Rachael J. Klein

Bone Marrow Transplantation Generates Immature Oocytes and Rescues Long-Term Fertility in a Preclinical Mouse Model of Chemotherapy-Induced Premature Ovarian Failure

PURPOSE Although early menopause frequently occurs in female cancer patients after chemotherapy (CTx), bone marrow (BM) transplantation (BMT) has been linked to an unexplained return of ovarian function and fertility in some survivors. Studies modeling this in mice have shown that BMT generates donor-derived oocytes in CTx-treated recipients. However, a subsequent report claimed that ovulated eggs are not derived from BM and that BM-derived oocytes reported previously are misidentified immune cells. This study was conducted to further clarify the impact of BMT on female reproductive function after CTx using a preclinical mouse model. METHODS Female mice were administered CTx followed by BMT using coat color-mismatched female donors. After housing with males, the number of pregnancies and offspring genotype were recorded. For cell tracking, BM from germline-specific green fluorescent protein-transgenic mice was transplanted into CTx-treated wild-type recipients. Immune cells were sorted from blood and analyzed for germline markers. RESULTS BMT rescued long-term fertility in CTx-treated females, but all offspring were derived from the recipient germline. Cell tracking showed that donor-derived oocytes were generated in ovaries of recipients after BMT, and two lines of evidence dispelled the claim that these oocytes are misidentified immune cells. CONCLUSION These data from a preclinical mouse model validate a testable clinical strategy for preserving or resurrecting ovarian function and fertility in female cancer patients after CTx, thus aligning with recommendations of the 2005 National Cancer Institute Breast Cancer Progress Review Group and Presidents Cancer Panel to prioritize research efforts aimed at improving the quality of life in cancer survivors.

Tumor Stromal-Derived Factor-1 Recruits Vascular Progenitors to Mitotic Neovasculature, where Microenvironment Influences Their Differentiated Phenotypes

Mechanisms underlying tumor vasculogenesis, the homing and engraftment of bone marrow-derived vascular progenitors, remain undefined. We hypothesized that tumor cell-secreted factors regulate vasculogenesis. We studied vasculogenic and nonvasculogenic intracranial murine gliomas. A PCR screen identified stromal-derived factor-1 (SDF-1/CXCL12) and vascular endothelial growth factor (VEGF) expression by vasculogenic glioma cells and spontaneously arising vasculogenic tumors in NF1+/-:Trp53+/- mice, but not by nonvasculogenic glioma cells. Enforced SDF-1, not VEGF, expression in nonvasculogenic cells caused vasculogenesis. Combined SDF-1 and VEGF expression augmented vasculogenesis over SDF-1 expression alone. Blocking SDF-1 receptor CXCR4 reduced short-term homing and long-term engraftment of vascular progenitors. Implanting tumor cells secreting SDF-1 was therefore necessary and sufficient to incorporate marrow-derived precursors into tumor endothelium. SDF-1 seemed to exert these effects by acting locally intratumorally and did not cause an efflux of marrow-derived progenitors into circulation. Tumor microenvironment determined additional fates of marrow-derived cells. Hypoxia, observed with ectopic s.c. murine tumors at levels approximating that of intracranial human glioblastoma, interacted with tumor-secreted SDF-1 to expand engrafted vascular progenitor differentiated phenotypes to include pericytes as well as endothelium. In contrast, less hypoxic orthotopic intracranial murine gliomas contained only marrow-derived endothelium without marrow-derived pericytes. Furthermore, we found that vasculogenesis is significant for tumors because it generates endothelium with a higher mitotic index than endothelium derived from local sources. Although CXCR4 blockade selectively targeted endothelium generated by vasculogenesis, completely inhibiting vessel formation may require combination therapy targeting locally derived and marrow-derived endothelium.

Differential CD146 expression on circulating versus tissue endothelial cells in rectal cancer patients: implications for circulating endothelial and progenitor cells as biomarkers for antiangiogenic therapy.

PURPOSE Circulating endothelial cells (CECs) and progenitor cells are currently evaluated as potential biomarkers of antiangiogenic therapy. CD146 is considered a panendothelial-specific marker, but its utility as a CEC marker in cancer patients remains unclear. PATIENTS AND METHODS We analyzed the expression of CD146 on mononuclear blood cells, primary tissue endothelial cells, and malignant and normal tissues by flow cytometric and immunohistochemical analyses. Furthermore, we measured the circulation kinetics of CD146+ cells before, and then 3 and 12 days after vascular endothelial growth factor (VEGF) antibody blockade by bevacizumab infusion in rectal cancer patients enrolled in a phase I trial. RESULTS In the peripheral blood of these cancer patients, over 90% of the CD146+ cells were CD45+ hematopoietic cells. CD146 expression was primarily detected on a subset of CD3+CD4+ lymphocytes, and was undetectable on CD34+CD133+CD45(dim) progenitor cells or CD31(bright)CD45- viable CECs. In contradistinction, CD146 was detectable in tissues on both cellular components of tumor vessel wall: CD31(bright)CD45- endothelial cells and alpha-SMA+ pericytes. Unlike viable CECs and progenitor cells, CD146+ cell concentration in the peripheral blood of cancer patients did not decrease during VEGF blockade. CONCLUSION CD146 is fairly homogeneously expressed on vascular endothelium but not on viable CECs or progenitor cells. The vast majority of CD146+ blood cells are lymphocytes, and VEGF blockade by bevacizumab did not significantly change their number in rectal cancer patients. These results underscore the need for further characterization and identification of new markers for CEC subpopulations for their development as biomarkers of antiangiogenic therapy.

Circulating CD34(+) progenitor cell frequency is associated with clinical and genetic factors.

Circulating blood CD34(+) cells consist of hematopoietic stem/progenitor cells, angiogenic cells, and endothelial cells. In addition to their clinical use in hematopoietic stem cell transplantation, CD34(+) cells may also promote therapeutic neovascularization. Therefore, understanding the factors that influence circulating CD34(+) cell frequency has wide implications for vascular biology in addition to stem cell transplantation. In the present study, we examined the clinical and genetic characteristics associated with circulating CD34(+) cell frequency in a large, community-based sample of 1786 Framingham Heart Study participants.Among subjects without cardiovascular disease (n = 1595), CD34(+) frequency was inversely related to older age, female sex, and smoking. CD34(+) frequency was positively related to weight, serum total cholesterol, and statin therapy. Clinical covariates accounted for 6.3% of CD34(+) variability. CD34(+) frequency was highly heritable (h(2) = 54%; P < .0001). Genome-wide association analysis of CD34(+) frequency identified suggestive associations at several loci, including OR4C12 (chromosome 11; P = 6.7 × 10(-7)) and ENO1 and RERE (chromosome 1; P = 8.8 × 10(-7)). CD34(+) cell frequency is reduced in older subjects and is influenced by environmental factors including smoking and statin use. CD34(+) frequency is highly heritable. The results of the present study have implications for therapies that use CD34(+) cell populations and support efforts to better understand the genetic mechanisms that underlie CD34(+) frequency.

Association of Colony-Forming Units With Coronary Artery and Abdominal Aortic Calcification

Background— Certain bone marrow-derived cell populations, called endothelial progenitor cells, have been reported to possess angiogenic activity. Experimental data suggest that depletion of these angiogenic cell populations may promote atherogenesis, but limited data are available on their relation to subclinical atherosclerotic cardiovascular disease in humans. Methods and Results— We studied 889 participants of the Framingham Heart Study who were free of clinically apparent cardiovascular disease (mean age, 65 years; 55% women). Participants underwent endothelial progenitor cell phenotyping with an early-outgrowth colony-forming unit assay and cell surface markers. Participants also underwent noncontrast multidetector computed tomography to assess the presence of subclinical atherosclerosis, as reflected by the burden of coronary artery calcification and abdominal aortic calcification. Across decreasing tertiles of colony-forming units, there was a progressive increase in median coronary artery calcification and abdominal aortic calcification scores. In multivariable analyses adjusting for traditional cardiovascular risk factors, each 1-SD increase in colony-forming units was associated with a ≈16% decrease in coronary artery calcification (P=0.02) and 17% decrease in abdominal aortic calcification (P=0.03). In contrast, neither CD34+/KDR+ nor CD34+ variation was associated with significant differences in coronary or aortic calcification. Conclusions— In this large, community-based sample of men and women, lower colony-forming unit number was associated with a higher burden of subclinical atherosclerosis in the coronary arteries and aorta. Decreased angiogenic potential could contribute to the development of atherosclerosis in humans.

Genetic and clinical correlates of early-outgrowth colony-forming units.

Background— Several bone marrow–derived cell populations may have angiogenic activity, including cells termed endothelial progenitor cells. Decreased numbers of circulating angiogenic cell populations have been associated with increased cardiovascular risk. However, few data exist from large, unselected samples, and the genetic determinants of these traits are unclear. Methods and Results— We examined the clinical and genetic correlates of early-outgrowth colony-forming units (CFUs) in 1799 participants of the Framingham Heart Study (mean age, 66 years; 54% women). Among individuals without cardiovascular disease (n=1612), CFU number was inversely related to advanced age (P=0.004), female sex (P=0.04), and triglycerides (P=0.008) and positively related to hormone replacement (P=0.008) and statin therapy (P=0.027) in stepwise multivariable analyses. Overall, CFU number was inversely related to the Framingham risk score (P=0.01) but not with prevalent cardiovascular disease. In genome-wide association analyses in the entire sample, polymorphisms were associated with CFUs at the MOSC1 locus (P=3.3×10−7) and at the SLC22A3-LPAL2-LPA locus (P=4.9×10−7), a previously replicated susceptibility locus for myocardial infarction. Furthermore, alleles at the SLC22A3-LPAL2-LPA locus that were associated with decreased CFUs were also related to increased risk of myocardial infarction (P=1.1×10−4). Conclusions— In a community-based sample, early-outgrowth CFUs are inversely associated with select cardiovascular risk factors. Furthermore, genetic variants at the SLC22A3-LPAL2-LPA locus are associated with both decreased CFUs and an increased risk of myocardial infarction. These findings are consistent with the hypothesis that decreased circulating angiogenic cell populations promote susceptibility to myocardial infarction.

Circulating CD31+ leukocyte frequency is associated with cardiovascular risk factors.

OBJECTIVES CD31 identifies a heterogeneous population of cells in the blood, consisting of mature leukocytes and platelets, as well as smaller numbers of endothelial and progenitor cells. Because unfractionated CD31+ blood cells have demonstrated angiogenic properties in vivo, we hypothesized that circulating CD31+ cells would be related to the presence of cardiovascular risk factors in humans. METHODS AND RESULTS We studied 1487 participants, free of cardiovascular disease, from the Framingham Offspring Study. Using anti-human CD31 and CD45 antibodies, distinct CD31+/CD45+ leukocyte populations were enumerated in blood samples by FACS analysis. We used linear regression analyses to investigate the relation of each cell phenotype with cardiovascular risk factors. We identified 3 distinct leukocyte populations: CD31-, CD31 dim, and CD31 bright cells. Using forward/side scatter analyses, CD31- and CD31 dim cells mapped to lymphoid gates while CD31 bright cells were monocytoid. In multivariable analyses, higher frequency of CD31 bright cells was associated with older age, male sex, HDL cholesterol, and CRP (all P < 0.01). In contrast, CD31 dim was inversely associated with age, male sex, CRP, and smoking (all P < 0.01). Framingham Risk Score was positively associated with CD31 bright frequency (P = 0.002), and negatively associated with CD31 dim frequency (P = 0.020). CONCLUSIONS CD31+ staining identifies 2 major leukocyte populations, CD31 bright and CD31 dim, which demonstrated significant and opposite associations with cardiovascular risk in humans. Further research is needed to define the biological and potential therapeutic roles of CD31+ subpopulations in vascular disease.

Analysis of the Hematopoietic Stem Cell Niche

Hematopoietic stem cells (HSCs) continuously replenish all blood cell lineages not only to maintain the normal rapid turnover of differentiated cells but also to respond to injury and stress. Cell-extrinsic mechanisms are critical determinants of the fine balance between HSC self-renewal and differentiation. The bone marrow microenvironment has emerged as a new area of intense study to identify which of its many components constitute the HSC niche and regulate HSC fate. While HSCs have been isolated, characterized and used in clinical practice for many years thanks to the development of very specific assays and technology (i.e., bone marrow transplants and fluorescence activated cell sorting), study of the HSC niche has evolved by combining experimental designs developed in different fields. In this unit we describe a collection of protocols spanning a wide range of techniques that can help every researcher tackling questions regarding the nature of the HSC niche.

Abstract 395: Host ephrinB2 regulates T-cells in tumor microenvironments

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: The tumor microenvironment consists of immune, stromal, and vascular cells. Proteins known to play roles in vascular development during embryogenesis also have been implicated in the control of leukocyte migration. The vascular patterning ligand ephrinB2, for example, influences T-cell migration in vitro. We therefore hypothesized that host ephrinB2 signaling regulates tumor immune microenvironments. Methods: EphrinB2+/lacZ mice were implanted with B16 melanoma, KR158 glioma, LLC lung, and EL4 T-lymphoma tumor cells. Macroscopic tumors were harvested and ephrinB2 expression assayed using x-gal staining. Mx1-cre/efnb2fl/fl mice were generated which allow for the post-natal deletion of ephrinB2 after pIpC treatment. Mx1-cre/efnb2+/+ mice served as controls. 1 million B16 or KR158 tumor cells were implanted in hindlimbs and tumor volume measured (V= 0.52\*L\*W2) at least twice weekly. Tumor tissues were stained with anti-CD3, F4/80, anti-CD34, and anti-NG2 antibodies. Experiments were repeated following T-cell depletion with i.p. injections of anti-CD4 and anti-CD8 antibodies 3x week. Finally, C57BL6 mice were transplanted with Mx1-cre/efnb2fl/fl or Mx1-cre/efnb2+/+ bone marrow and ephrinB2 was deleted after engraftment but prior to tumor growth experiments. Results: EphrinB2 was expressed in vascular structures all tumors. In addition we observed upregulation of ephrinB2 in microvascular structures in adjacent muscle during tumor invasion. Inducible deletion of host ephrinB2 significantly reduced B16 tumor growth (control tumor volume 2846 mm3 versus deleted tumor 869 mm3, p<0.01) and KR158 tumor growth (2028 mm3 versus 1403 mm3). EphrinB2 deletion did not affect CD34+ microvessel density, NG2+ pericyte or F4/80 macrophage infiltration. Rather, tumors grown in ephrinB2 deficient microenvironments demonstrated a significant increase in infiltrating CD3+ T-cells. In vivo depletion of T-cells abrogated the effect of ephrinB2 deletion on tumor growth delay demonstrating a role for immune cells in impairing tumor growth. EphrinB2 deletion in transplanted hematopoietic cells did not lead to tumor growth delay demonstrating a requirement for intact ephrinB2 signaling in vascular cells for T-cell regulation. Conclusion: EphrinB2 is upregulated in tumor vasculature and at sites of tumor invasion. Post-natal ephrinB2 deletion did not affect tumor angiogenesis but led to tumor growth delay via a T-cell mediated mechanism. These findings demonstrate a novel role for ephrinB2 specifically and vascular cells generally in regulating immune microenvironments. Therefore this study identifies a novel therapeutic strategy to increase the accumulation of T-cells in tumor tissues with relevance to combination immunotherapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 395. doi:1538-7445.AM2012-395