Chemoresistance to additive PARP/PI3K dual inhibition in triple-negative breast cancer cell lines is associated with adaptive stem cell-like prevalence.

Cancer stem-like cells (CSCs) are posited to exhibit specialized oncogenic capacity to drive malignancies. CSCs are distinguished by enhanced hallmarks of cancer, including apoptosis avoidance, phenotypic plasticity and aberrant growth pathway signaling. Standard-of-care chemotherapies targeted to rapidly cycling cells routinely fail to eliminate this resistant subpopulation, leading to disease recurrence and metastasis. Triple-negative breast cancer (TNBC), a highly aggressive subtype of breast cancer, is enriched for tumor-propagating CD44+/CD24-/low CSCs, which are poorly ablated by chemotherapeutics and are associated with poor prognosis. CD44 governs sustained PI3K signaling in breast cancer, which is essential for CSC maintenance. PI3K inhibition can elicit DNA damage and down-regulate BRCA1 expression, which in turn enhance the synthetic lethality of PARP inhibitors. Here, we examined a dual chemotherapeutic approach targeting these pathways by combining a pan-PI3K inhibitor (Buparlisib) and a PARP1 inhibitor (Olaparib) on a panel of TNBC cell lines with distinct mutational profiles and proportions of CSCs. We observed differential sensitivity to this dual inhibition strategy and varying cellular stress and resistance responses across eight TNBC lines. The dual chemotherapeutic effect is associated with a reduction in S-phase cells, an increased in apoptotic cells and elevated expression of cleaved PARP, indicating a provoked replicative stress response. We observed that PARP/PI3K inhibition efficacy was potentiated by repeated administration in some TNBC lines and identified critical treatment schedules, which further potentiated the dual chemotherapeutic effect. Dual inhibition induced small but significant increases in CSC relative abundance as marked by CD44+/CD24-/low or ALDH1+ cells and increased stress and survival signaling in multiple TNBC cell lines, suggesting this sub-population contributes to TNBC chemoresistance. These results suggest the additive effects of PARP and PI3K inhibition against CSC phenotypes may be enhanced by temporally-staged administration in TNBC cells.

Dysregulation of cell state dynamics during early stages of serous endometrial carcinogenesis.

Serous endometrial carcinoma (SEC) constitutes about 10% of endometrial carcinomas and is one of the most aggressive and lethal types of uterine cancer. Due to the rapid progression of SEC, early detection of this disease is of utmost importance. However, molecular and cellular dynamics during the pre-dysplastic stage of this disease remain largely unknown. Here, we provide a comprehensive census of cell types and their states for normal, pre-dysplastic, and dysplastic endometrium in a mouse model of SEC. This model is associated with inactivation of tumor suppressor genes Trp53 and Rb1 , whose pathways are altered frequently in SEC. We report that pre-dysplastic changes are characterized by an expanded and increasingly diverse immature luminal epithelial cell populations. Consistent with transcriptome changes, cells expressing the luminal epithelial marker TROP2 begin to substitute FOXA2+ cells in the glandular epithelium. These changes are associated with a reduction in number and strength of predicted interactions between epithelial and stromal endometrial cells. By using a multi-level approach combining single-cell and spatial transcriptomics paired with screening for clinically relevant genes in human endometrial carcinoma, we identified a panel of 44 genes suitable for further testing of their validity as early diagnostic and prognostic markers. Among these genes are known markers of human SEC, such as C DKN2A, and novel markers, such as OAS2 and OASL, members of 2-5A synthetase family that is essential for the innate immune response. In summary, our results suggest an important role of the luminal epithelium in SEC pathogenesis, highlight aberrant cell-cell interactions in pre-dysplastic stages, and provide a new platform for comparative identification and characterization of novel, clinically relevant prognostic and diagnostic markers and potential therapeutic modalities.

Combinatorial treatment rescues tumour-microenvironment-mediated attenuation of MALT1 inhibitors in B-cell lymphomas.

Activated B-cell-like diffuse large B-cell lymphomas (ABC-DLBCLs) are characterized by constitutive activation of nuclear factor κB driven by the B-cell receptor (BCR) and Toll-like receptor (TLR) pathways. However, BCR-pathway-targeted therapies have limited impact on DLBCLs. Here we used >1,100 DLBCL patient samples to determine immune and extracellular matrix cues in the lymphoid tumour microenvironment (Ly-TME) and built representative synthetic-hydrogel-based B-cell-lymphoma organoids accordingly. We demonstrate that Ly-TME cellular and biophysical factors amplify the BCR-MYD88-TLR9 multiprotein supercomplex and induce cooperative signalling pathways in ABC-DLBCL cells, which reduce the efficacy of compounds targeting the BCR pathway members Bruton tyrosine kinase and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1). Combinatorial inhibition of multiple aberrant signalling pathways induced higher antitumour efficacy in lymphoid organoids and implanted ABC-DLBCL patient tumours in vivo. Our studies define the complex crosstalk between malignant ABC-DLBCL cells and Ly-TME, and provide rational combinatorial therapies that rescue Ly-TME-mediated attenuation of treatment response to MALT1 inhibitors.

Cells expressing PAX8 are the main source of homeostatic regeneration of adult mouse endometrial epithelium and give rise to serous endometrial carcinoma.

Humans and mice have cyclical regeneration of the endometrial epithelium. It is expected that such regeneration is ensured by tissue stem cells, but their location and hierarchy remain debatable. A number of recent studies have suggested the presence of stem cells in the mouse endometrial epithelium. At the same time, it has been reported that this tissue can be regenerated by stem cells of stromal/mesenchymal or bone marrow cell origin. Here, we describe a single-cell transcriptomic atlas of the main cell types of the mouse uterus and epithelial subset transcriptome and evaluate the contribution of epithelial cells expressing the transcription factor PAX8 to the homeostatic regeneration and malignant transformation of adult endometrial epithelium. According to lineage tracing, PAX8+ epithelial cells are responsible for long-term maintenance of both luminal and glandular epithelium. Furthermore, multicolor tracing shows that individual glands and contiguous areas of luminal epithelium are formed by clonal cell expansion. Inactivation of the tumor suppressor genes Trp53 and Rb1 in PAX8+ cells, but not in FOXJ1+ cells, leads to the formation of neoplasms with features of serous endometrial carcinoma, one of the most aggressive types of human endometrial malignancies. Taken together, our results show that the progeny of single PAX8+ cells represents the main source of regeneration of the adult endometrial epithelium. They also provide direct experimental genetic evidence for the key roles of the P53 and RB pathways in the pathogenesis of serous endometrial carcinoma and suggest that PAX8+ cells represent the cell of origin of this neoplasm.

A defined N6-methyladenosine (m6A) profile conferred by METTL3 regulates muscle stem cell/myoblast state transitions.

Muscle-specific adult stem cells (MuSCs) are required for skeletal muscle regeneration. To ensure efficient skeletal muscle regeneration after injury, MuSCs must undergo state transitions as they are activated from quiescence, give rise to a population of proliferating myoblasts, and continue either to terminal differentiation, to repair or replace damaged myofibers, or self-renewal to repopulate the quiescent population. Changes in MuSC/myoblast state are accompanied by dramatic shifts in their transcriptional profile. Previous reports in other adult stem cell systems have identified alterations in the most abundant internal mRNA modification, N6-methyladenosine (m6A), conferred by its active writer, METTL3, to regulate cell state transitions through alterations in the transcriptional profile of these cells. Our objective was to determine if m6A-modification deposition via METTL3 is a regulator of MuSC/myoblast state transitions in vitro and in vivo. Using liquid chromatography/mass spectrometry we identified that global m6A levels increase during the early stages of skeletal muscle regeneration, in vivo, and decline when C2C12 myoblasts transition from proliferation to differentiation, in vitro. Using m6A-specific RNA-sequencing (MeRIP-seq), a distinct profile of m6A-modification was identified, distinguishing proliferating from differentiating C2C12 myoblasts. RNAi studies show that reducing levels of METTL3, the active m6A methyltransferase, reduced global m6A levels and forced C2C12 myoblasts to prematurely differentiate. Reducing levels of METTL3 in primary mouse MuSCs prior to transplantation enhanced their engraftment capacity upon primary transplantation, however their capacity for serial transplantation was lost. In conclusion, METTL3 regulates m6A levels in MuSCs/myoblasts and controls the transition of MuSCs/myoblasts to different cell states. Furthermore, the first transcriptome wide map of m6A-modifications in proliferating and differentiating C2C12 myoblasts is provided and reveals a number of genes that may regulate MuSC/myoblast state transitions which had not been previously identified.

A reference single-cell transcriptomic atlas of human skeletal muscle tissue reveals bifurcated muscle stem cell populations.

Single-cell RNA-sequencing (scRNA-seq) facilitates the unbiased reconstruction of multicellular tissue systems in health and disease. Here, we present a curated scRNA-seq dataset of human muscle samples from 10 adult donors with diverse anatomical locations. We integrated ~ 22,000 single-cell transcriptomes using Scanorama to account for technical and biological variation and resolved 16 distinct populations of muscle-resident cells using unsupervised clustering of the data compendium. These cell populations included muscle stem/progenitor cells (MuSCs), which bifurcated into discrete "quiescent" and "early-activated" MuSC subpopulations. Differential expression analysis identified transcriptional profiles altered in the activated MuSCs including genes associated with aging, obesity, diabetes, and impaired muscle regeneration, as well as long non-coding RNAs previously undescribed in human myogenic cells. Further, we modeled ligand-receptor cell-communication interactions and observed enrichment of the TWEAK-FN14 pathway in activated MuSCs, a characteristic signature of muscle wasting diseases. In contrast, the quiescent MuSCs have enhanced expression of the EGFR receptor, a recognized human MuSC marker. This work provides a new benchmark reference resource to examine human muscle tissue heterogeneity and identify potential targets in MuSC diversity and dysregulation in disease contexts.

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

How Biophysical Forces Regulate Human B Cell Lymphomas.

The role of microenvironment-mediated biophysical forces in human lymphomas remains elusive. Diffuse large B cell lymphomas (DLBCLs) are heterogeneous tumors, which originate from highly proliferative germinal center B cells. These tumors, their associated neo-vessels, and lymphatics presumably expose cells to particular fluid flow and survival signals. Here, we show that fluid flow enhances proliferation and modulates response of DLBCLs to specific therapeutic agents. Fluid flow upregulates surface expression of B cell receptors (BCRs) and integrin receptors in subsets of ABC-DLBCLs with either CD79A/B mutations or WT BCRs, similar to what is observed with xenografted human tumors in mice. Fluid flow differentially upregulates signaling targets, such as SYK and p70S6K, in ABC-DLBCLs. By selective knockdown of CD79B and inhibition of signaling targets, we provide mechanistic insights into how fluid flow mechanomodulates BCRs and integrins in ABC-DLBCLs. These findings redefine microenvironment factors that regulate lymphoma-drug interactions and will be critical for testing targeted therapies.

Injectable biomimetic liquid crystalline scaffolds enhance muscle stem cell transplantation.

Muscle stem cells are a potent cell population dedicated to efficacious skeletal muscle regeneration, but their therapeutic utility is currently limited by mode of delivery. We developed a cell delivery strategy based on a supramolecular liquid crystal formed by peptide amphiphiles (PAs) that encapsulates cells and growth factors within a muscle-like unidirectionally ordered environment of nanofibers. The stiffness of the PA scaffolds, dependent on amino acid sequence, was found to determine the macroscopic degree of cell alignment templated by the nanofibers in vitro. Furthermore, these PA scaffolds support myogenic progenitor cell survival and proliferation and they can be optimized to induce cell differentiation and maturation. We engineered an in vivo delivery system to assemble scaffolds by injection of a PA solution that enabled coalignment of scaffold nanofibers with endogenous myofibers. These scaffolds locally retained growth factors, displayed degradation rates matching the time course of muscle tissue regeneration, and markedly enhanced the engraftment of muscle stem cells in injured and noninjured muscles in mice.

High-resolution myogenic lineage mapping by single-cell mass cytometry.

Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation. We discovered two cell surface markers, CD9 and CD104, whose combined expression enabled in vivo identification and prospective isolation of stem and progenitor cells. Data analysis using the X-shift algorithm paired with single-cell force-directed layout visualization defined a molecular signature of the activated stem cell state (CD44+/CD98+/MyoD+) and delineated a myogenic trajectory during recovery from acute muscle injury. Our studies uncover the dynamics of skeletal muscle regeneration in vivo and pave the way for the elucidation of the regulatory networks that underlie cell-state transitions in muscle diseases and ageing.

Rejuvenation of the muscle stem cell population restores strength to injured aged muscles.

The elderly often suffer from progressive muscle weakness and regenerative failure. We demonstrate that muscle regeneration is impaired with aging owing in part to a cell-autonomous functional decline in skeletal muscle stem cells (MuSCs). Two-thirds of MuSCs from aged mice are intrinsically defective relative to MuSCs from young mice, with reduced capacity to repair myofibers and repopulate the stem cell reservoir in vivo following transplantation. This deficiency is correlated with a higher incidence of cells that express senescence markers and is due to elevated activity of the p38α and p38ß mitogen-activated kinase pathway. We show that these limitations cannot be overcome by transplantation into the microenvironment of young recipient muscles. In contrast, subjecting the MuSC population from aged mice to transient inhibition of p38α and p38ß in conjunction with culture on soft hydrogel substrates rapidly expands the residual functional MuSC population from aged mice, rejuvenating its potential for regeneration and serial transplantation as well as strengthening of damaged muscles of aged mice. These findings reveal a synergy between biophysical and biochemical cues that provides a paradigm for a localized autologous muscle stem cell therapy for the elderly.

Networks inferred from biochemical data reveal profound differences in toll-like receptor and inflammatory signaling between normal and transformed hepatocytes.

Systematic study of cell signaling networks increasingly involves high throughput proteomics, transcriptional profiling, and automated literature mining with the aim of assembling large scale interaction networks. In contrast, functional analysis of cell signaling usually focuses on a much smaller sets of proteins and eschews computation but focuses directly on cellular responses to environment and perturbation. We sought to combine these two traditions by collecting cell response measures on a reasonably large scale and then attempting to infer differences in network topology between two cell types. Human hepatocytes and hepatocellular carcinoma cell lines were exposed to inducers of inflammation, innate immunity, and proliferation in the presence and absence of small molecule drugs, and multiplex biochemical measurement was then performed on intra- and extracellular signaling molecules. We uncovered major differences between primary and transformed hepatocytes with respect to the engagement of toll-like receptor and NF-kappaB-dependent secretion of chemokines and cytokines that prime and attract immune cells. Overall, our results serve as a proof of principle for an approach to network analysis that is systematic, comparative, and biochemically focused. More specifically, our data support the hypothesis that hepatocellular carcinoma cells down-regulate normal inflammatory and immune responses to avoid immune editing.

Cytokine-associated drug toxicity in human hepatocytes is associated with signaling network dysregulation.

Idiosyncratic drug hepatotoxicity is a major problem in pharmaceutical development due to poor prediction capability of standard preclinical toxicity assessments and limited knowledge of its underlying mechanisms. Findings in animal models have shown that adverse effects of numerous drugs with idiosyncratic hepatotoxicity in humans can be reproduced in the presence of coincident inflammatory cytokine signaling. Following these observations, we have recently developed an in vitro drug/inflammatory cytokine co-treatment approach that can reproduce clinical drug hepatotoxicity signatures-particularly for idiosyncratic drugs-in cultured primary human hepatocytes. These observations have suggested that drug-induced stresses may interact with cytokine signaling to induce hepatic cytotoxicity, but the hepatocyte signaling mechanisms governing these interactions are poorly understood. Here, we collect high-throughput phosphoprotein signaling and cytotoxicity measurements in cultured hepatocytes, from multiple human donors, treated with combinations of hepatotoxic drugs (e.g. trovafloxacin, clarithromycin) and cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin-1 alpha, and interleukin-6). We demonstrate, through orthogonal partial least-squares regression (OPLSR) modeling of these signal-response data, that drug/cytokine hepatic cytotoxicity is integratively controlled by four key signaling pathways: Akt, p70 S6 kinase, MEK-ERK, and p38-HSP27. This modeling predicted, and experimental studies confirmed, that the MEK-ERK and p38-HSP27 pathways contribute pro-death signaling influences in drug/cytokine hepatic cytotoxicity synergy. Further, our four-pathway OPLSR model produced successful prediction of drug/cytokine hepatic cytotoxicities across different human donors, even though signaling and cytotoxicity responses were both highly donor-specific. Our findings highlight the critical role of kinase signaling in drug/cytokine hepatic cytotoxicity synergies and reveal that hepatic cytotoxicity responses are governed by multi-pathway signaling network balance.

Microfluidic concentration-enhanced cellular kinase activity assay.

In this paper, we reported a simple, disposable PDMS micro/nanofluidic preconcentration chip for in vitro concentration-enhanced cell kinase assays. Utilizing the preconcentration (electrokinetic trapping) directly from cell lysate (1 mM ATP) samples, we could achieve at least a 25-fold increase in reaction velocity and 65-fold enhancement in sensitivity. In addition, we shorten the assay time down to less than 10 min, with the sample volume requirements of down to approximately 5 cells. This device could be a generic and powerful tool for diagnostics and systems biology studies at the single-cell level, if properly optimized and integrated with the cell culture microdevices.

Three-kinase inhibitor combination recreates multipathway effects of a geldanamycin analogue on hepatocellular carcinoma cell death.

Multitarget compounds that act on a diverse set of regulatory pathways are emerging as a therapeutic approach for a variety of cancers. Toward a more specified use of this approach, we hypothesize that the desired efficacy can be recreated in terms of a particular combination of relatively more specific (i.e., ostensibly single target) compounds. We test this hypothesis for the geldanamycin analogue 17-Allylamino-17-demethoxygeldanamycin (17AAG) in hepatocellular carcinoma cells, measuring critical phosphorylation levels that indicate the kinase pathway effects correlating with apoptotic responsiveness of the Hep3B cell line in contrast to the apoptotic resistance of the Huh7 cell line. A principal components analysis (PCA) constructed from time course measurements of seven phosphoprotein signaling levels identified modulation of the AKT, IkappaB kinase, and signal transducer and activator of transcription 3 pathways by 17AAG treatment as most important for distinguishing these cell-specific death responses. The analysis correctly suggested from 17AAG-induced effects on these phosphoprotein levels that the FOCUS cell line would show apoptotic responsiveness similarly to Hep3B. The PCA also guided the inhibition of three critical pathways and rendered Huh7 cells responsive to 17AAG. Strikingly, in all three hepatocellular carcinoma lines, the three-inhibitor combination alone exhibited similar or greater efficacy to 17AAG. We conclude that (a) the PCA captures and clusters the multipathway phosphoprotein time courses with respect to their 17AAG-induced apoptotic responsiveness and (b) we can recreate, in a more specified manner, the cellular responses of a prospective multitarget cancer therapeutic.

Synergistic drug-cytokine induction of hepatocellular death as an in vitro approach for the study of inflammation-associated idiosyncratic drug hepatotoxicity.

Idiosyncratic drug hepatotoxicity represents a major problem in drug development due to inadequacy of current preclinical screening assays, but recently established rodent models utilizing bacterial LPS co-administration to induce an inflammatory background have successfully reproduced idiosyncratic hepatotoxicity signatures for certain drugs. However, the low-throughput nature of these models renders them problematic for employment as preclinical screening assays. Here, we present an analogous, but high-throughput, in vitro approach in which drugs are administered to a variety of cell types (primary human and rat hepatocytes and the human HepG2 cell line) across a landscape of inflammatory contexts containing LPS and cytokines TNF, IFN gamma, IL-1 alpha, and IL-6. Using this assay, we observed drug-cytokine hepatotoxicity synergies for multiple idiosyncratic hepatotoxicants (ranitidine, trovafloxacin, nefazodone, nimesulide, clarithromycin, and telithromycin) but not for their corresponding non-toxic control compounds (famotidine, levofloxacin, buspirone, and aspirin). A larger compendium of drug-cytokine mix hepatotoxicity data demonstrated that hepatotoxicity synergies were largely potentiated by TNF, IL-1 alpha, and LPS within the context of multi-cytokine mixes. Then, we screened 90 drugs for cytokine synergy in human hepatocytes and found that a significantly larger fraction of the idiosyncratic hepatotoxicants (19%) synergized with a single cytokine mix than did the non-hepatotoxic drugs (3%). Finally, we used an information theoretic approach to ascertain especially informative subsets of cytokine treatments for most highly effective construction of regression models for drug- and cytokine mix-induced hepatotoxicities across these cell systems. Our results suggest that this drug-cytokine co-treatment approach could provide a useful preclinical tool for investigating inflammation-associated idiosyncratic drug hepatotoxicity.

An inducible autocrine cascade regulates rat hepatocyte proliferation and apoptosis responses to tumor necrosis factor-alpha.

UNLABELLED: Tumor necrosis factor-alpha (TNF) is an inflammatory cytokine that induces context-dependent proliferation, survival, and apoptosis responses in hepatocytes. TNF stimulates and enhances growth factor-mediated hepatocyte proliferation and survival following partial hepatectomy, but also acts in concert with other inflammatory cytokines of the innate immune response during viral infection to induce apoptosis in hepatocytes. In other epithelial cell types, TNF has recently been shown to stimulate autocrine release of transforming growth factor-alpha (TGF-alpha) and interleukin-1 (IL-1) family ligands. Here, we examine the role of these autocrine ligands in modulating TNF-induced proliferation and apoptosis in primary hepatocytes. We show that TNF-induced hepatocyte proliferation is regulated by an inducible, coupled, and self-antagonizing autocrine cascade involving the pro-proliferative TGF-alpha and IL-1 receptor antagonist (IL-1ra) ligands and antiproliferative IL-1alpha/beta ligands. Moreover, cooperative stimulation of hepatocyte proliferation by combined TNF and TGF-alpha treatment is self-limited through antiproliferative autocrine IL-1alpha/beta feedback. We show that TNF potently induces apoptosis of adenovirus-infected hepatocytes in a manner similarly determined through the integrated activity of a coupled TGF-alpha-IL-1alpha/beta-IL-1ra autocrine cascade. Exogenous TGF-alpha can either enhance or diminish apoptosis in adenoviral vector-treated and TNF-treated hepatocytes, in a biphasic relationship also mediated by autocrine IL-1alpha/beta feedback. CONCLUSION: We demonstrate that TNF-induced hepatocyte proliferation and apoptosis are both governed by a self-antagonizing TGF-alpha-IL-1alpha/beta-IL-1ra autocrine cascade in vitro, and thus identify multiple molecular targets for control of TNF-regulated hepatocyte phenotypic responses related to liver regeneration and adenoviral gene therapy.