DEPLETION OF DENDRITIC CELLS DELAYS OVARIAN CANCER PROGRESSION BY BOOSTING ANTI-TUMOR IMMUNITY

Abstract

Dendritic cells (DCs) and cytokines that expand myeloid progenitors are widely used to treat cancer. Here we demonstrate that CD11c⁺DEC205⁺ DCs co-expressing α-Smooth Muscle Actin and VE-Cadherin home to perivascular areas in the ovarian cancer microenvironment and are required for the maintenance of tumor vasculature. Consequently, depletion of DCs in mice bearing established ovarian cancer by targeting different specific markers significantly delays tumor growth and enhances the effect of standard chemotherapies. Tumor growth restriction was associated with vascular apoptosis after DC ablation followed by necrosis, which triggered an anti-tumor immunogenic boost. Our findings provide a mechanistic rationale for selectively eliminating tumor-associated leukocytes to promote anti-tumor immunity while impeding tumor vascularization, and to develop more effective DC vaccines based on a better understanding of the tumor microenvironment.

INTRODUCTION

Despite significant advances in our understanding of the tumor cell cycle, the 5-year survival rate for ovarian carcinoma patients is still lower than 40%. As ovarian cancer cells express immunogenic determinants (1–4), immunotherapy offers great promise to complement standard treatments. However, therapies that induce protective immunity against melanoma have failed against ovarian carcinoma (5) due to an incomplete understanding of its peculiar microenvironment. Therefore, to develop effective immunotherapies against ovarian cancer, better knowledge of the immunosuppressive networks that operate in its atypical milieu is required.

Our previous work unveiled a mechanism whereby immature DCs contribute to ovarian cancer progression by acquiring a pro-angiogenic phenotype in response to VEGF (6–8). VE-Cadherin⁺ DCs are the most abundant leukocyte subset (>30% of total cells) in the microenvironment of ID8-Defb29/Vegf-a mouse ovarian tumor (6) as well as in human solid ovarian carcinoma samples, but not in human ascites (7). Unlike classical myeloid-derived suppressor cells (9) in other epithelial tumors (10, 11), most CD11b⁺ leukocytes in mouse ovarian cancer co-express CD11c and DEC205, supporting their DC lineage (6). Most importantly, although most ovarian tumor-infiltrating DEC205⁺CD11c⁺ are negative for co-stimulatory CD80 and exhibit low levels of MHC-II and CD86, their capacity to efficiently present antigens can be exposed under inflammatory conditions (6).

DCs are primarily viewed as orchestrators of peripheral tolerance under steady state conditions, but inducers of immunity during inflammation (12). Consequently, DCs are largely viewed as being crucial for anti-tumor immune responses in the context of cancer (13), and numerous DC-based vaccine trials have been attempted as a novel intervention against many cancer types. Moreover, cytokines are widely used to expand myeloid progenitors, including those that give rise to DCs, in cancer patients (14). In contrast, emerging studies suggest that DCs can also function as inducers of T cell tolerance under certain inflammatory conditions (11, 15–21).

Because of the abundance and immature phenotype of ovarian cancer DCs, we hypothesized that these cells could represent important contributors to tumor growth and therefore novel therapeutic targets. Here we show how the elimination of DCs dramatically delayed ovarian cancer progression. These effects were mediated by an anti-tumor immunogenic boost preceded by tumor necrosis. Our findings provide a rationale for manipulation of critical elements of the tumor microenvironment as anti-tumor therapy and to develop more effective DC-based vaccines based on a better understanding of their potential to support tumor development.

MATERIALS AND METHODS

Animals, tissues and treatments

Mice were purchased from the National Cancer Institute or Jackson Laboratories (Bar Harbor, ME). Animal experiments were approved by our Institutional Animal Care and Use Committee. Stage III–IV human ovarian carcinoma specimens were procured through Research Pathology Services at Dartmouth, under an approved protocol. Single cell suspensions were generated as we described (7).

We generated ID8-Defb29/Vegf-a flank or intraperitoneal tumors as described (6). Tumor volumes were calculated by the formula V = 0.5 (L × W2), where L is length and W is width. To generate ID8-luciferase, parental ID8 cells were transduced three times with pFB-neo-Luciferase retroviruses, and selected with 0.8mg/ml G418. For visualization of tumor burden, mice were injected with 0.2 ml of 15 mg/ml luciferin (Promega). After 10 min, animals were anaesthetized with isoflurane and imaged using the IVIS 200 system (Xenogen Corp., Alameda, CA). ID8-OVA cells were described (22).

The anti-DEC205 NLDC145 antibody, conjugated or not to ribosome-inactivating saporin, was provided by Celldex Therapeutics (Phillipsburg, NJ). Treatments started 1 day after tumor challenge.

To generate chimeric mice, we lethally irradiated congenic (CD45.1) mice (2 doses of 500 rads, 24 h apart). Four hours later, 2×10⁶ cells from the bone marrow of healthy (CD45.2) ITGAX-DTR-GFP were intravenously injected. After confirming that >95% of circulating cells were of donor origin, mice were intraperitoneally challenged with 2×10⁶ ID8-luciferase tumor cells 8 weeks after reconstitution. Treatments (150 ng of intraperitoneal DT every 3 days, or PBS) started 3 days later.

Generation of the anti-CD11c immunotoxin

The heavy- and light-chain variable region cDNA (VH and VL) of hamster anti-mouse CD11c antibody were cloned from N418 hybridoma (ATCC#HB224) (23), using the following degenerated primers: VH.Forward (5'-CAGATCCAGTTGGTGCAGTCTGG-3'); VH.Reverse (5'-CGAGGAGACTGTGACTGAGGT-3'); VL.Forward (5'-GACATCAAGATGACCCAGTC-3') and VL.Reverse (5'-CCGTTTCAGTTCCAGCTTGGTCCC-3'). The VH and VL were assembled with a (Gly4Ser)3 encoding linker to yield a full long single-chain fragment encoding the whole antibody variable region. The C-terminal end of the single-chain variable fragment (scFv) was fused to the hinge sequence of human CD8, followed by the sequence encoding for truncated Pseudomonas Exotoxin A (PE38; ATCC#67205). This fusion gene was then subcloned into a pET-28a(+) plasmid (Novagen, La Jolla, CA) for expression in E.coli BL21(DE3; Invitrogen, Carlsbad, CA). To produce immunotoxin, recombinant bacteria were harvested 15 h after induction of recombinant protein expression with 1 mM IPTG. Optimum conditions of denaturation, refolding and dyalysis from inclusion bodies have been published (24). We obtained a final amount of ~1 mg of immunotoxin after purification using a thiophilic resin according to manufacturer’s instructions (Clontech, Mountain View, CA). To generate a control compound, PE38 was subcloned into a pET-28a(+) plasmid without the scFv sequence, in frame with the His-tag, expressed in E.coli BL21 and purified using a standard Ni column.

ELISPOT

Peptides were from New England Peptide (Gardner, MA). Flat-bottomed, 96-well nitrocellulose-lined plates (Millipore, Bedford, MA) were coated with IFN-γ mAb (AN-18; eBioscience, San Diego, CA) and incubated overnight at 4°C. After washing with Coating Buffer (eBioscience), plates were blocked with 10% FBS serum for 2 h at 37°C. Effector and target cells were incubated together for 20 h in 10% FBS RPMI medium. After incubation, the plates were washed with 0.05% Tween 20 in PBS, and biotinylated secondary IFN-γ mAb (R4-6A2; eBioscience) was added. After incubation for 2 h at 37°C, the plates were washed and developed with Avidin-horseradish peroxidase (eBioscience) for 1 h at room temperature. After washing, fresh substrate (3-amino-9-ethyl carbazole, Sigma, St. Louis, MO) was added and the plates incubated for approx. 20 min.

Histology, flow cytometry and Bio-Plex

Biotinylated Tomato lectin (150 µg in 150 µl of PBS; Vector) was injected through the left ventricle and allowed to circulate. The vasculature was then perfused with 1% paraformaldehyde in PBS for 3 min. Cryosections of OCT-embedded tumors (8-µm thick) were immunostained as described (6). Biotinylated lectin was detected by rhodamine-streptavidin (Leinco, St. Louis, MO) or Alexa Fluor-350 (Molecular Probes, Carlsbad, CA). GFP signal was enhanced with a rabbit anti-GFP Alexa Fluor-488 antibody (Molecular Probes).

Flow cytometry was performed on a FACSCanto (BD Biosciences, San Jose, CA). (CD45⁺DEC205⁺VE-Cadherin⁺) DCs were sorted from mechanically dissociated human ovarian carcinoma specimens using a FACSAria sorter (BD Biosciences).

An allophycocyanin-labeled tetramer consisting of K^b folded with GQKMNAQAI peptide was provided by the NIH Tetramer Core Facility (Atlanta, GA).

Anti-mouse antibodies were specific for CD31 (MEC13.3), CD45 (30-F11), CD45.1 (A20), CD45.2 (104), CD25 (PC61), CD69 (H1.2F3), CD11c (HL3; all from BD Biosciences), VE-Cadherin (rabbit, Medsystems Diagnostics); NG2 (rabbit, Millipore), DEC205 (NLDC145; Serotec), MHC-II (NIMR-4, eBioscience), FoxP3 (Staining Buffer Set; eBioscience), and anti-Actin, α-Smooth Muscle-Cy3 (1A4; Sigma). Anti-human antibodies were specific for CD45 (HI30),DEC205 (MG38), CD3 (UCHT1), CD4 (OKT4), CD8 (RPA-T8), CD31 (M89D3), CD11b (ICRF44) and CD14 (M5E2), all from BD Biosciences.

Human VEGF-A, FGF (basic) and IL-8 and mouse Vegf-a, Fgf (basic) and CXCL1 were detected using a Human-27-Plex or a Mouse-9-Plex panel cytokine assays, respectively (Bio-Rad, Hercules, CA) with supernatants from human CD45⁺DEC205⁺ leukocytes sorted from dispersed ovarian carcinoma samples, or with mouse CD11c⁺DEC205⁺ cells sorted from tumor ascites, following manufacturer’s instructions.

RESULTS

Elimination of CD11c⁺ DCs decreases ovarian cancer growth

To investigate the contribution of CD11c⁺ DCs to ovarian tumor progression, we used CD11c-diphtheria toxin (DT) receptor-green fluorescent protein (GFP) transgenic mice (ITGAX-DTR-GFP). In this strain, CD11c⁺ DCs express the receptor of DT as well as traceable green fluorescence and can be transiently depleted (>92%) by DT administration, whereas DT administration has no effect on CD11c⁺ cells in wild-type mice (25, 26). Wild-type or ITGAX-DTR-GFP mice (n=6 per group; 2 independent experiments) were treated with either intraperitoneal DT or PBS and immediately challenged with subcutaneous ID8-Defb29/Vegf-a cells, an aggressive epithelial ovarian cancer line (6). As we have previously reported, these tumors are heavily infiltrated by DEC205⁺CD11c⁺MHC-II^low DCs (6, 8). Supporting the DC lineage of their human counterpart, most DEC205⁺ leukocytes infiltrating unselected human solid primary (n=3) or metastatic (n=5) ovarian carcinoma specimens were CD11b⁻, while expression of CD14, although more variable, was found on a minority of DEC205⁺ cells in most specimens analyzed (Fig. 1A and Suppl. Fig.1A). In contrast, and in agreement with previous reports (27), we found that most leukocytes in tumor ascites (n=3) were monocyte/macrophage cells (CD14⁺/CD11b⁺), underscoring the importance of microenvironmental factors (Fig. 1B and Suppl. Fig.1B). Most importantly, the temporary ablation of mouse CD11c⁺ DCs at the time of tumor injection resulted in 3-fold lower tumor size after two months (441.4±59 mm³ vs. 146.81±33.4 mm³; P<0.05; Wilcoxon test; Fig2A). Reduced tumor growth was not caused by an unknown anti-tumor effect of DT because tumor growth was not affected in wild-type littermates (Fig.2B).

Fig. 1. DEC205⁺CD11b⁻ DCs, but not monocyte/macrophages represent the most abundant leukocyte subset in the microenvironment of solid ovarian carcinoma specimens.

(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.

Fig. 2. Elimination of CD11c⁺ DCs abrogates tumor growth.

(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.

To determine the involvement of DCs in the progression of established tumors, we performed a temporary depletion of CD11c⁺ cells in mice bearing 10 day old flank ovarian tumors (n=10/group). This resulted in a 3-fold reduction in tumor size for five additional weeks of growth (Fig.2C; 713.2±65.1 mm³ vs. 254.1±137 mm³; P=0.03; Wilcoxon test). Thus, CD11c⁺ cells were also involved in the growth of established tumors.

Elimination of CD11c⁺ DCs results in tumor necrosis and increased survival

Histological analyses after DT administration demonstrated substantial necrosis in central areas of the tumor, compared to mice receiving PBS, and confirmed the elimination of CD11c⁺ (GFP⁺) cells from viable tumor locations (Fig.2D). Tumor necrosis became obvious after ~36 hours of treatment and reached a maximum at 48h (Fig.3A). As anti-tumor T cells require longer than 36h to become re-activated (28) it is unlikely that these initial anti-cancer effects were caused by an adaptive immune response. Because of the role of CD11c⁺ cells in ovarian cancer vascularization (6, 7), we hypothesized that impeded tumor vascularization accounted for the reduction in tumor growth. Correspondingly, preceding tumor cell death (24h after DT administration), we found expression of active caspase 3 (indicative of apoptosis) on ~40% of (CD31⁺) blood vessels in all tumors examined, while tumor samples from control mice only showed scattered caspase 3 staining on CD31⁻ cells (Fig.3B). Together, these data indicate that CD11c⁺ DCs acquire a pro-angiogenic phenotype in the tumor microenvironment that is important for the maintenance of tumor vasculature and therefore the progression of ovarian cancer.

Fig. 3. Elimination of CD11c⁺ DCs results in tumor necrosis.

(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.

However, although the ID8-Defb29/Vegf-a cell line induces aggressive tumors that mimic the physiopathology of human ovarian cancer (6) it is maximally engineered to attract immature DCs. To establish the therapeutic potential of constantly depleting CD11c⁺ DCs in hosts bearing orthopic (intraperitoneal) ovarian tumors that do not ectopically express Defb29 and Vegf-a, we reconstituted lethally irradiated congenic (CD45.1⁺) mice with bone marrow from (CD45.2⁺) ITGAX-DTR-GFP animals, which allows multiple injections of DT without compromising survival (26). Eight weeks after reconstitution, >95% of blood leukocytes were of donor origin in all mice (not shown). Animals (n=6/group; 2 independent experiments) were then challenged with parental ID8 cells, expressing luciferase for intravital monitoring of tumor progression. In treated mice, DCs were then depleted every 4 days with intraperitoneal DT, while control animals were injected with PBS. Depletion of CD11c⁺ cells resulted in a reduction of tumor burden (Fig.3C and Suppl. Fig. 2A) and induced a significant increase in survival (Fig.3D; P<0.01; log-rank test).

A novel anti-CD11c immunotoxin eliminates tumor-infiltrating DCs in non-transgenic hosts

To define the therapeutic potential of depleting DCs in non-transgenic tumor-bearing hosts, we generated an anti-CD11c immunotoxin (IT) by cloning the variable regions of the anti-mouse CD11c N418 hybridoma, fused to a Pseudomonas exotoxin-A lacking the cell-binding domain (29, 30). The IT was expressed and purified, along with the control truncated toxin lacking the single-chain antibody, which is harmless because it cannot get internalized.

Incubation of CD11c⁺ DCs sorted from tumor ascites with 15ng/ml of IT resulted in the death of ~70% of cells within 40h. Interestingly, the same dose of IT caused the death of only ~30% of bone marrow-derived DCs (Suppl. Fig.2B), while the intravenous administration of 20 µg of IT (n = 3) eliminated <40% of splenic CD11c⁺ cells after the same period (Suppl. Fig.2C). To test the effects of our IT in tumor-bearing hosts, we collected ascites from mice with ID8-Defb29/Vegf-a tumors (n=5), which contained ~30% of CD11c⁺ cells. Tumor ascites cells were CFSE-labeled and intraperitoneally injected (2×10⁶ cells/mouse) into healthy C57BL/6 animals (n=5/group). After 5 h, mice received an intraperitoneal injection of IT (0.33 mg/Kg body weight); or PBS (control). Two days later, CD11c⁺CFSE⁺ cells were found in peritoneal samples – but not in any other locations - in both groups, although the total was 5-fold lower in mice treated with the anti-CD11c IT compared to control animals (Suppl. Fig.2D). In contrast, CD11c⁻ CFSE⁺ cells were not decreased. Importantly, comparable proportions of CD11c⁺CFSE⁻ DCs were found in spleen and draining (mediastinal) lymph nodes samples from both groups. These data indicate that the anti-CD11c IT can predominantly deplete peritoneal ovarian cancer-infiltrating DCs with little effect on DCs from lymphatic compartments that may potentially elicit anti-tumor immunity.

Depletion of CD11c⁺ DCs delays tumor progression and enhances the effect of standard chemotherapies

Weekly intravenous injections of anti-CD11c IT induced a dramatic decrease in the growth of established ID8-Defb29/Vegf-a flank ovarian tumors (n=5/group) when treatment started at day 7 (6-fold; 109.8.0±65.1 mm³); or as late as day 21 (3.5-fold; 184.60±75.1 mm³), compared to mice treated with a truncated toxin (646.1±163.2 mm³; P<0.001 for both; Wilcoxon test; Fig.4A, left). As observed in transgenic mice, the depletion of CD11c⁺ DCs induced central necrosis within 48 h (Fig.4A, right). As a model for aggressive human ovarian carcinoma, we intraperitoneally injected ID8-Defb29/Vegf-a cells into wild-type mice (n≥6 mice/group; (6, 31). Mice with established tumors received the chemotherapeutic drug topotecan along with various doses of the anti-CD11c IT or truncated control. Topotecan or IT treatment alone induced a similar and significant 20% increase in the median lifespan (Fig.4B; P<0.05; log-rank test). No weight loss or animal death was detected throughout the treatments. Furthermore, the combination of topotecan and a daily treatment with IT for two weeks resulted in a further 18% increase in survival (P<0.05). Importantly, a continuous administration of IT following the administration of topotecan extended lifespan by 53%, compared to individual treatments (P<0.01). Corresponding results were observed at lower doses of IT (Suppl. Fig.2E).

Fig. 4. Elimination of DCs through CD11c or DEC205 targeting enhances survival.

(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.

Even in the absence of anti-tumor adaptive immunity, intravenous administration of anti-CD11c IT also impaired the growth of human ovarian carcinoma A2780 cells implanted in the flank of SCID mice (Suppl. Fig.2F; 2,267±398 mm³ vs. 832±250 mm³; n=5/group; P=0.03; Wilcoxon test). Although these effects can be only attributable to the impairment of tumor vasculature, central necrosis after DC depletion was observed, although less dramatic than in ID8-Defb29/Vegf-a tumors (Suppl. Fig.2G).

Suggesting the potential of using multiple targeting strategies, the beneficial effect of DC depletion was confirmed by administering an anti-CD11b saporin-based immunotoxin, which also induced a significant increase in survival, compared to saporin alone (Fig.4C; P<0.01).

Depletion of DEC205⁺ DCs induces comparable anti-tumor effects

To confirm that the impairment of tumor progression is only attributable to the elimination of the predominant CD11c⁺DEC205⁺ DC population, we used a different IT consisting of the DEC205-specific NLDC145 antibody, conjugated to ribosome-inactivating saporin. Although DEC-205 is expressed on other cells, this antibody primarily targets CD11c⁺MHC II⁺ DCs in vivo (32). We confirmed that the intraperitoneal administration of 0.25 mg/Kg body weight of anti-DEC205-saporin resulted in the elimination of >50% of splenic DCs, but not other leukocyte subsets, in healthy mice (Suppl. Fig.2H and unpublished observations). The same dose of anti-DEC205-saporin every 4 days in mice bearing intraperitoneal ID8-Defb29/Vegf-a ovarian cancer resulted in a >30% increase in the median lifespan, compared to mice injected with PBS (Fig.4D; P<0.01; log-rank test). In addition, control mice treated with the same regimen of unconjugated anti-DEC205 antibody also exhibited a significant (P<0.05; log-rank test) increase in survival compared to mice receiving PBS, although it was lower than that induced by anti-DEC205-saporin (Fig.4D; P<0.05; log-rank test). Together, these data confirm that the elimination of DCs and not other leukocytes from tumor-bearing hosts accounts for the anti-tumor effects.

Elimination of tumor DCs induces a boost in specific anti-tumor immunity

Although at the latest stage of cancer progression virtually all T lymphocytes in tumor ascites were not activated (CD69⁻) in either group, up to 40% of cytotoxic T cells from animals depleted of DEC205⁺ DCs were antigen experienced (CD44⁺), compared to 4% in control mice (Fig.5A). We surmised that the release of tumor antigens induced by the elimination of most DCs and the subsequent tumor disruption may enhance, rather than impair, the endogenous anti-tumor immune response. To test this hypothesis, C57BL/6 wild-type mice (n=4) were challenged with ID8-Defb29/Vegf-a tumors and, after 5 days of tumor progression, mice were depleted of DCs every 2 days with intraperitoneal injections of anti-CD11c IT (0.2 mg/Kg) for 2 weeks. One month after tumor challenge, before the occurrence of massive ascites, the elimination of DCs resulted in an increase in the proportion of activated (CD69⁺) CD8 T cells in peritoneal wash samples. Most importantly, the peritoneal cavity of mice depleted of CD11c⁺ DCs contained a ~6-fold higher proportion of cytotoxic T cells specifically recognizing an H-2D^b-restricted mesothelin epitope expressed by ID8 tumor cells, ranging from 2 to 5% of CD8 T cells (Fig.5B) (33).

Fig. 5. Elimination of DCs boosts anti-tumor immune responses.

(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).

To confirm the antigen-specificity of the immunogenic boost against a different antigen, we challenged CD8⁺ transgenic ovalbumin (OVA)-specific (OT-I) mice with OVA-expressing ID8 cells admixed with tumor ascites and injected them with IT or the truncated toxin one day later. As expected, the spleens of mice depleted of DCs exhibited a much higher frequency of T cells producing Interferon-γ in response to OVA (Fig.5C) and irradiated tumor cells (Fig.5D) in ELISPOT analysis, compared to spleens from control mice. Together, these data confirm that the elimination of CD11c⁺DEC205⁺ DCs from the tumor microenvironment, likely followed by the possible generation of new waves of DCs to a less suppressive milieu, dramatically enhanced, rather than abrogated, the specific anti-tumor immune response.

Tumor-infiltrating DCs acquire pericyte-like attributes

Vascular apoptosis and ensuing central necrosis resulting from the elimination of CD11c⁺ DCs supports our previous finding that tumor-infiltrating DCs are important for tumor vascularization (6). Confirming the massive recruitment of DCs by ovarian tumors, 85% of CD11c⁺ cells banding in the viable interface after density gradient centrifugation of mechanically dissociated ID8-Defb29/Vegf-a solid tumors co-expressed DEC205 (Suppl.Fig.3A). To further characterize their role in tumor blood vessel formation, we perfused the vasculature of flank tumor-bearing ITGAX-DTR-GFP mice (n=6/group) by intracardiac injection of biotinylated Tomato Lectin. All specimens contained structures assembled by GFP⁺ (CD11c⁺) cells, which primarily clustered around the tumor periphery, suggesting a permanent recruitment of these cells to the area of growth. Although these also represent areas of active angiogenesis, most peripheral CD11c⁺ structures were not perfused by lectin (Fig.6A, left and B, left) nor contained erythrocytes (not shown), indicating that they are assembled before being permeated by blood. In contrast, in central areas of the tumor >50% of perfusible blood vessels were surrounded by irregularly scattered GFP⁺ (CD11c⁺) leukocytes (Fig.6A, right). Although GFP fluorescence co-localized with intravascular lectin in selected structures inside the tumor (Fig.6B, center), most GFP⁺ cells in central areas were distributed in a pericyte-like pattern (near endothelial cells but in outer layers) or a perivascular pattern (near the vessel wall but not in contact with vessels) (Fig.6B, right). Surprisingly, virtually all CD45⁺CD11c⁺DEC205⁺ leukocytes in dispersed solid tumors or tumor ascites expressed α-Smooth Muscle Actin (SMA; Fig.6 D, and Suppl.Fig.3B and C) and NG2 (Suppl.Fig.3D, left), but not PDGF Receptor (Suppl.Fig.3D, right). As reported (6, 7), most DCs also co-expressed VE-Cadherin, which was confirmed by PCR (Suppl. Fig. 3B) and further supported by the presence of numerous tumor-infiltrating MHC-II⁺GFP⁺ leukocytes in mice transferred with transgenic VE-Cadherin-GFP bone marrow cells (Suppl. Fig. 3E). More importantly, CD45⁺CD11c⁺ DCs sorted from mouse tumor ascites secreted Vegf-a and CXCL1, while CD45⁺DEC205⁺ cells procured from dispersed stage III–IV human ovarian carcinoma specimens - but not CD4⁺ lymphocytes - secreted basic fibroblast growth factor (FGFb), VEGF-A and high levels of CXCL8 (Fig.6E). These factors are known to induce the recruitment and promote the survival of sprouted endothelial cells (34).

Fig. 6. DCs support tumor vascularization.

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.

DISCUSSION

Here we demonstrate that the depletion of DCs in mice bearing ovarian tumors with four different methods that target one of three DC markers (CD11c, DEC205 or CD11b) significantly delayed cancer progression. DC depletion activates multiple anti-tumor mechanisms. On the one hand, it causes vascular apoptosis followed by tumor necrosis, supporting the concept of a critical contribution of DCs to ovarian cancer vascularization (6). On the other hand, transient elimination of DCs paradoxically boosted the anti-tumor immune response, likely because the early disruption of tumor tissue makes available massive amounts of tumor antigens to undepleted or newly generated DCs. Therefore, our findings provide a mechanistic foundation to enable more effective biologic therapy for cancer patients targeting tumor-associated leukocytes and other critical elements of the tumor microenvironment to complement the standard “surgical debulking/chemotherapy” approach.

DCs are primarily viewed as professional antigen-presenting cells specialized in the stimulation of naïve T lymphocytes. Because our system only allows the depletion of most but not all DC subsets, and only in a temporary manner, our results do not challenge this dogma. Because at least 85% of CD11c⁺ cells in the solid tumor microenvironment co-expresses DEC205, these effects should be attributed to the elimination of DCs and not other non-DC leukocytes. It is likely that by “resetting the system”, newly generated DCs at lymphatic locations engulf these antigens and present them to naive T cells in a less tolerogenic milieu.

Ovarian tumor cell death takes place more rapidly after DC depletion than with standard anti-angiogenic strategies based on VEGF signaling neutralization and is preceded by vascular cell apoptosis, suggesting that tumor-infiltrating DCs are important for maintaining tumor blood vessels. The crucial role of different leukocyte subsets on neo-angiogenesis has been previously recognized (35–37). In ovarian cancer, CD11c⁺ cells home to the wall of most blood vessels within the tumor, where they express SMA and NG2, and secrete VEGF-A, FGF and IL-8, which are critical factors for the recruitment and support of sprouted endothelial cells. CD11c⁺ cells also assembled unperfused structures and express VE-Cadherin in areas of active angiogenesis, suggesting that they could be also important for initiating nascent tumor vessels. Although tumor-infiltrating DCs acquire endothelial-like and pericyte-like attributes in different tumor areas, we cannot claim with our current data that they turn into bona fide endothelial cells or canonical pericytes, as they retain leukocyte markers. Our results rather point to an alternative “scaffold” model, whereby CD11c⁺ phagocytes (8) generate tubular structures in the extracellular matrix, which are permeated by blood due to the leaky nature of tumor vasculature. Sprouted endothelial cells brought by the blood flow would then line these channels, displacing leukocytes to outer layers as the vessel matures. DCs could therefore provide paracrine as well as direct structural support for the survival, stabilization and branching of new blood vessels, thus becoming crucial players in ovarian cancer vascularization.

In summary, our results show how the elimination of pro-angiogenic DCs by targeting different DC-specific markers impairs ovarian cancer progression. Our findings provide a rationale for the design of more effective cancer interventions that incorporate the targeting of critical contributors to cancer progression different from tumor cells.