The reciprocity between radiotherapy and cancer immunotherapy

Abstract

The clinical success of immune checkpoint inhibitors in treating metastatic and refractory cancers has generated significant interest in investigating their role in treating locally advanced diseases, thus requiring them to be combined with standard treatments in hope to produce synergistic antitumor responses. Radiotherapy in particular, has long been hypothesized to have actions complementary to those of immune checkpoint blockade, and a growing body of evidence indicates that cancer immunotherapy may also have radiosensitizing effects, which would provide unique benefit for locoregional treatments. Recent studies have demonstrated that when immune cells are activated by immunotherapeutics, they can reprogram the tumor microenvironment in ways that may potentially increase the radiosensitivity of the tumor. In this review, we highlight the evidence that supports reciprocal interactions between cancer immunotherapy and radiotherapy, where in addition to the traditional notion that radiation serves to enhance the activation of antitumor immunity, an alternative scenario also exists in which T-cell activation by cancer immunotherapy may sensitize tumors to radiation treatment through mechanisms that include normalization of the tumor vasculature and tissue hypoxia. We describe the empirical observations from preclinical models that support such effects and discuss their implications for future research and trial design.

Introduction

Recent successes of immune checkpoint inhibitors for metastatic cancers have resulted in their increased investigation in treating locally advanced diseases, often in combination with other treatment modalities such as radiation. Radiation is the standard of care therapy for many types of cancer. Although ionizing radiation is classically known to induce tumor cell killing via direct or indirect damage to the cellular DNA, a growing list of evidences begins to suggest that radiation may also eliminate tumors via activation of local and/or systemic immune responses, particularly when it is combined with immune stimulating agents such as immune checkpoint inhibitors (Table 1) (1–8). Despite these exciting observations, the precise mechanism of how radiation and immunotherapy benefit one another remains unclear. Although ionizing radiation can induced immunological changes within the tumor microenvironment including facilitate tumor antigen release (9), increase effector T cell infiltration (10), and up-regulate MHC-1 molecule on tumor cells (11), recent evidences suggest that cancer immunotherapy such as immune checkpoint inhibitors may also have radiosensitization effects (12). The latter concept is especially important and clinically relevant because larger primary tumors often respond poorly to immunotherapies and require local therapies such as radiation. In this review, we summarize the evidences that support radiation’s immune stimulating effects and propose the mechanisms by which immunotherapies such as immune checkpoint blockade may serve as novel radiosensitizers. Finally, we discuss the implications of this concept for clinical studies and future trial design.

Table 1:

Selected studies using combination of radiotherapy and immunotherapy

Study Type	Disease	Sequence	RT	IT	Brief results	Reference
Clinical: stage III trial	Lung cancer	RT, IT	Definitive RT (54 to 66 Gy)	durvalumab	PFS improvement with durvalumab, similar side effects	56. Antonia et al. 2017
Clinical: stage I-II trial	Various	Concurrent	35 Gy in 10 fractions	GM-CSF	Abscopal responses in 27.6% patients	7. Golden et al. 2015
Clinical	Melanoma	IT, RT, IT	2850 cGy in 3 fractions over 7 days	ipilimumab	Abscopal effect, peripheral-blood immune cell changes	1. Postow et al. 2012
Clinical	Lung cancer	RT, IT, RT	Fractionation RT to primary and meta tumors	nivolumab	Abscopal effect	3. Schoenhals et al. 2016
Clinical	Pancreatic cancer	Concurrent	45 Gy in 15 fractions	GM-CSF	Abscopal effect, survival benefit	6. Shi et al. 2017
Clinical	Metastatic melanoma	IT, RT, IT	WBRT 30 Gy in 10 fractions	ipilimumab, pembrolizumab	Status improvement, long-term survival	4. Haymaker et al. 2017
Clinical	Various	Various	Various doses	anti-CTLA4, anti-PD-1/PD-L1	Induction immunotherapy begun more than 30 days before radiation resulted in longer OS	57. Samstein et al. 2017
Clinical	Brain metastasis	Various	Various, 18 – 30 Gy	ipilimumab, pembrolizumab, or nivolumab	Immunotherapy increased radiation necrosis	59. Martin et al. 2018
Preclinical	Melanoma	NA	15 Gy vs. 15 Gy in 3 fractions	NA	15 Gy single-dose generated more tumor-infiltrating T cells	62. Lugade et al. 2005
Preclinical	Colon cancer	RT, IT	10 Gy	T cell adoptive transfer	RT increased antigen presentation and enhanced IT efficacy	11. Reits et al. 2006
Preclinical	Melanoma	NA	20 Gy vs. 20 Gy in 4 fractions	NA	Immune response triggered by ablative radiation doses	61. Lee et al. 2009
Preclinical	Prostate cancer	NA	1 Gy × 10 vs. 10 Gy	NA	Multifraction radiation induced more DAMP release	66. Aryankalayil et al. 2014
Preclinical	Pancreatic cancer and melanoma	Various	20 Gy, 8 Gy	anti-CTLA4, anti-PD-L1	When combined with radiation, anti-CTLA4 and anti-PD-L1 promotes response through different mechanisms	9. Twyman-Saint Victor et al. 2015
Preclinical	Colon cancer	NA	30 Gy vs. 30 Gy in 10 fractions	NA	Ablative dose changed tumor immune microenvironment	10. Filatenkov et al. 2015
Preclinical	Breast cancer	RT, IT	6 Gy × 5	anti-TGF-beta, anti-PD-1	Anti-PD-1 prolonged survival of mice treated with RT and TGF-beta blockade	43. Vanpouille-Box et al. 2015
Preclinical	Pancreatic cancer	Concurrent	10 Gy	cyclic dinucleotides	STING activator and RT controlled both local and distant tumors synergistically	34. Baird et al. 2016
Preclinical	Colon cancer	IT, RT vs. RT, IT	20 Gy	anti-CTLA4	Anti-CTLA4 was most effective when given prior to radiation	55. Young et al. 2016
Preclinical	Colon cancer	IT, RT vs. RT, IT	20 Gy	anti-OX40	Anti-OX40 was more effective when given 1 day post radiation	55. Young et al. 2016
Preclinical	Colon and breast cancer	RT, IT	8 Gy × 3 vs. 20 Gy	anti-CTLA4	Anti-CTLA4 therapy only synergize with low dose radiation to induce an abscopal effect	65. Vanpouille-Box et al. 2017
Preclinical	Breast and colon cancer	IT, RT	0.5 Gy × 6	indoleamine 2,3-dioxygenase inhibitor	RT and IT induced rejection of both irradiated and non-irradiated tumors	8. Lu et al. 2018

Abbreviations: RT: radiotherapy; IT: immunotherapy; WBRT; whole brain radiotherapy; GM-CSF: Granulocyte-macrophage colony-stimulating factor; DAMP; Damage-associated molecular patterns; PFS: progression free survival; OS: overall survival; NA: not applicable.

Immune-modulating effects of radiation

Radiation increases antigen visibility

The ability to evade immune surveillance is one of the hallmarks of cancer (13). A tumor escapes from the host’s immune surveillance by several mechanisms, including evading detection (i.e., by downregulating MHC class I expression) and avoiding destruction (e.g., by creating an immunosuppressive tumor microenvironment) (14). However, radiation may render tumor cells antigens more visible to the immune system. Several mechanisms are involved in this “unmasking” process. First, radiation-induced DNA damage may lead to increased release of neoantigens by tumor cells for immune recognition (15). Given that tumor cells often accumulate DNA mutations as the result of DNA repair deficiencies, a higher burden of mutant nucleic acids and proteins has increased propensity for trigger immunogenic responses against tumors treated with immune checkpoint blockade (15–17). Ionizing radiation itself may also induce many DNA damages, including base modification, single strand break, and double strand break (18). These DNA alterations generated by radiation may act as a rich source of neoantigens that increase immune surveillance. In addition to modifying antigen release, radiation could also upregulate the expression of MHC molecules on cancer cells to facilitate presentation of tumor antigen to cytotoxic T cells. Activation of T cells is dependent on the recognition and binding of T cell receptor with peptide-bound MHC molecules. However, MHC-I is often downregulated by tumors to evade immune recognition (19). Radiation could increase the surface expression of MHC-I on tumor cells, making them more visible to cytotoxic T cells (11). Finally, radiation promotes the phagocytosis of damaged tumor cells by antigen-presenting cells, leading to increased priming of tumor-specific T cells. The pro-phagocytic effect of radiation is mediated by the release of damage-associated molecular pattern (DAMP) by tumor cells upon irradiation, including the translocation of the ER chaperone calreticulin, to the cell membrane. This immunogenic cell death process is also characterized by the release of high mobility group box 1 (HMGB1) and ATP, which together promotes tumor cell phagocytosis by macrophages and dendritic cells (20,21). These immune modulatory effects of radiation appear to be further augmented when it is combined with immune checkpoint blockers (22,23).

Radiation activates the cGAS-STING innate immune response

In addition to enhance the release and presentation of tumor-associated antigens, radiation may also activate antitumor immune responses by triggering the Stimulator of Interferon Genes (STING)-mediated DNA-sensing pathway (24). STING is part of the innate immune response that protects hosts from DNA pathogens (25) and is essential for host immunity (26). Radiation damage releases nuclear DNA into the cytosol (24). The presence of mutant DNA within the cytoplasma results in cyclin GMP-AMP (cGAMP), the product of cyclic GMP-AMP synthase (cGAS), to upregulate the transcription of type I interferon (IFN-I) genes via the STING-NFκB signaling pathway (27,28). Alternatively, STING and type I IFN activation can occur through ATM and IFI16 in a cGAS-independent manner (29). Type I IFN has important roles in regulating dentritic cell function and helper T-cell differentiation (30). The cGAS-STING pathway is essential for dendritic cells to sense the irradiated cancer cells and induce adaptive immune responses (31). However, the precise molecular mechanism governing STING activation in the presence of radiation is highly complex. A recent study found that both canonical and non-canonical NFκB signaling pathways were involved in radiation-induced type I IFN responses in dendritic cells (32). In murine colon cancer and melanoma models, the canonical NFκB pathway was required for the therapeutic effect of radiation, however, radiation also activated non-canonical NFκB pathway through cGAS-STING DNA sensing pathway to inhibit expression of IFN-β in dendritic cells. The inhibition of non-canonical NFκB pathway enhanced dendritic cell priming and the treatment efficacy of radiation (32). This study not only highlights the critical function of cGAS-STING in the immune responses after radiation, but also reveals the intriguing signal network downstream of cGAS-STING pathway.

Recognizing the importance of the STING pathway in antitumor immunity has led to several studies investigating the therapeutic potential of STING agonists in anticancer therapy. The direct activition of STING by agonists, either as monotherapy or in combination with radiation, has shown to induce both local and systemic antitumor immune response in murine cancer models (33,34). Currently, a phase I clinical trial (NCT02675439) is evaluating the safety and efficacy of a STING agonist, MIW815, for patients with advanced solid tumors or lymphoma. However, the role of STING in cancer immunotherapy remains controversial. A recent study showed that STING activation after radiation recruited myeloid-derived suppressor cells to the tumor, may in fact induce radioresistance and immunosuppression (35). Furthermore, the STING pathway may also promote immune tolerance by modulating the tumor microenvironment (36,37). Therefore, additional studies are warranted to investigate the biology and the role of the STING pathway in immune regulation within solid tumors.

Radiation modulates the tumor microenvironment

In addition to direct cancer cell killing, radiation may also reprogram the tumor microenvironment from an immunosuppressive to an immunostimulatory phenotype (38). Radiation can increase infiltration of CD8⁺ T cells while decreasing myeloid-derived suppressor cells, depending on the presence of cross-presenting dendritic cells and IFN-γ (10). Low-dose radiation promotes normalization of the tumor vasculature and polarization of M2-like macrophages toward an M1-like iNOS⁺ phenotype (39). The iNOS⁺ macrophages induce the expression of Th1 chemokines that recruit CD8⁺ and CD4⁺ T cells to the tumor, promoting T-cell mediated antitumor effects and prolonging survival in murine models of pancreatic cancer (39). On the other hand, radiation also stimulates secretion of cytokines that induce immunosuppression. For example, transforming growth factor-β (TGF-β) is upregulated shortly after radiation. TGF-β induces the suppression of CD8⁺ T cells and promotes regulatory T-cell transformation, causing immunosuppression in the tumor (20,40,41). In preclinical models, TGF-β neutralization regulated T-cells to promote anti-tumor immunity of dendritic cells (42). When combined with radiation, TGF-β neutralization increased IFN-γ-associated gene signatures and enhanced CD8⁺ T-cell response (43). Moreover, the combination of radiation and TGF-β neutralization controlled both local and distant tumor growth more effectively than did either treatment given alone (43).

Radiation regulates immune checkpoint expression

Radiation may also indirectly regulate the expression levels of immune checkpoint molecules on the surfaces of both cancer cells and immune cells within the tumor microenvironment through IFN-γ (12). Alternatively, a recent study suggested that radiation-induced DNA double-strand breakage upregulates the expression of PD-L1 on tumor cells via ATM/ATR/Chk1 kinases (44). Given that over-expression of PD-L1 within tumors is associated with improved responses to anti-PDL1 therapy (45), radiation may serve as an effective neoadjuvant treatment to increase the effectiveness of immune checkpoint blockade. However, the exact relationship between radiation and PDL1 induction has not been completely elucidated, and it remains to be seen whether it can be applied to difference tumors. The currently known mechanisms by which radiation enhances immunotherapy are summarized in Figure 1.

Figure 1. Mechanisms by which radiation enhances immunotherapy.

Cancer-specific peptides released from damaged cancer cells facilitate antigen uptake and presentation by dendritic cells. Radiation also decreases an anti-phagocytosis signal (CD47) and increases a pro-phagocytosis signal (calreticulin) to enhance calreticulin-LRP/CD91-mediated phagocytosis by macrophages, thereby increasing antigen presentation and priming of T cells. Upregulation of MHC-I expression on tumor cells after irradiation increases recognition of cancer cells by T cells. After radiation, damaged DNA is released from nucleus to cytosol, which triggers the cGAS-STING pathway to activate interferon gene transcription. Several proteins (such as TGF-β and HMGB1) secreted by cancer cells also modulate the immune microenvironment.

Radiosensitization by immune checkpoint blockades

Hypoxic tumor microenvironment induces radioresistance

The abnormal and dysfunctional nature of the tumor vasculature causes hypoxia, limits drug delivery, and contributes to an overall immunosuppressive microenvironment (46,47). Meanwhile, hypoxic microenvironments induce excessive secretion of proangiogenic signals, such as vascular endothelial growth factor (VEGF), resulting in rapid but abnormal tumor vessel formation. Hypoxia directly and indirectly induces radioresistance via several mechanisms. Firstly, the lack of oxygen within hypoxic tumors results in fewer DNA damages from the same dose of radiation as compared to well oxygenated ones (48,49). Additionally, hypoxia also drives radioresistance by accumulating and stabilizing hypoxia-inducible factor-1 (HIF1) (48,50). HIF1 activation increases the expression of key enzymes that drive glycolysis leading to the accumulation of lactate and pyruvate as well as the anti-oxidants glutathione and NADPH. The latter molecules scavenge reactive oxygen species (ROS) generated by radiation exposure to limit DNA damage (49). Lactate, on the other hand, affects immune cell maturation leading to immune escape and immunotherapy resistance (51). Lactate also upregulates the HIF1 pathway creating a futile cycle of radio- and immune resistance (49,52). Collectively, hypoxia is one of the major drivers of tumor radioresistance; therefore, targeting tumor hypoxia may increase tumor radiosensitivity.

Tumor vascular normalization and hypoxia reduction may sensitize tumors to radiation

Recent studies suggest that immune checkpoint blockade not only unleashes T cells to attack tumor cells but may also modulate the tumor microenvironment by normalizing tumor vessels (47,53,54). This novel interaction creates the potential opportunity for immunotherapy to reduce tumor hypoxia and increase radiosensitivity. One recent stud showed that cancer immunotherapy induced tumor-vascular normalization in a T-cell-dependent manner (54). Specifically, when murine breast and colon tumor models were treated by anti-CTLA4 or anti-PD1 agents, the responding tumors showed strong growth inhibition as well as increased vessel perfusion and decreased intratumoral hypoxia. These immunotherapies also normalized the tumor vasculature through the accumulation of IFN-γ producing CD8⁺ cells. Importantly, the extent of tumor vascular normalization by immune checkpoint inhibitor is strongly associated with its therapeutic efficacy (54). Neutralization of IFN-γ or CD8⁺ T cell depletion abrogated both the vascular normalization and antitumor effect of immune checkpoint blockade, suggesting that both processes are intricately related to one another. A related study using bioinformatics analysis also identified a correlation between immunostimulatory pathways and vascular normalization-related genes. This study found that type 1 T-helper (T_H1) cells were involved in the normalization of the tumor vasculature (53). Upon blockade of immune checkpoint, IFN-γ secretion of CD4⁺ T cells is prompted, which promotes vessel normalization and reduces hypoxia (53). Based on these evidences, it is highly plausible that the tumor vascular normalizing effects of immune checkpoint blockade not only create a feedback loop to reprogram the tumor immune microenvironment and enhance the efficacy of immunotherapy but also may sensitize tumors to radiation (Figure 2). With the combination of immune checkpoint inhibitors and radiation been tested in multiple clinical trials, this notion that cancer immunotherapy may promote radiosensitivity via normalization of tumor vasculature and hypoxia carries increased clinical significance. Detailed characterization of the intricate reciprocal relationships between radiation, vascular remodeling and immune regulation is critical to develop optimal combination immunotherapy-radiotherapy treatment for cancer patients.

Figure 2. Immunotherapy sensitizes tumors to radiation by modulating the tumor microenvironment.

Immunotherapy increases T-cell infiltration into tumors. The infiltrated T cells secrete cytokines such as IFN-γ, which normalize the tumor vasculature. This vessel normalization increases perfusion and oxygen supply in the tumor microenvironment, and therefore could sensitize tumors to radiation.

Implications for clinical translation

Optimizing the timing of radiation in combination with immunotherapy

Considering the complexities of the interplay between radiation and immunotherapy, careful planning as to the timing of radiation to be given with immunotherapy could be crucial to optimizing treatment efficacy (20). One preclinical study indicated that the best timing relative to radiation was different for anti-CTLA4 and anti-OX40, suggesting that the optimal treatment sequences could be specific to the type of immunotherapy (55). Interestingly, analysis of a randomized clinical trial (56) and a retrospective analysis of clinical data (57) suggest that patient outcome seems to be better if immunotherapy is given concurrently or begun soon after radiation, as compared with starting immunotherapy later after the radiation. This finding potentially reflects vascular normalization prompted by immunotherapy (53,54) and radiotherapy (39), which would initiate the feedback loop of vascular normalization and immune stimulation and thus potentiate both radiotherapy and immunotherapy. Mechanistically, the potential radiosensitizing function of immunotherapy would be effective only when it is administered concurrently with radiation or before radiation, to precondition the tumor microenvironment and thereby increase tumor radiosensitivity. Thus, synergistic effects of the two therapies may best be realized by alternating immunotherapy and radiotherapy, or vice versa. Whether this is true requires further preclinical and clinical investigations. Meanwhile, despite the favorablr antitumor effects in some patients, immunotherapy often induce adverse events and side effects, particular when combined with other treatment modalities such as radiation (58). Therefore, toxicity may be a hurdle for combining the two modalities, given the observed increase in adverse events from immunotherapy combined with radiation (59,60). The balance between benefits and risks needs further exploration in future clinical trials.

Optimizing the dose of radiation in combination with immunotherapy

The radiation dose and dosing schedule are other critical factors to consider (20) when given with immunotherapies. Conventionally fractionated radiation (i.e., once-daily 2-Gy fractions) mainly targets vulnerabilities in the tumor’s DNA damage repair and cell cycle arrest. Advances in radiotherapy techniques have enabled the delivery of large doses of radiation, also given daily, to similar or lower total doses in fewer fractions. The radiation dose per fraction not only affects direct cancer cell killing but also influences modulation of the tumor microenvironment. Compared with conventional fractionation, a single high dose of radiation may facilitate the maturation of antigen-presenting cells (61) and increase the infiltration of immune cells into the tumor (62). Low-dose radiation has been shown to normalize the tumor vasculature (39), but high-dose radiation could cause more vessel damage, thereby reducing blood flow and perfusion (63). Meanwhile, the lack of reoxygenation during the course of hypofractionated radiation therapy means that hypoxic tumors are more resistant to hypofractionated radiation (64). Theoretically, normalization of the tumor vasculature by immunotherapy may overcome the radioresistance of hypoxic tumors to hypofractionated radiation while maintaining its immune-stimulatory effects. However, the optimal dose of radiation to be given with immunotherapy is unknown, as some preclinical studies suggested that hypofractionated radiation led to a greater immune response (10,61,62) but others have found conventional fractionation to be more favorable when combined with immunotherapy (65,66). The intriguing interactions between immune cells and the tumor vasculature in response to different doses of radiation warrant further preclinical and clinical investigation.

Developing novel biomarkers of treatment response

Despite some remarkable successes in cancer immunotherapy, the response rates are still far from satisfying (67). Identification of biomarkers with which to predict and evaluate response to treatment is an unmet need for precision immunotherapy. Although several such markers have been tested, including DNA repair deficiencies (17,68), mutational burden (69), PDL1 expression (45), and the gut microbiome (70) have been tested, several limitations remain. Firstly, immunotherapy responses tend to emerge later than conventional therapies. In addition, tumor volume variations during immunotherapy do not necessarily reflect treatment responses (67,71). Thus novel markers are needed with which to evaluate responsiveness (or lack thereof) to immunotherapy early in the course of treatment, ideally even before tumor regression can be detected. Tumor-vessel normalization or increased perfusion are being explored as early markers of immunotherapy responsiveness (54). In a murine breast cancer model, tumor vessel perfusion, measured by 3D and color Doppler ultrasonography at 6 days after treatment with anti-CTLA4, indicated that high perfusion levels correlated strongly with tumor regression at day 14, with more than 90% sensitivity and specificity (54). Because tumor vessel perfusion can be visualized noninvasively by imaging early in the course of treatment, this may be a promising marker for predicting the efficacy of immunotherapy for a given patient (Figure 3). Whether this marker remains valid when radiation is added to the immunotherapy and whether it can be extended to clinical management require carefully designed exploratory and confirmatory studies of potential biomarkers for use in clinical trials.

Figure 3. Identification of novel biomarkers to guide radiation and immunotherapy treatment.

Pretreatment markers may be useful for identifying patients who may benefit from immunotherapy. For tumors that are resistant or not completely eradiated by immunotherapy, consolidation therapy such as radiation may be used. Noninvasive means to evaluate vascular-related changes within a solid tumor at earlier time points post immunotherapy treatments may further help guide clinical decision-making for personalized cancer care. CT, computed tomography; MRI, magnetic resonance imaging.

Can immunotherapy modulate intrinsic radiosensitivity of tumors?

Several lines of evidence suggest that pathways related to radiosensitivity may also modulate a tumor’s immunogenicity and predict its response to immunotherapies. For example, deficiencies in the DNA repair machinery could increase neoantigen release and trigger an immune response (16), and responsiveness to immunotherapy could also be predicted by the presence of these DNA repair deficiencies (15,68). To date, several common regulators of DNA repair and immune checkpoints have been discovered. PARP inhibitors have well-studied radiosensitizing effects (72), and interestingly they also upregulate PDL1 expression in breast cancer cell lines and animal models (73). An siRNA screen by Sato et al. showed that BRCA2 or Ku70/80 deletion was associated with enhanced PD-L1 expression after x-ray radiation of cancer cells (44). Another important DNA repair regulator, p53, was shown to regulate PD-L1 expression through miR-34 (74). In patients with ovarian cancer, expression levels of PD-1 and PD-L1 were found to be associated with BRCA1/2 and p53 mutation status (75). Growing evidence supports the notion that DNA repair deficiencies modulate tumor immune checkpoints, but whether immune checkpoint blockade conversely regulates DNA repair pathways is still unclear. This potential novel mechanism by which immunotherapy regulates the intrinsic radiosensitivity of tumors may prove to be a promising avenue for future investigation.

Conclusion

In summary, in addition to the orthodox understanding that radiation promotes the efficacy of immunotherapy, immunotherapy may also radiosensitize tumors to achieve better local control when combined with radiotherapy. Immune checkpoint blockers can promote normalization of tumor blood vessels, improve tissue perfusion, and decrease intratumoral hypoxia in a T cell-dependent manner through IFN-mediated signaling between T cells and endothelial cells (53,54). These changes within the local tumor microenvironment subsequently may sensitize the tumor to ionizing radiation by reducing tissue hypoxia, decreasing acidosis and improving the production of reactive oxygen species needed for radiation-induced tumor cell killing (49,51,53,54). In depth understanding of the reciprocal relationship between ionizing radiation and immune re-programming thus has significant implications for designing future clinical trials of combination immunotherapies. For example, incorporation of new biomarkers that correlate with intratumoral vascularity, and how radiation should be given in terms of dose and timing with respect to immunotherapy becomes highly important, especially considering the transient nature of immunotherapy-induced vascular normalization effects. With cancer immunotherapies play an ever-increasing role in the treatment of solid tumors, taking advantage of the mutual benefit between ionizing radiation and immunotherapies may open up new avenues for designing immune combination treatments to achieve improved therapeutic efficacy with minimal toxicity in cancer patients.