HXR9 Inhibits the HOX-PBX Cluster, Inducing Glioma Apoptosis and Cell Cycle Arrest

https://openworks.mdanderson.org/sumexp22/1066/thumbnail.jp

Validation of CyTOF Against Flow Cytometry for Immunological Studies and Monitoring of Human Cancer Clinical Trials

Flow cytometry is a widely applied approach for exploratory immune profiling and biomarker discovery in cancer and other diseases. However, flow cytometry is limited by the number of parameters that can be simultaneously analyzed, severely restricting its utility. Recently, the advent of mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system, allowing simultaneous interrogation of a large number of parameters. This is important for deep interrogation of immune responses and particularly when sample sizes are limited (such as in tumors). Our goal was to compare the accuracy and reproducibility of CyTOF against flow cytometry as a reliable analytic tool for human PBMC and tumor tissues for cancer clinical trials. We developed a 40+ parameter CyTOF panel and demonstrate that compared to flow cytometry, CyTOF yields analogous quantification of cell lineages in conjunction with markers of cell differentiation, function, activation, and exhaustion for use with fresh and viably frozen PBMC or tumor tissues. Further, we provide a protocol that enables reliable quantification by CyTOF down to low numbers of input human cells, an approach that is particularly important when cell numbers are limiting. Thus, we validate CyTOF as an accurate approach to perform high dimensional analysis in human tumor tissue and to utilize low cell numbers for subsequent immunologic studies and cancer clinical trials

The immunomodulatory role of tumor Syndecan-1 (CD138) on ex vivo tumor microenvironmental CD4+ T cell polarization in inflammatory and non-inflammatory breast cancer patients.

Herein, we aimed to identify the immunomodulatory role of tumor Syndecan-1 (CD138) in the polarization of CD4+ T helper (Th) subsets isolated from the tumor microenvironment of inflammatory breast cancer (IBC) and non-IBC patients. Lymphocytes and mononuclear cells isolated from the axillary tributaries of non-IBC and IBC patients during modified radical mastectomy were either stimulated with the secretome as indirect co-culture or directly co-cultured with control and Syndecan-1-silenced SUM-149 IBC cells. In addition, peripheral blood mononuclear cells (PBMCs) of normal subjects were used for the direct co-culture. Employing flow cytometry, we analyzed the expression of the intracellular IFN-γ, IL-4, IL-17, and Foxp3 markers as readout for basal and co-cultured Th1, Th2, Th17, and Treg CD4+ subsets, respectively. Our data revealed that IBC displayed a lower basal frequency of Th1 and Th2 subsets than non-IBC. Syndecan-1-silenced SUM-149 cells significantly upregulated only Treg subset polarization of normal subjects relative to controls. However, Syndecan-1 silencing significantly enhanced the polarization of Th17 and Treg subsets of non-IBC under both direct and indirect conditions and induced only Th1 subset polarization under indirect conditions compared to control. Interestingly, qPCR revealed that there was a negative correlation between Syndecan-1 and each of IL-4, IL-17, and Foxp3 mRNA expression in carcinoma tissues of IBC and that the correlation was reversed in non-IBC. Mechanistically, Syndecan-1 knockdown in SUM-149 cells promoted Th17 cell expansion via upregulation of IL-23 and the Notch ligand DLL4. Overall, this study indicates a low frequency of the circulating antitumor Th1 subset in IBC and suggests that tumor Syndecan-1 silencing enhances ex vivo polarization of CD4+ Th17 and Treg cells of non-IBC, whereby Th17 polarization is possibly mediated via upregulation of IL-23 and DLL4. These findings suggest the immunoregulatory role of tumor Syndecan-1 expression in Th cell polarization that may have therapeutic implications for breast cancer

Inflammatory breast cancer: Activation of the aryl hydrocarbon receptor and its target CYP1B1 correlates closely with Wnt5a/b-β-catenin signalling, the stem cell phenotype and disease progression

The aim of the present study was to evaluate the expression levels of the aryl hydrocarbon receptor (AHR) and its target gene CYP1B1 and to correlate their expression with Wnt5a/b-β-catenin, the CD44+/CD24(−/low) cancer stem cell (CSC) subset and factors associated with poor prognosis in inflammatory breast cancer (IBC) and non-IBC patients. The methods of analysis used were quantitative real-time PCR, western blotting, immunohistochemistry and flow cytometry. Compared to non-IBC tissues, IBC tissues exhibited the overexpression of AHR and its target gene/protein CYP1B1. AHR and CYP1B1 mRNA levels were associated with the poor clinical prognosis markers tumour grade, lymphovascular invasion, cell proliferation and lymph node metastasis. Furthermore, AHR expression correlated with the expression of Wnt5a/b and β-catenin signalling molecules, and Wnt5a mRNA expression was downregulated in the SUM149 human IBC cell line and the MDA-MB-231 non-IBC cell line upon inhibition of AHR. AHR gene knockout (CRISPR-Cas9) inhibits CYP1B1 and Wnt5a expression in the IBC cell line. The CD44+/CD24(−/low) subset was significantly correlated with the expression of AHR, CYP1B1, Wnt5a/b and β-catenin in IBC tissues. The overexpression of AHR and its target CYP1B1 correlated with the expression of Wnt5a/b and β-catenin, CSCs, and poor clinical prognostic factors of IBC. Thus, targeting AHR and/or its downstream target molecules CYP1B1 and Wnt5a/b may represent a therapeutic approach for IBC. Keywords: Inflammatory breast cancer, Aryl hydrocarbon receptor, CYP1B1, Wnt5a/b and β-catenin, CD44+/CD24(−/low) subset and lymphovascular invasio

Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

BACKGROUND: Inflammatory breast cancer (IBC), a particularly aggressive form of breast cancer, is characterized by cancer stem cell (CSC) phenotype. Due to a lack of targeted therapies, the identification of molecular markers of IBC is of major importance. The heparan sulfate proteoglycan Syndecan-1 acts as a coreceptor for growth factors and chemokines, modulating inflammation, tumor progression, and cancer stemness, thus it may emerge as a molecular marker for IBC. METHODS: We characterized expression of Syndecan-1 and the CSC marker CD44, Notch-1 & -3 and EGFR in carcinoma tissues of triple negative IBC (n = 13) and non-IBC (n = 17) patients using qPCR and immunohistochemistry. Impact of siRNA-mediated Syndecan-1 knockdown on the CSC phenotype of the human triple negative IBC cell line SUM-149 and HER-2-overexpressing non-IBC SKBR3 cells employing qPCR, flow cytometry, Western blotting, secretome profiling and Notch pharmacological inhibition experiments. Data were statistically analyzed using Student’s t-test/Mann-Whitney U-test or one-way ANOVA followed by Tukey’s multiple comparison tests. RESULTS: Our data indicate upregulation and a significant positive correlation of Syndecan-1 with CD44 protein, and Notch-1 & -3 and EGFR mRNA in IBC vs non-IBC. ALDH1 activity and the CD44(+)CD24(-/low) subset as readout of a CSC phenotype were reduced upon Syndecan-1 knockdown. Functionally, Syndecan-1 silencing significantly reduced 3D spheroid and colony formation. Intriguingly, qPCR results indicate downregulation of the IL-6, IL-8, CCL20, gp130 and EGFR mRNA upon Syndecan-1 suppression in both cell lines. Moreover, Syndecan-1 silencing significantly downregulated Notch-1, -3, -4 and Hey-1 in SUM-149 cells, and downregulated only Notch-3 and Gli-1 mRNA in SKBR3 cells. Secretome profiling unveiled reduced IL-6, IL-8, GRO-alpha and GRO a/b/g cytokines in conditioned media of Syndecan-1 knockdown SUM-149 cells compared to controls. The constitutively activated STAT3 and NFκB, and expression of gp130, Notch-1 & -2, and EGFR proteins were suppressed upon Syndecan-1 ablation. Mechanistically, gamma-secretase inhibition experiments suggested that Syndecan-1 may regulate the expression of IL-6, IL-8, gp130, Hey-1, EGFR and p-Akt via Notch signaling. CONCLUSIONS: Syndecan-1 acts as a novel tissue biomarker and a modulator of CSC phenotype of triple negative IBC via the IL-6/STAT3, Notch and EGFR signaling pathways, thus emerging as a promising therapeutic target for IBC.<br

Additional file 3: Figure S2. of Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

Flow cytometric analysis of Syndecan-1 expression in control and Syndecan-1 siRNA transfected SUM-149 cells. 500,000 cells were stained for isotype control mouse IgG1-PE and mouse anti-human Syndecan-1 (CD138)-PE and the cells were subjected to flow cytometry. Each plot shows mouse IgG-PE control (dotted line) and CD138-PE-stained cells (solid line). The median fluorescence intensity (MFI) of events is given for each peak. Data are a single experiment representative of three independent experiments. (ZIP 281 kb

Additional file 2: Figure S1. of Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

A representative image of tumor emboli, a unique feature for tissues of IBC patients showing a positive staining for CD44 and Syndecan-1. (ZIP 2337 kb

Additional file 1: Table S1. of Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

Primers sequences. (DOCX 18 kb

Tumor reactive γδ T cells contribute to a complete response to PD-1 blockade in a Merkel cell carcinoma patient

Abstract Immunotherapies targeting PD-1/PD-L1 are now widely used in the clinic to treat a variety of malignancies. While most of the research on T cell exhaustion and PD-1 blockade has been focused on conventional αβ T cells, the contribution of innate-like T cells such as γδ T cells to anti-PD-1/PD-L1 mediated therapy is limited. Here we show that tumor reactive γδ T cells respond to PD-1 blockade in a Merkel cell carcinoma (MCC) patient experiencing a complete response to therapy. We find clonally expanded γδ T cells in the blood and tumor after pembrolizumab treatment, and this Vγ2Vδ1 clonotype recognizes Merkel cancer cells in a TCR-dependent manner. Notably, the intra-tumoral γδ T cells in the MCC patient are characterized by higher expression of PD-1 and TIGIT, relative to conventional CD4 and CD8 T cells. Our results demonstrate that innate-like T cells could also contribute to an anti-tumor response after PD-1 blockade

Translational randomized phase II trial of cabozantinib in combination with nivolumab in advanced, recurrent, or metastatic endometrial cancer

Background Combining immunotherapy and antiangiogenic agents is a promising treatment strategy in endometrial cancer. To date, no biomarkers for response have been identified and data on post-immunotherapy progression are lacking. We explored the combination of a checkpoint inhibitor (nivolumab) and an antiangiogenic agent (cabozantinib) in immunotherapy-naïve endometrial cancer and in patients whose disease progressed on previous immunotherapy with baseline biopsy for immune profiling.Patients and methods In this phase II trial (ClinicalTrials.gov NCT03367741, registered December 11, 2017), women with recurrent endometrial cancer were randomized 2:1 to nivolumab with cabozantinib (Arm A) or nivolumab alone (Arm B). The primary endpoint was Response Evaluation Criteria in Solid Tumors-defined progression-free survival (PFS). Patients with carcinosarcoma or prior immune checkpoint inhibitor received combination treatment (Arm C). Baseline biopsy and serial peripheral blood mononuclear cell (PBMC) samples were analyzed and associations between patient outcome and immune data from cytometry by time of flight (CyTOF) and PBMCs were explored.Results Median PFS was 5.3 (90% CI 3.5 to 9.2) months in Arm A (n=36) and 1.9 (90% CI 1.6 to 3.4) months in Arm B (n=18) (HR=0.59, 90% CI 0.35 to 0.98; log-rank p=0.09, meeting the prespecified statistical significance criteria). The most common treatment-related adverse events in Arm A were diarrhea (50%) and elevated liver enzymes (aspartate aminotransferase 47%, alanine aminotransferase 42%). In-depth baseline CyTOF analysis across treatment arms (n=40) identified 35 immune-cell subsets. Among immunotherapy-pretreated patients in Arm C, non-progressors had significantly higher proportions of activated tissue-resident (CD103+CD69+) ɣδ T cells than progressors (adjusted p=0.009).Conclusions Adding cabozantinib to nivolumab significantly improved outcomes in heavily pretreated endometrial cancer. A subgroup of immunotherapy-pretreated patients identified by baseline immune profile and potentially benefiting from combination with antiangiogenics requires further investigation