Journal: Cancer Science

Article Title: Lenvatinib enhances antitumor immunity by promoting the infiltration of TCF1 + CD8 + T cells in HCC via blocking VEGFR2

doi: 10.1111/cas.15719

Figure Lengend Snippet: Effects of lenvatinib on infiltration of tcf1 + PD1 + CD8 + T cells in the TME and spleen in mice. A, Schematic of lenvatinib therapy. Hepa1‐6 cells were in situ inoculated into C57BL/6 mice on day 0, lenvatinib was intragastrically administered daily when the tumor volume reached 150 mm 3 on day 9, and tumor samples were collected for analysis on days 13 and 17. B, Proliferative activities (marked by expression of Ki‐67) of PD1 + CD8 + T cells from spleen and tumor in the negative control (NC) group and lenvatinib group were analyzed. Data were expressed as mean ± SEM ( n = 5). C, Phenotypes of exhausted PD1 + CD8 + T cells (marked by expression of TCF1 and TIM3) from tumor in the NC group and the lenvatinib group were analyzed. Data were expressed as mean ± SEM ( n = 5). D, The total number of tumor‐infiltrating CD8 + T cells and the number of pTex cells were detected. Data were expressed as mean ± SEM ( n = 5). E, Phenotypes of exhausted PD1 + CD8 + T cells (marked by expression of TCF1 and Ki‐67) from spleen in the NC group and the lenvatinib group were analyzed. Data were expressed as mean ± SEM ( n = 5). F, Tumor‐killing functions of intermediate Tex cells (marked by expression of granzyme B) from tumor in the NC group and the lenvatinib group were analyzed. Data were expressed as mean ± SEM ( n = 5). * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001

Article Snippet: Considering that lenvatinib is a multitarget inhibitor, we conducted in vitro experiments with antibodies targeted at each target to determine through which target lenvatinib affects the mTOR pathway (Figure ).

Techniques: In Situ, Expressing, Negative Control

Journal: Cancer Science

Article Title: Lenvatinib enhances antitumor immunity by promoting the infiltration of TCF1 + CD8 + T cells in HCC via blocking VEGFR2

doi: 10.1111/cas.15719

Figure Lengend Snippet: Lenvatinib increased the proportion of progenitor exhausted CD8 + T cells in vitro. A, Schematic diagram of logical door frame selection of exhausted CD8 + T cells was listed. Naïve CD8 + T cells were continuously stimulated by CD3 and CD28 in vitro for 6 d and cocultured with lenvatinib for 48 h. B, Proliferative activities (marked by CFSE) of PD1 + CD8 + T cells from the NC group and the lenvatinib group were analyzed. Data were expressed as mean ± SEM ( n = 5). C, Five‐day and seven‐day phenotypes of exhausted PD1 + CD8 + T cells (marked by expression of TCF1 and TIM3) from the NC group and the lenvatinib group were analyzed. Data were expressed as mean ± SEM ( n = 5). D, Tumor‐killing functions of intermediate Tex cells (marked by expression of granzyme B) from the NC group and the lenvatinib group were analyzed. Data were expressed as mean ± SEM ( n = 5). * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001

Article Snippet: Considering that lenvatinib is a multitarget inhibitor, we conducted in vitro experiments with antibodies targeted at each target to determine through which target lenvatinib affects the mTOR pathway (Figure ).

Techniques: In Vitro, Selection, Expressing

Journal: Cancer Science

Article Title: Lenvatinib enhances antitumor immunity by promoting the infiltration of TCF1 + CD8 + T cells in HCC via blocking VEGFR2

doi: 10.1111/cas.15719

Figure Lengend Snippet: Lenvatinib affects CD8 + T cell differentiation by affecting the PI3K/Akt/mTOR signaling pathway. A, GSEA barcode plot of the mTORC1 pathway in the NC group and the lenvatinib group derived from cultured cells in vitro. B, Heatmap of relative expression ( Z score) of the top 50 leading‐edge genes from GSEA of the mTORC1 signaling pathway. C, Phosphorylations of the mTOR pathway (marked by expression of p‐S6) of pTex and tTex cells in the NC group and the lenvatinib group were analyzed. Data were expressed as mean ± SEM ( n = 5). D, pTex and tTex cells’ activation status (marked by expression of Ki‐67), in the NC group and lenvatinib group and derived from cultured cells in vitro, was analyzed. Data were expressed as mean ± SEM ( n = 5). * p < 0.05; ** p < 0.01

Article Snippet: Considering that lenvatinib is a multitarget inhibitor, we conducted in vitro experiments with antibodies targeted at each target to determine through which target lenvatinib affects the mTOR pathway (Figure ).

Techniques: Cell Differentiation, Derivative Assay, Cell Culture, In Vitro, Expressing, Activation Assay

Journal: Cancer Science

Article Title: Lenvatinib enhances antitumor immunity by promoting the infiltration of TCF1 + CD8 + T cells in HCC via blocking VEGFR2

doi: 10.1111/cas.15719

Figure Lengend Snippet: Lenvatinib affects the PI3K/Akt/mTOR signaling pathway by blocking VEGFR2. A, Schematic diagram of the target of each drug. B, Phosphorylations of the mTOR pathway (marked by expression of p‐S6) of pTex and tTex cells in each group were analyzed. Data were expressed as mean ± SEM ( n = 5). C, In the different stages of CD8 + T cells, the expression of VEGFR2 was analyzed. Data were expressed as mean ± SEM ( n = 5). D, Phosphorylations of the mTOR pathway (marked by expression of p‐S6) of pTex and tTex T cells in each group were analyzed. Data were expressed as mean ± SEM ( n = 5). * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001

Article Snippet: Considering that lenvatinib is a multitarget inhibitor, we conducted in vitro experiments with antibodies targeted at each target to determine through which target lenvatinib affects the mTOR pathway (Figure ).

Techniques: Blocking Assay, Expressing

Journal: Cancer Science

Article Title: Lenvatinib enhances antitumor immunity by promoting the infiltration of TCF1 + CD8 + T cells in HCC via blocking VEGFR2

doi: 10.1111/cas.15719

Figure Lengend Snippet: Lenvatinib promotes the phosphorylation of the mTOR pathway by blocking VEGFR2 of PD1 + CD8 + T cells and enhances the tumor‐killing function of intermediate exhausted CD8 + T cells and amplification of pTex subsets. Schematic diagram showing that lenvatinib enhances production of progenitor exhausted T cells and then pTex produce more intermediate exhausted T cells with tumor‐killing function by promoting the phosphorylation of the mTOR pathway via blocking VEGFR2

Article Snippet: Considering that lenvatinib is a multitarget inhibitor, we conducted in vitro experiments with antibodies targeted at each target to determine through which target lenvatinib affects the mTOR pathway (Figure ).

Techniques: Phospho-proteomics, Blocking Assay, Amplification

Journal: Cancer Discovery

Article Title: A Targetable Developmental Program Coregulates Angiogenesis and Immune Evasion in Melanoma

doi: 10.1158/2159-8290.CD-24-1853

Figure Lengend Snippet: CD73 is a key mediator of the HOXD13-dependent T-cell exclusion phenotype. A, The NT5E locus displays different regulatory elements contacting each other and bound by HOXD13. Silencing of HOXD13 reduces the contacts around the NT5E promoter. B, Bar dot plot representing CD73 protein quantification in tumor (left) and blood vessel (right) areas of a TMA in which cores were segregated into HOXD13 -expressing ( n = 61) and HOXD13 -negative ( n = 39). An unpaired t test was conducted, and P values are displayed. C, Representative HOXD13 low and HOXD13 high cores (RNAScope, top) and CD73, SOX10, and CD31/CD34 (mIF, bottom). The white scale bar measures 200 μm. D, qRT-PCR of Nt5e (Cd73) mRNA expression in B16F10 OVA cells that are EMPTY or Cd73 overexpressing, incubated with siSCR or siHoxd13. A paired t test was performed, and P values are shown in the graphs. E, Relative cell growth of B16F10 OVA carrying Cd73 ectopic expression cocultured with OT-I T cells upon Hoxd13 KD. A two-way ANOVA was conducted between the conditions. P values are shown. F, YUMM1.7 EMPTY or HOXD13 OE tumor growth expressing an shSCR (control) or shCd73. Mixed-effect analysis with Tukey comparison was conducted. The P value of the comparison YUMM1.7 HOXD13 shSCR vs. shCd73 is displayed. G, Tumor growth measurements of YUMM1.7 HOXD13 syngeneic grafts upon vehicle (control), lenvatinib, etrumadenant, or lenvatinib + etrumadenant (combo). Mixed-effects analysis through Tukey multiple comparison was performed, and P values are shown in the curves’ graph. H, Representative mIF staining for DAPI, CD45, CD3, CD11b, CD31, and α-SMA in YUMM1.7 HOXD13 tumors upon vehicle, etrumadenant, lenvatinib, and combo treatments. Combo dramatically decreased intratumor vasculature (CD31, α-SMA) and increased immune cells, particularly macrophage infiltration (CD45, CD11b). White bars reflect 500 mm size. I, B16F10 tumor growth in syngeneic mice upon shNTC, shHoxd13, or treated with vehicle or combo ( n = 9 mice per condition). Mixed-effect Tukey comparison was used as a statistical study; P values are shown. J, mIF opal staining for fibrin, α-SMA, and CD31 in representative tumor areas of B16F10 tumors shNTC, shHoxd13, vehicle, and combo treated. The scale bar is a reference of 50 μm.

Article Snippet: For the etrumadenant and lenvatinib experiment, 8-week-old male mice C57BL/6J ( n = 10 per group) were injected intradermally in the flanks with 1 × 10 5 YUMM1.7 HOXD13 or B16F10, and after 3 days of grafting, mice were daily administered by oral gavage with 10 μg/g of lenvatinib (MedChemExpress, HY-10981) and/or 100 μg/g etrumadenant (MedChemExpress, HY-129393) or vehicle (95% corn oil + 5% DMSO).

Techniques: Expressing, RNAscope, Quantitative RT-PCR, Incubation, Control, Comparison, Staining

Journal: Cell Death & Disease

Article Title: TNF- α -induced LRG1 promotes angiogenesis and mesenchymal stem cell migration in the subchondral bone during osteoarthritis

doi: 10.1038/cddis.2017.129

Figure Lengend Snippet: LEN, a TNF- α production inhibitor, reduced LRG1 expression, alleviated the changes in the subchondral bone, and attenuated articular cartilage degeneration in ACLT. ( a and b ) Immunohistochemical analysis of LRG1 (top, a ), CD31 (middle, a ), TNF- α (bottom, a ), nestin (Top, b ) and osterix (bottom, b ) in tibial subchondral bone harvested 30 days after sham operation (sham), ACLT operation and treatment with vehicle (vehicle) or ACLT operation and treatment with LEN (LEN); n =5 per group. Scale bars, 20 μ m. ( c ) Safranin O and fast green staining in the articular cartilage of mice 60 days after surgery; n =5 per group. Scale bars, 50 μ m. ( d ) OARSI scores in c . ( e ) Immunohistochemical analysis of MMP13 (top) and LRG1 (bottom) in the articular cartilage of mice 30 days after surgery; n =5 per group. Scale bars, 20 μ m. All values are given as means±S.D. ** P <0.01 compared with the sham-operated group; # P <0.05 compared with the vehicle-treated ACLT group

Article Snippet: For the therapeutic experiment 3 days before surgery, we injected LEN (Selleck, Houston, TX, USA; 50 mg per kg body weight) or the equivalent volume of vehicle (DMSO and PBS) intraperitoneally daily for 30 or 60 days.

Techniques: Expressing, Immunohistochemical staining, Staining