BackgroundNatural killer (NK) cells have potent antitumor activities. Nevertheless, adoptive transfer therapy of NK cells has gained very limited success in patients with solid tumors as most infused NK cells remain circulating in the peripheral blood instead of entering tumor sites. Chemokines and their receptors play important roles in NK cell distribution. Enhancing chemokine receptors on immune cells to match and be driven to tumor-specific chemokines may improve the therapeutic efficacy of NK cells.MethodsThe CCR5-CCL5 axis is critical in NK cell homing to tumor sites. Thus, we analyzed CCR5 expression on NK cells from patients with cancer and healthy donors. We then upregulated CCR5 and CCL5 with lentiviruses and oncolytic viruses in NK and tumor cells, respectively. Animal experiments were also carried out to test the efficacy of the combination of oncolytic virus with NK cells.ResultsIn NK cells from patients with various solid tumors or healthy subjects, CCR5 was expressed at low levels before and after expansion in vitro. CCR5-engineered NK cells showed enhanced tumor infiltration and antitumor effects, but no complete regressions were noted in the in vivo tumor models. To further improve therapeutic efficacy, we constructed CCL5-expressing oncolytic vaccinia virus. In vitro data demonstrated that vaccinia virus can produce CCL5 in tumor cells while infectivity remained unaffected. Supernatants from tumor cells infected by CCL5-modified vaccinia virus enhanced the directional movement of CCR5-overexpressed NK cells but not green fluorescent protein (GFP)-expressing cells. More importantly, NK cells were resistant to the vaccinia virus and their functions were not affected after being in contact. In vivo assays demonstrated that CCL5-expressing vaccinia virus induced a greater accumulation of NK cells within tumor lesions compared with that of the prototype virus.ConclusionEnhancement of matched chemokines and chemokine receptors is a promising method of increasing NK cell homing and therapeutic effects. Oncolytic vaccinia viruses that express specific chemokines can synergistically augment the efficacies of NK cell-based therapy.

PurposeIntratumoral metabolic heterogeneity may increase the likelihood of treatment failure due to the presence of a subset of resistant tumor cells. Using a head and neck squamous cell carcinoma (HNSCC) xenograft model and a real-time fluorescence imaging approach, we tested the hypothesis that tumors are metabolically heterogeneous, and that tumor hypoxia alters patterns of glucose uptake within the tumor.Experimental DesignCal33 cells were grown as xenograft tumors (n = 16) in nude mice after identification of this cell line's metabolic response to hypoxia. Tumor uptake of fluorescent markers identifying hypoxia, glucose import, or vascularity was imaged simultaneously using fluorescent molecular tomography. The variability of intratumoral 2-deoxyglucose (IR800-2-DG) concentration was used to assess tumor metabolic heterogeneity, which was further investigated using immunohistochemistry for expression of key metabolic enzymes. HNSCC tumors in patients were assessed for intratumoral variability of 18F-fluorodeoxyglucose (18F-FDG) uptake in clinical PET scans.ResultsIR800-2-DG uptake in hypoxic regions of Cal33 tumors was 2.04 times higher compared to the whole tumor (p = 0.0001). IR800-2-DG uptake in tumors containing hypoxic regions was more heterogeneous as compared to tumors lacking a hypoxic signal. Immunohistochemistry staining for HIF-1α, carbonic anhydrase 9, and ATP synthase subunit 5β confirmed xenograft metabolic heterogeneity. We detected heterogeneous 18F-FDG uptake within patient HNSCC tumors, and the degree of heterogeneity varied amongst tumors.ConclusionHypoxia is associated with increased intratumoral metabolic heterogeneity. 18F-FDG PET scans may be used to stratify patients according to the metabolic heterogeneity within their tumors, which could be an indicator of prognosis.

Interferon regulatory factor 1 (IRF1) is a nuclear transcription factor which appears to have anti-tumor activity in human and mice. IRF1 can be induced by interferon-gamma (IFNγ). Although recent studies indicated that the IFNγ-PD-L1 axis played an important role during T cell exhaustion, and promoted tumorigenesis, the cell intrinsic role of IRF1 is not completely clear in this context. In this study, we investigated the intrinsic role of IRF1 during tumor progression. We generated several IRF1-deficient (IRF1-KO) murine tumor cell lines (CT26, MC38 and B16-F10) via CRISPR/Cas9-mediated genome editing, and compared their growth rates with wild type (WT) cells both in vitro and in vivo using syngeneic mouse tumor models. We found that the loss of IRF1 did not affect cell growth rates in vitro. However, the IRF1-KO cells showed significantly slower tumor growth than WT cells in mice models. Depletion of CD8+ T cell using antibody rescued the ability of IRF1-KO cells to grow tumors. Furthermore, analysis of tumor infiltrating T cells showed more activated CD8+ T cells and less exhausted T cells in IRF1-KO induced tumors than those induced by WT cells. To confirm whether these results were caused by IFNγ-IRF1-PD-L1 axis, we evaluated IFNγ-induced inhibitory ligands on tumor cells in both WT and IRF1 KO cell lines. We found that the expression levels of MHC class I were not affected by loss of IRF1, whereas, the induction of PD-L1 by IFNγ was abolished in IRF1 KO cells. In conclusion, intrinsic loss of IRF1 in tumor cells significantly reduces tumorigenesis in solid tumors. This phenomenon depends on the regulation of PD-L1 expression on tumor cells via IFNγ-IRF1-PD-L1 axis, and eventually affecting host anti-tumor immune responses, such as CD8+ T cells.

Invasive lobular breast carcinoma (ILC) is the most common special histological subtype of breast cancer, accounting for 10-15% of all cases, and characterized by the loss of adherens junctions through inactivating mutations in E-cadherin. Although currently existing genetically engineered mouse models of ILC faithfully capture its dyscohesive, single-file growth and expression of estrogen receptor alpha (ER), they do not fully recapitulate all aspects of human ILC such as unique sites of metastases and endocrine response. While recent work on human ILC cell lines has proven them useful for studying ILC in vitro, there is limited data on their growth as xenografts in mice and their fidelity as in vivo models. Using dual bioluminescent and fluorescent labeled ER-positive human ILC cell lines (MDA-MB-134, SUM44PE, MDA-MB-330, BCK4), herein we characterized their growth orthotopically and at secondary sites following spontaneous or experimental metastasis. Mammary fat pad xenografts gave rise to primary tumors with single-file infiltration and cytoplasmic translocation of p120-catenin, characteristic of human ILC. The tumor microenvironment exhibited deposition of collagen fibers and infiltration by fibroblasts and neutrophils. In this orthotopic model, we observed spontaneous metastases of most cell lines to bones, brain and ovaries, closely mirroring the clinical patterns of human ILC dissemination. In contrast, experimental metastases were only observed following intravenous or intracardiac xenografts of MDA-MB-134 cells. Interestingly, tail vein injections of this cell line lead to colonization of bones and lymph nodes but not of lungs, while intracardiac injections resulted in brain, bone and lymph node metastases. Importantly, we observed high ER expression in the primary tumors and the metastatic lesions, along with a significant response to the selective ER down-regulator fulvestrant in both the mammary fat pad and intracardiac models. Ongoing work focused on genomic and transcriptional analyses of primary tumors and metastases, as well as of cell lines isolated from these lesions, will reveal additional mechanistic insights into the biology of ILC dissemination. This is the first report of ER-positive and endocrine responsive human cell line xenografts faithfully representing unique ILC features such as ovarian metastases. These versatile models will serve as an invaluable pre-clinical platform for validating candidate ILC genetic drivers and testing novel therapeutics towards translation into the clinic. Citation Format: Nilgun Tasdemir, Laura Savariau, Julie Scott, Joseph D. Latoche, Weizhou Hou, Kyle Biery, Minji Chung, Emily A. Bossart, Sreeja Sreekumar, Daniel D. Brown, Azadeh Nasrazadani, Ye Qin, Jagmohan Hooda, Fangyuan Chen, Carlos A. Castro, Carolyn J. Anderson, Jennifer Atkinson, Peter C. Lucas, Nancy E. Davison, Adrian V. Lee, Steffi Oesterreich. ER alpha-positive human cell line xenograft models recapitulate metastatic dissemination and endocrine response of invasive lobular breast carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-002.