Local Delivery of SBRT and IL12 by mRNA Technology Overcomes Immunosuppressive Barriers to Eliminate Pancreatic Cancer.

The immunosuppressive milieu in pancreatic cancer (PC) is a significant hurdle to treatments, resulting in survival statistics that have barely changed in 5 decades. Here we present a combination treatment consisting of stereotactic body radiation therapy (SBRT) and IL-12 mRNA lipid nanoparticles delivered directly to pancreatic murine tumors. This treatment was effective against primary and metastatic models, achieving cures in both settings. IL-12 protein concentrations were transient and localized primarily to the tumor. Depleting CD4 and CD8 T cells abrogated treatment efficacy, confirming they were essential to treatment response. Single cell RNA sequencing from SBRT/IL-12 mRNA treated tumors demonstrated not only a complete loss of T cell exhaustion, but also an abundance of highly proliferative and effector T cell subtypes. SBRT elicited T cell receptor clonal expansion, whereas IL-12 licensed these cells with effector function. This is the first report demonstrating the utility of SBRT and IL-12 mRNA in PC. Statement of significance: This study demonstrates the use of a novel combination treatment consisting of radiation and immunotherapy in murine pancreatic tumors. This treatment could effectively treat local and metastatic disease, suggesting it may have the potential to treat a cancer that has not seen a meaningful increase in survival in 5 decades.

New insights into the responder/nonresponder divide in rectal cancer: Damage-induced Type I IFNs dictate treatment efficacy and can be targeted to enhance radiotherapy.

Rectal cancer ranks as the second leading cause of cancer-related deaths. Neoadjuvant therapy for rectal cancer patients often results in individuals that respond well to therapy and those that respond poorly, requiring life-altering excision surgery. It is inadequately understood what dictates this responder/nonresponder divide. Our major aim is to identify what factors in the tumor microenvironment drive a fraction of rectal cancer patients to respond to radiotherapy. We also sought to distinguish potential biomarkers that would indicate a positive response to therapy and design combinatorial therapeutics to enhance radiotherapy efficacy. To address this, we developed an orthotopic murine model of rectal cancer treated with short course radiotherapy that recapitulates the bimodal response observed in the clinic. We utilized a robust combination of transcriptomics and protein analysis to identify differences between responding and nonresponding tumors. Our mouse model recapitulates human disease in which a fraction of tumors respond to radiotherapy (responders) while the majority are nonresponsive. We determined that responding tumors had increased damage-induced cell death, and a unique immune-activation signature associated with tumor-associated macrophages, cancer-associated fibroblasts, and CD8+ T cells. This signature was dependent on radiation-induced increases of Type I Interferons (IFNs). We investigated a therapeutic approach targeting the cGAS/STING pathway and demonstrated improved response rate following radiotherapy. These results suggest that modulating the Type I IFN pathway has the potential to improve radiation therapy efficacy in RC.

CD73 and PD-L1 dual blockade amplifies antitumor efficacy of SBRT in murine PDAC models.

BACKGROUND: Stereotactic body radiotherapy (SBRT) induces immunogenic cell death, leading to subsequent antitumor immune response that is in part counterbalanced by activation of immune evasive processes, for example, upregulation of programmed cell death-ligand 1 (PD-L1) and adenosine generating enzyme, CD73. CD73 is upregulated in pancreatic ductal adenocarcinoma (PDAC) compared with normal pancreatic tissue and high expression of CD73 in PDACs is associated with increased tumor size, advanced stage, lymph node involvement, metastasis, PD-L1 expression and poor prognosis. Therefore, we hypothesized that blockade of both CD73 and PD-L1 in combination with SBRT might improve antitumor efficacy in an orthotopic murine PDAC model. METHODS: We assessed the combination of systemic blockade of CD73/PD-L1 and local SBRT on tumor growth in primary pancreatic tumors, and investigated systemic antitumor immunity using a metastatic murine model bearing both orthotopic primary pancreatic tumor and distal hepatic metastases. Immune response was quantified by flow cytometric and Luminex analyses. RESULTS: We demonstrated that blockade of both CD73 and PD-L1 significantly amplified the antitumor effect of SBRT, leading to superior survival. The triple therapy (SBRT+anti-CD73+anti-PD-L1) modulated tumor-infiltrating immune cells with increases of interferon-γ+CD8+ T cells. Additionally, triple therapy reprogramed the profile of cytokines/chemokines in the tumor microenvironment toward a more immunostimulatory phenotype. The beneficial effects of triple therapy are completely abrogated by depletion of CD8+ T cells, and partially reversed by depletion of CD4+ T cells. Triple therapy promoted systemic antitumor responses illustrated by: (1) potent long-term antitumor memory and (2) enhanced both primary and liver metastases control along with prolonged survival.

New insights into the responder/nonresponder divide in rectal cancer: Damage-induced Type I IFNs dictate treatment efficacy and can be targeted to enhance radiotherapy.

Rectal cancer ranks as the second leading cause of cancer-related deaths. Neoadjuvant therapy for rectal cancer patients often results in individuals that respond well to therapy and those that respond poorly, requiring life-altering excision surgery. It is inadequately understood what dictates this responder/nonresponder divide. Our major aim is to identify what factors in the tumor microenvironment drive a fraction of rectal cancer patients to respond to radiotherapy. We also sought to distinguish potential biomarkers that would indicate a positive response to therapy and design combinatorial therapeutics to enhance radiotherapy efficacy. To address this, we developed an orthotopic murine model of rectal cancer treated with short course radiotherapy that recapitulates the bimodal response observed in the clinic. We utilized a robust combination of transcriptomics and protein analysis to identify differences between responding and nonresponding tumors. Our mouse model recapitulates human disease in which a fraction of tumors respond to radiotherapy (responders) while the majority are nonresponsive. We determined that responding tumors had increased damage-induced cell death, and a unique immune-activation signature associated with tumor-associated macrophages, cancer-associated fibroblasts, and CD8 + T cells. This signature was dependent on radiation-induced increases of Type I interferons (IFNs). We investigated a therapeutic approach targeting the cGAS/STING pathway and demonstrated improved response rate following radiotherapy. These results suggest that modulating the Type I IFN pathway has the potential to improve radiation therapy efficacy in RC.

Radiation Therapy Exacerbates Tumor-Promoting Innervation and Nerve Signaling in Rectal Cancer.

PURPOSE: Many solid tumors present with perineural invasion (PNI), and innervation correlates with worsened prognosis. The effects that commonly administered therapies such as radiation therapy (RT) have on PNI status remain unknown. We investigated the contribution of RT on the nervous system and elucidated the implications that increased nerve signaling can have on tumor burden using our previously developed orthotopic murine model of rectal cancer (RC) and our targeted and clinically relevant short-course RT (SCRT) regimen. METHODS: Medical charts for patients with RC treated at the Wilmot Cancer Institute were obtained and PNI status was analyzed. Human data were accompanied by an orthotopic murine model of RC. Briefly, luciferase-expressing murine colon-38 (MC38-luc) tumor cells were injected orthotopically into the rectal wall of C57BL6 mice. Targeted SCRT (5 gray (Gy) per fraction for 5 consecutive fractions) was administered to the tumor. Intratumoral innervation was determined by immunohistochemistry (IHC), local norepinephrine (NE) concentration was quantified by enzyme-linked immunosorbent assay (ELISA), and ß2-adrenergic receptor (B2AR) expression was assessed by flow cytometry. Chronic NE signaling was mirrored by daily isoproterenol treatment, and the effect on tumor burden was determined by overall survival, presence of metastatic lesions, and tumor size. Isoproterenol signaling was inhibited by administration of propranolol. RESULTS: Human RC patients with PNI have decreased overall survival compared with patients without PNI. In our mouse model, SCRT induced the expression of genes involved in neurogenesis, increased local NE secretion, and upregulated B2AR expression. Treating mice with isoproterenol resulted in decreased overall survival, increased rate of metastasis, and reduced SCRT efficacy. Interestingly, the isoproterenol-induced decrease in SCRT efficacy could be abrogated by blocking the BAR through the use of propranolol, suggesting a direct role of BAR stimulation on impairing SCRT responses. CONCLUSIONS: Our results indicate that while SCRT is a valuable treatment, it is accompanied by adverse effects on the nervous system that may impede the efficacy of therapy and promote tumor burden. Therefore, we could speculate that therapies aimed at targeting this signaling cascade or impairing nerve growth in combination with SCRT may prove beneficial in future cancer treatment.

Combination of NKG2A and PD-1 Blockade Improves Radiotherapy Response in Radioresistant Tumors.

Radiotherapy (RT) is commonly employed to treat solid tumors. Immune checkpoint blockade of programmed cell death protein 1 (PD-1) and CTLA-4 improves survival in RT patients, yet many fail to respond to combination therapy. Natural killer group 2 (NKG2) family receptors, particularly inhibitory NKG2A and activating NKG2D, have emerged as promising therapeutic targets to improve antitumor T cell responses; thus, we examined how these receptors and their ligands (Qa-1b and retinoic acid early inducible 1 [Rae-1], respectively) regulate the RT response in C57BL/6 mice bearing syngeneic B16F10 melanoma and MC38 colorectal adenocarcinoma tumors. RT (15 Gy) transiently reduced B16F10 tumor burden, whereas MC38 tumors exhibited durable response to RT. Intratumoral NK and CD8 T cells expressed NKG2A and NKG2D in both models, which was unaltered by RT. In vitro/in vivo RT increased tumor/stromal cell Qa-1b and Rae-1 expression in both models, especially B16F10 tumors, but IFN-γ stimulation induced both Qa-1b and Rae-1 only in B16F10 tumors. NKG2A/Qa-1b inhibition alone did not improve RT response in either model, but combined RT and NKG2A/PD-1 blockade improved survival in the B16F10 model. Depletion experiments indicate that the triple therapy efficacy is CD8 T cell-dependent with negligible NK cell contribution. RNA sequencing of CD8 T cells from triple therapy-treated B16F10 tumors showed increased proliferative capacity compared with RT and PD-1 blockade alone. Our work demonstrates that RT modulates NKG2A ligand expression, which inhibits RT-induced T cell responses in tumors that fail to respond to combined RT and PD-1 blockade. These results provide a rationale for combining NKG2A blockade with immune checkpoint blockade therapies and RT to improve clinical response.