Journal: Cancer cell

Article Title: Patterns of transcription factor programs and immune pathway activation define four major subtypes of SCLC with distinct therapeutic vulnerabilities

doi: 10.1016/j.ccell.2020.12.014

Figure Lengend Snippet: Comparison between each SCLC cell line subtype of mean relative in vitro IC50 values for PARP inhibitors, nucleoside analogs, anti-folates, AURK inhibitors, and BCL2 inhibitors (A). Comparison if IC50 values for cisplatin (B) and the PARPi olaparib (C) among SCLC-A cell lines separated into high and low SLFN11 expression. IHC analysis of tumors representing each subtype (representative images for SCLC-A and –N; and n=1 each for SCLC-P and –I) for expression of BCL2, with associated H-score noted (D). Western blot showing expression of E-cadherin and Vimentin in H-841 cell line with addition of TGFβ (EMT inducer) and mocetinostat (E). Murine H841 flank cell line xenograft growth curves with vehicle or mocetinostat treatment (F). Mean expression of the cell surface protein encoding gene SSTR2 in multiple datasets (G-I) along with flow cytometry analysis of proportion of analyzed cells that express SSTR2 protein in subtyped cell lines (J). Sample sizes: n=62 cell lines (A, H), n=38 cell lines (B-C), n=8 mouse tumors per treatment arm 81 tumors (G), n=23 tumors (I), and n=18 cell lines (J). p-values are the result of one-way two-sided T-test (B-C) or one-way ANOVA (A, G-J). Error bars: +/− 1.5x interquartile range (B-C, G-J) or +/− SEM (F).

Article Snippet: Mouse monoclonal anti SSTR2 Alexa Fluor 594 conjugated , Novus Biological , Cat # IC4224T Clone 402038.

Techniques: Comparison, In Vitro, Expressing, Western Blot, Flow Cytometry

Journal: Cancer cell

Article Title: Patterns of transcription factor programs and immune pathway activation define four major subtypes of SCLC with distinct therapeutic vulnerabilities

doi: 10.1016/j.ccell.2020.12.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti SSTR2 Alexa Fluor 594 conjugated , Novus Biological , Cat # IC4224T Clone 402038.

Techniques: Virus, Recombinant, Methylation, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Development of anti-somatostatin receptors CAR T cells for treatment of neuroendocrine tumors

doi: 10.1136/jitc-2022-004854

Figure Lengend Snippet: Expression of SSTR2 and SSTR5 by NET cell lines. (A) WB of membrane extracts from CM, BON1, QGP1, H727 and CNDT2.5 cell lines. Na + /K + ATPase membrane expression was used as loading control. (B) Representative flow cytometry analysis of SSTR2 and SSTR5 expression in CM and BON1 cells as well as in HAP1 cells as negative control. (C) Representative confocal microscopy evaluation of SSTR2 and SSTR5 expression in CM and BON1 cells. For both flow cytometry and confocal microscopy experiments permeabilization procedures were omitted to allow detection of membrane SSTRs only. SSTRs, somatostatin receptors; WB, Western blot.

Article Snippet: The membrane expression of SSTR2 and SSTR5 was evaluated using human- (R&D Systems, cat#FAB4224A; R&D Systems, cat#IC4448G) or mouse-reactive (Novus Biologicals, cat#NB300-157SS; Novus Biologicals, cat#NB100-74540) Abs targeting extracellular epitopes of these receptors after omission of cell permeabilization procedures.

Techniques: Expressing, Membrane, Control, Flow Cytometry, Negative Control, Confocal Microscopy, Western Blot

Journal: Journal for Immunotherapy of Cancer

Article Title: Development of anti-somatostatin receptors CAR T cells for treatment of neuroendocrine tumors

doi: 10.1136/jitc-2022-004854

Figure Lengend Snippet: Expression of membrane SSTR2 and SSTR5 by flow cytometry across cell lines

Article Snippet: The membrane expression of SSTR2 and SSTR5 was evaluated using human- (R&D Systems, cat#FAB4224A; R&D Systems, cat#IC4448G) or mouse-reactive (Novus Biologicals, cat#NB300-157SS; Novus Biologicals, cat#NB100-74540) Abs targeting extracellular epitopes of these receptors after omission of cell permeabilization procedures.

Techniques: Expressing, Membrane, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Development of anti-somatostatin receptors CAR T cells for treatment of neuroendocrine tumors

doi: 10.1136/jitc-2022-004854

Figure Lengend Snippet: Anti-SSTR CAR T cells exhibit antigen-specific tumoricidal activity. (A) Anti-SSTR CARs endow human T lymphocytes with reactivity against SSTR-expressing targets. By in vitro BLI assay, CAR T cells induced cell death in up to 58% of Luc + NET cell lines as compared with UT T cells at an E:T ratio of 1:1. The percentage of specific tumor cell lysis was calculated as the ratio between CAR T cell and UT T cell antitumor activity using the formula: % lysis=1-(mean BLI signal in the presence of anti-SSTR CAR T cells/mean BLI signal in the presence of UT cells) x 100%. (B) In vitro BLI assay to evaluate the cytolytic activity of CAR T cells as compared with UT T cells according to increasing E:T ratios after 24 hours of coculture with NET cells. The degree of cytotoxicity induced by CAR T cells increased when the number of effector cells increased. (C) IFN-γ and (D) TNF-α release on 24 hours coculture of NET cells with CAR T cells or UT T cells at an E:T ratio of 1:1. Cytokine production was measured in culture supernatants by ELISA. CD3/CD28 T cell stimulation through TransAct was used for positive control experiments. (E) In vitro BLI assay to investigate the cytolytic activity of CAR T cells as compared with UT T cells against mutant CM cells and parental cells at an E:T ratio of 1:1. (F) Evaluation of the cytotoxic potential of CAR T cells against CM cells harboring wild-type or mutated SSTR2 and/or SSTR5 according to increasing E:T ratios after 24 hours of coculture. (G) IFN-γ and (H) TNF-α release on 24 hours coculture of CAR T cells or UT T cells with mutated or parental CM cells at an E:T ratio of 1:1. All experiments were carried out in technical triplicate using lymphocytes from three healthy donors. Mean values and standard errors are represented in figure. *P<0.05, **p<0.01. CAR, chimeric antigen receptor; E:T, effector:target; NET, neuroendocrine tumor; SSTR, somatostatin receptor; UT, untransduced.

Article Snippet: The membrane expression of SSTR2 and SSTR5 was evaluated using human- (R&D Systems, cat#FAB4224A; R&D Systems, cat#IC4448G) or mouse-reactive (Novus Biologicals, cat#NB300-157SS; Novus Biologicals, cat#NB100-74540) Abs targeting extracellular epitopes of these receptors after omission of cell permeabilization procedures.

Techniques: Activity Assay, Expressing, In Vitro, Lysis, Enzyme-linked Immunosorbent Assay, Cell Stimulation, Positive Control, Mutagenesis

Journal: Journal for Immunotherapy of Cancer

Article Title: Development of anti-somatostatin receptors CAR T cells for treatment of neuroendocrine tumors

doi: 10.1136/jitc-2022-004854

Figure Lengend Snippet: Anti-SSTR CAR T cells infiltrate human xenografts and murine SSTR-expressing organs. (A) Visualization of SSTR2 (UMB1 mAb) and SSTR5 (UMB5 mAb) within tumor xenografts by IHC. Magnification: ×20. Scale bar: 100 µm. (B) Explanted CM tumor xenografts as well as SSTR-expressing organs such as murine spleen, pancreas and brain were lysed and subjected to DNA extraction. The infiltration of CAR T cells was demonstrated by PCR using primers specific for the anti-SSTR CAR sequence. The purified CAR construct DNA was used for positive control experiments. Distilled water was used in negative control experiments. (C) FAM-labeled and HEX-labeled probes against the CAR sequence and the MKL2 reference gene, respectively, were employed in ddPCR experiments to quantify the infiltration of CM (blue dots) and BON1 xenografts (red dots) by anti-SSTR CAR T cells. An inverse correlation can be observed between CAR T cell infiltration and tumor bioluminescence increase relative to baseline. (D) Anti-SSTR CAR T cells recognize and kill the MIN6 cells, a murine NET cell line expressing SSTR2/5. By in vitro BLI, CAR T cells induced cell death in the 48% of Luc + MIN6 cells as compared with UT T cells at an E:T ratio of 1:1 after 72 hours of coculture. (E) IFN-γ release on 24 hours coculture of NET cells with CAR T cells or UT T cells at an E:T ratio of 1:1. (F) Histopathological analysis of human NET xenografts and murine pancreas and brain by H&E staining. Extensive necrosis foci could be identified within tumors (arrowhead). Representative microphotographs show the absence of necrosis or other tissue damages in the context of the murine pancreas and brain. Magnification: x20. Scale bar: 100 µm. **P<0.01. CAR, chimeric antigen receptor; ddPCR, droplet digital PCR; E:T, effector:target; IHC, immunohistochemistry; NET, neuroendocrine tumor; SSTR, somatostatin receptor; UT, untransduced.

Article Snippet: The membrane expression of SSTR2 and SSTR5 was evaluated using human- (R&D Systems, cat#FAB4224A; R&D Systems, cat#IC4448G) or mouse-reactive (Novus Biologicals, cat#NB300-157SS; Novus Biologicals, cat#NB100-74540) Abs targeting extracellular epitopes of these receptors after omission of cell permeabilization procedures.

Techniques: Expressing, DNA Extraction, Sequencing, Purification, Construct, Positive Control, Negative Control, Labeling, In Vitro, Staining, Digital PCR, Immunohistochemistry