Cancer-associated fibroblast-derived Dickkopf-1 suppresses NK cell cytotoxicity in breast cancer.

Breast cancer is poorly immunogenic, hence able to evade T cell recognition and respond poorly to immune checkpoint blockade. Breast cancer cells can also evade NK cell-mediated immune surveillance, but the mechanism remains enigmatic. Dickkopf-1 (DKK1) is a Wnt/b-catenin inhibitor, whose levels are increased in breast cancer patients and correlate with reduced overall survival. DKK1 is expressed by cancer-associated fibroblasts (CAFs) in orthotopic breast tumors and patient samples, and at higher levels by bone cells. While bone-derived DKK1 contributes to the systemic elevation of DKK1 in tumor-bearing mice, CAFs represent the primary source of DKK1 at the tumor site. Systemic or bone-specific DKK1 targeting reduces primary tumor growth. Intriguingly, specific deletion of CAF-derived DKK1 also limits breast cancer progression, regardless of its elevated levels in circulation and in the bone. DKK1 does not support tumor proliferation directly but rather suppresses the activation and tumoricidal activity of NK cells. Importantly, increased DKK1 levels and reduced number of cytotoxic NK cells are detected in breast cancer patients with progressive bone metastases compared to those with stable disease. Our findings indicate that DKK1 creates a tumor-supporting environment through the suppression of NK cells in breast cancer.

Determining Endosomal Toll-Like Receptors Gene Expression in NK Cells After Stimulation with Specific Agonists.

Poor knowledge is currently available about the biology of Toll-like receptors (TLRs) in natural killer (NK) cells. This is particularly due to the old belief that NK cells are unable to specifically eliminate microbes without presensitization. On the contrary, it has been clearly demonstrated that not only they can be activated through the engagement of Toll-like receptors (TLRs) by microbial molecules, but also that this interaction induces NK cells to release cytokines that, in turn, activate other cells of both innate and adaptive immunity. For this reason, immunotherapy based on local infusion of TLRs ligands is currently considered as a novel potential strategy to treat solid tumors. Here, we provide a protocol to efficiently stimulate NK cells via endosomal TLRs agonists and to determine endosomal TLRs gene expression level. This protocol can be used for in vitro investigation into endosomal TLRs function in NK cells under different conditions.

How the Immune System Responds to Allergy Immunotherapy.

IgE-mediated diseases represent a highly diversified and multifactorial group of disorders that can deeply impact the patients' quality of life. Currently, allergy immunotherapy (AIT) still remains the gold standard for the management of such pathologies. In this review, we comprehensively examine and discuss how AIT can affect both the innate and the adaptive immune responses at different cell levels and propose timing-scheduled alterations induced by AIT by hypothesizing five sequential phases: after the desensitization of effector non-lymphoid cells and a transient increase of IgE (phase 1), high doses of allergen given by AIT stimulate the shift from type 2/type 3 towards type 1 response (phase 2), which is progressively potentiated by the increase of IFN-γ that promotes the chronic activation of APCs, progressively leading to the hyperexpression of Notch1L (Delta4) and the secretion of IL-12 and IL-27, which are essential to activate IL-10 gene in Th1 and ILC1 cells. As consequence, an expansion of circulating memory Th1/Tr1 cells and ILC-reg characterizes the third phase addressed to antagonize/balance the excess of type 1 response (phase 3). The progressive increase of IL-10 triggers a number of regulatory circuits sustained by innate and adaptive immune cells and favoring T-cell tolerance (phase 4), which may also be maintained for a long period after AIT interruption (phase 5). Different administration approaches of AIT have shown a similar tailoring of the immune responses and can be monitored by timely, optimized biomarkers. The clinical failure of this treatment can occur, and many genetic/epigenetic polymorphisms/mutations involving several immunological mechanisms, such as the plasticity of immune responses and the induction/maintenance of regulatory circuits, have been described. The knowledge of how AIT can shape the immune system and its responses is a key tool to develop novel AIT strategies including the engineering of allergen or their epitopes. We now have the potential to understand the precise causes of AIT failure and to establish the best biomarkers of AIT efficacy in each phase of the treatment.

IL-1R8 silencing improves the anti-tumor function of freshly isolated human NK cells.

The inhibitory receptor interleukin-1 receptor 8 (IL-1R8) has been recently recognized to be expressed also by human natural killer (NK) cells. This study was aimed to design and optimize IL-1R8 silencing conditions in human NK cells to precisely establish the activity of such receptor in these cells. Electroporation of freshly isolated or IL-2-cultured NK cells with small interfering RNA (siRNA), resulted in a marked, even though variable, IL-1R8-silencing. Although the expression profile revealed downregulation of most genes involved in several intracellular pathways, some genes related to proliferation, expression of some chemokine receptors, antibody-dependent cell cytotoxicity and cytotoxic activity were upregulated in IL-1R8-silenced NK cells. Furthermore, upon IL-15 activation, the majority of genes involved in NK cell function were upregulated in IL-1R8-siRNA-compared with control-siRNA-transfected NK cells. More importantly, in agreement with these findings, the reduction of IL-1R8 gene expression levels resulted in enhanced expression of NK cell activation markers, production of cytokines and chemokines, and cytotoxic activity against several NK cell targets with different susceptibility to NK-mediated lysis. Similar results were obtained following stimulation with IL-18. All together these data, deeply impacting on the main effector functions of human NK cells, can lead to a better understanding of IL-1R8-mediated regulation on these cells and to the design of new strategies for improving NK cell-mediated anti-tumor responses.

Toll-like receptor 8 agonists improve NK-cell function primarily targeting CD56brightCD16- subset.

BACKGROUND: Toll-like receptors (TLRs) are pattern-recognition sensors mainly expressed in innate immune cells that directly recognize conserved pathogen structures (pathogen-associated molecular patterns-PAMPs). Natural killer (NK) cells have been described to express different endosomal TLRs triggered by RNA and DNA sequences derived from both viruses and bacteria. This study was addressed to establish which endosomal TLR could directly mediate NK activation and function after proper stimuli. It was also important to establish the most suitable TLR agonist to be used as adjuvant in tumor vaccines or in combined cancer immunotherapies. METHODS: We assessed endosomal TLR expression in total NK cells by using RT-qPCR and western blotting technique. In some experiments, we purified CD56brightCD16- and CD56dimCD16+ cells subsets by using NK Cell Isolation Kit Activation marker, cytokine production, CD107a expression and cytotoxicity assay were evaluated by flow cytometry. Cytokine release was quantified by ELISA. NK cells obtained from ovarian ascites underwent the same analyses. RESULTS: Although the four endosomal TLRs (TLR3, TLR7/8, and TLR9) were uniformly expressed on CD56brightCD16- and CD56dimCD16+ cell subsets, the TLR7/8 (R848), TLR3 (polyinosinic-polycytidylic acid, Poly I:C) and TLR9 (ODN2395) ligands promoted NK-cell function only in the presence of suboptimal doses of cytokines, including interleukin (IL)-2, IL-12, IL-15, and IL-18, produced in vivo by other environmental cells. We showed that R848 rather than TLR3 and TLR9 agonists primarily activated CD56brightCD16- NK cells by increasing their proliferation, cytokine production and cytotoxic activity. Moreover, we demonstrated that R848, which usually triggers TLR7 and TLR8 on dendritic cells, macrophages and neutrophils cells, activated CD56brightCD16- NK-cell subset only via TLR8. Indeed, specific TLR8 but not TLR7 agonists increased cytokine production and cytotoxic activity of CD56brightCD16- NK cells. Importantly, these activities were also observed in peritoneal NK cells from patients with metastatic ovarian carcinoma, prevalently belonging to the CD56brightCD16- subset. CONCLUSION: These data highlight the potential value of TLR8 in NK cells as a new target for immunotherapy in patients with cancer.

Actions of the NLRP3 and NLRC4 inflammasomes overlap in bone resorption.

Overwhelming evidence indicates that excessive stimulation of innate immune receptors of the NOD-like receptor (NLR) family causes significant damage to multiple tissues, yet the role of these proteins in bone metabolism is not well known. Here, we studied the interaction between the NLRP3 and NLRC4 inflammasomes in bone homeostasis and disease. We found that loss of NLRP3 or NLRC4 inflammasome attenuated osteoclast differentiation in vitro. At the tissue level, lack of NLRP3, or NLRC4 to a lesser extent, resulted in higher baseline bone mass compared to wild-type (WT) mice, and conferred protection against LPS-induced inflammatory osteolysis. Bone mass accrual in mutant mice correlated with lower serum IL-1ß levels in vivo. Unexpectedly, the phenotype of Nlrp3-deficient mice was reversed upon loss of NLRC4 as bone mass was comparable between WT mice and Nlrp3;Nlrc4 knockout mice. Thus, although bone homeostasis is perturbed to various degrees by the lack of NLRP3 or NLRC4, this tissue appears to function normally upon compound loss of the inflammasomes assembled by these receptors.

Regulation of the Immune System Development by Glucocorticoids and Sex Hormones.

Through the release of hormones, the neuro-endocrine system regulates the immune system function promoting adaptation of the organism to the external environment and to intrinsic physiological changes. Glucocorticoids (GCs) and sex hormones not only regulate immune responses, but also control the hematopoietic stem cell (HSC) differentiation and subsequent maturation of immune cell subsets. During the development of an organism, this regulation has long-term consequences. Indeed, the effects of GC exposure during the perinatal period become evident in the adulthood. Analogously, in the context of HSC transplantation (HSCT), the immune system development starts de novo from the donor HSCs. In this review, we summarize the effects of GCs and sex hormones on the regulation of HSC, as well as of adaptive and innate immune cells. Moreover, we discuss the short and long-term implications on hematopoiesis of sex steroid ablation and synthetic GC administration upon HSCT.

Constitutive activation of NF-κB inducing kinase (NIK) in the mesenchymal lineage using Osterix (Sp7)- or Fibroblast-specific protein 1 (S100a4)-Cre drives spontaneous soft tissue sarcoma.

Aberrant NF-κB signaling fuels tumor growth in multiple human cancer types including both hematologic and solid malignancies. Chronic elevated alternative NF-κB signaling can be modeled in transgenic mice upon activation of a conditional NF-κB-inducing kinase (NIK) allele lacking the regulatory TRAF3 binding domain (NT3). Here, we report that expression of NT3 in the mesenchymal lineage with Osterix (Osx/Sp7)-Cre or Fibroblast-Specific Protein 1 (FSP1)-Cre caused subcutaneous, soft tissue tumors. These tumors displayed significantly shorter latency and a greater multiple incidence rate in Fsp1-Cre;NT3 compared to Osx-Cre;NT3 mice, regardless of sex. Histological assessment revealed poorly differentiated solid tumors with some spindled patterns, as well as robust RelB immunostaining, confirming activation of alternative NF-κB. Even though NT3 expression also occurs in the osteolineage in Osx-Cre;NT3 mice, we observed no bony lesions. The staining profiles and pattern of Cre expression in the two lines pointed to a mesenchymal tumor origin. Immunohistochemistry revealed that these tumors stain strongly for alpha-smooth muscle actin (αSMA), although vimentin staining was uniform only in Osx-Cre;NT3 tumors. Negative CD45 and S100 immunostains precluded hematopoietic and melanocytic origins, respectively, while positive staining for cytokeratin 19 (CK19), typically associated with epithelia, was found in subpopulations of both tumors. Principal component, differential expression, and gene ontology analyses revealed that NT3 tumors are distinct from normal mesenchymal tissues and are enriched for NF-κB related biological processes. We conclude that constitutive activation of the alternative NF-κB pathway in the mesenchymal lineage drives spontaneous sarcoma and provides a novel mouse model for NF-κB related sarcomas.

Multi-tissue single-cell analysis deconstructs the complex programs of mouse natural killer and type 1 innate lymphoid cells in tissues and circulation.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s) are heterogenous innate lymphocytes broadly defined in mice as Lin-NK1.1+NKp46+ cells that express the transcription factor T-BET and produce interferon-γ. The ILC1 definition primarily stems from studies on liver and small intestinal populations. However, NK1.1+NKp46+ cells in the salivary glands, uterus, adipose, and other tissues exhibit nonuniform programs that differ from those of liver or intestinal ILC1s or NK cells. Here, we performed single-cell RNA sequencing on murine NK1.1+NKp46+ cells from blood, spleen, various tissues, and solid tumors. We identified gene expression programs of tissue-specific ILC1s, tissue-specific NK cells, and non-tissue-specific populations in blood, spleen, and other tissues largely corresponding to circulating cells. Moreover, we found that circulating NK cell programs were reshaped in tumor-bearing mice. Core programs of circulating and tumor NK cells paralleled conserved human NK cells signatures, advancing our understanding of the human NK-ILC1 spectrum.

TREM2 Modulation Remodels the Tumor Myeloid Landscape Enhancing Anti-PD-1 Immunotherapy.

Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer's disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2-/- mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1+ and CX3CR1+ macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy.

Osterix-Cre marks distinct subsets of CD45- and CD45+ stromal populations in extra-skeletal tumors with pro-tumorigenic characteristics.

Cancer-associated fibroblasts (CAFs) are a heterogeneous population of mesenchymal cells supporting tumor progression, whose origin remains to be fully elucidated. Osterix (Osx) is a marker of osteogenic differentiation, expressed in skeletal progenitor stem cells and bone-forming osteoblasts. We report Osx expression in CAFs and by using Osx-cre;TdTomato reporter mice we confirm the presence and pro-tumorigenic function of TdTOSX+ cells in extra-skeletal tumors. Surprisingly, only a minority of TdTOSX+ cells expresses fibroblast and osteogenic markers. The majority of TdTOSX+ cells express the hematopoietic marker CD45, have a genetic and phenotypic profile resembling that of tumor infiltrating myeloid and lymphoid populations, but with higher expression of lymphocytic immune suppressive genes. We find Osx transcript and Osx protein expression early during hematopoiesis, in subsets of hematopoietic stem cells and multipotent progenitor populations. Our results indicate that Osx marks distinct tumor promoting CD45- and CD45+ populations and challenge the dogma that Osx is expressed exclusively in cells of mesenchymal origin.

Therapy-Induced Senescence Drives Bone Loss.

Chemotherapy is important for cancer treatment, however, toxicities limit its use. While great strides have been made to ameliorate the acute toxicities induced by chemotherapy, long-term comorbidities including bone loss remain a significant problem. Chemotherapy-driven estrogen loss is postulated to drive bone loss, but significant data suggests the existence of an estrogen-independent mechanism of bone loss. Using clinically relevant mouse models, we showed that senescence and its senescence-associated secretory phenotype (SASP) contribute to chemotherapy-induced bone loss that can be rescued by depleting senescent cells. Chemotherapy-induced SASP could be limited by targeting the p38MAPK-MK2 pathway, which resulted in preservation of bone integrity in chemotherapy-treated mice. These results transform our understanding of chemotherapy-induced bone loss by identifying senescent cells as major drivers of bone loss and the p38MAPK-MK2 axis as a putative therapeutic target that can preserve bone and improve a cancer survivor's quality of life. SIGNIFICANCE: Senescence drives chemotherapy-induced bone loss that is rescued by p38MAPK or MK2 inhibitors. These findings may lead to treatments for therapy-induced bone loss, significantly increasing quality of life for cancer survivors.

Drug-Induced Senescent Multiple Myeloma Cells Elicit NK Cell Proliferation by Direct or Exosome-Mediated IL15 Trans-Presentation.

Treatment of multiple myeloma (MM) cells with sublethal doses of genotoxic drugs leads to senescence and results in increased NK cell recognition and effector functions. Herein, we demonstrated that doxorubicin- and melphalan-treated senescent cells display increased expression of IL15, a cytokine involved in NK cell activation, proliferation, and maturation. IL15 upregulation was evident at the mRNA and protein level, both in MM cell lines and malignant plasma cells from patients' bone marrow (BM) aspirates. However, IL15 was detectable as a soluble cytokine only in vivo, thus indicating a functional role of IL15 in the BM tumor microenvironment. The increased IL15 was accompanied by enhanced expression of the IL15/IL15RA complex on the membrane of senescent myeloma cells, allowing the functional trans-presentation of this cytokine to neighboring NK cells, which consequently underwent activation and proliferation. We demonstrated that MM cell-derived exosomes, the release of which was augmented by melphalan treatment in senescent cells, also expressed IL15RA and IL15, and their interaction with NK cells in the presence of exogenous IL15 resulted in increased proliferation. Altogether, our data demonstrated that low doses of chemotherapeutic drugs, by inducing tumor cell senescence and a senescence-associated secretory phenotype, promoted IL15 trans-presentation to NK cells and, in turn, their activation and proliferation, thus enhancing NK cell-tumor immune surveillance and providing new insights for the exploitation of senescence-based cancer therapies. Cancer Immunol Res; 6(7); 860-9. ©2018 AACR.

Bone matrix components activate the NLRP3 inflammasome and promote osteoclast differentiation.

The NLRP3 inflammasome senses a variety of signals referred to as danger associated molecular patterns (DAMPs), including those triggered by crystalline particulates or degradation products of extracellular matrix. Since some DAMPs confer tissue-specific activation of the inflammasomes, we tested the hypothesis that bone matrix components function as DAMPs for the NLRP3 inflammasome and regulate osteoclast differentiation. Indeed, bone particles cause exuberant osteoclastogenesis in the presence of RANKL, a response that correlates with NLRP3 abundance and the state of inflammasome activation. To determine the relevance of these findings to bone homeostasis, we studied the impact of Nlrp3 deficiency on bone using pre-clinical mouse models of high bone turnover, including estrogen deficiency and sustained exposure to parathyroid hormone or RANKL. Despite comparable baseline indices of bone mass, bone loss caused by hormonal or RANKL perturbations is significantly reduced in Nlrp3 deficient than in wild type mice. Consistent with the notion that osteolysis releases DAMPs from bone matrix, pharmacologic inhibition of bone resorption by zoledronate attenuates inflammasome activation in mice. Thus, signals originating from bone matrix activate the NLRP3 inflammasome in the osteoclast lineage, and may represent a bone-restricted positive feedback mechanism that amplifies bone resorption in pathologic conditions of accelerated bone turnover.

p38 MAPK differentially controls NK activating ligands at transcriptional and post-transcriptional level on multiple myeloma cells.

The mechanisms that regulate the expression of the NKG2D and DNAM-1 activating ligands are only partially known, but it is now widely established that their expression is finely regulated at transcriptional, post-transcriptional and post-translational level, and involve numerous stress pathways depending on the type of ligand, stressor, and cell context. We show that treatment of Multiple Myeloma (MM) cells with sub-lethal doses of Vincristine (VCR), an anticancer drug that inhibits the assembly of microtubules, stimulates the expression of NKG2D and DNAM-1 activating ligands, rendering these cells more susceptible to NK cell-mediated killing. Herein, we focused our attention on the identification of the signaling pathways leading to de novo surface expression of ULBP-1, and to MICA and PVR upregulation on VCR-treated MM cells, both at protein and mRNA levels. We found that p38MAPK differentially regulates drug-dependent ligand upregulation at transcriptional and post-transcriptional level. More specifically, we observed that ULBP-1 expression is attributable to both increased transcriptional activity mediated by ATM-dependent p53 activation, and enhanced mRNA stability; while the p38-activated E2F1 transcription factor regulates MICA and PVR mRNA expression. All together, our findings reveal a previously unrecognized activity of VCR as anticancer agent, and indicate that in addition to its established ability to arrest cell growth, VCR can also modulate the expression of NKG2D and DNAM-1 activating ligand on tumor cells and thus promoting NK cell-mediated immunosurveillance.

Natural killer cell recognition of in vivo drug-induced senescent multiple myeloma cells.

Recognition of tumor cells by the immune system is a key step in cancer eradication. Melphalan is an alkylating agent routinely used in the treatment of patients with multiple myeloma (MM), but at therapeutic doses it leads to an immunosuppressive state due to lymphopenia. Here, we used a mouse model of MM to investigate the ability of in vivo treatment with low doses of melphalan to modulate natural killer (NK) cell activity, which have been shown to play a major role in the control of MM growth. Melphalan treatment was able to enhance the surface expression of the stress-induced NKG2D ligands RAE-1 and MULT-1, and of the DNAM-1 ligand PVR (CD155) on MM cells, leading to better tumor cell recognition and killing by NK cells, as highlighted by NK cell increased degranulation triggered by melphalan-treated tumor cells. Remarkably, NK cell population was not affected by the melphalan dose used, but rather displayed activation features as indicated by CD107a and CD69 expression. Furthermore, we showed that low doses of melphalan fail to induce tumor cell apoptosis, but promote the in vivo establishment of a senescent tumor cell population, harboring high levels of the stress-induced ligands RAE-1 and PVR. Taken together our data support the concept of using chemotherapy in order to boost antitumor innate immune responses and report the possibility to induce cellular senescence of tumor cells in vivo.

Novel ERα positive breast cancer model with estrogen independent growth in the bone microenvironment.

Despite successful therapeutic options for estrogen receptor-α (ERα)+ breast cancer, resistance to endocrine therapy frequently occurs leading to tumor recurrence. In addition to intrinsic changes in the cancer cells, herein we demonstrate that tumor cell-microenvironment interactions can drive recurrence at specific sites. By using two ERα+ cell lines derived from spontaneous mammary carcinomas in STAT1-/- mice (SSM2, SSM3), we establish that the bone microenvironment offers growth advantage over primary site or lung in the absence of ovarian hormones. While SSM3 did not engraft at primary and skeletal locations in the absence of estrogen, SSM2 selectively grew in bone of ovariectomized mice and following administration of aromatase inhibitors. However, SSM2 growth remained hormone-dependent at extraskeletal sites. Unexpectedly, bone-residing SSM2 cells retained ERα expression and JAK2/STAT3 activation regardless of the hormonal status. These data position the bone microenvironment as a unique site for acquisition of tumor/estrogen independency and identify the first ERα+ hormone-independent tumor model in immunocompetent mice.

Dickkopf-related protein 1 (Dkk1) regulates the accumulation and function of myeloid derived suppressor cells in cancer.

Tumor-stroma interactions contribute to tumorigenesis. Tumor cells can educate the stroma at primary and distant sites to facilitate the recruitment of heterogeneous populations of immature myeloid cells, known as myeloid-derived suppressor cells (MDSCs). MDSCs suppress T cell responses and promote tumor proliferation. One outstanding question is how the local and distant stroma modulate MDSCs during tumor progression. Down-regulation of ß-catenin is critical for MDSC accumulation and immune suppressive functions in mice and humans. Here, we demonstrate that stroma-derived Dickkopf-1 (Dkk1) targets ß-catenin in MDSCs, thus exerting immune suppressive effects during tumor progression. Mice bearing extraskeletal tumors show significantly elevated levels of Dkk1 in bone microenvironment relative to tumor site. Strikingly, Dkk1 neutralization decreases tumor growth and MDSC numbers by rescuing ß-catenin in these cells and restores T cell recruitment at the tumor site. Recombinant Dkk1 suppresses ß-catenin target genes in MDSCs from mice and humans and anti-Dkk1 loses its antitumor effects in mice lacking ß-catenin in myeloid cells or after depletion of MDSCs, demonstrating that Dkk1 directly targets MDSCs. Furthermore, we find a correlation between CD15(+) myeloid cells and Dkk1 in pancreatic cancer patients. We establish a novel immunomodulatory role for Dkk1 in regulating tumor-induced immune suppression via targeting ß-catenin in MDSCs.

The human antibody fragment DIATHIS1 specific for CEACAM1 enhances natural killer cell cytotoxicity against melanoma cell lines in vitro.

Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1 malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1 melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1 melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro-expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell-mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin.

New Indole Tubulin Assembly Inhibitors Cause Stable Arrest of Mitotic Progression, Enhanced Stimulation of Natural Killer Cell Cytotoxic Activity, and Repression of Hedgehog-Dependent Cancer.

We designed 39 new 2-phenylindole derivatives as potential anticancer agents bearing the 3,4,5-trimethoxyphenyl moiety with a sulfur, ketone, or methylene bridging group at position 3 of the indole and with halogen or methoxy substituent(s) at positions 4-7. Compounds 33 and 44 strongly inhibited the growth of the P-glycoprotein-overexpressing multi-drug-resistant cell lines NCI/ADR-RES and Messa/Dx5. At 10 nM, 33 and 44 stimulated the cytotoxic activity of NK cells. At 20-50 nM, 33 and 44 arrested >80% of HeLa cells in the G2/M phase of the cell cycle, with stable arrest of mitotic progression. Cell cycle arrest was followed by cell death. Indoles 33, 44, and 81 showed strong inhibition of the SAG-induced Hedgehog signaling activation in NIH3T3 Shh-Light II cells with IC50 values of 19, 72, and 38 nM, respectively. Compounds of this class potently inhibited tubulin polymerization and cancer cell growth, including stimulation of natural killer cell cytotoxic activity and repression of Hedgehog-dependent cancer.