Figure 1.
NLGP modulates immunosuppressive cytokine milieu within TME.
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. *p<0.001, **p<0.05, in comparison to PBS treated tumor on day 15 and 20. B.1. 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.2. Densitometric analysis was performed in each case, ▪p<0.01. C.1. STAT3 and pSTAT3 levels were studied in total protein isolated from PBS and NLGP tumors (n = 3, in each case) by Western blot analysis, C.2. and data from three individual observations was analyzed by densitometric scanning, in comparison to PBS treated tumor on day 15 and 20, ▪p<0.01.
Figure 2.
NLGP normalizes protumor angiogenic and hypoxic TME.
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. A. 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. *p<0.001, **p<0.01, in comparison to PBS treated tumor on day 15 and 20. 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 C.1. 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 C.2. Densitometric analysis was performed in each case. Frozen sections of tumors from either PBS or NLGP treated mice were stained D.1. immunohistochemically with monoclonal antibodies, specific for VEGF, VEGFR1, VEGFR2, HIF1α and TGFβ and D.2. with fluorescence tagged anti-CD31 antibody E.1. 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. E.2. Densitometric analysis was performed in each case.
Figure 3.
NLGP downregulates suppressor cells within TME.
Single cell suspension was prepared from day 20 tumors of PBS and NLGP treated mice. A. Proportion of activated T cells B.1. CD4+CD25+ cells, B.2. Tregs, C. 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. *p<0.001, **p<0.05. D.1. Total protein was isolated from tumors at different days and level of Foxp3 was analyzed by immunoblot analysis. D.2. Densitometric analysis was performed in each case. E.1. Total RNA was also purified to assess the expression status of CTLA4 and IDO on different time points of tumor growth. E.2. Densitometric analysis was performed in each case.
Figure 4.
NLGP normalizes chemokine network within TME.
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, A.1. ccr5, ccl3, ccl4, ccl5, ccl8; B.1. cxcr3, cxcl9, cxcl10; C.1. cxcr4, cxcl12. A.2, B.2, C.2. Densitometric analysis was performed in each case.
Figure 5.
NLGP normalizes T cell functions within TME.
Lysates were prepared from tumors of PBS and NLGP treated mice, designated as PBS-TME and NLGP-TME. MNCs from normal mice were incubated with PBS-TME and NLGP-TME, prepared from tumors of different days for 120 hrs. A. percentage of CD8+CD69+ T cells was analyzed within different TME treated MNCs *p<0.01, **p<0.05. B. CD8+ T cells were then allowed to proliferate for 72 hrs and proliferation was determined by MTT assay. NLGP stimulation in MNCs was kept as control. *p<0.01. C. After 120 hrs of incubation with different TMEs, CD8+ T cells were purified by MACS. Cytotoxicity of these cells towards sarcoma 180 cells was assessed by LDH release assay. ▴p<0.01. D. Different growth factors (VEGF, TGFβ) and cytokines (IL-10, IL-6 and IL-12) in single or in combination were neutralized within PBS-TME (Tumor lysate prepared from tumor of day 20) using their respective antibodies. Splenic MNCs were then exposed to differentially neutralized PBS-TME and after 120 hrs incubation CD8+ T cells were purified by MACS. Cytotoxicity of these differentially exposed CD8+ T cells towards Sarcoma 180 cells was measured. •p<0.001, +p<0.01 in comparison to PBS-TME. E. CD8+ T cells purified in similar fashion as described in C and cultured for 48 hrs. Cell supernatants were used to measure IFNγ level by ELISA. NLGP stimulation in normal CD8+ T cells was kept as control. p = 0.0023. In every case, comparison was made between PBS-TME exposed T cells vs same exposed to NLGP-TME on day 15 and 20, ▴p<0.01, **p<0.05.
Figure 6.
NLGP protects CD8+ T cells from anergy within TME.
MNCs were isolated from normal mice and exposed to PBS-TME/NLGP-TME for 120 hrs and ionomycin for 48 hrs, as a positive control in vitro. A. Then CD8+ T cells were purified by MACS to isolate total RNA. RT-PCR analysis was performed for different anergy related genes, B.1. CD8+ T cells were directly purified from tumors of PBS and NLGP treated mice in in vivo condition and total RNA was isolated. Different anergy related genes were analyzed at transcriptional level by RT-PCR, B.2. Densitometric analysis was performed in each case. C. pNFAT and NFAT were analyzed at protein level, purified from CD8+ T cells as mentioned in A, by Western blotting D.1. MNCs were isolated from normal mice and exposed to PBS-TME and NLGP-TME for 120 hrs in vitro. Then CD8+ T cells were purified by MACS to isolate total RNA and protein. RT-PCR analysis was performed for Fas-R, cFlip, D.2. Densitometric analysis was performed in each case. E. and Western blot for Fas-R, cFlip, FasL, F. and activated Caspase3 and Caspase8. G. MNCs were purified from tumors of PBS and NLGP treated mice and assessed for FasR+CD8+ T cells by flow cytometry. *p<0.001.
Figure 7.
NLGP enhances T cell migration to TDLN and TIL to effectively kill tumors in vivo.
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, **p<0.001, *p = 0.024. 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 , *p = 0.009, **p = 0.008, ▪p = 0.005, in comparison to PBS treated tumor on day 20. C.1. 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. C.2. Densitometric analysis was performed in each case. D. 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 *p = 0.01, ▪p = 0.007, E. 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 Materials and Methods (n = 3). Mean tumor volume ± SD and survivability are presented, *p<0.001.
Table 1.
Primer sequences of genes related to chemokine receptor and ligands.
Table 2.
Primer sequences of various genes of cytokine, growth factors etc.
Table 3.
Primer sequences of apoptosis, anergy and AICD related genes.