Journal: bioRxiv

Article Title: Sulindac modulates the response of triple negative breast cancer to anti-PD-L1 immunotherapy

doi: 10.1101/2025.06.11.659159

Figure Lengend Snippet: (a-b) After MDA-MB-231-control or HCC70-control cells and MDA-MB-231-nsMase2 KD or HCC70-nsMase2 KD cells were treated with 20uM SS for 48h, total cell lysates were prepared with RIPA buffer and exosome from cell culture media were isolated and purified with ExoQuick-TC PLUS Exosome Purification Kit. Then exosome lysates were generated using RIPA buffer. Lastly, the intracellular PD-L1, nsMasae2 and exosomal PD-L1 were analyzed by Western blot.

Article Snippet: The murine breast cancer cell line 4T1 and E0771 and the human breast cancer cell lines MDA-MB-231, HCC70 and BT549 were purchased from ATCC (Manassas VA, USA).

Techniques: Control, Cell Culture, Isolation, Purification, Generated, Western Blot

Journal: Human Molecular Genetics

Article Title: An enhancer variant associated with breast cancer susceptibility in Black women regulates TNFSF10 expression and antitumor immunity in triple-negative breast cancer

doi: 10.1093/hmg/ddac168

Figure Lengend Snippet: Genotype-specific expression of TNFSF10 in TNBC cell lines from Black women. ( A ) The UCSC Genome Browser shows the locations of rs13074711 and TNFSF10 , along with ENCODE histone modification marks for H3K4Me1 and H3K27Ac of seven cell types and enhancer marks in HMEC. Arrows indicate the location of rs13074711 and a strong enhancer mark in HMEC. ( B ) DNA sequencing chromatograms are shown in three TNBC cell lines (HCC1569, HCC1806 and HCC70) from Black women. The arrow indicates the position of rs31074711 and Y represents TC heterozygote. ( C ) Expression of TNFSF10 mRNA was measured by qRT-PCR in three cell lines with or without IFN-β treatment (24 h). Results were normalized to RNA18S and fold induction is shown relative to untreated HCC1569 cells. ( D ) Representative images (top) and a summary graph (bottom) of western blot analysis of TNFSF10 protein expression in three cells with or without IFN-β treatment. Tublin was used as an endogenous loading control. All data represent the mean and SD of n = 3–4 biological replicates and are representative of at least two independent experiments.

Article Snippet: Breast cancer cell lines from humans (HCC1569, HCC1806 and HCC70) and mice (4 T1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Modification, DNA Sequencing, Quantitative RT-PCR, Western Blot, Control

Journal: Human Molecular Genetics

Article Title: An enhancer variant associated with breast cancer susceptibility in Black women regulates TNFSF10 expression and antitumor immunity in triple-negative breast cancer

doi: 10.1093/hmg/ddac168

Figure Lengend Snippet: Regulation of TNFSF10 expression by rs31074711 Alleles. ( A ) Depicted is CRISPR-Cas9 genome editing of rs13074711 from TC genotype to CC genotype through HDR. DNA sequencing chromatograms showed the shift from T to C, after genome editing in HCC1806 cells. ( B ) qRT-PCR analysis of TNFSF10 mRNA in HCC1806 cells with TC or CC genotype of rs31074711 that were treated with IFN-β for 1 day. Results were normalized to RNA18S and fold induction is shown relative to the untreated control with TC genotype. ( C ) Western blot analysis of TNFSF10 protein expression in HCC1806 cells with TC or CC genotype 1-day post IFN-β treatment. Tublin was used as an endogenous loading control. ( D ) HCC1806 cells with TC or CC genotypes were treated with IFN-β and subjected to the IncuCyte Live-Cell Imaging System. Apoptosis was quantified using green fluorescent signals from caspase-3/7–positive apoptotic cells normalized to cell density. All data represent the mean and SD of n = 3–6 biological replicates and are representative of at least two independent experiments. * * * P < 0.001.

Article Snippet: Breast cancer cell lines from humans (HCC1569, HCC1806 and HCC70) and mice (4 T1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, CRISPR, DNA Sequencing, Quantitative RT-PCR, Control, Western Blot, Live Cell Imaging

Journal: Human Molecular Genetics

Article Title: An enhancer variant associated with breast cancer susceptibility in Black women regulates TNFSF10 expression and antitumor immunity in triple-negative breast cancer

doi: 10.1093/hmg/ddac168

Figure Lengend Snippet: Correlation of TNFSF10 expression with anti-viral gene expression and regulation by cytokines. ( A ) Examples of key immune genes significantly correlated with TNFSF10 expression. Normalized RNA-seq data from TCGA-BRCA ( n = 1076) were analyzed for correlation. The values are presented as log 2 FPKM. ( B ) qRT-PCR analyses of TNFSF10, DDX58, IFIH1, OAS1, TNF, IL mRNA in HCC1806 cells that were treated with IFN-β, IFN-γ, or TNF-α for 1 day. ( C ) qRT-PCR analyses of DDX58, IFIH1, OAS1 mRNA in HCC1806 cells with TC or CC genotype of rs31074711 that were treated with IFN-β for 1 day. Results were normalized to RNA18S and fold induction is shown relative to untreated control cells. Data represent the mean and SD of n = 3–4 biological replicates and are representative of at least two independent experiments. * * * P < 0.001; * * * * P < 0.0001.

Article Snippet: Breast cancer cell lines from humans (HCC1569, HCC1806 and HCC70) and mice (4 T1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Control

Journal: Cell Reports Medicine

Article Title: Immune targeting of triple-negative breast cancer through a clinically actionable STING agonist-CAR T cell platform

doi: 10.1016/j.xcrm.2025.102198

Figure Lengend Snippet: Sustained tumor cell STING signaling promotes T cell migration (A) Representative IHC images of STING staining of a TMA ( n = 63). 0 = no tumor cell staining; 1+ = faint, 2+ = moderate, 3+ = strong staining in >10% of tumor cells. Scale bar, 50 μm. (B) Tumor cell STING staining in PTEN-null TNBCs. Scale bar, 50 μm. (C) Immunoblot of STING and other proteins across TNBC lines, including ADU-S100 (ADU; 50 μM, 3 h)-treated THP-1 monocytes to distinguish phosphorylated STING (pSTING). (D) Immunoblot of MDAMB231 parental cells, cells expressing scramble (Scr), or Rab7 knockout (KO) vectors (Sg1 and Sg2). (E) CXCL10 ELISA from conditioned medium (CM) of the indicated Scr or Rab7 KO cells treated with ADU at 50 μM for 48 h ( n = 4–8) or PBS control (ctrl). (F) Immunoblot of PTEN-null HCC70 cells treated with ADU at 50 μM for 3 h. (G) Immunoblot of MDAMB231 Scr or Rab7 KO cells treated with 50 μM ADU at the indicated time points. (H) Immunoblot of MDAMB231 Scr or Rab7 KO cells treated with 50 μM ADU for 24 h. (I) Heatmap showing log2 fold change (L2FC) of a cytokine/chemokine panel, normalized to untreated Scr cells ( n = 2–4). Red asterisk indicates values above assay in all conditions, black asterisk indicates above assay in treated conditions, using the upper limit for L2FC calculation. (J) CXCL10 ELISA in CM from the indicated cell lines with or without 50 μM ADU treatment at 48 h ( n = 2–4). (K) T cell migration assay schematic. (L) Representative images of CD8 + T cells (yellow) migrating toward MDAMB231 spheroids (Hoechst). Migrated CD8 + T cells were quantified after 48 h ( n = 9). Scale bar, 100 μm. (M) Representative images of CD8 + T cells (yellow) migrating toward Rab7 KO MDAMB231 spheroids (Hoechst). Migrated CD8 + T cells were quantified after 48 h ( n = 5–6). Scale bar, 100 μm. Quantitative data are represented as mean ± SEM. p values were calculated by one-way (J, L, and M) and two-way (E) ANOVA followed by Tukey’s post hoc test. ns, not significant. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: HEK 293T, human TNBC cell lines (HCC1143, HCC1806, MDAMB231, MDAMB468, HCC1937, HCC70), THP-1 cells, Jurkat cells and murine TNBC cell line 4T1 used in this study were obtained from American Type Culture Collection (ATCC).

Techniques: Migration, Staining, Western Blot, Expressing, Knock-Out, Enzyme-linked Immunosorbent Assay, Control, Cell Migration Assay

Journal: Cell Reports Medicine

Article Title: Immune targeting of triple-negative breast cancer through a clinically actionable STING agonist-CAR T cell platform

doi: 10.1016/j.xcrm.2025.102198

Figure Lengend Snippet: Toxicity of STING agonism to endogenous T cells mitigated by PEG marker delivery (A) Flow cytometry of CD45 + , CD3 + , CD4 + , and CD8 + T cells from a primary human TNBC explant treated with 50 μM ADU or PBS for 24 h. (B) Human CD8 + T cells isolated from PBMCs were treated with ADU for 24 h and analyzed by CellTiter-Glo assay ( n = 3). (C) Splenocytes from C57BL/6J mice were treated with ADU for 24 h. Viability of T cells was analyzed by Zombie NIR flow cytometry ( n = 2). (D) Human CD8 + T cells treated with CM for 24 h were collected at various time points from PEG+ADU as in C and analyzed by CTG assay ( n = 3). (E) Human CD8 + T cells treated with PBS controls or direct addition of 100 μg ADU versus PEG+100 μg ADU for 5 days and analyzed by CTG assay or then expanded in IL-2, IL-7, and IL-15, followed by CTG assay on day 10 ( n = 3). (F) Human CD8 + T cells were treated with PBS controls or with direct addition of 100 μg ADU versus PEG+100 μg ADU for 5 days; expanded in IL-2, IL-7, and IL-15; and counted every 2–3 days ( n = 3). (G) UMAP plot of TCR clonotype positivity from J (left). Pie charts show the percentage of cells with unique TCR clonotypes of frequency 1, ≥2, and ≥5 for PEG+PBS and PEG+ADU treatment (right). (H) UMAP plot of TCR clonotype positivity of PBMCs from PEG+PBS- and PEG+ADU-treated 4T1 mice (left). Pie charts show the percentage of cells with unique TCR clonotypes in PBMCs (right). Quantitative data are represented as mean ± SEM; p values were calculated by one-way ANOVA (B–E) or two-way ANOVA (F), followed by Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: HEK 293T, human TNBC cell lines (HCC1143, HCC1806, MDAMB231, MDAMB468, HCC1937, HCC70), THP-1 cells, Jurkat cells and murine TNBC cell line 4T1 used in this study were obtained from American Type Culture Collection (ATCC).

Techniques: Marker, Flow Cytometry, Isolation, Glo Assay, CTG Assay

Journal: Cell Reports Medicine

Article Title: Immune targeting of triple-negative breast cancer through a clinically actionable STING agonist-CAR T cell platform

doi: 10.1016/j.xcrm.2025.102198

Figure Lengend Snippet: 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.

Article Snippet: HEK 293T, human TNBC cell lines (HCC1143, HCC1806, MDAMB231, MDAMB468, HCC1937, HCC70), THP-1 cells, Jurkat cells and murine TNBC cell line 4T1 used in this study were obtained from American Type Culture Collection (ATCC).

Techniques: Western Blot, Flow Cytometry, Expressing, Control, Construct, Retroviral, Cell Culture, CTG Assay, Staining, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Migration

Journal: Cell Reports Medicine

Article Title: Immune targeting of triple-negative breast cancer through a clinically actionable STING agonist-CAR T cell platform

doi: 10.1016/j.xcrm.2025.102198

Figure Lengend Snippet: 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 × 10 6 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.

Article Snippet: HEK 293T, human TNBC cell lines (HCC1143, HCC1806, MDAMB231, MDAMB468, HCC1937, HCC70), THP-1 cells, Jurkat cells and murine TNBC cell line 4T1 used in this study were obtained from American Type Culture Collection (ATCC).

Techniques: Marker, Imaging, Injection, Cell Culture, Luciferase, Expressing, Dissection, Staining, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Immune targeting of triple-negative breast cancer through a clinically actionable STING agonist-CAR T cell platform

doi: 10.1016/j.xcrm.2025.102198

Figure Lengend Snippet: PEG biopsy marker-mediated local delivery of STING agonism and CAR T cells results in long-term durable control of TNBC growth (A) Orthotopically established HCC70 (non-labeled) tumors were treated with ADU+NanoLuc-labeled CAR T cells (4 × 10 6 ) via direct intratumoral injection, dual PEG delivery (PEG+ADU and PEG+CAR T), or the sd-PEG method ( n = 3–4 mice/group). Bioluminescent distribution of CAR T cells at the indicated time points is shown. (B) Total flux of bioluminescent signals from ADU+MSLN CAR T cell groups from (A) was quantified at the indicated time points. (C) Orthotopically established HCC70 tumors were implanted with PEG+PBS or PEG+ADU on day −7. On day 0, the indicated doses of NanoLuc-CD19/MSLN CAR T cells were injected into the PEG. MSLN2 = 2 × 10 6 MSLN CAR T cells; MSLN1 = 1 × 10 6 MSLN CAR T cells; MSLN0.5 = 0.5 × 10 6 MSLN CAR T cells. Mean tumor volume measurements are shown ( n = 3–4 mice/group). (D) Intratumoral CAR T cell expansion over time from (C). (E) Orthotopically established HCC70 tumors were treated via the sd-PEG method using 2 × 10 6 or 4 × 10 6 MSLN CAR T cells (MSLN2 or MSLN4) or 4 × 10 6 CD19 CAR T cells. Intratumoral CAR T cell expansion was monitored over 12 weeks (F) Quantification of tumor volume for each treatment group from (E) ( n = 5 mice/group). (G) Kaplan-Meier RFS curves ( n = 5 mice/group). Relapse was defined by tumor regrowth >500 mm 3 . (H) Circulating human IFNγ levels on days 7, 14, and 21 ( n = 5 mice/group). (I) Representative IHC images of MSLN staining of a PBS+CD19 control tumor versus three different relapsed tumors from the PBS+MSLN2 group from (F). Scale bar, 100 μm. Quantitative data are represented as mean ± SEM. p values were calculated by two-way ANOVA followed by Tukey’s post hoc test (C), Sidak’s post hoc test (F), and log rank Mantel-Cox test (G). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: HEK 293T, human TNBC cell lines (HCC1143, HCC1806, MDAMB231, MDAMB468, HCC1937, HCC70), THP-1 cells, Jurkat cells and murine TNBC cell line 4T1 used in this study were obtained from American Type Culture Collection (ATCC).

Techniques: Marker, Control, Labeling, Injection, Staining

Journal: Cell Reports Medicine

Article Title: Immune targeting of triple-negative breast cancer through a clinically actionable STING agonist-CAR T cell platform

doi: 10.1016/j.xcrm.2025.102198

Figure Lengend Snippet: 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 × 10 6 or 4 × 10 6 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 × 10 6 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.

Article Snippet: HEK 293T, human TNBC cell lines (HCC1143, HCC1806, MDAMB231, MDAMB468, HCC1937, HCC70), THP-1 cells, Jurkat cells and murine TNBC cell line 4T1 used in this study were obtained from American Type Culture Collection (ATCC).

Techniques: Control, Injection