Type 1 Innate Lymphoid Cells Limit the Antitumoral Immune Response.

Natural killer (NK) cells are known to be able to kill established tumor cell lines, but important caveats remain regarding their roles in the detection and elimination of developing primary tumors. Using a genetic model of selective ILC1 and NK cell deficiency, we showed that these cells were dispensable for tumor immunosurveillance and immunoediting in the MCA-induced carcinogenesis model. However, we were able to generate primary cell lines derived from MCA-induced tumors with graded sensitivity to NK1.1+ cells (including NK cells and ILC1). This differential sensitivity was associated neither with a modulation of intratumoral NK cell frequency, nor the capacity of tumor cells to activate NK cells. Instead, ILC1 infiltration into the tumor was found to be a critical determinant of NK1.1+ cell-dependent tumor growth. Finally, bulk tumor RNAseq analysis identified a gene expression signature associated with tumor sensitivity to NK1.1+ cells. ILC1 therefore appear to play an active role in inhibiting the antitumoral immune response, prompting to evaluate the differential tumor infiltration of ILC1 and NK cells in patients to optimize the harnessing of immunity in cancer therapies.

The Ability of the Nitric Oxide Synthases Inhibitor T1023 to Selectively Protect the Non-Malignant Tissues.

Previously, we showed that a nitric oxide synthase (NOS) inhibitor, compound T1023, induces transient hypoxia and prevents acute radiation syndrome (ARS) in mice. Significant efficacy (according to various tests, dose modifying factor (DMF)-1.6-1.9 against H-ARS/G-ARS) and safety in radioprotective doses (1/5-1/4 LD10) became the reason for testing its ability to prevent complications of tumor radiation therapy (RT). Research methods included studying T1023 effects on skin acute radiation reactions (RSR) in rats and mice without tumors and in tumor-bearing animals. The effects were evaluated using clinical, morphological and histological techniques as well as RTOG classification. T1023 administration prior to irradiation significantly limited the severity of acute RSR. This was due to a decrease in radiation alteration of the skin and underlying tissues, and the preservation of the functional activity of cell populations that are critical in the pathogenesis of radiation burn. The DMF values for T1023 for skin protection were 1.4-1.7. Moreover, its radioprotective effect was fully selective to normal tissues in RT models of solid tumors-T1023 reduced the severity of acute RSR and did not modify the antitumor effects of γ-radiation. The results indicate that T1023 can selectively protect the non-malignant tissues against γ-radiation due to hypoxic mechanism of action and potentiate opportunities of NOS inhibitors in RT complications prevention.

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.

The soluble glycoprotein NMB (GPNMB) produced by macrophages induces cancer stemness and metastasis via CD44 and IL-33.

In cancer, myeloid cells have tumor-supporting roles. We reported that the protein GPNMB (glycoprotein nonmetastatic B) was profoundly upregulated in macrophages interacting with tumor cells. Here, using mouse tumor models, we show that macrophage-derived soluble GPNMB increases tumor growth and metastasis in Gpnmb-mutant mice (DBA/2J). GPNMB triggers in the cancer cells the formation of self-renewing spheroids, which are characterized by the expression of cancer stem cell markers, prolonged cell survival and increased tumor-forming ability. Through the CD44 receptor, GPNMB mechanistically activates tumor cells to express the cytokine IL-33 and its receptor IL-1R1L. We also determined that recombinant IL-33 binding to IL-1R1L is sufficient to induce tumor spheroid formation with features of cancer stem cells. Overall, our results reveal a new paracrine axis, GPNMB and IL-33, which is activated during the cross talk of macrophages with tumor cells and eventually promotes cancer cell survival, the expansion of cancer stem cells and the acquisition of a metastatic phenotype.

Pharmacogenomics and Pharmacogenetics in Osteosarcoma: Translational Studies and Clinical Impact.

High-grade osteosarcoma (HGOS) is a very aggressive bone tumor which primarily affects adolescents and young adults. Although not advanced as is the case for other cancers, pharmacogenetic and pharmacogenomic studies applied to HGOS have been providing hope for an improved understanding of the biology and the identification of genetic biomarkers, which may impact on clinical care management. Recent developments of pharmacogenetics and pharmacogenomics in HGOS are expected to: i) highlight genetic events that trigger oncogenesis or which may act as drivers of disease; ii) validate research models that best predict clinical behavior; and iii) indicate genetic biomarkers associated with clinical outcome (in terms of treatment response, survival probability and susceptibility to chemotherapy-related toxicities). The generated body of information may be translated to clinical settings, in order to improve both effectiveness and safety of conventional chemotherapy trials as well as to indicate new tailored treatment strategies. Here, we review and summarize the current scientific evidence for each of the aforementioned issues in view of possible clinical applications.

Dual source hybrid spectral micro-CT using an energy-integrating and a photon-counting detector.

Preclinical micro-CT provides a hotbed in which to develop new imaging technologies, including spectral CT using photon counting detector (PCD) technology. Spectral imaging using PCDs promises to expand x-ray CT as a functional imaging modality, capable of molecular imaging, while maintaining CT's role as a powerful anatomical imaging modality. However, the utility of PCDs suffers due to distorted spectral measurements, affecting the accuracy of material decomposition. We attempt to improve material decomposition accuracy using our novel hybrid dual-source micro-CT system which combines a PCD and an energy integrating detector. Comparisons are made between PCD-only and hybrid CT results, both reconstructed with our iterative, multi-channel algorithm based on the split Bregman method and regularized with rank-sparse kernel regression. Multi-material decomposition is performed post-reconstruction for separation of iodine (I), gold (Au), gadolinium (Gd), and calcium (Ca). System performance is evaluated first in simulations, then in micro-CT phantoms, and finally in an in vivo experiment with a genetically modified p53fl/fl mouse cancer model with Au, Gd, and I nanoparticle (NP)-based contrasts agents. Our results show that the PCD-only and hybrid CT reconstructions offered very similar spatial resolution at 10% MTF (PCD: 3.50 lp mm-1; hybrid: 3.47 lp mm-1) and noise characteristics given by the noise power spectrum. For material decomposition we note successful separation of the four basis materials. We found that hybrid reconstruction reduces RMSE by an average of 37% across all material maps when compared to PCD-only of similar dose but does not provide much difference in terms of concentration accuracy. The in vivo results show separation of targeted Au and accumulated Gd NPs in the tumor from intravascular iodine NPs and bone. Hybrid spectral micro-CT can benefit nanotechnology and cancer research by providing quantitative imaging to test and optimize various NPs for diagnostic and therapeutic applications.

Melanocortin-4 receptor antagonist TCMCB07 ameliorates cancer- and chronic kidney disease-associated cachexia.

Cachexia, a devastating wasting syndrome characterized by severe weight loss with specific losses of muscle and adipose tissue, is driven by reduced food intake, increased energy expenditure, excess catabolism, and inflammation. Cachexia is associated with poor prognosis and high mortality and frequently occurs in patients with cancer, chronic kidney disease, infection, and many other illnesses. There is no effective treatment for this condition. Hypothalamic melanocortins have a potent and long-lasting inhibitory effect on feeding and anabolism, and pathophysiological processes increase melanocortin signaling tone, leading to anorexia, metabolic changes, and eventual cachexia. We used 3 rat models of anorexia and cachexia (LPS, methylcholanthrene sarcoma, and 5/6 subtotal nephrectomy) to evaluate efficacy of TCMCB07, a synthetic antagonist of the melanocortin-4 receptor. Our data show that peripheral treatment using TCMCB07 with intraperitoneal, subcutaneous, and oral administration increased food intake and body weight and preserved fat mass and lean mass during cachexia and LPS-induced anorexia. Furthermore, administration of TCMCB07 diminished hypothalamic inflammatory gene expression in cancer cachexia. These results suggest that peripheral TCMCB07 treatment effectively inhibits central melanocortin signaling and therefore stimulates appetite and enhances anabolism, indicating that TCMCB07 is a promising drug candidate for treating cachexia.

NKTR-214 immunotherapy synergizes with radiotherapy to stimulate systemic CD8+ T cell responses capable of curing multi-focal cancer.

BACKGROUND: High-dose radiotherapy (RT) is known to be immunogenic, but is rarely capable of driving clinically relevant abscopal antitumor immunity as monotherapy. RT is known to increase antigen presentation, type I/II interferon responses, and immune cell trafficking to irradiated tumors. Bempegaldesleukin (NKTR-214) is a CD122-preferential interleukin 2 (IL-2) pathway agonist that has been shown to increase tumor-infiltrating lymphocytes, T cell clonality, and increase PD-1 expression. NKTR-214 has increased drug half-life, decreased toxicity, and increased CD8+ T cell and natural killer cell stimulation compared with IL-2. METHODS: Animals bearing bilateral subcutaneous MCA-205 fibrosarcoma or CT26 colorectal tumors were treated with NKTR-214, RT, or combination therapy, and tumor growth of irradiated and abscopal lesions was assessed. Focal RT was delivered using a small animal radiation research platform. Peripheral and tumor-infiltrating immune phenotype and functional analyses were performed by flow cytometry. RNA expression profiling from both irradiated and abscopal lesions was performed using microarray. RESULTS: We demonstrate synergy between RT of a single tumor and NKTR-214 systemic therapy resulting in dramatically increased cure rates of mice bearing bilateral tumors compared with RT or NKTR-214 therapy alone. Combination therapy resulted in increased magnitude and effector function of tumor-specific CD8+ T cell responses and increased trafficking of these T cells to both irradiated and distant, unirradiated, tumors. CONCLUSIONS: Given the increasing role of hypofractionated and stereotactic body RT as standard of care treatments in the management of locally advanced and metastatic cancer, these data have important implications for future clinical trial development. The combination of RT and NKTR-214 therapy potently stimulates systemic antitumor immunity and should be evaluated for the treatment of patients with locally advanced and metastatic solid tumors.

Vaccine efficacy against primary and metastatic cancer with in vitro-generated CD103+ conventional dendritic cells.

BACKGROUND: Type 1 conventional dendritic cells (cDC1s) possess efficient antigen presentation and cross-presentation activity, as well as potent T cell priming ability. Tissue-resident cDC1s (CD103+ cDC1s in mice, CD141+ cDC1s in humans) are linked with improved tumor control, yet the efficacy of immunotherapy using this population is understudied. METHODS: We generated murine CD103+ cDC1s in vitro and examined their expression of cDC1-related factors, antigen cross-presentation activity, and accumulation in tumor-draining lymph nodes (TdLNs). The antitumor efficacy of the in vitro-generated CD103+ cDC1s was studied in murine melanoma and osteosarcoma models. We evaluated tumor responses on vaccination with CD103+ cDC1s, compared these to vaccination with monocyte-derived DCs (MoDCs), tested CD103+ cDC1 vaccination with checkpoint blockade, and examined the antimetastatic activity of CD103+ cDC1s. RESULTS: In vitro-generated CD103+ cDC1s produced cDC1-associated factors such as interleukin-12p70 and CXCL10, and demonstrated antigen cross-presentation activity on stimulation with the toll-like receptor 3 agonist polyinosinic:polycytidylic acid (poly I:C). In vitro-generated CD103+ cDC1s also migrated to TdLNs following poly I:C treatment and intratumoral delivery. Vaccination with poly I:C-activated and tumor antigen-loaded CD103+ cDC1s enhanced tumor infiltration of tumor antigen-specific and interferon-γ+ CD8+ T cells, and suppressed melanoma and osteosarcoma growth. CD103+ cDC1s showed superior antitumor efficacy compared with MoDC vaccination, and led to complete regression of 100% of osteosarcoma tumors in combination with CTLA-4 antibody-mediated checkpoint blockade. In vitro-generated CD103+ cDC1s effectively protected mice from pulmonary melanoma and osteosarcoma metastases. CONCLUSIONS: Our data indicate an in vitro-generated CD103+ cDC1 vaccine elicits systemic and long-lasting tumor-specific T cell-mediated cytotoxicity, which restrains primary and metastatic tumor growth. The CD103+ cDC1 vaccine was superior to MoDCs and enhanced response to immune checkpoint blockade. These results indicate the potential for new immunotherapies based on use of cDC1s alone or in combination with checkpoint blockade.

Multifaceted effects of soluble human CD6 in experimental cancer models.

BACKGROUND: CD6 is a lymphocyte surface co-receptor physically associated with the T-cell receptor (TCR)/CD3 complex at the center of the immunological synapse. There, CD6 assists in cell-to-cell contact stabilization and modulation of activation/differentiation events through interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), its main reported ligand. While accumulating evidence is attracting new interest on targeting CD6 for therapeutic purposes in autoimmune disorders, little is known on its potential in cancer. In an attempt to elucidate the in vivo relevance of blocking CD6-mediated interactions in health and disease, we explored the consequences of expressing high circulating levels of a soluble form CD6 (sCD6) as a decoy receptor. METHODS: High sCD6 serum levels were achieved by using transgenic C57BL/6 mice expressing human sCD6 under the control of lymphoid-specific transcriptional elements (shCD6LckEµTg) or wild type either transduced with hepatotropic adeno-associated virus coding for mouse sCD6 or undergoing repeated infusions of recombinant human sCD6 protein. Characterization of sCD6-induced changes was performed by ex vivo flow cytometry and functional analyses of mouse lymphoid organ cells. The in vivo relevance of those changes was explored by challenging mice with subcutaneous or metastatic tumors induced by syngeneic cancer cells of different lineage origins. RESULTS: Through a combination of in vitro and in vivo studies, we show that circulating sCD6 expression induces defective regulatory T cell (Treg) generation and function, decreased CD166/ALCAM-mediated tumor cell proliferation/migration and impaired galectin-induced T-cell apoptosis, supporting the fact that sCD6 modulates antitumor lymphocyte effector function and tumorigenesis. Accordingly, sCD6 expression in vivo resulted in delayed subcutaneous tumor growth and/or reduced metastasis on challenge of mice with syngeneic cancer cells. CONCLUSIONS: Evidence is provided for the disruption of CD6 receptor-ligand interactions as a feasible immunomodulatory approach in cancer.

Highly effective anti-tumor nanomedicines based on HPMA copolymer conjugates with pirarubicin prepared by controlled RAFT polymerization.

Here, we describe innovative synthesis of well-defined biocompatible N-(2-hydroxypropyl) methacrylamide (HPMA)-based polymer carriers and their drug conjugates with pirarubicin intended for controlled drug delivery and pH-triggered drug activation in tumor tissue. Polymer carrier synthesis was optimized to obtain well-defined linear HPMA-based polymer precursor with dispersity close to 1 and molar mass close to renal threshold with minimal synthesis steps. The developed synthesis enables preparation of tailored polymer nanomedicines with highly enhanced biological behavior in vivo, especially the biodistribution, urine elimination, tumor accumulation and anticancer activity. STATEMENT OF SIGNIFICANCE: The manuscript reports on novel synthesis and detailed physicochemical characterization and in vivo evaluation of well-defined biocompatible hydrophilic copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA) and their drug conjugates with pirarubicin enabling controlled drug delivery and pH-triggered drug activation in tumor tissue. Polymer carrier synthesis was optimized to obtain well-defined linear HPMA-based polymer precursor with minimal synthesis steps using controlled polymerization. Compared to previously published HPMA-based polymer drug conjugates whose polymer carriers were prepared by classical route via free radical polymerization, the newly prepared polymer drug conjugates exhibited enhanced biological behavior in vivo, especially the prolonged blood circulation, urine elimination, tumor accumulation and excellent anticancer activity. We believe that the newly prepared well-defined polymer conjugates could significantly enhance tumor therapy in humans.

Genome-wide CRISPR Screen to Identify Genes that Suppress Transformation in the Presence of Endogenous KrasG12D.

Cooperating gene mutations are typically required to transform normal cells enabling growth in soft agar or in immunodeficient mice. For example, mutations in Kras and transformation-related protein 53 (Trp53) are known to transform a variety of mesenchymal and epithelial cells in vitro and in vivo. Identifying other genes that can cooperate with oncogenic Kras and substitute for Trp53 mutation has the potential to lead to new insights into mechanisms of carcinogenesis. Here, we applied a genome-wide CRISPR/Cas9 knockout screen in KrasG12D immortalized mouse embryonic fibroblasts (MEFs) to search for genes that when mutated cooperate with oncogenic Kras to induce transformation. We also tested if mutation of the identified candidate genes could cooperate with KrasG12D to generate primary sarcomas in mice. In addition to identifying the well-known tumor suppressor cyclin dependent kinase inhibitor 2A (Cdkn2a), whose alternative reading frame product p19 activates Trp53, we also identified other putative tumor suppressors, such as F-box/WD repeat-containing protein 7 (Fbxw7) and solute carrier family 9 member 3 (Slc9a3). Remarkably, the TCGA database indicates that both FBXW7 and SLC9A3 are commonly co-mutated with KRAS in human cancers. However, we found that only mutation of Trp53 or Cdkn2a, but not Fbxw7 or Slc9a3 can cooperate with KrasG12D to generate primary sarcomas in mice. These results show that mutations in oncogenic Kras and either Fbxw7 or Slc9a3 are sufficient for transformation in vitro, but not for in vivo sarcomagenesis.

Evaluation of Antitumor Efficiency of High Intensity Radiation 169Yb Source on Experimental Sarcoma M-1.

Rats with sarcoma M-1 were exposed to high dose rate irradiation with 169Yb source. In 25 days after introduction of a trocar with sealed capsule with 169Yb source into the tumor, complete tumor regression was observed in 70% animals. The results suggest feasibility of using 169Yb source for high-dose rate brachytherapy and development of the personalized medicine approaches.

Nanotechnology-mediated immunochemotherapy with Ingenol-3-Mebutate for Systematic Anti-tumor Effects.

Cancer-Immunotherapy was the most exciting topic. However, either insensitivity due to singleness of therapeutic target or immune evasion leads to the failure of the treatment. Ingenol-3-mebutate (I3A) can inhibit cancer through synergy between immunotherapy and chemotherapy, however, the speculation and accurate mechanism haven't been confirmed in vivo limited by its hydrophobicity and pH-instability, which also hindered its clinical translation. Herein we developed a polymeric micelle with 'acidic core' provided by single alcoholic hydroxyl (-CH(CH3)-OH) encapsulating I3A (I3A-PM), which successfully overcome the aforementioned problems and reduce the toxicity in vivo. To test the synergy, S180 tumor-bearing mice were subjected to I3A-PM through intravenous and intratumoral administration, we found I3A-PM presented significant antitumor effect, and promoted Th1 polarization by upregulating the level of Th1 cytokines (IL-12, IL-2, IFN-γ and TNF-α), and accelerated the expansion of CD4+ and CD8+ T cells, meanwhile, I3A-PM depleted regulatory T cells, Th2 cytokine IL-6 through inhibiting TGF-ß signaling pathway. Furthermore, we appealed to virtual screening of tumor target, and found a new pathway of I3A as a TGF-ß receptor type I inhibitor to improve immunostimulatory effects. These results demonstrated I3A-PM as a promising nanoagent for cancer immunotherapy strategy. The synergistic therapeutic effects are encouraged to further evaluate in different cancer model compared with commercial products to facilitate research finding (I3A-PM) entering the clinic.

Loss of atrx cooperates with p53-deficiency to promote the development of sarcomas and other malignancies.

The SWI/SNF-family chromatin remodeling protein ATRX is a tumor suppressor in sarcomas, gliomas and other malignancies. Its loss of function facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells, while it also affects Polycomb repressive complex 2 (PRC2) silencing of its target genes. To further define the role of inactivating ATRX mutations in carcinogenesis, we knocked out atrx in our previously reported p53/nf1-deficient zebrafish line that develops malignant peripheral nerve sheath tumors and gliomas. Complete inactivation of atrx using CRISPR/Cas9 was lethal in developing fish and resulted in an alpha-thalassemia-like phenotype including reduced alpha-globin expression. In p53/nf1-deficient zebrafish neither peripheral nerve sheath tumors nor gliomas showed accelerated onset in atrx+/- fish, but these fish developed various tumors that were not observed in their atrx+/+ siblings, including epithelioid sarcoma, angiosarcoma, undifferentiated pleomorphic sarcoma and rare types of carcinoma. These cancer types are included in the AACR Genie database of human tumors associated with mutant ATRX, indicating that our zebrafish model reliably mimics a role for ATRX-loss in the early pathogenesis of these human cancer types. RNA-seq of p53/nf1- and p53/nf1/atrx-deficient tumors revealed that down-regulation of telomerase accompanied ALT-mediated lengthening of the telomeres in atrx-mutant samples. Moreover, inactivating mutations in atrx disturbed PRC2-target gene silencing, indicating a connection between ATRX loss and PRC2 dysfunction in cancer development.

Targeted Delivery of miRNA 155 to Tumor Associated Macrophages for Tumor Immunotherapy.

Tumor associated macrophages (TAMs) are important components residing in the tumor microenvironment. They are immunosuppressive and promote tumor progression. Targeting TAMs and reprogramming their phenotype may be a promising strategy that can restore antitumor immune responses. In this study, we developed a microRNA delivery system based on lipid-coated calcium phosphonate nanoparticles (CaP/miR@pMNPs) containing conjugated mannose and sterically shielded with a pH-responsive material. The nanocarrier could respond to the low pH in the tumor microenvironment and expose mannose to promote cellular internalization in TAMs. The carrier could reactivate TAMs and reprogram their functions, reverse the immunosuppressive tumor microenvironment, and inhibit tumor growth in a tumor-bearing mouse model. In summary, redirecting the polarization of TAMs is a potential therapeutic strategy for tumor immunotherapy.

Murine Models of Bone Sarcomas.

This chapter describes the procedures for inducing bone sarcoma in mice. Two models based on inoculation of cancer cells in paraosseous and intraosseous site will be described. In addition to providing technical aspects of anesthesia and surgical options, key information of cell preparation and postoperative follow-up will be discussed.

Nicotinamide Phosphoribosyltransferase Acts as a Metabolic Gate for Mobilization of Myeloid-Derived Suppressor Cells.

Cancer induces alteration of hematopoiesis to fuel disease progression. We report that in tumor-bearing mice the macrophage colony-stimulating factor elevates the myeloid cell levels of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD salvage pathway, which acts as negative regulator of the CXCR4 retention axis of hematopoietic cells in the bone marrow. NAMPT inhibits CXCR4 through a NAD/Sirtuin 1-mediated inactivation of HIF1α-driven CXCR4 gene transcription, leading to mobilization of immature myeloid-derived suppressor cells (MDSC) and enhancing their production of suppressive nitric oxide. Pharmacologic inhibition or myeloid-specific ablation of NAMPT prevented MDSC mobilization, reactivated specific antitumor immunity, and enhanced the antitumor activity of immune checkpoint inhibitors. Our findings identify NAMPT as a metabolic gate of MDSC precursor function, providing new opportunities to reverse tumor immunosuppression and to restore clinical efficacy of immunotherapy in patients with cancer. SIGNIFICANCE: These findings identify NAMPT as a metabolic gate of MDSC precursor function, providing new opportunities to reverse tumor immunosuppression and to restore clinical efficacy of immunotherapy in cancer patients.

The antitumor efficacy of monomeric disintegrin obtustatin in S-180 sarcoma mouse model.

Obtustatin, isolated from the Levantine Viper snake venom (Macrovipera lebetina obtusa -MLO), is the shortest known monomeric disintegrin shown to specifically inhibit the binding of the α1ß1 integrin to collagen IV. Its oncostatic effect is due to the inhibition of angiogenesis, likely through α1ß1 integrin inhibition in endothelial cells. To explore the therapeutic potential of obtustatin, we studied its effect in S-180 sarcoma-bearing mice model in vivo as well as in human dermal microvascular endothelial cells (HMVEC-D) in vitro, and tested anti-angiogenic activity in vivo using the chick embryo chorioallantoic membrane assay (CAM assay). Our in vivo results show that obtustatin inhibits tumour growth by 33%. The expression of vascular endothelial growth factor (VEGF) increased after treatment with obtustatin, but the level of expression of caspase 8 did not change. In addition, our results demonstrate that obtustatin inhibits FGF2-induced angiogenesis in the CAM assay. Our in vitro results show that obtustatin does not exhibit cytotoxic activity in HMVEC-D cells in comparison to in vivo results. Thus, our findings disclose that obtustatin might be a potential candidate for the treatment of sarcoma in vivo with low toxicity.

A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage.

OBJECTIVE: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. BACKGROUND: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. METHODS: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. RESULTS: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. CONCLUSION: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.