Correction: Gold(I) complexes based on six-membered phosphorus heterocycles as bio-active molecules against brain cancer.

Correction for 'Gold(i) complexes based on six-membered phosphorus heterocycles as bio-active molecules against brain cancer' by Saskia Roesch et al., Chem. Commun., 2020, DOI: 10.1039/d0cc05761d.

Gold(i) complexes based on six-membered phosphorus heterocycles as bio-active molecules against brain cancer.

π-Systems based on six-membered phosphorus heterocycles possess structural and electronic characteristics that clearly distinguish them from the rest of the organophosphorus molecules. However, their use in cancer therapy has been uninvestigated. In particular, glioblastoma is one of the most lethal brain tumors. The development of novel and more efficient drugs for the treatment of glioblastoma is thus crucial to battle this aggressive disease. Herein, we report a new family of gold(i) complexes based on six-membered phosphorus heterocycles as a promising tool to investigate brain cancer. We discovered that the latter complexes inhibit the proliferation, sensitize to apoptosis and hamper the migration of not only conventional but also stem-like glioblastoma cells. Our results unveil thus new research opportunities for the treatment of glioblastoma.

Cytotoxic T Cells and their Activation Status are Independent Prognostic Markers in Meningiomas.

PURPOSE: Clinically aggressive meningiomas (MGMs) are rare but treatment-resistant tumors in need for more effective therapies. Because tumor-infiltrating T lymphocytes (TILs) are essential for successful immunotherapy, we assessed TIL numbers and their activation status in primary (p-) and recurrent (r-) meningiomas and their impact on survival. EXPERIMENTAL DESIGN: Presence of TILs was analyzed in 202 clinically well-annotated cases (n = 123 pMGMs and n = 79 rMGMs) focusing on higher-grade meningiomas [n = 97 World Health Organization (WHO) °II, n = 62 WHO°III]. TILs were quantified by a semiautomated analysis on whole-tissue sections stained by multicolor immunofluorescence for CD3, CD8, FOXP3, and programmed cell death protein 1 (PD-1). RESULTS: Median T-cell infiltration accounted for 0.59% TILs per total cell count. Although there were no significant WHO°-dependent changes regarding helper (CD3+CD8-FOXP3-) and cytotoxic (CD3+CD8+FOXP3-) TILs in pMGMs, higher number of cytotoxic TILs were associated with an improved progression-free survival (PFS) independent of prognostic confounders. rMGMs were characterized by lower numbers of TILs in general, helper, and cytotoxic TILs. The additional analysis of their activation status revealed that a proportion of PD-1+CD8+ TILs within the TIL population was significantly decreased with higher WHO grade and in rMGMs. Furthermore, lower proportions of PD-1+CD8+ TILs were associated with inferior PFS in multivariate analyses, arguing for PD-1 as activation rather than exhaustion marker. CONCLUSIONS: We identified higher numbers of CD3+CD8+FOXP3- TILs and proportions of PD-1-expressing CD3+CD8+FOXP3- TILs as novel biomarkers for better survival. These findings might facilitate the selection of patients who may benefit from immunotherapy and argue in favor of an intervention in primary rather than recurrent tumors.

PET/CT Imaging of NSCLC with a αvß6 Integrin-Targeting Peptide.

PURPOSE: Targeted therapies are regarded as promising approaches to increase 5-year survival rate of non-small cell lung cancer (NSCLC) patients. Here, we investigated the clinical value of the αvß6 integrin-specific peptide SFITGv6 as a diagnostic reagent targeting NSCLC. METHODS: Affinity and binding properties of [125I]SFITGv6 or [177Lu]SFITGv6 for αvß6 integrin-expressing NSCLC cell lines were evaluated in cell culture experiments including competition, kinetic, internalization, and efflux. To confirm αvß6 integrin specificity in vivo small-animal positron emission tomography (PET) imaging using [68Ga]SFITGv6 as radiotracer and biodistribution of [177Lu]SFITGv6 in NCI-H2009 and NCI-H322 tumor-bearing mice was performed. Finally, to distinguish between benign and malignant lesions [68Ga]SFITGv6 was applied as radiotracer for PET/x-ray computed tomography (CT) imaging of NSCLC patients with unclear diagnosis upon routinely performed 2-deoxy-2-[18F]flouro-D-glucose ([18F]FDG)-PET/CT. The biodistribution of the SFITGv6-ligand in different organs and tumor lesions of NSCLC patients was quantified 1 h and 3 h after injection measuring standard uptake values (SUV)max. RESULTS: In vitro experiments revealed a significant time-dependent SFITGv6 binding of up to 33 % to αvß6 integrin-expressing the cell lines NCI-H2009, NCI-H322, NCI-H292, NCI-H358, and high affinity (IC50-mean 3.1 nM) to NCI-H2009 and NCI-H322. Moreover, a fast internalization of approximately 66 % by NCI-H2009 and NCI-H322 cells was observed. Small-animal PET imaging and biodistribution experiments of NCI-H2009 and NCI-H322 xenografts demonstrated an increased tumor-specific accumulation of SFITGv6 40 to 60 min after injection. Finally, PET/CT scans of NSCLC patients after [18F] FDG injection followed by [68Ga]SFITGv6 application revealed correlating images. Comparing the uptake of [68Ga]SFITGv6 and [18F] FDG both PET/CT-examinations presented with significantly increased SUVmax values in histologically proven NSCLC lesions, but a generally higher accumulation of [18F] FDG was noticed. CONCLUSIONS: Even if SFITGv6 demonstrates excellent affinity and specificity for αvß6 integrin-expressing NSCLC cell lines and several NSCLC xenografts [18F]FDG-PET/CT provides an advantage over [68Ga]SFITGv6-PET/CT for the diagnosis of NSCLC patients.

Expression of PD-1 by T Cells in Malignant Glioma Patients Reflects Exhaustion and Activation.

PURPOSE: Glioblastoma (GBM) is the most common primary malignant tumor in the central nervous system. Our recent preclinical work has suggested that PD-1/PD-L1 plays an important immunoregulatory role to limit effective antitumor T-cell responses induced by active immunotherapy. However, little is known about the functional role that PD-1 plays on human T lymphocytes in patients with malignant glioma.Experimental Design: In this study, we examined the immune landscape and function of PD-1 expression by T cells from tumor and peripheral blood in patients with malignant glioma. RESULTS: We found several differences between PD-1+ tumor-infiltrating lymphocytes (TIL) and patient-matched PD-1+ peripheral blood T lymphocytes. Phenotypically, PD-1+ TILs exhibited higher expression of markers of activation and exhaustion than peripheral blood PD-1+ T cells, which instead had increased markers of memory. A comparison of the T-cell receptor variable chain populations revealed decreased diversity in T cells that expressed PD-1, regardless of the location obtained. Functionally, peripheral blood PD-1+ T cells had a significantly increased proliferative capacity upon activation compared with PD-1- T cells. CONCLUSIONS: Our evidence suggests that PD-1 expression in patients with glioma reflects chronically activated effector T cells that display hallmarks of memory and exhaustion depending on its anatomic location. The decreased diversity in PD-1+ T cells suggests that the PD-1-expressing population has a narrower range of cognate antigen targets compared with the PD-1 nonexpression population. This information can be used to inform how we interpret immune responses to PD-1-blocking therapies or other immunotherapies.

Comparison of the RGD Motif-Containing αvß6 Integrin-Binding Peptides SFLAP3 and SFITGv6 for Diagnostic Application in HNSCC.

αvß6 integrin is overexpressed by several carcinomas and thus considered a target for diagnostic imaging and anticancer therapies. Recently, we presented the αvß6 integrin-binding peptide SFITGv6 as a novel potential tracer for imaging and targeted therapy of αvß6 integrin-positive carcinomas. Here, we analyzed the affinity and specificity of 5 native αvß6 integrin-specific binders in comparison to SFITGv6. Methods: Sunflower trypsin inhibitor 1 (SFTI1)-based peptides containing arginine-glycine-aspartic acid (RGD) motif-spanning octamers of fibronectin (SFFN1), tenascin C (SFTNC), vitronectin (SFVTN), and latency-associated peptides (LAP) 1 (SFLAP1) and 3 (SFLAP3) were synthesized, and their binding potential to αvß6 integrin-expressing head and neck squamous cell carcinoma (HNSCC) cell lines was evaluated. Subsequently, stability, affinity, and specificity were assessed in vitro using radio-high-pressure liquid chromatography, surface plasmon resonance assay, and binding experiments including competition, kinetics, internalization, and efflux. αvß6 integrin binding specificity was further evaluated by peptide histochemistry. Finally, in vivo binding properties were assessed using small-animal PET imaging and biodistribution experiments in HNSCC-bearing mice, and 68Ga-DOTA-SFLAP3 was applied for diagnostic PET/CT of an HNSCC patient. Results: When the newly designed peptides were compared, significant binding (>20%) to several HNSCC cell lines (HNO97, HNO399, and HNO223) and a fast internalization of up to 60% and 70% were observed for SFLAP3 (GRGDLGRL) and SFITGv6 (FRGDLMQL). In contrast, the other peptides displayed binding that was moderate (SFLAP1, 4.1%-12.1%) to marginal (SFFN1, SFTNC, and SFVTN, <1%) and were therefore excluded from further analysis. Notably, SFLAP3 exhibited improved affinity for αvß6 integrin (mean half-maximal inhibitory concentration, 3.5 nM; dissociation constant, 7.4). Moreover, small-animal PET imaging and biodistribution studies of HNSCC xenograft mice revealed an increased tumor-specific accumulation 30-60 min after injection of 68Ga-labeled or 177Lu-labeled DOTA-SFLAP3. Peptide staining further demonstrated binding specificity for SFLAP3 to HNSCC tumor cells. Finally, PET/CT scanning of an HNSCC patient showed specific SFLAP3 accumulation in the primary tumor lesion (SUVmax, 5.1) and in corresponding lymph node metastases (SUVmax, 4.1). Conclusion: SFLAP3 represents a promising tracer for prognostic assessment, diagnostic imaging, and possibly targeted therapy of αvß6 integrin-expressing tumors.

Identification of CRKII, CFL1, CNTN1, NME2, and TKT as Novel and Frequent T-Cell Targets in Human IDH-Mutant Glioma.

Purpose: Successful immunotherapies for IDHmut gliomas require better knowledge of T-cell target antigens. Here, we elucidated their antigen repertoire recognized by spontaneous T-cell responses using an unbiased proteomic approach.Experimental Design: Protein fractionations of tissue lysates from IDHmut gliomas (n = 4) were performed. Fractions were tested by IFNγ ELISpot assay for recognition through patients' T cells. Proteins of immunogenic fractions were identified by mass spectrometry and validated by in silico-predicted synthetic long peptides in patients of origin, additional IDHmut glioma patients (n = 16), and healthy donors (n = 13). mRNA and protein expression of immunogenic antigens was analyzed in tumor tissues and IDHmut glioma stem-like cells (GSC). HLA-A*02-restricted T-cell epitopes were functionally determined by short peptides and numbers of antigen-specific T cells by HLA-peptide tetramer analysis.Results: A total of 2,897 proteins were identified in immunogenic tumor fractions. Based on a thorough filter process, 79 proteins were selected as potential T-cell antigens. Twenty-six of these were recognized by the patients' T cells, and five of them (CRKII, CFL1, CNTN1, NME2, and TKT) in up to 56% unrelated IDHmut glioma patients. Most immunogenic tumor-associated antigens (TAA) were expressed in IDHmut gliomas and GSCs, while being almost absent in normal brain tissues. Finally, we identified HLA-A*02-restricted epitopes for CRKII, NME2, and TKT that were recognized by up to 2.82% of antigen-specific peripheral cytotoxic T cells in IDHmut glioma patients.Conclusions: By analyzing the repertoire of T-cell target antigens in IDHmut glioma patients, we identified five novel immunogenic TAAs and confirmed their expression on IDHmut tumors and GSCs. Clin Cancer Res; 24(12); 2951-62. ©2018 AACR.

When Immune Cells Turn Bad-Tumor-Associated Microglia/Macrophages in Glioma.

As a substantial part of the brain tumor microenvironment (TME), glioma-associated microglia/macrophages (GAMs) have an emerging role in tumor progression and in controlling anti-tumor immune responses. We review challenges and improvements of cell models and highlight the contribution of this highly plastic cell population to an immunosuppressive TME, besides their well-known functional role regarding glioma cell invasion and angiogenesis. Finally, we summarize first therapeutic interventions to target GAMs and their effect on the immunobiology of gliomas, focusing on their interaction with T cells.

Identification of a Novel ITGαvß6-Binding Peptide Using Protein Separation and Phage Display.

Purpose: Targeted therapies are regarded as promising approaches to increase 5-year survival rate of head and neck squamous cell carcinoma (HNSCC) patients.Experimental design: For the selection of carcinoma-specific peptides membrane proteome of HNO97 tumor cells fractionated by the ProteomeLab PF2D system and corresponding HNO97 cells were deployed for an alternating biopanning using a sunflower trypsin inhibitor1-based phage display (SFTI8Ph) library. Stability, binding properties and affinity of novel candidates were assessed in vitro using radio-HPLC, binding experiments and surface plasmon resonance assay (SPR), respectively. Subsequently, the affinity of the peptide was verified in situ by using peptide histochemistry, in vitro using flow cytometry, and in vivo by positron emissions tomography (PET/CT).Results: We identified a novel ITGαvß6 binding peptide (SFITGv6) containing the amino acid sequence FRGDLMQL. SFITGv6 provides stability over a period of 24 hours and demonstrates high affinity (KD = 14.8 nmol/L) for ITGαvß6 In HNO97 cells, a maximal uptake and internalization of up to 37.3% and 37.5%, respectively, was measured. Small-animal PET imaging and biodistribution studies of HNO97 xenografted Balb/c nu/nu mice showed tumor-specific accumulation of 68Ga- and 177Lu-labeled DOTA-SFITGv6, respectively, 30 to 60 minutes after injection. Moreover, peptide histochemistry revealed a strong and homogenous binding of biotin-labeled SFITGv6 to HNSCC tumors and breast- and lung cancer-derived brain metastases. Finally, first PET/CT scans of HNSCC and NSCLC patients displayed SFITGv6 accumulation specifically in tumors, but not in inflammatory lesions.Conclusions: Thus, SFITGv6 represents a novel powerful tracer for imaging and possibly for endoradiotherapy of ITGαvß6-positive carcinoma. Clin Cancer Res; 23(15); 4170-80. ©2017 AACR.

Identification of T cell target antigens in glioblastoma stem-like cells using an integrated proteomics-based approach in patient specimens.

Glioblastoma (GBM) is a highly aggressive brain tumor and still remains incurable. Among others, an immature subpopulation of self-renewing and therapy-resistant tumor cells-often referred to as glioblastoma stem-like cells (GSCs)-has been shown to contribute to disease recurrence. To target these cells personalized immunotherapy has gained a lot of interest, e.g. by reactivating pre-existing anti-tumor immune responses against GSC antigens. To identify T cell targets commonly presented by GSCs and their differentiated counterpart, we used a proteomics-based separation of GSC proteins in combination with a T cell activation assay. Altogether, 713 proteins were identified by LC-ESI-MS/MS mass spectrometry. After a thorough filtering process, 32 proteins were chosen for further analyses. Immunogenicity of corresponding peptides was tested ex vivo. A considerable number of these antigens induced T cell responses in GBM patients but not in healthy donors. Moreover, most of them were overexpressed in primary GBM and also highly expressed in recurrent GBM tissues. Interestingly, expression of the most frequent T cell target antigens could also be confirmed in quiescent, slow-cycling GSCs isolated in high purity by the DEPArray technology. Finally, for a subset of these T cell target antigens, an association between expression levels and higher T cell infiltration as well as an increased expression of positive immune modulators was observed. In summary, we identified novel immunogenic proteins, which frequently induce tumor-specific T cell responses in GBM patients and were also detected in vitro in therapy-resistant quiescent, slow-cycling GSCs. Stable expression of these T cell targets in primary and recurrent GBM support their suitability for future clinical use.

Transcriptomic analysis of aggressive meningiomas identifies PTTG1 and LEPR as prognostic biomarkers independent of WHO grade.

Meningiomas are frequent central nervous system tumors. Although most meningiomas are benign (WHO grade I) and curable by surgery, WHO grade II and III tumors remain therapeutically challenging due to frequent recurrence. Interestingly, relapse also occurs in some WHO grade I meningiomas. Hence, we investigated the transcriptional features defining aggressive (recurrent, malignantly progressing or WHO grade III) meningiomas in 144 cases. Meningiomas were categorized into non-recurrent (NR), recurrent (R), and tumors undergoing malignant progression (M) in addition to their WHO grade. Unsupervised transcriptomic analysis in 62 meningiomas revealed transcriptional profiles lining up according to WHO grade and clinical subgroup. Notably aggressive subgroups (R+M tumors and WHO grade III) shared a large set of differentially expressed genes (n=332; p<0.01, FC>1.25). In an independent multicenter validation set (n=82), differential expression of 10 genes between WHO grades was confirmed. Additionally, among WHO grade I tumors differential expression between NR and aggressive R+M tumors was affirmed for PTTG1, AURKB, ECT2, UBE2C and PRC1, while MN1 and LEPR discriminated between NR and R+M WHO grade II tumors. Univariate survival analysis revealed a significant association with progression-free survival for PTTG1, LEPR, MN1, ECT2, PRC1, COX10, UBE2C expression, while multivariate analysis identified a prediction for PTTG1 and LEPR mRNA expression independent of gender, WHO grade and extent of resection. Finally, stainings of PTTG1 and LEPR confirmed malignancy-associated protein expression changes. In conclusion, based on the so far largest study sample of WHO grade III and recurrent meningiomas we report a comprehensive transcriptional landscape and two prognostic markers.