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Protein synthesis supports robust immune responses. Nutrient competition and global cell stressors in the tumor microenvironment (TME) may impact protein translation in T cells and antitumor immunity. Using human and mouse tumors, we demonstrated here that protein translation in T cells is repressed in solid tumors. Reduced glucose availability to T cells in the TME led to activation of the unfolded protein response (UPR) element eIF2α (eukaryotic translation initiation factor 2 alpha). Genetic mouse models revealed that translation attenuation mediated by activated p-eIF2α undermines the ability of T cells to suppress tumor growth. Reprograming T-cell metabolism was able to alleviate p-eIF2α accumulation and translational attenuation in the TME, allowing for sustained protein translation. Metabolic and pharmacological approaches showed that proteasome activity mitigates induction of p-eIF2α to support optimal antitumor T-cell function, protecting from translation attenuation and enabling prolonged cytokine synthesis in solid tumors. Together, these data identify a new therapeutic avenue to fuel the efficacy of tumor immunotherapy. Significance: Proteasome function is a necessary cellular component for endowing T cells with tumor killing capacity by mitigating translation attenuation resulting from the unfolded protein response induced by stress in the tumor microenvironment.

Mechanisms of locally advanced rectal cancer (LARC) neoadjuvant chemoradiation resistance are poorly understood. Current research models to study this issue are incomplete and restricted by the limited supply of untreated tissue samples. Patient-derived xenografts (PDX) are commonly developed from surgical specimens and can serve as effective tools to investigate therapy. We previously demonstrated that the SNAI2/ miR-145 signaling axis enhanced the tumor-initiating cell (TIC) and chemotherapy resistance. We developed a unique PDX model from pre-neoadjuvant therapy LARC biopsy samples to study chemoradiation resistance. Further, we hypothesized that TICs isolated from LARC patient-derived xenografts would demonstrated increased SNAI2/ miR-145 signaling. Methods: Fresh LARC endoscopic biopsy samples were digested using Liberase DH to yield a dissociated single-cell solution. All in vivo experiments consisted of injecting cells with Matrigel subcutaneously into NSG NODSCID/IL2Rgnull immunocompromised mice. Xenograft (Passage 1, P1) single cells were resuspended in ALDEFLUOR assay buffer. ALDEFLUOR and EPCAM positive cells (ALDH+/EPCAM+) were then detected using a flow cytometer to identify the TICs. Using the negative control ALDH inhibitor, DEAB (1.5 μM), identify the ALDH+ cells compared with background. For limited dilution (LD) in vitro spheroid assay, 5000 EPCAM+/ALDH+ and ALDH- cells were cultured in ultra-low attachment plates in cancer stem-cell medium (DMEM-F12; 1X B-27, βEGF, βFGF, 1% penicillin/streptomycin, BSA, heparin, β-mercaptoethanol). For LD in vivo study, EPCAM+ ALDH sorted cells were implanted subcutaneously with Matrigel and closely observed. Droplet Digital PCR was conducted using the QX200 Droplet Digital PCR System. miR-145 PCR was performed using TaqMan MicroRNA Assays (Applied Biosystems) and normalized to RNU6B. Results: Our LARC PDX model had high outgrowth and engraftment rates of 70 and 85% respectively and maintained critical histologic features. Both LD in vitro and in vivo experiments demonstrated that EPCAM+/ALDH+ cells were highly tumorigenic suggestive of TICs (p Conclusions: The lack of novel strategies plays a central role in the inability to overcome therapeutic resistance in LARC. Our results demonstrate a novel model to investigate LARC and suggest that a novel miR-145 therapeutic strategy may be able to target the TIC in LARC patients. Citation Format: Ernest R. Camp, Cindy Wang, Katie Hurst, Lourdes M. Nogueira, Victoria J. Findlay. Improving rectal cancer therapy using a patient-derived tumor xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-043.