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Cancer Therapy: Preclinical |
Authors' Affiliations: Departments of 1 Pathology and 2 Medicine, Stanford University School of Medicine, Palo Alto, California and 3 Department of Surgery, Tokyo Medical University, Tokyo, Japan
Requests for reprints: Edgar G. Engleman, Stanford Blood Center, 3373 Hillview Avenue, Palo Alto, CA 94304-1204. Phone: 650-723-7960; Fax: 650-725-0592; E-mail: edengleman{at}stanford.edu.
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Abstract |
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Experimental Design: BALB/c or C57Bl/6 mice were injected s.c. with CT26 colorectal carcinoma cells and B16 melanoma cells, respectively, and following 10 to 12 days of tumor growth, the tumors were treated with PDT alone or PDT followed by IT-DC or IT-PBS. In other studies, tumors were established simultaneously in both lower flanks or in one flank and in the lungs, but only one flank was treated.
Results: Whereas neither PDT nor IT-DC alone was effective, PDT + IT-DC eradicated both CT26 and B16 tumors in a significant proportion of animals and prolonged the survival of mice of which the tumors were not cured. The spleens of mice treated with PDT + IT-DC contained tumor-specific cytotoxic and IFN-
-secreting T cells whereas the spleens of control groups did not. Moreover, adoptive transfer of splenocytes from successfully treated CT26 tumor-free mice protected naïve animals from a subsequent challenge with CT26, and this was mediated mainly by CD8 T cells. Most importantly, PDT plus IT-DC administered to one tumor site led to tumor regression at distant sites, including multiple lung metastases.
Conclusions: PDT + IT-DC induces potent systemic antitumor immunity in mice and should be evaluated in the treatment of human cancer.
Among the modalities that might be combined with DC-based immunotherapy are systemically administered antitumor drugs as well as locally targeted therapies such as radiation, radiofrequency ablation, and photodynamic therapy (PDT). PDT has been approved in the United States and other countries as an anticancer therapy, mainly for the palliative treatment of surgically inaccessible tumors. PDT involves the systemic administration of a photosensitizer that preferentially accumulates in transformed cells, followed by illumination of the tumor with a laser beam (12). In the presence of oxygen, the laser light activates the photosensitizer and initiates a complex photochemical reaction that generates cytotoxic intermediates. Tumor destruction after PDT results from direct cytotoxic effects as well as from the induction of a local inflammatory response (12). Thus, preclinical studies have shown that PDT not only mediates apoptotic and necrotic killing of tumor cells but also alters the tumor microenvironment through the release of proinflammatory cytokines such as tumor necrosis factor 
, interleukin (IL)-1 and IL-6 (12, 13).
On the basis of its unique mechanism of tumor destruction, PDT has the potential to create an environment at the tumor site that favors both tumor antigen loading and activation of DCs, key requirements for induction of antitumor immunity (14). Because most tumors lack an abundance of DCs, one way to potentially take advantage of this environment would be to inject a sufficient number of autologous DCs directly into PDT-treated tumors. Such a strategy alleviates the need to do in vitro loading of DCs with tumor antigens because the inflammatory milieu induces DC activation, which facilitates not only antigen acquisition and processing but also migration of the DCs to draining lymph nodes where they interact with a broad range of potential effector cells. In the current study, we evaluated the effect of combining PDT with intratumoral injection of syngeneic DCs (IT-DC) on two histologically distinct murine tumors, CT26 colon carcinoma and B16 melanoma. The results show that this combined treatment induces strong and durable tumor-specific immunity that results in the destruction not only of targeted tumors but also of tumors at distant sites.
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Materials and Methods |
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Cell lines. The murine CT26 colon carcinoma, B16 melanoma (F10 clone), MAD109 lung carcinoma, and EL-4 lymphoma cell lines used in this study were maintained in complete RPMI 1640 with 10% fetal bovine serum, penicillin G (100 units/mL), streptomycin (100 µg/mL), and L-glutamine (10 mmol/L).
DCs. Bone marrow–derived DCs were generated in the presence of granulocyte macrophage colony-stimulating factor and IL-4 for 7 days as previously described (15). Bone marrow cells were harvested from femurs and tibias, and after RBC lysis, the resulting cell suspension was incubated in complete RPMI 1640 containing recombinant murine granulocyte macrophage colony-stimulating factor (10 ng/mL; PeproTech, Inc., Rocky Hill, NJ) and recombinant murine IL-4 (10 ng/mL; PeproTech). On day 2, nonadherent granulocytes were gently removed and fresh medium with granulocyte macrophage colony-stimulating factor and IL-4 was added. On day 5, loosely adherent cells were dislodged and replated. On day 7 of culture, the unpulsed DCs were collected. The maturational status and percentage of DCs were verified by flow cytometry, and staining of three surface markers (CD11c, CD86, and class II antigen) showed the purity of DCs to be 
74%.
Photosensitizer and laser unit. ATX-S10 Na(II), a hydrophilic chlorin photosensitizer with an absorption maximum at 670 nm (16), was obtained from Photochemical Co. Ltd. (Okayama, Japan). A diode laser (Hamamatsu Photonics, Hamamatsu, Japan) was used as a light source for exciting ATX-S10 Na(II). The diode laser is a continuous-wave laser operating at 670-nm wavelength.
During the light irradiation, mice were anesthetized with ketamine (125 mg/kg: Vedco, Inc., St. Joseph, MO) and xylazine (25 mg/kg; Phoenix Pharmaceutical, Inc., St. Joseph, MO) and were restrained in a specially designed holder.
Combined PDT and IT-DC therapy of CT26 colon cancer. Preliminary studies with ATX-S10 Na(II) and a diode laser, were carried out on the basis of a published protocol (16) to identify a drug dose and laser setting for inhibition of growth of CT26 tumors in vivo without major local or systemic toxicity. CT26 tumor cells (106 per mouse) in 100 µL HBSS were injected into the lower right flank of BALB/c mice. On day 12, when the average tumor volume was 153.0 ± 11.5 mm3, the mice received an i.v. injection of ATX-S10 Na(II) (5 mg/kg body weight), followed 4 to 5 hours later by 150 J/cm2 laser irradiation of the tumor. DCs (1 x 106 per injection in 50 µL PBS) were injected into the tumor on days 13, 14, 15, and 17. The tumor volume was measured thrice a week using a caliper [tumor volume (mm3) = (longer diameter) x (shorter diameter)2 x 0.4]. Animals were sacrificed when the tumor diameter exceeded 20 mm or when there were signs of animal distress. Survival was recorded as the percentage of surviving animals on a given day. Surviving mice had no sign of tumor when experiments were terminated.
Combined PDT and IT-DC therapy of B16 melanoma. For the B16 melanoma tumor model, C57Bl/6 mice were inoculated s.c. with 5 x 105 tumor cells in the lower right flank. On day 10, mice with established tumors (average tumor volume, 80.0 ± 5.4 mm3) were treated with 150 J/cm2 laser irradiation to the tumor 4 to 5 hours after an ATX-S10 Na(II) (5 mg/kg body weight) injection and given intratumoral injections of DCs on days 11, 12, 13, and 15 (1 x 106 per injection in 50 µL PBS). The measurement of tumor volume and survival was as above.
ELISPOT assays. ELISPOT assays were done with an ELISpot mouse IFN- system (R&D Systems, Inc., Minneapolis, MN) according to the instructions of the manufacturer. Splenocytes were isolated 4 weeks after tumor inoculation. After lysis of RBC, splenocytes were resuspended at a final concentration of 5 x 105/mL and 100 µL of this suspension were then incubated at 37°C for 24 hours in ELISPOT plates coated with anti-IFN- with 100 µL medium with or without irradiated (30 Gy) stimulator cells (CT26, MAD109, B16, or EL-4).
Cytotoxicity assays. Cytotoxicity was measured by a standard chromium-51(51Cr) release assay. Splenocytes were harvested 4 weeks after tumor inoculation. After lysis of RBC, splenocytes (1 x 106/mL) were stimulated in vitro by irradiated (100 Gy) tumor cells (1 x 105/mL) at 37°C for 5 days in the presence of 10 units/mL IL-2. Following culture, splenocytes were separated from dead cells and debris with Lympholyte-M cell separation media (Cederlane Laboratories, Inc., Hornby, Ontario, Canada). Target cells were labeled with 51Cr (200 µCi/5 x 106 cells) for 1 hour at 37°C, washed, and then incubated in U-bottomed wells with effector cells at various effector-to-target cell ratios at 37°C for 4 hours. Spontaneous release and maximum release were determined by incubating target cells in medium alone or in 1% SDS, respectively. Spontaneous release was always <20% of maximum. Radioactivity was counted in a liquid scintillation counter and the percentage of specific target cell lysis was calculated with the formula [(E – S) / (T – S)] x 100, where E is the average experimental release, S is the average spontaneous release, and T is the average maximal release.
Adoptive transfer of splenocytes. To determine whether lymphocytes induced by PDT + IT-DC could protect naïve animals from a tumor challenge, BALB/c mice were inoculated s.c. with CT26 cells and treated with PDT + IT-DC as before. Four weeks later, the splenocytes were harvested and 1 x 107 cells were infused i.v. into naïve mice. Control groups of mice received splenocytes from CT26 tumor-bearing mice treated with either IT-PBS alone, IT-DC alone, or PDT + IT-PBS. One day later, these mice were s.c. challenged with a lethal number (1 x 106) of CT26 cells and monitored for tumor volume and survival.
To analyze the role of specific T-cell subsets in tumor protection, splenocytes were harvested from inoculated tumor-free mice treated with PDT + IT-DC, and CD4+ or CD8+ T cells were depleted by using CD4 (L3T4) or CD8a (Ly-2) coupled microbeads and magnetic cell sorting (Miltenyi Biotec, Inc., Auburn, CA). Splenocytes, CD4+ T-cell–depleted splenocytes, or CD8+ T-cell–depleted splenocytes (1 x 107) were infused i.v. into groups of five naïve mice. Control mice received splenocytes from naïve mice. One day later, the mice were s.c. challenged with a lethal number (1 x 106) of CT26 cells and monitored for tumor volume and survival. These depletion conditions were validated by flow cytometry analysis using anti-CD4 (L3T4)-FITC and CD8a (Ly-2)-phycoerythrin monoclonal antibodies (PharMingen, San Diego, CA). The percent depletion of CD4+ and CD8+ cells was 97% and 93%, respectively.
Secondary tumor challenge. To determine the persistence of tumor-specific immunity in the mice treated by PDT + IT-DC, at day 60 after first tumor inoculation, mice showing complete regression of CT26 tumors were given a second s.c. tumor challenge (1 x 106 CT26) in the left lower flank (contralateral to the first injection site). These mice, as well as a control group of naïve mice that were inoculated with 1 x 106 CT26 tumor cells, were monitored for tumor size and survival.
Effect of PDT + IT-DC on contralateral tumors. To determine whether PDT + IT-DC treatment of one s.c. tumor affected an established contralateral s.c. tumor, CT26 cells (1 x 106) were injected s.c. into both lower flanks of BALB/c mice on day 0. On day 12, tumor-bearing mice were either untreated or treated with combined PDT + IT-DC (using the protocol above) into the tumor on the right side but not into the tumor on the left side. The bilateral tumor-bearing mice were followed for tumor volume on both flanks.
Effect of PDT + IT-DC on multiple distant tumors. BALB/c mice were inoculated s.c. with 1 x 106 CT26 tumor cells on day 0. On day 5, these mice were injected i.v. with 1 x 106 CT26 tumor cells. PDT + IT-DC was administered on day 12 (as above) to the s.c. tumor alone. On day 21 after i.v. tumor inoculation, mice were euthanized and lungs were harvested and fixed with Bouin's solution (Sigma-Aldrich, St. Louis, MO).
Statistical analysis. Differences between groups were analyzed using unpaired two-tailed Student's t test. Survival curves were plotted by the Kaplan-Meier method and comparisons among groups in the survival data were calculated by log-rank test.
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40 days after treatment (Fig. 2C).
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production by host-derived T cells and systemic antitumor immunity has been shown in other studies (18, 19). Using ELISPOT assays, we evaluated whether treatment of CT26 tumor-bearing mice with PDT + IT-DC could elicit tumor-specific IFN--secreting T cells. As shown in Fig. 3A
, splenocytes from mice treated with PDT + IT-DC contained significantly more tumor-specific IFN--secreting cells than splenocytes from other groups (P < 0.05, versus other groups). Moreover, this cytokine was not secreted spontaneously or in response to MAD109 cells, which are irrelevant syngeneic murine lung tumor cells, indicating that the observed response was immunologically specific to CT26 tumor cells. In additional studies, we analyzed the splenocytes of the different treatment groups for the presence of CT26-specific CTLs. Figure 3B shows that such cells were present in the PDT + IT-DC group but not in other groups (P < 0.05, versus all other groups). There was no killing of a syngeneic lung cancer cell line (MAD109), indicating that the CTLs are CT26 tumor specific (data not shown).
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-secreting cells compared with splenocytes from control groups (P < 0.05). In addition, as shown in Fig. 4B, PDT + IT-DC induced tumor-specific CTLs, as indicated by the presence of such cells in treated but not control animals. No lysis of a syngeneic lymphoma cell line (EL-4) was observed, indicating those CTLs are B16 tumor specific (data not shown).
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Discussion |
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Although relatively little work has been done to evaluate the combination of PDT and IT-DC, several previous studies have shown the benefit of combining IT-DC with chemotherapy or radiotherapy (20–26). In contrast to chemotherapy or radiotherapy, PDT is not associated with systemic toxicity. Moreover, PDT renders murine tumors more immunogenic than tumors treated with UV or ionizing irradiation, or frozen and thawed tumors (27). Recently, Jalili et al. (28) reported that PDT in combination with IT-DC had little or no effect on s.c. CT26 tumors but inhibited the growth of contralateral tumors. By contrast, we observed dramatic effects on both local and distant tumors despite the fact that treatment was begun 12 days after inoculation of mice with a higher tumor dose than that studied by Jalili et al. One possible explanation for this surprising result is that we injected the same DC number four times as opposed to twice in their study. Another difference in our two studies is that Jalili et al. used the hematoprophyrin derivative, profimersodium (Photofrin), as a photosensitizer whereas we used ATX-S10 Na(II). Although Photofrin is widely used clinically, its potency is limited by weak absorbance at the shorter range of the red region of the spectrum. In addition, Photofrin is not a pure substance but a mixture of hematoporphyrin monomers, dimers, oligomers, and their dehydration products, and these products are associated with long-lasting skin photosensitivity (12). In contrast, ATX-S10 Na(II) is a homogeneous agent that preferentially accumulates in tumor tissues and is eliminated from normal tissues within 24 to 48 hours after injection. Moreover, its absorption maximum lies at 670 nm, which is longer than that of Photofrin (630 nm) and enables deeper tissue penetration (16). Using ATX-S10 Na(II) as a photosensitizer, we showed that PDT in combination with IT-DC inhibits the growth of two histologically distinct murine tumors. Interestingly, treatment of B16 tumors with combined PDT + IT-DC was at least as effective as it was for CT26, despite the poor immunogenicity of B16 and its well-documented resistance to PDT alone (29).
Our studies clearly show that PDT + IT-DC induces systemic antitumor immunity as well as tumor-specific immunologic memory. In the B16 model, the observation of white hair in untreated sites of mice, of which the tumors had been eradicated following PDT + IT-DC, suggests that treatment induced a systemic immune response against one or more shared antigens present in normal melanocytes as well as B16 tumors. In the CT26 model, PDT + IT-DC treatment of a single s.c. tumor resulted in regression of both contralateral as well as multiple pulmonary tumors. The cells responsible for mediating tumor regression were cytotoxic T cells as indicated by both in vitro cytotoxicty assays and the observation that naïve animals were protected against tumors by adoptively transferred splenocytes from successfully treated tumor-bearing mice. Depletion of specific T-cell subsets (CD4 or CD8) in the adoptively transferred splenocytes indicated the CD8 T cells are the major effector cells induced by the PDT + IT-DC treatment.
Critical to the systemic antitumor effect of PDT + IT-DC is the capture of tumor-associated antigens by DCs as well as DC activation. Whether necrotic or apoptotic tumor cells serve as the superior source of tumor-associated antigens is controversial (30–34). Our data (not shown) strongly suggest that PDT, which induces both apoptosis and necrosis of tumors (12, 28), causes DCs to take up and process tumor antigen released by the dying tumor cells, mature, and become activated in situ and then cross-prime T cells against tumor-derived antigens. Interestingly, PDT alone had little or no effect on the growth of B16 tumors and analysis of such tumors following their treatment with PDT revealed a much smaller percentage of apoptotic and necrotic cells than in identically treated CT26 tumors (data not shown). Because PDT + IT-DC was effective as a treatment for both tumors, it seems likely that a relatively small number of dead or dying tumor cells can provide the necessary antigens required for DC-mediated induction of antitumor immunity. Furthermore, because it is known that PDT stimulates the expression of inflammatory cytokines such as tumor necrosis factor , IL-1, and IL-6 (12, 13), perhaps the presence of such factors in the tumor microenvironment played a critical role in the induction of DC maturation.
In summary, the data presented in this report indicate that PDT + IT-DC results in potent systemic antitumor immunity and regression of tumors including tumors at sites distant from the treated site. Based on these findings, this novel regimen may prove beneficial in the treatment of patients with advanced metastatic disease as well as in the neoadjuvant setting before resection of tumors known to have a high recurrence rate.
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Acknowledgments |
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Footnotes |
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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.
Note: H. Saji and W. Song contributed equally to this work.
Received 9/12/05; revised 1/ 5/06; accepted 1/19/06.
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References |
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and tumor necrosis factor have a role in tumor regressions mediated by murine CD8+ tumor-infiltrating lymphocytes. J Exp Med 1991;173:647–58.This article has been cited by other articles:
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  I. Melero, A. Arina, O. Murillo, J. Dubrot, C. Alfaro, J. L. Perez-Gracia, M. Bendandi, and S. Hervas-Stubbs Immunogenic Cell Death and Cross-Priming Are Reaching the Clinical Immunotherapy Arena Clin. Cancer Res., April 15, 2006; 12(8): 2385 - 2389. [Full Text] [PDF]
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); PDT combined with intratumoral PBS (n = 9; 
); and PDT combined with IT-DC (n = 9; 
). A, mean tumor volume (mm3) for treatment groups [mean tumor volume = (longer diameter) x (shorter diameter)2 x 0.4]. Points, mean; bars, SE. *, P < 0.05, PDT + IT-DC versus other treatments. B, survival of mice recorded as the percentage of surviving animals on a given day. Surviving mice had no sign of tumor when the experiment was terminated. C, CT26 tumor rechallenge of tumor-free mice after PDT + IT-DC. Mice that had received CT26 inoculation followed by PDT + IT-DC were rechallenged s.c. with a lethal number (1 x 106) of CT26 tumor cells (n = 4; 
). Points, average tumor volume (mm3) of five mice per group; bars, SE. B, role of CD4 and CD8 T-cell subsets in protection against CT26 tumors. Splenocytes were harvested from tumor-free mice treated with PDT + IT-DC. CD4+ or CD8+ T cells in splenocytes were depleted by using CD4 (L3T4) or CD8a (Ly-2) microbead magnetic cell sorting. After magnetic selection, whole splenocytes (