Valproic acid reprograms the metabolic aberration of cisplatin treatment via ALDH modulation in triple-negative breast cancer cells.

We recently demonstrated that the histone deacetylase inhibitor valproic acid (VPA) reprograms the cisplatin-induced metabolome of triple-negative breast cancer (TNBC) cells, including a shift in hexose levels. Accordingly, here, we tested the hypothesis that VPA alters glucose metabolism in correlation with cisplatin sensitivity. Two TNBC cell lines, MDA-MB-231 (a cisplatin-resistant line) and MDA-MB-436 (a cisplatin-sensitive line), were analyzed. The glycolysis and oxidative metabolism were measured using the Glycolysis Stress Test kit. The expression of aldehyde dehydrogenases (ALDHs), enzymes linked to drug resistance, was investigated by Western blot and real-time PCR analyses. We additionally studied the influence of ALDH inhibition by disulfiram on the viability of MDA-MB-231 cells and on a TNBC patient-derived organoid system. Cisplatin treatment reduced the extracellular acidification rate in MDA-MB-436 cells but not MDA-MB-231 cells, whereas VPA addition increased the extracellular acidification rate in both cell lines. VPA further reduced the oxygen consumption rate of cisplatin-treated MDA-MB-436 cells, which correlated with cell cycle alterations. However, in MDA-MB-231 cells, the cell cycle distribution did not change between cisplatin/VPA-cisplatin treatments. In both cell lines, VPA increased the expression of ALDH isoform and ALDH1A1 expression. However, only in MDA-MB-231 cells, VPA synergized with cisplatin to augment this effect. Disulfiram sensitized the cells to the cytotoxic effects of the VPA-cisplatin combination. Furthermore, the disulfiram-VPA-chemotherapy combination was most effective in TNBC organoids. Our results show that ALDH overexpression may act as one mechanism of cellular resistance to VPA in TNBC and that its inhibition may enhance the therapeutic efficacy of VPA-chemotherapeutic drug combinations.

Identification of vascular cues contributing to cancer cell stemness and function.

Glioblastoma stem cells (GSCs) reside close to blood vessels (BVs) but vascular cues contributing to GSC stemness and the nature of GSC-BVs cross talk are not fully understood. Here, we dissected vascular cues influencing GSC gene expression and function to perfusion-based vascular cues, as well as to those requiring direct GSC-endothelial cell (EC) contacts. In light of our previous finding that perivascular tumor cells are metabolically different from tumor cells residing further downstream, cancer cells residing within a narrow, < 60 µm wide perivascular niche were isolated and confirmed to possess a superior tumor-initiation potential compared with those residing further downstream. To circumvent reliance on marker expression, perivascular GSCs were isolated from the respective locales based on their relative state of quiescence. Combined use of these procedures uncovered a large number of previously unrecognized differentially expressed GSC genes. We show that the unique metabolic milieu of the perivascular niche dominated by the highly restricted zone of mTOR activity is conducive for acquisition of GSC properties, primarily in the regulation of genes implicated in cell cycle control. A complementary role of vascular cues including those requiring direct glioma/EC contacts was revealed using glioma/EC co-cultures. Outstanding in the group of glioma cells impacted by nearby ECs were multiple genes responsible for maintaining GSCs in an undifferentiated state, a large fraction of which also relied on Notch-mediated signaling. Glioma-EC communication was found to be bidirectional, evidenced by extensive Notch-mediated EC reprogramming by contacting tumor cells, primarily metabolic EC reprogramming.

Alleviation of acute radiation-induced bone marrow failure in mice with human fetal placental stromal cell therapy.

PURPOSE: Selected placental mesenchymal stromal cells isolated from the fetal mesenchymal placental tissues (f-hPSCs) were tested as cell therapy of lethal acute radiation syndrome (ARS) with bone marrow regeneration and induced extramedullary hematopoiesis. METHODS AND MATERIALS: f-hPSCs were isolated from the chorionic plate of human placentae and further expanded in regular culture conditions. 2 × 106 f-hPSCs were injected on days 1 and 4 to 8-Gy total body irradiated (TBI) C3H mice, both intramuscularly and subcutaneously. Pre-splenectomized TBI mice were used to test the involvement of extramedullary spleen hematopoiesis in the f-hPSC-induced hematopoiesis recovery in the TBI mice. Weight and survival of the mice were followed up within the morbid period of up to 23 days following irradiation. The role of hematopoietic progenitors in the recovery of treated mice was evaluated by flow cytometry, blood cell counts, and assay of possibly relevant growth factors. RESULTS AND CONCLUSIONS: The survival rate of all groups of TBI f-hPSC-treated mice at the end of the follow-up was dramatically elevated from < 10% in untreated to ~ 80%, with a parallel regain of body weight, bone marrow (BM) recovery, and elevated circulating progenitors of blood cell lineages. Blood erythropoietin levels were elevated in all f-hPSC-treated mice. Extramedullary splenic hematopoiesis was recorded in the f-hPSC-treated mice, though splenectomized mice still had similar survival rate. Our findings suggest that the indirect f-hPSC life-saving therapy of ARS may also be applied for treating other conditions with a failure of the hematopoietic system and severe pancytopenia.

Hippocampal neural stem cells facilitate access from circulation via apical cytoplasmic processes.

Blood vessels (BVs) are considered an integral component of neural stem cells (NSCs) niches. NSCs in the dentate gyrus (DG(have enigmatic elaborated apical cellular processes that are associated with BVs. Whether this contact serves as a mechanism for delivering circulating molecules is not known. Here we uncovered a previously unrecognized communication route allowing exclusive direct access of blood-borne substances to hippocampal NSCs. BBB-impermeable fluorescent tracer injected transcardially to mice is selectively uptaken by DG NSCs within a minute, via the vessel-associated apical processes. These processes, measured >30 nm in diameter, establish direct membrane-to-membrane contact with endothelial cells in specialized areas of irregular endothelial basement membrane and enriched with vesicular activity. Doxorubicin, a brain-impermeable chemotherapeutic agent, is also readily and selectively uptaken by NSCs and reduces their proliferation, which might explain its problematic anti-neurogenic or cognitive side-effect. The newly-discovered NSC-BV communication route explains how circulatory neurogenic mediators are 'sensed' by NSCs.

Isolation of Tumor Cells Based on Their Distance from Blood Vessels.

Differential exposure of tumor cells to microenvironmental cues greatly impacts cell phenotypes, raising a need for position based sorting of tumor cells amenable to multiple OMICs and functional analyses. One such key determinant of tumor heterogeneity in solid tumors is its vasculature. Proximity to blood vessels (BVs) profoundly affects tumor cell phenotypes due to differential availability of oxygen, gradient exposure to blood-borne substances and inputs by angiocrine factors. To unravel the whole spectrum of genes, pathways and phenotypes impacted by BVs and to determine spatial domains of vascular influences, we developed a methodology for sorting tumor cells according to their relative distance from BVs. The procedure exemplified here using glioblastoma (GBM) model is based on differential uptake of intra-venously injected, freely-diffusing fluorescent dye that allows separation of stroma-free tumor cells residing in different, successive microenvironments amenable for subsequent OMICs and functional analyses. This reliable, easy to use, cost effective strategy can be extended to all solid tumors to study the impact of vasculature or the lack of it.

Intra-Tumoral Metabolic Zonation and Resultant Phenotypic Diversification Are Dictated by Blood Vessel Proximity.

Differential exposure of tumor cells to blood-borne and angiocrine factors results in diverse metabolic microenvironments conducive for non-genetic tumor cell diversification. Here, we harnessed a methodology for retrospective sorting of fully functional, stroma-free cancer cells solely on the basis of their relative distance from blood vessels (BVs) to unveil the whole spectrum of genes, metabolites, and biological traits impacted by BV proximity. In both grafted mouse tumors and natural human glioblastoma (GBM), mTOR activity was confined to few cell layers from the nearest perfused vessel. Cancer cells within this perivascular tier are distinguished by intense anabolic metabolism and defy the Warburg principle through exercising extensive oxidative phosphorylation. Functional traits acquired by perivascular cancer cells, namely, enhanced tumorigenicity, superior migratory or invasive capabilities, and, unexpectedly, exceptional chemo- and radioresistance, are all mTOR dependent. Taken together, the study revealed a previously unappreciated graded metabolic zonation directly impacting the acquisition of multiple aggressive tumor traits.

VEGF expands erythropoiesis via hypoxia-independent induction of erythropoietin in noncanonical perivascular stromal cells.

Insufficient erythropoiesis due to increased demand is usually met by hypoxia-driven up-regulation of erythropoietin (Epo). Here, we uncovered vascular endothelial growth factor (VEGF) as a novel inducer of Epo capable of increasing circulating Epo under normoxic, nonanemic conditions in a previously unrecognized reservoir of Epo-producing cells (EPCs), leading to expansion of the erythroid progenitor pool and robust splenic erythropoiesis. Epo induction by VEGF occurs in kidney, liver, and spleen in a population of Gli1+SMA+PDGFRß+ cells, a signature shared with vascular smooth muscle cells (VSMCs) derived from mesenchymal stem cell-like progenitors. Surprisingly, inhibition of PDGFRß signaling, but not VEGF signaling, abrogated VEGF-induced Epo synthesis. We thus introduce VEGF as a new player in Epo induction and perivascular Gli1+SMA+PDGFRß+ cells as a previously unrecognized EPC reservoir that could be harnessed for augmenting Epo synthesis in circumstances such as chronic kidney disease where production by canonical EPCs is compromised.

Trained Memory of Human Uterine NK Cells Enhances Their Function in Subsequent Pregnancies.

Natural killer cells (NKs) are abundant in the human decidua, regulating trophoblast invasion and angiogenesis. Several diseases of poor placental development are associated with first pregnancies, so we thus looked to characterize differences in decidual NKs (dNKs) in first versus repeated pregnancies. We discovered a population found in repeated pregnancies, which has a unique transcriptome and epigenetic signature, and is characterized by high expression of the receptors NKG2C and LILRB1. We named these cells Pregnancy Trained decidual NK cells (PTdNKs). PTdNKs have open chromatin around the enhancers of IFNG and VEGFA. Activation of PTdNKs led to increased production and secretion of IFN-γ and VEGFα, with the latter supporting vascular sprouting and tumor growth. The precursors of PTdNKs seem to be found in the endometrium. Because repeated pregnancies are associated with improved placentation, we propose that PTdNKs, which are present primarily in repeated pregnancies, might be involved in proper placentation.

Hyperglycemia Impairs Neutrophil Mobilization Leading to Enhanced Metastatic Seeding.

Preexisting diabetes is a risk factor for the development of multiple types of cancer. Additionally, diabetic patients face a poorer prognosis when diagnosed with cancer. To gain insight into the effects of hyperglycemia, a hallmark of diabetes, on tumor growth and metastatic progression, we combined mouse models of cancer and hyperglycemia. We show that while hyperglycemia attenuates primary tumor growth, it concomitantly increases metastatic seeding in a distant organ. We further show that the increase in metastatic seeding is due to impaired secretion of granulocyte colony-stimulating factor (G-CSF) and impaired neutrophil mobilization. Normalizing blood glucose levels using insulin rescues neutrophil recruitment and tumor growth and concomitantly reduces metastatic seeding. These results provide links among hyperglycemia-induced changes in neutrophil mobilization, primary tumor growth, and metastatic progression. Furthermore, our observations highlight the importance of normalizing blood glucose levels in hyperglycemic cancer patients.

VEGF regulates relative allocation of Isl1+ cardiac progenitors to myocardial and endocardial lineages.

A fundamental issue in organogenesis is how dichotomous fate decisions are made securing proper allocation of multipotent progenitors to their respective descendants. Previous lineage tracing analyses showing Isl1+/VEGFR2+ cardiac progenitors in the second heart field give rise to both endocardium and myocardium suggest VEGF plays a role in this fate decision, conceivably promoting an endocardial fate. Isl1+ multipotent progenitors and lineage-committed descendants thereof were visualized and quantified within their transition zone in the outflow tract. Forced VEGF expression during the critical E8.5-E10.5 interval tilted the balance between myocardial- and endocardial-committed progenitors towards the latter, culminating in generation of surplus endocardium developing at the expense of a much thinner myocardium. Experiments ruled-out that surplus endocardium is due to VEGF-induced endocardial proliferation and that reduced myocardium is due to myocardial apoptosis. Inducing VEGF after most Isl1+ progenitors have been exhausted had no effect on the normal endocardia/myocardial ratio but instead produced an unrelated coronary phenotype. Together, these results uncover a novel role for VEGF in controlling proper allocation of Isl1+ cardiac progenitors to their respective descending lineages.

Human and mouse VEGFA-amplified hepatocellular carcinomas are highly sensitive to sorafenib treatment.

UNLABELLED: Death rates from hepatocellular carcinoma (HCC) are steadily increasing, yet therapeutic options for advanced HCC are limited. We identify a subset of mouse and human HCCs harboring VEGFA genomic amplification, displaying distinct biologic characteristics. Unlike common tumor amplifications, this one seems to work via heterotypic paracrine interactions; stromal VEGF receptors (VEGFR), responding to tumor VEGF-A, produce hepatocyte growth factor (HGF) that reciprocally affects tumor cells. VEGF-A inhibition results in HGF downregulation and reduced proliferation, specifically in amplicon-positive mouse HCCs. Sorafenib-the first-line drug in advanced HCC-targets multiple kinases, including VEGFRs, but has only an overall mild beneficial effect. We found that VEGFA amplification specifies mouse and human HCCs that are distinctly sensitive to sorafenib. FISH analysis of a retrospective patient cohort showed markedly improved survival of sorafenib-treated patients with VEGFA-amplified HCCs, suggesting that VEGFA amplification is a potential biomarker for HCC response to VEGF-A-blocking drugs. SIGNIFICANCE: Using a mouse model of inflammation-driven cancer, we identified a subclass of HCC carrying VEGFA amplification, which is particularly sensitive to VEGF-A inhibition. We found that a similar amplification in human HCC identifies patients who favorably responded to sorafenib-the first-line treatment of advanced HCC-which has an overall moderate therapeutic efficacy.

On-site education of VEGF-recruited monocytes improves their performance as angiogenic and arteriogenic accessory cells.

Adult neovascularization relies on the recruitment of monocytes to the target organ or tumor and functioning therein as a paracrine accessory. The exact origins of the recruited monocytes and the mechanisms underlying their plasticity remain unclear. Using a VEGF-based transgenic system in which genetically tagged monocytes are conditionally summoned to the liver as part of a VEGF-initiated angiogenic program, we show that these recruited cells are derived from the abundant pool of circulating Ly6C(hi) monocytes. Remarkably, however, upon arrival at the VEGF-induced organ, but not the naive organ, monocytes undergo multiple phenotypic and functional changes, endowing them with enhanced proangiogenic capabilities and, importantly, with a markedly increased capacity to remodel existing small vessels into larger conduits. Notably, monocytes do not differentiate into long-lived macrophages, but rather appear as transient accessory cells. Results from transfers of presorted subpopulations and a novel tandem transfer strategy ruled out selective recruitment of a dedicated preexisting subpopulation or onsite selection, thereby reinforcing active reprogramming as the underlying mechanism for improved performance. Collectively, this study uncovered a novel function of VEGF, namely, on-site education of recruited "standard" monocytes to become angiogenic and arteriogenic professional cells, a finding that may also lend itself for a better design of angiogenic therapies.

Diclofenac inhibits tumor growth in a murine model of pancreatic cancer by modulation of VEGF levels and arginase activity.

BACKGROUND: Diclofenac is one of the oldest anti-inflammatory drugs in use. In addition to its inhibition of cyclooxygenases (COX), diclofenac potently inhibits phospholipase A(2) (PLA(2)), thus yielding a broad anti-inflammatory effect. Since inflammation is an important factor in the development of pancreatic tumors we explored the potential of diclofenac to inhibit tumor growth in mice inoculated with PANCO2 cells orthotopically. METHODOLOGY/PRINCIPAL FINDINGS: We found that diclofenac treatment (30 mg/kg/bw for 11 days) of mice inoculated with PANC02 cells, reduced the tumor weight by 60%, correlating with increased apoptosis of tumor cells. Since this effect was not observed in vitro on cultured PANCO2 cells, we theorized that diclofenac beneficial treatment involved other mediators present in vivo. Indeed, diclofenac drastically decreased tumor vascularization by downregulating VEGF in the tumor and in abdominal cavity fluid. Furthermore, diclofenac directly inhibited vascular sprouting ex vivo. Surprisingly, in contrast to other COX-2 inhibitors, diclofenac increased arginase activity/arginase 1 protein content in tumor stroma cells, peritoneal macrophages and white blood cells by 2.4, 4.8 and 2 fold, respectively. We propose that the subsequent arginine depletion and decrease in NO levels, both in serum and peritoneal cavity, adds to tumor growth inhibition by malnourishment and poor vasculature development. CONCLUSION/SIGNIFICANCE: In conclusion, diclofenac shows pronounced antitumoral properties in pancreatic cancer model that can contribute to further treatment development. The ability of diclofenac to induce arginase activity in tumor stroma, peritoneal macrophages and white blood cells provides a tool to study a controversial issue of pro-and antitumoral effects of arginine depletion.

VEGF-induced adult neovascularization: recruitment, retention, and role of accessory cells.

Adult neovascularization relies on the recruitment of circulating cells, but their angiogenic roles and recruitment mechanisms are unclear. We show that the endothelial growth factor VEGF is sufficient for organ homing of circulating mononuclear myeloid cells and is required for their perivascular positioning and retention. Recruited bone marrow-derived circulating cells (RBCCs) summoned by VEGF serve a function distinct from endothelial progenitor cells. Retention of RBCCs in close proximity to angiogenic vessels is mediated by SDF1, a chemokine induced by VEGF in activated perivascular myofibroblasts. RBCCs enhance in situ proliferation of endothelial cells via secreting proangiogenic activities distinct from locally induced activities. Precluding RBCCs strongly attenuated the proangiogenic response to VEGF and addition of purified RBCCs enhanced angiogenesis in excision wounds. Together, the data suggest a model for VEGF-programmed adult neovascularization highlighting the essential paracrine role of recruited myeloid cells and a role for SDF1 in their perivascular retention.

Specific induction of tie1 promoter by disturbed flow in atherosclerosis-prone vascular niches and flow-obstructing pathologies.

Nonlaminar flow is a major predisposing factor to atherosclerosis. Yet little is known regarding hemodynamic gene regulation in disease-prone areas of the vascular tree in vivo. We have determined spatial patterns of expression of endothelial cell receptors in the arterial tree and of reporter gene constructs in transgenic animals. In this study we show that the endothelial cell-specific receptor Tie1 is induced by disturbed flow in atherogenic vascular niches. Specifically, tie1 expression in the adult is upregulated in vascular bifurcations and branching points along the arterial tree. It is often confined to a single ring of endothelial cells functioning as sphincters and hence experiencing the steepest gradient in shear stress. In aortic valves, tie1 is asymmetrically induced only in endothelial cells encountering changes in flow direction. Disturbance of laminar flow by a surgical interposition of a vein into an artery led to induction of tie1, specifically in the region where the differently sized vessels adjoin. In pathological settings, tie1 expression is specifically induced in areas of disturbed flow because of the emergence of aneurysms and, importantly, in endothelial cells precisely overlying atherosclerotic plaques. Hemodynamic features of atherosclerotic lesion-prone regions, recreated in vitro with the aid of a flow chamber with a built-in step, corroborated an upregulated tie1 promoter activity only in cells residing where flow separation and recirculation take place. These defined promoter elements might be harnessed for targeting gene expression to atherosclerotic lesions.

Cysteine-scanning mutagenesis reveals a conformationally sensitive reentrant pore-loop in the glutamate transporter GLT-1.

Removal of glutamate from the synaptic cleft by (Na(+) + K(+))-coupled transporters prevents neurotoxicity due to elevated concentrations of the transmitter. These transporters exhibit an unusual topology, including two reentrant loops. Reentrant loop II plays a pivotal role in coupling ion and glutamate fluxes. Here we used cysteine-scanning mutagenesis of the GLT-1 transporter to test the idea that this loop undergoes conformational changes following sodium and substrate binding. 15 of 22 consecutive single cysteine mutants in the stretch between Gly-422 and Ser-443 exhibited 30-100% of the transport activity of the cysteine-less transporter when expressed in HeLa cells. The transport activity of 11 of the 15 active mutants including five consecutive residues in the ascending limb was inhibited by small hydrophilic methanethiosulfonate reagents. The sensitivity of seven cysteine mutants, including A438C and S440C, to the reagents was significantly reduced by sodium ions, but the opposite was true for A439C. The non-transportable analogue dihydrokainate protected at almost all positions throughout the loop, and at two of the positions, the analogue protected even in the absence of sodium. Our results indicate that reentrant loop II forms part of an aqueous pore, the access of which is blocked by the glutamate analogue dihydrokainate, and that sodium influences the conformation of this pore-loop.

Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesis.

Sodium- and potassium-coupled transporters clear the excitatory neurotransmitter glutamate from the synaptic cleft. Their function is essential for effective glutamatergic neurotransmission. Glutamate transporters have an unusual topology, containing eight membrane-spanning domains and two reentrant loops of opposite orientation. We have introduced pairwise cysteine substitutions in several structural elements of the GLT-1 transporter. A complete inhibition of transport by Cu(II)(1,10-phenanthroline)(3) is observed in the double mutants A412C/V427C and A364C/S440C, but not in the corresponding single mutants. No inhibition is observed in more then 20 other double cysteine mutants. The Cu(II)(1,10-phenanthroline)(3) inhibition can be partly prevented by the nontransportable glutamate analogue dihydrokainate. Treatment with dithiothreitol restores much of the transport activity. Moreover, micromolar concentrations of cadmium ions reversibly inhibit transport catalyzed by A412C/V427C and A364C/S440C double mutants, but not by the corresponding single mutants. Inhibition by Cu(II)(1,10-phenanthroline)(3) and by cadmium is only observed when the cysteine pairs are introduced in the same polypeptide. Therefore, in both cases the proximity appears to be intra- rather than intermolecular. Positions 364 and 440 are located on reentrant loop I and II, respectively. Our results suggest that these two loops, previously shown to be essential for glutamate transport, come in close proximity.