Article Figures & Data
Figures
- Figure 1.
Expression of FKN in neuroblastoma. The gene expression of chemokines and chemokine receptors was determined in 68 human neuroblastoma (NB) samples (A) and correlated with the expression of MYCN oncogene in these tumors (B). A, results indicate expression of chemokines in the majority of neuroblastomas and the presence of FKN (CX3CL1), as well as SDF1, in almost all of the tumors investigated. Expression of chemokine receptors was virtually absent in neuroblastoma samples analyzed here, with exception of the FKN receptors CX3CR1 and CXCR4. B, the amplification of MYCN in these tumor samples inversely correlated with the expression of the FKN gene as shown by box plots of MYCN amplification and FKN gene expression (*, P < 0.05, t test). Expression of the FKN protein by NXS2 cells as secreted (C) and membrane-bound (D) protein was determined by sandwich ELISA and flow cytometry, respectively. C, columns, mean FKN secretion rates (ng/mL/24 h) obtained from triplicate experiments; bars, SD. 1, NXS2-FKN (third-generation subclone); 2, NXS2-FKN bulk culture; 3, NXS2 mock-transfected cells; 4, NXS2 parental cells. Asterisks, nondetectable levels of FKN. D, the presence of the membrane-bound FKN protein was quantified by flow cytometry. Black, NXS2 wild-type cells; yellow, NXS2 mock-transfected cells; green, NXS2-FKN bulk culture; red, NXS2-FKN third-generation subclone.
- Figure 2.
FKN-mediated chemotaxis. Chemotactic activity of FKN was determined both in vivo and in vitro. A, FKN-mediated chemotaxis was assessed in a Boyden chamber assay in vitro. The total number of transmigrated cells was determined microscopically. Columns, mean percent of transmigrated cells from triplicate experiments; bars, SD. 1, serum-free NXS2-mock supernatant (negative control); 2, recombinant murine FKN 12.5 ng/mL; 3, 20 ng/mL; 4, 50 ng/mL; 5, serum-free NXS2-FKN supernatant; 6, serum-free NXS2-FKN supernatant plus 2 μg/mL anti-mFKN mAb (M18), indicating that there is an additional migration-inducing bioactivity in the supernatant equivalent to 20 ng/mL FKN, assuming 100% efficiency of blockade by the mFKN mAb. *, P < 0.01, experimental groups versus control groups. B and C, cryosections of primary tumors induced with NXS2 cells (wild-type, mock, and NXS2-FKN) were stained with mAbs specific for CD45 (pan leukocyte marker) and for CD4 and CD8 (T-cell subpopulations). B, number of tumor infiltrating cells quantified by counting the total number of infiltrating cells per high-power field at ×400. Columns, mean of 10 high-power fields; bars, SD. *, P < 0.01, between mice receiving NXS2-FKN cells and all control groups. C, photographs of representative areas within distinct primary tumors (×400). Black arrows, infiltrating cells with characteristic red membrane staining.
- Figure 3.
Leukocyte adhesion and secretion of IFN-γ induced by FKN. Adhesion induced by the membrane-bound form of FKN was determined by coincubation of splenocytes and NXS2-FKN cells. A, adhesion between NXS2-FKN cells and splenocytes after 12 h of incubation. Pictures were taken at ×400. Arrows, splenocytes adherent to NXS2-FKN cells. B, the percentage of splenocytes adherent to NXS2 cells (wild-type, mock, and NXS2-FKN) was determined by counting 10 high-power fields. *, P < 0.01, between NXS2-FKN cells and all control groups. C, secretion of IFN-γ from NK cells was determined by coincubation of NK cells with NXS2 cells (wild-type, mock, and NXS2-FKN) for 36 h. IFN-γ was measured in culture supernatant by ELISA. Columns, mean of triplicate cultures; bars, SD. ND, nondetectable. *, P < 0.05, between NXS2-FKN cells and all control groups.
- Figure 4.
Antitumor effect of targeted IL-2 against neuroblastoma expressing FKN. Antitumor effects of IL-2 targeted to the GD2-positive neuroblastoma microenvironment expressing FKN was determined on primary tumors (A) and liver metastases (B and C). Mice (n = 6) were inoculated s.c. with 2 × 106 NXS2 cells (mock and NXS2-FKN). Treatment with noncurative doses (5 × 5 μg i.v.) of targeted IL-2 (ch14.18-IL-2) and nonspecific control (ch225-IL-2) was started on day 5. A, primary tumor growth was monitored over time by microcaliper measurements. *, P < 0.05, between mice receiving ch14.18-IL-2 and all control groups. One day after removal of primary tumors (day 18), mice received a lethal challenge of NXS2 wild-type cells (1 × 105, i.v.; B and C). After 3 wk, liver metastasis was determined by assessment of the percentage of liver surfaces covered by fused metastases (C) and by measuring liver weights (B). Points, mean; bars, SD. *, P < 0.05, between the group of mice treated with ch14.18-IL-2 and all control groups.
- Figure 5.
Effect of T-cell and NK cell depletion on targeted IL-2 therapy of FKN-expressing neuroblastoma. Mice (n = 6) were inoculated s.c. with 2 × 106 NXS2 cells (mock and NXS2-FKN) and treatment with noncurative doses (5 × 5 μg i.v.) of targeted IL-2 (ch14.18-IL-2) was started on day 5. In vivo depletion of CD4+ and CD8+ T cells, as well as NK cells, during the effector phase was accomplished as described. Arrows, time points of in vivo depletion. Primary tumor growth was monitored over time by microcaliper measurements. Points, mean; bars, SD. *, P < 0.05; **, P < 0.01, between the nondepleted group and all control groups.
- Figure 6.
T-cell activation and cytolytic response following ch14.18-IL-2 therapy of neuroblastoma expressing FKN. Splenocytes were isolated from mice bearing FKN-producing neuroblastoma tumors treated with specific ch14.18-IL-2 and nonspecific ch225-IL-2 as described in Fig. 5 and subjected to in vitro stimulation (4 d, 50 Gy irradiated NXS2 wild-type). A and B, cytotoxicity was determined by 51Cr release assay at varying effector-to-target (E:T) ratios (left to right: 200:1, 100:1, 50:1, and 25:1). MHC class I–restricted lysis (A) was determined in the absence (▴) and presence (▪) of anti–MHC class I antibody (anti–H-2Kk, 25 μg/mL). Cytolytic response induced by NK cells (B) was measured with YAC-1 target cells. Points, mean of triplicate experiments; bars, SD. Error bars not displayed are covered by symbols. C, intracellular cytokine production (IFN-γ, TNF-α) and the expression of T-cell activation markers (CD25, CD69) by T-cell subpopulations (CD4, CD8) were determined by two-color flow cytometry. Columns, mean percent relative increase over naïve control splenocytes obtained in triplicate experiments; bars, SD. **, P < 0.01, between the group of mice treated with ch14.18-IL-2 and control groups.
Volume 67, Issue 5
