Shear wave elastography can stratify rectal cancer response to short-course radiation therapy.

Rectal cancer is a deadly disease typically treated using neoadjuvant chemoradiotherapy followed by total mesorectal excision surgery. To reduce the occurrence of mesorectal excision surgery for patients whose tumors regress from the neoadjuvant therapy alone, conventional imaging, such as computed tomography (CT) or magnetic resonance imaging (MRI), is used to assess tumor response to neoadjuvant therapy before surgery. In this work, we hypothesize that shear wave elastography offers valuable insights into tumor response to short-course radiation therapy (SCRT)-information that could help distinguish radiation-responsive from radiation-non-responsive tumors and shed light on changes in the tumor microenvironment that may affect radiation response. To test this hypothesis, we performed elastographic imaging on murine rectal tumors (n = 32) on days 6, 10, 12, 16, 18, 20, 23, and 25 post-tumor cell injection. The study revealed that radiation-responsive and non-radiation-responsive tumors had different mechanical properties. Specifically, radiation-non-responsive tumors showed significantly higher shear wave speed SWS (p < 0.01) than radiation-responsive tumors 11 days after SCRT. Furthermore, there was a significant difference in shear wave attenuation (SWA) (p < 0.01) in radiation-non-responsive tumors 16 days after SCRT compared to SWA measured just one day after SCRT. These results demonstrate the potential of shear wave elastography to provide valuable insights into tumor response to SCRT and aid in exploring the underlying biology that drives tumors' responses to radiation.

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.

Toll-like receptor 7/8 agonist R848 alters the immune tumor microenvironment and enhances SBRT-induced antitumor efficacy in murine models of pancreatic cancer.

BACKGROUND: Stereotactic body radiotherapy (SBRT) has been increasingly used as adjuvant therapy in pancreatic ductal adenocarcinoma (PDAC), and induces immunogenic cell death, which leads to the release of tumor antigen and damage-associated molecular patterns. However, this induction often fails to generate sufficient response to overcome pre-existing tumor microenvironment (TME) immunosuppression. Toll-like receptor (TLR) 7/8 ligands, such as R848, can amplify the effect of tumor vaccines, with recent evidence showing its antitumor effect in pancreatic cancer by modulating the immunosuppressive TME. Therefore, we hypothesized that the combination of R848 and SBRT would improve local and systemic antitumor immune responses by potentiating the antitumor effects of SBRT and reversing the immunosuppressive nature of the PDAC TME. METHODS: Using murine models of orthotopic PDAC, we assessed the combination of intravenous TLR7/8 agonist R848 and local SBRT on tumor growth and immune response in primary pancreatic tumors. Additionally, we employed a hepatic metastatic model to investigate if the combination of SBRT targeting only the primary pancreatic tumor and systemic R848 is effective in controlling established liver metastases. RESULTS: We demonstrated that intravenous administration of the TLR7/8 agonist R848, in combination with local SBRT, leads to superior tumor control compared with either treatment alone. The combination of R848 and SBRT results in significant immune activation of the pancreatic TME, including increased tumor antigen-specific CD8+ T cells, decreased regulatory T cells, and enhanced antigen-presenting cells maturation, as well as increased interferon gamma, granzyme B, and CCL5 along with decreased levels of interleukin 4 (IL-4), IL-6, and IL-10. Importantly, the combination of SBRT and systemic R848 also resulted in similar immunostimulatory changes in liver metastases, leading to improved metastatic control. CD8+ T cell depletion studies highlighted the necessity of these effector cells at both the local and hepatic metastatic sites. T cell receptor (TCR) clonotype analysis indicated that systemic R848 not only diversified the TCR repertoire but also conditioned the metastatic foci to facilitate entry of CD8+ T cells generated by SBRT therapy. CONCLUSIONS: These findings suggest that systemic administration of TLR7/8 agonists in combination with SBRT may be a promising avenue for metastatic PDAC 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.

Bladder Cancer Extracellular Vesicles Elicit a CD8 T Cell-Mediated Antitumor Immunity.

Tumor-derived extracellular vesicles (TEVs) play crucial roles in mediating immune responses, as they carry and present functional MHC-peptide complexes that enable them to modulate antigen-specific CD8+ T-cell responses. However, the therapeutic potential and immunogenicity of TEV-based therapies against bladder cancer (BC) have not yet been tested. Here, we demonstrated that priming with immunogenic Extracellular Vesicles (EVs) derived from murine MB49 BC cells was sufficient to prevent MB49 tumor growth in mice. Importantly, antibody-mediated CD8+ T-cell depletion diminished the protective effect of MB49 EVs, suggesting that MB49 EVs elicit cytotoxic CD8+ T-cell-mediated protection against MB49 tumor growth. Such antitumor activity may be augmented by TEV-enhanced immune cell infiltration into the tumors. Interestingly, MB49 EV priming was unable to completely prevent, but significantly delayed, unrelated syngeneic murine colon MC-38 tumor growth. Cytokine array analyses revealed that MB49 EVs were enriched with pro-inflammatory factors that might contribute to increasing tumor-infiltrating immune cells in EV-primed MC-38 tumors. These results support the potential application of TEVs in personalized medicine, and open new avenues for the development of adjuvant therapies based on patient-derived EVs aimed at preventing disease progression.

Development of an Orthotopic Murine Model of Rectal Cancer in Conjunction With Targeted Short-Course Radiation Therapy.

PURPOSE: Orthotopic tumors more closely recapitulate human cancers than do ectopic models; however, precision targeting of such internal tumors for radiation therapy (RT) without inducing systemic toxicity remains a barrier. We developed an innovative murine orthotopic rectal tumor model where the insertion of clinical grade titanium fiducial clips on opposing sides of the rectal tumor allowed for targeted administration of short-course radiation therapy (SCRT). With this novel approach, clinically relevant RT regimens can be administered to orthotopic tumors to explore the biology and efficacy of radiation alone or as a combination therapy in a murine model that closely recapitulates human disease. METHODS AND MATERIALS: Murine Colon 38-luciferase tumor cells were injected into the rectal wall of syngeneic mice, and fiducial clips were applied to demarcate the tumor. An SCRT regimen consisting of 5 consecutive daily doses of 5 Gy delivered by an image-guided conformal small animal irradiator was administered 9 days after implantation. Tumor burden and survival were monitored along with histological and flow cytometric analyses on irradiated versus untreated tumors at various time points. RESULTS: SCRT administered to orthotopic rectal tumors resulted in a reduction in tumor burden and enhanced overall survival with no apparent signs of systemic toxicity. This treatment paradigm resulted in significant reductions in tumor cellularity and increases in fibrosis and hyaluronic acid production, recapitulating the SCRT-induced effects observed in human cancers. CONCLUSIONS: We have established a means to target murine orthotopic rectal tumors using fiducial markers with a fractionated and clinically relevant SCRT schedule that results in an RT response similar to what is observed in human rectal cancer. We also validated our model through examining various parameters associated with human cancer that are influenced by irradiation. This model can be used to further explore RT doses and scheduling, and to test combinatorial therapies.

Modulation of the Human Pancreatic Ductal Adenocarcinoma Immune Microenvironment by Stereotactic Body Radiotherapy.

PURPOSE: Stereotactic body radiotherapy (SBRT) is an emerging treatment modality for pancreatic ductal adenocarcinoma (PDAC), which can effectively prime cytotoxic T cells by inducing immunogenic tumor cell death in preclinical models. SBRT effects on human PDAC have yet to be thoroughly investigated; therefore, this study aimed to characterize immunomodulation in the human PDAC tumor microenvironment following therapy. EXPERIMENTAL DESIGN: Tumor samples were obtained from patients with resectable PDAC. Radiotherapy was delivered a median of 7 days prior to surgical resection, and sections were analyzed by multiplex IHC (mIHC), RNA sequencing, and T-cell receptor sequencing (TCR-seq). RESULTS: Analysis of SBRT-treated tumor tissue indicated reduced tumor cell density and increased immunogenic cell death relative to untreated controls. Radiotherapy promoted collagen deposition; however, vasculature was unaffected and spatial analyses lacked evidence of T-cell sequestration. Conversely, SBRT resulted in fewer tertiary lymphoid structures and failed to lessen or reprogram abundant immune suppressor populations. Higher percentages of PD-1+ T cells were observed following SBRT, and a subset of tumors displayed more clonal T-cell repertoires. CONCLUSIONS: These findings suggest that SBRT augmentation of antitumor immunogenicity may be dampened by an overabundance of refractory immunosuppressive populations, and support the continued development of SBRT/immunotherapy combination for human PDAC.

Coadministration of a Clinically Relevant Dexamethasone Dosage With Ablative Radiation Therapy Reduces Peripheral Lymphocytes But Does Not Alter In Vivo Intratumoral Lymphocyte Phenotype or Inhibit Efficacy of Radiation Therapy in a Murine Colorectal Tumor Model.

PURPOSE: Dexamethasone is commonly given during radiation therapy (RT) to manage toxicities. Our study examines if dexamethasone coadministration with RT inhibits the RT-induced antitumor T cell response in mouse. METHODS AND MATERIALS: Intramuscularly implanted MC38 tumors were irradiated with 15 Gy after establishing for 7 days. Tumor bearing mice were administered dexamethasone using multiple schedules and doses. Peripheral lymphocyte reduction was monitored by complete blood count and intratumoral and tumor draining lymph node (tdLN) populations by flow cytometry. Effector phenotype and function of ex vivo stimulated tumor-infiltrating lymphocytes (TILs) and naïve splenocytes as well as in vivo TILs with or without dexamethasone were monitored by flow cytometry and ELISA. RESULTS: Long course high dose, short course high dose, and short course human equivalent dose dexamethasone reduced peripheral lymphocytes yet did not inhibit survival after irradiation. Short course high dose administration decreased TIL and tdLN lymphocyte activation as well as tdLN mass but did not affect TIL frequencies or change tdLN cell population composition. Dexamethasone inhibited effector function of ex vivo stimulated naïve splenocytes and TILs, but magnitude of IFN-γ secretion was consistently higher in TILs regardless of dexamethasone dose. In vivo analysis of TILs after irradiation and HE dexamethasone treatment showed that TILs had a similar effector phenotype compared with vehicle controls. CONCLUSIONS: Dexamethasone reduces blood and tdLN lymphocytes. Dexamethasone also suppresses TIL activation/effector function yet does not affect survival in irradiated MC38 tumor bearing mice, which depend on RT-induced immune responses for therapy efficacy. Additional study in human subjects is warranted.

Preliminary indications that recombinant human IL-16 attracts and stimulates lymphocytes of the amphibian, Xenopus laevis implying an ancestral role for CD4 as a cytokine receptor.

The D1 domain of the CD4 co-receptor interacts with MHC class II during Helper CD4+ Th-cell activation and effector function in all gnathostomes but the sequence and structure of this region are not well conserved through phylogeny. Conversely, the proximal D4 domain of CD4 is the binding site of the cytokine IL-16 and is highly conserved, allowing for promiscuous binding of IL-16 to CD4 between disparate gnathostomes. We report here that recombinant human IL-16 (rhIL-16) bound to Xenopus lymphocytes to allow separation on a magnetic column. Incubation with rhIL-16 resulted in an increased expression of MHC class II mRNA by Xenopus CD8- cells more than by CD8+ cells. An in vivo assay demonstrated that rhIL-16 can recruit lymphocytes of Xenopus frogs. Our data suggest that a subset of Xenopus laevis lymphocytes express a CD4 homolog on their surface that is capable of binding IL-16. These results imply that CD4 most likely arose from a primordial cytokine receptor.

Microspheres Encapsulating Immunotherapy Agents Target the Tumor-Draining Lymph Node in Pancreatic Ductal Adenocarcinoma.

INTRODUCTION: The tumor-draining lymph node (TDLN) plays a role in tumor immunity. Intratumorally administered microspheres (MS) that encapsulate immunomodulatory agents have emerged as a treatment strategy capable of causing profound changes in the tumor microenvironment (TME) and eliciting potent antitumor effects. We hypothesized that local delivery of MS to the TME may also drain to and therefore target the TDLN to initiate antitumor immune responses. METHODS: Fluorescent MS were injected into orthotopically implanted murine pancreatic tumors, and tissues were examined by whole-mount microscopy and imaging flow cytometry. The role of the TDLN was investigated for mice treated with intratumoral interleukin-12 (IL-12)-encapsulated MS in combination with stereotactic body radiotherapy (SBRT) by cytokine profile and TDLN ablation. RESULTS: Fluorescent AF-594 MS delivered intratumorally were detected in the tumor, peritumoral lymphatics, and the TDLN 2 h after injection. Phagocytic cells were observed with internalized fluorescent MS. SBRT + IL-12 MS-induced upregulation of Th1 and antitumor factors IL-12, IFN-γ, CXCL10, and granzyme B in the TDLN, and excision of the TDLN partially abrogated treatment efficacy. CONCLUSIONS: Our results demonstrate that intratumorally administered MS not only target the TME, but also drain to the TDLN. Furthermore, MS encapsulated with a potent antitumor cytokine, IL-12, induce an antitumor cytokine profile in the TDLN, which is essential for treatment efficacy.

Stereotactic Body Radiation and Interleukin-12 Combination Therapy Eradicates Pancreatic Tumors by Repolarizing the Immune Microenvironment.

Over 80% of pancreatic ductal adenocarcinoma (PDA) patients are diagnosed with non-resectable late-stage disease that lacks effective neoadjuvant therapies. Stereotactic body radiation therapy (SBRT) has shown promise as an emerging neoadjuvant approach for treating PDA, and here, we report that its combination with local interleukin-12 (IL-12) microsphere (MS) immunotherapy results in marked tumor reduction and cures in multiple preclinical mouse models of PDA. Our findings demonstrate an increase of intratumoral interferon gamma (IFNγ) production following SBRT/IL-12 MS administration that initiates suppressor cell reprogramming and a subsequent increase in CD8 T cell activation. Furthermore, SBRT/IL-12 MS therapy results in the generation of systemic tumor immunity that is capable of eliminating established liver metastases, providing a rationale for follow-up studies in advanced metastatic disease.

Adrenergic Receptor Signaling Regulates the Response of Tumors to Ionizing Radiation.

While ionizing radiation is a major form of cancer therapy, radioresistance remains a therapeutic obstacle. We have previously shown that the mandated housing temperature for laboratory mice (∼22°C) induces mild, but chronic, cold stress resulting in increased circulating norepinephrine, which binds to, and triggers activation of, beta-adrenergic receptors (ß-AR) on tumor and immune cells. This adrenergic signaling increases tumor cell intrinsic resistance to chemotherapy and suppression of the anti-tumor immune response. These findings led us to hypothesize that adrenergic stress signaling increases radioresistance in tumor cells in addition to suppressing T-cell-mediated anti-tumor immunity, thus suppressing the overall sensitivity of tumors to radiation. We used three strategies to test the effect of adrenergic signaling on responsiveness to radiation. For one strategy, mice implanted with CT26 murine colon adenocarcinoma were housed at either 22°C or at thermoneutrality (30°C), which reduces physiological adrenergic stress. For a second strategy, we used a ß-AR antagonist ("beta blocker") to block adrenergic signaling in mice housed at 22°C. In either case, tumors were then irradiated with a single 6 Gy dose and the response was compared to mice whose adrenergic stress signaling was not reduced. For the third strategy, we used an in vitro approach in which several different tumor cell lines were treated with a ß-AR agonist and irradiated, and cell survival was then assessed by clonogenic assay. Overall, we found that adrenergic stress significantly impaired the anti-tumor efficacy of radiation by inducing tumor cell resistance to radiation-induced cell killing and by suppression of anti-tumor immunity. Treatment using beta blockers is a promising strategy for increasing the anti-tumor efficacy of radiotherapy.