Melanoma growth is reduced in <i>Cd34^−/−</i> animals at day 14, with CD34 expression on vasculature.

<p>A, Average tumor mass 14 days after subcutaneous injection of 5×10⁵ B16F1-OVA cells. (Pooled from three experiments, <i>Cd34^+/+</i> n = 12; <i>Cd34^−/−</i> n = 15). B, Average number of lung metastases 12 days after intravenous injection of 3×10⁵ B16F1-OVA cells. (<i>Cd34^+/+</i> n = 5, <i>Cd34^−/−</i> n = 6, *represents p<0.05; Error bars = SEM). C, Representative photomicrographs from s.c.-injected tumor sections stained for CD34 and CD31 or carbocyanine fluorescence, as indicated (CD31-red, CD34-blue, carbocyanine-green).</p

Reduced subcutaneous tumor size at day 14 correlates with loss of CD34 on non-hematopoietic cells.

<p>A, Average tumor mass and B, volume at day 14 in <i>Cd34^+/+</i> or <i>Cd34^−/−</i> animals reconstituted with wildtype Ly5.1 bone marrow. (Pooled from two experiments, <i>Cd34^+/+</i> n = 13, <i>Cd34^−/−</i> n = 14). C, Average tumor mass at day 14, in reciprocal reconstitutions of wildtype Ly5.1 animals with <i>Cd34^+/+</i> or <i>Cd34^−/−</i> bone marrow (n = 9, *represents p<0.05; Error bars = SEM).</p

Increased tumor cell extravasation in <i>Cd34^−/−</i> animals.

<p>A, CMFDA-labeled B16F1 cells (green) were coinjected <i>i.v.</i> with fluorescent dextran (red) and lungs were removed after 4 hours, fixed, sectioned and imaged. Representative sections are shown. Tumor cells were quantified as extravasated (green – solid arrows), or within the microvasculature (yellow – hollow arrows). Scale bar, 50 µm, inset = 2×zoom. B, Proportion (%) of tumor cells extravasated per section (n = 5 lungs per genotype, >5 sections per lung, *represents p<0.05; Error bars = SEM).</p

Increased carbocyanine leakage and altered vessel morphology in subcutaneously-injected tumors from <i>Cd34^−/−</i> animals.

<p>Carbocyanine fluorescence intensity quantified A, proximal to CD31⁺ vessels (at distance 0 µm) and B, with increasing distance from the nearest vessel (CD31⁺), in day 14 tumors. C, Average distance of each pixel within the tumor tissue from the nearest CD31⁺ staining pixel. D, Perfused vessels (%), corresponding to the percentage of CD31⁺ pixels colocalized with carbocyanine staining. For carbocyanine data, one outlier per genotype was removed prior to analysis. (n = 5 for carbocyanine, n = 6 for average distance, *represents p<0.05; **represents p<0.01; Error bars = SEM). Representative CT images of tumors excised 14 days post-injection and perfused with Microfil contrast reagent from E, <i>Cd34^+/+</i> and F, <i>Cd34^−/−</i> mice. Scale bars, 2 mm (n = 3).</p

Mast cell tumor infiltration is reduced in <i>Cd34^−/−</i> mice.

<p>Toluidine blue staining of A, peri-tumoral mast cells in tissues surrounding tumors and B, intra-tumoral mast cells within tumor tissues. (arrows, mast cells; dotted line, tumor boundary). Mast cell counts in <i>s.c.-</i>injected tumors from <i>Cd34^+/+</i> and <i>Cd34^−/−</i> animals at C, day 14 (pooled from three experiments, <i>Cd34^+/+</i> n = 13, <i>Cd34^−/−</i> n = 16) and D, day 19 (pooled from three experiments, n = 13). Counts in tumors from E, <i>Cd34^+/+</i> or <i>Cd34^−/−</i> animals reconstituted with wildtype Ly5.1 marrow at day 14 (pooled from three experiments, Ly5.1 into <i>Cd34^+/+</i> n = 13, Ly5.1 into <i>Cd34^−/−</i> n = 14) and F, in wildtype Ly5.1 animals reconstituted with either <i>Cd34^+/+</i> or <i>Cd34^−/−</i> bone marrow at day 19 (pooled from three experiments, <i>Cd34^+/+</i> into Ly5.1 n = 11, <i>Cd34^−/−</i> into Ly5.1 n = 13). (*represents p<0.05; Error bars = SEM).</p

CD34 function in the tumor microenvironment.

<p>Model schematic of CD34 function in the tumor microenvironment, highlighting effects on vascular integrity, associated with decreased tumor growth at an early time-point (day 14) and immune cell accumulation, associated with increased tumor growth at a later time-point (day 19) in <i>Cd34^−/−</i> mice.</p

Tumor size in <i>Cd34^−/−</i> surpasses <i>Cd34^+/+</i> by day 19, due to hematopoietic loss of CD34.

<p>A, Average <i>s.c.-</i>injected tumor masses 19 days post-injection in <i>Cd34^+/+</i> and <i>Cd34^−/−</i> animals. (Pooled from three experiments, <i>Cd34^+/+</i> n = 15, <i>Cd34^−/−</i> n = 16). B, Average tumor mass at day 19 in wildtype Ly5.1 animals reconstituted with either <i>Cd34^+/+</i> or <i>Cd34^−/−</i> bone marrow. (Pooled from two experiments, <i>Cd34^+/+</i> into Ly5.1 n = 11, <i>Cd34^−/−</i> into Ly5.1 n = 13). (*represents p<0.05; Error bars = SEM).</p

Localized diacylglycerol-dependent stimulation of Ras and Rap1 during phagocytosis

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcgamma receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcgamma receptor-mediated phagocytosis to enhance the innate immune response.  </p