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Arming Tumor-Associated Macrophages to Reverse Epithelial Published OnlineFirst August 15, 2019; DOI: 10.1158/0008-5472.CAN-19-1246 Cancer Tumor Biology and Immunology Research Arming Tumor-Associated Macrophages to Reverse Epithelial Cancer Progression Hiromi I.Wettersten1,2,3, Sara M.Weis1,2,3, Paulina Pathria1,2,Tami Von Schalscha1,2,3, Toshiyuki Minami1,2,3, Judith A. Varner1,2, and David A. Cheresh1,2,3 Abstract Tumor-associated macrophages (TAM) are highly expressed it engaged macrophages but not natural killer (NK) cells to within the tumor microenvironment of a wide range of cancers, induce antibody-dependent cellular cytotoxicity (ADCC) of where they exert a protumor phenotype by promoting tumor avb3-expressing tumor cells despite their expression of the cell growth and suppressing antitumor immune function. CD47 "don't eat me" signal. In contrast to strategies designed Here, we show that TAM accumulation in human and mouse to eliminate TAMs, these findings suggest that anti-avb3 tumors correlates with tumor cell expression of integrin avb3, represents a promising immunotherapeutic approach to redi- a known driver of epithelial cancer progression and drug rect TAMs to serve as tumor killers for late-stage or drug- resistance. A monoclonal antibody targeting avb3 (LM609) resistant cancers. exploited the coenrichment of avb3 and TAMs to not only eradicate highly aggressive drug-resistant human lung and Significance: Therapeutic antibodies are commonly engi- pancreas cancers in mice, but also to prevent the emergence neered to optimize engagement of NK cells as effectors. In of circulating tumor cells. Importantly, this antitumor activity contrast, LM609 targets avb3 to suppress tumor progression in mice was eliminated following macrophage depletion. and enhance drug sensitivity by exploiting TAMs to trigger Although LM609 had no direct effect on tumor cell viability, ADCC. Introduction and "classical" subtypes of glioblastoma that are addicted to aberrant signaling from integrin avb3, making them highly Integrin avb3 is a marker of angiogenic blood vessels in sensitive to agents, including cilengitide, that disrupt the path- cancer (1, 2), and its expression on tumor cells has been linked way (9). Thus, it is tempting to speculate that cilengitide would to cancer progression, drug resistance, and epithelial-to- have been able to meet the survival endpoints for a more defined mesenchymal transition (EMT; refs. 3, 4). As such, two primary subset of glioblastoma patients. drugs targeting avb3 have advanced to clinical testing, including a The monoclonal anti-avb3 antibody etaracizumab has been monoclonal antibody (etaracizumab) and a cyclic peptide antag- tested in patients with several types of solid tumors, including a onist (cilengitide). These agents, although safe, produced only small cohort of patients with metastatic renal cancer where it was modest clinical activity in the patient populations tested. able to prolong stable disease for 3 patients (10). Despite a good Cilengitide, a selective cyclic peptide inhibitor of integrins avb3 safety profile, etaracizumab did not slow disease progression and avb5, produced encouraging results in phase I/II trials for in patients with sarcoma, prostate, or colorectal cancers (10). patients with advanced glioblastoma multiforme (5, 6), yet Interestingly, this antibody was tested in a phase II trial ultimately failed to meet overall survival endpoints in phase (NCT00072930) for patients with metastatic prostate cancer in III (7). A subsequent correlative IHC study revealed that higher combination with standard-of-care docetaxel, prednisone, and avb3 protein levels were associated with improved survival for zoledronic acid. Notably, steroids such as prednisone have been patients treated with cilengitide (8), suggesting that certain sub- associated with inferior immunotherapy responses (11), whereas sets of patients may be particularly sensitive to this drug. In fact, zoledronic acid can block the activity of M2 macrophages (12). we recently identified a subset of tumors within the "proneural" Similarly, etaracizumab was tested in a phase II trial for melano- ma in combination with the standard-of-care dacarbazine (13). In this trial, the overall survival for patients treated with etaracizu- 1 Department of Pathology, University of California, San Diego, La Jolla, mab was 12.6 months, whereas patients treated with etaracizu- California. 2Moores Cancer Center, University of California, San Diego, La Jolla, California. 3Sanford Consortium for Regenerative Medicine, University of mab plus dacarbazine had a worse overall survival (9.4 months), California, San Diego, La Jolla, California. compared with historical data for dacarbazine alone (6– 10 months; ref. 13). In addition to acting as a cytotoxic agent Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). for tumor cells, dacarbazine also produces immunosuppressive effects within the tumor microenvironment, including inhibition Corresponding Author: David A. Cheresh, University of California, San Diego, of M2 macrophage activity (14). Together, these clinical trial 2880 Torrey Pines Scenic Drive, Room 4003, La Jolla, CA 92037-0695. Phone: 858-822-2232; Fax: 858-534-8329; E-mail: [email protected] results suggest that the monoclonal antibody etaracizumab may improve survival in the context of immune effector cells. Cancer Res 2019;79:5048–59 Monoclonal antibodies have emerged as a class of anticancer doi: 10.1158/0008-5472.CAN-19-1246 therapeutics to not only directly kill tumor cells, but also manip- Ó2019 American Association for Cancer Research. ulate anticancer immune responses (15). The activity of several 5048 Cancer Res; 79(19) October 1, 2019 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst August 15, 2019; DOI: 10.1158/0008-5472.CAN-19-1246 Arming Tumor-Associated Macrophages for Cancer Therapy FDA-approved anticancer antibodies such as anti-CD20 (Ritux- obtained from ClodronateLiposome.com. Captisol (Cydex) was imab) and anti-EGFR (Cetuximab) has been attributed to their diluted in water at 6%. Erlotinib (Selleckchem, OSI-744) was ability to induce antibody-dependent cellular cytotoxicity diluted in DMSO for in vitro or Captisol for in vivo experiments. (ADCC; refs. 16, 17). This process is triggered when antibodies Anti-avb3 antibody, LM609, was produced as previously bind to tumor cell antigens and are simultaneously recognized by described (21). Batch-to-batch activity is confirmed by adhesion Fcg receptors on immune effector cells, including natural killer assays. Antibodies are listed in Supplementary Table S1. (NK) cells, monocytes, macrophages, neutrophils, and dendritic cells (DC; ref. 18). Gene expression analysis using public databases Although not present in early stage disease, integrin avb3 mRNA expression in The Cancer Genome Atlas (TCGA) data- becomes enriched during epithelial cancer progression, in partic- sets was used to analyze the correlation between ITGB3 and ular as tumors lose sensitivity to standard of care (19). As such, immune cell type scores, calculated as previously described (22) integrin avb3 represents an ideal tumor antigen to mount an using cBioPortal. Gene sets for immune cell markers are listed in attack against. Here, we show that avb3 on epithelial cancers Supplementary Table S2. positively correlates with the accumulation of tumor-associated macrophages (TAM) within the tumor microenvironment, Correlation analysis of ITGB3 and immune cell types using immune cells that typically lead to tumor progression and NanoString nCounter immune suppression. Interestingly, TAMs armed with an anti- Ten preexisting, deidentified lung adenocarcinoma frozen tis- þ avb3 antibody can be activated to kill the highly aggressive avb3 sue biopsies were obtained from the Moores UCSD Cancer Center þ CD47 epithelial cancer cells via ADCC and thereby suppress Biorepository. mRNA was extracted using the RNeasy Mini Kit cancer progression and drug resistance. Our work reveals a poten- (Qiagen, 74104). The quality of extracted mRNA was tested using tially valuable subpopulation of patients who might be particu- Agilent Bioanalyzer (Agilent). Expression of mRNAs involved in larly sensitive to the effects of an avb3-targeted antibody, namely immune cell activities was analyzed using nCounter PanCancer those with advanced epithelial cancers whose tumors have Immune Profiling Panel (NanoString). become coenriched for integrin avb3 and TAMs. Protein analysis IHC staining. IHC staining was performed on formalin-fixed, Materials and Methods paraffin-embedded slides using the VECTASTAIN Elite ABC HRP Statistical analysis Kit (Vector, PK-6100), ImmPRESS Excel Staining Kit (Vector, MP- Testing for normality was performed using the Shapiro–Wilk 7602), and ImmPRESS HRP anti-rat IgG, mouse adsorbed (per- test. The Student t test, Mann–Whitney U test, one-sample t test, or oxidase) polymer detection kit (Vector, MP-7444). Slides were ANOVA was performed to compare independent sample groups. imaged on a NanoZoomer Slide Scanning System (Hamamatsu), Pearson or Spearman correlation was used to measure correlation and the area fraction for each protein with respect to tumor tissue of multiple groups. Excel (Microsoft) and SPSS (IBM Analytics) was calculated utilizing ImageJ (NIH; ref. 23). Integrin b3 levels were utilized for analysis. on tumor cells for cancer microarray slides were analyzed blindly, and the tissues were categorized into
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