Noninvasive therapy of brain cancer using a unique systemic delivery methodology with a cancer terminator virus.

Primary, glioblastoma, and secondary brain tumors, from metastases outside the brain, are among the most aggressive and therapeutically resistant cancers. A physiological barrier protecting the brain, the blood-brain barrier (BBB), functions as a deterrent to effective therapies. To enhance cancer therapy, we developed a cancer terminator virus (CTV), a unique tropism-modified adenovirus consisting of serotype 3 fiber knob on an otherwise Ad5 capsid that replicates in a cancer-selective manner and simultaneously produces a potent therapeutic cytokine, melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24). A limitation of the CTV and most other viruses, including adenoviruses, is an inability to deliver systemically to treat brain tumors because of the BBB, nonspecific virus trapping, and immune clearance. These obstacles to effective viral therapy of brain cancer have now been overcome using focused ultrasound with a dual microbubble treatment, the focused ultrasound-double microbubble (FUS-DMB) approach. Proof-of-principle is now provided indicating that the BBB can be safely and transiently opened, and the CTV can then be administered in a second set of complement-treated microbubbles and released in the brain using focused ultrasound. Moreover, the FUS-DMB can be used to deliver the CTV multiple times in animals with glioblastoma  growing in their brain thereby resulting in a further enhancement in survival. This strategy permits efficient therapy of primary and secondary brain tumors enhancing animal survival without promoting harmful toxic or behavioral side effects. Additionally, when combined with a standard of care therapy, Temozolomide, a further increase in survival is achieved. The FUS-DMB approach with the CTV highlights a noninvasive strategy to treat brain cancers without surgery. This innovative delivery scheme combined with the therapeutic efficacy of the CTV provides a novel potential translational therapeutic approach for brain cancers.

Cytoplasmic-delivery of polyinosine-polycytidylic acid inhibits pancreatic cancer progression increasing survival by activating Stat1-CCL2-mediated immunity.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer without effective therapies and with poor prognosis, causing 7% of all cancer-related fatalities in the USA. Considering the lack of effective therapies for this aggressive cancer, there is an urgent need to define newer and more effective therapeutic strategies. Polyinosine-polycytidylic acid (pIC) is a synthetic double-stranded RNA (dsRNA) which directly activates dendritic cells and natural killer cells inhibiting tumor growth. When pIC is delivered into the cytoplasm using polyethyleneimine (PEI), pIC-PEI, programmed-cell death is induced in PDAC. Transfection of [pIC]PEI into PDAC cells inhibits growth, promotes toxic autophagy and also induces apoptosis in vitro and in vivo in animal models. METHODS: The KPC transgenic mouse model that recapitulates PDAC development in patients was used to interrogate the role of an intact immune system in vivo in PDAC in response to [pIC]PEI. Antitumor efficacy and survival were monitored endpoints. Comprehensive analysis of the tumor microenvironment (TME) and immune cells, cytokines and chemokines in the spleen, and macrophage polarization were analyzed. RESULTS: Cytosolic delivery of [pIC]PEI induces apoptosis and provokes strong antitumor immunity in vivo in immune competent mice with PDAC. The mechanism underlying the immune stimulatory properties of [pIC]PEI involves Stat1 activation resulting in CCL2 and MMP13 stimulation thereby provoking macrophage polarization. [pIC]PEI induces apoptosis via the AKT-XIAP pathway, as well as macrophage differentiation and T-cell activation via the IFNγ-Stat1-CCL2 signaling pathways in PDAC. In transgenic tumor mouse models, [pIC]PEI promotes robust and profound antitumor activity implying that stimulating the immune system contributes to biological activity. The [pIC]PEI anti-PDAC effects are enhanced when used in combination with a standard of care (SOC) treatment, that is, gemcitabine. CONCLUSIONS: In summary, [pIC]PEI treatment is non-toxic toward normal pancreatic cells while displaying strong cytotoxic and potent immune activating activities in PDAC, making it an attractive therapeutic when used alone or in conjunction with SOC therapeutic agents, potentially providing a safe and effective treatment protocol with translational potential for the effective therapy of PDAC.

MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis.

Bone metastasis is a frequent and incurable consequence of advanced prostate cancer (PC). An interplay between disseminated tumor cells and heterogeneous bone resident cells in the metastatic niche initiates this process. Melanoma differentiation associated gene-9 (mda-9/Syntenin/syndecan binding protein) is a prometastatic gene expressed in multiple organs, including bone marrow-derived mesenchymal stromal cells (BM-MSCs), under both physiological and pathological conditions. We demonstrate that PDGF-AA secreted by tumor cells induces CXCL5 expression in BM-MSCs by suppressing MDA-9-dependent YAP/MST signaling. CXCL5-derived tumor cell proliferation and immune suppression are consequences of the MDA-9/CXCL5 signaling axis, promoting PC disease progression. mda-9 knockout tumor cells express less PDGF-AA and do not develop bone metastases. Our data document a previously undefined role of MDA-9/Syntenin in the tumor and microenvironment in regulating PC bone metastasis. This study provides a framework for translational strategies to ameliorate health complications and morbidity associated with advanced PC.

CellNeighborEX: deciphering neighbor-dependent gene expression from spatial transcriptomics data.

Cells have evolved their communication methods to sense their microenvironments and send biological signals. In addition to communication using ligands and receptors, cells use diverse channels including gap junctions to communicate with their immediate neighbors. Current approaches, however, cannot effectively capture the influence of various microenvironments. Here, we propose a novel approach to investigate cell neighbor-dependent gene expression (CellNeighborEX) in spatial transcriptomics (ST) data. To categorize cells based on their microenvironment, CellNeighborEX uses direct cell location or the mixture of transcriptome from multiple cells depending on ST technologies. For each cell type, CellNeighborEX identifies diverse gene sets associated with partnering cell types, providing further insight. We found that cells express different genes depending on their neighboring cell types in various tissues including mouse embryos, brain, and liver cancer. Those genes are associated with critical biological processes such as development or metastases. We further validated that gene expression is induced by neighboring partners via spatial visualization. The neighbor-dependent gene expression suggests new potential genes involved in cell-cell interactions beyond what ligand-receptor co-expression can discover.

Tumor heterogeneity: An oncogenic driver of PDAC progression and therapy resistance under stress conditions.

Pancreatic ductal adenocarcinoma (PDAC) is a clinically challenging disease usually diagnosed at advanced or metastasized stage. By this year end, there are an expected increase in 62,210 new cases and 49,830 deaths in the United States, with 90% corresponding to PDAC subtype alone. Despite advances in cancer therapy, one of the major challenges combating PDAC remains tumor heterogeneity between PDAC patients and within the primary and metastatic lesions of the same patient. This review describes the PDAC subtypes based on the genomic, transcriptional, epigenetic, and metabolic signatures observed among patients and within individual tumors. Recent studies in tumor biology suggest PDAC heterogeneity as a major driver of disease progression under conditions of stress including hypoxia and nutrient deprivation, leading to metabolic reprogramming. We therefore advance our understanding in identifying the underlying mechanisms that interfere with the crosstalk between the extracellular matrix components and tumor cells that define the mechanics of tumor growth and metastasis. The bilateral interaction between the heterogeneous tumor microenvironment and PDAC cells serves as another important contributor that characterizes the tumor-promoting or tumor-suppressing phenotypes providing an opportunity for an effective treatment regime. Furthermore, we highlight the dynamic reciprocating interplay between the stromal and immune cells that impact immune surveillance or immune evasion response and contribute towards a complex process of tumorigenesis. In summary, the review encapsulates the existing knowledge of the currently applied treatments for PDAC with emphasis on tumor heterogeneity, manifesting at multiple levels, impacting disease progression and therapy resistance under stress.

Neighbor-specific gene expression revealed from physically interacting cells during mouse embryonic development.

Development of multicellular organisms is orchestrated by persistent cell-cell communication between neighboring partners. Direct interaction between different cell types can induce molecular signals that dictate lineage specification and cell fate decisions. Current single-cell RNA-seq technology cannot adequately analyze cell-cell contact-dependent gene expression, mainly due to the loss of spatial information. To overcome this obstacle and resolve cell-cell contact-specific gene expression during embryogenesis, we performed RNA sequencing of physically interacting cells (PIC-seq) and assessed them alongside similar single-cell transcriptomes derived from developing mouse embryos between embryonic day (E) 7.5 and E9.5. Analysis of the PIC-seq data identified gene expression signatures that were dependent on the presence of specific neighboring cell types. Our computational predictions, validated experimentally, demonstrated that neural progenitor (NP) cells upregulate Lhx5 and Nkx2-1 genes, when exclusively interacting with definitive endoderm (DE) cells. Moreover, there was a reciprocal impact on the transcriptome of DE cells, as they tend to upregulate Rax and Gsc when in contact with NP cells. Using individual cell transcriptome data, we formulated a means of computationally predicting the impact of one cell type on the transcriptome of its neighboring cell types. We have further developed a distinctive spatial-t-distributed stochastic neighboring embedding to display the pseudospatial distribution of cells in a 2-dimensional space. In summary, we describe an innovative approach to study contact-specific gene regulation during embryogenesis.

Cell Competition in Carcinogenesis.

The majority of human cancers evolve over time through the stepwise accumulation of somatic mutations followed by clonal selection akin to Darwinian evolution. However, the in-depth mechanisms that govern clonal dynamics and selection remain elusive, particularly during the earliest stages of tissue transformation. Cell competition (CC), often referred to as 'survival of the fittest' at the cellular level, results in the elimination of less fit cells by their more fit neighbors supporting optimal organism health and function. Alternatively, CC may allow an uncontrolled expansion of super-fit cancer cells to outcompete their less fit neighbors thereby fueling tumorigenesis. Recent research discussed herein highlights the various non-cell-autonomous principles, including interclonal competition and cancer microenvironment competition supporting the ability of a tumor to progress from the initial stages to tissue colonization. In addition, we extend current insights from CC-mediated clonal interactions and selection in normal tissues to better comprehend those factors that contribute to cancer development.

IFI27 transcription is an early predictor for COVID-19 outcomes, a multi-cohort observational study.

Purpose: Robust biomarkers that predict disease outcomes amongst COVID-19 patients are necessary for both patient triage and resource prioritisation. Numerous candidate biomarkers have been proposed for COVID-19. However, at present, there is no consensus on the best diagnostic approach to predict outcomes in infected patients. Moreover, it is not clear whether such tools would apply to other potentially pandemic pathogens and therefore of use as stockpile for future pandemic preparedness. Methods: We conducted a multi-cohort observational study to investigate the biology and the prognostic role of interferon alpha-inducible protein 27 (IFI27) in COVID-19 patients. Results: We show that IFI27 is expressed in the respiratory tract of COVID-19 patients and elevated IFI27 expression in the lower respiratory tract is associated with the presence of a high viral load. We further demonstrate that the systemic host response, as measured by blood IFI27 expression, is associated with COVID-19 infection. For clinical outcome prediction (e.g., respiratory failure), IFI27 expression displays a high sensitivity (0.95) and specificity (0.83), outperforming other known predictors of COVID-19 outcomes. Furthermore, IFI27 is upregulated in the blood of infected patients in response to other respiratory viruses. For example, in the pandemic H1N1/09 influenza virus infection, IFI27-like genes were highly upregulated in the blood samples of severely infected patients. Conclusion: These data suggest that prognostic biomarkers targeting the family of IFI27 genes could potentially supplement conventional diagnostic tools in future virus pandemics, independent of whether such pandemics are caused by a coronavirus, an influenza virus or another as yet-to-be discovered respiratory virus.

Flower lose, a cell fitness marker, predicts COVID-19 prognosis.

Risk stratification of COVID-19 patients is essential for pandemic management. Changes in the cell fitness marker, hFwe-Lose, can precede the host immune response to infection, potentially making such a biomarker an earlier triage tool. Here, we evaluate whether hFwe-Lose gene expression can outperform conventional methods in predicting outcomes (e.g., death and hospitalization) in COVID-19 patients. We performed a post-mortem examination of infected lung tissue in deceased COVID-19 patients to determine hFwe-Lose's biological role in acute lung injury. We then performed an observational study (n = 283) to evaluate whether hFwe-Lose expression (in nasopharyngeal samples) could accurately predict hospitalization or death in COVID-19 patients. In COVID-19 patients with acute lung injury, hFwe-Lose is highly expressed in the lower respiratory tract and is co-localized to areas of cell death. In patients presenting in the early phase of COVID-19 illness, hFwe-Lose expression accurately predicts subsequent hospitalization or death with positive predictive values of 87.8-100% and a negative predictive value of 64.1-93.2%. hFwe-Lose outperforms conventional inflammatory biomarkers and patient age and comorbidities, with an area under the receiver operating characteristic curve (AUROC) 0.93-0.97 in predicting hospitalization/death. Specifically, this is significantly higher than the prognostic value of combining biomarkers (serum ferritin, D-dimer, C-reactive protein, and neutrophil-lymphocyte ratio), patient age and comorbidities (AUROC of 0.67-0.92). The cell fitness marker, hFwe-Lose, accurately predicts outcomes in COVID-19 patients. This finding demonstrates how tissue fitness pathways dictate the response to infection and disease and their utility in managing the current COVID-19 pandemic.

Cell competition in intratumoral and tumor microenvironment interactions.

Tumors are complex cellular and acellular environments within which cancer clones are under continuous selection pressures. Cancer cells are in a permanent mode of interaction and competition with each other as well as with the immediate microenvironment. In the course of these competitive interactions, cells share information regarding their general state of fitness, with less-fit cells being typically eliminated via apoptosis at the hands of those cells with greater cellular fitness. Competitive interactions involving exchange of cell fitness information have implications for tumor growth, metastasis, and therapy outcomes. Recent research has highlighted sophisticated pathways such as Flower, Hippo, Myc, and p53 signaling, which are employed by cancer cells and the surrounding microenvironment cells to achieve their evolutionary goals by means of cell competition mechanisms. In this review, we discuss these recent findings and explain their importance and role in evolution, growth, and treatment of cancer. We further consider potential physiological conditions, such as hypoxia and chemotherapy, that can function as selective pressures under which cell competition mechanisms may evolve differently or synergistically to confer oncogenic advantages to cancer.

Cell Competition Boosts Clonal Evolution and Hypoxic Selection in Cancer.

The comparison of fitness between cells leads to the elimination of less competent cells in the presence of more competent neighbors via cell competition (CC). This phenomenon has been linked with several cancer-related genes and thus may play an important role in cancer. Various processes are involved in the regulation of tumor initiation and growth, including tumor hypoxia, clonal stem cell selection, and immune cell response, all of which have been recently shown to have a potential connection with the mechanisms involved in CC. This review aims to unravel the relation between these processes and competitive cell interactions and how this affects disease progression.

Cell Competition Spurs Selection of Aggressive Cancer Cells.

Within heterogeneous tumors, cancer cells are constantly interacting. Cell competition (CC) is a fitness-based selection mechanism that results in increased proliferation of discrete populations at the expense of their less fit neighbors. CC-based selection of fit cells may also drive selection of the most aggressive cancer cells.

Cell competition in tumor evolution and heterogeneity: Merging past and present.

In many cases, cancers are difficult to eliminate because they develop resistance to a primary chemotherapy or targeted therapy. Tumors grow into diverse cell subpopulations, increasing the ability to resist elimination. The phenomenon of 'cell competition' describes our body's natural surveillance system to optimize tissue fitness by forcing viable but aberrant cells to undergo cell death. Cell competition is not simply comparison of cell division potential. Competition factors signal for 'loser' cell elimination and 'winner' cell dominance. New evidence demonstrates it is possible to restrict cancer growth by strengthening the cell fitness of surrounding healthy tissue via anti-apoptotic pathways. Hence, cell competition provides strong conceptual explanation for oncogenesis, tumor growth and suppression. Tumor heterogeneity is a hallmark of many cancers and establishes gradients in which competitive interactions are able to occur among tumor cell subpopulations as well as neighboring stromal tissue. Here we review cellular/molecular competition pathways in the context of tumor evolution, heterogeneity and response to interventions. We propose strategies to exploit these mediators and design novel broad-spectrum therapeutic approaches that eliminate cancer and enhance fitness of neighboring tissue to improve patient outcomes.

HIF-transcribed p53 chaperones HIF-1α.

Chronic hypoxia is associated with a variety of physiological conditions such as rheumatoid arthritis, ischemia/reperfusion injury, stroke, diabetic vasculopathy, epilepsy and cancer. At the molecular level, hypoxia manifests its effects via activation of HIF-dependent transcription. On the other hand, an important transcription factor p53, which controls a myriad of biological functions, is rendered transcriptionally inactive under hypoxic conditions. p53 and HIF-1α are known to share a mysterious relationship and play an ambiguous role in the regulation of hypoxia-induced cellular changes. Here we demonstrate a novel pathway where HIF-1α transcriptionally upregulates both WT and MT p53 by binding to five response elements in p53 promoter. In hypoxic cells, this HIF-1α-induced p53 is transcriptionally inefficient but is abundantly available for protein-protein interactions. Further, both WT and MT p53 proteins bind and chaperone HIF-1α to stabilize its binding at its downstream DNA response elements. This p53-induced chaperoning of HIF-1α increases synthesis of HIF-regulated genes and thus the efficiency of hypoxia-induced molecular changes. This basic biology finding has important implications not only in the design of anti-cancer strategies but also for other physiological conditions where hypoxia results in disease manifestation.

Flower isoforms promote competitive growth in cancer.

In humans, the adaptive immune system uses the exchange of information between cells to detect and eliminate foreign or damaged cells; however, the removal of unwanted cells does not always require an adaptive immune system1,2. For example, cell selection in Drosophila uses a cell selection mechanism based on 'fitness fingerprints', which allow it to delay ageing3, prevent developmental malformations3,4 and replace old tissues during regeneration5. At the molecular level, these fitness fingerprints consist of combinations of Flower membrane proteins3,4,6. Proteins that indicate reduced fitness are called Flower-Lose, because they are expressed in cells marked to be eliminated6. However, the presence of Flower-Lose isoforms at a cell's membrane does not always lead to elimination, because if neighbouring cells have similar levels of Lose proteins, the cell will not be killed4,6,7. Humans could benefit from the capability to recognize unfit cells, because accumulation of damaged but viable cells during development and ageing causes organ dysfunction and disease8-17. However, in Drosophila this mechanism is hijacked by premalignant cells to gain a competitive growth advantage18. This would be undesirable for humans because it might make tumours more aggressive19-21. It is unknown whether a similar mechanism of cell-fitness comparison is present in humans. Here we show that two human Flower isoforms (hFWE1 and hFWE3) behave as Flower-Lose proteins, whereas the other two isoforms (hFWE2 and hFWE4) behave as Flower-Win proteins. The latter give cells a competitive advantage over cells expressing Lose isoforms, but Lose-expressing cells are not eliminated if their neighbours express similar levels of Lose isoforms; these proteins therefore act as fitness fingerprints. Moreover, human cancer cells show increased Win isoform expression and proliferate in the presence of Lose-expressing stroma, which confers a competitive growth advantage on the cancer cells. Inhibition of the expression of Flower proteins reduces tumour growth and metastasis, and induces sensitivity to chemotherapy. Our results show that ancient mechanisms of cell recognition and selection are active in humans and affect oncogenic growth.

Cell competition in development: information from flies and vertebrates.

Cell competition is a biological mechanism conserved from Drosophila to vertebrates wherein neighboring cells compare their relative fitness status resulting in the elimination of less fit cells by those with higher fitness. This is an active process that is essential for embryonic and organ development, tissue homeostasis, delay of ageing and in various disease models such as cancer. Recent research is beginning to unravel the various mechanisms of cell competition and the sensing of fitness status. Fitness fingerprints, death receptors, mechanical cell competition and a set of unknown genetic or signaling pathways are emerging as important pathways governing the mechanisms for cell to compare their relative fitness levels. In this review we are providing an account of recent advances which help summarize the mechanisms of operation and growing role of cell competition in regulation of oncogenesis in vertebrates.

The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53.

p53 is an important tumor-suppressor protein that is mutated in more than 50% of cancers. Strategies for restoring normal p53 function are complicated by the oncogenic properties of mutant p53 and have not met with clinical success. To counteract mutant p53 activity, a variety of drugs with the potential to reconvert mutant p53 to an active wildtype form have been developed. However, these drugs are associated with various negative effects such as cellular toxicity, nonspecific binding to other proteins, and inability to induce a wildtype p53 response in cancer tissue. Here, we report on the effects of a curcumin analog, HO-3867, on p53 activity in cancer cells from different origins. We found that HO-3867 covalently binds to mutant p53, initiates a wildtype p53-like anticancer genetic response, is exclusively cytotoxic toward cancer cells, and exhibits high anticancer efficacy in tumor models. In conclusion, HO-3867 is a p53 mutant-reactivating drug with high clinical anticancer potential.

Synthesis and Biological Evaluation of Curcumin-Nitroxide-Based Molecular Hybrids as Antioxidant and Anti-Proliferative Agents.

BACKGROUND: Natural products and their derivatives are widely used to treat cancer and other diseases associated with ROS- and RNS-induced damages. METHODS: A series of paramagnetic modified curcumin analogs and 3,5-diarylidene-piperidones (DAP) have been designed, synthesized, and characterized on their anti-proliferative and antioxidant activity. RESULTS: Biological characterization of the new compounds supported the earlier results that incorporation of a nitroxide moiety or its precursor into curcumin or diarylidenylpiperidone (DAP) scaffolds resulted in anti-proliferative effect toward cancerous cell-lines in case of aryl hydroxy and/or methoxy substituent containing derivatives, suggesting their potential for targeted therapeutic applications. In case of basic side chain derivatives, nitroxide incorporation gave unambiguous results, however in tendency the more accessible DAP derivatives had stronger anti-proliferative effect. In most cases, the nitroxide incorporation increased the TEAC value (proton and electron donation capability) of DAP derivatives. CONCLUSIONS: Among the compounds synthesized and investigated the spin-labeled curcumin and 3,5-bis(4-hydroxy-3-methoxybenzylidene)piperidin-4-one derivatives were the most effective antiproliferative and antioxidant derivatives.

Oxygen regulates molecular mechanisms of cancer progression and metastasis.

Oxygen is the basic molecule which supports life and it truly is "god's gift to life." Despite its immense importance, research on "oxygen biology" has never received the light of the day and has been limited to physiological and biochemical studies. It seems that in modern day biology, oxygen research is summarized in one word "hypoxia." Scientists have focused on hypoxia-induced transcriptomics and molecular-cellular alterations exclusively in disease models. Interestingly, the potential of oxygen to control the basic principles of biology like homeostatic maintenance, transcription, replication, and protein folding among many others, at the molecular level, has been completely ignored. Here, we present a perspective on the crucial role played by oxygen in regulation of basic biological phenomena. Our conclusion highlights the importance of establishing novel research areas like oxygen biology, as there is great potential in this field for basic science discoveries and clinical benefits to the society.

p53's choice of myocardial death or survival: Oxygen protects infarct myocardium by recruiting p53 on NOS3 promoter through regulation of p53-Lys(118) acetylation.

Myocardial infarction, an irreversible cardiac tissue damage, involves progressive loss of cardiomyocytes due to p53-mediated apoptosis. Oxygenation is known to promote cardiac survival through activation of NOS3 gene. We hypothesized a dual role for p53, which, depending on oxygenation, can elicit apoptotic death signals or NOS3-mediated survival signals in the infarct heart. p53 exhibited a differential DNA-binding, namely, BAX-p53RE in the infarct heart or NOS3-p53RE in the oxygenated heart, which was regulated by oxygen-induced, post-translational modification of p53. In the infarct heart, p53 was heavily acetylated at Lys(118) residue, which was exclusively reversed in the oxygenated heart, apparently regulated by oxygen-dependent expression of TIP60. The inhibition of Lys(118) acetylation promoted the generation of NOS3-promoting prosurvival form of p53. Thus, oxygenation switches p53-DNA interaction by regulating p53 core-domain acetylation, promoting a prosurvival transcription activity of p53. Understanding this novel oxygen-p53 survival pathway will open new avenues in cardioprotection molecular therapy.