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Acute treatment with TMZ induces cell cycle arrest and senescence in glioma cells. (A) The U87 cells stably expressing GFP-LC3 were treated with 100 mM TMZ for 3 h, followed by growth in the drug-free medium (DFM) for the indicated time. Time zero (D0) represents 3 h after treatment. (B) The Western blot for the indicated proteins on D3, D5, and D7 after DFM replating; LC, loading control, Coomassie blue stained membrane. (C) The cell cycle distribution. Top, representative plots; bottom left, quantification of the percentage of sub-G 1 , G 1 , S, G 2 /M and hyperdiploid cells; bottom right, percentage of multinucleated cells, assessed by the direct counting of cells with 2 or more nuclei with a normal area on D7. (D) Cumulative population doubling (CPD) of the cells treated as in (A). (E) Representative images of the SA-b-gal staining; the numbers represent the quantification of the percentage of b-gal-positive cells (mean § SEM); scale bar D 80 mm. (F) The NMA contour plot of the TMZ-treated cells over time. The nuclear areas (NA) were defined based on the average of the normal nuclei, and the thresholds were determined by the mean area § 2 SD (NA1), 2 SD to 4 SD from the mean (NA2) and more than 4 SD from the mean (NA3). NII represents the index of nuclear irregularity, as a measurement of nuclear morphometric alterations that occur over time. The values on the heat map legends represent the number of cells. *P < 0.05; **P < 0.01, ***P < 0.001 in relation to the control.
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Autophagy and senescence have been described as central features of cell biology, but the interplay between these mechanisms remains obscure. Using a therapeutically relevant model of DNA damage-induced senescence in human glioma cells, we demonstrated that acute treatment with temozolomide induces DNA damage, a transitory activation of PRKAA/AMPK-...
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... treatment with TMZ induced long-term senescence U87 glioma cells stably expressing the autophagy marker GFP-LC3 (GFP fused to MAP1LC3A, microtubule-associated protein 1 light chain 3 a) were treated with 100 mM TMZ for 3 h, followed by replating the cells in drug-free medium (DFM) (Fig. 1A). The phosphorylated form of H2AFX at Ser139 (com- monly termed g-H2AFX), an indicator of DDR activation, was transiently increased with a peak at day 3 (D3); this was accom- panied by a gradual increase in the phosphorylated form of CDC2 (Tyr15), which inhibits the activity of the CCNB1- CDK1 complex at G 2 /M, and an induction of the ...
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... phosphorylated form of CDC2 (Tyr15), which inhibits the activity of the CCNB1- CDK1 complex at G 2 /M, and an induction of the CDK inhibi- tor CDKN1A/p21. This signaling is indicative of the activation of the G 2 /M checkpoint, which is corroborated by the decrease of both HIST1H3A/C histone Ser10 phosphorylation and the CCND1 (cyclin D1) levels (Fig. 1B). As expected, TMZ pro- duced an accumulation of cells at G 2 /M, peaking on D3; this was followed by a gradual increase in the hyperdiploid and multi- nucleated cells (Fig. 1C). The cumulative population doubling (CPD) indicated that the acute TMZ treatment led to a stabiliza- tion of the cell number, suggesting permanent cell growth ...
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... of the activation of the G 2 /M checkpoint, which is corroborated by the decrease of both HIST1H3A/C histone Ser10 phosphorylation and the CCND1 (cyclin D1) levels (Fig. 1B). As expected, TMZ pro- duced an accumulation of cells at G 2 /M, peaking on D3; this was followed by a gradual increase in the hyperdiploid and multi- nucleated cells (Fig. 1C). The cumulative population doubling (CPD) indicated that the acute TMZ treatment led to a stabiliza- tion of the cell number, suggesting permanent cell growth arrest (Fig. 1D). The CPD profile suggested the beginning of senescence, which was corroborated by an increase in the percent- age of cells positively marked with the ...
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... 1B). As expected, TMZ pro- duced an accumulation of cells at G 2 /M, peaking on D3; this was followed by a gradual increase in the hyperdiploid and multi- nucleated cells (Fig. 1C). The cumulative population doubling (CPD) indicated that the acute TMZ treatment led to a stabiliza- tion of the cell number, suggesting permanent cell growth arrest (Fig. 1D). The CPD profile suggested the beginning of senescence, which was corroborated by an increase in the percent- age of cells positively marked with the senescence-associated b-galactosidase (SA-b-Gal C cells) ( Fig. 1 E) and an increase in the percentage of cells with large and regular nuclei, a morpho- logical feature of senescent cells ...
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... cumulative population doubling (CPD) indicated that the acute TMZ treatment led to a stabiliza- tion of the cell number, suggesting permanent cell growth arrest (Fig. 1D). The CPD profile suggested the beginning of senescence, which was corroborated by an increase in the percent- age of cells positively marked with the senescence-associated b-galactosidase (SA-b-Gal C cells) ( Fig. 1 E) and an increase in the percentage of cells with large and regular nuclei, a morpho- logical feature of senescent cells (Fig. S1A); as observed through the nuclear morphometric analysis (NMA) technique. 34 Interest- ingly, when NMA was analyzed as a contour plot, it was possible to observe a dynamic distribution of the nuclei over time in 3 well-defined regions, as described in the legend of Fig. 1. ...
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... (Fig. 1D). The CPD profile suggested the beginning of senescence, which was corroborated by an increase in the percent- age of cells positively marked with the senescence-associated b-galactosidase (SA-b-Gal C cells) ( Fig. 1 E) and an increase in the percentage of cells with large and regular nuclei, a morpho- logical feature of senescent cells (Fig. S1A); as observed through the nuclear morphometric analysis (NMA) technique. 34 Interest- ingly, when NMA was analyzed as a contour plot, it was possible to observe a dynamic distribution of the nuclei over time in 3 well-defined regions, as described in the legend of Fig. 1. The nuclear area (NA) from the TMZ-treated cells progressed from ...
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... large and regular nuclei, a morpho- logical feature of senescent cells (Fig. S1A); as observed through the nuclear morphometric analysis (NMA) technique. 34 Interest- ingly, when NMA was analyzed as a contour plot, it was possible to observe a dynamic distribution of the nuclei over time in 3 well-defined regions, as described in the legend of Fig. 1. The nuclear area (NA) from the TMZ-treated cells progressed from NA1 to NA3, which is characteristic of senescent cells, through the intermediary state, NA2. On D7, only a few cells remained that had a nuclear area of nonsenescent cells (NA1) or that were in the intermediary region NA2 (Fig. 1F and Fig. ...
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... 3 well-defined regions, as described in the legend of Fig. 1. The nuclear area (NA) from the TMZ-treated cells progressed from NA1 to NA3, which is characteristic of senescent cells, through the intermediary state, NA2. On D7, only a few cells remained that had a nuclear area of nonsenescent cells (NA1) or that were in the intermediary region NA2 (Fig. 1F and Fig. ...
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... (Ser9), was gradually reduced, remaining low on D7. MAPK14/p38, an important sensor of cell damage (including genomic injury), had increased levels of phosphoryla- tion on D3 and D5 ( Fig. 2A). It is important to note that the phosphorylation of MAPK14, PRKAA, and ULK1 follows kinet- ics that resemble the primary DNA damage that was produced (Fig. 1B), whereas the remaining proteins had slower and longer lasting post-translational modifications. These data are support- ive of proautophagic signaling after DNA damage, involving the activation of the PRKAA-ULK1 axis and the suppression of the AKT-MTORC1 ...
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... TMZ led to an increase in the proportion of GFP- LC3 C cells (Fig. 2B). The proportion of acridine orange (AO)- positive cells increased until D4, followed by a sudden increase in the mean level of AO staining with a concomitant reduction in the proportion of AOC cells (Fig. 2C, Fig. S1C and D). Autophagy induction was also confirmed by western blot for the LC3-I to LC3-II conversion and the levels of the SQSTM1/p62 protein, which is degraded during autophagy (Fig. 2D). These data confirm that acute DNA damage triggers autophagy, peak- ing on approximately D3 and D4 followed by a gradual reduc- tion. Despite the high levels ...
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... this heterogeneous response in individual cells, at a population level, a significantly larger nuclear area was observed in the few cells that were already bgal-C on D3 in relation to the Figure 2. Acute treatment with TMZ induces autophagy accompanied by early AMPK-ULK1 axis activation and long lasting AKT-PI3K-MTOR suppression. (A) The U87 cells were treated as described in Figure 1A, and western blots for the indicated proteins and phosphoproteins were performed; LC, loading control, comassie blue stained membrane. (B) The GFP-LC3 dot formation assay; the numbers depict the percentage of cells with at least 5 GFP-LC3 dots (mean § SEM); *P < 0.05, **P < 0.01 and ***P < 0.001; scale bar D 20 mm. ...
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... The GFP-LC3 dot formation assay; the numbers depict the percentage of cells with at least 5 GFP-LC3 dots (mean § SEM); *P < 0.05, **P < 0.01 and ***P < 0.001; scale bar D 20 mm. (C) Acridine orange (AO) staining: the graph shows the percentage of AO-pos- itive cells versus the AO intensity of the positive population in relation to the control over time (to see the data of AO intensity and the percentage of cells in a separate manner, see The ATP levels of the cells treated with TMZ as in Figure 1A; *P < 0.05 and **P < 0.01 in relation to the control, which was considered 100%. ...
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... single-cell analysis of the GFP-LC3, nuclear, and cell areas after TMZ treatment. (A) Contour plots of the cell and nuclear areas in relation to the GFP-LC3 area after treatment as described in Figure 1A. The circles mark the populations based on regions with high density that emerged from the plots of nuclear area (nP, top) and cell area (cP, bottom) in relation to GFP-LC3; the dashed lines mark the thresholds of NA1, NA2, and NA3, as indicated in Figure 1F. ...
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... Contour plots of the cell and nuclear areas in relation to the GFP-LC3 area after treatment as described in Figure 1A. The circles mark the populations based on regions with high density that emerged from the plots of nuclear area (nP, top) and cell area (cP, bottom) in relation to GFP-LC3; the dashed lines mark the thresholds of NA1, NA2, and NA3, as indicated in Figure 1F. with faster and transient kinetics ( Fig. 2B-D; Fig. 3B), while senescence markers increased with slower and sustained kinetics (Fig. 1A, C, D and F; Fig. 3B). ...
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... on regions with high density that emerged from the plots of nuclear area (nP, top) and cell area (cP, bottom) in relation to GFP-LC3; the dashed lines mark the thresholds of NA1, NA2, and NA3, as indicated in Figure 1F. with faster and transient kinetics ( Fig. 2B-D; Fig. 3B), while senescence markers increased with slower and sustained kinetics (Fig. 1A, C, D and F; Fig. 3B). Autophagy decreased before senescence became dominant in the population, which was observed through the inverse correlation between the variation of the cell or nuclear areas and the variation of the LC3 area from D3 to D4.5 in the population. This inverse correlation was also high when considering data from D3 to D7 (Fig. 5D, bottom ...
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... D3, some nuclei of the TMZ-treated cells were already in the NA2 and NA3 regions, but by D7, almost all the nuclei were in the NA3 population (Fig. 1D). The inhibition of autophagy with either BafA1 or 3MA kept the nuclei in the NA1 and NA2 populations (Fig. 6D), in agreement with the reduction in the proportion of bGal C cells (Fig. 6E and F). Finally, the treatment with 3MA reduced the number of cells from D3 onwards, likely due to the induction of apoptosis, which was not observed ...
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... cells with nuclei in the NA2 region on D3 (Fig. 1F) responded differently according to their autophagic status: high LC3 cells increased their average nuclear area from D3 to D4.5, whereas low LC3 cells maintained the same area or decreased their nuclear area, i.e., underwent apoptosis (Fig. 8A). Therefore, we wondered whether activating and blocking autophagy at dif- ferent times ...
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... our data in a graph of red fluor PI (propidium Iodide)-area per red fluor (PI)-width. Thus, after a first gate of cells in a graph of forward scatter by side scatter to remove cell debris and cell death, we eliminated the cells with a high PtdIns- width (i.e., the doublets) and only the singlets were evaluated for the cell cycle determination (Fig. ...
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... An irreversible cell division arrest characterizes senescence and is a characteristic cellular process in aging. However, senescent cells remain metabolically active and can influence the cell microenvironment [43]. Cell senescence is induced by chemical agents, such as genotoxic and reactive oxygen species, and biological processes, such as DNA damage, telomere shortening, activation of oncogenes, and tumor suppressor genes [44]. ...
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... Accordingly, when senescence is induced together with autophagy in response to radiation, it becomes more challenging to determine which process is likely to dictate the ultimate cell fate. This complexity has been proposed by Filippi-Chiela et al., who demonstrated the induction of autophagy, followed by the induction of senescence, in glioma cells exposed to temozolomide, as is the case of many other reports in the literature [73]. However, at the single-cell analysis level, Filippi-Chiela et al. showed that autophagy and senescence were induced in a significantly heterogeneous manner despite being triggered simultaneously in mass cultures [73]. ...
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Background and Objectives: The development of radioresistance is a fundamental barrier to successful glioblastoma therapy. Autophagy is thought to play a role in facilitating the DNA repair of DNA damage foci in radiation-exposed tumor cells, thus, potentially contributing to their restoration of proliferative capacity and development of resistance in vitro. However, the effect of autophagy inhibitors on DNA damage repair is not fully clear and requires further investigation. Materials and Methods: In this work, we utilized M059K (DNA-PKcs proficient) and M059J (DNA-PKcs deficient) glioma cell lines to investigate the role of autophagy inhibitors in the DNA repair of radiation-induced DNA damage. Cell viability following radiation was determined by trypan blue exclusion in both cell lines. Cell death and autophagy assays were performed to evaluate radiation-induced cell stress responses. DNA damage was measured as based on the intensity of phosphorylated γ-H2AX, a DNA double-stranded breaks (DSBs) marker, in the presence or absence of autophagy inhibitors. Results: The cell viability assay showed that M059J cells were more sensitive to the same dose of radiation (4 Gy) than M059K cells. This observation was accompanied by an elevation in γ-H2AX formation in M059J but not in M059K cells. In addition, the DAPI/TUNEL and Senescence-associated β-galactosidase (SA-β-gal) staining assays did not reveal significant differences in apoptosis and/or senescence induction in response to radiation, respectively, in either cell line. However, acridine orange staining demonstrated clear promotion of acidic vesicular organelles (AVOs) in both cell lines in response to 4 Gy radiation. Moreover, DNA damage marker levels were found to be elevated 72 h post-radiation when autophagy was inhibited by the lysosomotropic agent bafilomycin A1 (BafA1) or the PI3K inhibitor 3-methyl adenine (3-MA) in M059K cells. Conclusions: The extent of the DNA damage response remained high in the DNA-PKcs deficient cells following exposure to radiation, indicating their inability to repair the newly formed DNA-DSBs. On the other hand, radioresistant M059K cells showed more DNA damage response only when autophagy inhibitors were used with radiation, suggesting that the combination of autophagy inhibitors with radiation may interfere with DNA repair efficiency.
... The function of the p38α-MAPK pathway is to relay, amplify and integrate a variety of extracellular stresses, such as radiotherapy, chemotherapy, hypoxia and hunger, thereby regulating the genomic and physiological response of cells to their environment [61]. It has been reported that acute treatment with TMZ induces DNA damage and transitory activation of MAPK14/p38α [62,63]. In addition, activation of the MAPK pathway has been associated with poor survival in GBM patients during the TMZ era [64]. ...
Background:
Resistance to temozolomide (TMZ) is a major obstacle to preventing glioblastoma (GBM) recurrence after surgery. Although long noncoding RNAs (lncRNAs) play a variety of roles in GBM, the lncRNAs that regulate TMZ resistance have not yet been clearly elucidated. This study aims to identify lncRNAs that may affect TMZ treatment sensitivity and to explore novel therapeutic strategies to overcome TMZ resistance in GBM.
Methods:
LncRNAs associated with TMZ resistance were identified using the Cancer Cell Line Encyclopedia (CCLE) and Genomics of Drug Sensitivity in Cancer (GDSC) datasets. Quantitative real-time PCR (qRT-PCR) was used to determine the expression of PDIA3P1 in TMZ-resistant and TMZ-sensitive GBM cell lines. Both gain-of-function and loss-of-function studies were used to assess the effects of PDIA3P1 on TMZ resistance using in vitro and in vivo assays. Glioma stem cells (GSCs) were used to determine the effect of PDIA3P1 on the GBM subtype. The hypothesis that PDIA3P1 promotes proneural-to-mesenchymal transition (PMT) was established using bioinformatics analysis and functional experiments. RNA pull-down and RNA immunoprecipitation (RIP) assays were performed to examine the interaction between PDIA3P1 and C/EBPβ. The posttranslational modification mechanism of C/EBPβ was verified using ubiquitination and coimmunoprecipitation (co-IP) experiments. CompuSyn was leveraged to calculate the combination index (CI), and the antitumor effect of TMZ combined with nefllamapimod (NEF) was validated both in vitro and in vivo.
Results:
We identified a lncRNA, PDIA3P1, which was upregulated in TMZ-resistant GBM cell lines. Overexpression of PDIA3P1 promoted the acquisition of TMZ resistance, whereas knockdown of PDIA3P1 restored TMZ sensitivity. PDIA3P1 was upregulated in MES-GBM, promoted PMT progression in GSCs, and caused GBMs to be more resistant to TMZ treatment. Mechanistically, PDIA3P1 disrupted the C/EBPβ-MDM2 complex and stabilized the C/EBPβ protein by preventing MDM2-mediated ubiquitination. Expression of PDIA3P1 was upregulated in a time- and concentration-dependent manner in response to TMZ treatment, and TMZ-induced upregulation of PDIA3P1 was mediated by the p38α-MAPK signaling pathway. NEF is a small molecule drug that specifically targets p38α with excellent blood-brain barrier (BBB) permeability. NEF blocked TMZ-responsive PDIA3P1 upregulation and produced synergistic effects when combined with TMZ at specific concentrations. The combination of TMZ and NEF exhibited excellent synergistic antitumor effects both in vitro and in vivo.
Conclusion:
PDIA3P1 promotes PMT by stabilizing C/EBPβ, reducing the sensitivity of GBM cells to TMZ treatment. NEF inhibits TMZ-responsive PDIA3P1 upregulation, and NEF combined with TMZ provides better antitumor effects.
... Different from IR, when autophagy is inhibited in early stages using 3-MA or via BECN1 knockdown, TMZ has its antitumoral effect suppressed (antagonizing with apoptosis), while the inhibition with b-A1 or ATG4C knockdown sensitizes the tumor cells by inducing apoptosis [287][288][289][290]. TMZ-induced senescence requires autophagy, and inhibition of autophagy though 3-MA treatment impairs TMZ-mediated senescence [287,291]. TMZ-induced DSBs directly activate ATM, the upstream kinase of the DDR, which is a component of the ATM/AMPK/ULK1 cascade [289,292] (Figure 6). Inhibition of AMPK, a protein involved in the initiation of the autophagosome formation by interacting with mammalian autophagy-initiating kinase ULK1, augments the cytotoxicity of GBM cells [293]. ...
Simple Summary
Glioblastoma is a type of brain cancer that remains incurable. Despite multiple past and ongoing preclinical studies and clinical trials, involving adjuvants to the conventional therapy and based on molecular targeting, no relevant benefit for patients’ survival has been achieved so far. The current first-line treatment regimen is based on ionizing radiation and the monoalkylating compound, temozolomide, and has been administered for more than 15 years. Glioblastoma is extremely resistant to most agents due to a mutational background that elicits quick response to insults and adapts to microenvironmental and metabolic changes. Here, we present the most recent evidence concerning the molecular features and their alterations governing pathways involved in GBM response to the standard radio-chemotherapy and discuss how they collaborate with acquired GBM’s resistance.
Abstract
Glioblastoma multiforme (GBM) is a brain tumor characterized by high heterogeneity, diffuse infiltration, aggressiveness, and formation of recurrences. Patients with this kind of tumor suffer from cognitive, emotional, and behavioral problems, beyond exhibiting dismal survival rates. Current treatment comprises surgery, radiotherapy, and chemotherapy with the methylating agent, temozolomide (TMZ). GBMs harbor intrinsic mutations involving major pathways that elicit the cells to evade cell death, adapt to the genotoxic stress, and regrow. Ionizing radiation and TMZ induce, for the most part, DNA damage repair, autophagy, stemness, and senescence, whereas only a small fraction of GBM cells undergoes treatment-induced apoptosis. Particularly upon TMZ exposure, most of the GBM cells undergo cellular senescence. Increased DNA repair attenuates the agent-induced cytotoxicity; autophagy functions as a pro-survival mechanism, protecting the cells from damage and facilitating the cells to have energy to grow. Stemness grants the cells capacity to repopulate the tumor, and senescence triggers an inflammatory microenvironment favorable to transformation. Here, we highlight this mutational background and its interference with the response to the standard radiochemotherapy. We discuss the most relevant and recent evidence obtained from the studies revealing the molecular mechanisms that lead these cells to be resistant and indicate some future perspectives on combating this incurable tumor.
... Autophagy response Cancer (glioma, U87) High levels of autophagy favors cell senescence over apoptosis in response to DNA damage. [27] Cancer (Neuroblastoma, H4) High levels of autophagy favors staurosporine survival. [41] Bacteria death and resistance ...
... Considering the response of cancer cells to therapy, single-cell tracking revealed that: (i) autophagy and senescence are not interdependent mechanisms, while high levels of autophagy suppress apoptosis in glioblastoma cells (U87 cells) treated with temozolomide [27]; (ii) acute monocytic leukemia cells (MOLM3 cells) in S or G2 phase die faster in response to vincristine or daunorubicin [28]; (iii) after DNA damage induced by the radiomimetic neocarzinostatin, breast cancer cells (MCF7 cells) with sustained ERK activation evade G2 arrest and enter mitosis [29]; (iv) melanoma cells (A375 and WM983B cells) that quickly reactivate ERK pathway resist to vemurafenib [4]; (v) cisplatin affects the binding of HMGB1 and SMAD3 transcription factors to DNA, favoring the resistance of lung cancer cells (A549 cells) to therapy [30]. ...
... Therefore, as cell plasticity in cancer is important to adapt to changes in the microenvironment or in response to therapy, tracking single cells is key for understanding cell plasticity during tumor formation or in response to therapy. This may allow an optimized design of therapies combining modulators of signaling pathways or cellular mechanisms [27,32], and the discovery of new players involved in tumor progression [3]. Single-cell tracking also has the potential to reveal new targets for site-directed anti-cancer therapies as well as new players for preclinical models development or early diagnosis in cancer. ...
Tracking individual cells has allowed a new understanding of cellular behavior in human health and disease by adding a dynamic component to the already complex heterogeneity of single cells. Technically, despite countless advances, numerous experimental variables can affect data collection and interpretation and need to be considered. In this review, we discuss the main technical aspects and biological findings in the analysis of the behavior of individual cells. We discuss the most relevant contributions provided by these approaches in clinically relevant human conditions like embryo development, stem cells biology, inflammation, cancer and microbiology, along with the cellular mechanisms and molecular pathways underlying these conditions. We also discuss the key technical aspects to be considered when planning and performing experiments involving the analysis of individual cells over long periods. Despite the challenges in automatic detection, features extraction and long-term tracking that need to be tackled, the potential impact of single-cell bioimaging is enormous in understanding the pathogenesis and development of new therapies in human pathophysiology.
