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Neem leaf glycoprotein prophylaxis transduces immune dependent stop signal for tumor angiogenic switch within tumor microenvironment.

Banerjee S, Ghosh T, Barik S, Das A, Ghosh S, Bhuniya A, Bose A, Baral R - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunoregulation and Immunodiagnostics, Chittaranjan National Cancer Institute (CNCI), Kolkata, India.

ABSTRACT
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.

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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.
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pone-0110040-g001: 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.

Mentions: ‘Dormant’ tumor requires both angiogenic switch and immune escape to proceed towards malignancy [7], [9]. As prophylaxis with neem leaf preparation, precursor of NLGP, previously reported to be associated with significant immune-mediated tumor growth restriction [16], [29], here, we intended to study how NLGP prophylaxis regulates pathological tumor angiogenesis. Consistent with our previous results prophylactic NLGP administration (4×) significantly restricts Ehrlich's carcinoma and B16 melanoma tumor growth (Figure 1a). Repeated investigations confirmed 4 immunizations with NLGP are required for optimum immune activation [15]–[18], [27]–[29]. Angiogenic profiles were studied in mice after establishment of tumor (in between day 21 to 32) (Figure 1A.1 and A.2) and visual observations suggested a significant decrease in heavy, very thick, thick blood vessels, while thin blood vessels were retained substantially in NLGP pretreated carcinoma and melanoma tumor bearing mice group compared to PBS controls (Figure 1A.3). Additionally, histological analysis of tumor sections demonstrated less number of blood vessels with more regularized pattern in NLGP pretreated tumors than PBS mice. This regularized pattern of blood vessels is further evidenced by downregulation of CD31, a marker of VECs (Figure 1B). Correlating angiogenic profile with tumor volume revealed that normalized angiogenesis associated with NLGP prophylaxis represents restricted tumor growth, whereas, chaotic angiogenesis is correlated well with bigger tumor volume (Table 1; Figure 1C). Therefore, these data furnish evidences that NLGP can normalize tumor vasculature by decreasing only the thick and ‘tortured’ blood vessels while retaining the more compact thin blood vessels within tumor to maintain the optimum interstitial pressure and vaccine mediated immune benefits.


Neem leaf glycoprotein prophylaxis transduces immune dependent stop signal for tumor angiogenic switch within tumor microenvironment.

Banerjee S, Ghosh T, Barik S, Das A, Ghosh S, Bhuniya A, Bose A, Baral R - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4229107&req=5

pone-0110040-g001: 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.
Mentions: ‘Dormant’ tumor requires both angiogenic switch and immune escape to proceed towards malignancy [7], [9]. As prophylaxis with neem leaf preparation, precursor of NLGP, previously reported to be associated with significant immune-mediated tumor growth restriction [16], [29], here, we intended to study how NLGP prophylaxis regulates pathological tumor angiogenesis. Consistent with our previous results prophylactic NLGP administration (4×) significantly restricts Ehrlich's carcinoma and B16 melanoma tumor growth (Figure 1a). Repeated investigations confirmed 4 immunizations with NLGP are required for optimum immune activation [15]–[18], [27]–[29]. Angiogenic profiles were studied in mice after establishment of tumor (in between day 21 to 32) (Figure 1A.1 and A.2) and visual observations suggested a significant decrease in heavy, very thick, thick blood vessels, while thin blood vessels were retained substantially in NLGP pretreated carcinoma and melanoma tumor bearing mice group compared to PBS controls (Figure 1A.3). Additionally, histological analysis of tumor sections demonstrated less number of blood vessels with more regularized pattern in NLGP pretreated tumors than PBS mice. This regularized pattern of blood vessels is further evidenced by downregulation of CD31, a marker of VECs (Figure 1B). Correlating angiogenic profile with tumor volume revealed that normalized angiogenesis associated with NLGP prophylaxis represents restricted tumor growth, whereas, chaotic angiogenesis is correlated well with bigger tumor volume (Table 1; Figure 1C). Therefore, these data furnish evidences that NLGP can normalize tumor vasculature by decreasing only the thick and ‘tortured’ blood vessels while retaining the more compact thin blood vessels within tumor to maintain the optimum interstitial pressure and vaccine mediated immune benefits.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunoregulation and Immunodiagnostics, Chittaranjan National Cancer Institute (CNCI), Kolkata, India.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus