Modular dual-color BiAD sensors for locus-specific readout of epigenome modifications in single cells.

Dynamic changes in the epigenome at defined genomic loci play crucial roles during cellular differentiation and disease development. Here, we developed dual-color bimolecular anchor detector (BiAD) sensors for high-sensitivity readout of locus-specific epigenome modifications by fluorescence microscopy. Our BiAD sensors comprise an sgRNA/dCas9 complex as anchor and double chromatin reader domains as detector modules, both fused to complementary parts of a split IFP2.0 fluorophore, enabling its reconstitution upon binding of both parts in close proximity. In addition, a YPet fluorophore is recruited to the sgRNA to mark the genomic locus of interest. With these dual-color BiAD sensors, we detected H3K9me2/3 and DNA methylation and their dynamic changes upon RNAi or inhibitor treatment with high sensitivity at endogenous genomic regions. Furthermore, we showcased locus-specific H3K36me2/3 readout as well as H3K27me3 and H3K9me2/3 enrichment on the inactive X chromosome, highlighting the broad applicability of our dual-color BiAD sensors for single-cell epigenome studies.

Repeat DNA methylation is modulated by adherens junction signaling.

Through its involvement in gene transcription and heterochromatin formation, DNA methylation regulates how cells interact with their environment. Nevertheless, the extracellular signaling cues that modulate the distribution of this central chromatin modification are largely unclear. DNA methylation is highly abundant at repetitive elements, but its investigation in live cells has been complicated by methodological challenges. Utilizing a CRISPR/dCas9 biosensor that reads DNA methylation of human α-satellite repeats in live cells, we here uncover a signaling pathway linking the chromatin and transcriptional state of repetitive elements to epithelial adherens junction integrity. Specifically, we find that in confluent breast epithelial cell monolayers, α-satellite repeat methylation is reduced by comparison to low density cultures. This is coupled with increased transcriptional activity at repeats. Through comprehensive perturbation experiments, we identify the junctional protein E-cadherin, which links to the actin cytoskeleton, as a central molecular player for signal relay into the nucleus. Furthermore, we find that this pathway is impaired in cancer cells that lack E-cadherin and are not contact-inhibited. This suggests that the molecular connection between cell density and repetitive element methylation could play a role in the maintenance of epithelial tissue homeostasis.

Learning Dynamics from Multicellular Graphs with Deep Neural Networks.

The inference of multicellular self-assembly is the central quest of understanding morphogenesis, including embryos, organoids, tumors, and many others. However, it has been tremendously difficult to identify structural features that can indicate multicellular dynamics. Here we propose to harness the predictive power of graph-based deep neural networks (GNN) to discover important graph features that can predict dynamics. To demonstrate, we apply a physically informed GNN (piGNN) to predict the motility of multi-cellular collectives from a snapshot of their positions both in experiments and simulations. We demonstrate that piGNN is capable of navigating through complex graph features of multicellular living systems, which otherwise can not be achieved by classical mechanistic models. With increasing amounts of multicellular data, we propose that collaborative efforts can be made to create a multicellular data bank (MDB) from which it is possible to construct a large multicellular graph model (LMGM) for general-purposed predictions of multicellular organization.

Golgi screen identifies the RhoGEF Solo as a novel regulator of RhoB and endocytic transport.

The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase-activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy-based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP-RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking.

Proteasomal turnover of the RhoGAP tumor suppressor DLC1 is regulated by HECTD1 and USP7.

The Rho GTPase activating protein Deleted in Liver Cancer 1 (DLC1) is frequently downregulated through genetic and epigenetic mechanisms in various malignancies, leading to aberrant Rho GTPase signaling and thus facilitating cancer progression. Here we show that in breast cancer cells, dysregulation of DLC1 expression occurs at the protein level through rapid degradation via the ubiquitin-proteasome system. Using mass spectrometry, we identify two novel DLC1 interaction partners, the ubiquitin-ligase HECTD1 and the deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7). While DLC1 protein expression was rapidly downregulated upon pharmacological inhibition of USP7, siRNA-mediated knockdown of HECTD1 increased DLC1 protein levels and impaired its degradation. Immunofluorescence microscopy analyses revealed that the modulation of HECTD1 levels and USP7 activity altered DLC1 abundance at focal adhesions, its primary site of action. Thus, we propose opposing regulatory mechanisms of DLC1 protein homeostasis by USP7 and HECTD1, which could open up strategies to counteract downregulation and restore DLC1 expression in cancer.

Triple Targeting of HER Receptors Overcomes Heregulin-mediated Resistance to EGFR Blockade in Colorectal Cancer.

Current treatment options for patients with advanced colorectal cancers include anti-EGFR/HER1 therapy with the blocking antibody cetuximab. Although a subset of patients with KRAS WT disease initially respond to the treatment, resistance develops in almost all cases. Relapse has been associated with the production of the ligand heregulin (HRG) and/or compensatory signaling involving the receptor tyrosine kinases HER2 and HER3. Here, we provide evidence that triple-HER receptor blockade based on a newly developed bispecific EGFR×HER3-targeting antibody (scDb-Fc) together with the HER2-blocking antibody trastuzumab effectively inhibited HRG-induced HER receptor phosphorylation, downstream signaling, proliferation, and stem cell expansion of DiFi and LIM1215 colorectal cancer cells. Comparative analyses revealed that the biological activity of scDb-Fc plus trastuzumab was sometimes even superior to that of the combination of the parental antibodies, with PI3K/Akt pathway inhibition correlating with improved therapeutic response and apoptosis induction as seen by single-cell analysis. Importantly, growth suppression by triple-HER targeting was recapitulated in primary KRAS WT patient-derived organoid cultures exposed to HRG. Collectively, our results provide strong support for a pan-HER receptor blocking approach to combat anti-EGFR therapy resistance of KRAS WT colorectal cancer tumors mediated by the upregulation of HRG and/or HER2/HER3 signaling.

A bivalent, bispecific Dab-Fc antibody molecule for dual targeting of HER2 and HER3.

Dual targeting of surface receptors with bispecific antibodies is attracting increasing interest in cancer therapy. Here, we present a novel bivalent and bispecific antagonistic molecule (Dab-Fc) targeting human epidermal growth factors 2 and 3 (HER2 and HER3) derived from the Db-Ig platform, which was developed for the generation of multivalent and multispecific antibody molecules. Dab-Fc comprises the variable domains of the anti-HER2 antibody trastuzumab and the anti-HER3 antibody 3-43 assembled into a diabody-like structure stabilized by CH1 and CL domains and further fused to a human γ1 Fc region. The resulting Dab-Fc 2 × 3 molecule retained unhindered binding to both antigens and was able to bind both antigens sequentially. In cellular experiments, the Dab-Fc 2 × 3 molecule strongly bound to different tumor cell lines expressing HER2 and HER3 and was efficiently internalized. This was associated with potent inhibition of the proliferation and migration of these tumor cell lines. Furthermore, IgG-like pharmacokinetics and anti-tumoral activity were demonstrated in a xenograft tumor model of the gastric cancer cell-line NCI-N87. These results illustrate the suitability of our versatile Db-Ig platform technology for the generation of bivalent bispecific molecules, which has been successfully used here for the dual targeting of HER2 and HER3.

Inhibition of Tumor Cell Growth and Cancer Stem Cell Expansion by a Bispecific Antibody Targeting EGFR and HER3.

The frequent activation of HER3 signaling as a resistance mechanism to EGFR-targeted therapy has motivated the development of combination therapies that block more than one receptor tyrosine kinase. Here, we have developed a novel tetravalent, bispecific single-chain diabody-Fc fusion protein targeting EGFR and HER3 (also known as ErbB3) that integrates the antigen-binding sites of a humanized version of cetuximab as well as a recently developed anti-HER3 antibody, IgG 3-43. This bispecific antibody combines the binding and neutralizing properties of the parental antibodies, as observed in biochemical and in vitro two-dimensional and three-dimensional cell culture assays, and gave rise to long-lasting growth suppression in a subcutaneous xenograft head and neck tumor model. In triple-negative breast cancer (TNBC) cell lines, treatment with the bispecific antibody inhibited the proliferation and oncosphere formation efficiency driven by HER3 signaling. In an orthotopic MDA-MB-468 tumor model, this translated into antitumor effects superior to those obtained by the parental antibodies alone or in combination and was associated with a reduced number of cells with stem-like properties. These findings demonstrate that the bispecific antibody efficiently blocks not only TNBC proliferation, but also the survival and expansion of the cancer stem cell population, holding promise for further preclinical development.

miR-149 Suppresses Breast Cancer Metastasis by Blocking Paracrine Interactions with Macrophages.

Paracrine activation of cells contained in the tumor microenvironment promotes tumor progression and metastasis. In breast cancer, malignant cells recruit and educate macrophages into a M2 tumor-promoting phenotype that supports the metastatic spread of cancer cells. Here, we show that miR-149 functions as a metastasis-suppressing microRNA in breast cancer cells by limiting colony-stimulating factor-1 (CSF1)-dependent recruitment and M2 polarization of macrophages. In lymph node-positive, triple-negative breast cancer (TNBC) tissues, low miR-149 expression correlated with macrophage infiltration and reduced patient survival. By directly targeting CSF1, miR-149 expression in TNBC cell lines (MDA-MB-231 and BT-549) inhibited the recruitment of human monocytic THP-1 cells and primary human macrophages. Furthermore, in macrophages cocultured with MDA-MB-231 cells expressing miR-149, epidermal growth factor (EGF) and amphiregulin expression levels were strongly reduced, resulting in reduced EGF receptor activation in the cancer cells. In vivo, lung metastases developing from orthotopic MDA-MB-231 tumors were reduced by 75% by miR-149 expression, and this was associated with impaired M2 macrophage infiltration of the primary tumors. These data suggest that miR-149 downregulation functionally contributes to breast tumor progression by recruiting macrophages to the tumor and facilitating CSF1 and EGF receptor cross-talk between cancer cells and macrophages. SIGNIFICANCE: These findings contribute to the understanding of tumor-stroma interactions by showing that miR-149 downregulation in TNBC enhances reciprocal growth factor signaling between macrophages and cancer cells, which promotes tumor progression and metastasis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/6/1330/F1.large.jpg.

The GEF-H1/PKD3 signaling pathway promotes the maintenance of triple-negative breast cancer stem cells.

Protein kinase D3 (PKD3) is upregulated in triple-negative breast cancer (TNBC) and associated with cell proliferation and metastasis development but its precise pro-oncogenic function is unknown. Here we show that PKD3 is required for the maintenance of the TNBC stem cell population. The depletion of PKD3 in MDA-MB-231 cells reduced the cancer stem cell frequency in vitro and tumor initiation potential in vivo. We further provide evidence that the RhoGEF GEF-H1 is upstream of PKD3 activation in TNBC stem cells. Most importantly, pharmacological PKD inhibition in combination with paclitaxel synergistically decreased oncosphere and colony formation efficiency in vitro and tumor recurrence in vivo. Based on our results we propose that targeting the GEF-H1/PKD3 signaling pathway in combination with chemotherapy might provide an effective therapeutic option for TNBC.

Direct impact of cisplatin on mitochondria induces ROS production that dictates cell fate of ovarian cancer cells.

Patients with high-grade serous ovarian cancer (HGSC) frequently receive platinum-based chemotherapeutics, such as cisplatin. Cisplatin binds to DNA and induces DNA-damage culminating in mitochondria-mediated apoptosis. Interestingly, mitochondrial DNA is critically affected by cisplatin but its relevance in cell death induction is scarcely investigated. We find that cisplatin sensitive HGSC cell lines contain higher mitochondrial content and higher levels of mitochondrial ROS (mtROS) than cells resistant to cisplatin induced cell death. In clonal sub-lines from OVCAR-3 mitochondrial content and basal oxygen consumption rate correlate with sensitivity to cisplatin induced apoptosis. Mitochondria are in two ways pivotal for cisplatin sensitivity because not only knock-down of BAX and BAK but also the ROS scavenger glutathione diminish cisplatin induced apoptosis. Mitochondrial ROS correlates with mitochondrial content and reduction of mitochondrial biogenesis by knock-down of transcription factors PGC1α or TFAM attenuates both mtROS induction and cisplatin induced apoptosis. Increasing mitochondrial ROS by inhibition or knock-down of the ROS-protective uncoupling protein UCP2 enhances cisplatin induced apoptosis. Similarly, enhancing ROS by high-dose ascorbic acid or H2O2 augments cisplatin induced apoptosis. In summary, mitochondrial content and the resulting mitochondrial capacity to produce ROS critically determine HGSC cell sensitivity to cisplatin induced apoptosis. In line with this observation, data from the human protein atlas (www.proteinatlas.org) indicates that high expression of mitochondrial marker proteins (TFAM and TIMM23) is a favorable prognostic factor in ovarian cancer patients. Thus, we propose mitochondrial content as a biomarker for the response to platinum-based therapies. Functionally, this might be exploited by increasing mitochondrial content or mitochondrial ROS production to enhance sensitivity to cisplatin based anti-cancer therapies.

Spatiotemporal Control of Intracellular Membrane Trafficking by Rho GTPases.

As membrane-associated master regulators of cytoskeletal remodeling, Rho GTPases coordinate a wide range of biological processes such as cell adhesion, motility, and polarity. In the last years, Rho GTPases have also been recognized to control intracellular membrane sorting and trafficking steps directly; however, how Rho GTPase signaling is regulated at endomembranes is still poorly understood. In this review, we will specifically address the local Rho GTPase pools coordinating intracellular membrane trafficking with a focus on the endo- and exocytic pathways. We will further highlight the spatiotemporal molecular regulation of Rho signaling at endomembrane sites through Rho regulatory proteins, the GEFs and GAPs. Finally, we will discuss the contribution of dysregulated Rho signaling emanating from endomembranes to the development and progression of cancer.

DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes.

Cancer cells degrade the extracellular matrix through actin-rich protrusions termed invadopodia. The formation of functional invadopodia requires polarized membrane trafficking driven by Rho GTPase-mediated cytoskeletal remodeling. We identify the Rho GTPase-activating protein deleted in liver cancer 3 (DLC3; also known as STARD8) as an integral component of the endosomal transport and sorting machinery. We provide evidence for the direct regulation of RhoB by DLC3 at endosomal membranes to which DLC3 is recruited by interacting with the sorting nexin SNX27. In TGF-ß-treated MCF10A breast epithelial cells, DLC3 knockdown enhanced metalloproteinase-dependent matrix degradation, which was partially rescued by RhoB co-depletion. This was recapitulated in MDA-MB-231 breast cancer cells in which early endosomes demonstrated aberrantly enriched F-actin and accumulated the metalloproteinase MT1-MMP (also known as MMP14) upon DLC3 knockdown. Remarkably, Rab4 (herein referring to Rab4A) downregulation fully rescued the enhanced matrix degradation of TGF-ß-treated MCF10A and MDA-MB-231 cells. In summary, our findings establish a novel role for DLC3 in the suppression of MT1-MMP-dependent matrix degradation by inactivating RhoB signaling at endosomal membranes. We propose that DLC3 function is required to limit endosomal actin polymerization, Rab4-dependent recycling of MT1-MMP and, consequently, matrix degradation mediated by invadopodial activity.

Spatial Rho regulation: Molecular mechanisms controlling the GAP protein DLC3.

The spatial regulation of cellular Rho signaling by GEF and GAP proteins and the molecular mechanisms controlling the Rho regulators themselves are still incompletely understood. We previously reported that the poorly characterized RhoGAP protein DLC3 localizes to cell-cell adhesions and Rab8-positive membrane tubules. However, it was unclear how DLC3 is targeted to these subcellular sites to execute its functions. In our recent work, protein partners of DLC3 were identified by mass spectrometry, identifying the basolateral polarity protein Scribble as a scaffold for DLC3 at cell-cell contacts. We found that the PDZ-mediated interaction of DLC3 and Scribble is essential for junctional DLC3 recruitment and its role as a local regulator of RhoA-ROCK signaling controlling adherens junction integrity and Scribble localization. Furthermore, DLC3 and Scribble depletion interfered with polarized lumen formation in a 3D model of cyst morphogenesis, emphasizing the relevance of both proteins in epithelial polarity. These findings reveal a new mechanism for spatial Rho regulation at adherens junctions in polarized epithelial cells and highlight the necessity to investigate DLC3 localization and function also in cellular contexts that require cell junction remodeling.

The tumor suppressor protein DLC1 maintains protein kinase D activity and Golgi secretory function.

Many newly synthesized cellular proteins pass through the Golgi complex from where secretory transport carriers sort them to the plasma membrane and the extracellular environment. The formation of these secretory carriers at the trans-Golgi network is promoted by the protein kinase D (PKD) family of serine/threonine kinases. Here, using mathematical modeling and experimental validation of the PKD activation and substrate phosphorylation kinetics, we reveal that the expression level of the PKD substrate deleted in liver cancer 1 (DLC1), a Rho GTPase-activating protein that is inhibited by PKD-mediated phosphorylation, determines PKD activity at the Golgi membranes. RNAi-mediated depletion of DLC1 reduced PKD activity in a Rho-Rho-associated protein kinase (ROCK)-dependent manner, impaired the exocytosis of the cargo protein horseradish peroxidase, and was associated with the accumulation of the small GTPase RAB6 on Golgi membranes, indicating a protein-trafficking defect. In summary, our findings reveal that DLC1 maintains basal activation of PKD at the Golgi and Golgi secretory activity, in part by down-regulating Rho-ROCK signaling. We propose that PKD senses cytoskeletal changes downstream of DLC1 to coordinate Rho signaling with Golgi secretory function.

A Rho signaling network links microtubules to PKD controlled carrier transport to focal adhesions.

Protein kinase D (PKD) is a family of serine/threonine kinases that is required for the structural integrity and function of the Golgi complex. Despite its importance in the regulation of Golgi function, the molecular mechanisms regulating PKD activity are still incompletely understood. Using the genetically encoded PKD activity reporter G-PKDrep we now uncover a Rho signaling network comprising GEF-H1, the RhoGAP DLC3, and the Rho effector PLCε that regulate the activation of PKD at trans-Golgi membranes. We further show that this molecular network coordinates the formation of TGN-derived Rab6-positive transport carriers delivering cargo for localized exocytosis at focal adhesions.

m6A RNA Degradation Products Are Catabolized by an Evolutionarily Conserved N6-Methyl-AMP Deaminase in Plant and Mammalian Cells.

N6-methylated adenine (m6A) is the most frequent posttranscriptional modification in eukaryotic mRNA. Turnover of RNA generates N6-methylated AMP (N6-mAMP), which has an unclear metabolic fate. We show that Arabidopsis thaliana and human cells require an N6-mAMP deaminase (ADAL, renamed MAPDA) to catabolize N6-mAMP to inosine monophosphate in vivo by hydrolytically removing the aminomethyl group. A phylogenetic, structural, and biochemical analysis revealed that many fungi partially or fully lack MAPDA, which coincides with a minor role of N6A-RNA methylation in these organisms. MAPDA likely protects RNA from m6A misincorporation. This is required because eukaryotic RNA polymerase can use N6-mATP as a substrate. Upon abrogation of MAPDA, root growth is slightly reduced, and the N6-methyladenosine, N6-mAMP, and N6-mATP concentrations are increased in Arabidopsis. Although this will potentially lead to m6A misincorporation into RNA, we show that the frequency is too low to be reliably detected in vivo. Since N6-mAMP was severalfold more abundant than N6-mATP in MAPDA mutants, we speculate that additional molecular filters suppress the generation of N6-mATP. Enzyme kinetic data indicate that adenylate kinases represent such filters being highly selective for AMP versus N6-mAMP phosphorylation. We conclude that a multilayer molecular protection system is in place preventing N6-mAMP accumulation and salvage.

Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope formed by domain III and IV.

Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) has emerged as relevant target for antibody-mediated tumor therapy. Here, we describe a novel human antibody, IgG 3-43, recognizing a unique epitope formed by domain III and parts of domain IV of the extracellular region of HER3, conserved between HER3 and mouse ErbB3. An affinity of 11 nM was determined for the monovalent interaction. In the IgG format, the antibody bound recombinant bivalent HER3 with subnanomolar affinity (KD = 220 pM) and HER3-expressing tumor cells with EC50 values in the low picomolar range (27 - 83 pM). The antibody competed with binding of heregulin to HER3-expressing cells, efficiently inhibited phosphorylation of HER3 as well as downstream signaling, and induced receptor internalization and degradation. Furthermore, IgG 3-43 inhibited heregulin-dependent proliferation of several HER3-positive cancer cell lines and heregulin-independent colony formation of HER2-overexpressing tumor cell lines. Importantly, inhibition of tumor growth and prolonged survival was demonstrated in a FaDu xenograft tumor model in SCID mice. These findings demonstrate that by binding to the membrane-proximal domains III and IV involved in ligand binding and receptor dimerization, IgG 3-43 efficiently inhibits activation of HER3, thereby blocking tumor cell growth both in vitro and in vivo.

ATF6ß-based fine-tuning of the unfolded protein response enhances therapeutic antibody productivity of Chinese hamster ovary cells.

The dynamics of protein folding and secretion are key issues in improving the productivity and robustness of Chinese hamster ovary (CHO) producer cells. High recombinant protein secretion in CHO producer clones triggers the activation of the unfolded protein response (UPR), an intracellular response to the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER). We previously reported that the human microRNA (miRNA) miR-1287 enhances productivity in IgG-expressing CHO cells (CHO-IgG). Here, through next-generation sequencing (NGS), we identified the activating transcription factor 6 beta (ATF6ß), a repressor of the pro-survival and UPR promoting factor ATF6α, as a direct target gene of miR-1287 in CHO-IgG cells. We show that the transient depletion of ATF6ß resulted in enhanced specific productivity comparable to that of miR-1287-expressing CHO-IgG cells. Strikingly, stable ATF6ß knockdown in CHO-IgG cells significantly improved antibody titer and viable cell density under fed-batch conditions. This was associated with the elevated expression of the UPR genes glucose-regulated protein 78 (GRP78), homocysteine inducible ER protein with ubiquitin like domain 1 (Herpud1) and CCAAT/enhancer-binding protein homologous protein (CHOP). We hence demonstrate that ATF6ß-based cell line engineering is a promising strategy to improve the productivity of CHO producer cells by activating an optimally balanced UPR program. Biotechnol. Bioeng. 2017;114: 1310-1318. © 2017 Wiley Periodicals, Inc.

Applying phasor approach analysis of multiphoton FLIM measurements to probe the metabolic activity of three-dimensional in vitro cell culture models.

Fluorescence lifetime imaging microscopy (FLIM) can measure and discriminate endogenous fluorophores present in biological samples. This study seeks to identify FLIM as a suitable method to non-invasively detect a shift in cellular metabolic activity towards glycolysis or oxidative phosphorylation in 3D Caco-2 models of colorectal carcinoma. These models were treated with potassium cyanide or hydrogen peroxide as controls, and epidermal growth factor (EGF) as a physiologically-relevant influencer of cell metabolic behaviour. Autofluorescence, attributed to nicotinamide adenine dinucleotide (NADH), was induced by two-photon laser excitation and its lifetime decay was analysed using a standard multi-exponential decay approach and also a novel custom-written code for phasor-based analysis. While both methods enabled detection of a statistically significant shift of metabolic activity towards glycolysis using potassium cyanide, and oxidative phosphorylation using hydrogen peroxide, employing the phasor approach required fewer initial assumptions to quantify the lifetimes of contributing fluorophores. 3D Caco-2 models treated with EGF had increased glucose consumption, production of lactate, and presence of ATP. FLIM analyses of these cultures revealed a significant shift in the contribution of protein-bound NADH towards free NADH, indicating increased glycolysis-mediated metabolic activity. This data demonstrate that FLIM is suitable to interpret metabolic changes in 3D in vitro models.