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Background We investigated the role of A2B-adenosine receptor (A2BAR) in regulating immunosuppressive metabolic stress in the tumor microenvironment (TME). Novel A2BAR antagonist PBF-1129 was tested for anti-tumor activity in animals and evaluated for safety and immunological efficacy in a phase-I clinical trial in non-small-cell lung cancer (NSCLC) patients. Methods Anti-tumor efficacy of A2BAR antagonists and impact on metabolic and immune TME was evaluated in lung, melanoma, colon, breast and EGFR-inducible transgenic cancer models. Employing Electron Paramagnetic Resonance, we assessed changes in TME metabolic parameters including pO2, pH, and inorganic phosphate (Pi) during tumor growth and evaluated the immunological effects of PBF-1129, including its pharmacokinetics, safety, and toxicity in NSCLC patients. Results Levels of metabolic stress correlated with tumor growth, metastasis, and immunosuppression. Tumor interstitial Pi emerged as a correlative and cumulative measure of TME stress and immunosuppression. A2BAR inhibition alleviated metabolic stress, downregulated expression of adenosine-generating ecto-nucleotidases, increased expression of adenosine deaminase (ADA), decreased tumor growth and metastasis, increased IFN-γ-production and enhanced the efficacy of anti-tumor therapies following combination regimens in animal models (anti-PD-1 vs. anti-PD-1 plus PBF-1129 treatment hazard ratio [HR] = 11.74, 95% CI = 3.35 to 41.13, n = 10, P Conclusions Data identify A2BAR as a valuable therapeutic target to modify metabolic and immune TME to reduce immunosuppression, enhance the efficacy of immunotherapies, and support clinical application of PBF-1129 in combination therapies.

Activation of Notch signaling in hematopoietic cells by tumors contributes to immune escape. T-cell defects in tumors can be reversed by treating tumor-bearing mice with multivalent forms of the Notch receptor ligand DLL-1, but the immunologic correlates of this effect have not been elucidated. Here, we report mechanistic insights along with the efficacy of combinational treatments of multivalent DLL-1 with oncoprotein targeting drugs in preclinical mouse models of lung cancer. Systemic DLL-1 administration increased T-cell infiltration into tumors and elevated numbers of CD44+CD62L+CD8+ memory T cells while decreasing the number of regulatory T cells and limiting tumor vascularization. This treatment was associated with upregulation of Notch and its ligands in tumor-infiltrating T cells enhanced expression of T-bet and phosphorylation of Stat1/2. Adoptive transfer of T cells from DLL1-treated tumor-bearing immunocompetent hosts into tumor-bearing SCID-NOD immunocompromised mice attenuated tumor growth and extended tumor-free survival in the recipients. When combined with the EGFR-targeted drug erlotinib, DLL-1 significantly improved progression-free survival by inducing robust tumor-specific T-cell immunity. In tissue culture, DLL1 induced proliferation of human peripheral T cells, but lacked proliferative or clonogenic effects on lung cancer cells. Our findings offer preclinical mechanistic support for the development of multivalent DLL1 to stimulate antitumor immunity. Cancer Res; 75(22); 4728–41. ©2015 AACR.

Primers used for qRT-PCR in the study.

Clustered DLL1 triggers Notch downstream target genes in cancer cells.

The environment of the clinical microbiology laboratory is rapidly changing. Testing methodologies based upon organism growth on an array of liquid and solid media are being replaced by newer methods that enhance the rate of organism identification as well as increase the sensitivity and specificity by which identification occurs. A majority of these new techniques are based upon nucleic hybridization and polymerase-chain reaction technology and range from identification of single-organisms or organism families to multiplexed syndromic panels, which can concurrently examine for the presence of numerous suspect organisms based upon the symptoms exhibited by the patient. In addition, the clinical microbiology laboratory now has access to a level of automation thus far only seen in the chemistry and hematology sections of the clinical laboratory. These transitions have been repeatedly shown to enhance the level of patient care when properly implemented into the laboratory workflow. Conversely, with the rapid encroachment of these new technologies comes potential downfalls, including cost and challenges with training laboratory staff. Collectively, the clinical microbiology laboratory is coming into a new era of technology and patient care that will bring about dramatic changes to conventional testing and organism identification.

Background Notch intercellular communication instructs tissue-specific T-cell development and function. In this study, we explored the roles of dendritic cell (DC)-expressed Notch ligands in the regulation of T-cell effector function. Methods We generated mice with CD11c lineage-specific deletion of Notch Delta-like ligand (Dll)1 and Jagged (Jag)2. Using these genetically-ablated mice and engineered pharmacological Notch ligand constructs, the roles of various Delta-like and Jagged ligands in the regulation of T-cell-mediated immunity were investigated. We assessed tumor growth, mouse survival, cytokine production, immunophenotyping of myeloid and lymphoid populations infiltrating the tumors, expression of checkpoint molecules and T-cell function in the experimental settings of murine lung and pancreatic tumors and cardiac allograft rejection. Correlative studies were also performed for the expression of NOTCH ligands, NOTCH receptors and PD-1 on various subsets of myeloid and lymphoid cells in tumor-infiltrating immune cells analyzed from primary human lung cancers. Results Mice with CD11c lineage-specific deletion of Notch ligand gene Dll1, but not Jag2, exhibited accelerated growth of lung and pancreatic tumors concomitant with decreased antigen-specific CD8+T-cell functions and effector-memory (Tem) differentiation. Increased IL-4 but decreased IFN-γ production and elevated populations of T-regulatory and myeloid-derived suppressor cells were observed in Dll1-ablated mice. Multivalent clustered DLL1-triggered Notch signaling overcame DC Dll1 deficiency and improved anti-tumor T-cell responses, whereas the pharmacological interference by monomeric soluble DLL1 construct suppressed the rejection of mouse tumors and cardiac allograft. Moreover, monomeric soluble JAG1 treatment reduced T-regulatory cells and improved anti-tumor immune responses by decreasing the expression of PD-1 on CD8+Tem cells. A significant correlation was observed between DC-expressed Jagged and Delta-like ligands with Tem-expressed PD-1 and Notch receptors, respectively, in human lung tumor-infiltrates. Conclusion Our data show the importance of specific expression of Notch ligands on DCs in the regulation of T-cell effector function. Thus, strategies incorporating selectively engineered Notch ligands could provide a novel approach of therapeutics for modulating immunity in various immunosuppressive conditions including cancer. Electronic supplementary material The online version of this article (10.1186/s40425-019-0566-4) contains supplementary material, which is available to authorized users.

EGFR tyrosine kinase inhibitors cause dramatic responses in EGFR-mutant lung cancer, but resistance universally develops. The involvement of β-catenin in EGFR TKI resistance has been previously reported, however, the precise mechanism by which β-catenin activation contributes to EGFR TKI resistance is not clear. Here, we show that EGFR inhibition results in the activation of β-catenin signaling in a Notch3-dependent manner, which facilitates the survival of a subset of cells that we call “adaptive persisters”. We previously reported that EGFR-TKI treatment rapidly activates Notch3, and here we describe the physical association of Notch3 with β-catenin, leading to increased stability and activation of β-catenin. We demonstrate that the combination of EGFR-TKI and a β-catenin inhibitor inhibits the development of these adaptive persisters, decreases tumor burden, improves recurrence free survival, and overall survival in xenograft models. These results supports combined EGFR-TKI and β-catenin inhibition in patients with EGFR mutant lung cancer., Treatment of EGFR mutant non-small cell lung cancer (NSCLC) often develops resistance to EGFR TKIs. In this study, the authors discover a non-canonical activation of β-catenin signaling through Notch3 as a mechanism of adaptation to and resistance to EGFR TKI treatment in NSCLC.

Over the course of nearly 150 years, the clinical laboratory has diagnosed infectious diseases and identified their causative agents using a variety of approaches. These approaches can be broadly placed into three categories: biochemical, or growth-based methods, molecular and genomic diagnostics, and biomarker and serological detection of blood components. The principle of the biochemical approach is based on isolating an unknown microorganism before conducting a series of growth-based and preformed enzyme detection tests to determine an identification and subsequent antimicrobial susceptibilities. The molecular approach is the newest diagnostic approach utilized by the laboratory and is based on detection of the genetic components of an unknown organism, either isolated or directly in a clinical specimen. There are a variety of molecular techniques, with the polymerase chain reaction serving as the bases of most currently available methods. The detection of infection and inflammatory indicators, as well as serological molecules, have been utilized for diagnostic purposes nearly as long as growth-based identification methods. The biomarker approach to infectious disease diagnosis has primarily occurred outside of the traditional microbiology department, usually within chemistry and hematology where large scale automated instruments provide rapid results. With this focus series we present a concise review, with a brief history, of these different approaches by describing their underlying methodology and advantages and disadvantages, while highlighting specific examples of each, and the internal and external factors which influenced their development.

Tissue oxygenation, extracellular acidity and tissue reducing capacity are among crucial parameters of tumor microenvironment (TME) of significant importance for tumor pathophysiology. In this paper we demonstrate the complementary application of particulate lithium octa-n-butoxy-naphthalocyanine (LiNc-BuO) and soluble nitroxide (NR) paramagnetic probes for monitoring of these TME parameters using electron paramagnetic resonance (EPR) technique. Two different types of therapeutic interventions were studied: hypothermia and systemic administration of metabolically active drug. In summary, the results demonstrate utility of EPR technique for noninvasive concurrent longitudinal monitoring of physiologically relevant chemical parameters of TME in a mouse xenograft tumor models including that under therapeutic intervention.

Activation of Notch signaling in hematopoietic cells by tumors contributes to immune escape. T-cell defects in tumors can be reversed by treating tumor-bearing mice with multivalent forms of the Notch receptor ligand DLL-1, but the immunologic correlates of this effect have not been elucidated. Here, we report mechanistic insights along with the efficacy of combinational treatments of multivalent DLL-1 with oncoprotein targeting drugs in preclinical mouse models of lung cancer. Systemic DLL-1 administration increased T-cell infiltration into tumors and elevated numbers of CD44+CD62L+CD8+ memory T cells while decreasing the number of regulatory T cells and limiting tumor vascularization. This treatment was associated with upregulation of Notch and its ligands in tumor-infiltrating T cells enhanced expression of T-bet and phosphorylation of Stat1/2. Adoptive transfer of T cells from DLL1-treated tumor-bearing immunocompetent hosts into tumor-bearing SCID-NOD immunocompromised mice attenuated tumor growth and extended tumor-free survival in the recipients. When combined with the EGFR-targeted drug erlotinib, DLL-1 significantly improved progression-free survival by inducing robust tumor-specific T-cell immunity. In tissue culture, DLL1 induced proliferation of human peripheral T cells, but lacked proliferative or clonogenic effects on lung cancer cells. Our findings offer preclinical mechanistic support for the development of multivalent DLL1 to stimulate antitumor immunity. Cancer Res; 75(22); 4728–41. ©2015 AACR.

Noninvasive in vivo assessment of chemical tumor microenvironment (TME) parameters such as oxygen (pO2), extracellular acidosis (pHe), and concentration of interstitial inorganic phosphate (Pi) may provide unique insights into biological processes in solid tumors. In this work, we employ a recently developed multifunctional trityl paramagnetic probe and electron paramagnetic resonance (EPR) technique for in vivo concurrent assessment of these TME parameters in various mouse models of cancer. While the data support the existence of hypoxic and acidic regions in TME, the most dramatic differences, about 2-fold higher concentrations in tumors vs. normal tissues, were observed for interstitial Pi - the only parameter that also allowed for discrimination between non-metastatic and highly metastatic tumors. Correlation analysis between [Pi], pO2, pHe and tumor volumes reveal an association of high [Pi] with changes in tumor metabolism and supports different mechanisms of protons and Pi accumulation in TME. Our data identifies interstitial inorganic phosphate as a new TME marker for tumor progression. Pi association with tumor metabolism, buffer-mediated proton transport, and a requirement of high phosphorus content for the rapid growth in the “growth rate hypothesis” may underline its potential role in tumorigenesis and tumor progression.

The objective of this study was to assess the variability of quantitative streaking between and within groups of laboratory professionals, students, and an automated spiral plater. In today9s clinical laboratories, advances in technology present an ever-changing landscape that mandates adaptations and the microbiology lab is no exception. Despite these advancements, one of the most quintessential manual techniques employed by laboratory professionals and taught to clinical laboratory science and technician students is quantitative streaking of bacterial cultures. Although few, there are studies detailing the accuracy of a 0.001 mL calibrated loop, perhaps the most common tool for quantitative streaking; however, there has been a lack of work addressing the variability associated with laboratory personnel9s individual techniques and inherent variability. Our study analyzed the number of bacterial colony forming units (CFU)/mL that resulted from sequential plating by our control groups and automatic spiral plater from a common sample. The sample was a dilution of bacteria in saline from an initial 0.5 McFarland standard (approximation of 1.5x108 CFU/mL). Preliminary data indicates that in most instances there were significant differences seen (via ANOVA and Tukey post hoc tests; p

Summary Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.

We demonstrated in human and mouse studies that tumor-induced modulation of Notch ligand expression and Notch signaling in hematopoietic compartment contributes to tumor immune escape. Down-regulation of delta-like ligands (DLL) leads to defects in T cell development and T helper (Th1) cell differentiation with the prevalence of regulatory T cell (Treg) generation. To determine the roles of Notch ligands in antigen-presenting dendritic cells in regulation of antitumor immune responses we generated a set of lineage-specific knock-out mice lacking one of the Notch ligands in CD11c+ dendritic cells (DC). We are developing and testing a set of reagents for clinical application for ligand-specific activation or inhibition of Notch signaling to stimulate or inhibit, respectively, various types of immune responses for applications in oncology and immune diseases. Mice with DLL1 insufficiency in DC demonstrated remarkably accelerated growth of Lewis lung carcinoma (LLC) tumor, and reduced survival compared to wild type animals. This associated with impaired anti-tumor immune responses indicated by the decreased tumor infiltration by IFNγ-producing T cells. Jagged2 knockout did not cause any significant alterations. Notch ligand expression in antigen-presenting cells was identified as a “checkpoint” regulating the type of immune response. Data reveal that expression of Notch ligands by antigen-presenting cells is an important immune response specifying mechanism and that ligand-specific Notch signaling could be a valuable therapeutic target. Reagents for the pharmacological modulation of immune responses based on Notch ligand constructs is proposed. Our cell-based study showed that pharmacological activation of Notch ligands required multivalent receptor-ligand interaction, whereas soluble ligands acted as competitive Notch inhibitors. We have generated reagents that comprise specific domains of the DLL1 in multivalent or monovalent form. Therapeutic inhibition of Notch by monovalent DLL1-based reagent accelerated LLC tumor growth and attenuated T cell-mediated anti-tumor immune response. In a heart transplantation mouse model, monovalent DLL1 reagent significantly prolonged allograft survival by inhibiting Th1 effector and memory T cell differentiation. Multivalent forms of DLL1 effectively stimulated Notch signaling in T cell cultures and enhanced IFNγ production, whereas monovalent reagent had opposite effects. Pharmacological up-regulation of DLL1-mediated Notch signaling with multivalent forms of ligand represents an efficient strategy for the enhancement of anti-tumor immunity and targeting multiple mechanisms of tumor growth. Monovalent DLL1 forms could be utilized for therapeutic inhibition of Th1 responses in autoimmune diseases and organ transplantation. Reagents based on the mono- and multivalent forms of Notch ligands can be efficiently utilized for therapeutic modulation of Notch signaling. Note: This abstract was not presented at the meeting. Citation Format: Elena I. Tchekneva, Anneliese E. Antonucci, Irina Chekneva, Nicholas Long, Jason V. Evans, Anwari Akhter, David P. Carbone, Thomas Magliery, Mikhail M. Dikov. NOTCH ligand-based therapeutics for immunomodulation in cancer and organ transplantation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1624. doi:10.1158/1538-7445.AM2017-1624

Platinum-based combination is a standard of care treatment for lung cancer patients. Accumulating evidence indicates that aquaporins are emerging targets in cancer. Aquaporin 11 (AQP11), a non-ubiquitous member of aquaporins family, is an endoplasmic reticulum (ER)-specific water channel mostly present in epithelial cells and is implicated in the maintenance of ER homeostasis. In our recent publications, we demonstrated that under the stress the reduction of AQP11 expression is a pro-apoptotic factor in normal epithelial cells. We revealed that under normal conditions AQP11 is heavily S-nitrosylated forming functionally important multimeric structures (> 1000 kDa), whereas cisplatin treatment interferes with AQP11 causing its structural and post-translational modification. We hypothesized that if tumor cells express AQP11 then cells with lower levels of AQP11 are not protected from cisplatin-associated ER stress and develop higher sensitivity to drug treatment. In normal lung AQP11 is expressed at low levels. We evaluated the expression of AQP11 in lung cancer cells to determine whether the AQP11 expression correlates with cisplatin sensitivity and whether lower AQP11 expression in lung tumor associates with positive overall survival in patients with lung adenocarcinoma. A strong link between the level of AQP11 protein expression and cell sensitivity to CP was detected using MTT assay. Screening lung cancer cell lines we found that all tested cancer cell lines expressed AQP11 protein at various levels. Western blot of stress markers showed that cisplatin-treated cells with higher AQP11 expression had lowered cellular and ER stress. Reduction of AQP11 expression using shRNA AQP11 silencing in cispaltin-resistant A549 and HCC827 cell lines, resulted in the significantly increased up to 3-fold sensitivity to cisplatin compared with control shRNA transfected or parental non-transfected cells (p AQP11 gene is located within oncogenic 11q13-q14 amplicon, which is a common aberration in solid tumors associated with poor prognosis. The AQP11 is a pro-survival factor protecting tumor cells from cisplatin-induced stress. Low AQP11 expression associates with higher OS in lung adenocarcinoma patients and with higher cispaltin sensitivity in lung cancer cell. The ER stress aggravation via interference with AQP11 could become a prospective therapeutic approach in anti-tumor treatment. Citation Format: Jason V. Evans, Anwari Akhter, David P. Carbone, Mikhail M. Dikov, Elena E. Tchekneva. Expression of aquaporin 11 as a potential predictor of cisplatin sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5468. doi:10.1158/1538-7445.AM2017-5468

Cyclin D1 dysregulation and differential inactivation of p16INK4a and Rb have been observed in human lung cancer. In chemically induced mouse lung tumors, the p16INK4a gene is a target of inactivation, and Rb is reduced at the mRNA level (Northern blot) although similar at the protein level (Western blot) when compared to normal lung tissues. The expression of cyclin D1, cdk4, p16INK4a, and Rb protein was examined by immunohistochemistry in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse lung tumors. Immunohistochemical staining revealed exclusive nuclear staining of both cyclin D1 and cdk4 that was light to moderate in normal mouse lung tissues, but intense in lung adenomas and adenocarcinomas. Western blot analysis confirmed the increased expression of cyclin D1 and cdk4 in lung tumors compared to normal lung. Immunohistochemical analyses of lung tumors showed focal areas which lacked p16INK4a staining. Expression of p16INK4a, as determined by RT-PCR, was variable in lung tumors. Mutations in p16INK4a were not found by SSCP analysis. Immunohistochemical analyses of normal lung tissues showed intense staining for Rb protein in alveolar epithelial cells and in other lung cell types; however, in the lung tumors the staining intensity was reduced and the distribution was altered. Expression of Rb was detected in normal lung tissues but was barely detectable by Northern blot hybridization in lung tumors. Western blot analysis indicated the presence of both hypophosphorylated and hyperphosphorylated Rb protein in lung tumors and in normal lung tissues. These results suggest that alterations in the cell cycle proteins, cyclin D1, cdk4, p16INK4a, and Rb, may play a role in the acquisition of autonomous growth by adenomas. Furthermore, they demonstrate the importance of immunohistochemical studies to examine expression in tissues that contain multiple cell types, such as the lung, and in tumors that by nature are heterogeneous.

The actin regulatory protein cortactin is involved in multiple signaling pathways impinging on the cortical actin cytoskeleton. Cortactin is phosphorylated by ERK1/2 and Src family tyrosine kinases, resulting in neuronal Wiskott Aldrich Syndrome protein (N-WASp) activation and enhanced actin related protein (Arp)2/3-mediated actin nucleation. Cortactin migrates as an 80/85 kDa doublet when analyzed by SDS-PAGE. Phosphorylation by ERK1/2 is associated with conversion of the 80 kDa to the 85 kDa form, postulated to occur by inducing a conformational alteration that releases the carboxyl-terminal SH3 domain from autoinhibition. Our recent analysis of the 80–85 kDa cortactin “shift” in tumor cells indicates that while ERK1/2 phosphorylation is associated with the 85 kDa shift, this phosphorylation event is not required for the shift to occur, nor does ERK1/2 phosphorylation appreciably alter global cortactin confirmation. These data indicate that additional factors besides ERK1/2 phosphorylation contribute to generating and/or maintaining the activated 85 kDa cortactin form in stimulated cells.

Elevated Src kinase activity is linked to the progression of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Src regulates HNSCC proliferation and tumor invasion, with the Src-targeted small molecule inhibitor saracatinib displaying potent anti-invasive effects in preclinical studies. However, the pro-invasive cellular mechanism(s) perturbed by saracatinib are unclear. The anti-proliferative and anti-invasive effects of saracatinib on HNSCC cell lines were therefore investigated in pre-clinical cell and mouse model systems. Saracatinib treatment inhibited growth, cell cycle progression and transwell Matrigel invasion in HNSCC cell lines. Dose-dependent decreases in Src activation and phosphorylation of the invasion-associated substrates focal adhesion kinase, p130 CAS and cortactin were also observed. While saracatinib did not significantly impact HNSCC tumor growth in a mouse orthotopic model of tongue squamous cell carcinoma, impaired perineural invasion and cervical lymph node metastasis was observed. Accordingly, saracatinib treatment displayed a dose-dependent inhibitory effect on invadopodia formation, extracellular matrix degradation and matrix metalloprotease 9 activation. These results suggest that inhibition of Src kinase by saracatinib impairs the pro-invasive activity of HNSCC by inhibiting Src substrate phosphorylation important for invadopodia formation and associated matrix metalloprotease activity.