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. 2005 Jun 1;105(11):4500-7.
doi: 10.1182/blood-2004-08-3210. Epub 2005 Feb 8.

The splenic microenvironment is a source of proangiogenesis/inflammatory mediators accelerating the expansion of murine erythroleukemic cells

Yuval Shaked  1 , Dave Cervi, Manuela Neuman, Limor Chen, Giannoula Klement, Crystal R Michaud, Mehran Haeri, Brian J Pak, Robert S Kerbel, Yaacov Ben-David
Affiliations

The splenic microenvironment is a source of proangiogenesis/inflammatory mediators accelerating the expansion of murine erythroleukemic cells

Yuval Shaked et al. Blood. .

Abstract

The stromal compartments of hematopoietic organs (eg, spleen) are known to influence the viability and growth of diseased hematopoietic progenitors. Here we have used Friend murine leukemia virus (F-MuLV)-induced erythroleukemia to investigate factors of the splenic microenvironment that may make it fertile for the expansion and survival of malignant erythroblasts. We found that splenectomized, erythroleukemic mice exhibited extended survival compared with age-matched sham controls. In vitro, the proliferation of primary erythroleukemic cells cocultured with leukemic-derived splenic adherent cells or their conditioned media was found to be significantly higher than that observed in cocultures with healthy-derived adherent splenic cells. Cytokine protein arrays revealed that F-MuLV-infected splenocytes secreted elevated levels of interleukin-6 (IL-6), vascular endothelial growth factor-A (VEGF-A), macrophage chemoattractant protein-5 (MCP-5), soluble tumor necrosis factor receptor-1 (sTNFR1), IL-12p70, tumor necrosis factor-alpha (TNF-alpha), and IL-2 over normal splenocytes. Medium supplemented with both VEGF-A and MCP-5 could sustain proliferation of primary erythroleukemic cells in vitro, and significant proliferative suppression was observed upon addition of neutralizing antibodies to either of these factors. Furthermore, in vivo administration of a neutralizing antibody to VEGF-A extended survival times of erythroleukemic mice in comparison with controls. These findings suggest that VEGF-A and MCP-5 are potentially pivotal paracrine mediators occurring within the diseased splenic microenvironment capable of promoting disease acceleration and expansion of erythroleukemic blasts.

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Figures

Figure 1.
Figure 1.
Survival analysis of splenectomized and sham erythroleukemic mice. Age-matched BALB/c neonates were infected with F-MuLV according to “Materials and methods.” Splenectomy (□) and sham (▪) procedures were performed 4 weeks later followed by daily observations over the duration of the study. All deceased mice and those required to be killed according to institutional guidelines were documented according to date of death and plotted according to the method of Kaplan-Meier. Significance between splenectomy and sham groups was calculated using a Mann-Whitney U test with 2-sided P values and significant differences reflecting P < .05. Dotted horizontal line represents median survival.
Figure 2.
Figure 2.
Analysis of primary erythroleukemic cell growth cocultured with LDSACs, HDSACs, or their conditioned media. (A) Primary erythroleukemic (suspension) cells, seeded at a density of 5 × 104/mL, were cocultured with adherent cells derived in vitro either from healthy controls or mice infected with F-MuLV and presenting with Friend disease. Scoring of live cells occurred every 24 hours by trypan blue exclusion over a 72-hour time course with significance considered between LDSAC and HDSAC cocultures to reflect P < .05. (B) Primary erythroleukemic cells, seeded at a density of 1 × 104/mL, were cultured in conditioned medium previously used to grow LDSACs (○) versus HDSACs (□), hence LDSAC- and HDSAC-derived media, respectively. Their respective controls (•, ▪) represent similar experiments conducted in control media. Scoring of live cells occurred every 24 hours by trypan blue exclusion over a 72-hour time course with significance considered between different growth conditions to reflect P < .05 (*). Error bars indicate ± standard deviation.
Figure 3.
Figure 3.
Analysis of F-MuLV integration within the host genome. Genomic DNA from LDSACs (1), leukemic suspension cells (2), HDSACs (3), normal suspension cells (4), NIH-3T3 cells as negative control (5), HB60 leukemic cells as positive control (6), and 1 kbp ladder (7) was extracted. Friend virus env primers were used to amplify the integration site of the virus's env as described in “Materials and methods.”
Figure 4.
Figure 4.
Cytokine profiling of LDSAC- and HDSAC-conditioned growth medium. Mouse cytokine antibody arrays were performed using TransSignal RayBio Mouse Cytokine Antibody Array Kit (MA6050) according to the manufacturer's instructions. Membranes were incubated with LDSAC- or HDSAC-derived medium for 24 hours. Blots were developed and subsequently treated according to “Materials and methods.” Autoradiography was then performed, and intensities were measured by densitometric analysis. Following normalization of the data, with positive controls set at 100% and negative controls at 0, data were plotted and significance considered among the tested cytokines to reflect P < .05. GCSF indicates granulocyte colony stimulating factor; GM-CSF, granulocyte macrophage colony stimulating factor; INF, interferon; and IL, interleukin. Error bars indicate ± standard deviation.
Figure 5.
Figure 5.
In vitro analysis of angiogenesis-potentiated overgrowth of primary erythroleukemic cells. (A) Using the cytokine array findings as a reference, primary erythroleukemic cells, seeded at a density of 1 × 104/mL, were cultured in standard medium with the addition of exogenous VEGF-A, MCP-5, IL-6 (□); VEGF-A and MCP-5 (▪); IL-6 and VEGF-A (○); or media alone (•) as per “Materials and methods.” (B) In separate experiments, erythroleukemic cells were cultured in LDSAC-conditioned media (•) with the addition of neutralizing antibodies to VEGF-A (□), MCP-5 (○), and TNF-α (▪), or in various combinations; only combined inhibition of VEGF-A and MCP-5 (▴) is shown for simplicity. Scoring of live cells occurred every 24 hours by trypan blue exclusion over a 72-hour time course with significance considered between all tested groups to reflect P < .05 (*). Error bars represent ± standard deviation.
Figure 6.
Figure 6.
Flow cytometry, immunohistochemistry, and casting analyses of the endothelial cell composition in LDSAC and HDSAC populations. Representative flow cytometry evaluation of endothelial cells either from LDSAC or HDSAC cultures in the late stage of Friend disease. (A) The chosen region (R1) from the total cell population, negative for CD45, either from HDSAC or LDSAC adherent cell populations. (B) The percentage of gated R1 cells that are positive for flk-1 (M1) in the HDSAC population. (C) The percentage of gated R1 cells that are positive for fetal liver kinase-1 (flk-1) (M1) in the LDSAC population. Immunohistochemistry of VE-cadherin stained with 3′3-diaminobenzidine DAB (red) and counterstained with hematoxylin for blood vessel detection was performed on healthy and erythroleukemic spleen sections as described in “Materials and methods” (D,F, respectively). Casting images were performed according to “Materials and methods” on healthy and erythroleukemic spleens in late-stage disease (E,G, respectively).
Figure 7.
Figure 7.
Survival analysis of erythroleukemic mice treated with neutralizing antibody against VEGF-A. Erythroleukemic mice were randomized and divided into 3 groups: (1) control untreated group (solid line); (2) group treated with goat IgG as a control (data not shown); or (3) a group treated with a neutralizing antibody against VEGF-A (dotted line) for 2 weeks. Mice were monitored on a daily basis and killed according to institutional guidelines, and survival was assessed by the method of Kaplan-Meier. Dotted horizontal lines represent median survival.
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References

    1. Liotta LA, Kohn EC. The microenvironment of the tumour-host interface. Nature. 2001;411: 375-379. - PubMed
    1. Wernert N. The multiple roles of tumour stroma. Virchows Arch. 1997;430: 433-443. - PubMed
    1. Litwin C, Leong KG, Zapf R, Sutherland H, Naiman SC, Karsan A. Role of the microenvironment in promoting angiogenesis in acute myeloid leukemia. Am J Hematol. 2002;70: 22-30. - PubMed
    1. Rafii S, Mohle R, Shapiro F, Frey BM, Moore MA. Regulation of hematopoiesis by microvascular endothelium. Leuk Lymphoma. 1997;27: 375-386. - PubMed
    1. Goldman JM, Nolasco I. The spleen in myeloproliferative disorders. Clin Haematol. 1983;12: 505-516. - PubMed

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