NLGP normalizes chemokine network within TME.

<p>Total RNA was isolated from tumors of PBS and NLGP treated mice. cDNA was prepared from RNA and PCR was performed for different chemokine related genes, <b>A.1.</b> ccr5, ccl3, ccl4, ccl5, ccl8; <b>B.1.</b> cxcr3, cxcl9, cxcl10; <b>C.1.</b> cxcr4, cxcl12. <b>A.2, B.2, C.2.</b> Densitometric analysis was performed in each case.</p

NLGP enhances T cell migration to TDLN and TIL to effectively kill tumors <i>in vivo</i>.

<p>A. MNCs were isolated from normal mice and exposed to PBS-TME and NLGP-TME for 120 hrs, along with a set of unexposed cells. Then CD8⁺ T cells were purified by MACS. Purified cells were labeled with CFSE and injected into three groups of mice having tumors of identical volume. After 24 hrs migration of CD8⁺CFSE⁺ cells were detected in TDLN and TIL, **<i>p</i><0.001, *<i>p = </i>0.024. <b>B.</b> Depletion of CD8 impairs NLGP mediated TME normalization. Tumor tissue lysates, representing TME from either NLGP or CD8 depleted NLGP treated mice (n = 4 in each case) were assessed for IFNγ, IL-2, IL-12, IL-6, TGFβ and IL-10 by ELISA. Cytokines were quantitated as pg/mg of tumor tissue ± SE , *<i>p</i> = 0.009, **<i>p</i> = 0.008, ^▪<i>p = </i>0.005, in comparison to PBS treated tumor on day 20. <b>C.1.</b> Total RNA was isolated from tumor of PBS and NLGP treated mice (n = 4 in each case) to analyze genes of IFNγ, IL-2, IL-12, IL-6, TGFβ and IL-10 by RT-PCR. <b>C.2.</b> Densitometric analysis was performed in each case. <b>D.</b> MNCs were isolated from normal mice and exposed to NLGP-TME and CD8 depleted NLGP-TME for 120 hrs. Then CD8⁺ T cells were purified by MACS and T-Cell proliferation, IFNγ release and cytotoxicity towards Sarcoma180 were measured *<i>p</i> = 0.01, ^▪<i>p = </i>0.007, <b>E.</b> MNCs were isolated from normal mice and exposed to PBS-TME, NLGP-TME and CD8 depleted NLGP-TME for 120 hrs, along with a set of unexposed cells. Then CD8⁺ T cells were purified by MACS and activated CD8⁺ T cells (1×10⁷ cells) were adoptively transferred through tail vein into four groups of tumor bearing mice (n = 6 in each group) once weekly as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066501#s4" target="_blank">Materials and Methods</a> (n = 3). Mean tumor volume ± SD and survivability are presented, *<i>p</i><0.001.</p

Primer sequences of various genes of cytokine, growth factors etc.

<p>Primer sequences of various genes of cytokine, growth factors etc.</p

Primer sequences of genes related to chemokine receptor and ligands.

<p>Primer sequences of genes related to chemokine receptor and ligands.</p

NLGP normalizes protumor angiogenic and hypoxic TME.

<p>Sarcoma 180 tumor tissue (100 mg) harvested from Swiss albino mice was lysed by freeze-thaw technique in 1 ml PBS supplemented with a cocktail of protease inhibitors. <b>A.</b> Tumor tissue lysates, representing TME from either PBS or NLGP treated mice (n = 6 in each case) were assessed for TGFβ and VEGF by ELISA. Cytokines were quantitated as pg/mg of tissue ± SE. *<i>p<</i>0.001, **<i>p<</i>0.01, in comparison to PBS treated tumor on day 15 and 20. <b>B.</b> Total protein was isolated from PBS and NLGP treated tumors (n = 3 in each case) to assess TGFβ, VEGF, HIF1α, VEGFR1, VEGFR2 and β-actin by Western blot analysis <b>C.1.</b> Total RNA was isolated from tumors of PBS and NLGP treated mice (n = 3 in each case) to analyze genes, like, VEGF, HIF1α, VEGFR1, VEGFR2 and TGFβ at transcriptional level by RT-PCR <b>C.2.</b> Densitometric analysis was performed in each case. Frozen sections of tumors from either PBS or NLGP treated mice were stained <b>D.1.</b> immunohistochemically with monoclonal antibodies, specific for VEGF, VEGFR1, VEGFR2, HIF1α and TGFβ and <b>D.2.</b> with fluorescence tagged anti-CD31 antibody <b>E.1.</b> Total RNA from tumors of PBS and NLGP treated mice was used to determine the status of perforin, granzyme B on day 15 and 20 of tumor inoculation. <b>E.2.</b> Densitometric analysis was performed in each case.</p

NLGP downregulates suppressor cells within TME.

<p>Single cell suspension was prepared from day 20 tumors of PBS and NLGP treated mice. <b>A.</b> Proportion of activated T cells <b>B.1.</b> CD4⁺CD25⁺ cells, <b>B.2.</b> Tregs, <b>C.</b> and MDSCs within tumors from these two groups of mice was determined flow cytometrically after labeling cells with fluorescence tagged CD8, CD69, CD4, CD25 and CD11b, GR1 antibodies. *<i>p</i><0.001, **<i>p</i><0.05. <b>D.1.</b> Total protein was isolated from tumors at different days and level of Foxp3 was analyzed by immunoblot analysis. <b>D.2.</b> Densitometric analysis was performed in each case. <b>E.1.</b> Total RNA was also purified to assess the expression status of CTLA4 and IDO on different time points of tumor growth. <b>E.2.</b> Densitometric analysis was performed in each case.</p

Neem Leaf Glycoprotein Prophylaxis Transduces Immune Dependent Stop Signal for Tumor Angiogenic Switch within Tumor Microenvironment

<div><p>We have reported that prophylactic as well as therapeutic administration of neem leaf glycoprotein (NLGP) induces significant restriction of solid tumor growth in mice. Here, we investigate whether the effect of such pretreatment (25µg/mice; weekly, 4 times) benefits regulation of tumor angiogenesis, an obligate factor for tumor progression. We show that NLGP pretreatment results in vascular normalization in melanoma and carcinoma bearing mice along with downregulation of CD31, VEGF and VEGFR2. NLGP pretreatment facilitates profound infiltration of CD8⁺ T cells within tumor parenchyma, which subsequently regulates VEGF-VEGFR2 signaling in CD31⁺ vascular endothelial cells to prevent aberrant neovascularization. Pericyte stabilization, VEGF dependent inhibition of VEC proliferation and subsequent vascular normalization are also experienced. Studies in immune compromised mice confirmed that these vascular and intratumoral changes in angiogenic profile are dependent upon active adoptive immunity particularly those mediated by CD8⁺ T cells. Accumulated evidences suggest that NLGP regulated immunomodulation is active in tumor growth restriction and normalization of tumor angiogenesis as well, thereby, signifying its clinical translation.</p></div

NLGP modulates immunosuppressive cytokine milieu within TME.

<p>A. Sarcoma 180 tumor tissues (100 mg) harvested from Swiss albino mice was lysed by freeze-thaw technique in 1 ml PBS supplemented with a cocktail of protease inhibitors. Tumor tissue lysates, representing TME from either PBS or NLGP treated mice (n = 6 in each case) were assessed for IFNγ, IL-2, IL-12, IL-6 and IL-10 by ELISA. Cytokines were quantitated as pg/mg of tumor tissue ± SE. *<i>p</i><0.001, **<i>p</i><0.05, in comparison to PBS treated tumor on day 15 and 20. <b>B.1.</b> Total RNA was isolated from tumor of PBS and NLGP treated mice (n = 6 in each case) to analyze genes of IFNγ, IL-2, IL-12, IL-6 and IL-10 by RT-PCR. <b>B.2.</b> Densitometric analysis was performed in each case, ▪<i>p</i><0.01. <b>C.1.</b> STAT3 and <i>p</i>STAT3 levels were studied in total protein isolated from PBS and NLGP tumors (n = 3, in each case) by Western blot analysis, <b>C.2.</b> and data from three individual observations was analyzed by densitometric scanning, in comparison to PBS treated tumor on day 15 and 20, ▪<i>p</i><0.01.</p

Primer sequences of apoptosis, anergy and AICD related genes.

<p>Primer sequences of apoptosis, anergy and AICD related genes.</p

Primer sequences of various cytokine genes studied.

<p>Primer sequences of various cytokine genes studied.</p