TCM alters cytoplasmic rheology.

<p>The time-dependent ensemble averaged MSDs of 100 nm particles embedded in the cytoplasm of MSCs and 3T3 fibroblasts were converted to frequency-dependent elastic (G′, solid lines) and visous (G″, dashed lines) moduli using a custom algorithm written for Matlab software. The ensemble-averaged frequency-dependent viscoelasticities of MSCs (A–E, left) and 3T3 fibroblasts (F–J, right) prior to (A,F) and 30 minutes (B,G), 1 hour (C,H), 2 hours (D,I), or 3 hours (E,J) after treatment with TCM. The cytoplasm of MSCs became predominantly elastic within 60 minutes; whereas, 3T3 fibroblasts required 3 hours to undergo a similar change.</p

Multiple particle tracking microrheology.

<p>The ensemble averaged mean squared displacements (<2(Δt)>>) of 100 nm particles embedded in the cytoplasm of TCM-treated MSCs (A) and 3T3 fibroblasts (B) were evaluated from 0–3 hours. For both cell lines, treatment with TCM reduced the rate of cytoplasmic particle transport in a time-dependent manner. Fluorescent image of 100 nm particles (green) in the cytoplasm of a MSC, which was fixed and stained with phalloidin (red) and DAPI (blue) (C). The phase angle, δ = arctan (G″(ω))/G′(ω)), was used to characterize the viscoelastic nature of the cytoplasm over the course of the experiment (D). The viscoelastic nature of MSCs and 3T3 fibroblasts were similar initially and 3 hours after TCM-treatment; however, MSCs responded much more rapidly to TCM with a 4-fold reduction in δ within 60 minutes. (scale bar = 10 µm)</p

Effect of TCM on Cell Migration.

<p>Tranwell assays were used to measure the migration of MSCs and 3T3 fibroblasts through 3 µm- (A) and 8 µm- (B) pore transwell inserts toward CM or TCM. The average number of cells per image (n = 9), collected with a 10×-objective, was reported. TCM significantly increased MSC migration, compared to CM, through 3 µm pores within 3 hours and 8 µm pores within 2 hours; however, fibroblast migration was only increased through 8 µm pores within 3 hours. MSCs and 3T3 fibroblasts were then treated with CM or TCM for 1 hour and allowed to migrate through 8 µm-pore transwell inserts toward CM or TCM for 3 hours (C). Pre-treatment with TCM resulted in synergistic effects on chemotactic migration for both cell types.</p

Characterization of bone marrow isolated MSCs and fibroblasts.

<p>Phenotypic analysis was performed by flow cytometry was performed on adherent bone marrow cells and Swiss 3T3 fibroblasts with positive populations in red given with S.E.M.(A). Purified MSCs differentiated into adipocytes (B) and osteoblasts (C) within 3 weeks in lineage-specific differentiation media as shown both my staining and RT-PCR (scale bar = 100 µm).</p

Enhanced Adhesion of Stromal Cells to Invasive Cancer Cells Regulated by Cadherin 11

Cancer-associated fibroblasts (CAFs) are known to promote tumor growth and metastasis; however their differential accumulation in invasive and noninvasive tumors is not fully understood. We hypothesized that differences in cell adhesion may contribute to this phenomenon. To test this, we analyzed the adhesion of CAF-precursor fibroblasts and mesenchymal stem cells to invasive and noninvasive cancers originating from the the breast, ovaries, and prostate. In all cases, stromal cells preferentially adhered to more invasive cancer cells. Modulating integrin and cadherin binding affinities with calcium chelation revealed that adhesion was independent of integrin activity but required cadherin function. Invasive cancer cells had increased expression of mesenchymal markers cadherin 2 and 11 that localized with stromal cell cadherin 11, suggesting that these molecules are involved in stromal cell engraftment. Blockade of cadherin 11 on stromal cells inhibited adhesion and may serve as a target for metastatic disease

MSCs reorganize their cytoskeleton in response to tumor-secreted soluble factors.

<p>(A) Confocal micrographs of CM and TCM-treated MSCs (a–c) and 3T3 fibroblasts (d–e) stained with Phalloidin (F-actin, red), anti-α-tubulin (microtubules, green), and DAPI (nucleus, blue). The shape and cytoskeletal organization of CM-treated MSCs (a) and CM- (d) and TCM- (e) treated Swiss 3T3 fibroblasts were similar (24 hours after CM or TCM addition); whereas, TCM-treated MSCs were elongated with extended cytoskeletal filaments (b–c). MSC elongation increased between 12- (b) and 24- (c) hours, indicating that cytoskeletal changes may be progressive. Cytoskeletal parameters (B–D) were determined by analysis of confocal images with a custom MATLAB routine. The cell (B) and nuclear (D) shape factors were used to characterize the circularity of an elliptical outline of the cell or nucleus, respectively, with a shape factor of 1 indicating a perfect circle. The stress fiber factor (C) was used to characterize the density of actin stress fibers per cell area. Cytoskeletal changes observed in TCM-treated MSCs (b–c) were confirmed using the cytoskeletal parameters (B–D), which indicated dramatic reductions in cell and nuclear shape factors and stress fiber densities. (scale bars = 10 µm)</p

Nuclear CXCR4 Expression in Prostate Cancer Cell Lines.

<p><b><i>A</i></b>, Normal prostate epithelial (RWPE1) and PCa (PC3, DU145, 22RV1) cells were stimulated with SDF1α (100 ng/ µl) prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls. The bar graphs are quantitative results of the band density representing expression of CXCR4 in each fraction. Data were mean <u>+</u>S.E. from three independent experiments. *, P<0.05. <b><i>B</i></b>, Immunocytochemistry of PCa cell lines for CXCR4. PCa cells were stimulated with SDF1α (100 ng/ µl), fixed with methanol, blocked then incubated with an antibody mixture containing a mouse anti-CXCR4 monoclonal antibody and a rabbit polyclonal anti-Lamin A/C antibody, followed by secondary mixture containing a Cy3-conjugated anti-mouse antibody and FITC-conjugated anti-rabbit antibody. Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at excitation 488 nm for FITC and 543 nm for Cy3. Confocal images demonstrating the plasma membrane and cytosolic localization of CXCR4 (red), intact nuclear membrane (green), and nuclear-associated localization of CXCR4 (yellow/orange) are shown. Small arrows indicate co-localization of CXCR4 with the nucleus (yellow/orange). Scale bars represent 50 µm.</p

Nuclear CXCR4 was Functional at the Nucleus.

<p><b><i>A</i></b>, Representative light images of whole cells and isolated nuclei confirmed the integrity of nuclear isolation at 20× magnification. <b><i>B</i></b>, Whole cells were treated with SDF1α prior to isolating and lysing intact nuclei. Nuclei lysates (1 mg) were immunoprecipitated with anti-CXCR4 and separated by SDS-PAGE. Immunocomplexes were probed for G_αi (first row) or CXCR4 antibody (second row), respectively. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo1, nuclear) were used as markers for fractionation purity and as loading controls. <b><i>C</i></b>, PC3 nuclei were isolated, incubated with FluoForte dye Ca²⁺ probe, followed by incubation with AMD3100 or pertussis toxin (PTX) for 1 hr, then stimulated with SDF1α for 30 min. An increase in fluorescent-bound Ca²⁺ was measured on a microplate reader at ex = 490 nm/em = 525 nm.</p

A Putative Functional NLS within CXCR4.

<p><b><i>A</i></b>, GFP-CXCR4 fusion protein localized similar to endogenous CXCR4. CXCR4-pEGFPN1 transfected PC3 cells were stimulated with SDF1α, fixed with methanol, blocked then incubated with a mouse anti-CXCR4 monoclonal antibody, followed by a Cy3-conjugated anti-mouse secondary antibody. Nuclei were stained with DAPI (blue). Images were taken at 40× maginification using Axiovision software 4.8.2 with a Zeiss Axio Imager.z1 fluorescence microscope at ex = 470 nm for FITC, ex = 358 nm for DAPI and ex = 551 nm for Cy3. Images demonstrate the co-localization (yellow) of endogenous CXCR4 (red) with GFP-tagged CXCR4 (green). <b><i>B</i></b>, Localization analysis of wild type CXCR4 (CXCR4-pEGFPN1), NLS-mutant of CXCR4 (pEGFPN1-CXCR4<b>R146A</b>,) and deleted NLS of CXCR4 (CXCR4<b>ΔNLS</b>) by immunocytochemistry in PC3 cells. Nuclei were stained with propidium iodide (red) and CXCR4 was detected as the fusion protein GFP-CXCR4 (green). Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at ex = 488 nm for FITC and ex = 543 nm for Cy3. Scale bars represent 50 µm. <b><i>C</i></b>, Transfected cells were stimulated with SDF1α prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-GFP to detect the fusion protein GFP-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls.</p

Immunohistochemical (IHC) Staining of Prostate Tissues for CXCR4.

<p><b><i>A</i></b>, A human prostate tissue array, ranging from normal to high-grade prostate cancer, was evaluated by IHC for CXCR4 expression using standard methods. Samples were evaluated at magnification 40X, using a Q-Imaging camera of Olympus BX51 Microscope with Bioquant® Image Analysis Software (RtmBometrics). Normal prostate tissues demonstrated slightly weak or undetectable brown staining for CXCR4 (positive cells<5%), and no CXCR4 expression in the nucleus. Representative low grade prostate tissue (grade 2, stage II, T₂N₀M₀, adenocarcinoma) demonstrated random/focal positive staining for CXCR4 in the nucleus (positive cells >11%, but less than 50%), indicating low expression of CXCR4. Representative high grade metastatic prostate tissue (grade 4, stage IV, T₄N₁M₁, adenocarcinoma) demonstrated diffuse/intense staining (positive cells >50%), indicating high expression for CXCR4 in the nucleus. Scale bar represents 50 µm. <b><i>B</i></b>, CXCR4 IgG2B mouse monoclonal antibody was evaluated for specificity to CXCR4 protein by western blot analysis in PC3 (CXCR4 positive) or 293T (CXCR4 null) cell lines. <b><i>C</i></b>, CXCR4 antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation for CXCR4 and western blot analysis for CXCR4. <b><i>D</i></b>, CXCR4 IgG2B antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation with Fibronectin IgG2B mouse monoclonal antibody (unrelated isotype control) and western blot analysis for CXCR4; expression of Fibronectin protein was confirmed by western blot analysis. Beta-actin was used as a loading control.</p