balb c mice  (ATCC)

Journal: Genes & Diseases

Article Title: NRG4 suppresses breast cancer metastasis via ERBB4-YAP1-mediated down-regulation of MMPs

doi: 10.1016/j.gendis.2025.101691

Figure Lengend Snippet: Obesity promotes breast cancer metastasis. (A) The HFD-induced MMTV-PyMT mouse model. (B) Body weight changes in HFD-induced MMTV-PyMT mice ( n = 6 for each group). (C) Obesity increases the weight of primary mammary tumors and the number of lung metastases of MMTV-PyMT mice. (D – F) Representative microscopic images of lung tissues (D), relative mRNA levels (normalized to Gapdh) of the EMT-related genes and Ctgf gene in the 4th pair of mammary tumors (E) and metastatic lung tissues (F) of MMTV-PyMT mice ( n = 6) fed with a HFD. (G) Model of the HFD-induced 4T1 breast intra-tumoral model. (H) Body weight changes in the HFD-induced 4T1 breast intra-tumoral model ( n = 6 for each group). (I) Obesity increases primary tumor weight and the number of metastases in lung and liver tissues after the inoculation of 4T1 cells in mice. (J – M) Representative microscopic images of lung tissues (J), relative mRNA levels (normalized to Gapdh) of the EMT-related genes and Ctgf gene in the primary tumors (K), lung tissues (L), and liver tissues (M) of the 4T1 tumor-bearing mice ( n = 6) fed with a high-fat diet. The data were presented as mean ± standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. n.s., no significance.

Article Snippet: 4T1, MDA-MB-231, MDA-MB-453, MCF10A, MCF7, T47D, BT549, MDA-MB-468, and HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Standard Deviation

Journal: Genes & Diseases

Article Title: NRG4 suppresses breast cancer metastasis via ERBB4-YAP1-mediated down-regulation of MMPs

doi: 10.1016/j.gendis.2025.101691

Figure Lengend Snippet: NRG4, secreted by iWAT, possesses the potential to inhibit the invasion and metastasis of breast cancer. (A – C) Coculture of pre- or post-differentiated iWAT cells with breast cancer cells (A), followed by transwell (B) and qPCR experiments (C) for assessing cell migration. (D) Protein mass spectrometry analysis of conditioned media from pre- or post-differentiated C3H10T1/2 cells was carried out to identify commonly secreted proteins, such as NRG4. (E) qPCR analysis of secreted protein expression in the primary iWAT cells isolated from HFD-fed- and NCD-fed mice. (F) ELISA was performed to detect the NRG4 content in iWAT from mice with different metabolic statuses and the culture media from primary iWAT cells and C3H10T1/2 cells at various stages of differentiation. (G) Single-cell transcriptomic data of subcutaneous adipose tissues showed that NRG4 was expressed in adipocytes. (H) Single-cell transcriptomic data from circulating tumor cells (CTCs) of 14 TNBC patients indicated that NRG4 had the strongest negative correlation with tumor cell invasion and metastasis. (I – L) The correlation between the expression of NRG4 in serum (I, J) or adipose tissue (K, L) and the number of tumor metastases in the lung and liver in 4T1 tumor-bearing mice. The data were presented as mean ± standard deviation. ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: 4T1, MDA-MB-231, MDA-MB-453, MCF10A, MCF7, T47D, BT549, MDA-MB-468, and HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Mass Spectrometry, Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Single Cell, Standard Deviation

Journal: Genes & Diseases

Article Title: NRG4 suppresses breast cancer metastasis via ERBB4-YAP1-mediated down-regulation of MMPs

doi: 10.1016/j.gendis.2025.101691

Figure Lengend Snippet: NRG4 inhibits the cell migration and metastasis of breast cancer. (A) qPCR analysis of NRG4 expression in different tissues of mice fed a chow diet revealed high expression of NRG4 in adipose tissue. (B) TCGA data revealed a significant decrease in NRG4 expression in invasive breast cancer tissue compared with adjacent tissue. (C) Immunohistochemistry analysis demonstrated a significant decrease in NRG4 expression in late-stage breast cancer tissue. (D) Schematic diagram of the model of obese mice bearing breast tumors treated with rNRG4. (E, F) rNRG4 treatment did not affect the weight of primary tumors (E) but inhibited lung metastases of 4T1 tumor-bearing mice ( n = 6) (F). (G) HE staining was used to detect the morphology of primary tumors and lung tissues. (H) qPCR analysis showed that the expression of migration-related genes in primary tumor cells was inhibited by rNRG4 ( n = 6). (I) Immunoblotting analysis showed that the protein expression of migration-related genes in lung tissue was inhibited by rNRG4 after tumor metastasis. (J) Transwell experiments demonstrated the inhibition of cell migration by rNRG4. (K, L) qPCR and Immunoblotting analysis showed the expression of migration-related genes inhibited by rNRG4 in breast cancer cells. The data were presented as mean ± standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. n.s., no significance.

Article Snippet: 4T1, MDA-MB-231, MDA-MB-453, MCF10A, MCF7, T47D, BT549, MDA-MB-468, and HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Expressing, Immunohistochemistry, Staining, Western Blot, Inhibition, Standard Deviation

Journal: Genes & Diseases

Article Title: NRG4 suppresses breast cancer metastasis via ERBB4-YAP1-mediated down-regulation of MMPs

doi: 10.1016/j.gendis.2025.101691

Figure Lengend Snippet: NRG4 facilitates the interaction between pERBB4 and pYAP1 to hinder the nuclear translocation of YAP1. (A) Co-IP was used to enrich for ERBB4-bound proteins, followed by silver staining of differential bands and protein mass spectrometry for identifying 162 binding proteins in 4T1 cells. (B) The number of ERBB4 binding proteins in LC/MS assays upon rNRG4 treatment for 24 h. (C) STRING analysis revealed the interaction between ERBB4 and YAP1. (D) Western blotting analysis after nuclear-cytoplasmic fractionation revealed increased pERBB4 and pYAP1 in the cytoplasm and reduced YAP1 expression in the nucleus after rNRG4 treatment. (E) Co-IP of ERBB4 in the cytoplasm of 4T1 cells treated with rNRG4, followed by immunoblotting. (F) qPCR analysis of the mRNA expression levels of EMT-related genes in MDA-MB-231 cells overexpressing YAP5SA in the presence of NRG4. (G) qPCR analysis of the expression levels of Tead subunits in 4T1 cells and MDA-MB-231 cells. (H) qPCR analysis of the mRNA expression levels of Tead1 in 4T1 cells and MDA-MB-231 cells treated with 100 ng/mL rNRG4. (I) Co-IP results demonstrated the decrease of TEAD1 and YAP1 binding by rNRG4 treatment. The data were presented as mean ± standard deviation. ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: 4T1, MDA-MB-231, MDA-MB-453, MCF10A, MCF7, T47D, BT549, MDA-MB-468, and HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Translocation Assay, Co-Immunoprecipitation Assay, Silver Staining, Mass Spectrometry, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Fractionation, Expressing, Standard Deviation

Journal: Genes & Diseases

Article Title: NRG4 suppresses breast cancer metastasis via ERBB4-YAP1-mediated down-regulation of MMPs

doi: 10.1016/j.gendis.2025.101691

Figure Lengend Snippet: NRG4 suppresses breast cancer metastasis by inhibiting the transcription of MMPs. (A – D) RNA sequencing data from 4T1 cells treated with rNRG4 revealed a significant decrease in the extracellular matrix composition. (E) The heatmap showing commonly dysregulated MMP genes following rNRG4 treatment in 4T1 cells. (F) Volcano plot of the differentially expressed genes and labeled MMPs. (G) qPCR analysis of the dose-dependent impact of rNRG4 on MMP9 and MMP12 expression. (H) qPCR analysis of the effect of the general MMP inhibitor ilomastat on EMT induced by ERBB4 deletion. (I) qPCR analysis of the effect of siMMP9 on ERBB4 deletion-induced EMT. The data were presented as mean ± standard deviation. ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: 4T1, MDA-MB-231, MDA-MB-453, MCF10A, MCF7, T47D, BT549, MDA-MB-468, and HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: RNA Sequencing, Labeling, Expressing, Standard Deviation

Journal: Materials Today Bio

Article Title: Expanding the toolbox of bioorthogonal activation of photosensitizers for precise photodynamic therapy through transition metal-mediated deallylation

doi: 10.1016/j.mtbio.2026.102797

Figure Lengend Snippet: (a) Fluorescence confocal images of HeLa, 4T1, MCF-7, and NIH 3T3 cells after incubation with Pro-BDP-3 (5.0 μM) for 2 h with or without further incubation with RuL2 or RuL3 (2.5 μM) for a further 4 h (red fluorescence; λ ex = 633 nm, λ em = 650–900 nm). The cells being incubated with BDP-COOH (5.0 μM) for 2 h were used as the positive control. The cell nuclei were stained with Hoechst (1.0 μM) for 15 min (blue fluorescence; λ ex = 405 nm, λ em = 420–500 nm). Scale bar = 20 μm. (b) Corresponding mean red fluorescence intensities quantified by ImageJ. Data are reported as the mean ± standard error of the mean (SEM) for three independent experiments (∗∗∗∗p < 0.0001). (c) Fluorescence confocal images of HeLa, 4T1, MCF-7, and NIH 3T3 cells after the aforementioned treatments and further incubation with H 2 DCFDA (10 μM) for 30 min, followed by light irradiation (λ > 610 nm, 25.8 mW/cm 2 ) for 8 min to give a total fluence of 12 J/cm 2 (green fluorescence; λ ex = 488 nm, λ em = 493–550 nm). Scale bar = 20 μm. (d) Corresponding mean green fluorescence intensities of DCF quantified by ImageJ. Data are reported as the mean ± SEM for three independent experiments (∗∗∗∗p < 0.0001). (e) Dark and photo (λ > 610 nm, 25.8 mW/cm 2 , 12 J/cm 2 ) cytotoxicity of BDP-COOH , Pro-BDP-3 , RuL2 , Pro-BDP-3 + RuL2 , RuL3 , and Pro-BDP-3 + RuL3 against HeLa, 4T1, MCF-7, and NIH 3T3 cells. The cells were incubated with BDP-COOH , Pro-BDP-3 , RuL2 , or RuL3 for 2 h. For Pro-BDP-3 + RuL2 and Pro-BDP-3 + RuL3 , the cells were first incubated with Pro-BDP-3 for 2 h and then with RuL2 or RuL3 (0.5 equiv.) for a further 4 h. Data are expressed as the mean ± SEM of three independent experiments, each performed in quadruplicate. (f) Photocytotoxicity of these agents at 5.0 μM and the combination treatments at 5.0 μM of Pro-BDP-3 against the four cell lines. The rightmost figure compiles the results for Pro-BDP-3 + RuL3 (∗∗∗∗p < 0.0001). Data are expressed as the mean ± SEM of three independent experiments, each performed in quadruplicate. (g) Live/dead cell viability assay using calcein-AM and PI. The cells were treated as described above, followed by incubation with calcein-AM (1 μM) and PI (2 μM) in binding buffer (2 mL) at 37 °C for 30 min. The live cells were indicated by the green fluorescence of calcein-AM (λ ex = 488 nm, λ em = 493–550 nm), while the dead cells were indicated by the red fluorescence of PI (λ ex = 561 nm, λ em = 600–800 nm). Scale bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The HeLa human cervical cancer cells (ATCC, CCL-2), 4T1 murine mammary carcinoma cells (ATCC, CRL-2539), MCF-7 human breast cancer cells (ATCC, HTB-22), and NIH 3T3 murine embryonic fibroblast cells were maintained in Dulbecco's modified Eagle's medium (DMEM, ThermoFisher, cat. no. 11965092) supplemented with fetal calf serum (10 %) and penicillin-streptomycin (100 unit/mL and 100 μg/mL, respectively).

Techniques: Fluorescence, Incubation, Positive Control, Staining, Irradiation, Viability Assay, Binding Assay

Journal: Journal of Bone Oncology

Article Title: Doxorubicin enhances adipogenesis in an FGF2-dependent manner and induces a tumour-promoting secretory phenotype

doi: 10.1016/j.jbo.2026.100754

Figure Lengend Snippet: Tumour cell proliferation is increased by culture with or media transfer from doxorubicin-treated adipogenic differentiated MSC and is dependent in part on FGF2. A) Proliferation of PC3 cells was assessed at 24 h, 48 h and 72 h following I) treatment with conditioned media from doxorubicin-treated BMA or II) direct seeding on top of doxorubicin-treated BMA. B) Proliferation of PC3 following direct co-culture with day 12 adipogenic differentiated MSC that were treated at day 9 of adipogenic differentiation with vehicle control or 25 nM or 50 nM doxorubicin. PC3 cells were enumerated at 24 h, 48 h and 72 h following addition to adipogenic differentiated MSC. Proliferation of PC3 treated with 30% conditioned media isolated at C) day 10 or D) day 12 from differentiated MSC treated at day 9 of adipogenic differentiation with vehicle control or 25 nM doxorubicin. E) Proliferation of PC3 in response to treatment with 30% conditioned media isolated at day 10 from adipogenic differentiating MSC following treatment at day 9 with 25 nM doxorubicin and siRNA targeting FGF2 or non-targeting control siRNA. F) Conditioned media was collected at day 10 from differentiating MSC/BMA cells treated with vehicle or 25 nM doxorubicin at day 9 of the differentiation process. K562 or 4T1 tumour cells were treated with 30% adipocyte conditioned media and were imaged and counted 48 h after addition of conditioned media. Mean cell count normalized to vehicle control is presented. In all graphs, mean cell count is normalized to cell count at 0 h prior to addition of conditioned media. Graphs show the mean ± SEM, using the statistical test two-way ANOVA in Figure B) and one-way ANOVA in Figure C), D) and E), (*p < 0.05, ***p < 0.001; ****p < 0.0001), n = 3 biological replicates each with 3 technical replicates.

Article Snippet: PC3 prostate tumour cells (ATCC CRL-1435), 4T1 breast tumour cells (CRL-2539) and K562 leukemia tumour cells (CCL-243) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Co-Culture Assay, Control, Isolation, Cell Characterization

Journal: Materials Today Bio

Article Title: Ultrasound-triggered carrier-free nanoprodrugs activate cGAS-STING pathway to enhance tumor-targeting chemo-immunotherapy

doi: 10.1016/j.mtbio.2026.102858

Figure Lengend Snippet: The characterizations of in vitro cellular uptake and sonodynamic effects. (A and B) Cellular uptake behaviors of Cy5-labeled PBSN38-CUR in 4T1 cells. Characterization of (C and D) intracellular ROS generation, ( E ) Ca 2+ concentration, and (F) mitochondrial function (JC-1) after various treatments. US irradiation (frequency: 3 MHz, intensity: 1.5 W cm −2 , duty cycle: 50 %, duration time: 1 min). Scale bar: 100 μm. SN38-equal: 10 μM. The scale bar of D is 50 μm. Data were expressed as mean ± standard deviation of biological replicates (n = 3). One-way analysis of variance (ANOVA) followed by the post-Tukey's multiple comparisons test was used to compare multiple groups.

Article Snippet: The 4T1 cell line (murine breast cancer cells, RRID: CVCL_0125) was purchased from the American Type Culture Collection (ATCC) and is free of mycoplasma contamination.

Techniques: In Vitro, Labeling, Concentration Assay, Irradiation, Standard Deviation

Journal: Materials Today Bio

Article Title: Ultrasound-triggered carrier-free nanoprodrugs activate cGAS-STING pathway to enhance tumor-targeting chemo-immunotherapy

doi: 10.1016/j.mtbio.2026.102858

Figure Lengend Snippet: The characterizations of in vitro sonodynamic effects and cGAS-STING pathway activation. 4T1 cells were inoculated and incubated with PBSN38-CUR for 24 h, followed by characterization of (A) cytotoxicity, (B - D) apoptosis, and DNA damage after US irradiation (frequency: 3 MHz, intensity: 1.5 W cm −2 , duty cycle: 50 %, duration time: 1 min). Scale bar: 100 μm. (E) Release behavior of cellular dsDNA after various treatments. (F) Expression of STING, TBK1, and IRF3 proteins and their corresponding phosphorylated proteins after different treatments. ( G ) Maturation characterization of DCs after various treatments. Data were expressed as mean ± standard deviation of biological replicates (n = 3). One-way analysis of variance (ANOVA) followed by the post-Tukey's multiple comparisons test was used to compare multiple groups.

Article Snippet: The 4T1 cell line (murine breast cancer cells, RRID: CVCL_0125) was purchased from the American Type Culture Collection (ATCC) and is free of mycoplasma contamination.

Techniques: In Vitro, Activation Assay, Incubation, Irradiation, Expressing, Standard Deviation

Journal: Materials Today Bio

Article Title: Ultrasound-triggered carrier-free nanoprodrugs activate cGAS-STING pathway to enhance tumor-targeting chemo-immunotherapy

doi: 10.1016/j.mtbio.2026.102858

Figure Lengend Snippet: Evaluation of in vivo tumor growth inhibition by PBSN38-CUR. (A) Schematic illustration of the therapeutic schedule in a 4T1 murine breast cancer model. (B) Tumor volume change curves of the mice following various treatments. The 4T1 tumors harvested after the therapeutic evaluation were photographed and weighed (C and D) . Scale bar: 20 mm. Data were expressed as mean ± standard deviation of biological replicates (n = 5). Tumor-bearing mice were intravenously injected with PBSN38-CUR at an SN38-equivalent dose of 4 mg kg −1 and then treated without or with US irradiation (3 MHz, 1.5 W cm −2 , 50 % duty cycle, 10 min) 4 h after intravenous injection. (E) Representative images of H&E and TUNEL staining in 4T1 tumor sections after different treatments. Scale bar: 100 μm. (F) Characterization of tumoral DC maturation and CD8 + and Foxp3 + T cell infiltration following various treatments. (G) Serum inflammatory cytokine secretion from mice after various treatments. (H) H&E staining of lung tissue collected from mice 15 days after various treatments. (I) Tumor volume change curves of the mice after PBSN38-CUR and anti-PD-L1 antibody combination treatments. The 4T1 tumors harvested at the end of the therapeutic evaluation were photographed and weighed (J and K) . Scale bar: 20 mm. Data were expressed as mean ± standard deviation of biological replicates (n = 5). One-way analysis of variance (ANOVA) followed by the post-Tukey's multiple comparisons test was used to compare multiple groups.

Article Snippet: The 4T1 cell line (murine breast cancer cells, RRID: CVCL_0125) was purchased from the American Type Culture Collection (ATCC) and is free of mycoplasma contamination.

Techniques: In Vivo, Inhibition, Standard Deviation, Injection, Irradiation, TUNEL Assay, Staining

Journal: Molecular Therapy Oncology

Article Title: Virus-based IFNγ gene delivery and photodynamic therapy cooperate to remodel the tumor microenvironment and suppress breast cancer

doi: 10.1016/j.omton.2025.201124

Figure Lengend Snippet: The effect of chlorin e6 (Ce6) photodynamic treatment on cancer cells (A) Viability assay of 4T1 cancer cells treated with different concentrations of Ce6 (0–3 μg/mL) and irradiation intensities (1–5 J/cm 2 ) ( n = 4); (B) Ce6/LED treatment (0–10 μg/mL, 5 J/cm 2 ) induced production of reactive oxygen species (ROS) measured by fluorometry of ROS-sensitive H 2 DCFDA dye ( n = 3); (C) flow cytometry analysis of Ce6 uptake, phosphatidylserine (PS) externalization detected by staining with Annexin V, and surface exposure of the immunogenic cell death marker calreticulin (CRT) after Ce6/LED treatment (3 μg/mL, 3 J/cm 2 ) in a monolayer 4T1 cell culture model ( n = 3); (D) flow cytometry analysis of Ce6 uptake, PS externalization detected by staining with Annexin V, and surface exposure of CRT after Ce6/LED treatment (0–5 μg/mL, 3 J/cm 2 ) in a 4T1 spheroid cell culture model ( n = 3). In (C) and (D), CRT exposure is shown as the percentage of CRT-positive cells. (E) GFP fluorescence intensity of 4T1/GFP spheroids following Ce6/LED treatment, measured by fluorometry. ( Left ) 4T1/GFP spheroid was irradiated twice ( n = 16); ( right ) 4T1/GFP spheroid was irradiated 7 times ( n = 12). RFU, relative fluorescence units. Data are presented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Murine mammary gland adenocarcinoma 4T1 cells (ATCC CRL-2539) were cultured in DMEM-GlutaMAX (Cat. No. 31966-021; Gibco, Life Technologies, Thermo Fischer Scientific, USA) supplemented with 10% fetal bovine serum (FBS; Cat. No. FBS-HI-12A; Capricorn Scientific) and 40 μg/mL gentamicin (Cat. No. 00-0442; Sopharma, Bulgaria).

Techniques: Viability Assay, Irradiation, Flow Cytometry, Staining, Marker, Cell Culture, Fluorescence

Journal: Molecular Therapy Oncology

Article Title: Virus-based IFNγ gene delivery and photodynamic therapy cooperate to remodel the tumor microenvironment and suppress breast cancer

doi: 10.1016/j.omton.2025.201124

Figure Lengend Snippet: Phagocytosis of Ce6/LED-treated (5 μg/mL, 3 J/cm 2 ) 4T1 cells by M0, M1, and M2 macrophages 24h after the addition of macrophages Bone marrow-derived macrophages were labeled with CellTracker Blue CMAC Dye, and 4T1 cancer cells were labeled with CellTracker Green CMFDA Dye. (A) Flow cytometry analysis showing representative staining of CMAC (macrophages) on the x axis and CMFDA (4T1 cancer cells) on the y axis. (B) Quantitative analysis. Phagocytosis was quantified by flow cytometry as the percentage of double-positive (CMAC + CMFDA + ) cells in macrophage (CMAC + ) population. Ctrl, 4T1 cells not treated with Ce6, irradiated with NIR LED. Data are presented as mean ± SD ( n = 3). ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Murine mammary gland adenocarcinoma 4T1 cells (ATCC CRL-2539) were cultured in DMEM-GlutaMAX (Cat. No. 31966-021; Gibco, Life Technologies, Thermo Fischer Scientific, USA) supplemented with 10% fetal bovine serum (FBS; Cat. No. FBS-HI-12A; Capricorn Scientific) and 40 μg/mL gentamicin (Cat. No. 00-0442; Sopharma, Bulgaria).

Techniques: Derivative Assay, Labeling, Flow Cytometry, Staining, Irradiation

Journal: Molecular Therapy Oncology

Article Title: Virus-based IFNγ gene delivery and photodynamic therapy cooperate to remodel the tumor microenvironment and suppress breast cancer

doi: 10.1016/j.omton.2025.201124

Figure Lengend Snippet: Combination of Ce6/LED and alphavirus treatment (A) ROS production in 4T1 cells after infection with SFV/Luc and SFV/IFNγ at different multiplicities of infection (MOI), 6 and 20 h after infection ( n = 3). (B) Viability assay of 4T1 cells treated with a combination of Ce6/LED (0–5 μg/mL, 5 J/cm 2 ) and SFV/RFP (MOI 5) infection in 2D conditions ( n = 3). (C) Cell Titer-Glo 3D viability tests after applying Ce6/LED (5 μg/mL, 5 J/cm 2 ) and SFV/IFNγ (5×10 5 iu) combined therapies. Strategy I: Spheroids were first treated with Ce6/LED and infected with SFV/IFNγ the next day; Strategy II: Spheroids were first infected with SFV/IFNγ and treated with Ce6/LED after 48h; Strategy III: Spheroids were infected with SFV/IFNγ and treated with Ce6/LED immediately after infection ( n = 3). (D) Confocal microscopy of spheroids: 4T1 spheroid treated with Ce6/LED (5 μg/mL, 5 J/cm 2 ); 4T1 spheroid infected with SFV/GFP (5×10 5 iu); both therapies applied together as in Strategy I. Scale bars in figures represent 100 μm. The mean fluorescence intensity for each group is illustrated as a histogram. (E) SFV/GFP or SFV/Luc infection (5×10 5 iu) efficiency of 4T1 cell spheroids after Ce6/LED treatment as in Strategy I (5 μg/mL, 5 J/cm 2 ). Total GFP fluorescence intensity of 4T1 spheroids treated with Ce6/LED and subsequently infected with virus SFV/GFP was measured by confocal microscopy ( n = 3). In a parallel experiment, luciferase activity was assessed in 4T1 spheroids treated with Ce6/LED and subsequently infected with virus SFV/Luc ( n = 3). Ctrl, untreated 4T1 cells or spheroids. RFU, relative fluorescence units. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Murine mammary gland adenocarcinoma 4T1 cells (ATCC CRL-2539) were cultured in DMEM-GlutaMAX (Cat. No. 31966-021; Gibco, Life Technologies, Thermo Fischer Scientific, USA) supplemented with 10% fetal bovine serum (FBS; Cat. No. FBS-HI-12A; Capricorn Scientific) and 40 μg/mL gentamicin (Cat. No. 00-0442; Sopharma, Bulgaria).

Techniques: Infection, Viability Assay, Confocal Microscopy, Fluorescence, Virus, Luciferase, Activity Assay

Journal: Molecular Therapy Oncology

Article Title: Virus-based IFNγ gene delivery and photodynamic therapy cooperate to remodel the tumor microenvironment and suppress breast cancer

doi: 10.1016/j.omton.2025.201124

Figure Lengend Snippet: Combined anti-tumor treatment in vivo (A) Distribution of Ce6 in mice 1.5, 4, and 18 h upon intravenous administration. Levels of Ce6 were quantified by IVIS in excised organs and using fluorometry in blood serum ( n = 3). (B) Combined anti-tumor treatment in mouse 4T1 breast cancer model. ( Top ) Experimental design of the combined therapy. Tumor-bearing mice received photodynamic therapy (PDT), consisting of intravenous Ce6 administration followed by tumor-specific irradiation with near-infrared (NIR) light 1.5 h post injection of the Ce6 in combination with intratumoral injection of SFV/Luc or SFV/IFNγ vectors at respective time points. The experiment included six groups: (1) control (PBS, PDT untreated tumors), (2) PDT, (3) SFV/Luc, (4) SFV/IFNγ, (5) PDT + SFV/Luc, and (6) PDT + SFV/IFNγ. Weight of excised tumors after treatment ( n = 6). (C) Flow cytometry analysis of tumor-infiltrating dendritic cells (DCs), natural killer cells (NKs), and T cells ( n = 6). (D) Flow cytometry analysis of tumor-infiltrating immune cell populations ( n = 6). Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Murine mammary gland adenocarcinoma 4T1 cells (ATCC CRL-2539) were cultured in DMEM-GlutaMAX (Cat. No. 31966-021; Gibco, Life Technologies, Thermo Fischer Scientific, USA) supplemented with 10% fetal bovine serum (FBS; Cat. No. FBS-HI-12A; Capricorn Scientific) and 40 μg/mL gentamicin (Cat. No. 00-0442; Sopharma, Bulgaria).

Techniques: In Vivo, Irradiation, Injection, Control, Flow Cytometry

Journal: bioRxiv

Article Title: Targeting phosphodiesterase 10A disrupts MAPK signaling pathways in the tumor microenvironment to unleash antitumor immunity

doi: 10.64898/2026.02.26.708383

Figure Lengend Snippet: (A) RAS activity measured by RAS pull-down assay. Western blot shows GTP-bound RAS in tumor cells, with signal normalized to total RAS. (B) WB analysis of phosphorylated cRaf and AKT in 4T1 cells following ADT-030 treatment. Total cRaf and AKT are shown as loading controls. (C) WB analysis of phosphorylated JNK, c-JUN, ERK1/2, and cleaved caspase-3 in 4T1 cells after ADT-030 treatment. β-actin serves as a loading control. (D) Representative flow cytometry profiles of p-AKT, p-S6, and cleaved caspase-3 in DMSO- and ADT-030–treated 4T1 cells. Mean fluorescence intensities (MFI) are summarized in bar graphs. *, P < 0.05; ***, P < 0.001. (E) Volcano plot of bulk RNA-seq analysis of 4T1 cells treated with ADT-030 or DMSO for 16h. Each dot represents one gene. Red dots indicate upregulated genes, and blue dots indicate downregulated genes. (F) Heat map of selected differentially expressed genes in ADT-030-treated versus control 4T1 cells. Genes shown include regulators of tumor growth, RAS and Wnt signaling, and immune-related pathways..

Article Snippet: Murine tumor cell lines 4T1, A20, B16F10, CT26, EL4, EG7.OVA and MC38 were purchased from ATCC.

Techniques: Activity Assay, Pull Down Assay, Western Blot, Control, Flow Cytometry, Fluorescence, RNA Sequencing

Journal: bioRxiv

Article Title: Targeting phosphodiesterase 10A disrupts MAPK signaling pathways in the tumor microenvironment to unleash antitumor immunity

doi: 10.64898/2026.02.26.708383

Figure Lengend Snippet: Pathway enrichment analysis was performed for 4T1 bulk RNA-seq. Pathway enrichment analysis was performed on differentially expressed genes from 4T1 cells treated with ADT-030 or DMSO. Bar plots show the most significantly upregulated and downregulated pathways. Gene ratio indicates the proportion of genes in each pathway. Color indicates adjusted p-values.

Article Snippet: Murine tumor cell lines 4T1, A20, B16F10, CT26, EL4, EG7.OVA and MC38 were purchased from ATCC.

Techniques: RNA Sequencing

Journal: bioRxiv

Article Title: Targeting phosphodiesterase 10A disrupts MAPK signaling pathways in the tumor microenvironment to unleash antitumor immunity

doi: 10.64898/2026.02.26.708383

Figure Lengend Snippet: The experimental timeline is shown in the schema. 4T1 cells were orthotopically implanted into the mammary fat pads of female mice, while other tumor cell lines were implanted subcutaneously into the rear flanks. When tumors became palpable (∼5 mm), mice were treated daily by oral gavage with vehicle or ADT-030 (150 mg/kg). (A) Tumor growth curves for each treatment group across tumor models. Data are shown as mean ± SEM of tumor area over time after treatment with number of mice in each group indicated. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (B) Schematic of the timeline for plasma and tissue collection for LC–MS quantification of ADT-030. (C) Plasma concentrations of ADT-030 in tumor-bearing mice. Plasma from untreated naïve mice serves as a control. Data are shown as mean ± SEM. (D) ADT-030 concentrations in the indicated organs. Tissues from untreated 4T1-bearing mice were included as controls. Data are shown as mean ± SEM.

Article Snippet: Murine tumor cell lines 4T1, A20, B16F10, CT26, EL4, EG7.OVA and MC38 were purchased from ATCC.

Techniques: Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Control

Journal: bioRxiv

Article Title: Targeting phosphodiesterase 10A disrupts MAPK signaling pathways in the tumor microenvironment to unleash antitumor immunity

doi: 10.64898/2026.02.26.708383

Figure Lengend Snippet: (A) ADT-030 prolongs survival in an experimental 4T1 metastasis model. Schematic shows the treatment timeline. In brief, mice injected with 4T1.luci cells via i.v. were treated with either vehicle or ADT-030 for 21 days, starting 3 days after tumor inoculation. Representative BLI showing tumor burden at the indicated time points is shown. Kaplan-Meier survival curves are shown in (B). (C) Schema depicting the experimental design to assess the effect of ADT-030 on tumor colonization. Mice were injected intravenously with 4T1.luci cells and treated with vehicle or ADT-030 beginning shortly after tumor inoculation. Tumor burden in live mice and in isolated lungs was quantified by BLI. (D, E) Representative BLI images of tumor signal in mice and lungs acquired 2 h after tumor inoculation (D) and after 7 days of treatment (E). Quantification of photon flux (photons/s) is shown as mean ± SEM in bar graphs.. ns, not significant; **, P < 0.01; ****, P < 0.0001.

Article Snippet: Murine tumor cell lines 4T1, A20, B16F10, CT26, EL4, EG7.OVA and MC38 were purchased from ATCC.

Techniques: Injection, Isolation

Journal: bioRxiv

Article Title: Targeting phosphodiesterase 10A disrupts MAPK signaling pathways in the tumor microenvironment to unleash antitumor immunity

doi: 10.64898/2026.02.26.708383

Figure Lengend Snippet: (A-C) Detection of ICD markers. Tumor cells were treated with DMSO or ADT-030 for 24 hours. Cell surface expression of CRT was detected by FACS, and MFI values are summarized in (A) as mean ± SEM. Extracellular ATP and HMGB1 levels from the indicated cells were measured and results shown as mean +/- SEM are summarized in (B) and (C), respectively. (D) ADT-030-treated tumor cells promote DC maturation. The schema depicts the experimental setup in which BMDCs were co-cultured with ADT-030 or DMSO pre-treated 4T1.GFP cells for 48 hrs. Uptake of the GFP signal by DCs and DC activation markers (MHC-II, CD80 and CD86) were assessed by FACS. MFIs are summarized as mean ± SEM in bar graphs. (E, F) cDC1-dependent priming of tumor-specific CD8⁺ T cells. The schema depicts the experimental procedures (E). EG7.OVA tumor cells were s.c. implanted to the flanks of WT or Batf3KO mice. Mice with palpable tumors were treated with vehicle or ADT-030. Peripheral blood was collected 10 days after treatment to detect OVA-specific CD8+ T cells by OVA-Kb tetramer. Representative dot plots are shown in (F). Numbers of the gated region demark percent of tetramer-positive cells in CD8+ T cell population. Results shown as mean +/- SEM are summarized in the bar graph. (G) ADT-030 efficacy in mice diminishes in the absence of cDC1. As depicted in the schema, EL4 or KPC cells were s.c. implanted to WT or Batf3KO mice. Mice with palpable tumors were treated daily with vehicle or ADT-030 daily till the endpoint. Tumor growth curves are shown as mean ± SEM, with the number of mice per group indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Murine tumor cell lines 4T1, A20, B16F10, CT26, EL4, EG7.OVA and MC38 were purchased from ATCC.

Techniques: Expressing, Cell Culture, Activation Assay

Journal: bioRxiv

Article Title: Targeting phosphodiesterase 10A disrupts MAPK signaling pathways in the tumor microenvironment to unleash antitumor immunity

doi: 10.64898/2026.02.26.708383

Figure Lengend Snippet: The schema depicts the workflow for scRNA-seq analysis of lung samples from the 4T1 experimental metastasis model. (A) t-SNE visualization of immune cell subsets among CD45⁺ cells in the lungs defined based on transcriptional profiles. (B) t-SNE plots showing annotated immune cell populations in lung samples across the indicated treatment conditions. The relative proportions of immune cell subsets among CD45⁺ cells are summarized in stacked bar plots. (C) Dot plots depicting signaling pathways in lung neutrophils that are differentially regulated by ADT-030 in tumor-bearing versus tumor-free mice. (D) Dot plots comparing the expression of interferon-α and interferon-γ response gene signatures in lung neutrophils from vehicle- and ADT-030-treated mice.

Article Snippet: Murine tumor cell lines 4T1, A20, B16F10, CT26, EL4, EG7.OVA and MC38 were purchased from ATCC.

Techniques: Protein-Protein interactions, Expressing

Journal: bioRxiv

Article Title: Targeting phosphodiesterase 10A disrupts MAPK signaling pathways in the tumor microenvironment to unleash antitumor immunity

doi: 10.64898/2026.02.26.708383

Figure Lengend Snippet: Heatmap showing the top 10 marker genes defining each immune cell cluster identified by scRNA-seq of CD45.1⁺ cells isolated from the lungs in the 4T1 experimental metastasis model. Columns represent individual cells and rows represent genes. Cells are grouped by cluster, with the top 10 most significant marker genes displayed for each cluster.

Article Snippet: Murine tumor cell lines 4T1, A20, B16F10, CT26, EL4, EG7.OVA and MC38 were purchased from ATCC.

Techniques: Marker, Isolation

Journal: bioRxiv

Article Title: Targeting phosphodiesterase 10A disrupts MAPK signaling pathways in the tumor microenvironment to unleash antitumor immunity

doi: 10.64898/2026.02.26.708383

Figure Lengend Snippet: (A) Schema depicting the workflow for assessing neutrophil sensitivity to ADT-030 ex vivo. Peripheral blood was collected via tail vein from mice bearing advanced 4T1 tumors (>10 mm). White blood cells were cultured and treated with DMSO or increasing concentrations of ADT-030 for 16 h, followed by assessment of apoptosis by FACS. (B) Representative dot plots showing apoptosis in myeloid cells and CD8⁺ T cells. Numbers indicate the percentage of apoptotic cells within the gated populations. (C) Quantification of apoptotic myeloid cells and CD8⁺ T cells under each culture condition, presented as mean ± SD (n = 6 replicates). (D) Schema depicting the in vivo assessment of apoptosis in circulating immune cells. Mice bearing established orthotopic 4T1 tumors were treated with vehicle or ADT-030 for 7 days. Peripheral blood was collected for apoptosis analysis by FACS. Blood from tumor-free naïve mice served as controls. (E) Representative dot plots showing apoptosis in circulating myeloid cells and CD8⁺ T cells. Numbers indicate the percentage of apoptotic cells. (F) Summary of apoptotic cell frequencies in myeloid cells and CD8⁺ T cells under the indicated treatment conditions. (G) Representative histograms showing expression of p-ERK, p-p38, p-p53, and cCas3 in circulating myeloid cells from vehicle- or ADT-030-treated mice. MFI values are summarized in bar graphs (mean ± SEM). (H) Bar graphs summarizing MFI values of the indicated markers in circulating CD8⁺ T cells. ns, not significant; . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Murine tumor cell lines 4T1, A20, B16F10, CT26, EL4, EG7.OVA and MC38 were purchased from ATCC.

Techniques: Ex Vivo, Cell Culture, In Vivo, Expressing