The enumeration of EpCAM-positive circulating tumor cells (CTCs) has allowed estimation of overall metastatic burden in breast cancer patients. However, a thorough understanding of CTCs associated with breast cancer brain metastasis (BCBM) is necessary for early identification and evaluation of treatment response to BCBM. Here we report that BCBM CTCs is enriched in a distinct sub-population of cells identifiable by their biomarker expression and mutational content. Deriving from a comprehensive analysis of CTC transcriptomes, we discovered a unique “circulating tumor cell gene signature” that is distinct from primary breast cancer tissues. Further dissection of the circulating tumor cell gene signature identified signaling pathways associated with BCBM CTCs that may have roles in potentiating BCBM. This study proposes CTC biomarkers and signaling pathways implicated in BCBM that may be used either as a screening tool for brain micro-metastasis detection or for making rational treatment decisions and monitoring therapeutic response in patients with BCBM.

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.

Uncovering CTCs phenotypes offer the promise to dissect their heterogeneity related to metastatic competence. CTC survival rates are highly variable and this can lead to many questions as yet unexplored properties of CTCs responsible for invasion and metastasis vs dormancy. We isolated CTC subsets from peripheral blood of patients diagnosed with or without breast cancer brain metastasis. CTC subsets were selected for EpCAM negativity but positivity for CD44+/CD24− stem cell signature; along with combinatorial expression of uPAR and int β1, two markers directly implicated in breast cancer dormancy mechanisms. CTC subsets were cultured in vitro generating 3D CTC tumorspheres which were interrogated for biomarker profiling and biological characteristics. We identified proliferative and invasive properties of 3D CTC tumorspheres distinctive upon uPAR/int β1 combinatorial expression. The molecular characterization of uPAR/int β1 CTC subsets may enhance abilities to prospectively identify patients who may be at high risk of developing BCBM.

Mature mammalian sperm contain a complex population of RNAs some of which might regulate spermatogenesis while others probably play a role in fertilization and early development. Due to this limited knowledge, the biological functions of sperm RNAs remain enigmatic. Here we report the first characterization of the global transcriptome of the sperm of fertile stallions. The findings improved understanding of the biological significance of sperm RNAs which in turn will allow the discovery of sperm-based biomarkers for stallion fertility. The stallion sperm transcriptome was interrogated by analyzing sperm and testes RNA on a 21,000-element equine whole-genome oligoarray and by RNA-seq. Microarray analysis revealed 6,761 transcripts in the sperm, of which 165 were sperm-enriched, and 155 were differentially expressed between the sperm and testes. Next, 70 million raw reads were generated by RNA-seq of which 50% could be aligned with the horse reference genome. A total of 19,257 sequence tags were mapped to all horse chromosomes and the mitochondrial genome. The highest density of mapped transcripts was in gene-rich ECA11, 12 and 13, and the lowest in gene-poor ECA9 and X; 7 gene transcripts originated from ECAY. Structural annotation aligned sperm transcripts with 4,504 known horse and/or human genes, rRNAs and 82 miRNAs, whereas 13,354 sequence tags remained anonymous. The data were aligned with selected equine gene models to identify additional exons and splice variants. Gene Ontology annotations showed that sperm transcripts were associated with molecular processes (chemoattractant-activated signal transduction, ion transport) and cellular components (membranes and vesicles) related to known sperm functions at fertilization, while some messenger and micro RNAs might be critical for early development. The findings suggest that the rich repertoire of coding and non-coding RNAs in stallion sperm is not a random remnant from spermatogenesis in testes but a selectively retained and functionally coherent collection of RNAs.

Impaired acrosomal reaction (IAR) of sperm causes male subfertility in humans and animals. Despite compelling evidence about the genetic control over acrosome biogenesis and function, the genomics of IAR is as yet poorly understood, providing no molecular tools for diagnostics. Here we conducted Equine SNP50 Beadchip genotyping and GWAS using 7 IAR–affected and 37 control Thoroughbred stallions. A significant (P<6.75E-08) genotype–phenotype association was found in horse chromosome 13 in FK506 binding protein 6 (FKBP6). The gene belongs to the immunophilins FKBP family known to be involved in meiosis, calcium homeostasis, clathrin-coated vesicles, and membrane fusions. Direct sequencing of FKBP6 exons in cases and controls identified SNPs g.11040315G>A and g.11040379C>A (p.166H>N) in exon 4 that were significantly associated with the IAR phenotype both in the GWAS cohort (n = 44) and in a large multi-breed cohort of 265 horses. All IAR stallions were homozygous for the A-alleles, while this genotype was found only in 2% of controls. The equine FKBP6 was exclusively expressed in testis and sperm and had 5 different transcripts, of which 4 were novel. The expression of this gene in AC/AG heterozygous controls was monoallelic, and we observed a tendency for FKBP6 up-regulation in IAR stallions compared to controls. Because exon 4 SNPs had no effect on the protein structure, it is likely that FKBP6 relates to the IAR phenotype via regulatory or modifying functions. In conclusion, FKBP6 was considered a susceptibility gene of incomplete penetrance for IAR in stallions and a candidate gene for male subfertility in mammals. FKBP6 genotyping is recommended for the detection of IAR–susceptible individuals among potential breeding stallions. Successful use of sperm as a source of DNA and RNA propagates non-invasive sample procurement for fertility genomics in animals and humans.

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Systemic metastasis is the major cause of death from melanoma, the most lethal form of skin cancer. Although most patients with melanoma exhibit a substantial gap between onset of primary and metastatic tumors, signaling mechanisms implicated in the period of metastatic latency remain unclear. We hypothesized that melanoma circulating tumor cells (CTC) home to and reside in the bone marrow during the asymptomatic phase of disease progression. Using a strategy to deplete normal cell lineages (Lin), we isolated CTC-enriched cell populations from the blood of patients with metastatic melanoma, verified by the presence of putative CTCs characterized by melanoma-specific biomarkers and upregulated gene transcripts involved in cell survival and prodevelopment functions. Implantation of Lin population in NSG mice (CTC-derived xenografts, i.e., CDX), and subsequent transcriptomic analysis of bone marrow-resident tumor cells (BMRTC) versus CTC identified protein ubiquitination as a significant regulatory pathway of BMRTC signaling. Selective inhibition of USP7, a key deubiquinating enzyme, arrested BMRTCs in bone marrow locales and decreased systemic micrometastasis. This study provides first-time evidence that the asymptomatic progression of metastatic melanoma can be recapitulated using patient-isolated CTCs. Furthermore, these results suggest that USP7 inhibitors warrant further investigation as a strategy to prevent progression to overt clinical metastasis. These findings provide insights into mechanism of melanoma recurrence and propose a novel approach to inhibit systematic metastatic disease by targeting bone marrow-resident tumor cells through pharmacological inhibition of USP7. http://cancerres.aacrjournals.org/content/canres/78/18/5349/F1.large.jpg .

SummarySporulation initiation in Bacillus subtilis is controlled by the phosphorylated form of the master regulator Spo0A which controls transcription of a multitude of sporulation genes. In this study, we investigated the importance of temporal dynamics of phosphorylated Spo0A (Spo0A∼P) accumulation by rewiring the network controlling its phosphorylation. We showed that simultaneous induction of KinC, a kinase that can directly phosphorylate Spo0A, and Spo0A itself from separately controlled inducible promoters can efficiently trigger sporulation even under nutrient rich conditions. However, the sporulation efficiency in this artificial two-component system was significantly impaired when KinC and/or Spo0A induction was too high. Using mathematical modelling, we showed that gradual accumulation of Spo0A∼P is essential for the proper temporal order of the Spo0A regulon expression, and that reduction in sporulation efficiency results from the reversal of that order. These insights led us to identify premature repression of DivIVA as one possible explanation for the adverse effects of accelerated accumulation of Spo0A∼P on sporulation. Moreover, we found that positive feedback resulting from autoregulation of the native spo0A promoter leads to robust control of Spo0A∼P accumulation kinetics. Thus we propose that a major function of the conserved architecture of the sporulation network is controlling Spo0A activation dynamics.