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The enumeration of circulating tumor cells has long been regarded as an attractive diagnostic tool,as circulating tumor cells are thought to reflect aggressiveness of the tumor and may assist in therapeutic decisions in patients with solid malignancies. However,implementation of this assay into clinical routine has been cumbersome,as a validated test was not available until recently. Circulating tumor cells are rare events which can be detected specifically only by using a combination of surface and intracellular markers,and only recently a number of technical advances have made their reliable detection possible. Most of these new techniques rely on a combination of an enrichment and a detection step. This review addresses the assays that have been described so far in the literature,including the enrichment and detection steps and the markers used in these assays. We have focused on breast cancer as most clinical studies on CTC detection so far have been done in these patients. View Publication

α-Lipoic acid (LA) is a thiol with antioxidant properties that protects against oxidative stress-induced apoptosis. LA is absorbed from the diet,taken up by cells and tissues,and subsequently reduced to dihydrolipoic acid (DHLA). Recently,DHLA has been used as the hydrophilic nanomaterial preparations,and therefore,determination of its bio-safety profile is essential. In this article,we show that DHLA (50-100 μM) induces apoptotic processes in mouse embryonic stem cells (ESC-B5),but exerts no injury effects at treatment dosages below 50 μM. Higher concentrations of DHLA (50-100 μM) directly increased the reactive oxygen species (ROS) content in ESC-B5 cells,along with a significant increase in cytoplasmic free calcium and nitric oxide (NO) levels,loss of mitochondrial membrane potential (MMP),activation of caspases-9 and -3,and cell death. Pretreatment with NO scavengers suppressed the apoptotic biochemical changes induced by 100 μM DHLA and promoted the gene expression levels of p53 and p21 involved in apoptotic signaling. Our results collectively indicate that DHLA at concentrations of 50-100 μM triggers apoptosis of ESC-B5 cells,which involves both ROS and NO. Importantly,at doses of less than 50 μM (0-25 μM),DHLA does not exert hazardous effects on ESC-B5 cell properties,including viability,development and differentiation. These results provide important information in terms of dosage safety and biocompatibility of DHLA to facilitate its further use as a precursor for biomaterial preparation. View Publication

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation,a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation,as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data,possibly due to their crosstalk with Notch Signaling. Overall,this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs. View Publication

The sub-population of tumor cells termed 'cancer stem cells' (CSCs) possess the capability to generate tumors,undergo epithelial-mesenchymal transition (EMT) and are implicated in metastasis,making treatments to specifically target CSCs an attractive therapeutic strategy. Tumor hypoxia plays a key role in regulating EMT and cancer stem cell function. Carbonic anhydrase IX (CAIX) is a hypoxia-inducible protein that regulates cellular pH to promote cancer cell survival and invasion in hypoxic microenvironments and is a biomarker of poor prognosis for breast cancer metastasis and survival. Here,we demonstrate that inhibition of CAIX expression or activity with novel small-molecule inhibitors in breast cancer cell lines,or in primary metastatic breast cancer cells,results in the inhibition of breast CSC expansion in hypoxia. We identify the mTORC1 axis as a critical pathway downstream of CAIX in the regulation of cancer stem cell function. CAIX is also required for expression of EMT markers and regulators,as well as drivers of 'stemness',such as Notch1 and Jagged1 in isolated CSCs. In addition,treatment of mice bearing orthotopic breast tumors with CAIX-specific small-molecule inhibitors results in significant depletion of CSCs within these tumors. Furthermore,combination treatment with paclitaxel results in enhanced tumor growth delay and eradication of lung metastases. These data demonstrate that CAIX is a critical mediator of the expansion of breast CSCs in hypoxic niches by sustaining the mesenchymal and 'stemness' phenotypes of these cells,making CAIX an important therapeutic target for selectively depleting breast CSCs. View Publication

Prominin-1 (CD133) is commonly used to isolate stem and progenitor cells from the developing and adult nervous system and to identify cancer stem cells in brain tumors. However,despite extensive characterization of Prominin-1(+) precursor cells from the adult subventricular zone,no information about the expression of Prominin-1 by precursor cells in the subgranular zone (SGZ) of the adult hippocampus has been available. We show here that Prominin-1 is expressed by a significant number of cells in the SGZ of adult mice in vivo and ex vivo,including postmitotic astrocytes. A small subset of Prominin-1(+) cells coexpressed the nonspecific precursor cell marker Nestin as well as GFAP and Sox2. Upon fluorescence-activated cell sorting,only Prominin-1/Nestin double-positive cells fulfilled the defining stem cell criteria of proliferation,self-renewal,and multipotentiality as assessed by a neurosphere assay. In addition,isolated primary Prominin-1(+) cells preferentially migrated to the neurogenic niche in the SGZ upon transplantation in vivo. Finally,despite its expression by various stem and progenitor cells,Prominin-1 turned out to be dispensable for precursor cell proliferation in vitro and in vivo. Nevertheless,a net decrease in hippocampal neurogenesis,by ∼30% was found in Prominin-1 knock-out mice,suggesting other roles in controlling adult hippocampal neurogenesis. Remarkably,an upregulation of Prominin-2 was detected in Prominin-1-deficient mice highlighting a potential compensatory mechanism,which might explain the lack of severe symptoms in individuals carrying mutations in the Prom1 gene. View Publication

Aldehyde dehydrogenase (ALDH) has been identified in stem cells from both normal and cancerous tissues. This study aimed to evaluate the potential of ALDH as a universal brain tumour initiating cell (BTIC) marker applicable to primary brain tumours and their biological role in maintaining stem cell status. Cells from various primary brain tumours (24paediatric and 6 adult brain tumours) were stained with Aldefluor and sorted by flow cytometry. We investigated the impact of ALDH expression on BTIC characteristics in vitro and on tumourigenic potential in vivo. Primary brain tumours showed universal expression of ALDH,with 0.3-28.9% of the cells in various tumours identified as ALDH(+). The proportion of CD133(+) cells within ALDH(+) is higher than ALDH cells. ALDH(+) cells generate neurospheres with high proliferative potential,express neural stem cell markers and differentiate into multiple nervous system lineages. ALDH(+) cells tend to show high expression of induced pluripotent stem cell-related genes. Notably,targeted knockdown of ALDH1 by shRNA interference in BTICs potently disturbed their self-renewing ability. After 3months,ALDH(+) cells gave rise to tumours in 93% of mice whereas ALDH cells did not. The characteristic pathology of mice brain tumours from ALDH(+) cells was similar to that of human brain tumours,and these cells are highly proliferative in vivo. Our data suggest that primary brain tumours contain distinct subpopulations of cells that have high expression levels of ALDH and BTIC characteristics. ALDH might be a potential therapeutic target applicable to primary brain tumours. View Publication

Recent advances in developmental biology and stem cell technology have led to the engineering of functional organs in a dish. However,the limited size of these organoids and absence of a large circulatory system poses limits to its clinical translation. To overcome these issues,decellularized whole kidney scaffolds with native microstructure and extracellular matrix (ECM) are employed for kidney bioengineering,using human-induced pluripotent-stem-cell-derived renal progenitor cells and endothelial cells. To demonstrate ECM-guided cellular assembly,the present work is focused on generating the functional unit of the kidney,the glomerulus. In the repopulated organ,the presence of endothelial cells broadly upregulates the expression level of genes related to renal development. When the cellularized native scaffolds are implanted in SCID mice,glomeruli assembly can be achieved by co-culture of the renal progenitors and endothelial cells. These individual glomerular units are shown to be functional in the context of the whole organ using a simulated bio-reactor set-up with urea and creatinine excretion and albumin reabsorption. Our results indicate that the repopulation of decellularized native kidney using clinically relevant,expandable patient-specific renal progenitors and endothelial cells may be a viable approach for the generation of a functional whole kidney. View Publication

While distinct stem cell phenotypes follow global changes in chromatin marks,single-cell chromatin technologies are unable to resolve or predict stem cell fates. We propose the first such use of optical high content nanoscopy of histone epigenetic marks (epi-marks) in stem cells to classify emergent cell states. By combining nanoscopy with epi-mark textural image informatics,we developed a novel approach,termed EDICTS (Epi-mark Descriptor Imaging of Cell Transitional States),to discern chromatin organizational changes,demarcate lineage gradations across a range of stem cell types and robustly track lineage restriction kinetics. We demonstrate the utility of EDICTS by predicting the lineage progression of stem cells cultured on biomaterial substrates with graded nanotopographies and mechanical stiffness,thus parsing the role of specific biophysical cues as sensitive epigenetic drivers. We also demonstrate the unique power of EDICTS to resolve cellular states based on epi-marks that cannot be detected via mass spectrometry based methods for quantifying the abundance of histone post-translational modifications. Overall,EDICTS represents a powerful new methodology to predict single cell lineage decisions by integrating high content super-resolution nanoscopy and imaging informatics of the nuclear organization of epi-marks. View Publication

Specialized immune cell subsets are involved in autoimmune disease,cancer immunity,and infectious disease through a diverse range of functions mediated by overlapping pathways and signals. However,subset-specific responses may not be detectable in analyses of whole blood samples,and no efficient approach for profiling cell subsets at high throughput from small samples is available. We present a low-input microfluidic system for sorting immune cells into subsets and profiling their gene expression. We validate the system's technical performance against standard subset isolation and library construction protocols and demonstrate the importance of subset-specific profiling through in vitro stimulation experiments. We show the ability of this integrated platform to identify subset-specific disease signatures by profiling four immune cell subsets in blood from patients with systemic lupus erythematosus (SLE) and matched control subjects. The platform has the potential to make multiplexed subset-specific analysis routine in many research laboratories and clinical settings. View Publication

SummaryTumor-infiltrating regulatory T cells (TI-Tregs) elicit immunosuppressive effects in the tumor microenvironment (TME) leading to accelerated tumor growth and resistance to immunotherapies against solid tumors. Here,we demonstrate that poly-(ADP-ribose)-polymerase-11 (PARP11) is an essential regulator of immunosuppressive activities of TI-Tregs. Expression of PARP11 correlates with TI-Treg cell numbers and poor responses to immune checkpoint blockade (ICB) in human patients with cancer. Tumor-derived factors including adenosine and prostaglandin E2 induce PARP11 in TI-Tregs. Knockout of PARP11 in the cells of the TME or treatment of tumor-bearing mice with selective PARP11 inhibitor ITK7 inactivates TI-Tregs and reinvigorates anti-tumor immune responses. Accordingly,ITK7 decelerates tumor growth and significantly increases the efficacy of anti-tumor immunotherapies including ICB and adoptive transfer of chimeric antigen receptor (CAR) T cells. These results characterize PARP11 as a key driver of TI-Treg activities and a major regulator of immunosuppressive TME and argue for targeting PARP11 to augment anti-cancer immunotherapies. Graphical abstract Highlights•Tumor-derived factors upregulate PARP11 in the tumor-infiltrating Treg cells•PARP11 supports the immunosuppressive properties of Treg cells•Pharmacologic inhibition of PARP11 inactivates intratumoral Treg cells•PARP11 inhibitor augments the efficacy of immunotherapies Basavaraja et al. demonstrate that induction of PARP11 in the intratumoral regulatory T (Treg) cells is required for their regulatory functions and contributes to the immunosuppressive tumor microenvironment. The selective inhibitor of PARP11 ITK7 inactivates tumor Treg cells and improves the efficacy of immunotherapies against tumors. View Publication

AbstractBackgroundNatural killer (NK) cells’ unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However,NK cells have demonstrated only moderate responses against cancer in clinical trials.MethodsAdvanced genome engineering may thus be used to unlock their full potential. Multiplex genome editing with CRISPR/Cas9 base editors (BEs) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here,we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations.ResultsWe observed highly efficient single and multiplex base editing,resulting in significantly enhanced NK cell function in vitro and in vivo. Next,we combined multiplex BE with non-viral TcBuster transposon-based integration to generate interleukin-15 armored CD19 chimeric antigen receptor (CAR)-NK cells with significantly improved functionality in a highly suppressive model of Burkitt’s lymphoma both in vitro and in vivo.ConclusionsThe use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell-based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated. View Publication

Cohesin plays a crucial role in the organization of topologically-associated domains (TADs),which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here,we discover that the histone demethylase PHF2 associates with RAD21,a core subunit of cohesin,to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably,the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient,active replication origins,and can act as boundaries to separate adjacent domains. Accordingly,PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore,we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation,and that PHF2’s histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops,where cohesin represses dormant replication origins directly or indirectly,to sustain DNA replication in NSC. View Publication