Microfluidic isolation of breast cancer circulating tumor cells from microvolumes of mouse blood.

Liquid biopsy has shown significant research and clinical implications in cancer. Particularly, the isolation of circulating tumor cells (CTCs) in preclinical studies can provide crucial information about disease progression and therefore may guide treatment decisions. Microfluidic isolation systems have played a considerable role in CTC isolation for cancer studies, disease diagnosis, and prognosis. CTCs are often studied using preclinical animal models such as xenografts or syngeneic models. However, most isolation systems are tested on human cell lines and human blood, whereas less validation studies are done on preclinical samples such as CTCs from mouse models. Here, we demonstrate and evaluate a complete workflow of a sized-based inertial microfluidic device to isolate CTCs from blood using exclusively mouse blood and mouse cancer cell lines. We then incorporate the cytospin, a commonly used method for enumeration of small number of cells in a glass slide to quantify the total cell yield of our workflow.

Cell-Autonomous versus Systemic Akt Isoform Deletions Uncovered New Roles for Akt1 and Akt2 in Breast Cancer.

Studies in three mouse models of breast cancer identified profound discrepancies between cell-autonomous and systemic Akt1- or Akt2-inducible deletion on breast cancer tumorigenesis and metastasis. Although systemic Akt1 deletion inhibits metastasis, cell-autonomous Akt1 deletion does not. Single-cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing survival and mobilization of tumor-associated neutrophils. Importantly, either systemic or neutrophil-specific Akt1 deletion is sufficient to inhibit metastasis of Akt-proficient tumors. Thus, Akt1-specific inhibition could be therapeutic for breast cancer metastasis regardless of primary tumor origin. Systemic Akt2 deletion does not inhibit and exacerbates mammary tumorigenesis and metastasis, but cell-autonomous Akt2 deletion prevents breast cancer tumorigenesis by ErbB2. Elevated circulating insulin level induced by Akt2 systemic deletion hyperactivates tumor Akt, exacerbating ErbB2-mediated tumorigenesis, curbed by pharmacological reduction of the elevated insulin.

6mer seed toxicity in tumor suppressive microRNAs.

Many small-interfering (si)RNAs are toxic to cancer cells through a 6mer seed sequence (positions 2-7 of the guide strand). Here we performed an siRNA screen with all 4096 6mer seeds revealing a preference for guanine in positions 1 and 2 and a high overall G or C content in the seed of the most toxic siRNAs for four tested human and mouse cell lines. Toxicity of these siRNAs stems from targeting survival genes with C-rich 3'UTRs. The master tumor suppressor miRNA miR-34a-5p is toxic through such a G-rich 6mer seed and is upregulated in cells subjected to genotoxic stress. An analysis of all mature miRNAs suggests that during evolution most miRNAs evolved to avoid guanine at the 5' end of the 6mer seed sequence of the guide strand. In contrast, for certain tumor-suppressive miRNAs the guide strand contains a G-rich toxic 6mer seed, presumably to eliminate cancer cells.

Small interfering RNAs based on huntingtin trinucleotide repeats are highly toxic to cancer cells.

Trinucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington's disease (HD). Pathology is caused by protein and RNA generated from the TNR regions including small siRNA-sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR are toxic to cancer cells by targeting genes that contain long reverse complementary TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the six members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG TNR-based siRNAs induce cell death in vitro in all tested cancer cell lines and slow down tumor growth in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR-based siRNAs as a novel form of anticancer reagents.

DISE: A Seed-Dependent RNAi Off-Target Effect That Kills Cancer Cells.

Off-target effects (OTEs) represent a significant caveat for RNAi caused by substantial complementarity between siRNAs and unintended mRNAs. We now discuss the existence of three types of seed-dependent OTEs (sOTEs). Type I involves unintended targeting through the guide strand seed of an siRNA. Type II is caused by the activity of the seed on the designated siRNA passenger strand when loaded into the RNA-induced silencing complex (RISC). Both type I and II sOTEs will elicit unpredictable cellular responses. By contrast, in sOTE type III the guide strand seed preferentially targets essential survival genes resulting in death induced by survival gene elimination (DISE). In this Opinion article, we discuss DISE as a consequence of RNAi that may preferentially affect cancer cells.

Many si/shRNAs can kill cancer cells by targeting multiple survival genes through an off-target mechanism.

Over 80% of multiple-tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3'UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer-deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that specific toxic RNAi-active sequences present in the genome can kill cancer cells.

CD95/Fas Increases Stemness in Cancer Cells by Inducing a STAT1-Dependent Type I Interferon Response.

Stimulation of CD95/Fas drives and maintains cancer stem cells (CSCs). We now report that this involves activation of signal transducer and activator of transcription 1 (STAT1) and induction of STAT1-regulated genes and that this process is inhibited by active caspases. STAT1 is enriched in CSCs in cancer cell lines, patient-derived human breast cancer, and CD95high-expressing glioblastoma neurospheres. CD95 stimulation of cancer cells induced secretion of type I interferons (IFNs) that bind to type I IFN receptors, resulting in activation of Janus-activated kinases, activation of STAT1, and induction of a number of STAT1-regulated genes that are part of a gene signature recently linked to therapy resistance in five primary human cancers. Consequently, we identified type I IFNs as drivers of cancer stemness. Knockdown or knockout of STAT1 resulted in a strongly reduced ability of CD95L or type I IFN to increase cancer stemness. This identifies STAT1 as a key regulator of the CSC-inducing activity of CD95.

Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1.

The directed movement of cells is critical for numerous developmental and disease processes. A developmentally reiterated form of migration is radial intercalation; the process by which cells move in a direction orthogonal to the plane of the tissue from an inner layer to an outer layer. We use the radial intercalation of cells into the skin of Xenopus laevis embryos as a model to study directed cell migration within an epithelial tissue. We identify a novel function for both the microtubule-binding protein CLAMP and members of the microtubule-regulating Par complex during intercalation. Specifically, we show that Par3 and aPKC promote the apical positioning of centrioles, whereas CLAMP stabilizes microtubules along the axis of migration. We propose a model in which the Par complex defines the orientation of apical migration during intercalation and in which subcellular localization of CLAMP promotes the establishment of an axis of microtubule stability required for the active migration of cells into the outer epithelium.

Immobilization techniques in the fabrication of nanomaterial-based electrochemical biosensors: a review.

The evolution of 1st to 3rd generation electrochemical biosensors reflects a simplification and enhancement of the transduction pathway. However, in recent years, modification of the transducer with nanomaterials has become increasingly studied and imparts many advantages. The sensitivity and overall performance of enzymatic biosensors has improved tremendously as a result of incorporating nanomaterials in their fabrication. Given the unique and favorable qualities of gold nanoparticles, graphene and carbon nanotubes as applied to electrochemical biosensors, a consolidated survey of the different methods of nanomaterial immobilization on transducer surfaces and enzyme immobilization on these species is beneficial and timely. This review encompasses modification of enzymatic biosensors with gold nanoparticles, carbon nanotubes, and graphene.