Opposing tumor-cell-intrinsic and -extrinsic roles of the IRF1 transcription factor in antitumor immunity.

Type I interferon (IFN-I) and IFN-γ foster antitumor immunity by facilitating T cell responses. Paradoxically, IFNs may promote T cell exhaustion by activating immune checkpoints. The downstream regulators of these disparate responses are incompletely understood. Here, we describe how interferon regulatory factor 1 (IRF1) orchestrates these opposing effects of IFNs. IRF1 expression in tumor cells blocks Toll-like receptor- and IFN-I-dependent host antitumor immunity by preventing interferon-stimulated gene (ISG) and effector programs in immune cells. In contrast, expression of IRF1 in the host is required for antitumor immunity. Mechanistically, IRF1 binds distinctly or together with STAT1 at promoters of immunosuppressive but not immunostimulatory ISGs in tumor cells. Overexpression of programmed cell death ligand 1 (PD-L1) in Irf1-/- tumors only partially restores tumor growth, suggesting multifactorial effects of IRF1 on antitumor immunity. Thus, we identify that IRF1 expression in tumor cells opposes host IFN-I- and IRF1-dependent antitumor immunity to facilitate immune escape and tumor growth.

CCL21-DC in situ vaccination in murine NSCLC overcomes resistance to immunotherapy and generates systemic tumor-specific immunity.

BACKGROUND: Despite recent advances in immunotherapy, many patients with non-small cell lung cancer (NSCLC) do not respond to immune checkpoint inhibitors (ICI). Resistance to ICI may be driven by suboptimal priming of antitumor T lymphocytes due to poor antigen presentation as well as their exclusion and impairment by the immunosuppressive tumor microenvironment (TME). In a recent phase I trial in patients with NSCLC, in situ vaccination (ISV) with dendritic cells engineered to secrete CCL21 (CCL21-DC), a chemokine that facilitates the recruitment of T cells and DC, promoted T lymphocyte tumor infiltration and PD-L1 upregulation. METHODS: Murine models of NSCLC with distinct driver mutations (KrasG12D/P53+/-/Lkb1-/- (KPL); KrasG12D/P53+/- (KP); and KrasG12D (K)) and varying tumor mutational burden were used to evaluate the efficacy of combination therapy with CCL21-DC ISV plus ICI. Comprehensive analyses of longitudinal preclinical samples by flow cytometry, single cell RNA-sequencing (scRNA-seq) and whole-exome sequencing were performed to assess mechanisms of combination therapy. RESULTS: ISV with CCL21-DC sensitized immune-resistant murine NSCLCs to ICI and led to the establishment of tumor-specific immune memory. Immunophenotyping revealed that CCL21-DC obliterated tumor-promoting neutrophils, promoted sustained infiltration of CD8 cytolytic and CD4 Th1 lymphocytes and enriched progenitor T cells in the TME. Addition of ICI to CCL21-DC further enhanced the expansion and effector function of T cells both locally and systemically. Longitudinal evaluation of tumor mutation profiles revealed that CCL21-DC plus ICI induced immunoediting of tumor subclones, consistent with the broadening of tumor-specific T cell responses. CONCLUSIONS: CCL21-DC ISV synergizes with anti-PD-1 to eradicate murine NSCLC. Our data support the clinical application of CCL21-DC ISV in combination with checkpoint inhibition for patients with NSCLC.

Design, Synthesis, and Biological Evaluation of Novel Quercetin Derivatives as PPAR-γ Partial Agonists by Modulating Epithelial-Mesenchymal Transition in Lung Cancer Metastasis.

Epithelial-to-mesenchymal transition (EMT) is responsible for driving metastasis of multiple cancer types including lung cancer. Peroxisome proliferator-activated receptor (PPAR)-γ, a ligand-activated transcription factor, controls expression of variety of genes involved in EMT. Although several synthetic compounds act as potent full agonists for PPAR-γ, their long term application is restricted due to serious adverse effects. Therefore, partial agonists involving reduced and balanced PPAR-γ activity are more effective and valued. A previous study discerned the efficacy of quercetin and its derivatives to attain favorable stabilization with PPAR-γ. Here this work is extended by synthesizing five novel quercetin derivatives (QDs) namely thiosemicarbazone (QUETSC)) and hydrazones (quercetin isonicotinic acid hydrazone (QUEINH), quercetin nicotinic acid hydrazone (QUENH), quercetin 2-furoic hydrazone (QUE2FH), and quercetin salicyl hydrazone (QUESH)) and their effects are analyzed in modulating EMT in lung cancer cell lines via PPAR-γ partial activation. QDs-treated A549 cells diminish cell proliferation strongly at nanomolar concentration compared to NCI-H460 cells. Of the five screened derivatives, QUETSC, QUE2FH, and QUESH exhibit the property of partial activation as compared to the overexpressive level of rosiglitazone. Consistently, these QDs also suppress EMT process by markedly downregulating the levels of mesenchymal markers (Snail, Slug, and zinc finger E-box binding homeobox 1) and concomitant upregulation of epithelial marker (E-cadherin).

Mechanistic insight into the protective and pathogenic immune-responses against SARS-CoV-2.

COVID-19 is a severe respiratory illness that has emerged as a devasting health problem worldwide. The disease outcome is heterogeneous, which is most likely dependent on the immunity of an individual. Asymptomatic and mildly/moderate symptomatic (non-severe) patients likely develop an effective early immune response and clear the virus. However, severe symptoms dominate due to a failure in the generation of an effective and specific early immune response against SARS-CoV-2. Moreover, a late surge in pathogenic inflammation involves dysregulated innate and adaptive immune responses leading to local and systemic tissue damage and the emergence of severe disease symptoms. In this review, we describe the potential mechanisms of protective and pathogenic immune responses in "mild/moderate" and "severe" symptomatic SARS-CoV-2 infected people, respectively, and discuss the immune components that are currently targeted for therapeutic intervention.

Lung cancer immunotherapy: progress, pitfalls, and promises.

Lung cancer is the primary cause of mortality in the United States and around the globe. Therapeutic options for lung cancer treatment include surgery, radiation therapy, chemotherapy, and targeted drug therapy. Medical management is often associated with the development of treatment resistance leading to relapse. Immunotherapy is profoundly altering the approach to cancer treatment owing to its tolerable safety profile, sustained therapeutic response due to immunological memory generation, and effectiveness across a broad patient population. Different tumor-specific vaccination strategies are gaining ground in the treatment of lung cancer. Recent advances in adoptive cell therapy (CAR T, TCR, TIL), the associated clinical trials on lung cancer, and associated hurdles are discussed in this review. Recent trials on lung cancer patients (without a targetable oncogenic driver alteration) reveal significant and sustained responses when treated with programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) checkpoint blockade immunotherapies. Accumulating evidence indicates that a loss of effective anti-tumor immunity is associated with lung tumor evolution. Therapeutic cancer vaccines combined with immune checkpoint inhibitors (ICI) can achieve better therapeutic effects. To this end, the present article encompasses a detailed overview of the recent developments in the immunotherapeutic landscape in targeting small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Additionally, the review also explores the implication of nanomedicine in lung cancer immunotherapy as well as the combinatorial application of traditional therapy along with immunotherapy regimens. Finally, ongoing clinical trials, significant obstacles, and the future outlook of this treatment strategy are also highlighted to boost further research in the field.

Angiogenic stimulation strategies in bone tissue regeneration.

Current tissue engineering strategies in bone repair and regeneration have limitations regarding tissue rejection, insufficient blood supply, and tissue integration. Specific host response results in isolation, degeneration, and subsequent loss of function of the implanted/scaffold biomaterial. Therefore, strategies to increase the interplay between angiogenesis and complex bone tissue formation are required to develop fully functional vascularized bone tissue. Angiogenesis is essential for oxygen/nutrient supply, waste removal, endothelial/stem cell homing, and the release of mitogenic/angiogenic/osteogenic factors. Hence, the challenge lies in understanding the complex interdependence of angiogenesis with neo-bone formation. Therefore, recent bone tissue regeneration strategies have focused on biomaterial development concerning induction of neovascularization and subsequent angiogenesis. Scaffold architecture (macro/micro/nano) scales, culture conditions (3-Dimension, hypoxia, etc), stimuli-dependent delivery of angiofactors, and gene delivery may significantly modulate vascularization in tissue-engineered products. Therefore, the current review discusses the key mechanisms/steps involved in defining the relationship between angiogenic and osteogenic factors. The recent strategies incorporating the above understanding in the development of bone tissue-engineered constructs are also deliberated. Eventually, these strategies may give the potential way forward to develop a bioengineered, vascularized bone tissue construct for implant applications.

Drug repurposing-an emerging strategy in cancer therapeutics.

Cancer is a complex disease affecting millions of people around the world. Despite advances in surgical and radiation therapy, chemotherapy continues to be an important therapeutic option for the treatment of cancer. The current treatment is expensive and has several side effects. Also, over time, cancer cells develop resistance to chemotherapy, due to which there is a demand for new drugs. Drug repurposing is a novel approach that focuses on finding new applications for the old clinically approved drugs. Current advances in the high-dimensional multiomics landscape, especially proteomics, genomics, and computational omics-data analysis, have facilitated drug repurposing. The drug repurposing approach provides cheaper, effective, and safe drugs with fewer side effects and fastens the process of drug development. The review further delineates each repurposed drug's original indication and mechanism of action in cancer. Along with this, the article also provides insight upon artificial intelligence and its application in drug repurposing. Clinical trials are vital for determining medication safety and effectiveness, and hence the clinical studies for each repurposed medicine in cancer, including their stages, status, and National Clinical Trial (NCT) identification, are reported in this review article. Various emerging evidences imply that repurposing drugs is critical for the faster and more affordable discovery of anti-cancerous drugs, and the advent of artificial intelligence-based computational tools can accelerate the translational cancer-targeting pipeline.

Autophagy and EMT in cancer and metastasis: Who controls whom?

Metastasis consists of hallmark events, including Epithelial-Mesenchymal Transition (EMT), angiogenesis, initiation of inflammatory tumor microenvironment, and malfunctions in apoptosis. Autophagy is known to play a pivotal role in the metastatic process. Autophagy has pulled researchers towards it in recent times because of its dual role in the maintenance of cancer cells. Evidence states that cells undergoing EMT need autophagy in order to survive during migration and dissemination. Additionally, it orchestrates EMT markers in certain cancers. On the other side of the coin, autophagy plays an oncosuppressive role in impeding early metastasis. This review aims to project the interrelationship between autophagy and EMT. Targeting EMT via autophagy as a useful strategy is discussed in this review. Furthermore, for the first time, we have covered the possible reciprocating roles of EMT and autophagy and its consequences in cancer metastasis.

Statistical parametrization of cell cytoskeleton reveals lung cancer cytoskeletal phenotype with partial EMT signature.

Epithelial-mesenchymal Transition (EMT) is a multi-step process that involves cytoskeletal rearrangement. Here, developing and using an image quantification tool, Statistical Parametrization of Cell Cytoskeleton (SPOCC), we have identified an intermediate EMT state with a specific cytoskeletal signature. We have been able to partition EMT into two steps: (1) initial formation of transverse arcs and dorsal stress fibers and (2) their subsequent conversion to ventral stress fibers with a concurrent alignment of fibers. Using the Orientational Order Parameter (OOP) as a figure of merit, we have been able to track EMT progression in live cells as well as characterize and quantify their cytoskeletal response to drugs. SPOCC has improved throughput and is non-destructive, making it a viable candidate for studying a broad range of biological processes. Further, owing to the increased stiffness (and by inference invasiveness) of the intermediate EMT phenotype compared to mesenchymal cells, our work can be instrumental in aiding the search for future treatment strategies that combat metastasis by specifically targeting the fiber alignment process.

Current advances in the use of exosomes, liposomes, and bioengineered hybrid nanovesicles in cancer detection and therapy.

Three major approaches of cancer therapy can be enunciated as delivery of biotherapeutics, tumor image analysis, and immunotherapy. Liposomes, artificial fat bubbles, are long known for their capacity to encapsulate a diverse range of bioactive molecules and release the payload in a sustained, stimuli-responsive manner. They have already been widely explored as a delivery vehicle for therapeutic drugs as well as imaging agents. They are also extensively being used in cancer immunotherapy. On the other hand, exosomes are naturally occurring nanosized extracellular vesicles that serve an important role in cell-cell communication. Importantly, the exosomes also have proven their capability to carry an array of active pharmaceuticals and diagnostic molecules to the tumor cells. Exosomes, being enriched with tumor antigens, have numerous immunomodulatory effects. Much to our intrigue, in recent times, efforts have been directed toward developing smart, bioengineered, exosome-liposome hybrid nanovesicles, which are augmented by the benefits of both vesicular systems. This review attempts to summarize the contemporary developments in the use of exosome and liposome toward cancer diagnosis, therapy, as a vehicle for drug delivery, diagnostic carrier for tumor imaging, and cancer immunotherapy. We shall also briefly reflect upon the recent advancements of the exosome-liposome hybrids in cancer therapy. Finally, we put forward future directions for the use of exosome/liposome and/or hybrid nanocarriers for accurate diagnosis and personalized therapies for cancers.

Advances in targeting the WNT/ß-catenin signaling pathway in cancer.

WNT/ß-catenin signaling orchestrates various physiological processes, including embryonic development, growth, tissue homeostasis, and regeneration. Abnormal WNT/ß-catenin signaling is associated with various cancers and its inhibition has shown effective antitumor responses. In this review, we discuss the pathway, potential targets for the development of WNT/ß-catenin inhibitors, available inhibitors, and their specific molecular interactions with the target proteins. We also discuss inhibitors that are in clinical trials and describe potential new avenues for therapeutically targeting the WNT/ß-catenin pathway. Furthermore, we introduce emerging strategies, including artificial intelligence (AI)-assisted tools and technology-based actionable approaches, to translate WNT/ß-catenin inhibitors to the clinic for cancer therapy.

Emerging role and promise of nanomaterials in organoid research.

Organoids are 3D stem cell-derived self-organization of cells. Organoid bioengineering helps recreate and tailor their architecture in vitro to generate mini organ-like properties, providing the opportunity to study fundamental cell behavior in heterogeneous populations and as a tool to model various diseases. Nanomaterials (NMs) are becoming indispensable in regenerative medicine and in developing treatment modalities for various diseases. Therefore, organoid-NM interactions are set to gain traction for the development of advanced diagnostics and therapeutics. Here, we discuss the interactions of NMs with distinctive organoid types, organoid matrices, trafficking and cargo delivery, organs-on-a-chip, bioprinting, downstream therapeutic implications, and future approaches.

The actin cytoskeleton: Morphological changes in pre- and fully developed lung cancer.

Actin, a primary component of the cell cytoskeleton can have multiple isoforms, each of which can have specific properties uniquely suited for their purpose. These monomers are then bound together to form polymeric filaments utilizing adenosine triphosphate hydrolysis as a source of energy. Proteins, such as Arp2/3, VASP, formin, profilin, and cofilin, serve important roles in the polymerization process. These filaments can further be linked to form stress fibers by proteins called actin-binding proteins, such as α-actinin, myosin, fascin, filamin, zyxin, and epsin. These stress fibers are responsible for mechanotransduction, maintaining cell shape, cell motility, and intracellular cargo transport. Cancer metastasis, specifically epithelial mesenchymal transition (EMT), which is one of the key steps of the process, is accompanied by the formation of thick stress fibers through the Rho-associated protein kinase, MAPK/ERK, and Wnt pathways. Recently, with the advent of "field cancerization," pre-malignant cells have also been demonstrated to possess stress fibers and related cytoskeletal features. Analytical methods ranging from western blot and RNA-sequencing to cryo-EM and fluorescent imaging have been employed to understand the structure and dynamics of actin and related proteins including polymerization/depolymerization. More recent methods involve quantifying properties of the actin cytoskeleton from fluorescent images and utilizing them to study biological processes, such as EMT. These image analysis approaches exploit the fact that filaments have a unique structure (curvilinear) compared to the noise or other artifacts to separate them. Line segments are extracted from these filament images that have assigned lengths and orientations. Coupling such methods with statistical analysis has resulted in development of a new reporter for EMT in lung cancer cells as well as their drug responses.

Emergence of Cardiac Glycosides as Potential Drugs: Current and Future Scope for Cancer Therapeutics.

Cardiac glycosides are natural sterols and constitute a group of secondary metabolites isolated from plants and animals. These cardiotonic agents are well recognized and accepted in the treatment of various cardiac diseases as they can increase the rate of cardiac contractions by acting on the cellular sodium potassium ATPase pump. However, a growing number of recent efforts were focused on exploring the antitumor and antiviral potential of these compounds. Several reports suggest their antitumor properties and hence, today cardiac glycosides (CG) represent the most diversified naturally derived compounds strongly recommended for the treatment of various cancers. Mutated or dysregulated transcription factors have also gained prominence as potential therapeutic targets that can be selectively targeted. Thus, we have explored the recent advances in CGs mediated cancer scope and have considered various signaling pathways, molecular aberration, transcription factors (TFs), and oncogenic genes to highlight potential therapeutic targets in cancer management.

Advances in the Fabrication of Scaffold and 3D Printing of Biomimetic Bone Graft.

The need for bone grafts is tremendous, and that leads to the use of autograft, allograft, and bone graft substitutes. The biology of the bone is quite complex regarding cellular composition and architecture, hence developing a mineralized connective tissue graft is challenging. Traditionally used bone graft substitutes including metals, biomaterial coated metals and biodegradable scaffolds, suffer from persistent limitations. With the advent and rise of additive manufacturing technologies, the future of repairing bone trauma and defects seems to be optimistic. 3D printing has significant advantages, the foremost of all being faster manipulation of various biocompatible materials and live cells or tissues into the complex natural geometries necessary to mimic and stimulate cellular bone growth. The advent of new-generation bioprinters working with high-precision, micro-dispensing and direct digital manufacturing is aiding in ground-breaking organ and tissue printing, including the bone. The future bone replacement for patients holds excellent promise as scientists are moving closer to the generation of better 3D printed bio-bone grafts that will be safer and more effective. This review aims to summarize the advances in scaffold fabrication techniques, emphasizing 3D printing of biomimetic bone grafts.

Novel Kras-mutant murine models of non-small cell lung cancer possessing co-occurring oncogenic mutations and increased tumor mutational burden.

Conditional genetically engineered mouse models (GEMMs) of non-small cell lung cancer (NSCLC) harbor common oncogenic driver mutations of the disease, but in contrast to human NSCLC these models possess low tumor mutational burden (TMB). As a result, these models often lack tumor antigens that can elicit host adaptive immune responses, which limits their utility in immunotherapy studies. Here, we establish Kras-mutant murine models of NSCLC bearing the common driver mutations associated with the disease and increased TMB, by in vitro exposure of cell lines derived from GEMMs of NSCLC [KrasG12D (K), KrasG12DTp53-/-(KP), KrasG12DTp53+/-Lkb1-/- (KPL)] to the alkylating agent N-methyl-N-nitrosourea (MNU). Increasing the TMB enhanced host anti-tumor T cell responses and improved anti-PD-1 efficacy in syngeneic models across all genetic backgrounds. However, limited anti-PD-1 efficacy was observed in the KPL cell lines with increased TMB, which possessed a distinct immunosuppressed tumor microenvironment (TME) primarily composed of granulocytic myeloid-derived suppressor cells (G-MDSCs). This KPL phenotype is consistent with findings in human KRAS-mutant NSCLC where LKB1 loss is a driver of primary resistance to PD-1 blockade. In summary, these novel Kras-mutant NSCLC murine models with known driver mutations and increased TMB have distinct TMEs and recapitulate the therapeutic vulnerabilities of human NSCLC. We anticipate that these immunogenic models will facilitate the development of innovative immunotherapies in NSCLC.

COVID-19 and gut immunomodulation.

The disease coronavirus disease 2019 (COVID-19) is a severe respiratory illness that has emerged as a devastating health problem worldwide. The disease outcome is heterogeneous, and severity is likely dependent on the immunity of infected individuals and comorbidities. Although symptoms of the disease are primarily associated with respiratory problems, additional infection or failure of other vital organs are being reported. Emerging reports suggest a quite common co-existence of gastrointestinal (GI) tract symptoms in addition to respiratory symptoms in many COVID-19 patients, and some patients show just the GI symptoms. The possible cause of the GI symptoms could be due to direct infection of the epithelial cells of the gut, which is supported by the fact that (1) The intestinal epithelium expresses a high level of angiotensin-converting enzyme-2 and transmembrane protease serine 2 protein that are required for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into the cells; (2) About half of the severe COVID-19 patients show viral RNA in their feces and various parts of the GI tract; and (3) SARS-CoV-2 can directly infect gut epithelial cells in vitro (gut epithelial cells and organoids) and in vivo (rhesus monkey). The GI tract seems to be a site of active innate and adaptive immune responses to SARS-CoV-2 as clinically, stool samples of COVID-19 patients possess proinflammatory cytokines (interleukin 8), calprotectin (neutrophils activity), and immunoglobulin A antibodies. In addition to direct immune activation by the virus, impairment of GI epithelium integrity can evoke immune response under the influence of systemic cytokines, hypoxia, and changes in gut microbiota (dysbiosis) due to infection of the respiratory system, which is confirmed by the observation that not all of the GI symptomatic patients are viral RNA positive. This review comprehensively summarizes the possible GI immunomodulation by SARS-CoV-2 that could lead to GI symptoms, their association with disease severity, and potential therapeutic interventions.