RESUMO
Understanding how signaling networks are regulated offers valuable insights into how cells and organisms react to internal and external stimuli and is crucial for developing novel strategies to treat diseases. To achieve this, it is necessary to delineate the intricate interactions between the nodes in the network, which can be accomplished by measuring the activities of individual nodes under perturbation conditions. To facilitate this, we have recently developed a biosensor barcoding technique that enables massively multiplexed tracking of numerous signaling activities in live cells using genetically encoded fluorescent biosensors. In this chapter, we detail how we employed this method to reconstruct the EGFR signaling network by systematically monitoring the activities of individual nodes under perturbations.
Assuntos
Técnicas Biossensoriais , Transdução de Sinais , Técnicas Biossensoriais/métodos , Humanos , Receptores ErbB/metabolismo , Receptores ErbB/genéticaRESUMO
Liver metastasis (LM) confers poor survival and therapy resistance across cancer types, but the mechanisms of liver-metastatic organotropism remain unknown. Here, through in vivo CRISPR-Cas9 screens, we found that Pip4k2c loss conferred LM but had no impact on lung metastasis or primary tumor growth. Pip4k2c-deficient cells were hypersensitized to insulin-mediated PI3K/AKT signaling and exploited the insulin-rich liver milieu for organ-specific metastasis. We observed concordant changes in PIP4K2C expression and distinct metabolic changes in 3,511 patient melanomas, including primary tumors, LMs and lung metastases. We found that systemic PI3K inhibition exacerbated LM burden in mice injected with Pip4k2c-deficient cancer cells through host-mediated increase in hepatic insulin levels; however, this circuit could be broken by concurrent administration of an SGLT2 inhibitor or feeding of a ketogenic diet. Thus, this work demonstrates a rare example of metastatic organotropism through co-optation of physiological metabolic cues and proposes therapeutic avenues to counteract these mechanisms.
Assuntos
Neoplasias Hepáticas , Proteínas Proto-Oncogênicas c-akt , Humanos , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases , Transdução de Sinais , Insulina , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Coronavirus Disease 2019 (COVID-19) has been the most severe public health challenge in this century. Two years after its emergence, the rapid development and deployment of effective COVID-19 vaccines have successfully controlled this pandemic and greatly reduced the risk of severe illness and death associated with COVID-19. However, due to its ability to rapidly evolve, the SARS-CoV-2 virus may never be eradicated, and there are many important new topics to work on if we need to live with this virus for a long time. To this end, we hope to provide essential knowledge for researchers who work on the improvement of future COVID-19 vaccines. In this review, we provided an up-to-date summary for current COVID-19 vaccines, discussed the biological basis and clinical impact of SARS-CoV-2 variants and subvariants, and analyzed the effectiveness of various vaccine booster regimens against different SARS-CoV-2 strains. Additionally, we reviewed potential mechanisms of vaccine-induced severe adverse events, summarized current studies regarding immune correlates of protection, and finally, discussed the development of next-generation vaccines.
Assuntos
COVID-19 , Vacinas Virais , COVID-19/prevenção & controle , Vacinas contra COVID-19/efeitos adversos , Humanos , SARS-CoV-2/genética , Eficácia de VacinasRESUMO
We recently developed a biosensor barcoding approach for highly multiplexed tracking of molecular activities in live cells. In this protocol, we detail the labeling of cells expressing different genetically encoded fluorescent biosensors with a pair of barcoding proteins and parallel imaging. Signals from cells with the same barcodes are then pooled together to obtain the dynamics of the corresponding biosensor activity. We describe the steps involved in cell barcoding, image acquisition, and analysis by deep learning models. For complete details on the use and execution of this protocol, please refer to Yang et al. (2021).
Assuntos
Técnicas Biossensoriais , Técnicas Biossensoriais/métodos , ProteínasRESUMO
Genetically encoded fluorescent biosensors are powerful tools for monitoring biochemical activities in live cells, but their multiplexing capacity is limited by the available spectral space. We overcome this problem by developing a set of barcoding proteins that can generate over 100 barcodes and are spectrally separable from commonly used biosensors. Mixtures of barcoded cells expressing different biosensors are simultaneously imaged and analyzed by deep learning models to achieve massively multiplexed tracking of signaling events. Importantly, different biosensors in cell mixtures show highly coordinated activities, thus facilitating the delineation of their temporal relationship. Simultaneous tracking of multiple biosensors in the receptor tyrosine kinase signaling network reveals distinct mechanisms of effector adaptation, cell autonomous and non-autonomous effects of KRAS mutations, as well as complex interactions in the network. Biosensor barcoding presents a scalable method to expand multiplexing capabilities for deciphering the complexity of signaling networks and their interactions between cells.
Assuntos
Técnicas Biossensoriais/métodos , Células/ultraestrutura , Microscopia de Fluorescência/métodos , Análise de Célula Única/métodos , Linhagem Celular Tumoral , HumanosRESUMO
Immunotherapy for cervical cancer should target high-risk human papillomavirus types 16 and 18, which cause 50% and 20% of cervical cancers, respectively. Here, we describe the construction and characterization of the pBI-11 DNA vaccine via the addition of codon-optimized human papillomavirus 18 (HPV18) E7 and HPV16 and 18 E6 genes to the HPV16 E7-targeted DNA vaccine pNGVL4a-SigE7(detox)HSP70 (DNA vaccine pBI-1). Codon optimization of the HPV16/18 E6/E7 genes in pBI-11 improved fusion protein expression compared to that in DNA vaccine pBI-10.1 that utilized the native viral sequences fused 3' to a signal sequence and 5' to the HSP70 gene of Mycobacterium tuberculosis Intramuscular vaccination of mice with pBI-11 DNA better induced HPV antigen-specific CD8+ T cell immune responses than pBI-10.1 DNA. Furthermore, intramuscular vaccination with pBI-11 DNA generated stronger therapeutic responses for C57BL/6 mice bearing HPV16 E6/E7-expressing TC-1 tumors. The HPV16/18 antigen-specific T cell-mediated immune responses generated by pBI-11 DNA vaccination were further enhanced by boosting with tissue-antigen HPV vaccine (TA-HPV). Combination of the pBI-11 DNA and TA-HPV boost vaccination with PD-1 antibody blockade significantly improved the control of TC-1 tumors and extended the survival of the mice. Finally, repeat vaccination with clinical-grade pBI-11 with or without clinical-grade TA-HPV was well tolerated in vaccinated mice. These preclinical studies suggest that the pBI-11 DNA vaccine may be used with TA-HPV in a heterologous prime-boost strategy to enhance HPV 16/18 E6/E7-specific CD8+ T cell responses, either alone or in combination with immune checkpoint blockade, to control HPV16/18-associated tumors. Our data serve as an important foundation for future clinical translation.IMPORTANCE Persistent expression of high-risk human papillomavirus (HPV) E6 and E7 is an obligate driver for several human malignancies, including cervical cancer, wherein HPV16 and HPV18 are the most common types. PD-1 antibody immunotherapy helps a subset of cervical cancer patients, and its efficacy might be improved by combination with active vaccination against E6 and/or E7. For patients with HPV16+ cervical intraepithelial neoplasia grade 2/3 (CIN2/3), the precursor of cervical cancer, intramuscular vaccination with a DNA vaccine targeting HPV16 E7 and then a recombinant vaccinia virus expressing HPV16/18 E6-E7 fusion proteins (TA-HPV) was safe, and half of the patients cleared their lesions in a small study (NCT00788164). Here, we sought to improve upon this therapeutic approach by developing a new DNA vaccine that targets E6 and E7 of HPV16 and HPV18 for administration prior to a TA-HPV booster vaccination and for application against cervical cancer in combination with a PD-1-blocking antibody.
Assuntos
Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/genética , Neoplasias do Colo do Útero/prevenção & controle , Vacinas de DNA/genética , Animais , Anticorpos Monoclonais/administração & dosagem , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Feminino , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/imunologia , Papillomavirus Humano 16/efeitos dos fármacos , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/efeitos dos fármacos , Papillomavirus Humano 18/imunologia , Humanos , Imunização Secundária/métodos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/mortalidade , Vacinas contra Papillomavirus/administração & dosagem , Vacinas contra Papillomavirus/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Análise de Sobrevida , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/mortalidade , Vacinação/métodos , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vaccinia virus/química , Vaccinia virus/imunologiaRESUMO
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a new type of coronavirus that causes the Coronavirus Disease 2019 (COVID-19), which has been the most challenging pandemic in this century. Considering its high mortality and rapid spread, an effective vaccine is urgently needed to control this pandemic. As a result, the academia, industry, and government sectors are working tightly together to develop and test a variety of vaccines at an unprecedented pace. In this review, we outline the essential coronavirus biological characteristics that are important for vaccine design. In addition, we summarize key takeaways from previous vaccination studies of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), highlighting the pros and cons of each immunization strategy. Finally, based on these prior vaccination experiences, we discuss recent progress and potential challenges of COVID-19 vaccine development.