NLGP mediated vascular normalization is not due to CD8⁺ T cell mediated apoptosis of CD31⁺ cells.

<p>Mice were inoculated with B16 melanoma cells (2×10⁵ cells/mice) to grow tumor. After reaching the tumor volume to a considerable size (1372 mm³ approximately), tumor was harvested and CD31⁺ VECs were isolated by flow sorting. CD8⁺ T lymphocytes were isolated from NLGP or PBS pretreated (4×) mice by MACS purification and CD8⁺ T cells were co-cultured with the CD31⁺ VECs. <b>A.</b> Cytotoxicity was measured by LDH release assay. NLGP pretreated C57BL/6 mice were inoculated with B16 melanoma cells as mentioned earlier. <b>B.1.</b> As tumor reached a considerable volume (1372 mm³ approximately), tumors were harvested, single cells prepared and stained with anti-CD31 antibody along with either Annexin V or Propidium Iodide (PI)<b>.</b> Representative figures of Annexin-V and PI⁺ cells from CD31 gated population. <b>B.2.</b> Bar diagram showing % positive cells and MFI. <b>C.</b> Cell lysates prepared from carcinoma and melanoma tumors of PBS and NLGP pretreated mice were used to quantitate the level of VEGF by ELISA. Cytokines were measured as pg/mg of tissue ± SE and Mean ± SD of 3 individual observations are presented in bar diagram. *<i>p</i><0.01. <b>D.1.</b> Obtained cells as mentioned in <b>B.1</b>, were stained for CD31, along with Ki67. Gated CD31⁺ population was assessed for Ki67 staining using Flowjo software and presented in histogram. <b>D.2.</b> Cryo-sections obtained from tumors of NLGP and PBS pretreated mice were stained with fluorescence labeled anti-CD31 (red) and anti-Ki67 (green) antibodies, along with DAPI (blue). Representative figures from 3 separate sets of experiments are presented. <b>D.3.</b> PBS and NLGP pretreated tumor bearing mice were injected with BrdU within tumor and sacrificed after 48 hours. Single cells were prepared to check BrdU staining after gating the CD31 population, as shown in a representative figure. <b>E.1, E.2.</b> PBMC were isolated from both PBS and NLGP treated tumor bearing mice and cultured with EC cells (2×10⁵ cells) for 24 hours and cell free supernatant were measured in pg/ml for VEGF (E.1) and IFNγ (E.2) by ELISA. Cytokines were quantitated as pg/ml ± SE. *<i>p</i><0.001, **<i>p</i><0.01. <b>E.3.</b> Flow sorted CD31⁺ ECs isolated from tumor microenvironment were cultured with the above mentioned supernatants (NLGP-PBMC+EC vs PBS-PBMC+EC) for 48 hours and assessed for the EC proliferation flow cytometrically after Ki67 labeling.