Thyroid-stimulating hormone receptor (TSHR) as a target for imaging differentiated thyroid cancer.

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 89Zr (t1/2 = 78.4 h, ß+ =22.7%) to produce [89Zr]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 [89Zr]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 [89Zr]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 [89Zr]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.

Preclinical safety evaluation of continuous UV-A lighting in an operative setting.

BACKGROUND: Germicidal ultraviolet (UV-C) light has been shown as an effective modality for disinfection in laboratory settings and in the operative room. Traditionally, short-wavelength UV-C devices, which have previously been shown to cause DNA damage, are utilized only for disinfection in pre- and post-operative settings and are not continuously active during operations. Continuous use of intraoperative UV light has potential to decrease pathogens and subsequent surgical site infections (SSIs), which arise in approximately 5-15% of operative cases. SSIs are a significant determinant of patient morbidity, readmission rates, and overall cost. Therefore, a method of UV light disinfection with a low risk of DNA damage is needed so that greater antimicrobial protection can be afforded to patients during the entirety of their surgical procedures. A new disinfection device that harnesses longer-wavelength UV-A light to disinfect the surgical field throughout the entirety of the procedure, including pre- and post-operation has been developed. METHODS: This study aimed to determine if UV-A light administered intraoperatively was safe, as defined by the minimal presence of DNA damage and safe amounts of reflection upon medical personnel. Using in vitro models, we examined the differential impacts of UV-C and UV-A light on DNA damage and repair pathways. In a murine model, we looked at the production of DNA damage photoproduction in relation to UV-A versus UV-C exposure. RESULTS: Our results show UV-A light does not induce a significant amount of DNA damage at the cellular or tissue level. Furthermore, a preclinical porcine study showed that surgical personnel were exposed to safe levels of UV-A irradiance from an overhead UV-A light used during an operation. The amount of UV-A transmitted through surgical personal protective equipment (PPE) also remained within safe levels. CONCLUSIONS: In conclusion, we found that UV-A may be safe for intraoperative use.

Somatostatin Receptor Type 2 and Thyroid-Stimulating Hormone Receptor Expression in Oncocytic Thyroid Neoplasms: Implications for Prognosis and Treatment.

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. χ2 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.

Voltage-Gated Sodium Channel NaV1.7 Inhibitors with Potent Anticancer Activities in Medullary Thyroid Cancer Cells.

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 NaV1.7 is uniquely expressed in aggressive MTC and not expressed in normal thyroid cells and tissues. We establish the druggability of NaV1.7 in MTC by identifying a novel inhibitor (SV188) and investigate its mode of binding and ability to inhibit INa current in NaV1.7. The whole-cell patch-clamp studies of the SV188 in the NaV1.7 channels expressed in HEK-293 cells show that SV188 inhibited the INa current in NaV1.7 with an IC50 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 IC50 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 NaV1.7. In addition, SV188 at 3 µM significantly inhibited the migration of MZ-CRC-1 and TT cells by 27% and 57%, respectively.

High-Throughput Analysis to Identify Activators of Notch Signaling.

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.

A hitchhiker's guide to cancer models.

Cancer is a complex and uniquely personal disease. More than 1.7 million people in the United States are diagnosed with cancer every year. As the burden of cancer grows, so does the need for new, more effective therapeutics and for predictive tools to identify optimal, personalized treatment options for every patient. Cancer models that recapitulate various aspects of the disease are fundamental to making advances along the continuum of cancer treatment from benchside discoveries to bedside delivery. In this review, we use a thought experiment as a vehicle to arrive at four broad categories of cancer models and explore the strengths, weaknesses, opportunities, and threats for each category in advancing our understanding of the disease and improving treatment strategies.

Under-Representation of Racial Groups in Genomics Studies of Gastroenteropancreatic Neuroendocrine Neoplasms.

Not all populations are poised to benefit from advancing genomics in gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN), as genomics have focused on White patients. This study aimed to evaluate racial populations represented in genomic studies of GEP-NENs and to provide evidence of differential genomic findings between racial groups in GEP-NENs. Manuscripts analyzing DNA, RNA, or DNA methylation in GEP-NENs were queried using PUBMED and EMBASE. NIH race/ethnicity term frequency was then determined by Natural Language Processing, followed by manual evaluation of tumor types and subjects by racial group. IHC of institutional tissue micro-arrays and analysis of AACR GENIE data analyzed was performed to determine mutational differences between Black and White pancreatic NEN (pNEN) patients. 313 manuscripts conducted the requisite genomic analyses, 16 of which included subject race data. Race data were included in 13/184 DNA, 4/107 RNA, and 1/54 DNA Methylation analyses. These studies included 89% White subjects (n = 2032), 5.8% Asian subjects (n = 132), 4.0% "Other" subjects (n = 93), and 1.2% Black subjects (n = 27). No Native American/Alaska Native, Native Hawaiian/Pacific Islander, or ethnically Hispanic/Latinx subjects were represented. There were significant differences in MEN1 mutations among Black and White patients in immunohistochemical (13:40) and GENIE data (24:268 patients per group, respectively), with 9 additional genes differentially mutated in the GENIE dataset. Genomic sequencing data for GEP-NENs is almost racially homogenous. Differences in pNEN genomics may exist between racial groups, highlighting a need for diversity in future genomic analyses of GEP-NENs to understand the putative influence of interracial genomic variation on GEP-NEN prevention, diagnosis, and therapy. Significance: There is little diversity in genomic studies of GEP-NENs, which may exhibit clinically impactful variation in their tumor biology among racial groups. Improved diversity in such studies is imperative for understanding this variation and its potential impacts on disease prevention, diagnosis, therapeutic targeting, and clinical outcomes.

Cdk5 drives formation of heterogeneous pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous population of neoplasms that arise from hormone-secreting islet cells of the pancreas and have increased markedly in incidence over the past four decades. Non-functional PanNETs, which occur more frequently than hormone-secreting tumors, are often not diagnosed until later stages of tumor development and have poorer prognoses. Development of successful therapeutics for PanNETs has been slow, partially due to a lack of diverse animal models for pre-clinical testing. Here, we report development of an inducible, conditional mouse model of PanNETs by using a bi-transgenic system for regulated expression of the aberrant activator of Cdk5, p25, specifically in ß-islet cells. This model produces a heterogeneous population of PanNETs that includes a subgroup of well-differentiated, non-functional tumors. Production of these tumors demonstrates the causative potential of aberrantly active Cdk5 for generation of PanNETs. Further, we show that human PanNETs express Cdk5 pathway components, are dependent on Cdk5 for growth, and share genetic and transcriptional overlap with the INS-p25OE model. The utility of this model is enhanced by the ability to form tumor-derived allografts. This new model of PanNETs will facilitate molecular delineation of Cdk5-dependent PanNETs and the development of new targeted therapeutics.

Development of an arteriolar niche and self-renewal of breast cancer stem cells by lysophosphatidic acid/protein kinase D signaling.

Breast cancer stem cells (BCSCs) are essential for cancer growth, metastasis and recurrence. The regulatory mechanisms of BCSC interactions with the vascular niche within the tumor microenvironment (TME) and their self-renewal are currently under extensive investigation. We have demonstrated the existence of an arteriolar niche in the TME of human BC tissues. Intriguingly, BCSCs tend to be enriched within the arteriolar niche in human estrogen receptor positive (ER+) BC and bi-directionally interact with arteriolar endothelial cells (ECs). Mechanistically, this interaction is driven by the lysophosphatidic acid (LPA)/protein kinase D (PKD-1) signaling pathway, which promotes both arteriolar differentiation of ECs and self-renewal of CSCs likely via differential regulation of CD36 transcription. This study indicates that CSCs may enjoy blood perfusion to maintain their stemness features. Targeting the LPA/PKD-1 -CD36 signaling pathway may have therapeutic potential to curb tumor progression by disrupting the arteriolar niche and effectively eliminating CSCs.

Notch Signaling in Vascular Endothelial Cells, Angiogenesis, and Tumor Progression: An Update and Prospective.

The Notch signaling pathway plays an essential role in a wide variety of biological processes including cell fate determination of vascular endothelial cells and the regulation of arterial differentiation and angiogenesis. The Notch pathway is also an essential regulator of tumor growth and survival by functioning as either an oncogene or a tumor suppressor in a context-dependent manner. Crosstalk between the Notch and other signaling pathways is also pivotal in tumor progression by promoting cancer cell growth, migration, invasion, metastasis, tumor angiogenesis, and the expansion of cancer stem cells (CSCs). In this review, we provide an overview and update of Notch signaling in endothelial cell fate determination and functioning, angiogenesis, and tumor progression, particularly in the development of CSCs and therapeutic resistance. We further summarize recent studies on how endothelial signaling crosstalk with the Notch pathway contributes to tumor angiogenesis and the development of CSCs, thereby providing insights into vascular biology within the tumor microenvironment and tumor progression.

Notch Signaling in Thyroid Cancer.

Thyroid cancer is the most common malignancy of the endocrine system with a steadily rising incidence. The term "thyroid cancer" encompasses a spectrum of subtypes, namely papillary thyroid cancer, follicular thyroid cancer, anaplastic thyroid cancer, and medullary thyroid cancer. Each subtype differs histopathologically and in degrees of cellular differentiation, which may be in part due to signaling of the Notch pathway. The Notch pathway is an evolutionarily conserved signal transduction mechanism that regulates cell proliferation, differentiation, survival, stem cell maintenance, embryonic and adult development, epithelial-mesenchymal transition, and angiogenesis. Its role in cancer biology is controversial, as it has been shown to play both an oncogenic and tumor-suppressive role in many different types of cancers. This discordance holds true for each subtype of thyroid cancer, indicating that Notch signaling is likely cell type and context dependent. Whether oncogenic or not, Notch signaling has proven to be significantly involved in the tumorigenesis of thyroid cancer and has thus earned interest as a therapeutic target. Advancement in the understanding of Notch signaling in thyroid cancer holds great promise for the development of novel treatment strategies to benefit patients.

A growth model of neuroendocrine tumor surrogates and the efficacy of a novel somatostatin-receptor-guided antibody-drug conjugate: Perspectives on clinical response?

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.

Overexpression of somatostatin receptor type 2 in neuroendocrine tumors for improved Ga68-DOTATATE imaging and treatment.

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.

Pulmonary Carcinoid Surface Receptor Modulation Using Histone Deacetylase Inhibitors.

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 [18F]-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.