Journal: Nature Communications

Article Title: Two-dimensional coordination risedronate-manganese nanobelts as adjuvant for cancer radiotherapy and immunotherapy

doi: 10.1038/s41467-024-53084-w

Figure Lengend Snippet: a Primary and ( b ) distant tumor growth curves of CT26 colorectal bilateral tumor-bearing mice treated with Saline, Saline + RT, RMn-NBs + RT, RMn-NBs + RT + αCD8a and RMn-NBs + RT + αPD-L1. The doses of Mn 2+ and RANa are 5.5 mg kg −1 and 30.5 mg kg −1 , respectively. X-ray irradiation is performed after intravenous injection of RMn-NBs (10 mM × 0.2 mL) for 6 h. Treatments are performed with 3 fractions (total 6 Gy) on days 0, 3, and 6, and only primary tumors receive RT (black arrow). αPD-L1 (10 mg kg −1 ) and αCD8a (10 mg kg −1 ) are intraperitoneally injected respectively after RT for 6 h, and treatments are performed on days 0, 3, 6, and 9 (red arrow, n = 6 mice). c Primary and ( d ) distant tumor weights from mice with various treatments ( n = 6 mice). The percentages of CD3 + CD4 + T cells infiltrated within ( e ) primary and ( f ) distant tumor tissues detected by FCM ( n = 6 mice). The percentages of CD3 + CD8 + T cells infiltrated within ( g ) primary and ( h ) distant tumor tissues detected by FCM ( n = 6 mice). The percentages of FoxP3 + T cells (Tregs) among CD3 + CD4 + T cells infiltrated within ( i ) primary and ( j ) distant tumor tissues detected by FCM ( n = 6 mice). Relative content of IFN-γ in the ( k ) primary and ( l ) distant tumor tissues detected by ELISA kit ( n = 6 mice). All data are shown as mean ± SD. Statistical analysis is performed using two-tailed Student’s t -test for comparing two groups, and one-way ANOVA analysis of variance for multiple groups. p > 0.05 represent nonsignificance, while p < 0.05 indicate statistical significance. Source data are provided as a file.

Article Snippet: APC anti-mouse CD80 Antibody [16-10A1] (Cat# 104713, 1.0 μg per million cells in 100 μL volume), PE anti-mouse CD86 Antibody [GL-1] (Cat# 105007, 0.25 μg per million cells in 100 μL volume), FITC anti-mouse CD11c Antibody [N418] (Cat# 117306, 0.25 μg per million cells in 100 μL volume), APC anti-mouse CD3 Antibody [17A2] (Cat# 100236, 0.5 μg per million cells in 100 μL volume), PE anti-mouse CD4 Antibody [GK1.5] (Cat# 100408, 0.25 μg per million cells in 100 μL volume), FITC anti-mouse CD8a Antibody [53–6.7] (Cat# 100706, 1.0 μg per million cells in 100 μL volume), Alexa Fluor® 488 anti-mouse FoxP3 antibody [MF-14] (Cat# 126406, 0.25 µg per million cells in 100 µL volume), FoxP3 Fix/Perm buffer (4×) (Cat# 421401), APC anti-mouse H-2Kb Antibody [AF6-88.5] (Cat# 116506, 1.0 μg per million cells in 100 μL volume), PE anti-mouse CD3 Antibody [17A2] (Cat# 100205, 0.5 µg per million cells in 100 µL volume), APC anti-mouse/human CD44 Antibody [IM7] (Cat# 103011, 0.25 µg per million cells in 100 µL volume), PerCP/Cyanine5.5 anti-mouse CD62L Antibody [MEL-14] (Cat# 104432, 0.25 µg per million cells in 100 µL volume), PE anti-mouse F4/80 Antibody [BM8] (Cat# 123110, 1.0 µg per million cells in 100 µL volume), PerCP/Cyanine5.5 anti-mouse/human CD11b Antibody [M1-70] (Cat# 101227, 0.25 µg per million cells in 100 µL volume), True-Nuclear™ transcription factor buffer set (Cat# 421403), Purified anti-mouse IFN-γ Antibody [R4-6A2] (Cat# 505702, 2.0 μg mL −1 ) and Ultra-LEAF™ Purified anti-mouse CD8a Antibody [53–6.7] (Cat# 100764, 10.0 mg kg −1 ) are purchased from BioLegend (USA).

Techniques: Saline, Irradiation, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Two-dimensional coordination risedronate-manganese nanobelts as adjuvant for cancer radiotherapy and immunotherapy

doi: 10.1038/s41467-024-53084-w

Figure Lengend Snippet: a Tumor growth curves of B16F10-OVA tumor-bearing C57BL/6 mice treated with Saline, Saline + RT, RMn-NBs + RT, RMn-NBs + RT + αCD8a and RMn-NBs + RT + αPD-L1. Treatments are performed with 3 fractions on days 0, 2, and 4 (black arrow). αPD-L1 and αCD8a are intraperitoneally injected respectively after RT for 6 h (red arrow, n = 6 mice). b Tumor weights from mice with various treatments ( n = 6 mice). c F4/80 + and CD11b + macrophages in B16F10-OVA tumor-bearing C57BL/6 mice with different treatments detected by FCM ( n = 6 mice). d FCM analysis of CD11c + H2Kb-SIINFEKL + DCs in tumor-draining lymph nodes from mice treated with Saline, Saline + RT, RMn-NBs + RT, RMn-NBs + RT + αCD8a and RMn-NBs + RT + αPD-L1 ( n = 6 mice). e Mature DCs (CD80 + and CD86 + gated on CD11c + ) in B16F10-OVA tumor-bearing C57BL/6 mice with different treatments detected by FCM ( n = 6 mice). f The percentages of CD3 + CD4 + T cells infiltrated within tumor tissues detected by FCM ( n = 6 mice). g The percentages of CD3 + CD8 + T cells infiltrated within tumor tissues detected by FCM ( n = 6 mice). h Percentages of effector memory T cells (T EM ) in the spleen analyzed by FCM. The spleens in different groups are collected on day 10 ( n = 6 mice). i The quantification of antigen-specific T cells by IFN-γ ELISpot ( n = 3 mice). j The representative images of antigen-specific T cells by IFN-γ ELISpot. This experiment is repeated twice independently with similar results. All data are shown as mean ± SD. Statistical analysis is performed using two-tailed Student’s t -test for comparing two groups, and one-way ANOVA analysis of variance for multiple groups. p > 0.05 represent nonsignificance, while p < 0.05 indicate statistical significance. Source data are provided as a file.

Article Snippet: APC anti-mouse CD80 Antibody [16-10A1] (Cat# 104713, 1.0 μg per million cells in 100 μL volume), PE anti-mouse CD86 Antibody [GL-1] (Cat# 105007, 0.25 μg per million cells in 100 μL volume), FITC anti-mouse CD11c Antibody [N418] (Cat# 117306, 0.25 μg per million cells in 100 μL volume), APC anti-mouse CD3 Antibody [17A2] (Cat# 100236, 0.5 μg per million cells in 100 μL volume), PE anti-mouse CD4 Antibody [GK1.5] (Cat# 100408, 0.25 μg per million cells in 100 μL volume), FITC anti-mouse CD8a Antibody [53–6.7] (Cat# 100706, 1.0 μg per million cells in 100 μL volume), Alexa Fluor® 488 anti-mouse FoxP3 antibody [MF-14] (Cat# 126406, 0.25 µg per million cells in 100 µL volume), FoxP3 Fix/Perm buffer (4×) (Cat# 421401), APC anti-mouse H-2Kb Antibody [AF6-88.5] (Cat# 116506, 1.0 μg per million cells in 100 μL volume), PE anti-mouse CD3 Antibody [17A2] (Cat# 100205, 0.5 µg per million cells in 100 µL volume), APC anti-mouse/human CD44 Antibody [IM7] (Cat# 103011, 0.25 µg per million cells in 100 µL volume), PerCP/Cyanine5.5 anti-mouse CD62L Antibody [MEL-14] (Cat# 104432, 0.25 µg per million cells in 100 µL volume), PE anti-mouse F4/80 Antibody [BM8] (Cat# 123110, 1.0 µg per million cells in 100 µL volume), PerCP/Cyanine5.5 anti-mouse/human CD11b Antibody [M1-70] (Cat# 101227, 0.25 µg per million cells in 100 µL volume), True-Nuclear™ transcription factor buffer set (Cat# 421403), Purified anti-mouse IFN-γ Antibody [R4-6A2] (Cat# 505702, 2.0 μg mL −1 ) and Ultra-LEAF™ Purified anti-mouse CD8a Antibody [53–6.7] (Cat# 100764, 10.0 mg kg −1 ) are purchased from BioLegend (USA).

Techniques: Saline, Injection, Enzyme-linked Immunospot, Two Tailed Test

Journal: Nature Communications

Article Title: Two-dimensional coordination risedronate-manganese nanobelts as adjuvant for cancer radiotherapy and immunotherapy

doi: 10.1038/s41467-024-53084-w

Figure Lengend Snippet: a Schematic illustration of in situ vaccination mediated by RMn-NB-sensitized RT. Created in BioRender. Luo, Z. (2024) BioRender.com/n85g329. b Tumor growth curves, ( c ) isolated tumor photographs, and ( d ) tumor weights of BALB/c mice immunized with Saline + RT and RMn-NBs + RT-treated CT26 tumor cells, respectively ( n = 6 mice). e Percentages of T EM in the spleen analyzed by FCM. The spleens in different groups are collected on day 30 ( n = 6 mice). f Tumor growth curves of 4T1 breast tumor-bearing mice treated with Saline, Saline + RT, RMn-NBs + RT, RMn-NBs + RT + αCD8a and RMn-NBs + RT + αPD-L1 ( n = 6 mice). The doses of Mn 2+ and RANa are 5.5 mg kg −1 and 30.5 mg kg −1 , respectively. X-ray irradiation is performed after intravenous injection of RMn-NBs (10 mM × 0.2 mL) for 6 h. Treatments are performed with 3 fractions (total 6 Gy) on days 0, 3, and 6. αCD8a (10 mg kg −1 ) and αPD-L1 (10 mg kg −1 ) are respectively administered via intraperitoneal injection after RT for 6 h, and treatments are performed on days 0, 3, and 6 ( n = 6 mice). g Weights of 4T1 breast tumor after various treatments ( n = 6 mice). h Survival curves of 4T1 breast tumor-bearing mice ( n = 6 mice). i Quantification of metastatic lesions in the lungs ( n = 6 mice). j Average size of each nodule in the lungs ( n = 6 mice). All data are shown as mean ± SD. Statistical analysis is performed using two-tailed Student’s t -test for comparing two groups, and one-way ANOVA analysis of variance for multiple groups. p > 0.05 represent nonsignificance, while p < 0.05 indicate statistical significance. Source data are provided as a file.

Article Snippet: APC anti-mouse CD80 Antibody [16-10A1] (Cat# 104713, 1.0 μg per million cells in 100 μL volume), PE anti-mouse CD86 Antibody [GL-1] (Cat# 105007, 0.25 μg per million cells in 100 μL volume), FITC anti-mouse CD11c Antibody [N418] (Cat# 117306, 0.25 μg per million cells in 100 μL volume), APC anti-mouse CD3 Antibody [17A2] (Cat# 100236, 0.5 μg per million cells in 100 μL volume), PE anti-mouse CD4 Antibody [GK1.5] (Cat# 100408, 0.25 μg per million cells in 100 μL volume), FITC anti-mouse CD8a Antibody [53–6.7] (Cat# 100706, 1.0 μg per million cells in 100 μL volume), Alexa Fluor® 488 anti-mouse FoxP3 antibody [MF-14] (Cat# 126406, 0.25 µg per million cells in 100 µL volume), FoxP3 Fix/Perm buffer (4×) (Cat# 421401), APC anti-mouse H-2Kb Antibody [AF6-88.5] (Cat# 116506, 1.0 μg per million cells in 100 μL volume), PE anti-mouse CD3 Antibody [17A2] (Cat# 100205, 0.5 µg per million cells in 100 µL volume), APC anti-mouse/human CD44 Antibody [IM7] (Cat# 103011, 0.25 µg per million cells in 100 µL volume), PerCP/Cyanine5.5 anti-mouse CD62L Antibody [MEL-14] (Cat# 104432, 0.25 µg per million cells in 100 µL volume), PE anti-mouse F4/80 Antibody [BM8] (Cat# 123110, 1.0 µg per million cells in 100 µL volume), PerCP/Cyanine5.5 anti-mouse/human CD11b Antibody [M1-70] (Cat# 101227, 0.25 µg per million cells in 100 µL volume), True-Nuclear™ transcription factor buffer set (Cat# 421403), Purified anti-mouse IFN-γ Antibody [R4-6A2] (Cat# 505702, 2.0 μg mL −1 ) and Ultra-LEAF™ Purified anti-mouse CD8a Antibody [53–6.7] (Cat# 100764, 10.0 mg kg −1 ) are purchased from BioLegend (USA).

Techniques: In Situ, Isolation, Saline, Irradiation, Injection, Two Tailed Test

Journal: iScience

Article Title: Tumor-infiltrating mast cells confer resistance to immunotherapy in pancreatic cancer

doi: 10.1016/j.isci.2024.111085

Figure Lengend Snippet: Blocking mast cell function improves the anti-tumor efficacy of anti-OX40 in both syngeneic allograft and autochthonous KPC model (A) Experimental design and (B) survival curves of KPC-GEMM mice. Combination therapy with anti-OX40 and cromolyn significantly extended survival in KPC mice compared with control ( p = 0.0007); anti-OX40 alone had no survival benefit ( p = 0.1318); cromolyn significantly increased survival ( p = 0.0388); and combination therapy with anti-OX40 and cromolyn resulted in longer survival than did cromolyn alone ( p = 0.0435). (C–F) Immunohistochemical analysis of CD8 infiltration (C and D) and granzyme B expression (E and F) showed little infiltration of effector T cells into the tumor microenvironment in control mice (black sets in C–F) and after treatment with cromolyn (green sets in C–F), slightly increased with the treatment with anti-OX40 (blue sets in C–F), however, a dramatic increase was found in mice treated with agonist anti-OX40 and cromolyn (red sets in C–F). (D and F) Quantification of CD8 and granzyme B positive cells—average from 5 representative high-power fields. A one-way ANOVA, followed by the Fisher’s least significant difference (LSD) multiple comparison test, shows the significances by ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001; ∗∗∗∗: p < 0.0001.

Article Snippet: Purified Rat Anti-Mouse CD8a , BD PharmingenTM , Cat#553027; RRID: AB_394565.

Techniques: Blocking Assay, Cell Function Assay, Control, Immunohistochemical staining, Expressing, Comparison

Journal: iScience

Article Title: Tumor-infiltrating mast cells confer resistance to immunotherapy in pancreatic cancer

doi: 10.1016/j.isci.2024.111085

Figure Lengend Snippet:

Article Snippet: Purified Rat Anti-Mouse CD8a , BD PharmingenTM , Cat#553027; RRID: AB_394565.

Techniques: Purification, Recombinant, Software, RNA Sequencing Assay

Journal: iScience

Article Title: Enniatin A inhibits the chaperone Hsp90 and unleashes the immune system against triple-negative breast cancer

doi: 10.1016/j.isci.2023.108308

Figure Lengend Snippet: EnnA antitumor activity against E0771 tumors is mediated by CD8 + T cells (A) Immunofluorescence analysis showing that EnnA induces recruitment of CD8β + T cells to the E0771 tumor site and increases tumor infiltration compared to control. The scale bar represents 300 μm for 4x and 20 μm for 40x. (B) Average of 180 cells from three different fields of control and EnnA-treated tumors (n = 3). Data are represented in the percentage of mean ± S.E.M. (C) Immunofluorescence analysis of 4T1 tumors treated with EnnA (10 mg/kg/48 h) or 17-AAG (25 mg/kg/48 h) stained with anti-CD45 and anti-CD8α. The scale bar represents 50 μm. (D) Average of 250 cells from three different fields of control and EnnA-treated tumors (n = 3). Data are represented in the percentage of mean ± S.E.M. (E) Immunofluorescence analysis of E0771 tumors from control and EnnA-treated animals showing that CD8β+ T cells also express granzyme b. The scale bar represents 300 μm for 4x and 50 μm for 20x. (F) Average of 135 cells from three different fields of control and EnnA-treated tumors (n = 3). (G) Flow cytometric analysis of cells from tumors treated with EnnA or DMSO. Data are represented in the percentage of mean ± S.E.M. (H) Depletion of CD8 + T cells from mice using a monoclonal antibody (InVivoMAb anti-mouse CD8α, clone 53–6.7) abrogates antitumor activity of EnnA. Data are represented in the percentage of mean ± S.E.M. n represents the number of mice per group. (I) EnnA treatment increases the number of CD8 + T cells in spleens, which is abrogated by an anti-CD8 antibody. Data are represented as mean ± S.E.M. ∗p < 0.05; ∗∗p < 0.01 by one-way ANOVA or unpaired tailed Student’s t test and are a representation of at least two independent studies.

Article Snippet: Purified anti-mouse CD8a , BioLegend , Cat# 100801; RRID: AB_312762.

Techniques: Activity Assay, Immunofluorescence, Control, Staining

Journal: iScience

Article Title: Enniatin A inhibits the chaperone Hsp90 and unleashes the immune system against triple-negative breast cancer

doi: 10.1016/j.isci.2023.108308

Figure Lengend Snippet: CX3CR1 chemoattraction pathway and PD-L1 pathways are modulated by EnnA treatment (A) Flow cytometry analysis of CX3CR1 surface expression on E0771 cells treated with 7.5 μM EnnA or DMSO control. Data are represented as a mean ± S.E.M. (B) Flow cytometry analysis of CX3CR1 surface expression on cell suspensions from E0771 tumors treated with EnnA or control. Data are represented as a mean ± S.E.M. (C) The CX3CR1 chemoattraction pathway is important for EnnA’s antitumor efficacy. 10 × 10 4 E0771 cells were injected into the mammary fat pad of seven-week-old female mice. EnnA and JMS-17-2 groups were injected intraperitoneally with 10 mg/kg of EnnA or JMS-17-2 every 48 h, and the control group (DMSO) received vehicle (mixture of 15% DMSO and 20% cremophor in PBS). The EnnA + JMS-17-2 group received the compounds at alternate 48-h time points. Tumor volume was measured as treatment progressed. n represents the number of mice per group. Data are represented as a mean ± S.E.M. (D and E) Western blot analysis for PD-L1 expression in cell extracts from indicated cell lines treated with various concentrations of EnnA or 17-AAG. DMSO is used as control. (F) Immunofluorescence analysis for PD-L1 expression in E0771 tumors from animals treated with DMSO control or EnnA. The scale bar represents 20 μm. (G) The corrected total cell fluorescence (CTCF) intensity was calculated from 100 PD-L1+ cells from three different microscopic fields of control and EnnA-treated tumors. (H) Western blot analysis of PD-L1 complexes isolated from E0771 cells by immunoprecipitation using anti-PD-L1 antibody. PD-L1 expression was induced with 10 ng/mL interferon-gamma for 24 h. Cells were treated with 10 μM EnnA or DMSO for an extra 5 h. Rat IgG was used as a control. (I) Cells were treated as in H. After immunoprecipitation, part of the PD-L1 resin was stripped (with stripping) from endogenous interacting proteins using 250 mM KCl and then incubated with purified Hsp90. The other fraction was kept as is (No stripping) to visualize the endogenous Hsp90 binding to PD-L1. Mouse IgG was used as control. (J) 10x10 4 E0771 cells were injected into 4 th mammary fat pad of seven-week-old female B6 mice. Tumors were allowed to grow to about 50 mm 3 before starting treatment. EnnA (10 mg/kg) and DMSO were injected every 48 h. Anti-PD-L1 (InVivoMAb anti-mouse PD-L1 (B7-H1) (BioXCell)) was injected intraperitoneally twice a week at 200 μg/mouse. n represents the number of mice per group. (K) Model for EnnA’s antitumor activity. EnnA inhibits Hsp90, causing degradation of its oncogenic client proteins and autophagy. EnnA also causes exposure of calreticulin and Hsp90 on the surface of cancer cells, leading to cancer antigen cross-presentation to DCs and activation of CD8 + T cells and their mobilization through the CX3CR1 pathway to the tumor site. CD8 + T cell suppression by cancer cells is reduced by downregulation of PD-L1 expression. Data are represented as a mean ± S.E.M. ∗∗p < 0.01; ∗∗∗∗p < 0.0001 by one-way ANOVA or unpaired tailed Student’s t test.

Article Snippet: Purified anti-mouse CD8a , BioLegend , Cat# 100801; RRID: AB_312762.

Techniques: Flow Cytometry, Expressing, Control, Injection, Western Blot, Immunofluorescence, Fluorescence, Isolation, Immunoprecipitation, Stripping Membranes, Incubation, Purification, Binding Assay, Activity Assay, Activation Assay

Journal: iScience

Article Title: Enniatin A inhibits the chaperone Hsp90 and unleashes the immune system against triple-negative breast cancer

doi: 10.1016/j.isci.2023.108308

Figure Lengend Snippet:

Article Snippet: Purified anti-mouse CD8a , BioLegend , Cat# 100801; RRID: AB_312762.

Techniques: Affinity Purification, Purification, Recombinant, Proliferation Assay, Software

Journal: Heliyon

Article Title: Endogenous CCL21-Ser deficiency reduces B16–F10 melanoma growth by enhanced antitumor immunity

doi: 10.1016/j.heliyon.2023.e19215

Figure Lengend Snippet: List of reagent and resource used in this study.

Article Snippet: Six-week-old Ccl21a- KO mice were injected intraperitoneally with 100 μg of GoInVivo purified anti-mouse CD8 mAb (clone 53–6.7, BioLegend) or allogeneic isotype immunoglobulin (control Ig) three days prior to B16–F10 injection.

Techniques: Concentration Assay, Plasmid Preparation

Journal: Heliyon

Article Title: Endogenous CCL21-Ser deficiency reduces B16–F10 melanoma growth by enhanced antitumor immunity

doi: 10.1016/j.heliyon.2023.e19215

Figure Lengend Snippet: Analysis of tumor-infiltrated T cell subsets in wild-type and Ccl21a -deficient mice. The B16–F10-derived tumor generated in WT and Ccl21a -KO mice were harvested two weeks after injection. The tumor-infiltrated T cell subset was analyzed by flow cytometry. (A) The lymphocyte subset was distinguished by the forward and side scatter profiles, and the proportion of CD8 + or CD4 + cells in the CD3 + cell population was analyzed. (B) The percentage ( n = 9 in WT and n = 8 in KO) of CD3 + cells in the tumor. Each dot represents an individual sample. The data were analyzed by Mann-Whitney U test (**, P < 0.01). Red lines represent the median. (C) The percentage of CD8 + or CD4 + cells in the CD3 + cell fraction ( n = 6). The data are shown as mean ± SD analyzed by Student's t- test (*, P < 0.05). (D) The percentage of naïve (CD44 − CD62L + ) or activated (CD44 + CD62L − ) T cells in the CD4 + (WT: n = 11, KO: n = 16) and the CD8 + cell subsets (WT: n = 9, KO: n = 8) were analyzed. The data are shown as mean ± SD analyzed by Student's t -test (*, P < 0.05, NS: Not Significant).

Article Snippet: Six-week-old Ccl21a- KO mice were injected intraperitoneally with 100 μg of GoInVivo purified anti-mouse CD8 mAb (clone 53–6.7, BioLegend) or allogeneic isotype immunoglobulin (control Ig) three days prior to B16–F10 injection.

Techniques: Derivative Assay, Generated, Injection, Flow Cytometry, MANN-WHITNEY

Journal: Heliyon

Article Title: Endogenous CCL21-Ser deficiency reduces B16–F10 melanoma growth by enhanced antitumor immunity

doi: 10.1016/j.heliyon.2023.e19215

Figure Lengend Snippet: The effect of intratumoral CD8 + T cells on melanoma growth. (A) The effect of CD8 + T cell depletion on tumor weight in the Ccl21a -KO mice. Anti-CD8 antibody or control immunoglobulin (control Ig) was administered to the Ccl21a -KO mice ( n = 3) three days before, on days 0 and 7 of transplantation. The percentage of CD8 + cells and tumor weight in each mouse two weeks after transplantation was measured. (B) The expression of tumor-reactive T cell marker molecules (PD-1, Ki-67, IFN-γ, TRP2 tetramer) in the intratumoral CD8 + T cells. Results are representative of two biological repeats.

Article Snippet: Six-week-old Ccl21a- KO mice were injected intraperitoneally with 100 μg of GoInVivo purified anti-mouse CD8 mAb (clone 53–6.7, BioLegend) or allogeneic isotype immunoglobulin (control Ig) three days prior to B16–F10 injection.

Techniques: Control, Transplantation Assay, Expressing, Marker

Journal: Heliyon

Article Title: Endogenous CCL21-Ser deficiency reduces B16–F10 melanoma growth by enhanced antitumor immunity

doi: 10.1016/j.heliyon.2023.e19215

Figure Lengend Snippet: Changes in T cell subsets in the tumor-draining LNs of Ccl21a -KO mice. The number and percentage of T cells in the tumor-draining inguinal LNs of tumor-bearing or control mice were analyzed by flow cytometry. (A) The number of CD3 + , CD4 + , and CD8 + cells in the inguinal LNs of control or tumor-bearing mice ( n = 9). (B) The percentage of CD4 + or CD8 + T cells in the CD3 + cell subset. (C) Naïve (CD62L high CD44 lo ), effector (CD62L lo CD44 mid ), and effector memory (CD62L lo CD44 high , Tem) T cell percentage of CD4 + and CD8 + T cells in WT and Ccl21a -KO mice of tumor-bearing mice ( n = 10). The data are shown as mean ± SD analyzed by Student's t- test (*, P < 0.05, NS: Not Significant).

Article Snippet: Six-week-old Ccl21a- KO mice were injected intraperitoneally with 100 μg of GoInVivo purified anti-mouse CD8 mAb (clone 53–6.7, BioLegend) or allogeneic isotype immunoglobulin (control Ig) three days prior to B16–F10 injection.

Techniques: Control, Flow Cytometry

Journal: Heliyon

Article Title: Endogenous CCL21-Ser deficiency reduces B16–F10 melanoma growth by enhanced antitumor immunity

doi: 10.1016/j.heliyon.2023.e19215

Figure Lengend Snippet: The effect of CD8 + T cells in B16–F10-bearing Ccl21a -KO mice on in vitro melanoma growth. Splenocytes prepared form no tumor control or B16–F10-bearing wild-type or Ccl21a -KO mice two weeks after B16–F10 transplantation were cocultured with or without B16–F10 cells for three days. (A) The frequencies of activated CD8 + T cells (CD62L − CD44 mid and CD62L − CD44 hi ) with B16–F10 tumor ( n = 12, 22, 20, 18, 8, 8) or without B16–F10 tumor ( n = 11, 6, 20, 15, 8, 6) were analyzed by flow cytometry. Statistical significance was defined by Mann-Whitney U test. NS: Not Significant *: P < 0.05 (B) Effector activity of spleen-derived CD8 + T cells was evaluated by the bioluminescence derived from the target B16–F10 cell viability. The luminescence values is expressed as 100% of the maximum value when responder cells are not added. *: P < 0.05 (C) Bystander cytotoxicity with KO-derived CD8 + T cells was analyzed using LLC as target cells ( n = 3).

Article Snippet: Six-week-old Ccl21a- KO mice were injected intraperitoneally with 100 μg of GoInVivo purified anti-mouse CD8 mAb (clone 53–6.7, BioLegend) or allogeneic isotype immunoglobulin (control Ig) three days prior to B16–F10 injection.

Techniques: In Vitro, Control, Transplantation Assay, Flow Cytometry, MANN-WHITNEY, Activity Assay, Derivative Assay

Journal: Heliyon

Article Title: Endogenous CCL21-Ser deficiency reduces B16–F10 melanoma growth by enhanced antitumor immunity

doi: 10.1016/j.heliyon.2023.e19215

Figure Lengend Snippet: Tregs in the tumor-draining LNs and B16–F10-derived tumors of WT and Ccl21a -KO mice. The number and percentage of tumor-infiltrated Tregs in B16–F10-derived tumors in WT and Ccl21a -KO mice were analyzed by flow cytometry. Each plot represents an individual sample. (A) The number of Tregs in the tumor-draining inguinal LNs of control (n = 9) or tumor-bearing mice (WT: n = 10, KO: n = 9, mean ± SD). The data are shown as mean ± SD analyzed by Student's t -test (*, P < 0.05, NS: Not Significant). Each dot represents an individual sample. (B) The percentage of CD4 + lymphocyte subset (left) and tumor-infiltrated Tregs of CD4 + cells (right) in tumor-bearing WT and Ccl21a -KO mice (WT: n = 10, KO: n = 11). The data are shown as Mann-Whitney U test (*, P < 0.05, NS: Not Significant). Red lines represent the median. (C) A scatter plot of mouse tumor weight versus intratumoral Treg/activated CD8 ratio (WT: n = 9, KO: n = 8). (D) The percentage of PD-1 + (WT: n = 11, KO: n = 9), Tim-3 + (WT: n = 11, KO: n = 9), and TIGIT + (WT: n = 14, KO: n = 12) of tumor-infiltrated Tregs. The data are shown as Mann-Whitney U test (*, P < 0.05, NS: Not Significant).

Article Snippet: Six-week-old Ccl21a- KO mice were injected intraperitoneally with 100 μg of GoInVivo purified anti-mouse CD8 mAb (clone 53–6.7, BioLegend) or allogeneic isotype immunoglobulin (control Ig) three days prior to B16–F10 injection.

Techniques: Derivative Assay, Flow Cytometry, Control, MANN-WHITNEY

Journal: Cell reports

Article Title: SMYD3 represses tumor-intrinsic interferon response in HPV-negative squamous cell carcinoma of the head and neck

doi: 10.1016/j.celrep.2023.112823

Figure Lengend Snippet: (A) C57BL/6 mice were injected with MOC1 cells in the right flank, and, once they reached an average tumor volume of 0.05cm 3 (day 6), treatment with Smyd3 or control ASOs was initiated and continued up to day 26 post tumor implantation. Mice were sacrificed and tumors were resected and formalin-fixed. Top left: average tumor volumes, control ASO 25 mg/kg, n = 4; Smyd3 ASO 25 mg/kg, n = 5; control ASO 50 mg/kg, n = 5; Smyd3 ASO 50 mg/kg, n = 5. Top right: percentage CD8 + T cell infiltration (CD8 IHC) assessed on MOC1 sections of tumors treated with control or Smyd3 ASOs. Data are represented as mean ± SEM, Student’s t test, *p = 0.03; NS, non-significant. Bottom left, representative examples of IHC for control or Smyd3 ASOs in MOC1 tumors. Scale bar, 100 μm. Bottom right: IHC for CD8 in MOC1 tumors treated with control or Smyd3 ASOs. Scale bar, 100 μm. (B) Left: average tumor volumes of MOC1 tumors treated with PBS (n = 10), control ASOs at 25 mg/kg (n = 10) and Smyd3 ASOs at 25 mg/kg (n = 10) and 12.5 mg/kg (n = 10). Treatment was initiated once tumors reached an average of 0.1 cm 3 . Differences among all groups were non-significant. Middle: average weight of mice per treatment group (day 38 post-tumor implantation). Right, qRT-PCR for Smyd3 mRNA of MOC1 tumors treated as indicated (n = 5 per group). Data are represented as mean ± SEM. Unpaired t test with Welch’s correction, *p < 0.05, ***p < 0.001. (C) Multicolor flow cytometry of MOC1 tumors treated with PBS, control ASOs at 25 mg/kg, and Smyd3 ASOs at 25 mg/kg and at 12.5 mg/kg (day 39 post tumor implantation, n = 5 per group). Data are represented as mean ± SEM. Unpaired t test between control ASO at 25 mg/kg and Smyd3 ASO at 12.5 mg/kg, *p < 0.05. (D) Top: average tumor volumes of flank MOC1 tumors in C57BL/6 mice treated with control ASOs plus isotype immunoglobulin (Ig) G, Smyd3 ASOs plus isotype IgG, control ASOs plus anti-PD-1, and Smyd3 ASOs plus anti-PD-1 (n = 8 mice per group). Data are represented as mean ± SEM. Unpaired t test, **p = 0.004. Bottom: hairline growth curves of flank MOC1 tumors treated with control ASOs plus anti-PD-1 and Smyd3 ASOs plus anti-PD-1. Similar results were obtained in two independent experiments.

Article Snippet: Mouse anti-CD8A , SinoBiological , Cat# 50389-T26.

Techniques: Injection, Tumor Implantation, Quantitative RT-PCR, Flow Cytometry

Journal: Cell reports

Article Title: SMYD3 represses tumor-intrinsic interferon response in HPV-negative squamous cell carcinoma of the head and neck

doi: 10.1016/j.celrep.2023.112823

Figure Lengend Snippet: (A) SMYD3 mRNA expression levels are higher in HPV-negative HNSCC tumors compared to normal squamous epithelium (TCGA). Wilcoxon rank-sum test, ***p < 0.001. (B) Representative images of IHC staining for SMYD3 in normal squamous epithelium, dysplastic epithelium, and HPV-negative HNSCC sections. Red arrows show examples of SMYD3-stained nuclear speckles. Scale bar, 10 μm. (C) Distribution of IHC scores among normal squamous epithelium, dysplastic epithelium and HPV-negative HNSCC tumors. Kruskal-Wallis test, ***p < 0.001. (D) Median IHC score for SMYD3 in normal squamous epithelium, dysplastic epithelium, and HPV-negative HNSCC tumors. Dunn test for multiple comparisons, *p < 0.05, **p < 0.01. (E) Scatterplots of protein abundance showing correlations for SMYD3, UHRF1, and CD8A. Correlations were conducted in 108 HPV-negative HNSCC tumor samples of the CPTAC. R , Pearson correlation coefficient. (F) Representative images of IHC in HPV-negative HNSCC tumor sections with high SMYD3/low CD8A or low SMYD3/high CD8A staining (left, scale bar, 100 μm) and high SMYD3/high UHRF1 or low SMYD3/low UHRF1 staining (right, scale bar, 50 μm).

Article Snippet: Mouse anti-CD8A , SinoBiological , Cat# 50389-T26.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Cell reports

Article Title: SMYD3 represses tumor-intrinsic interferon response in HPV-negative squamous cell carcinoma of the head and neck

doi: 10.1016/j.celrep.2023.112823

Figure Lengend Snippet: (A) Boxplot showing the correlation between baseline SMYD3 (left), UHRF1 (middle), and CD8A (right) mRNA levels and clinical to pathological downstaging after one dose of neoadjuvant pembrolizumab in 20 oral cavity, treatment-naive, HPV-negative HNSCC patients. Sixteen patients without and four patients with clinical to pathological downstaging. Wilcoxon test for SMYD3 , p = 0.022; for UHRF1 , p = 0.49; for CD8A , p = 0.09. (B and C) Kaplan-Meier OS (n = 61 patients) (B) and PFS curve (n = 54) (C) in HPV-negative HNSCC patients of the CPTAC. Patients with higher SMYD3/UHRF1 protein levels at baseline had significantly worse OS and PFS.

Article Snippet: Mouse anti-CD8A , SinoBiological , Cat# 50389-T26.

Techniques:

Journal: Cell reports

Article Title: SMYD3 represses tumor-intrinsic interferon response in HPV-negative squamous cell carcinoma of the head and neck

doi: 10.1016/j.celrep.2023.112823

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-CD8A , SinoBiological , Cat# 50389-T26.

Techniques: Recombinant, Staining, Purification, Negative Control, CRISPR, Plasmid Preparation, Software, Blocking Assay