Journal: BMC Cancer

Article Title: Targeting tissue factor on tumour cells and angiogenic vascular endothelial cells by factor VII-targeted verteporfin photodynamic therapy for breast cancer in vitro and in vivo in mice

doi: 10.1186/1471-2407-10-235

Figure Lengend Snippet: TF is expressed on tumour cells and VEGF-stimulated HUVECs but not on unstimulated HUVECs . A . TF expression was detected on breast cancer cell lines but not on CHO-K1 cells by flow cytometry using mouse Icon (mfVII/hIgG1 Fc) protein. B . Selective expression of TF on VEGF-stimulated HUVECs but not on unstimulated HUVECs was observed and photographed under confocal microscope. HUVECs were stained for TF expression by mouse Icon or anti-HTF antibody (FITC, green) and then by anti-human CD31 PE (red) to verify that TF-expressing cells were of vascular endothelial origin. Results in A and B are representative of two experiments.

Article Snippet: Chinese Hamster Ovary K1 (CHO-K1, ATCC) cells were grown in F12 medium, murine breast cancer EMT6 and human embryonic kidney 293 cells (kindly provided by Dr. Albert Deisseroth during his tenure at Yale University) in DMEM and human breast cancer MDA-MB-231 cells in RPMI1640 medium supplemented with 10% heat inactivated fetal bovine serum (FBS) (Invitrogen) and 1:100 antibiotics (Sigma).

Techniques: Expressing, Flow Cytometry, Microscopy, Staining

Journal: BMC Cancer

Article Title: Targeting tissue factor on tumour cells and angiogenic vascular endothelial cells by factor VII-targeted verteporfin photodynamic therapy for breast cancer in vitro and in vivo in mice

doi: 10.1186/1471-2407-10-235

Figure Lengend Snippet: fVII targeting improves the selectivity of VP PDT . A . Incubation time of fVII-VP and free VP affected the effect of fVII-tPDT and ntPDT (2 μM, 689 nm laser at 60 J/cm 2 ) on killing MDA-MB-231 cancer cells. However, VP alone without laser irradiation (VP alone) did not have an effect on killing the cancer cells. B . Comparison of fVII-tPDT and ntPDT (689 nm laser at 60 J/cm 2 ) for side effects on non-TF-expressing CHO-K1 cells as a normal cell line. C . VP PDT (fVII-tPDT or ntPDT with 0.5 or 5 μM VP at 60 J/cm 2 ) for the non-TF expressing 293 line as a normal cell line control. D and E . fVII-tPDT and ntPDT (689 nm laser at 36 J/cm 2 ) for HUVEC with or without induction of TF expression by VEGF, as seen in Figure 2B. Results in A-D are representative of two experiments.

Article Snippet: Chinese Hamster Ovary K1 (CHO-K1, ATCC) cells were grown in F12 medium, murine breast cancer EMT6 and human embryonic kidney 293 cells (kindly provided by Dr. Albert Deisseroth during his tenure at Yale University) in DMEM and human breast cancer MDA-MB-231 cells in RPMI1640 medium supplemented with 10% heat inactivated fetal bovine serum (FBS) (Invitrogen) and 1:100 antibiotics (Sigma).

Techniques: Incubation, Irradiation, Comparison, Expressing, Control

Journal: Cancer Research

Article Title: A Combinatorial CRISPR–Cas9 Screen Identifies Ifenprodil as an Adjunct to Sorafenib for Liver Cancer Treatment

doi: 10.1158/0008-5472.can-21-1017

Figure Lengend Snippet: Figure 2. NMDAR inhibitor synergizes with sorafenib and lenvatinib in reducing viability and self-renewal ability of HCC cells. A–I, Cell viability of combined treatment of NMDAR inhibitor (ifenprodil or MK-801) and FLT4/FGFR inhibitor(s) (infigratinib, erdafitinib, SAR131675,sorafenib, or lenvatinib) in MHCC97L (A–F), Hep3B (G), Huh7 (H), and HepG2 (I) cells. Cells were treated with the indicated drug pair at multiple doses for two days. Cell viability was measured by the MTT assay. Synergy was determined based on the Bliss independence and HSA models for each drug pair. Dose combinationswith a 95% lower confidence bound of the estimated excess over Bliss scores that is greater than 0 (see Supplementary Fig. S5) are in bold. Data are mean SD from biological replicates (n ¼ 3). J, The workflow of the limiting dilution spheroid formation assay. Cells preseeded in spheroid growth medium were treated with ifenprodil and sorafenib. The number of tumor spheres formed was counted after 10 days. K–N, Combined treatment of ifenprodil and sorafenib suppressed the ability of HCC cells to form tumor spheres. Limiting dilution spheroid formation assays were performed with MHCC97L cells treated with 10 mmol/L ifenprodil þ 5 mmol/L sorafenib (K), Hep3B cells treated with 5 mmol/L ifenprodil þ 5 mmol/Lsorafenib (L), and Huh7 and HepG2 cells treated with 10 mmol/Lifenprodilþ 10 mmol/L sorafenib (M–N), for 10 days.Stem cell frequency and 95% confidence intervals were calculated. Data were collected from three biological replicates. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001; n.s., nonsignificant.

Article Snippet: Ifenprodil and sorafenib were purchased from Selleckchem (#S4091) and LC Laboratories (#S-8502), respectively.

Techniques: MTT Assay, Tube Formation Assay

Journal: Cancer Research

Article Title: A Combinatorial CRISPR–Cas9 Screen Identifies Ifenprodil as an Adjunct to Sorafenib for Liver Cancer Treatment

doi: 10.1158/0008-5472.can-21-1017

Figure Lengend Snippet: Figure 3. Combined treatment of ifenprodil and sorafenib upregulates UPR, suppresses WNT signaling, and triggers cell-cycle arrest in HCC cells. A, Log2-fold change values [Log2(FC)] among differentially expressed genes (DEG) identified by RNA-seq. Log2(FC) of differentially expressed genes found in at least one treatment condition (IFEN, 10 mmol/L ifenprodil; SOR, 5 mmol/L sorafenib; and I þ S, 10 mmol/L ifenprodil and 5 mmol/L sorafenib; treated for 24 hours) was compared with DMSO-treated control in the MHCC97L cell line. Differentially expressed genes are ordered from the lowest to the highest Log2(FC) in each condition, respectively. Data was collected from three biological replicates. B, Log2(FC) among significantly differentially expressed Gene Ontology (GO) groups identified in the RNA-seq data. Log2(FC) of differentially expressed genes belonging to the GO categories were found to be marked up-/downregulated in the I þ S treatment. Highlighted genes were subjected to further validations. C, Heatmap summary of GO enrichment analysis. Median Log2(FC) of differentially expressed genes (left), number of differentially expressed genes (middle), and the log10 (FDR; right) of the GO categories among the top hits in GO enrichment analysis are shown. Here the top-hit child GO categories are collapsed into the parent GO, and only the parent ones are plotted. (Continued on the following page.)

Article Snippet: Ifenprodil and sorafenib were purchased from Selleckchem (#S4091) and LC Laboratories (#S-8502), respectively.

Techniques: RNA Sequencing, Control

Journal: Cancer Research

Article Title: A Combinatorial CRISPR–Cas9 Screen Identifies Ifenprodil as an Adjunct to Sorafenib for Liver Cancer Treatment

doi: 10.1158/0008-5472.can-21-1017

Figure Lengend Snippet: Figure 4. Ifenprodil treatment enhances the efficacy of sorafenib in HCC patient-derived organoids. A, Combined treatment of ifenprodil and sorafenib synergistically inhibited the growth of multiple HCC patient-derived organoids. Dissociated cells of HCC-HK P1, HCC#23, HCC#10, and HCC-HK P2 organoids were seeded in complete culture medium. Three daysafter cell seeding when the cells formed small organoids, multiple doses of ifenprodil and sorafenib were added for additional 3 days.Cell viability was measured by CellTiter-Glo assays after the treatment. Synergy was determined based on the Bliss independence and HSA models for each drug pair. Dose combinations with a 95% lower confidence bound of the estimated excess over Bliss score that is greater than 0 (see Supplementary Fig. S15) are in bold. Data are mean SD from biological replicates (n ¼ 4). B, Ifenprodil in combination with sorafenib increased the level of UPR-related proteins (including phosphorylated IRE1- alpha and CHOP) and p21Cip1 and decreased the level of CDK2 and WNT signaling in HCC-HK P1 and HCC#23 organoids. HCC-HK P1 and HCC#23 organoids were treated with 20 mmol/L ifenprodil and 8 mmol/L sorafenib for 3 days. WNT signaling (bottom) was measured using a lentiviral-based TOPFlash reporter. Data are mean SD from biological replicates (n ¼ 4 for HCC-HK P1; n ¼ 3 for HCC#23). C, Ifenprodil in combination with sorafenib suppressed the cell self-renewal ability of HCC organoids. Limiting dilution spheroid formation assays were performed with the organoids treated with 20 mmol/L ifenprodil and 8 mmol/L sorafenib for 3 days. Stem cell frequency and 95% confidence intervals were calculated. Data were collected from three biological replicates. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001; n.s., nonsignificant.

Article Snippet: Ifenprodil and sorafenib were purchased from Selleckchem (#S4091) and LC Laboratories (#S-8502), respectively.

Techniques: Derivative Assay

Journal: Cancer Research

Article Title: A Combinatorial CRISPR–Cas9 Screen Identifies Ifenprodil as an Adjunct to Sorafenib for Liver Cancer Treatment

doi: 10.1158/0008-5472.can-21-1017

Figure Lengend Snippet: Figure 5. Treatment with ifenprodil in combination with sorafenib suppresses tumor formation in xenograft models. A, Combined treatment of ifenprodil and sorafenib suppressed tumor growth of MHCC97L-derived xenograft in mice. MHCC97L cells were subcutaneously in- jected in nude mice and treated with 20 mg/kg ifenprodil, 28 mg/kg sorafenib, or combi- nation of ifenprodil and sorafenib for 21 days. Images of ex vivo–resected tumors, tumor sections stained with hematoxylin and eosin, tumor size change, xenograft weight at end- point, and body weight change during treat- ment are shown (n ¼ 5 per treatment group). Scale bars in resected tumor and hematoxylin and eosin staining images are 1 cm and 100 mm, respectively. B and C, Combined treatment of ifenprodil and sorafenib suppressed tumor growth of two patient-derived xenografts in mice. PDXs were subcutaneously injected in NOD/SCID mice and treated with 20 mg/kg ifenprodil, 28 mg/kg sorafenib, or combina- tion of ifenprodil and sorafenib for 21 days. Representative images of ex vivo–resected tumors, tumor size change, xenograft weight at endpoint, and body weight change during treatment are shown (n ¼ 14 for PDX1 and n ¼ 8 for PDX2 per group). Scale bars in resected tumor and hematoxylin and eosin staining images are 1 cm and 100 mm, respec- tively. Arrows indicate the areas with signs of apoptosis. , P < 0.05; , P < 0.01; , P < 0.0001; n.s., nonsignificant.

Article Snippet: Ifenprodil and sorafenib were purchased from Selleckchem (#S4091) and LC Laboratories (#S-8502), respectively.

Techniques: Derivative Assay, Ex Vivo, Staining, Injection

Journal: Cancer Research

Article Title: A Combinatorial CRISPR–Cas9 Screen Identifies Ifenprodil as an Adjunct to Sorafenib for Liver Cancer Treatment

doi: 10.1158/0008-5472.can-21-1017

Figure Lengend Snippet: Figure 6. Treatment with ifenprodil in combina- tion with sorafenib suppresses tumor- initiating cell frequency in xenograft models. A, IHC images of Lgr5 and Axin2 staining on tissues harvested from the resected MHCC97L-derived (top) and patient-derived (bottom) xenografts treated with DMSO, ifen- prodil, sorafenib, or combination of ifenprodil and sorafenib. Scale bar, 100 mm. Bar chart represents the intensity of indicated signal from five random field under a light microscope at 200 magnification. B, Ifenprodil in combination with sorafenib sup- pressed the cell self-renewal ability of xenografts. The xenograft residuals from MHCC97L-derived and patient- derived models were dissociated and seeded for limiting dilution spheroid formation assays. Stem cell frequency and 95% confidence intervals were calculated. C, Combined treatment of ifenprodil and sorafenib decreased the repropagation capability of HCC cells in vivo. Residual tumor cells were har- vested from the drug(s)-treated PDXs, and 10,000, 5,000, 1,000, and 500 cells were transplanted into secondary NOD/SCID mouse recipients (n ¼ 6 per group). Average tumor latency and incidence were recorded, and tumor-initiating cell frequency was calculated. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001; n.s., nonsignificant.

Article Snippet: Ifenprodil and sorafenib were purchased from Selleckchem (#S4091) and LC Laboratories (#S-8502), respectively.

Techniques: Staining, Derivative Assay, Light Microscopy, In Vivo

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: EGCG promoted retention of YAP1 in the cytoplasm by activating the Hippo pathway and promoting autophagy in breast cancer cells. (A) Natural small‐molecule compounds predicted to activate the Hippo pathway in breast cancer cell lines. (B) Bioinformatics analysis of pathways related to EGCG. (C) WB analysis was performed to examine expression of Hippo pathway components and YAP1 target genes ( CTGF and CYR61 ) in MCF7 and MDA‐MB‐231 cells treated with EGCG of various concentrations for 6 h. (D) Expression levels of YAP1 and p‐YAP1 were determined in the nucleus and cytoplasm of MCF7 and MDA‐MB‐231 cells via WB analysis. LaminB1 and β‐actin were used as extraction controls for the nucleus and cytoplasm, respectively. (E‐F) Expression levels of the autophagy markers LC3 and p62 were detected by WB in MCF7 and MDA‐MB‐231 cells after treatment with EGCG of various concentrations or for different lengths of time. (G) IF staining images showing LC3 fluorescence puncta and quantitative analysis of MCF7 and MDA‐MB‐231 cells treated with EGCG (50 µg/mL, 6 h). DAPI labelled with blue fluorescent signal was used to mark the nucleus, while green fluorescent signal was used to label LC3. (H) Autophagic structures (indicated with red arrows) were detected with TEM in MCF7 and MDA‐MB‐231 cells treated with EGCG (50 µg/ml, 6 h). Data are from three independent experiments and are shown as the mean ± standard deviation. ** P < 0.01 (Student's t ‐test). Abbreviations: YAP1, Yes1‐associated transcriptional regulator; EGCG, epigallocatechin gallate; WB, Western blotting; MST1, macrophage stimulating 1; p‐MST1, phosphorylated‐macrophage stimulating 1; MOB1A: MOB kinase activator 1A; p‐MOB1A: phosphorylated‐MOB1A; CTGF, connective tissue growth factor; CYR61, cysteine rich angiogenic inducer 61; LC3, microtubule‐associated protein 1 light chain 3; SQSTM1/p62, sequestosome 1; IF, immunofluorescence staining; DAPI, 4',6‐diamidino‐2‐phenylindole; TEM, transmission electron microscopy.

Article Snippet: Antibodies for YAP1 (13584‐1‐AP, 66900‐1‐Ig; 1:1000 for WB, 1:200 for immunohistochemistry [IHC], 1:100 for IF), NEDD4 like E3 ubiquitin protein ligase (NEDD4L, 13690‐1‐AP, 1:1000 for WB, 1:400 for IHC), sequestosome 1 (SQSTM1/p62, 18420‐1‐AP, 1:1000 for WB, 1:50 for IHC) and CYR61 (26689‐1‐AP, 1:1000 for WB, 1:50 for IHC) were purchased from Proteintech Group (Rosemont, IL, USA).

Techniques: Expressing, Extraction, Staining, Fluorescence, Standard Deviation, Western Blot, Immunofluorescence, Transmission Assay, Electron Microscopy

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: Cytoplasmic YAP1 promoted autophagic cell death in vivo. (A) Tumor‐bearing nude mice from different treatment groups ( n = 6 per group). (B) Tumors taken from mice in different treatment groups. (C) The weight of tumors from mice in each treatment group. (D) The tumor volume from mice in each treatment group over time. (E) WB analysis was performed to detect the expression of YAP1, p‐YAP1, the autophagy markers LC3 and p62, and YAP1 target genes CTGF and CYR61 in tumor tissues. (F) IHC staining of LC3, p62, YAP1, CTGF, and CYR61 in tumor tissues from mice in different treatment groups. (G) Autophagic structures (indicated with red arrows) in tumor tissues from the control and EGCG treatment groups were detected with TEM. Data are shown as the mean ± standard deviation. *** P < 0.001 (Student's t ‐test and one‐way ANOVA). Abbreviations: EGCG, epigallocatechin gallate; LC3, microtubule‐associated protein 1 light chain 3; SQSTM1/p62, sequestosome 1; WB, Western blotting; YAP1, Yes1‐associated transcriptional regulator; CTGF, connective tissue growth factor; CYR61, cysteine rich angiogenic inducer 61; IHC, immunohistochemistry; TEM, transmission electron microscopy; ANOVA, analysis of variance.

Article Snippet: Antibodies for YAP1 (13584‐1‐AP, 66900‐1‐Ig; 1:1000 for WB, 1:200 for immunohistochemistry [IHC], 1:100 for IF), NEDD4 like E3 ubiquitin protein ligase (NEDD4L, 13690‐1‐AP, 1:1000 for WB, 1:400 for IHC), sequestosome 1 (SQSTM1/p62, 18420‐1‐AP, 1:1000 for WB, 1:50 for IHC) and CYR61 (26689‐1‐AP, 1:1000 for WB, 1:50 for IHC) were purchased from Proteintech Group (Rosemont, IL, USA).

Techniques: In Vivo, Expressing, Immunohistochemistry, Control, Standard Deviation, Western Blot, Transmission Assay, Electron Microscopy