Tumor metastases and relapse are the major causes of morbidity and mortality in cancer. Although surgery, chemotherapy and/or radiation therapy can typically control primary tumor growth, metastatic and relapsing tumors are often inaccessible or resistant to these treatments. An adaptive immune response can be generated during these conventional treatments of the primary tumor, and presumably both the primary tumor and secondary metastases share many of the same or similar antigenic characteristics recognized by the immune system. Thus, when established, this response should be able to control metastatic growth and tumor relapse. This review summarizes the mechanisms by which antitumor immune responses are generated, and recent findings supporting the hypothesis that many therapies targeting primary tumors can generate antitumor adaptive immune responses to prevent metastases and tumor relapse.
Conventional treatments such as surgery and chemotherapy are effective means of eliminating or reducing primary tumor growth, but have not proved effective in eradicating metastases. Because metastatic disease may not respond similarly to chemotherapies used in treating the primary tumor,1 recent research has focused on the importance of generating an antitumor immune response to control metastatic disease. Presumably, both the primary tumor and secondary metastases share many of the same or similar antigenic characteristics recognized by the immune system; yet, there has been no clear strategy formulated in which a patient's own tumor tissues is used to generate an antitumor adaptive immune response for the eradication of metastases and prevention of relapse. Once tumors metastasize, curing the disease is difficult and rare. This review summarizes recent findings supporting the hypothesis that targeting primary tumors with both conventional and new therapies can potentially generate antitumor adaptive immune responses that prevent metastases and relapse.
Due to the vast number of genetic changes associated with carcinogenesis, tumors express many neo-antigens and mutated antigens. Indeed, much evidence exists to indicate that many cancers are antigenic and thus recognizable by the adaptive immune system.2 Mere recognition by adaptive immunity, however, is insufficient, given that these antigenic cancers rarely regress spontaneously. Effective antitumor immunity depends on both the presence of tumor-reactive lymphocyte precursors and means by which these precursor lymphocytes can be activated. Most T cells with high affinity to tissue-specific antigens are deleted in the thymus or later tolerized in the peripheral tissues.3, 4, 5 Thus, overcoming tumor-associated immune-suppressive mechanisms to induce potent antitumor immunity is the first step for effective cancer immunotherapy.5, 6, 7 Many mechanisms are involved in tumor-associated immune suppression of naive and previously activated T cells. First, tumors create an environment insufficient for expansion and maturation of effector tumor-specific T cells. Second, effector T cells must be capable of reaching the site of the malignancy, and many tumors have physical and chemical barriers preventing lymphocyte infiltration. Finally, tumors downregulate receptors or find other means to prevent lymphocytes within the tumor microenvironment from appropriately executing effector functions to destroy cancer cells. Multiple mechanisms employed by tumors to hinder the immune response at each of these steps have been identified.
The current strategies for cancer immunotherapy
Active immunization and adoptive T-cell transfer therapy are currently the main cancer immunotherapy strategies used. Both of these strategies are designed to overcome deficiencies in priming of tumor antigen-specific T cells in cancer-bearing hosts. Cancer vaccines rely on immunization of patients with antigenic peptides, proteins or DNA expressed by the tumor directly or delivered by some other vehicle such as dendritic cells (DCs) or virus. Despite its relative simplicity and safety, vaccine treatments have shown very limited success.8 Although tetramer or ELISPOT assays revealed the generation of in vivo antitumor T cells in vaccinated patients,9, 10, 11 clinical responses observed from these trials were few.8 This was consistent with observations in murine models where the presence of large numbers of antigen-specific T cells was insufficient to mediate tumor regression in tumor-bearing mice.12, 13 There are a multitude of explanations for this observation: an inadequate number or avidity of immune cells, an inability of lymphocytes to migrate into the tumor microenvironment, or a reduced activation of quiescent or precursor lymphocytes may all prevent successful vaccination. In addition, tolerance mechanisms induced by the tumor, including the lack of costimulation leading to anergy induction and/or active suppressive mechanisms, create a great barrier to productive vaccination. These obstacles must be overcome if cancer vaccines are to be effective in mediating cancer regression.
Adoptive transfer therapies in which tumor-infiltrating lymphocytes are isolated from the tumor, or peripheral blood in some cases, expanded in an antigen-specific way, and adoptively transferred back into the patient,14 have been effective in a small number of highly selected melanoma patients.15 Although many tumor-specific and -associated antigens have been identified, few of these antigens in human tumors have been confirmed to be rejecting antigens. In addition, the rejecting antigen may vary greatly among different tumors. Moreover, the best adjuvants for ex vivo expansion of cytotoxic T cells are still uncertain. Therefore, the limited knowledge regarding the rejecting tumor antigens for T-cell expansion and the potential inability to physically isolate or expand these T cells, is preventing this therapy from being applied to a wide variety of cancers. As a result, understanding how immune responses are generated in vivo during conventional antitumor therapies and how to enhance these responses, is of great importance.
Conventional treatments on primary tumors induce immune responses
The first antibody approved for cancer treatment was Rituximab in 1997. Now, there are more than 10 antibodies approved by the Food and Drug Administration (FDA) for cancer therapy.16 The current dogma is that antibodies inhibit tumor growth by oncogenic signal blockade and/or antibody-dependent cellular cytotoxicity (ADCC).16 Many of these antibodies target the oncogenic receptor on the surface of the tumor cell, such as epidermal growth factor receptor in lung, head and neck, and colorectal cancer or human epidermal growth factor receptor 2 (HER2) in breast cancer. Usually, inhibition of tumor growth occurs by inducing oncogenic blockade through competition with the natural ligands, impairing the receptor oncogenic signal pathway, or inducing tumor cell apoptosis. Another important effect of antibodies is ADCC mediated through Fc–Fc receptor (FcR) interactions, which may induce FcR+ cells such as natural killer cells and macrophages to kill tumor cells. In cancer patients, FcR polymorphisms directly affect therapeutic responses to antibodies,17 and in mice lacking FcR, cancer therapeutic antibodies lose their effect on tumor growth.18 Recently, it was reported that the apoptosis-inducing ability of an antibody is greatly dependent upon the FcR expressed on host cells.19
Most conclusions regarding antibody effector mechanisms were drawn from in vitro cell culture studies and xenograft tumor models, both of which ignore adaptive immunity. Thus, until recently, determining whether antibodies can induce adaptive immunity against the tumor and whether this is essential for the therapeutic effect was unclear. Now, however, transgenic mice expressing oncogenes and mouse tumor cell lines expressing human oncogenic genes have been developed, both of which allow for the investigation of the role of adaptive immunity in cancer therapy.20
Indeed, the number of studies demonstrating that antibody therapy can induce both cellular and humoral immunity is rapidly increasing. For instance, Trastuzumab treatment can induce antitumor cytotoxic T lymphocytes (CTLs) in patients.21 Similarly, it was demonstrated that a mouse anti-epidermal growth factor receptor antibody could control metastasis in a CD4 and CD8 T cell-dependent manner.22 However, the mechanisms by which antibody therapy generates activate adaptive immunity has remained largely unclear. Our current findings demonstrated that anti-neu antibody treatment induced antitumor adaptive immunity and this is essential for the therapeutic effect in a syngenic mouse tumor model. This study revealed an essential role for the adaptive immune system in the therapeutic effect of antibody treatment on HER2/neu tumors. Given the fact that the antibody mediated blockade of oncogenic signals and the induction of ADCC had been previously demonstrated, we proposed that both of these outcomes lead to a significant release of danger signals (such as High-mobility group protein 1 (HMGB1)) to activate DCs and promote cytokine production. Furthermore, FcR-mediated signaling and phagocytosis could induce additional cytokine and danger signal production, leading to MyD88-enhanced cross-presentation for more effective activation of the adaptive immune system for enhanced tumor control. Most importantly, the adaptive immunity induced by targeting primary tumors could protect from tumor rechallenge which is indicative of a response to metastatic disease and relapse.23 Recently, it was reported that the therapeutic effect of anti-HER2/neu did not require CD4+ T cells, but did depend on both type I and type II interferon.24 In other models of oncogenic blockade, however, it was reported that tumor regression did require CD4+ T cells.25 Thus, the detailed mechanism of CD4+ T-cell activation and the role of these cells in antibody-mediated tumor regression are unclear.
Total control or elimination of tumor metastases by antibody alone is rare. Most antibodies only partially delay the disease process. Thus, improving the efficiency of antibody therapies is a great challenge in this field of study. Many companies have developed different strategies to increase the ability of oncogenic blockade and ADCC. We think another important aspect is enhancing the adaptive immunity trigged by antibody therapy through a combination of immune modulators. For instance, it has been shown that the therapeutic effect is greatly improved when antibodies are conjugated to immune modulators.26, 27
Local ablative radiation therapy (RT)
The antitumor effect of RT depends on the dose and treatment schedule. Our review focused on the use of ablative RT, during which a high dose of radiation is precisely delivered to small tumors. The current mechanistic explanation for the clinical efficacy of local ablative RT centers on the induction of lethal DNA damage directly to tumor cells or tumor-associated stroma. Although RT has traditionally been viewed as immunosuppressive due to the inherent sensitivity of lymphocytes to radiation-induced death, RT has been demonstrated to enhance tumor-specific immune responses.28, 29 Ionizing radiation increases production of inflammatory cytokines, such as tumor-necrosis factor (TNF), IL-1α and IL-6, by human tumor cells in vitro.30, 31 Local irradiation of a single tumor site can reduce the size of non-irradiated metastases that are located at a distant site, a phenomenon known as the ‘abscopal effect’, which is mediated by the immune system. In addition, radiation can modulate the peptide repertoire and enhance major histocompatibility complex class I expression by tumor cells, and alter their phenotype resulting in heightened susceptibility to T-cell killing.32, 33
Regarding the specific immune cell types involved in these processes, RT can induce more lymphocytes trafficking to tumor sites and draining lymph node, which includes CD4+, CD8+, CD11b+ and CD11c+ cell populations.28 Moreover, Apetoh et al. demonstrated that RT also increases DC maturation and cross-presentation.34 In this study, the authors revealed that RT induced the release of HMGB1, which in turn activated DCs through the TLR4–MyD88 signal pathway leading to a strong antitumor CTL response. Collectively, these data suggest that the antitumor effect may act through DC cross-presentation to activate T cells. Constant with this hypothesis, our studies clearly demonstrated that RT generates strong CD8+ T cell-dependent immunity leading to tumor reduction and reduced relapse of the primary tumor. Furthermore, we observed that type I interferons produced by tumor-infiltrating myeloid cells are required to endow tumor-infiltrated DCs with T-cell cross-priming capacity and are essential for tumor reduction following RT.35 Impressively, when combined with immune therapy, RT is more effective at eradicating metastasis than either therapy alone.29 Therefore, proper localized RT given at appropriate doses and scheduling may tip the balance in favor of antitumor immunity both through endogenous priming mechanisms and in combination with immunotherapy.29
Chemotherapy was designed to kill fast-dividing tumor cells by targeting essential metabolic steps, such as DNA replication and transcription. This therapy has been very successful in reducing tumor burden and is the standard treatment for many types of cancer. Although chemotherapy is often viewed as a strategy that specifically affects tumor cells, accumulating evidence indicates that these cytotoxic drugs also affect cells of the immune system. For instance, our data revealed that administration chemotherapy in conjunction with anti-neu antibody treatment abrogated resistance to tumor rechallenge.23 In other models, however, tumor cell death induced by cytotoxic drugs induced antitumor immunity, which is critical for the therapeutic effect of chemotherapy.34, 36
Therefore, the effects of chemotherapy extend beyond inducing tumor cell death. Of special note is a study by Obeid et al., demonstrating that the translocation of calreticulin determines the immunogenicity of dying tumors induced by cytotoxic drugs.37 Anthracyclins induce the translocation of calreticulin to the cell surface, which will enhance the phagocytosis of tumor cell by DCs and induce antitumor immunity. This process can be suppressed by blockade or knockdown of calreticulin. However, other DNA replication targeting drugs, such as mitomycin C and etoposide, were unable to induce a strong antitumor immune response since they could not induce the translocation of calreticulin. In addition, if supplied with exogenous calreticulin during mitomycin C and etoposide treatment, the immunogenicity of the dying tumor cell was restored and antitumor immunity was induced. The same research group also showed that the danger signal molecules, such as HMGB1, released by dying tumor cells could activate DCs through TLR4–MyD88 signal pathway on DCs, which is essential for processing and cross-presentation of antigen from dying tumor cells. Most importantly, breast cancer patients carrying a TLR4 loss-of-function allele relapse more quickly after chemotherapy than those carrying the normal TLR4 allele. This observation provides insights into designing more effective chemotherapy strategies for different patients based on the polymorphisms and mutation of genes.34
Chemotherapy also influences the constitution of the tumor microenvironment. Data from both preclinical studies and clinical trails highlight that some chemotherapeutic drugs reduce regulatory T-cell (Treg) numbers, although the detailed mechanism of this action remains unknown. These drugs include cyclophosphamide (Cy),38, 39, 40 fludarabine41, 42 and gemcitabine,43 among which, Cy is the best described. In mouse models, it was reported that Cy depletes CD4+CD25+ Tregs,38 and in vitro studies revealed that Cy reduces the suppressive function of Tregs by decreasing the expression of the transcription factors GITR and FoxP3.39 In addition, data from a recent clinical trail showed a decrease in Treg numbers in peripheral blood after low-dose Cy treatment. Very importantly, this study reported that a combination of Cy and fludarabine with total-body irradiation prior to adoptive CTL immunotherapy for melanoma, increased objective response rates from 50% to 70%.44 However, the mechanism by which low doses of Cy or other chemotherapy drugs selectively deplete Tregs, remains unclear. A recent study by Zhao et al. suggested that the low adenosine triphosphate (ATP) levels in Tregs may account for the selective depletion of this population. Compared to other cell types, Tregs express low intracellular ATP due to the downregulation of miR-142-3p and upregulation of CD39. Low levels of ATP affect the activity of glutamate cysteine ligase, which is required for glutathione synthesis. Since conjugation with glutathione is an important route for detoxification from Cy therapy, the low intracellular level of ATP will eventually cause the selective cytotoxic effect on Tregs.45
It is conceivable that some chemotherapies can trigger danger signals to bridge innate and adaptive immunity. In addition, different doses of chemotherapeutic drugs have distinct effects in tumor and autoimmune disease models.40 Therefore, future studies examining how different cytotoxic drugs affect the immune system are necessary. Understanding the proper timing and dosing of chemotherapy for a combination with immunotherapy will be necessary to optimize synergy between these therapies resulting in better antitumor activity.
Targeted immunotherapy to primary tumor tissues can generate immunity against distal tumor tissues
Creating lymphoid tissue inside the tumor to improve recognition
Although cancer cells express mutated or unique proteins recognizable as foreign antigens by the immune system,2 malignant cells are surrounded by non-malignant stroma to form a complex multicellular ‘organ’ resembling self. Thus, the induction of immunity against normal, non-mutated differentiation antigens expressed by tumors resembles autoimmunity. It has been reported recently that the organized tertiary lymphoid structure (TLS) is necessary and sufficient to induce autoimmune destruction of pancreatic islets.46 Indeed, de novo organization of a TLS is known to precede the development of a number of human autoimmune diseases and animal models.47, 48 These observations suggest that lymphoid neogenesis within the target tissue may have a critical role in initiating and maintaining immune responses against persistent antigens. TLSs are not supplied by afferent lymph vessels and are not encapsulated, which implies that they are directly exposed to signals such as stimulating antigens and cytokines from the environment. This unique structure could potentially result in unrestricted access of DCs and lymphocytes to the TLS, favoring immune activation.
Clues to understanding the signals that lead to TLS formation come from the study of signaling pathways involved in secondary lymphoid tissue organogenesis. Studies in mutant mice and blocking experiments have identified a key requirement for TNF family members, mainly lymphotoxin α1β2 (LTα1β2), and to some extent, TNF, in the development and organization of lymph nodes and spleen microarchitecture.49, 50, 51, 52 Binding of LTα1β2 and TNF to their respective receptors, LTβR and TNFR1, induce the expression of chemokines and adhesion molecules, which directly mediate lymphocyte migration and homing.53 The first evidence that TLS formation could involve the same signaling pathways that regulate lymphoid organogenesis came from studies of transgenic mice.48 The ectopic expression of LTα and LTβ in pancreatic islets induced the formation of in situ TLSs.54, 55, 56, 57 Extrapolating from this, stimulation of LTβR or TNFR expressed by tumor stromal cells might promote the formation of lymphoid-like structures inside the tumor tissues for its destruction.
Systemic TNFR signaling is of course too toxic, as evidenced by other systemic TNF treatments.58 Soluble LTα can signal through the TNFR resulting in the upregulation of chemokines. To avoid systemic toxic effects, recombinant LTα has been conjugated with a tumor-specific antibody to be delivered specifically to the tumor tissue.59 Targeting of recombinant LTα to the tumor elicits an efficient immune response associated with induction of peripheral lymphoid-like tissue containing L-selectin+ naive T cells and major histocompatibility complex class II+ antigen-presenting cells.59 Secondary lymphoid tissue chemokine (SLC or CCL21) is among the chemokines controlled by LTβR and TNFR signaling.53 It is normally expressed in high endothelial venules and in T-cell zones of spleen and lymph nodes and strongly attracts naive T cells and DCs.60 The expression of SLC inside the tumor resulted in a substantial, sustained influx of T cells within the mass as well as retention of DCs at the tumor site. By recruiting T cells and DCs, SLC in the tumor may lead to extranodal priming and inhibition of tumor growth.61
Besides, LTα1β2, LTβR is activated by LIGHT, another member of the TNF family (a name derived from: homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes).62 LIGHT is a ligand that signals through two receptors: LTβR expressed on stromal cells and herpesvirus entry mediator expressed on T cells.62, 63 LIGHT is predominantly expressed on lymphoid tissues, especially on the surface of activated DCs and T cells. Signaling via its receptor herpesvirus entry mediator, LIGHT acts as a strong costimulatory molecule for T-cell activation.62, 64, 65 Our data indicate that the interactions between LIGHT and LTβR restore lymphoid structures in the spleen of LTα−/− mice.66
In vivo data demonstrate that LIGHT, signaling through two receptors, mediates a microenvironment sufficient to break immunological tolerance to self-antigens. First, ectopic expression of LIGHT in the pancreatic islets resulted in the formation of lymphoid-like structures, and was necessary and sufficient for pancreatic islet destruction.46 In addition, sustained expression of LIGHT on T cells leads to their activation, migration into peripheral tissues and the establishment of lymphoid-like structures in situ.66, 67 LIGHT also acts as a strong costimulatory molecule for T-cell activation, possibly by binding to the herpesvirus entry mediator on T cells.64 Therefore, LIGHT is an ideal candidate for delivery inside the tumor to create TLSs, recruit T cells and subsequently expand antigen-specific clones already inside the tumor. In addition to the cross-presentation pathway, tumor-reactive T cells can be activated via direct-presentation pathway in the presence of antigens and costimulation. Indeed, LIGHT ectopically expressed in the tumor can effectively recruit and activate naive T cells. The expression of LIGHT in the tumor environment induces an infiltration of naive T lymphocytes that correlates with an upregulation of both chemokine production and expression of adhesion molecules inside the tumor.68 These experiments demonstrate that introduction of the lymphoid-like structure into the tumor stroma can be highly effective in enhancing antigen recognition and may be an effective strategy for cancer immunotherapy.
Generation of TLS within the tumor allows naive T cells to be primed within the tumor itself, and this has several advantages. First, the efficiency and specificity of priming will be increased due to the heightened tumor antigen load in situ relative to that collected in the draining lymphoid tissues.69 Second, a broader repertoire of tumor-specific naive T cells is recruited to the site of tumor antigens, leading to a more comprehensive response.59, 70 It has been demonstrated that some tumor antigens may not be efficiently cross-presented due to the antigen bias in T-cell cross-priming.71 Furthermore, no additional migration steps are required for CTLs to reach the site of effector function, which leads to the appearance of activated tumor-reactive T cells in a short period of time. In support of this reasoning, we demonstrated in our experimental system using the tumor cell line Ag104Ld–LIGHT that certain antigens are presented to and activate naive T cells within the tumor via a direct presentation mechanism.71 The same tumor antigen would have otherwise been missed by the host immune response, had draining lymphoid tissues been the sole location, and cross-presentation the only means, of activation. Finally, T-cell responses may react more readily to the shifting tumor antigen expression profile in situ. T-cell stimulation in the absence of costimulation can induce anergy and apoptosis of antigen-specific T cells.6, 72, 73 Costimulation has also been shown to greatly enhance tumor-specific T-cell function during the effector phase.74 Earlier studies show that LIGHT provides CD28-independent costimulation,64 which may be essential for the selective and effective activation, expansion and maintenance of tumor-specific T cells among infiltrating naive T cells in Ag104Ld–LIGHT tumors. Thus, the introduction of a lymphoid-like milieu, including a combination of chemokines, adhesion molecules and costimulatory properties inside tumor tissues, may allow LIGHT to be a potent antitumor immunostimulatory molecule.
Generation of CTLs in the LIGHT-mediated tumor environment for treatment of metastases
Generation of a potent antitumor adaptive immune response can reduce the primary tumor burden, and help control the metastatic and relapsing tumors. We have shown that expression of LIGHT in the tumor microenvironment can lead to rejection of both the local and distal sites. In an effort to develop more clinically relevant approaches, adenovirus vectors expressing LIGHT (Ad-LIGHT) have been constructed to deliver LIGHT into the tumor tissue. The advantages of adenovirus are: (i) high production of a non-replicable virus; (ii) ease of manufacture; (iii) activation of innate immunity; (iv) an ability to express its carrying gene in undividing cells; and (v) readily expression in most tumor cell lines. Administration of Ad-LIGHT into the tumor tissue leads to the complete rejection or significant delay of aggressive murine tumors.75 Local treatment with Ad-LIGHT initiated priming of tumor-specific CD8+ T cells directly in the primary tumor, with subsequent exit of CTLs which homed to distal tumors to elicit immune-mediated eradication of spontaneous metastases.75 This strategy is an example that the generation of immune responses in primary tumor tissues prior to surgical resection can produce tumor-specific effector T cells sufficient to eradicate distant metastases in a CD8-dependent fashion.
Although local RT treatment cannot completely clear established tumors or established metastasis, additional immunotherapy can amplify or sustain immune responses. To test this hypothesis, we have added Ad-LIGHT therapy after RT or antibody treatment and observed more profound immunity against tumor, including established metastasis.29 Similarly, antibody treatment can trigger an immune response that can be amplified by Ad-LIGHT treatment leading to a response superior to the single treatment. Most importantly, complete regression of the primary tumor and heightened resistance to a secondary challenge are observed, supporting the role this therapy has in treating relapse.23
There is accumulating evidence that adaptive immunity generated by antitumor therapy is correlated with good prognosis. Thus, generating protective antitumor immunity should be an important consideration during drug screening and therapy strategy development (Figure 1). On the one hand, more potent strategies targeting the primary tumor are necessary. This includes improved target accuracy and broadened cytotoxicity on various tumor cell types, including tumor stem cells. This would not only reduce the tumor burden in patients, but supply more dying tumor cells and danger signals create to a resource of powerful antigenic and stimulatory signals. On the other hand, it is necessary to explore the mechanism by which current antitumor therapy influence the host immune system. Based on the observations outlined in this review, some conventional treatments can enhance antitumor immunity and decrease immune suppression in the tumor microenvironment, which break tolerance and facilitate subsequent immunotherapy leading to long-term protection. For instance, conventional treatments such as tumor resection followed by radiation and chemotherapy, may prevent effective immune recognition of cancers due to the loss of a major source of antigens and damage to preexisting CTLs by radiation and chemotherapy. Thus, new strategies focused on utilizing the primary tumor as the site of CTL priming prior to surgical resection could offer a great advantage. The future of antitumor therapies requires redesigning of conventional treatment and further evaluation on how to best combine these therapies with immunotherapy to achieve the best therapeutic effect ultimately resulting in the control of metastatic disease and tumor relapse.
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Yang, X., Mortenson, E. & Fu, YX. Targeting and utilizing primary tumors as live vaccines: changing strategies. Cell Mol Immunol 9, 20–26 (2012). https://doi.org/10.1038/cmi.2011.49
- tumor vaccine