a bioinformatics perspective, novel computational methodology has been introduced for integrating and mapping continuous data with cross-sectional data. This establishes the foundation for developing predictive models and in silico tracking and prediction of tumor progression

Chemical synthesis of cell-permeable apoptotic peptides from in vivo produced proteins.

In vivo synthesis of peptides by bacterial expression has developed into a reliable alternative to solid-phase peptide synthesis. A significant drawback of in vivo methods is the difficulty with which gene products can be modified post-translationally. Here, we present a method for the facile modification of peptides generated in bacterial hosts after cyanogen bromide cleavage at C-terminal methionines. Reaction of the resulting homoserine lactones with propargylamine allows efficient and selective modification with a wide variety of chemicals such as fluorescent dyes, biotin derivatives, polyprenyls, lipids, polysaccharides, or peptides. Attachment of the cell penetrating peptide octa-arginine (R(8)) to peptides derived from the proapoptotic tumor suppressor Bak BH3 led to efficient cellular uptake and subsequent cytochrome c release from mitochondria, culminating in induction of apoptosis similar to that observed with peptides linked to R(8) via the peptide backbone. These results highlight the significant potential for use of such tools in live cells.

Flow-based cytometric analysis of cell cycle via simulated cell populations.

We present a new approach to the handling and interrogating of large flow cytometry data where cell status and function can be described, at the population level, by global descriptors such as distribution mean or co-efficient of variation experimental data. Here we link the "real" data to initialise a computer simulation of the cell cycle that mimics the evolution of individual cells within a larger population and simulates the associated changes in fluorescence intensity of functional reporters. The model is based on stochastic formulations of cell cycle progression and cell division and uses evolutionary algorithms, allied to further experimental data sets, to optimise the system variables. At the population level, the in-silico cells provide the same statistical distributions of fluorescence as their real counterparts; in addition the model maintains information at the single cell level. The cell model is demonstrated in the analysis of cell cycle perturbation in human osteosarcoma tumour cells, using the topoisomerase II inhibitor, ICRF-193. The simulation gives a continuous temporal description of the pharmacodynamics between discrete experimental analysis points with a 24 hour interval; providing quantitative assessment of inter-mitotic time variation, drug interaction time constants and sub-population fractions within normal and polyploid cell cycles. Repeated simulations indicate a model accuracy of +/-5%. The development of a simulated cell model, initialized and calibrated by reference to experimental data, provides an analysis tool in which biological knowledge can be obtained directly via interrogation of the in-silico cell population. It is envisaged that this approach to the study of cell biology by simulating a virtual cell population pertinent to the data available can be applied to "generic" cell-based outputs including experimental data from imaging platforms.

Macrophage Plasticity and Function in the Lung Tumour Microenvironment Revealed in 3D Heterotypic Spheroid and Explant Models.

In non-small cell lung cancer (NSCLC), stroma-resident and tumour-infiltrating macrophages may facilitate an immunosuppressive tumour microenvironment (TME) and hamper immunotherapeutic responses. Analysis of tumour-associated macrophage (TAM) plasticity in NSCLC is largely lacking. We established a novel, multi-marker, dual analysis approach for assessing monocyte-derived macrophage (Mφ) polarisation and M1/M2 phenotypic plasticity. We developed a flow cytometry-based, two-marker analysis (CD64 and CD206) of CD14+ cells. The phenotype and immune function of in vitro-induced TAMs was studied in a heterotypic spheroid and tumour-derived explant model of NSCLC. Heterotypic spheroids and NSCLC explants skewed Mφs from an M1- (CD206loCD64hi) to M2-like (CD206hiCD64lo) phenotype. Lipopolysaccharide (LPS) and IFNγ treatment reversed M2-like Mφ polarisation, indicating the plasticity of Mφs. Importantly, antigen-specific CD8+ T cell responses were reduced in the presence of tumour explant-conditioned Mφs, but not spheroid-conditioned Mφs, suggesting explants are likely a more relevant model of the immune TME than cell line-derived spheroids. Our data indicates the importance of multi-marker, functional analyses within Mφ subsets and the advantages of the ex vivo NSCLC explant model in immunomodulation studies. We highlight the plasticity of the M1/M2 phenotype using the explant model and provide a tool for studying therapeutic interventions designed to reprogram M2-like Mφ-induced immunosuppression.