Journal: JCI Insight

Article Title: Targeting CD46 for both adenocarcinoma and neuroendocrine prostate cancer

doi: 10.1172/jci.insight.121497

Figure Lengend Snippet: (A) Confocal microscopy study of YS5 IgG1 internalization into mCRPC lines (DU145, LNCaP-C4-2B, and PC3M) and primary human T cells. Colocalization between the macropinocytosis marker ND70 and YS5 IgG1 is analyzed in a single confocal slice. The rate of colocalization, calculated as the percentage of double-positive intracellular spots relative to antibody-positive intracellular spots, was 93.75% (15 of 16) for DU145, 94.44% (LNCaP-C4-2B), and 92.68% (38 of 41) for PC3M. There was no internalization and no colocalization for primary T cells. In the above experiment, the BPH-1 cell line was used as a negative control (no YS5 binding and no fluorescence signal; data not shown). Nuc, nuclei. Scale bars: 30 μm. (B) In vitro tumor killing assay by CD46 ADC. YS5-MC-vc-PAB-MMAF was incubated with a panel of mCRPC lines (LNCaP-C4-2B, DU145, and PC3M) and control normal human cells (BPH-1, CD3+ T cells, and CD14-depleted PBMCs, and the normal human primary prostate epithelial cell line HPrEpC). A nonbinding human IgG1 was conjugated to MMAF and used as a control. EC50 values were 275 ± 98 pM for LNCaP-C4-2B; 1.13 ± 0.45 nM for DU145; and 607 ± 172 pM for PC3M. No cytotoxic effects were observed on the panel of control normal cells studied across the ADC concentration (conc.) range.

Article Snippet: DU145, LNCaP-C4-2B, and PC3M-luc cells (5,000 per well) were grown overnight in glass chamber slides (Thermo Fisher Scientific, Lab-Tek) at 37°C, incubated with 20 μg/ml YS5 IgG1 for the indicated time periods (4 and 24 hours) with Texas red–conjugated 70-kDa neutral dextran ND70-TR (a macropinocytosis marker; Thermal Fisher Scientific, Life Technologies), washed with PBS, fixed with 4% paraformaldehyde (PFA), and permeabilized with PBS containing 0.1% Triton X-100 and 1% BSA.

Techniques: Confocal Microscopy, Marker, Negative Control, Binding Assay, Fluorescence, In Vitro, Incubation, Concentration Assay

Journal: JCI Insight

Article Title: Targeting CD46 for both adenocarcinoma and neuroendocrine prostate cancer

doi: 10.1172/jci.insight.121497

Figure Lengend Snippet: (A–E) CD46 is highly expressed on the surface of the NEPC cell line H660, and H660 is sensitive to CD46 ADC. (F–H) Upregulation of CD46 and enhanced sensitivity to CD46 ADC by ASI-resistant mCRPC cells. (A) FACS analysis of CD46 and PSMA expression on H660. (B) Antigen density (surface copy number per cell) for CD46 and PSMA on H660 cell (520,430 ± 52,020 for CD46 vs. 23,733 ± 8,591 for PSMA). ***P < 0.01 (P = 0.0007), Student’s t test, unpaired, 2-tailed; triplicate. (C) Western blot analysis showed CD46 protein expression as well as the neuroendocrine marker neuron-specific enolase (NSE). (D) YS5 IgG1 is internalized by H660 cells via macropinocytosis (colocalization with ND70) during 4-hour incubation at 37°C. The rate of colocalization was 83.33% (5/6). Scale bars: 30 μm. (E) CD46 ADC potently kills the neuroendocrine cancer line H660 in vitro (EC50 796 ± 460 pM). (F–H) CD46 antigen density increases in LNCaP-C4-2B cells following incubation with abiraterone (F) and enzalutamide (G) for 7 days, rising from 163,548 ± 6,402 before treatment to 336,690 ± 11,064 and 365,058 ± 42,168, respectively. ***P < 0.05 (P = 0.0002) (F) and **P < 0.01 (P = 0.0091) (G); Student’s t test, unpaired, 2-tailed; triplicate. The increased CD46 antigen density leads to increased sensitivity to CD46 ADC (H), with EC50 decreasing by 17-fold (226 ± 103 pM vs. 13 ± 1.8 pM). LNCaP C4-2B CD46 ADC: LNCaP-C4-2B cells treated with CD46 ADC. LNCaP C4-2B/Abi CD46 ADC: LNCaP-C4-2B cells with prior exposure to abiraterone were treated with CD46 ADC. LNCaP C4-2B Ctrl IgG ADC: LNCaP-C4-2B cells treated with the control ADC (Ctrl IgG-MC-vc-PAB-MMAF). LNCaP C4-2B/Abi Ctrl IgG ADC: LNCaP-C4-2B cells with prior exposure to abiraterone were treated with the control ADC (Ctrl IgG-MC-vc-PAB-MMAF).

Article Snippet: DU145, LNCaP-C4-2B, and PC3M-luc cells (5,000 per well) were grown overnight in glass chamber slides (Thermo Fisher Scientific, Lab-Tek) at 37°C, incubated with 20 μg/ml YS5 IgG1 for the indicated time periods (4 and 24 hours) with Texas red–conjugated 70-kDa neutral dextran ND70-TR (a macropinocytosis marker; Thermal Fisher Scientific, Life Technologies), washed with PBS, fixed with 4% paraformaldehyde (PFA), and permeabilized with PBS containing 0.1% Triton X-100 and 1% BSA.

Techniques: Expressing, Western Blot, Marker, Incubation, In Vitro