Supplementary Data from SC68896, a Novel Small Molecule Proteasome Inhibitor, Exerts Antiglioma Activity <i>In vitro</i> and <i>In vivo</i>

Supplementary Data from SC68896, a Novel Small Molecule Proteasome Inhibitor, Exerts Antiglioma Activity In vitro and In vivo</i

The final size of regenerating splenic tissue is determined by the number of LTβR expressing cells at distant sites.

(A) Weight of splenic regenerates eight weeks of implantation into 4 types of recipients: i) WT mice that expressed LTβR in spleen (+) as well in other tissues (+); ii) splenectomized WT mice that expressed LTβR not in the spleen (-) but only in other tissues (+); iii) LTβR deficient mice that received WT splenic implants 8 weeks earlier, therefore expressing LTβR only within the regenerated splenic tissue (+) but not in other tissues (-); iv) LTβR deficient mice expressing LTβR neither in the spleen (-) nor in other tissues (-). Indicated are means and standard deviation (n = 5–9, ** = p < 0.01). (B) Cell number of splenic regenerates eight weeks of implantation into 4 types of recipients. Indicated are means and standard deviation (n = 5–9, ** = p < 0.01). (C) Indicated is the weight of the splenic implant and the weight of the resulting regenerate 8 weeks after implantation. There was no correlation between both parameters (Spearman-ρ = -0.1). The broken line indicates the mean. Each dot represents one animal. These experiments were 2 times independently performed.</p

The two faces of the LTβR signaling pathway.

After LTα1β2 expressed by activated B and T cell binds to LTβR, stromal cells up-regulate the secretion of chemokines (e.g. CCL19, CCL21, CXCL13) and the expression of adhesion molecules (e.g. ICAM-1, VCAM-1, MAdCAM-1). This further attracts LTα1β2 expressing cells and establishes a positive feed-back loop that is essential for the development and maintenance of secondary lymphoid organs (left side, green). The present study now indicates that ligation of the LTβR also leads to production of growth inhibitory factor(s) which upon binding to unknown receptors expressed by not yet characterized cells might counteract the positive feed-back loop. Interference with stromal cell turnover and/or angiogenesis might be possible targets (right side, red). When reaching a threshold level, LTβR dependent supportive and suppressive activities are in balance and secondary lymphoid organs homeostasis is achieved.</p

LTβR dependent growth suppressive activity also affects fully developed splenic tissue.

<p>(A) WT recipients without prior splenectomy were either sham operated or received WT or LTβR^-/- splenic implants. Eight weeks later weight (left side) and cell number (right side) of the endogenous spleen was determined. Only WT splenic regenerates significantly reduced weight and cell number of the endogenous WT spleen. Indicated are means and standard deviation (n = 4–11, * = p < 0.05, ** = p < 0.01). (B) LTβR^-/- recipients without prior splenectomy were either sham operated or received WT or LTβR^-/- splenic implants. Eight weeks later weight (left side) and cell number (right side) of the endogenous spleen was determined. Only WT splenic regenerates significantly reduced weight and cell number of the endogenous LTβR^-/- deficient spleen. Indicated are means and standard deviation (n = 5–8, ** = p < 0.01). These experiments were 2 times independently performed.</p

LTβR expression by non-splenic tissues suppresses growth of regenerating splenic tissue.

<p>(A) Macroscopic appearance of splenic regenerates 8 weeks after implantation of wild-type splenic tissue (WT) into wild-type recipients (WT), LTβR deficient splenic tissue (LTβR^-/-) into WT recipients, and WT splenic tissue into LTβR deficient recipients (LTβR^-/-). (B) Weight (left side) and cell number (right side) of splenic regenerates. Indicated are means and standard deviation (n = 4–9, ** = p < 0.01). (C) Microscopic appearance of splenic regenerates. Cryostat sections were stained by immunohistochemistry for T cells (brown; TCRβ⁺) and B cells (blue; B220⁺). Red pulp (RP), T-cell zone (T), and B-cell zone (B) are well developed except in the LTβR^-/- into WT combination where T and B cells are intermixed and only the red pulp (RP) is clearly recognizable (bar: 100 μm). This experiment was 3 times independently performed.</p

The splenic transplantation model.

<p>After removal of the endogenous spleen the recipient (WT or LTβR^-/-) receives a splenic implant which is composed of two pieces representing 50% of a WT or LTβR deficient spleen. The implanted splenic tissue first becomes necrotic because the blood supply is lacking. Then, formation of splenic tissue starts and within eight weeks splenic regeneration is completed.</p

Identification of LTβR regulated proteins by two-dimensional differential gel electrophoresis and mass spectrometry.

<p>(A) 2D-DIGE experiment performed with splenic stroma of LTβR^-/- (Cy3, green) and WT (Cy5, red) mice. The Cy2 channel is masked (representative gel of three replicas). The encircled area is shown in (B). (B) Gel section showing four spots with an increase in volume ratio when LTβR^-/- (left side) and WT (right side) splenic stroma was compared. Mass spectrometry identified the four spots as four isoforms of MFAP4 (representative gel of three replicas). (C) Indicated is the MFAP4 abundance in splenic stroma of LTβR^-/- spleen, LTβR^-/- spleen from a donor which received a WT splenic implant 8 weeks earlier (LTβR^-/- + WT), and WT spleen. The different 2D-DIGE experiments were compared quantitatively by summing up the volume ratios of the four isoforms of MFAP4 and relating it to the abundance of MFAP4 in LTβR^-/- splenic stroma which was set to one. The abundance of MFAP4 increased from LTβR^-/- splenic stroma to LTβR^-/- splenic stroma obtained from mice with a WT splenic regenerate and further to WT splenic stroma. Indicated are means and standard deviation (n = 3–6). This experiment was 2 times independently performed. (D) Western blot analysis of MFAP4 from splenic stroma lysates of LTβR^-/- mice, LTβR^-/- mice which received a WT splenic implant 8 weeks earlier (LTβR^-/- + WT), and WT mice. Three different sets of experiments are shown.</p

Architecture and cellular structure of WT spleen and WT into LTβR^-/- splenic regenerates are comparable.

<p>(A) The compartment sizes of normal WT spleens were analyzed and compared to that of splenic regenerates (WT→LTβR^-/-) 8 weeks after implantation. Indicated is the size of individual splenic zones as percent of total section area (n = 6–7 animals per group; RP: red pulp, MZ: marginal zone, WP: white pulp, T: T-cell zone, B: B-cell zone, T/B: mixed T/B-cell zone). (B) Frequency of leukocyte subsets in normal WT spleens and splenic regenerates (WT→LTβR^-/-) as determined by flow cytometry (n = 5–8 animals per group; B: B cells, IgD^- B: memory B cells, T: T cells; CD8: CD8⁺ T cells, CD4: CD4⁺ T cells, CD25: CD25⁺CD4⁺ -regulatory- T cells). (C) Expression of cytokines and chemokines in normal WT spleens and splenic regenerates (WT→ LTβR^-/-). Shown is the relative expression level normalized to the housekeeping gene (MLN 51) and relative to WT (fold expression change). The bars represent the mean values ± SD. Mann-Whitney <i>U</i> test was used to indicate a significant difference between WT spleens and splenic regenerates (*p < 0.05; ***p < 0.001).</p

MFAP4 does not suppress the regeneration of splenic implants.

<p>(A) WT and MFAP4 deficient recipients were splenectomized and then received a WT splenic implant. Eight weeks later the weight of the splenic regenerate was determined. No difference was seen between WT and MFAP4 deficient recipients. Indicated are means and standard deviation (n = 6). (B) WT and MFAP4 deficient recipients without prior splenectomy received a WT splenic implant. Eight weeks later the weight of the splenic regenerate was determined. No difference was seen between WT and MFAP4 deficient recipients. In addition, the suppressive activity of the endogenous spleen was similar in both groups (compare weights to the weights under A). Indicated are means and standard deviation (n = 6). These experiments were 2 times independently performed.</p