Radiolabeled HRG homes to the perivascular area and to inflammatory cells.

<p>Micro-autoradiography of ¹²⁵I-mHRG in the spleen and T241 fibrosarcomas (Tumor) tissue at 15 min post injection of radiolabeled HRG. A. Panels show tumor and spleen tissues from mice injected with PBS (left) or ¹²⁵I-mHRG (middle and right). Immunohistochemical staining with CD31 antibodies show endothelial cells colocalized with ¹²⁵I-mHRG in the middle panels (arrows). Panels to the right show the retention of ¹²⁵I-mHRG alone. B. Panels as above but immunohistochemical staining with CD45 antibodies to identify leukocytes. Arrowheads in the middle panel indicate colocalization of CD45-positive leukocytes and ¹²⁵I-mHRG. Panels to the right show the retention of ¹²⁵I-mHRG alone.</p

Biodistribution of ¹²⁵I-mHRG in normal and tumor-bearing C57Bl/6 mice.

<p>Uptake is expressed as % ID/g and presented as an average value from 4 animals ± S.D.</p><p>“Naive” and “Tumor”, indicate healthy mice and mice challenged with subcutaneous T241 fibrosarcomes, respectively.</p>§<p>) Data for intestines with content and carcass are presented as %ID per whole sample.</p><p>*) Significant (<i>p</i><0.05) difference between normal and tumor bearing mice at this time point.</p>#<p>) Significant (<i>p</i><0.05) difference between hHRG and mHRG at this time point.</p>‡<p>) Significant (<i>p</i><0.05) difference between tumor and muscle at this time point.</p><p>Biodistribution of ¹²⁵I-mHRG in normal and tumor-bearing C57Bl/6 mice.</p

Comparative biodistribution of ¹²⁵I-hHRG and ¹²⁵I-albumin in female C57Bl/6 mice.

<p>Uptake is expressed as % ID/g and presented as an average value from 4 animals ± standard deviation.</p><p>§) Data for intestines with content and carcass are presented as %ID per whole sample.</p><p>* Significant (<i>p</i><0.05) difference between hHRG and albumin at this time point.</p><p>Comparative biodistribution of ¹²⁵I-hHRG and ¹²⁵I-albumin in female C57Bl/6 mice.</p

Purification and bioactivity of radiolabeled HRG.

<p>A. Coomassie brilliant blue-stained SDS-PAGE showing purified mHRG and hHRG protein (0.5 µg and 4 µg/lane). B. SDS-PAGE analysis of freshly radiolabelled ¹²⁵I-mHRG (top) and after incubation in murine plasma at 37°C for 15 min (middle) and 3 h (bottom) visualized using Cyclone Storage Phosphor system. C. Binding of ¹²⁵I-hHRG and ¹²⁵I-mHRG to heparin-coated plastic <i>in vitro</i> in the presence (blocked) and absence (unblocked) of unlabeled HRG; *p<0.05; Student's t test. D. Inhibition of human umbilical vein endothelial cell migration towards VEGFA by non-radioactive iodinated hHRG and mHRG (HRG-I). *p<0.05; **p<0.01; Student's t test.</p

Unusually rapid biodistribution of radiolabeled HRG.

<p>A. Blood kinetics of ¹²⁵I-albumin, ¹²⁵I-hHRG and ¹²⁵I-mHRG in C57BL/6 mice. (n = 4/time point). B. Biodistribution of ¹²⁵I-hHRG in selected C57BL/6 mouse organs. C. Percentage of radioactivity in blood plasma, associated with a high molecular-weight fraction (>5 kDa). D. Biodistribution of ¹²⁵I-mHRG in selected organs of naive C57BL/6 mice. E. Liver uptake of ¹²⁵I-mHRG and ¹²⁵I-mHRG. F. Biodistribution of ¹²⁵I-mHRG in selected organs of T241 fibrosarcoma-bearing C57BL/6 mice. G. TCA-precipitable ¹²⁵I-radioactivity in plasma after 2 h of circulation in naive and tumor-bearing mice injected with ¹²⁵I-mHRG.</p

Schematic outline of HRG's interaction with its receptor on mononuclear phagocytes.

<p>HRG is shown produced in the liver, distributed in the circulation bound to mononuclear phagocytes which home to sites of inflammation. Binding of HRG to the HRG receptor (HRGR) leads internalization, degradation and thereby regulation of HRG turnover.</p

Binding of radiolabeled HRG to U937 cells.

<p>A. Binding of ¹²⁵I-mHRG to differentiated U937 cells (treated with 1a, 25(OH)₂D₃ (VitD3) for 24 h) was competed with a 10-fold concentration of unlabeled (cold) HRG on cells treated or not with heparitinase. *; p<0.05, Student's t-test. ns; not significant. B. Binding of ¹²⁵I-mHRG to undifferentiated or differentiated U937 cells (treated with 1a, 25(OH)₂D₃ (VitD3) for 24 h) was competed with a 10-fold concentration of unlabeled (cold) HRG. All cells were treated with heparitinase. *; p<0.05, Student's t-test. ns; not significant.</p

Stable HRG levels in livers and plasma from T241 fibrosarcoma-bearing mice and plasma in CRC patients.

<p>ELISA showing HRG levels in plasma (A) and livers (B) from naïve and T241 fibrosarcoma-bearing mice terminated at early (day 11 after inoculation) or late (day 21) stages. C. ELISA showing HRG levels in the plasma of healthy individuals (n = 66) and patients with CRC stage 1–4 (n = 146). p = 0.103, t-test.</p

HRG detected by IHC of CRC tumor tissue arrays.

<p>A. Scoring of HRG IHC signals associated with inflammatory cells in CRC arrays from strong to no signal. Statistical analysis; p<0.05 was considered significant. The number, n, of biopsies were; normal = 10, adenoma = 10, stage 1 = 20, stage 2 = 20, stage 3 = 20, lymph node metastasis = 10, distant metastasis = 10. B. Scoring of HRG IHC signals associated with vessels, as above. C. Upper and middle row of panels: Representative images of the HRG IHC signals from the indicated categories at 20× magnification. Lower row of panels: Representative images of the HRC IHC signals in CRC at 60× magnification. Arrows indicate typical vessel-associated HRG signals in normal colorectal tissue (left) and in inflammatory cells in normal tissue (middle) and in stage 2 CRC (right).</p

Decreased HRG turnover with anti-CSF1 antibody treatment.

<p>A. Number of CD115⁺ and CD68⁺ cells in livers of mice at day 7 of treatment of C57BL/6 mice with anti-CSF1 neutralizing antibody as compared to treatment with isotype-matched control. n = 4 for each treatment. **p<0.01, or as indicated. Student's t-test. B. Morphology of CD115⁺ cells (red) in the liver with anti-CSF1 antibody treatment or with isotype-matched control IgG antibody treatment for 7 days. Blue indicates nuclei stained with Hoechst 33342. Bar; 100 µm. C. Levels of endogenous HRG in blood in mice treated with the anti-CSF1 antibody or an isotype-matched control at different time points. N = 4 for each treatment. **p<0.01, Student's t-test. D. Liver <i>hrg</i> transcript levels at day 7 of anti-CSF1 or control IgG treatment. E. Blood kinetics of ¹²⁵I-mHRG after tail vein injection of C57BL/6 mice and circulation for 15 min or 60 min, performed at day 7 of treatment with anti-CSF1 antibody or isotype-matched control (n = 4/time point). *p<0.05, Student's t-test.</p