bt474 cell lines  (ATCC)

Journal: ImmunoTargets and Therapy

Article Title: Directed-Complement Activation as a Novel Immunotherapeutic Approach for HER2-Breast Cancer

doi: 10.2147/ITT.S517584

Figure Lengend Snippet: CoMiX induce complement activation on BT474 tumor cells. ( A ) Flow cytometry analysis of C3b/iC3b deposition detected with mouse anti-human C3b mAb and a secondary goat anti-mouse IgG Ab conjugated with AF647, ( B ) membrane attack complex (MAC) formation was analyzed using anti-C5b9 mAb followed by PE-conjugated anti-mouse IgG pAb. MAC-formation was highest when CoMiX-Fc and CoMiX-FHR4 were combined, and ( C ) CDC on BT474 tumor cells incubated with 15 µg/well of multimeric immunotherapeutic complexes. The percentage of dead cells was calculated by dividing the number of live/dead-positive (dead) cells with the total number of analyzed cells. Data are presented as mean values ±SD of n = 3 independent experiments. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey’s test. ( D ) A linear correlation between C3b deposition (MFI) and the percentage of dead cells was observed (15 µg/well applied). Consistent with C3b deposition and MAC-formation, CoMiX-Fc and CoMiX-FHR4 significantly increased the percentage of dead cells compared to control multimers and Abs. ( E and F ) CoMiX-FHR4 activate the alternative complement pathway, whereas CoMiX-Fc facilitate classical pathway activation. C3b deposition ( E ) and CDC ( F ) of BT474 tumor cells incubated with saturating concentrations (15 µg/well) of CoMiX and control mAbs, individually or in combinations. 25% NHS diluted in either GVB ++ (Orange bars) or GVB + buffer (green bars) was added for 30 minutes at 37°C. Data are presented as mean values ±SD of n = 3 independent experiments. Statistical analysis was performed using a two-way ANOVA test, assessing the GVB ++ and GVB + conditions for each of the molecules. All comparisons between GVB ++ and GVB + reached statistical significance (p < 0.0001). * p < 0.05 **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: We thus explored the effects of CoMiX FHR4/V H H(P) and its combination with CoMiX FHR4/V H H(T) ( ) on xenografts of a trastuzumab-resistant BT474 cell line (ATCC-CRL-3247).

Techniques: Activation Assay, Flow Cytometry, Membrane, Incubation, Control

Journal: ImmunoTargets and Therapy

Article Title: Directed-Complement Activation as a Novel Immunotherapeutic Approach for HER2-Breast Cancer

doi: 10.2147/ITT.S517584

Figure Lengend Snippet: CoMiX-Fc enables NK cell activation and BT474 phagocytosis mediated by M2 macrophages. ( A ) HER2-positive BT474 tumor cells incubated with 15 μg/well of CoMiX were co-incubated with NK92humCD cells. The expression of the CD107a degranulation marker was analyzed by flow cytometry. ( B ) The intracellular accumulation of the cytokine IFN-γ was likewise analyzed by flow cytometry. Data are presented as mean values ±SD of the triplicates of one representative experiment of three independent experiments. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey’s test. ( C ) Complement-dependent macrophage-mediated phagocytosis of human BT474 cells. CFSE-stained BT474 tumor cells were incubated with controls, CoMiX, trastuzumab + pertuzumab or their combination with 25% C5-deficient human serum to prevent lysis. The percentage of phagocytic macrophages was measured. Data are presented as mean values ± SD out of n = 3 series of 10 confocal images for each condition. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey’s test. ( D ) Left: Confocal microscopy images of BT474 phagocytosis by M2 macrophages when incubated with CoMiX V H H(T)/Fc. The white arrows show the phagocytic macrophages (red) having engulfed BT474 cell(s) that harbor a green or yellowish color. Scale bar represents 50 µm. Right: Details of phagocytic macrophage (red) at higher magnification (60× oil immersion). Scale bar represents 10 µm. ( E ) Immunofluorescent staining of tumor sections collected 1 or 6 hours after injection of CoMiX-FHR4 (upper panel), CoMiX-Fc (intermediate panel), PBS (lower panel) and the controls V H H(T) and trastuzumab + pertuzumab (C3 staining 6 hours after injection). CoMiX were visualized with either a rabbit anti-His mAb followed by the goat anti-rabbit IgG Fc AF568- or a goat anti-human IgG AF647-conjugated antibody. Complement activation was visualized using the polyclonal rabbit anti-C3d antibody followed by AF568-conjugated anti-rabbit IgG. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: We thus explored the effects of CoMiX FHR4/V H H(P) and its combination with CoMiX FHR4/V H H(T) ( ) on xenografts of a trastuzumab-resistant BT474 cell line (ATCC-CRL-3247).

Techniques: Activation Assay, Incubation, Expressing, Marker, Flow Cytometry, Staining, Lysis, Confocal Microscopy, Injection

Journal: ImmunoTargets and Therapy

Article Title: Directed-Complement Activation as a Novel Immunotherapeutic Approach for HER2-Breast Cancer

doi: 10.2147/ITT.S517584

Figure Lengend Snippet: CoMiX FHR4/V H H(T) and FHR4/V H H(P) exert their anti-tumor effect on trastuzumab-resistant BT474 cells. ( A ) Trastuzumab-resistant BT474 cells were injected into the mammary fat pads of female BALB/c nude mice. When the tumor volume reached ~60 mm 3 , the mice were injected with 100 µg of CoMiX-FHR4, trastuzumab, or PBS, as described in . The tumors were measured every second or third day until day 37 of the study or until meeting the endpoint of 10,000 mm 3 . ( B ) Immunofluorescent stainings of NK cells in trastuzumab-resistant BT474 tumor xenografts performed just after the end of the treatment (at D+11). Cryosections were stained with a mouse NKp46/NCR1 antibody, revealed using a donkey anti-rat AF568-conjugated pAb, and analyzed by confocal microscope (lens X40) monitored by the Nikon NIS-Elements software. One representative image and its magnified view are shown for tumors treated with combined CoMiX-FHR4 [FHR4/V H H(T) + FHR4/V H H(P)] (upper left panels), tumors treated with CoMiX FHR4/V H H(P) (lower left panels), tumors treated with trastuzumab (upper right panels), and tumors treated with PBS (mock, lower right panels).

Article Snippet: We thus explored the effects of CoMiX FHR4/V H H(P) and its combination with CoMiX FHR4/V H H(T) ( ) on xenografts of a trastuzumab-resistant BT474 cell line (ATCC-CRL-3247).

Techniques: Injection, Staining, Microscopy, Software

Journal: Journal of Nuclear Medicine

Article Title: Preirradiation of Spheroids with 225 Ac-Trastuzumab Improves Penetration of 225 Ac-Liposomes and MIRDcell Predictions of Responses to Drug Cocktails

doi: 10.2967/jnumed.124.269273

Figure Lengend Snippet: Penetration data in BT474 spheroids (200-µm radius) for Alexa Fluor 647-NHS-trastuzumab ( ), CFDA-SE liposomes ( ), and CFDA-SE liposomes after preirradiating spheroids with 6.5 kBq/mL 225 Ac-trastuzumab ( ). Shown are time-integrated average concentrations as function of distance from centers of spheroids (A) and correlated mean decays per cell using activity concentrations of 6.875 kBq/mL for all drugs (calculated with MIRDcell-Ã) (B). Distances beyond 200 µm represent medium.

Article Snippet: The spheroids comprised the human epidermal growth factor receptor 2–positive human BT474 breast cancer cell line, obtained from the American Type Culture Collection.

Techniques: Liposomes, Activity Assay

Journal: Journal of Nuclear Medicine

Article Title: Preirradiation of Spheroids with 225 Ac-Trastuzumab Improves Penetration of 225 Ac-Liposomes and MIRDcell Predictions of Responses to Drug Cocktails

doi: 10.2967/jnumed.124.269273

Figure Lengend Snippet: MIRDcell calculated mean decays per cell attributed to liposomes in BT474 spheroids based on experimental CFDA-SE liposome penetration data for (100% 225 Ac-liposome case from Howe et al. ) and (50% 225 Ac-liposome, 50% 225 Ac-trastuzumab case from newly retrieved data), and extrapolated/averaged (weighted) liposome penetration data for (70% 225 Ac-liposome, 30% 225 Ac-trastuzumab case) and (30% 225 Ac-liposome, 70% 225 Ac-trastuzumab case).

Article Snippet: The spheroids comprised the human epidermal growth factor receptor 2–positive human BT474 breast cancer cell line, obtained from the American Type Culture Collection.

Techniques: Liposomes

Journal: Journal of Nuclear Medicine

Article Title: Preirradiation of Spheroids with 225 Ac-Trastuzumab Improves Penetration of 225 Ac-Liposomes and MIRDcell Predictions of Responses to Drug Cocktails

doi: 10.2967/jnumed.124.269273

Figure Lengend Snippet: Comparison of BT474 experimental outgrowths taken from Howe et al. to MIRDcell (version 4.14)-predicted SFs as bar plot (A), linear regression plot (B), and Bland–Altman plot (C) (95% limit of agreement, −0.048 to 0.095). Predicted SFs shown were based on 225 Ac-trastuzumab in spheroids with no 225 Ac daughters present and 225 Ac-liposomes in spheroids with 225 Ac daughters present. 225 Ac-trastuzumab and 225 Ac-liposomes in medium both had 225 Ac daughters present. Bars on abscissa of panel A and legend for panels B and C are labeled to denote percentage of activity corresponding to 225 Ac-liposomes and 225 Ac-trastuzumab. For example, “L-100, A-0” is 100% 225 Ac-liposomes and 0% activity on trastuzumab antibodies. Error bars for experimental outgrowths correspond to SD. Final prediction uses penetration data for CFDA-SE liposomes after spheroids are exposed to 6.5 kBq/mL 225 Ac-trastuzumab for 50%/50% case; 70% and 30% 225 Ac-liposome cases use extrapolated/averaged penetration data shown in . For CFDA-SE liposomes, published penetration data that were used for old prediction did not involve pretreatment 225 Ac-trastuzumab. Only final predictions are shown in panels B and C.

Article Snippet: The spheroids comprised the human epidermal growth factor receptor 2–positive human BT474 breast cancer cell line, obtained from the American Type Culture Collection.

Techniques: Comparison, Liposomes, Labeling, Activity Assay

Journal: Journal of Nuclear Medicine

Article Title: Preirradiation of Spheroids with 225 Ac-Trastuzumab Improves Penetration of 225 Ac-Liposomes and MIRDcell Predictions of Responses to Drug Cocktails

doi: 10.2967/jnumed.124.269273

Figure Lengend Snippet: Mean absorbed dose due to 225 Ac-trastuzumab (antibodies) and 225 Ac-liposomes (liposomes) in BT474 spheroids and medium for cells at various radial depths within 200-µm-radius spheroids (top). Below each graph, corresponding MIRDcell 3D slice representation of spheroid is shown for equatorial slices of spheroids. Dark red dots in equatorial slices represent living cells, whereas pink dots represent dead cells.

Article Snippet: The spheroids comprised the human epidermal growth factor receptor 2–positive human BT474 breast cancer cell line, obtained from the American Type Culture Collection.

Techniques: Liposomes

Journal: Journal of Nuclear Medicine

Article Title: Preirradiation of Spheroids with 225 Ac-Trastuzumab Improves Penetration of 225 Ac-Liposomes and MIRDcell Predictions of Responses to Drug Cocktails

doi: 10.2967/jnumed.124.269273

Figure Lengend Snippet: MIRDcell (version 4.16) AI–predicted minimum number of decays necessary to achieve target SF < 0.0001 in BT474 spheroids treated with 225 Ac-trastuzumab (antibodies), 225 Ac-liposomes (liposomes), or combination thereof. 225 Ac daughters were assumed to migrate away from 225 Ac-trastuzumab and to remain near 225 Ac-liposomes within spheroid. MIRDcell AI was limited to maximum molar activity of 10 6 GBq/mol for both drugs and lower limit of 1.17 × 10 5 GBq/mol for antibodies only. No bar appearing for antibodies indicates that 225 Ac-trastuzumab alone could not achieve target SF. When optimized molar activities for combined therapy and liposome-only therapy were checked in Monte Carlo–based MIRDcell simulation, zero cell survivors were achieved for both therapies although liposomes required more total decays to achieve same result. “Liposome with preirradiation” indicates use of penetration profiles after preirradiation with 225 Ac-trastuzumab as in 50%/50% case, and “Liposome without preirradiation” indicates use of liposome penetration profile without preirradiation as in 100% 225 Ac-liposomes case.

Article Snippet: The spheroids comprised the human epidermal growth factor receptor 2–positive human BT474 breast cancer cell line, obtained from the American Type Culture Collection.

Techniques: Liposomes, Activity Assay