Loss of cancer cell-derived ADAM15 alters the tumor microenvironment in colorectal tumors.

Tumor progression and response to treatment are highly affected by interactions between cancer cells and the tumor microenvironment (TME). Many of the soluble factors and signaling receptors involved in this crosstalk are shed by a disintegrin and metalloproteinases (ADAMs). Upregulation of ADAM15 has been linked to worse survival in cancer patients and a tumor-promoting function both in vitro and in murine cancer models. Although ADAM15 has been involved in cell-cell and cell-extracellular matrix interactions, its role in the crosstalk between cancer cells and the TME in vivo remains unexplored. Therefore, we aimed to understand how ADAM15 regulates the cell composition of the TME and how it affects tumor progression. Here, we showed an upregulation of ADAM15 in tumor tissues from rectal cancer patients. Subcutaneous injection of wildtype and ADAM15-knockout CT26 colon cancer cells in syngeneic mice confirmed the protumorigenic role of ADAM15. Profiling of tumors revealed higher immune cell infiltration and cancer cell apoptosis in the ADAM15-deficient tumors. Specifically, loss of ADAM15 led to a reduced number of granulocytes and higher infiltration of antigen-presenting cells, including dendritic cells and macrophages, as well as more T cells. Using in vitro assays, we confirmed the regulatory effect of ADAM15 on macrophage migration and identified ADAM15-derived CYR61 as a potential molecular mediator of this effect. Based on these findings, we speculate that targeting ADAM15 could increase the infiltration of immune cells in colorectal tumors, which is a prerequisite for effective immunotherapy.

Genomic profiling of newly diagnosed glioblastoma patients and its potential for clinical utility - a prospective, translational study.

Glioblastoma (GBM) is an incurable brain tumor for which new treatment strategies are urgently needed. Next-generation sequencing of GBM has most often been performed retrospectively and on archival tissue from both diagnostic and relapse surgeries with limited knowledge of clinical information, including treatment given. We sought to investigate the genomic composition prospectively in treatment-naïve patients, searched for possible targetable aberrations, and investigated for prognostic and/or predictive factors. A total of 108 newly diagnosed GBM patients were included. Clinical information, progression-free survival, and overall survival (OS) were noted. Tissues were analyzed by whole-exome sequencing, single nucleotide polymorphism (SNP) and transcriptome arrays, and RNA sequencing; assessed for mutations, fusions, tumor mutational burden (TMB), and chromosomal instability (CI); and classified into GBM subgroups. Each genomic report was discussed at a multidisciplinary tumor board meeting to evaluate for matching trials. From 111 consecutive patients, 97.3% accepted inclusion in this study. Eighty-six (77%) were treated with radiation therapy/temozolomide (TMZ) and adjuvant TMZ. One NTRK2 and three FGFR3-TACC3 fusions were identified. Copy number alterations in GRB2 and SMYD4 were significantly correlated with worse median OS together with known clinical variables like age, performance status, steroid dose, and O6-methyl-guanine-DNA-methyl-transferase status. Patients with CI-median or TMB-high had significantly worse median OS compared to CI-low/high or TMB-low/median. In conclusion, performing genomic profiling at diagnosis enables evaluation of genomic-driven therapy at the first progression. Furthermore, TMB-high or CI-median patients had worse median OS, which can support the possibility of offering experimental treatment already at the first line for this group.

VEGF-C sustains VEGFR2 activation under bevacizumab therapy and promotes glioblastoma maintenance.

Background: Glioblastoma ranks among the most lethal cancers, with current therapies offering only palliation. Paracrine vascular endothelial growth factor (VEGF) signaling has been targeted using anti-angiogenic agents, whereas autocrine VEGF/VEGF receptor 2 (VEGFR2) signaling is poorly understood. Bevacizumab resistance of VEGFR2-expressing glioblastoma cells prompted interrogation of autocrine VEGF-C/VEGFR2 signaling in glioblastoma. Methods: Autocrine VEGF-C/VEGFR2 signaling was functionally investigated using RNA interference and exogenous ligands in patient-derived xenograft lines and primary glioblastoma cell cultures in vitro and in vivo. VEGF-C expression and interaction with VEGFR2 in a matched pre- and post-bevacizumab treatment cohort were analyzed by immunohistochemistry and proximity ligation assay. Results: VEGF-C was expressed by patient-derived xenograft glioblastoma lines, primary cells, and matched surgical specimens before and after bevacizumab treatment. VEGF-C activated autocrine VEGFR2 signaling to promote cell survival, whereas targeting VEGF-C expression reprogrammed cellular transcription to attenuate survival and cell cycle progression. Supporting potential translational significance, targeting VEGF-C impaired tumor growth in vivo, with superiority to bevacizumab treatment. Conclusions: Our results demonstrate VEGF-C serves as both a paracrine and an autocrine pro-survival cytokine in glioblastoma, promoting tumor cell survival and tumorigenesis. VEGF-C permits sustained VEGFR2 activation and tumor growth, where its inhibition appears superior to bevacizumab therapy in improving tumor control.

Assessment of Quantitative and Allelic MGMT Methylation Patterns as a Prognostic Marker in Glioblastoma.

Methylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is a predictive and prognostic marker in newly diagnosed glioblastoma patients treated with temozolomide but how MGMT methylation should be assessed to ensure optimal detection accuracy is debated. We developed a novel quantitative methylation-specific PCR (qMSP) MGMT assay capable of providing allelic methylation data and analyzed 151 glioblastomas from patients receiving standard of care treatment (Stupp protocol). The samples were also analyzed by immunohistochemistry (IHC), standard bisulfite pyrosequencing, and genotyped for the rs1690252 MGMT promoter single nucleotide polymorphism. Monoallelic methylation was observed more frequently than biallelic methylation, and some cases with monoallelic methylation expressed the MGMT protein whereas others did not. The presence of MGMT methylation was associated with better overall survival (p = 0.006; qMSP and p = 0.002; standard pyrosequencing), and the presence of the protein was associated with worse overall survival (p = 0.009). Combined analyses of qMSP and standard pyrosequencing or IHC identified additional patients who benefited from temozolomide treatment. Finally, low methylation levels were also associated with better overall survival (p = 0.061; qMSP and p = 0.02; standard pyrosequencing). These data support the use of both MGMT methylation and MGMT IHC but not allelic methylation data as prognostic markers in patients with temozolomide-treated glioblastoma.

Urokinase-Type Plasminogen Activator Receptor as a Potential PET Biomarker in Glioblastoma.

UNLABELLED: Glioblastoma is one of the most malignant types of human cancer, and the prognosis is poor. The development and validation of novel molecular imaging biomarkers has the potential to improve tumor detection, grading, risk stratification, and treatment monitoring of gliomas. The aim of this study was to explore the potential of PET imaging of the urokinase-type plasminogen activator receptor (uPAR) in glioblastoma. METHODS: The uPAR messenger RNA expression of tumors from 19 glioblastoma patients was analyzed, and a cell culture derived from one of these patients was used to establish an orthotopic xenograft model of glioblastoma. Tumor growth was monitored using bioluminescence imaging. Five to six weeks after inoculation, all mice were scanned with small-animal PET/CT using two new uPAR PET ligands ((64)Cu-NOTA-AE105 and (68)Ga-NOTA-AE105) and, for comparison, O-(2-(18)F-fluoroethyl)-l-tyrosine ((18)F-FET). One MRI scan was obtained for each mouse to confirm tumor location. The uPAR specificity of (64)Cu-NOTA-AE105 was confirmed by alignment of hematoxylin- and eosin-stained and uPAR immunohistochemistry-stained slides of the brain with the activity distribution as determined using autoradiography. RESULTS: uPAR expression was found in all 19 glioblastoma patient tumors, and high expression of uPAR correlated with decreased overall survival (P = 0.04). Radiolabeling of NOTA-AE105 with (64)Cu and (68)Ga was straightforward, resulting in a specific activity of approximately 20 GBq/µmol and a radiochemical purity of more than 98% for (64)Cu-NOTA-AE105 and more than 97% for (68)Ga-NOTA-AE105. High image contrast resulting in clear tumor delineation was found for both (68)Ga-NOTA-AE105 and (64)Cu-NOTA-AE105. Absolute uptake in tumor was higher for (18)F-FET (3.5 ± 0.8 percentage injected dose [%ID]/g) than for (64)Cu-NOTA-AE105 (1.2 ± 0.4 %ID/g) or (68)Ga-NOTA-AE105 (0.4 ± 0.1 %ID/g). A similar pattern was observed in background brain tissue, where uptake was 1.9 ± 0.1 %ID/g for (18)F-fluorothymidine, compared with 0.05 ± 0.01 %ID/g for (68)Ga-NOTA-AE105 and 0.11 ± 0.02 %ID/g for (64)Cu-NOTA-AE105. The result was a significantly higher tumor-to-background ratio for both (68)Ga-NOTA-AE105 (7.6 ± 2.1, P < 0.05) and (64)Cu-NOTA-AE105 (10.6 ± 2.3, P < 0.01) than for (18)F-FET PET (1.8 ± 0.3). Autoradiography of brain slides confirmed that the accumulation of (64)Cu-NOTA-AE105 corresponded well with uPAR-positive cancer cells. CONCLUSION: On the basis of our translational study, uPAR PET may be a highly promising imaging biomarker for glioblastoma. Further clinical exploration of uPAR PET in glioblastoma is therefore justified.

Targeting transcriptional addictions in small cell lung cancer with a covalent CDK7 inhibitor.

Small cell lung cancer (SCLC) is an aggressive disease with high mortality, and the identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library, we observe that SCLC is sensitive to transcription-targeting drugs, in particular to THZ1, a recently identified covalent inhibitor of cyclin-dependent kinase 7. We find that expression of super-enhancer-associated transcription factor genes, including MYC family proto-oncogenes and neuroendocrine lineage-specific factors, is highly vulnerability to THZ1 treatment. We propose that downregulation of these transcription factors contributes, in part, to SCLC sensitivity to transcriptional inhibitors and that THZ1 represents a prototype drug for tailored SCLC therapy.

The use of longitudinal 18F-FET MicroPET imaging to evaluate response to irinotecan in orthotopic human glioblastoma multiforme xenografts.

OBJECTIVES: Brain tumor imaging is challenging. Although 18F-FET PET is widely used in the clinic, the value of 18F-FET MicroPET to evaluate brain tumors in xenograft has not been assessed to date. The aim of this study therefore was to evaluate the performance of in vivo 18F-FET MicroPET in detecting a treatment response in xenografts. In addition, the correlations between the 18F-FET tumor accumulation and the gene expression of Ki67 and the amino acid transporters LAT1 and LAT2 were investigated. Furthermore, Ki67, LAT1 and LAT2 gene expression in xenograft and archival patient tumors was compared. METHODS: Human GBM cells were injected orthotopically in nude mice and 18F-FET uptake was followed by weekly MicroPET/CT. When tumor take was observed, mice were treated with CPT-11 or saline weekly. After two weeks of treatment the brain tumors were isolated and quantitative polymerase chain reaction were performed on the xenograft tumors and in parallel on archival patient tumor specimens. RESULTS: The relative tumor-to-brain (T/B) ratio of SUV max was significantly lower after one week (123 ± 6%, n = 7 vs. 147 ± 6%, n = 7; p = 0.018) and after two weeks (142 ± 8%, n = 5 vs. 204 ± 27%, n = 4; p = 0.047) in the CPT-11 group compared with the control group. Strong negative correlations between SUV max T/B ratio and LAT1 (r = -0.62, p = 0.04) and LAT2 (r = -0.67, p = 0.02) were observed. In addition, a strong positive correlation between LAT1 and Ki67 was detected in xenografts. Furthermore, a 1.6 fold higher expression of LAT1 and a 23 fold higher expression of LAT2 were observed in patient specimens compared to xenografts. CONCLUSIONS: 18F-FET MicroPET can be used to detect a treatment response to CPT-11 in GBM xenografts. The strong negative correlation between SUV max T/B ratio and LAT1/LAT2 indicates an export transport function. We suggest that 18F-FET PET may be used for detection of early treatment response in patients.

Single agent- and combination treatment with two targeted suicide gene therapy systems is effective in chemoresistant small cell lung cancer cells.

BACKGROUND: Transcriptional targeted suicide gene (SG) therapy driven by the insulinoma-associated 1 (INSM1) promoter makes it possible to target suicide toxin production and cytotoxicity exclusively to small cell lung cancer (SCLC) cells and tumors. It remains to be determined whether acquired chemoresistance, as observed in the majority of SCLC patients, desensitizes SCLC cells to INSM1 promoter-driven SG therapy. METHODS: A panel of SCLC cell lines resistant to clinically relevant chemotherapeutics was characterized regarding the expression of proteins involved in response to chemotherapy and regarding INSM1 promoter activity. Sensitivity towards INSM1 promoter-driven SG therapy was tested using different systems: Yeast cytosine deaminase-uracil phosphoribosyl transferase (YCD-YUPRT) in combination with the prodrug 5-fluorocytosine (5-FC) or Escherichia coli nitroreductase (NTR) together with the bromomustard prodrug SN27686. RESULTS: The chemoresistant cell lines displayed heterogeneous expression profiles of molecules involved in multidrug resistance, apoptosis and survival pathways. Despite this, the INSM1 promoter activity was found to be unchanged or increased in SCLC chemoresistant cells and xenografts compared to chemosensitive variants. INSM1 promoter-driven SG therapy with YCD-YUPRT/5-FC or NTR/SN27686, was found to induce high levels of cytotoxicity in both chemosensitive and chemoresistant SCLC cells. Moreover, the combination of INSM1 promoter-driven YCD-YUPRT/5-FC therapy and chemotherapy, as well as the combination of INSM1 promoter-driven YCD-YUPRT/5-FC and NTR/SN27686 therapy, was observed to be superior to single agent therapy in chemoresistant SCLC cells. CONCLUSIONS: Collectively, the present study demonstrates that targeted SG therapy is a potent therapeutic approach for chemoresistant SCLC patients, with the highest efficacy achieved when applied as combination SG therapy or in combination with standard chemotherapy.