megashortscript t7 transcription kit  (Thermo Fisher)

Journal: Cancer Research

Article Title: Targeting Intracellular Innate RNA-Sensing Systems Overcomes Resistance to CAR T-cell Therapy in Solid Tumors

doi: 10.1158/0008-5472.CAN-24-3425

Figure Lengend Snippet: Therapeutic stimulation of tumor cell–intrinsic RIG-I/MAVS signaling sensitizes tumor cells to CAR T-cell–mediated apoptosis. A–E, WT or Mavs − / − B16-EpCAM tumor cells were transfected with 3pRNA 24 hours prior to exposure to anti-EpCAM CAR T cells or UTD control T cells. A and B, Tumor cells were sorted 24 hours after coculture for bulk RNA extraction. Relative expression of Puma and Bid ( A ) as well as Fas and Trailr2 ( B ) in B16-EpCAM cells. Data are normalized to unstim. B16 cells. C–E, Apoptosis induction ( C ), cleavage of caspase-3 ( D ), and viability ( E ) in B16-EpCAM cells by flow cytometry. All data are mean ± SEM of n = 3 to 6 replicates. F, B16-EpCAM cells were treated with 3pRNA as described above and were subsequently cocultured with either WT or Mavs − / − CAR T cells. Apoptosis induction in B16-EpCAM cells by flow cytometry is presented as mean ± SEM of n = 3 to 4 replicates. G, Different EGFR-expressing human tumor cell lines were transfected with 3pRNA for 24 hours prior to exposure to EGFR CAR or UTD T cells. Apoptosis induction in tumor cells by flow cytometry is presented as mean ± SEM of n = 3 replicates. All data are representative of or pooled from at least two independent experiments. Unstim., unstimulated.

Article Snippet: For specific activation of RIG-I/MAVS, double-stranded in vitro -transcribed 3pRNA (sense, 5′- UCA AAC AGU CCU CGC AUG CCU AUA GUG AGU CG -3′) was synthesized using the 3pRNA synthesis MEGAshortscript T7 transcription kit (Thermo Fisher Scientific) as described previously ( ).

Techniques: Transfection, Control, RNA Extraction, Expressing, Flow Cytometry

Journal: Cancer Research

Article Title: Targeting Intracellular Innate RNA-Sensing Systems Overcomes Resistance to CAR T-cell Therapy in Solid Tumors

doi: 10.1158/0008-5472.CAN-24-3425

Figure Lengend Snippet: Tumor-intrinsic MAVS signaling induces susceptibility to CAR T-cell killing via soluble factors and auto-/paracrine IFN signaling that can spread to bystander tumor cells. A, B16-EpCAM cells were pretreated with anti-IFNaR1 blocking antibody before 3pRNA transfection and subsequent coculture with anti-EpCAM CAR or UTD T cells. Apoptosis induction in tumor cells after 24 hours was determined by flow cytometry. Data are mean ± SEM of n = 3 replicates. B, WT or Mavs − / − B16-EpCAM cells were either directly transfected with 3pRNA or exposed to conditioned culture medium from 3pRNA-activated WT B16-EpCAM cells. Relative expression of Puma , Bid , and Ifnb1 in bystander B16-EpCAM cells was determined by RT-PCR. Data were normalized to unstimulated (unstim.) B16 cells and are mean ± SEM of n = 3 replicates. C, WT or Mavs − / − B16-EpCAM cells were exposed to anti-EpCAM CAR or UTD T cells in the presence of conditioned culture medium from 3pRNA-activated WT B16-EpCAM cells. Apoptosis induction by flow cytometry is presented as mean ± SEM of n = 6 replicates. All data are representative of or pooled from at least two independent experiments.

Article Snippet: For specific activation of RIG-I/MAVS, double-stranded in vitro -transcribed 3pRNA (sense, 5′- UCA AAC AGU CCU CGC AUG CCU AUA GUG AGU CG -3′) was synthesized using the 3pRNA synthesis MEGAshortscript T7 transcription kit (Thermo Fisher Scientific) as described previously ( ).

Techniques: Blocking Assay, Transfection, Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Research

Article Title: Targeting Intracellular Innate RNA-Sensing Systems Overcomes Resistance to CAR T-cell Therapy in Solid Tumors

doi: 10.1158/0008-5472.CAN-24-3425

Figure Lengend Snippet: Therapeutically activated tumor-intrinsic RIG-I/MAVS signaling imprints a cytolytic phenotype on CAR T cells. A and B, B16-EpCAM tumor cells were transfected with 3pRNA prior to exposure to anti-EpCAM CAR T cells or UTD control T cells. Expression of CD69, IFNγ, and granzyme B ( A ) and Fas ligand (FasL; B ) on T cells was measured by flow cytometry 24 hours after coculture. Data are mean ± SEM of n = 6 replicates. C, Anti-EpCAM CAR or UTD T cells were cocultured with 3pRNA-treated WT or Mavs − / − B16-EpCAM for 24 hours before rechallenge with steady-state WT B16-EpCAM cells. Apoptosis induction in initial and rechallenge B16 cells was determined by flow cytometry. All data are mean ± SEM of n = 6 replicates. D, Anti-EpCAM CAR T cells were exposed to conditioned culture medium from RIG-I–treated B16-EpCAM cells. Expression of CD25, PD-1, CD69, and granzyme B on CAR T cells is presented as mean ± SEM of n = 3 replicates. E, Apoptosis induction in 3pRNA-treated B16-EpCAM cells 24 hours after coculture with WT or Ifnar1 − / − anti-EpCAM CAR or UTD T cells. Data are mean ± SEM of n = 4 replicates. F, Heatmap showing the relative release of cytokines and chemokines from B16-EpCAM, Panc02-EpCAM, and MG846-MSLN cells in response to RIG-I activation. All data are representative of or pooled from at least two independent experiments. Unstim., unstimulated. C, Created in BioRender. Soliman, N. (2025) https://BioRender.com/l0n2a28 .

Article Snippet: For specific activation of RIG-I/MAVS, double-stranded in vitro -transcribed 3pRNA (sense, 5′- UCA AAC AGU CCU CGC AUG CCU AUA GUG AGU CG -3′) was synthesized using the 3pRNA synthesis MEGAshortscript T7 transcription kit (Thermo Fisher Scientific) as described previously ( ).

Techniques: Transfection, Control, Expressing, Flow Cytometry, Activation Assay