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

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

Primer list^*.

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

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

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 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

Study of association and molecular analysis of human papillomavirus in breast cancer of Indian patients: Clinical and prognostic implication

<div><p>Objectives</p><p>Human papillomavirus (HPV) causes tumors primarily Cervical cancer. Recently, inconsistent reports came up in Breast cancer (BC) too. In India, despite treatment 70,218 BC patients die each year. So, we explored the association of HPV, if any, with BC prognosis in Indian pre-therapeutic (PT) and Neo-adjuvant chemotherapy (NACT) patients with subsequent analysis of HPV profile.</p><p>Methods</p><p>HPV prevalence was checked and analysis of physical status, copy number, genome variation, promoter methylation and expression (mRNA and protein) of the prevalent subtype was done.</p><p>Results</p><p>High prevalence of HPV was observed in both PT (64.0%) and NACT (71.0%) cases with significant association with younger (20–45 yrs) PT patients. Interestingly, HPV infection was significantly increased from adjacent normal breast (9.5%, 2/21), fibro adenomas (30%, 3/10) to tumors (64.8%, 203/313) samples. In both PT and NACT cases, HPV16 was the most prevalent subtype (69.0%) followed by HPV18 and HPV33. Survival analysis illustrated hrHPV infected PT patients had worst prognosis. So, detailed analysis of HPV16 profile was done which showed Europian-G350 as the most frequent HPV16 variant along with high rate of integration. Moreover, low copy number and hyper-methylation of P97 early promoter were concordant with low HPV16 E6 and E7 mRNA and protein expression. Notably, four novel variations (KT020838, KT020840, KT020841 and KT020839) in the LCR region and two (KT020836 and KT020837) in the E6 region were identified for the first time along with two novel E6^E7*I (KU199314) and E6^E7*II (KU199315) fusion transcript variants.</p><p>Conclusion</p><p>Thus, significant association of hrHPV with prognosis of Indian BC patients led to additional investigation of HPV16 profile. Outcomes indicated a plausible role of HPV in Indian BC patients.</p></div

Analysis of HPV16 physical status in breast tumor samples and MCF7 breast cancer cell line.

<p>Representative agarose gel of physical status of HPV16 genome at <b>(a)</b> E2A region. <b>(b)</b> E2B region and <b>(c)</b> E2C region <b>(d)</b> Histogram represent significant high frequency of integrated viral genome in both pre-therapeutic and NACT samples (p≤0.01). Frequency of integration at three regions of the E2 gene in <b>(e)</b> pre-therapeutic cases and <b>(f)</b> NACT samples. [Here M: 100bp marker, NC represent Negative control with no DNA, +Ve represent Episomal control where HPV16 plasmid was used. SiHa is the HPV16 positive Cervical cancer cell line used as Integration control for E2A and E2B region while Episomal control for E2C region]</p

Immunohistochemical detection of E6 and E7 protein of HPV16 in pre-therapeutic breast tumor tissues.

<p><b>(b) & (e)</b> Representative immunohistochemical staining of E6 and E7 in HPV16 positive samples. <b>(c) & (f)</b> Representative immunohistochemical staining in HPV negative sample <b>(a) & (d)</b> Immunohistochemical staining with out primary antibody represented Negative control (NC). [Magnification of tissue samples is 20X and for inset, magnification is 40X, Scale bars = 50 μm].</p

Comparative HPV infection in adjacent normal breast, fibro adenomas, benign phyllode and breast tumors.

<p>Comparative HPV infection in adjacent normal breast, fibro adenomas, benign phyllode and breast tumors.</p