small molecule cxcr4 inhibitor plerixafor mozobiltm  (Genzyme)

Journal: Cell Death Discovery

Article Title: CXCR7 signaling promotes breast cancer survival in response to mesenchymal stromal stem cell-derived factors

doi: 10.1038/s41420-019-0169-3

Figure Lengend Snippet: a Hierarchical clustering based on differentially expressed genes between tumor cell lines that exhibited growth advantage (MCF7, FaDu, MDA-MB-231, and PC-3) compared to those that did not exhibit growth advantage (HT-29 and MDA-MB-468). b Bar chart depicting the expression of CXCR7 and CXCR4 on the indicated tumor cell lines. c Effect of inhibition of CXCR4 (using WZ811) or inhibition of CXCR7 on tumor cell growth in the presence of recombinant CXCL12 (SDF1) or hBMSC-derived CM. Data are presented as mean ± S.E.M. from three experiments

Article Snippet: Small-molecule inhibitor targeting CXCR4 (WZ811) was purchased from Selleckchem Inc. (Houston, TX) and was used at 1.0 μM.

Techniques: Expressing, Inhibition, Recombinant, Derivative Assay

Journal: Breast Cancer Research and Treatment

Article Title: Adipose stem cell crosstalk with chemo-residual breast cancer cells: implications for tumor recurrence

doi: 10.1007/s10549-018-05103-w

Figure Lengend Snippet: Adipose stem cells (ASCs) Migrate toward Conditioned Media (CM) from Chemo-Residual TNBC Cells. A CM was prepared by growing chemo-residual SUM159 TNBC cells in serum-free media for 48 h. The ability of primary human ASCs (Zenbio) to migrate toward this CM (Chemo-residual CM), FBS (+ Control) or serum-free media (− Control) was evaluated in a 15 h transwell assay. Total number of migrated cells from 5 representative fields (100 × magnification) was determined for each well, and mean cell number from triplicate wells (± SD) was calculated. Significance was determined by two tailed t test (**** p < 0.0001). Similar results were obtained in at least three independent trials. B Representative fields (100 ×) showing migration of ASCs towards chemo-residual TNBC CM in a 15 h transwell assay. C CXCR4 expression was assessed by incubating ASCs with CXCR4 antibody (blue line) or control IgG (red line), followed by FITC-conjugated secondary antibody. CXCR4 cell surface expression was determined by flow cytometry. D RNA was isolated from untreated and chemo-residual SUM159 cells and subjected to SDF-1 alpha and GAPDH real-time PCR. SDF-1 alpha gene expression relative to GAPDH is presented for both cell lines

Article Snippet: We also showed that a CXCR4 small molecule inhibitor (AMD3100; EMD Milipore) reduced ASC migration toward chemo-residual cells by two-fold (Fig. B).

Techniques: Control, Transwell Assay, Two Tailed Test, Migration, Expressing, Flow Cytometry, Isolation, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Breast Cancer Research and Treatment

Article Title: Adipose stem cell crosstalk with chemo-residual breast cancer cells: implications for tumor recurrence

doi: 10.1007/s10549-018-05103-w

Figure Lengend Snippet: CXCR4 and SDF-1α Neutralizing Antibodies Block ASC Migration toward chemo-residual TNBC cell Conditioned Media. A Human ASC migration toward chemo-residual TNBC cell CM (prepared as described in Fig. ) was measured in the presence of a CXCR4-neutralizing Ab (EMDMillipore, 10 µg/mL), an SDF1α -neutralizing antibody (R&D Systems, 10 µg/mL), or control IgG (R&D Systems, 10 µg/mL). Chemotaxis was measured as in Fig. . The right panels show representative fields of migrated ASCs in the presence of the indicated antibodies. Significance was measured with a two tailed t test (*** p < 0.0005). B The ability of a CXCR4 small molecule inhibitor (AMD3100; EMD Millipore, 50 µg/mL) to block ASC migration toward chemo-residual TNBC cell CM was tested in a 15 h transwell assay. The bottom panels are representative fields of migrated ASCs ± AMD3100. Significance was measured with a two tailed t test (*** p < 0.0005). C The ability of ASCs to migrate toward chemo-residual TNBC cell CM was tested in a 15 h transwell assay in the presence of the indicated concentrations of CXCR4 small molecule inhibitor (AMD3100). Significance was measured using a two tailed t test (** p < 0.005; *** p < 0.0005)

Article Snippet: We also showed that a CXCR4 small molecule inhibitor (AMD3100; EMD Milipore) reduced ASC migration toward chemo-residual cells by two-fold (Fig. B).

Techniques: Blocking Assay, Migration, Control, Chemotaxis Assay, Two Tailed Test, Transwell Assay

Journal: Breast Cancer Research and Treatment

Article Title: Adipose stem cell crosstalk with chemo-residual breast cancer cells: implications for tumor recurrence

doi: 10.1007/s10549-018-05103-w

Figure Lengend Snippet: ASC cross-talk with chemo-residual TNBC cells- implications for tumor recurrence. A Chemo-residual TNBC cells secrete the chemokine SDF1α, resulting in recruitment of ASCs, which express the SDF1α chemokine receptor CXCR4, to the tumor site. B ASCs secrete FGF2, which binds to an FGF2 receptor on chemo-residual tumor cells, initiating signaling that drives tumor cell proliferation. Considering that chemo-residual tumor cells can remain dormant in patients for months to years, this ASC/chemo-residual tumor cell cross-talk likely contributes to tumor recurrence in patients post-chemotherapy treatment

Article Snippet: We also showed that a CXCR4 small molecule inhibitor (AMD3100; EMD Milipore) reduced ASC migration toward chemo-residual cells by two-fold (Fig. B).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Early pancreatic cancer lesions suppress pain through CXCL12-mediated chemoattraction of Schwann cells

doi: 10.1073/pnas.1606909114

Figure Lengend Snippet: SC in human PDAC harbor the chemokine receptors CXCR4 and CXCR7, whereas PanIN lesions and established human PCC lines contain CXCL12. (A–C) Coimmunolabeling of nerves in PDAC tissues with the SC markers S100/p75NTR demonstrates the presence of CXCR4 and CXCR7 in nerves (A and B) and also in intrapancreatic ganglia (C). The asterisks indicate the immunostained perineural inflammatory cells. (D and E) In normal human pancreas the chemokine receptors are only faintly detectable in SC. The white arrow points to the nerve embedded in the exocrine tissue. (F) However, several nerves in PDAC tissues exhibited immunoreactivity for CXCR4 and CXCR7. (G and H) Immunolabeling of PDAC tissues against CXCL12 and colabeling with the PCC marker CK19 revealed PanIN lesions (G) and PCC (H) as the major source of this chemokine in PDAC. The blue arrows point to nerves that are invaded by CXCL12-expressing cancer cells. (I) Accordingly, the mean immunoreactivity of PDAC tissues for CXCL12 (Left) and the mean tissue CXCL12 mRNA expression (Right) was prominently greater in PDAC than in NP. For the mRNA expression, the levels of NP tissues were normalized to 1 (Mann–Whitney u test). (J) Human PCC lines synthesize substantial amounts of CXCL12 as detected via ELISA. Experiments were repeated three times. (Scale bars: A–F, 40 μm; G–H, 100 μm.)

Article Snippet: In the assays with hSC, these cells were pretreated with the CXCR4 small-molecule inhibitor AMD3100 (0.5 μg/mL or 1.25 μg/mL) (Tocris) or mouse anti-CXCR7 neutralizing antibody (10 μg/mL or 30 μg/mL) (MBL) for 30 min at 37 °C in serum-free medium and subsequently resuspended in the ECM gel for the assay.

Techniques: Immunolabeling, Marker, Expressing, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Early pancreatic cancer lesions suppress pain through CXCL12-mediated chemoattraction of Schwann cells

doi: 10.1073/pnas.1606909114

Figure Lengend Snippet: Semiquantitative analysis of CXCL12, CXCR4, and CXCR7 immunoreactivity in NP and in human PDAC

Article Snippet: In the assays with hSC, these cells were pretreated with the CXCR4 small-molecule inhibitor AMD3100 (0.5 μg/mL or 1.25 μg/mL) (Tocris) or mouse anti-CXCR7 neutralizing antibody (10 μg/mL or 30 μg/mL) (MBL) for 30 min at 37 °C in serum-free medium and subsequently resuspended in the ECM gel for the assay.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Early pancreatic cancer lesions suppress pain through CXCL12-mediated chemoattraction of Schwann cells

doi: 10.1073/pnas.1606909114

Figure Lengend Snippet: Basal and regulated expression of CXCR4 and CXCR7 in SC. (A and B) Primary hSC were found to express CXCR4 and CXCR7 via immunofluorescence (A) or via immunoblotting (B) in simultaneous comparison with human PCC lines and one human colon cancer cell line (HCT116). (C, Left) hSC were cocultured with the SU86.86 PCC line, and at 24 h their CXCR4 content was compared with that of monocultured hSC via immunoblotting. (Right) Newborn rat DRG that contain SC were also cocultured with SU86.86 or T3M4 PCC and at 48 h of coculture exhibited higher CXCR4 content than monocultured DRG (unpaired t test). (D, Upper Left) hSC that were exposed to increasing periods of hypoxia (0–48 h) exhibited both the 42-kDa native CXCR7 isoform and another larger isoform that also was present in Jurkat T cells. (Lower Left) At the mRNA level, 2- or 12-h hypoxia exposure led to significant up-regulation of CXCR7 in hSC, with a similar tendency for CXCR4. However, CXCL12 expression in hSC was not influenced by hypoxia. (Right) Hypoxia exposure enhanced CXCL12 expression in SU86.86 and T3M4 PCC lines. Accordingly, the addition of the supernatant of hypoxia-treated (6 h) PCC lines to the medium of hSC enhanced the hSC transmigration in Boyden chambers (Mann–Whitney u test). (E, Left) hSC were placed in the upper chamber of a Boyden Transwell chamber and were supplied with increasing doses of recombinant human CXCL12. The number of transmigrating hSC was determined and expressed as percent of control. (Center and Right) Similarly, T3M4 or MiaPaCa-2 human PCC were placed in the lower chamber, and hSC in the upper compartment were pretreated with the CXCR4 inhibitor AMD3100 or with a CXCR7-blocking antibody (9C4). The solvent for AMD3100 or a nonimmunized mouse IgG1 antibody was used as control (unpaired t test). Experiments were repeated three times.

Article Snippet: In the assays with hSC, these cells were pretreated with the CXCR4 small-molecule inhibitor AMD3100 (0.5 μg/mL or 1.25 μg/mL) (Tocris) or mouse anti-CXCR7 neutralizing antibody (10 μg/mL or 30 μg/mL) (MBL) for 30 min at 37 °C in serum-free medium and subsequently resuspended in the ECM gel for the assay.

Techniques: Expressing, Immunofluorescence, Western Blot, Comparison, Transmigration Assay, MANN-WHITNEY, Recombinant, Control, Blocking Assay, Solvent

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Early pancreatic cancer lesions suppress pain through CXCL12-mediated chemoattraction of Schwann cells

doi: 10.1073/pnas.1606909114

Figure Lengend Snippet: Impact of CXCR4/CXCR7 on hSC migration to PCC in a 3D migration assay. (A–C) hSC pretreated with AMD3100 or CXCR7-blocking antibody were resuspended in an ECM drop, placed next to PCC, and recoded via live-cell imaging. Although solvent- or IgG1-treated (Control) hSC rapidly extend protrusions and migrate toward PCC (which do not possess any affinity to hSC) (A), hSC in which CXCR4 or CXCR7 was inhibited did not demonstrate any 3D migration, as evidenced by their prominently reduced FMI (B and C). (Magnification: A, 100×.) (D and E) In comparison, the pretreatment of the human PCC lines T3M4 or SU86.86 with AMD3100 or CXCR7-blocking antibody did not reduce their migration toward DRG neurons (Mann–Whitney u test for the FMI, unpaired t test for the remaining parameters). Experiments were repeated three times.

Article Snippet: In the assays with hSC, these cells were pretreated with the CXCR4 small-molecule inhibitor AMD3100 (0.5 μg/mL or 1.25 μg/mL) (Tocris) or mouse anti-CXCR7 neutralizing antibody (10 μg/mL or 30 μg/mL) (MBL) for 30 min at 37 °C in serum-free medium and subsequently resuspended in the ECM gel for the assay.

Techniques: Migration, Blocking Assay, Live Cell Imaging, Solvent, Control, Comparison, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Early pancreatic cancer lesions suppress pain through CXCL12-mediated chemoattraction of Schwann cells

doi: 10.1073/pnas.1606909114

Figure Lengend Snippet: Glia-specific knockout of CXCR4 or CXCR7 abrogates SC migration to cancer cells. (A) GFAP-CreERT2 mice were interbred with CXCR4lox/lox or CXCR7lox/lox mice to generate GFAP-CreERT2;CXCR4lox/lox (n = 3) or GFAP-CreERT2;CXCR7lox/lox (n = 3) mice. The DRG of these mice were explanted. (B) After 4-OHT supplementation of their medium, SC of dissociated DRG from GFAP-CreERT2;CXCR4ox/lox or GFAP-CreERT2;CXCR7lox/lox (collectively, KO) mice were compared with SC of dissociated DRG from CXCR4ox/lox or CXCR7lox/lox (NR) mice for their migration to PDAC cells. (C) The addition of 4-OHT significantly impaired the migration of SC from KO animals to human PCC as compared with SC from NR mice; this difference was not seen in the absence of 4-OHT (unpaired t test). Experiments were repeated three times.

Article Snippet: In the assays with hSC, these cells were pretreated with the CXCR4 small-molecule inhibitor AMD3100 (0.5 μg/mL or 1.25 μg/mL) (Tocris) or mouse anti-CXCR7 neutralizing antibody (10 μg/mL or 30 μg/mL) (MBL) for 30 min at 37 °C in serum-free medium and subsequently resuspended in the ECM gel for the assay.

Techniques: Knock-Out, Migration

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Early pancreatic cancer lesions suppress pain through CXCL12-mediated chemoattraction of Schwann cells

doi: 10.1073/pnas.1606909114

Figure Lengend Snippet: FMI of SC that grow out of DRG from SC-specific CXCR4- or CXCR7-KO mice

Article Snippet: In the assays with hSC, these cells were pretreated with the CXCR4 small-molecule inhibitor AMD3100 (0.5 μg/mL or 1.25 μg/mL) (Tocris) or mouse anti-CXCR7 neutralizing antibody (10 μg/mL or 30 μg/mL) (MBL) for 30 min at 37 °C in serum-free medium and subsequently resuspended in the ECM gel for the assay.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Early pancreatic cancer lesions suppress pain through CXCL12-mediated chemoattraction of Schwann cells

doi: 10.1073/pnas.1606909114

Figure Lengend Snippet: Reciprocal relationship between CXCR4 and CXCR7 expression in hSC. siRNA-mediated silencing of CXCR4 in hSC resulted in the suppression of both CXCR4 and CXCR7 expression, whereas siRNA-mediated silencing of CXCR7 in hSC up-regulated CXCR4 expression to ca. 300% of control.

Article Snippet: In the assays with hSC, these cells were pretreated with the CXCR4 small-molecule inhibitor AMD3100 (0.5 μg/mL or 1.25 μg/mL) (Tocris) or mouse anti-CXCR7 neutralizing antibody (10 μg/mL or 30 μg/mL) (MBL) for 30 min at 37 °C in serum-free medium and subsequently resuspended in the ECM gel for the assay.

Techniques: Expressing, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Early pancreatic cancer lesions suppress pain through CXCL12-mediated chemoattraction of Schwann cells

doi: 10.1073/pnas.1606909114

Figure Lengend Snippet: siRNA sequences

Article Snippet: In the assays with hSC, these cells were pretreated with the CXCR4 small-molecule inhibitor AMD3100 (0.5 μg/mL or 1.25 μg/mL) (Tocris) or mouse anti-CXCR7 neutralizing antibody (10 μg/mL or 30 μg/mL) (MBL) for 30 min at 37 °C in serum-free medium and subsequently resuspended in the ECM gel for the assay.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Early pancreatic cancer lesions suppress pain through CXCL12-mediated chemoattraction of Schwann cells

doi: 10.1073/pnas.1606909114

Figure Lengend Snippet: Active CXCL12 signaling in the pancreas diminishes pain by suppressing spinal glial activity. (A) Abdominal mechanosensitivity to von Frey hairs was compared in KC (n = 3) and KC12lox/+ (n = 4) mice. The number of abdominal retractions when the abdomen was touched was quantified and compared with wild-type C57BL6/J mice. The enhanced SC activity in KC mice was associated with the suppression of abdominal retractions and thus less mechanosensitivity (unpaired t test). (B) The immunoreactivity of tumor tissue and tumor nerves for CXCR4 and CXCR7 was compared in PDAC patients with (n = 10) and without pain (n = 11). The neural immunoreactivity for CXCR7 was prominently lower in patients with painful disease (unpaired t test). (C) In the spinal cord of KC and KC12lox/+ mice, the proportion of activated dorsal horn astrocytes (p75NTR+ proportioned to GFAP+ cells) and microglia (p-p38+ proportioned to Iba-1+ cells) was compared via immunolabeling of the thoracic spinal segments 8–11 (three sections per spinal cord). Diminished CXCL12-signaling was associated with enhanced astrocyte and microglia activity in the dorsal horn (unpaired t test). The white arrows point toward double-stained astrocytes or microglia. (Scale bars: 100 μm.)

Article Snippet: In the assays with hSC, these cells were pretreated with the CXCR4 small-molecule inhibitor AMD3100 (0.5 μg/mL or 1.25 μg/mL) (Tocris) or mouse anti-CXCR7 neutralizing antibody (10 μg/mL or 30 μg/mL) (MBL) for 30 min at 37 °C in serum-free medium and subsequently resuspended in the ECM gel for the assay.

Techniques: Activity Assay, Immunolabeling, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Early pancreatic cancer lesions suppress pain through CXCL12-mediated chemoattraction of Schwann cells

doi: 10.1073/pnas.1606909114

Figure Lengend Snippet: Primary and secondary antibodies

Article Snippet: In the assays with hSC, these cells were pretreated with the CXCR4 small-molecule inhibitor AMD3100 (0.5 μg/mL or 1.25 μg/mL) (Tocris) or mouse anti-CXCR7 neutralizing antibody (10 μg/mL or 30 μg/mL) (MBL) for 30 min at 37 °C in serum-free medium and subsequently resuspended in the ECM gel for the assay.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Early pancreatic cancer lesions suppress pain through CXCL12-mediated chemoattraction of Schwann cells

doi: 10.1073/pnas.1606909114

Figure Lengend Snippet: Histology scores for CXCR4 immunostaining

Article Snippet: In the assays with hSC, these cells were pretreated with the CXCR4 small-molecule inhibitor AMD3100 (0.5 μg/mL or 1.25 μg/mL) (Tocris) or mouse anti-CXCR7 neutralizing antibody (10 μg/mL or 30 μg/mL) (MBL) for 30 min at 37 °C in serum-free medium and subsequently resuspended in the ECM gel for the assay.

Techniques: