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 normalizes tumor vasculature.

<p>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. <b>A.1.</b> Tumor growth curve till day 27 is presented. *<i>p</i><0.01. <b>A.2.</b> Mice were sacrificed and their angiogenic profile was studied and presented in photographs and bar diagrams (Mean±SD of pixel values). *<i>p</i><0.01. <b>A.3.</b> Differentially dilated angiogenic vessels as shown in a representative figure (<i>inset</i>) were counted from NLGP and PBS treated mice and presented in bar diagram. *<i>p</i><0.05; **<i>p</i><0.001. <b>B.</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. <b>C.</b> Mean index of tumor angiogenesis is presented in bar diagram. **<i>p</i><0.001.</p

Primer sequences of various cytokine genes studied.

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

NLGP mediated normalization of tumor vasculature is dependent on CD8⁺ T cells.

<p>B16 melanoma tumors were harvested from both PBS and NLGP pretreated C57BL/6 mice. <b>A.1.</b> Immune infiltration within tumors from PBS and NLGP treated mice were assessed histologically (H&E). <b>A.2.</b> Status of CD8⁺ T cells in blood was assessed by flow cytometry. <b>A.3.</b> Bar diagram shows the status of CD8⁺ T cells within carcinoma and melanoma tumors. *<i>p</i><0.01. <b>B.1.</b> Schematic presentation of control and CD8⁺ T cell depletion in either PBS or NLGP pretreated mice. <b>B.2.</b> Status of CD8⁺ T cells in all four mice groups (PBS, NLGP, PBS-CD8 dep and NLGP CD8 dep) were presented with representative figures. <b>C.</b> Representative picture of tumors, tumor growth curve and angiogenesis of PBS and NLGP pretreated mice with or without CD8⁺ T cell depletion. <i>p</i><0.001. <b>D.1.</b> 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, <i>cd31 and vegf</i> at transcriptional level by RT-PCR and <b>D.2.</b> densitometric analysis of band intensities from 3 individual observations (Mean ± SD) is presented. *<i>p</i><0.001, **<i>p</i><0.01. <b>D.3.</b> 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.</p

MITA^* relating tumor volume and angiogenesis in NLGP pretreated mice.

<p>*Mean index of tumor angiogenesis. Mean is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110040#pone-0110040-g001" target="_blank">Figure 1C</a></p><p>MITA^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110040#nt101" target="_blank">*</a>} relating tumor volume and angiogenesis in NLGP pretreated mice.</p

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.</p

Neem leaf glycoprotein prevents post-surgical sarcoma recurrence in Swiss mice by differentially regulating cytotoxic T and myeloid-derived suppressor cells

<div><p>Post-surgical tumor recurrence is a common problem in cancer treatment. In the present study, the role of neem leaf glycoprotein (NLGP), a novel immunomodulator, in prevention of post-surgical recurrence of solid sarcoma was examined. Data suggest that NLGP prevents tumor recurrence after surgical removal of sarcoma in Swiss mice and increases their tumor-free survival time. In NLGP-treated tumor-free mice, increased cytotoxic CD8⁺ T cells and a decreased population of suppressor cells, especially myeloid-derived suppressor cells (MDSCs) was observed. NLGP-treated CD8⁺ T cells showed greater cytotoxicity towards tumor-derived MDSCs and supernatants from the same CD8⁺ T cell culture caused upregulation of FasR and downregulation of cFLIP in MDSCs. To elucidate the role of CD8⁺ T cells, specifically in association with the downregulation in MDSCs, CD8⁺ T cells were depleted <i>in vivo</i> before NLGP immunization in surgically tumor removed mice and tumor recurrence was noted. These mice also exhibited increased MDSCs along with decreased levels of Caspase 3, Caspase 8 and increased cFLIP expression. In conclusion, it can be stated that NLGP, by activating CD8⁺ T cells, down regulates the proportion of MDSCs. Accordingly, suppressive effects of MDSCs on CD8⁺ T cells are minimized and optimum immune surveillance in tumor hosts is maintained to eliminate the residual tumor mass appearing during recurrence.</p></div

Primer list^*.

<p>Primer list^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175540#t001fn001" target="_blank">*</a>}.</p

CD8⁺ T cells play an important role in NLGP mediated prevention of tumor recurrence.

<p>(A1) Status of CD8⁺ T cells in PBS and NLGP immunized mice after tumor inoculation (n = 9). (B1) Percent positive CD8⁺ T cells in PBS and NLGP immunized mice after surgery (n = 9). A representative figure in both cases is shown in right upper corner panel (A2, B2). (C) Expression of CD69 on CD8⁺ T cells in post-surgical PBS- and NLGP-treated mice (n = 9). (D) Flow cytometric analysis of Granzyme B on CD8⁺ T cells in post-surgical PBS- and NLGP-treated mice (n = 9). Bar diagrams along with representative figures in right panel are shown (C,D). (E) Experimental design showing sarcoma inoculation, CD8⁺ T cell depletion, NLGP immunization and blood collection. (F) Circulating CD8⁺ T cell status following <i>in vivo</i> depletion of same cells. (G) Table showing number of recurrent tumor bearing and tumor free mice. (H) Tumor growth curve of recurrent tumor bearing mice in CD8⁺ T cell depleted NLGP immunized mice (n = 6). (I) Survivability curve in NLGP-treated post-surgery mice with or without CD8⁺ T cell depletion (n = 6). (J) RT-PCR analysis of the expression of IFNγ, Perforin and Granzyme B gene expression profile in partial CD8⁺ T cell depleted post-surgery NLGP-treated mice. The bar diagram represents the mean ± SD of three individual observations from each group at each time point (**<i>p</i><0.001,*<i>p</i><0.01).</p

CD8⁺ T cells downregulate MDSCs in Fas dependent pathway.

<p>(A) Percentage of Annexin V-PI⁺ MDSCs within the blood of PBS, NLGP, CD8⁺ T cell depleted NLGP immunized mice (n = 6). (B) Flow cytometric assessment of Gr1⁺FasR⁺ MDSCs in post-surgery PBS-, NLGP-treated mice with or without CD8⁺ T cell depletion. (C) Expression of FasL within CD8⁺ T cells in mice with tumor surgery in PBS and NLGP immunized mice. (D) Flow cytometric assessment of Caspase 3 within Gr1⁺ MDSCs in PBS, NLGP and CD8 depleted NLGP immunized mice. (E) Protein level expression of Caspase 3, Caspase 8 and cFLIP within MDSCs from PBS, NLGP and CD8 depleted NLGP immunized surgically tumor removed mice. (n = 6, in each group). (F) Experimental design with MDSCs and CD8⁺ T cells. (G1) Expression of FasL within NLGP-treated CD8⁺ T cells. (G2) Expression of cFLIP and FasR within MDSCs in the presence and absence of supernatants from NLGP-treated CD8⁺ T cells, with or without IFNγ neutralization. (H) Assessment of the cytotoxic potential of NLGP-treated CD8⁺ T cells towards tumor-derived MDSCs, in the presence of Brefeldin A and Concanamycin A. (**<i>p</i><0.001,*<i>p</i><0.01). (n = 3, in each group). Bar diagrams along with representative figures are present in each case (A-C).</p