Journal: International Journal of Molecular Medicine

Article Title: RAS-stimulated release of exosomal miR-494-3p promotes the osteolytic bone metastasis of breast cancer cells

doi: 10.3892/ijmm.2023.5287

Figure Lengend Snippet: Exosomes derived from MDA-MB-231 cells stimulate RANKL-induced osteoclastogenesis in vitro . (A) Representative transmission electron microscopy images of exosomes derived from MDA-MB-231 cells. Scale bar, 500 nm (left panel) and 100 nm (right panel). (B) Nanoparticle tracking analysis of exosomes derived from MDA-MB-231 cells. (C) Western blot analysis of whole cell lysates (WC) and exosomes (EXOs) prepared from MDA-MB-231 cells. (D) BMMs were treated with exosomes derived from MDA-MB-231 cells in the presence of RANKL and M-CSF for 4 days. Representative TRAP staining images and quantification of TRAP-positive multinucleated cells. * P<0.05 and *** P<0.001. (E) BMMs were treated with exosomes derived from MDA-MB-231 cells treated with vehicle (Con) or the pan RAS inhibitor, salirasib (10 µ M), in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and quantification of TRAP-positive multinucleated cells. ** P<0.01. (F) BMMs were treated with exosomes derived from MCF-7 cells transfected with control vector (Con) or K-RASV12 vector in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and number of TRAP-positive multinucleated cells. *** P<0.001. (G) BMMs were treated with exosomes derived from T47D cells transfected with control vector (Con), K-RASV12, H-RASV12 , or N-RASV12 vector in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and number of TRAP-positive multinucleated cells. * P<0.05 and *** P<0.001. (H) BMMs were treated with exosomes derived from control vector (Con) or HER2 vector-transfected T47D cells in the presence of RANKL and M-CSF for 6 days. * P<0.05 and **** P<0.0001. RANKL, receptor activator of nuclear factor-κB ligand; BMMs, bone marrow-derived macrophages; TRAP, tartrate-resistant acid phosphatase; M-CSF, macrophage colony-stimulating factor; EXOs, exosomes; ALIX, apoptosis-linked gene 2-interacting protein X; HSP, heat shock protein; TSG101, tumor susceptibility 101; GM130, Golgi matrix protein 130; HER2, human epidermal growth factor receptor 2.

Article Snippet: The HER2 expression vector (cat. no. HG10004-UT) was purchased from Sino Biological.

Techniques: Derivative Assay, In Vitro, Transmission Assay, Electron Microscopy, Western Blot, Staining, Transfection, Plasmid Preparation

Journal: International Journal of Molecular Medicine

Article Title: RAS-stimulated release of exosomal miR-494-3p promotes the osteolytic bone metastasis of breast cancer cells

doi: 10.3892/ijmm.2023.5287

Figure Lengend Snippet: Identification of osteoclastogenic miRNAs in exosomes induced by RAS activation. (A) Expression levels of 28 miRNAs in exosomes derived from MCF-7 cells were determined using RT-qPCR. The results were normalized to U6 snRNA. (B) The expression levels of 11 selected miRNAs in exosomes derived from control or KRASV12 vector-transfected MCF-7 cells were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05 and ** P<0.01. (C) Cellular expression levels of 11 selected miRNAs in MCF-7 cells transfected with the control or K-RASV12 vector were determined using RT-qPCR. The results were normalized to U6 snRNA. (D) The expression levels of eight selected miRNAs in exosomes derived from MDA-MB-231 cells treated with the control or salirasib (10 µ M) were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05 and ** P<0.01. (E) The expression levels of seven selected miRNAs in exosomes derived from T47D cells transfected with the control vector (Con) or N-RASV12 vector were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05, ** P<0.01 and **** P<0.0001. (F) Representative images of TRAP-positive osteoclasts in BMMs transfected with the indicated miRNAs. BMMs transfected with the indicated miRNAs (20 nM each) were stimulated with RANKL and M-CSF for 4 days. NC, miRNA mimic negative control. ** P<0.01. (G) The expression levels of miR-494-3p, miR-1915-3p, miR-4508 and miR-6869-5p in sera derived from patients with HER2-positive breast cancer (n=19) and triple-negative breast cancer (n=15). The results were normalized to U6 snRNA. * P<0.05, *** P<0.001 and **** P<0.0001. RT-qPCR, reverse transcription-quantitative PCR; M-CSF, macrophage colony-stimulating factor; HER2, human epidermal growth factor receptor 2; TNBC, triple-negative breast cancer; BMMs, bone marrow-derived macrophages.

Article Snippet: The HER2 expression vector (cat. no. HG10004-UT) was purchased from Sino Biological.

Techniques: Activation Assay, Expressing, Derivative Assay, Quantitative RT-PCR, Plasmid Preparation, Transfection, Negative Control, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: Potent Antitumor Activity of Liposomal Irinotecan in an Organoid- and CRISPR-Cas9-Based Murine Model of Gallbladder Cancer

doi: 10.3390/cancers11121904

Figure Lengend Snippet: Mutant ERBB2 cooperates with loss of p53 and leads to papillary GBC in recipient mice. ( A ) Top: Schematic of human ERBB2, indicating the location of two point mutants (S310F and V777L). Bottom: retroviral vector used to transduce organoids, that had been treated with an sgp53-containing plasmid (px459) to induce loss of p53. ( B ) Immunofluorescence for ERBB2 (top) and phospho-ERBB2 (bottom) on organoids harboring the indicated genetic alterations. ( C ) Tumor volumes 36 days after s.c. implantation of the respective organoids into recipient mice. All mice transplanted with sgp53;ERBB2 S310F - and sgp53;ERBB2 V777L organoids exhibited tumor development, whereas sgp53;empty vector- and sgp53;ERBB2 wildtype organoids did not give rise to tumors over a four-month observation period. There was no significant difference in the tumor burden of mice transplanted with sgp53;ERBB2 S310F - and sgp53;ERBB2 V777L organoids ( p = 0.999). ( D ) Mice transplanted with sgp53;ERBB2 S310F - and sgp53;ERBB2 V777L organoids reached endpoint criteria with a median survival of 79.5 days and 58.5 days, respectively. ( E ) H&E and IHC for CK19 and EGFP on tumors generated with sgp53;ERBB2 S310 - and sgp53;ERBB2 V777L organoids.

Article Snippet: The pMSCV- ERBB2 -IRES- EGFP was a gift from Martine Roussel (Addgene, Watertown, MA, USA, plasmid #91888).

Techniques: Mutagenesis, Retroviral, Plasmid Preparation, Transduction, Immunofluorescence, Generated

Journal: bioRxiv

Article Title: Decoupling individual host response and immune cell engager cytotoxic potency

doi: 10.1101/2024.06.22.600188

Figure Lengend Snippet: ADCC assay and dependence of potency (EC 50 ) on bispecific antibody (bsAb) and NK cell donor. A. Schematic representation of co-culture assay mixing adherent tumour target cells (MCF7-WT, SKBR3 or MCF7-HER2+) and primary human NK cells from 15 donors in the presence of bsAb. Six bsAb anti-HER2 × CD16 constructs are tested, based on 3 anti-HER2 (Nanobodies CE4, CA5 or C7b) and 2 anti-CD16 (Nanobodies C21 or C28). B. Example of lysis fraction vs bsAb concentration measured on the MCF7-HER2+ target cell line and bispecific Ab C7b-21, donor A . Data was fitted with Eq. (1) (black line). C. Result of Hill fit for all conditions. Data are normalized using the fitting parameters Min, Max and EC 50 and compared to the normalized Hill function c/ (1 + c ) with c in nM units (black line). Raw residuals are shown below. D. EC 50 for each target cell line and bsAb. Each point is the median on the donors, with the bar representing 95% percentile interval. bsAbs are ranked according to the median value for SKBR3 cell line. E. EC 50 for each target cell line and donor. Each point is the median on the bsAbs, with the bar representing 95% percentile interval. Donors are ranked according to the median value for SKBR3 cell line.

Article Snippet: To obtain them, MCF7 cells were electroporated with 1 µ g of DNA plasmid HER2-GFP from Sino biologicals (ref HG10004-ACG) with Nucle-ofector 2b device (Lonza), and selected by antibiotic hygromycine.

Techniques: ADCC Assay, Co-culture Assay, Construct, Lysis, Concentration Assay

Journal: bioRxiv

Article Title: Decoupling individual host response and immune cell engager cytotoxic potency

doi: 10.1101/2024.06.22.600188

Figure Lengend Snippet: Physical model for bsAb dependent cell-mediated cytotoxicity. A. Reaction scheme for the two-step binding of bispecific antibody L at the immune synapse on membrane receptors R 1 (tumour side, HER2) and R 2 (effector side, CD16). K 1 , K 2 , K S 1 , K S 2 are dissociation constants. B-C. The equilibrium density σ of bridging bsAbs as a function of bulk bsAb concentration c given by the solution of . Each colored curve has been obtained for a different value of the cooperativity parameter α varied from 0.01 to 10. Other parameters K 1 , K 2 , ∑ 1 , ∑ 2 correspond to Target MCF7-WT and antibody C7b-21 (B) or Target SKBR3 and antibody CE4-28 (C) (values in Tabs. S1 and S2). The horizontal line in (B,C) represents an example of σ threshold value for the NK cell cytotoxic response (here σ 50 = 0.1 molec/ µ m 2 ). Together with the value of the cooperativity (here α = 0.01), it sets the value of c 50 , such that σ ( c 50 ) = σ 50 . The maximum density σ * is found for .

Article Snippet: To obtain them, MCF7 cells were electroporated with 1 µ g of DNA plasmid HER2-GFP from Sino biologicals (ref HG10004-ACG) with Nucle-ofector 2b device (Lonza), and selected by antibiotic hygromycine.

Techniques: Binding Assay, Membrane, Concentration Assay

Journal: bioRxiv

Article Title: Decoupling individual host response and immune cell engager cytotoxic potency

doi: 10.1101/2024.06.22.600188

Figure Lengend Snippet: Correlation of best-fit parameters of the multiscale model between cell lines. A. MCF7-WT and SKBR3. B. MCF7-WT and MCF7-HER2+. C. SKBR3 and MCF7-HER2+. P: Pearson coefficient. S: Spearman coefficient. n: number of points. Dashed lines indicate x = y .

Article Snippet: To obtain them, MCF7 cells were electroporated with 1 µ g of DNA plasmid HER2-GFP from Sino biologicals (ref HG10004-ACG) with Nucle-ofector 2b device (Lonza), and selected by antibiotic hygromycine.

Techniques: