(Gated on CD45⁺ cells) (A) FACS analysis of a mechanically dissociated metastatic stage III ovarian carcinoma specimen. These data are representative of 3 primary and 5 metastatic stage III–IV samples (see also Suppl. Fig.1). (B) FACS analysis of the same tumor and its matching tumor ascites.

(A) ITGAX-DTR-GFP mice (n=6/group; 2 independent experiments) were challenged with a subcutaneous injection of 10⁷ ID8-Defb29/Vegf-a cells in 200 µl Matrigel and immediately received 4 ng/g body weight DT in PBS (top) or PBS (bottom). Tumors were removed at 2 months. (B) Administration of DT did not result in reduced tumor growth in wild-type mice. (C) ITGAX-DTR-GFP mice were subcutaneously inoculated with 10⁷ ID8-Defb29/Vegf-a cells (in 200 µl Matrigel). After 10 days, mice received 2 ng/g DT in PBS (ITGAX + DT) or a similar volume of PBS (ITGAX+PBS). (D) DT (Depleted), but not PBS (Control) administration eliminates tumor CD11c⁺ DCs (GFP⁺) within 48 h. Magnification is included. Data are representative of 3 independent experiments.

(A) Tumor necrosis is obvious 36h after the administration of DT and becomes massive at 48h. N, Necrosis (×40). (B) ITGAX-DTR-GFP mice (n=4) bearing established flank ID8-Defb29/Vegf-a tumors received 4 ng/g body weight DT in PBS (24h, right), or a similar volume of PBS (control, left). Tumors were removed after 24h and stained with PE-labeled anti-CD31 and biotinylated anti-active caspase 3 antibodies (both from BD Pharmingen), plus Alexa-Fluor-350 (×200). (C) In reconstituted mice, depletion of CD11c⁺ DCs every 4 days for 2 months (DT) decreased tumor burden, compared to animals injected with PBS. Data are representative of two independent experiments. (D) Constant depletion of CD11c⁺ DCs (DT) increases in the survival of reconstituted ovarian ID8 tumor-bearing mice, compared to reconstituted mice injected with PBS (Control). Pooled data from two independent experiments (n=12/group, total) are displayed.

(A) FACS analysis of CD44 and CD69 in tumor ascites from tumor-bearing mice injected with the antiDEC205 IT (Depleted) or PBS (Control). (B) FACS analysis of peritoneal wash samples from tumor-bearing mice injected with the antiCD11c IT (Depleted) or PBS (Control) one month after tumor challenge. (C) OT-I mice (n=4) were intraperitoneally injected with 10⁶ OVA-expressing ID8 cells mixed with 10⁷ ID8-Defb29/Vegf-a tumor ascites cells (containing 30% CD45⁺CD11⁺VE-Cadherin⁺ DCs) and received 0.5 mg/Kg of IT (IT) or truncated toxin (Ctrl) one day later. After seven days, mice received again 10⁷ ID8-Defb29/Vegf-a tumor ascites cells i.p., followed by a new DC depletion or control treatment at day 8. Splenocytes were collected at day 11 and stimulated for 7 days with OVA (Sigma; 1 µg/ml). Interferon-γ ELISPOT was performed against bone marrow-derived DCs pulsed with OVA (1 µg/ml; 10:1, splenocyte:DC ratio). (D) Depletion of CD11c⁺ DCs in OT-I transgenic mice also enhanced the amount of interferon-γ-producing splenocytes responding to DCs pulsed for 4 h. with gamma and UV irradiated OVA-ID8 tumor cells (10:1, DC:ID8 ratio).

Tumor-bearing ITGAX-DTR-GFP mice were perfused with an intracardiac injection of biotinylated Tomato Lectin at different times after tumor inoculation (A) (left) Unperfused (white arrows) and blood transporting (yellow arrows) structures assembled by CD11c⁺ (GFP⁺) cells mixed in the growing edge of all specimens analyzed (×200). (right) In central areas of the tumor, CD11c⁺ (GFP⁺) leukocytes are irregularly scattered on the wall of most neovessels (×400). (B) Confocal microscopy analysis of the different kinds of structures assembled by CD11c⁺ (GFP⁺) DCs (×600). (left) Another example of a different unperfused GFP⁺ structure. (center) Co-localization of Tomato Lectin and GFP⁺ cells in selected blood vessels. (right; projection of stack images) CD11c⁺ cells in big vascular arrangements were predominantly found in an abluminal second layer. (C) FACS analysis of SMA and VE-Cadherin expression on CD11c⁺ DCs from tumor ascites. (D) CD45⁺DEC205⁺ DCs (hDEC205⁺) or CD3⁺CD4⁺ lymphocytes (hCD3⁺CD4⁺) were FACS sorted from eight unselected human ovarian carcinoma suspensions. CD45⁺CD11c⁺ DCs (mCD11c⁺) were sorted from the ascites of ID8-Defb29/Vegf-a tumor-bearing mice. Cells (10⁶/µl) were stimulated for 4 h. with PMA/ionomycin (50ng/l µg/ml). Cytokines were determined by Bioplex analysis.

(A) (left) Depletion of CD11c⁺ cells with 4 weekly intravenous administrations of anti-CD11c immunotoxin in established flank ovarian tumor-bearing animals (n=5/group; 100 µg/Kg after day 7 or 21) decreases tumor growth, compared to mice receiving a truncated toxin. (right) Formation of necrotic cavities (N) encapsulated by a viable ring (highlighted) in treated mice (IT), compared to mice receiving the harmless truncated IT (Control; ×400). (B) Survival curves of mice bearing intraperitoneal tumors and treated with either: 0.33 mg/Kg/day of truncated toxin (Truncated) or IT (IT) for two weeks, starting at day 3; 7 mg/Kg of topotecan at day 7 (Topotecan); 7 mg/Kg topotecan at day 7 plus 0.33 mg/Kg/day IT for two weeks (IT+Top); and the same regime of topotecan (day 7) plus IT (daily from day 3) administered until mice developed ascites (IT+Topb). (C) Intraperitoneal ovarian carcinoma-bearing mice (n=6/group), depleted of CD11b⁺ leukocytes by 4 weekly intraperitoneal administrations of anti-CD11b-saporin immunotoxin (90 µg/Kg) survived longer than mice receiving the same dose of unconjugated saporin (both from Advanced Targeting Systems, San Diego, CA). (D) Intraperitoneal ovarian carcinoma-bearing mice (n=6/group), depleted of DEC205⁺ DCs by intraperitoneal administrations of anti-DEC205-saporin immunotoxin (DEC205SAP; Celldex, 0.25 mg/Kg) survived longer than animals receiving the same amount of unconjugated antibody (DEC205), which in turn exhibited an increase in lifespan compared to animals injected with PBS.