Journal: Biomedicines

Article Title: Exploring CAR-PBMCs: A Novel Strategy Against EGFR-Positive Tumor Cells.

doi: 10.3390/biomedicines13020264

Figure Lengend Snippet: Figure 1. Schematic representation of anti-EGFR CAR lentiviral LentiONE expression vector (GEGTech). The CAR construct contains an anti-EGFR scFv fused to a CD8a hinge and trans- membrane domain, a 4-1BB intracellular, and a CD3ζ signaling domain. SP—signaling peptide leader sequence. The CAR is expressed from a PGK promoter, while the GFP (green fluorescent protein) reporter gene is expressed from a miniCMV promoter, part of the same bidirectional promoter unit. 5′,3′-LTR—long tandem repeats; Ψ—RNA packaging signal; cPPT—central polypurine tract; polyA—polyadenylation signal; WPRE—woodchuck hepatitis promoter regulatory element.

Article Snippet: CAR expression assessment was performed by labeling the transduced cells with biotinylated EGFR (R&D Systems, Minneapolis, MN, USA, cat. #BAF231), followed by APC-conjugated streptavidin (BioLegend, San Diego, CA, USA, cat. #405207), according to the manufacturer’s protocol, and subsequent flow cytometric analysis.

Techniques: Expressing, Plasmid Preparation, Construct, Membrane, Sequencing

Journal: Biomedicines

Article Title: Exploring CAR-PBMCs: A Novel Strategy Against EGFR-Positive Tumor Cells.

doi: 10.3390/biomedicines13020264

Figure Lengend Snippet: Figure 3. (A) Transduction efficiency assessed by the flow cytometry analysis of GFP-expressing cells at 3 days post-transduction. In the TNK-polarized group (stimulated with cytokines and beads), a higher transduction efficiency was observed (83 ± 8% vs. 26 ± 9%, p = 0.001). (B,C) Dot plot graphs of CAR-NK-polarized cells (B) and CAR-TNK-polarized cells (C); cells were treated with biotinylated EGFR and streptavidin-APC. The upper right quadrant (UR) indicates the population of CAR+ cells (FITC-positive and APC-positive); **—p < 0.01.

Article Snippet: CAR expression assessment was performed by labeling the transduced cells with biotinylated EGFR (R&D Systems, Minneapolis, MN, USA, cat. #BAF231), followed by APC-conjugated streptavidin (BioLegend, San Diego, CA, USA, cat. #405207), according to the manufacturer’s protocol, and subsequent flow cytometric analysis.

Techniques: Transduction, Flow Cytometry, Expressing

Journal: Biomedicines

Article Title: Exploring CAR-PBMCs: A Novel Strategy Against EGFR-Positive Tumor Cells.

doi: 10.3390/biomedicines13020264

Figure Lengend Snippet: Figure 4. (A) Dot-plot analysis of MICA/B expression in MDA-MB468 (red) cells vs. SK-BR3 (blue) cells showed a similar expression profile. (B) The MDA-MB468 cell line (red) exhibited a higher EGFR expression compared to SK-BR3 cells (blue). (C) CD1d expression in both cell types is reduced, but with a slight increase in the MDA-MB468 cell line. (D) CD95 (Fas) expression in MDA-MB468 cells (red) vs. SK-BR3 (blue) exhibited a similar expression profile in both cell types.

Article Snippet: CAR expression assessment was performed by labeling the transduced cells with biotinylated EGFR (R&D Systems, Minneapolis, MN, USA, cat. #BAF231), followed by APC-conjugated streptavidin (BioLegend, San Diego, CA, USA, cat. #405207), according to the manufacturer’s protocol, and subsequent flow cytometric analysis.

Techniques: Expressing

Journal: Biomedicines

Article Title: Exploring CAR-PBMCs: A Novel Strategy Against EGFR-Positive Tumor Cells.

doi: 10.3390/biomedicines13020264

Figure Lengend Snippet: Figure 5. Short-term cytotoxic activity of anti-EGFR CAR transduced cells compared to untransduced cells against calcein-labeled target MDA-MB-468 cells (A), SK-BR3 cells (B), K562 cells (C), and allogeneic PBMCs (D). The transduction significantly increased the effector cells’ cytotoxic activity, which correlated with EGFR expression levels in EGFR-expressing tumor cell lines MDA-MB-468 (E:T = 10:1—34 ± 4% vs. 5 ± 1%, p < 0.001; E:T = 5:1—25 ± 2% vs. 3 ± 1%, p < 0.05; E:T = 1:1—10 ± 0.5% vs. 1 ± 0.5%, p < 0.05) and SK-BR-3 (E:T = 10:1—24 ± 2% vs. 6 ± 1%, p < 0.05; E:T = 5:1—17 ± 0.2% vs. 5 ± 0.2%, p < 0.001; E:T = 1:1—3 ± 1% vs. 2 ± 1%, p = 0.28), but without statistical significance in EGFR-negative, NK- sensitive K562 cells, nor allogeneic PBMCs (E:T = 1:1—3.58 ± 2.53% vs. 2.74 ± 1.90%, p = 0.66; E:T = 5:1— 0.79 ± 0.26% vs. 0.84 ± 0.62%, p = 0.90; E:T = 10:1—0.99 ± 0.79% vs. 1.06 ± 0.53%, p = 0.90); ns—not significant (p > 0.05); **—p < 0.01; *** p < 0.001.

Article Snippet: CAR expression assessment was performed by labeling the transduced cells with biotinylated EGFR (R&D Systems, Minneapolis, MN, USA, cat. #BAF231), followed by APC-conjugated streptavidin (BioLegend, San Diego, CA, USA, cat. #405207), according to the manufacturer’s protocol, and subsequent flow cytometric analysis.

Techniques: Activity Assay, Labeling, Transduction, Expressing

Journal: Biomedicines

Article Title: Exploring CAR-PBMCs: A Novel Strategy Against EGFR-Positive Tumor Cells.

doi: 10.3390/biomedicines13020264

Figure Lengend Snippet: Figure 12. (A) RTCA analysis of EGFR-expressing MDA-MB468 cells incubated for 24 h with transduced anti-EGFR CAR cells (red) and untransduced cells (blue). (B) RTCA analysis of the EGFR positive SK-BR3 cell line, incubated for 24 hours with CAR-expressing cells (red) and untransduced PBMCs (blue). In the first 2 h, a rapid decline in cell index was observed in both cell lines treated with CAR cells (red). However, this effect was more pronounced in the MDA-MB-468 group. A poor decline was also observed in the untransduced group (blue), which may account for non-specific lysis. The control group experienced constant proliferation (black).

Article Snippet: CAR expression assessment was performed by labeling the transduced cells with biotinylated EGFR (R&D Systems, Minneapolis, MN, USA, cat. #BAF231), followed by APC-conjugated streptavidin (BioLegend, San Diego, CA, USA, cat. #405207), according to the manufacturer’s protocol, and subsequent flow cytometric analysis.

Techniques: Expressing, Incubation, Lysis, Control

Journal: Biomedicines

Article Title: Exploring CAR-PBMCs: A Novel Strategy Against EGFR-Positive Tumor Cells.

doi: 10.3390/biomedicines13020264

Figure Lengend Snippet: Figure 13. (A) Cell index slopes for MDA-MB468 cells incubated with CAR-expressing and un- transduced cells (UnTD). (B) Cell index slopes for SK-BR3 cells treated with CAR-expressing cells and untransduced (UnTD) cells. The slopes of the cell indices (CI slope) were more negative in the CAR-treated target cells compared to untransduced cells. In the MDA-MB-468 control cells (A), the slope had an overall positive value, suggestive for cell proliferation, compared to a negative value in the SK-BR3 control group, which may reflect the differences in EGFR expression as a mechanism for cell survival, as well as an increased expression of the non-specific activator molecule MICA/B in SK-BR3. The CI slopes for tumor cells incubated with CAR-PBMCs were comparable, with net negative values (MDA-MB468: −0.076 vs. SK-BR3: −0.074, p = 0.1714).

Article Snippet: CAR expression assessment was performed by labeling the transduced cells with biotinylated EGFR (R&D Systems, Minneapolis, MN, USA, cat. #BAF231), followed by APC-conjugated streptavidin (BioLegend, San Diego, CA, USA, cat. #405207), according to the manufacturer’s protocol, and subsequent flow cytometric analysis.

Techniques: Incubation, Expressing, Control