Journal: Nature Communications

Article Title: Regulatory T cells crosstalk with tumor cells and endothelium through lymphotoxin signaling

doi: 10.1038/s41467-024-54874-y

Figure Lengend Snippet: a Transwell assay of mouse cancer cell lines. Mouse melanoma B16F10 ( n = 4), mouse breast cancer 410 ( n = 3), mouse sarcoma KPI30 ( n = 4), mouse ovarian cancer ID8 ( n = 4), and mouse mammary adenocarcinoma 66.1 ( n = 4) cells pretreated with 20 μM each of nciLT, ciLT, nciLT combined with ciLT, or CP for 1 h at 37 o C; or treated with 2 μg/mL agonist anti-mouse LTβR Ab (3C8) for 1 h at 37 o C, washed and loaded for TEM across mouse LECs toward 200 μM S1P for 16 h. b Transwell assay of human cancer cell lines. Human melanoma A375 ( n = 4), human breast cancer MDA-MB-231 ( n = 3), human lung cancer A549 ( n = 4) cells pretreated with 20 μM each of nciLT, hciLT, nciLT combined with hciLT, or CP for 1 h at 37 o C; or treated with 100 ng/mL human LTβR ligand huLTαβ for 1 h at 37 o C, washed and loaded for TEM across human LECs toward 200 μM S1P for 16 h. c Transwell TEM assay of mouse or human sarcoma cells toward medium or 200 μM S1P crossing the 8 μm Boyden chamber coated with or without mouse or human LECs, respectively. d Representative images ( n = 4) of cell migration into the area of the defect after scratching confluent B16F10 monolayers treated with indicated blocking peptides. Migration into the area of cell defect was measured after 16 h. Magnification 10x; scale bar 450 μm. e Representative images ( n = 4) of mouse breast cancer 4T1-eGFP migration into the area of cell defect at 0 and 16 h after treatment with indicated peptides. Area recovery was measured with time-lapse microscopy. Each peptide-treated group was compared to the nontreated using a paired two-tailed t -test, * p = 0.0181, **** p < 0.0001. Magnification 10x; scale bar 220 μm. f Time-lapse microscopy of WT B16F10-GFP pretreated with peptides as in ( a – c ). Samples run in triplicates. g TEM of WT or LTβR −/− B16F10 across transwell coated with or without mouse LECs toward 200 μM S1P for 16 h. One representative of three images was taken. 20x magnification. Representative of 2 ( e – g) or 3 ( a – d ) independent experiments. Mean ± SEM. **** p < 0.0001 by one-way ANOVA. Source data are provided as a Source Data file.

Article Snippet: The mouse melanoma B16F10-eGFP and mouse breast cancer 4T1-eGFP from Imanis Life Sciences (Rochester, MN), B16F10 and Human melanoma A375 from the American Type Culture Collection. were cultured in DMEM with GlutaMAX (4.5 g/L glucose, Invitrogen) containing 10% FCS (Gemini, CA) and 1% Penicillin/Streptomycin (Invitrogen).

Techniques: Transwell Assay, Migration, Blocking Assay, Time-lapse Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Regulatory T cells crosstalk with tumor cells and endothelium through lymphotoxin signaling

doi: 10.1038/s41467-024-54874-y

Figure Lengend Snippet: a – d Bulk RNA-Seq analysis of B16F10 (2 independent samples per group). Genes are downregulated by nciLT ( a ) or ciLT ( c ) and regulated by NIK ( a ) or IKKβ ( c )-deficiency. WT or CRISPR/Cas9 NIK KO B16F10 pretreated with nciLT for 1 h followed by 6 h anti-LTβR (3C8) stimulation ( a , b ). WT or CRISPR/Cas9 IKKβ KO B16F10 treated with ciLT for 1 h prior to 1 h LTβR stimulation ( c , d ). Selected genes confirmed by RT-PCR for nonclassical ( b ) and classical ( d ) regulation. Relative gene expressions are normalized to HPRT gene expression. Samples are triplicated in three independent experiments. Mean ± SEM. *** p = 0.0001, **** p < 0.0001 by one-way ANOVA. e – g Immunoblots for p100/p52 in human A357 (left panel of e , g ) and mouse B16F10 (right panel of e , f ) melanoma cells cocultured with various dose of human or mouse Tregs, Teffs, or LTα-deficient (LTα −/− ) Tregs as indicated for 5 h. A375 pretreated with 20 μM CP, nciLT, or hciLT for 1 h prior to coculturing with human T cells ( g ). Representative blots are shown from three independent experiments. Quantification of the protein expression of P52 normalized to p65. Uncropped gels are provided in the Source Data file. Mean ± SEM. *** p = 0.0002, **** p < 0.0001 by one-way ANOVA. h B16F10 pretreated with 20 μM CP, nciLT, or ciLT for 1 h, washed, and cocultured with Tregs for 6 h, washed and grown for 48 h. One representative of three images taken from each group is shown. ** p = 0.0036, *** p = 0.0005 by one-way ANOVA. i – m Migration of B16F10 cocultured with Tregs or Teffs with various ratio ( i ), Compared to B16F10 only, *** p = 0.0006 (1:2), *** p = 0.0002 (2:2), ****p < 0.0001. Migration of B16F10 cocultured with WT (**** p < 0.0001) or LTα -/- Tregs ( j , k ) as indicated for 6 h, washed, and migrated across transwell coated with LECs (**** p < 0.0001) or without LEC toward 200 μM S1P for 16 h ( j ). B16F10 pretreated with indicated peptides before coculturing with WT or LTα −/− Tregs and TEM as in ( h , k ). *** p = 0.0003. TEM of breast cancer 4T1 cells ( l ) or B cell lymphoma M12.4 cells ( m ) cocultured with WT or LTα −/− Tregs with various ratios. n , o 0.5 × 10 6 WT or LTα-deficient (LTα −/− ) Tregs intravenously transferred to LTβRKO (LTβR −/− ) C57BL/6 mice which were subcutaneously inoculated with 0.5 × 10 6 WT B16F10. Scheme of experimental setup and tumor growth ( n ). p value of tumor growth ( n = 5 mice) determined by two-way ANOVA Tukey’s multiple comparisons test. * p = 0.0394, *** p = 0.0008, **** p < 0.0001. At day 14, tumors ( n = 5) analyzed for CXCL1 (**** p < 0.0001), CXCL10 (*** p = 0.0002), and CCL5 (*** p = 0.0004) expression. Data representative of 3 ( b , d , e – m ) and 2 ( n , o ) independent experiments. b , d , e – i : Mean ± SEM. **** p < 0.0001 by one-way ANOVA. Source data are provided as a Source Data file.

Article Snippet: The mouse melanoma B16F10-eGFP and mouse breast cancer 4T1-eGFP from Imanis Life Sciences (Rochester, MN), B16F10 and Human melanoma A375 from the American Type Culture Collection. were cultured in DMEM with GlutaMAX (4.5 g/L glucose, Invitrogen) containing 10% FCS (Gemini, CA) and 1% Penicillin/Streptomycin (Invitrogen).

Techniques: RNA Sequencing Assay, CRISPR, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Migration

Journal: Nature Communications

Article Title: Regulatory T cells crosstalk with tumor cells and endothelium through lymphotoxin signaling

doi: 10.1038/s41467-024-54874-y

Figure Lengend Snippet: a Peptide-pretreated LEC cocultured with Tregs or Teffs (LEC: T cell = 1: 2) for 16 h, followed by removal of T cells, and then LEC cocultured with B16F10-GFPs (LEC: B16F10 = 1:1) for 48 h. Representative images of B16F10-GFPs in triplicated wells analyzed by EVOS microscopy. b , c Transwell assay, B16F10-GFPs migrated across WT or LTβR −/− LECs pretreated with or without Tregs as in ( a , b ). B16F10 or 4T1 cells migrated across LECs pretreated with WT or LTα −/− Tregs for 16 h ( c ). d Heatmap of gene array analysis of mouse primary dermal LECs treated with nciLT and ciLT. LECs were treated with 20 μM nciLT or ciLT for 30 min prior to LTβR stimulation with anti-LTβR Ab (3C8) for 4 h. Three independent samples per group. e Regulated genes selected for real-time PCRs. Mouse LECs pretreated with 20 μM indicated peptides for 30 min, washed and treated with or without anti-LTβR Ab (3C8) for 6 h. Triplicated samples were measured in three independent experiments. * p = 0.03, **** p < 0.0001 by one-way ANOVA. f Immunohistochemistry of intercellular VE-cadherin in lymphatic vessels (LV) or cultured LECs pretreated with WT or LTα −/− Tregs for 16 h. One representative of four images of each group. Magnification 60x; scale bar:14 μm (LV) and 7 μm (LEC). g – i Intravenous transfer of WT or LTα −/− Tregs (5 × 10 5 ) to WT C57BL/6 mice subcutaneously inoculated with LTβRKO B16F10 (5 × 10 5 ). Scheme of experimental setup and tumor growth ( g ). p value of tumor growth ( n = 6) determined by two-way ANOVA Tukey’s multiple comparisons test. ** p = 0.0017, **** p < 0.0001. At day 14, tumors ( n = 4) were analyzed for CXCL1 and CXCL10 expression in LECs by flow cytometry analysis ( h ); One representative of 6 images of FLRT2 expression in tumor LECs by Immunohistochemistry ( i ). Magnification 20x; scale bar:42 μm. j – m Intradermal transfer of B16F10 cells (5 × 10 5 ) to WT or LTβR −/− C57BL6 mice. p value of tumor growth ( n = 8) calculated by two-way ANOVA, Sidak’s multiple comparisons test, * p = 0.0362, **** p < 0.0001 ( j ). One representative of eight images of tumor (day 20) blood (CD31 + Lyve-1 − ) and lymphatic (Lyve-1 + ) vessels by immunohistochemistry (magnification 20x; scale bar 40 μm) ( k ), mouse survival (10 mice each group) ( l ) p value is calculated by log-rank (Mantel–Cox) test, * p = 0.025. and intratumoral Foxp3 + CD25 + Tregs (*** p = 0.0002) in 6 tumors (day 13) from WT or LTβR −/− C57BL6 mice by flow cytometry ( m ). n Lung colonization assessed at 3 weeks after tail vein injection of 3 × 10 5 B16F10 cells with or without primary mouse LECs (1 × 10 5 ) to WT or LTβR -/- C57BL6 mice ( n = 8 per group, five representative lung images per group shown). ** p = 0.0023, *** p = 0.0009. Representative of 3 ( a – c , e , f ) and 2 ( g – n ) independent experiments shown. a – n Mean ± SEM. **** p < 0.0001 by one-way ANOVA ( a – c , e , f , h , i , n ) or unpaired two-tailed t -test ( k , m ). Source data are provided as a Source Data file.

Article Snippet: The mouse melanoma B16F10-eGFP and mouse breast cancer 4T1-eGFP from Imanis Life Sciences (Rochester, MN), B16F10 and Human melanoma A375 from the American Type Culture Collection. were cultured in DMEM with GlutaMAX (4.5 g/L glucose, Invitrogen) containing 10% FCS (Gemini, CA) and 1% Penicillin/Streptomycin (Invitrogen).

Techniques: Microscopy, Transwell Assay, Immunohistochemistry, Cell Culture, Expressing, Flow Cytometry, Injection, Two Tailed Test