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In many aggressive cancers, such as glioblastoma multiforme, progression is enabled by local immunosuppression driven by the accumulation of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). However, the mechanistic details of how Tregs and MDSCs are recruited in various tumors are not yet well understood. Here we report that macrophages and microglia within the glioma microenvironment produce CCL2, a chemokine that is critical for recruiting both CCR4+ Treg and CCR2+Ly-6C+ monocytic MDSCs in this disease setting. In murine gliomas, we established novel roles for tumor-derived CCL20 and osteoprotegerin in inducing CCL2 production from macrophages and microglia. Tumors grown in CCL2-deficient mice failed to maximally accrue Tregs and monocytic MDSCs. In mixed-bone marrow chimera assays, we found that CCR4-deficient Treg and CCR2-deficient monocytic MDSCs were defective in glioma accumulation. Furthermore, administration of a small-molecule antagonist of CCR4 improved median survival in the model. In clinical specimens of glioblastoma multiforme, elevated levels of CCL2 expression correlated with reduced overall survival of patients. Finally, we found that CD163-positive infiltrating macrophages were a major source of CCL2 in glioblastoma multiforme patients. Collectively, our findings show how glioma cells influence the tumor microenvironment to recruit potent effectors of immunosuppression that drive progression. Cancer Res; 76(19); 5671–82. ©2016 AACR.

Combined use of new molecular diagnostic and imaging methods could improve care of individual patients with rare cancers. A 57-year-old developed apocrine adenocarcinoma of the left axilla with regional lymph node metastasis detected at initial surgery. After adjuvant regional radiotherapy, metastases developed and over the subsequent 7 years he received serial systemic therapy with 11 different regimens. Expression of 128 proteins/phosphoproteins was evaluated in 8 tissue samples from diagnosis, progressive adenopathy at 45 months, brain metastases at 60 months, and abdominal metastases at 63 months by CLIA-standardized reverse phase phosphoprotein arrays (RPPA). Tumor burden was estimated by quantifying the volume of individual lesions with semi-automated segmentation algorithms on serially collected CT images. Next-generation sequencing (NGS) revealed a PIK3CA K111T mutation, prompting treatment with sirolimus 28 months after diagnosis. Seventeen months later, new neoplastic adenopathy emerged. NGS revealed a new PIK3CA M610V mutation. Semiquantitative scoring of RPPA demonstrated relative increase in AKT (from 0.72 to 0.9) and mTOR (from 0.55 to 0.8) pathway activation. The relative estimated tumor burden (RETB) was 21,145 mm3. Over 6 months of docetaxel therapy the RETB decreased to 12,077 mm3. The patient subsequently received pembrolizumab with increased RETB (71,308 mm3). Gemcitabine appeared to stabilize the RETB (73,168 mm3), but brain metastases emerged. The patient underwent craniotomy, began paclitaxel therapy, and then RETB declined to 35,623 mm3. But new symptomatic abdominal metastases prompted surgical resection. The patient then received the PI3K inhibitor taselisib; however, after an initial decrease, RETB rose to 31,208 mm3. At this point, the patient began bicalutamide therapy. NGS of the primary mass and the initial metastatic adenopathy revealed no amplification of the androgen receptor (AR). But RPPA demonstrated inversion of the ratio of measured phosphorylated AR S81 to AR S650, suggesting primarily cytoplasmic AR in tumor through 45 months but nuclear AR in samples resected at 60 and 63 months. Immunohistochemistry revealed 3+ nuclear AR expression in the latter metastatic tissue. After 9 months of bicalutamide, enzalutamide and leuprolide were administered for 7 months (final RETB = 1,555 mm3). Treatment continued beyond the study period. Tumor burden assessment by new CT imaging measurement methods, combined with RPPA could improve adaptive, responsive, therapy for patients with rare tumors. We identified the phenotypic evolution of androgen-driven growth of apocrine adenocarcinoma after cytotoxic and PI3K-mTOR inhibitor therapy. Protein-based diagnostics revealed an effective treatment strategy in late metastatic disease that was not indicated by NGS testing. Citation Format: Erik Dvergsten, Anthony Serritella, Danielle Kimble, Malcom McIver, Sanja Karovic, Vasiliki Thomeas-McEwing, Hao Yang, Manish R. Sharma, Thomas P. Conrads, Mariaelena Pierobon, Peter Pytel, Binsheng Zhao, Lawrence H. Schwartz, Emanuel F. Petricoin, Russell Szmulewitz, Michael L. Maitland. Longitudinal proteomic assessment of patient with metastatic apocrine adenocarcinoma reveals evolutionary selection for androgen-receptor-dependence and therapeutic response [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 5451.

Introduction. Glioblastoma multiforme (GBM) is the most common primary central nervous system tumor in adults. The median survival after surgical intervention alone is approximately 6 months and the addition of radiation and chemotherapy (like Temozolomide, TMZ) can only extend this up to 14.6 months. Other treatment options are therefore needed for better disease management and improved survival. We ran an Affymetrix analysis on the Rembrandt Database to determine the role of metalloproteinase expression in the biology of GBMs. High expression of MT1-MMP (MMP14) correlated with a poor patient survival. MMP14, as other metalloproteinases, is a cellular based proteolytic endopeptidase that plays a role in extracellular matrix (ECM) and basic membrane (BM) degradation. In glioma, expression of MMP14 is correlated with tumor invasiveness and associated with the CD133+ phenotype. Inhibition of MMP14 could therefore offer a potential target for the therapy of GBM. Experimental methods. Expression of MMP14 was determined in 100 primary and recurrent gliomas by immunohistochemistry. We performed quantitative RT-PCR and Western blotting analysis of MMP14 expression to investigate the effect of radiation and TMZ on U87 and U251 glioma cells. We used the Ingenuity Software to analyze the relationship between MMP14 and proteins responsible for angiogenesis and cell proliferation. Anti-angiogenic effect of MMP14 knockdown was determined by tube formation assay and angiogenic cytokine array (RayBio). Finally, treatment of MMP14 knockdown of glioma cells with temozolomide and ionizing radiation was analyzed by MTT assay. Results. Genetic silencing of MMP14 leads to inhibition of glioma cell proliferation and cell cycle arrest. We also show that silencing of MMP14 results in downregulation of angiogenic markers at the mRNA and protein level. Furthermore, we have found that inhibition of MMP14 expression in glioma cells improves the therapeutic effect of temozolomide and ionizing radiation. Conclusions. This study suggests that silencing of MMP14 via RNAi technology improves therapeutic effect of TMZ and ionizing radiation on glioma cell proliferation. Therefore, inhibition of MMP14 provides a new target for treatment of gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 880. doi:1538-7445.AM2012-880

Aim: The present study was aimed to investigate whether KISS1 suppressor protein could be used as a prognostic marker in development of breast cancer metastases. Methods: To test whether breast cancer brain metastases (BMHB) have altered expression of KISS1, we created a TMA (tissue microarray) recipient block containing 136 specimens: 103 specimens of primary breast cancer cells (invasive ductal carcinoma, IDC), 7 specimens from draining lymph nodes (LN), 39 brain metastases and 14 samples of normal breast tissue (NBreast). Each core was duplicated. Sections from this block were stained with antibodies that recognize KISS1. Immunoassays were graded based on: A) intensity of immunoreactive cells staining (Negative, 0% of the cells have staining; 1+, 5-20% of total cells have staining (Mild); 2+, 20-50% of total cells have staining (Moderate); 3+, 50% or more of total cells have staining(High)) and B) score calculated by ACIS system (Chromavision/Clarient, Aliso Viejo, CA). Positive staining in the present study was defined as visibility at low magnification (10x) and was confirmed by two pathologists. To validate the IHC findings, relative expression of KISS1 was evaluated by real time PCR technique. Results: Based on TMA expression results, we found that breast cancer metastases exhibited statistically significant less KISS1 expression vs. primary specimens (Kruskal-Wallis p value = 0.011). In validation set, 84.2% of IDC samples had either moderate (38/76) or high (26/76) expression of KISS1 protein whereas weak (negative or mild) expression was observed in 15.8% cases (12/76). In contrast, metastatic cases demonstrated weak expression in 100% cases (19/19). The real time PCR data confirmed IHC findings. We analyzed the correlation between the level of KISS1 expression and breast cancer prognosis based on the IHC data. The group of patients with high expression of KISS1 protein (1x and more) was found to have a trend toward slower disease progression. Those patients developed brain metastases 51.0+15.0 (CI: 17.7-84.3) months after tumor resection whereas the group of patients with no KISS1 expression had metastases after only 27.0+4.93 (CI: 15.39-38.69) months. Conclusions: These results suggest that high level of KISS1 protein expression is advantageous for breast cancer patients and that low levels correlate with metastases towards secondary organs such as brain. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1439. doi:10.1158/1538-7445.AM2011-1439