CXCL1 mediates obesity-associated adipose stromal cell trafficking and function in the tumour microenvironment.

White adipose tissue (WAT) overgrowth in obesity is linked with increased aggressiveness of certain cancers. Adipose stromal cells (ASCs) can become mobilized from WAT, recruited by tumours and promote cancer progression. Mechanisms underlying ASC trafficking are unclear. Here we demonstrate that chemokines CXCL1 and CXCL8 chemoattract ASC by signalling through their receptors, CXCR1 and CXCR2, in cell culture models. We further show that obese patients with prostate cancer have increased epithelial CXCL1 expression. Concomitantly, we observe that cells with ASC phenotype are mobilized and infiltrate tumours in obese patients. Using mouse models, we show that the CXCL1 chemokine gradient is required for the obesity-dependent tumour ASC recruitment, vascularization and tumour growth promotion. We demonstrate that αSMA expression in ASCs is induced by chemokine signalling and mediates the stimulatory effects of ASCs on endothelial cells. Our data suggest that ASC recruitment to tumours, driven by CXCL1 and CXCL8, promotes prostate cancer progression.

Treatment of obesity as a potential complementary approach to cancer therapy.

Obesity has long been recognized as a risk factor for diabetes and cardiovascular disease. Recent epidemiological data also associate obesity with cancer risk and progression. For this reason, a combination treatment of obesity along with treatment of the cancer itself may improve patient survival and well-being. As the molecular pathways linking obesity and cancer become better understood, new potential therapy targets are surfacing. In this article, we summarize the mechanisms proposed to account for the obesity-cancer association and discuss approaches to manipulation of adipose tissue as potential interventions aimed at cancer prevention or supplemental therapy.

Stromal progenitor cells from endogenous adipose tissue contribute to pericytes and adipocytes that populate the tumor microenvironment.

Epidemiologic studies associate cancer with obesity, but the pathophysiologic connections remain obscure. In this study, we show that obesity facilitates tumor growth in mice irrespective of concurrent diet, suggesting a direct effect of excess white adipose tissue (WAT). When transplanted into mice, adipose stromal cells (ASC) can serve as perivascular adipocyte progenitors that promote tumor growth, perhaps helping explain the obesity-cancer link. In developing this hypothesis, we showed that ASCs are expanded in obesity and that they traffic from endogenous WAT to tumors in several mouse models of cancer. Strikingly, a comparison of circulating and tumor-infiltrating cell populations in lean, and obese mice revealed that cancer induces a six-fold increase of ASC frequency in the systemic circulation. We obtained evidence that ASCs mobilized in this way can be recruited into tumors, where they can be incorporated into blood vessels as pericytes and they can differentiate into adipocytes in an obesity-dependent manner. Extending this evidence, we found that increased tumor vascularization (reflected by changes in tumor vascular morphology and a two-fold increase in vascular density) was associated with intratumoral adipocytes and elevated proliferation of neighboring malignant cells. Taken together, our results suggest that ASCs recruited from endogenous adipose tissue can be recruited by tumors to potentiate the supportive properties of the tumor microenvironment.

Hematopoietic stem cell characterization and isolation.

Hematopoietic stem cells (HSCs) are defined by the capabilities of multi-lineage differentiation and long-term self-renewal. Both these characteristics contribute to maintain the homeostasis of the system and allow the restoration of hematopoiesis after insults, such as infections or therapeutic ablation. Reconstitution after lethal irradiation strictly depends on a third, fundamental property of HSCs: the capability to migrate under the influence of specific chemokines. Directed by a chemotactic compass, after transplant HSCs find their way to the bone marrow, where they eventually home and engraft. HSCs represent a rare population that primarily resides in the bone marrow with an estimated frequency of 0.01% of total nucleated cells. Separating HSCs from differentiated cells that reside in the bone marrow has been the focus of intense investigation for years. In this chapter, we will describe in detail the strategy routinely used by our laboratory to purify murine HSCs, by exploiting their antigenic phenotype (KSL), combined with the physiological capability to efficiently efflux the vital dye Hoechst 33342, generating the so-called Side Population, or SP.

The orphan nuclear receptor Nurr1 restricts the proliferation of haematopoietic stem cells.

Successful haematopoiesis requires long-term retention of haematopoietic stem cells (HSCs) in a quiescent state. The transcriptional regulation of stem cell quiescence, especially by factors with specific functions in HSCs, is only beginning to be understood. Here, we demonstrate that Nurr1, a nuclear receptor transcription factor, has such a regulatory role. Overexpression of Nurr1 drives early haematopoietic progenitors into quiescence. When stem cells overexpressing Nurr1 are transplanted into lethally irradiated mice, they localize to the bone marrow, but do not contribute to regeneration of the blood system. Furthermore, the loss of only one allele of Nurr1 is sufficient to induce HSCs to enter the cell cycle and proliferate. Molecular analysis revealed an association between Nurr1 overexpression and upregulation of the cell-cycle inhibitor p18 (also known as INK4C), suggesting a mechanism by which Nurr1 could regulate HSC quiescence. Our findings provide critical insight into the transcriptional control mechanisms that determine whether HSCs remain dormant or enter the cell cycle and begin to proliferate.

Hematopoietic fingerprints: an expression database of stem cells and their progeny.

Hematopoietic stem cells (HSCs) continuously regenerate the hematologic system, yet few genes regulating this process have been defined. To identify candidate factors involved in differentiation and self-renewal, we have generated an expression database of hematopoietic stem cells and their differentiated progeny, including erythrocytes, granulocytes, monocytes, NK cells, activated and naive T cells, and B cells. Bioinformatic analysis revealed HSCs were more transcriptionally active than their progeny and shared a common activation mechanism with T cells. Each cell type also displayed unique biases in the regulation of particular genetic pathways, with Wnt signaling particularly enhanced in HSCs. We identified approximately 100-400 genes uniquely expressed in each cell type, termed lineage "fingerprints." In overexpression studies, two of these genes, Zfp 105 from the NK cell lineage, and Ets2 from the monocyte lineage, were able to significantly influence differentiation toward their respective lineages, demonstrating the utility of the fingerprints for identifying genes that regulate differentiation.

Regulating gene expression using self-inactivating lentiviral vectors containing the mifepristone-inducible system.

Methods to regulate gene expression in vitro and in vivo are currently areas of intense research. The present study, therefore, was designed to determine the efficacy of transgene expression using the GeneSwitch mifepristone-regulatable system within the context of an integrating HIV-1 vector. Lentiviral transfer plasmids expressing the red (DsRed2) and green fluorescent protein (EGFP) markers were constructed for in vitro assessment on the basal and mifepristone-induced cell activation levels by FACS analyses. In our design, efficient cell activation and transgene expression were found using a binary lentivector system i.e., the trans-activator, Switch, and the inducible promoter-transgene expression cassette were cloned into separate vectors. Note that the Switch trans-activator performed optimally when cloned into the reverse-orientation, but the inducible promoter containing lentivector did not appear to be dependent upon the orientation within the lentivector backbone. This binary lentivector system resulted in tightly regulated transgene expression, with low basal cell activation in the absence of mifepristone (MFP). Upon induction, a 41- to 275-fold increase in the number of DsRed2- and EGFP-positive cells were detected (n=3). To determine the inducing ability of the GeneSwitch, we cloned the human alpha(1)-antitrypsin cDNA into the optimal lentiviral vector and transduced HeLa and Huh7 cells at increasing lentivector doses as determined by p24 Gag ELISA. We found that MFP could induce the expression of hAAT protein in HeLa cells from 310 to 15,000 ng hAAT/10(6) cells/24 h, which was a 48-fold induction. Similar results were observed in huH7 cells. In all, this study demonstrates that the GeneSwitch system can be designed within the context of a lentiviral vector for in vitro gene transfer, and this may also provide a viable method for temporally regulating gene expression for therapeutic applications in vivo or ex vivo.