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.

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.