Journal: Journal for immunotherapy of cancer

Article Title: Understanding the dynamics of TKI-induced changes in the tumor immune microenvironment for improved therapeutic effect.

doi: 10.1136/jitc-2024-009165

Figure Lengend Snippet: Figure 1 TKI therapy alters the tumor immune microenvironment. (A) Representative mIF images of pretreatment tumor and resected samples analyzed for immune-related biomarkers. (B) Densities (cells/mm2) of CD8+, CD8+GB+, CD8+PD-1+, CD163+, CD68+, and CD163+CD68+ by mIF quantification in paired pretreatment tumor samples and resected tumors. (C) Cell viability CCK-8 assay for cells treated with TKIs (osimertinib: 10 nM, lorlatinib: 10 nM), activated T cells (1:1 ratio to cancer cells), or the combination. (D) T cell-mediated cancer cell-killing assay. PC-9 and H3122 cells co-cultured with activated T cells for 48 hours with or without TKIs (osimertinib: 10 nM, lorlatinib: 10 nM) were subjected to crystal violet staining. Ratio of cancer cells to T cells: 1:1. (E) Ki67 incorporation assay on PC-9 and H3122 cells treated as indicated. Activated T cells (1:1 ratio to cancer cells) or TKIs (osimertinib: 10 nM, lorlatinib: 10 nM) were added to the culture medium for 48 hours. Cells were then counterstained with DAPI. (F) PC-9 cells were injected into mice (n=3 mice per group) on day 0, hu-PBMCs were injected into mice via the tail vein on day 7, and osimertinib was administered as indicated. (G) Macroscopic appearance of tumors after drug application for 4 weeks. (H) The tumor weight (g) for each mouse is shown. *p<0.05, **p<0.01. ns, no significance. (I) Immunofluorescence staining with an antibody against CD8 to detect T cells and antibodies against CD68 and CD206 to detect macrophages in TKI-resistant non-small cell lung cancer tissues (11 cases of EGFR-TKI resistance, 5 cases of ALK-TKI resistance). Scale bar: 50 µm. ALK, anaplastic lymphoma kinase; DAPI, 4′,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; ALKi,anaplastic lymphoma kinase inhibitor; EGFRi,epidermal growth factor receptor inhibitor; hu-PBMC,human-Peripheral blood mononuclear cell; mIF, multiplex immunofluorescence; PD-1, programmed cell death protein 1; TKI, tyrosine kinase inhibitor.

Article Snippet: Then, the cells were fixed and incubated overnight with Ki67 (#M00254- 8, Boster, Wuhan, China).

Techniques: CCK-8 Assay, Cell Culture, Staining, Injection, Immunofluorescence, Multiplex Assay

Journal: Journal for immunotherapy of cancer

Article Title: Understanding the dynamics of TKI-induced changes in the tumor immune microenvironment for improved therapeutic effect.

doi: 10.1136/jitc-2024-009165

Figure Lengend Snippet: Figure 6 Aspirin enhances the antitumor immunity response. (A) PC-9 cells were injected into mice (n=3 mice per group) on day 0, hu-PBMCs were injected into the tail veins of mice on day 7, and osimertinib/osimertinib plus aspirin was administered as indicated. (B) Macroscopic appearance of tumors after drug application for 4 weeks. The tumor weight (g) of each mouse is shown. *p<0.05, ***p<0.001. (C) Cell viability CCK-8 assay for cells treated with aspirin (200 µM), activated T cells (1:2 ratio to cancer cells), or the combination for 48 hours. Data are shown as the mean±SEM. *p<0.01. (D) T cell-mediated cancer cell-killing assay. PC-9OR and HCC827OR cells co-cultured in the indicated groups for 48 hours were subjected to crystal violet staining. Ratio of cancer cells to T cells: 2:1. (E) Ki67 incorporation assay on PC-9OR and HCC827OR cells treated as indicated. Activated T cells (1:2 ratio to cancer cells) or aspirin (200 µM) were added to the culture medium for 48 hours. Cells were then counterstained with DAPI. (F) Flow cytometry analysis of the exhaustion- and activation-related molecule Foxp3 in activated T cells co-cultured with the indicated cells (ratio of cancer cells to T cells: 1:1) for 48 hours with or without aspirin (200 µM). (G) PD-L1 levels in total protein extracts from indicated cells treated with aspirin for 48 hours, analyzed by western blotting. (H) LAMC2 levels in total protein extracts from the indicated cells treated with aspirin for 48 hours, analyzed by western blotting. (I) Immunofluorescence staining with an antibody against LAMC2 in PC-9 xenografts treated with osimertinib or osimertinib plus aspirin. (J) Schematic diagram of a T-cell chemotaxis assay directly regulated by the treatment of PC-9OR cells with aspirin (200 µM). Representative images of infiltrating T cells stained with CFSE dye. (K) Representative images of the immunofluorescence staining of CD68 (red), CD206 (green), and DAPI (blue) in mouse tumor tissue sections. (L) Immunofluorescence staining with antibodies against CD8 (red) and LAMC2 (green) in non-small cell lung cancer tissues treated with osimertinib or osimertinib plus aspirin. Scale bar: 50 µm.hu-PBMC,human-Peripheral blood mononuclear cell; S.C,Subcutaneous; CFSE, carboxyfluorescein diacetate succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; Foxp3, forkhead box P3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LAMC2, laminin subunit γ−2; PBS, phosphate-buffered saline; PD-L1, programmed death ligand 1.

Article Snippet: Then, the cells were fixed and incubated overnight with Ki67 (#M00254- 8, Boster, Wuhan, China).

Techniques: Injection, CCK-8 Assay, Cell Culture, Staining, Flow Cytometry, Activation Assay, Western Blot, Immunofluorescence, Chemotaxis Assay, Saline

Journal: Cell stem cell

Article Title: Melanocyte Stem Cell Activation and Translocation Initiate Cutaneous Melanoma in Response to Ultraviolet Exposure

doi: 10.1016/j.stem.2017.09.001

Figure Lengend Snippet: (A) MCSC translocation by topical TPA treatment shown by tdTomato lineage tracing. (B) Experimental scheme. (C) Macroscopic phenotypes in TBP mice with/without topical TPA treatment, and quantification of epidermal pigmentation. n = 10 TPA-treated control, n = 7 non-TPA TBP and n = 7 TPA-treated TBP mice. (D) Histological phenotypes of TPA-induced melanoma during quiescent MCSC, telogen period, and co-immunostaining for Sox10, a melanocyte stem cell/melanoma marker, with Ki67, a marker of proliferation. (E) Experimental scheme. Dex., dexamethasone. (F) Relative number of MCSC migration was quantified via tdTomato lineage tracing in TT mice, compared to the total number of hair follicles (≥ 500 hair follicles / each). Mean ± SEM, n = 10 / group. (G) Experimental scheme. (H) Macroscopic phenotypes with the quantification of epidermal pigmentation. n = 6 low-dose (80 mJ cm−2) UVB, n = 10 per each group (UVB only or UVB + Dex.). Data are represented as Mean ± SEM. Also see Figures S5 and S6.

Article Snippet: Rabbit monoclonal anti-Ki-67 , Rockland , Cat#900-C01-B38; RRID:AB_2142230.

Techniques: Translocation Assay, Immunostaining, Marker, Migration

Journal: Cell stem cell

Article Title: Melanocyte Stem Cell Activation and Translocation Initiate Cutaneous Melanoma in Response to Ultraviolet Exposure

doi: 10.1016/j.stem.2017.09.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-Ki-67 , Rockland , Cat#900-C01-B38; RRID:AB_2142230.

Techniques: Plasmid Preparation, Recombinant, DNA Ligation, SYBR Green Assay, BIA-KA, Ab Array, Software, CRISPR

Journal: Cells

Article Title: Cistanoside F Ameliorates Lipid Accumulation and Enhances Myogenic Differentiation via AMPK-Dependent Signaling in C2C12 Myotubes.

doi: 10.3390/cells14120874

Figure Lengend Snippet: Figure 4. Expression of PGC-1α, MMP, and ROS in two adipogenic models after Cis adminis- tration. (A,B) PGC-1α expression. (C,D) Immunofluorescence of TMRE, the statistic was eval- uated by fluorescence spectroscopy, at λexcitation = 550 nm and λemission = 575 nm. (E,G) Im- munofluorescence of JC-1, statistic was evaluated by fluorescence spectroscopy, JC-1 monomer at λexcitation = 490 nm and λemission = 530 nm and JC-1 aggregate at λexcitation = 525 nm and λemission = 590 nm. (F,H) Immunofluorescence of ROS, the statistic was evaluated by fluorescence spectroscopy, at λexcitation = 488 nm and λemission = 525 nm. n = 6. ## p < 0.01, compared with the vehicle group; * p < 0.05, ** p < 0.01, compared with the model group.

Article Snippet: Antibodies against UCP1, CPT1b, ACC2, Desmin, and PGC-1α were purchased from Boster Biotech (Wuhan, China; #M00255; #A03558; #A03668; #PB9105; #BM4898; 1:1000).

Techniques: Expressing, Immunofluorescence, Fluorescence, Spectroscopy

Journal: Cells

Article Title: An In Vitro System for Evaluating Molecular Targeted Drugs Using Lung Patient-Derived Tumor Organoids

doi: 10.3390/cells8050481

Figure Lengend Snippet: Phase-contrast and confocal images of three lung F-PDOs. ( a ) Phase-contrast images of RLUN5, RLUN16, and RLUN21 were obtained using a ×4 objective. Scale bar: 200 µm. The upper panels show each F-PDO immediately after passage and the lower panels show each F-PDO before passage. ( b ) Confocal images of RLUN5, RLUN16, and RLUN21 were obtained using a ×10 objective. Scale bar: 100 µm. Ki67-expression images for RLUN5, RLUN16, and RLUN21, prepared using an anti-Ki67 antibody (green). DNA stained with DAPI (blue). ( c – e ) Confocal imaging data analyzed with NoviSight. ( c ) Box-and-whisker plot of the cell cluster volumes. ( d ) Box-and-whisker plot of the cell density expressed as the cell number/cell volume in a cell cluster. ( e ) Box-and-whisker plot of the Ki67-positive ratio expressed as 100× the number of positive cells/the number of total cells in a cluster.

Article Snippet: Immunofluorescence staining of F-PDOs was performed using an anti-Ki-67 antibody (1:250 dilution) and 4′,6-diamidino-2-phenylindole (DAPI) after fixation in phosphate buffer solution containing 4% paraformaldehyde (PFA; Fujifilm Wako Pure Chemical, Ltd.) and 1% Triton X-100 (Fujifilm Wako Pure Chemical, Ltd.).

Techniques: Expressing, Staining, Imaging, Whisker Assay

Journal: Cells

Article Title: An In Vitro System for Evaluating Molecular Targeted Drugs Using Lung Patient-Derived Tumor Organoids

doi: 10.3390/cells8050481

Figure Lengend Snippet: Dependency of F-PDO proliferation on epidermal growth factor (EGF). ( a ) Growth rate of RLUN5, RLU16, and RLUN21 in the presence or absence of EGF. Each F-PDO was seeded in a 96-well, round-bottomed, ultra-low-attachment microplate (Corning, Inc.). Twenty-four hours after seeding, EGF was added into the wells at a final concentration of 100 ng/mL. The wells without EGF were also set up to confirm the dependency of cell growth on EGF. One hour (culture start time) or 144 h later, the amount of ATP was measured as described in . Growth rates were calculated by dividing the amount of ATP in each well at 144 h by that at the culture start time. ( b ) Phase-contrast images of RLUN21 cultured with EGF (upper panels) or without EGF (lower panels) for six days. The images were obtained using a ×5 objective. Scale bar: 100 µm. ( c ) Images of Ki67 expression in RLUN21 cultured with EGF were obtained using an anti-Ki67 antibody (green). DNA stained with DAPI (blue). Magnification: ×10. Scale bar: 100 µm. Box-and-whisker plot of the Ki67-positive ratio ( d ) and cell density ( e ) of RLUN21 cells cultured with EGF.

Article Snippet: Immunofluorescence staining of F-PDOs was performed using an anti-Ki-67 antibody (1:250 dilution) and 4′,6-diamidino-2-phenylindole (DAPI) after fixation in phosphate buffer solution containing 4% paraformaldehyde (PFA; Fujifilm Wako Pure Chemical, Ltd.) and 1% Triton X-100 (Fujifilm Wako Pure Chemical, Ltd.).

Techniques: Concentration Assay, Cell Culture, Expressing, Staining, Whisker Assay

Journal: Oncotarget

Article Title: AXL knockdown gene signature reveals a drug repurposing opportunity for a class of antipsychotics to reduce growth and metastasis of triple-negative breast cancer

doi: 10.18632/oncotarget.26725

Figure Lengend Snippet: ( A ) Schematic showing the treatment of nude mice bearing mammary fat pad xenografts of MDA-MB-231-Luc cells. ( B , C ) Treatments of mice with MDA-MB-231-Luc mammary fat pad grafts with the antipsychotics reduce tumor growth. Tumor diameters were measured before the beginning of the treatments (7 days after the graft) to quantify the fold increase overtime. To make the growth curve, the tumor diameters were measured twice a week. ( * p = 0.0242, * p = 0.0197, * p = 0.0344). ( n = 8) Data are represented as mean ± SEM. ( D ) Tumors treated with THZ, FLZ and TFP showed reduced proliferation. Immunohistochemistry detection of Ki67 using DAB staining was done on primary tumors and Hematoxylin was used as a counterstain to reveal the nuclei. ( *** p < 0.0001, ** p = 0.0067, ** p = 0.0043) ( n = 5) Scale bar: 50μm Data are represented as mean ± SEM. ( E ) Treatments with phenothiazines in vivo increased apoptosis shown by TUNEL fluorescent staining with DAPI as a counterstain for the nuclei. ( * p = 0.0415, *** p < 0.0001, *** p = 0.0003) ( n = 5) Scale bar: 50 μm Data are represented as mean ± SEM. ( F , G ) Treatments with THZ, FLZ and TFP reduce the metastatic progression of MDA-MB-231-Luc xenografts to the lungs. Representative images of lungs showing metastases. 31 days after tumor graft, at the end point of the growth experiments, mice were injected with Luciferin and lungs were dissected and imaged for bioluminescence signal to quantify the metastases. ( *** p < 0.0001). Data are represented as mean ± SEM.

Article Snippet: The sections were consequently incubated with a primary antibody against Ki67 (1:250, Medicorp), a biotin-conjugated anti-rabbit IgG secondary antibody (1:1000, BA-1000 Vector Laboratories) and a Streptavidin-HRP ternary antibody (1:1000, BD Pharmingen).

Techniques: Immunohistochemistry, Staining, In Vivo, TUNEL Assay, Injection

Journal: Cancer Medicine

Article Title: Targeting stanniocalcin‐1‐expressing tumor cells elicits efficient antitumor effects in a mouse model of human lung cancer

doi: 10.1002/cam4.3852

Figure Lengend Snippet: Evaluation of antitumor effects by targeting STC‐1‐expressing lung cancer cells by the UPRT/5‐FU system in vivo. (A) Establishment of a human lung cancer xenograft model using nude mice. The size of the tumor formed by P STC‐1 ‐UPRT‐PC‐9 (filled circle) or P STC‐1 ‐Null‐PC‐9 (open circle) in mice ( n = 8 for each group) was measured without 5‐FU treatment. All data represent the means ±S.D. values. This experiment was repeated independently three times with reproducible result, and a representative result is shown. (B) Tumor growth was evaluated without 5‐FU treatment. Macroscopic findings (left) and weight (right) of collected tumors. The bar graph and overlaid dots represent the means ±S.D. values and measured values respectively. This experiment was repeated independently three times with reproducible result, and a representative result is shown. (C) Histopathologic findings of collected tumors formed by P STC‐1 ‐UPRT‐PC‐9 or P STC‐1 ‐Null‐PC‐9. Representative microscopic images of hematoxylin and eosin staining (upper) and anti‐Ki‐67 immunohistochemistry (middle). Scale bar: 500 µm (scale bar in inset: 100 µm). Representative microscopic images of fluorescence in a tumor section (bottom) Scale bar: 50 µm. (D) RT‐PCR for detection of UPRT mRNA in tumor mass formed by P STC‐1 ‐UPRT‐PC‐9 or P STC‐1 ‐Null‐PC‐9. Bands of β‐actin served as control. (E) Enhanced antitumor effects by systemic administration of 5‐FU. Every second day from day 5 through day 17 (seven times total), 5‐FU (1.0 mg/kg body weight) was administered intraperitoneally into tumor‐bearing mice. The tumor size of P STC‐1 ‐UPRT‐PC‐9 (filled circle) or P STC‐1 ‐Null‐PC‐9 (open circle) in mice ( n = 8 for each group) was measured. All data represent the means ±S.D. values. This experiment was repeated independently three times with reproducible result, and a representative result is shown. (F) Tumor growth was evaluated after 5‐FU treatment. Macroscopic findings (left) and weight (right) of collected tumors. The bar graph and overlaid dots represent the means ±S.D. values and measured values respectively. This experiment was repeated independently three times with reproducible result, and a representative result is shown. p ‐values were the results of Mann–Whitney U ‐test

Article Snippet: Thereafter, sections were reacted with an anti‐Ki‐67 antibody (LS‐B6433) (LifeSpan BioSciences Inc.) at a dilution of 1:200 in blocking solution for 24 h r at 4°C.

Techniques: Expressing, In Vivo, Staining, Immunohistochemistry, Fluorescence, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Frontiers in Cellular Neuroscience

Article Title: Testosterone-induced adult neurosphere growth is mediated by sexually-dimorphic aromatase expression

doi: 10.3389/fncel.2015.00253

Figure Lengend Snippet: Female and male NSPCs were grown as neurospheres without sex hormones for 4 days then allowed to adhere to a laminin substrate and expose to either 10 nM testosterone (T), 17β-estradiol (E 2 ), progesterone (P 4 ) for 20 min before Ki67/nestin double label fluorescent immunocytochemistry was performed (A, female control neurosphere shown). The proportion of DAPI/nestin-labeled cells expressing Ki67 per neurosphere was increased by 20 min of either T, E 2 or P 4 compared to controls in both females (B) and males (C) . Female and male neurospheres maintained without sex hormones were differentiated on a laminin substrate for 72 h ± 10 nM T, E 2 or P 4 when βIII-tubulin (red) immunocytochemistry (D) showed that while T, P 4 and E 2 increased the proportion of neuronal differentiation compared to controls, E 2 was more potent compared to T and P 4 in females (E) and males (F) . Results show mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.005 compared to control, # p < 0.05 compared to T and P 4 , one-way ANOVA with Tukey’s multiple comparisons test. Scale bar = 50 μM.

Article Snippet: Cells were then washed in ice-cold 0.1 M phosphate buffered saline (PBS) then fixed in 4% paraformaldehyde (PFA) on ice for 20 min. Immunocytochemistry was then performed; cells were blocked in 0.1 M PBS containing 5% normal donkey serum (Millipore) and 0.1% triton-X100 (Sigma-Aldrich) for 60 min at 20°C then incubated with primary antibodies, rabbit anti-Ki67 (1:300, DKSH Australia Pty Ltd., VIC, Australia), mouse anti-nestin (1:300, R and D Systems, MN, USA) for 3 h, washed in PBS then incubated in secondary antibodies, Alexa fluor 488-conjugated donkey anti-rabbit (1:400, Molecular Probes, Life Technologies) and Alexa fluor 555-conjugated donkey anti-mouse (1:400, Molecular Probes) then counterstained with 4′,6-Diamidino-2-Phenylindole (1:2000, DAPI, Sigma-Aldrich) for 10 min and coverslipped in Fluoromount (DAKO, North Sydney, NSW, Australia).

Techniques: Immunocytochemistry, Labeling, Expressing