Reconstructing Signaling Networks Using Biosensor Barcoding.

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.

Arginine-linked HPV-associated E7 displaying bacteria-derived outer membrane vesicles as a potent antigen-specific cancer vaccine.

BACKGROUND: Bacteria-based cancer therapy have demonstrated innovative strategies to combat tumors. Recent studies have focused on gram-negative bacterial outer membrane vesicles (OMVs) as a novel cancer immunotherapy strategy due to its intrinsic properties as a versatile carrier. METHOD: Here, we developed an Human Papillomavirus (HPV)-associated E7 antigen displaying Salmonella-derived OMV vaccine, utilizing a Poly(L-arginine) cell penetrating peptide (CPP) to enhance HPV16 E7 (aa49-67) H-2 Db and OMV affinity, termed SOMV-9RE7. RESULTS: Due to OMV's intrinsic immunogenic properties, SOMV-9RE7 effectively activates adaptive immunity through antigen-presenting cell uptake and antigen cross-presentation. Vaccination of engineered OMVs shows immediate tumor suppression and recruitment of infiltrating tumor-reactive immune cells. CONCLUSION: The simplicity of the arginine coating strategy boasts the versatility of immuno-stimulating OMVs that can be broadly implemented to personalized bacterial immunotherapeutic applications.

Self-organizing glycolytic waves fuel cell migration and cancer progression.

Glycolysis has traditionally been thought to take place in the cytosol but we observed the enrichment of glycolytic enzymes in propagating waves of the cell cortex in human epithelial cells. These waves reflect excitable Ras/PI3K signal transduction and F-actin/actomyosin networks that drive cellular protrusions, suggesting that localized glycolysis at the cortex provides ATP for cell morphological events such as migration, phagocytosis, and cytokinesis. Perturbations that altered cortical waves caused corresponding changes in enzyme localization and ATP production whereas synthetic recruitment of glycolytic enzymes to the cell cortex enhanced cell spreading and motility. Interestingly, the cortical waves and ATP levels were positively correlated with the metastatic potential of cancer cells. The coordinated signal transduction, cytoskeletal, and glycolytic waves in cancer cells may explain their increased motility and their greater reliance on glycolysis, often referred to as the Warburg effect.

Salmonella immunotherapy engineered with highly efficient tumor antigen coating establishes antigen-specific CD8+ T cell immunity and increases in antitumor efficacy with type I interferon combination therapy.

Bacteria-based cancer therapy employs various strategies to combat tumors, one of which is delivering tumor-associated antigen (TAA) to generate specific immunity. Here, we utilized a poly-arginine extended HPV E7 antigen (9RE7) for attachment on Salmonella SL7207 outer membrane to synthesize the bacterial vaccine Salmonella-9RE7 (Sal-9RE7), which yielded a significant improvement in the amount of antigen presentation compared to the previous lysine-extended antigen coating strategy. In TC-1 tumor mouse models, Sal-9RE7 monotherapy decreased tumor growth by inducing E7 antigen-specific immunity. In addition, pairing Sal-9RE7 with adjuvant Albumin-IFNß (Alb-IFNß), a protein cytokine fusion, the combination significantly increased the antitumor efficacy and enhanced immunogenicity in the tumor microenvironment (TME). Our study made a significant contribution to personalized bacterial immunotherapy via TAA delivery and demonstrated the advantage of combination therapy.

Targeting Ras signaling excitability in cancer cells through combined inhibition of FAK and PI3K.

The Ras/PI3K/ERK signaling network is frequently mutated in various human cancers including cervical cancer and pancreatic cancer. Previous studies showed that the Ras/PI3K/ERK signaling network displays features of excitable systems including propagation of activity waves, all-or-none responses, and refractoriness. Oncogenic mutations lead to enhanced excitability of the network. A positive feedback loop between Ras, PI3K, the cytoskeleton, and FAK was identified as a driver of excitability. In this study, we investigated the effectiveness of targeting signaling excitability by inhibiting both FAK and PI3K in cervical and pancreatic cancer cells. We found that the combination of FAK and PI3K inhibitors synergistically suppressed the growth of select cervical and pancreatic cancer cell lines through increased apoptosis and decreased mitosis. In particular, FAK inhibition caused downregulation of PI3K and ERK signaling in cervical cancer but not pancreatic cancer cells. Interestingly, PI3K inhibitors activated multiple receptor tyrosine kinases (RTKs), including insulin receptor and IGF-1R in cervical cancer cells, as well as EGFR, Her2, Her3, Axl, and EphA2 in pancreatic cancer cells. Our results highlight the potential of combining FAK and PI3K inhibition for treating cervical and pancreatic cancer, although appropriate biomarkers for drug sensitivity are needed, and concurrent targeting of RTKs may be required for resistant cells.

Imaging and analysis for simultaneous tracking of fluorescent biosensors in barcoded cells.

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).

Deciphering cell signaling networks with massively multiplexed biosensor barcoding.

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.

Development of DNA Vaccine Targeting E6 and E7 Proteins of Human Papillomavirus 16 (HPV16) and HPV18 for Immunotherapy in Combination with Recombinant Vaccinia Boost and PD-1 Antibody.

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.

An Excitable Ras/PI3K/ERK Signaling Network Controls Migration and Oncogenic Transformation in Epithelial Cells.

The Ras/PI3K/extracellular signal-regulated kinases (ERK) signaling network plays fundamental roles in cell growth, survival, and migration and is frequently activated in cancer. Here, we show that the activities of the signaling network propagate as coordinated waves, biased by growth factor, which drive actin-based protrusions in human epithelial cells. The network exhibits hallmarks of biochemical excitability: the annihilation of oppositely directed waves, all-or-none responsiveness, and refractoriness. Abrupt perturbations to Ras, PI(4,5)P2, PI(3,4)P2, ERK, and TORC2 alter the threshold, observations that define positive and negative feedback loops within the network. Oncogenic transformation dramatically increases the wave activity, the frequency of ERK pulses, and the sensitivity to EGF stimuli. Wave activity was progressively enhanced across a series of increasingly metastatic breast cancer cell lines. The view that oncogenic transformation is a shift to a lower threshold of excitable Ras/PI3K/ERK network, caused by various combinations of genetic insults, can facilitate the assessment of cancer severity and effectiveness of interventions.

Publisher Correction: Integrating chemical and mechanical signals through dynamic coupling between cellular protrusions and pulsed ERK activation.

The original version of this Article contained an error in the spelling of the author Jr-Ming Yang, which was incorrectly given as J.-Ming Yang. This has now been corrected in both the PDF and HTML versions of the Article.

Publisher Correction: Integrating chemical and mechanical signals through dynamic coupling between cellular protrusions and pulsed ERK activation.

In the original version of this Article, the label "RTK" in Figure 6a was inadvertently changed to "RTE". This has now been corrected in the PDF and HTML versions of the Article.

Integrating chemical and mechanical signals through dynamic coupling between cellular protrusions and pulsed ERK activation.

The Ras-ERK signaling pathway regulates diverse cellular processes in response to environmental stimuli and contains important therapeutic targets for cancer. Recent single cell studies revealed stochastic pulses of ERK activation, the frequency of which determines functional outcomes such as cell proliferation. Here we show that ERK pulses are initiated by localized protrusive activities. Chemically and optogenetically induced protrusions trigger ERK activation through various entry points into the feedback loop involving Ras, PI3K, the cytoskeleton, and cellular adhesion. The excitability of the protrusive signaling network drives stochastic ERK activation in unstimulated cells and oscillations upon growth factor stimulation. Importantly, protrusions allow cells to sense combined signals from substrate stiffness and the growth factor. Thus, by uncovering the basis of ERK pulse generation we demonstrate how signals involved in cell growth and differentiation are regulated by dynamic protrusions that integrate chemical and mechanical inputs from the environment.

Inhibition of ovarian tumor cell invasiveness by targeting SYK in the tyrosine kinase signaling pathway.

Cell motility and invasiveness are prerequisites for dissemination, and largely account for cancer mortality. We have identified an actionable kinase, spleen tyrosine kinase (SYK), which is keenly tightly associated with tumor progression in ovarian cancer. Here, we report that active recombinant SYK directly phosphorylates cortactin and cofilin, which are critically involved in assembly and dynamics of actin filament through phosphorylation signaling. Enhancing SYK activity by inducing expression of a constitutively active SYK mutant, SYK130E, increased growth factor-stimulated migration and invasion of ovarian cancer cells, which was abrogated by cortactin knockdown. Similarly, SYK inhibitors significantly decreased invasion of ovarian cancer cells across basement membrane in real-time transwell assays and in 3D tumor spheroid models. SYK inactivation by targeted gene knockout or by small molecule inhibition reduced actin polymerization. Collectively, this study reported a new mechanism by which SYK signaling regulates ovarian cancer cell motility and invasiveness, and suggest a target-based strategy to prevent or suppress the advancement of ovarian malignancies.

NKCC1 Regulates Migration Ability of Glioblastoma Cells by Modulation of Actin Dynamics and Interacting with Cofilin.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. The mechanisms that confer GBM cells their invasive behavior are poorly understood. The electroneutral Na+-K+-2Cl- co-transporter 1 (NKCC1) is an important cell volume regulator that participates in cell migration. We have shown that inhibition of NKCC1 in GBM cells leads to decreased cell migration, in vitro and in vivo. We now report on the role of NKCC1 on cytoskeletal dynamics. We show that GBM cells display a significant decrease in F-actin content upon NKCC1 knockdown (NKCC1-KD). To determine the potential actin-regulatory mechanisms affected by NKCC1 inhibition, we studied NKCC1 protein interactions. We found that NKCC1 interacts with the actin-regulating protein Cofilin-1 and can regulate its membrane localization. Finally, we analyzed whether NKCC1 could regulate the activity of the small Rho-GTPases RhoA and Rac1. We observed that the active forms of RhoA and Rac1 were decreased in NKCC1-KD cells. In summary, we report that NKCC1 regulates GBM cell migration by modulating the cytoskeleton through multiple targets including F-actin regulation through Cofilin-1 and RhoGTPase activity. Due to its essential role in cell migration NKCC1 may serve as a specific therapeutic target to decrease cell invasion in patients with primary brain cancer.

Gß Regulates Coupling between Actin Oscillators for Cell Polarity and Directional Migration.

For directional movement, eukaryotic cells depend on the proper organization of their actin cytoskeleton. This engine of motility is made up of highly dynamic nonequilibrium actin structures such as flashes, oscillations, and traveling waves. In Dictyostelium, oscillatory actin foci interact with signals such as Ras and phosphatidylinositol 3,4,5-trisphosphate (PIP3) to form protrusions. However, how signaling cues tame actin dynamics to produce a pseudopod and guide cellular motility is a critical open question in eukaryotic chemotaxis. Here, we demonstrate that the strength of coupling between individual actin oscillators controls cell polarization and directional movement. We implement an inducible sequestration system to inactivate the heterotrimeric G protein subunit Gß and find that this acute perturbation triggers persistent, high-amplitude cortical oscillations of F-actin. Actin oscillators that are normally weakly coupled to one another in wild-type cells become strongly synchronized following acute inactivation of Gß. This global coupling impairs sensing of internal cues during spontaneous polarization and sensing of external cues during directional motility. A simple mathematical model of coupled actin oscillators reveals the importance of appropriate coupling strength for chemotaxis: moderate coupling can increase sensitivity to noisy inputs. Taken together, our data suggest that Gß regulates the strength of coupling between actin oscillators for efficient polarity and directional migration. As these observations are only possible following acute inhibition of Gß and are masked by slow compensation in genetic knockouts, our work also shows that acute loss-of-function approaches can complement and extend the reach of classical genetics in Dictyostelium and likely other systems as well.

Evolutionarily conserved coupling of adaptive and excitable networks mediates eukaryotic chemotaxis.

Numerous models explain how cells sense and migrate towards shallow chemoattractant gradients. Studies show that an excitable signal transduction network acts as a pacemaker that controls the cytoskeleton to drive motility. Here we show that this network is required to link stimuli to actin polymerization and chemotactic motility and we distinguish the various models of chemotaxis. First, signalling activity is suppressed towards the low side in a gradient or following removal of uniform chemoattractant. Second, signalling activities display a rapid shut off and a slower adaptation during which responsiveness to subsequent test stimuli decline. Simulations of various models indicate that these properties require coupled adaptive and excitable networks. Adaptation involves a G-protein-independent inhibitor, as stimulation of cells lacking G-protein function suppresses basal activities. The salient features of the coupled networks were observed for different chemoattractants in Dictyostelium and in human neutrophils, suggesting an evolutionarily conserved mechanism for eukaryotic chemotaxis.

An excitable signal integrator couples to an idling cytoskeletal oscillator to drive cell migration.

It is generally believed that cytoskeletal activities drive random cell migration, whereas signal transduction events initiated by receptors regulate the cytoskeleton to guide cells. However, we find that the cytoskeletal network, involving SCAR/WAVE, Arp 2/3 and actin-binding proteins, is capable of generating only rapid oscillations and undulations of the cell boundary. The signal transduction network, comprising multiple pathways that include Ras GTPases, PI(3)K and Rac GTPases, is required to generate the sustained protrusions of migrating cells. The signal transduction network is excitable, exhibiting wave propagation, refractoriness and maximal response to suprathreshold stimuli, even in the absence of the cytoskeleton. We suggest that cell motility results from coupling of 'pacemaker' signal transduction and 'idling motor' cytoskeletal networks, and various guidance cues that modulate the threshold for triggering signal transduction events are integrated to control the mode and direction of migration.

Interaction of motility, directional sensing, and polarity modules recreates the behaviors of chemotaxing cells.

Chemotaxis involves the coordinated action of separable but interrelated processes: motility, gradient sensing, and polarization. We have hypothesized that these are mediated by separate modules that account for these processes individually and that, when combined, recreate most of the behaviors of chemotactic cells. Here, we describe a mathematical model where the modules are implemented in terms of reaction-diffusion equations. Migration and the accompanying changes in cellular morphology are demonstrated in simulations using a mechanical model of the cell cortex implemented in the level set framework. The central module is an excitable network that accounts for random migration. The response to combinations of uniform stimuli and gradients is mediated by a local excitation, global inhibition module that biases the direction in which excitability is directed. A polarization module linked to the excitable network through the cytoskeleton allows unstimulated cells to move persistently and, for cells in gradients, to gradually acquire distinct sensitivity between front and back. Finally, by varying the strengths of various feedback loops in the model we obtain cellular behaviors that mirror those of genetically altered cell lines.