Current Trends in Neoantigen-Based Cancer Vaccines.

Cancer immunotherapies are treatments that use drugs or cells to activate patients' own immune systems against cancer cells. Among them, cancer vaccines have recently been rapidly developed. Based on tumor-specific antigens referred to as neoantigens, these vaccines can be in various forms such as messenger (m)RNA and synthetic peptides to activate cytotoxic T cells and act with or without dendritic cells. Growing evidence suggests that neoantigen-based cancer vaccines possess a very promising future, yet the processes of immune recognition and activation to relay identification of a neoantigen through the histocompatibility complex (MHC) and T-cell receptor (TCR) remain unclear. Here, we describe features of neoantigens and the biological process of validating neoantigens, along with a discussion of recent progress in the scientific development and clinical applications of neoantigen-based cancer vaccines.

Niclosamide Revitalizes Sorafenib through Insulin-like Growth Factor 1 Receptor (IGF-1R)/Stemness and Metabolic Changes in Hepatocellular Carcinoma.

Sorafenib is the first approved systemic targeting agent for advanced HCC; however, when used alone, drug resistance can result in considerably reduced efficacy. Here, we demonstrate that niclosamide, an antihelminthic agent approved by the US Food and Drug Administration, can be repurposed to increase sorafenib sensitivity in sorafenib-resistant HCC cells. We generated sorafenib-resistant HCC cell lines (HepG2215_R and Hep3B_R) with elevated IGF-1R levels and strong properties in terms of stemness and epithelial-mesenchymal transition. Niclosamide was found to increase sorafenib sensitivity effectively in both cell lines and their organoids. The underlying mechanism involves the modulation of cancer stemness, IGF-1R/p-IGF1R/OCT4, and metabolic changes. The combination of sorafenib and niclosamide, but not linsitinib, effectively suppressed the IGF-1R/OCT4 expressions, yielded a synergistic combination index (CI), and attenuated stemness-related properties such as secondary tumor sphere formation and cell migration in sorafenib-resistant HCC cells. Notably, niclosamide significantly suppressed the sorafenib-induced IGF-1R phosphorylation prompted by IGF-1 treatment. Niclosamide effectively downregulated the sorafenib-induced gene expression associated with glycolysis (GLUT1, HK2, LDHA, and PEPCK), stemness (OCT4), and drug resistance (ABCG2) and enhanced the ability of sorafenib to reduce the mitochondrial membrane potential in vitro. The synergistic effect of a combination of niclosamide and sorafenib in vivo was further demonstrated by the decreased tumor size and tumor volume resulting from apoptosis regulation. Our results suggest that niclosamide can enhance sorafenib sensitivity in sorafenib-resistant HCC cells through IGF-1R/stemness regulation and metabolic changes. Our findings highlight a practical clinical strategy for enhancing sorafenib sensitivity in HCC.

On the peptide binding affinity changes in population-specific HLA repertoires to the SARS-CoV-2 variants Delta and Omicron.

OBJECTIVE: To investigate the changes of Spike protein-HLA binding affinity profiles between the Wuhan strain and two dominant variants, the Delta and the Omicron strains, among the Taiwanese, the British and the Russian populations. METHODS: The HLA frequencies and the HLA-peptide binding affinity profiles in the T-CoV database were combined to conduct the study. We focused on the public alleles in the three populations (HLA-A, HLA-B, HLA-C, HLA-DRB1, and/or HLA-DPA1/DPB1 alleles) and the altered peptides of the spike protein (compared to the Wuhan strain) in the Delta G/478K·V1 (B.1.617.2 + AY.1 + AY.2) and the Omicron (BA.1) strains. RESULTS: For the Delta strain, tight bindings of the altered peptides to the HLA alleles decrease in all three populations and almost vanish in the Taiwanese population. For the Omicron strain, tight bindings are mostly preserved for both HLA classes and in the Taiwanese and the British populations, with a slight reduction in HLA class II in the Taiwanese (1.4%), while the Russian population preserves a relatively high fraction of tight bindings for both HLA classes. CONCLUSION: We comprehensively reported the changes in the HLA-associated SARS-CoV-2 Spike protein peptide binding profiles among the Taiwanese, the British, and the Russian populations. Further studies are needed to understand the immunological mechanisms and the clinical value of our findings.

Multiomic characterization and drug testing establish circulating tumor cells as an ex vivo tool for personalized medicine.

Matching the treatment to an individual patient's tumor state can increase therapeutic efficacy and reduce tumor recurrence. Circulating tumor cells (CTCs) derived from solid tumors are promising subjects for theragnostic analysis. To analyze how CTCs represent tumor states, we established cell lines from CTCs, primary and metastatic tumors from a mouse model and provided phenotypic and multiomic analyses of these cells. CTCs and metastatic cells, but not primary tumor cells, shared stochastic mutations and similar hypomethylation levels at transcription start sites. CTCs and metastatic tumor cells shared a hybrid epithelial/mesenchymal transcriptome state with reduced adhesive and enhanced mobilization characteristics. We tested anti-cancer drugs on tumor cells from a metastatic breast cancer patient. CTC responses mirrored the impact of drugs on metastatic rather than primary tumors. Our multiomic and clinical anti-cancer drug response results reveal that CTCs resemble metastatic tumors and establish CTCs as an ex vivo tool for personalized medicine.

Self-Sustained Regulation or Self-Perpetuating Dysregulation: ROS-dependent HIF-YAP-Notch Signaling as a Double-Edged Sword on Stem Cell Physiology and Tumorigenesis.

Organ development, homeostasis, and repair often rely on bidirectional, self-organized cell-niche interactions, through which cells select cell fate, such as stem cell self-renewal and differentiation. The niche contains multiplexed chemical and mechanical factors. How cells interpret niche structural information such as the 3D topology of organs and integrate with multiplexed mechano-chemical signals is an open and active research field. Among all the niche factors, reactive oxygen species (ROS) have recently gained growing interest. Once considered harmful, ROS are now recognized as an important niche factor in the regulation of tissue mechanics and topology through, for example, the HIF-YAP-Notch signaling pathways. These pathways are not only involved in the regulation of stem cell physiology but also associated with inflammation, neurological disorder, aging, tumorigenesis, and the regulation of the immune checkpoint molecule PD-L1. Positive feedback circuits have been identified in the interplay of ROS and HIF-YAP-Notch signaling, leading to the possibility that under aberrant conditions, self-organized, ROS-dependent physiological regulations can be switched to self-perpetuating dysregulation, making ROS a double-edged sword at the interface of stem cell physiology and tumorigenesis. In this review, we discuss the recent findings on how ROS and tissue mechanics affect YAP-HIF-Notch-PD-L1 signaling, hoping that the knowledge can be used to design strategies for stem cell-based and ROS-targeting therapy and tissue engineering.

Niche Laminin and IGF-1 Additively Coordinate the Maintenance of Oct-4 Through CD49f/IGF-1R-Hif-2α Feedforward Loop in Mouse Germline Stem Cells.

The mechanism on how extracellular matrix (ECM) cooperates with niche growth factors and oxygen tension to regulate the self-renewal of embryonic germline stem cells (GSCs) still remains unclear. Lacking of an appropriate in vitro cell model dramatically hinders the progress. Herein, using a serum-free culture system, we demonstrated that ECM laminin cooperated with hypoxia and insulin-like growth factor 1 receptor (IGF-1R) to additively maintain AP activity and Oct-4 expression of AP+GSCs. We found the laminin receptor CD49f expression in d2 testicular GSCs that were surrounded by laminin. Laminin and hypoxia significantly increased the GSC stemness-related genes, including Hif-2α, Oct-4, IGF-1R, and CD49f. Cotreatment of IGF-1 and laminin additively increased the expression of IGF-IR, CD49f, Hif-2α, and Oct-4. Conversely, silencing IGF-1R and/or CD49f decreased the expression of Hif-2α and Oct-4. The underlying mechanism involved CD49f/IGF1R-(PI3K/AKT)-Hif-2α signaling loop, which in turn maintains Oct-4 expression, symmetric self-renewal, and cell migration. These findings reveal the additive niche laminin/IGF-IR network during early GSC development.

Cell-extracellular matrix interactions in the fluidic phase direct the topology and polarity of self-organized epithelial structures.

INTRODUCTION: In vivo, cells are surrounded by extracellular matrix (ECM). To build organs from single cells, it is generally believed that ECM serves as scaffolds to coordinate cell positioning and differentiation. Nevertheless, how cells utilize cell-ECM interactions for the spatiotemporal coordination to different ECM at the tissue scale is not fully understood. METHODS: Here, using in vitro assay with engineered MDCK cells expressing H2B-mCherry (nucleus) and gp135/Podocalyxin-GFP (apical marker), we show in multi-dimensions that such coordination for epithelial morphogenesis can be determined by cell-soluble ECM interaction in the fluidic phase. RESULTS: The coordination depends on the native topology of ECM components such as sheet-like basement membrane (BM) and type I collagen (COL) fibres: scaffold formed by BM (COL) facilitates a close-ended (open-ended) coordination that leads to the formation of lobular (tubular) epithelium. Further, cells form apicobasal polarity throughout the entire lobule/tubule without a complete coverage of ECM at the basal side, and time-lapse two-photon scanning imaging reveals the polarization occurring early and maintained through the lobular expansion. During polarization, gp135-GFP was converged to the apical surface collectively in the lobular/tubular structures, suggesting possible intercellular communications. Under suspension culture, the polarization was impaired with multi-lumen formation in the tubules, implying the importance of ECM biomechanical microenvironment. CONCLUSION: Our results suggest a biophysical mechanism for cells to form polarity and coordinate positioning at tissue scale, and in engineering epithelium through cell-soluble ECM interaction and self-assembly.

Transcriptome network analyses in human coronavirus infections suggest a rational use of immunomodulatory drugs for COVID-19 therapy.

The recent outbreak of coronavirus disease 2019 (COVID-19) by SARS-CoV-2 has led to uptodate 24.3 M cases and 0.8 M deaths. It is thus in urgent need to rationalize potential therapeutic targets against the progression of diseases. An effective, feasible way is to use the pre-existing ΔORF6 mutant of SARS-CoV as a surrogate for SARS-CoV-2, since both lack the moiety responsible for interferon antagonistic effects. By analyzing temporal profiles of upregulated genes in ΔORF6-infected Calu-3 cells, we prioritized 55 genes and 238 ligands to reposition currently available medications for COVID-19 therapy. Eight of them are already in clinical trials, including dexamethasone, ritonavir, baricitinib, tofacitinib, naproxen, budesonide, ciclesonide and formoterol. We also pinpointed 16 drug groups from the Anatomical Therapeutic Chemical classification system, with the potential to mitigate symptoms of SARS-CoV-2 infection and thus to be repositioned for COVID-19 therapy.

The M1/M2 spectrum and plasticity of malignant pleural effusion-macrophage in advanced lung cancer.

BACKGROUND: Malignant pleural effusion (MPE)-macrophage (Mφ) of lung cancer patients within unique M1/M2 spectrum showed plasticity in M1-M2 transition. The M1/M2 features of MPE-Mφ and their significance to patient outcomes need to be clarified; furthermore, whether M1-repolarization could benefit treatment remains unclear. METHODS: Total 147 stage-IV lung adenocarcinoma patients undergoing MPE drainage were enrolled for profiling and validation of their M1/M2 spectrum. In addition, the MPE-Mφ signature on overall patient survival was analyzed. The impact of the M1-polarization strategy of patient-derived MPE-Mφ on anti-cancer activity was examined. RESULTS: We found that MPE-Mφ expressed both traditional M1 (HLA-DRA) and M2 (CD163) markers and showed a wide range of M1/M2 spectrum. Most of the MPE-Mφ displayed diverse PD-L1 expression patterns, while the low PD-L1 expression group was correlated with higher levels of IL-10. Among these markers, we identified a novel two-gene MPE-Mφ signature, IL-1ß and TGF-ß1, representing the M1/M2 tendency, which showed a strong predictive power in patient outcomes in our MPE-Mφ patient cohort (N = 60, p = 0.013) and The Cancer Genome Atlas Lung Adenocarcinoma dataset (N = 478, p < 0.0001). Significantly, ß-glucan worked synergistically with IFN-γ to reverse the risk signature by repolarizing the MPE-Mφ toward the M1 pattern, enhancing anti-cancer activity. CONCLUSIONS: We identified MPE-Mφ on the M1/M2 spectrum and plasticity and described a two-gene M1/M2 signature that could predict the outcome of late-stage lung cancer patients. In addition, we found that "re-education" of these MPE-Mφ toward anti-cancer M1 macrophages using clinically applicable strategies may overcome tumor immune escape and benefit anti-cancer therapies.

Parylene-Based Porous Scaffold with Functionalized Encapsulation of Platelet-Rich Plasma and Living Stem Cells for Tissue Engineering Applications.

A scaffold was fabricated to synergistically encapsulate living human adipose-derived stem cells (hASCs) and platelet-rich plasma (PRP) based on a vapor-phase sublimation and deposition process. During the process, ice templates were prepared using sterile water as the solvent and were used to accommodate the sensitive living cells and PRP molecules. Under controlled processing conditions, the ice templates underwent vapor sublimation to evaporate water molecules, while at the same time, vapor-phase deposition of poly-p-xylylene (Parylene, USP Class VI highly biocompatible) occurred to replace the templates, and the final construction yielded a scaffold with Parylene as the matrix, with simultaneously encapsulated living hASCs and PRP molecules. Evaluation of the fabricated synergistic scaffold for the proliferation activities toward the encapsulated hASCs indicated significant augmentation of cell proliferation contributed by the PRP ingredients. In addition, osteogenic activity in the early stage by alkaline phosphatase expression and later stage with calcium mineralization indicated significant enhancement toward osteogenetic differentiation of the encapsulated hASCs, which were guided by the PRP molecules. By contrast, examinations of adipogenic activity by lipid droplet formation revealed an inhibition of adipogenesis with decreased intracellular lipid accumulation, and a statistically significant downregulation of adipogenic differentiation was postulated for the scaffold products when compared to the osteogenetic results and the control experiments. The reported fabrication method featured a clean and simple process to construct scaffolds that combined delicate living hASCs and PRP molecules inside the structure. The resultant synergistic scaffold and the selected commercially available hASCs and PRP are emerging as tissue engineering tools that provide multifunctionality for tissue repair and regeneration.

Scalable Multilayer Cell Collector to Capture Circulating Tumor Cells with an Unlimited Volume Capacity.

Circulating tumor cells (CTCs) have been suggested as the precursors of metastatic cancer. CTC-based characterization has thus been used to monitor tumor status before the onset of metastasis and has shown to be an independent factor. The low abundance of CTCs, however, makes it challenging to employ CTC as a clinical routine, thus making it impossible to address tumor heterogeneity. Here, we present a cell collection prototype for an efficient capture of CTCs from a large volume of body fluids such as blood. An antibody-PEG modified multilayer matrix column is engineered and connected to an apheresis-based circulation system. This setup allows us to capture CTCs repetitively from an unlimited sample volume through the circulation system, thereby increasing the capture count. Compared to conventional CTC capturing devices where the sample handling is generally limited to 1-10 mL, our collector is able to handle a wide range of fluidic sample (40-2000 mL) at a high flow rate (400 mL/min). By processing 90 min in circulation, we obtained an average capture efficiency of at least 75% for the colorectal cancer cell line HCT116 spiked in either 40-200 mL of buffer solution or 40 mL of a whole blood sample. This result highlights a possibility to construct personalized CTC libraries through high-throughput CTC collection for the study of tumor heterogeneity in precision medicine.

Multiphoton structured thin-plane imaging with a single optical path.

Optical sectioning has become an indispensable technique for high-speed volumetric imaging in the past decade. Here we present a novel optical-sectioning method that produces a thin plane of illumination by exploiting the spatial and temporal properties of multiphoton excitation. Critically, the illumination and detection share the same optical path, as in a conventional epi-fluorescence microscope configuration. Therefore, the imaged sample can be prepared as for standard fluorescence microscopy. Our method also leads to a laterally structured illumination pattern, and this feature can be utilized in structured illumination microscopy to further enhance the imaging performance. We show an example of such an approach, which achieves axial resolution finer than confocal microscopy. We also demonstrate the potential of the new method for biological applications by performing three-dimensional imaging of living Caenorhabditis elegans.

Fiber-bundle illumination: realizing high-degree time-multiplexed multifocal multiphoton microscopy with simplicity.

High-degree time-multiplexed multifocal multiphoton microscopy was expected to provide a facile path to scanningless optical-sectioning and the fast imaging of dynamic three-dimensional biological systems. However, physical constraints on typical time multiplexing devices, arising from diffraction in the free-space propagation of light waves, lead to significant manufacturing difficulties and have prevented the experimental realization of high-degree time multiplexing. To resolve this issue, we have developed a novel method using optical fiber bundles of various lengths to confine the diffraction of propagating light waves and to create a time multiplexing effect. Through this method, we experimentally demonstrate the highest degree of time multiplexing ever achieved in multifocal multiphoton microscopy (~50 times larger than conventional approaches), and hence the potential of using simply-manufactured devices for scanningless optical sectioning of biological systems.

Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery.

Neurite initiation is the first step in neuronal development and occurs spontaneously in soft tissue environments. Although the mechanisms regulating the morphology of migratory cells on rigid substrates in cell culture are widely known, how soft environments modulate neurite initiation remains elusive. Using hydrogel cultures, pharmacologic inhibition, and genetic approaches, we reveal that paxillin-linked endocytosis and adhesion are components of a bistable switch controlling neurite initiation in a substrate modulus-dependent manner. On soft substrates, most paxillin binds to endocytic factors and facilitates vesicle invagination, elevating neuritogenic Rac1 activity and expression of genes encoding the endocytic machinery. By contrast, on rigid substrates, cells develop extensive adhesions, increase RhoA activity and sequester paxillin from the endocytic machinery, thereby delaying neurite initiation. Our results highlight paxillin as a core molecule in substrate modulus-controlled morphogenesis and define a mechanism whereby neuronal cells respond to environments exhibiting varying mechanical properties.

A Predictive Model for Yeast Cell Polarization in Pheromone Gradients.

Budding yeast cells exist in two mating types, a and α, which use peptide pheromones to communicate with each other during mating. Mating depends on the ability of cells to polarize up pheromone gradients, but cells also respond to spatially uniform fields of pheromone by polarizing along a single axis. We used quantitative measurements of the response of a cells to α-factor to produce a predictive model of yeast polarization towards a pheromone gradient. We found that cells make a sharp transition between budding cycles and mating induced polarization and that they detect pheromone gradients accurately only over a narrow range of pheromone concentrations corresponding to this transition. We fit all the parameters of the mathematical model by using quantitative data on spontaneous polarization in uniform pheromone concentration. Once these parameters have been computed, and without any further fit, our model quantitatively predicts the yeast cell response to pheromone gradient providing an important step toward understanding how cells communicate with each other.

Stage-Dependent Axon Transport of Proteasomes Contributes to Axon Development.

Axon extension at the growing tip requires elevated local protein supply, with a capability sustainable over long axons in varying environments. The exact mechanisms, however, remain elusive. Here we report that axon-promoting factors elicited a retrograde transport-dependent removal of proteasomes from nascent axon terminals, thereby increasing protein stability at axon tips. Such an effect occurred through phosphorylation of a dynein-interacting proteasome adaptor protein Ecm29. During the transition from immature neurites to nascent axons in cultured hippocampal neurons, live-cell imaging revealed a significant increase of the retrograde axonal transport of fluorescently labeled 20S proteasomes. This retrograde proteasome transport depended on neuron stage and increased with axon lengths. Blockade of retrograde transport caused accumulation of proteasomes, reduction of axon growth, and attenuation of growth-associated Par6 at the axon tip of newly polarized neurons. Our results delineate a regulatory mechanism that controls proteasome abundance via preferential transport required for axon development in newborn neurons.

Computational design of co-assembling protein-DNA nanowires.

Biomolecular self-assemblies are of great interest to nanotechnologists because of their functional versatility and their biocompatibility. Over the past decade, sophisticated single-component nanostructures composed exclusively of nucleic acids, peptides and proteins have been reported, and these nanostructures have been used in a wide range of applications, from drug delivery to molecular computing. Despite these successes, the development of hybrid co-assemblies of nucleic acids and proteins has remained elusive. Here we use computational protein design to create a protein-DNA co-assembling nanomaterial whose assembly is driven via non-covalent interactions. To achieve this, a homodimerization interface is engineered onto the Drosophila Engrailed homeodomain (ENH), allowing the dimerized protein complex to bind to two double-stranded DNA (dsDNA) molecules. By varying the arrangement of protein-binding sites on the dsDNA, an irregular bulk nanoparticle or a nanowire with single-molecule width can be spontaneously formed by mixing the protein and dsDNA building blocks. We characterize the protein-DNA nanowire using fluorescence microscopy, atomic force microscopy and X-ray crystallography, confirming that the nanowire is formed via the proposed mechanism. This work lays the foundation for the development of new classes of protein-DNA hybrid materials. Further applications can be explored by incorporating DNA origami, DNA aptamers and/or peptide epitopes into the protein-DNA framework presented here.

Self-repairing symmetry in jellyfish through mechanically driven reorganization.

What happens when an animal is injured and loses important structures? Some animals simply heal the wound, whereas others are able to regenerate lost parts. In this study, we report a previously unidentified strategy of self-repair, where moon jellyfish respond to injuries by reorganizing existing parts, and rebuilding essential body symmetry, without regenerating what is lost. Specifically, in response to arm amputation, the young jellyfish of Aurelia aurita rearrange their remaining arms, recenter their manubria, and rebuild their muscular networks, all completed within 12 hours to 4 days. We call this process symmetrization. We find that symmetrization is not driven by external cues, cell proliferation, cell death, and proceeded even when foreign arms were grafted on. Instead, we find that forces generated by the muscular network are essential. Inhibiting pulsation using muscle relaxants completely, and reversibly, blocked symmetrization. Furthermore, we observed that decreasing pulse frequency using muscle relaxants slowed symmetrization, whereas increasing pulse frequency by lowering the magnesium concentration in seawater accelerated symmetrization. A mathematical model that describes the compressive forces from the muscle contraction, within the context of the elastic response from the mesoglea and the ephyra geometry, can recapitulate the recovery of global symmetry. Thus, self-repair in Aurelia proceeds through the reorganization of existing parts, and is driven by forces generated by its own propulsion machinery. We find evidence for symmetrization across species of jellyfish (Chrysaora pacifica, Mastigias sp., and Cotylorhiza tuberculata).

Second messenger signaling for neuronal polarization: cell mechanics-dependent pattern formation.

Neuronal polarization is a critical step in the neuronal morphogenesis. Despite the identification of several evolutionarily conserved factors for neural polarization, the exact mechanisms by which cells initiate and maintain polarity remain to be characterized. Here, we review the recent progress on the roles of second messengers, specifically the cyclic nucleotides and membrane-associated phospholipids, in the initiation, propagation, and integration of polarization signals, and propose an inhibitor-free model for neural polarization. The characteristic features of neuron polarization include the formation of single axon and multiple dendrites. These features involve chemical and mechanical mechanisms such as reaction-diffusion and tug-of-war, by which second messengers can act in concert to initiate and stabilize the cellular asymmetry. Nevertheless, biochemical factors eliciting the long-range inhibition remain ambiguous. Thus, we provide a simple, inhibitor-free model that can incorporate known cytochemical and cytomechanical factors, and produce features of neuronal polarization in environments provided with minimized extracellular regulators.

Multiscale mechanobiology: mechanics at the molecular, cellular, and tissue levels.

Mechanical force is present in all aspects of living systems. It affects the conformation of molecules, the shape of cells, and the morphology of tissues. All of these are crucial in architecture-dependent biological functions. Nanoscience of advanced materials has provided knowledge and techniques that can be used to understand how mechanical force is involved in biological systems, as well as to open new avenues to tailor-made bio-mimetic materials with desirable properties.In this article, we describe models and show examples of how force is involved in molecular functioning, cell shape patterning, and tissue morphology.