Journal: Journal of Translational Medicine

Article Title: Hypoxic migrasomes drive colorectal cancer liver metastasis by mediating CD5L + macrophage efferocytosis via NRP2/PROX1 axis

doi: 10.1186/s12967-025-07485-0

Figure Lengend Snippet: Phagocytic activity and immunofluorescence validation of CD5L⁺ macrophages in CRC liver metastases following migrasome treatment. ( A ) UMAP blot showing the expression of migrasome marker TSPAN4 in myeloid subsets. ( B ) Boxplot showing efferocytosis scores across the 10 identified myeloid cell subtypes. ( C ) Violin plots depicting the expression of efferocytosis markers CD300B, MERTK, and CD300D across 10 distinct myeloid cell subtypes. ( D ) UMAP plots displaying the expression patterns of three efferocytosis-associated marker genes specifically enriched in CD5L⁺ macrophages. ( E ) Immunofluorescence staining of tumor tissues from MC38-tumor bearing mice showing colocalization of CD163, CD5L, and the migrasome marker MERTK in both treatment groups. Increased MERTK expression is observed in the hypoxic group, indicating enhanced migrasome targeting of CD5L⁺ macrophages

Article Snippet: Samples were incubated with primary antibodies, CD5L (1:500, 17224-1-AP, Proteintech), CD163 (1:200, ab182422, Abcam), NRP2 (1:250, #3366, CST), PROX1(1:250, sc-81983, Santa cruz), MERTK (200 μg/mL, sc-365499, Santa Cruz).

Techniques: Activity Assay, Immunofluorescence, Biomarker Discovery, Expressing, Marker, Staining

Journal: Journal of Translational Medicine

Article Title: Hypoxic migrasomes drive colorectal cancer liver metastasis by mediating CD5L + macrophage efferocytosis via NRP2/PROX1 axis

doi: 10.1186/s12967-025-07485-0

Figure Lengend Snippet: Migrasomal NRP2 is required for CRC-induced CD5L⁺ macrophage differentiation and efferocytosis. ( A ) RT-qPCR analysis confirming efficient knockdown of NRP2 in MC38 cells under hypoxia. ( B ) Western blot analysis confirming efficient knockdown of NRP2 in MC38 cells under hypoxia. ( C ) Flow cytometry analysis of CD5L⁺ macrophage proportion after treatment with control or NRP2-deficient hypoxic migrasomes from MC38 cells. ( D ) Immunofluorescence assay of efferocytosis by CD5L⁺ macrophages following treatment with control or NRP2-deficient hypoxic migrasomes. F4/80 (green) labels macrophages; PI (red) labels apoptotic tumor cells. Scare bar: 50 μm. ( E ) Quantification of mRNA expression of efferocytosis receptors (MERTK, TYRO3, OLR1, CD36, AXL, and TIM3) in macrophages treated with control or NRP2-deficient migrasomes by RT-qPCR. ( F ) Quantification of protein expression of efferocytosis receptors (MERTK, TYRO3, OLR1, CD36, AXL, and TIM3) in macrophages treated with control or NRP2-deficient migrasomes by Western blot. * p < 0.05, ** p < 0.01

Article Snippet: Samples were incubated with primary antibodies, CD5L (1:500, 17224-1-AP, Proteintech), CD163 (1:200, ab182422, Abcam), NRP2 (1:250, #3366, CST), PROX1(1:250, sc-81983, Santa cruz), MERTK (200 μg/mL, sc-365499, Santa Cruz).

Techniques: Quantitative RT-PCR, Knockdown, Western Blot, Flow Cytometry, Control, Immunofluorescence, Expressing

Journal: Journal of Translational Medicine

Article Title: Hypoxic migrasomes drive colorectal cancer liver metastasis by mediating CD5L + macrophage efferocytosis via NRP2/PROX1 axis

doi: 10.1186/s12967-025-07485-0

Figure Lengend Snippet: NRP2–PROX1 interaction promotes CD5L⁺ macrophage differentiation and enhances efferocytosis. ( A ) Co-immunoprecipitation (Co-IP) assays showing that NRP2 interacts with PROX1 in macrophages under normoxic and hypoxic migrasome-treated conditions. ( B ) Immunofluorescence co-localization images confirming the spatial association between NRP2 (green) and PROX1 (red) in macrophages. Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. ( C ) Flow cytometry analysis showing the proportion of CD5L⁺ macrophages following NRP2 overexpression and/or PROX1 knockdown. ( D ) RT-qPCR analysis of efferocytosis-related genes (AXL, MERTK, and TYRO3) in macrophages with indicated treatments. ( E ) Immunofluorescence staining of F4/80⁺ macrophages (green) engulfing PI-labeled apoptotic MC38 debris (red). Knockdown of PROX1 suppressed efferocytic activity and attenuated the NRP2-induced enhancement. Scale bar, 50 μm. ( F ) Representative fluorescence images and quantification of transwell assay. Fluorescently labeled CRC cells were co-cultured with macrophages overexpressing NRP2, MERTK-knockdown macrophages, or macrophages with combined NRP2 overexpression and MERTK knockdown, and CRC cell transmigration was assessed using a transwell assay. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Samples were incubated with primary antibodies, CD5L (1:500, 17224-1-AP, Proteintech), CD163 (1:200, ab182422, Abcam), NRP2 (1:250, #3366, CST), PROX1(1:250, sc-81983, Santa cruz), MERTK (200 μg/mL, sc-365499, Santa Cruz).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Flow Cytometry, Over Expression, Knockdown, Quantitative RT-PCR, Staining, Labeling, Activity Assay, Fluorescence, Transwell Assay, Cell Culture, Transmigration Assay

Journal: Clinical and Translational Medicine

Article Title: Chromium (VI)‐induced ALDH1A1/EGF axis promotes lung cancer progression

doi: 10.1002/ctm2.1136

Figure Lengend Snippet: Cr(VI)‐induced overexpression of ALDH1A1 maintains self‐renewal of CrT/TICs. (A) Cell sorting for ALDH1A1 High and ALDH1A1 Low CrT cells. CrT cells were stained with ALDEFLUOR kit and PI. ALDH1A1 High cells: AF top 10%, PI (–); ALDH1A1 Low cells: AF bottom 10%, PI (–). AF: ALDEFLUOR Fluorescence. (B) ALDH1A1 High and ALDH1A1 Low CrT cells were lysed for immunoblot analyses with the indicated antibodies. (C) Reactive oxygen species (ROS) levels were detected by DCFH‐DA staining in ALDH1A1 Low and ALDH1A1 High CrT cells exposed with or without Cr (VI). Data represent the mean ± SD of triplicate experiments. ** p < .001. (D) In vitro limiting dilution assays on ALDH1A1 High and ALDH1A1 Low CrT cells. ** p < .001. (E) Tumoursphere formation assays using ALDH1A1 High and ALDH1A1 Low CrT cells. (F) CrT/TICs with or without Dox‐inducible ALDH1A1 shRNA were treated with or without Dox and lysed for immunoblot analyses with the indicated antibodies. (G) In vitro limiting dilution assays on CrT/TICs cells with or without doxycycline (Dox)‐inducible ALDH1A1 shRNA. ** p < .001. (H) Tumoursphere formation assays using CrT/TICs with or without Dox‐inducible ALDH1A1 shRNA. (I) CrT/TICs with Dox‐inducible ALDH1A1 shRNA were subcutaneously implanted in the left side of mice. (J) CrT/TICs with Dox‐inducible ALDH1A1 shRNA were orthotopically implanted in the lung of mice. (Top) Representative BLIs of lung orthotopic tumours with or without Dox treatment for 50 days. (Bottom) Quantification of BLIs every 10 days. Data are presented as the mean ± SD from five mice. ** P < .001. (K) Kaplan–Meier survival curves for indicated mice. (L) Immunohistochemical (IHC) staining was performed with antibody against ALDH1A1. Scale bar, 20 μm. (M) IHC staining was performed with antibodies against Ki‐67, CD133, and CD44. Scale bar, 20 μm. (N) ALDH1A1 activity were detected in CrT/TICs with the indicated concentration of A37. Data represent the mean ± SD of triplicate experiments. * p < .01, *** p < .0001. (O) In vitro limiting dilution assays on CrT cells treated with or without A37 (50 μM). *** p < .0001. (P) Tumoursphere formation assays using CrT cells treated with or without A37 (50 μM). (Q) CrT/TICs were subcutaneously implanted in the left side of mice. (R) CrT/TICs were orthotopically implanted in the lung of mice. (Top) Representative BLIs of lung orthotopic tumours with or without A37 treatment for 50 days. (Bottom) Quantification of BLIs every 10 days. Data are presented as the mean ± SD from five mice. ** P < .001. (S) Kaplan–Meier survival curves for indicated mice. (T) IHC staining was performed with antibodies against Ki‐67, CD133, and CD44. Scale bar, 20 μm

Article Snippet: DACH1 siRNA (sc‐77089), ABCB5 siRNA (sc‐89856), MERTK siRNA (sc‐37127), KLF4 siRNA (sc‐35480), SOX2 siRNA (sc‐38408), EGF siRNA (sc‐39416), and ALDH1A1 siRNA (sc‐41442) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Over Expression, FACS, Staining, Fluorescence, Western Blot, In Vitro, shRNA, Immunohistochemical staining, Immunohistochemistry, Activity Assay, Concentration Assay

Journal: Clinical and Translational Medicine

Article Title: Chromium (VI)‐induced ALDH1A1/EGF axis promotes lung cancer progression

doi: 10.1002/ctm2.1136

Figure Lengend Snippet: Cr(VI) induces ALDH1A1 expression through KLF4. (A) CrT cells transfected with siRNAs (50 nM) targeting KLF4, DACH1, ABCB5, MERTK, SOX2 or EGF for 72 h and were lysed for immunoblot analyses with the indicated antibodies. (B) CrT cells transfected with or without EGF siRNA (50 nM, 72 h) were lysed for ELISA analyses for detecting secreted EGF levels in the culturing media. (C) CrT cells transfected with siRNAs (50 nM) targeting KLF4, DACH1, ABCB5, MERTK, SOX2 or EGF for 72 h and were lysed for qRT‐PCR analysis of ALDH1A1 mRNA expression levels. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (D) ALDH1A1 High and ALDH1A1 Low CrT cells were lysed for immunoblot analyses with the indicated antibodies. (E) ALDH1A1 Low CrT cells transfected with or without Flag‐KLF4 for 72 h were lysed for immunoblot analysis with the indicated antibodies. ALDH1A1 Low CrT cells transfected with or without Flag‐ALDH1A1 were lysed for immunoblot analyses with the indicated antibodies. (F) ALDH1A1 Low CrT cells transfected with or without KLF4 siRNA (50 nM) for 72 h were lysed for immunoblot analysis with the indicated antibodies. ALDH1A1 High CrT cells transfected with or without ALDH1A1 siRNA were lysed for immunoblot analyses with the indicated antibodies. (G) Schematic image represents the KLF4 binding sequence within the ALDH1A1 transcriptional regulation region. (H) Luciferase reporter assays were performed in BEAS‐2B and CrT cells transfected with pGL‐3.0 vector containing ALDH1A1 WT or mutant promoter. Data represent the mean ± SD of triplicate experiments. ** p < .001. (I) CrT cells with or without KLF4 depletion and BEAS‐2B cells with or without expression of Flag‐KLF4 were transfected with a luciferase reporter gene under the control of the ALDH1A1 promoter for 24 h. Luciferase reporter assays were performed. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (J) BEAS‐2B cells, CrT cells, and CrT/TICs were used for ChIP‐qPCR analysis of the ALDH1A1 promoter with the indicated antibody. Data are presented as the mean ± SD of triplicate experiments. * P < .01, ** P < .001. (K) CrT cells with or without KLF4 depletion were used for the detection of ALDH1A1 activity by flow cytometry. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (L) Tumoursphere formation assays using ALDH1A1 High CrT cells transfected with or without KLF4 siRNA. (M) In vitro limiting dilution assays on ALDH1A1 High CrT cells transfected with or without KLF4 siRNA. ** p < .001. (N) In vitro limiting dilution assays on ALDH1A1 Low CrT cells transfected with or without Flag‐KLF4. ** p < .001. (O) Tumoursphere formation assays using ALDH1A1 Low CrT cells transfected with or without Flag‐KLF4

Article Snippet: DACH1 siRNA (sc‐77089), ABCB5 siRNA (sc‐89856), MERTK siRNA (sc‐37127), KLF4 siRNA (sc‐35480), SOX2 siRNA (sc‐38408), EGF siRNA (sc‐39416), and ALDH1A1 siRNA (sc‐41442) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Binding Assay, Sequencing, Luciferase, Plasmid Preparation, Mutagenesis, Control, ChIP-qPCR, Activity Assay, Flow Cytometry, In Vitro

Journal: Clinical and Translational Medicine

Article Title: Chromium (VI)‐induced ALDH1A1/EGF axis promotes lung cancer progression

doi: 10.1002/ctm2.1136

Figure Lengend Snippet: CrT/TIC‐secreted EGF activates EGFR signalling and promotes LUSC cell growth. (A) HCC95 and H226 cells incubated with a conditioned medium or co‐cultured with the indicated cells were lysed for immunoblot analysis with the indicated antibodies; HCC95 and H226 cells co‐cultured with CrT/TICs transfected with or without KLF4 siRNA were lysed for immunoblot analyses with the indicated antibodies. (B) HCC95 and H226 cells incubated with CrT/TIC‐derived conditioned medium for 12 h in the presence or the absence of human recombinant truncated EGF or EGF L26G were lysed for immunoblot analysis with the indicated antibodies; HCC95 and H226 cells co‐cultured with CrT/TICs transfected with or without KLF4 siRNA were lysed for immunoblot analyses with the indicated antibodies. (C) HCC95 and H226 cells incubated with CrT/TIC‐derived conditioned medium for 12 h in the presence or the absence of EGF‐neutralising antibodies were lysed for immunoblot analyses with the indicated antibodies. (D) HCC95 and H226 cells co‐cultured with CrT/TICs with or without ALDH1A1 depletion were lysed for immunoblot analyses with the indicated antibodies. (E) HCC95 and H226 cells co‐cultured with CrT/TICs transfected with or without KLF4 siRNA were lysed for immunoblot analyses with the indicated antibodies. (F) HCC95 and H226 cells co‐cultured with CrT/TICs pretreated with or without A37 were lysed for immunoblot analyses with the indicated antibodies. (G) HCC95 and H226 cells incubated with conditional medium derived from ALDH1A1 Low CrT or ALDH1A1 High CrT were lysed for immunoblot analyses with the indicated antibodies. (H) Growth curves of HCC95 and H226 cells cultured with BEAS‐2B‐, CrT‐, and CrT/TIC‐derived conditioned medium. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (I) Growth curves of HCC95 and H226 cells cultured with CrT/TIC‐derived conditioned medium pretreated with truncated EGF or EGF L26G. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (J) Growth curves for the HCC95 and H226 cells cultured with CrT/TICs‐derived conditional medium pretreated with or without anti‐EGF antibody. Data represent the mean ± SD of triplicate experiments. ** p < .001. (K) Growth curves of HCC95 and H226 cells cultured with the indicated conditioned medium derived from CrT/TICs with or without ALDH1A1 depletion. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (L) Growth curves of HCC95 and H226 cells cultured with the indicated conditioned medium derived from CrT/TICs with or without A37 treatment. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (M) HCC95 and H226 cells co‐cultured with CrT/TICs pretreated with or without U0126 were lysed for immunoblot analyses with the indicated antibodies. (N) HCC95 and H226 cells co‐cultured with CrT/TICs pretreated with or without PD98 were lysed for immunoblot analyses with the indicated antibodies. (O) Growth curves of HCC95 and H226 cells with or without U0126 treatment cultured with the indicated conditioned medium derived from CrT/TICs. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (P) Growth curves of HCC95 and H226 cells with or without PD98 treatment cultured with the indicated conditioned medium derived from CrT/TICs. Data are presented as the mean ± SD of triplicate experiments. ** P < .001.

Article Snippet: DACH1 siRNA (sc‐77089), ABCB5 siRNA (sc‐89856), MERTK siRNA (sc‐37127), KLF4 siRNA (sc‐35480), SOX2 siRNA (sc‐38408), EGF siRNA (sc‐39416), and ALDH1A1 siRNA (sc‐41442) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Incubation, Cell Culture, Western Blot, Transfection, Derivative Assay, Recombinant

Journal: Clinical and Translational Medicine

Article Title: Chromium (VI)‐induced ALDH1A1/EGF axis promotes lung cancer progression

doi: 10.1002/ctm2.1136

Figure Lengend Snippet: ALDH1A1 inhibition increases the anti‐tumour effects of gemcitabine. (A) HCC95 or H226 cells (2 × 10 ⁶ ) mixed with or without CrT/TICs (1 × 10 ³ ) that stably expressed Dox‐inducible ALDH1A1 shRNA were orthotopically transplanted in the lungs of mice. After 5 days, mice were intraperitoneally injected with A37 or Dox triplicate times per week. Representative BLIs of orthotopic tumours and quantification of BLIs every 5 days are shown. (B) Kaplan–Meier survival curves for indicated mice. (C) IHC staining was performed with the indicated antibodies. Scale bar, 50 μm. (D) Tumour tissues were collected and homogenate. Immunoblot was conducted with indicated antibodies. (E) Representative TUNEL staining (green) and corresponding DAPI nuclear staining (blue) for indicated cells. (F) Drug treatment regimen for administration of A37 and gemcitabine (GEM). (G) HCC95 or H226 cells (2 × 10 ⁶ ) mixed with or without CrT/TICs (1 × 10 ³ ) were orthotopically transplanted in the lungs of mice. After 5 days, mice were treated with GEM. Representative BLIs of orthotopic tumours and quantification of BLIs every 14 days are shown. (H) Kaplan–Meier survival curves for indicated mice. MST, median survival time. (I) IHC staining was performed with anti‐Ki‐67. Scale bar, 50 μm. (J) Tumour tissues were collected and homogenate. Immunoblot was conducted with indicated antibodies. (K) Representative TUNEL staining (green) and corresponding DAPI nuclear staining (blue) for indicated cells

Article Snippet: DACH1 siRNA (sc‐77089), ABCB5 siRNA (sc‐89856), MERTK siRNA (sc‐37127), KLF4 siRNA (sc‐35480), SOX2 siRNA (sc‐38408), EGF siRNA (sc‐39416), and ALDH1A1 siRNA (sc‐41442) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Inhibition, Stable Transfection, shRNA, Injection, Immunohistochemistry, Western Blot, TUNEL Assay, Staining