Tumor-associated Macrophage Markers Research Articles

Fc fragment of immunoglobulin G receptor IIa (FCGR2A) as a new potential prognostic biomarker of esophageal squamous cell carcinoma.

Fc fragment of immunoglobulin G receptor IIa (FCGR2A) as a new potential prognostic biomarker of esophageal squamous cell carcinoma.

Identification and Validation of m6A-Related lncRNA Signature as Potential Predictive Biomarkers in Breast Cancer.

The metastasis and poor prognosis are still regarded as the main challenge in the clinical treatment of breast cancer (BC). Both N6-methyladenosine (m6A) modification and lncRNAs play vital roles in the carcinogenesis and evolvement of BC. Considering the unknown association of m6A and lncRNAs in BC, this study therefore aims to discern m6A-related lncRNAs and explore their prognostic value in BC patients. Firstly, a total of 6 m6A-related lncRNAs were screened from TCGA database and accordingly constructed a prognostic-predicting model. The BC patients were then divided into high-risk and low-risk groups dependent on the median cutoff of risk score based on this model. Then, the predictive value of this model was validated by the analyses of cox regression, Kaplan-Meier curve, ROC curve, and the biological differences in the two groups were validated by PCA, KEGG, GSEA, immune status as well as in vitro assay. Finally, we accordingly constructed a risk prognostic model based on the 6 identified m6A-related lncRNAs, including Z68871.1, AL122010.1, OTUD6B-AS1, AC090948.3, AL138724.1, EGOT. Interestingly, the BC patients were divided into the low-risk and high-risk groups with different prognoses according to the risk score. Notably, the risk score of the model was an excellent independent prognostic factor. In the clinical sample validation, m6A regulatory proteins were differentially expressed in patients with different risks, and the markers of tumor-associated macrophages and m6A regulators were co-localized in high-risk BC tissues. This well-validated risk assessment tool based on the repertoire of these m6A-related genes and m6A-related lncRNAs, is of highly prognosis-predicting ability, and might provide a supplemental screening method for precisely judging BC prognosis.

7S,15R-Dihydroxy-16S,17S-Epoxy-Docosapentaenoic Acid, a Novel DHA Epoxy Derivative, Inhibits Colorectal Cancer Stemness through Repolarization of Tumor-Associated Macrophage Functions and the ROS/STAT3 Signaling Pathway.

Colorectal cancer is a highly malignant cancer that is inherently resistant to many chemotherapeutic drugs owing to the complicated tumor-supportive microenvironment (TME). Tumor-associated macrophages (TAM) are known to mediate colorectal cancer metastasis and relapse and are therefore a promising therapeutic target. In the current study, we first confirmed the anti-inflammatory effect of 7S,15R-dihydroxy-16S,17S-epoxy-docosapentaenoic acid (diHEP-DPA), a novel DHA dihydroxy derivative synthesized in our previous work. We found that diHEP-DPA significantly reduced lipopolysaccharide (LPS)-induced inflammatory cytokines secretion of THP1 macrophages, IL-6, and TNF-α. As expected, diHEP-DPA also modulated TAM polarization, as evidenced by decreased gene and protein expression of the TAM markers, CD206, CD163, VEGF, and TGF-β1. During the polarization process, diHEP-DPA treatment decreased the concentration of TGF-β1, IL-1β, IL-6, and TNF-α in culture supernatants via inhibiting the NF-κB pathway. Moreover, diHEP-DPA blocked immunosuppression by reducing the expression of SIRPα in TAMs and CD47 in colorectal cancer cells. Knowing that an inflammatory TME largely serves to support epithelial-mesenchymal transition (EMT) and cancer stemness, we tested whether diHEP-DPA acted through polarization of TAMs to regulate these processes. The intraperitoneally injected diHEP-DPA inhibited tumor growth when administered alone or in combination with 5-fluorouracil (5-FU) chemotherapy in vivo. We further found that diHEP-DPA effectively reversed TAM-conditioned medium (TCCM)-induced EMT and enhanced colorectal cancer stemness, as evidenced by its inhibition of colorectal cancer cell migration, invasion and expression of EMT markers, as well as cancer cell tumorspheres formation, without damaging colorectal cancer cells. DiHEP-DPA reduced the population of aldehyde dehydrogenase (ALDH)-positive cells and expression of colorectal stemness marker proteins (CD133, CD44, and Sox2) by modulating TAM polarization. Additionally, diHEP-DPA directly inhibited cancer stemness by inducing the production of reactive oxygen species (ROS), which, in turn, reduced the phosphorylation of nuclear signal transducer and activator of transcription 3 (STAT3). These data collectively suggest that diHEP-DPA has the potential for development as an anticancer agent against colorectal cancer.

Abstract SY07-02: Gaining biologic insights into glioblastoma using proteomics

Abstract Glioblastoma (GBM) is the most common type of primary malignant tumor in adults and contributes disproportionately to cancer morbidity and mortality. Understanding its molecular pathogenesis is crucial for improving diagnosis and treatment. Integrated analysis of genomic, proteomic, post-translational modification and metabolomics data on 99-treatment naïve GBMs provided insights to GBM biology. Multiple key findings emerged that were not known from traditional genomic analyses: (1) Analysis of protein phosphorylation identified key signaling intermediates in the RTK/RAS path-way common to multiple RTK genomic alterations (PTPN11 and PLCG1), potentially offering common therapeutic targets for different oncogenic drivers in GBM. (2) Phosphoproteomics also identified potential druggable targets based on kinase-substrate pathway analysis, as well as novel phosphoprotein targets associated with the regulation of telomere length by ATRX in IDH mutants. (3) TP53 protein abundance in GBM is regulated by protein/phosphoprotein effectors (4) Four immune GBM subtypes exist, characterized by distinct immune cell population differences – Immune High and Immune Low phenotypes in GBM were driven by tumor-associated macrophage markers, and associated with distinct epigenetic modifications and histone acetylation patterns. (5) The mesenchymal subtype displays EMT signatures specific in tumor cells, distinct from infiltrating immune cells. (6) Histone H2B acetylation and immune-low GBM was driven largely by BRDs, CREBBP, and EP300. (7) Identification of key metabolic changes in IDH mutants facilitating the accumulation of onco-metabolite 2-HG were also associated with lipidomic changes. This work identifies additional therapeutic channels for GBM and novel information useful for more accurate stratification patients for effective treatment. Citation Format: Jill S. Barnholtz-Sloan. Gaining biologic insights into glioblastoma using proteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY07-02.

Contacts with Macrophages Promote an Aggressive Nanomechanical Phenotype of Circulating Tumor Cells in Prostate Cancer.

Aggressive tumors of epithelial origin shed cells that intravasate and become circulating tumor cells (CTC). The CTCs that are able to survive the stresses encountered in the bloodstream can then seed metastases. We demonstrated previously that CTCs isolated from the blood of prostate cancer patients display specific nanomechanical phenotypes characteristic of cell endurance and invasiveness and patient sensitivity to androgen deprivation therapy. Here we report that patient-isolated CTCs are nanomechanically distinct from cells randomly shed from the tumor, with high adhesion as the most distinguishing biophysical marker. CTCs uniquely coisolated with macrophage-like cells bearing the markers of tumor-associated macrophages (TAM). The presence of these immune cells was indicative of a survival-promoting phenotype of "mechanical fitness" in CTCs based on high softness and high adhesion as determined by atomic force microscopy. Correlations between enumeration of macrophages and mechanical fitness of CTCs were strong in patients before the start of hormonal therapy. Single-cell proteomic analysis and nanomechanical phenotyping of tumor cell-macrophage cocultures revealed that macrophages promoted epithelial-mesenchymal plasticity in prostate cancer cells, manifesting in their mechanical fitness. The resulting softness and adhesiveness of the mechanically fit CTCs confer resistance to shear stress and enable protective cell clustering. These findings suggest that selected tumor cells are coached by TAMs and accompanied by them to acquire intermediate epithelial/mesenchymal status, thereby facilitating survival during the critical early stage leading to metastasis. SIGNIFICANCE: The interaction between macrophages and circulating tumor cells increases the capacity of tumor cells to initiate metastasis and may constitute a new set of blood-based targets for pharmacologic intervention.

The Crosstalk between Tumor Stroma Components and ESR1 Mutant Breast Cancer Cells Remodels Tumor Microenvironment and Enhances Tumor Growth and Progression

Background Cancer is a genetic disease characterized by the progressive accumulation of mutations that randomly occur in key cancer-related genes such as ESR1, encoding Estrogen Receptor Alpha (ERα). ESR1 mutations (ESR1m) are rarely detected in primary tumors, but they have been largely found in metastatic breast cancers (BCs). Particularly, mutations in the ligand-binding domain of ERα may represent one mechanism of clinical acquired resistance after long-term adjuvant endocrine therapy and are associated with worse clinical outcome. Several studies reported that the complex interactions between the tumor cell niche and its surrounding microenvironment (stroma) are crucial for tumor development and metastasis and influence therapeutic response. The contribution of the tumor stroma in potentiating malignant phenotype of mutated BC cells is one of the most investigated issues. Thus, we evaluated the functional interactions between BC cells expressing the most frequent ESR1m, Y537S, and surrounding stroma and vice versa. Methods As experimental models we used human parental MCF-7 and Y537S CRISPR BC cells. We ran a microarray analysis to compare genomic profiling of the two BC cell lines and qRT-PCR to confirm these data. We performed co-culture experiments employing the most representative tumor stroma components such as the human monocyte cell line, THP-1, cancer-associated fibroblasts (CAFs), and pre-adipocytes derived from a mouse embryo, 3T3L1. We tested the effects of the different tumor stroma components on the behavior of BC cells and vice versa. Results Microarray analysis revealed that mutant BC cells exhibited higher mRNA levels of several genes encoding different mediators of tumor-stroma interactions compared to parental counterpart. These data have been confirmed with qRT-PCR assays. Conditioned media (CM) derived from CAFs, macrophages and mature adipocytes increased the growth, migration and invasion of both tested BC cells, even though to a higher extent in the mutant clones. Vice versa, CM-derived from both BC cells contributed to: i) the dedifferentiation of adipocytes into pre-adipocytes featured by the reduction in lipid droplets together with the decreased mRNA expression level of adipocyte markers, ii) the higher CAF activation, as revealed by an altered morphology, an increased FAP mRNA expression, and enhanced migratory capabilities, iii) the enhanced recruitment of monocytes displaying tumor associated macrophage markers. It is worth to mention how these events were emphasized in the presence of CM-derived from mutant cells. Conclusions Our results showed how mutant BC cells educate tumor stroma to further potentiate its own malignant phenotype. In the era of precision medicine, our findings lead to identifying novel drugs able to target stromal components and cancer cells to be both implemented in the therapeutic strategies of BC treatment, particularly in the occurrence of ESR1 mutations.

ARG1 mRNA Level Is a Promising Prognostic Marker in Head and Neck Squamous Cell Carcinomas.

Head and neck squamous cell carcinomas (HNSCC) can be induced by smoking or alcohol consumption, but a growing part of cases relate to a persistent high-risk papillomavirus (HPV) infection. Viral etiology has a beneficial impact on the prognosis, which may be explained by a specific immune response. Tumor associated macrophages (TAMs) represent the main immune population of the tumor microenvironment with a controversial influence on the prognosis. In this study, the level, phenotype, and spatial distribution of TAMs were evaluated, and the expression of TAM-associated markers was compared in HPV positive (HPV+) and HPV negative (HPV−) tumors. Seventy-three formalin and embedded in paraffin (FFPE) tumor specimens were examined using multispectral immunohistochemistry for the detection of TAM subpopulations in the tumor parenchyma and stroma. Moreover, the mRNA expression of TAM markers was evaluated using RT-qPCR. Results were compared with respect to tumor etiology, and the prognostic significance was evaluated. In HPV− tumors, we observed more pro-tumorigenic M2 in the stroma and a non-macrophage arginase 1 (ARG1)-expressing population in both compartments. Moreover, higher mRNA expression of M2 markers—cluster of differentiation 163 (CD163), ARG1, and prostaglandin-endoperoxide synthase 2 (PTGS2)—was detected in HPV− patients, and of M1 marker nitric oxide synthase 2 (NOS2) in HPV+ group. The expression of ARG1 mRNA was revealed as a negative prognostic factor for overall survival of HNSCC patients.

Aryl Hydrocarbon Receptor Signaling Controls CD155 Expression on Macrophages and Mediates Tumor Immunosuppression.

Crosstalk between costimulatory and coinhibitory ligands are a prominent node of immune cell regulation. Mounting evidence points toward a critical role for CD155, the poliovirus receptor, in suppressing T cell function, particularly in cancer. However, relative to other known costimulatory/coinhibitory ligands (e.g., CD86, CD80, PD-L1), the physiological functions of CD155 and the mechanisms controlling its expression remain unclear. We discovered that CD155 expression is coregulated with PD-L1 on tumor-associated macrophages, is transcriptionally regulated by persistently active aryl hydrocarbon receptor (AhR), and can be targeted for suppression via AhR inhibition in vivo. Therapeutic inhibition of AhR reversed tumor immunosuppression in an immune competent murine tumor model, and markers of AhR activity were highly correlated with tumor-associated macrophage markers in human glioblastomas. Thus, CD155 functions within a broader, AhR-controlled macrophage activation phenotype that can be targeted to reverse tumor immunosuppression.

Survivin drives tumor-associated macrophage reprogramming: a novel mechanism with potential impact for obesity

PurposeRecent studies point to adipose-derived stem cells (ASCs) as a link between obesity and cancer. We aimed to determine whether survivin, which is highly secreted by ASCs from subjects with obesity, might drive a pro-tumoral phenotype in macrophages.MethodsThe effect of ASC conditioned medium on the macrophage phenotype was assessed by expression studies. Survivin intracellular localization and internalization were examined by subcellular fractionation and immunofluorescence, respectively. Loss- and gain-of-function studies were performed using adenoviral vectors, and gene expression patterns, migration and invasion capacities of cancer cells were examined. Heterotypic cultures of ASCs, macrophages and cancer cells were established to mimic the tumor microenvironment. Survivin-blocking experiments were used to determine the impact of survivin on both macrophages and cancer cells. Immunohistochemical analysis of survivin was performed in macrophages from ascitic fluids of cancer patients and healthy controls.ResultsWe found that obese-derived ASCs induced a phenotypic switch in macrophages characterized by the expression of both pro- and anti-inflammatory markers. Macrophages were found to internalize extracellular survivin, generating hybrid macrophages with a tumor-associated phenotype that included secretion of survivin. Exogenous expression of survivin in macrophages generated a similar phenotype and enhanced the malignant characteristics of cancer cells by a mechanism dependent on survivin phosphorylation at threonine 34. Survivin secreted by both ASCs from subjects with obesity and tumor-associated macrophages synergistically boosted the malignancy of cancer cells. Importantly, survivin was mainly detected in ascites-associated macrophages from patients with a malignant diagnosis.ConclusionOur data indicate that survivin may serve as a molecular link between obesity and cancer and as a novel marker for tumor-associated macrophages.

Combining UBR5 and CD163+ tumor-associated macrophages better predicts prognosis of clear cell renal cell carcinoma patients.

Identification of reliable postoperative indicators for accurately evaluating prognosis of clear cell renal cell carcinoma (ccRCC) patients remains an important clinical issue. This study determined the prognostic value of UBR5 expression in ccRCC patients by combining with CD163+ tumor-associated macrophages (TAMs) and the established clinical parameters. The expression of UBR5 was analyzed in ccRCC patients from TCGA databases. A total of 310 ccRCC patients were randomly divided into the training and validation cohorts at a 3:2 or 1:1 ratio, and immunohistochemistry (IHC) and statistical analyses were performed to examine the prognostic value of UBR5 and CD163+ TAMs. UBR5 expression was commonly downregulated in human ccRCC specimens, which was associated with TNM stage, SSIGN, WHO/ISUP Grading and poor prognosis of ccRCC patients. In addition, UBR5 expression was negatively correlated with CD163 expression (a TAM marker) in ccRCC tissues, and combining expressions of UBR5 and CD163 better predicted worse overall survival and progression-free survival of ccRCC patients. Even after multivariable adjustment, UBR5, CD163, TNM stage and SSIGN appeared to be independent risk factors. By time-dependent c-index analysis, the integration of intratumoral UBR5 and CD163 achieved higher c-index value than UBR5, CD163, TNM stage or SSIGN alone in predicting ccRCC patients' prognosis. Moreover, the incorporation of both UBR5 and CD163 into the clinical indicators TNM stage or SSIGN exhibited highest c-index value. Integrating intratumoral UBR5 and CD163+ TAMs with the current clinical parameters achieves better accuracy in predicting ccRCC patients' postoperative prognosis.

Neoadjuvant Chemotherapy Induces IL34 Signaling and Promotes Chemoresistance via Tumor-Associated Macrophage Polarization in Esophageal Squamous Cell Carcinoma.

The tumor microenvironment (TME) plays a key role in the efficacy of neoadjuvant chemotherapy (NAC) in solid tumors including esophageal squamous cell carcinoma (ESCC). However, the TME profile of ESCC treated with NAC is not fully understood. In this study, we investigated the effect of NAC on the TME especially tumor-associated macrophages (TAM), the important immunosuppressive components of the TME, in ESCC. We quantified the expression of CD163, a crucial marker of TAM, in pretherapeutic biopsy and surgically resected ESCC specimens from patients who received NAC (n = 33) or did not receive NAC (n = 12). We found that NAC dramatically increased the expression of CD163 on TAMs in ESCC. Colony-stimulating factor 1 (CSF-1) and IL34 are crucial cytokines that recruit monocytes into tumor sites and differentiate them into TAMs. Interestingly, NAC significantly upregulated the expression of IL34 but not CSF-1 on tumor cells, and the frequencies of CD163+ TAMs were significantly correlated with IL34 expression in ESCC after NAC. The expression of IL34 in NAC-nonresponsive patients was significantly higher than that in NAC-responsive patients, and patients with IL34-high ESCC exhibited worse prognosis as compared with patients with IL34-low ESCC. We also demonstrated that 5-fluorouracil (5-FU)/cisplatin preferentially increased mRNA expression of IL34 on human ESCC cell lines. Human peripheral blood monocytes co-cultured with ESCC cells treated with 5-FU/cisplatin increased the expression of CD163, which was attenuated by the treatment with CSF-1R inhibitors. These data suggest that IL34 expression by NAC shifts the TME toward CD163+ TAM-rich immunosuppressive and chemo-insensitive microenvironment in ESCC. IMPLICATIONS: The blockade of IL34 signaling may offer a novel therapeutic strategy against chemoresistance in ESCC by inhibiting M2-TAM polarization.

Prospective Analyses of Lifestyle Factors Related to Energy Balance and Ovarian Cancer Risk by Infiltration of Tumor-Associated Macrophages.

Lifestyle factors related to energy balance have been associated with ovarian cancer risk and influence the tumor immune microenvironment, including tumor-associated macrophages (TAM). However, no studies have assessed whether these factors differentially impact ovarian cancer risk by TAM densities. We conducted a prospective analysis in the Nurses' Health Studies to examine the associations of physical activity, sitting time, and a food-based empirical dietary inflammatory pattern (EDIP) score with invasive epithelial ovarian cancer risk by TAM density assessed by immunohistochemistry. We considered density of CD68 (marker of total TAMs) and CD163 (marker of pro-carcinogenic M2-type TAMs), and their ratios. We used multivariable Cox proportional hazards regression to calculate hazard ratios (HR) and 95% confidence intervals (CI) of exposures with risk of ovarian tumors with high versus low TAMs, including analyses stratified by body mass index. Analyses included 312 incident ovarian cancer cases with TAM measurements. Physical activity, sitting time, and EDIP score were not differentially associated with ovarian cancer risk by TAM densities (P heterogeneity > 0.05). Among overweight and obese women, higher EDIP score was associated with increased risk of CD163 low-density tumors (HR comparing extreme tertiles, 1.57; 95% CI, 0.88-2.80; P trend = 0.01), but not CD163 high-density tumors (comparable HR, 1.16; 95% CI, 0.73-1.86; P trend = 0.24), though this difference was not statistically significant (P heterogeneity = 0.22). We did not observe differential associations between lifestyle factors and ovarian cancer risk by TAM densities. Future investigations examining the interplay between other ovarian cancer risk factors and the tumor immune microenvironment may help provide insight into ovarian cancer etiology.

Fusion of Invasive Tumor Cells with Infiltrating Macrophages Fuels Epithelial-Mesenchymal Transition and Adaptive Immune Evasion

Heterotypic interaction between tumor cells and adjacent stromal cells mediates tumor development. However, how tumor heterogeneity commits tumors to the malignant transformation and evasion of immunity against metastasis is poorly understood. Here, we have investigated the fusogenicity of human invasive glioblastoma, triple negative breast cancer and gallbladder cancer cells that are all characterized by mesenchymal cell plasticity. These cells displayed the rigorous ability to fuse with macrophages and augment epithelial-mesenchymal transition (EMT), transforming the fused cells into highly invasive hybrids. YKL-40 (Chitinase-3-like-1), known to promote inflammation and serve as an EMT marker, was essential and sufficient for both cell fusion and the invasiveness of tumor cells that express EMT and tumor-associated macrophage markers. Intriguingly, differential gene profiling of single clones from the hybrids demonstrated that YKL-40 and immune checkpoint protein B7-2 (CD86) were elevated and functioned to independently suppress anti-tumor immune factor levels of CD8 + -cytotoxic T lymphocytes (CTL); thus resulting in escape of immune surveillance. YKL-40 and B7-2 dual shRNA abrogated YKL-40-mediated cell fusion and restored CTL anti-tumor immunity, compromising tumor development in xenografts. Clinically, we found tumor hybrids were present in mesenchymal types of glioblastoma, gallbladder cancer and breast cancer. In addition, YKL-40 expression in glioblastoma was correlated with decreased disease-free survival in patients. Collectively, these data offer novel cellular and molecular mechanisms underpinning immune evasion and tumor malignancy, and suggest a new immunotherapeutic intervention strategy by targeting both YKL-40 and B7-2 in cancer.

Single-cell image analysis reveals a protective role for microglia in glioblastoma.

BackgroundMicroglia and tumor-associated macrophages (TAMs) constitute up to half of the total tumor mass of glioblastomas. Despite these myeloid populations being ontogenetically distinct, they have been largely conflated. Recent single-cell transcriptomic studies have identified genes that distinguish microglia from TAMs. Here we investigated whether the translated proteins of genes enriched in microglial or TAM populations can be used to differentiate these myeloid cells in immunohistochemically stained human glioblastoma tissue.MethodsTissue sections from resected low-grade, meningioma, and glioblastoma (grade IV) tumors and epilepsy tissues were immunofluorescently triple-labeled for Iba1 (pan-myeloid marker), CD14 or CD163 (preferential TAM markers), and either P2RY12 or TMEM119 (microglial-specific markers). Using a single-cell-based image analysis pipeline, we quantified the abundance of each marker within single myeloid cells, allowing the identification and analysis of myeloid populations.ResultsP2RY12 and TMEM119 successfully discriminated microglia from TAMs in glioblastoma. In contrast, CD14 and CD163 expression were not restricted to invading TAMs and were upregulated by tumor microglia. Notably, a higher ratio of microglia to TAMs significantly correlated with increased patient survival.ConclusionsWe demonstrate the validity of previously defined microglial-specific genes P2RY12 and TMEM119 as robust discriminators of microglia and TAMs at the protein level in human tissue. Moreover, our data suggest that a higher proportion of microglia may be beneficial for patient survival in glioblastoma. Accordingly, this tissue-based method for myeloid population differentiation could serve as a useful prognostic tool.

Thrombospondin-4 mediates hyperglycemia- and TGF-beta-induced inflammation in breast cancer.

Inflammation drives the growth of tumors and is an important predictor of cancer aggressiveness. CD68, a marker of tumor-associated macrophages (TAM), is routinely used to aid in prognosis and treatment choices for breast cancer patients. We report that thrombospondin-4 (TSP-4) mediates breast cancer inflammation and growth in mouse models in response to hyperglycemia and TGF-beta by increasing TAM infiltration and production of inflammatory signals in tumors. Analysis of breast cancers and noncancerous tissue specimens from hyperglycemic patients revealed that levels of TSP-4 and of macrophage marker CD68 are upregulated in diabetic tissues. TSP-4 was colocalized with macrophages in cancer tissues. Bone-marrow-derived macrophages (BMDM) responded to high glucose and TGF-beta by upregulating TSP-4 production and expression, as well as the expression of inflammatory markers. We report a novel function for TSP-4 in breast cancer: regulation of TAM infiltration and inflammation. The results of our study provide new insights into regulation of cancer growth by hyperglycemia and TGF-beta and suggest TSP-4 as a potential therapeutic target.

BIOM-20. PROTEOMICS PROVIDES POTENTIAL THERAPEUTIC APPROACHES TO GLIOBLASTOMA TREATMENT: RESULTS FROM THE CLINICAL PROTEOMICS TUMOR ANALYSIS CONSORTIUM (CPTAC)

Abstract Glioblastoma (GBM) is the most common type of primary malignant tumor in adults and contributes disproportionately to cancer morbidity and mortality. The ability to augment current knowledge based on the genome and transcriptome with information regarding protein abundance and post-translational modifications with small molecule and lipid abundance has provided novel biological insights into GBM tumorigenesis. In this presentation we will discuss results from proteogenomic and metabolomic investigations of prospectively recruited, newly diagnosed, treatment-naive glioblastomas (GBMs; N=99). Multiple key findings emerged that were not known from traditional genomic analyses as implemented in most currently available datasets. Analysis of protein phosphorylation identified key signaling intermediates in the RTK/RAS pathway common to multiple RTK genomic alterations, potentially offering common therapeutic targets for different oncogenic drivers in GBM. Phosphoproteomics also identified potential druggable targets based on kinase-substrate pathway analysis, as well as novel phosphoprotein targets associated with the regulation of telomere length by ATRX in IDH mutants. Identification of distinct Immune High and Immune Low phenotypes in GBM from whole genome interrogation of the proteome were driven by tumor-associated macrophage markers, and associated with distinct epigenetic modifications and histone acetylation patterns. Identification of key metabolic changes in IDH mutants facilitating the accumulation of oncometabolite 2-HG were also associated with lipidomic changes. Taken all together this work identifies additional therapeutic channels for GBM and novel information useful for more accurate stratification patients for effective treatment.

Abstract 2046: Inhibiting IDH mutations in low-grade glioma alters cellular function and the immune environment

Abstract Somatic mutations in the genes encoding the metabolic enzymes isocitrate dehydrogenase (IDH) 1 and 2 occur in many cancers, including glioma, cholangiocarcinoma, and acute myeloid leukemia. Mutant IDH (mIDH) proteins have a gain-of-function enzyme activity, catalyzing the reduction of alpha-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (2-HG). 2-HG competitively inhibits α-KG-dependent enzymes, leading to epigenetic dysregulation and tumorigenesis. TET2 is an α-KG-dependent enzyme mediating the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which leads to DNA demethylation. In mIDH tumors, TET2 activity is inhibited by 2-HG, leading to DNA hypermethylation. In addition, IDH mutations are associated with immune evasion in gliomas. IDH mutations are common in low-grade gliomas (LGG; WHO grade 2/3) and include mutations in IDH1 (~80%) and IDH2 (~4%). Ivosidenib (AG-120, IVO) is a first-in-class, oral inhibitor of mIDH1, and vorasidenib (AG-881, VOR) is an oral, potent, brain-penetrant inhibitor of both mIDH1 and mIDH2. In an ongoing perioperative study (NCT03343197), IVO and VOR demonstrated brain penetrance and >90% suppression of 2-HG in resected mIDH1 gliomas after pre-operative treatment for approximately 4 weeks. In this analysis, we examined the cellular mechanisms underlying mIDH inhibition in LGG using resected tumor tissues collected following treatment with IVO and VOR. Optimal 2-HG suppression (defined by post-treatment 2-HG levels equal to or lower than the upper range of IDH wild-type tumor 2-HG levels) in IVO- or VOR-treated samples (n=17) led to a ~2-fold increase in 5hmC compared with untreated controls and banked reference samples (n=35; P=0.04). Furthermore, immunohistochemistry (IHC) analysis of the proliferation marker Ki-67 showed a ~3-fold decrease in the percentage of Ki-67-positive cells in IVO- or VOR-treated samples with optimal 2-HG suppression (n=21) compared with untreated controls (n=5; P=0.02). Tumor immune microenvironment analyses were also conducted. Comparing IVO- or VOR-treated samples (n=21) with untreated controls and banked reference samples (n=33), RNAseq analysis showed a ~2-fold decrease in the expression of tumor-associated macrophage markers CD68 (P=0.01) and CD163 (P=0.03) in mIDH oligodendrogliomas. In addition, IHC analysis from paired pre-treatment (archival) and post-treatment samples showed an increase of CD3+ T-cell infiltration (P=0.03) and CD8+ T-cell infiltration (P=0.01) in IVO- or VOR-treated samples with optimal 2-HG suppression (n=21). Single-cell RNAseq was conducted for a subset of samples, and the results will be presented. Overall, these data suggest that mIDH inhibition decreases DNA hypermethylation and tumor cell proliferation, and activates the immune microenvironment in mIDH LGG. In conclusion, these data elucidate the mechanisms underlying mIDH inhibition and support further development of mIDH inhibitors in mIDH LGG. Page 1 of 1 Citation Format: Min Lu, Ingo K. Mellinghoff, Aaron Diaz, Jennie W. Taylor, Sung Choe, Ania Tassinari, Dongwei Zhu, Katie Sellers, Kha Le, Feng Tai, Islam Hassan, Shuchi S. Pandya, Lori Steelman, Bin Wu. Inhibiting IDH mutations in low-grade glioma alters cellular function and the immune environment [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 2046.

The standard form of CD44 as a marker for invasion of encapsulated papillary carcinoma of the breast.

Encapsulated papillary carcinoma (EPC), a rare variant of papillary carcinoma of the breast, is regarded as a transition form between carcinoma in situ and invasive carcinoma. Here, we tried to identify differences in immunohistochemical phenotype between 10 EPCs with invasive properties (EPC with invasion) and 17 non-invasive EPCs (EPC). We immunohistochemically assessed the expression of hormone receptors, matrix metalloproteinase (MMP) 2 and MMP9, vascular endothelial growth factor (VEGF), CD31, and D2-40, markers of tumor-associated macrophages (CD163, CD206), Ki-67 and stem cell markers (CD44 and CD24). The frequency of MMP9-positive cases and the number of tumor-associated macrophages infiltrating into the fibrous capsule were significantly higher in EPC with invasion than in EPC. The expression of the standard form of CD44 (CD44s) was significantly stronger in EPC with invasion than in EPC (P = 0.0036) and was correlated with MMP2 expression and M2-like macrophage infiltration. A multivariate logistic model analysis showed that CD44s expression in tumor cell and infiltration of CD163 positive macrophage in EPC capsule showed an independent odds ratio for invasion of EPC. Thus, CD44s may be a potential marker predicting invasive potential of EPC and could play an important role in progression to the invasive phase of EPC.

Overexpression of PREX1 in oral squamous cell carcinoma indicates poor prognosis.

Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger (P-Rex) proteins control many fundamental cellular functions including cell migration, actin cytoskeletal rearrangement and adhesion in many cancers. However, P-Rex1 expression and its prognostic effect and possible clinical value are not clearly elucidated in human oral squamous cell carcinoma (OSCC). Here, OSCC tissue microarrays were used to verify the expression levels of P-Rex1, coinhibitory immune checkpoints and tumor associated macrophage (TAM) markers, and to analyze the relationship between PREX1 expression levels and clinicopathological characteristics in OSCC. The study found that P-Rex1 expression was elevated in OSCC compared to dysplasia and normal mucosa (P < 0.0001). In addition, patients who expressed high PREX1 had a poorer prognosis than those who expressed low PREX1 (P = 0.0070). Furthermore, positive correlations were found between P-Rex1 expression and the immune checkpoints PD-L1, Galectin-9 and B7-H4, and the TAM markers CD68, CD206 and CD163. In short, these findings implicated that overexpression of P-Rex1 may predict a poor prognosis in human OSCC.

Prognosis of Macrophage Density in the Absence of Neutrophils in Differentiated Thyroid Cancer

BackgroundDespite the advances in treatment of differentiated thyroid cancer (DTC), predicting prognosis remains a challenge. Immune cells in the tumor microenvironment may provide an insight to predicting recurrence. Therefore, the objective of this study was to investigate the association of tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) with recurrence in DTC and to identify serum cytokines that correlate with the presence of these immune cells in the tumor. Materials and methodsForty-two DTC tissues from our institutional neoplasia repository were stained for immunohistochemistry markers for TAMs and TANs. In addition, cytokine levels were analyzed from these patients from preoperative blood samples. TAM and TAN staining were compared with clinical data and serum cytokine levels. ResultsNeither TAM nor TAN scores alone correlated with tumor size, the presence of lymph node metastases, multifocal tumors, lymphovascular or capsular invasion, or the presence of BRAFV600E mutation (all P > 0.05). There was no association with recurrence-free survival (RFS) in TAN density (mean RFS, 169.1 versus 148.1 mo, P = 0.23) or TAM density alone (mean RFS, 121.3 versus 205.2 mo, P = 0.54). However, when scoring from both markers were combined, patients with high TAM density and TAN negative scores had significantly lower RFS (mean RFS, 50.7 versus 187.3 mo, P = 0.04) compared with the remaining cohort. Patients with high TAM/negative TAN tumors had significantly lower serum levels of interleukin 12p70, interleukin 8, tumor necrosis factor alpha, and tumor necrosis factor beta. ConclusionsIn DTCs, high density of TAMs in the absence of TANs is associated with worse outcome. Assessment of multiple immune cell types and serum cytokines may predict outcomes in DTC.