Journal: Cancers

Article Title: Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8 + T Cells in Immunotherapy-Resistant and Metastatic Cancers

doi: 10.3390/cancers14061596

Figure Lengend Snippet: PKC-θ is enriched in the nuclei of dysfunctional CD8+ T cells isolated from stage IV metastatic cancers. ( A ) Quantification of PKC-θ-Thr568p Fn/c in CD8+ T cells or CSV+ CTCs isolated from immunotherapy-responsive (complete response, CR; partial response PR) or resistant (PD, progressive disease) melanoma patients defined using RECIST 1.1 criteria. The Fn/c for PKC-θ-Thr568p was quantified by ASI digital pathology and ImageJ-Fiji ( n ≥ 20 cells per group). ( B ) Quantification of PKC-θ-Thr568p Fn/c in CD8+ T cells or CSV+ CTCs isolated from healthy donors (HD), stage IV metastatic breast cancer patients (Mets), or stage IV breast cancer patients with brain metastases (Brain Mets). The Fn/c for PKC-θ-Thr568p was quantified by ASI digital pathology and ImageJ-Fiji ( n ≥ 40 cells per group). ( C ) The amino acid sequence of ZEB1 indicating the top eight peptides for peptide phosphorylation by PKC-θ. The top peptide sequences are displayed in a table with the mean signal intensity (2 SD above the mean was considered a positive phosphorylation event). ( D ) Top peptides positive for phosphorylation and their overlap with the ZEB1 amino acid sequence as well as the structure of ZEB1, adapted from . ( E ) Duolink ® proximity ligation assay (PLA) for PKC-θ and ZEB1 in CD8+PD1+ T cells isolated from immunotherapy-resistant or responder melanoma patients. Representative images are shown for PKC-θ/ZEB1, scale bar represents 10 µM. Graphs represent the PLA signal intensity of the Duolink ® assay; data represent n ≥ 100 cells/sample. Graphs plot the percentage of PLA signal positive cells out of total cells for (A). Data represent n ≥ 100 cells/sample. ( F ) FFPE sections from primary breast cancers ( n = 6 patients, >500 cells counted per patient) or breast cancer brain metastases ( n = 20 patients, >500 cells counted per patient) were processed for high-resolution microscopy using the BondRX platform. FFPE sections were fixed and immunofluorescence microscopy performed probing with primary antibodies targeting CD8, PKC-θ (T53p), and ZEB1 with DAPI. Plots represent the % population of CD8+ T cells positive for PKC-θ and ZEB1 out of total CD8+ T cells. Example images are shown with 20 µM scale bar. ( G ) CD8+ cells were isolated from melanoma patient liquid biopsies (responder = complete response (CR) or resistant, where primary = primary resistance, secondary = secondary resistance, PD = progression of disease) and stimulated with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore (CI) and pre-clinically screened with either vehicle control or nPKC-i2. Samples were then fixed and immunofluorescence microscopy performed with primary antibodies targeting ZEB1, PKC-θ, and CD8. Representative images for each dataset are shown in . Graph represents the mean TNFI for PKC-θ and ZEB1 measured using ImageJ to select the nucleus minus background ( n > 20 cells/sample). ( H ) Plot profiles for each cohort for ZEB1 and PKC-θ are also depicted (red = ZEB1, green = PKC-θ) with the Pearson correlation coefficient (PCC) used to quantify colocalization between fluorophore-tagged proteins indicated and plotted. −1 = inverse of colocalization; 0 = no colocalization; +1 = perfect colocalization. Statistical significance is denoted by ns (not significant), ** p ≤ 0.005 and **** p ≤ 0.0001.

Article Snippet: Human MCF-7 breast cancer cells were activated with phorbol 12-myristate 13-acetate (PMA; 0.65 ng/mL) (Sigma-Aldrich, St. Louis, MO, USA) or PMA and transforming growth factor β (TGF-β; 20 ng/mL) (R & D Systems, Minneapolis, MN, USA) for 60 h to induce EMT and mesenchymal-like MCF-7 cells.

Techniques: Isolation, Sequencing, Proximity Ligation Assay, Microscopy, Immunofluorescence