In recent years, it has become apparent that immunoregulatory processes influence cancer development. The key players in tumor progression are mainly present in the microenvironment of the tumor and the draining lymph nodes. Interventions aimed at shifting tumor-promoting actions toward effective tumor-eradicating immunity are thus foremost required locally. As immune-modulating therapy has been shown to cause many adverse side effects when administered systemically, we strongly advocate the further development of local treatment for cancer immunotherapy.
The concept of tumor immune surveillance, the potential of the immune system to keep the formation and outgrowth of malignant cells in check, has been described as early as 1891, by William Coley, and has waned and recurred in scientific publications several times. The experimental evidence supporting the concept of immune surveillance is still increasing (see recent reviews by Swann and Smyth1 and Vesely et al.2) and was recently extended by Schreiber and colleagues who described the association of tumor cells and lymphocytes actively inhibiting the formation and progression of transformed cells and ultimately causing selective evolution of tumor cells that can evade the immune response, a phenomenon called cancer immunoediting.3, 4 Based on this knowledge, many tumor intervention treatments have been designed and studied involving immunotherapy in both preclinical models and clinical trials with varying successes and pitfalls.
Lymph nodes (LNs) are organs composed of lymphoid cells that occupy strategic positions throughout the body and play a pivotal role in the immune system. LNs act as sentinels within the system, filtering the afferent lymph and bringing together cells of the innate and adaptive immune system to interact, which in acute infections generally leads to robust priming of naïve T cells. Tumor-draining LNs (TDLNs) have a dubious position as they can induce antitumor T-cell responses but are at the same time under the direct influence of the often immunity-dampening tumor microenvironment and can act as routes for malignant cells toward distant organ metastasis.5–7 Because of this controversial role, the surgical removal of sentinel LNs in cancer patients has been a matter of debate for decades.8
The potential for targeted immunotherapy to the tumor area and more specifically to the TDLN has been brought forward in recent years. In this review, we would like to discuss the latest insights into tumor immune therapy and the strategies and advantages of local targeting.
Balancing Induction and Suppression of Tumor Immunity
The growth of a tumor often coincides with both the stimulation of antitumor T-cell responses and in parallel the unwanted induction of immune suppression. Both processes take place mainly in the tumor area and TDLNs. This balance between T-cell priming and suppression is one of the key aspects in disease prognosis.9
In mice, the critical importance of the adaptive immune system, especially of T cells, to prevent cancer development was proven by sophisticated experimental tumor models involving the ablation of the specific regulators of the adaptive immune system.10–12 In cancer patients, tumor-associated T-cell responses have been analyzed and found to be correlated with improved prognosis.13–18 Tumor antigens can be presented by antigen-presenting cells (APCs), such as dendritic cells (DCs), within the tumor or are first carried from the tumor by tumor cells or APCs traveling through lymphatic channels to become crosspresented in LNs to T cells.19, 20 The APCs in the tumor mass and TDLNs frequently display a certain level of maturation owing to the presence of endogenous danger signals from the growing tumor, such as heat shock proteins and uric acid from decaying tumor cells. Compared to pathogenic infections, however, the APC maturation signals are poorer, which can lead to inadequate T-cell priming.21–24 Especially, the lack of costimulatory signals (e.g., CD80/86) has been linked to dysfunctional T cells.25 Likely, also the lower production of the proinflammatory cytokines interleukin-12 (IL-12) and interferon-γ (IFN-γ)21, 22, 24 contributes to the low state of T-cell activation to tumor antigens. This phenomenon of inadequate T-cell activation has led to different nomenclatures for these T cells including anergic T cells, division-arrested T cells, incompletely differentiated T cells, dysfunctional T cells and tolerized T cells. In tumor settings, anergic T cells are characterized by inadequate effector function such as the lack of cytolytic molecules (e.g., perforin or granzyme B), expression of low levels of IFN-γ and a division arrest phenotype, which all contribute to reduced capacity to kill tumor cells.20, 26–30 Besides lower “quality” of T-cell priming by APCs, both animal models and human studies show that TDLNs also harbor lower numbers of DCs. Nevertheless, tumor-specific T cells with full cytotoxic capacity have been described with respect to phenotype and function,16, 27, 31 suggesting that transformed cells express tumor antigens that can elicit proper T-cell activation, providing hope for immunotherapeutic strategies.
Immune suppression within the tumor microenvironment and TDLNs is characterized by an unfavorable mixture of immunosuppressive cytokines, growth factors and various immunosuppressive cell populations. Well-studied suppressive cytokines, produced by tumors or tumor-associated macrophages, are IL-10, transforming growth factor-β (TGF-β), vascular endothelial growth factor and IL-6. Chemokines, such as CCL2 and CXCL8, secreted by monocytes and tumor-associated macrophages, cause tumor progression by attraction of myeloid-derived suppressor cells (MDSCs) and tumor angiogenesis. Effector T-cell suppression is mediated by regulatory T cells (Tregs), MDSCs and tolerogenic DCs.22, 23, 32 A special type of factor that inhibits the induction of proinflammatory immune responses is indoleamine 2,3 dioxygenase (IDO). Expression of IDO by plasmacytoid DCs and some types of tumor cells causes inhibition of T-cell proliferation by enzymatic degradation of tryptophan, leading to tryptophan starvation. This can also lead to conversion of CD4+ T cells to Tregs in TDLNs.33
The increased presence of Tregs in TDLNs compared to nondraining LNs has been well established in both animal models and cancer patients, where accumulation of Tregs in TDLNs of colorectal cancer patients and not in tumor or peripheral blood is correlated with disease progression.23, 32, 34, 35 Treg accumulation in tumor bearing animals can result from either proliferation of natural, thymic-differentiated Tregs or conversion of naïve CD4+ T cells into Tregs. The mechanisms of Treg suppression are not fully understood yet, but can include IL-2 deprivation, expression of CTLA-4 and secretion of the suppressive cytokines IL-10 and/or TGF-β. Recent publications showed that Tregs can also limit DC, NK and CD8+ T-cell numbers by direct granzyme B and perforin-dependent killing in TDLNs.33, 36
An accidental factor that might contribute to cancer progression in elderly patients is deleterious alteration of the immune system owing to aging.37 This phenomenon, also called immunosenescence, is characterized by loss of immunocompetence (by, e.g., reduced T-cell proliferation) which limits immune resistance not only to tumors but also to pathogens such as influenza virus, respiratory syncytial virus, pneumococci and tuberculosis bacilli. Therapies designed in animal models to boost the immune system against tumors may be imperfect in elderly patients, because of this phenomenon, and more vigorous therapies or different strategies may be necessary. Accordingly, it is interesting to note that Belloni et al. recently reported age-dependent differences in side effects to systemic anti-IL-10 receptor antibodies. IL-10 inhibition caused high mortality in older animals, whereas no mortality was observed in young animals. As the majority of cancer patients are elderly, these results imply that systemically blocking the IL-10 receptor should be evaluated carefully.38
Local Immune Therapy: Targeting the Tumor Microenvironment and Draining LNs
Adverse side effects of systemic treatment
Recent reports describe the dangers of toxic side effects of systemic immune-activating treatments, emphasizing the need for more targeted therapies (Fig. 1). Together with the growing evidence defining the local suppressive effects of the tumor microenvironment and the unique position of the tumor draining LNs, this calls for exploring the potential of immune intervention strategies that act mainly locally.
Many different strategies have been proposed to reactivate the TDLN-resident anergic T cells, and overcome tumor-induced immune suppression, some of which specifically target the tumor, tumor draining area and/or TDLN. Many of these strategies were first described as systemically applied immunostimulatory strategies in experimental models and later in clinical trials. Numerous preclinical studies have described that such systemic therapies can overcome T-cell anergy, either by directly activating DCs (using toll-like receptor [TLR] ligands or agonistic CD40 antibody), blocking inhibitory signals (blockade of CTLA-4, PD-1 or TGF-β) or addition of proinflammatory cytokines (IL-12, IFN-α or IL-2),39, 40 resulting in tumor eradication. Clinical trials, however, did not show a similar success rate in clearing tumors as observed in some animal models. Frequently, the relative dose of immune stimulating reagents used in rodents is higher than the maximum tolerated dose used in humans (corrected for body weight). Immunologists using animal models are often less focused on side effects than on efficacy. However, more researchers are starting to become aware that in order for preclinical animal models to be more representative to the human situation, lower doses of immune stimulating agents should be used, and toxic side effects in animal models should be meticulously analyzed.38, 41, 42
Moreover, systemic activation of the immune system can cause serious toxicity as shown in a number of clinical trials and animal studies. An example is the catastrophic clinical trial with the CD28 superagonist TGN1412.43 Indeed, potent systemic activation of the entire immune system is unadvisable, and should be applied with utmost caution. In many other studies, adverse events caused by systemic immune activation were dose limiting and hence hampered the efficiency. Agonistic antibodies against CD40 and the cytokines IL-12 and IL-2 have all been described to have potent effects in enhancing the antitumor T-cell response, but all have caused severe toxicity in patients after systemic administration.44–46
Also granulocyte macrophage (GM)-colony-stimulating factor (CSF) administration, which is not directly immune activating and therefore contains a lower risk of causing toxicity, has been shown to cause adverse effects when injected systemically. Serafini et al. published a paper in which data were presented, showing the increase in MDSC in mice treated with high-dose systemic GM-CSF, causing an impaired immune response.47
Specific targeting of the tumor microenvironment
One way of reducing systemic side effects is to target exclusively the tumor lymphoid drainage area. Already decades ago it was reported that perilymphatic injections of IL-2 in head-and-neck squamous carcinoma patients yielded promising results, but such local treatment studies have not been extensively pursued.48, 49 Later studies revealed that local injection of CpG, a toll-like receptor 9 ligand (TLR9) enhances DC maturation and migration to TDLNs.42, 50–52 When compared to other administration routes, local injection was superior in DC maturation, T-cell priming and tumor eradication, in a preclinical model.52 In a clinical trial, CpG was administered intradermally directly adjacent to the scar of melanoma resection, before the sentinel lymph node (SLN) resection, and the immune response was analyzed in the SLN and peripheral blood mononuclear cells (PBMCs). Patients tolerated this therapy well and displayed higher numbers of DCs in the SNL associated with upregulation of costimulatory molecules, increased release of proinflammatory cytokines and reduction in immunosuppressive Treg frequencies.53 Fifty percent of these patients had a measurable proinflammatory T-cell response against melanoma-specific tumor antigens in the SLN and in 40% of the patients tumor-specific T-cell responses were also found in blood.54 In another clinical trial, intratumoral injection of CpG was combined with low-dose, local irradiation. An increase in tumor-specific T cells was detected in PBMCs of patients, and objective responses were noted.55
Induction of inflammation in the TDLN area leads to upregulation of chemotactic molecules like CCR7 on DCs and CCL21 on lymphatic endothelial cells, which in turn leads to enhanced migration of DCs to the LN.56, 57 The influx of mature DCs into the LN causes the LN to increase in size and cellularity, called reactive LN. The inflammatory state of the reactive LN influences the activation of T cells as described recently57. Especially important for memory recall responses, T cells that had developed in the presence of a reactive LN had a significant quantitative advantage over T cells in mice without a reactive LN.58 In animal models and clinical trials, genetically engineered tumor cells secreting GM-CSF, CTLA-4 blocking antibody or CCL20 (a DC-attracting chemokine) have been studied as local treatment. By injecting the irradiated tumor cells close to the tumor, they serve as antigen- and antibody-secreting depot to the TDLN, and cause activation of effective antitumor T-cell responses and tumor eradication, with lower treatment-associated toxicity than upon systemic administration.59, 60
Previously we reported that targeting the tumor-draining area with a low dose of agonistic CD40 antibody in a slow-release formulation overcomes tumor-induced immune suppression and induces excellent systemic tumor-specific T-cell responses capable of killing metastatic malignant cells located elsewhere in the body60. Local therapy, therefore, can thus lead to systemic responses, with only a fraction of the toxic side effects.61 Anti-CD40 has also been described to alter the tumor stroma, resulting in tumor eradication by macrophages.62
We envisage an important role for slow-release formulations in targeting immune-stimulating agents to the TDLNs, because they keep the tumor-draining area, or regional basin, in a proinflammatory status for a prolonged period of time, allowing the T-cell response to fully develop and the immune suppression to remain blocked. In addition, the concentration of an immune stimulatory agent remains high only locally and not systemically, thereby preventing undesirable side effects and unspecific overstimulation. Slow-release formulations such as mineral oils (e.g., Montanide), have been studied for their efficiency in delivering immune-modulating antibodies (such as anti-CD40) to the TDLN with strong systemic antitumor responses as a result, but no systemic toxicity.61 The discovery of several new sustained release systems, such as PLGA- and dextran-based micro- and nanoparticles, opens up possibilities for targeted treatments which can be explored for tumor immunotherapy.63, 64
Because of the decrease of systemic side effects, the targeted approach for delivery of immunotherapy lends itself without difficulty for combined use with other cancer treatments, like adoptive T-cell transfers and chemotherapy which has been described to have immune-enhancing properties.65, 66
Another aspect that strengthens the use of local immunotherapy lies in the fact that many immunosuppressive mechanisms that inhibit tumor-specific T-cell responses, as described before, are not uniquely operable in the tumor microenvironment, but are mechanisms that have evolved to keep the immune system from attacking self-tissue. Interfering with these interactions on a systemic scale, therefore, is risky. Not surprisingly, examples of systemic immunostimulatory tumor immunotherapy causing severe autoimmunity are abundant.40, 67, 68
Potential hurdles for local immunotherapy
As discussed above, local immunotherapies have clear advantages over systemic treatments; however, there are factors that could potentially be unfavorable to a local administration of immune-modulating agents. Recent studies have shown that elevated levels of MDSCs are present in cancer patients and tumor-bearing mice. As these cells are described to incite systemic suppression, rather than local suppression, targeting of the TDLN is not likely to overcome suppression by these cells.69, 70 Several studies mentioned in this review describe that local targeting can overcome suppression by activating robust antitumor T-cell responses that eradicate distant tumors. However, systemic suppression by MDSCs was not analyzed in these studies, and might have been weak.
Targeting TDLNs might cause the practical problem of inaccessibility of a draining node, as in several types of cancer TDLNs are not always easily accessible. New approaches are being studied, however, that could overcome this problem such as delivery of nanoparticles coupled to tumor–antigen-specific antibodies, which can be injected systemically but, deliver their immunomodulating content selectively into the tumor from where it will eventually drain to the TDLNs.42, 50
The tumor microenvironment and especially the TDLNs are the key locations for important antitumor immunological processes, and therefore the quintessential targets for immune-modulating therapies in solid tumor-bearing subjects. As both priming of tumor-specific T-cell responses and immune suppression occur in this area, local therapies designed to balance this equilibrium toward more effective antitumor T-cell responses will be most efficient. Whether tumor eradication is most efficiently achieved by promoting the stimulation of DCs presenting tumor antigens, enhancing tumor antigen presentation, directly activating tumor-specific T cells, abolishing immune suppressive pathways or a combination of these, remains to be defined experimentally and clinically. Notably, as most of the tumor immunotherapy strategies harbor the risk of causing serious toxicity and/or autoimmunity, targeting specifically the TDLNs and/or the tumor microenvironment instead of systemic administration should be a focus of future immunotherapeutic strategies.
This work was supported by a grant from the Dutch Cancer Society (UL 2004-3016, M.F. Fransen) and by a Marie Curie fellowship from the European Commission (to R.A.). C.J. Melief has been employed part-time by ISA Pharmaceuticals.