Arrayed molecular barcoding identifies TNFSF13 as a positive regulator of acute myeloid leukemia-initiating cells.

Dysregulation of cytokines in the bone marrow (BM) microenvironment promotes acute myeloid leukemia (AML) cell growth. Due to the complexity and low throughput of in vivo stem-cell based assays, studying the role of cytokines in the BM niche in a screening setting is challenging. Here, we developed an ex vivo cytokine screen using 11 arrayed molecular barcodes, allowing for a competitive in vivo readout of leukemia-initiating capacity. With this approach, we assessed the effect of 114 murine cytokines on MLL-AF9 AML mouse cells and identified the tumor necrosis factor ligand superfamily member 13 (TNFSF13) as a positive regulator of leukemia-initiating cells. By using Tnfsf13-/- recipient mice, we confirmed that TNFSF13 supports leukemia initiation also under physiological conditions. TNFSF13 was secreted by normal myeloid cells but not by leukemia mouse cells, suggesting that mature myeloid BM cells support leukemia cells by secreting TNFSF13. TNFSF13 supported leukemia cell proliferation in an NF-κB-dependent manner by binding TNFRSF17 and suppressed apoptosis. Moreover, TNFSF13 supported the growth and survival of several human myeloid leukemia cell lines, demonstrating that our findings translate to human disease. Taken together, using arrayed molecular barcoding, we identified a previously unrecognized role of TNFSF13 as a positive regulator of AML-initiating cells. The arrayed barcoded screening methodology is not limited to cytokines and leukemia, but can be extended to other types of ex vivo screens, where a multiplexed in vivo read-out of stem cell functionality is needed.

The effect of environmental chemicals on the tumor microenvironment.

Potentially carcinogenic compounds may cause cancer through direct DNA damage or through indirect cellular or physiological effects. To study possible carcinogens, the fields of endocrinology, genetics, epigenetics, medicine, environmental health, toxicology, pharmacology and oncology must be considered. Disruptive chemicals may also contribute to multiple stages of tumor development through effects on the tumor microenvironment. In turn, the tumor microenvironment consists of a complex interaction among blood vessels that feed the tumor, the extracellular matrix that provides structural and biochemical support, signaling molecules that send messages and soluble factors such as cytokines. The tumor microenvironment also consists of many host cellular effectors including multipotent stromal cells/mesenchymal stem cells, fibroblasts, endothelial cell precursors, antigen-presenting cells, lymphocytes and innate immune cells. Carcinogens can influence the tumor microenvironment through effects on epithelial cells, the most common origin of cancer, as well as on stromal cells, extracellular matrix components and immune cells. Here, we review how environmental exposures can perturb the tumor microenvironment. We suggest a role for disrupting chemicals such as nickel chloride, Bisphenol A, butyltins, methylmercury and paraquat as well as more traditional carcinogens, such as radiation, and pharmaceuticals, such as diabetes medications, in the disruption of the tumor microenvironment. Further studies interrogating the role of chemicals and their mixtures in dose-dependent effects on the tumor microenvironment could have important general mechanistic implications for the etiology and prevention of tumorigenesis.

Diffusion-weighted MRI in pretreatment prediction of response to neoadjuvant chemotherapy in patients with breast cancer.

PURPOSE: To evaluate the accuracy of the apparent diffusion coefficient (ADC) provided by diffusion-weighted imaging (DWI) in predicting the response to neoadjuvant chemotherapy (NACT) at baseline in patients according to their breast tumour phenotypes. MATERIALS & METHODS: This retrospective study was approved by our institutional review board. One hundred eighteen consecutive women with locally advanced breast cancer who had undergone NACT followed by breast surgery were included. DWI was performed at 1.5 T less than 2 weeks before NACT. We studied the correlation between pretreatment ADC and response in pathology after surgery according to immunohistochemical features and intrinsic subtypes (luminal A, luminal B, HER2-enriched, and triple-negative tumours). RESULTS: After surgery, the pathologist recognized 24 complete responders (CRps) and 94 non-complete responders (NCRps). No difference was identified between the pretreatment ADCs of the CRp and NCRp patients. There were differences in pretreatment ADCs among the luminal A (1.001 ± 0.143 × 10(-3) mm(2)/s), luminal B (0.983 ± 0.150 × 10(-3) mm(2)/s), HER2-enriched (1.132 ± 0.216 × 10(-3) mm(2)/s), and triple-negative (1.168 ± 0.245 × 10(-3) mm(2)/s; P = 0.0003) tumour subtypes. In triple-negative tumours, the pretreatment ADC was higher in NCRp (1.060 ± 0.143 × 10(-3) mm(2)/s) than in CRp patients (1.227 ± 0.271 × 10(-3) mm(2)/s; P = 0.047). CONCLUSION: Pretreatment ADC can predict the response of breast cancer to NACT if tumour subtypes are considered. Key Points • Apparent diffusion coefficient helps clinicians to assess patients with breast cancer. • Pretreatment ADC is related to tumour grade and hormone receptor status. • Pretreatment ADC is lower in luminal A and B than in triple-negative tumours. • Pretreatment ADC is higher in complete than in non-complete responders to neoadjuvant chemotherapy.

[BMP and cancer: the Yin and Yang of stem cells]. / BMP et cancer: le Yin et le Yang des cellules souches.

In a normal context, bone morphogenetic proteins (BMPs), members of the TGFß superfamily, are key players in adult stem cell biology. They are involved in the control of the overall functional and phenotypic properties of the stem cell population (self-renewal, proliferation, differentiation, apoptosis, quiescence, etc.). They can act directly on the stem cell or through its microenvironment, contributing to the tight balance of this system. In the tumorigenic context, alterations of the BMP signalling are involved in the deregulation of the interaction between stem cells and their microenvironment and, as such, participate to the different steps of the transformation process.

CCL22-based peptide vaccines induce anti-cancer immunity by modulating tumor microenvironment.

CCL22 is a macrophage-derived immunosuppressive chemokine that recruits regulatory T cells through the CCL22:CCR4 axis. CCL22 was shown to play a key role in suppressing anti-cancer immune responses in different cancer types. Recently, we showed that CCL22-specific T cells generated from cancer patients could kill CCL22-expressing tumor cells and directly influence the levels of CCL22 in vitro. The present study aimed to provide a rationale for developing a CCL22-targeting immunotherapy. Vaccination with CCL22-derived peptides induced CCL22-specific T-cell responses in both BALB/c and C57BL/6 mice, assessed by interferon-γ secretion ex vivo. Anti-tumor efficacy of the peptides was evaluated in mouse models engrafted with syngeneic tumor models showing a reduced tumor growth and prolonged survival of the treated mice. Vaccination induced changes in the cellular composition of immune cells that infiltrated the tumor microenvironment assessed with multicolor flow cytometry. In particular, the infiltration of CD8+ cells and M1 macrophages increased, which increased the CD8/Treg and the M1/M2 macrophage ratio. This study provided preclinical evidence that targeting CCL22 with CCL22 peptide vaccines modulated the immune milieu in the tumor microenvironment. This modulation led to an augmentation of anti-tumor responses. This study provided a rationale for developing a novel immunotherapeutic modality in cancer.

The CD10 enzyme is a key player to identify and regulate human mammary stem cells.

The major components of the mammary ductal tree are an inner layer of luminal cells, an outer layer of myoepithelial cells, and a basement membrane that separates the ducts from the underlying stroma. Cells in the outer layer express CD10, a zinc-dependent metalloprotease that regulates the growth of the ductal tree during mammary gland development. To define the steps in the human mammary lineage at which CD10 acts, we have developed an in vitro assay for human mammary lineage progression. We show that sorting for CD10 and EpCAM cleanly separates progenitors from differentiated luminal cells and that the CD10-high EpCAM-low population is enriched for early common progenitor and mammosphere-forming cells. We also show that sorting for CD10 enriches sphere-forming cells from other tissue types, suggesting that it may provide a simple tool to identify stem or progenitor populations in tissues for which lineage studies are not currently possible. We demonstrate that the protease activity of CD10 and the adhesion function of beta1-integrin are required to prevent differentiation of mammary progenitors. Taken together, our data suggest that integrin-mediated contact with the basement membrane and cleavage of signaling factors by CD10 are key elements in the niche that maintains the progenitor and stem cell pools in the mammary lineage.

Arrayed Molecular Barcoding of Leukemic Stem Cells.

Functional screens on cancer cells using compound or protein libraries are usually performed in vitro. However, to assess the effects on leukemia stem cells (LSCs) in a screening setting, methodologies that allow for a high-throughput in vivo readout of leukemia-initiating activity are needed. One experimental approach to solve this issue is to genetically label, also referred to as "barcoding," the leukemia cells in an arrayed format prior to exposing them to separate experimental conditions. The cells can then be pooled and injected into mice for competitive readout of leukemia-initiating activity. Here, we describe a procedure for combining lentiviral arrayed molecular barcoding of leukemia cells with next-generation sequencing, to enable screens on leukemia cells ex vivo followed by an in vivo competitive readout of LSC function. This methodology can also be applied to other model systems in which a competitive in vivo readout of cells is needed.

Arginase 1-Based Immune Modulatory Vaccines Induce Anticancer Immunity and Synergize with Anti-PD-1 Checkpoint Blockade.

Expression of the L-arginine catabolizing enzyme arginase 1 (ARG1) is a central immunosuppressive mechanism mediated by tumor-educated myeloid cells. Increased activity of ARG1 promotes the formation of an immunosuppressive microenvironment and leads to a more aggressive phenotype in many cancers. Intrinsic T-cell immunity against ARG1-derived epitopes in the peripheral blood of cancer patients and healthy subjects has previously been demonstrated. To evaluate the antitumor efficacy of ARG1-derived peptide vaccines as a monotherapy and as a combinational therapy with checkpoint blockade, different in vivo syngeneic mouse tumor models were utilized. To evaluate the antitumor effects, flow cytometry analysis and IHC were performed on tumors, and ELISPOT assays were performed to characterize immune responses. We show that ARG1-targeting therapeutic vaccines were able to activate endogenous antitumor immunity in several in vivo syngeneic mouse tumor models and to modulate the cell composition of the tumor microenvironment without causing any associated side effects or systemic toxicity. ARG1-targeting vaccines in combination with anti-PD-1 also resulted in increased T-cell infiltration, decreased ARG1 expression, reduced suppressive function of tumor-educated myeloid cells, and a shift in the M1/M2 ratio of tumor-infiltrating macrophages. These results indicated that the induced shift toward a more proinflammatory microenvironment by ARG1-targeting immunotherapy favors effective tumor control when combined with anti-PD-1 checkpoint blockade. Our data illustrate the ability of ARG1-based immune modulatory vaccination to elicit antigen-specific immunosurveillance and imply the feasibility of this novel immunotherapeutic approach for clinical translation.

CXCR4 Signaling Has a CXCL12-Independent Essential Role in Murine MLL-AF9-Driven Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is defined by an accumulation of immature myeloid blasts in the bone marrow. To identify key dependencies of AML stem cells in vivo, here we use a CRISPR-Cas9 screen targeting cell surface genes in a syngeneic MLL-AF9 AML mouse model and show that CXCR4 is a top cell surface regulator of AML cell growth and survival. Deletion of Cxcr4 in AML cells eradicates leukemia cells in vivo without impairing their homing to the bone marrow. In contrast, the CXCR4 ligand CXCL12 is dispensable for leukemia development in recipient mice. Moreover, expression of mutated Cxcr4 variants reveals that CXCR4 signaling is essential for leukemia cells. Notably, loss of CXCR4 signaling in leukemia cells leads to oxidative stress and differentiation in vivo. Taken together, our results identify CXCR4 signaling as essential for AML stem cells by protecting them from differentiation independent of CXCL12 stimulation.

Agonistic targeting of TLR1/TLR2 induces p38 MAPK-dependent apoptosis and NFκB-dependent differentiation of AML cells.

Acute myeloid leukemia (AML) is associated with poor survival, and there is a strong need to identify disease vulnerabilities that might reveal new treatment opportunities. Here, we found that Toll-like receptor 1 (TLR1) and TLR2 are upregulated on primary AML CD34+CD38- cells relative to corresponding normal bone marrow cells. Activating the TLR1/TLR2 complex by the agonist Pam3CSK4 in MLL-AF9-driven human AML resulted in induction of apoptosis by p38 MAPK-dependent activation of Caspase 3 and myeloid differentiation in a NFκB-dependent manner. By using murine Trp53-/-MLL-AF9 AML cells, we demonstrate that p53 is dispensable for Pam3CSK4-induced apoptosis and differentiation. Moreover, murine AML1-ETO9a-driven AML cells also were forced into apoptosis and differentiation on TLR1/TLR2 activation, demonstrating that the antileukemic effects observed were not confined to MLL-rearranged AML. We further evaluated whether Pam3CSK4 would exhibit selective antileukemic effects. Ex vivo Pam3CSK4 treatment inhibited murine and human leukemia-initiating cells, whereas murine normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected. Consistent with these findings, primary human AML cells across several genetic subtypes of AML were more vulnerable for TLR1/TLR2 activation relative to normal human HSPCs. In the MLL-AF9 AML mouse model, treatment with Pam3CSK4 provided proof of concept for in vivo therapeutic efficacy. Our results demonstrate that TLR1 and TLR2 are upregulated on primitive AML cells and that agonistic targeting of TLR1/TLR2 forces AML cells into apoptosis by p38 MAPK-dependent activation of Caspase 3, and differentiation by activating NFκB, thus revealing a new putative strategy for therapeutically targeting AML cells.

BMP2, a key to uncover luminal breast cancer origin linked to pollutant effects on epithelial stem cells niche.

Chronic exposure of epithelial cells to high levels of bone morphogenetic protein 2 (BMP2) has recently been demonstrated to initiate stem cell transformation toward a luminal tumor-like phenotype through a BMP2-BMPR1B-dependent mechanism. Carcinogen-driven deregulation of the stem cell niche could therefore represent a driving force to promote transformation and dictate the ultimate breast tumor subtype.

Disequilibrium of BMP2 levels in the breast stem cell niche launches epithelial transformation by overamplifying BMPR1B cell response.

Understanding the mechanisms of cancer initiation will help to prevent and manage the disease. At present, the role of the breast microenvironment in transformation remains unknown. As BMP2 and BMP4 are important regulators of stem cells and their niches in many tissues, we investigated their function in early phases of breast cancer. BMP2 production by tumor microenvironment appeared to be specifically upregulated in luminal tumors. Chronic exposure of immature human mammary epithelial cells to high BMP2 levels initiated transformation toward a luminal tumor-like phenotype, mediated by the receptor BMPR1B. Under physiological conditions, BMP2 controlled the maintenance and differentiation of early luminal progenitors, while BMP4 acted on stem cells/myoepithelial progenitors. Our data also suggest that microenvironment-induced overexpression of BMP2 may result from carcinogenic exposure. We reveal a role for BMP2 and the breast microenvironment in the initiation of stem cell transformation, thus providing insight into the etiology of luminal breast cancer.

A protocol to quantify mammary early common progenitors from long-term mammosphere culture.

Here we describe a protocol established in our laboratory to quantify early common progenitors/stem cells grown as spheres in non-adherent culture conditions. This protocol is based on the combination of two functional tests: the mammosphere assay to maintain and enrich early mammary progenitors/stem cells, and the epithelial colony-forming cells (E-CFC) assay to identify and quantify further differentiated epithelial progenitors. Primary spheres mainly contain progenitors and rare stem/early common progenitor cells while secondary and tertiary spheres contain progenitor cells derived from the early common progenitor/stem cell population maintained through passages and partially differentiated. Spheres are enzymatically and mechanically dissociated; the derived cells are subsequently plated on irradiated NIH-3T3 fibroblasts for further processing, as in the E-CFC assay. The principle of this assay is to quantify the number of epithelial colonies generated by cells present in the different sequential spheres. This assay has therefore been named ECP-DC, standing for Early Common Progenitor-Derived Colonies assay.

Determinants of hypertension control in a large French population of treated hypertensive subjects.

Only a minority of all hypertensives is well controlled in the population. In order to assess the proportion of well controlled hypertensives and the factors associated with hypertension control in France, we designed an observational cross-sectional epidemiological study in a population of 4702 treated adult hypertensives selected by general practitioners: EPISTRAT. This hypertensive treated population presented the following characteristics (mean+/-standard deviation): age: 60+/-12 years; blood pressure: 151+/-16/87+/-10 mmHg; men: 58%; body mass index: 27+/-5 kg/m2; diabetes mellitus: 12%; subjects in secondary cardiovascular prevention: 14%. Half of the patients presented two or more CV risk factors in addition to hypertension. Forty-eight per cent of the subjects were treated with antihypertensive monotherapy, 31% with bitherapy and 21% with more than two drugs. Patients with controlled hypertension (<140/90 mmHg) represented "only" 18% of the population. Multivariate analysis showed that male gender and advanced age were the two main variables independently associated with poor blood pressure control. Finally, the majority of patients experienced at least one antihypertensive treatment modification, mainly for insufficient therapeutic effect. In conclusion, this study has shown poor blood pressure control in a primary care-recruited population, especially in males and in the elderly.