The overexpression of folate receptors (FR) on many human cancers has led to the development of folate-linked drugs for the imaging and therapy of FR-expressing cancers. In a recent phase I clinical trial of late-stage renal cell carcinoma patients, folate was exploited to deliver an immunogenic hapten, fluorescein, to FR+ tumor cells in an effort to render the cancer cells more immunogenic. Although >50% of the patients showed prolonged stable disease, all patients eventually progressed, suggesting that the folate-hapten immunotherapy was insufficient by itself to treat the cancer. In an effort to identify a companion therapy that might augment the folate-hapten immunotherapy, we explored coadministration of two approved cancer drugs that had been previously shown to also stimulate the immune system. We report that sunitinib and axitinib (VEGF receptor inhibitors that simultaneously mitigate immune suppression) synergize with the folate-hapten–targeted immunotherapy to reduce tumor growth in three different syngeneic murine tumor models. We further demonstrate that the combination therapy not only enhances tumor infiltration of CD4+ and CD8+ effector cells, but surprisingly reduces tumor neovasculogenesis more than predicted. Subsequent investigation of the mechanism for this unexpected suppression of neovasculogenesis revealed that it is independent of elimination of any tumor cells, but instead likely derives from a reduction in the numbers of FR+ tumor-associated macrophages and myeloid-derived suppressor cells, that is, immunosuppressive cells that release significant quantities of VEGF. These data suggest that a reduction in stromal cells of myeloid origin can inhibit tumor growth by suppressing neovasculogenesis. Mol Cancer Ther; 16(3); 461–8. ©2016 AACR.

Folic acid, a vitamin required for the synthesis of nucleotide bases, methylation of DNA, and the posttranslational modification of G proteins, enters cells via either the reduced folate carrier, the proton-coupled folate transporter, or a folate receptor (FR; refs. 1–4). Although the reduced folate carrier and proton-coupled folate transporter are expressed in virtually all cells, the FR is present and accessible primarily on activated macrophages, the proximal tubule cells of the kidney, and certain cancers of epithelial origin, including malignancies of the ovary, endometrium, kidney, lung, and breast (5–8). Because folate-linked imaging and therapeutic agents can only enter cells via the FR, their uptake in normal tissues is highly limited, allowing folate to be used as a targeting ligand to deliver attached drugs to cancers and sites of inflammation (9–14).

The first folate-targeted therapy to enter human clinical trials involved the delivery of a highly immunogenic hapten (fluorescein) to FR-overexpressing tumor cells. In this strategy, the patient was first vaccinated against fluorescein by immunization with keyhole limpet hemocyanin (KLH) linked to fluorescein. After establishing the presence of a high anti-fluorescein antibody titer, the patient was injected with folate-fluorescein to decorate the surfaces of FR-expressing cancer cells with the immunogenic hapten, thereby marking the malignant cells for elimination by the immune system (15–19). Although the phase I human clinical data in advanced renal cell carcinoma patients (>75% stage 4) with bulky disease revealed no complete responses and few partial responses, >50% of the patients showed prolonged stable disease (20, 21), suggesting some degree of tumor suppression. Unfortunately, when more potent immune stimulants, IFNα and IL2, were later added to the immunotherapy to further stimulate immune activity, influenza-like symptoms triggered by the cytokine treatments emerged, discouraging further clinical testing of the combination therapy (20). Nevertheless, the prolonged stable disease in the majority of patients motivated further exploration of less toxic companion therapies that might augment the ability of folate-fluorescein to label and stimulate removal of FR+ cancers.

In this paper, we examine the efficacy of combining the aforementioned folate-fluorescein immunotherapy with inhibitors of vascular endothelial growth factor receptor (VEGFR). Our reasons for exploring this combination lies not only in the observation that VEGFR inhibitors suppress unregulated neovascularization (22–24), but they also enhance tumor immunogenicity (25, 26). Thus, sunitinib, a VEGFR inhibitor, has not only been shown to reduce tumor neovascularization, but also to decrease immunosuppressive Treg and myeloid-derived suppressor cell (MDSC) populations in tumor-bearing animals (26, 27). Because sunitinib is approved for treatment of multiple malignancies, we elected to examine whether a combination of a VEGFR inhibitor and the folate-hapten therapy might exceed the efficacies of either therapy alone. In this paper, we report significant synergy between the VEGFR inhibitors and the folate-targeted immunotherapy, not only in their abilities to suppress tumor growth, but also in their capacities to reduce angiogenesis.

Antibodies and reagents

Folic acid, KLH, carboxymethylcellulose (CMC), Tween 20, and female Balb/c serum were purchased from Sigma Aldrich. BSA conjugated to fluorescein (BSA-FITC), folate-EDA-fluorescein (Folate-FITC), and the GPI-0100 adjuvant was kindly provided by Endocyte, Inc. The structure of folate-FITC has previously been published (28). Sunitinib malate and axitinib free base were purchased from LC Laboratories. Disposable PD-10 desalting columns were obtained from GE Healthcare Bio-Sciences. The biotin-conjugated goat anti-mouse IgG2a antibody and streptavidin–HRP conjugate for ELISA antibody titer analysis was manufactured by Caltag Laboratories. The Shandon Cryomatrix resin was purchased from Thermo Scientific. All fluorescently labeled antibodies for flow cytometry and confocal microscopy were obtained from either BioLegend or eBioscience, Inc. The special folate-deficient diet on which animals in treatment studies were maintained was purchased from Harlan Laboratories.

Cell lines and culture

L1210A cells were a generous gift from Dr. Gerrit Jansen, Department of Rheumatology at the University Hospital Vrije Universiteid (Amsterdam, the Netherlands) in 2012. M109 cells were kindly provided by Dr. Alberto Gabizon, Hadassah-Hebrew University Medical Center (Jerusalem, Israel) in 2012. Both L1210A (lymphocytic leukemia) and M109 (lung cancer) cells were selected for high FR expression and have been shown to maintain these elevated FR levels through multiple passages. M109 cells were harvested by digestion of solid tumor xenografts grown in Balb/c mice and cryopreserved at passage 0 or 1. When desired, cell lines were cultured in folate-deficient RPMI1640 medium (Invitrogen) supplemented with 10% heat-inactivated FBS (Sigma Aldrich), penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37°C in a humidified atmosphere containing 5% CO2. Adherent M109 cells were passaged in a monolayer, while L1210A cells were grown in suspension before implantation into syngeneic mice. Renca (renal cell carcinoma) cells were used as a FR-negative control.

Animals and tumor models

All procedures conducted on animals were carried out in accordance with protocols approved by the Purdue Animal Care and Use Committee. M109 and Renca tumors were grown in 5- to 7-week-old female Balb/c mice (Harlan Laboratories), whereas L1210A tumors were implanted in 5- to 7-week-old female DBA/2 mice (The Jackson Laboratory). For M109 tumor implantation, frozen cells at early passage were thawed and allowed to grow to confluence. On the day of tumor injection, M109 or Renca cells were collected and suspended in folate-deficient RPMI1640 medium containing 1% syngeneic female Balb/c serum. Mice were injected with 1 × 106 cells subcutaneously, and therapy was usually initiated between day 10 and 14, when tumors had reached 50–75 mm3. Similar procedures were followed for L1210A tumor growth, only cells were suspended in PBS immediately prior to implantation and treatment began in 7–10 days when tumors had reached 50–75 mm3. Tumor growth was monitored at 48 hours intervals by measurement of size with laboratory calipers and tumor volume was calculated using a standard two-dimensional tumor volume equation; that is, the length of the longest axis of each tumor (L) and the length of the axis perpendicular to it (W) were recorded in millimeters and the corresponding tumor volume in mm3 was determined by solving 0.5(L × W2).

Immunization with KLH-FITC

Animals were vaccinated with a KLH labeled with fluorescein in order to elicit an anti-fluorescein antibody response. The KLH–FITC conjugate was synthesized by reaction of KLH protein with excess FITC according to previously published procedures (15). The resulting KLH–FITC conjugate was purified on a PD-10 desalting column followed by dialysis in PBS at 4°C. KLH-FITC stock solutions were stored in the dark at −20°C until use in immunization cocktails.

Female Balb/c and DBA/2 mice were vaccinated with 35 μg KLH-FITC in 50 μg GPI-0100 adjuvant formulated in sterile saline every 2 weeks for 6 weeks by subcutaneous injection alternating between the base of tail and back of the neck. Blood samples from immunized mice were collected by submandibular puncture one week after the second and third vaccinations and analyzed for anti-fluorescein titer by ELISA, as described previously (15).

Formulation of drugs for in vivo administration

Human cancer patients generally take sunitinib or axitinib orally (29, 30). Therefore, in order to mimic human treatment procedures, sunitinib malate was formulated in a CMC suspension (0.5% carboxymethylcellulose, 1.8% sodium chloride, 0.4% Tween 80, and 0.9% benzyl alcohol, pH 6) and administered to mice by oral gavage. Axitinib was dosed similarly, but was formulated in a different CMC suspension (0.5% CMC in deionized water acidified to pH 2–3). Both drug formulations were prepared freshly every week and stored in the dark at 4°C. The suspensions were shaken vigorously to ensure even drug distribution before dosing. Folate-FITC stock solutions supplied by Endocyte, Inc. were diluted to the desired concentration in sterile PBS, aliquoted, and stored in the dark at −20°C. Aliquots were thawed completely on the day of treatment and mice in the appropriate groups were injected subcutaneously with 100 μL of the diluted conjugate.

Combination therapy studies

The day of tumor cell implantation was designated as day 0 for all experiments. Once tumors had reached approximately 50–75 mm3, mice were randomized into different treatment groups: PBS control, folate-FITC immunotherapy, sunitinib or axitinib therapy alone, or the combination of folate-FITC plus sunitinib or axitinib. Mice in the PBS control group were injected daily with 100 μL of sterile PBS (subcutaneously) and mice treated with folate-FITC alone were injected with 500 nmol/kg on a 5 days on 2 days off schedule (subcutaneously). Mice in the sunitinib alone or axitinib alone groups were gavaged daily with 20 mg/kg sunitinib or 15 mg/kg axitinib, respectively. All mice treated with the combination of folate-FITC plus sunitinib or axitinib were dosed as described above for each of the individual components. Treatments were administered continually until the PBS control tumors reached 1,000 to 1,500 mm3, at which point mice were euthanized and tumors resected for further analysis. To reduce serum folate levels to concentrations comparable with folate levels in humans, all mice were placed on a special folate-deficient diet 1 week following the second immunization.

Flow cytometric analysis of isolated spleen cells

At the conclusion of each study, all animals were euthanized by CO2 asphyxiation, and their tumors and spleens were harvested, washed in PBS, and weighed. Each tumor was then snap-frozen for immunofluorescence staining. For analysis of immune cells in the spleens, spleens were gently mashed and pressed through a 40-μm cell strainer, after which the strainer was carefully washed with PBS to collect any residual cells. Red blood cells were then removed using lysis buffer (Sigma Aldrich) and the remaining splenocytes were suspended in labeling buffer (PBS containing 1% BSA). Fc receptors were blocked using a commercially available Fc blocker (BD Biosciences) to reduce nonspecific labeling. Blocked cells were then incubated for 1 hour at 4°C with the following antibody combinations: APC or PE-conjugated anti-mouse F4/80 for macrophages; PE-conjugated anti-mouse CD3 and FITC-conjugated anti-mouse CD4 or FITC-conjugated anti-mouse CD8 for CD4+ and CD8+ T cells, respectively; and APC-conjugated anti-mouse CD11b and PE-conjugated anti-mouse GR-1 for MDSCs. Following labeling, cells were washed with cold labeling buffer and analyzed on a Becton Dickinson FACSCalibur instrument using CellQuest software. At least 1 × 105 cells were counted from each sample. All flow cytometry data were analyzed on FlowJo software.

Cryopreservation and confocal imaging of tumors

Each resected tumor was embedded in Shandon Cryomatrix resin and snap frozen by partial submersion in liquid nitrogen. The frozen tumor tissues were then sectioned (10 μm) using a Shandon SME Cryotome cryostat (GMI, Inc.) and mounted on to polylysine-coated Superfrost microscopy slides (Thermo Fisher Scientific). For confocal imaging, the slides were allowed to equilibrate at room temperature for approximately 1 hour before fixation and permeabilization in ice-cold acetone (10 minutes). After washing in PBS, the tissue sections were incubated in PBS-Tween containing 1% BSA to block any nonspecific binding followed by washing in PBS (2× 2 min). Individual slides were then incubated with a 1:50 dilution of AlexaFluor 647 conjugated rat anti-mouse CD31 antibody (BioLegend) in a dark humidified chamber for 1 hour at room temperature. The stained slides were washed (3× 5 minutes in PBS), dried, and mounted with cover slips before examination under an Olympus FV1000 inverted confocal laser scanning microscope using a Plan Apo 10× objective. All fluorescence images were obtained under identical conditions and unlabeled tissue sections used as controls were treated in the same manner as labeled slides. Confocal microscopy data were analyzed on FLUOVIEW Viewer software. The confocal images of tumor sections stained with CD31 were also analyzed for vascular density by calculating the percentage of each image's surface that was occupied by a colored pixel.

Statistical analysis

Graphing was performed using GraphPad Prism software. Statistical analyses were performed using the PROC MIXED procedure of SAS statistical software version 9.3. Data were analyzed by two-tailed ANOVA and differences were considered statistically significant at P < 0.05. Pairwise comparisons were performed using a Tukey–Kramer adjustment.

Induction of anti-FITC antibody titer in mice

Mice were immunized three times at 2-week intervals with a KLH–FITC conjugate formulated in GPI-0100 adjuvant. Blood was collected following both the second and third immunization, and antibody titers were assessed to ensure development of a robust anti-FITC immune response. As shown in Supplementary Fig. S1, a strong anti-FITC antibody titer was induced in all immunized groups, and subsequent titer analysis at completion of each study further established maintenance of the titer during the treatment period.

Sunitinib synergizes with folate-hapten–mediated immunotherapy in L1210A tumor-bearing mice

VEGFR inhibitors have been observed to not only downregulate angiogenesis, but also inhibit the immunosuppressive activities of both MDSCs and regulatory T cells (Tregs; ref. 26). To test whether these immunomodulatory properties might augment the potency of our receptor-targeted hapten immunotherapy, it was necessary to grow FR-expressing tumors in immunocompetent mice. For this purpose, L1210A tumors were implanted subcutaneously in DBA/2 mice and the mice were treated with either sunitinib, folate-hapten immunotherapy, or a combination of the two. As seen in Fig. 1, the PBS-treated DBA/2 mice grew tumors that expanded to 1,000 mm3 in <3 weeks. Surprisingly, neither the moderate doses of sunitinib nor folate-targeted immunotherapy used in this study exerted a significant effect on this rapid tumor expansion. In contrast, a combination of the same doses of sunitinib- and folate-targeted immunotherapy caused a decrease in tumor volume (Fig. 1A). These data imply that some type of synergy must exist between the tumor-targeted hapten therapy and VEGFR inhibitor therapy.

Figure 1.

A, Effect of folate-FITC (500 nmol/L/kg, 5 days/week) and/or sunitinib (20 mg/kg daily) therapy on the growth of L1210A tumors in DBA/2 mice. Treatments started on day 7 when tumors reached 50–75 mm3. B, Average spleen weight. Data are mean ± SEM (n = 5–9).

Figure 1.

A, Effect of folate-FITC (500 nmol/L/kg, 5 days/week) and/or sunitinib (20 mg/kg daily) therapy on the growth of L1210A tumors in DBA/2 mice. Treatments started on day 7 when tumors reached 50–75 mm3. B, Average spleen weight. Data are mean ± SEM (n = 5–9).

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Because L1210A cells derive from a murine lymphocytic leukemia that commonly metastasizes to the spleen, it was also instructive to examine whether the spleen weights of each treatment group might differ as an indication of tumor cell metastasis (31). As shown in Fig. 1B, spleens became similarly enlarged in the PBS-, sunitinib-, and folate-FITC–treated animals, but were relatively small in the combination therapy animals. Isolation of extractable cells from these spleens demonstrated that their increase in size derived exclusively from accumulation of metastatic L1210A cells. These data suggest that metastasis of tumor cells to the spleen is suppressed by the combination therapy but not by either individual therapy.

Sunitinib synergizes with folate-hapten–mediated immunotherapy in the M109 tumor-bearing mice

To determine whether the above hypothesized synergy between the folate-hapten therapy and anti-VEGFR therapy might also apply to other tumor types, a second FR+ tumor was similarly examined in syngeneic mice. As seen in Fig. 2, M109 tumor-bearing mice treated with either sunitinib or folate-FITC alone responded only mildly to the individual therapy, resulting in moderately retarded tumor growth. However, mice treated with both folate-FITC and sunitinib again maintained a significantly slower tumor growth rate than either monotherapy group alone. Combination therapy resulted in a 77% reduction in tumor volume and a 55% reduction in tumor weight compared with PBS-treated controls (P < 0.05), which supports the hypothesis that a combination of folate-hapten plus anti-VEGFR therapy results in augmentation of each drug's individual efficacy.

Figure 2.

A, Effect of folate-FITC (500 nmol/kg, 5 days/week) and/or sunitinib (20 mg/kg daily) therapy on the growth of M109 tumors in Balb/c mice. B, Average weights of excised tumors from M109 tumor-bearing Balb/c mice dosed with the indicated therapies. Data are mean ± SEM (n = 5–7). Means that do not share a letter are significantly different, P < 0.05.

Figure 2.

A, Effect of folate-FITC (500 nmol/kg, 5 days/week) and/or sunitinib (20 mg/kg daily) therapy on the growth of M109 tumors in Balb/c mice. B, Average weights of excised tumors from M109 tumor-bearing Balb/c mice dosed with the indicated therapies. Data are mean ± SEM (n = 5–7). Means that do not share a letter are significantly different, P < 0.05.

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Axitinib synergizes with folate-hapten–mediated immunotherapy in L1210A tumor-bearing mice

To determine whether other angiogenesis inhibitors might similarly synergize with the folate-hapten immunotherapy, we explored the use of a more selective VEGFR inhibitor (axitinib) than sunitinib in combination with our FR-targeted immunotherapy. For this purpose, L1210A tumors were again implanted in DBA/2 mice and the affected mice were treated with axitinib (15 mg/kg), folate-targeted immunotherapy, or the combination of the two. As shown in Fig. 3, axitinib, like sunitinib, combined with folate-FITC immunotherapy to significantly slow tumor growth and prolong survival of all treated mice. The average tumor volume in the axitinib alone treated group at the end of the study (day 20) was 53% of the average tumor volume of the PBS-treated mice. Mice treated with axitinib plus folate-FITC, however, displayed significantly smaller tumor volumes (23% of controls, P < 0.05).

Figure 3.

Effect of folate-FITC and/or axitinib therapy on the growth of L1210A tumors in DBA/2 mice. Data are mean ± SEM (n = 6). Means that do not share a letter are significantly different, P < 0.05.

Figure 3.

Effect of folate-FITC and/or axitinib therapy on the growth of L1210A tumors in DBA/2 mice. Data are mean ± SEM (n = 6). Means that do not share a letter are significantly different, P < 0.05.

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Immune effector and suppressor cell levels are altered in combination therapy–treated mice

Studies reported in the literature propose several mechanisms for the known abilities of angiogenesis inhibitors to downregulate immunosuppressive MDSCs and Tregs (22, 25, 26). To determine whether the observed augmentation of folate-FITC immunotherapy might arise from a reduction in either of these immune cell types, spleen cells were isolated from all mice in the above studies and analyzed for different immune cell populations by flow cytometry. Although no differences in Treg numbers could be detected among treatment groups (data not shown), CD4+ and CD8+ T cells were found to be elevated in the combination therapy groups in both L1210A and M109 tumor models (Fig. 4A and B). Moreover, MDSC numbers were reduced in the spleens of M109 tumor-bearing mice treated with the combination therapy (Fig. 4C), although no difference could be observed in the L1210A model. Because immunosuppressive T cells constitute only a fraction of the total T-cell population (usually <3%; ref. 32), the significant increase in both CD4+ and CD8+ T cells argues that the combination therapy augments the cellular immune response against the tumor.

Figure 4.

A, CD4+ and CD8+ T-cell populations in the spleens of L1210A tumor-bearing DBA/2 mice and (B) M109 tumor-bearing Balb/c mice. Isolated splenocytes stained with florescent antibodies against CD3, CD4, and CD8, were analyzed by flow cytometry. C, Percent of MDSC found in the spleens isolated from M109 tumor-bearing mice dosed with the indicated therapies. Isolated splenocytes labeled with fluorescent antibodies against CD11b and GR-1 were analyzed by flow cytometry. A total of 1 × 105 cells were counted from each sample. Data are mean ± SEM (n = 5–7). Means that do not share a letter are significantly different, P < 0.05 (#P = 0.06).

Figure 4.

A, CD4+ and CD8+ T-cell populations in the spleens of L1210A tumor-bearing DBA/2 mice and (B) M109 tumor-bearing Balb/c mice. Isolated splenocytes stained with florescent antibodies against CD3, CD4, and CD8, were analyzed by flow cytometry. C, Percent of MDSC found in the spleens isolated from M109 tumor-bearing mice dosed with the indicated therapies. Isolated splenocytes labeled with fluorescent antibodies against CD11b and GR-1 were analyzed by flow cytometry. A total of 1 × 105 cells were counted from each sample. Data are mean ± SEM (n = 5–7). Means that do not share a letter are significantly different, P < 0.05 (#P = 0.06).

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Synergistic inhibition of tumor vascular growth is observed in combination therapy–treated mice

Because a proposed mechanism of antitumor activity of angiogenesis inhibitors derives from their retardation of neovascularization, we anticipated a reduction in tumor blood vessel density in sunitinib- and axitinib-treated animals, but no significant diminution in other treatment groups. To ascertain the extent of inhibition of neovasculogenesis, we stained tumor sections from mice in each of the treatment groups with an antibody to CD31 (i.e., an established endothelial cell marker), and examined the vascular density/distribution by confocal microscopy. In contrast to expectations, sunitinib alone had little effect on blood vessel density in either of the tumor models, whereas the combination therapy showed a dramatic reduction in CD31 staining in both L1210A and M109 tumors (Fig. 5, top and center). Indeed, quantitative analysis of CD31 staining in each image showed the vascular density of combination treated tumors to be approximately half that of tumors treated with either folate-FITC immunotherapy or sunitinib alone. These data indicate that folate-hapten therapy synergizes with VEGFR inhibitors to augment downregulation of the tumor vasculature. However, as initially anticipated, tumors treated solely with axitinib showed a significant reduction in tumor vasculature staining (∼57% of control), and the combination of axitinib plus immunotherapy further decreased this vascular density to 37% of the PBS-treated control. Representative images of anti-CD31-stained tumors derived from each treatment group as well as the corresponding graphs of a computer analysis of the vascular density are shown in Fig. 5.

Figure 5.

Fluorescent staining and imaging of the vasculature within tumors harvested from treated mice. Top row shows images obtained from solid L1210A tumors treated with PBS, folate-FITC, sunitinib, or a combination of folate-FITC and sunitinib. The center row represents images obtained from solid M109 tumors treated with PBS, folate-FITC, sunitinib, or a combination of folate-FITC and sunitinib. The bottom row shows images collected from stained L1210A tumors collected from mice treated with PBS, folate-FITC, axitinib, or a combination of folate-FITC and axitinib. Frozen tumors were sectioned and stained with an anti-CD31 antibody labeled with AlexaFluor 647 and imaged under an Olympus FV1000 confocal microscope using a 10× objective. Error bars, SEM. Means that do not share a letter are significantly different, P < 0.05.

Figure 5.

Fluorescent staining and imaging of the vasculature within tumors harvested from treated mice. Top row shows images obtained from solid L1210A tumors treated with PBS, folate-FITC, sunitinib, or a combination of folate-FITC and sunitinib. The center row represents images obtained from solid M109 tumors treated with PBS, folate-FITC, sunitinib, or a combination of folate-FITC and sunitinib. The bottom row shows images collected from stained L1210A tumors collected from mice treated with PBS, folate-FITC, axitinib, or a combination of folate-FITC and axitinib. Frozen tumors were sectioned and stained with an anti-CD31 antibody labeled with AlexaFluor 647 and imaged under an Olympus FV1000 confocal microscope using a 10× objective. Error bars, SEM. Means that do not share a letter are significantly different, P < 0.05.

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The unanticipated observation that folate-hapten immunotherapy can augment the ability of VEGFR inhibitors to suppress tumor neovascularization raised the question of whether inhibition of neovasculogenesis might constitute the predominant mechanism accounting for their synergistic effect on tumor growth. To test this hypothesis, FR-negative Renca tumors were implanted in Balb/c mice and treated with folate-hapten immunotherapy, sunitinib, or a combination of the two. In this tumor model, any impact of the folate-targeted immunotherapy would have to be exerted on the FR+ tumor-infiltrating macrophages, because all Renca tumor cells are FR-negative. As shown in Fig. 6A, inhibition of tumor growth was minimal in both sunitinib and folate-hapten monotherapies. In contrast, the combination therapy caused a dramatic decrease in tumor growth, suggesting that the mechanism of synergy does not rely on any direct effect of folate-FITC on the cancer cells. Importantly, anti-CD31-stained sections of the same Renca tumors revealed that their vascular density was lower in the combination than monotherapy-treated animals (Fig. 6B). These data further argue that suppression of neovascularization does not depend on prior destruction of the cancer cells, but rather that a reduction in the vasculature can independently inhibit tumor growth. Based on all of the above data, it is not unreasonable to posit that downregulation of neovasculogenesis may constitute the primary mechanism of tumor growth inhibition in the combination therapy group.

Figure 6.

A, Effect of folate-FITC and/or sunitinib therapy on the weight of FR Renca tumors in Balb/c mice (n = 2–3). Individual data points and means (horizontal lines) are shown. B, Quantification of CD31 fluorescent staining and imaging of the vasculature within Renca tumors harvested from mice treated with PBS, folate-FITC, sunitinib, or a combination of folate-FITC and sunitinib. Data are from five tumor sections. Individual data points and means (horizontal lines) are shown. C, Representative images of CD31 staining for above tumors. Frozen tumors were sectioned and stained with an anti-CD31 antibody labeled with AlexaFluor 647 and imaged under an Olympus FV1000 confocal microscope using a 10× objective.

Figure 6.

A, Effect of folate-FITC and/or sunitinib therapy on the weight of FR Renca tumors in Balb/c mice (n = 2–3). Individual data points and means (horizontal lines) are shown. B, Quantification of CD31 fluorescent staining and imaging of the vasculature within Renca tumors harvested from mice treated with PBS, folate-FITC, sunitinib, or a combination of folate-FITC and sunitinib. Data are from five tumor sections. Individual data points and means (horizontal lines) are shown. C, Representative images of CD31 staining for above tumors. Frozen tumors were sectioned and stained with an anti-CD31 antibody labeled with AlexaFluor 647 and imaged under an Olympus FV1000 confocal microscope using a 10× objective.

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As noted above, the folate-hapten–targeted immunotherapy showed significant promise in human clinical trials, but failed to yield complete responses probably due to inadequate activation of the immune system (21). Attempts to augment the immunotherapy's potency by coadministration of cytokines also failed due to unacceptable toxicities (20). In an effort to identify a less toxic and more effective companion therapy, we explored coadministration of VEGFR inhibitors which are known to activate immune effector cells (22, 33, 34). We found a strong synergy between the VEGFR inhibitors and the folate-hapten immunotherapy, where the combination therapy significantly augmented suppression of tumor vascularization, reduction of MDSCs, and recruitment of CD4+ and CD8+ T cells.

One of the more interesting results from this study was the observed synergy in suppression of tumor neovasculogenesis. Although reduction in blood vessel formation by the two VEGFR inhibitors (i.e., sunitinib and axitinib) was anticipated, the strong augmentation of this reduction by the FR-targeted immunotherapy was not. Contemplation of plausible mechanisms raised the possibility that the immunotherapy-mediated elimination of tumor-associated myeloid cells [i.e., tumor-associated macrophages (TAM) and MDSCs] will simultaneously reduce the levels of growth factors that they produce. Thus, recent examination of FR beta expression in a large number of human tumor samples reveals that virtually all tumor types are characterized by significant infiltration of FR+ TAMs and MDSCs (35). Because these tumor-associated myeloid cells are known to release VEGF, FGF, and many other angiogenic factors (36–39), immune-mediated elimination of these TAMs/MDSCs should significantly reduce the tumor's supply of cytokines that impact tumor angiogenesis. Together with the blockade of VEGF receptor signaling by sunitinib and axitinib (22, 24, 29), the observed synergy of the combination therapy in suppressing vasculogenesis should probably have been predicted. If the same reduction in vascular density can lower the probability of tumor metastasis, then the mechanism underpinning the observed synergy in the combination therapy's ability to eliminate L1210A cell metastasis to the spleen might also be explained.

Taken together, our data suggest that multiple mechanisms may contribute to the efficacy of the combination therapy comprised of the folate-hapten immunotherapy plus VEGFR inhibitors. Not only can the targeted therapy lead to antibody opsonization and immune cell–mediated destruction of FR+ cancer cells (16–18), but the concurrent elimination of FR+ tumor-associated macrophages and MDSCs could reduce both the immunosuppressive microenvironment and growth of new blood vessels within the tumor. Because many human cancer cells do not express FR, but virtually all human tumors contain large numbers of FR+ myeloid cells (35, 40, 41), the combination therapy described here could conceivably prove useful in controlling many human tumor types.

P.S. Low is a Chief Scientific Officer in Endocyte, Inc. and also reports receiving a commercial research grant from Endocyte, Inc. No potential conflicts of interest were disclosed by the other authors.

Conception and design: N.A. Bandara, Y. Lu, P.S. Low

Development of methodology: N.A. Bandara, Y. Lu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N.A. Bandara, C.D. Bates, E.K. Hoylman

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N.A. Bandara, C.D. Bates

Writing, review, and/or revision of the manuscript: N.A. Bandara, P.S. Low

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E.K. Hoylman

Study supervision: P.S. Low

We thank Endocyte, Inc. for providing folate-FITC (EC17) and GPI-0100. We are grateful to Dr. Leroy Wheeler for advice during design of flow cytometry experiments and to Dr. Ahmad Gheith for assistance in interpretation of confocal images of CD31-stained tumor tissues. The authors gratefully acknowledge the flow cytometry resources from the Purdue University Center for Cancer Research, NIH grant P30 CA023168, and the imaging facilities of the Bindley Bioscience Center, a core facility of the NIH-funded Indiana Clinical and Translational Sciences Institute.

This work was supported in part by a grant from Endocyte, Inc. (awarded to P.S. Low) and NIH grant P30 CA023168.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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