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: 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: iScience

Article Title: DACIT device for axon cancer cell interaction testing in 2D and 3D

doi: 10.1016/j.isci.2025.114557

Figure Lengend Snippet: Time-lapse recording of 4T1 cancer cells spreading in the presence of axons, low-speed recording (4 images/hour, 20 h). Scale bars, 50 μm.

Article Snippet: Authenticated, mycoplasma-free 4T1 cells were obtained from ATCC.

Techniques:

Journal: iScience

Article Title: DACIT device for axon cancer cell interaction testing in 2D and 3D

doi: 10.1016/j.isci.2025.114557

Figure Lengend Snippet: Two-dimensional (2D) assays probing neuronal or cancer cell behavior, done in DACIT (A) Stills from a time-lapse recording of GCaMP6f-expressing adult DRG neurons plated in the neuronal compartment of DACIT. Scale bars, 50 μm. (B) GCaMP6f signal over time, as measured in neuronal soma without additional stimulation (gray background) and after stimulation with capsaicin (Cap, 1 μM, orange background) and KCl (50 mM, blue background). The violin plot (right) shows the mean and quartiles of the relative number of responsive sensory neurons (Δ F / F 0 > 3) from 10 fields of view in two independent experiments, done with different primary cultures. Statistical analysis was performed using the Mann-Whitney test; ∗ p < 0.05. (C) Standard experimental timeline of 2D assays in DACIT. Cancer cells were plated once axons populated the axonal compartment. (D) 4T1 breast cancer cells (magenta, F-actin; green, E-cadherin) plated on 2D gelatin interact with axons (white, anti-PGP9.5) in the axonal compartment of DACIT. Scale bars, 25 μm. (E) Schematic of the gelatin degradation assay. Cancer cells (magenta) are plated on a fluorescent gelatin layer (green). Invadopodia assembly and maturation is followed by local degradation of the gelatin, visible as black spots in a fluorescent gelatin layer. (F) Gelatin degradation puncta (black) in human breast cancer BT-549 cells (magenta, F-actin) cultured in the axonal compartment of DACIT. The yellow dashed box outlines the zoomed-in area (right). F-actin-positive invadopodia (yellow arrows, upper) colocalize with gelatin degradation puncta (magenta arrows, lower). Scale bars, 25 μm. (G) Degradation areas per cell are similar in DACIT and in 48-well plate. Measurements were done in breast cancer cells MDA-MB-231. Data from 30 fields of view per condition are shown. Two experiments were analyzed for DACIT and three experiments for 48-well plate. Statistical test: Mann-Whitney test shows no significance, ns.

Article Snippet: Authenticated, mycoplasma-free 4T1 cells were obtained from ATCC.

Techniques: Expressing, MANN-WHITNEY, Degradation Assay, Cell Culture

Journal: iScience

Article Title: DACIT device for axon cancer cell interaction testing in 2D and 3D

doi: 10.1016/j.isci.2025.114557

Figure Lengend Snippet: 3D Spheroid invasion assay in DACIT (A) Experimental timeline of 3D spheroid invasion assays in DACIT. Tumor spheroids were added after axons reached the axonal compartment. (B) Representative images of 4T1 spheroids embedded in a collagen: Matrigel mix. Spheroid invasion was monitored in the presence of DMSO (control) or GM6001 (25 μM) at 2 h post-embedding (Day 0), 24 h (Day 1), and 48 h (Day 2). Scale bars, 200 μm. Graphs show the mean ± SEM relative spheroid area (C) and minimum-to-maximum circularity (D) of nine spheroids per treatment from two biologically independent experiments. Statistical differences (∗ p < 0.05 compared with control) were determined using an unpaired, two-tailed Mann-Whitney test. (E) Top (left) and side (right) views of tumor spheroids (4T1-mScarlet, magenta) embedded in a 3D ECM mix of collagen: Matrigel (blue) in the presence of neurons (yellow), fixed 24 h after invasion. Scale bars, 200 μm. (F) Brightfield image of a spheroid embedded in 3D ECM, 24 h post-embedding. Microgrooves are visible on the left side of the image. Scale bars, 200 μm. (G) Maximum projection of invasive strands from a 4T1-mScarlet 3D spheroid (magenta), 24 h post-embedding, interacting with axons (yellow, PGP9.5). Scale bars, 100 μm. (H) 3D Imaris reconstruction and (I) single z-slice of an invasive strand in the spheroid (magenta) interacting with axons (yellow). Scale bars, 50 μm.

Article Snippet: Authenticated, mycoplasma-free 4T1 cells were obtained from ATCC.

Techniques: Invasion Assay, Control, Two Tailed Test, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: Targeting ENO1 reprograms macrophage polarization to trigger antitumor immunity and improves the therapeutic effect of radiotherapy

doi: 10.1038/s41419-026-08416-7

Figure Lengend Snippet: A A total of 2.5 × 10 5 CT26 cells were subcutaneously injected into BALB/c mice for 11 days and then intraperitoneally administered anti-ENO1 antibodies (HuL001, 20 mg/kg) five times at three-day intervals ( n = 5). The tumor volume was recorded every three days. * p < 0.05. Two-Way ANOVA test ( n = 5). B The resected tumors from the CT26 tumor-bearing mice were analyzed by immunohistochemical analysis. ** p < 0.01. Unpaired t test ( n = 5). C A total of 2.5 × 10 5 CT26 cells were intravenously injected into BALB/c mice for 11 days and then intraperitoneally administered anti-ENO1 antibodies (HuL001, 40 mg/kg) five times at three-day intervals ( n = 5). The tumor volume was recorded every three days. The resected lung tissues were analyzed by HE staining. *** p < 0.001. Unpaired t test ( n = 5). D 4T1 cells (5 × 105) were intravenously injected into BALB/c mice, which were then intraperitoneally administered anti-ENO1 antibodies (HuL001, 40 mg/kg) on the indicated days (n = 5). The tumor volume was recorded every three days. * p < 0.05. Two-Way ANOVA test ( n = 5). E The resected tumors from 4T1-bearing mice were analyzed by HE staining. ** p < 0.01. Unpaired t test ( n = 5). F The resected lung tissues from 4T1-bearing mice were analyzed by HE staining, immunofluorescence staining and immunohistochemical analysis. ** p < 0.01 and *** p < 0.001. Unpaired t test ( n = 5). G The resected liver tissues from 4T1-bearing mice were analyzed by HE staining, immunofluorescence staining and immunohistochemical analysis. *** p < 0.001. Unpaired t test ( n = 5). H The density of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors from CT26- and 4T1-bearing mice was evaluated by immunofluorescence staining ( n = 3). I The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors from CT26-bearing mice. * p < 0.05 and *** p < 0.001. Unpaired t test ( n = 5). J The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors from 4T1-bearing mice. * p < 0.05 and *** p < 0.001. Unpaired t test ( n = 5). K The DEGs between HuL001- and Veh-treated resected CT26 tumors are shown in a volcano plot ( n = 2). L Compared with those in Veh.-treated resected tumors, the number of glycolysis-related gene sets significantly decreased in HuL001-treated resected tumors ( n = 2).

Article Snippet: The mouse colon cancer cell line CT26, mouse breast cancer cell line 4T1, human CRC cell lines (SW480, SW620, HCT116, LoVo and HT29) and human breast cancer cell lines (MDA-MB-468, BT-20, HS578T and SUM-159) were obtained from the ATCC.

Techniques: Injection, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Targeting ENO1 reprograms macrophage polarization to trigger antitumor immunity and improves the therapeutic effect of radiotherapy

doi: 10.1038/s41419-026-08416-7

Figure Lengend Snippet: A A total of 5 × 10 5 CT26 cells were subcutaneously injected into the left legs of BALB/c mice for 5 days and then intraperitoneally administered with anti-ENO1 antibodies (HuL001, 40 mg/kg) or clodronate liposomes (50 μL/mouse) on the indicated days ( n = 5). Local radiotherapy was given on Day 10. The tumor volume was recorded every three days. * p < 0.05. Two-Way ANOVA test ( n = 4). B The densities of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors were analyzed by immunofluorescence staining. The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors. * p < 0.05 and *** p < 0.001. One-Way ANOVA test ( n = 3). C 4T1 cells (5 ×10 4 ) were subcutaneously injected into the left legs of BALB/c mice for 4 days and then intraperitoneally administered anti-ENO1 antibodies (HuL001, 20 mg/kg) six times on the indicated days ( n = 5). Local radiotherapy was given on Days 10 and 12. The tumor volume was recorded every three days. * p < 0.05 and *** p < 0.001. Two-Way ANOVA test ( n = 4). D The resected tumors were weighed on Day 40. * p < 0.05. One-Way ANOVA test ( n = 4). E The densities of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors were analyzed by immunofluorescence staining. F The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors. * p < 0.05. One-Way ANOVA test ( n = 3). G The frequencies of M1 (CD11c + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) and M2 (CD1206 + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) tumor-infiltrating macrophages were analyzed by flow cytometry. * p < 0.05. One-Way ANOVA test ( n = 3-4). H The quantification of the M1/M2 ratio is shown. * p < 0.05. One-Way ANOVA test ( n = 3). I A representative image of flow cytometric analysis of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells. J The frequency of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells was analyzed by flow cytometry. * p < 0.05. One-Way ANOVA test ( n = 3-4). K The density of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells is shown. * p < 0.05. One-Way ANOVA test ( n = 3-4). L The density of IFNγ + (IFNγ hi CD8 + CD3 + CD45 + 7AAD - ) T cells is shown ( n = 3-4). * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3-4). M The proposed mechanism of TGFβ1/TGFβR/Smad3/PRMT5-mediated ENO1 translocation for lactate release via MCT4.

Article Snippet: The mouse colon cancer cell line CT26, mouse breast cancer cell line 4T1, human CRC cell lines (SW480, SW620, HCT116, LoVo and HT29) and human breast cancer cell lines (MDA-MB-468, BT-20, HS578T and SUM-159) were obtained from the ATCC.

Techniques: Injection, Liposomes, Immunofluorescence, Staining, Flow Cytometry, Translocation Assay