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Abstract Recent advances in spatial transcriptomics (ST) techniques provide valuable insights into cellular interactions within the tumor microenvironment (TME). However, most analytical tools lack consideration of histological features and rely on matched single-cell RNA sequencing data, limiting their effectiveness in TME studies. To address this, we introduce the Morphology-Enhanced Spatial Transcriptome Analysis Integrator (METI), an end-to-end framework that maps cancer cells and TME components, stratifies cell types and states, and analyzes cell co-localization. By integrating spatial transcriptomics, cell morphology, and curated gene signatures, METI enhances our understanding of the molecular landscape and cellular interactions within the tissue. We evaluate the performance of METI on ST data generated from various tumor tissues, including gastric, lung, and bladder cancers, as well as premalignant tissues. We also conduct a quantitative comparison of METI with existing clustering and cell deconvolution tools, demonstrating METI’s robust and consistent performance.

Summary: Orofacial clefts (OFCs) are among the most common human congenital birth defects. Previous multiethnic studies have identified dozens of associated loci for both cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP). Although several nearby genes have been highlighted, the “casual” variants are largely unknown. Here, we developed DeepFace, a convolutional neural network model, to assess the functional impact of variants by SNP activity difference (SAD) scores. The DeepFace model is trained with 204 epigenomic assays from crucial human embryonic craniofacial developmental stages of post-conception week (pcw) 4 to pcw 10. The Pearson correlation coefficient between the predicted and actual values for 12 epigenetic features achieved a median range of 0.50–0.83. Specifically, our model revealed that SNPs significantly associated with OFCs tended to exhibit higher SAD scores across various variant categories compared to less related groups, indicating a context-specific impact of OFC-related SNPs. Notably, we identified six SNPs with a significant linear relationship to SAD scores throughout developmental progression, suggesting that these SNPs could play a temporal regulatory role. Furthermore, our cell-type specificity analysis pinpointed the trophoblast cell as having the highest enrichment of risk signals associated with OFCs. Overall, DeepFace can harness distal regulatory signals from extensive epigenomic assays, offering new perspectives for prioritizing OFC variants using contextualized functional genomic features. We expect DeepFace to be instrumental in accessing and predicting the regulatory roles of variants associated with OFCs, and the model can be extended to study other complex diseases or traits.

Abstract Perturbations in gene regulation during palatogenesis can lead to cleft palate, which is among the most common congenital birth defects. Here, we perform single-cell multiome sequencing and profile chromatin accessibility and gene expression simultaneously within the same cells (n = 36,154) isolated from mouse secondary palate across embryonic days (E) 12.5, E13.5, E14.0, and E14.5. We construct five trajectories representing continuous differentiation of cranial neural crest-derived multipotent cells into distinct lineages. By linking open chromatin signals to gene expression changes, we characterize the underlying lineage-determining transcription factors. In silico perturbation analysis identifies transcription factors SHOX2 and MEOX2 as important regulators of the development of the anterior and posterior palate, respectively. In conclusion, our study charts epigenetic and transcriptional dynamics in palatogenesis, serving as a valuable resource for further cleft palate research.

Longstanding evidence implicate glioma stem-like cells as the main drivers contributing toward glioblastoma (GBM) therapy resistance and tumor recurrence. Although oncolytic herpes simplex virus (oHSV) viral therapy is a promising biological therapy recently approved for melanoma (in the United States and Europe) and GBM (in Japan); however, the impact of this therapy on GBM stem-like cells (GSCs) is understudied. Here we show that post-oHSV virotherapy activated AKT signaling results in an enrichment of GSC signatures in glioma, which mimics the enrichment in GSC observed after radiation treatment. We also uncovered that a second-generation oncolytic virus armed with PTEN-L (oHSV-P10) decreases this by moderating IL6/JAK/STAT3 signaling. This ability was retained in the presence of radiation treatment and oHSV-P10-sensitized intracranial GBM to radiotherapy. Collectively, our findings uncover potential mechanisms to overcome GSC-mediated radiation resistance via oHSV-P10.

The incidence of bladder cancer and patient survival vary greatly among different populations, but the influence of the associated molecular features and evolutionary processes on its clinical treatment and prognostication remains unknown. Here, we analyze the genomic architectures of 505 bladder cancer patients from Asian/Black/White populations. We identify a previously unknown association between AHNAK mutations and activity of the APOBEC‐a mutational signature, the activity of which varied substantially across populations. All significantly mutated genes but only half of arm‐level somatic copy number alterations (SCNAs) are enriched with clonal events, indicating large‐scale SCNAs as rich sources of bladder cancer clonal diversities. The prevalence of TP53 and ATM clonal mutations as well as the associated burden of SCNAs is significantly higher in Whites/Blacks than in Asians. We identify a trans‐ancestry prognostic subtype of bladder cancer characterized by enrichment of non‐muscle‐invasive patients and muscle‐invasive patients with good prognosis, increased CREBBP/FGFR3/HRAS/NFE2L2 mutations, decreased intra‐tumor heterogeneity and genome instability, and an activated tumor microenvironment.

Background Mammalian cells have developed multiple intracellular mechanisms to defend against viral infections. These include RNA-activated protein kinase (PKR), cyclic GMP-AMP synthase and stimulation of interferon genes (cGAS-STING) and toll-like receptor-myeloid differentiation primary response 88 (TLR-MyD88). Among these, we identified that PKR presents the most formidable barrier to oncolytic herpes simplex virus (oHSV) replication in vitro.Methods To elucidate the impact of PKR on host responses to oncolytic therapy, we generated a novel oncolytic virus (oHSV-shPKR) which disables tumor intrinsic PKR signaling in infected tumor cells.Results As anticipated, oHSV-shPKR resulted in suppression of innate antiviral immunity and improves virus spread and tumor cell lysis both in vitro and in vivo. Single cell RNA sequencing combined with cell-cell communication analysis uncovered a strong correlation between PKR activation and transforming growth factor beta (TGF-ß) immune suppressive signaling in both human and preclinical models. Using a murine PKR targeting oHSV, we found that in immune-competent mice this virus could rewire the tumor immune microenvironment to increase the activation of antigen presentation and enhance tumor antigen-specific CD8 T cell expansion and activity. Further, a single intratumoral injection of oHSV-shPKR significantly improved the survival of mice bearing orthotopic glioblastoma. To our knowledge, this is the first report to identify dual and opposing roles of PKR wherein PKR activates antivirus innate immunity and induces TGF-ß signaling to inhibit antitumor adaptive immune responses.Conclusions Thus, PKR represents the Achilles heel of oHSV therapy, restricting both viral replication and antitumor immunity, and an oncolytic virus that can target this pathway significantly improves response to virotherapy.

The oncolytic effect of virotherapy derives from the intrinsic capability of the applied virus in selectively infecting and killing tumor cells. Although oncolytic viruses of various constructions have been shown to efficiently infect and kill tumor cells in vitro, the efficiency of these viruses to exert the same effect on tumor cells within tumor tissues in vivo has not been extensively investigated. Here we report our studies using single-cell RNA sequencing to comprehensively analyze the gene expression profile of tumor tissues following herpes simplex virus 2-based oncolytic virotherapy. Our data revealed the extent and cell types within the tumor microenvironment that could be infected by the virus. Moreover, we observed changes in the expression of cellular genes, including antiviral genes, in response to viral infection. One notable gene found to be upregulated significantly in oncolytic virus-infected tumor cells was Gadd45g, which is desirable for optimal virus replication. These results not only help reveal the precise infection status of the oncolytic virus in vivo but also provide insight that may lead to the development of new strategies to further enhance the therapeutic efficacy of oncolytic virotherapy.

Understanding the molecular mechanisms of coronavirus disease 2019 (COVID-19) pathogenesis and immune response is vital for developing therapies. Single-cell RNA sequencing has been applied to delineate the cellular heterogeneity of the host response toward COVID-19 in multiple tissues and organs. Here, we review the applications and findings from over 80 original COVID-19 single-cell RNA sequencing studies as well as many secondary analysis studies. We describe that single-cell RNA sequencing reveals multiple features of COVID-19 patients with different severity, including cell populations with proportional alteration, COVID-19-induced genes and pathways, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in single cells, and adaptation of immune repertoire. We also collect published single-cell RNA sequencing datasets from original studies. Finally, we discuss the limitations in current studies and perspectives for future advance. Public summary: • Single-cell RNA sequencing is a powerful biotechnology in COVID-19 research • It reveals multiple features of SARS-CoV-2 pathogenesis and host immune response • Single-cell immune profiling characterizes the adaptive immune response in COVID-19 • Analysis of B cell receptors accelerates the neutralizing antibody identification

Drug response in cancer patients vary dramatically due to inter- and intra-tumor heterogeneity and transcriptome context plays a significant role in shaping the actual treatment outcome. Here, the authors develop a deep variational autoencoder model to compress gene signatures into latent vectors and accurately impute drug response.

Abstract Background Psychiatric disorders such as schizophrenia (SCZ), bipolar disorder (BIP), major depressive disorder (MDD), attention deficit-hyperactivity disorder (ADHD), and autism spectrum disorder (ASD) are often related to brain development. Both shared and unique biological and neurodevelopmental processes have been reported to be involved in these disorders. Methods In this work, we developed an integrative analysis framework to seek for the sensitive spatiotemporal point during brain development underlying each disorder. Specifically, we first identified spatiotemporal gene co-expression modules for four brain regions three developmental stages (prenatal, birth to 11 years old, and older than 13 years), totaling 12 spatiotemporal sites. By integrating GWAS summary statistics and the spatiotemporal co-expression modules, we characterized the risk genes and their co-expression partners for five disorders. Results We found that SCZ and BIP, ASD and ADHD tend to cluster with each other and keep a distance from other psychiatric disorders. At the gene level, we identified several genes that were shared among the most significant modules, such as CTNNB1 and LNX1, and a hub gene, ATF2, in multiple modules. Moreover, we pinpointed two spatiotemporal points in the prenatal stage with active expression activities and highlighted one postnatal point for BIP. Further functional analysis of the disorder-related module highlighted the apoptotic signaling pathway for ASD and the immune-related and cell-cell adhesion function for SCZ, respectively. Conclusion Our study demonstrated the dynamic changes of disorder-related genes at the network level, shedding light on the spatiotemporal regulation during brain development.

Abstract Background Docosahexaenoic acid (DHA) is essential for human diet. However, high production cost of DHA using C. cohnii makes it currently less competitive commercially, which is mainly caused by low DHA productivity. In recent years, repeated fed-batch strategies have been evaluated for increasing the production of many fermentation products. The reduction in terms of stability of culture system was one of the major challenges for repeated fed-batch fermentation. However, the possible mechanisms responsible for the decreased stability of the culture system in the repeated fed-batch fermentation are so far less investigated, restricting the efforts to further improve the productivity. In this study, a repeated fed-batch strategy for DHA production using C. cohnii M-1-2 was evaluated to improve DHA productivity and reduce production cost, and then the underlying mechanisms related to the gradually decreased stability of the culture system in repeated fed-batch culture were explored through LC– and GC–MS metabolomic analyses. Results It was discovered that glucose concentration at 15–27 g/L and 80% medium replacement ratio were suitable for the growth of C. cohnii M-1-2 during the repeated fed-batch culture. A four-cycle repeated fed-batch culture was successfully developed and assessed at the optimum cultivation parameters, resulting in increasing the total DHA productivity by 26.28% compared with the highest DHA productivity of 57.08 mg/L/h reported using C. cohnii, including the time required for preparing seed culture and fermentor. In addition, LC– and GC–MS metabolomics analyses showed that the gradually decreased nitrogen utilization capacity, and down-regulated glycolysis and TCA cycle were correlated with the decreased stability of the culture system during the long-time repeated fed-batch culture. At last, some biomarkers, such as Pyr, Cit, OXA, FUM, l-tryptophan, l-threonine, l-leucine, serotonin, and 4-guanidinobutyric acid, correlated with the stability of culture system of C. cohnii M-1-2 were identified. Conclusions The study proved that repeated fed-batch cultivation was an efficient and energy-saving strategy for industrial production of DHA using C. cohnii, which could also be useful for cultivation of other microbes to improve productivity and reduce production cost. In addition, the mechanisms study at metabolite level can also be useful to further optimize production processes for C. cohnii and other microbes.

Background and Objective: Corticosteroid resistance is a major barrier to chronic obstructive pulmonary disease (COPD), but the exact mechanism of corticosteroid resistance in COPD has been less well studied.Methods: The microarray dataset GSE11906, which includes genomic and clinical data on COPD, was downloaded from the Gene Expression Omnibus (GEO) database, and the differentially expressed genes (DEGs) were identified using R software. Gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes (KEGG) were utilized to enrich and analyze the gene cohort related to the response to steroid hormones, respectively. The Connectivity Map (CMap) database was used to screen corticosteroid resistance-related drugs that might exert a potential therapeutic effect. STRING was used to construct a protein-protein interaction (PPI) network of the gene cohort, and the CytoHubba plug-in of Cytoscape was used to screen the hub genes in the PPI network. The expression levels of hub genes in cigarette smoke extract (CSE)-stimulated bronchial epithelial cells were assayed by quantitative real-time PCR and western blotting.Results: Twenty-one genes were found to be correlated with the response to steroid hormones. In the CMap database, 32 small-molecule compounds that might exert a therapeutic effect on corticosteroid resistance in COPD were identified. Nine hub genes were extracted from the PPI network. The expression levels of the BMP4, FOS, FN1, EGFR, and SPP1 proteins were consistent with the microarray data obtained from molecular biology experiments. Scopoletin significantly restrained the increases in the levels of AKR1C3, ALDH3A1, FN1 and reversed the decreases of phosphorylated GR and HDAC2 caused by CSE exposure.Conclusion: The BMP4, FOS, FN1, EGFR, and SPP1 genes are closely correlated with CSE-induced glucocorticoid resistance in airway epithelial cells. Scopoletin may be a potential drug for the treatment of glucocorticoid resistance caused by CSE.

Background Although oncolytic virotherapy has shown substantial promises as a new treatment modality for many malignancies, further improvement on its therapeutic efficacy will likely bring more clinical benefits. One plausible way of enhancing the therapeutic effect of virotherapy is to enable it with the ability to concurrently engage the infiltrating immune cells to provide additional antitumor mechanisms. Here, we report the construction and evaluation of two novel chimeric molecules (bispecific chimeric engager proteins, BiCEP and trispecific chimeric engager protein, TriCEP) that can engage both natural killer (NK) and T cells with tumor cells for enhanced antitumor activities.Methods BiCEP was constructed by linking orthopoxvirus major histocompatibility complex class I-like protein, which can selectively bind to NKG2D with a high affinity to a mutant form of epidermal growth factor (EGF) that can strongly bind to EGF receptor. TriCEP is similarly constructed except that it also contains a modified form of interleukin-2 that can only function as a tethered form. As NKG2D is expressed on both NK and CD8+ T cells, both of which can thus be engaged by BiCEP and TriCEP.Results Both BiCEP and TriCEP showed the ability to engage NK and T cells to kill tumor cells in vitro. Coadministration of BiCEP and TriCEP with an oncolytic herpes simplex virus enhanced the overall antitumor effect. Furthermore, single-cell RNA sequencing analysis revealed that TriCEP not only engaged NK and T cells to kill tumor cells, it also promotes the infiltration and activation of these important immune cells.Conclusions These novel chimeric molecules exploit the ability of the oncolytic virotherapy in altering the tumor microenvironment with increased infiltration of important immune cells such as NK and T cells for cancer immunotherapy. The ability of BiCEP and TriCEP to engage both NK and T cells makes them an ideal choice for arming an oncolytic virotherapy.

Clear cell renal cell carcinoma (ccRCC) is a urogenital cancer with a well-defined genetic landscape. Here, the authors analyse the clonal architecture of ccRCC patients from three populations, and find prognostic subtypes linked to immune infiltrates and clonal architecture.

Abstract Background Genome-wide association studies (GWAS) have been successful in identifying disease-associated genetic variants. Recently, an increasing number of GWAS summary statistics have been made available to the research community, providing extensive repositories for studies of human complex diseases. In particular, cross-trait associations at the genetic level can be beneficial from large-scale GWAS summary statistics by using genetic variants that are associated with multiple traits. However, direct assessment of cross-trait associations using susceptibility loci has been challenging due to the complex genetic architectures in most diseases, calling for advantageous methods that could integrate functional interpretation and imply biological mechanisms. Results We developed an analytical framework for systematic integration of cross-trait associations. It incorporates two different approaches to detect enriched pathways and requires only summary statistics. We demonstrated the framework using 25 traits belonging to four phenotype groups. Our results revealed an average of 54 significantly associated pathways (ranged between 18 and 175) per trait. We further proved that pathway-based analysis provided increased power to estimate cross-trait associations compared to gene-level analysis. Based on Fisher’s Exact Test (FET), we identified a total of 24 (53) pairs of trait-trait association at adjusted p FET

Summary: 53BP1 plays a central role in dictating DNA repair choice between non-homologous end joining (NHEJ) and homologous recombination (HR), which is important for the sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPis) of BRCA1-deficient cancers. In this study, we show that FOXK1 associates with 53BP1 and regulates 53BP1-dependent functions. FOXK1-53BP1 interaction is significantly enhanced upon DNA damage during the S phase in an ATM/CHK2-dependent manner, which reduces the association of 53BP1 with its downstream factors RIF1 and PTIP. Depletion of FOXK1 impairs DNA repair and induces compromised cell survival upon DNA damage. Overexpression of FOXK1 diminishes 53BP1 foci formation, which leads to resistance to PARPis and elevation of HR in BRCA1-deficient cells and decreased telomere fusion in TRF2-depleted cells. Collectively, our findings demonstrate that FOXK1 negatively regulates 53BP1 function by inhibiting 53BP1 localization to sites of DNA damage, which alters the DSB-induced protein complexes centering on 53BP1 and thus influences DNA repair choice.

Abstract Background Photosynthetic cyanobacteria have attracted a significant attention as promising chassis to produce renewable fuels and chemicals due to their capability to utilizing solar energy and CO2. Notably, the enhancing supply of key precursors like malonyl-CoA would benefit the production of many bio-compounds. Nevertheless, the lacking of genetic tools in cyanobacteria, especially the knockdown strategies for essential pathways, has seriously restricted the attempts to re-direct carbon flux from the central carbohydrate metabolism to the synthesis of bioproducts. Results Aiming at developing new genetic tools, two small RNA regulatory tools are reported for the model cyanobacterium Synechocystis sp. PCC6803, based on paired termini RNAs as well as the exogenous Hfq chaperone and MicC scaffold (Hfq-MicC) previously developed in Escherichia coli. Both regulatory tools functioned well in regulating exogenous reporter gene lacZ and endogenous glgC gene in Synechocystis sp. PCC6803, achieving a downregulation of gene expression up to 90% compared with wildtype. In addition, the Hfq-MicC tool was developed to simultaneously regulate multiple genes related to essential fatty acids biosynthesis, which led to decreased fatty acids content by 11%. Furthermore, aiming to re-direct the carbon flux, the Hfq-MicC tool was utilized to interfere the competing pathway of malonyl-CoA, achieving an increased intracellular malonyl-CoA abundance up to 41% (~ 698.3 pg/mL/OD730 nm) compared to the wildtype. Finally, the Hfq-MicC system was further modified into an inducible system based on the theophylline-inducible riboswitch. Conclusions In this study, two small RNA regulatory tools for manipulating essential metabolic pathways and re-directing carbon flux are reported for Synechocystis sp. PCC6803. The work introduces efficient and valuable metabolic regulatory strategies for photosynthetic cyanobacteria.

One contributor’s name was missing in the original version of the authorship of the paper [...]

Although cisplatin is very effective as a treatment strategy in triple-negative breast cancer (TNBC), it has unwarranted outcomes owing to recurrence, chemoresistance and neurotoxicity. There is critically important to find new, effective and safe therapeutics for TNBC. We determined if SP-receptor antagonism in combination with cisplatin may serve as a novel, more efficacious and safer therapeutic option than existing therapies for TNBC. We used a neuronal cell line (PC12) and two TNBC cell lines (Sum 185 and Sum 159) for these studies. We determined that the levels of cells expressing the high-affinity SP-receptor (neurokinin 1 receptor (NK1R)), as determined by flow-cytometry was significantly elevated in response to cisplatin in all three cells. We determined that treatment with aprepitant, an SP-receptor antagonist decreased cisplatin-induced, loss of viability (studied by MTT assay), production of reactive oxygen species (by DCFDA assay) and apoptosis (by flow-cytometry) in PC12 cells while it was increased in the two TNBC cells. Furthermore, we demonstrated that important genes associated with metastases, inflammation, chemoresistance and cell cycle progression are attenuated by SP-receptor antagonism in the TNBC cell line, Sum 185. These studies implicate that SP-receptor antagonism in combination with cisplatin may possibly serve as a novel, more efficacious and safer therapeutic option than existing therapies for TNBC.

Crohn’s Disease (CD) is one of the predominant forms of inflammatory bowel disease (IBD). A combination of genetic and non-genetic risk factors have been reported to contribute to the development of CD. Many high-throughput omics studies have been conducted to identify disease associated risk variants that might contribute to CD, such as genome-wide association studies (GWAS) and next generation sequencing studies. A pressing need remains to prioritize and characterize candidate genes that underlie the etiology of CD. In this study, we collected a comprehensive multi-dimensional data from GWAS, gene expression, and methylation studies and generated transcriptome-wide association study (TWAS) data to further interpret the GWAS association results. We applied our previously developed method called mega-analysis of Odds Ratio (MegaOR) to prioritize CD candidate genes (CDgenes). As a result, we identified consensus sets of CDgenes (62–235 genes) based on the evidence matrix. We demonstrated that these CDgenes were significantly more frequently interact with each other than randomly expected. Functional annotation of these genes highlighted critical immune-related processes such as immune response, MHC class II receptor activity, and immunological disorders. In particular, the constitutive photomorphogenesis 9 (COP9) signalosome related genes were found to be significantly enriched in CDgenes, implying a potential role of COP9 signalosome involved in the pathogenesis of CD. Finally, we found some of the CDgenes shared biological functions with known drug targets of CD, such as the regulation of inflammatory response and the leukocyte adhesion to vascular endothelial cell. In summary, we identified highly confident CDgenes from multi-dimensional evidence, providing insights for the understanding of CD etiology.

Microbial small RNAs (sRNAs) play essential roles against many stress conditions in cyanobacteria. However, little is known on their regulatory mechanisms on biofuels tolerance. In our previous sRNA analysis, a trans-encoded sRNA Nc117 was found involved in the tolerance to ethanol and 1-butanol in Synechocystis sp. PCC 6803. However, its functional mechanism is yet to be determined. In this study, functional characterization of sRNA Nc117 was performed. Briefly, the exact length of the trans-encoded sRNA Nc117 was determined to be 102 nucleotides using 3′ RACE, and the positive regulation of Nc117 on short chain alcohols tolerance was further confirmed. Then, computational target prediction and transcriptomic analysis were integrated to explore the potential targets of Nc117. A total of 119 up-regulated and 116 down-regulated genes were identified in nc117 overexpression strain compared with the wild type by comparative transcriptomic analysis, among which the upstream regions of five genes were overlapped with those predicted by computational target approach. Based on the phenotype analysis of gene deletion and overexpression strains under short chain alcohols stress, one gene slr0007 encoding D-glycero-alpha-D-manno-heptose 1-phosphate guanylyltransferase was determined as a potential target of Nc117, suggesting that the synthesis of LPS or S-layer glycoprotein may be responsible for the tolerance enhancement. As the first reported trans-encoded sRNA positively regulating biofuels tolerance in cyanobacteria, this study not only provided evidence for a new regulatory mechanism of trans-encoded sRNA in cyanobacteria, but also valuable information for rational construction of high-tolerant cyanobacterial chassis.

Intratumoral infiltration of myeloid-derived suppressor cells (MDSCs) is known to promote neoplastic growth by inhibiting the tumoricidal activity of T cells. However, direct interactions between patient-derived MDSCs and circulating tumors cells (CTCs) within the microenvironment of blood remain unexplored. Dissecting interplays between CTCs and circulatory MDSCs by heterotypic CTC/MDSC clustering is critical as a key mechanism to promote CTC survival and sustain the metastatic process. We characterized CTCs and polymorphonuclear-MDSCs (PMN-MDSCs) isolated in parallel from peripheral blood of metastatic melanoma and breast cancer patients by multi-parametric flow cytometry. Transplantation of both cell populations in the systemic circulation of mice revealed significantly enhanced dissemination and metastasis in mice co-injected with CTCs and PMN-MDSCs compared to mice injected with CTCs or MDSCs alone. Notably, CTC/PMN-MDSC clusters were detected in vitro and in vivo either in patients’ blood or by longitudinal monitoring of blood from animals. This was coupled with in vitro co-culturing of cell populations, demonstrating that CTCs formed physical clusters with PMN-MDSCs; and induced their pro-tumorigenic differentiation through paracrine Nodal signaling, augmenting the production of reactive oxygen species (ROS) by PMN-MDSCs. These findings were validated by detecting significantly higher Nodal and ROS levels in blood of cancer patients in the presence of naïve, heterotypic CTC/PMN-MDSC clusters. Augmented PMN-MDSC ROS upregulated Notch1 receptor expression in CTCs through the ROS-NRF2-ARE axis, thus priming CTCs to respond to ligand-mediated (Jagged1) Notch activation. Jagged1-expressing PMN-MDSCs contributed to enhanced Notch activation in CTCs by engagement of Notch1 receptor. The reciprocity of CTC/PMN-MDSC bi-directional paracrine interactions and signaling was functionally validated in inhibitor-based analyses, demonstrating that combined Nodal and ROS inhibition abrogated CTC/PMN-MDSC interactions and led to a reduction of CTC survival and proliferation. This study provides seminal evidence showing that PMN-MDSCs, additive to their immuno-suppressive roles, directly interact with CTCs and promote their dissemination and metastatic potency. Targeting CTC/PMN-MDSC heterotypic clusters and associated crosstalks can therefore represent a novel therapeutic avenue for limiting hematogenous spread of metastatic disease.