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

NLGP mediated downregulation of regulatory cells is CD8⁺ T cell dependent.

<p>(A) Flow cytometric assessment of the status of TAMs (CD11b⁺F4/80⁺), DC2s (CD11c⁺IL-10⁺), Tregs (CD4⁺CD25⁺Foxp3⁺) and MDSCs (Gr1⁺CD11b⁺) in pre- and post-surgical S180 tumor bearing mice (n = 6). (B) Status of regulatory cells (TAMs, DC2s, Tregs, MDSCs) in post-surgery PBS, NLGP, CD8⁺ T cell depleted NLGP immunized mice (n = 6). (C) RT-PCR analysis to assess the expression of suppressive molecules present in MDSCs in surgically tumor removed PBS, NLGP and CD8⁺ T cell depleted NLGP immunized cohorts (n = 6). (D) Gene expression profile of molecules responsible for MDSC’s differentiation in NLGP and CD8⁺ T cell depleted NLGP immunized surgically tumor removed mice (n = 6). (E) RT-PCR analysis of S100A8 and S1001A9 molecules responsible for MDSCs trafficking in PBS, NLGP and CD8⁺ T cell depleted NLGP immunized surgically tumor removed mice (n = 6). (F) Status of CD8⁺ Ki67⁺ T cells after co culture with MDSCs isolated from PBS, NLGP, CD8+ T cell depleted NLGP mice. Representative figures along with bar diagram showing mean relative expression of three individual mice in each group are presented. (**<i>p</i><0.001,*<i>p</i><0.01).</p

Primer list^*.

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

Recurrent tumor growth and survival of Swiss mice with post-surgery NLGP treatment.

<p>(A) Experimental design showing sarcoma inoculation, NLGP treatment and blood collection. (B) Recurrent tumor growth curve in pre- and post-surgery phases of mice with or without NLGP treatment (n = 9). (C) Representative photographs of tumor free and tumor bearing mice in the NLGP and PBS groups, respectively, in the post-surgical period. (D) Survival of mice undergoing surgery followed by NLGP treatment (n = 9) (**<i>p</i><0.001).</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

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

Table_1_High monocytic MDSC signature predicts multi-drug resistance and cancer relapse in non-Hodgkin lymphoma patients treated with R-CHOP.xlsx

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.</p

DataSheet_1_High monocytic MDSC signature predicts multi-drug resistance and cancer relapse in non-Hodgkin lymphoma patients treated with R-CHOP.pdf

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.</p

Table_2_High monocytic MDSC signature predicts multi-drug resistance and cancer relapse in non-Hodgkin lymphoma patients treated with R-CHOP.xlsx

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.</p

Table_3_High monocytic MDSC signature predicts multi-drug resistance and cancer relapse in non-Hodgkin lymphoma patients treated with R-CHOP.xls

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.</p