Figure 1.
NLGP normalizes tumor vasculature.
Swiss and C57BL/6 mice were pretreated with NLGP (25µg) once a week for four weeks in total followed by inoculation of EC (1×106 cells/mice) and B16 melanoma cells (2×105 cells/mice) subcutaneously. A.1. Tumor growth curve till day 27 is presented. *p<0.01. A.2. Mice were sacrificed and their angiogenic profile was studied and presented in photographs and bar diagrams (Mean±SD of pixel values). *p<0.01. A.3. Differentially dilated angiogenic vessels as shown in a representative figure (inset) were counted from NLGP and PBS treated mice and presented in bar diagram. *p<0.05; **p<0.001. B. Angiogenic blood vessels within tumors were studied by routine histology after H&E staining and CD31+ VECs were studied by immunofluorescence staining. Representative figures in each case are presented. C. Mean index of tumor angiogenesis is presented in bar diagram. **p<0.001.
Table 1.
MITA* relating tumor volume and angiogenesis in NLGP pretreated mice.
Figure 2.
NLGP normalizes tumor microenvironment by downregulating expression of VEGF, VEGFR2 and CD31.
B16 melanoma tumors (100 mm3) harvested from either PBS or NLGP pretreated C57BL/6 mice representing tumor microenvironment were used for different analysis. A.1. Representative presentation of mRNA expression levels of vegf, vegfr1, vegfr2, cd31 by RT-PCR analysis (n = 3). A.2. Densitometric analysis of three individual observations is presented with Mean ± SD. *p<0.05; **p<0.01. B.1. Another portion (100 mg) of tumors was lysed by freeze-thaw cycles in PBS used for Western Blotting to check the expression of various angiogenic proteins. Representative presentation of expression levels of different molecules as mentioned by Western blot analysis (n = 3) is shown. B.2. Densitometric analysis of three individual observations and Mean ± SD are presented. *p<0.05; **p<0.01. C.1. Immunohistochemistry with monoclonal antibodies, specific for VEGF, VEGFR1, VEGFR2 and CD31 and C.2. NG2 were detected on tumor sections. Arrows showed the pericyte coverage on endothelial cell lining on blood vessels. D. Fluorescence tagged monoclonal antibodies, specific for NG2+ (green) and CD31+ (Red) cells were used to study tumor vasculature. Nuclear staining was performed by DAPI.
Figure 3.
NLGP mediated normalization of angiogenesis is absent in immunocompromised mice.
Schematic presentation of NLGP prophylaxis in A.1. normal mice, A.2. and A.3. cyclosporine treated/immunocompromised and A.4. athymic nude mice. B.1.-B.4. Representative photographs of murine tumors with B.1. PBS, NLGP, B.2. NLGP-Cyclosporin pretreatment, B.3. NLGP pretreatment with adoptive transfer of immune cells and B.4. NLGP and PBS pretreatment (athymic nude mice). C.1.-C.4. Tumor growth curve presenting Mean ± SD. *p<0.001, **p<0.01, in comparison to NLGP group with other above mentioned group and D.1.-D.4. Angiogenic profile of mice with D.1. PBS, NLGP, D.2. NLGP-Cyclosporin pretreatment, D.3. NLGP pretreatment with adoptive transfer of immune cells and D.4. NLGP and PBS pretreatment (athymic nude mice). E. Immunohistochemical detection of VEGF, VEGFR2 and CD31 in tumor sections as mentioned in A.1–A.3.
Figure 4.
NLGP mediated normalization of tumor vasculature is dependent on CD8+ T cells.
B16 melanoma tumors were harvested from both PBS and NLGP pretreated C57BL/6 mice. A.1. Immune infiltration within tumors from PBS and NLGP treated mice were assessed histologically (H&E). A.2. Status of CD8+ T cells in blood was assessed by flow cytometry. A.3. Bar diagram shows the status of CD8+ T cells within carcinoma and melanoma tumors. *p<0.01. B.1. Schematic presentation of control and CD8+ T cell depletion in either PBS or NLGP pretreated mice. B.2. Status of CD8+ T cells in all four mice groups (PBS, NLGP, PBS-CD8 dep and NLGP CD8 dep) were presented with representative figures. C. Representative picture of tumors, tumor growth curve and angiogenesis of PBS and NLGP pretreated mice with or without CD8+ T cell depletion. p<0.001. D.1. Total RNA was isolated from tumors of PBS, NLGP, PBS-CD8 depleted group (PBS-CD8 dep) and NLGP-CD8 depleted mice (NLGP-CD8-Dep) group (n = 3 in each case) to analyze genes, like, cd31 and vegf at transcriptional level by RT-PCR and D.2. densitometric analysis of band intensities from 3 individual observations (Mean ± SD) is presented. *p<0.001, **p<0.01. D.3. Immunohistochemical analysis of tumors obtained from PBS and NLGP pretreated mice with or without CD8 depletion were performed using monoclonal antibodies, specific for CD31, VEGF and VEGFR2.
Figure 5.
NLGP mediated vascular normalization is not due to CD8+ T cell mediated apoptosis of CD31+ cells.
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. A. Cytotoxicity was measured by LDH release assay. NLGP pretreated C57BL/6 mice were inoculated with B16 melanoma cells as mentioned earlier. B.1. 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). Representative figures of Annexin-V and PI+ cells from CD31 gated population. B.2. Bar diagram showing % positive cells and MFI. C. 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. *p<0.01. D.1. Obtained cells as mentioned in B.1, were stained for CD31, along with Ki67. Gated CD31+ population was assessed for Ki67 staining using Flowjo software and presented in histogram. D.2. 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. D.3. 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. E.1, E.2. 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. *p<0.001, **p<0.01. E.3. 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.
Figure 6.
NLGP mediated vascular normalization has no adverse effect on normal wound healing process.
Swiss mice were pretreated with NLGP (25µg) and PBS once in a week for four weeks and 4 mm3 wound was made on the back of both groups of mice. A. Diameter of wounds was measured every two days and percentages of wound closure were calculated and data presented as Mean ± SD of 6 individual observations. B. Histological sections of wound beds were stained with H&E and assessed microscopically. Representative figures are presented. C. Cryo-sections of wound beds were stained with fluorescence labeled anti-CD31 (red) and anti-NG2 (green) antibodies along with DAPI.
Table 2.
Primer sequences of various cytokine genes studied.