Nanoparticle-enhanced proton beam immunoradiotherapy drives immune activation and durable tumor rejection.

The combination of radiation therapy (RT) and immunotherapy has emerged as a promising treatment option in oncology. Historically, x-ray radiation (XRT) has been the most commonly used form of RT. However, proton beam therapy (PBT) is gaining recognition as a viable alternative, as it has been shown to produce similar outcomes to XRT while minimizing off-target effects. The effects of PBT on the antitumor immune response have only just begun to be described, and to our knowledge no studies to date have examined the effect of PBT as part of a combinatorial immunoradiotherapeutic strategy. Here, using a 2-tumor model of lung cancer in mice, we show that PBT in tandem with an anti-PD1 antibody substantially reduced growth in both irradiated and unirradiated tumors. This was accompanied by robust activation of the immune response, as evidenced by whole-tumor and single-cell RNA sequencing showing upregulation of a multitude of immune-related transcripts. This response was further significantly enhanced by the injection of the tumor to be irradiated with NBTXR3 nanoparticles. Tumors of mice treated with the triple combination exhibited increased infiltration and activation of cytotoxic immune cells. This triple combination eradicated both tumors in 37.5% of the treated mice and showed robust long-term immunity to cancer.

NLRP3 agonist enhances radiation-induced immune priming and promotes abscopal responses in anti-PD1 resistant model.

Radiotherapy (XRT), a well-known activator of the inflammasome and immune priming, is in part capable of reversing resistance to anti-PD1 treatment. The NLRP3 inflammasome is a pattern recognition receptor which is activated by both exogenous and endogenous stimuli, leading to a downstream inflammatory response. Although NLRP3 is typically recognized for its role in exacerbating XRT-induced tissue damage, the NLRP3 inflammasome can also yield an effective antitumor response when used in proper dosing and sequencing with XRT. However, whether NLRP3 agonist boosts radiation-induced immune priming and promote abscopal responses in anti-PD1 resistant model is still unknown. Therefore, in this study, we paired intratumoral injection of an NLRP3 agonist with XRT to stimulate the immune system in both wild type (344SQ-P) and anti-PD1 resistant (344SQ-R) murine-implanted lung adenocarcinoma models. We found that the combination of XRT + NLPR3 agonist enhanced the control of implanted lung adenocarcinoma primary as well as secondary tumors in a radiological dose-dependent manner, in which 12Gyx3 fractions of stereotactic XRT was better than 5Gyx3, while 1Gyx2 did not improve the NLRP3 effect. Survival and tumor growth data also showed significant abscopal response with the triple therapy (12Gyx3 + NLRP3 agonist + α-PD1) in both 344SQ-P and 344SQ-R aggressively growing models. Multiple pro-inflammatory cytokines (IL-1b, IL-4, IL-12, IL-17, IFN-γ and GM-CSF) were elevated in the serum of mice treated with XRT + NLRP3 or triple therapy. The Nanostring results showed that NLRP3 agonist is capable of increasing antigen presentation, innate function, and T-cell priming. This study can be of particular importance to treat patients with immunologically-cold solid tumors whom are also refractory to prior checkpoint treatments.

NLRP3 agonist enhances radiation-induced immune priming and promotes abscopal responses in anti-PD1 resistant model.

Radiotherapy (XRT), a well-known activator of the inflammasome and immune priming, is in part capable of reversing resistance to anti-PD1 treatment. Although NLRP3 is typically observed for its role in exacerbating XRT-induced tissue damage, the NLRP3 inflammasome can also be protective and augment the effect of XRT when used in proper dosing and sequencing. However, whether NLRP3 agonist boosts radiation-induced immune priming and promote abscopal responses in anti-PD1 resistant model is still unknown. Therefore, in this study, we paired intratumoral injection of an NLRP3 agonist with XRT to stimulate the immune system in both wild type (344SQ-P) and anti-PD1 resistant (344SQ-R) murine-implanted lung adenocarcinoma models. We found that the combination of XRT + NLPR3 agonist enhanced control of implanted lung adenocarcinoma primary as well as secondary tumors in a radiological dose-dependent manner, in which 12Gy x 3 fractions of stereotactic XRT was better than 5Gy x 3, while 1Gy x 2 did not improve the NLRP3 effect. Survival and tumor growth data also showed significant abscopal response with the triple therapy (12Gyx3 + NLRP3 agonist + α-PD1) in both 344SQ-P and 344SQ-R aggressively growing models. Multiple pro-inflammatory cytokines (IL-1b, IL-4, IL-12, IL-17, IFN-γ and GM-CSF) were elevated in the serum of mice treated with XRT + NLRP3 or triple therapy. The Nanostring results showed that NLRP3 agonist is capable of increasing antigen presentation, innate function, and T-cell priming. This study can be of particular importance to treat patients with immunologically-cold solid tumors whom are also refractory to prior checkpoint treatments.

Radiation Therapy Modulates Tumor Physical Characteristics to Reduce Intratumoral Pressure and Enhance Intratumoral Drug Delivery and Retention.

Purpose: High intratumoral pressure, caused by tumor cell-to-cell interactions, interstitial fluid pressure, and surrounding stromal composition, plays a substantial role in resistance to intratumoral drug delivery and distribution. Radiation therapy (XRT) is commonly administered in conjunction with different intratumoral drugs, but assessing how radiation can reduce pressure locally and help intratumoral drug administration and retention is important. Methods and Materials: 344SQ-parental or 344SQ-anti-programmed cell death protein 1-resistant lung adenocarcinoma cells were established in 129Sv/Ev mice, and irradiated with either 1 Gy × 2, 5 Gy × 3, 8 Gy × 3, 12 Gy × 3, or 20 Gy × 1. Intratumoral pressure was measured every 3 to 4 days after XRT. Contrast dye was injected into the tumors 3- and 6-days after XRT, and imaged to measure drug retention. Results: In the 344SQ-parental model, low-dose radiation (1 Gy × 2) created an early window of reduced intratumoral pressure 1 to 3 days after XRT compared with untreated control. High-dose stereotactic radiation (12 Gy × 3) reduced intratumoral pressure 3 to 12 days after XRT, and 20 Gy × 1 showed a delayed pressure reduction on day 12. Intermediate doses of radiation did not significantly affect intratumoral pressure. In the more aggressive 344SQ-anti-programmed cell death protein 1-resistant model, low-dose radiation reduced pressure 1 to 5 days after XRT, and 12 Gy × 3 reduced pressure 1 to 3 days after XRT. Moreover, both 1 Gy × 2 and 12 Gy × 3 significantly improved drug retention 3 days after XRT; however, there was no significance detected 6 days after XRT. Lastly, a histopathologic evaluation showed that 1 Gy × 2 reduced collagen deposition within the tumor, and 12 Gy × 3 led to more necrotic core and higher extracellular matrix formation in the tumor periphery. Conclusions: Optimized low-dose XRT, as well as higher stereotactic XRT regimen led to a reduction in intratumoral pressure and increased drug retention. The findings from this work can be readily translated into the clinic to enhance intratumoral injections of various anticancer agents.

Systemic AAV9.BVES delivery ameliorates muscular dystrophy in a mouse model of LGMDR25.

Limb-girdle muscular dystrophy type R25 (LGMDR25) is caused by recessive mutations in BVES encoding a cAMP-binding protein, characterized by progressive muscular dystrophy with deteriorating muscle function and impaired cardiac conduction in patients. There is currently no therapeutic treatment for LGMDR25 patients. Here we report the efficacy and safety of recombinant adeno-associated virus 9 (AAV9)-mediated systemic delivery of human BVES driven by a muscle-specific promoter MHCK7 (AAV9.BVES) in BVES-knockout (BVES-KO) mice. AAV9.BVES efficiently transduced the cardiac and skeletal muscle tissues when intraperitoneally injected into neonatal BVES-KO mice. AAV9.BVES dramatically improved body weight gain, muscle mass, muscle strength, and exercise performance in BVES-KO mice regardless of sex. AAV9.BVES also significantly ameliorated the histopathological features of muscular dystrophy. The heart rate reduction was also normalized in BVES-KO mice under exercise-induced stress following systemic AAV9.BVES delivery. Moreover, intravenous AAV9.BVES administration into adult BVES-KO mice after the disease onset also resulted in substantial improvement in body weight, muscle mass, muscle contractility, and stress-induced heart rhythm abnormality. No obvious toxicity was detected. Taken together, these results provide the proof-of-concept evidence to support the AAV9.BVES gene therapy for LGMDR25.

Bone morphogenetic proteins, activins, and growth and differentiation factors in tumor immunology and immunotherapy resistance.

The TGF-ß superfamily is a group of secreted polypeptides with key roles in exerting and regulating a variety of physiologic effects, especially those related to cell signaling, growth, development, and differentiation. Although its central member, TGF-ß, has been extensively reviewed, other members of the family-namely bone morphogenetic proteins (BMPs), activins, and growth and differentiation factors (GDFs)-have not been as thoroughly investigated. Moreover, although the specific roles of TGF-ß signaling in cancer immunology and immunotherapy resistance have been extensively reported, little is known of the roles of BMPs, activins, and GDFs in these domains. This review focuses on how these superfamily members influence key immune cells in cancer progression and resistance to treatment.

NBTXR3 improves the efficacy of immunoradiotherapy combining nonfucosylated anti-CTLA4 in an anti-PD1 resistant lung cancer model.

The efficacy of immunoradiotherapy consisting of radiation therapy and immune checkpoint blockade relies on effectively promoting the systemic antitumor immune response's activation while simultaneously reducing local factors favoring immune suppression. We previously demonstrated that NBTXR3, a nanoparticle radioenhancer, significantly improved immune responses in a murine anti-PD1-resistant metastatic lung cancer model. We hypothesize that radioactivated-NBTXR3 addition to anti-PD1 and a second-generation anti-CTLA4 could improve treatment effectiveness. To test this hypothesis, we inoculated mice with 344SQR cells in the right and left legs to establish primary and secondary tumors. The primary tumors were intratumorally injected with NBTXR3 nanoparticles on day 7, followed by three fractions of 12 Gy radiation on days 8, 9, and 10. The secondary tumors received two fractions of 1Gy radiation on days 13 and 14. Multiple rounds of anti-PD1, anti-CTLA4 or nonfucosylated anti-CTLA4 were given to the mice. Immune profiling of the tumors revealed that the combination of NBTXR3 with immunoradiotherapy significantly upregulated the activities of a wide range of antitumor immune pathways and reduced the abundance of regulatory suppressor T cells. This combination effectively eradicated the primary and secondary tumors and increased animal survival to 75%. Remarkably, previously treated with NBTXR3-containing treatment, the survivor mice exhibited a long-lasting antitumor memory immune response. This data provides compelling evidence of the efficacy of NBTXR3 to synergize with the immunoradiotherapy approach when combined with an anti-PD1 and multiple checkpoints such as a second generation anti-CTLA4 and show the potential for clinical uses of antitumor immunomodulatory effects of NBTXR3.

Pulsed radiotherapy to mitigate high tumor burden and generate immune memory.

Radiation therapy (XRT) has a well-established role in cancer treatment. Given the encouraging results on immunostimulatory effects, radiation has been increasingly used with immune-check-point inhibitors in metastatic disease, especially when immunotherapy fails due to tumor immune evasion. We hypothesized that using high-dose stereotactic radiation in cycles (pulses) would increase T-cell priming and repertoire with each pulse and build immune memory in an incremental manner. To prove this hypothesis, we studied the combination of anti-CTLA-4 and Pulsed radiation therapy in our 344SQ non-small cell lung adenocarcinoma murine model. Primary and secondary tumors were bilaterally implanted in 129Sv/Ev mice. In the Pulsed XRT group, both primary and secondary tumors received 12Gyx2 radiation one week apart, and blood was collected seven days afterwards for TCR repertoire analysis. As for the delayed-Pulse group, primary tumors received 12Gyx2, and after a window of two weeks, the secondary tumors received 12Gyx2. Blood was collected seven days after the second cycle of radiation. The immunotherapy backbone for both groups was anti-CTLA-4 antibody to help with priming. Treatment with Pulsed XRT + anti-CTLA-4 led to significantly improved survival and resulted in a delayed tumor growth, where we observed enhanced antitumor efficacy at primary tumor sites beyond XRT + anti-CTLA-4 treatment group. More importantly, Pulsed XRT treatment led to increased CD4+ effector memory compared to single-cycle XRT. Pulsed XRT demonstrated superior efficacy to XRT in driving antitumor effects that were largely dependent on CD4+ T cells and partially dependent on CD8+ T cells. These results suggest that combinatorial strategies targeting multiple points of tumor immune evasion may lead to a robust and sustained antitumor response.

High Plus Low Dose Radiation Strategy in Combination with TIGIT and PD1 Blockade to Promote Systemic Antitumor Responses.

Tumors deploy various immune-evasion mechanisms that create a suppressive environment and render effector T-cells exhausted and inactive. Therefore, a rational utilization of checkpoint inhibitors may alleviate exhaustion and may partially restore antitumor functions. However, in high-tumor-burden models, the checkpoint blockade fails to maintain optimal efficacy, and other interventions are necessary to overcome the inhibitory tumor stroma. One such strategy is the use of radiotherapy to reset the tumor microenvironment and maximize systemic antitumor outcomes. In this study, we propose the use of anti-PD1 and anti-TIGIT checkpoint inhibitors in conjunction with our novel RadScopal technique to battle highly metastatic lung adenocarcinoma tumors, bilaterally established in 129Sv/Ev mice, to mimic high-tumor-burden settings. The RadScopal approach is comprised of high-dose radiation directed at primary tumors with low-dose radiation delivered to secondary tumors to improve the outcomes of systemic immunotherapy. Indeed, the triple therapy with RadScopal + anti-TIGIT + anti-PD1 was able to prolong the survival of treated mice and halted the growth of both primary and secondary tumors. Lung metastasis counts were also significantly reduced. In addition, the low-dose radiation component reduced TIGIT receptor (PVR) expression by tumor-associated macrophages and dendritic cells in secondary tumors. Finally, low-dose radiation within triple therapy decreased the percentages of TIGIT+ exhausted T-cells and TIGIT+ regulatory T-cells. Together, our translational approach provides a new treatment alternative for cases refractory to other checkpoints and may bring immunotherapy into a new realm of systemic disease control.

Pulsed Radiation Therapy to Improve Systemic Control of Metastatic Cancer.

Radiation therapy (RT) is emerging as an interventional modality in the cancer-immunity cycle, augmenting the activation of an adaptive immune response against tumors. RT, particularly in combination with immunotherapy, can enhance immune memory effects and shape the tumor-directed T-cell populations. However, a single cycle of RT delivered to a limited number of polymetastatic lesions is rarely sufficient to achieve systemic control. We hypothesize that several rounds of RT, akin to several rounds of immunotherapeutic drugs, is likely to provide greater clinical benefit to patients with metastatic disease. We propose that the repeated exposure to tumor antigens released by "pulsed-RT" (i.e., treating 2-4 tumor lesions with 3 irradiation cycles given one month apart) may amplify the adaptive immune response by expanding the tumor-specific T-cell receptor repertoire, the production of high-affinity tumor antibodies, and the generation of memory lymphocytes and thereby improve immune control of systemic disease.

Novel Use of Low-Dose Radiotherapy to Modulate the Tumor Microenvironment of Liver Metastases.

Despite multiple therapeutic approaches, the presence of liver metastases carries a guarded prognosis, urgently necessitating further clinical and scientific research to develop curative interventions. The liver is an immunoprivileged organ that suppresses the effectiveness of immunotherapies in patients with hepatic metastases. Cancer immunotherapies have been successfully bolstered by low-dose radiotherapy (LDRT), which is capable of reprogramming the tumor microenvironment (TME) from an immunosuppressive to an immunostimulatory one. Likewise, LDRT may be able to revoke the immune privilege enjoyed by the liver, permitting successful immunotherapies there. Here, we first review challenges that face the treatment of liver metastases. We next outline emerging preclinical and clinical evidence supporting enhanced systemic tumor control of LDRT in the context of cancer immunotherapy. Finally, we will discuss the rationale of combining liver-directed LDRT with immunostimulatory strategies to overcome immune resistance and achieve better clinical response. This notion is supported by a recent case study in which a patient who had progressed following T cell therapy experienced a complete response after LDRT to the liver.