Supplementary Fig. S4 Tagging of the endogenous locus of Ki67 with EYFP. Sequence coding mVenu... more Supplementary Fig. S4 Tagging of the endogenous locus of Ki67 with EYFP. Sequence coding mVenus-P2A-Neomycin was PCR amplified from the eFlut-mVenus-Neomycin plasmid generously donated by Galit Lahav (Harvard University) and used to insert flanking regions of about 500 nt, amplified with the indicated primers. This was used for homologous recombination in the CRISPR-Cas9 targeted C-terminal region of Ki67 gene.
Supplementary Fig. S5  Growth characteristics of individual treated colonies. a, GR of all col... more Supplementary Fig. S5  Growth characteristics of individual treated colonies. a, GR of all colonies used in Fig. 1e. Mean {plus minus} SD in red. b. Pairwise correlations among the three GRs. c. Mean {plus minus} variance of GR2 of U251 glioma colonies according to the number of cells in CS1 or all colonies. Growth rate of colonies with 1 and 2 cells were different from all growth rates (ANOVA, Tukey's multiple comparison post-hoc test). d, Distribution of the number of colonies in each bin for Fig. 1e. e, GR of the indicated cell lines at high and low density. f, CVP and g, hypothesis plot of the glioma cell line A172wt (WT), GFP-tagged alone (GFP) or in the presence of 100 fold excess of untagged cells (GFP 1:100).
Supplementary Fig. S3Analysis of the desynchronization of colonies of cells expressing FastFUC... more Supplementary Fig. S3Analysis of the desynchronization of colonies of cells expressing FastFUCCI. a, FUCCI simulation for a single, synchronized colony with 4 cells. At a given time point, all cells in the colony will all either be green or not. b, simulating multiple colonies gives rise to multimodal distributions of green cell percentage in colonies. c, adding a phase shift parameter allows cells in each colony to be out of sync, producing percentages of green cells in each colony other than 0% or 100% for a given observation point. d, by modifying the simulation parameters, various frequency distributions of the percentage of green cells can be obtained for several colonies. Note that a, b and c represent three specific time steps of a full simulation run, which must walk all cells around the cell cycle at least once, and produced a final distribution considering all evaluated time steps.
Supplementary Fig. S7  Comparison of Dynafit results with growth rate. a, GR and DynaFit Cumul... more Supplementary Fig. S7  Comparison of Dynafit results with growth rate. a, GR and DynaFit Cumulative Hypothesis plot results b. GR2 versus CS1-GR2 c. GR3 versus CS1-GR2 d. GR3 versus CS1-GR3 of untreated (green), treated only with cytotoxic drugs (red) and the combination of epigenetic modulators and cytotoxic drugs (blue).
Supplementary Fig. S2 DynaFit implementations. a, data collected from Supplementary Fig. S1c i... more Supplementary Fig. S2 DynaFit implementations. a, data collected from Supplementary Fig. S1c is given as input to the two DynaFit apps. The Python bootstrap app and the R predictive modeling app are based on different analytical strategies. b, Colony Variance Plot formed by the Python app and c, its hypothesis plot. d, Colony Variance Plot formed by the R app and e, its hypothesis plot.
Cancer cells have heterogeneous fitness, and this heterogeneity stems from genetic and epigenetic... more Cancer cells have heterogeneous fitness, and this heterogeneity stems from genetic and epigenetic sources. Here, we sought to assess the contribution of asymmetric mitosis (AM) and time on the variability of fitness in sister cells. Around one quarter of sisters had differences in fitness, assessed as the intermitotic time (IMT), from 330 to 510 min. Phenotypes related to fitness, such as ERK activity (herein referring to ERK1 and ERK2, also known as MAPK3 and MAPK1, respectively), DNA damage and nuclear morphological phenotypes were also asymmetric at mitosis or turned asymmetric over the course of the cell cycle. The ERK activity of mother cell was found to influence the ERK activity and the IMT of the daughter cells, and cells with ERK asymmetry at mitosis produced more offspring with AMs, suggesting heritability of the AM phenotype for ERK activity. Our findings demonstrate how variabilities in sister cells can be generated, contributing to the phenotype heterogeneities in tumor...
Heterogeneity is a pervasive feature of cancer, and understanding the sources and regulatory mech... more Heterogeneity is a pervasive feature of cancer, and understanding the sources and regulatory mechanisms underlying heterogeneity could provide key insights to help improve the diagnosis and treatment of cancer. In this review, we discuss the origin of heterogeneity in the phenotype of individual cancer cells. Genotype–phenotype (G–P) maps are widely used in evolutionary biology to represent the complex interactions of genes and the environment that lead to phenotypes that impact fitness. Here, we present the rationale of an extended G–P (eG–P) map with a cone structure in cancer. The eG–P cone is formed by cells that are similar at the genome layer but gradually increase variability in the epigenome, transcriptome, proteome, metabolome, and signalome layers to produce large variability at the phenome layer. Experimental evidence from single-cell-omics analyses supporting the cancer eG–P cone concept is presented, and the impact of epimutations and the interaction of cancer and tumor...
Lung cancer is the most frequent type of cancer and the leading cause of cancer-related mortality... more Lung cancer is the most frequent type of cancer and the leading cause of cancer-related mortality worldwide. This study aimed to develop erlotinib (ELB)-loaded poly(ε-caprolactone) nanocapsules (NCELB) and evaluated their in vitro cytotoxicity in A549 cells. The formulation was characterized in relation to hydrodynamic diameter (171 nm), polydispersity index (0.076), zeta potential (− 8 mV), drug content (0.5 mg.mL−1), encapsulation efficiency (99%), and pH (6.0). NCELB presented higher cytotoxicity than ELB in solution against A549 cells in the MTT and LIVE/DEAD cell viability assays after 24 h of treatment. The main mechanism of cytotoxicity of NCELB was the induction of apoptosis in A549 cells. Further, a significant decrease in A549 colony formation was verified after NCELB treatment in comparison with the unencapsulated drug treatment. The reduction in clonogenic capacity is very relevant as it can reduce the risk of tumor recurrence and metastasis. In conclusion, erlotinib-loaded PCL nanocapsules are promising nanoparticles carriers to increase the efficacy of ELB in lung cancer treatment.
Several phenotypes that impact the capacity of cancer cells to survive and proliferate are dynami... more Several phenotypes that impact the capacity of cancer cells to survive and proliferate are dynamic. Here we used the number of cells in colonies as an assessment of fitness and devised a novel method called Dynamic Fitness Analysis (DynaFit) to measure the dynamics in fitness over the course of colony formation. DynaFit is based on the variance in growth rate of a population of founder cells compared with the variance in growth rate of colonies with different sizes. DynaFit revealed that cell fitness in cancer cell lines, primary cancer cells, and fibroblasts under unhindered growth conditions is dynamic. Key cellular mechanisms such as ERK signaling and cell-cycle synchronization differed significantly among cells in colonies after 2 to 4 generations and became indistinguishable from randomly sampled cells regarding these features. In the presence of cytotoxic agents, colonies reduced their variance in growth rate when compared with their founder cell, indicating a dynamic nature i...
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, Jan 22, 2017
Breast cancer is a global public health problem. For some subtypes, such as Claudin-low, the prog... more Breast cancer is a global public health problem. For some subtypes, such as Claudin-low, the prognosis is poorer and the treatment is still a challenge. Pyrazoles are an important class of heterocyclic compounds and are promising anticancer agents based on their chemical properties. The present study was aimed not only at testing pyrazoles previously prepared by our research group in two breast cancer cell lines characterized by intermediated response to conventional chemotherapy but also at analyzing the possible synergistic effect of these pyrazoles associated with doxorubicin. Four 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H pyrazoles were tested for the first time in MCF-7 and MDA-MB-231 culture cells. The pyrazoles with best results in cytotoxicity were used in combination with doxorubicin and compared with this drug alone as standard. The synergic effect was analyzed using Combination Index method. In addition, cell death and apoptosis assays were carried out. Two pyrazoles with...
Nanostructured drug delivery systems have been extensively studied, mainly for applications in ca... more Nanostructured drug delivery systems have been extensively studied, mainly for applications in cancer therapy. The advantages of these materials include protection against drug degradation and improvement in both the relative solubility of poorly water soluble drugs as in targeting of therapy, due to the enhanced permeability and retention effect on tumor sites. In this work, we evaluate the antitumor activity of tretinoin-loaded lipid core nanocapsules (TT-LNC) in a tretinoin-resistant breast cancer cell-line, MDA-MB- 231, as well as the synergistic effect of combination of this treatment with 5-FU or DOXO. The inhibition of cell growth was assayed by MTT reduction. Live/Dead assay and DAPI staining evaluated cytotoxicity. Apoptosis was evaluated by Annexin V-PE/7AAD and the effect of chronic exposure was evaluated by colony formation assay. TT-LNC reduced the cell viability even at lower concentrations (1μM) and displayed synergistic effect with 5-FU or DOXO on cytotoxicity and co...
Bladder cancer is a genitourinary malignant disease common worldwide. Current chemotherapy is oft... more Bladder cancer is a genitourinary malignant disease common worldwide. Current chemotherapy is often limited mainly due to toxicity and drug resistance. Thus, there is a continued need to discover new therapies. Recently evidences shows that pyrazoline derivatives are promising antitumor agents in many types of cancers, but there are no studies with bladder cancer. In order to find potent and novel chemotherapy drugs for bladder cancer, a series of pyrazoline derivatives 2a-2d were tested for their antitumor activity in two human bladder cancer cell lines 5647 and T24. The MTT assay showed that the compounds 1-thiocarbamoyl-3,5-diphenyl-4,5-dihydro-1H-pyrazole (2a) and 1-thiocarbamoyl-5-(4-chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole (2c) decrease the cell viability of 5637 cells. Molecular modeling indicated that these compounds had a good oral bioavailability and low toxicities. Clonogenic assay and flow cytometric analysis were used to assess colony formation, apoptosis induction and cell cycle distribution. Overall, our results suggest that pyrazoline 2a and 2c, with the substituents hydrogen and chlorine respectively, may decrease cell viability and colony formation of bladder cancer 5637 cell line by inhibition of cell cycle progression, and for pyrazoline 2a, by induction of apoptosis. As indicated by the physicochemical properties of these compounds, the steric factor influences the activity. Therefore, these pyrazoline derivatives can be considered promising anticancer agents for the treatment of bladder cancer.
Herein we report the use of ultrasonic irradiation (US) in the synthesis of six new semi-syntheti... more Herein we report the use of ultrasonic irradiation (US) in the synthesis of six new semi-synthetic selenium-containing chrysin derivatives by a simple and effective methodology utilizing CuI as catalyst, in good to excellent yields (60-89%). It was observed that US accelerates the reaction compared to conventional heating with excellent selectivity for diselenylated products. Compounds were tested for their antioxidant and anticancer activities in vitro and it was observed that the presence of selenium in the A-ring of chrysin enhanced both antioxidant and anticancer properties. Semi-synthetic 6,8-bis(o-tolylselanyl)-chrysin 3b has the best radical scavenging activity of DPPH (Imax: 39.79µM) and ABTS(+) (IC50: 6.5µM) radicals. Similarly, in the Reactive Species (RS) assay, 3b showed high antioxidant activity in mice cortex (IC50: 5.67µM), whereas 6,8-bis(p-anisoylselanyl)-chrysin 3c was the more active in the hippocampus (IC50: 5.63µM). The Se-chrysins were effective in prevention o...
Supplementary Fig. S4 Tagging of the endogenous locus of Ki67 with EYFP. Sequence coding mVenu... more Supplementary Fig. S4 Tagging of the endogenous locus of Ki67 with EYFP. Sequence coding mVenus-P2A-Neomycin was PCR amplified from the eFlut-mVenus-Neomycin plasmid generously donated by Galit Lahav (Harvard University) and used to insert flanking regions of about 500 nt, amplified with the indicated primers. This was used for homologous recombination in the CRISPR-Cas9 targeted C-terminal region of Ki67 gene.
Supplementary Fig. S5  Growth characteristics of individual treated colonies. a, GR of all col... more Supplementary Fig. S5  Growth characteristics of individual treated colonies. a, GR of all colonies used in Fig. 1e. Mean {plus minus} SD in red. b. Pairwise correlations among the three GRs. c. Mean {plus minus} variance of GR2 of U251 glioma colonies according to the number of cells in CS1 or all colonies. Growth rate of colonies with 1 and 2 cells were different from all growth rates (ANOVA, Tukey's multiple comparison post-hoc test). d, Distribution of the number of colonies in each bin for Fig. 1e. e, GR of the indicated cell lines at high and low density. f, CVP and g, hypothesis plot of the glioma cell line A172wt (WT), GFP-tagged alone (GFP) or in the presence of 100 fold excess of untagged cells (GFP 1:100).
Supplementary Fig. S3Analysis of the desynchronization of colonies of cells expressing FastFUC... more Supplementary Fig. S3Analysis of the desynchronization of colonies of cells expressing FastFUCCI. a, FUCCI simulation for a single, synchronized colony with 4 cells. At a given time point, all cells in the colony will all either be green or not. b, simulating multiple colonies gives rise to multimodal distributions of green cell percentage in colonies. c, adding a phase shift parameter allows cells in each colony to be out of sync, producing percentages of green cells in each colony other than 0% or 100% for a given observation point. d, by modifying the simulation parameters, various frequency distributions of the percentage of green cells can be obtained for several colonies. Note that a, b and c represent three specific time steps of a full simulation run, which must walk all cells around the cell cycle at least once, and produced a final distribution considering all evaluated time steps.
Supplementary Fig. S7  Comparison of Dynafit results with growth rate. a, GR and DynaFit Cumul... more Supplementary Fig. S7  Comparison of Dynafit results with growth rate. a, GR and DynaFit Cumulative Hypothesis plot results b. GR2 versus CS1-GR2 c. GR3 versus CS1-GR2 d. GR3 versus CS1-GR3 of untreated (green), treated only with cytotoxic drugs (red) and the combination of epigenetic modulators and cytotoxic drugs (blue).
Supplementary Fig. S2 DynaFit implementations. a, data collected from Supplementary Fig. S1c i... more Supplementary Fig. S2 DynaFit implementations. a, data collected from Supplementary Fig. S1c is given as input to the two DynaFit apps. The Python bootstrap app and the R predictive modeling app are based on different analytical strategies. b, Colony Variance Plot formed by the Python app and c, its hypothesis plot. d, Colony Variance Plot formed by the R app and e, its hypothesis plot.
Cancer cells have heterogeneous fitness, and this heterogeneity stems from genetic and epigenetic... more Cancer cells have heterogeneous fitness, and this heterogeneity stems from genetic and epigenetic sources. Here, we sought to assess the contribution of asymmetric mitosis (AM) and time on the variability of fitness in sister cells. Around one quarter of sisters had differences in fitness, assessed as the intermitotic time (IMT), from 330 to 510 min. Phenotypes related to fitness, such as ERK activity (herein referring to ERK1 and ERK2, also known as MAPK3 and MAPK1, respectively), DNA damage and nuclear morphological phenotypes were also asymmetric at mitosis or turned asymmetric over the course of the cell cycle. The ERK activity of mother cell was found to influence the ERK activity and the IMT of the daughter cells, and cells with ERK asymmetry at mitosis produced more offspring with AMs, suggesting heritability of the AM phenotype for ERK activity. Our findings demonstrate how variabilities in sister cells can be generated, contributing to the phenotype heterogeneities in tumor...
Heterogeneity is a pervasive feature of cancer, and understanding the sources and regulatory mech... more Heterogeneity is a pervasive feature of cancer, and understanding the sources and regulatory mechanisms underlying heterogeneity could provide key insights to help improve the diagnosis and treatment of cancer. In this review, we discuss the origin of heterogeneity in the phenotype of individual cancer cells. Genotype–phenotype (G–P) maps are widely used in evolutionary biology to represent the complex interactions of genes and the environment that lead to phenotypes that impact fitness. Here, we present the rationale of an extended G–P (eG–P) map with a cone structure in cancer. The eG–P cone is formed by cells that are similar at the genome layer but gradually increase variability in the epigenome, transcriptome, proteome, metabolome, and signalome layers to produce large variability at the phenome layer. Experimental evidence from single-cell-omics analyses supporting the cancer eG–P cone concept is presented, and the impact of epimutations and the interaction of cancer and tumor...
Lung cancer is the most frequent type of cancer and the leading cause of cancer-related mortality... more Lung cancer is the most frequent type of cancer and the leading cause of cancer-related mortality worldwide. This study aimed to develop erlotinib (ELB)-loaded poly(ε-caprolactone) nanocapsules (NCELB) and evaluated their in vitro cytotoxicity in A549 cells. The formulation was characterized in relation to hydrodynamic diameter (171 nm), polydispersity index (0.076), zeta potential (− 8 mV), drug content (0.5 mg.mL−1), encapsulation efficiency (99%), and pH (6.0). NCELB presented higher cytotoxicity than ELB in solution against A549 cells in the MTT and LIVE/DEAD cell viability assays after 24 h of treatment. The main mechanism of cytotoxicity of NCELB was the induction of apoptosis in A549 cells. Further, a significant decrease in A549 colony formation was verified after NCELB treatment in comparison with the unencapsulated drug treatment. The reduction in clonogenic capacity is very relevant as it can reduce the risk of tumor recurrence and metastasis. In conclusion, erlotinib-loaded PCL nanocapsules are promising nanoparticles carriers to increase the efficacy of ELB in lung cancer treatment.
Several phenotypes that impact the capacity of cancer cells to survive and proliferate are dynami... more Several phenotypes that impact the capacity of cancer cells to survive and proliferate are dynamic. Here we used the number of cells in colonies as an assessment of fitness and devised a novel method called Dynamic Fitness Analysis (DynaFit) to measure the dynamics in fitness over the course of colony formation. DynaFit is based on the variance in growth rate of a population of founder cells compared with the variance in growth rate of colonies with different sizes. DynaFit revealed that cell fitness in cancer cell lines, primary cancer cells, and fibroblasts under unhindered growth conditions is dynamic. Key cellular mechanisms such as ERK signaling and cell-cycle synchronization differed significantly among cells in colonies after 2 to 4 generations and became indistinguishable from randomly sampled cells regarding these features. In the presence of cytotoxic agents, colonies reduced their variance in growth rate when compared with their founder cell, indicating a dynamic nature i...
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, Jan 22, 2017
Breast cancer is a global public health problem. For some subtypes, such as Claudin-low, the prog... more Breast cancer is a global public health problem. For some subtypes, such as Claudin-low, the prognosis is poorer and the treatment is still a challenge. Pyrazoles are an important class of heterocyclic compounds and are promising anticancer agents based on their chemical properties. The present study was aimed not only at testing pyrazoles previously prepared by our research group in two breast cancer cell lines characterized by intermediated response to conventional chemotherapy but also at analyzing the possible synergistic effect of these pyrazoles associated with doxorubicin. Four 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H pyrazoles were tested for the first time in MCF-7 and MDA-MB-231 culture cells. The pyrazoles with best results in cytotoxicity were used in combination with doxorubicin and compared with this drug alone as standard. The synergic effect was analyzed using Combination Index method. In addition, cell death and apoptosis assays were carried out. Two pyrazoles with...
Nanostructured drug delivery systems have been extensively studied, mainly for applications in ca... more Nanostructured drug delivery systems have been extensively studied, mainly for applications in cancer therapy. The advantages of these materials include protection against drug degradation and improvement in both the relative solubility of poorly water soluble drugs as in targeting of therapy, due to the enhanced permeability and retention effect on tumor sites. In this work, we evaluate the antitumor activity of tretinoin-loaded lipid core nanocapsules (TT-LNC) in a tretinoin-resistant breast cancer cell-line, MDA-MB- 231, as well as the synergistic effect of combination of this treatment with 5-FU or DOXO. The inhibition of cell growth was assayed by MTT reduction. Live/Dead assay and DAPI staining evaluated cytotoxicity. Apoptosis was evaluated by Annexin V-PE/7AAD and the effect of chronic exposure was evaluated by colony formation assay. TT-LNC reduced the cell viability even at lower concentrations (1μM) and displayed synergistic effect with 5-FU or DOXO on cytotoxicity and co...
Bladder cancer is a genitourinary malignant disease common worldwide. Current chemotherapy is oft... more Bladder cancer is a genitourinary malignant disease common worldwide. Current chemotherapy is often limited mainly due to toxicity and drug resistance. Thus, there is a continued need to discover new therapies. Recently evidences shows that pyrazoline derivatives are promising antitumor agents in many types of cancers, but there are no studies with bladder cancer. In order to find potent and novel chemotherapy drugs for bladder cancer, a series of pyrazoline derivatives 2a-2d were tested for their antitumor activity in two human bladder cancer cell lines 5647 and T24. The MTT assay showed that the compounds 1-thiocarbamoyl-3,5-diphenyl-4,5-dihydro-1H-pyrazole (2a) and 1-thiocarbamoyl-5-(4-chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole (2c) decrease the cell viability of 5637 cells. Molecular modeling indicated that these compounds had a good oral bioavailability and low toxicities. Clonogenic assay and flow cytometric analysis were used to assess colony formation, apoptosis induction and cell cycle distribution. Overall, our results suggest that pyrazoline 2a and 2c, with the substituents hydrogen and chlorine respectively, may decrease cell viability and colony formation of bladder cancer 5637 cell line by inhibition of cell cycle progression, and for pyrazoline 2a, by induction of apoptosis. As indicated by the physicochemical properties of these compounds, the steric factor influences the activity. Therefore, these pyrazoline derivatives can be considered promising anticancer agents for the treatment of bladder cancer.
Herein we report the use of ultrasonic irradiation (US) in the synthesis of six new semi-syntheti... more Herein we report the use of ultrasonic irradiation (US) in the synthesis of six new semi-synthetic selenium-containing chrysin derivatives by a simple and effective methodology utilizing CuI as catalyst, in good to excellent yields (60-89%). It was observed that US accelerates the reaction compared to conventional heating with excellent selectivity for diselenylated products. Compounds were tested for their antioxidant and anticancer activities in vitro and it was observed that the presence of selenium in the A-ring of chrysin enhanced both antioxidant and anticancer properties. Semi-synthetic 6,8-bis(o-tolylselanyl)-chrysin 3b has the best radical scavenging activity of DPPH (Imax: 39.79µM) and ABTS(+) (IC50: 6.5µM) radicals. Similarly, in the Reactive Species (RS) assay, 3b showed high antioxidant activity in mice cortex (IC50: 5.67µM), whereas 6,8-bis(p-anisoylselanyl)-chrysin 3c was the more active in the hippocampus (IC50: 5.63µM). The Se-chrysins were effective in prevention o...
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