Journal: Pharmaceuticals

Article Title: Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

doi: 10.3390/ph6030295

Figure Lengend Snippet: List of aptamers used in this study.

Article Snippet: Recombinant PSMA was prepared by diluting 2 μg recombinant human PSMA (4234-ZN-010) from R&D Systems (Minneapolis, MN, USA) in 500 μL of 50 mM pH 7.5 Tris buffer.

Techniques: In Vitro, Inhibition, Activity Assay

Journal: Pharmaceuticals

Article Title: Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

doi: 10.3390/ph6030295

Figure Lengend Snippet: Fluorescence microscopy. ( A ) Direct fluorescence method. FAM labeled anti-PSMA RNA aptamer (A9g) was incubated with either PC3(PSMA+) or PC3(PSMA-) cells. A high salt wash step was performed to remove unbound or surface bound RNA. Internalized RNA was visualized using fluorescence microscopy. A scrambled, non-internalizing aptamer (Scr) was used as a negative control in these experiments. Florescence images were overlaid with DAPI and P/C (phase contrast) channels. Arrows indicate perinuclear localization of internalized A9g aptamer. ( B ) Antibody amplification method. FAM-labeled anti-TrkB RNA aptamer (C4-3) was incubated with either TrkB expressing or non-expressing HEK293 cells at 37 °C. FAM-labeled control aptamer (Scr) was used as a control for specificity. Unbound and surface bound RNA was removed as described above. Internalized RNA signal was amplified by incubation with an anti-FITC antibody and Alexa488 secondary antibody. Vehicle treated cells (No RNA) or cells subjected to incubation with antibodies alone (Ab control) were used as controls.

Article Snippet: Recombinant PSMA was prepared by diluting 2 μg recombinant human PSMA (4234-ZN-010) from R&D Systems (Minneapolis, MN, USA) in 500 μL of 50 mM pH 7.5 Tris buffer.

Techniques: Fluorescence, Microscopy, Labeling, Incubation, Negative Control, Amplification, Expressing, Control

Journal: Pharmaceuticals

Article Title: Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

doi: 10.3390/ph6030295

Figure Lengend Snippet: Plate-reader assay to assess aptamer binding and internalization. For the binding experiments, cells were fixed to inhibit active transport before incubation with the RNA aptamers. Live cells were used for the internalization experiments. ( A ) Binding ( left ) and internalization ( right ) of A9g into PSMA-expressing prostate cancer cells. ( B ) Binding ( left ) and internalization ( right ) of E1 aptamer into rat HER2-expressing mammary carcinoma cells. Cells only (no RNA) controls were carried out for each condition. Fluorescence was measured using an Analyst HT plate reader. (*, p < 0.001).

Article Snippet: Recombinant PSMA was prepared by diluting 2 μg recombinant human PSMA (4234-ZN-010) from R&D Systems (Minneapolis, MN, USA) in 500 μL of 50 mM pH 7.5 Tris buffer.

Techniques: Binding Assay, Incubation, Expressing, Fluorescence

Journal: Pharmaceuticals

Article Title: Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

doi: 10.3390/ph6030295

Figure Lengend Snippet: Evaluation of aptamer binding and internalization by flow cytometry. ( A ) Cell-specific binding of a human HER2 aptamer-Qdot conjugate. Cell lines expressing HER2 receptor (N202.1E-hHER2 and SKBR3) and HER2 non-expressing cell line (N202.1E) were incubated for 45min at 37 °C with human HER2 aptamer conjugated to Qdots (605nm). Cell-specific aptamer binding was evaluated by flow cytometry (upper panel). Quantification of specific fluorescence signal is shown in the middle panel bar graph. Cell surface human HER2 receptor expression in N202.1E, N202.1E(hHER2) and SKBR-3 cells (grey for isotype control, colored histograms for anti-HER2 Ab) (lower panel). ( B ) Measurement of A9g cell-internalization. PSMA-positive cells were incubated with FAM-A9g aptamer for 30 min at either 4 °C (left top panel) or 37 °C (right top panel). Cells were then washed with either DPBS or a High Salt (DPBS plus 0.5M NaCl) wash for 5min at 4 °C. The high salt wash step removes any unbound or surface bound aptamer. Bound and/or internalized aptamers were subsequently visualized using flow cytometry. *, internalized aptamer fraction (middle right panel). Fluorescence intensity quantified in the bar graph (**, p < 0.005).

Article Snippet: Recombinant PSMA was prepared by diluting 2 μg recombinant human PSMA (4234-ZN-010) from R&D Systems (Minneapolis, MN, USA) in 500 μL of 50 mM pH 7.5 Tris buffer.

Techniques: Binding Assay, Flow Cytometry, Expressing, Incubation, Fluorescence, Control

Journal: Pharmaceuticals

Article Title: Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

doi: 10.3390/ph6030295

Figure Lengend Snippet: Quantitative and ultrasensitive internalization method ( “QUSIM” ). ( A ) 96-well microplate NIR Odyssey imager scan of serial dilutions for binding aptamer-NIR conjugate (A9g-NIR) and mutant, non-binding sequence conjugate (A9g.6-NIR) ( upper panel ) and standard curves with linear regression for RNA aptamer quantification ( lower panel ). ( B ) Quantification of the amount of aptamer-NIR internalized into PC3(PSMA+) cells vs. PC3(PSMA–) cells. ( C ) Time-dependent cell uptake of binding aptamer (A9g-NIR) vs. non-binding (mutant) aptamer (A9g.6-NIR). ( D ) Kinetics of specific A9g internalization using one-phase association curve fit (R 2 = 0.9924, k = 0.542 min −1 , half-time = 1.278 min).

Article Snippet: Recombinant PSMA was prepared by diluting 2 μg recombinant human PSMA (4234-ZN-010) from R&D Systems (Minneapolis, MN, USA) in 500 μL of 50 mM pH 7.5 Tris buffer.

Techniques: Binding Assay, Mutagenesis, Sequencing

Journal: Pharmaceuticals

Article Title: Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

doi: 10.3390/ph6030295

Figure Lengend Snippet: RNA-RIP assay. (A) Schematic of A9g-saporin conjugate internalization and RIP effect leading to cell death. (B) Dose dependent response of A9g-saporin and control, A9g.6-saporin, conjugates in PC3(PSMA+) ( left ) and PC3(PSMA–) cells ( right ).

Article Snippet: Recombinant PSMA was prepared by diluting 2 μg recombinant human PSMA (4234-ZN-010) from R&D Systems (Minneapolis, MN, USA) in 500 μL of 50 mM pH 7.5 Tris buffer.

Techniques: Control

Journal: bioRxiv

Article Title: Pooled screening identifies combinatorial CAR signaling domains for next-generation CAR-M immunotherapies

doi: 10.1101/2025.02.16.638489

Figure Lengend Snippet: (A) Schematic representation of the pro-phagocytic module screening strategy. (B) Correlation between log2 fold change and −log10 rho scores for pro-phagocytic CAR variant screens. Data represent 18 biological replicates across six NaSDC lentivirus batches. (C) FACS-based quantification of PC3-PSMA tumor cell phagocytosis by CAR-Ms (n = 4 biological replicates). (D) Schematic representation of the pro-inflammatory module screening strategy. (E) Correlation between log2 fold change and −log10 rho scores for pro-inflammatory CAR variant screens. Data represent 15 biological replicates across five NaSDC lentivirus batches. (F) Relative mRNA expression of TNF , IL6 and IL1B in CAR-Ms cocultured with PSMA positive target tumor cells (n = 4 biological replicates). (G) Schematic representation of the pro-infiltrating module screening strategy. (H) Correlation between log2 fold change and −log10 rho scores for pro-infiltrating CAR variant screens. Data represent 9 biological replicates across three NaSDC lentivirus batches. (I) Relative mRNA expression of MMP family genes in CAR-Ms cocultured with PSMA positive target tumor cells (n = 4 biological replicates). Dashed lines in ( C , F , I ) indicate −log10(0.05) (horizontal) and log2 fold change of truncated CARs (CARΔ) (vertical). Data in (C) are presented as mean ± s.e.m. and analyzed using one-way ANOVA followed by Dunnett’s multiple-comparison test.

Article Snippet: Biotinylated human PSMA proteins (ACROBiosystems) were added to the beads at a concentration sufficient to occupy 25% of the binding sites.

Techniques: Variant Assay, Expressing, Comparison

Journal: bioRxiv

Article Title: Pooled screening identifies combinatorial CAR signaling domains for next-generation CAR-M immunotherapies

doi: 10.1101/2025.02.16.638489

Figure Lengend Snippet: (A) Description of CAR variants used for individual characterization. (B) FACS-based quantification of PC3-PSMA tumor cell phagocytosis by UTD and CAR-Ms (n = 4 biological replicates). (C) Flow cytometric histograms showing surface expression of CD80 and CD86 in UTD and CAR-Ms cocultured with PSMA positive tumor cells. (D) Flow cytometric histograms of reactive oxygen species (ROS) levels in UTD and CAR-Ms cocultured with PSMA positive tumor cells. DCF, 2’-7’dichlorofluorescein. (E) Relative mRNA expression of TNF and IL6 in UTD and CAR-Ms cocultured with PSMA positive tumor cells (n = 4 biological replicates). (F) Relative mRNA expression of MMP11 and MMP13 in UTD and CAR-Ms cocultured with PSMA positive tumor cells (n = 4 biological replicates). Data in ( B) are presented as mean ± s.e.m. and analyzed using one-way ANOVA followed by Dunnett’s multiple-comparison. Data in ( C , D) are representative of three independent experiments. Dashed lines ( C , D ) represent the mean fluorescence intensity (MFI) for UTD macrophages.

Article Snippet: Biotinylated human PSMA proteins (ACROBiosystems) were added to the beads at a concentration sufficient to occupy 25% of the binding sites.

Techniques: Expressing, Comparison, Fluorescence

Journal: bioRxiv

Article Title: Pooled screening identifies combinatorial CAR signaling domains for next-generation CAR-M immunotherapies

doi: 10.1101/2025.02.16.638489

Figure Lengend Snippet: (A) Schematic illustration of the humanized immune system TME model. PC3-PSMA tumors were established bilaterally, followed by PBMC engraftment on day 7. On day 14, 5 × 10 6 UTD or CAR-Ms were injected IT. Tumors were harvested on day 18 for scRNA-seq. SC, subcutaneously. IV, intravenously. IT, intratumorally. (B) Proportions of cell types from scRNA-seq (left) and tSNE-based clustering of all sequenced cells (right). B, B cell. T, T cell. Mac, macrophage. (C) Dimensionality reduction (tSNE) and clustering for macrophages. Data are colored by treatment conditions. (D) Density plots within tSNE maps depicting the distribution of CAR-expressing cells. (E) AUC scores of M1 and M2 gene sets in the indicated macrophage clusters. (F) Feature plots showing expression levels of selected genes. (G) MFI of HLA-ABC and HLA-DR in UTD and CAR-Ms after 24 h coculture with PSMA positive tumor cells (n = 3 biological replicates). (H) AUC scores of T cell cytotoxicity and exhaustion gene sets in the indicated T cell clusters. Unless specified otherwise, data are presented as mean ± s.e.m. and analyzed using the two-tailed Student’s t-test.

Article Snippet: Biotinylated human PSMA proteins (ACROBiosystems) were added to the beads at a concentration sufficient to occupy 25% of the binding sites.

Techniques: Injection, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Pooled screening identifies combinatorial CAR signaling domains for next-generation CAR-M immunotherapies

doi: 10.1101/2025.02.16.638489

Figure Lengend Snippet: (A) Specific killing of PC3-PSMA cells by UTD and COMB7 CAR-Ms at varying E/T ratios. Data represent two independent experiments from two human donors. (B) Specific killing of PC3-PSMA cells by UTD, CD247 CAR-Ms or COMB7 CAR-Ms at an E/T ratio of 3/1. Data collected from two human donors. (C-D) Specific killing of HER2 + SKOV3 or GPC3 + HepG2 cells by UTD or COMB7 CAR-Ms at an E/T ratio of 3/1 (n = 6 biological replicates). (E) NCG mice were IP injected with 1 × 10 6 PC3-PSMA cells, followed by treatment with PBS, 5 × 10 6 UTD, or 5 × 10 6 COMB7 CAR-Ms one day later. (F) Overall survival for mice in each treatment group (n = 6 for PBS, n = 5 for UTD and COMB7). (G) Tumor burden measured by bioluminescence (total flux). Data shown are individual values (n = 6 for PBS, n = 5 for UTD and COMB7). Unless specified otherwise, data are presented as mean ± s.e.m. and analyzed using the two-tailed Student’s t-test.

Article Snippet: Biotinylated human PSMA proteins (ACROBiosystems) were added to the beads at a concentration sufficient to occupy 25% of the binding sites.

Techniques: Injection, Two Tailed Test