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Thyroid cancer is the most common endocrine cancer, with differentiated thyroid cancers (DTCs) accounting for 95% of diagnoses. While most DTC patients are diagnosed and treated with radioiodine (RAI), up to 20% of DTC patients become RAI refractory (RAI-R). RAI-R patients have significantly reduced survival rates compared to patients who remain RAI-avid. This study explores [ 89 Zr]Zr-TR1402 as a thyroid-stimulating hormone receptor (TSHR)-targeted PET radiopharmaceutical for DTC. [ 89 Zr]Zr-TR1402 was synthesized with a molar activity of 25.9 MBq/nmol by conjugating recombinant human TSH (rhTSH) analogue TR1402 to chelator p-SCN-Bn-deferoxamine (DFO) in a molar ratio of 3:1 (DFO/TR1402) and radiolabeling with 89 Zr ( t 1/2 = 78.4 h, β + = 22.7%). As TSHR is absent in commonly available DTC-derived cell lines, TSHR was reintroduced via stable transduction by delivering a lentivirus containing the full-length coding region of the human TSHR gene. Receptor-mediated uptake of [ 89 Zr]Zr-TR1402 was evaluated in vitro in stably transduced TSHR+ and wild-type TSHR- DTC cell lines. In vivo PET imaging was performed on Days 1-3 postinjection in male and female athymic nude mice bearing TSHR+ and TSHR- xenografts, along with ex vivo biodistribution on Day 3 postinjection. In vitro uptake of 1 nM [ 89 Zr]Zr-TR1402 was significantly higher in TSHR+ THJ529T ( P < 0.0001) and FTC133 ( P < 0.01) cells than in TSHR- THJ529T and FTC133 cells. This uptake was shown to be specific in both TSHR+ THJ529T ( P < 0.0001) and TSHR+ FTC133 ( P < 0.0001) cells by blocking uptake with 250 nm DFO-TR1402. In vivo PET imaging showed accumulation of [ 89 Zr]Zr-TR1402 in TSHR+ tumors, which was the highest on Day 1. In the male FTC133 xenograft model, ex vivo biodistribution confirmed a significant difference ( P < 0.001) in uptake between FTC133+ (1.3 ± 0.1%ID/g) and FTC133- (0.8 ± 0.1%ID/g) tumors. A significant difference ( P < 0.05) in uptake was also seen in the male THJ529T xenograft model between THJ529T+ (1.8 ± 0.6%ID/g) and THJ529T- (0.8 ± 0.4%ID/g) tumors. The in vitro and in vivo accumulation of [ 89 Zr]Zr-TR1402 in TSHR-expressing DTC cell lines support the continued preclinical optimization of this approach.

This review focuses on the expression and function of voltage-gated sodium channel subtype Na V 1.7 in various cancers and explores its impact on the metastasis driving cell functions such as proliferation, migration, and invasiveness. An overview of its structural characteristics, drug binding sites, inhibitors and their likely mechanisms of action are presented. Despite the lack of clarity on the precise mechanism by which Na V 1.7 contributes to cancer progression and metastasis; many studies have suggested a connection between Na V 1.7 and proteins involved in multiple signaling pathways such as PKA and EGF/EGFR-ERK1/2. Moreover, the functional activity of Na V 1.7 appears to elevate the expression levels of MACC1 and NHE-1, which are controlled by p38 MAPK activity, HGF/c-MET signaling and c-Jun activity. This cascade potentially enhances the secretion of extracellular matrix proteases, such as MMPs which play critical roles in cell migration and invasion activities. Furthermore, the Na V 1.7 activity may indirectly upregulate Rho GTPases Rac activity, which is critical for cytoskeleton reorganization, cell adhesion, and actin polymerization. The relationship between Na V 1.7 and cancer progression has prompted researchers to investigate the therapeutic potential of targeting Na V 1.7 using inhibitors. The positive outcome of such studies resulted in the discovery of several inhibitors with the ability to reduce cancer cell migration, invasion, and tumor growth underscoring the significance of Na V 1.7 as a promising pharmacological target for attenuating cancer cell proliferation and metastasis. The research findings summarized in this review suggest that the regulation of Na V 1.7 expression and function by small molecules and/or by genetic engineering is a viable approach to discover novel therapeutics for the prevention and treatment of metastasis of cancers with elevated Na V 1.7 expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Pukkanasut, Jaskula-Sztul, Gomora and Velu.)

Background: Of the half a million cases of thyroid cancer diagnosed annually, 95% are differentiated thyroid cancers. Although clinical guidelines recommend surgical resection followed by radioactive iodine ablation, loss of sodium-iodine symporter expression causes up to 20% of differentiated thyroid cancers to become radioactive iodine refractory. For patients with radioactive iodine refractory disease, there is an urgent need for new diagnostic and therapeutic approaches. We evaluated the thyroid-stimulating hormone receptor as a potential target for imaging of differentiated thyroid cancer., Methods: We immunostained tissue microarrays containing 52 Hurthle cell carcinomas to confirm thyroid-stimulating hormone receptor expression. We radiolabeled chelator deferoxamine conjugated to recombinant human thyroid-stimulating hormone analog superagonist TR1402 with 89 Zr (t 1/2  = 78.4 h, β +  =22.7%) to produce [ 89 Zr]Zr-TR1402. We performed in vitro uptake assays in high-thyroid-stimulating hormone receptor and low-thyroid-stimulating hormone receptor-expressing THJ529T and FTC133 thyroid cancer cell lines. We performed in vivo positron emission tomography/computed tomography and biodistribution studies in male athymic nude mice bearing thyroid-stimulating hormone receptor-positive THJ529T tumors., Results: Immunohistochemical analysis revealed 62% of patients (27 primary and 5 recurrent) were thyroid-stimulating hormone receptor membranous immunostain positive. In vitro uptake of 1nM [ 89 Zr]Zr-TR1402 was 38 ± 17% bound/mg in thyroid-stimulating hormone receptor-positive THJ529T thyroid cancer cell lines compared to 3.2 ± 0.5 in the low-expressing cell line (P < .01), with a similar difference seen in FTC133 cell lines (P < .0001). In vivo and biodistribution studies showed uptake of [ 89 Zr]Zr-TR1402 in thyroid-stimulating hormone receptor-expressing tumors, with a mean percentage of injected dose/g of 1.9 ± 0.4 at 3 days post-injection., Conclusion: Our observation of thyroid-stimulating hormone receptor expression in tissue microarrays and [ 89 Zr]Zr-TR1402 accumulation in thyroid-stimulating hormone receptor-positive thyroid cancer cells and tumors suggests thyroid-stimulating hormone receptor is a promising target for imaging of differentiated thyroid cancer., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Somatostatin receptor type 2 (SSTR2) and thyroid-stimulating hormone receptor (TSHR) display variable expression in primary thyroid tumors and have been implicated as theranostic targets. This study was designed to explore the differential expression of SSTR2 and TSHR in oncocytic (Hurthle cell) carcinoma (OC) vs oncocytic adenoma (OA). We performed a retrospective review for oncocytic neoplasms treated at our institution from 2012 to 2019. Formalin-fixed paraffin-embedded tissue blocks were used for tissue microarray construction. Tissue microarray blocks were cut into 5-μm sections and stained with anti-SSTR2 and anti-TSHR antibodies. Immunostains were analyzed by 3 independent pathologists. χ 2 and logistic regression analysis were used to analyze clinical and pathologic variables. Sixty-seven specimens were analyzed with 15 OA and 52 OC. The mean age was 57 years, 61.2% were women, and 70% were White. SSTR2 positivity was noted in 2 OA (13%) and 15 OC (28%; 10 primary, 4 recurrent, and 1 metastatic) (P = .22). TSHR positivity was noted in 11 OA (73%) and 32 OC (62%; 31 primary and 1 metastatic) (P = .40). Those who presented with or developed clinical recurrence/metastasis were more likely to be SSTR2-positive (50% vs 21%; P = .04) and TSHR-negative (64.3% vs 28.9%; P = .02) than primary OC patients. Widely invasive OC was more likely to be SSTR2-positive compared to all other OC subtypes (minimally invasive and angioinvasive) (P = .003). For all patients with OC, TSHR positivity was inversely correlated with SSTR2 positivity (odds ratio, 0.12; CI, 0.03-0.43; P = .006). This relationship was not seen in the patients with OA (odds ratio, 0.30; CI, 0.01-9.14; P = .440). Our results show that recurrent/metastatic OC was more likely to be SSTR2-positive and TSHR-negative than primary OC. Patients with OC displayed a significant inverse relationship between SSTR2 and TSHR expression that was not seen in patients with OA. This may be a key relationship that can be used to prognosticate and treat OCs., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Our results from quantitative RT-PCR, Western blotting, immunohistochemistry, and the tissue microarray of medullary thyroid cancer (MTC) cell lines and patient specimens confirm that VGSC subtype Na V 1.7 is uniquely expressed in aggressive MTC and not expressed in normal thyroid cells and tissues. We establish the druggability of Na V 1.7 in MTC by identifying a novel inhibitor ( SV188 ) and investigate its mode of binding and ability to inhibit I Na current in Na V 1.7. The whole-cell patch-clamp studies of the SV188 in the Na V 1.7 channels expressed in HEK-293 cells show that SV188 inhibited the I Na current in Na V 1.7 with an IC 50 value of 3.6 µM by a voltage- and use-dependent blockade mechanism, and the maximum inhibitory effect is observed when the channel is open. SV188 inhibited the viability of MTC cell lines, MZ-CRC-1 and TT, with IC 50 values of 8.47 μM and 9.32 μM, respectively, and significantly inhibited the invasion of MZ-CRC-1 cells by 35% and 52% at 3 μM and 6 μM, respectively. In contrast, SV188 had no effect on the invasion of TT cells derived from primary tumor, which have lower basal expression of Na V 1.7. In addition, SV188 at 3 μM significantly inhibited the migration of MZ-CRC-1 and TT cells by 27% and 57%, respectively.

The cross talk between cancer cells and endothelial cells (ECs) within the tumor microenvironment plays a critical role in tumor progression, recurrence, and cancer stemness. Here, we present a protocol containing two in vitro approaches to study such interactions. We first describe an indirect co-culture system to study the regulation of stemness markers in cancer cells by secreted factors from ECs. We then detail a direct co-culture system to study juxtracrine communications between the cell types. For complete details on the use and execution of this protocol, please refer to Sewell-Loftin et al. 1 and Guo et al. 2 ., Competing Interests: Declaration of interests M.K.S-.L. performs consulting work for CerFlux, Inc., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pancreatic neuroendocrine tumors (pNETs) are extremely diverse and highly vascularized neoplasms that arise from endocrine cells in the pancreas. The pNETs harbor a subpopulation of stem cell-like malignant cells, known as cancer stem cells (CSCs), which contribute to intratumoral heterogeneity and promote tumor maintenance and recurrence. In this study, we demonstrate that CSCs in human pNETs co-express protein kinase PKD1 and CD44. We further identify PKD1 signaling as a critical pathway in the control of CSC maintenance in pNET cells. PKD1 signaling regulates the expression of a CSC- and EMT-related gene signature and promotes CSC self-renewal, likely leading to the preservation of a subpopulation of CSCs at an intermediate EMT state. This suggests that the PKD1 signaling pathway may be required for the development of a unique CSC phenotype with plasticity and partial EMT. Given that the signaling networks connected with CSC maintenance and EMT are complex, and extend through multiple levels of regulation, this study provides insight into signaling regulation of CSC plasticity and partial EMT in determining the fate of CSCs. Inhibition of the PKD1 pathway may facilitate the elimination of specific CSC subsets, thereby curbing tumor progression and metastasis.

Growth factors in tumor environments are regulators of cell survival and metastasis. Here, we reveal the dichotomy between TGF-β superfamily growth factors BMP and TGF-β/activin and their downstream SMAD effectors. Gene expression profiling uncovers SOX2 as a key contextual signaling node regulated in an opposing manner by BMP2, -4, and -9 and TGF-β and activin A to impact anchorage-independent cell survival. We find that SOX2 is repressed by BMPs, leading to a reduction in intraperitoneal tumor burden and improved survival of tumor-bearing mice. Repression of SOX2 is driven by SMAD1-dependent histone H3K27me3 recruitment and DNA methylation at SOX2's promoter. Conversely, TGF-β, which is elevated in patient ascites, and activin A can promote SOX2 expression and anchorage-independent survival by SMAD3-dependent histone H3K4me3 recruitment. Our findings identify SOX2 as a contextual and contrastingly regulated node downstream of TGF-β members controlling anchorage-independent survival and metastasis in ovarian cancers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The Notch pathway regulates many cellular functions in a context-dependent manner. Depending on the cell type, either the activation or inhibition of Notch signaling can influence many processes such as cellular proliferation, specification, differentiation, and survival. The activation of Notch signaling has been shown to have therapeutic advantages in some cancers, thus having a method to identify Notch-activating compounds is needed. In this chapter we outline a method for high-throughput analysis of potential Notch pathway activators in a pancreatic neuroendocrine tumor cell line as an example. We also include the steps for subsequent validation of results and preclinical testing., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Few models exist for studying neuroendocrine tumors (NETs), and there are mounting concerns that the currently available array of cell lines is not representative of NET biology. The lack of stable patient-derived NET xenograft models further limits the scientific community's ability to make conclusions about NETs and their response to therapy in patients. To address these limitations, we propose the use of an ex vivo 3D flow-perfusion bioreactor system for culturing and studying patient-derived NET surrogates. Herein, we demonstrate the utility of the bioreactor system for culturing NET surrogates and provide methods for evaluating the efficacy of therapeutic agents on human NET cell line xenograft constructs and patient-derived NET surrogates. We also demonstrate that patient-derived NET tissues can be propagated using the bioreactor system and investigate the near-infrared (NIR) dye IR-783 for its use in monitoring their status within the bioreactor. The results indicate that the bioreactor system and similar 3D culture models may be valuable tools for culturing patient-derived NETs and monitoring their response to therapy ex vivo ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Herring, Jang, Whitt, Goliwas, Aburjania, Dudeja, Ren, Berry, Bibb, Frost, Chen, Rose and Jaskula-Sztul.)

Neuroendocrine (NE) tumors include a diverse spectrum of hormone-secreting neoplasms that arise from the endocrine and nervous systems. Current chemo- and radio-therapies have marginal curative benefits. The goal of this study was to develop an innovative antibody-drug conjugate (ADC) to effectively treat NE tumors (NETs). First, we confirmed that somatostatin receptor 2 (SSTR2) is an ideal cancer cell surface target by analyzing 38 patient-derived NET tissues, 33 normal organs, and three NET cell lines. Then, we developed a new monoclonal antibody (mAb, IgG1, and kappa) to target two extracellular domains of SSTR2, which showed strong and specific surface binding to NETs. The ADC was constructed by conjugating the anti-SSTR2 mAb and antimitotic monomethyl auristatin E. In vitro evaluations indicated that the ADC can effectively bind, internalize, release payload, and kill NET cells. Finally, the ADC was evaluated in vivo using a NET xenograft mouse model to assess cancer-specific targeting, tolerated dosage, pharmacokinetics, and antitumor efficacy. The anti-SSTR2 ADC exclusively targeted and killed NET cells with minimal toxicity and high stability in vivo. This study demonstrates that the anti-SSTR2 ADC has a high-therapeutic potential for NET therapy., (© 2020. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Gamma secretase inhibitors (GSIs), initially developed as Alzheimer's therapies, have been repurposed as anticancer agents given their inhibition of Notch receptor cleavage. The success of GSIs in preclinical models has been ascribed to induction of cancer stem-like cell differentiation and apoptosis, while also impairing epithelial-to-mesenchymal transition and sensitizing cells to traditional chemoradiotherapies. The promise of these agents has yet to be realized in the clinic, however, as GSIs have failed to demonstrate clinical benefit in most solid tumors with the notable exceptions of CNS malignancies and desmoid tumors. Disappointing clinical performance to date reflects important questions that remain to be answered. For example, what is the net impact of these agents on antitumor immune responses, and will they require concurrent targeting of tumor-intrinsic compensatory pathways? Addressing these limitations in our current understanding of GSI mechanisms will undoubtedly facilitate their rational incorporation into combinatorial strategies and provide a valuable tool with which to combat Notch-dependent cancers. In the present review, we provide a current understanding of GSI mechanisms, discuss clinical performance to date, and suggest areas for future investigation that might maximize the utility of these agents. IMPLICATIONS FOR PRACTICE: The performance of gamma secretase inhibitors (GSIs) in clinical trials generally has not reflected their encouraging performance in preclinical studies. This review provides a current perspective on the clinical performance of GSIs across various solid tumor types alongside putative mechanisms of antitumor activity. Through exploration of outstanding gaps in knowledge as well as reasons for success in certain cancer types, the authors identify areas for future investigation that will likely enable incorporation of GSIs into rational combinatorial strategies for superior tumor control and patient outcomes., (© 2020 AlphaMed Press.)

Surgical resection is the only cure for neuroendocrine tumors (NETs). However, widespread metastases have already occured by the time of initial diagnosis in many cases making complete surgical removal impossible. We developed a recombinant heavy-chain receptor binding domain (rHCR) of botulinum neurotoxin type A that can specifically target synaptic vesicle 2 (SV2), a surface receptor abundantly expressed in multiple neuroendocrine tumors. Expression of neuroendocrine differentiation markers chromogranin A (CgA) and achaete-scute complex 1 (ASCL1) were signficantly reduced when treated with rHCR. rHCR conjugated to the antimitotic agent monomethyl auristatin E (MMAE) significantly suppressed proliferation of pancreatic carcinoid (BON) and medullary thyroid cancer cells (MZ) at concentrations of 500 and 300 nM respectively, while no growth suppression was observed in pulmonary fibroblasts and cortical neuron control cell lines. In vivo, rHCR-MMAE significantly reduced tumor volume in mouse xenografts with no observed adverse effects. These data suggest recombinant HCR (rHCR) of BoNT/A preferentially targets neuroendocrine cancer without the neurotoxicity of the full BoNT/A and that SV2 is a specific and promising target for delivering drugs to neuroendocrine tumors.

Neuroendocrine (NE) cancers arise from cells within the neuroendocrine system. Chemotherapies and endoradiotherapy have been developed, but their clinical efficacy is limited. The objective of this study was to develop a dual-targeted extracellular vesicles (EV)-delivered combined therapies to treat NE cancer. Specifically, we produced EV in stirred-tank bioreactors and surface tagged both anti-somatostatin receptor 2 (SSTR 2) monoclonal antibody (mAb) and anti-C-X-C motif chemokine receptor 4 (CXCR4) mAb to generate mAbs-EV. Both live-cell confocal microscopy imaging and In Vivo Imaging System (IVIS) imaging confirmed that mAbs-EV specifically targeted and accumulated in NE cancer cells and NE tumor xenografts. Then the highly potent natural cytotoxic marine compound verrucarin A (Ver-A) with IC 50 of 2.2-2.8 nM and microtubule polymerization inhibitor mertansine (DM1) with IC 50 of 3.1-4.2 nM were packed into mAbs-EV. The in vivo maximum tolerated dose study performed in non-tumor-bearing mice indicated minimal systemic toxicity of mAbs-EV-Ver-A/DM1. Finally, the in vivo anticancer efficacy study demonstrated that the SSTR2/CXCR4 dual-targeted EV-Ver-A/DM1 is more effective to inhibit NE tumor growth than the single targeting and single drug. The results from this study could expand the application of EV to targeting deliver the combined potent chemotherapies for cancer treatment.

In an effort to discover viable systemic chemotherapeutic agents for neuroendocrine tumors (NETs), we screened a small library of 18 drug-like compounds obtained from the Velu lab against pulmonary (H727) and thyroid (MZ-CRC-1 and TT) neuroendocrine tumor-derived cell lines. Two potent lead compounds (DHN-II-84 and DHN-III-14) identified from this screening were found to be analogs of the natural product makaluvamine. We further characterized the antitumor activities of these two compounds using pulmonary (H727), thyroid (MZ-CRC-1) and pancreatic (BON) neuroendocrine tumor cell lines. Flow cytometry showed a dose-dependent increase in apoptosis in all cell lines. Induction of apoptosis with these compounds was also supported by the decrease in myeloid cell leukemia-1 (MCL-1) and X-chromosome linked inhibitor of apoptosis (XIAP) detected by Western blot. Compound treatment decreased NET markers chromogranin A (CgA) and achaete-scute homolog 1 (ASCL1) in a dose-dependent manner. Moreover, the gene expression analysis showed that the compound treatment reduced c-Kit proto-oncogene expression in the NET cell lines. Induction of apoptosis could also have been caused by the inhibition of c-Kit expression, in addition to the known mechanisms such as damage of DNA by topoisomerase II inhibition for this class of compounds. In summary, makaluvamine analogs DHN-II-84 and DHN-III-14 induced apoptosis, decreased neuroendocrine tumor markers, and showed promising antitumor activity in pulmonary, thyroid, and pancreatic NET cell lines, and hold potential to be developed as an effective treatment to combat neuroendocrine tumors.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants that are metabolized to carcinogenic dihydrodiol epoxides (PAHDE) by cytochrome P450 1B1 (CYP1B1). This metabolism occurs in bone marrow (BM) mesenchymal stem cells (MSC), which sustain hematopoietic stem and progenitor cells (HSPC). In BM, CYP1B1-mediated metabolism of 7, 12-dimethylbenz[a]anthracene (DMBA) suppresses HSPC colony formation within 6 h, whereas benzo(a)pyrene (BP) generates protective cytokines. MSC, enriched from adherent BM cells, yielded the bone marrow stromal, BMS2, cell line. These cells express elevated basal CYP1B1 that scarcely responds to Ah receptor (AhR) inducers. BMS2 cells exhibit extensive transcriptome overlap with leptin receptor positive mesenchymal stem cells (Lepr+ MSC) that control the hematopoietic niche. The overlap includes CYP1B1 and the expression of HSPC regulatory factors (Ebf3, Cxcl12, Kitl, Csf1 and Gas6). MSC are large, adherent fibroblasts that sequester small HSPC and macrophage in the BM niche (Graphic abstract). High basal CYP1B1 expression in BMS2 cells derives from interactions between the Ah-receptor enhancer and proximal promoter SP1 complexes, boosted by autocrine signaling. PAH effects on BMS2 cells model Lepr+MSC niche activity. CYP1B1 metabolizes DMBA to PAHDE, producing p53-mediated mRNA increases, long after the in vivo HSPC suppression. Faster, direct p53 effects, favored by stem cells, remain possible PAHDE targets. However, HSPC regulatory factors remained unresponsive. BP is less toxic in BMS2 cells, but, in BM, CYP1A1 metabolism stimulates macrophage cytokines (Il1b > Tnfa> Ifng) within 6 h. Although absent from BMS2 and Lepr+MSC, their receptors are highly expressed. The impact of this cytokine signaling in MSC remains to be determined., Competing Interests: Declaration of Competing Interest The authors acknowledge no conflicts of interest with these studies., (Copyright © 2020. Published by Elsevier Inc.)

Disparities in cancer patient responses have prompted widespread searches to identify differences in sensitive vs. nonsensitive populations and form the basis of personalized medicine. This customized approach is dependent upon the development of pathway-specific therapeutics in conjunction with biomarkers that predict patient responses. Here, we show that Cdk5 drives growth in subgroups of patients with multiple types of neuroendocrine neoplasms. Phosphoproteomics and high throughput screening identified phosphorylation sites downstream of Cdk5. These phosphorylation events serve as biomarkers and effectively pinpoint Cdk5-driven tumors. Toward achieving targeted therapy, we demonstrate that mouse models of neuroendocrine cancer are responsive to selective Cdk5 inhibitors and biomimetic nanoparticles are effective vehicles for enhanced tumor targeting and reduction of drug toxicity. Finally, we show that biomarkers of Cdk5-dependent tumors effectively predict response to anti-Cdk5 therapy in patient-derived xenografts. Thus, a phosphoprotein-based diagnostic assay combined with Cdk5-targeted therapy is a rational treatment approach for neuroendocrine malignancies., Competing Interests: The authors declare no competing interest.

Background: Neuroendocrine tumors are found throughout the body, including the pancreas. These tumors are phenotypically and genetically heterogeneous and can be difficult to accurately image using current imaging standards. However, positron emission tomography/computed tomography with radiolabeled somatostatin analogs has shown clinical success because many neuroendocrine tumors overexpress somatostatin receptor subtype 2. Unfortunately, patients with poorly differentiated neuroendocrine tumors often have a diminished level of somatostatin receptor subtype 2. We found that histone deacetylase inhibitors can upregulate the functional expression of somatostatin receptor subtype 2., Methods: We evaluated the effect of histone deacetylase inhibitors on somatostatin receptor subtype 2 expression at the mRNA and protein level in neuroendocrine tumor cell lines. The effect of histone deacetylase inhibitors on surface somatostatin receptor subtype 2 was also investigated by fluorescence-activated cell sorting analysis. Changes in somatostatin receptor subtype 2 expression in neuroendocrine tumor xenografts after treatment were imaged using Ga68-DOTATATE positron emission tomography/computed tomography., Results: The functional increase of somatostatin receptor subtype 2 in neuroendocrine tumors after histone deacetylase inhibitor treatment was confirmed through in vitro experiments and small animal Ga68-DOTATATE positron emission tomography/computed tomography imaging. Histone deacetylase inhibitors increased somatostatin receptor subtype 2 transcription and protein expression in neuroendocrine tumor cell lines. Small animal Ga68-DOTATATE positron emission tomography/computed tomography imaging confirmed the enhancement of radiopeptide uptake after histone deacetylase inhibitor administration., Conclusion: This study demonstrates a new method to potentially improve imaging and treatments that target somatostatin receptor subtype 2 in neuroendocrine tumors., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Exosomes hold great potential to deliver therapeutic reagents for cancer treatment due to its inherent low antigenicity. However, several technical barriers, such as low productivity and ineffective cancer targeting, need to be overcome before wide clinical applications. The present study aims at creating a new biomanufacturing platform of cancer-targeted exosomes for drug delivery. Specifically, a scalable, robust, high-yield, cell line based exosome production process is created in a stirred-tank bioreactor, and an efficient surface tagging technique is developed to generate monoclonal antibody (mAb)-exosomes. The in vitro characterization using transmission electron microscopy, NanoSight, and western blotting confirm the high quality of exosomes. Flow cytometry and confocal laser scanning microscopy demonstrate that mAb-exosomes have strong surface binding to cancer cells. Furthermore, to validate the targeted drug delivery efficiency, romidepsin, a histone deacetylase inhibitor, is loaded into mAb-exosomes. The in vitro anti-cancer toxicity study shows high cytotoxicity of mAb-exosome-romidepsin to cancer cells. Finally, the in vivo study using tumor xenograft animal model validates the cancer targeting specificity, anti-cancer efficacy, and drug delivery capability of the targeted exosomes. In summary, new techniques enabling targeted exosomes for drug delivery are developed to support large-scale animal studies and to facilitate the translation from research to clinics., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: As patient-derived xenografts and other preclinical models of neuroendocrine tumors for testing personalized therapeutics are lacking, we have developed a perfused, 3D bioreactor model to culture tumor surrogates from patient-derived neuroendocrine tumors. This work evaluates the duration of surrogate culture and surrogate response to a novel antibody-drug conjugate., Methods: Twenty-seven patient-derived neuroendocrine tumors were cultured. Histologic sections of a pancreatic neuroendocrine tumor xenograft (BON-1) tumor were assessed for SSTR2 expression before tumor implantation into 2 bioreactors. One surrogate was treated with an antibody-drug conjugate composed of an anti-mitotic Monomethyl auristatin-E linked to a somatostatin receptor 2 antibody. Viability and therapeutic response were assessed by pre-imaging incubation with IR-783 and the RealTime-Glo AnnexinV Apoptosis and Necrosis Assay (Promega Corporation, Madison, WI) over 6 days. A primary human pancreatic neuroendocrine tumor was evaluated similarly., Results: Mean surrogate growth duration was 34.8 days. Treated BON-1 surrogates exhibited less proliferation (1.2 vs 1.9-fold) and greater apoptosis (1.5 vs 1.1-fold) than controls, whereas treated patient-derived neuroendocrine tumor bioreactors exhibited greater degrees of apoptosis (13- vs 9-fold) and necrosis (2.5- vs 1.6-fold)., Conclusion: Patient-derived neuroendocrine tumor surrogates can be cultured reliably within the bioreactor. This model can be used to evaluate the efficacy of antibody-guided chemotherapy ex vivo and may be useful for predicting clinical responses., (Published by Elsevier Inc.)

Pulmonary carcinoids are a type of neuroendocrine tumor (NET) accounting for 1-2% of lung cancer cases. Currently, Positron Emission Tomography (PET)/CT based on the radiolabeled sugar analogue [ 18 F]-FDG is used to diagnose and stage pulmonary carcinoids, but is suboptimal due to low metabolic activity in these tumors. A new technique for pulmonary carcinoid imaging, using PET/CT with radiolabeled somatostatin analogs that specifically target somatostatin receptor subtype 2 (SSTR2), is becoming more standard, as many tumors overexpress SSTR2. However, pulmonary carcinoid patients with diminished SSTR2 expression are not eligible for this imaging or any type of SSTR2-specific treatment. We have found that histone deacetylase (HDAC) inhibitors can upregulate the expression of SSTR2 in pulmonary carcinoid cell lines. In this study, we used a non-cytotoxic dose of HDAC inhibitors to induce pulmonary carcinoid SSTR2 expression in which we confirmed in vitro and in vivo. A non-cytotoxic dose of the HDAC inhibitors: thailandepsin A (TDP-A), romidepsin (FK228), suberoylanilide hydroxamic acid (SAHA), AB3, and valproic acid (VPA) were administered to promote SSTR2 expression in pulmonary carcinoid cell lines and xenografts. This SSTR2 upregulation technique using HDAC inhibitors could enhance radiolabeled somatostatin analog-based imaging and the development of potential targeted treatments for pulmonary carcinoid patients with marginal or diminished SSTR2 expression.

Background: Certain human carcinomas have demonstrated a distinct expression of somatostatin receptors. Data on somatostatin receptor expression in follicular thyroid cancer and anaplastic thyroid cancer has been limited and conflicting. This study seeks to characterize somatostatin receptor expression in follicular thyroid cancer and anaplastic thyroid cancer and to assess the effects of somatostatin analogues., Methods: Anaplastic thyroid cancer (Hth7 and 8505C) and follicular thyroid cancer (FTC-236) (Sigma-Aldrich, St. Louis, MO) cells were cultured. Capillary immunoblotting and reverse transcription polymerase chain reaction (RT-PCR) were used to determine the basal expression of protein and mRNA of SSTR1-SSTR5. Cells were treated with the somatostatin analogues octreotide, pasireotride (SOM230), and KE-108 for 48h. IC 50 was determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and cell proliferation was measured by viable cell count. Presence of SSTR2 was assessed by immunohistochemistry., Results: Immunoblotting analysis demonstrated that most cell lines expressed SSTR1-SSTR3 and SSTR5 in varying degrees. Reverse transcription polymerase chain reaction analysis showed that mRNA expression for SSTR2 and SSTR3 correlated with protein expression. MTT assays showed that KE-108 and SOM230 were able to inhibit cell proliferation. Tissue microarray (TMA) showed that SSTR2 was highly expressed in human tissues of aggressive thyroid carcinomas., Conclusion: Follicular thyroid cancer and anaplastic thyroid cancer express SSTR1-3 and SSTR5 in distinct fashions both at a message and protein level. Our results suggest that somatostatin receptors are still a relevant and promising drug target against non-medullary thyroid cancers., (Published by Elsevier Inc.)

Antibody-drug conjugate (ADC) is a class of targeted cancer therapies that combine the advantages of monoclonal antibody (mAb)'s specific targeting and chemotherapy's potent cytotoxicity. The therapeutic effect of ADC is significantly affected by its bioproduction process. This study aims to develop an effective ADC production process using anti-HER2 mAb-drug as a model therapeutic. First, a high titer (>2 g/L) of mAb was produced by Chinese hamster ovary cells from fed-batch cell culture. Both live-cell confocal microscopy imaging and flow cytometry analysis demonstrated that the produced mAb and ADC had strong and specific binding to HER2+ cell line BT474. Second, various conjugation conditions of mAb and drug, including linker selection, ratio of drug and mAb, and conjugation approaches, were investigated to improve the production yield and product quality. Finally, the ADC structure and biological quality were evaluated by SDS-PAGE and anti-breast cancer toxicity study, respectively. The ADC with integral molecular structure and high cytotoxicity (IC50 of 1.95 nM) was produced using the optimized production process. The robust bioproduction process could guide the development of ADC-based biopharmaceuticals., Competing Interests: The authors have declared that no competing interests exist.

Background: Medullary thyroid cancer portends poor survival once liver metastasis occurs. We hypothesize that Notch3 overexpression in medullary thyroid cancer liver metastasis will decrease proliferation and growth of the tumor., Methods: TT cells were modified genetically to overexpress Notch3 in the presence of doxycycline, creating the TT-Notch3 cell line. Mice were injected intrasplenically with either TT-Notch3 or control vector TT-TRE cells. Each cell line had 3 treatment groups: control with 12 weeks of standard chow, early DOX with doxycycline chow at day 0 and for 70 days thereafter, and late DOX with doxycycline chow at 8 weeks. Each animal underwent micro-computed tomography to evaluate for tumor formation and tumor quantification was performed. Animals were killed at 12 weeks, and the harvested liver was stained with Ki-67, hematoxylin and eosin, and Notch3., Results: Induction of Notch3 did not prevent formation of medullary thyroid cancer liver metastases as all mice in the early DOX group developed tumors. However, induction of Notch after medullary thyroid cancer liver tumor formation decreased tumor size, as seen on micro-computed tomography scans (late DOX group). This translated to a 37-fold decrease in tumor volume (P = .001). Notch3 overexpression also resulted in decreased Ki-67 index (P = .038). Moreover, Notch3 induction led to increased areas of neutrophil infiltration and necrosis on hematoxylin and eosin staining of the tumors CONCLUSION: Notch3 overexpression demonstrates an antiproliferative effect on established metastatic medullary thyroid cancer liver tumors and is a potential therapeutic target in treatment., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Notch signaling is minimally active in neuroendocrine (NE) cancer cells. While histone deacetylase inhibitors (HDACi) suppress NE cancer growth by inducing Notch, the molecular mechanism underlying this interplay has not yet been defined. NE cancer cell lines BON, H727, and MZ-CRC-1 were treated with known HDACi Thailadepsin-A (TDP-A) and valproic acid (VPA), and Notch1 mRNA expression was measured with RT-PCR. Truncated genomic fragments of the Notch1 promotor region fused with luciferase reporter were used to identify the potential transcription factor (TF) binding site. The key regulatory TF was identified with the electrophoretic mobility shift assay (EMSA). The effect of HDACi on Notch1 level was determined before and after silencing the TF. TDP-A and VPA induced Notch1 mRNA in a dose-dependent manner. A functional DNA motif at -80 to -52 from the Notch1 start codon responsible for the HDACi-dependent Notch1 induction was identified. Mutation of this core sequence failed to induce luciferase activity despite HDACi treatment. EMSA showed the greatest gel shift with AP-1 in nuclear extracts. Knockdown of AP-1 significantly attenuated the effect of HDACi on Notch1 induction. Interestingly, AP-1 transfection did not alter Notch1 level, suggesting that AP-1 is necessary but insufficient for HDACi activation of Notch1. Therefore, AP-1 is the TF that binds to a specific transcription-binding site within the Notch1 promotor region to trigger Notch1 transcription. Elucidating the HDACi activation mechanism may lead to the development of novel therapeutic options against NE cancers and facilitate the identification of clinical responders and prevent adverse effects., (© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Novel therapies for neuroendocrine (NE) cancers are desperately needed as they frequently present as metastatic disease and cause debilitating symptoms by secreting excessive hormones. Induction of Notch isoforms has a tumor suppressive effect in NE cancer cell lines, and we have observed that histone deacetylase inhibitors (HDACi) potently activate Notch. In this study, we describe the potential for Burkholderia thailandensis -derived class I HDACi thailandepsin A (TDP-A) as a Notch activator and therapeutic agent against NE cancer. IC 50 for TDP-A was determined to be 4-6 nM in NE cancer cell lines (BON, MZ-CRC-1, and TT) without cytotoxicity to lung fibroblasts. The binding characteristics of TDP-A to its target HDAC1 was examined using bioluminescence resonance energy transfer (BRET). Western blot and flow cytometry analysis showed that TDP-A induces cell cycle arrest and apoptosis in a dose-dependent manner. TDP-A dose-dependently activated the Notch pathway as measured by increasing functional CBF1-luciferase reporter signal and mRNA and protein expressions of Notch isoforms, which were attenuated by pretreatment with γ-secretase inhibitor DAPT. Furthermore, TDP-A lead to changes in expression level of downstream targets of Notch pathway and reduced expression of NE cancer markers. An in vivo study demonstrated that TDP-A suppressed NE cancer progression. These results show that TDP-A, as a Notch activator, is a promising agent against NE cancers., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Background: Thyroid tumorigenesis is characterized by a progressive loss of differentiation exhibited by a range of disease variants. The Notch receptor family (1-4) regulates developmental progression in both normal and cancerous tissues. This study sought to characterize the third Notch isoform (Notch3) across the various differentiated states of thyroid cancer, and determine its clinical impact., Methods: Notch3 expression was analyzed in a tissue microarray of normal and pathologic thyroid biopsies from 155 patients. The functional role of Notch3 was then investigated by upregulating its expression in a follicular thyroid cancer (FTC) cell line., Results: Notch3 expression regressed across decreasingly differentiated, increasingly malignant thyroid specimens, correlated with clinicopathological attributes reflecting poor prognosis, and independently predicted survival following univariate and multivariate analyses. Overexpression of the active Notch3 intracellular domain (NICD3) in a gain-of-function FTC line led to functional activation of centromere-binding protein 1, while increasing thyroid-specific gene transcription. NICD3 induction also reduced tumor burden in vivo and initiated the intrinsic apoptotic cascade, alongside suppressing cyclin and B-cell lymphoma 2 family expression., Conclusions: Loss of Notch3 expression may be fundamental to the process of dedifferentiation that accompanies thyroid oncogenesis. Conversely, activation of Notch3 in thyroid cancer exerts an antiproliferative effect and restores elements of a differentiated phenotype. These findings provide preclinical rationale for evaluating Notch3 as a disease prognosticator and therapeutic target in advanced thyroid cancer. Cancer 2017;123:769-82. © 2016 American Cancer Society., (© 2016 American Cancer Society.)