Journal: Biomolecules and Biomedicine

Article Title: Decitabine suppresses tumor growth by activating mouse mammary tumor virus and interferon-β pathways

doi: 10.17305/bb.2025.12852

Figure Lengend Snippet: DAC treatment, tumor growth/metastasis assessments, and MMTV expression readouts in murine tumor models. (A) Tumor growth curves for 4T1 (BALB/c) and MC38 (C57BL/6) tumors treated with decitabine (DAC) or vehicle (PBS); n ═ 5 mice per group. Individual trajectories are shown with group medians overlaid ( x -axis: Days after inoculation; y -axis: Tumor volume, mm 3 ); (B) Tumor growth rates derived as linear-regression slopes of the curves in (A). Points indicate group medians with 95% CIs; P values are annotated; (C) Final tumor mass across strains and treatments: 4T1 in BALB/c and NU/J; MC38 in C57BL/6 and NU/J. n ═ 5 per group except 4T1 in NU/J ( n ═ 9). Medians with 95% CIs; (D) Lung metastasis of 4T1 tumors quantified as log10 colony counts from lung cell cultures. n ═ 5 for BALB/c; n ═ 9 for NU/J. Medians with 95% CIs; (E) MMTV env and pol RNA in 4T1 tumors (BALB/c) measured by qRT-PCR and displayed as log10 relative quantity (PBS n ═ 6; DAC n ═ 5); (F) MMTV env and pol RNA in MC38 tumors (C57BL/6) measured by qRT-PCR ( n ═ 5 per group); (G) Immunoblot of MMTV Env in 4T1 tumors: Lanes 1–5 PBS; lanes 6–10 DAC. Bands near ∼80 kDa and ∼45 kDa are shown; GAPDH (37 kDa) is the loading control. Bar plot summarizes band intensities per lane; (H) Scatter plot of tumor mass versus Env band intensity for the 10 4T1 tumors in (G); five PBS and five DAC samples. Spearman correlation statistics are annotated in the panel. Abbreviations: DAC: Decitabine (5-aza-2ʹ-deoxycytidine); PBS: Phosphate-buffered saline; MMTV: Mousemammary tumor virus; env/pol: MMTV genes; qRT-PCR: Quantitative reverse-transcription PCR; kDa: Kilodalton; CI: Confidence interval; BALB/c = B/c; C57BL/6 = B/6; NU/J: Nude mice; n: Number of mice.

Article Snippet: The 4T1 murine mammary cancer cell line (American Type Culture Collection; STR authenticated) was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Western Blot, Control, Saline, Virus, Reverse Transcription

Journal: Biomolecules and Biomedicine

Article Title: Decitabine suppresses tumor growth by activating mouse mammary tumor virus and interferon-β pathways

doi: 10.17305/bb.2025.12852

Figure Lengend Snippet: MMTV knockdown reduces sensitivity of 4T1 cells and tumors to DAC treatment. (A) Effect of MMTV knockdown and DAC treatment on proliferation of 4T1 cells. Cell viability was determined by NucleoCounter analysis after DAC exposure; (B) Effect of MMTV knockdown on DAC-mediated growth inhibition of 4T1 cells in vitro . Percent inhibition was calculated relative to untreated controls; (C) Impact of MMTV knockdown and DAC on tumor growth kinetics of 4T1 tumors in BALB/c mice. Tumor volumes were monitored over time following inoculation and treatment with PBS or DAC; (D) Growth rate analysis of 4T1 tumors in BALB/c mice after MMTV knockdown and DAC treatment. Pairwise comparisons were performed between PBS- and DAC-treated groups, as well as between control and knockdown tumors; (E) Quantification of MMTV env transcript levels in control and knockdown tumors with or without DAC treatment. Relative expression was determined by qRT-PCR; (F) Quantification of MMTV pol transcript levels in control and knockdown tumors with or without DAC treatment. Relative expression was determined by qRT-PCR. Values represent group medians with 95% confidence intervals. n ═ 4 for in vitr o assays (A and B); n ═ 9 for control tumors, n ═ 10 for KD1, and n ═ 5 for KD3 in vivo experiments (C and D); n ═ 4–6 for transcript quantification (E and F). Abbreviations: MMTV: Mouse mammary tumor virus; DAC: Decitabine; PBS: Phosphate-buffered saline; KD: Knockdown; qRT-PCR: Quantitative reverse transcription polymerase chain reaction.

Article Snippet: The 4T1 murine mammary cancer cell line (American Type Culture Collection; STR authenticated) was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS).

Techniques: Knockdown, Inhibition, In Vitro, Control, Expressing, Quantitative RT-PCR, In Vivo, Virus, Saline, Reverse Transcription, Polymerase Chain Reaction

Journal: Biomolecules and Biomedicine

Article Title: Decitabine suppresses tumor growth by activating mouse mammary tumor virus and interferon-β pathways

doi: 10.17305/bb.2025.12852

Figure Lengend Snippet: IFN-β mediates the antitumor effect of DAC and reciprocally regulates MMTV expression. (A) DAC induces transcriptional upregulation of Ifnb1 in MC38 cells. Expression was quantified by qRT-PCR; (B) DAC treatment increases IFN-β and IRF7 protein levels in 4T1 tumors. BALB/c mice were treated daily for 5 days and every other day thereafter. Protein abundance was analyzed by immunoblotting. Normalized band intensities are shown below the blots; (C) MMTV knockdown reduces IFN-β protein expression in 4T1 cells. Western blot analysis of KD1–KD3 knockdown lines compared with control; (D) Knockdown of Ifnb1 increases MMTV Env expression in 4T1 cells. Western blot showing reciprocal regulation of IFN-β and MMTV Env; (E) Effect of IFN-α on the proliferation of Ifnb1 knockdown cell lines; (F) Effect of Ifnb1 knockdown and DAC treatment on 4T1 cell proliferation. Viable cell counts were determined by NucleoCounter; (G) Ifnb1 knockdown reduces sensitivity of 4T1 cells to DAC in vitro . Percent inhibition was calculated relative to untreated controls; (H) Tumor growth kinetics of control and Ifnb1 knockdown 4T1 tumors in BALB/c mice treated with PBS or DAC. Tumor volumes were measured longitudinally after inoculation; (I) Growth rate analysis of 4T1 tumors following Ifnb1 knockdown and DAC treatment. Pairwise comparisons included untreated and DAC-treated groups, as well as untreated tumors from control vs KD1. Values represent medians with 95% confidence intervals. n ═ 4 for in vitro proliferation assays (A, E–G); n ═ 6–7 for control and KD1, and n ═ 10 for KD2 in vivo studies (H and I). Abbreviations: DAC: Decitabine; IFN: Interferon; IFN-β: Interferon beta; IFN-α: Interferon alpha; IRF7: Interferon regulatory factor 7; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; MMTV: Mouse mammary tumor virus; Env: Envelope protein; KD: Knockdown; PBS: Phosphate-buffered saline; qRT-PCR: Quantitative reverse transcription polymerase chain reaction.

Article Snippet: The 4T1 murine mammary cancer cell line (American Type Culture Collection; STR authenticated) was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS).

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Western Blot, Knockdown, Control, In Vitro, Inhibition, In Vivo, Virus, Saline, Reverse Transcription, Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism

doi: 10.1038/s41467-025-56637-9

Figure Lengend Snippet: a Schematic representation of the macrophage loading with HFt and subsequent transfer to cancer cells. Created in BioRender. Taciak, B. (2024) https://BioRender.com/s50b255 . b Snapshots from the movie recorded under the confocal microscopy (Supplementary Video ) in 15 min where BMDM macrophages loaded with HFt-FITC transfer it to EMT6 cancer cell labeled with red CellTrace. c Representative flow cytometry scatter plot of macrophages with internalized HFt-AF488 and MDA-MB-231 breast cancer cells labeled with CellTrace (CellTrace Far Red—APC). d and ( e ) Flow cytometry quantification of HFt-AF488 transfer from hMDM and (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to various human cancer cell lines at 24 h following co-culture at 2:1 ratio (macrophages: cancer cells). Data are presented as mean ± SEM from n = 3 different donors (hMDM). f Representative confocal microscopy images of HFt-AF488 (green) transfer from hMDM (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to MDA‑MB‑231 breast cancer cells (blue) after 24 h of co-culture. Scale bar = 10 µm. Wheat Germ Agglutinin (WGA), Alexa Fluor-555 Conjugate (red) was used to visualize the cell membrane. g Comparative analysis of HFt-AF488 transfer from hMDM of different polarization states—M0, M1 (stimulated with LPS, 100 ng/ml), and M2 (stimulated with IL-4, 20 ng/ml)—to MDA-MB-231 breast cancer cells. hMDM were pre-loaded with HFt-AF488 (500 μg/ml) for 1 h at 37 °C, followed by co-culture with MDA-MB-231 cells for 4 and 24 h. Control conditions included co-cultures of M0, M1, and M2 hMDM with MDA-MB-231 cells without HFt-AF488. Data are presented as mean ± SEM from n = 3 different donors (hMDM). h and ( i ) Flow cytometry quantification of HFt-AF488 transfer from iPSC-derived macrophages (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to various human cancer cell lines at 24 h following co-culture at 2:1 ratio (macrophages: cancer cells). Data are presented as mean ± SEM from n = 3 (A549, SK-OV-3) or n = 4 (DU145, MDA-MB-231, SW1353) independent replicates. j Representative confocal microscopy images of HFt-AF488 (green) transfer from iPSC-derived macrophages (loaded with HFt-AF-647 at concentration 500 µg/ml for 1 h at 37 °C) to MDA‑MB‑231 breast cancer cells (blue) after 24 h of co-culture. Scale bar = 10 µm. Wheat Germ Agglutinin (WGA), Alexa Fluor-555 Conjugate (red) was used to visualize the cell membrane. k Flow cytometry analysis of Tfn-AF647 transfer from RAW 264.7 and THP-1 macrophages (loaded with Tfn-AF-647 at concentration 0.2 mg/ml for 1 h at 37 °C) to cancer cells (EMT6 and MDA-MB-231, respectively) after 24 h of co-culture. Data are presented as mean ± SEM from n = 3 independent replicates. l Flow cytometry analysis of HFt-AF488 transfer from THP1-derived macrophages (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to various cancer cell lines after 4 h co-culture. Data are presented as mean ± SEM from n = 3 independent replicates. m Correlation of the percentage of HFt-AF488-positive recipient cells from human cell lines shown in ( l ) to their CD71 (TfR1) receptor expression assessed by flow cytometry. n Western blot analysis showing TfR1 gene knockdown efficiency in MDA-MB-231 cells using two different siRNA sequences and scramble as a control. o–q Flow cytometry analysis of AF488 fluorescence in MDA-MB-231 cancer cells with TfR1 gene knockdown using two different siRNA sequences, cells transfected with negative control (Scramble) siRNA or untreated cells (Control). Effect on ( o ) Tfn-AF488 and ( p ) HFt-AF488 uptake from medium and ( q ) HFt-AF488 transfer from THP-1 macrophages in 4 h co-culture was calculated relative to Scramble. Data are presented as mean ± SEM from n = 3 independent replicates. r and ( s ) Flow cytometry quantification of HFt-AF488 transfer from RAW 264.7 or THP-1 macrophages (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to ( r ) EMT6 or ( s ) MDA-MB-231 breast cancer cells (respectively) at 24 h following direct, or Transwell membrane-separated co-culture system (macrophages seeded on Transwell insets). Co-culture of macrophages without HFt-AF488 and cancer cells [co-culture HFt(−)] was used as a control. Data are presented as mean ± SEM from n = 3 independent replicates. Statistical analysis was performed using one-way ANOVA with post-hoc Tukey HSD test. For all panels, * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: Human leukemia monocytic cell line THP-1 (ATCC TIB-202), human breast cancer cell line MDA-MB-231 (ATCC HTB-26), human colon cancer cell line LoVo (ATCC CCL-229), human embryonic kidney cell line HEK-293 (ATCC CRL-1573), Chinese hamster ovary cell line CHO-K1 (ATCC CCL-61), human glioblastoma cell line U-87 MG (ATCC HTB-14), murine macrophage cell line RAW 264.7 (ATCC TIB-71), murine mammary cancer cell lines EMT6 (ATCC CRL-2755), EMT6-Fluc-Puro (Imanis, CL154), and 4T1 (ATTC CRL-2539) were obtained from the American Type Culture Collection (ATCC; www.atcc.org ).

Techniques: Confocal Microscopy, Labeling, Flow Cytometry, Concentration Assay, Co-Culture Assay, Membrane, Control, Derivative Assay, Expressing, Western Blot, Knockdown, Fluorescence, Transfection, Negative Control

Journal: Nature Communications

Article Title: Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism

doi: 10.1038/s41467-025-56637-9

Figure Lengend Snippet: a Representative confocal microscopy images captured after 24 h co-culture of donor hMDM labeled with CellVue Claret Far Red dye (red) and loaded with HFt-AF488 (green) and MDA-MB-231 cancer cells labeled with CellTrace Violet dye (blue). Cells fixed after 24 h were additionally stained with anti-CD63 antibody (yellow). Co-localization of AF488, CellVue, and CD63 signals both in macrophages and in cancer cells are pointed out by the arrowheads. Scale bar = 20 µM (5 µm in the zoomed region) . b and ( c ) Comparison of HFt-AF488 signal in MDA-MB-231 cancer cells when co-cultured with THP-1 macrophages for 24 h or incubated with conditioned-media or microvesicles (MV) isolated from THP-1 carrying HFt-AF488 identical to the cells used in co-culture (equivalent to the number of macrophages used in co-culture (150k) or concentrated (5 µg)). Data are presented as mean ± SEM from n = 3 independent replicates. d Typical MV markers found in MVs isolated from THP-1 macrophages by western blot analysis. e–g Flow cytometry analysis of AF488 fluorescence in EMT6 cancer cells following 4 h co-culture with HFt-AF488-loaded RAW 264.7 macrophages in the presence of ( e ) DMA, ( f ) Piceatannol, ( g ) Wortmannin or respective vehicle (Control). Data presented as mean ± SEM relative to control from n = 3 ( f ) or 4 ( e , g ) independent replicates. h and ( i ) Flow cytometry analysis of AF488 fluorescence in ( h ) MDA-MB-231 and ( i ) EMT6 cancer cells with CLTC gene knockdown using two different siRNA sequences relative to fluorescence in cells transfected with negative control (Scramble) siRNA after 4 h co-culture with THP-1 or RAW 264.7 macrophages, respectively, loaded with HFt-AF488. Data are presented as mean ± SEM from n = 3 independent replicates. j and ( k ) Knockdown efficiency in ( j ) MDA-MB-231 and ( k ) EMT6 cancer cells was confirmed with western blot analysis. l Representative ImageStream images and ( m ) phalloidin-AF555 fluorescence quantification analysis showing F-actin accumulation at the cell-cell contact site following 2 h co-culture of hMDM and MDA-MB-231 cancer cells (from images recorded by imaging flow cytometry, data are presented as mean ± SEM from n = 3 independent replicates). The one-way ANOVA and Tukey HSD post-hoc tests were used for statistical analysis. Schematic illustration of analyzed image areas created in BioRender. Taciak, B. (2024) https://BioRender.com/r16l469 . Scale bar = 7 µm. n and ( o ) Flow cytometry quantification of AF488 fluorescence in EMT6 cells following 4 h co-culture with RAW 264.7 macrophages in the presence of actin polymerization inhibitors ( n ) Latrunculin B and ( o ) Cytochalasin D or vehicle (Control). Data are presented as mean ± SEM from n = 3 independent replicates. p and ( q ) Representative histograms showing CD11b ( p ) expression in hMDM but not MDA-MB-231 cells and ICAM-1 ( q ) expression in MDA-MB-231 and HMDM cells. Representative ImageStream images showing ( r ) ICAM-1 but not ( s ) CD11b accumulation at the cell-cell contact site following 4 h co-culture of hMDM and MDA-MB-231 cancer cells and fluorescence quantification ( t and u ) (from images recorded by imaging flow cytometry, data are presented as mean ± SEM from n = 4 ( t ) and n = 3 ( u ) independent replicates). The one-way ANOVA and Tukey HSD post-hoc test were used for statistical analysis. Scale bar = 7 µm. v and ( w ) Western blot analysis showing ICAM-1 expression in hMDM ( v ) or MDA-MB-231 cells ( w ) either untransfected named as control or transfected with one of the following siRNA sequences: scramble siRNA (siScr), no. 1 siRNA targeting ICAM-1 at 72 h after transfection. x and ( y ) Flow cytometry quantification of HFt-AF488 transfer from hMDM loaded with HFt-AF488 to MDA-MB-231 cancer cells after ICAM-1 gene knockdown in MDA-MB-231 ( x ) or hMDM ( y ) cells using siRNA (siICAM-1) after 48 h coculture. For comparison, cells treated with a negative scramble control siRNA (Scramble) were used. z Flow cytometry quantification of HFt-AF488 transfer from hMDM pre-incubated with ICAM-1 blocking antibodies and co-cultured with MDA-MB-231 cancer cells (anti-ICAM-1 blocking antibodies were used during the co-culture) for 4 h. x–z Data are presented as mean ± SEM from n = 3 biologically independent replicates. An unpaired t -test was used for statistical analysis in panels ( n , o , x ), a paired t -test in panel ( y ), and one-way ANOVA with Dunnett’s post-hoc test in panel ( z ). For all panels, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: Human leukemia monocytic cell line THP-1 (ATCC TIB-202), human breast cancer cell line MDA-MB-231 (ATCC HTB-26), human colon cancer cell line LoVo (ATCC CCL-229), human embryonic kidney cell line HEK-293 (ATCC CRL-1573), Chinese hamster ovary cell line CHO-K1 (ATCC CCL-61), human glioblastoma cell line U-87 MG (ATCC HTB-14), murine macrophage cell line RAW 264.7 (ATCC TIB-71), murine mammary cancer cell lines EMT6 (ATCC CRL-2755), EMT6-Fluc-Puro (Imanis, CL154), and 4T1 (ATTC CRL-2539) were obtained from the American Type Culture Collection (ATCC; www.atcc.org ).

Techniques: Confocal Microscopy, Co-Culture Assay, Labeling, Staining, Comparison, Cell Culture, Incubation, Isolation, Western Blot, Flow Cytometry, Fluorescence, Control, Knockdown, Transfection, Negative Control, Imaging, Expressing, Blocking Assay

Journal: Nature Communications

Article Title: Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism

doi: 10.1038/s41467-025-56637-9

Figure Lengend Snippet: a Schematic representation of the treatment schedule employed in the in vivo mouse EMT6 breast cancer metastasis to the lungs study: vehicle control group (PBS control), standard of care group (Doxorubicin) and treatment group (autologous MDC-735) administered intravenously. Created in BioRender. Taciak, B. (2024) https://BioRender.com/m50e375 . b Mean bioluminescence curve of mice treated with MDC-735 therapy compared to the control groups. c Effect of mouse MDC-735 and doxorubicin on lung colonization in an EMT6 breast cancer mouse model. d Effect of MDC-735 (generated from BMDM) and doxorubicin on lung weight in EMT6 breast cancer mouse model. b–d Data are presented as mean ± SEM from n = 7 mice in MDC-735 and PBS, n = 8 in Doxorubicin group. d Additionally n = 3 Naïve mice were used as a control. e Schematic representation of the treatment schedule employed in the in vivo EMT6 breast metastasis to the lungs study: vehicle control group (PBS control) and treatment groups (allogeneic MDC-735 loaded with HFt-735 at 0.25 mg/ml and 0.50 mg/ml). Created in BioRender. Taciak, B. (2024) https://BioRender.com/p63h677 . f Mean bioluminescence curve of mice treated with MDC-735 therapy compared to the control group. g Effect of allogeneic mouse MDC-735 on lung colonization in an EMT6 breast cancer mouse model. f and ( g ) Data are presented as mean ± SEM from n = 17 mice in PBS control group and n = 15 in each of the MDC-735 group. h Schematic representation of the treatment schedule employed in the in vivo mouse MB49 bladder cancer study: vehicle control group, isotype control group, Anti-PD-1 antibody, treatment group (allogeneic MDC-735) and combination group (MDC-735 + Anti-PD-1 antibody). Created in BioRender. Taciak, B. (2024) https://BioRender.com/z27l686 . i Kaplan–Meier survival curve of mice treated with MDC-735 therapy compared to the control groups. j The statistical significance of the survival was analyzed using the pairwise log-rank tests. k Effect of MDC-735 and Anti-PD-1 on tumor volume in an MB49 bladder cancer mouse model. Data are presented as mean ± SEM. l Tumor volume progression over time with different treatments of MB49 bladder cancer mouse model. k and ( l ) n = 10 mice per group. m Schematic representation of the treatment schedule employed in the in vivo mouse SCC7 squamous cell carcinoma study: vehicle control group, isotype control group, Anti-PD-1 antibody, treatment group (allogeneic MDC-735), and combination group (MDC-735 + Anti-PD-1 antibody). Created in BioRender. Taciak, B. (2024) https://BioRender.com/m43v541 . n Kaplan–Meier survival curve of mice treated with MDC-735 therapy compared to the control groups. o The statistical significance of the survival was analyzed using the pairwise log-rank tests. p Effect of MDC-735 and Anti-PD-1 on tumor volume in a SCC7 squamous cell carcinoma mouse model. Data are presented as mean ± SEM. q Tumor volume progression over time with different treatments of SCC7 squamous cell carcinoma mouse model. p and ( q ) n = 9 mice in Vehicle + Isotype control and Anti-PD-1 groups and n = 10 in MDC-735 and MDC-735 + Anti-PD-1 groups. The one-way ANOVA with Tukey’s post-hoc test was used for statistical analysis in ( c , d , g ), Welch’s t -test with Bonferroni multiple comparison correction in ( b , f , p ), one-way ANOVA with Dunnett’s post-hoc test in ( k ). For all panels * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Source data are provided as a Source Data file.

Article Snippet: Human leukemia monocytic cell line THP-1 (ATCC TIB-202), human breast cancer cell line MDA-MB-231 (ATCC HTB-26), human colon cancer cell line LoVo (ATCC CCL-229), human embryonic kidney cell line HEK-293 (ATCC CRL-1573), Chinese hamster ovary cell line CHO-K1 (ATCC CCL-61), human glioblastoma cell line U-87 MG (ATCC HTB-14), murine macrophage cell line RAW 264.7 (ATCC TIB-71), murine mammary cancer cell lines EMT6 (ATCC CRL-2755), EMT6-Fluc-Puro (Imanis, CL154), and 4T1 (ATTC CRL-2539) were obtained from the American Type Culture Collection (ATCC; www.atcc.org ).

Techniques: In Vivo, Control, Generated, Comparison

Journal: Nature Communications

Article Title: Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism

doi: 10.1038/s41467-025-56637-9

Figure Lengend Snippet: a Histopathological examination of different types of tissue stained with Hematoxylin and Eosin (H&E) in mice with breast metastasis to the lungs (EMT6) treated with allogeneic mouse MDC-735: vehicle control group (PBS control) and treatment groups (MDC-735 loaded with HFt-735 at 0.25 mg/ml and 0.50 mg/ml). Representative images of n = 4 samples in PBS and n = 3 samples per MDC-735 group. Scale bar = 20 µm. b Histopathological examination of different types of tissue stained with Hematoxylin and Eosin (H&E) in mice dosed subcutaneously with MDC-735 ( n = 3). 28 days later animals were euthanized, and liver, lung, spleen, and skin tissue resected and processed to FFPE, 4 µM sections were sectioned and H&E stained. Scale bar = 300 µm (8x), 350 µm (10x) or 250 µm (20x). c and ( d ) Comparison of normal cells vs. cancer cell line viability in co-culture with MDC-735. Bar plots show the viability of various types of normal cells and the EMT6 cancer cell ( c ) and the LN-229 cancer cell line ( d ) after 48-h co-culture with MDC-735. Viability was measured as the number of live cells relative to the control condition (no macrophages). Data represents the mean ± SEM from n = 3 independent replicates. Statistical analysis was performed using one-way ANOVA and post hoc Tukey HSD test; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.001. Source data are provided as a Source Data file.

Article Snippet: Human leukemia monocytic cell line THP-1 (ATCC TIB-202), human breast cancer cell line MDA-MB-231 (ATCC HTB-26), human colon cancer cell line LoVo (ATCC CCL-229), human embryonic kidney cell line HEK-293 (ATCC CRL-1573), Chinese hamster ovary cell line CHO-K1 (ATCC CCL-61), human glioblastoma cell line U-87 MG (ATCC HTB-14), murine macrophage cell line RAW 264.7 (ATCC TIB-71), murine mammary cancer cell lines EMT6 (ATCC CRL-2755), EMT6-Fluc-Puro (Imanis, CL154), and 4T1 (ATTC CRL-2539) were obtained from the American Type Culture Collection (ATCC; www.atcc.org ).

Techniques: Staining, Control, Comparison, Co-Culture Assay