High monocytic MDSC signature predicts multi-drug resistance and cancer relapse in non-Hodgkin lymphoma patients treated with R-CHOP

IntroductionNon-Hodgkin Lymphoma (NHL) is a heterogeneous lymphoproliferative malignancy with B cell origin. Combinatorial treatment of rituximab, cyclophsphamide, hydroxydaunorubicin, oncovin, prednisone (R-CHOP) is the standard treatment regimen for NHL, yielding a complete remission (CR) rate of 40-50%. Unfortunately, considerable patients undergo relapse after CR or initial treatment, resulting in poor clinical implications. Patient’s response to chemotherapy varies widely from static disease to cancer recurrence and later is primarily associated with the development of multi-drug resistance (MDR). The immunosuppressive cells within the tumor microenvironment (TME) have become a crucial target for improving the therapy efficacy. However, a better understanding of their involvement is needed for distinctive response of NHL patients after receiving chemotherapy to design more effective front-line treatment algorithms based on reliable predictive biomarkers.MethodsPeripheral blood from 61 CD20+ NHL patients before and after chemotherapy was utilized for immunophenotyping by flow-cytometry at different phases of treatment. In-vivo and in-vitro doxorubicin (Dox) resistance models were developed with murine Dalton’s lymphoma and Jurkat/Raji cell-lines respectively and impact of responsible immune cells on generation of drug resistance was studied by RT-PCR, flow-cytometry and colorimetric assays. Gene silencing, ChIP and western blot were performed to explore the involved signaling pathways.ResultsWe observed a strong positive correlation between elevated level of CD33+CD11b+CD14+CD15- monocytic MDSCs (M-MDSC) and MDR in NHL relapse cohorts. We executed the role of M-MDSCs in fostering drug resistance phenomenon in doxorubicin-resistant cancer cells in both in-vitro, in-vivo models. Moreover, in-vitro supplementation of MDSCs in murine and human lymphoma culture augments early expression of MDR phenotypes than culture without MDSCs, correlated well with in-vitro drug efflux and tumor progression. We found that MDSC secreted cytokines IL-6, IL-10, IL-1β are the dominant factors elevating MDR expression in cancer cells, neutralization of MDSC secreted IL-6, IL-10, IL-1β reversed the MDR trait. Moreover, we identified MDSC secreted IL-6/IL-10/IL-1β induced STAT1/STAT3/NF-κβ signaling axis as a targeted cascade to promote early drug resistance in cancer cells.ConclusionOur data suggests that screening patients for high titre of M-MDSCs might be considered as a new potential biomarker and treatment modality in overcoming chemo-resistance in NHL patients

Neem leaf glycoprotein optimizes effector and regulatory functions within tumor microenvironment to intervene therapeutically the growth of B16 melanoma in C57BL/6 mice

Therapy with neem leaf glycoprotein (NLGP) inhibits murine B16-melanoma in vivo and improves survivability. Studies on tumor-microenvironment (TME) from NLGP treated mice (NLGP-TME) suggests that anti-tumor effect is directly associated with enhanced CD8+T cell activity, dominance of type 1 cytokines/chemokine network with downregulation of suppressive cellular functions. NLGP-TME educated CD8+T cells showed higher perforin and granzymeB expression with greater in vitro cytotoxicity against B16 melanoma. These CD8+T cells showed proportionally lower FasR expression, denotes prevention from activation induced cell death by NLGP. Accumulated evidences strongly suggest NLGP influenced normalized TME allows CD8+T cells to perform optimally to inhibit melanoma growth

Normalization of tumor microenvironment by neem leaf glycoprotein potentiates effector T cell functions and therapeutically intervenes in the growth of mouse sarcoma.

We have observed restriction of the murine sarcoma growth by therapeutic intervention of neem leaf glycoprotein (NLGP). In order to evaluate the mechanism of tumor growth restriction, here, we have analyzed tumor microenvironment (TME) from sarcoma bearing mice with NLGP therapy (NLGP-TME, in comparison to PBS-TME). Analysis of cytokine milieu within TME revealed IL-10, TGFβ, IL-6 rich type 2 characters was switched to type 1 microenvironment with dominance of IFNγ secretion within NLGP-TME. Proportion of CD8(+) T cells was increased within NLGP-TME and these T cells were protected from TME-induced anergy by NLGP, as indicated by higher expression of pNFAT and inhibit related downstream signaling. Moreover, low expression of FasR(+) cells within CD8(+) T cell population denotes prevention from activation induced cell death. Using CFSE as a probe, better migration of T cells was noted within TME from NLGP treated mice than PBS cohort. CD8(+) T cells isolated from NLGP-TME exhibited greater cytotoxicity to sarcoma cells in vitro and these cells show higher expression of cytotoxicity related molecules, perforin and granzyme B. Adoptive transfer of NLGP-TME exposed T cells, but not PBS-TME exposed cells in mice, is able to significantly inhibit the growth of sarcoma in vivo. Such tumor growth inhibition by NLGP-TME exposed T cells was not observed when mice were depleted for CD8(+) T cells. Accumulated evidences strongly suggest NLGP mediated normalization of TME allows T cells to perform optimally to inhibit the tumor growth

Neem leaf glycoprotein activates CD8(+) T cells to promote therapeutic anti-tumor immunity inhibiting the growth of mouse sarcoma.

In spite of sufficient data on Neem Leaf Glycoprotein (NLGP) as a prophylactic vaccine, little knowledge currently exists to support the use of NLGP as a therapeutic vaccine. Treatment of mice bearing established sarcomas with NLGP (25 µg/mice/week subcutaneously for 4 weeks) resulted in tumor regression or dormancy (Tumor free/Regressor, 13/24 (NLGP), 4/24 (PBS)). Evaluation of CD8(+) T cell status in blood, spleen, TDLN, VDLN and tumor revealed increase in cellular number. Elevated expression of CD69, CD44 and Ki67 on CD8(+) T cells revealed their state of activation and proliferation by NLGP. Depletion of CD8(+) T cells in mice at the time of NLGP treatment resulted in partial termination of tumor regression. An expansion of CXCR3(+) and CCR5(+) T cells was observed in the TDLN and tumor, along with their corresponding ligands. NLGP treatment enhances type 1 polarized T-bet expressing T cells with downregulation of GATA3. Treg cell population was almost unchanged. However, T∶Treg ratios significantly increased with NLGP. Enhanced secretion/expression of IFNγ was noted after NLGP therapy. In vitro culture of T cells with IL-2 and sarcoma antigen resulted in significant enhancement in cytotoxic efficacy. Consistently higher expression of CD107a was also observed in CD8(+) T cells from tumors. Reinoculation of sarcoma cells in tumor regressed NLGP-treated mice maintained tumor free status in majority. This is correlated with the increment of CD44(hi)CD62L(hi) central memory T cells. Collectively, these findings support a paradigm in which NLGP dynamically orchestrates the activation, expansion, and recruitment of CD8(+) T cells into established tumors to operate significant tumor cell lysis

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

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 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 genes related to chemokine receptor and ligands.

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

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