Imaging and quantification of the tumor microenvironment of triple negative breast cancer using TPEF and scanning laser optical tomography.

Triple-negative breast cancer is an aggressive subtype of breast cancer that has a poor five-year survival rate. The tumor's extracellular matrix is a major compartment of its microenvironment and influences the proliferation, migration and the formation of metastases. The study of such dependencies requires methods to analyze the tumor matrix in its native form. In this work, the limits of SHG-microscopy, namely limited penetration depth, sample size and specificity, are addressed by correlative three-dimensional imaging. We present the combination of scanning laser optical tomography (SLOT) and multiphoton microscopy, to depict the matrix collagen on different scales. Both methods can be used complementarily to generate full-volume views and allow for in-depth analysis. Additionally, we explore the use of SHG as a contrast mechanism for complex samples in SLOT. It was possible to depict the overall collagen structure and specific fibers using marker free imaging on different scales. An appropriate sample preparation enables the fixation of the structures while simultaneously conserving the fluorescence of antibody staining. We find that SHG is a suitable contrast mechanism to depict matrix collagen even in complex samples and using SLOT. The insights presented here shall further facilitate the study of the tumor extracellular matrix by correlative 3d imaging.

Comparison of Serum and Urine as Sources of miRNA Markers for the Detection of Ovarian Cancer.

Ovarian cancer is the second most fatal gynecological cancer. Early detection, which could be achieved through widespread screening, has not yet had an impact on mortality. The aim of our pilot study was to investigate the expression of miRNAs analyzed by a human miRNA microarray chip in urine and serum of patients with ovarian cancer. We analyzed three serum and three urine samples from healthy donors and five serum and five urine samples from patients with ovarian cancer taken at first diagnosis, before any treatment. We selected the seven miRNAs with the highest expression fold change in the microarray chip (cancer vs. control) in urine and serum, for validation by qPCR. We were able to validate two of the seven miRNAs in serum. In contrast to these findings, we were able to validate all of the top seven miRNAs identified in urine using qPCR. The top seven miRNAs in urine identified by microarray chip showed significantly greater differences in expression between patients with ovarian cancer and healthy donors compared to serum. Based on our finding, we can suggest that urine as a biomaterial is more suitable than serum for miRNA profiling by microarray chip in the search for new biomarkers in ovarian cancer.

IRAK2 Downregulation in Triple-Negative Breast Cancer Cells Decreases Cellular Growth In Vitro and Delays Tumour Progression in Murine Models.

Breast cancer stem cells (BCSCs) are responsible for tumour recurrence and therapy resistance. We have established primary BCSC cultures from human tumours of triple-negative breast cancer (TNBC), a subgroup of breast cancer likely driven by BCSCs. Primary BCSCs produce xenografts that phenocopy the tumours of origin, making them an ideal model for studying breast cancer treatment options. In the TNBC cell line MDA-MB-468, we previously screened kinases whose depletion elicited a differentiation response, among which IRAK2 was identified. Because primary BCSCs are enriched in IRAK2, we wondered whether IRAK2 downregulation might affect cellular growth. IRAK2 was downregulated in primary BCSCs and MDA-MB-468 by lentiviral delivery of shRNA, causing a decrease in cellular proliferation and sphere-forming capacity. When orthotopically transplanted into immunocompromised mice, IRAK2 knockdown cells produced smaller xenografts than control cells. At the molecular level, IRAK2 downregulation reduced NF-κB and ERK phosphorylation, IL-6 and cyclin D1 expression, ERN1 signalling and autophagy in a cell line-dependent way. Overall, IRAK2 downregulation decreased cellular aggressive growth and pathways often exploited by cancer cells to endure stress; therefore, IRAK2 may be considered an interesting target to compromise TNBC progression.

Tumor Necrosis Factor-α (TNFα) Stimulate Triple-Negative Breast Cancer Stem Cells to Promote Intratumoral Invasion and Neovasculogenesis in the Liver of a Xenograft Model.

TNBC represents the most aggressive breast cancer subtype. Although cancer stem cells (CSCs) are a minor fraction of all cancer cells, they are highly cancerous when compared to their non-stem counterparts, playing a major role in tumor recurrence and metastasis. Angiogenic stimuli and the tumor environment response are vital factors in cancer metastasis. However, the causes and effects of tumor angiogenesis are still poorly understood. In this study, we demonstrate TNFα effects on primary triple-negative breast cancer stem cells (BCSCs). TNFα stimulation increased the mesenchymality of BCSCs in an intermediate epithelial-to-mesenchymal transition (EMT) state, enhanced proliferation, self-renewal, and invasive capacity. TNFα-treatment elicited BCSC signaling on endothelial networks in vitro and increased the network forming capacity of the endothelial cells. Our findings further demonstrate that TNFα stimulation in BCSCs has the ability to instigate distinct cellular communication within the tumor microenvironment, inducing intra-tumoral stromal invasion. Further, TNFα-treatment in BCSCs induced a pre-metastatic niche through breast-liver organ crosstalk by inducing vascular cell adhesion molecule-1 (VCAM-1) enriched neovasculogenesis in the liver of tumor-bearing mice. Overall, TNFα is an important angiogenic target to be considered in breast cancer progression to attenuate any angiogenic response in the tumor environment that could lead to secondary organ metastasis.

Stability of circulating microRNAs in serum.

There is a strong body of evidence by several translational studies which demonstrate the potential of circulating miRNAs as a potential biomarker in oncology. However, recent reports documented varying stability of these small RNA molecules in serum samples. The aim of our pilot study was to evaluate the stability of miRNAs in serum in relation to food intake and sample storage. Serum miRNA expression levels of 16 different miRNAs from 8 healthy volunteers were quantified by real-time PCR. 4 samples from each donor were analysed-2 samples (fasting, in the morning and after food intake, at noon) were analysed within 24h and 2 samples (fasting and after food intake, at noon) were stored at -80°C for 14 days and subsequently analysed. Student´s t-test was used to determine significant differences. The detectability of the distinct miRNA as a surrogate for the stability of these small RNA molecules was slightly altered by the storage conditions, but only a miRNA 22-3p, out of the analysed 16 miRNAs, shows significant lower dCq expression (3.821 vs. 4.530; p<0,01) by qPCR dependent on storage conditions (-80°C vs. 4°C). However, miRNA levels were not affected by food intake. The difference between samples taken in the morning (fasting) and at noon (after a normal meal) did not show any significant differences. MiRNAs can be considered to be a relatively stable tool in laboratory diagnostics, but clearly every new assay needs thorough evaluation. The stability of miRNAs documented here in healthy volunteers shows their potential in the search for innovative biomarkers in oncology.

Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I.

SMIP004-7 is a small molecule inhibitor of mitochondrial respiration with selective in vivo anti-cancer activity through an as-yet unknown molecular target. We demonstrate here that SMIP004-7 targets drug-resistant cancer cells with stem-like features by inhibiting mitochondrial respiration complex I (NADH:ubiquinone oxidoreductase, complex I [CI]). Instead of affecting the quinone-binding site targeted by most CI inhibitors, SMIP004-7 and its cytochrome P450-dependent activated metabolite(s) have an uncompetitive mechanism of inhibition involving a distinct N-terminal region of catalytic subunit NDUFS2 that leads to rapid disassembly of CI. SMIP004-7 and an improved chemical analog selectively engage NDUFS2 in vivo to inhibit the growth of triple-negative breast cancer transplants, a response mediated at least in part by boosting CD4+ and CD8+ T cell-mediated immune surveillance. Thus, SMIP004-7 defines an emerging class of ubiquinone uncompetitive CI inhibitors for cell autonomous and microenvironmental metabolic targeting of mitochondrial respiration in cancer.

EGFR activity addiction facilitates anti-ERBB based combination treatment of squamous bladder cancer.

Recent findings suggested a benefit of anti-EGFR therapy for basal-like muscle-invasive bladder cancer (MIBC). However, the impact on bladder cancer with substantial squamous differentiation (Sq-BLCA) and especially pure squamous cell carcinoma (SCC) remains unknown. Therefore, we comprehensively characterized pure and mixed Sq-BLCA (n = 125) on genetic and protein expression level, and performed functional pathway and drug-response analyses with cell line models and isolated primary SCC (p-SCC) cells of the human urinary bladder. We identified abundant EGFR expression in 95% of Sq-BLCA without evidence for activating EGFR mutations. Both SCaBER and p-SCC cells were sensitive to EGFR tyrosine kinase inhibitors (TKIs: erlotinib and gefitinib). Combined treatment with anti-EGFR TKIs and varying chemotherapeutics led to a concentration-dependent synergism in SCC cells according to the Chou-Talalay method. In addition, the siRNA knockdown of EGFR impaired SCaBER viability suggesting a putative "Achilles heel" of Sq-BLCA. The observed effects seem Sq-BLCA-specific since non-basal urothelial cancer cells were characterized by poor TKI sensitivity associated with a short-term feedback response potentially attenuating anti-tumor activity. Hence, our findings give further insights into a crucial, Sq-BLCA-specific role of the ERBB signaling pathway proposing improved effectiveness of anti-EGFR based regimens in combination with chemotherapeutics in squamous bladder cancers with wild-type EGFR-overexpression.

Chemotherapeutic Stress Influences Epithelial-Mesenchymal Transition and Stemness in Cancer Stem Cells of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by the absence of estrogen and progesterone receptors (ER, PR) and lacking an overexpression of human epidermal growth factor receptor 2 (HER2). Apart from this lack of therapeutic targets, TNBC also shows an increased capacity for early metastasis and therapy resistance. Currently, many TNBC patients receive neoadjuvant chemotherapy (NACT) upon detection of the disease. With TNBC likely being driven at least in part by a cancer stem-like cell type, we wanted to evaluate the response of primary cancer stem cells (CSCs) to standard chemotherapeutics. Therefore, we set up a survival model using primary CSCs to mimic tumor cells in patients under chemotherapy. Breast cancer stem cells (BCSCs) were exposed to chemotherapeutics with a sublethal dose for six days. Surviving cells were allowed to recover in culture medium without chemotherapeutics. Surviving and recovered cells were examined in regard to proliferation, migratory capacity, sphere forming capacity, epithelial-mesenchymal transition (EMT) factor expression at the mRNA level, and cancer-related microRNA (miRNA) profile. Our results indicate that chemotherapeutic stress enhanced sphere forming capacity of BCSCs, and changed cell morphology and EMT-related gene expression at the mRNA level, whereas the migratory capacity was unaffected. Six miRNAs were identified as potential regulators in this process.

Fine Tuning Antibody Conjugation Methods using SNAP-tag Technology.

BACKGROUND: Targeted imaging and therapy (theranostics) is a promising approach for the simultaneous improvement of cancer diagnosis, prognosis and management. Therapeutic and imaging reagents are coupled to tumor-targeting molecules such as antibodies, providing a basis for truly personalized medicine. However, the development of antibody-drug conjugates with acceptable pharmaceutical properties is a complex process and several parameters must be optimized, such as the controlled conjugation method and the drug-to-antibody ratio. OBJECTIVE: The major aim of this work is to address fundamental key challenges for the development of versatile technology platform for generating homogenous immunotheranostic reagent. METHOD: We conjugated the theranostics reagent IRDye700dx to a recombinant antibody fusion protein containing a self-labeling protein (SNAP-tag) which provides a unique reaction site. RESULTS: The resulting conjugate was suitable for the imaging of cancer cells expressing the epidermal growth factor receptor and demonstrated potent phototherapeutic and imaging activities against them. CONCLUSION: Here, we describe a simple, rapid and robust site-directed labeling method that can be used to generate homogeneous immunoconjugate with defined pharmacological properties.

Detection and Specific Elimination of EGFR+ Ovarian Cancer Cells Using a Near Infrared Photoimmunotheranostic Approach.

PURPOSE: Targeted theranostics is an alternative strategy in cancer management that aims to improve cancer detection and treatment simultaneously. This approach combines potent therapeutic and diagnostic agents with the specificity of different cell receptor ligands in one product. The success of antibody drug conjugates (ADCs) in clinical practice has encouraged the development of antibody theranostics conjugates (ATCs). However, the generation of homogeneous and pharmaceutically-acceptable ATCs remains a major challenge. The aim of this study is to detect and eliminate ovarian cancer cells on-demand using an ATC directed to EGFR. METHODS: An ATC with a defined drug-to-antibody ratio was generated by the site-directed conjugation of IRDye®700 to a self-labeling protein (SNAP-tag) fused to an EGFR-specific antibody fragment (scFv-425). RESULTS: In vitro and ex vivo imaging showed that the ATC based on scFv-425 is suitable for the highly specific detection of EGFR+ ovarian cancer cell, human tissues and ascites samples. The construct was also able to eliminate EGFR+ cells and human ascites cells with IC50 values of 45-66 nM and 40-90 nM, respectively. CONCLUSION: Our experiments provide a framework to create a versatile technology platform for the development of ATCs for precise detection and treatment of ovarian cancer cells.

Photoimmunotheranostic agents for triple-negative breast cancer diagnosis and therapy that can be activated on demand.

Triple-negative breast cancer (TNBC) is a heterogeneous disease in which the tumors do not express estrogen receptor (ER), progesterone receptor (PgR) or human epidermal growth factor receptor 2 (HER2). Classical receptor-targeted therapies such as tamoxifen or trastuzumab are therefore unsuitable and combinations of surgery, chemotherapy and/or radiotherapy are required. Photoimmunotheranostics is a minimally invasive approach in which antibodies deliver nontoxic photosensitizers that emit light to facilitate diagnosis and produce cytotoxic reactive oxygen species to induce apoptosis and/or necrosis in cancer cells. We developed a panel of photoimmunotheranostic agents against three TNBC-associated cell surface antigens. Antibodies against epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM) and chondroitin sulfate proteoglycan 4 (CSPG4) were conjugated to the highly potent near-infrared imaging agent/photosensitizer IRDye®700DX phthalocyanine using SNAP-tag technology achieving clear imaging in both breast cancer cell lines and human biopsies and highly potent phototherapeutic activity with IC50values of 62-165 nM against five different cell lines expressing different levels of EGFR, EpCAM and CSPG4. A combination of all three reagents increased the therapeutic activity against TNBC cells by up to 40%.