Journal: NPJ Precision Oncology

Article Title: Single-organoid analysis reveals clinically relevant treatment-resistant and invasive subclones in pancreatic cancer

doi: 10.1038/s41698-023-00480-y

Figure Lengend Snippet: a Bar plot representation of the progression-free survival (PFS) in months per patient. b NDR results upon treatment with gemcitabine-paclitaxel highlighting the 3 distinct responses. Error bars indicate the standard deviation. c Representative brightfield/fluorescence (Cytotox Green) images of PDAC052, PDAC060 and PDAC082 indicating the presence of persistent PDAC organoid clones (black circle). One representative image was selected for visualization out of the two technical replicates. Scale bar=100 µm d Single organoid dose response based on cell death (green area/brightfield area) labeled as fraction affected and area (brightfield) of PDAC052, PDAC060 and PDAC082 treated with gemcitabine-paclitaxel (400 nM:80 nM). Dark grey region (Fraction affected <0.15) indicates resistant, middle grey region sensitive (Fraction affected 0.15-0.34) and light grey highly sensitive (Fraction affected >0.34) PDAC organoid clones. Bubble size correlates with the organoid area. e Relative fraction of resistant, sensitive and highly sensitive PDAC organoids for each patient treated with gemcitabine-paclitaxel (400 nM:80 nM). f Dynamic quantification of single-organoid responses treated with gemcitabine-paclitaxel (400 nM:80 nM) or FOLFIRINOX (20 µM 5-FU:0.0625 µM SN38:2.5 µM Oxaliplatin). The single organoid data was combined from 2 technical replicates.

Article Snippet: Cytotox Green (60 nM / well, Sartorius), Staurosporine (2 µM), 5-Fluorouracil (5-FU), SN38 (active metabolite Irinotecan), gemcitabine and paclitaxel (MedChemExpress) were dissolved in DMSO.

Techniques: Standard Deviation, Fluorescence, Clone Assay, Labeling

Journal: NPJ Precision Oncology

Article Title: Single-organoid analysis reveals clinically relevant treatment-resistant and invasive subclones in pancreatic cancer

doi: 10.1038/s41698-023-00480-y

Figure Lengend Snippet: a – c Spearman rank correlation of the % sensitive, % resistant and ratio % sensitive/resistant PDAC clones (gemcitabine-paclitaxel; 400 nM: 80 nM) with the PFS. d . Correlation of % ratio sensitive/resistant with the initial clinical response to gemcitabine-paclitaxel. Responses were based on available radiological CT protocols. Good= tumor regression and/or PFS > 10 months, Mixed= minor tumor regression, Bad= no tumor regression and/or fast disease progression. e NDR signatures indicating the NDR value (left y-axis) and % cell death (right y-axis) upon treatment with gemcitabine-paclitaxel (400 nM:80 nM) or FOLFIRINOX (4 µM 5-FU:0.0125 µM SN38:0.5 µM Oxaliplatin). f Representative CT-scans before and after treatment with gemcitabine/nab-paclitaxel or FOLFIRINOX (8 cycles). Red contours indicate the tumor margins. g Overview of the clinical characteristics of patient PDAC044, PDAC052, PDAC060, PDAC068 and PDAC087. h Comparison of the implemented readouts, suggesting a potential clinical application of combining the NDR readout with the developed single-organoid analysis.

Article Snippet: Cytotox Green (60 nM / well, Sartorius), Staurosporine (2 µM), 5-Fluorouracil (5-FU), SN38 (active metabolite Irinotecan), gemcitabine and paclitaxel (MedChemExpress) were dissolved in DMSO.

Techniques: Clone Assay, Comparison

Journal: Oncotarget

Article Title: Akt inhibition improves irinotecan treatment and prevents cell emergence by switching the senescence response to apoptosis

doi:

Figure Lengend Snippet: A. HCT116 (left) and LS174T (right) cells have been treated with sn38 at the indicated concentrations and clonogenic assays were used to evaluate cell survival after 8-10 days of culture ( n = 5 +/− sd, 1 ng/ml = 2.5 nM). B. LS174T and HCT116 cells have been treated with sn38 (5 ng/ml or 12.7 nM) for the indicated time, total cell extracts were then prepared and p21waf1 expression was evaluated by western blot (lanes 1-6, n = 4). Following sn38 treatment, the percentage of senescent cells was evaluated as the number of cells expressing SA-Δgal activity ( n = 4 +/− sd). C. , D. LS174T (C) and HCT116 (D) cells have been treated with sn38 (5 ng/ml or 12.7 nM) for the indicated time, total cell extracts were then prepared and Akt activation was evaluated by western blot ( n = 4).

Article Snippet: To induce persistent cell generation, LS174T cells were treated for 4 days with sn38 (5 ng/ml or 12.7 nM; Tocris Bioscience 2684) in 3% FBS, washed with PBS and then restimulated with fresh 10% FBS for 7 days.

Techniques: Expressing, Western Blot, Activity Assay, Activation Assay

Journal: Oncotarget

Article Title: Akt inhibition improves irinotecan treatment and prevents cell emergence by switching the senescence response to apoptosis

doi:

Figure Lengend Snippet: A. HCT116 and LS174T cells were treated as above with sn38 (5 ng/ml or 12.7 nM), GSK690693 (20 μM) or Akti ½ (10 μM) for 72h. Flow cytometry experiments were then performed to quantify the percentage of cells in each phase of cell cycle ( n = 4 +/− sd). B. Cells were treated as above and apoptosis was evaluated by FACS analysis and the detection of the active form of caspase 3 ( n = 3 +/− sd). C. Akt was downregulated by RNA interference, the next day LS174T cells were treated with sn38 (5 ng/ml or 12.7 nM), total cell extracts were then prepared and the expression of the cleaved caspase-3 was evaluated by western blot ( n = 3). D. HCT116 and HCT116 p21−/− cells were treated with sn38 (5 ng/ml or 12.7 nM) for the indicated time and the percentage of cells presenting a subG1 content was evaluated by FACS analysis ( n = 5 +/− sd). E. p21waf1 was downregulated by RNA interference, the next day HCT116 cells were treated with sn38 (5 ng/ml or 12.7 nM) for 48 hours, total cell extracts were then prepared and the expression of the cleaved caspase-3 was evaluated by western blot ( n = 3). F. p53 expression was downregulated by RNA interference, LS174T cells were treated as above and the expression of the cleaved caspase-3 was evaluated ( n = 3).

Article Snippet: To induce persistent cell generation, LS174T cells were treated for 4 days with sn38 (5 ng/ml or 12.7 nM; Tocris Bioscience 2684) in 3% FBS, washed with PBS and then restimulated with fresh 10% FBS for 7 days.

Techniques: Flow Cytometry, Expressing, Western Blot

Journal: Oncotarget

Article Title: Akt inhibition improves irinotecan treatment and prevents cell emergence by switching the senescence response to apoptosis

doi:

Figure Lengend Snippet: A. The indicated cell lines were treated with different concentrations of GSK690693 and clonogenic assays were used to evaluate cell survival after 8-10 days of culture. IC50 values were calculated by counting the number of surviving colonies. B. LS174T and HCT116 cells have been treated with sn38 (5 ng/ml or 12.7 nM), GSK690693 (20μM) or both drug for 8hr. Akt and GSK3Δ expression and phosphorylation have been evaluated by western blot ( n = 3). C. Following treatment with sn38 (0.25 ng/ml or 0.63 nM), GSK690693 (1μM for LS174T cells and 7μM for HCT116 cells), Akti 1/2 inhibitor (4μM), clonogenic assays were used to evaluated long term cell death after 7-10 days of culture ( n = 8 +/− sd). D. LS174T and HCT116 cells have been treated with sn38 (5 ng/ml or 12.7 nM) in the presence or absence of GSK690693 (20 μM) or Akti 1/2 (10 μM) as indicated for 72hr. MTT assays were then used to evaluate short time effects on cell viability ( n = 4 +/− sd).

Article Snippet: To induce persistent cell generation, LS174T cells were treated for 4 days with sn38 (5 ng/ml or 12.7 nM; Tocris Bioscience 2684) in 3% FBS, washed with PBS and then restimulated with fresh 10% FBS for 7 days.

Techniques: Expressing, Phospho-proteomics, Western Blot

Journal: Oncotarget

Article Title: Akt inhibition improves irinotecan treatment and prevents cell emergence by switching the senescence response to apoptosis

doi:

Figure Lengend Snippet: A. Experimental procedure to generate persistent LS174T cells named PLC in this study: following sn38 treatment (5 ng/ml or 12.7 nM, 96 hrs), cells have been stimulated with 10% FBS for 7 days to reinduce cell growth. Representative images showing PLC heterogeneity are shown on the right, illustrating the presence of dividing (PLD) clones within senescent cells (PLS) stained with Δ-galactosidase. B. Following DAPI staining and flow cytometry analysis, PLC cells were gated according to low (PLD) or high (PLS) FSC/SSC values. Ki67 expression was evaluated in each subpopulation. Cell percentages are presented on the right side ( n = 5 +/− sd). C. pAkt Ser473 phosphorylation was analyzed by flow cytometry in PLC. Cells were gated according to low and high FSC/SSC values and the corresponding pAkt Ser473 expression was evaluated in PLD and PLS ( n = 4 +/− sd). Representative images are shown on the right. D. PLD and PLS cells have been cell sorted by flow cytometry according to low or high FSC/SSC values, as described in . Akt phosphorylation has been evaluated by western blot in the indicated cells ( n = 3). E. , F. PLC were generated in the presence or absence of GSK690693 added at different times during emergence, either at the same time as sn38 (condition 1) or at the time of release in 10% serum (condition 2). Emerging clones were counted using crystal violet staining and compared to PLC generated in the absence of Akt inhibition ( n = 6 +/− sd). G. In the same conditions, PLD and PLS cells were gated according to low and high FSC/SSC values and the amount of proliferating cells was evaluated in PLD cells using Ki67 expression ( n = 6 +/− sd).

Article Snippet: To induce persistent cell generation, LS174T cells were treated for 4 days with sn38 (5 ng/ml or 12.7 nM; Tocris Bioscience 2684) in 3% FBS, washed with PBS and then restimulated with fresh 10% FBS for 7 days.

Techniques: Clone Assay, Staining, Flow Cytometry, Expressing, Phospho-proteomics, Western Blot, Generated, Inhibition

Journal: Oncotarget

Article Title: Akt inhibition improves irinotecan treatment and prevents cell emergence by switching the senescence response to apoptosis

doi:

Figure Lengend Snippet: A. LS174T and HCT116 cells have been treated with sn38 (5 ng/ml or 12.7 nM) in the presence or absence of GSK690693 (20 μM) and Akti 1/2 (10 μM) as indicated for 72 hrs and the percentage of Δ-galactosidase-positive cells was evaluated ( n = 4 +/−sd). B. LS174T and HCT116 cells have been treated as above, total cell extracts were prepared and p21waf1 expression was evaluated by western blot ( n = 5). C. Akt expression was down-regulated by RNA interference, the next day LS174T cells were treated with sn38 during 72 hours, total cell extracts were then prepared and p21waf1 and Akt expression were evaluated ( n = 3). D. Akt expression was down-regulated by RNA interference, the next day LS174T cells were treated with sn38 during 72 hours and the percentage of Δ-galactosidase-positive cells was evaluated ( n = 4 +/− sd). E. HCT116 cells were treated with sn38 in the presence or absence of GSK690693 and Akti 1/2 for 72 hrs. PML staining was then evaluated by immunofluorescence. The average of fluorescence intensity per cell has been quantified ( n = 3). F. LS174T (bottom panel) and HCT116 (top panel) cells have been treated as above, total cell extracts were prepared and cyclin D1 expression was evaluated by western blot ( n = 3).

Article Snippet: To induce persistent cell generation, LS174T cells were treated for 4 days with sn38 (5 ng/ml or 12.7 nM; Tocris Bioscience 2684) in 3% FBS, washed with PBS and then restimulated with fresh 10% FBS for 7 days.

Techniques: Expressing, Western Blot, Staining, Immunofluorescence, Fluorescence

Journal: Oncotarget

Article Title: Akt inhibition improves irinotecan treatment and prevents cell emergence by switching the senescence response to apoptosis

doi:

Figure Lengend Snippet: A. HCT116 and HCT116 p21−/− cells have been treated with sn38 (5 ng/ml or 12.7 nM) and the percentage of Δ-Galactosidase positive cells was evaluated after 72 hrs ( n = 4 +/− sd). B. Emergence was evaluated in HCT116 and HCT116 p21−/− cells following sn38 treatment. Note that in comparison with LS174T cells, emergence occurred after more than 14 days in this cell line. Clones were then counted using crystal violet staining ( n = 5 +/− sd). C. p21waf1 expression was downregulated by RNA interference, in LS174T cells. PLC were then generated as above and emergence was evaluated by crystal violet staining ( n = 5 +/− sd). D. LS174T cells were treated with sn38 (5 ng/ml or 12.7 nM) for the indicated time, total cell extracts were then prepared and p53 activation was evaluated by western blot with the indicated antibodies ( n = 5). E. p53 expression was downregulated by RNA interference, the next day LS174T cells were treated with sn38 for 48 hours, total cell extracts were then prepared and p21waf1 and p53 expressions were evaluated ( n = 3). F. p53 expression was down-regulated by RNA interference, PLC were generated and cell emergence was evaluated as compared to cells transfected with a control siRNA. Representative images of crystal violet staining are shown ( n = 5 +/− sd).

Article Snippet: To induce persistent cell generation, LS174T cells were treated for 4 days with sn38 (5 ng/ml or 12.7 nM; Tocris Bioscience 2684) in 3% FBS, washed with PBS and then restimulated with fresh 10% FBS for 7 days.

Techniques: Comparison, Clone Assay, Staining, Expressing, Generated, Activation Assay, Western Blot, Transfection, Control

Journal: Oncotarget

Article Title: Akt inhibition improves irinotecan treatment and prevents cell emergence by switching the senescence response to apoptosis

doi:

Figure Lengend Snippet: A. , B. Cells have been treated with sn38, GSK690693 or Akti 1/2 inhibitor as above for 72 hr, total cell extracts were then prepared and Mcl-1 (A), Bcl-xL (A) and Noxa (B) expressions were evaluated by western blot ( n = 3). C. Cells have been treated with sn38 and GSK690693 as above for 72 hr, total cell extracts were then prepared and the interaction between Mcl-1 and Noxa was evaluated by co-immunoprecipitation. An antibody directed against the Gal4 protein was used as a control ( n = 4). D. Noxa was downregulated by RNA interference, the next day HCT116 cells were treated with sn38 and GSK690693 for 48 hours, total cell extracts were prepared and the expression of the active form of caspase 3 was evaluated ( n = 3). E. Noxa or Mcl-1 expressions were downregulated by RNA interference, persistent HCT116 cells were then generated and cell emergence was evaluated by crystal violet staining after 10-15 days ( n = 5 +/−sd).

Article Snippet: To induce persistent cell generation, LS174T cells were treated for 4 days with sn38 (5 ng/ml or 12.7 nM; Tocris Bioscience 2684) in 3% FBS, washed with PBS and then restimulated with fresh 10% FBS for 7 days.

Techniques: Western Blot, Immunoprecipitation, Control, Expressing, Generated, Staining

Journal: Oncotarget

Article Title: Akt inhibition improves irinotecan treatment and prevents cell emergence by switching the senescence response to apoptosis

doi:

Figure Lengend Snippet: A. LS174T cells have been treated with sn38 (5 ng/mL or 12.7 nM) for 96 hrs (lane 2), serum was added (lane 3) in the absence or presence of GSK690693 (20μM, lane 3) or Akti 1/2 (10μM, lane 4) for 3 days in 10% serum. B. In these experimental conditions, total cell extracts were prepared, and p21waf1 expression was then evaluated by western blot ( n = 4). C. In the same condition, the percentage of Δ-galactosidase-positive cells was evaluated ( n = 4, +/− sd). D. In these experimental conditions, total cell extracts were prepared, and p21waf1 expression was then evaluated by western blot ( n = 4).

Article Snippet: To induce persistent cell generation, LS174T cells were treated for 4 days with sn38 (5 ng/ml or 12.7 nM; Tocris Bioscience 2684) in 3% FBS, washed with PBS and then restimulated with fresh 10% FBS for 7 days.

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: Akt inhibition improves irinotecan treatment and prevents cell emergence by switching the senescence response to apoptosis

doi:

Figure Lengend Snippet: In response to sn38, a small fraction of cells escapes the senescence suppressive arrest and emerges as an heterogeneous and more transformed population (see also reference ). Akt is activated during the early step of this response and in the emergent dividing cells. Its presence favors cell emergence and its inhibition consequently improves sn38 efficacy by reducing the senescence suppressive arrest and activating apoptosis instead. We speculate that these emergent cells are responsible of treatment failure and disease relapse. Therefore, sequential Akt targeting should be considered in the future to improve the treatment of irinotecan-refractory colorectal cancers through the induction of apoptosis.

Article Snippet: To induce persistent cell generation, LS174T cells were treated for 4 days with sn38 (5 ng/ml or 12.7 nM; Tocris Bioscience 2684) in 3% FBS, washed with PBS and then restimulated with fresh 10% FBS for 7 days.

Techniques: Transformation Assay, Inhibition

Journal: Cancer research and treatment

Article Title: The Synergistic Effect of PARP Inhibitors and Irinotecan in Small Cell Lung Cancer Cells.

doi: 10.4143/crt.2024.728

Figure Lengend Snippet: Fig. 3. PARP inhibitors in combination with irinotecan had synergistic effects on in vitro SCLC

Article Snippet: Small cell lung cancer cells were seeded in 96-well plates at a proper density and allowed to stabilize for 24 h. Drugs including olaparib (Selleckchem, cat. no. S1060), talazoparib (Selleckchem, cat. no. S7048), venadaparib (provided by Idience Co., Ltd), and irinotecan (Selleckchem, cat. no. S1198), were used from stock solutions dissolved in DMSO and added at screening concentrations up to 10 μM.

Techniques: In Vitro

Journal: Cancer research and treatment

Article Title: The Synergistic Effect of PARP Inhibitors and Irinotecan in Small Cell Lung Cancer Cells.

doi: 10.4143/crt.2024.728

Figure Lengend Snippet: Fig. 4. PARP inhibitors combined with irinotecan increase apoptotic signaling in SCLC cells.

Article Snippet: Small cell lung cancer cells were seeded in 96-well plates at a proper density and allowed to stabilize for 24 h. Drugs including olaparib (Selleckchem, cat. no. S1060), talazoparib (Selleckchem, cat. no. S7048), venadaparib (provided by Idience Co., Ltd), and irinotecan (Selleckchem, cat. no. S1198), were used from stock solutions dissolved in DMSO and added at screening concentrations up to 10 μM.

Techniques:

Journal: Cancer research and treatment

Article Title: The Synergistic Effect of PARP Inhibitors and Irinotecan in Small Cell Lung Cancer Cells.

doi: 10.4143/crt.2024.728

Figure Lengend Snippet: Fig. 5. PARP inhibitors combined with irinotecan increase DNA damage. The effects of PARP

Article Snippet: Small cell lung cancer cells were seeded in 96-well plates at a proper density and allowed to stabilize for 24 h. Drugs including olaparib (Selleckchem, cat. no. S1060), talazoparib (Selleckchem, cat. no. S7048), venadaparib (provided by Idience Co., Ltd), and irinotecan (Selleckchem, cat. no. S1198), were used from stock solutions dissolved in DMSO and added at screening concentrations up to 10 μM.

Techniques:

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Tumour regression and improved gastrointestinal tolerability from controlled release of SN-38 from novel polyoxazoline-modified dendrimers.

doi: 10.1016/j.jconrel.2016.12.034

Figure Lengend Snippet: Figure 1. (A) In vitro release of SN-38 in PBS. (B) In vitro plasma release (Wistar rat). (C) In vivo

Article Snippet: Irinotecan was purchased from CarboSynth (UK).

Techniques: In Vitro, Clinical Proteomics, In Vivo

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Tumour regression and improved gastrointestinal tolerability from controlled release of SN-38 from novel polyoxazoline-modified dendrimers.

doi: 10.1016/j.jconrel.2016.12.034

Figure Lengend Snippet: Figure 2. Efficacy and gastrointestinal toxicity of Dend-SN38 conjugates (2a-c) versus irinotecan

Article Snippet: Irinotecan was purchased from CarboSynth (UK).

Techniques:

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Tumour regression and improved gastrointestinal tolerability from controlled release of SN-38 from novel polyoxazoline-modified dendrimers.

doi: 10.1016/j.jconrel.2016.12.034

Figure Lengend Snippet: Figure 4. Tumour pharmacokinetics and pharmacodynamic effects of irinotecan and DEND-38

Article Snippet: Irinotecan was purchased from CarboSynth (UK).

Techniques: Drug discovery

Journal: Cell Reports Medicine

Article Title: Histone lysine demethylase 4 family proteins maintain the transcriptional program and adrenergic cellular state of MYCN-amplified neuroblastoma

doi: 10.1016/j.xcrm.2024.101468

Figure Lengend Snippet:

Article Snippet: Irinotecan , MedChem Express , HY-16562A.

Techniques: Recombinant, Modification, Synthesized, Saline, Transfection, Apoptosis Assay, Purification, Transgenic Assay, Control, Expressing, Plasmid Preparation, Software

Journal: Cell Death & Disease

Article Title: Inhibition of mitochondrial fission activates glycogen synthesis to support cell survival in colon cancer

doi: 10.1038/s41419-023-06202-3

Figure Lengend Snippet: A Sh-Ctrl and sh-Drp1 PT130 cells were cultured in regular growth media or glucose-free medium for 48 h. The percentage of cell survival were obtained by normalizing the number of cells survived in glucose-free media to that of regular growth media. Data were presented as mean ± SD ( n = 6, ** p < 0.01 and *** p < 0.001). B Sh-Ctrl and sh-Drp1 PT130 cells were cultured in low glucose or glucose-free media for 24 h. Representative confocal images were obtained from cells stained with the anti-glycogen antibody. Scale Bar, 10 μm. C The relative fluorescence intensity of glycogen staining was quantified using ImageJ fluorescence analyzer. Data were presented as mean ± SD ( n = 20, * p < 0.05, ** p < 0.01 and **** p < 0.0001). D Sh-Ctrl and sh-Drp1 PT130 cells were treated with irinotecan, DAB or combinations of both agents for 48 h. Cells treated with DMSO were used as control. The relative of cell survival were obtained by normalizing to cells treated with DMSO. Data were presented as mean ± SD ( n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001). E Results from our study demonstrate that disruption of mitochondrial dynamics as a consequence of silencing Drp1 increases glucose uptake and AMPK-dependent transcriptional activation of GYS1. Subsequently, GYS1-mediated glycogen accumulation serves as a compensatory survival mechanism to protect colon cancer cells from glucose starvation and chemotherapy treatment. Thus, co-targeting glycogenolysis may provide a novel strategy to sensitize Drp1 knockdown cells to chemotherapy.

Article Snippet: The following reagents were obtained from commercial sources: 1,4-dideoxy-1,4-imino-D-Arabinitol hydrochloride (DAB) was from Cayman Chemical (#20939), irinotecan hydrochloride and Compound C (AMPK inhibitor) were from MilliporeSigma (I1406 and 171260, respectively); control siRNA and siRNA targeting GYS1 was obtained from Santa Cruz Biotechnology.

Techniques: Cell Culture, Staining, Fluorescence, Control, Disruption, Activation Assay, Knockdown