There is increasing evidence of the potential for radiation therapy to generate antitumor immune responses. The mechanisms of this immune-activating potential include actions on tumor cells such as immunogenic cell death and phenotypic change. Radiation modulates tumor cell surface expression of cell death receptors, tumor-associated antigens and adhesion molecules. This process of immunomodulation sensitizes tumor cells to immune-mediated killing. Radiation also affects immune compartments, including antigen-presenting cells, cytotoxic T lymphocytes and humoral immunity, leading to specific antitumor immune responses. Recognizing the importance of immunity as a potentiator of response to radiation leads to rational augmentation of antitumor immunity by combining radiation and immunotherapy. Targeted immunotherapy manipulates the immune system in a way that best synergizes with radiation. This article discusses the ability of radiation monotherapy to induce antitumor immunity, with a focus on the effect of radiation on antigen-presenting cells and cytotoxic T lymphocytes. We define two important responses generated by tumor cells, immunogenic cell death and immunomodulation, both of which are radiation dose-dependent. In conclusion, we describe the translation of several combination therapies from the preclinical to the clinical setting and identify opportunities for further exploration.

To understand the impact of clinically relevant radiation therapy (RT) on tumor immune gene expression and to utilize the changes that occur during treatment to improve cancer treatment outcome, we examined how immune response genes are modulated in prostate cancer cells of varying p53 status. LNCaP (p53 wild-type), PC3 (p53 null) and DU145 (p53 mutant) cells received a 10 Gy single dose or 1 Gy × 10 multifractionated radiation dose to simulate hypofractionated and conventionally fractionated prostate radiotherapy. Total RNA was isolated 24 h after multifractionated radiation treatment and single-dose treatments and subjected to microarray analysis and later validated by RT-PCR. RT-PCR was utilized to identify total-dose inflection points for significantly upregulated genes in response to multifractionated radiation therapy. Radiation-induced damage-associated molecular pattern molecules (DAMPs) and cytokine analyses were performed using bioluminescence and ELISA. Multifractionated doses activated immune response genes more robustly than single-dose treatment, with a relatively larger number of immune genes upregulated in PC3 compared to DU145 and LNCaP cells. The inflection point of multifractionated radiation-induced immune genes in PC3 cells was observed in the range of 8–10 Gy total radiation dose. Although both multifractionated and single-dose radiation-induced proinflammatory DAMPs and positively modulated the cytokine environment, the changes were of higher magnitude with multifractionated therapy. The findings of this study together with the gene expression data suggest that cells subjected to multifractionated radiation treatment would promote productive immune cell–tumor cell interactions.

Radiation is a potent immune-modulator that elicits cell death upon tumor, stromal and angiogenic compartments of tumor microenvironment. Here, we test a novel approach of high-dose radiation delivery using three dimensional volume based lattice radiation therapy (LRT) to understand the impact of different volume irradiation in eliciting both local and metastatic/distant tumor control through modulation of tumor immune micro-environment. To study such effects of LRT, tumors were implanted in both hind legs of C57BL/6 mice using Lewis lung carcinoma 1 (LLC1) cells. Mice were divided into five groups: untreated; partial tumor volume groups included two 10% vertices, one 20% vertex and one 50% vertex of the total tumor volume; and 100% open-field irradiation. Tumors implanted in the left flank were irradiated with a single dose of 20 Gy while the tumors in the right flank were unirradiated. Tumor growth and regression as well as immune responses (such as Th1 and Th2; T-cell infiltration) were determined after radiation treatment. Results demonstrated that both 100% open-field irradiation and 20% volume irradiation (in two 10% volumes) resulted in significant growth delay in the irradiated tumor. Further, all types of radiation exposures, partial or 100% volume, demonstrated distal effectiveness, however, 20% volume irradiation (in two 10% volumes) and 50% tumor volume irradiation led to maximum growth delay. Mice treated with partial tumor volume radiation induced a robust IFN-γ and Th1 response when compared to whole-tumor irradiation and down-modulated Th2 functions. The presence of increased CD3 cells and TRAIL in partially irradiated tumor volumes correlated well with tumor growth delay. Further, serum obtained from any of the LRT treated mice caused growth inhibition of endothelial cells when compared to serum obtained from either untreated or open-field irradiated groups. These results indicate that high-dose partial volume irradiation can cause an improved distant effect than the total tumor volume irradiation through activating the host immune system.

The goal of this study was to determine whether a combination of local tumor irradiation and autologous T-cell transplantation can effectively treat metastatic 4T1 breast cancer in mice. BALB/c mice were injected subcutaneously with luciferase-labeled 4T1 breast tumor cells and allowed to grow for 21 days, at which time metastases appeared in the lungs. Primary tumors were treated at that time with 3 daily fractions of 20 Gy of radiation each. Although this approach could eradicate primary tumors, tumors in the lungs grew progressively. We attempted to improve efficacy of the radiation by adding autologous T-cell infusions. Accordingly, T cells were purified from the spleens of tumor-bearing mice after completion of irradiation and cryopreserved. Cyclophosphamide was administered thereafter to induce lymphodepletion, followed by T-cell infusion. Although the addition of cyclophosphamide to irradiation did not improve survival or reduce tumor progression, the combination of radiation, cyclophosphamide and autologous T-cell infusion induced durable remissions and markedly improved survival. We conclude that the combination of radiation and autologous T-cell infusion is an effective treatment for metastatic 4T1 breast cancer.

The main role of the immune system is to restore tissue homeostasis when altered by pathogenic processes, including neoplastic transformation. Immune-mediated tumor rejection has been recognized as an extrinsic tumor suppressor mechanism that tumors need to overcome to progress. By the time a tumor becomes clinically apparent it has successfully escaped immune control by establishing an immunosuppressive microenvironment. Ionizing radiation applied locally to a tumor alters these tumor-host interactions. Accumulating evidence indicates that standard therapeutic doses of radiation have the potential to recover tumor immunogenicity and convert the tumor into an in situ personalized vaccine. Radiotherapy induces an immunogenic tumor cell death promoting cross-presentation of tumor-derived antigens by dendritic cells to T cells. In addition, radiotherapy stimulates chemokine-mediated recruitment of effector T cells to the tumor, and cellular recognition and killing by T cells that is facilitated by upregulation of major histocompatibility antigens, NKG2D ligands, adhesion molecules and death receptors. Despite these effects, radiotherapy alone is only rarely capable of generating enough proinflammatory signals to sufficiently overcome suppression, as it can also activate immunosuppressive factors. However, our group and others have shown that when combined with targeted immunotherapy agents radiotherapy significantly contributes to a therapeutically effective anti-tumor immune response. To illustrate this partnership between radiation and immunotherapy we will discuss as an example our experience in preclinical models and the molecular mechanisms identified. Additionally, the clinical translation of these combinations will be discussed.

An accumulating body of evidence demonstrates that radiation therapy can generate adaptive immune responses that contribute to tumor control. However, in the absence of additional immune therapy, the adaptive immune response is insufficient to prevent tumor recurrence or affect distant disease. It has been shown in multiple models that tumor-infiltrating myeloid cells exhibit alternative activation phenotypes and are able to suppress adaptive immune responses, and recent data suggests that the myeloid response in tumors treated with cytotoxic therapy limits tumor control. We hypothesized that tumor myeloid cells inhibit the adaptive immune response after radiation therapy through expression of the enzyme arginase I. Using a myeloid cell-specific deletion of arginase I in mice, we demonstrate an improved tumor control after radiation therapy. However, tumors still recurred despite the conditional knockdown of arginase I. Since multiple alternative factors may combine to inhibit adaptive immunity, we propose that targeting macrophage differentiation may be a more effective strategy than targeting individual suppressive pathways.

Hepatocellular carcinoma (HCC) often presents as a diffuse or multifocal tumor making it difficult to control by surgery or radiation. Radio-inducible herpes simplex virus thymidine kinase (HSV-TK) gene therapy has been shown to enhance local tumor control after radiation therapy (RT), while limiting the expression of the transgene in the irradiated tumor tissues. To prevent liver tumor recurrence and control systemic disease while limiting the potential bystander toxicity of HSV-TK therapy, we proposed to stimulate endogenous dendritic cell (DC) proliferation with systemic adenovirus Flt3 ligand (Adeno-Flt3L) gene therapy, followed by primary tumor radiation therapy combined with a radio-inducible HSV-TK gene therapy. We hypothesized that adenovirus-expressing Flt3L gene therapy will stimulate DC proliferation, allowing the upregulated DCs to locally harness tumor antigens released from HSV-TK/RT-treated HCC cells, thereby converting irradiated tumors to an autologous in situ tumor vaccine in mice with primary liver tumors. To test this hypothesis, an expression vector of HSV-TK was constructed under the control of a radio-inducible promoter early-growth response (Egr-TK) and a recombinant adenovirus-expressing human Flt3L was constructed. The Adeno-Flt3L [10⁹ plaque forming units (pfu)] was administered intravenously on days 1 and 8 after radiation therapy. The murine hepatoma cell line (BNL1ME) was stably transfected by Egr-TK or Egr-Null (encoding no therapeutic gene). Palpable tumors in BALB/c mice were treated with a localized dose of 25 Gy of radiation followed by ganciclovir (GCV, 100 mg/kg, 14 days). Four treatment cohorts were compared: Egr-Null/GCV RT Adeno-LacZ; Egr-Null/GCV RT Adeno-Flt3L; Egr-TK/GCV RT Adeno-LacZ; and Egr-TK/GCV RT Adeno-Flt3L. There was no primary tumor regression in the Egr-Null tumors after radiation therapy alone. In contrast, Egr-TK tumors had nearly complete tumor regression for 3 weeks after radiation therapy (P < 0.01), however, long-term follow-up demonstrated primary tumor recurrence and death secondary to pulmonary metastasis. Flt3L expression was confirmed by serum bioassay (mean = 88 ng/mL) in these animals and Western blotting of tissue culture medium in Adeno-Flt3L-infected BaF/huFlt3L cells. Radiation therapy with Adeno-Flt3L gene therapy effectively retarded primary tumor growth when compared to radiation therapy alone. The trimodality therapy (Egr-TK/GCV RT Adeno-Flt3L) was the most efficacious with 40% complete tumor regression (>100 days) and <20% pulmonary metastases, indicating the development of sustained antitumor immune response. These studies provide a rationale for triple modality therapies with radiation-inducible HSV-TK gene therapy and Adeno-Flt3L when used in combination with primary tumor radiation therapy for improved local and systemic control of HCC.

Diffuse hepatocellular carcinoma (HCC) is a lethal disease that radiation therapy (RT) currently has a limited role in treating because of the potential for developing fatal radiation-induced liver disease. However, recently diffuse HCC, “radio-inducible suicide gene therapy” has been shown to enhance local tumor control and residual microscopic disease within the liver for diffuse HCC, by using a combination of chemoactivation and molecular radiosensitization. We have demonstrated that the addition of recombinant adenovirus-expressing human Flt3 ligand (Adeno-Flt3L) after radio-inducible suicide gene therapy induced a Th1-biased, immune response and enhanced tumor control in an ectopic model of HCC. We hypothesized that sequential administration of recombinant adenovirus-expressing CD40L (Adeno-CD40L) could further potentiate the efficacy of our trimodal therapy with RT HSV-TK Adeno-Flt3L. We examined our hypothesis in an orthotopic model of diffuse HCC using BNL1ME A.7R.1 (BNL) cells in Balb/c mice. BNL murine hepatoma cells (5 × 10⁴) transfected with an expression vector of HSV-TK under the control of a radiation-inducible promoter were injected intraportally into BALB/cJ mice. Fourteen days after the HCC injection, mice were treated with a 25 Gy dose of radiation to the whole liver, followed by ganciclovir (GCV) treatment and systemic adenoviral cytokine gene therapy (Flt3L or CD40L or both). Untreated mice died in 27 ± 4 days. Radiation therapy alone had a marginal effect on survival (median = 35 ± 7 days) and the addition of HSV-TK/GCV gene therapy improved the median survival to 47 ± 6 days. However, the addition of Adeno-Flt3L to radiation therapy and HSV-TK/GCV therapy significantly (P = 0.0005) increased survival to a median of 63 ± 20 days with 44% (7/16) of the animals still alive 116 days after tumor implantation. The curative effect of Flt3L was completely abolished when using immunodeficient nude mice or mice depleted for CD4, CD8 and natural killer cells. The addition of Adeno-CD40L further improved the median survival of animals to 80 ± 15 days and this effect was abolished only when using anti-CD8 antibodies. Chromium-51 (⁵¹Cr) release assay showed cytotoxic T lymphocyte (CTL) activation, suggesting efficient dendritic cell (DC) activation with CTL activation after the treatment. Furthermore, when surviving mice were rechallenged with BNL-ETK cells on the foot pad, RT HSV-TK/GCV Flt3L CD40L-treated mice developed a small tumor on day 56 but the tumor eventually disappeared after 105 days. Mice treated with RT HSV-TK/GCV Flt3L showed a slowed tumor growth curve compared with untreated mice. Therefore, combination therapy using Flt3L to induce DC proliferation and CD40L to enhance DC maturation holds great promise for immunomodulation of radiation therapy to enhance HCC tumor control and prevent progression of disease in patients with diffuse HCC.

The last decade has witnessed significant progress in the field of cancer immunotherapy. This has, in part, been driven by a growing recognition that elements of the innate immune response can be harnessed to induce robust immunity against tumor-associated targets. Nonetheless, as clinically effective immunotherapy for the majority of cancers remains a distant goal, attention has shifted toward multimodality approaches to cancer therapy, sometimes combining novel immunotherapeutics and conventional therapeutics. The traditional view of radiation therapy as immunosuppressive has been challenged, prompting a re-evaluation of its potential as an adjunct to, or even a component of immunotherapy. Radiation therapy may enhance expression of tumor-associated antigens, induce targeting of tumor stroma, diminish regulatory T-cell activity and activate effectors of innate immunity such as dendritic cells through Toll-like receptor (TLR)-dependent mechanisms. Here, we review recent progress in the field of dendritic cell-based immunotherapy, evidence for radiation-induced antitumor immunity and TLR signaling and the results of efforts to rationally integrate radiation into dendritic cell-based immunotherapy strategies.

Radiotherapy is a successful treatment modality for localized cancer. Our group has been exploring radiotherapy in combination with immunotherapy (radioimmunotherapy) to enhance systemic antitumor responses. Previously, we have shown that when local radiotherapy was combined with monoclonal antibodies (mAbs) (that enable T-cell responses by engaging costimulation [anti (α)-CD137] and blocking coinhibition [α-PD-1], up to 100% of mice bearing established syngeneic AT-3 mammary tumors were cured, but single modality treatments were not curative. Here, we investigated the molecular mechanisms underlying responses to this radioimmunotherapy approach. We observed that inhibition of signaling through the mammalian target of rapamycin (mTOR) pathway during the first 10 days of treatment severely impaired the curative effect of radioimmunotherapy, at least in part by reducing MHC class I expression on tumor cells, reducing dendritic cell (DC) activation status and CD8 T-cell function. This data indicates that the efficacy of this type of radioimmunotherapy approach involves mTOR signaling and therefore, mTOR inhibitory drugs may impede the efficacy of similar radioimmunotherapy approaches in humans.

Recent advances indicate that new therapeutic strategies for the treatment of malignancies will be realized from combined radiation treatment and immune checkpoint modulation. Numerous biophysical properties must be considered for effective biologic development, including affinity, selectivity, oligomeric state and valency. High-resolution structural characterization contributes to our understanding of these properties and can lead to the realization of proteins with unique in vitro activities and novel in vivo therapeutic functions. In this article we focus on the importance of these factors for new potential biologics and consider these in the context of combination therapies with physical modalities, including radiation therapy. In particular, we examine the consequences of altered avidities and subset-specific ligand density on the rational modification of biological function in the immunoglobulin and tumor necrosis factor superfamilies and for new optimized combination therapies.

Despite major advances in the treatment of ovarian cancer over the past two decades, it is still an incurable disease and requires the development of better treatment strategies. In recent years, we have developed a greater understanding of tumor immunology and the interactions between tumors and the immune system. This has led to the emergence of cancer immunotherapy as the fourth treatment modality in cancer. In this article, we address the principles of immunotherapy and different approaches that have been investigated over the past decade and discuss the future of immune therapy in ovarian cancer.

As the immunotherapy of cancer comes of age, adding immunotherapeutic agents to radiation therapy has the potential to improve the outcomes for patients with a wide variety of malignancies. Despite the enormous potential of such combination therapy, laboratory data has been lacking and there is little guidance for pursuing novel treatment strategies. Animal models have significant limitation in combining radiation therapy with immunotherapy and some of the limitations of preclinical models are discussed in this article. In addition to the preclinical challenges, radiation therapy and immunotherapy combinations may have overlapping toxicities, and for both types of therapy, early and late manifestations of toxicity are possible. Given these risks, special attention should be given to the design of the specific Phase I clinical trial that is chosen. In this article, we describe several Phase I design possibilities that may be employed, including the 3 3 design (also known as the cohort of 3 design), the continual reassessment method (CRM), and the time-to-event continual reassessment method (TITE-CRM). Efficacy end points for further development of combination therapy must be based on multiple factors, including disease type, stage of disease, the setting of therapy and the goal of therapy. While the designs for future clinical trials will vary, it is clear that these two successful modalities of therapy can and should be combined for the benefit of cancer patients.