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Epithelial-mesenchymal transition (EMT) and its reverse mechanism, mesenchymal-epithelial transition (MET), are evolutionarily conserved mechanisms initially identified in studies of early metazoan development. EMT may even have been established in choanoflagellates, the closest unicellular relative of Metazoa. These crucial morphological transitions operate during body plan formation and subsequently in organogenesis. These findings have prompted an increasing number of investigators in biomedicine to assess the importance of such mechanisms that drive epithelial cell plasticity in multiple diseases associated with congenital disabilities and fibrosis, and, most importantly, in the progression of carcinoma. EMT and MET also play crucial roles in regenerative medicine, notably by contributing epigenetic changes in somatic cells to initiate reprogramming into stem cells and their subsequent differentiation into distinct lineages. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGF-β) signaling regulates cell-specific programs involved in embryonic development, wound-healing, and immune homeostasis. Yet, during tumor progression, these TGF-β-mediated programs are altered, leading to epithelial cell plasticity and a reprogramming of epithelial cells into mesenchymal lineages through epithelial-to-mesenchymal transition (EMT), a critical developmental program in morphogenesis and organogenesis. These changes, in turn, lead to enhanced carcinoma cell invasion, metastasis, immune cell differentiation, immune evasion, and chemotherapy resistance. Here, we discuss EMT as one of the critical programs associated with carcinoma cell plasticity and the influence exerted by TGF-β on carcinoma status and function. We further explore the composition of carcinoma and other cell populations within the tumor microenvironment, and consider the relevant outcomes related to the programs associated with cancer treatment resistance. [ABSTRACT FROM AUTHOR]

The Neural Crest, a transient epithelium in vertebrate embryos, is the source of putative stem cells known to give rise to neuronal, glial and endocrine components of the peripheral (sensory, autonomic and enteric) nervous system (PNS) and pigment cells in the skin. The Neural Crest is also widely believed to be the source of mesectodermal derivatives (skeletogenic, odontogenic, connective tissue and smooth muscle mesenchyme) in the vertebrate head [see ( Bronner and LeDouarin, 2012; Le Douarin, 2012; Le Douarin and Kalcheim, 1999 ); see also ( Hörstadius, 1950; Weston, 1970 )]. This conventional understanding of the broad developmental potential of the Neural Crest has been challenged over the past few years ( Breau et al., 2008; Lee et al., 2013a, 2013b; Weston et al., 2004 ), based on recognition that the definition of the embryonic epithelia that comprise the Neural Crest may be imprecise. Indeed, the definition of the embryonic tissues understood to constitute the Neural Crest has changed considerably since it was first described by Wilhelm His 150 years ago ( His, 1868 ). Today, the operational definition of the Neural Crest is inconsistent and functionally ambiguous. We believe that more precise definitions of the embryonic tissues involved in Neural Crest development would be useful to understand (1) the range of cellular phenotypes that actually segregate from it, (2) when this lineage diversification occurs, and (3) how diversification is regulated. In this idiosyncratic review, we aim to explain our concerns with the current definitions in this field, and in the chiastic words of Samuel Johnson (1781), “… make new things familiar and familiar things new”. 1 1 Samuel Johnson׳s words were paraphrased by Mardy Grothe [drmardy.com] from Johnson׳s Lives of the Poets [1781]. Discussing Alexander Pope׳s poem, The Rape of the Lock , Johnson wrote, “In this work are exhibited in a very high degree the two most engaging powers of an author: New things are made familiar, and familiar things are made new.” Then, we will try to distinguish the developmental events crucial to the regulation of Neural Crest development at both cranial and trunk axial levels of vertebrate embryos, and address some of the implicit assumptions that underlie the conventional interpretation of experimental results on the origin and fates of Neural Crest-derived cells. We hope our discussion will resolve some ambiguities regarding both the range of derivatives in the Neural Crest lineage and the conventional understanding that cranial mesectodermal derivatives share a common Neural Crest-derived lineage precursor with components of the PNS. [ABSTRACT FROM AUTHOR]

Epithelial--mesenchymal transition (EMT) can be induced by several pleiotropically activated transcription factors, including the zincfinger E-box-binding protein Zeb1. Mechanisms regulating Zeb1 expression have been partly uncovered, showing a critical role for the miR-200 family members. In the present study, we show that Zeb1 is regulated by the Arf GTPase-activating protein (GAP) Git2. Following the loss of Git2, we found that miR-146a maturation is enhanced, which in turn promotes the expression of Zeb1 and induction of EMT. Furthermore, we found that Cnot6L, a validated target of miR-146a, affects the stability of Zeb1 mRNA through its deadenylase activity. Our results present evidence for a new role for loss of Git2 in promoting EMT through a novel regulatory pathway. [ABSTRACT FROM AUTHOR]

Epithelial-mesenchymal transition (EMT) is a crucial, evolutionarily conserved process that occurs during development and is essential for shaping embryos. Also implicated in cancer, this morphological transition is executed through multiple mechanisms in different contexts, and studies suggest that the molecular programs governing EMT, albeit still enigmatic, are embedded within developmental programs that regulate specification and differentiation. As we review here, knowledge garnered from studies of EMT during gastrulation, neural crest delamination and heart formation have furthered our understanding of tumor progression and metastasis. INSETS: Development: the big picture;Box 1. Glossary;Box 2. Heart EndMT. [ABSTRACT FROM AUTHOR]

Classical cadherins are single-pass transmembrane proteins mediating adhesive interactions between animal cells. As such, they play key roles during morphogenetic movements, in cell sorting and in tissue integrity. Being positioned at the cell–cell interface, cadherins are most likely important players in mechanotransduction pathways. This review briefly outlines our current understanding of the biochemical and biophysical basis for various adhesive properties of cadherins and the ensuing intercellular adhesive strength and specificity. We summarize the attempts to explain cadherin specificity from their ultrastructural features and their adhesive behavior at the single molecule level. The role of cadherin clusters and cooperative binding is then reviewed. Lastly, we consider the attempts to understand the link between local stress and the adhesive properties of cadherin-mediated junctions. [ABSTRACT FROM AUTHOR]

The epithelial to mesenchymal transition (EMT) plays crucial roles in the formation of the body plan and in the differentiation of multiple tissues and organs. EMT also contributes to tissue repair, but it can adversely cause organ fibrosis and promote carcinoma progression through a variety of mechanisms. EMT endows cells with migratory and invasive properties, induces stem cell properties, prevents apoptosis and senescence, and contributes to immunosuppression. Thus, the mesenchymal state is associated with the capacity of cells to migrate to distant organs and maintain stemness, allowing their subsequent differentiation into multiple cell types during development and the initiation of metastasis. [ABSTRACT FROM AUTHOR]

Epithelial–mesenchymal transition is an indispensable mechanism during morphogenesis, as without mesenchymal cells, tissues and organs will never be formed. However, epithelial-cell plasticity, coupled to the transient or permanent formation of mesenchyme, goes far beyond the problem of cell-lineage segregation. Understanding how mesenchymal cells arise from an epithelial default status will also have a strong impact in unravelling the mechanisms that control fibrosis and cancer progression. [ABSTRACT FROM AUTHOR]

The epithelial–mesenchymal transition (EMT) is a fundamental process governing morphogenesis in multicellular organisms. This process is also reactivated in a variety of diseases including fibrosis and in the progression of carcinoma. The molecular mechanisms of EMT were primarily studied in epithelial cell lines, leading to the discovery of transduction pathways involved in the loss of epithelial cell polarity and the acquisition of a variety of mesenchymal phenotypic traits. Similar mechanisms have also been uncovered in vivo in different species, showing that EMT is controlled by remarkably well-conserved mechanisms. Current studies further emphasise the critical importance of EMT and provide a better molecular and functional definition of mesenchymal cells and how they emerged >500 million years ago as a key event in evolution. [Copyright &y& Elsevier]

Cell-adhesion molecules play a major role in morphogenesis and organogenesis. In vertebrates, a significant fraction of genes encode cell-adhesion molecules. Multiple signal-transduction pathways have been described that modulate the adhesion process. These pathways have been studied in great detail for cadherins and integrins — two major adhesion systems controlling cell–cell and cell–substrate interactions. Recent findings confirm that a given cell-adhesion molecule can be implicated at different stages of development in processes as diverse as cell positioning, tissue patterning and compartmentalization, axon guidance and synaptogenesis. Clearly, a wide variety of new biophysical techniques and genomic approaches will permit analysis of the roles of adhesive interactions in development to be addressed with far greater precision. [Copyright &y& Elsevier]

Cell adhesion is a key physiological event tightly coupled to other major cellular processes coordinating morphogenesis and histogenesis. Cell-to-cell and cell-to-extracellular matrix adhesion regulates the social behavior of cells in developing embryos and in the adult. These two adhesion systems also play a critical role in pathogenesis. In vertebrates, more than 3% of genes are thought to encode adhesion molecules. The largest cell adhesion molecule superfamily is that related to N-CAM, the members of which characteristically contain immunoglobulin domains. Cadherins, which also possess Ig domains, constitute another important superfamily with different properties. Integrins are major receptors for many extracellular matrix components. This review describes the structure and function of these adhesion systems and their impact in cancer invasion and metastasis. To cite this article: J.P. Thiery, C. R. Physique 4 (2003). [Copyright &y& Elsevier]

Without epithelial-mesenchymal transitions, in which polarized epithelial cells are converted into motile cells, multicellular organisms would be incapable of getting past the blastula stage of embryonic development. However, this important developmental programme has a more sinister role in tumour progression. Epithelial-mesenchymal transition provides a new basis for understanding the progression of carcinoma towards dedifferentiated and more malignant states. [ABSTRACT FROM AUTHOR]

Simple Summary: This review describes mechanisms driving epithelial plasticity in carcinoma mediated by transforming growth factor beta (TGF-β) signaling. Plasticity in carcinoma is frequently induced through epithelial–mesenchymal transition (EMT), an evolutionary conserved process in the development of multicellular organisms. The review explores the multifaceted functions of EMT, particularly focusing on the intermediate stages, which provide more adaptive responses of carcinoma cells in their microenvironment. The review critically considers how different intermediate or hybrid EMT stages confer carcinoma cells with stemness, refractoriness to therapies, and ability to execute all steps of the metastatic cascade. Finally, the review provides examples of therapeutic interventions based on the EMT concept. Epithelial cell plasticity, a hallmark of carcinoma progression, results in local and distant cancer dissemination. Carcinoma cell plasticity can be achieved through epithelial–mesenchymal transition (EMT), with cells positioned seemingly indiscriminately across the spectrum of EMT phenotypes. Different degrees of plasticity are achieved by transcriptional regulation and feedback-loops, which confer carcinoma cells with unique properties of tumor propagation and therapy resistance. Decoding the molecular and cellular basis of EMT in carcinoma should enable the discovery of new therapeutic strategies against cancer. In this review, we discuss the different attributes of plasticity in carcinoma and highlight the role of the canonical TGFβ receptor signaling pathway in the acquisition of plasticity. We emphasize the potential stochasticity of stemness in carcinoma in relation to plasticity and provide data from recent clinical trials that seek to target plasticity. [ABSTRACT FROM AUTHOR]

Patients with head and neck squamous cell carcinoma (HNSCC) are at a high risk of developing recurrence and secondary cancers. This study evaluates the prognostic and surveillance utilities of circulating tumour cells (CTCs) in HNSCC. A total of 154 HNSCC patients were recruited and followed up for 4.5 years. Blood samples were collected at baseline and follow-up. CTCs were isolated using a spiral microfluid device. Recurrence and death due to cancer were assessed during the follow-up period. In patients with HNSCC, the presence of CTCs at baseline was a predictor of recurrence (OR = 8.40, p < 0.0001) and death (OR= ∞, p < 0.0001). Patients with CTCs at baseline had poor survival outcomes (p < 0.0001). Additionally, our study found that patients with CTCs in a follow-up appointment were 2.5 times more likely to experience recurrence or death from HNSCC (p < 0.05) prior to their next clinical visit. Our study highlights the prognostic and monitoring utilities of CTCs' in HNSCC patients. Early identification of CTCs facilitates precise risk assessment, guiding treatment choices and ultimately enhancing patient outcomes. [ABSTRACT FROM AUTHOR]

RNA interactomes and their diversified functionalities have recently benefited from critical methodological advances leading to a paradigm shift from a conventional conception on the regulatory roles of RNA in pathogenesis. However, the dynamic RNA interactomes in adenoma-carcinoma sequence of human CRC remain unexplored. The coexistence of adenoma, cancer, and normal tissues in colorectal cancer (CRC) patients provides an appropriate model to address this issue. Here, we adopted an RNA in situ conformation sequencing technology for mapping RNA-RNA interactions in CRC patients. We observed large-scale paired RNA counts and identified some unique RNA complexes including multiple partners RNAs, single partner RNAs, non-overlapping single partner RNAs. We focused on the antisense RNA OIP5-AS1 and found that OIP5-AS1 could sponge different miRNA to regulate the production of metabolites including pyruvate, alanine and lactic acid. Our findings provide novel perspectives in CRC pathogenesis and suggest metabolic reprogramming of pyruvate for the early diagnosis and treatment of CRC. [ABSTRACT FROM AUTHOR]

• Chimeric transcripts are critical for diagnosis or prognosis and could constitute effective therapeutic targets. • Although fresh tissues are the major source for the identification of fusion transcripts, formalin-fixed paraffin-embedded (FFPE) has also been widely used to identify fusion transcripts. • RNA sequencing data generated from FFPE tissues for gene expression analysis and fusion transcript detection can be reproducible when considering certain criteria discussed in this article. Chimeric transcripts are critical for diagnosis or prognosis and could constitute effective therapeutic targets. Fresh tissues are the major source for the identification of these fusion transcripts. The quality and quantity of the extracted RNA directly affect fusion transcript discovery. Formalin-fixed paraffin-embedded (FFPE) tissues allow long-time preservation of tumor histology for microscopic evaluation; however, no provision has been made for either the type of fixative or embedding procedure used for preserving RNA. Nonetheless, the widespread use of these FFPE tissues in translational and clinical research prompts to overcome these issues. RNA is, by nature, of reduced quality and amount in these FFPE tissues. Therefore, attempts should be taken to minimize the limitations of FFPE tissues as a widely available source of fusion transcript identification. In this review, we describe approaches allowing fusion transcript identification from FFPE tissues using RNA sequencing techniques. [ABSTRACT FROM AUTHOR]

The kinetics of degradation of calf thymus DNA by the DNAase from Helix aspersa has been investigated in its middle and terminal phase by determining both hyperchromic shift and average chain length of the digest. The results obtained are practically identical with those previously reported for acid DNAase from hog spleen. In contrast, the distribution of the isostichs in snail DNAase digests is significantly different from those found for spleen acid DNAase digests of similar sizes. The 3′phosphate and 5′-hydroxy terminal nucleotides were found not to vary in their compositions in the average size range 34 to 10 and 70 to 12, respectively. In the 3′-phosphate terminal position, deoxyadenosine is by far predominant (78%) and deoxycytidine almost absent (less than 1%); deoxyguanosinc and thymidine form 6% and 16%, respectively, of the terminals. In the 5′-hydroxy terminal position deoxyguanosine is predominant (45%), followed by deoxycytidine (31%), thymidine (14%) and deoxyadenosine (10%); in the 5′-hydroxy penultimate position thymidine forms 38% of the nucleotides, deoxyguanosine 24%, deoxy-adenosine 21% and deoxycytidine 17%. [ABSTRACT FROM AUTHOR]

The kinetics of the middle and terminal phases of calf thymus DNA degradation by spleen acid DNAase has been investigated in detail with the main purpose of providing a background for a study of the specificity of the enzyme and its use in investigations of nucleotide sequences in DNA. During the middle phase the ultraviolet absorption, the acid solubility and the reciprocal average degree of polymerization of the hydrolysate, &Pmacr;n-1 increase linearly with digestion time, while the elution profile of the DEAE-cellulose-urea chromatogram is progressively shifted to the left. Empirical relationships between &Pmacr;n-1 and hyperchromic shift have been established. Extrapolation of &Pmacr;n-1 to zero digestion time indicates that bond splitting proceeds at a faster, yet decreasing rate during the early part of digestion. The beginning of the terminal phase is characterized by a sudden slow down of the reaction rate; this phenomenon has been shown to be essentially due to the melting of double-stranded DNA fragments; the single-stranded fragments so originated arc a poor substrate for the enzyme, in spite of the fact that they still contain a large number of sequences which can be spit. During the terminal phase, the ultraviolet absorption, the acid solubility and &Pmacr;n-1 increase at a progressively slower rate until an end-point is practically reached. Investigations on the liberation of mono- and dinucleotides, the base composition and termini of isostichs and the effects of digestion temperature and of dephosphorylation on the isostich patterns are also reported. [ABSTRACT FROM AUTHOR]

The kinetics of liberation of 3′-phosphate terminal and of 5′-hydroxy terminal and penultimate nucleotides (termini) by spleen acid DNAase was investigated using as substrates calf thymus DNA, three bacterial DNAs of different base composition, and poly-[d(A-T] · d(A-T)]. The relative amounts of the nucleotides deoxyguanosine, deoxyadenosine, thymidine and deoxycytidine in the termini released by the enzyme from all DNAs were found to be constant in the terminal phase of digestion. In contrast, some variations were seen during the middle phase, particularly in the amount of 3′-phosphate terminal deoxyguanosine, which decreased with digestion time, specially in the calf thymus DNA digest. This phenomenon appears to he due to an initial accumulation of breaks in some nucleotide sequences which are preferentially split at the beginning of digestion, possibly by a diplotomic mechanism, with predominant release of 3′-phosphate deoxyguanosine. In contrast, no change in the composition of 3′-phosphate terminals released from poly[d(A-T) · d(A-T)] took place in the 40–15 average size range; deoxyadenosine and thymidine formed 80% and 20%, respectively, of these terminals. The composition of the 3′-phosphate terminal nucleotides was very little or not at all affected by changes in the ionic environment of the incubation mixture, whereas it was significantly changed in degradations of native DNA digested at neutrality and low ionic strength, or of heat-denatured DNA. Linear relationships were found to hold, at all degradation stages investigated here, between the relative amounts of termini liberated from different bacterial DNAs and their (dG + dC) contents. In contrast, the termini obtained from calf thymus DNA were released in relative amounts which did not fit the relationships established for bacterial DNAs. [ABSTRACT FROM AUTHOR]

Simple Summary: Expression of the transcription factor Slug/SNAI2 is associated with the epithelial–mesenchymal transition (EMT) and is correlated with poorer disease-free survival in colorectal cancer (CRC). In order to decipher the basis for the Slug-mediated aggressive phenotype, we conducted RNAseq experiments with a panel of HT-29 CRC cells expressing different levels of Slug, both in vitro and in tumor models. Osteopontin (OPN), a mediator associated with tumor progression in different tumor types, was among the top upregulated genes in both cells and tumors and was the most overexpressed gene coding for a secreted protein. We further show that Slug is a direct regulator of osteopontin via binding to the OPN promoter. Interestingly, Slug expression and osteopontin secretion were correlated in vitro, as well as in tumor models, suggesting that liquid biopsies may be useful in estimating the aggressiveness phenotype of the tumor. In colorectal cancer (CRC), disease-related death is closely linked to tumor aggressiveness and metastasis. Gene expression profiling of patient tumors has suggested that a more mesenchymal phenotype, present in about one-fourth of all patients, is associated with increased aggressiveness. Accordingly, the mesenchymal transcription factor Slug/SNAI2 has been associated with decreased disease-free survival. To decipher the basis for the Slug-mediated phenotype, we conducted RNAseq experiments with a panel of HT-29 CRC cells expressing different levels of Slug, both in vitro and in tumor models. The results show that osteopontin, a secreted pleotropic protein involved in multiple steps of colorectal cancer progression, was highly upregulated by Slug in vitro, as well as in vivo. We further show that Slug is a direct regulator of osteopontin at the promoter level. The levels of secreted osteopontin were correlated with Slug expression, thereby linking the tumor phenotype to a biomarker available by liquid biopsies. The results also suggest that osteopontin neutralization may attenuate at least some of the Slug-mediated functions. [ABSTRACT FROM AUTHOR]

Epithelial-mesenchymal transition (EMT) is involved in the formation of the body plan, tissue remodeling, and cancer progression. Two recent reports in Science () have decisively advanced our understanding of EMT. Par6 was identified as a key player in the control of tight junction (TJ) stability. This new study provides further insight into the protein networks involved in topologically regulated control of epithelial cell polarity and plasticity. [Copyright &y& Elsevier]

Background & Aims: The class I‐ phosphatidylinositol‐3 kinases (PI3Ks) signalling is dysregulated in almost all human cancers whereas the isoform‐specific roles remain poorly investigated. We reported that the isoform δ (PI3Kδ) regulated epithelial cell polarity and plasticity and recent developments have heightened its role in hepatocellular carcinoma (HCC) and solid tumour progression. However, its role in cholangiocarcinoma (CCA) still lacks investigation. Approach & Results: Immunohistochemical analyses of CCA samples reveal a high expression of PI3Kδ in the less differentiated CCA. The RT‐qPCR and immunoblot analyses performed on CCA cells stably overexpressing PI3Kδ using lentiviral construction reveal an increase of mesenchymal and stem cell markers and the pluripotency transcription factors. CCA cells stably overexpressing PI3Kδ cultured in 3D culture display a thick layer of ECM at the basement membrane and a wide single lumen compared to control cells. Similar data are observed in vivo, in xenografted tumours established with PI3Kδ‐overexpressing CCA cells in immunodeficient mice. The expression of mesenchymal and stemness genes also increases and tumour tissue displays necrosis and fibrosis, along with a prominent angiogenesis and lymphangiogenesis, as in mice liver of AAV8‐based‐PI3Kδ overexpression. These PI3Kδ‐mediated cell morphogenesis and stroma remodelling were dependent on TGFβ/Src/Notch signalling. Whole transcriptome analysis of PI3Kδ using the cancer cell line encyclopedia allows the classification of CCA cells according to cancer progression. Conclusions: Overall, our results support the critical role of PI3Kδ in the progression and aggressiveness of CCA via TGFβ/src/Notch‐dependent mechanisms and open new directions for the classification and treatment of CCA patients. [ABSTRACT FROM AUTHOR]

The nucleosome remodeling and histone deacetylase (NuRD) complex physically associates with BCL11B to regulate murine T‐cell development. However, the function of NuRD complex in mature T cells remains unclear. Here, we characterize the fate and metabolism of human T cells in which key subunits of the NuRD complex or BCL11B are ablated. BCL11B and the NuRD complex bind to each other and repress natural killer (NK)‐cell fate in T cells. In addition, T cells upregulate the NK cell‐associated receptors and transcription factors, lyse NK‐cell targets, and are reprogrammed into NK‐like cells (ITNKs) upon deletion of MTA2, MBD2, CHD4, or BCL11B. ITNKs increase OPA1 expression and exhibit characteristically elongated mitochondria with augmented oxidative phosphorylation (OXPHOS) activity. OPA1‐mediated elevated OXPHOS enhances cellular acetyl‐CoA levels, thereby promoting the reprogramming efficiency and antitumor effects of ITNKs via regulating H3K27 acetylation at specific targets. In conclusion, our findings demonstrate that the NuRD complex and BCL11B cooperatively maintain T‐cell fate directly by repressing NK cell‐associated transcription and indirectly through a metabolic‐epigenetic axis, providing strategies to improve the reprogramming efficiency and antitumor effects of ITNKs. Synopsis: While BCL11b is known to bind the NuRD complex and be required for T cell development, the function of these factors in mature T cells has so far been unclear. This work demonstrates how the NuRD complex and BCL11B together maintain T‐cells from reprogramming into NK cells. T cells are reprogrammed into NK‐like cells upon deletion of MTA2, MBD2, or CHD4.BCL11B associates with the NuRD complex to repress OPA1 expression in T cells.OPA1‐mediated mitochondrial fusion facilitates ITNK reprogramming and cytotoxicity by increasing OXPHOS.Acetyl‐CoA elevated by OXPHOS promotes NK‐cell‐associated genes expression and antitumor effects in ITNKs by regulating H3K27 acetylation. [ABSTRACT FROM AUTHOR]

A recent paper reports that circulating tumor cells (CTCs) from metastatic breast cancer patients exhibit heterogeneous epithelial and mesenchymal phenotypes and that CTCs display higher frequencies of partial or full-blown mesenchymal phenotype than carcinoma cells within primary tumors. Mesenchymal-like CTCs are also elevated in patients who are refractory to therapy. [Copyright &y& Elsevier]

The epithelial-to-mesenchymal transition (EMT), a key process in morphogenesis, is often driven by repressing expression of adherens junction components, such as E-cadherin. In this issue of Developmental Cell, uncover an alternative mechanism in the Drosophila embryonic gut that promotes EMT via Serpent, a GATA transcriptional repressor of the apicobasal polarity gene crumbs. [ABSTRACT FROM AUTHOR]

Can tumor progression be understood in terms of the redeployment of mechanisms used in embryonic development? The identification of Twist, a bHLH transcription factor, as a protein linked to metastases in breast cancers would suggest so. [ABSTRACT FROM AUTHOR]

Summary: Neural crest cells disperse throughout the embryonic head to generate diverse cell types of two classes: non-ectomesenchymal (including melanocytes, peripheral neurons and glia) and ectomesenchymal (skeletogenic, odontogenic, cartilaginous and connective tissue cell fates). In contrast to cranial neural crest, trunk neural crest of amniotes generates only non-ectomesenchymal cell types. Anamniote trunk neural crest, however, has been assumed to generate derivatives of both classes, including osteoblasts of dermal skeletal elements, which includes scales and fin rays. Through genetic lineage tracing in zebrafish, we present the first test of this assumption and find that trunk neural crest does not generate fin osteoblasts; rather, these derive from a late emerging population of paraxial mesoderm. Similarly we show that the mineralising cells of the scales are mesodermally derived, with no contribution from neural crest. Our data suggest that trunk/tail exoskeletal structures evolved through deployment of mesodermally derived mesenchyme, rather than neural crest. [ABSTRACT FROM AUTHOR]

The article discusses a 2022 study on a minority population of LMO2+ breast cancer cells that integrate into the vasculature and initiate metastasis. Topics covered include the reduction of lung metastasis by LMO2 knockdown in human breast tumors, and the binding of LMO2 to STAT3 that is required for the latter's activation. Also noted is the identification of cells with angiogenic features that establishes the LMO2-STAT3 signaling axis as a therapeutic target in breast cancer metastasis.

Summary: Recent studies of carcinoma progression reveal the distinct routes of dissemination of discrete carcinoma cell populations and suggest that melanoma cell dissemination is linked to differentiation rather than stemness status. [Copyright &y& Elsevier]

Multimodal analysis of circulating tumour cells (CTCs) has the potential to provide remarkable insight for cancer development and metastasis. CTCs and CTC clusters investigation using single-cell analysis, enables researchers to gain crucial information on metastatic mechanisms and the genomic alterations responsible for drug resistance, empowering treatment, and management of cancer. Despite a plethora of CTC isolation technologies, careful attention to the strengths and weaknesses of each method should be considered in order to isolate these rare cells. Here, we provide an overview of cutting-edge technologies used for single-cell isolation and analysis of CTCs. Additionally, we highlight the biological features, clinical application, and the therapeutic potential of CTCs and CTC clusters using single-cell analysis platforms for cancer management. In-depth characterisation of circulating tumour cells (CTCs) has shown promise in diagnosis and management of cancer patients in a noninvasive manner. However, rarity of CTCs poses critical challenges for isolating and analysing them. CTC analysis provides insights into tumour heterogeneity beyond genomic aberrations that are not found in circulating tumour DNA (ctDNA). Single-cell molecular analysis of CTCs offers a new prognostic approach to identification of targeted therapy resistance mechanisms. Choice of technology for isolation and analysis of CTCs mainly depends on cell-loss, study cost per CTC, and workflow complexity. Current technologies suffer from high analysis costs and cell-loss during handling of the low number of CTCs. Additionally, different physical and biological features of CTC clusters often lead to difficulties in analysing them via current commercial platforms. [ABSTRACT FROM AUTHOR]

Background: Local recurrence and metastasis remain the major causes of death in head and neck cancer (HNC) patients. Circulating tumour cells (CTCs) are shed from primary and metastatic sites into the circulation system and have been reported to play critical roles in the metastasis and recurrence of HNC. Here, we explored the use of CTCs to predict the response to treatment and disease progression in HNC patients. Methods: Blood samples were collected at diagnosis from HNC patients (n = 119). CTCs were isolated using a spiral microfluidic device and were identified using immunofluorescence staining. Correlation of baseline CTC numbers to 13-week PET-CT data and multidisciplinary team consensus data were conducted. Results: CTCs were detected in 60/119 (50.4%) of treatment naïve HNC patients at diagnosis. Baseline CTC numbers were higher in stage III vs. stage I-II p16-positive oropharyngeal cancers (OPCs) and other HNCs (p = 0.0143 and 0.032, respectively). In addition, we found that baseline CTC numbers may serve as independent predictors of treatment response, even after adjusting for other conventional prognostic factors. CTCs were detected in 10 out of 11 patients exhibiting incomplete treatment responses. Conclusions: We found that baseline CTC numbers are correlated with treatment response in patients with HNC. The expression level of cell-surface vimentin (CSV) on CTCs was significantly higher in patients with persistent or progressive disease, thus providing additional prognostic information for stratifying the risk at diagnosis in HNC patients. The ability to detect CTCs at diagnosis allows more accurate risk stratification, which in the future may be translated into better patient selection for treatment intensification and/or de-intensification strategies. [ABSTRACT FROM AUTHOR]

The stem cells involved in formation of the complex human body are epithelial cells that undergo apicobasal polarization and form a hollow lumen. Epithelial plasticity manifests as epithelial to mesenchymal transition (EMT), a process by which epithelial cells switch their polarity and epithelial features to adopt a mesenchymal phenotype. The connection between the EMT program and acquisition of stemness is now supported by a substantial number of reports, although what discriminates these two processes remains largely elusive. In this study, based on 3D organoid culture of hepatocellular carcinoma (HCC)-derived cell lines and AAV8-based protein overexpression in the mouse liver, we show that activity modulation of isoform δ of phosphoinositide 3-kinase (PI3Kδ) controls differentiation and discriminates between stemness and EMT by regulating the transforming growth factor β (TGFβ) signaling. This study provides an important tool to control epithelial cell fate and represents a step forward in understanding the development of aggressive carcinoma. Overexpression of isoform δ of phosphoinositide 3-kinase (PI3Kδ) promotes stemness in hepatocellular carcinoma cell lines and mouse liver, whereas its inhibition promotes EMT, suggesting a key role for PI3Kδ in epithelial plasticity. [ABSTRACT FROM AUTHOR]

Simple Summary: Prostate cancer (PCa) is notoriously difficult to diagnose owing to the lack of reliable biomarkers and the invasiveness of obtaining a tissue biopsy from the prostate. As an alternative, we developed a liquid biopsy technique, based on isolating tumor cells from semen samples via a microfluidic device. To optimize the device, we first attempted to recover PCa cells from semen samples spiked with PCa cell lines, achieving an average efficiency of >87% cell recovery at the chosen flow rate. We then transitioned to a clinical setting using semen samples from PCa patients. The yield of isolated clinical PCa cells varied between 67 and 307 cells per mL of semen (in 15 cancer patients). These cells were stained and compared to the standard prognostic parameters such as Gleason score and PSA serum level. This study presents a potential liquid biopsy technique to augment the existing diagnosis and prognosis of PCa. Prostate cancer (PCa) diagnosis is primarily based on prostate-specific antigen (PSA) testing and prostate tissue biopsies. However, PSA testing has relatively low specificity, while tissue biopsies are highly invasive and have relatively low sensitivity at early stages of PCa. As an alternative, we developed a technique of liquid biopsy, based on isolation of circulating tumor cells (CTCs) from seminal fluid (SF). The recovery of PCa cells from SF was demonstrated using PCa cell lines, achieving an efficiency and throughput as high as 89% (±3.8%) and 1.7 mL min−1, respectively, while 99% (±0.7%) of sperm cells were disposed of. The introduced approach was further tested in a clinical setting by collecting and processing SF samples of PCa patients. The yield of isolated CTCs measured as high as 613 cells per SF sample in comparison with that of 6 cells from SF of healthy donors, holding significant promise for PCa diagnosis. The correlation analysis of the isolated CTC numbers with the standard prognostic parameters such as Gleason score and PSA serum level showed correlation coefficient values at 0.40 and 0.73, respectively. Taken together, our results show promise in the developed liquid biopsy technique to augment the existing diagnosis and prognosis of PCa. [ABSTRACT FROM AUTHOR]

Cadherins control intercellular adhesion in most metazoans. In vertebrates, intercellular adhesion differs considerably between cadherins of type-I and type-II, predominantly due to their different extracellular regions. Yet, intercellular adhesion critically depends on actomyosin contractility, in which the role of the cadherin extracellular region is unclear. Here, we dissect the roles of the Extracellular Cadherin (EC) Ig-like domains by expressing chimeric E-cadherin with E-cadherin and cadherin-7 Ig-like domains in cells naturally devoid of cadherins. Using cell-cell separation, cortical tension measurement, tissue stretching and migration assays, we show that distinct EC repeats in the extracellular region of cadherins differentially modulate epithelial sheet integrity, cell-cell separation forces, and cell cortical tension with the Cdc42 pathway, which further differentially regulate epithelial tensile strength, ductility, and ultimately collective migration. Interestingly, dissipative processes rather than static adhesion energy mostly dominate cell-cell separation forces. We provide a framework for the emergence of epithelial phenotypes from cell mechanical properties dependent on EC outside-in signaling. [ABSTRACT FROM AUTHOR]

The neural crest is a multipotent stem cell population that arises from the dorsal aspect of the neural tube and generates both nonectomesenchymal (melanocytes, peripheral neurons and glia) and ectomesenchymal (skeletogenic, odontogenic, cartilaginous and connective tissue) derivatives. In amniotes, only cranial neural crest generates both classes, with trunk neural crest restricted to nonectomesenchyme. By contrast, it has been suggested that anamniotes might generate derivatives of both classes at all axial levels, with trunk neural crest generating fin osteoblasts, scale mineral-forming cells and connective tissue cells; however, this has not been fully tested. The cause and evolutionary significance of this cranial/trunk dichotomy, and its absence in anamniotes, are debated. Recent experiments have disputed the contribution of fish trunk neural crest to fin osteoblasts and scale mineral-forming cells. This prompted us to test the contribution of anamniote trunk neural crest to fin connective tissue cells. Using genetics-based lineage tracing in zebrafish, we find that these fin mesenchyme cells derive entirely from the mesoderm and that neural crest makes no contribution. Furthermore, contrary to previous suggestions, larval fin mesenchyme cells do not generate the skeletogenic cells of the adult fin, but persist to form fibroblasts associated with adult fin rays. Our data demonstrate that zebrafish trunk neural crest does not generate ectomesenchymal derivatives and challenge long-held ideas about trunk neural crest fate. These findings have important implications for the ontogeny and evolution of the neural crest. [ABSTRACT FROM AUTHOR]

The article focuses on cell adhesion and polarity during epithelial-mesenchymal transition (EMT). EMT leads to the sequential destabilization of cell-cell adhesive junctions and the regulatory mechanism that controls cell polarity. The highly polarized epithelial cells are located above a layer of basement membrane, and comprise a complex network of extracellular matrix (ECM). Epithelial cells connect to the basement membrane through integrins, and bind to peptides found on collagens.

FGF-1 and FGF-2 are pleiotropic growth factors for many cell types, operating through the activation of specific transmembrane FGF receptors (FGFRs). The role of these factors in tumor progression was investigated, with specific discrimination between their autocrine and non autocrine cellular activity. The rat bladder carcinoma NBT-II cells were engineered to produce FGF-1 or 18 kDa FGF-2 in the presence or absence of their specific receptor. Non-autocrine cells that produced FGF-1 or FGF-2 but lacked FGFRs were epithelial and reminiscent of the parental NBT-II cells. Whilst autocrine cells, which both constitutively produced and secreted the growth factor and expressed FGFRs, had a highly invasive mesenchymal phenotype. Correspondingly, the autocrine cells were highly tumorigenic in vivo compared to the parental and non-autocrine cells, which correlated with the increased production of uPAR and active uPA and increased in vitro invasive potential. Although all cells produced VEGF, only tumors derived from cells that produced FGF-1 or FGF-2 were highly vascularized, suggesting that these two growth factors could be involved in the angiogenic process by activating host endothelial cells. As a result of activation of the FGFR in autocrine cells, changes in cell morphology and an increase in the invasive and tumorigenic properties were observed, however no in vitro or in vivo differential functions between FGF-1 and FGF-2 could be identified in this system. In conclusion, our data demonstrates that rapid tumor development is not dependent upon increased tumor vascularization, suggesting that ‘basal’ angiogenesis, probably mediated by VEGF, is sufficient to support tumor growth.Oncogene (2002) 21, 8128–8139. doi:10.1038/sj.onc.1205935 [ABSTRACT FROM AUTHOR]

Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), are believed to play key roles in facilitating the metastatic cascade. Metastatic lesions often exhibit a similar epithelial-like state to that of the primary tumour, in particular, by forming carcinoma cell clusters via E-cadherin-mediated junctional complexes. However, the factors enabling mesenchymal-like micrometastatic cells to resume growth and reacquire an epithelial phenotype in the target organ microenvironment remain elusive. In this study, we developed a workflow using image-based cell profiling and machine learning to examine morphological, contextual and molecular states of individual breast carcinoma cells (MDA-MB-231). MDA-MB-231 heterogeneous response to the host organ microenvironment was modelled by substrates with controllable stiffness varying from 0.2kPa (soft tissues) to 64kPa (bone tissues). We identified 3 distinct morphological cell types (morphs) varying from compact round-shaped to flattened irregular-shaped cells with lamellipodia, predominantly populating 2-kPa and >16kPa substrates, respectively. These observations were accompanied by significant changes in E-cadherin and vimentin expression. Furthermore, we demonstrate that the bone-mimicking substrate (64kPa) induced multicellular cluster formation accompanied by E-cadherin cell surface localisation. MDA-MB-231 cells responded to different substrate stiffness by morphological adaptation, changes in proliferation rate and cytoskeleton markers, and cluster formation on bone-mimicking substrate. Our results suggest that the stiffest microenvironment can induce MET. Author summary: The epithelial-mesenchymal transition (EMT), a process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal cells, is believed to play the key role in the initiation of the metastatic cascade. The ability of mesenchymal cells to survive, transform and establish colonies in distant organs is well known but not well understood. To gain insight into this process, we developed a workflow using machine learning and image analysis of cells to examine how morphology (or physical form and structure) and biochemical properties of individual (triple negative) breast carcinoma cells vary in response to their interaction with a substrate of variable stiffness. We found that mesenchymal breast cancer cells responded to different substrate stiffness by morphological adaptation, changes in proliferation rate and cytoskeleton markers such as cell-cell adhesion protein E-cadherin. Surprisingly, the cell properties on the stiffest tested substrate that mimicked bone stiffness changed dramatically–they formed multicellular clusters with distinct E-cadherin localisation at the cell-cell adhesion surfaces. Our results suggest that the stiffest microenvironment can induce cell transitioning from mesenchymal to epithelial cell phenotype. [ABSTRACT FROM AUTHOR]

Innovative diagnostics are becoming an essential component in personalized cancer medicine. These diagnostics are increasingly based on cell-free nucleic acids and membrane vesicles. Isolating and sequencing cell free circulating DNA (cfDNA) in plasma may progressively substitute tumor biopsies. A small albeit now detectable fraction of cfDNA correspond to circulating tumor DNA (ctDNA). In this review, we describe the pre-analytical procedures for collecting ctDNA from plasma, since these procedures should be optimized within laboratories depending on the available infrastructures. We also provide an overview of the technological breakthrough in ctDNA Isolation for instance digital PCR methods and next generation sequencing techniques and discuss their key challenges. The clinical implementations of liquid biopsy and more specifically ctDNA in cancer management are reviewed. We predict in the near future, ctDNA will be used more routinely to guide cancer treatment and provide a new approach to personalize treatment in precision medicine. [ABSTRACT FROM AUTHOR]

Epithelial–mesenchymal transition (EMT) is a critical biological process allowing epithelial cells to de-differentiate into mesenchymal cells. Orchestrated signaling pathways cooperatively induce EMT and effect physiological, sometimes pathological outcomes. Traditional Chinese Medicine (TCM) has been clinically prescribed for thousands of years and recent studies have found that TCM therapies can participate in EMT regulation. In this review, the historical discovery of EMT will be introduced, followed by a brief overview of its major roles in development and diseases. The second section will focus on EMT in organ fibrosis and tissue regeneration. The third section discusses EMT-induced cancer metastasis, and details how EMT contribute to distant dissemination. Finally, new EMT players are described, namely microRNA, epigenetic modifications, and alternative splicing. TCM drugs that affect EMT proven through an evidence-based research approach will be presented in each section. [ABSTRACT FROM AUTHOR]

Epithelial to mesenchymal transition (EMT) is a complex plastic and reversible cellular process that has critical roles in diverse physiological and pathological phenomena. EMT is involved in embryonic development, organogenesis and tissue repair, as well as in fibrosis, cancer metastasis and drug resistance. In recent years, the ability to edit the genome using the clustered regularly interspaced palindromic repeats (CRISPR) and associated protein (Cas) system has greatly contributed to identify or validate critical genes in pathway signaling. This review delineates the complex EMT networks and discusses recent studies that have used CRISPR/Cas technology to further advance our understanding of the EMT process. [ABSTRACT FROM AUTHOR]

Biological barriers are multicellular structures that precisely regulate the transport of ions, biomolecules, drugs, cells, and other organisms. Transendothelial/epithelial electrical resistance (TEER) is a label-free method for predicting the properties of biological barriers. Understanding the mechanisms that control TEER significantly enhances our knowledge of the physiopathology of different diseases and aids in the development of new drugs. Measuring TEER values within microphysiological systems called organ-on-a-chip devices that simulate the microenvironment, architecture, and physiology of biological barriers in the body provides valuable insight into the behavior of barriers in response to different drugs and pathogens. These integrated systems should increase the accuracy, reproducibility, sensitivity, resolution, high throughput, speed, cost-effectiveness, and reliable predictability of TEER measurements. Implementing advanced micro and nanoscale manufacturing techniques, surface modification methods, biomaterials, biosensors, electronics, and stem cell biology is necessary for integrating TEER measuring systems with organ-on-chip technology. This review focuses on the applications, advantages, and future perspectives of integrating organ-on-a-chip technology with TEER measurement methods for studying biological barriers. After briefly reviewing the role of TEER in the physiology and pathology of barriers, standard techniques for measuring TEER, including Ohm's law and impedance spectroscopy, and commercially available devices are described. Furthermore, advances in TEER measurement are discussed in multiple barrier-on-a-chip system models representing different organs. Finally, we outline future trends in implementing advanced technologies to design and fabricate nanostructured electrodes, complicated microfluidic chips, and membranes for more advanced and accurate TEER measurements. Applications of TEER measurements for studying development, permeability, and response to biological behavior. The routine TEER measurement, including Ohm's low and impedance spectroscopy-based methods, can be developed using advanced micro and nanofabrication methods. A variety of advantages, including implementing biological shear stress, nanostructured and surface-modified membranes, microelectrodes, cell types, advanced electrodes, biosensors, and arrays, can be implemented in the in vitro measuring of TEER inside barriers designed in organ-on-chips. [Display omitted] • Report on state-of-the-art advances in TEER measurement in biological barriers-on-chips (organ-on-chip). • Review of latest applications of advanced techniques and strategies for increasing the quality of TEER measurements. • Highlight challenges in TEER measurement with sensitive electrodes, microfluidics, stem cells, and membranes. [ABSTRACT FROM AUTHOR]

Abstract Background Previous molecular subtyping for bladder carcinoma (BLCA) involved <450 samples, with diverse classifications. Objective To identify molecular subtypes by curating a large BLCA dataset. Design, setting, and participants Gene expression publicly available were combined and reanalyzed. The dataset contained 2411 unique tumors encompassing non-muscle-invasive (NMIBC) and muscle-invasive BLCA (MIBC). Subtypes were reproduced on The Cancer Genome Atlas, UROMOL, and IMvigor210. Intervention Subtypes were assigned by gene expression. Outcome measurements and statistical analysis Kaplan-Meier analyses were performed for subtype-clinical outcome correlations; Chi-square/Fisher exact tests were used for subtype-clinicopathological parameters associations. Results and limitations We identified six molecular subtypes with different overall survival (OS) and molecular features. Subtype Neural-like (median OS, 87 mo) is prevalent in MIBC and characterized by high WNT/β-catenin signaling. HER2-like (107.7 mo) is distributed evenly across NMIBC and MIBC, with higher ERBB2 amplification and signaling. Papillary-like (>135 mo), an NMIBC subtype enriched in urothelial differentiation genes, shows a high frequency of actionable FGFR3 mutations, amplifications, and FGFR3 - TACC3 fusion. Luminal-like (91.7 mo), predominantly NMIBC, has higher MAPK signaling and more KRAS and KMT2 C/D mutations than other subtypes. Mesenchymal-like (MES; 86.6 mo) and Squamous-cell carcinoma-like (SCC; 20.6 mo) are predominant in MIBC. MES is high in AXL signaling, whereas SCC has elevated PD1, CTLA4 signaling, and macrophage M2 infiltration. About 20% of NMIBCs show MIBC subtype traits and a lower 5-yr OS rate than Papillary-like NMIBC (81% vs 96%). The main limitations of our study are the incomplete clinical annotation, and the analyses were based on transcriptome subset due to comparisons across gene expression quantification technologies. Conclusions BLCA can be stratified into six molecular subtypes. NMIBC, with a high risk of progression, displays the molecular features of MIBC. Patient summary Biomarkers are urgently needed to guide patient treatment selection and avoid unnecessary toxicities in those who fail to respond. We believe molecular subtyping is a promising way to tailor disease management for those who will benefit most. Take Home Message A gene expression analysis for over 2000 bladder carcinomas shows six potential clinically relevant molecular subtypes with distinct pathways and targetable vulnerability. Non–muscle-invasive bladder cancer with a high risk of progression displays molecular features of muscle-invasive disease. [ABSTRACT FROM AUTHOR]

With the advent of high-throughput technologies leading to big data generation, increasing number of gene signatures are being published to predict various features of diseases such as prognosis and patient survival. However, to use these signatures for identifying therapeutic targets, use of additional bioinformatic tools is indispensible part of research. Here, we have generated a pipeline comprised of nearly 15 bioinformatic tools and enrichment statistical methods to propose and validate a drug combination strategy from already approved drugs and present our approach using published pan-cancer epithelial–mesenchymal transition (EMT) signatures as a case study. We observed that histone deacetylases were critical targets to tune expression of multiple epithelial versus mesenchymal genes. Moreover, SRC and IKBK were the principal intracellular kinases regulating multiple signaling pathways. To confirm the anti-EMT efficacy of the proposed target combination in silico , we validated expression of targets in mesenchymal versus epithelial subtypes of ovarian cancer. Additionally, we inhibited the pinpointed proteins in vitro using an invasive lung cancer cell line. We found that whereas low-dose mono-therapy failed to limit cell dispersion from collagen spheroids in a microfluidic device as a metric of EMT, the combination fully inhibited dissociation and invasion of cancer cells toward cocultured endothelial cells. Given the approval status and safety profiles of the suggested drugs, the proposed combination set can be considered in clinical trials. [ABSTRACT FROM AUTHOR]

Tumor biopsy is the gold standard for the assessment of clinical biomarkers for treatment. However, tumors change dynamically in response to therapy, and there remains a need for a more representative biomarker that can be assayed over the course of treatment. Circulating tumor cells (CTCs) may provide clinically important and comprehensive tumoral information that is predictive of treatment response and outcome. Blood samples were processed for CTCs from 56 patients using the ClearCell FX system. Captured cells were phenotyped for CTC clusters and markers for immunotherapy (PD‐L1) CTC chromosomal architecture (ALK, EGFR). CTCs were isolated in 11/23 (47.8%) of head and neck cancer (HNC) patients and 17/33 (51.5%) of non‐small‐cell lung cancer (NSCLC) patients. CTCs were determined to be PD‐L1‐positive in 6/11 (54.4%) HNC and 11/17 (64.7%) NSCLC cases, respectively. 3D chromosomal DNA FISH for ALK and EGFR molecular targets showed better resolution than in 2D when imaging CTCs. HNC CTC‐positive patients had shorter progression‐free survival (PFS) (hazard ratio[HR]: 4.946; 95% confidence internal[CI]:1.571‐15.57; P = 0.0063), and PD‐L1‐positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011‐26.33; P = 0.0485). In the advanced stage NSCLC patient cohort, PFS was not found to be associated with CTCs prior to therapy ([HR]:2.246; 95% [CI]:0.9565‐5.273; P = 0.0632), nor the presence of PD‐L1 expression ([HR]:1.646; 95% [CI]:0.5128‐5.283; P = 0.4023). This study demonstrated that CTCs are predictive of poorer outcomes in HNC and provides distinct and separate utility for CTCs in HNC and NSCLC, which may be more representative of the disease burden and overall survival than the parameters used to measure them. 3D DNA FISH of circulating tumor cells demonstrates a higher resolution for genomic aberrations compared to 2D imaging modalities. [ABSTRACT FROM AUTHOR]

Protein-based cancer therapies are considered an alternative to conventional anticancer regimens, providing multifunctional properties while showing low toxicity. However, its widespread use is limited by absorption and instability issues, resulting in higher dosage requirements and a prolonged onset of bioactivity to elicit the desired response. Here, we developed a non-invasive antitumor treatment using designed ankyrin repeat protein (DARPin)-anticancer protein-conjugate that specifically targets the cancer biomarker, epithelial cell adhesion molecule (EpCAM). The DARPin-anticancer proteins bind to EpCAM-positive cancer cells and improve the in vitro anticancer efficacy by over 100-folds within 24 h, where the DARPin-tagged human lactoferrin fragment (drtHLF4) IC50 value is within the nanomolar range. Orally administered drtHLF4 was readily absorbed into the systemic flow of the HT-29 cancer murine model, exerting its anticancer effect on other tumors in the host body. Orally administered drtHFL4 cleared HT29-colorectal tumors using a single dose, whereas intratumoral injection cleared HT29-subcutaneous tumors within three doses. This approach addresses the limitations of other protein-based anticancer treatments by providing a non-invasive anticancer therapy with improved potency and tumor-specificity. [Display omitted] • DARPin tagged-anticancer protein increased specificity, efficacy & rapid onset against EpCAM (+) cancer cells. • DARPin tagged protein exert over 1000-fold increase in the anticancer activity compared to unmodified proteins. • Orally fed DARPin tagged-lactoferrin fragment (drtHLF4) is readily absorbed by the host via the gastrointestinal tract. [ABSTRACT FROM AUTHOR]

We report that CD47 was upregulated in different EMT-activated human breast cancer cells versus epithelial MCF7 cells. Overexpression of SNAI1 or ZEB1 in epithelial MCF7 cells activated EMT and upregulated CD47 while siRNA-mediated targeting of SNAI1 or ZEB1 in mesenchymal MDA-MB-231 cells reversed EMT and strongly decreased CD47. Mechanistically, SNAI1 and ZEB1 upregulated CD47 by binding directly to E-boxes in the human CD47 promoter. TCGA and METABRIC data sets from breast cancer patients revealed that CD47 correlated with SNAI1 and Vimentin. At functional level, different EMT-activated breast cancer cells were less efficiently phagocytosed by macrophages vs. MCF7 cells. The phagocytosis of EMT-activated cells was rescued by using CD47 blocking antibody or by genetic targeting of SNAI1, ZEB1 or CD47. These results provide a rationale for an innovative preclinical combination immunotherapy based on PD-1/PD-L1 and CD47 blockade along with EMT inhibitors in patients with highly aggressive, mesenchymal, and metastatic breast cancer. [ABSTRACT FROM AUTHOR]