Neem leaf glycoprotein binding to Dectin-1 receptors on dendritic cell induces type-1 immunity through CARD9 mediated intracellular signal to NFκB.

BACKGROUND: A water-soluble ingredient of mature leaves of the tropical mahogany 'Neem' (Azadirachta indica), was identified as glycoprotein, thus being named as 'Neem Leaf Glycoprotein' (NLGP). This non-toxic leaf-component regressed cancerous murine tumors (melanoma, carcinoma, sarcoma) recurrently in different experimental circumstances by boosting prime antitumor immune attributes. Such antitumor immunomodulation, aid cytotoxic T cell (Tc)-based annihilation of tumor cells. This study focused on identifying and characterizing the signaling gateway that initiate this systemic immunomodulation. In search of this gateway, antigen-presenting cells (APCs) were explored, which activate and induce the cytotoxic thrust in Tc cells. METHODS: Six glycoprotein-binding C-type lectins found on APCs, namely, MBR, Dectin-1, Dectin-2, DC-SIGN, DEC205 and DNGR-1 were screened on bone marrow-derived dendritic cells from C57BL/6 J mice. Fluorescence microscopy, RT-PCR, flow cytometry and ELISA revealed Dectin-1 as the NLGP-binding receptor, followed by verifications through RNAi. Following detection of ß-Glucans in NLGP, their interactions with Dectin-1 were explored in silico. Roles of second messengers and transcription factors in the downstream signal were studied by co-immunoprecipitation, western blotting, and chromatin-immunoprecipitation. Intracellularization of FITC-coupled NLGP was observed by processing confocal micrographs of DCs. RESULTS: Considering extents of hindrance in NLGP-driven transcription rates of the cytokines IL-10 and IL-12p35 by receptor-neutralization, Dectin-1 receptors on dendritic cells were found to bind NLGP through the ligand's peripheral ß-Glucan chains. The resulting signal phosphorylates PKCδ, forming a trimolecular complex of CARD9, Bcl10 and MALT1, which in turn activates the canonical NFκB-pathway of transcription-regulation. Consequently, the NFκB-heterodimer p65:p50 enhances Il12a transcription and the p50:p50 homodimer represses Il10 transcription, bringing about a cytokine-based systemic-bias towards type-1 immune environment. Further, NLGP gets engulfed within dendritic cells, possibly through endocytic activities of Dectin-1. CONCLUSION: NLGP's binding to Dectin-1 receptors on murine dendritic cells, followed by the intracellular signal, lead to NFκB-mediated contrasting regulation of cytokine-transcriptions, initiating a pro-inflammatory immunopolarization, which amplifies further by the responding immune cells including Tc cells, alongside their enhanced cytotoxicity. These insights into the initiation of mammalian systemic immunomodulation by NLGP at cellular and molecular levels, may help uncovering its mode of action as a novel immunomodulator against human cancers, following clinical trials.

Evolvability and emergence of tumor heterogeneity as a space-time function.

The loss of control of cell proliferation, apoptosis regulation and contact inhibition leads to tumor development. While benign tumors are restricted to their primary space, i.e. where these tumors first originate, the metastatic tumors not only disseminate- facilitated by hypoxia-driven neovascularization- to distant secondary sites but also show substantial changes in metabolism, tissue architectures, gene expression profiles and immune phenotypes. All these alterations result in radio-, chemo- and immune-resistance rendering these metastatic tumor cells refractory to therapy. Since the beginning of the transformation, these factors- which influence each other- are incorporated to the developing and metastasizing tumor. As a result, the complexities in the heterogeneity of tumor progressively increase. This space-time function in the heterogeneity of tumors is generated by various conditions and factors at the genetic as well as microenvironmental levels, for example, endogenous retroviruses, methylation and epigenetic dysregulation that may be etiology-specific, cancer associated inflammation, remodeling of the extracellular matrix and mesenchymal cell shifted functions. On the one hand, these factors may cause de-differentiation of the tumor cells leading to cancer stem cells that contribute to radio-, chemo- and immune-resistance and recurrence of tumors. On the other hand, they may also enhance the heterogeneity under specific microenvironment-driven proliferation. In this editorial, we intend to underline the importance of heterogeneity in cancer progress, its evaluation and its use in correlation with the tumor evolution in a specific patient as a field of research for achieving precise patient-tailored treatments and amelioration of diagnostic (monitoring) tools and prognostic capacity.

Toll-like receptor 2 selectively modulates Ras isoforms expression in Leishmania major infection.

Leishmania infection of macrophages results in altered Ras isoforms expression and Toll-like receptor-2 (TLR2) expression and functions. Therefore, we examined whether TLR2 would selectively alter Ras isoforms' expression in macrophages. We observed that TLR2 ligands- Pam3CSK4, peptidoglycan (PGN), and FSL- selectively modulated the expression of Ras isoforms in BALB/c-derived elicited macrophages. Lentivirally-expressed TLR1-shRNA significantly reversed this Ras isoforms expression profile. TLR2-deficient L. major-infected macrophages and the lymph node cells from the L. major-infected mice showed similarly reversed Ras isoforms expression. Transfection of the macrophages with the siRNAs for the adaptors- Myeloid Differentiation factor 88 (MyD88) and Toll-Interleukin-1 Receptor (TIR) domain-containing adaptor protein (TIRAP)- or Interleukin-1 Receptor-Associated Kinases (IRAKs)- IRAK1 and IRAK4- significantly inhibited the L. major-induced down-regulation of K-Ras, and up-regulation of N-Ras and H-Ras, expression. The TLR1/TLR2-ligand Pam3CSK4 increased IL-10 and TGF-ß expression in macrophages. Pam3CSK4 upregulated N-Ras and H-Ras, but down-regulated K-Ras, expression in C57BL/6 wild-type, but not in IL-10-deficient, macrophages. IL-10 or TGF-ß signaling inhibition selectively regulated Ras isoforms expression. These observations indicate the specificity of the TLR2 regulation of Ras isoforms and their selective modulation by MyD88, TIRAP, and IRAKs, but not IL-10 or TGF-ß, signaling.

NLGP regulates RGS5-TGFß axis to promote pericyte-dependent vascular normalization during restricted tumor growth.

Altered RGS5-associated intracellular pericyte signaling and its abnormal crosstalk with endothelial cells (ECs) result chaotic tumor-vasculature, prevent effective drug delivery, promote immune-evasion and many more to ensure ultimate tumor progression. Moreover, the frequency of lethal-RGS5high  pericytes within tumor was found to increase with disease progression, which signifies the presence of altered cell death pathway within tumor microenvironment (TME). In this study, we checked whether and how neem leaf glycoprotein (NLGP)-immunotherapy-mediated tumor growth restriction is associated with modification of pericytes' signaling, functions and its interaction with ECs. Analysis of pericytes isolated from tumors of NLGP treated mice suggested that NLGP treatment promotes apoptosis of NG2+ RGS5high -fuctionally altered pericytes by downregulating intra-tumoral TGFß, along with maintenance of more matured RGS5neg  pericytes. NLGP-mediated inhibition of TGFß within TME rescues binding of RGS5 with Gαi and thereby termination of PI3K-AKT mediated survival signaling by downregulating Bcl2 and initiating pJNK mediated apoptosis. Limited availability of TGFß also prevents complex-formation between RGS5 and Smad2 and rapid RGS5 nuclear translocation to mitigate alternate immunoregulatory functions of RGS5high  tumor-pericytes. We also observed binding of Ang1 from pericytes with Tie2 on ECs in NLGP-treated tumor, which support re-association of pericytes with endothelium and subsequent vessel stabilization. Furthermore, NLGP-therapy- associated RGS5 deficiency relieved CD4+  and CD8+ T cells from anergy by regulating 'alternate-APC-like' immunomodulatory characters of tumor-pericytes. Taken together, present study described the mechanisms of NLGP's effectiveness in normalizing tumor-vasculature by chiefly modulating pericyte-biology and EC-pericyte interactions in tumor-host to further strengthen its translational potential as single modality treatment.

COVID-19 imparted immune manifestation can be combated by NLGP: Lessons from cancer research.

SARS-CoV-2 easily infects human monocytes, macrophages and possibly dendritic cells (DCs), causing dysfunctions of these important antigen presenting cells (APCs). Observed DC dysfunctions facilitate improper antigen presentation, which obviously results T cell anergy, exhaustion and apoptosis, thus, may be contributing significantly in SARS-CoV-2 infection associated lymphopenia. Neem Leaf Glycoprotein or NLGP has enormous role in altered DC functions, thereby, offering optimum T cell mediated cytotoxicity, as experienced from cancer system. Such NLGP guided correction of altered DCs might also be effective to generate proper SARS-CoV-2-specific effector and central memory T cells.

TLR induced IL-27 plays host-protective role against B16BL6 melanoma in C57BL/6 mice.

Elicitation of the tumor-eliminating immune response is a major challenge, as macrophages- constituting a major component of solid tumor mass- play important roles in development, maintenance and tumor regression. The macrophage-expressed Toll-Like Receptors (TLRs) enhance macrophage function and their ability to activate T cells via secretion of cytokines, which may help in tumor regression. IL-27, a member of the IL-12 family of cytokines, is shown to exhibit anti-tumor and anti-angiogenic activities. Herein, we developed B16BL6 melanoma model in C75BL/6 mouse to dissect the crosstalk between TLRs and IL-27 in tumors. We report existence of a novel TLR- IL-27 feed-forward loop, whereby TLRs and IL-27 up-regulated each other's expression, which we found perturbed during melanoma tumorigenesis. Intra-tumoral injection of Imiquimod, a TLR7/8 ligand, reduced the tumor burden; the anti-tumor effect was reversed upon IL-27 and IL-27R silencing by intra-tumorally administered, lentivirally expressed IL-27 and IL-27R shRNA. The reduced tumor growth was accompanied by significantly fewer Treg cells but increased IFN-γ and granzyme B expression by CD8+ T cells. These data indicate the preventative role for TLR-induced IL-27 in aggressive and highly invasive melanoma.

Tumor activated platelets induce vascular mimicry in mesenchymal stem cells and aid metastasis.

Extent of metastasis influences activation of platelets in tumor-microenvironment. Activated platelets potentiate mesenchymal-stem-cells (MSCs) to migrate in secondary metastatic sites without participation in process of invasion. Presence of higher percentage of MSCs along with activated-platelets induces formation of vascular-mimicry (VM). The pathophysiology, VM, has already been reported in multiple types of cancer including lung, ovary, melanoma etc. and related to poor-prognosis. Interaction of MSCs with platelets in cell-to-cell contact dependent manner is essential for their migration, thereby, VM. Evidences are obtained suggesting that under influence of tumor-associated-activated-platelets, expressions of vimentin, ve-cadherin are increased, along with decrease in e-cadherin on CD105+ MSCs in both mRNA and protein levels that may help in formation of vessel like structure in VM. Adoptive transfer of MSCs along with tumor-activated-platelets causes greater B16 melanoma metastasis at lungs in comparison to MSCs with non-activated platelets. Presence of CD105+Vimentin+ MSCs in vessel like structure in the metastatic lung confirms the involvement of platelet-activated-MSCs in VM, thereby, in metastasis.

Macrophages in tumor: An inflammatory perspective.

Inflammation is a part of carefully co-ordinated healing immune exercise to eliminate injurious stimuli. However, in substantial number of cancer types, it contributes in shaping up of robust tumor microenvironment (TME). Solid TME promotes infiltration of tumor associated macrophages (TAMs) that contributes to cancer promotion. TAMs are functionally heterogeneous and display an extraordinary degree of plasticity, which allow 'Switching' of macrophages into an 'M2', phenotype, linked with immunosuppression, advancement of tumor angiogenesis with metastatic consequences. In contrary to the classical M1 macrophages, these M2 TAMs are high-IL-10, TGF-ß secreting-'anti-inflammatory'. In this review, we will discuss the modes of infiltration and switching of TAMs into M2 anti-inflammatory state in the TME to promote immunosuppression and inflammation-driven cancer.

PKCζ mediated anti-proliferative effect of C2 ceramide on neutralization of the tumor microenvironment and melanoma regression.

Immunotherapy, which has advantages over chemotherapy due to lesser toxicity and higher specificity, is on the rise to treat cancer. Recently, pro-apoptotic glycolipid, ceramide has emerged as a key regulator in cancer immunotherapy. The present study elucidated the potential anti-melanoma efficacy of cell-permeable, exogenous C2 ceramide on cell death and amelioration of tumor microenvironment (TME). We, for the first time, demonstrated that C2 ceramide triggered apoptosis of melanoma cells by augmenting PKCζ along with pro-inflammatory cytokines and signaling factors. C2 ceramide showed a PKCζ-mediated tumor-suppressive role in melanoma without exhibiting hepatotoxicity and nephrotoxicity. Moreover, PKCζ was revealed as one of the key regulators of Akt and ceramide during C2 ceramide-mediated apoptosis. C2 ceramide was effective in repolarization of M2 macrophage phenotype and reduction of angiogenic factors such as VEGF, VEGFR1, VEGFR2, HIF1α. Interestingly, PKCζ knockdown attenuated C2 ceramide-mediated inhibition of melanoma progression. Restoration of the Th1 type TME by C2 ceramide enhanced cytotoxic T cell-mediated killing of melanoma cells. Altogether, the study unraveled that C2 ceramide-induced PKCζ was associated with favorable immune cell functioning in TME leading to melanoma regression. Thus, our findings explored a novel mechanistic insight into C2 ceramide as a promising immunotherapeutic agent in melanoma treatment.

Fluoxetine triggers selective apoptosis in inflammation-induced proliferating (Ki-67high ) thymocytes.

Inappropriate functioning of the immune system is observed during sustained systemic inflammation, which might lead to immune deficiencies, autoimmune disorders and cancer. Primary lymphoid organs may progress to a deregulated proliferative state in response to inflammatory signals in order to intensify host defense mechanisms and exacerbate an inflammatory niche. Fluoxetine, a selective serotonin reuptake inhibitor, has recently been projected as an anti-inflammatory agent. This study had been designed to evaluate the potential novel role of fluoxetine in reversing inflammation-induced immune dysfunction. Lipopolysaccharide (LPS) administration in Swiss albino mice potentiated a systemic inflammatory response, along with increased proliferation of thymocytes and peripheral blood mononuclear cells, as evident from increased Ki-67 expression. The proliferative changes in the immune system were mainly associated with increased phosphorylation of PI3k, AKT and IκB along with elevated NFκB-p65 nuclear translocation. The Ki-67high thymocytes obtained from LPS administered mice demonstrated significantly low p53 nuclear activity, which was established to be mediated by NFκB through reduced nuclear translocation of p53 during LPS-induced proliferative conditions, thereby blocking p53-dependent apoptosis. Fluoxetine supplementation not only reversed the proinflammatory condition, but also induced selective apoptosis in the proliferation-dictated Ki-67high thymocytes possibly by modulating the hypothalamus-pituitary-adrenal axis and inducing glucocorticoid receptor activation and apoptosis in these proliferation-biased immune cells, authenticating a novel antiproliferative role of an established drug.

Activation of D2 Dopamine Receptors in CD133+ve Cancer Stem Cells in Non-small Cell Lung Carcinoma Inhibits Proliferation, Clonogenic Ability, and Invasiveness of These Cells.

Lung carcinoma is the leading cause of cancer-related death worldwide, and among this cancer, non-small cell lung carcinoma (NSCLC) comprises the majority of cases. Furthermore, recurrence and metastasis of NSCLC correlate well with CD133+ve tumor cells, a small population of tumor cells that have been designated as cancer stem cells (CSC). We have demonstrated for the first time high expression of D2 dopamine (DA) receptors in CD133+ve adenocarcinoma NSCLC cells. Also, activation of D2 DA receptors in these cells significantly inhibited their proliferation, clonogenic ability, and invasiveness by suppressing extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT, as well as down-regulation of octamer-binding transcription factor 4 (Oct-4) expression and matrix metalloproteinase-9 (MMP-9) secretion by these cells. These results are of significance as D2 DA agonists that are already in clinical use for treatment of other diseases may be useful in combination with conventional chemotherapy and radiotherapy for better management of NSCLC patients by targeting both tumor cells and stem cell compartments in the tumor mass.

Tumor promoting role of anti-tumor macrophages in tumor microenvironment.

Recent advances in tumor biology demand detailed analysis of the complex interaction of tumor cells with their adjacent microenvironment (tumor stroma) to understand the various mechanisms involved in tumor growth and metastasis. Mononuclear phagocytes or macrophages, a type of innate immune cells, defend the organism against infection and injury. On the otherhand, tumor associated macrophages (TAMs) constitute a significant part of the tumor-infiltrating immune cells, have been linked to the growth, angiogenesis, and metastasis of a variety of cancers, most likely through polarization of TAMs to the M2 (alternative) phenotype. Clinical and experimental evidences have shown that cancer tissues with high infiltration of TAMs are associated with poor patient prognosis and resistance to therapies, thus, targeting of TAMs in tumors is considered as a promising immunotherapeutic strategy. Depletion of M2 TAMs or 're-education' of them as anti-tumor effectors might contribute significantly to the search of new modalities in anti-cancer treatments. Basic questions on the factors responsible for homing of macrophages in tumors, mechanism of conversion of M1 to M2 TAMs, their functionality and, finally, the possible ways to target M2 TAMs are discussed.

Vanadium(III)-l-cysteine enhances the sensitivity of murine breast adenocarcinoma cells to cyclophosphamide by promoting apoptosis and blocking angiogenesis.

Various epidemiological and preclinical studies have already established the cancer chemopreventive potential of vanadium-based compounds. In addition to its preventive efficacy, studies have also indicated the abilities of vanadium-based compounds to induce cell death selectively toward malignant cells. Therefore, the objective of the present investigation is to improve the therapeutic efficacy and toxicity profile of an alkylating agent, cyclophosphamide, by the concurrent use of an organovanadium complex, vanadium(III)-l-cysteine. In this study, vanadium(III)-l-cysteine (1 mg/kg body weight, per os) was administered alone as well as in combination with cyclophosphamide (25 mg/kg body weight, intraperitoneal) in concomitant and pretreatment schedule in mice bearing breast adenocarcinoma cells. The results showed that the combination treatment significantly decreased the tumor burden and enhanced survivability of tumor-bearing mice through generation of reactive oxygen species in tumor cells. These ultimately led to DNA damage, depolarization of mitochondrial membrane potential, and apoptosis in tumor cells. Further insight into the molecular pathway disclosed that the combination treatment caused upregulation of p53 and Bax and suppression of Bcl-2 followed by the activation of caspase cascade and poly (ADP-ribose) polymerase cleavage. Administration of vanadium(III)-l-cysteine also resulted in significant attenuation of peritoneal vasculature and sprouting of the blood vessels by decreasing the levels of vascular endothelial growth factor A and matrix metalloproteinase 9 in the ascites fluid of tumor-bearing mice. Furthermore, vanadium(III)-l-cysteine significantly attenuated cyclophosphamide-induced hematopoietic, hepatic, and genetic damages and provided additional survival advantages. Hence, this study suggested that vanadium(III)-l-cysteine may offer potential therapeutic benefit in combination with cyclophosphamide by augmenting anticancer efficacy and diminishing toxicity to the host.

A Novel Approach of Synthesizing and Evaluating the Anticancer Potential of Silver Oxide Nanoparticles in vitro.

BACKGROUND: Development of novel strategies to kill cancer by sparing normal cells is of utmost importance. Apart from their known antimicrobial activity, only limited information has been recorded regarding the antitumor potential of biocompatible silver oxide nanoparticles (AgONPs). There is a need to evaluate the anticancer potential of biocompatible AgONPs in vitro. METHODS: A new approach of utilizing the leaf extract of Excoecaria agallocha was used to synthesize AgONPs. This was then characterized by ultraviolet-visible spectrophotometry, nanoparticle-tracking analysis, and ζ-potential analysis. Cytotoxicity and apoptotic potential were evaluated with an MTT assay and an annexin V-binding assay against the murine melanoma (B16F10), murine colon cancer (CT26), murine lung adenocarcinoma (3LL), and murine Ehrlich ascites carcinoma (EAC) cell lines. Cellular localization of AgONPs was evaluated on fluorescence microscopy. RESULTS: UV peaks at 270 and 330 nm indicated the formation of nanoparticles (NPs) and the NP-tracking analyzer revealed them to have a size of 228 nm. AgONPs exerted initial cytotoxicity, specifically against all the experimental malignant cells by sparing the normal cell lines. Moreover, AgONPs exert apoptosis equally on all the malignant cells in vitro and ex vivo. This cytotoxicity possibly occurs via the nuclear translocation of AgONPs as analyzed in B16F10 cells. CONCLUSIONS: AgONPs utilizing natural sources would be a new medicinal approach against a broad spectrum of malignancy.

Tumor-associated mesenchymal stem cells inhibit naïve T cell expansion by blocking cysteine export from dendritic cells.

Mesenchymal stem cells (MSCs) represent an important cellular constituent of the tumor microenvironment, which along with tumor cells themselves, serve to regulate protective immune responses in support of progressive disease. We report that tumor MSCs prevent the ability of dendritic cells (DC) to promote naïve CD4(+) and CD8(+) T cell expansion, interferon gamma secretion and cytotoxicity against tumor cells, which are critical to immune-mediated tumor eradication. Notably, tumor MSCs fail to prevent DC-mediated early T cell activation events or the ability of responder T cells to produce IL-2. The immunoregulatory activity of tumor MSCs is IL-10- and STAT3-dependent, with STAT3 repressing DC expression of cystathionase, a critical enzyme that converts methionine-to-cysteine. Under cysteine-deficient priming conditions, naïve T cells exhibit defective cellular metabolism and proliferation. Bioinformatics analyses as well as in vitro observations suggest that STAT3 may directly bind to a GAS-like motif within the cystathionase promoter (-269 to -261) leading to IL-10-STAT3 mediated repression of cystathionase gene transcription. Our collective results provide evidence for a novel mechanism of tumor MSC-mediated T cell inhibition within tumor microenvironment.

Tumor-derived vascular pericytes anergize Th cells.

Immune evasion within the tumor microenvironment supports malignant growth and is also a major obstacle for successful immunotherapy. Multiple cellular components and soluble factors coordinate to disrupt protective immune responses. Although stromal cells are well-known for their parenchymal supportive roles in cancer establishment and progression, we demonstrate for the first time, to our knowledge, that tumor-derived vascular pericytes negatively influence CD4(+) T cell activation and proliferation, and promote anergy in recall response to Ag by CD4(+)CD44(+) T cells via regulator of G protein signaling 5- and IL-6-dependent pathways. Our data support a new specific role for tumor-derived pericytes in the immune evasion paradigm within the tumor microenvironment and suggest the targeting of these cell populations in the context of successful immunotherapeutics for the treatment of cancer.

The immunomodulatory impact of naturally derived neem leaf glycoprotein on the initiation progression model of 4NQO induced murine oral carcinogenesis: a preclinical study.

Introduction: Murine tumor growth restriction by neem leaf glycoprotein (NLGP) was established in various transplanted models of murine sarcoma, melanoma and carcinoma. However, the role of NLGP in the sequential carcinogenic steps has not been explored. Thus, tongue carcinogenesis in Swiss mice was induced by 4-nitroquinoline-1-oxide (4NQO), which has close resemblance to human carcinogenesis process. Interventional role of NLGP in initiation-promotion protocol established during 4NQO mediated tongue carcinogenesis in relation to systemic immune alteration and epithelial-mesenchymal transition (EMT) is investigated. Methods: 4NQO was painted on tongue of Swiss mice every third day at a dose of 25µl of 5mg/ml stock solution. After five consecutive treatment with 4NQO (starting Day7), one group of mice was treated with NLGP (s.c., 25µg/mice/week), keeping a group as PBS control. Mice were sacrificed in different time-intervals to harvest tongues and studied using histology, immunohistochemistry, flow-cytometry and RT-PCR on different immune cells and EMT markers (e-cadherin, vimentin) to elucidate their phenotypic and secretory status. Results: Local administration of 4NQO for consecutive 300 days promotes significant alteration in tongue mucosa including erosion in papillae and migration of malignant epithelial cells to the underlying connective tissue stroma with the formation of cell nests (exophytic-hyperkeratosis with mild dysplasia). Therapeutic NLGP treatment delayed pre-neoplastic changes promoting normalization of mucosa by maintaining normal structure. Flow-cytometric evidences suggest that NLGP treatment upregulated CD8+, IFNγ+, granzyme B+, CD11c+ cells in comparison to 4NQO treated mice with a decrease in Ki67+ and CD4+FoxP3+ cells in NLGP treated cohort. RT-PCR demonstrated a marked reduction of MMP9, IL-6, IL-2, CD31 and an upregulation in CCR5 in tongues from 4NQO+NLGP treated mice in comparison to 4NQO treated group. Moreover, 4NQO mediated changes were associated with reduction of e-cadherin and simultaneous up-regulation of vimentin expression in epithelium that was partially reversed by NLGP. Discussion: Efficacy of NLGP was tested first time in sequential carcinogenesis model and proved effective in delaying the initial progression. NLGP normalizes type 1 immunity including activation of the CD8+T effector functions, reduction of regulatory T cell functions, along with changes in EMT to make the host systemically alert to combat the carcinogenic threat.

Neem Leaf Glycoprotein Disrupts Exhausted CD8+ T-Cell-Mediated Cancer Stem Cell Aggression.

Targeting exhausted CD8+ T-cell (TEX)-induced aggravated cancer stem cells (CSC) holds immense therapeutic potential. In this regard, immunomodulation via Neem Leaf Glycoprotein (NLGP), a plant-derived glycoprotein immunomodulator is explored. Since former reports have proven immune dependent-tumor restriction of NLGP across multiple tumor models, we hypothesized that NLGP might reprogram and rectify TEX to target CSCs successfully. In this study, we report that NLGP's therapeutic administration significantly reduced TEX-associated CSC virulence in in vivo B16-F10 melanoma tumor model. A similar trend was observed in in vitro generated TEX and B16-F10/MCF7 coculture setups. NLGP rewired CSCs by downregulating clonogenicity, multidrug resistance phenotypes and PDL1, OCT4, and SOX2 expression. Cell cycle analysis revealed that NLGP educated-TEX efficiently pushed CSCs out of quiescent phase (G0G1) into synthesis phase (S), supported by hyper-phosphorylation of G0G1-S transitory cyclins and Rb proteins. This rendered quiescent CSCs susceptible to S-phase-targeting chemotherapeutic drugs like 5-fluorouracil (5FU). Consequently, combinatorial treatment of NLGP and 5FU brought optimal CSC-targeting efficiency with an increase in apoptotic bodies and proapoptotic BID expression. Notably a strong nephron-protective effect of NLGP was also observed, which prevented 5FU-associated toxicity. Furthermore, Dectin-1-mediated NLGP uptake and subsequent alteration of Notch1 and mTOR axis were deciphered as the involved signaling network. This observation unveiled Dectin-1 as a potent immunotherapeutic drug target to counter T-cell exhaustion. Cumulatively, NLGP immunotherapy alleviated exhausted CD8+ T-cell-induced CSC aggravation. Implications: Our study recommends that NLGP immunotherapy can be utilized to counter ramifications of T-cell exhaustion and to target therapy elusive aggressive CSCs without evoking toxicity.

Terminally Exhausted CD8+ T Cells Resistant to PD-1 Blockade Promote Generation and Maintenance of Aggressive Cancer Stem Cells.

SIGNIFICANCE: Cross-talk with TTEX CD8+ T cells mediated by the VEGFR2 axis induces aggressive properties in cancer stem cells to promote tumor progression.

Cancer stem cell-immune cell crosstalk in breast tumor microenvironment: a determinant of therapeutic facet.

Breast cancer (BC) is globally one of the leading killers among women. Within a breast tumor, a minor population of transformed cells accountable for drug resistance, survival, and metastasis is known as breast cancer stem cells (BCSCs). Several experimental lines of evidence have indicated that BCSCs influence the functionality of immune cells. They evade immune surveillance by altering the characteristics of immune cells and modulate the tumor landscape to an immune-suppressive type. They are proficient in switching from a quiescent phase (slowly cycling) to an actively proliferating phenotype with a high degree of plasticity. This review confers the relevance and impact of crosstalk between immune cells and BCSCs as a fate determinant for BC prognosis. It also focuses on current strategies for targeting these aberrant BCSCs that could open avenues for the treatment of breast carcinoma.