Dendritic cells (DCs) are attractive candidates for innovative cancer immunotherapy by virtue of their potential to function as professional antigen-presenting cells for initiating cellular immune responses. In this study, we evaluated a possible synergy of conventional chemotherapy together with intratumoral injection of syngeneic bone marrow-derived DCs for the treatment of preexisting tumors. Using murine CT26 colon adenocarcinoma cells (parental or modified to express β-galactosidase as a model tumor antigen) to produce s.c. tumors in syngeneic BALB/c mice, the data demonstrate that direct injections of DCs at the tumor site result in partial eradication of established tumors. Strikingly, the addition of systemic chemotherapy (cyclophosphamide) combined with local intratumoral injection of DCs led to complete tumor regression in the treated animals. The tumor-free mice were able to resist a repeat challenge with the same tumor, suggesting that the animals had acquired long term antitumor immunity. Supporting evidence for the paradigm of systemic chemotherapy and intratumoral administration of DCs was obtained using melanoma B16 syngeneic tumor treated with Adriamycin plus DCs. These novel findings raise the possibility of using this potent strategy of combined intratumoral injections of DCs and systemic chemotherapy for cancer treatment.
DCs3 function as antigen-presenting cells in the induction of T lymphocyte-mediated immune responses restricted to complementing the MHC (1, 2, 3). In vivo, DCs can take up and process antigens derived from bacteria, proteins, viruses, and cells and present these antigens to T cells for initiating immunity (1, 2, 3, 4). Although it is widely accepted that many murine and human tumors carry immunogenic epitopes on their surface as potential targets that can be recognized by the host immune system, failure of host defenses to mount an appropriate response to these antigens allows uncontrolled tumor growth in cancer patients (5, 6). The reasons that the immune system does not effectively destroy tumor cells are not fully understood but are linked to low immunogenicity of the tumor and/or defects in the host immune system, such as defective function and number of T cells and antigen-presenting cells (7, 8). As strategies to overcome host tolerance to tumors, several approaches have been developed in which syngeneic DCs are armed with tumor-specific antigens as cancer vaccines. The basic concept is to boost the process of tumor antigen-specific sensitization of T cells, enhancing antigen-specific responses against the tumor in vivo (9, 10). Examples of loading DCs to initiate both protective and therapeutic antitumor responses include: (a) DCs pulsed with defined peptides, proteins, or tumor cell lysate; (b) DCs genetically modified to express tumor antigens; and (c) DCs fused with tumor cells (11, 12, 13, 14, 15, 16, 17, 18). In all of these approaches, there is a requirement for ex vivo manipulation of the DCs to acquire and present tumor-specific antigens.
On the basis of the emerging evidence that in vitro physical interaction between DCs and tumor cells is fundamental to the induction of therapeutic immunity, and that DCs are capable of ingesting apoptotic tumor cells and acquiring tumor-associated antigens to induce class I-restricted CTLs in vitro (19, 20, 21), we hypothesized that intratumoral administration of bone marrow-derived DCs, in the context of chemotherapy to induce tumor cell death, may be a strategy to enhance the presentation of tumor-specific antigens to the immune system. This approach may induce tumor immunity with consequent suppression of tumor growth without the requirement for in vitro interaction of DCs with tumor cells/antigens.
To evaluate this hypothesis, we used several well-characterized syngeneic murine tumor models to test the antitumor potential of intratumoral DC injection, with or without chemotherapy. The data demonstrate that DCs can act as therapeutic reagents when injected into the tumor site, with consequent delay of tumor growth mediated by tumor-specific immune responses. Far more dramatic antitumor effects, however, are observed when the intratumoral administration of DCs is combined with systemic chemotherapy, resulting in marked tumor suppression with persistent antitumor immune memory.
MATERIALS AND METHODS
Female BALB/c (H-2d) and C57Bl/6 mice, 6–8 weeks of age, were purchased from The Jackson Laboratory (Bar Harbor, ME). The animals were housed under specific pathogen-free conditions according to NIH guidelines.
CT26.WT (H-2d) is a clone of the N-nitroso-N-methylurethane-induced BALB/c undifferentiated colon adenocarcinoma cell line. CT26.CL25 is a clone of CT26.WT that has been transduced with the Escherichia coli βgal gene (both cell lines provided by N. P. Restifo, National Cancer Institute, Bethesda, MD; Ref. 22). B16 is a C57Bl/6-derived melanoma (American Type Culture Collection, Rockville, MD). C3, a tumor cell line, was generated by transfecting C57Bl/6 mouse embryonal fibroblasts with plasmids containing the entire genome of the human papilloma virus type 16 (gift of M. Vierboom, University of Leiden, Leiden, the Netherlands; Ref. 23). The SVBalb (H-2d) fibroblast cell line (provided by Dr. L. Gooding, Emory University, Atlanta, GA), syngeneic to BALB/c, was used as an in vitro stimulator of cells in the CTL assay (24).
CT26.WT cells were cultured in complete RPMI-1640 medium with 10% fetal bovine serum, 2 mm l-glutamine, 100 μg/ml streptomycin, and 100 units/ml penicillin. CT26.CL25 cells were maintained in the same medium plus 400 μg/ml G418 (Life Technologies, Inc., Gaithersburg, MD). SVBalb and B16 cells were grown in DMEM containing 10% FCS and antibiotics. C3 cells were cultured in Iscove’s modified Dulbecco medium supplemented with 8% FCS, penicillin, and 2-mercaptoethanol.
Both CTX and MMC were from Sigma Chemical Co. (St. Louis, MO), and 5-FU was from SoloPak Laboratories (Elk Grove Village, IL). ADR was from Gensia Laboratories, Ltd. (Irvine, CA).
Primary DCs were obtained from mouse bone marrow precursors as described previously (18, 25). Briefly, murine bone marrow cells were harvested from femurs and tibias and then plated in complete RPMI 1640 containing recombinant murine GM-CSF (500 units/ml; Sigma Chemical Co.) and recombinant murine IL-4 (20 ng/ml; Genzyme, Farmington, MA). On day 2, nonadherent granulocytes were gently removed, and fresh medium with GM-CSF and IL-4 was added. On day 4, loosely adherent cells were dislodged and replated. On days 5–7 of culture, immature DCs, as nonadherent proliferating aggregates, were collected. Functional analysis of the bone marrow-derived DCs was performed, and the maturation status and percentage of DCs were verified by flow cytometry (18), with three surface markers (CD11b, CD44, and class II antigen IAd) showing the purity of DCs to be ≥72%.
Combined Intratumoral DCs and Chemotherapy.
Preliminary studies were carried out using four antitumor agents (5-FU, CTX, ADR, and MMC) to control the growth of the model tumors (CT26.WT, CT26.CL25, B16, and C3) with different doses and schedules to determine the drug dose for inhibition of tumor growth in vivo without major systemic toxicity. Mitomycin and 5-FU were not found to be useful for the tumors evaluated and were not assessed further.
For the CT26.CL25 or CT26.WT tumor models, 105 cells/mouse in 100 μl of in HBSS were implanted into BALB/c mice in the lower right flank. On day 11, the average tumor sizes of CT26.CL25 and CT26.WT were 27.8 ± 3.8 and 22.0 ± 2.1 mm2, respectively. The mice then received i.p. injections of CTX (150 mg/kg body weight); injections were repeated 2 days later with the same dose. Four injections of DCs (each injection was 106 cells/mouse in 30 μl of HBSS) were given into the tumor on days 12, 13, 14, and 18. The experimental groups included: group 1, untreated controls; group 2, intratumoral injections of DC alone; group 3, CTX treatment alone; group 4, combined CTX and intratumoral DC injections; and group 5, combined CTX and DCs that were injected s.c. in the left flank, a site distant to the tumor. For all animal experiments, the tumor size was measured biweekly using a caliper and expressed as the product of the maximal perpendicular diameters (mm2). Animals were sacrificed when the tumor diameter exceeded 15 mm, when there was tumor ulceration, or when there were other 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 the experiment was terminated. All experiments were performed at least twice with reproducible outcomes using individual treatment groups of five mice for each treatment.
For the melanoma B16 tumor model, naive C57Bl/6 mice were inoculated s.c. with 2 × 105 B16 cells in the lower right flank. On day 9, mice with established tumors (average tumor size, 25.7 ± 4.6 mm2) were administered ADR i.p. treatment on 3 consecutive days (3 mg/kg body weight each day) and received injections of DCs four times at days 10, 11, 12, and 16 (each injection 106 cells/mouse in 30 μl of HBSS). Four groups (untreated controls, DC alone, ADR alone, and ADR plus intratumoral DC injections) were included. The measurement of tumor size and survival was as described above.
The mice bearing C3 tumors were treated by intratumoral injections of DCs using 106 C3 tumor cells and intratumoral DC injections on days 13, 14, 15, and 20 (on day 13, average tumor size was 21.6 ± 4.2 mm2). For this tumor, the DCs completely protected the mice, and additional experiments with chemotherapy were not carried out.
To determine whether the CT26.CL25 tumor-free mice that survived the combined intratumoral injection of bone marrow DCs and chemotherapy with CTX were associated with tumor-specific CTLs, the spleens were removed from these mice, and the splenocytes (2 × 106/ml) were plated with irradiated (5000 rads) syngeneic SVBalb cells (2 × 105/ml), pulsed previously with a synthetic βgal peptide (1 μm). After 5 days of coculture, the in vitro restimulated viable splenocytes were examined for specific cell lysis using a standard 51Cr release assay against a variety of target cell lines, including syngeneic CT26.WT, βgal-expressing CT26.CL25, and βgal peptide-pulsed CT26.WT, as well as SVBalb used as a negative control. The peptide used in the CTL studies was the nanomer TPHPARIGL (residues 876–885) for βgal (26), synthesized (Biosynthesis, Lewisville, TX) to a purity of >99% as determined by high pressure liquid chromatography and amino acid analysis. The 51Cr release assay was performed as described previously (27).
Secondary Tumor Challenge in Tumor-free Mice.
To determine the persistence of tumor-specific immunity in the mice treated by combination therapy, at day 60 after first tumor inoculation, five mice showing complete regression of CT26.CL25 tumors were given a second s.c. tumor challenge. To generate s.c. tumors, CT26.CL25 and CT26.WT tumor cells (105 cells each in 100 μl of HBSS) were implanted into BALB/c mice on the right and left lower flanks, respectively. The mice were followed for survival and measurement of tumor size, as compared with the control group, both CT26.CL25 and CT26.WT tumors were inoculated (105 cells, each in 100 μl of HBSS on opposite flanks of naive mice).
Adoptive CTL Transfer for Tumor Prevention.
To determine whether the tumor-specific CTLs induced by bone marrow-derived DCs via intratumoral injections acquiring tumor antigens in vivo were responsible for complete regression of preestablished s.c. tumors, βgal-specific CTLs were generated from tumor-free mice as described above and then i.v. infused into naive mice (3 × 106 CTLs/mouse). One day later, these mice were s.c. challenged with 105 CT26.CL25 and monitored for tumor size and survival.
Regression of Second Preestablished Tumor by Chemotherapy and Local DC Injection.
To evaluate whether multiple preestablished tumors could be cured after systemic chemotherapy and local DC injection into one of the tumors, CT26.CL25 tumors (105 cells for each side) were s.c. implanted in both lower flanks of BALB/c mice. Eleven days later, tumor-bearing mice were either untreated or treated with combined CTX and intratumoral injection of naive DCs only into the tumor on the right side but not into the tumor on the left side, following the same dose schedule as described above. The bilateral tumor-bearing mice were followed for tumor size on both flanks.
The tumor growth data are presented as mean ± SE. Comparisons among groups of tumor growth were made using the unpaired two-tailed Student’s t test. Comparisons among groups in the survival data were made using the log-rank test (28).
Overview of the DC + Chemotherapy Models.
To determine whether direct administration of syngeneic DCs to tumors in the context of concomitant chemotherapy suppresses growth of preexisting tumors, four well-characterized tumor models (colon CT26.CL25 and CT26.WT, melanoma B16, and sarcoma C3) were evaluated. The starting point of treatment for this study was an average tumor size ranging from 21 to 28 mm2. As described below, intratumoral injections of immature DCs alone or chemotherapy alone to CT26.CL25, CT26.WT, and B16 tumors had minimal effects. In contrast, when equivalent numbers of DCs were delivered into mice with sarcoma C3 tumors (which grow slower than B16 and CT26 tumors and express high levels of human papilloma virus), complete eradication of all preestablished C3 tumors was observed in the absence of chemotherapy (not shown). In this context, the C3 tumors in BALB/c mice were not evaluated further.
The selected antitumor agent for the combination treatment of CT26.WT and CT26.CL25 cells was CTX, because it was the most effective drug among the four drugs tested (CTX, 5-FU, MMC, and ADR) for delaying CT26 colon tumor growth in vivo (not shown). In contrast, for the B16 tumors, the most effective chemotherapy was ADR (not shown).
Combination Treatment with Chemotherapy and Intratumor Administration of DCs.
To test the hypothesis that systemic administration of chemotherapeutic agents together with intratumoral administration of DCs will control tumor growth, treatment with chemotherapy was initiated when the tumor was of appropriate size, and local administration of DCs were started 1 day after the first dose of chemotherapy.
For the CT26.CL25 colon carcinoma tumors in BALB/c mice, the tumors grew rapidly in the control (untreated) mice, as well as in the mice treated with DCs alone (Fig. 1, A and B). Although treatment with systemic CTX alone (Fig. 1,A) or systemic CTX together with DCs in the opposite flank (Fig. 1,B) resulted in partial suppression of tumor growth, the combination of systemic CTX + DCs administered directly to the tumor resulted in complete suppression of tumor growth (P < 0.03, all observations of CTX + DCs compared with other groups; Fig. 1, A and B). Consistent with the complete suppression of tumor growth in the CTX + DC treated mice, the CTX + DC treated mice had 100% survival (Fig. 1,C). In contrast, there was reduced survival for all of the control groups (P < 0.01, all observations of CTX + DCs compared with other groups; Fig. 1 C).
A similar pattern was observed with the CT26.WT tumors in BALB/c mice (Fig. 2). Whereas the CTX + DC therapy resulted in complete suppression of tumor growth (Fig. 2,A), CTX alone (Fig. 2,A), or DC alone, or CTX + DCs in the opposite flank (Fig. 2,B) gave only partial suppression compared with the control (P < 0.04, all observations of CTX + DCs compared with other groups; Fig. 2, A and B). Consistent with the complete suppression of tumor growth in the CTX + DC treated mice, the CTX + DC treated mice had 100% survival (Fig. 2,C). In contrast, there was reduced survival for all of the control groups (P < 0.01, all observations of CTX + DCs compared with other groups; Fig. 2 C).
Finally, assessment of survival in the B16 tumors in C57Bl/6 mice showed that the combination of DC + ADR markedly enhanced survival compared with ADR alone, or DC alone compared with the control (untreated) mice (P < 0.001, all observations of ADR + DCs compared with other groups; Fig. 3).
Persistent Antitumor Memory after Combined Chemotherapy and Intratumor Administration of DCs.
To further understand the mechanism responsible for complete regression of preestablished s.c. tumors and to evaluate the memory of antitumor immunity in vivo after combined chemotherapy and administration of DCs, the following were evaluated using the CT26.CL25 model: the generation of antitumor CTLs; a second lethal challenge of the same tumor to the surviving mice; and adoptive transfer of the tumor-specific CTLs. The CT26.CL25 tumor cells not only carry the artificial βgal gene as a model tumor antigen but also its parent antigens (29). Splenocytes from CT26.CL25 tumor-free mice were harvested and stimulated in vitro with βgal peptide and then measured for specific cell lysis against a panel of target cells in a standard 51Cr release assay. Significant lysis of βgal-expressing CT26.CL25 target cells or βgal peptide-pulsed SVBalb target cells by the splenocytes of the surviving mice was observed, indicating that the cellular immune response contained CTLs specific against βgal (Fig. 4). Moreover, the CT26.CL25 tumor-free mice that survived after the combined CTX + DC therapy were s.c. rechallenged bilaterally with CT26.WT and CT26.CL25 tumor cells. The results show that these mice resisted lethal doses of not only CT26.CL25s but also CT26.WT tumor cells (Fig. 5), further indicating that the tumor protection was mediated by persistent antitumor immunity specific for antigens relevant to the CT26 tumor per se. It is of interest that the CT26.CL25 CTX + DC-treated tumor-free mice also had protective immunity against subsequent challenge with the nontransfected parental tumor line (CT26.WT), suggesting that the intratumor-injected DCs presented in vivo both βgal and parent antigens, resulting in long-lasting memory against these tumors. Furthermore, CT26.WT tumor-free mice treated with the combination therapy survived a second challenge of CT26.WT and CT26.CL25 (not shown), demonstrating that the observed effect of established immunological memory is not only dependent on the βgal antigen but likely also on other unidentified antigens of CT26 tumors.
Consistent with the concept that the CTX + DC therapy induced specific antitumor immunity, the βgal-specific CTLs generated from CTX + DC-treated tumor-free mice were transferred to mice and the mice rechallenged with CT26.CL25 tumor cells. Mice receiving CTLs from DC- and CTX-treated mice, but not from CTL-negative mice, were protected from a subsequent lethal tumor challenge with the CT26.CL25 cells (Fig. 6).
To exclude the possibility that locally released toxic factors and/or changes in the microenvironment as a result of exogenous DCs added in large amounts might have caused tumor regression, bilateral tumors (CT26.CL25) were inoculated in both lower flanks. The right tumor was considered as primary tumor and was treated by DC injection in combination with systemic chemotherapy (CTX), whereas the secondary tumor on the left side was untouched. Both tumors stopped growing, regressed, and disappeared simultaneously (Fig. 7), indicating that the antitumor suppression was systemic rather than local. This observation may have significant clinical implications because it suggests that treatment of a single tumor with the intratumoral DC injection, and systemic chemotherapy might be sufficient to generate effective therapeutic immunity against metastatic cancer.
A number of studies have demonstrated that antitumor immunity can be achieved by generating DCs in vitro from proliferating hematopoietic precursors, loading the DCs ex vivo with tumor-specific antigens and reinfusing the DCs back to the recipient host (11, 12, 13, 14, 15, 16, 17, 18). In contrast, the present study demonstrates the effectiveness of an antitumor strategy that is similar but eliminates the step of loading the DCs ex vivo with tumor-specific antigens. We achieved this by combining intratumor administration of DCs with systemic administration of chemotherapy. Using a model of murine flank tumors syngeneic to BALB/c and C57Bl/6, the data demonstrate that the combination of systemic chemotherapy plus direct intratumor administration of syngeneic DCs results in complete suppression of tumor growth. Importantly, the combined therapy induced the generation of tumor-specific cytotoxic T cells that were able to protect animals from tumor challenge, and that the chemotherapy + DC-treated animals were able to resist repeat challenge with the same tumor, suggesting that the mice had acquired long-term antitumor immunity. These observations suggest that it is not necessary to identify specific tumor antigens for immunotherapy using DCs. Instead, conventional cancer therapies, such as chemotherapy, may cause sufficient death of tumor cells by providing antigens to DCs in the milieu of the tumor.
Immunotherapy with DCs.
The state of maturation of DCs can be controlled in vitro by the culture conditions used to produce DCs. Immature DCs are produced by culturing DC precursors in the presence of GM-CSF and IL-4, whereas further maturation of DCs can be achieved by longer culture or subsequent addition of cytokines such as tumor necrosis factor-α or CD40 ligand (1, 2, 30). Because immature DCs have phagocytic activity and perform effective capture of exogenous antigens (1, 2), the DCs used in the present study were generated by 6-day exposure to GM-CSF and IL-4. The hypothesis underlying the use of these immature DCs is that the DCs will reside in the tumor for a short period of time, where they take up and efficiently process antigens from dying and apoptotic cells (19, 21). Once antigens have been captured, antigen-bearing DCs rapidly undergo a functional maturation process and become very potent antigen-presenting cells, with down-regulated processing capacity and increased surface expression of MHC and costimulatory molecules (1, 2). There is increasing evidence that the tumor antigens endocytosed by bone marrow-derived DCs are introduced not only into the MHC class II but also class I processing pathway to cross-prime naive T cells for developing potent immunity (3, 19, 31, 32). In addition, there is evidence that antigens can be transferred from antigen-bearing DCs to naive DCs in vivo and that antigen-pulsed DCs act synergistically with these DCs in the production of primary T-cell proliferation to antigens (33).
In the context of the central role of DCs in presenting MHC-restricted tumor antigens, a variety of strategies have been developed to use DCs in immunotherapy against tumors (11, 12, 13, 14, 15, 16, 17, 18, 34, 35). Although the molecular identification of the tumor-specific antigens would greatly expand the use of DC-based tumor vaccine strategies, most tumor-specific antigens remain unknown and may vary between different individuals. Antigen identification, a fundamental requirement for directly pulsing defined peptides and proteins with DCs for tumor vaccination, can be bypassed by approaches that arm the DCs with the full antigenic spectrum of the tumor by using the tumor cell itself as a source of antigens. Strategies to accomplish this include pulsing DCs with tumor-derived lysates or tumor mRNA, or by fusing tumors with DCs in vitro, or coculturing tumor cells with DCs in vitro (11, 12, 13, 14, 15, 16, 17, 18). These strategies can induce CTLs directed specifically against the tumor but require the in vitro step of exposing the DCs to the tumor. One alternative approach is to attract functional DCs to the tumor site, resulting in an immune response attributable to the short-term contact of DCs and tumor cells in vivo (35). Another is the strategy used in the present study, i.e., to take the DCs generated in vitro and directly administer the DCs into the tumor, bringing the DCs and tumor into close contact. Although there are data that blood and tumor-draining lymph node DCs are impaired in cancer patients because of a systemic effect of the tumor on DCs (36), functional DCs prepared from bone marrow or peripheral blood-derived precursors appear to escape this impairment (37). In experimental animal models, reports from other groups have showed that mice given i.v. injections of 106 DCs develop a weak protective cellular immunity (15), whereas the administration of 5 × 105 bone marrow-derived DCs in combination with a vaccine results in eradication of tumors (38).
A number of mechanisms have been proposed to explain the failure in cancer patients of chemotherapy alone as a sole or follow-up treatment after surgery. A major limitation is acquired drug resistance, which may occur because of drug-targeted gene amplification and mutation in some tumor cells which escape the cytotoxic effect of the drug and lead to tumor recurrence (39). Similarly, there are many ways in which tumors can escape recognition and clearance by the immune system. The reasons that the host is unable to trigger the recruitment and activation of DCs at the tumor site of rapidly growing tumors may include secretion of immunosuppressive factors such as tumor necrosis factor-β, fas ligand, and IL-10 to inhibit the host’s immune system, and lack of enough systemic functional DCs to achieve a local critical mass in the tumor-bearing host (40). In this context, the injection of in vitro generated DCs directly into the tumor may be able to break an operational state of tumor tolerance and trigger tumor regression, resulting in the development of effective endogenous immunity against cancer. The positive results from the present study show that the apparent limitation of DC function in tumor-bearing hosts can be overcome if sufficient autologous DCs at the tumor site are provided in the context of systemic chemotherapy.
The results in the present study have clinical implications. Because chemotherapy is effective in controlling the rate of tumor growth and causing tumor cells to undergo apoptosis, local DC-based therapy may be synergistic with chemotherapy in enhancing antitumor host responses. On the basis of the fact that complete tumor regression and long-lasting tumor immunity was observed in the present study, we suggest that the same strategy could be applied to treat other tumors by DCs combined with effective drugs. However, before undertaking such treatments, the routes and doses of administration of DCs and drugs need to be optimized. We predict that intratumoral DC injections combined with other efficient cancer treatments such as radiotherapy, antiangiogenesis agents, or prodrug strategies may be made more effective by virtue of increasing local toxic effects against tumors with minimal systemic immunosuppression. However, we do not have direct evidence showing how, after chemotherapy, the increased dying tumor cells release tumor antigens to be taken up by DCs in vivo. This putative mechanism remains hypothetical and will be researched in additional studies.
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.
These studies were supported, in part, by Grant R01 CA75192; the Will Rogers Memorial Fund, Los Angeles, CA; and GenVec, Inc., Gaithersburg MD.
The abbreviations used are: DC, dendritic cell; βgal, β-galactosidase; CTX, cyclophosphamide; MMC, mitomycin; 5-FU, 5-fluorouracil; ADR, Adriamycin; GM-CSF, granulocyte/macrophage-colony stimulating factor; IL, interleukin.
We thank N. Mohamed for help in preparing the manuscript.