Journal: Molecular Cancer

Article Title: LMTK3 regulation of EV biogenesis and cargo sorting promotes tumour growth by reducing monocyte infiltration and driving pro-tumourigenic macrophage polarisation in breast cancer

doi: 10.1186/s12943-025-02346-2

Figure Lengend Snippet: Overexpression of LMTK3 increases average EV size, alters EV protein cargo, and changes the EV subpopulation distribution. a . Western blot for LMTK3 in LMTK3-overexpressing cells compared to control (pCMV6 empty vector) cells. GAPDH is shown as a loading control ( n = 3). b . Western blot of cell lysate and EV fractions showing enrichment of the EV markers, CD9 and CD81, presence of HSP70 and depletion of GM130 in EVs compared to cell lysates ( n = 3). c . Summary data showing mean EV size. d . NTA showing concentration of EVs. e–g . Representative transmission electron micrographs showing EVs from control and LMTK3-overexpressing breast cancer cells. Scale bars = 500 nm (12,000 x) and 100 nm (30,000 x). h . Quantification of ( e–g ). 500 EVs were measured for each condition. A representative distribution of EV size is shown. Median is shown with a solid line, and quartiles are shown with dotted lines. i . Proteomic analysis of EVs from control and T47D LMTK3-overexpressing cells. Volcano plot showing EV protein cargo changes as log 2 (fold change) of LMTK3 EVs/Control EVs. Significantly upregulated hits are shown in red and downregulated hits in blue (Welch’s t test, p < 0.05). j , k . Western blotting of T47D ( j ) and MDA-MB-231 ( k ) cell lysate and EV fractions for control (Ct) and LMTK3-overexpressing cells. PSAT1, LDHB, CD63 and TSG101 were validated as hits from the proteomics analysis. CD9 and HSP70 were used as positive EV markers. GM130 was used as a negative EV marker ( n = 3). l . Quantification of PSAT1 and LDHB levels in LMTK3 EVs relative to control EVs compared to HSP70. m . Quantification of CD63 and TSG101 levels in EVs relative to control EVs compared to HSP70. n . EV diameter for control and LMTK3 cells as measured by SP-IRIS. An increase in EV size is seen when LMTK3 is overexpressed for all three capture antibodies. o . The percentage abundance of particles positive for CD63, CD81 and CD9. CD63 positive particles were downregulated when LMTK3 was overexpressed. Graphs show the mean ± SEM of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; two-tailed unpaired Student’s t-test) ( c , d , l , m , n , o )

Article Snippet: In experiments involving LMTK3 inhibition, C28 (MedChemExpress, HY-153896) was used at a final concentration of 10 μM, diluted in DMSO.

Techniques: Over Expression, Western Blot, Control, Plasmid Preparation, Concentration Assay, Transmission Assay, Marker, Two Tailed Test

Journal: Molecular Cancer

Article Title: LMTK3 regulation of EV biogenesis and cargo sorting promotes tumour growth by reducing monocyte infiltration and driving pro-tumourigenic macrophage polarisation in breast cancer

doi: 10.1186/s12943-025-02346-2

Figure Lengend Snippet: LMTK3 directly phosphorylates Rab7 at Ser72. a . Immunofluorescence images of LMTK3 co-localisation with Rab7 in T47D and MDA-MB-231 cells. Scale bars = 10 µm and 1 µm (zoom). Images were taken with the LSM880 with Airyscan and a 63 × objective lens. b . Graph showing mean Pearson’s correlation coefficient (r) for co-localisation between LMTK3 and Rab7 ( n = 50 cells). c . Co-immunoprecipitation western blot showing interaction between LMTK3 and Rab7 in T47D LMTK3 cells. IgG is used as a negative control. Anti-FLAG (top) or anti-Rab7 (bottom) antibody was used to capture FLAG-LMTK3 and Rab7 respectively from cell lysates ( n = 3). d . Western blot showing overexpression of LMTK3 leads to an increase in phospho Rab7 (serine 72). Tubulin was used as a loading control (n = 3). e . Quantification of intensity of pRab7/Rab7 in T47D and MDA-MB-231. Graphs show the mean ± SEM of three independent experiments (* p < 0.05, ** p < 0.01; two-tailed unpaired Student’s t-test). f. Western blot showing a decrease in Rab7 Ser72 phosphorylation following LMTK3 inhibition by C28. Tubulin is shown as a loading control ( n = 3). g . Quantification of intensity of pRab7/Rab7 in T47D and MDA-MB-231. h . Western blot showing overexpression of LMTK3 in C28-treated cells restores Rab7 phosphorylation, showing specificity of C28 ( n = 3). i . Quantification of ( h ). j . In vitro kinase assay of Rab7 and HSP27 (positive control) as substrates. An autoradiogram was produced to show phosphorylated proteins. 1 µM C28 was used to inhibit LMTK3 kinase activity. k . Western blot showing Rab7 phosphorylation by LMTK3 at Ser72. Following an in vitro kinase assay, proteins were separated by SDS PAGE. Western blot for phosphorylated Rab7 (Ser72) is presented ( n = 3). l . Western blot showing overexpression of Rab7 mutant constructs in T47D and MDA-MB-231. Tubulin was used as a loading control ( n = 3). m . Nanoparticle tracking analysis showing Rab7 S72E overexpression results in an increase in average EV size in T47D and MDA-MB-231 cells. n . Nanoparticle tracking analysis showing concentration of EVs from Rab7 mutant T47D and MDA-MB-231 cells. o , p . EVs were collected from the Rab7 mutant cell lines. Cell lysates and EVs were subjected to SDS-PAGE followed by western blotting. Figure showing a representative western blot showing PSAT1 levels in Rab7 mutant T47D ( o ) and MDA-MB-231 ( p ) cells. GM130 was used as a cellular marker and a negative EV marker. HSP70 and CD9 were used as positive EV markers ( n = 3). q . Quantification of ( o , p ). Graphs show the mean ± SEM of at least three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA, followed by Tukey’s multiple comparisons test)

Article Snippet: In experiments involving LMTK3 inhibition, C28 (MedChemExpress, HY-153896) was used at a final concentration of 10 μM, diluted in DMSO.

Techniques: Immunofluorescence, Immunoprecipitation, Western Blot, Negative Control, Over Expression, Control, Two Tailed Test, Phospho-proteomics, Inhibition, In Vitro, Kinase Assay, Positive Control, Produced, Activity Assay, SDS Page, Mutagenesis, Construct, Concentration Assay, Marker

Journal: Molecular Cancer

Article Title: LMTK3 regulation of EV biogenesis and cargo sorting promotes tumour growth by reducing monocyte infiltration and driving pro-tumourigenic macrophage polarisation in breast cancer

doi: 10.1186/s12943-025-02346-2

Figure Lengend Snippet: Rab7 phosphorylation by LMTK3 increases MVB and ILV size. a . Western blot showing EGFR degradation in control and LMTK3 overexpressing T47D. Cells were collected at 0, 30, 60, 120 and 180 min after treatment with EGF. GAPDH is shown as a loading control ( n = 3). b . Quantification of ( a ). *, p < 0.05, **, p < 0.01 (two-way ANOVA with Bonferroni’s multiple comparisons test). c . Representative electron micrographs of control and LMTK3 MDA-MB-231. Figure shows MVBs containing ILVs. Images taken at 10,000 × magnification. Scale bars = 200 nm, at least 50 structures were measured per condition from at least 30 fields of view, 3 biological replicates were performed. d , e . Quantification of ( c ). f . Western blot showing Rab7 knockout cells and re-expression of Rab7 wild-type or Rab7 S72 A mutant ( n = 3). g . T47D Rab7 wild-type or Rab7 S72 A cells stably expressing CD63-RFP with and without LMTK3 overexpression were imaged by confocal microscopy to assess changes in MVB diameter. Scale bars = 10 µm or 1 µm (zoom), n = 500 MVBs. h . Quantification of MVB diameter. Graphs show the mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA, followed by Tukey’s multiple comparisons test)

Article Snippet: In experiments involving LMTK3 inhibition, C28 (MedChemExpress, HY-153896) was used at a final concentration of 10 μM, diluted in DMSO.

Techniques: Phospho-proteomics, Western Blot, Control, Knock-Out, Expressing, Mutagenesis, Stable Transfection, Over Expression, Confocal Microscopy

Journal: Molecular Cancer

Article Title: LMTK3 regulation of EV biogenesis and cargo sorting promotes tumour growth by reducing monocyte infiltration and driving pro-tumourigenic macrophage polarisation in breast cancer

doi: 10.1186/s12943-025-02346-2

Figure Lengend Snippet: Immune cell infiltration is altered by LMTK3 abundance. a . RNA sequencing data and TIMER2.0 infiltration data were used to compare LMTK3 abundance with infiltration of different immune cell populations. Graph shows the mean ± SEM infiltration scores in LMTK3 low vs LMTK3 high samples (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; unpaired two-tailed Student’s t-test). b . Changes in the levels of three M1 and three M2 macrophage markers in breast cancer clinical samples, displayed as mRNA expression z-scores relative to all samples (log RNA Seq V2 RSEM). Q1 (lowest quartile of LMTK3 expression; blue) and Q4 (highest quartile of LMTK3 expression; red) are shown. Graphs show the mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; two-tailed unpaired Student’s t-test), n = 4,715. c . Schematic diagram of the spheroid infiltration assay. d . Representative images of T47D tumour spheroids with infiltrating monocytes stained in green. e . Quantification of ( d ). Data are shown from three biological repeats. Graphs show the mean ± SEM of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA, followed by Tukey’s multiple comparisons test). f , g . PBMC-derived monocytes were treated with PBS, control EVs or LMTK3 EVs. They were then co-cultured with T47D tumour spheroids and imaged after 24 h. f . Representative images of tumour spheroids with infiltrating monocytes. g . Quantification of ( f ). h . THP-1 monocytes were treated with either control EVs or LMTK3 EVs. Proteomic analysis revealed changes in the protein expression of the monocytes. Figure shows a volcano plot of the proteomic analysis. X axis shows log 2 (fold change) of LMTK3 EV-treated THP-1/Control EV-treated THP-1. Significant upregulated hits are shown in red and downregulated hits in blue. Welch’s t test was used to calculate p values. Dotted line indicates p = 0.05. i. Volcano plot showing proteomics data of control and LMTK3 EV-treated monocytes displaying only proteins involved in M1 or M2 polarisation. Red points are associated with M2 polarisation and blue points are associated with M1 polarisation. j . RT-qPCR showing changes in macrophage markers following EV treatment. Graphs show the mean ± SEM of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA, followed by Tukey’s multiple comparisons test)

Article Snippet: In experiments involving LMTK3 inhibition, C28 (MedChemExpress, HY-153896) was used at a final concentration of 10 μM, diluted in DMSO.

Techniques: RNA Sequencing, Two Tailed Test, Expressing, Staining, Derivative Assay, Control, Cell Culture, Quantitative RT-PCR

Journal: Molecular Cancer

Article Title: LMTK3 regulation of EV biogenesis and cargo sorting promotes tumour growth by reducing monocyte infiltration and driving pro-tumourigenic macrophage polarisation in breast cancer

doi: 10.1186/s12943-025-02346-2

Figure Lengend Snippet: LMTK3 EVs promote PSAT1 packaging in EVs, causing an upregulation of PHGDH in monocytes. a . Western blot of monocytes treated with PBS, control EVs or LMTK3 EVs. PHGDH was confirmed as upregulated in monocytes treated with LMTK3 EVs. GAPDH is used as a loading control (n = 3). b . Quantification of ( a ). c . Western blot showing PHGDH overexpression in THP-1 monocytes compared to control monocytes ( n = 3). d . T47D tumour spheroid infiltration assay using control and PHGDH overexpressing monocytes. Monocytes were stained with a green lipophilic tracer. Representative images are shown. e . Quantification of ( d ). Graphs show the mean ± SEM of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; as measured by two-tailed unpaired Student’s t-test). f . RT-qPCR of PHGDH overexpressing monocytes differentiated into macrophages showing upregulation of CD206 and downregulation of CD86 compared to control cells. g . A plasmid containing doxycycline-inducible PSAT1 was transfected into MDA-MB-231 and a stable cell line was generated. EVs were collected from the cells with and without doxycycline (24 h). The figure shows a western blot for PSAT1 in the cell lysates and EVs for PSAT1. CD81 and CD9 are used as positive EV markers and an EV loading control. GM130 is used as a negative EV marker and cell lysate loading control ( n = 3). h . THP-1 monocytes were treated with EVs from cells with Dox-inducible PSAT1 overexpression. EVs from MDA-MB-231 cells without doxycycline were used as a control treatment. Vinculin was used as a loading control. Figure is representative of three biological repeats (n = 3). i. Quantification of ( h ). j . T47D tumour spheroid infiltration assay was carried out using monocytes treated with either PBS, EVs from Dox-inducible PSAT1 MDA-MB-231 without doxycycline or EVs from Dox-inducible PSAT1 MDA-MB-231 with doxycycline. Figure shows representative images from the spheroid infiltration experiment using monocytes treated with EVs from PSAT1 overexpressing cells with and without doxycycline, or PBS. k . Quantification of ( i ). Graphs show the mean ± SEM of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA, followed by Tukey’s multiple comparisons test)

Article Snippet: In experiments involving LMTK3 inhibition, C28 (MedChemExpress, HY-153896) was used at a final concentration of 10 μM, diluted in DMSO.

Techniques: Western Blot, Control, Over Expression, Staining, Two Tailed Test, Quantitative RT-PCR, Plasmid Preparation, Transfection, Stable Transfection, Generated, Marker

Journal: Molecular Cancer

Article Title: LMTK3 regulation of EV biogenesis and cargo sorting promotes tumour growth by reducing monocyte infiltration and driving pro-tumourigenic macrophage polarisation in breast cancer

doi: 10.1186/s12943-025-02346-2

Figure Lengend Snippet: LMTK3 alters monocyte behaviour and macrophage polarisation in vivo. a . Schematic diagram showing the experimental procedure where mice were injected with breast cancer cells that were allowed to form tumours. Tumours were then measured and analysed by flow cytometry and IHC. b . Western blot showing LMTK3 overexpression in MMTV-Neu mouse mammary carcinoma cells. GAPDH is shown as a loading control (n = 3). c. Western blot showing overexpression of LMTK3 in MMTV-Neu cells results in upregulation of PSAT1 in EVs. HSP70 was used as a loading control ( n = 3). d . Tumours were measured twice per week with callipers. Tumour volume is displayed in mm 3 (two-way ANOVA with Bonferroni’s multiple comparisons test). e . Tumour weights on day 32. Graphs show the mean ± SEM * p < 0.05, ** p < 0.01, *** p < 0.001; two-tailed unpaired Student’s t-test. n = 8 tumours (control) and n = 9 tumours (LMTK3). f . IHC analysis of ki-67 positive nuclei. Representative images are shown. Scale bar = 100 µm. g . Quantification of ( f ). Graph shows the mean ± SEM as measured by two-tailed unpaired Student’s t-test. h . Tumours were dissociated and stained for CD45, CD11b and F4/80. Triple-positive cells were counted and normalised to tumour weights. Graph shows number of macrophages per gram of tumour. Graph shows the mean ± SEM; two-tailed unpaired Student’s t-test. n = 8 control and 9 LMTK3 tumours. i. Representative flow cytometry of cells indicating M1 and M2-like macrophages in dissociated tumours. j , k . Flow cytometry showing percentage of M1- ( j ) and M2-like ( k ) macrophages in tumours. Graphs show the mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired Student’s t-test

Article Snippet: In experiments involving LMTK3 inhibition, C28 (MedChemExpress, HY-153896) was used at a final concentration of 10 μM, diluted in DMSO.

Techniques: In Vivo, Injection, Flow Cytometry, Western Blot, Over Expression, Control, Two Tailed Test, Staining

Journal: Molecular Cancer

Article Title: LMTK3 regulation of EV biogenesis and cargo sorting promotes tumour growth by reducing monocyte infiltration and driving pro-tumourigenic macrophage polarisation in breast cancer

doi: 10.1186/s12943-025-02346-2

Figure Lengend Snippet: LMTK3 EVs increase tumour growth and promote an immunosuppressive phenotype in infiltrating macrophages. a . Schematic diagram showing mice were injected with 4T1 cells to form tumours. Mice were then randomised and treated with 50 μg of EVs three times per week. Tumours were analysed with flow cytometry. b , c . Tumour growth curves showing tumour volume in mm 3 for mice treated with T47D EVs ( b ) and MDA-MB-231 EVs ( c ) (two-way ANOVA with Bonferroni’s multiple comparisons test). d,e . Graphs show the final tumour weights (grams) for 4T1 mouse models treated with T47D ( d ) or MDA-MB-231 ( e ) EVs. Graphs show mean ± SEM * p < 0.05, ** p < 0.01; two-tailed unpaired Student’s t-test. n = 8 tumours per condition. f , g . IHC analysis of 4T1 tumours for ki-67 positive nuclei. Representative images are shown. Scale bar = 50 µm . h , i . Quantification of ( f , g ). Graph shows the mean ± SEM, * p < 0.05 as measured by two-tailed unpaired Student’s t-test. j-m . Flow cytometry showing percentage of M1- ( j , k ) and M2-like ( l , m ) macrophages in tumours. Graphs show the mean ± SEM *p < 0.05; two-tailed unpaired Student’s t-test. n . Proposed model showing LMTK3 overexpression in breast cancer cells results in increased serine 72 phosphorylation of Rab7. This alters MVB trafficking, resulting in increased packaging of PSAT1 into EVs. These EVs are taken up by monocytes, causing an upregulation of PHGDH, resulting in an M2-like phenotype in monocyte-derived macrophages and reduced infiltration

Article Snippet: In experiments involving LMTK3 inhibition, C28 (MedChemExpress, HY-153896) was used at a final concentration of 10 μM, diluted in DMSO.

Techniques: Injection, Flow Cytometry, Two Tailed Test, Over Expression, Phospho-proteomics, Derivative Assay