Fig 2: TNBC tumor cell STING signaling primes response to CAR T cell therapy(A) Immunoblot of mesothelin (MSLN) in PTEN WT (HCC1143, HCC1806, and MDAMB231) and PTEN-null (MDAMB468, HCC1937, and HCC70) TNBC cell lines.(B) Flow cytometry of MSLN expression in HCC1806 and HCC70 using HCC1143 as a control (representative of 2 independent experiments).(C) Schematic of CAR constructs with retroviral and lentiviral vectors. SP, signal peptide; ScFv, single-chain variable fragment; EC, extracellular; TM, transmembrane.(D and E) Cell lines were co-cultured in 2D with CD19 or MSLN CAR T cells for 24 h at indicated effector:target (E:T) ratios and assessed by CTG assay (n = 3). Data are representative of three independent experiments.(F and G) Representative images of HCC1806 and HCC70 co-cultured as 3D spheroids with CD19 or MSLN CAR T cells in a microfluidics device for 72 h. E:T ratio of 1:1. Staining as indicated for live and dead cells. Scale bar, 100 μm.(H) Quantification of 3D tumor spheroid cell death using DRAQ7/Hoechst staining (n = 2–6). Ctrl indicates tumor cells only.(I) CM from 3D co-culture was analyzed for IFNγ, granzyme B, and TNF-α by ELISA (n = 2–6).(J) Representative images of MSLN CAR T cell migration with untreated HCC70 and HCC1806 spheroids utilizing the 3D microfluidics device. MSLN CAR T cells fully migrate toward HCC70 (PTEN null) by 48 h, in contrast to HCC1806 cells (PTEN WT), denoted by a black arrow. E:T ratio of 1:1. Live and dead cells were stained as indicated. Scale bar, 100 μm. Quantification of migrated CAR T cells and percent tumor cell death are shown (n = 5–6).(K) Representative images of MSLN CAR T cell migration with HCC1806 Scr and Rab7 KO spheroids utilizing the 3D microfluidic device after 48 h. E:T ratio of 1:1. Scale bar, 100 μm. Spheroids were pretreated with 50 μM ADU or ctrl (PBS) for 24 h before loading. Quantification of migrated CAR T cells and percent tumor cell death are shown (n = 4–7). CM was analyzed for IFNγ and granzyme B by ELISA (n = 3–5).Quantitative data are represented as mean ± SEM. p values were calculated by two-way ANOVA followed by Sidak’s post hoc test (D and E), one-way (K) and two-way (H and I) ANOVA followed by Tukey’s post hoc test, and unpaired Student’s t test (J). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig 3: STING agonist followed by CAR T cell delivery via the sd-PEG method results in effective control of distant TNBC growth(A) Increase in circulating human CD3+ T cells after ADU priming (n = 5 mice/group).(B) Schematic of the dual tumor model.(C and D) Tumors were established orthotopically and in the ipsilateral flank, followed by 2 × 106 or 4 × 106 MSLN CAR T cell or control CD19 CAR T cell treatment with or without ADU as per the sd-PEG method. Mean tumor volume measurements of primary and flank tumors are shown (n = 3–4 mice/group).(E) Intratumoral CAR T cell expansion at the primary and flank tumor sites.(F and G) Kaplan-Meier survival curves from both experiments (n = 7–8 mice/group).(H) Schematic of the metastasis prevention model.(I) Tumors were established orthotopically and treated with 2 × 106 MSLN CAR T cells or control CD19 CAR T cells via the sd-PEG method; 7 days later, HCC70 cells were injected into the ipsilateral flank. Mean tumor volume measurements of primary and flank tumors are shown (n = 4 mice/group).(J) Kaplan-Meier survival curves (n = 4 mice/group).Quantitative data are represented as mean ± SEM. p values were calculated by two-way ANOVA followed by Tukey’s post hoc test (C and D) or Sidak’s post hoc test (I) and log rank Mantel-Cox test (F, G, and J). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig 4: PEG biopsy marker-mediated local delivery of CAR T cells to target TNBC(A) Bioluminescent imaging of a PEG marker soaked with PBS or NanoLuc-MSLN CAR T cells.(B) Bioluminescent imaging of a hydrated PEG marker injected with PBS or NanoLuc-MSLN CAR T cells.(C) Schematic of MSLN CAR T cells soaked into various biopsy markers and then co-cultured with firefly luciferase expressing HCC70 spheroids (ffLUC-HCC70).(D and E) ffLUC-HCC70 spheroids co-cultured with PEG markers soaked (D) or injected (E) with B-cell maturation antigen (BCMA) or MSLN CAR T cells at the indicated E:T ratios. Tumor cell death was determined by luciferase assay at 24 h (n = 3–4).(F and G) Orthotopically established HCC70 tumors were implanted with PEG soaked with PBS or 4 × 106 MSLN CAR T cells. CD3 IHC was performed on day 14 (F) and day 42 (G). Scale bar, 100 μm.(H) HCC70 tumor with an adjacent PEG. Dissection shows an intact PEG marker with a visible metal clip.(I) Orthotopically established HCC70 tumors were implanted with PEG+PBS or PEG+100 μg ADU, and 7 days later, PEG markers were extracted and cocultured with MSLN CAR T cells.(J) Quantification of MSLN CAR T cell viability from (I) using CTG (n = 2–3).(K) Schematic of the sequential delivery PEG (sd-PEG) method. Established tumors are implanted with PEG+PBS or PEG+ADU, followed 7 days later by CAR T cell injection into the PEG.(L) Representative IHC images of human CD3 staining on day 2 using the sd-PEG method with 50,000 MSLN CAR T cells (left). Scale bar, 100 μm. Quantification of CD3+ cells per high-power field is shown (right) (n = 4–5 mice/group).(M and N) Digested tumors were also analyzed for human CD3 by flow cytometry on day 2. Quantification of CD3+ cells is shown (n = 5 mice/group).Quantitative data are represented as mean ± SEM. p values were calculated by two-way ANOVA followed by Sidak’s post hoc test (D and E), one-way ANOVA followed by Tukey’s post hoc test (J), and unpaired Student’s t test (L and N). ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.