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Representative 3D-rendering of segmented MCF-7, MDA-MB-468 and MDA-MB-231 xenografts following PET/CT image acquisition 3 h after 89Zr-DFO-ZHER3:8698 injection.

Purpose: Recent studies have highlighted a role of HER3 in HER2-driven cancers (e.g., breast cancer), implicating the upregulation of the receptor in resistance to HER-targeted therapies and Hsp90 inhibitors (e.g., AUY922). Therefore, we have developed an affibody-based PET radioconjugate that quantitatively assesses HER3 changes induced by Hsp90 inhibition in vivo.Experimental Design: ZHER3:8698 affibody molecules were conjugated via the C-terminus cysteine to DFO-maleimide for 89Zr radiolabeling. The probe was characterized in vitro and in vivo in a panel of human breast cell lines and xenograft models with varying HER3 receptor levels. In addition, the radioconjugate was investigated as a tool to monitor the outcome of AUY922, an Hsp90 inhibitor, in an MCF-7 xenograft model.Results: We demonstrated that 89Zr-DFO-ZHER3:8698 can track changes in receptor expression in HER3-positive xenograft models and monitor the outcome of AUY922 treatment. Our in vitro findings showed that MCF-7 cells, which are phenotypically different from BT474, develop resistance to treatment with AUY922 through HER3/IGF-1Rβ–mediated signaling. Of note, the lack of response in vitro due to HER3 recovery was confirmed in vivo using 89Zr-DFO-ZHER3:8698–based imaging. Upon AUY922 treatment, higher radioconjugate uptake was detected in treated MCF-7 xenografts, correlating with an AUY922-induced HER3 upregulation concomitant with an increase in IGF-1Rβ expression.Conclusions: These data underline the potential of HER3-based PET imaging to noninvasively provide information about HER3 expression and to identify patients not responding to targeted therapies due to HER3 recovery. Clin Cancer Res; 24(8); 1853–65. ©2018 AACR.

Materials and Methods Fig. S1. Time course of AUY922 treatment in BT-474 and MCF-7 cells. Fig. S2. Effects of AUY922 and PPP treatment in low and high HER2-expressing breast cancer cell lines. Fig. S3. Effect of HRG stimulation in AUY922 and PPP treated MCF-7 cells. Fig. S4. AUY922 and PPP treatment effect on the proliferative and invasive phenotype of BT-474 and MCF-7 breast cancer cells. Fig. S5. HPLC chromatograms of purified DFO-affibody conjugates. Fig. S6. MALDI-MS spectra of purified DFO-ZHER3:8698 (A) and DFO-ZTAQ (B). Fig. S7. Representative radio-ITLC of the crude radiolabelling mixture of 89Zr-DFO-ZHER3:8698. Fig. S8. Representative radio-ITLC of the 89Zr-DFO-ZHER3:8698 serum stability at 0, 3 and 24 h. Fig. S9. In vivo 89Zr-DFO-ZHER3:8698 tumour uptake quantification by image analysis. Fig. S10. Ex vivo biodistribution of 89Zr-DFO-ZHER3:8698 following AUY922 treatment in MCF-7 xenografts. Fig. S11. Microvessel density analysis in response to AUY922 treatment in MCF-7 xenografts. Fig. S12. Tumour volume estimation in response to AUY922 treatment in MCF-7 xenografts. Fig. S13. AUY922 treatment response monitoring in BT-474 xenografts by 89Zr-DFO-ZHER3:8698 PET/CT imaging. Fig. S14. Texture analysis of 89Zr-DFO-ZHER3:8698 PET imaging data following AUY922 treatment in MCF-7 xenografts. Fig. S15. Correlation between %ID/g ratios obtained from 89Zr-DFO-ZHER3:8698 PET images and HER3 protein expression per control and AUY922-treated MCF-7 xenografts. Fig. S16. HER3 intra-tumoral heterogeneity highlighted by histopathological analysis of control and AUY922-treated MCF-7 xenografts. Fig. S17. HER2-HER3 interaction in BT-474 and MCF-7 cells following AUY922 treatment. Fig. S18. Scheme illustrating the proposed effect of AUY922-induced Hsp90 inhibition in MCF-7 breast cancer xenografts. Table S1. Densitometric analysis of Western blots related to the treatment of BT-474, MCF-7, MCF-7 HER2+++ (-dox), and MCF-7 HER2+ (+dox) following 48 h treatment with AUY922 and PPP alone, or in combination. Table S2. Biodistribution results for 89Zr-DFO-ZHER3:8698 and 89Zr-DFO-ZTAQ (3 µg, 7.2-8.1 MBq/mouse) at 3 h after injection. Table S3. Biodistribution results for 89Zr-DFO-ZHER3:8698 at 3 h and 24 h after injection in MCF-7 and MDA-MB-468 tumour-bearing mice. Table S4. Tumour/organ ratios for 89Zr-DFO-ZHER3:8698 at 3 h and 24 h after injection in MCF-7 and MDA-MB-468 tumour-bearing mice. Table S5. HER3 and IGF1R co-occurrence in breast invasive carcinoma samples. Table S6. HER3 and IGF1R alterations lead to a shorter overall survival in breast invasive carcinoma samples. Table S7. HER3 and IGF1R alterations lead to a shorter disease free survival in breast invasive carcinoma samples. Table S8. HER3 and IGF1R co-occurrence in HER2 up-regulated breast invasive carcinoma samples.

Chronic lymphocytic leukemia (CLL) remains incurable despite B-cell receptor–targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate that is activated by unmethylated cytosine guanine dinucleotide–DNA. Here, we show that plasma from patients with CLL contains significantly more unmethylated DNA than plasma from healthy control subjects (P < .0001) and that cell-free DNA levels correlate with the prognostic markers CD38, β2-microglobulin, and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (hazard ratio, 4.0; P = .003). We also show that TLR9 expression was associated with in vitro CLL cell migration (P < .001), and intracellular endosomal TLR9 strongly correlated with aberrant surface expression (sTLR9; r = 0.9). In addition, lymph node–derived CLL cells exhibited increased sTLR9 (P = .016), and RNA-sequencing of paired sTLR9hi and sTLR9lo CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility, and inflammation in sTLR9hi cells. Mechanistically, a TLR9 agonist, ODN2006, promoted CLL cell migration (P < .001) that was mediated by p65 NF-κB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NOD/Shi-scid/IL-2Rγnull mouse xenograft model. Finally, we showed that dual targeting of TLR9 and Bruton’s tyrosine kinase (BTK) was strongly synergistic (median combination index, 0.2 at half maximal effective dose), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease.

The retention and re-migration of Chronic Lymphocytic Leukemia cells into cytoprotective and proliferative lymphoid niches is thought to contribute to the development of resistance, leading to subsequent disease relapse. The aim of this study was to elucidate the molecular processes that govern CLL cell migration to elicit a more complete inhibition of tumor cell migration. We compared the phenotypic and transcriptional changes induced in CLL cells using two distinct models designed to recapitulate the peripheral circulation, CLL cell migration across an endothelial barrier, and the lymph node interaction between CLL cells and activated T cells. Initially, CLL cells were co-cultured with CD40L-expressing fibroblasts and exhibited an activated B-cell phenotype, and their transcriptional signatures demonstrated the upregulation of pro-survival and anti-apoptotic genes and overrepresentation of the NF-κB signaling pathway. Using our dynamic circulating model, we were able to study the transcriptomics and miRNomics associated with CLL migration. More than 3000 genes were altered when CLL cells underwent transendothelial migration, with an overrepresentation of adhesion and cell migration gene sets. From this analysis, an upregulation of the FAK signaling pathway was observed. Importantly, PTK2 (FAK) gene expression was significantly upregulated in migrating CLL cells (PTK2 Fold-change = 4.9). Here we demonstrate that TLR9 agonism increased levels of p-FAK (p ≤ 0.05), which could be prevented by pharmacological inhibition of FAK with defactinib (p ≤ 0.01). Furthermore, a reduction in CLL cell migration and invasion was observed when FAK was inhibited (p ≤ 0.0001), supporting a role for FAK in both CLL migration and tissue invasion. When taken together, our data highlights the potential for combining FAK inhibition with current targeted therapies as a more effective treatment regime for CLL.

In this study, we evaluated an NF-κB inducing kinase (NIK) inhibitor, CW15337, in primary chronic lymphocytic leukemia (CLL) cells, CLL and multiple myeloma (MM) cell lines and normal B- and T-lymphocytes. Basal NF-κB subunit activity was characterized using an enzyme linked immunosorbent assay (ELISA), and the effects of NIK inhibition were then assessed in terms of cytotoxicity and the expression of nuclear NF-κB subunits following monoculture and co-culture with CD40L-expressing fibroblasts, as a model of the lymphoid niche. CW15337 induced a dose-dependent increase in apoptosis, and nuclear expression of the non-canonical NF-κB subunit, p52, was correlated with sensitivity to CW15337 (p = 0.01; r2 = 0.39). Co-culture on CD40L-expressing cells induced both canonical and non-canonical subunit expression in nuclear extracts, which promoted in vitro resistance against fludarabine and ABT-199 (venetoclax) but not CW15337. Furthermore, the combination of CW15337 with fludarabine or ABT-199 showed cytotoxic synergy. Mechanistically, CW15337 caused the selective inhibition of non-canonical NF-κB subunits and the transcriptional repression of BCL2L1, BCL2A1 and MCL1 gene transcription. Taken together, these data suggest that the NIK inhibitor, CW15337, exerts its effects via suppression of the non-canonical NF-κB signaling pathway, which reverses BCL2 family-mediated resistance in the context of CD40L stimulation.

There is an urgent need to develop therapeutic approaches that can increase the response rate to immuno-oncology agents. Photoimmunotherapy has recently been shown to generate anti-tumour immunological responses by releasing tumour-associated antigens from ablated tumour cell residues, thereby enhancing antigenicity and adjuvanticity. Here, we investigate the feasibility of a novel HER2-targeted affibody-based conjugate (ZHER2:2395-IR700) selectively to induce cancer cell death in vitro and in vivo. The studies in vitro confirmed the specificity of ZHER2:2395-IR700 binding to HER2-positive cells and its ability to produce reactive oxygen species upon light irradiation. A conjugate concentration- and light irradiation-dependent decrease in cell viability was also demonstrated. Furthermore, light-activated ZHER2:2395-IR700 triggered all hallmarks of immunogenic cell death, as defined by the translocation of calreticulin to the cell surface, and the secretion of ATP, HSP70/90 and HMGB1 from dying cancer cells into the medium. Irradiating a co-culture of immature dendritic cells (DCs) and cancer cells exposed to light-activated ZHER2:2395-IR700 enhanced DC maturation, as indicated by augmented expression of CD86 and HLA-DR. In SKOV-3 xenografts, the ZHER2:2395-IR700-based phototherapy delayed tumour growth and increased median overall survival. Collectively, our results strongly suggest that ZHER2:2395-IR700 is a promising new therapeutic conjugate that has great potential to be applicable for photoimmunotherapy-based regimens.

In head and neck squamous cell cancer, the human epidermal growth factor receptor 1 (EGFR) is the dominant signaling molecule among all members of the family. So far, cetuximab is the only approved anti-EGFR monoclonal antibody used for the treatment of head and neck squamous cell cancer, but despite the benefits of adding it to standard treatment regimens, attempts to define a predictive biomarker to stratify patients for cetuximab treatment have been unsuccessful. We hypothesized that imaging with EGFR-specific radioligands may facilitate noninvasive measurement of EGFR expression across the entire tumor burden and allow for dynamic monitoring of cetuximab-mediated changes in receptor expression. Methods: EGFR-specific Affibody molecule (ZEGFR:03115) was radiolabeled with 89Zr and 18F. The radioligands were characterized in vitro and in mice bearing subcutaneous tumors with varying levels of EGFR expression. The protein dose for imaging studies was assessed by injecting 89Zr-deferoxamine-ZEGFR:03115 (2.4–3.6 MBq, 2 μg) either together with or 30 min after increasing amounts of unlabeled ZEGFR:03115 (1, 5, 10, 15, and 20 μg). PET images were acquired at 3, 24, and 48 h after injection, and the image quantification data were correlated with the biodistribution results. The EGFR expression and biodistribution of the tracer were assessed ex vivo by immunohistochemistry, Western blot, and autoradiography. To downregulate the EGFR level, treatment with cetuximab was performed, and 18F-aluminium fluoride-NOTA-ZEGFR:03115 (12 μg, 1.5–2 MBq/mouse) was used to monitor receptor changes. Results: In vivo studies demonstrated that coinjecting 10 μg of nonlabeled molecules with 89Zr-deferoxamine-ZEGFR:03115 allows for clear tumor visualization 3 h after injection. The radioconjugate tumor accumulation was EGFR-specific, and PET imaging data showed a clear differentiation between xenografts with varying EGFR expression levels. A strong correlation was observed between PET analysis, ex vivo estimates of tracer concentration, and receptor expression in tumor tissues. Additionally, 18F-aluminium fluoride-NOTA-ZEGFR:03115 could measure receptor downregulation in response to EGFR inhibition. Conclusion: ZEGFR:03115-based radioconjugates can assess different levels of EGFR level in vivo and measure receptor expression changes in response to cetuximab, indicating a potential for assessment of adequate treatment dosing with anti-EGFR antibodies.

In non-invasive anticancer photodynamic therapy (PDT), a nontoxic photosensitizer (PS), which is activated by visible light, is used as a magic bullet that selectively destroys cancer cells. Recently, we described the combined therapy of 5-aminolevulinic acid (ALA-PDT) with thiosemicarbazone (TSC), i.e. an iron-chelating agent. This resulted in a strong synergistic effect. Herein, we investigated a novel strategy using a combination of PDT consist of the xenobiotic-porphyrin type PS with TSC. We observed a synergistic effect for all of the pairs of TSC-PS. This approach can be rationalized by the fact that both chlorin and TSC can affect the generation of reactive oxygen species (ROS). In order to elucidate the plausible mechanism of action, we also combined the investigated PSs with DFO, which forms complexes that are redox inactive. We detected a slight antagonism or additivity for this combination. This may suggest that the ability of an iron chelator (IC) to participate in the production of ROS and the generation of oxidative stress is important.

Glioblastoma (GBM) is a primary neuroepithelial tumor of the central nervous system, characterized by an extremely aggressive clinical phenotype. Patients with GBM have a poor prognosis and only 3–5% of them survive for more than 5 years. The current GBM treatment standards include maximal resection followed by radiotherapy with concomitant and adjuvant therapies. Despite these aggressive therapeutic regimens, the majority of patients suffer recurrence due to molecular heterogeneity of GBM. Consequently, a number of potential diagnostic, prognostic, and predictive biomarkers have been investigated. Some of them, such as IDH mutations, 1p19q deletion, MGMT promoter methylation, and EGFRvIII amplification are frequently tested in routine clinical practice. With the development of sequencing technology, detailed characterization of GBM molecular signatures has facilitated a more personalized therapeutic approach and contributed to the development of a new generation of anti-GBM therapies such as molecular inhibitors targeting growth factor receptors, vaccines, antibody-based drug conjugates, and more recently inhibitors blocking the immune checkpoints. In this article, we review the exciting progress towards elucidating the potential of current and novel GBM biomarkers and discuss their implications for clinical practice.

Glyphosate and atrazine are the most intensively used herbicides in the United States. Although there is abundant spatial and temporal information on atrazine occurrence at regional scales, there are far fewer data for glyphosate, and studies that compare the two herbicides are rare. We investigated temporal patterns in glyphosate and atrazine concentrations measured weekly during the 2013 growing season in 100 small streams in the Midwestern United States. Glyphosate was detected in 44% of samples (method reporting level 0.2 μg/L); atrazine was detected above a threshold of 0.2 μg/L in 54% of samples. Glyphosate was detected more frequently in 12 urban streams than in 88 agricultural streams, and at concentrations similar to those in streams with high agricultural land use (> 40% row crop) in the watershed. In contrast, atrazine was detected more frequently and at higher concentrations in agricultural streams than in urban streams. The maximum concentration of glyphosate measured at most urban sites exceeded the maximum atrazine concentration, whereas at agricultural sites the reverse was true. Measurement at a 2-day interval at 8 sites in northern Missouri revealed that transport of both herbicide compounds appeared to be controlled by spring flush, that peak concentration duration was brief, but that peaks in atrazine concentrations were of longer duration than those of glyphosate. The 2-day sampling also indicated that weekly sampling is unlikely to capture peak concentrations of glyphosate and atrazine.

1 Background 1 The Data Lifecycle Model 2 Primary Model Elements 2 Cross-Cutting Model Elements 3 Data Management Roles and Responsibilities in Research Projects 3 Summary 4 Acknowledgments 4 References Cited 4

We have determined this item to be in the public domain according to US copyright law through information in the bibliographic record and/or US copyright renewal records. The digital version is available for all educational uses worldwide. Please contact HathiTrust staff at hathitrust-help@umich.edu with any questions about this item., Great Britain--Administrative and political division., (LCCN)10006789., (OCoLC)7010833., 2436107., Sdr-ia-srlf2436107., JN566 .O44., Http://hdl.handle.net/2027/uc2.ark:/13960/t89g5j19x., Http://hdl.handle.net/2027/uc2.ark:/13960/t2s46jc0p.