Unirradiated Tumors Research Articles

2-Hexyl-4-Pentylenic Acid (HPTA) Stimulates the Radiotherapy-induced Abscopal Effect on Distal Tumor through Polarization of Tumor-associated Macrophages

ObjectiveThe aim of this study was to explore the effects of 2-hexyl-4-pentylenic acid (HPTA) in combination with radiotherapy (RT) on distant unirradiated breast tumors. MethodsUsing a rat model of chemical carcinogen (7,12-dimethylbenz[a]anthracene,DMBA)-induced breast cancer, tumor volume was monitored and treatment response was evaluated by performing HE staining, immunohistochemistry, immunofluorescence, qRT-PCR, and western blot analyses. ResultsThe results demonstrated that HPTA in combination with RT significantly delayed the growth of distant, unirradiated breast tumors. The mechanism of action included tumor-associated macrophage (TAM) infiltration into distant tumor tissues, M1 polarization, and inhibition of tumor angiogenesis by IFN-γ. ConclusionThe results suggest that the combination of HPTA with RT has an abscopal effect on distant tumors via M1-polarized TAMs, and HPTA may be considered as a new therapeutic for amplifying the efficacy of local RT for non-targeted breast tumors.

156: Stereotactic Body Radiotherapy (SBRT) for the Palliation of Unirradiated Mucosal Head and Neck Tumours

156: Stereotactic Body Radiotherapy (SBRT) for the Palliation of Unirradiated Mucosal Head and Neck Tumours

Radiation Therapy Enhanced by NBTXR3 Nanoparticles Overcomes Anti-PD1 Resistance and Evokes Abscopal Effects

Radiation combined with PD1 blockade offers significant treatment benefits in several tumor types; however, anti-PD1 resistance precludes such benefits in many cases. Here we attempted to overcome anti-PD1 resistance by combining localized radiation with a radioenhancing nanoparticle (NBTXR3) and systemic anti-PD1 treatment to achieve abscopal effects in an anti-PD1-resistant mouse model of lung cancer. Female 129Sv/Ev mice were inoculated with 344SQ anti-PD1-resistant (344SQR) or anti-PD1-sensitive (344SQP) metastatic lung cancer cells in the right leg on day 0 ("primary" tumor) and the left leg on day 4 ("secondary" tumor). Primary tumors were injected intratumorally with NBTXR3 on day 7 and were irradiated with 12 Gy on days 8, 9, and 10. Mice were given 6 intraperitoneal injections of anti-PD1. T cell receptor repertoire was analyzed in tumor samples with RNA sequencing, infiltration of CD8 T cells with immunohistochemical staining, and activities of various immune pathways with NanoString analysis. The triple combination of NBTXR3 with localized radiation and systemic anti-PD1 significantly delayed the growth of both irradiated and unirradiated tumors in both 344SQP and 344SQR tumor models. NBTXR3 remodeled the immune microenvironment of unirradiated tumors by triggering the activation of various immune pathways, increasing the number of CD8+ T cells, and modifying the T cell receptor repertoire in the 344SQR tumor model. The ability of NBTXR3 to evoke significant abscopal effects in both anti-PD1-sensitive and anti-PD1-resistant lung cancers could open the possibility of its use for treating patients with metastatic lung cancer regardless of sensitivity (or resistance) to immunotherapies.

Valproic acid triggers radiation-induced abscopal effect by modulating the unirradiated tumor immune microenvironment in a rat model of breast cancer.

An abscopal effect occurs when localized radiotherapy causes the regression of tumors distant from the irradiated site. However, such a clinically detectable abscopal effect from radiotherapy alone is rare. This study investigated whether valproic acid ([VPA], a histone deacetylase inhibitor [HDACi]) treatment can stimulate radiation-induced abscopal effect. We used 7,12-dimethylbenz[a]anthracene, a typical environmental carcinogen, to establish a rat model with multiple breast tumors. Only one tumor received 8 Gy fractionated doses of X-rays (2 Gy daily fractions over four days) and 200mg/kg VPA was administered intraperitoneally. We monitored the growth of both irradiated and unirradiated tumors after treatments. The unirradiated tumor was collected for hematoxylin and eosin (HE) staining, immunohistochemistry (IHC) (CD8, Granzyme B, Cleaved Caspase-3, BrdU, Ki67, F4/80 and CD68), double immunofluorescence (F4/80 and CD86), Western blot (Cleaved Caspase-3) and qRT-PCR (CD86, CD163, IL-1β, IL-6, IL-12, IL-23, IL-10, TGF-β) analysis. We found ionizing radiation (IR) + VPA treatment inhibited both irradiated and unirradiated tumor growth as compared to IR alone. Such observe abscopal effect was mediated by the recruitment of activated CD8+ T cells into the unirradiated tumor sites, which released Granzyme B to cause tumor cell apoptosis. Furthermore, IR + VPA treatment led to macrophages infiltration into the unirradiated tumor sites and polarization to M1 phenotype, resulted in increased levels of pro-inflammatory cytokines such as IL-1β and IL-12, and decreased levels of anti-inflammatory cytokines such as IL-10 and TGF-β. Our data supports the proposition that VPA may be a potential therapeutic candidate to trigger radiation-induced abscopal effect by modulating the unirradiated tumor immune microenvironment.

Abstract PO-040: Integration of anti-TIGIT and anti-Lag3 with NBTXR3-mediated immunoradiation therapy improves abscopal effect and induces long-term memory against cancer

Abstract Introduction: TIGIT and Lag3 are inhibitory receptors expressed on cytotoxic CD8+ T cells and NK cells and directly inhibit the activation and proliferation of these cells. We proposed that blockade of TIGIT and Lag3 could improve antitumor immune response in a mouse model of anti-PD1-resistant mice. Methods: 129Sv/Ev mice were inoculated with 50,000 anti-PD1-resistant 344SQR cells in the right leg on day 0 (primary tumor) and with 50,000 cells in the left leg on day 4 (secondary tumor). Primary tumors were injected with NBTXR3 (radioenhancer nanoparticles) on day 7 and irradiated with 12 Gy on days 8, 9, and 10. Anti-PD1 (200 mg), anti-Lag3 (200 mg), and anti-TIGIT (200 mg) were given to mice by intraperitoneal injections on days 5, 8, 11, 14, 21, 28, 35, and 42. On day 21, primary tumors, secondary tumors, and blood samples were harvested and analyzed with flow cytometry to evaluate changes in immune cell populations. Mice in which tumors were completely eradicated were re-challenged with another 50,000 344SQR cells in the right flank at least two months post radiation; no further treatment was given to these mice, and tumor growth was monitored. Results: The addition of anti-TIGIT, anti-Lag3, or anti-TIGIT+anti-LAG3 to NBTXR3+XRT+anti-PD1 therapy significantly improved control of both primary and secondary tumors, and the addition of anti-TIGIT plus anti-LAG3 also led to fewer spontaneous lung metastases. The addition of either anti-TIGIT or anti-Lag3 to NBTXR3+XRT+anti-PD1 extended the mouse survival time relative to NBTXR3+XRT+anti-PD1. None of the 8 mice in either the NBTXR3+XRT+anti-PD1+anti-TIGIT group or the NBTXR3+XRT+anti-PD1+anti-Lag3 group survived more than 32 days; in contrast, 3 of the 8 mice that received NBTXR3+XRT+anti-PD1+anti-TIGIT+anti-Lag3 survived until the end of the experiment. These surviving mice were found to have developed memory against 344SQR cells, and no further tumor growth was observed after re-challenge. Flow cytometry analysis showed that adding anti-TIGIT+anti-Lag3 to NBTXR3+XRT+anti-PD1 increased the percentages of proliferating CD8+ T cells in primary tumors, secondary tumors, and blood, and increased the percentage of NK cells in the secondary tumors. Conclusions: Blockade of TIGIT and Lag3 with NBTXR3+XRT+anti-PD1 improved CD8+ T-cell proliferation, augmented the antitumor response at both irradiated and unirradiated (abscopal) tumors, and induced potent long-term antitumor memory in mice. Citation Format: Yun Hu, Sebastien Paris, Hampartsoum Barsoumian, Duygu Sezen, Ke Wen He, Mark Wasley, Fatemeh Masrorpour, Peng Liang Yang, Nahum Puebla-Osorio, Maria Angelica Cortez, James Welsh. Integration of anti-TIGIT and anti-Lag3 with NBTXR3-mediated immunoradiation therapy improves abscopal effect and induces long-term memory against cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-040.

Maintaining manganese in tumor to activate cGAS-STING pathway evokes a robust abscopal anti-tumor effect

Radiotherapy (RT)-induced DNA damage leaked into cytosol can elicit host antitumor immune response. However, such response rate is unpromising due to limited cyclic GMP-AMP synthase (cGAS) recognition of cytosolic DNA, which could be digested inherently by host DNases. Here we show that synchronizing Mn2+ delivery with accumulated cytosolic DNA after RT can promote the activation of cGAS-STING pathway, thereby enhancing RT-induced antitumor immunity. Intratumoral Mn2+ injection immediately after RT cannot enhance RT, while intratumoral Mn2+ injection 24 h after RT can. Direct-injected Mn2+ can be metabolized out from tumor in minutes while RT-induced DNA damage need cells mitotic progression for up to 24 h to accumulate into cytosol. Alginate can maintain Mn2+ in tumor for up to 24 h due to it can chelate divalent cations. When the release profile of Mn2+ is controlled by alginate (Alg) and synchronized with the accumulation of RT-induced DNA damage, over 90% inhibition rate can be obtained even in the unirradiated tumor, and survival time is significantly extended. This synchronizing strategy provides a simple and novel approach to effectively activate cGAS-STING pathway in tumor and promote RT-induced immunity.

Multicellular Spheroids as In Vitro Models of Oxygen Depletion During FLASH Irradiation

The differential response of normal and tumor tissues to ultrahigh-dose-rate radiation (FLASH) has raised new hope for treating solid tumors but, to date, the mechanism remains elusive. One leading hypothesis is that FLASH radiochemically depletes oxygen from irradiated tissues faster than it is replenished through diffusion. The purpose of this study was to investigate these effects within hypoxic multicellular tumor spheroids through simulations and experiments. Physicobiological equations were derived to model (1) the diffusion and metabolism of oxygen within spheroids; (2) its depletion through reactions involving radiation-induced radicals; and (3) the increase in radioresistance of spheroids, modeled according to the classical oxygen enhancement ratio and linear-quadratic response. These predictions were then tested experimentally in A549 spheroids exposed to electron irradiation at conventional (0.075 Gy/s) or FLASH (90 Gy/s) dose rates. Clonogenic survival, cell viability, and spheroid growth were scored postradiation. Clonogenic survival of 2 other cell lines was also investigated. The existence of a hypoxic core in unirradiated tumor spheroids is predicted by simulations and visualized by fluorescence microscopy. Upon FLASH irradiation, this hypoxic core transiently expands, engulfing a large number of well-oxygenated cells. In contrast, oxygen is steadily replenished during slower conventional irradiation. Experimentally, clonogenic survival was around 3-fold higher in FLASH-irradiated spheroids compared with conventional irradiation, but no significant difference was observed for well-oxygenated 2-dimensional cultured cells. This differential survival is consistent with the predictions of the computational model. FLASH irradiation of spheroids resulted in a dose-modifying factor of around 1.3 for doses above 10 Gy. Tumor spheroids can be used as a model to study FLASH irradiation invitro. The improved survival of tumor spheroids receiving FLASH radiation confirms that ultrafast radiochemical oxygen depletion and its slow replenishment are critical components of the FLASH effect.

Tumor size-dependent abscopal effect of polydopamine-coated all-in-one nanoparticles for immunochemo-photothermal therapy of early- and late-stage metastatic cancer

Metastatic cancer is a persistent clinical enigma, which requires combination of several treatment modules. Here, we developed an all-in-one nanomedicine strategy to systemically co-deliver photosensitive, chemotherapeutic, and immunomodulating agents for effective immunochemo-photothermal therapy (PTT) to inhibit both primary tumor and distal metastatic tumor. Two types of polydopamine (dp)-coated nanoparticles (NPs) (N/PGEM/dp-5 and N/PGEM/dp-16) co-loaded with gemcitabine (GEM) and NLG919, a potent indoleamine-2, 3-dioxygenase (IDO) inhibitor, were prepared. N/PGEM/dp-16 NPs with a thicker dp coating layer showed higher photothermal conversion ability, more favorable biodistribution profile and better tumor inhibition effect compared to N/PGEM/dp-5 NPs with a thinner coating layer. Combination with laser irradiation further enhanced the tumor inhibition effect of N/PGEM/dp-16 NPs. In an “early metastatic” pancreatic cancer PANC02 model with small distal tumors, introduction of NLG and dp coating improved the inhibition effect on both primary and distal tumors. Compared to N/PGEM/dp-16, N/PGEM/dp-16 plus laser irradiation further enhanced the inhibition effect on primary tumor, but didn't improve the abscopal antitumor effect. When the initial volume of distal tumor was sufficiently large in a “late metastasis” model, a more dramatic abscopal antitumor effect was achieved, resulting in a significant growth inhibition of both primary tumor and the unirradiated distal tumor. Furthermore, laser irradiation can amplify the immunochemo-NPs-mediated innate and adaptive immune responses in both tumors. This work demonstrated a distal tumor-size dependent abscopal effect, and provided a perspective for future design of more effective immunochemo-PTT nano-formulations for early- and late-stage metastatic tumors.

Evaluation of Radiotherapy-Induced Systemic Antitumor Effects in Mice Bearing 4T1 Mouse Breast Cancer Cells.

Background: Recently, several clinical studies have reported that combination treatments of radiation therapy (RT) and immunotherapy in patients with multiple lesions can improve tumor regression at a distance from the irradiated site, known as the abscopal effect. However, when RT and immunotherapy are concurrently applied, it is hard to distinguish the pure systemic effects of RT from those of the immunotherapy drug. In this preclinical study, the authors investigated the systemic antitumor effects of RT alone according to fraction dose size and splitting schedules. Materials and Methods: 4T1 mouse breast cancer cells were implanted into the right and left sides of mammary gland fat pads of BALB/c mice, followed by irradiation with 6 Gy × 3, 8 Gy × 2, and 13 Gy × 1 fractions when the right-side tumors were palpable. Results: The different irradiation schedules produced similar antitumor effects in irradiated right-side tumors and unirradiated left-side tumors. However, 8 Gy × 2 and 13 Gy × 1 fractions exhibited better antimetastatic potential than that from irradiation using 6 Gy × 3 fractions. Furthermore, 8 Gy × 2 and 13 Gy × 1 fractions produced higher expressions of HMGB1 and lower expressions of the proinflammatory cytokines, IFN-γ, TNF-α, IL-6, and IL-1β, from the irradiated tumor tissues. Conclusions: These findings suggest that 8 Gy × 2 and 13 Gy × 1 fractions can provide better systemic antitumor effects than 6 Gy × 3 fractions. The authors hope these results provide clues to optimize RT dose regimens to make the abscopal effect clinically more relevant in future combination treatments.

Biological Guided Carbon-Ion Microporous Radiation to Tumor Hypoxia Area Triggers Robust Abscopal Effects as Open Field Radiation.

Recently, a growing number of studies focus on partial tumor irradiation to induce the stronger non-target effects. However, the value of partial volume carbon ion radiotherapy (CIRT) targeting hypoxic region of a tumor under imaging guidance as well as its effect of inducing radiation induced abscopal effects (RIAEs) have not been well investigated. Herein, we developed a technique of carbon ion microporous radiation (CI-MPR), guided by 18F-FMISO PET/computerized tomography (CT), for partial volume radiation targeting the hypoxia area of a tumor and investigated its capability of inducing abscopal effects. Tumor-bearing mice were inoculated subcutaneously with breast cancer 4T1 cells into the flanks of both hind legs of mouse. Mice were assigned to three groups: group I: control group with no treatment; group II: carbon ion open field radiation (CI-OFR group) targeting the entire tumor; group III: partial volume carbon ion microporous radiation (CI-MPR group) targeting the hypoxia region. The tumors on the left hind legs of mice were irradiated with single fraction of 20 Gy of CIRT. Mice treated with CI-MPR or CI-OFR showed that significant growth delay on both the irradiated and unirradiated of tumor as compared to the control groups. Tumor regression of left tumor irradiated with CI-OFR was more prominent as compared to the tumor treated with CI-MPR, while the regression of the unirradiated tumor in both CI-MPR and CI-OFR group was similar. Biological-guided CIRT using the newly developed microporous technique targeting tumor hypoxia region could induce robust abscopal effects similar to CIRT covering the entire tumor.

Combination of radiotherapy and suppression of Tregs enhances abscopal antitumor effect and inhibits metastasis in rectal cancer

BackgroundDistant metastasis is the major cause of mortality in patients with locally advanced rectal cancer (LARC) following neoadjuvant chemoradiotherapy. Local radiotherapy can trigger an abscopal response to metastatic tumor cells....

Radiation Dose Escalation is Crucial in Anti-CTLA-4 Antibody Therapy to Enhance Local and Distant Antitumor Effect in Murine Osteosarcoma.

We previously reported that a combination of 10 Gy of X-ray irradiation and dual immune checkpoint blockade with anti-CTLA-4 (C4) and anti-PD-L1 antibodies produced a significant shrinkage of irradiated and unirradiated tumors (abscopal effect) and prolonged overall survival. However, the optimal radiation delivery regimen combined with single immune checkpoint blockade of C4 for inducing a maximum systemic antitumor response still remains unclear, particularly for patients with osteosarcoma. We used syngeneic C3H mice that were subcutaneously injected with LM8 osteosarcoma cells into both legs. C4 was administered three times, and one side of the tumor was irradiated by X-ray beams. The optimal radiation dose required to induce the abscopal effect was explored with a focus on the induction of the type-I interferon pathway. Radiation delivered in a single fraction of 10 Gy, 4.5 Gy × 3 fractions (fx), and 2 Gy × 8 fx with C4 failed to produce significant inhibition of unirradiated tumor growth compared with monotherapy with C4. Dose escalation to 16 Gy in a single fraction, or the equivalent hypofractionated dose of 8 Gy × 3 fx, which significantly increased secretion of IFN-β in vitro, produced a dramatic regression of both irradiated and unirradiated tumors and prolonged overall survival in combination with C4. Furthermore, irradiation at 16 Gy in both a single fraction and 8 Gy × 3 fx diminished regulatory T cells in the unirradiated tumor microenvironment. These results suggest that total dose escalation of radiation is crucial in C4 therapy to enhance the antitumor response in both local and distant tumors and prolonged overall survival regardless of fractionation for osteosarcoma.

Tumor Size Matters-Understanding Concomitant Tumor Immunity in the Context of Hypofractionated Radiotherapy with Immunotherapy.

The purpose of this study was to determine the dynamic contributions of different immune cell subsets to primary and abscopal tumor regression after hypofractionated radiation therapy (hRT) and the impact of anti-PD-1 therapy. A bilateral syngeneic FSA1 fibrosarcoma model was used in immunocompetent C3H mice, with delayed inoculation to mimic primary and microscopic disease. The effect of tumor burden on intratumoral and splenic immune cell content was delineated as a prelude to hRT on macroscopic T1 tumors with 3 fractions of 8 Gy while microscopic T2 tumors were left untreated. This was performed with and without systemic anti-PD-1. Immune profiles within T1 and T2 tumors and in spleen changed drastically with tumor burden in untreated mice with infiltrating CD4+ content declining, while the proportion of CD4+ Tregs rose. Myeloid cell representation escalated in larger tumors, resulting in major decreases in the lymphoid:myeloid ratios. In general, activation of Tregs and myeloid-derived suppressor cells allow immunogenic tumors to grow, although their relative contributions change with time. The evidence suggests that primary T1 tumors self-regulate their immune content depending on their size and this can influence the lymphoid compartment of T2 tumors, especially with respect to Tregs. Tumor burden is a major confounding factor in immune analysis that has to be taken into consideration in experimental models and in the clinic. hRT caused complete local regression of primary tumors, which was accompanied by heavy infiltration of CD8+ T cells activated to express IFN-γ and PD-1; while certain myeloid populations diminished. In spite of this active infiltrate, primary hRT failed to generate the systemic conditions required to cause abscopal regression of unirradiated microscopic tumors unless PD-1 blockade, which on its own was ineffective, was added to the RT regimen. The combination further increased local and systemically activated CD8+ T cells, but few other changes. This study emphasizes the subtle interplay between the immune system and tumors as they grow and how difficult it is for local RT, which can generate a local immune response that may help with primary tumor regression, to overcome the systemic barriers that are generated so as to effect immune regression of even small abscopal lesions.

Anti-PD-L1 antibody direct activation of macrophages contributes to a radiation-induced abscopal response in glioblastoma.

Most glioblastomas recur near prior radiation treatment sites. Future clinical success will require achieving and optimizing an "abscopal effect," whereby unirradiated neoplastic cells outside treatment sites are recognized and attacked by the immune system. Radiation combined with anti-programmed cell death ligand 1 (PD-L1) demonstrated modest efficacy in phase II human glioblastoma clinical trials, but the mechanism and relevance of the abscopal effect during this response remain unknown. We modified an immune-competent, genetically driven mouse glioma model (forced platelet derived growth factor [PDGF] expression + phosphatase and tensin homolog loss) where a portion of the tumor burden is irradiated (PDGF) and another unirradiated luciferase-expressing tumor (PDGF + luciferase) is used as a readout of the abscopal effect following systemic anti-PD-L1 immunotherapy. We assessed relevance of tumor neoepitope during the abscopal response by inducing expression of epidermal growth factor receptor variant III (EGFRvIII) (PDGF + EGFRvIII). Statistical tests were two-sided. Following radiation of one lesion, anti-PD-L1 immunotherapy enhanced the abscopal response to the unirradiated lesion. In PDGF-driven gliomas without tumor neoepitope (PDGF + luciferase, n = 8), the abscopal response occurred via anti-PD-L1 driven, extracellular signal-regulated kinase-mediated, bone marrow-derived macrophage phagocytosis of adjacent unirradiated tumor cells, with modest survival implications (median survival 41 days vs radiation alone 37.5 days, P = 0.03). In PDGF-driven gliomas with tumor neoepitope (PDGF + EGFRvIII, n = 8), anti-PD-L1 enhanced abscopal response was associated with macrophage and T-cell infiltration and increased survival benefit (median survival 36 days vs radiation alone 28 days, P = 0.001). Our results indicate that anti-PD-L1 immunotherapy enhances a radiation- induced abscopal response via canonical T-cell activation and direct macrophage activation in glioblastoma.

Bioactive Polysaccharide Nanoparticles Improve Radiation-Induced Abscopal Effect through Manipulation of Dendritic Cells.

Radiotherapy was considered to induce an abscopal effect initiated through antigen release and presented by dendritic cells (DC), while the immunosuppressive tumor microenvironment (TEM) attenuated the effects. Herein, we utilized bioactive polysaccharides extracted from the natural herb Astragalus membranaceus and developed polysaccharide nanoparticles (ANPs) that can reverse TEM and, accordingly, enhance the radiation-induced abscopal effect. ANP showed ability to prolong the survival rate of tumor-bearing mice. In addition, ANP dramatically inhibited the growth of the primary tumor subjected to radiation as well as the secondary tumor distant from the primary lesion. Mechanistic study demonstrated that an ANP-induced immune response was mainly reflected by DC activation, represented by phenotypic maturation and enhanced antigen presentation through the TLR4 signaling pathway. Mature DC induced by ANP migrated to the tumor-draining lymph node and initiated T-cell expansion. Specifically, DC activation was successfully translated into an increase in CD4+ T/Treg and CD8+ T/Treg ratios within both primary (irradiated) and secondary (unirradiated) tumors. Our results also indicated that the systemic antitumor immune response and immune memory were enhanced with the increase in IFN-γ production and effector memory T-cell population. Our work provided a novel strategy to facilitate the incorporation of immunoactive macromolecules purified from natural herbs into modern nanotechnology in the era of immunotherapy.

Tumor-reprogrammed resident T cells resist radiation to control tumors

Successful combinations of radiotherapy and immunotherapy depend on the presence of live T cells within the tumor; however, radiotherapy is believed to damage T cells. Here, based on longitudinal in vivo imaging and functional analysis, we report that a large proportion of T cells survive clinically relevant doses of radiation and show increased motility, and higher production of interferon gamma, compared with T cells from unirradiated tumors. Irradiated intratumoral T cells can mediate tumor control without newly-infiltrating T cells. Transcriptomic analysis suggests T cell reprogramming in the tumor microenvironment and similarities with tissue-resident memory T cells, which are more radio-resistant than circulating/lymphoid tissue T cells. TGFβ is a key upstream regulator of T cell reprogramming and contributes to intratumoral Tcell radio-resistance. These findings have implications for the design of radio-immunotherapy trials in that local irradiation is not inherently immunosuppressive, and irradiation of multiple tumors might optimize systemic effects of radiotherapy.

Abstract 526: Tumor-draining lymph node irradiation reduces tumor-infiltrating stem-like CD8+T-cells and abrogates the abscopal effect

Abstract Purpose: PD-1 and PD-L1 antagonists are efficacious because cancer induces T-cell exhaustion via upregulation of PD-L1 and persistent antigen exposure. Our lab, has shown that the proliferative burst following αPD-L1 therapy of exhausted CD8+PD-1+T-cells is restricted to a “stem-like” CD8+T-cell subset in a murine chronic viral infection model. The role of these stem-like CD8+T-cells in malignancies and whether these cells respond to other immuno-stimulation including radiotherapy (RT) is unknown. RT’s immuno-stimulation includes acting as an in-situ vaccine by liberating cryptic tumor neo-antigens, generating a potent anti-tumor CD8+T-cell response, synergizing with αPD-1/L1 and leading to control at distant sites of disease outside the radiation field (abscopal effect). In initial studies, we found these stem-like CD8+T-cells in B16F10 tumors and at high frequencies in the tumor-draining lymph nodes (TDLN) of immunocompetent mice. Here, we investigated: (1) the impact of tumor-directed RT on this stem-like CD8+T-cell population in the tumor and TDLN in the context of an abscopal response, and (2) due to the high frequency of stem-like CD8+T-cells in the TDLN, whether depletion of lymphocytes from the TDLN via RT influenced the abscopal effect. Experimental Design: We developed a preclinical model of oligo-metastatic melanoma to evaluate the role of stem-like CD8+ T-cells and the TDLN in the abscopal effect. This was done with unilateral tumor-directed RT with or without TDLN-directed RT. This was also done in the presence and absence of αPD-L1. Results: Tumor-directed RT improved local tumor control and induced an abscopal response with a concomitant increase in tumor infiltrating tumor-specific stem-like CD8+T-cells. Tumor-specific stem-like CD8+T-cells were also observed in the TDLN of tumors on both sides. Importantly, the tumor-directed RT increased tumor-specific T-cell proliferation in the TDLNs bilaterally despite only being targeted at one tumor. Given this robust proliferative response and the high frequency of stem-like CD8+T-cells in the TDLN, we next evaluated the role of the TDLN in mediating the abscopal effect. We found that the abscopal effect is impaired if the TDLN is lymphocyte depleted with TDLN-directed RT in the presence or absence of αPD-L1. Additionally, the tumor-directed RT mediated increase in stem-like T-cells in the irradiated and unirradiated tumor was abrogated with TDLN-directed RT. Conclusion: Our results demonstrate slowed distant tumor growth following irradiation of a local site and that this correlates with an increase in tumor-infiltrating stem-like CD8+T-cells which is reduced with TDLN-directed RT. These data suggest a critical role for both the stem-like CD8+T-cells and the TDLN in mediating the abscopal effect. Citation Format: Zachary S. Buchwald, Tahseen H. Nasti, Christiane S. Eberhardt, Andreas Wieland, David Lawson, Walter Curran, Rafi Ahmed, Mohammad K. Khan. Tumor-draining lymph node irradiation reduces tumor-infiltrating stem-like CD8+T-cells and abrogates the abscopal effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 526.

Irradiation increases the immunogenicity of lung cancer cells and irradiation-based tumor cell vaccine elicits tumor-specific T cell responses in vivo.

Background: During the past decades, great efforts have been built to develop lung cancer vaccines. Whole tumor cell lysate (TCL) are ideal sources of antigens for cancer vaccine design, which however have limited efficacy due to insufficient immunogenicity. Recently, radiotherapy has been closely related to immunotherapy. Numerous studies have demonstrated the regulatory effect of irradiation (IR) on tumor immune response.Purpose: To explore the immunogenicity modulation effect of IR on lung cancer cells.Methods: RNA-sequence and qPCR assay was used to evaluate the change of tumor antigens expression after repeated X rays radiation on A549 cells. Vaccine based on TCL of irradiated Lewis lung cancer cells (IR-LLC) was established; therapeutic effect of TCL (IR-LLC) was examined in xenografted tumor model of mice. Flow cytometry was conducted to evaluate the rate of immune cells in spleen; ELISA was used to detect the level of cytokines in plasma. Immunohistochemistry was performed to evaluate the infiltrations of T-cell in tumor tissues; TIMER analysis was used to explore the correlations between tumor antigen expressions and the abundances of immune infiltrates.Results: IR upregulated the expression of tumor antigens in A549 cells. Compared to the control group and unirradiated tumor cell vaccine, TCL(IR-LLC) had a significantly stronger anti-tumor effect in mice bearing with LLC xenografts. TCL(IR-LLC) significantly increased matured DCs and total CD4+ T cells but downregulated Tregs and PD-1+ CD8+ T cells in mice spleen; TCL(IR-LLC) vaccine upregulated the level of IFN-γ and IL-4 while decreased IL-10 in serum; increased infiltrations of CD4+ T-cells and CD8+ T-cells were observed in the tumor issues of mice immunized with TCL(IR-LLC). Tumor antigens including FN1, MFGE8, MMP2, MYL9 may contribute to the enhanced T-cell response.Conclusion: This study confirmed the immunogenicity modulation effect of IR in NSCLC cells, indicating IR might be an effective strategy to enhance the anti-tumor immunity of cancer cell vaccine.

Carbon ion irradiation enhances the antitumor efficacy of dual immune checkpoint blockade therapy both for local and distant sites in murine osteosarcoma.

Carbon ion radiotherapy has been utilized even for X-ray resistant tumors. However, control of distant metastasis remains a major challenge in carbon ion irradiation. We investigated whether carbon ion irradiation combined with dual immune checkpoint blockade therapy (anti-PD-L1 and anti-CTLA-4 antibodies [P1C4]) provides anti-tumor efficacy for both local and distant sites. A mouse osteosarcoma cell line (LM8) was inoculated into both hind legs of C3H mice assigned to four groups: no treatment (NoTX), P1C4, 5.3 Gy of carbon ion irradiation to one leg (Cion), and combination (Comb) groups. In the Comb group, tumor growth delay was observed not only in the irradiated tumors but also in the unirradiated tumors. Notably, a complete response of unirradiated tumors was observed in 64% of mice in the Comb group, while only 20% of mice in the P1C4 group showed a complete response. Significant activation of immune cells was observed in the Comb group, with an increase in CD8+/GzmB+ tumor-infiltrating lymphocytes (TILs) in the irradiated tumor, and of CD8+/GzmB+ and CD4+ TILs in the unirradiated tumor, respectively. Depletion of CD8 abolished the tumor growth delay in unirradiated tumors in mice treated by Cion and P1C4. Overall survival was significantly prolonged in the Comb group. HMGB-1 release from irradiated tumors was significantly increased after Cion both in vitro and in vivo. These data suggest that carbon ion therapy enhances P1C4 efficacy against osteosarcoma in both the primary tumor and distant metastases mediated by immune activation.

Interim Safety and Toxicity Analysis of a Prospective Phase II Randomized Trial of Checkpoint Blockade Immunotherapy Combined with Stereotactic Body Radiation Therapy in Advanced Metastatic Disease

Checkpoint blockade immunotherapy (CBI) targeting the PD-1 and CTLA-4 pathways is revolutionizing oncology. However, objective response rates for single agent CBI in solid malignancies remain low and combinatorial strategies are required to improve outcomes. Our group has pioneered the use of radiation (RT) and stereotactic body radiation therapy (SBRT) in combination with anti-PD-1 CBI. We previously demonstrated in tumor models that RT+ CBI can enhance antigen specific immune responses and improve local and systemic tumor control. Nevertheless, the clinical benefit of SBRT+CBI remains unclear. Our primary objective was to conduct a clinical trial to test the hypothesis that SBRT + CBI increases objective response rates (ORR) compared to CBI alone. Here we present an interim analysis. We designed and opened an investigator initiated Phase II study (ClinicalTrials.gov NCT02843165) which randomizes patients (1:1) with advanced metastatic disease to receive investigators choice of anti-PD-1/PD-L1 CBI versus anti-PD-1/PD-L1 CBI combined with concurrent SBRT. SBRT was started 1-21 days after C1D1 of CBI and was prescribed at 9.5Gy x 3 fractions delivered to 1-3 non-CNS tumor foci consecutively or every other day. The study is powered to detect a 33% relative improvement in ORR with a total anticipated enrollment of 146. Safety and Toxicity was analyzed prospectively for all patients using CTCAE 4.0. ORR are measured from unirradiated tumors using RECIST 1.1 criteria. Three research blood draws are obtained to analyze immune correlates. We performed an interim analysis of safety and toxicity among all patients and between treatment groups. We have enrolled a total of 35 patients at this time. There were a total of 126 CTCAE toxicity events observed with 67 (53.2%) Low grade (CTCAE 1-2), and 59 (46.8%) High grade (CTCAE 3-5) events with a mean followup of 5.1 months. The most common toxicities were fatigue, nausea, pain, and rash. The most common SBRT treatment sites were lung, liver, adrenal gland, and soft tissue. Experimental treatment attributions are as follows: 18 events (14.3%) Unrelated, 44 events (34.9%) Unlikely, 48 events (38.1%) Possible, and 13 events (10.3%) Probable or Definite. There were 7 (5.5%) high grade events attributed as probably or definitely related to study: one grade 4 fatigue, one grade 3 weight loss, one grade 3 rash, and two grade 3 electrolyte abnormalities, and two grade 3 acute kidney injury. When comparing between CBI and CBI+SBRT treatment groups there was no significant difference in any grade of toxicity at this time. Our interim analysis suggests that SBRT to lung, liver, adrenal and soft tissues prescribed at 9.5Gy x 3 fractions combined with concurrent anti-PD-1/PD-L1 CBI appears to be safe and well tolerated in patients with advanced metastatic disease. Continued analysis of safety and toxicity, ORR, and immune correlates between randomized treatment groups is ongoing.