Heterogeneity usually restricts conventional mass spectrometry to molecular weights less than around a megadalton. As a single-particle technique, charge detection mass spectrometry (CDMS) overcomes this limitation. In CDMS, the mass-to-charge (m/z) ratio and charge are measured simultaneously for individual ions, giving a direct mass measurement for each ion. Recent applications include the analysis of viruses, virus-like particles, vaccines, heavily glycosylated proteins, and gene therapy vectors.

Plant resistance (R) genes are members of large gene families with significant within and between species variation. It has been hypothesised that a variety of processes have shaped R gene evolution and the evolution of R gene specificity. In this review, we illustrate the main mechanisms that generate R gene diversity and provide examples of how they can change R gene specificity. Next, we explain which evolutionary mechanisms are at play and how they determine the fate of new R gene alleles and R genes. Finally, we place this in a larger context by comparing the diversity and evolution of R gene specificity within and between species scales.

The apoplast comprises the intercellular space between cell membranes, includes the xylem, and extends to the rhizoplane and the outer surfaces of the plant. The apoplast plays roles in different biological processes including plant immunity. This highly specialised space is often the first place where pathogen recognition occurs, and this then triggers the immune response. The immune response in the apoplast involves different mechanisms that restrict pathogen infection. Among these responses, secretion of different molecules like proteases, proteins related to immunity, small RNAs and secondary metabolites play important and often additive or synergistic roles. In addition, production of reactive oxygen species occurs to cause direct deleterious effects on the pathogen as well as reinforce the plant's immune response by triggering modifications to cell wall composition and providing additional defence signalling capabilities. The pool of available sugar in the apoplast also plays a role in immunity. These sugars can be manipulated by both interactors, pathogens gaining access to nutrients whilst the plant's responses restrict the pathogen's access to nutrients. In this review, we describe the latest findings in the field to highlight the importance of the apoplast in plant-pathogen interactions and plant immunity. We also indicate where new discoveries are needed.

Transcriptional reprogramming is an integral part of plant immunity. Tight regulation of the immune transcriptome is essential for a proper response of plants to different types of pathogens. Consequently, transcriptional regulators are proven targets of pathogens to enhance their virulence. The plant immune transcriptome is regulated by many different, interconnected mechanisms that can determine the rate at which genes are transcribed. These include intracellular calcium signaling, modulation of the redox state, post-translational modifications of transcriptional regulators, histone modifications, DNA methylation, modulation of RNA polymerases, alternative transcription inititation, the Mediator complex and regulation by non-coding RNAs. In addition, on their journey from transcription to translation, mRNAs are further modulated through mechanisms such as nuclear RNA retention, storage of mRNA in stress granules and P-bodies, and post-transcriptional gene silencing. In this review, we highlight the latest insights into these mechanisms. Furthermore, we discuss some emerging technologies that promise to greatly enhance our understanding of the regulation of the plant immune transcriptome in the future.

Nonhost resistance (NHR) is a plant immune response that prevents many microorganisms in the plant's environment from pathogenicity against the plant. Since successful pathogens have adapted to overcome the immune systems of their host, the durable nature of NHR has potential in the management of plant disease. At present, there is genetic and molecular evidence that the underlying molecular mechanisms of NHR are similar to the plant immune responses that occur in host plants following infection by adapted pathogens. We consider that the molecular basis of NHR is multilayered, conferred by physicochemical barriers and defense responses that are induced following molecular recognition events. Moreover, the relative contribution of each component may depend on evolutionary distances between host and nonhost plants of given pathogen species. This mini-review has focused on the current knowledge of plant NHR, especially the recognition of non-adapted pathogens by nonhost plants at the cellular level. Recent gains in understanding the roles of plasma membrane-localized pattern-recognition receptors (PRRs) and the cytoplasmic nucleotide-binding leucine-rich repeat receptors (NLRs) associated with these processes, as well as the genes involved, are summarized. Finally, we provide a theoretical perspective on the durability of receptor-mediated NHR and its practical potential as an innovative strategy for crop protection against pathogens.

Plants utilise cell-surface immune receptors (functioning as pattern recognition receptors, PRRs) and intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) to detect pathogens. Perception of pathogens by these receptors activates immune signalling and resistance to infections. PRR- and NLR-mediated immunity have primarily been considered parallel processes contributing to disease resistance. Recent studies suggest that these two pathways are interdependent and converge at multiple nodes. This review summarises and provides a perspective on these convergent points.

To perceive pathogen threats, plants utilize both plasma membrane-localized and intracellular receptors. Nucleotide-binding domain leucine-rich repeat containing (NLR) proteins are key receptors that can recognize pathogen-derived intracellularly delivered effectors and activate downstream defense. Exciting recent findings have propelled our understanding of the various recognition and activation mechanisms of plant NLRs. Some NLRs directly bind to effectors, but others can perceive effector-induced changes on targeted host proteins (guardees), or non-functional host protein mimics (decoys). Such guarding strategies are thought to afford the host more durable resistance to quick-evolving and diverse pathogens. Here, we review classic and recent examples of indirect effector recognition by NLRs and discuss strategies for the discovery and study of new NLR-decoy/guardee systems. We also provide a perspective on how executor NLRs and helper NLRs (hNLRs) provide recognition for a wider range of effectors through sensor NLRs and how this can be considered an expanded form of indirect recognition. Furthermore, we summarize recent structural findings on NLR activation and resistosome formation upon indirect recognition. Finally, we discuss existing and potential applications that harness NLR indirect recognition for plant disease resistance and crop resilience.

Recognition by plant receptors of microbe-associated molecular patterns (MAMPs) and pathogenicity effectors activates immunity. However, before evolving the capacity of perceiving and responding to MAMPs and pathogenicity factors, plants, like animals, must have faced the necessity to protect and repair the mechanical wounds used by pathogens as an easy passage into their tissue. Consequently, plants evolved the capacity to react to damage-associated molecular patterns (DAMPs) with responses capable of functioning also in the absence of pathogens. DAMPs include not only primarily cell wall (CW) fragments but also extracellular peptides, nucleotides and amino acids that activate both local and long-distance systemic responses and, in some cases, prime the subsequent responses to MAMPs. It is conceivable that DAMPs and MAMPs act in synergy to activate a stronger plant immunity and that MAMPs exploit the mechanisms and transduction pathways traced by DAMPs. The interest for the biology and mechanism of action of DAMPs, either in the plant or animal kingdom, is expected to substantially increase in the next future. This review focuses on the most recent advances in DAMPs biology, particularly in the field of CW-derived DAMPs.

To fight off diverse pathogens and pests, the plant immune system must recognize these invaders; however, as plant immune receptors evolve to recognize a pathogen, the pathogen often evolves to escape this recognition. Plant-pathogen co-evolution has led to the vast expansion of a family of intracellular immune receptors-nucleotide-binding domain and leucine-rich repeat proteins (NLRs). When an NLR receptor recognizes a pathogen ligand, it activates immune signaling and thus initiates defense responses. However, in contrast with the model of NLRs acting individually to activate resistance, an emerging paradigm holds that plants have complex receptor networks where the large repertoire of functionally specialized NLRs function together to act against the large repertoire of rapidly evolving pathogen effectors. In this article, we highlight key aspects of immune receptor networks in plant NLR biology and discuss NLR network architecture, the advantages of this receptor network system, and the evolution of the NLR network in asterid plants.

Plant immunity is triggered following the perception of pathogen-derived molecules by plant receptor proteins. Two protein families, membrane-localized receptor-like kinases (RLK) and intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, play key roles in pathogen perception and in the initiation of downstream signaling cascades that lead to defense responses. In addition to RLKs and NLRs, recent research has identified additional protein families that function as plant resistance (R) proteins. In particular, the botanical tribe Triticeae, which includes the globally important crop species wheat and barley, has played a significant role in the discovery of 'unconventional' R proteins. In this review, we will summarize the current knowledge on unconventional R genes in Triticeae and the proteins they encode. The knowledge on unconventional R proteins will not only broaden our understanding of plant-pathogen interactions but also have great implications for disease resistance breeding in crops.

Plants host a multipart immune signalling network to ward off pathogens. Pathogen attack upon plant tissues can often lead to an amplified state of (induced) defence against subsequent infections in distal tissues; this is known as systemic acquired resistance (SAR). The interaction of plants with beneficial microbes of the rhizosphere microbiome can also lead to an induced resistance in above-ground plant tissues, known as induced systemic resistance. Second messengers such as calcium (Ca2+), reactive oxygen species (ROS), and nitric oxide (NO) are necessary for cell-to-cell signal propagation during SAR and show emergent roles in the mediation of other SAR metabolites. These include the lysine-derived signals pipecolic acid (Pip) and N-hydroxypipecolic acid (NHP), which are key signalling metabolites in SAR. Emerging evidence additionally pinpoints plant volatiles as modulators of defence signalling within and between plants. Plant volatile organic compounds (VOCs) such as monoterpenes can promote SAR by functioning through ROS. Furthermore, plant-derived and additionally also microbial VOCs can target both salicylic acid and jasmonic acid signalling pathways in plants and modulate defence against pathogens. In this review, an overview of recent findings in induced defence signalling, with a particular focus on newer signalling molecules and how they integrate into these networks is discussed.

The phytohormones salicylic acid (SA) and jasmonic acid (JA) are major players in plant immunity. Numerous studies have provided evidence that SA- and JA-mediated signaling interact with each other (SA-JA crosstalk) to orchestrate plant immune responses against pathogens. At the same time, SA-JA crosstalk is often exploited by pathogens to promote their virulence. In this review, we summarize our current knowledge of molecular mechanisms for and modulations of SA-JA crosstalk during pathogen infection.

Pathogenic fungi use diverse infection strategies to obtain nutrients from plants. Biotrophic fungi feed only on living plant tissue, whereas necrotrophic fungi kill host cells to extract nutrients. To prevent disease, plants need to distinguish between pathogens with different life cycles, as a successful defense against a biotroph, which often involves programmed cell-death around the site of infection, is not an appropriate response to some necrotrophs. Plants utilize a vast collection of extracellular and intracellular receptors to detect the signatures of pathogen attack. In turn, pathogens are under strong selection to mask or avoid certain receptor responses while enhancing or manipulating other receptor responses to promote virulence. In this review, we focus on the plant receptors involved in resistance responses to fungal pathogens and highlight, with examples, how the infection strategy of fungal pathogens can determine if recognition responses are effective at preventing disease.

Enhancing plant resistance against pests and diseases by priming plant immunity is an attractive concept for crop protection because it provides long-lasting broad-spectrum protection against pests and diseases. This review provides a selected overview of the latest advances in research on the molecular, biochemical and epigenetic drivers of plant immune priming. We review recent findings about the perception and signalling mechanisms controlling the onset of priming by the plant stress metabolite β-aminobutyric acid. In addition, we review the evidence for epigenetic regulation of long-term maintenance of priming and discuss how stress-induced reductions in DNA hypomethylation at transposable elements can prime defence genes. Finally, we examine how priming can be exploited in crop protection and articulate the opportunities and challenges of translating research results from the Arabidopsis model system to crops.

Systemic acquired resistance (SAR), a type of long-distance immunity in plants, provides long-lasting resistance to a broad spectrum of pathogens. SAR is thought to involve the rapid generation and systemic transport of a mobile signal that prepares systemic parts of the plant to better resist future infections. Exploration of the molecular mechanisms underlying SAR have identified multiple mobile regulators of SAR in the last few decades. Examination of the relationship among several of these seemingly unrelated molecules depicts a forked pathway comprising at least two branches of equal importance to SAR. One branch is regulated by the plant hormone salicylic acid (SA), and the other culminates (based on current knowledge) with the phosphorylated sugar derivative, glycerol-3-phosphate (G3P). This review summarizes the activities that contribute to pathogen-responsive generation of SA and G3P and the components that regulate their systemic transport during SAR.

Plants deploy extracellular and intracellular immune receptors to sense and restrict pathogen attacks. Rapidly evolving pathogen effectors play crucial roles in suppressing plant immunity but are also monitored by intracellular nucleotide-binding, leucine-rich repeat immune receptors (NLRs), leading to effector-triggered immunity (ETI). Here, we review how NLRs recognize effectors with a focus on direct interactions and summarize recent research findings on the signalling functions of NLRs. Coiled-coil (CC)-type NLR proteins execute immune responses by oligomerizing to form membrane-penetrating ion channels after effector recognition. Some CC-NLRs function in sensor-helper networks with the sensor NLR triggering oligomerization of the helper NLR. Toll/interleukin-1 receptor (TIR)-type NLR proteins possess catalytic activities that are activated upon effector recognition-induced oligomerization. Small molecules produced by TIR activity are detected by additional signalling partners of the EDS1 lipase-like family (enhanced disease susceptibility 1), leading to activation of helper NLRs that trigger the defense response.

Adult-plant resistance (APR) is a type of genetic resistance in cereals that is effective during the later growth stages and can protect plants from a range of disease-causing pathogens. Our understanding of the functions of APR-associated genes stems from the well-studied wheat-rust pathosystem. Genes conferring APR can offer pathogen-specific resistance or multi-pathogen resistance, whereby resistance is activated following a molecular recognition event. The breeding community prefers APR to other types of resistance because it offers broad-spectrum protection that has proven to be more durable. In practice, however, deployment of new cultivars incorporating APR is challenging because there is a lack of well-characterised APRs in elite germplasm and multiple loci must be combined to achieve high levels of resistance. Genebanks provide an excellent source of genetic diversity that can be used to diversify resistance factors, but introgression of novel alleles into elite germplasm is a lengthy and challenging process. To overcome this bottleneck, new tools in breeding for resistance must be integrated to fast-track the discovery, introgression and pyramiding of APR genes. This review highlights recent advances in understanding the functions of APR genes in the well-studied wheat-rust pathosystem, the opportunities to adopt APR genes in other crops and the technology that can speed up the utilisation of new sources of APR in genebank accessions.

Plant immunity is crucial to plant health but comes at an expense. For optimal plant growth, tight immune regulation is required to prevent unnecessary rechannelling of valuable resources. Pattern- and effector-triggered immunity (PTI/ETI) represent the two tiers of immunity initiated after sensing microbial patterns at the cell surface or pathogen effectors secreted into plant cells, respectively. Recent evidence of PTI-ETI cross-potentiation suggests a close interplay of signalling pathways and defense responses downstream of perception that is still poorly understood. This review will focus on controls on plant immunity through phosphorylation, a universal and key cellular regulatory mechanism. Rather than a complete overview, we highlight "what's new in protein kinase/phosphatase signalling" in the immunity field. In addition to phosphoregulation of components in the pattern recognition receptor (PRR) complex, we will cover the actions of the major immunity-relevant intracellular protein kinases/phosphatases in the 'signal relay', namely calcium-regulated kinases (e.g. calcium-dependent protein kinases, CDPKs), mitogen-activated protein kinases (MAPKs), and various protein phosphatases. We discuss how these factors define a phosphocode that generates cellular decision-making 'logic gates', which contribute to signalling fidelity, amplitude, and duration. To underscore the importance of phosphorylation, we summarize strategies employed by pathogens to subvert plant immune phosphopathways. In view of recent game-changing discoveries of ETI-derived resistosomes organizing into calcium-permeable pores, we speculate on a possible calcium-regulated phosphocode as the mechanistic control of the PTI-ETI continuum.

Herbivore-induced plant volatiles regulate defenses in undamaged neighboring plants. Understanding the mechanisms by which plant volatiles are taken up, perceived, and translated into canonical defense signaling pathways is an important frontier of knowledge. Volatiles can enter plants through stomata and the cuticle. They are likely perceived by membrane-associated receptors as well as intracellular receptors. The latter likely involves metabolization and transport across cell membranes by volatile transporters. Translation of volatiles into defense priming and induction typically involves mitogen-activated protein kinases (MAPKs), WRKY transcription factors, and jasmonates. We propose that the broad range of molecular processes involved in volatile signaling will likely result in substantial spatiotemporal and ontogenetic variation in plant responsiveness to volatiles, with important consequences for plant-environment interactions.

The specific recognition of pathogen effectors by intracellular nucleotide-binding domain and leucine-rich repeat receptors (NLRs) is an important component of plant immunity. NLRs have a conserved modular architecture and can be subdivided according to their signaling domain that is mostly a coiled-coil (CC) or a Toll/Interleukin1 receptor (TIR) domain into CNLs and TNLs. Single NLR proteins are often sufficient for both effector recognition and immune activation. However, sometimes, they act in pairs, where two different NLRs are required for disease resistance. Functional studies have revealed that in these cases one NLR of the pair acts as a sensor (sNLR) and one as a helper (hNLR). The genes corresponding to such resistance protein pairs with one-to-one functional co-dependence are clustered, generally with a head-to-head orientation and shared promoter sequences. sNLRs in such functional NLR pairs have additional, non-canonical and highly diverse domains integrated in their conserved modular architecture, which are thought to act as decoys to trap effectors. Recent structure-function studies on the Arabidopsis thaliana TNL pair RRS1/RPS4 and on the rice CNL pairs RGA4/RGA5 and Pik-1/Pik-2 are unraveling how such protein pairs function together. Focusing on these model NLR pairs and other recent examples, this review highlights the distinctive features of NLR pairs and their various fascinating mode of action in pathogen effector perception. We also discuss how these findings on NLR pairs pave the way toward improved plant disease resistance.

Perception of non-self molecules known as microbe-associated molecular patterns (MAMPs) by host pattern recognition receptors (PRRs) activates plant pattern-triggered immunity (PTI). Pathogen infections often trigger the release of modified-self molecules, termed damage- or danger-associated molecular patterns (DAMPs), which modulate MAMP-triggered signaling to shape the frontline of plant immune responses against infections. In the context of advances in identifying MAMPs and DAMPs, cognate receptors, and their signaling, here, we focus on the most recent breakthroughs in understanding the perception and role of non-self and modified-self patterns. We highlight the commonalities and differences of MAMPs from diverse microbes, insects, and parasitic plants, as well as the production and perception of DAMPs upon infections. We discuss the interplay between MAMPs and DAMPs for emerging themes of the mutual potentiation and attenuation of PTI signaling upon MAMP and DAMP perception during infections.

Nucleotide-binding and leucine-rich repeat receptors (NLRs) are intracellular plant immune receptors that recognize pathogen effectors secreted into the plant cell. Canonical NLRs typically contain three conserved domains including a central nucleotide binding (NB-ARC) domain, C-terminal leucine-rich repeats (LRRs) and an N-terminal domain. A subfamily of plant NLRs contain additional noncanonical domain(s) that have potentially evolved from the integration of the effector targets in the canonical NLR structure. These NLRs with extra domains are thus referred to as NLRs with integrated domains (NLR-IDs). Here, we first summarize our current understanding of NLR-ID activation upon effector binding, focusing on the NLR pairs Pik-1/Pik-2, RGA4/RGA5, and RRS1/RPS4. We speculate on their potential oligomerization into resistosomes as it was recently shown for certain canonical plant NLRs. Furthermore, we discuss how our growing understanding of the mode of action of NLR-ID continuously informs engineering approaches to design new resistance specificities in the context of rapidly evolving pathogens.

Phytohormones are essential for all aspects of plant growth, development, and immunity; however, it is the interplay between phytohormones, as they dynamically change during these processes, that is key to this regulation. Hormones have traditionally been split into two groups: growth-promoting and stress-related. Here, we will discuss and show that all hormones play a role in plant defence, regardless of current designation. We highlight recent advances in our understanding of the complex phytohormone networks with less focus on archetypal immunity-related pathways and discuss protein and transcription factor signalling hubs that mediate hormone interplay.

Learning and memory are fundamental but highly complex functions of the brain. They rely on multiple mechanisms including the processing of sensory information, memory formation, maintenance of short- and long-term memory, memory retrieval and memory extinction. Recent experiments provide strong evidence that, besides neurons, astrocytes crucially contribute to these higher brain functions. However, the complex interplay of astrocytes and neurons in local neuron-glia assemblies is far from being understood. Although important basic cellular principles that govern and link neuronal and astrocytic cellular functions have been established, additional mechanisms clearly continue to emerge. In this short essay, we first review current technologies allowing the experimenter to explore the role of astrocytes in behaving animals, with focus on spatial memory. We then discuss astrocytic signaling mechanisms and their role in learning and memory. We also reveal gaps in our knowledge that currently prevent a comprehensive understanding of how astrocytes contribute to acquisition, storage and retrieval of memory by modulating neuronal signaling in local circuits.

Cancer stemness, defined as the self-renewal and tumor-initiation potential of cancer stem cells (CSCs), is a cancer biology property featuring activation of CSC signaling networks. Canonical WNT signaling through Frizzled and LRP5/6 receptors is transmitted to the β-catenin-TCF/LEF-dependent transcription machinery to up-regulate MYC, CCND1, LGR5, SNAI1, IFNG, CCL28, CD274 (PD-L1) and other target genes. Canonical WNT signaling causes expansion of rapidly cycling CSCs and modulates both immune surveillance and immune tolerance. In contrast, noncanonical WNT signaling through Frizzled or the ROR1/2 receptors is transmitted to phospholipase C, Rac1 and RhoA to control transcriptional outputs mediated by NFAT, AP-1 and YAP-TEAD, respectively. Noncanonical WNT signaling supports maintenance of slowly cycling, quiescent or dormant CSCs and promotes epithelial-mesenchymal transition via crosstalk with TGFβ (transforming growth factor-β) signaling cascades, while the TGFβ signaling network induces immune evasion. The WNT signaling network orchestrates the functions of cancer-associated fibroblasts, endothelial cells and immune cells in the tumor microenvironment and fine-tunes stemness in human cancers, such as breast, colorectal, gastric and lung cancers. Here, WNT-related cancer stemness features, including proliferation/dormancy plasticity, epithelial-mesenchymal plasticity and immune-landscape plasticity, will be discussed. Porcupine inhibitors, β-catenin protein-protein interaction inhibitors, β-catenin proteolysis targeting chimeras, ROR1 inhibitors and ROR1-targeted biologics are investigational drugs targeting WNT signaling cascades. Mechanisms of cancer plasticity regulated by the WNT signaling network are promising targets for therapeutic intervention; however, further understanding of context-dependent reprogramming trajectories might be necessary to optimize the clinical benefits of WNT-targeted monotherapy and applied combination therapy for patients with cancer.

Cancer stem cells (CSCs) have been believed to be one driving force for tumor progression and drug resistance. Despite the significance of biochemical signaling in malignancy, highly malignant tumor cells or CSCs exhibit lower cellular stiffness than weakly malignant cells or non-CSCs, which are softer than their healthy counterparts, suggesting the inverse correlation between cell stiffness and malignancy. Recent years have witnessed the rapid accumulation of evidence illustrating the reciprocity between cell cytoskeleton/mechanics and CSC functions and the potential of cellular stiffness for specific targeting of CSCs. However, a systematic understanding of tumor cell mechanics and their role in CSCs and tumor progression is still lacking. The present review summarizes the recent progress in the alterations of tumor cell cytoskeleton and stiffness at different stages of tumor progression and recapitulates the relationship between cellular stiffness and CSC functions. The altered cell mechanics may mediate the mechanoadaptive responses that possibly empower CSCs to survive and thrive during metastasis. Furthermore, we highlight the possible impact of tumor cell mechanics on CSC malignancy, which may potentiate low cell stiffness as a mechanical marker for CSC targeting.

Cancer stem cells (CSCs) are considered as a small population of cells with stem-like properties within the tumor bulk, and are largely responsible for tumor recurrence, metastasis, and therapy resistance. CSCs share critical features with embryonic stem cells (ESCs). The pluripotent transcription factors (TFs) and developmental signaling pathways of ESCs are invariably hijacked by CSCs termed 'oncofetal drivers' in many cancers, which are rarely detectable in adult tissues. The unique expression pattern makes oncofetal proteins ideal therapeutic targets in cancer treatment. Therefore, elucidation of oncofetal drivers in cancers is critical for the development of effective CSCs-directed therapy. In this review, we summarize the common pluripotent TFs such as OCT4, SOX2, NANOG, KLF4, MYC, SALL4, and FOXM1, as well as the development signaling including Wnt/β-catenin, Hedgehog (Hh), Hippo, Notch, and TGF-β pathways of ESCs and CSCs. We also describe the newly identified oncofetal proteins that drive the self-renewal, plasticity, and therapy-resistance of CSCs. Finally, we explore how the clinical implementation of targeting oncofetal drivers, including small-molecule inhibitors, vaccines, antibodies, and CAR-T (chimeric antigen receptor T cell) can facilitate the development of CSCs-directed therapy.

Cancer stem cells (CSCs) are subpopulations of cancer cells within the tumor bulk that have emerged as an attractive therapeutic target for cancer therapy. Accumulating evidence has shown the critical involvement of protein kinase signaling pathways in driving tumor development, cancer relapse, metastasis, and therapeutic resistance. Given that protein kinases are druggable targets for cancer therapy, tremendous efforts are being made to target CSCs with kinase inhibitors. In this review, we summarize the current knowledge and overview of the roles of protein kinases in various signaling pathways in CSC regulation and drug resistance. Furthermore, we provide an update on the preclinical and clinical studies for the use of kinase inhibitors alone or in combination with current therapies for effective cancer therapy. Despite great premises for the use of kinase inhibitors against CSCs, further investigations are needed to evaluate their efficiencies without any adverse effects on normal stem cells.

Cancer stem cells (CSCs) are a subpopulation of tumor cells with self-renewal ability. Increasing evidence points to the critical roles of CSCs in tumorigenesis, metastasis, therapy resistance, and cancer relapse. As such, the elimination of CSCs improves cancer treatment outcomes. However, challenges remain due to limited understanding of the molecular mechanisms governing self-renewal and survival of CSCs. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screening has been increasingly used to identify genetic determinants in cancers. In this primer, we discuss the progress made and emerging opportunities of coupling advanced CRISPR screening systems with CSC models to reveal the understudied vulnerabilities of CSCs.

Cancer stem cells (CSCs) are a small population of cells in human malignancies that resemble the biology of human pluripotent stem cells. CSCs are closely related to the critical hallmarks in human cancers, ranging from oncogenesis to disease progression, therapeutic resistance, and overall outcome. Mutations in isocitrate dehydrogenase (IDH) were recently identified as founder mutations for human cancers. An increasing amount of evidence indicates that IDH mutations are closely related to the establishment and maintenance of CSCs. Biosynthesis of oncometabolite, metabolic reprogramming, and epigenetic shifts establish distinctive molecular signatures in IDH-mutated CSCs. Additionally, IDH mutation and IDH-related pathways could be valuable molecular targets to impact the CSC components in human cancers and to improve the disease outcome.

Cancer stem cells (CSCs) are a subgroup of tumor cells, possessing the abilities of self-renewal and generation of heterogeneous tumor cell lineages. They are believed to be responsible for tumor initiation, metastasis, as well as chemoresistance in human malignancies. MicroRNAs (miRNAs) are small noncoding RNAs that play essential roles in various cellular activities including CSC initiation and CSC-related properties. Mature miRNAs with ∼22 nucleotides in length are generated from primary miRNAs via its precursors by miRNA-processing machinery. Extensive studies have demonstrated that mature miRNAs modulate CSC initiation and stemness features by regulating multiple pathways and targeting stemness-related factors. Meanwhile, both miRNA precursors and miRNA-processing machinery can also affect CSC properties, unveiling a new insight into miRNA function. The present review summarizes the roles of mature miRNAs, miRNA precursors, and miRNA-processing machinery in regulating CSC properties with a specific focus on the related molecular mechanisms, and also outlines the potential application of miRNAs in cancer diagnosis, predicting prognosis, as well as clinical therapy.

Cancer stem cells (CSCs) are subpopulation of cells within the tumor bulk, which leads to tumor recurrence and therapeutic resistance. Identification of specific CSC targets for detection and efficient cancer therapy are the major hurdles in this research field. In this decade, basic researchers and clinicians made every effort to overcome these challenges to target CSCs using different approaches. This special issue includes a varied collection of review articles with comprehensive discussion on the complexity of CSC heterogeneity, signaling pathways regulating the behaviors of CSCs, the therapeutic resistance mechanism of CSCs, and therapeutic targets against CSCs. These review articles shed light on current advances in understanding of CSC biology.

The administration of tyrosine kinase inhibitors (TKIs) for the treatment of advanced-stage patients is common in hepatocellular carcinoma (HCC). However, therapy resistance is often encountered, and its emergence eventually curtails long-term clinical benefits. Cancer stem cells (CSCs) are essential drivers of tumor recurrence and therapy resistance; thus, the elucidation of key hallmarks of resistance mechanisms of liver CSC-driven HCC may help improve patient outcomes and reduce relapse. The present review provides a comprehensive summary of the intrinsic and extrinsic mechanisms of TKI resistance in liver CSCs, which mediate treatment failure, and discusses potential strategies to overcome TKI resistance from a preclinical perspective.

Intratumoral heterogeneity can exist along multiple axes: Cancer stem cells (CSCs)/non-CSCs, drug-sensitive/drug-tolerant states, and a spectrum of epithelial-hybrid-mesenchymal phenotypes. Further, these diverse cell-states can switch reversibly among one another, thereby posing a major challenge to therapeutic efficacy. Therefore, understanding the origins of phenotypic plasticity and heterogeneity remains an active area of investigation. While genomic components (mutations, chromosomal instability) driving heterogeneity have been well-studied, recent reports highlight the role of non-genetic mechanisms in enabling both phenotypic plasticity and heterogeneity. Here, we discuss various processes underlying phenotypic plasticity such as stochastic gene expression, chromatin reprogramming, asymmetric cell division and the presence of multiple stable gene expression patterns ('attractors'). These processes can facilitate a dynamically evolving cell population such that a subpopulation of (drug-tolerant) cells can survive lethal drug exposure and recapitulate population heterogeneity on drug withdrawal, leading to relapse. These drug-tolerant cells can be both pre-existing and also induced by the drug itself through cell-state reprogramming. The dynamics of cell-state transitions both in absence and presence of the drug can be quantified through mathematical models. Such a dynamical systems approach to elucidating patterns of intratumoral heterogeneity by integrating longitudinal experimental data with mathematical models can help design effective combinatorial and/or sequential therapies for better clinical outcomes.

Acute myeloid leukemia (AML) is a heterogeneous disease of impaired myeloid differentiation and a caricature of normal hematopoiesis. Leukemic stem cells (LSCs) are responsible for long-term clonal propagation in AML just as hematopoietic stem cells (HSCs) sustain lifelong hematopoiesis. LSCs are often resistant to standard chemotherapy and are responsible for clinical relapse. Although AML is highly heterogeneous, determinants of stemness are prognostic for AML patient survival and can predict AML drug sensitivity. Therefore, one way to overcome challenges preventing efficacious treatment outcomes is to target LSC stemness. Metabolomic and lipidomic studies of serum and cells from AML patients are emerging to complement genomic, transcriptomic, epigenetic, and proteomic data sets to characterize and stratify AML. Recent studies have shown the value of fractionating LSCs versus blasts when characterizing metabolic pathways and implicate the importance of lipid balance to LSCs function. As more extensive metabolic studies coupled to functional in vivo assays are conducted on highly purified HSCs, bulk AML, and LSCs, the similarities and differences in lipid homeostasis in stem-like versus more mature AML subtypes as well as from normal HSCs are emerging. Here, we discuss the latest findings from studies of lipid function in LSCs, with a focus on sphingolipids (SLs) as stemness/lineage fate mediators in AML, and the balance of fatty acid anabolism and catabolism fueling metabolic flexibility and drug resistance in AML. We also discuss how designing successful strategies to target lipid vulnerabilities and improve AML patient survival should take into consideration the hierarchical nature of AML.

Cancer progression is characterized and driven by gradual loss of a differentiated phenotype and gain of stem cell-like features. In prostate cancer (PCa), androgen receptor (AR) signaling is important for cancer growth, progression, and emergence of therapy resistance. Targeting the AR signaling axis has been, over the decades, the mainstay of PCa therapy. However, AR signaling at the transcription level is reduced in high-grade cancer relative to low-grade PCa and loss of AR expression promotes a stem cell-like phenotype, suggesting that emergence of resistance to AR-targeted therapy may be associated with loss of AR signaling and gain of stemness. In the present mini-review, we first discuss PCa from the perspective of an abnormal organ with increasingly deregulated differentiation, and discuss the role of AR signaling during PCa progression. We then focus on the relationship between prostate cancer stem cells (PCSCs) and AR signaling. We further elaborate on the current methods of using transcriptome-based stemness-enriched signature to evaluate the degree of oncogenic dedifferentiation (cancer stemness) in pan-cancer datasets, and present the clinical significance of scoring transcriptome-based stemness across the spectrum of PCa development. Our discussions highlight the importance to evaluate the dynamic changes in both stem cell-like features (stemness score) and AR signaling activity across the PCa spectrum.