Mechanisms of Sorafenib Resistance in HCC Culture Relate to the Impaired Membrane Expression of Organic Cation Transporter 1 (OCT1).

Introduction: Sorafenib, an FDA-approved drug for advanced hepatocellular carcinoma (HCC) treatment, encounters resistance in many patients. Deciphering the mechanisms underlying sorafenib resistance is crucial for devising alternative strategies to overcome it. Aim: This study aimed to investigate sorafenib resistance mechanisms using a diverse panel of HCC cell lines. Methods: HCC cell lines were subjected to continuous sorafenib treatment, and stable cell lines (Huh 7.5 and Huh 7PX) exhibiting sustained growth in its presence were isolated. The investigation of drug resistance mechanisms involved a comparative analysis of drug-targeted signal transduction pathways (EGFR/RAF/MEK/ERK/Cyclin D), sorafenib uptake, and membrane expression of the drug uptake transporter. Results: HCC cell lines (Huh 7.5 and Huh 7PX) with a higher IC50 (10µM) displayed a more frequent development of sorafenib resistance compared to those with a lower IC50 (2-4.8µM), indicating a potential impact of IC50 variation on initial treatment response. Our findings reveal that activated overexpression of Raf1 kinases and impaired sorafenib uptake, mediated by reduced membrane expression of organic cation transporter-1 (OCT1), contribute to sorafenib resistance in HCC cultures. Stable expression of the drug transporter OCT1 through cDNA transfection or adenoviral delivery of OCT1 mRNA increased sorafenib uptake and successfully overcame sorafenib resistance. Additionally, consistent with sorafenib resistance in HCC cultures, cirrhotic liver-associated human HCC tumors often exhibited impaired membrane expression of OCT1 and OCT3. Conclusion: Intrinsic differences among HCC cell clones, affecting sorafenib sensitivity at the expression level of Raf kinases, drug uptake, and OCT1 transporters, were identified. This study underscores the potential of HCC tumor targeted OCT1 expression to enhance sorafenib treatment response.

Assessing Adequacy: A Meta-Analysis of Rapid Onsite Evaluation of Thyroid Nodules.

INTRODUCTION: Fine-needle aspiration (FNA) is the standard form of preoperative evaluation of thyroid nodule cytological status. A significant number FNAs are classified as inadequate for interpretation, requiring a repeat FNA which is potentially avoidable, costly, and delays treatment. To address these concerns and maximize first-time FNA adequacy, rapid onsite evaluation (ROSE) of FNA specimens was introduced. Our study aims to determine the impact of ROSE on FNA adequacy. METHODS: PubMed, Embase, and Web of Science were searched for primary articles assessing the adequacy of ROSE in thyroid nodules. RESULTS: A total of 17 studies were included for a total of 24,649 thyroid nodes. Thirteen thousand two hundred fifteen (53.6%) thyroid nodules were assessed utilizing ROSE and 11,434 (46.4%) were not. Pooled adequacy increased significantly from 76% without ROSE to 92% with rose (P = 0.001). Use of ROSE increased the odds of adequate FNA by 22% (risk ratio (RR) = 1.22, 95% confidence interval (CI) = 1.12-1.32). At institutions with less than 85% effective diagnostic adequacy without ROSE, the risk for diagnostic adequacy increased by 28% with ROSE implementation (RR = 1.28, 95% CI = 1.20-1.37). In contrast, in studies reported from institutions with an effective diagnostic rate greater than 85% without the use of ROSE, the diagnostic adequacy only increased by 5% with ROSE implementation (RR = 1.05, 95% CI = 1.03-1.06). CONCLUSIONS: The use of ROSE during first-time FNA of thyroid nodules can significantly improve adequacy, especially at institutions with baseline high inadequacy rates. Implementation of ROSE can reduce repeat FNAs and its associated consequences.

Survival Outcomes of Medullary Thyroid Cancer With and Without Amyloid Deposition.

OBJECTIVE: Amyloid deposition within tumor stroma is a distinctive histologic feature of medullary thyroid cancer (MTC). However, its prognostic significance remains uncertain. We aimed to elucidate the impact of amyloid status on survival outcomes in a large cohort. METHODS: The Surveillance, Epidemiology, and End Results registry was queried to identify patients diagnosed with MTC from 2000 to 2019. Patients with amyloid-positive (International Classification of Diseases for Oncology, third edition code 8345/3) and amyloid negative (International Classification of Diseases for Oncology, third edition code 8510/3) tumors were analyzed. Overall and disease-specific survival were compared between matched cohorts using Kaplan-Meier and Cox proportional hazards analyses. RESULTS: Of the 2526 MTC patients, 511 of which were amyloid-positive and 2015 that were amyloid negative. Amyloid-positive patients displayed lower T stage (T3/4: 28% vs 85%, P < .001) and less extrathyroidal extension (11.3% vs 81.6%, P < .001). No difference in distant metastasis rate was observed between groups (14.5% vs 14.4%, P = .98). However, amyloid-positive patients showed a tendency for distal lymph node metastasis (1.2% vs 0.3%, P = .020). On univariate analysis, amyloid-positive status showed comparable overall survival times (mean 172.2 vs 177.8 months, P = .17), but a trend toward worse cancer-specific survival (hazard ratios [HR] = 1.31, 95% CI = 0.99-1.71, P = .051). After adjusting for covariates, amyloid deposition did not independently predict overall (HR = 1.15, 95% CI = 0.91-1.47, P = .25) or cancer-specific survival (HR = 1.30, 95% CI = 0.96-1.77, P = .09). Initiating therapy later than 1 month following diagnosis was associated with worse overall survival (HR = 1.25, 95% CI = 1.02-1.54, P = .029). CONCLUSIONS: The presence of amyloid in MTC paradoxically associates with lower T stage yet exhibits a trend toward worse cancer-specific mortality. Amyloid deposition alone does not independently influence prognosis. Delayed treatment adversely impacted overall survival.

Matrix Metalloproteinase 9/microRNA-145 Ratio: Bridging Genomic and Immunological Variabilities in Thyroid Cancer.

Matrix metalloproteinase 9 (MMP9) and microRNA-145 (miR-145) have emerged as essential biomarkers in thyroid cancer progression and metastasis. However, their combined evaluation and clinical utility as a unified prognostic marker across diverse thyroid cancer subgroups remain unexplored. We investigated the diagnostic and prognostic value of the MMP9/miR-145 ratio in thyroid cancer, hypothesizing it may overcome inter-patient heterogeneity and serve as a versatile biomarker regardless of genetic mutations or autoimmune status. MMP9 and miR-145 expressions were analyzed in 175 paired papillary thyroid cancer (PTC) and normal tissues. Plasma levels were assessed perioperatively and longitudinally over 12-18 months in 86 matched PTC patients. The associations with clinicopathological parameters and patient outcomes were evaluated. MMP9 was upregulated, and miR-145 downregulated in cancer tissues, with a median MMP9/miR-145 ratio 17.6-fold higher versus controls. The tissue ratio accurately diagnosed thyroid malignancy regardless of BRAF mutation or Hashimoto's thyroiditis status, overcoming genetic and autoimmune heterogeneity. A high preoperative circulating ratio predicted aggressive disease features, including lymph node metastasis, extrathyroidal extension, progression/relapse, and recurrence. Although the preoperative plasma ratio was elevated in patients with unfavorable outcomes, it had limited utility for post-surgical monitoring. In conclusion, the MMP9/miR-145 ratio is a promising biomarker in PTC that bridges genetic and immunological variabilities, enhancing preoperative diagnosis and prognostication across diverse patient subgroups. It accurately stratifies heterogenous cases by aggressiveness. The longitudinal trends indicate decreasing applicability for post-thyroidectomy surveillance. Further large-scale validation and protocol standardization can facilitate clinical translation of the MMP9/miR-145 ratio to guide personalized thyroid cancer management.

Genomic Breakpoint Characterization and Transcriptome Analysis of Metastatic, Recurrent Desmoplastic Small Round Cell Tumor.

Desmoplastic small round cell tumor (DSRCT) is a rare pediatric cancer caused by the EWSR1-WT1 fusion oncogene. Despite initial response to chemotherapy, DSRCT has a recurrence rate of over 80% leading to poor patient prognosis with a 5-year survival rate of only 15-25%. Owing to the rarity of DSRCT, sample scarcity is a barrier in understanding DSRCT biology and developing effective therapies. Utilizing a novel pair of primary and recurrent DSRCTs, we present the first map of DSRCT genomic breakpoints and the first comparison of gene expression alterations between primary and recurrent DSRCT. Our genomic breakpoint map includes the lone previously published DSRCT genomic breakpoint, the breakpoint from our novel primary/recurrent DSRCT pair, as well as the breakpoints of five available DSRCT cell lines and five additional DSRCTs. All mapped breakpoints were unique and most breakpoints included a 1-3 base pair microhomology suggesting microhomology-mediated end-joining as the mechanism of translocation fusion and providing novel insights into the etiology of DSRCT. Through RNA-sequencing analysis, we identified altered genes and pathways between primary and recurrent DSRCTs. Upregulated pathways in the recurrent tumor included several DNA repair and mRNA splicing-related pathways, while downregulated pathways included immune system function and focal adhesion. We further found higher expression of the EWSR1-WT1 upregulated gene set in the recurrent tumor as compared to the primary tumor and lower expression of the EWSR1-WT1 downregulated gene set, suggesting the EWSR1-WT1 fusion continues to play a prominent role in recurrent tumors. The identified pathways including upregulation of DNA repair and downregulation of immune system function may help explain DSRCT's high rate of recurrence and can be utilized to improve the understanding of DSRCT biology and identify novel therapies to both help prevent recurrence and treat recurrent tumors.

Diagnostic Performance of Afirma and Interpace Diagnostics Genetic Testing in Indeterminate Thyroid Nodules: A Single Center Study.

Indeterminate thyroid nodules (ITN) represent 20-30% of biopsied nodules, with a 10-60% risk of malignancy. Molecular testing can stratify the risk of malignancy among ITNs, and subsequently reduce the need for unnecessary diagnostic surgery. We aimed to assess the performance of these molecular tests at a single institution. Patients with Bethesda III, IV, and V nodules with Afirma and Interpace Diagnostics genetic testing data from November 2013 to November 2021 were included. Three cohorts were formed, including GSC + XA, ThyGeNEXT + ThyraMIR, and GSC + GEC. Statistical analysis determined the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic odds ratio (DOR), and accuracy of each type of testing. The PPV of nodules undergoing genetic testing by ThyGeNEXT + ThyraMIR (45.00%, 95%CI: 28.28-62.93%, p = 0.032) and GSC + XA (57.14%, 95%CI: 29.32-81.08%, p < 0.001) were superior to that of GEC + GSC (30.72%, 95%CI: 26.83-34.90%). The NPV was above 85% in all cohorts, suggesting overall suitable rule-out tests. The Afirma platform (GSC + XA) had the highest NPV at 96.97%. The overall accuracy for nodules undergoing ThyGeNEXT + ThyraMIR was 81.42% (95%CI: 73.01-88.11%, p < 0.001). A total of 230 patients underwent thyroidectomy, including less than 60% of each of the ThyGeNEXT + ThyraMIR and GSC + XA cohorts. Specifically, only 25% of patients in the GSC + XA cohort underwent surgery, considerably decreasing the rate of unnecessary surgical intervention. Sub-group analysis, including only patients with surgical pathology, found that PPV tended to be higher in the GSC + XA cohort, at 66.67% (95%CI: 37.28-87.06%), as compared to the ThyGeNEXT + ThyraMIR cohort, at 52.94% (95%CI: 35.25-69.92%). The Afirma genetic testing platform GSC + XA outperformed the other platforms with regards to both PPV and NPV and decreased the rate of surgery in patients with ITNs by 75%, significantly preventing unnecessary surgical intervention.

Chromatin-Accessible miRNA Regulons Driving Thyroid Tumorigenesis and Progression.

BACKGROUND: Although papillary thyroid cancer can remain indolent, associated lymph node metastases and recurrence rates are approximately 50% and 20%, respectively. Omics-based medicine has led to the discovery of predictive biomarkers that can be used to predict tumor progression and clinical outcomes. We aimed to develop a noninvasive omics-driven blood test to allow accurate risk stratification and help tailor individual patient treatment plans. STUDY DESIGN: RNA sequencing (seq) and microRNA analysis of The Cancer Genome Atlas and Gene Expression Omnibus datasets were employed to identify an epigenetic prognostic panel. Integrated bulk assay for transposase-accessible chromatin-seq and RNA-seq experiments confirmed the results. Sixty-two paired tumor and adjacent control thyroid tissues and 67 blood samples (62 papillary thyroid cancer and 5 controls) were analyzed for validation using sequencing and real-time polymerase chain reaction and correlated to clinical outcomes. A liposome-exosome fusion clustered regularly interspaced short palindromic repeats (CRISPR)-fluorescent detection system miRNA assay was developed. A predictive risk nomogram was generated and tested for performance. RESULTS: Our miRNA panel (miR-146b-5p and miR-221-3p) from tissue and blood was associated with aggressive features and was located within accessible chromatin regions. The miRNA risk score and prognostic nomogram showed higher accuracy in predicting lymph node metastases (miR-146b: area under the curve [AUC] 0.816, sensitivity 76.9%; miR-221: AUC 0.740, sensitivity 79.5%) and recurrence (miR-146b: AUC 0.921, sensitivity 75.0%; miR-221: AUC 0.756, sensitivity 70.0%; p < 0.001) than staging and American Thyroid Association risk stratification. CRISPR-based miRNA assays showed upregulation in the blood of cancer cohorts. CONCLUSIONS: CRISPR-based detection of miR-146b and miR-221 in the blood of thyroid cancer patients is a reliable and noninvasive tool for real-time assessment and prognostication that has great potential to provide a direct impact on the care of these patients.

A miRNA-Based Prognostic Model to Trace Thyroid Cancer Recurrence.

Papillary thyroid carcinomas (PTCs) account for most endocrine tumors; however, screening and diagnosing the recurrence of PTC remains a clinical challenge. Using microRNA sequencing (miR-seq) to explore miRNA expression profiles in PTC tissues and adjacent normal tissues, we aimed to determine which miRNAs may be associated with PTC recurrence and metastasis. Public databases such as TCGA and GEO were utilized for data sourcing and external validation, respectively, and miR-seq results were validated using quantitative real-time PCR (qRT-PCR). We found miR-145 to be significantly downregulated in tumor tissues and blood. Deregulation was significantly related to clinicopathological features of PTC patients including tumor size, lymph node metastasis, TNM stage, and recurrence. In silico data analysis showed that miR-145 can negatively regulate multiple genes in the TC signaling pathway and was associated with cell apoptosis, proliferation, stem cell differentiation, angiogenesis, and metastasis. Taken together, the current study suggests that miR-145 may be a biomarker for PTC recurrence. Further mechanistic studies are required to uncover its cellular roles in this regard.

Salt-Inducible Kinase 1 is a potential therapeutic target in Desmoplastic Small Round Cell Tumor.

Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive malignant cancer caused by a chromosomal translocation t(11;22)(p13;q12) that produces an oncogenic transcription factor, EWSR1-WT1. EWSR1-WT1 is essential for the initiation and progression of DSRCT. However, the precise mechanism by which EWSR1-WT1 drives DSRCT oncogenesis remains unresolved. Through our integrative gene expression analysis, we identified Salt Inducible Kinase 1 (SIK1) as a direct target of EWSR1-WT1. SIK1 as a member of the AMPK related kinase is involved in many biological processes. We showed that depletion of SIK1 causes inhibition of tumor cell growth, similar to the growth inhibition observed when EWSR1-WT1 is depleted. We further showed that silencing SIK1 leads to cessation of DNA replication in DSRCT cells and inhibition of tumor growth in vivo. Lastly, combined inhibition of SIK1 and CHEK1with small molecule inhibitors, YKL-05-099 and prexasertib, respectively, showed enhanced cytotoxicity in DSRCT cells compared to inhibition of either kinases alone. This work identified SIK1 as a new potential therapeutic target in DSRCT and the efficacy of SIK1 inhibition may be improved when combined with other intervention strategies.

Experimental Validation of Novel Glypican 3 Exosomes for the Detection of Hepatocellular Carcinoma in Liver Cirrhosis.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) developing in the context of preexisting cirrhosis is characterized by impaired autophagy that results in increased exosome release. This study was conducted to determine whether circulating exosomes expressing glypican 3 (GPC3) could be utilized as a biomarker for HCC detection and treatment response in patients with cirrhosis. METHODS: Immunohistochemistry was performed to assess p62 and GPC3 expression in the lesion and adjacent tissue from cirrhosis with HCC. GPC3-enriched exosomes were captured by an enzyme-linked immunosorbent assay (ELISA). The diagnostic specificity of serum exosome-derived GPC3 (eGPC3) was determined using samples obtained from malignancy-free controls, malignancy-free cirrhotics, cirrhotics with confirmed HCC, and patients with a non-HCC malignancy. The performance of eGPC3 was validated using serum samples of HCC patients received chemotherapy. RESULTS: We found that the expression of p62 and GPC3 was significantly increased in HCC tissues compared to adjacent cirrhotic liver. Impaired autophagy and exosome shedding were confirmed in HCC cell lines. Mass spectroscopic analysis revealed that GPC3 was enriched in exosomes isolated from HCC cell lines. An affinity ELISA assay was developed that specifically captures GPC3 positive exosomes in the serum. Total exosome concentration and eGPC3 were significantly elevated in cirrhotic patients with HCC as compared to the reference control groups. Furthermore, decreases in post-treatment exosome concentration and eGPC3 levels were more closely correlated with response to locoregional chemotherapy compared to change in serum AFP in HCC patients awaiting liver transplantation. CONCLUSION: We developed an affinity exosome capture assay to detect GPC3 enriched exosomes. Our preliminary assessment shows that GPC3 positive exosomes can be used for HCC detection and prediction of treatment outcomes.

MicroRNA-Based Risk Score for Predicting Tumor Progression Following Radioactive Iodine Ablation in Well-Differentiated Thyroid Cancer Patients: A Propensity-Score Matched Analysis.

To identify molecular markers that can accurately predict aggressive tumor behavior at the time of surgery, a propensity-matching score analysis of archived specimens yielded two similar datasets of DTC patients (with and without RAI). Bioinformatically selected microRNAs were quantified by qRT-PCR. The risk score was generated using Cox regression and assessed using ROC, C-statistic, and Brier-score. A predictive Bayesian nomogram was established. External validation was performed, and causal network analysis was generated. Within the eight-year follow-up period, progression was reported in 51.5% of cases; of these, 48.6% had the T1a/b stage. Analysis showed upregulation of miR-221-3p and miR-222-3p and downregulation of miR-204-5p in 68 paired cancer tissues (p < 0.001). These three miRNAs were not differentially expressed in RAI and non-RAI groups. The ATA risk score showed poor discriminative ability (AUC = 0.518, p = 0.80). In contrast, the microRNA-based risk score showed high accuracy in predicting tumor progression in the whole cohorts (median = 1.87 vs. 0.39, AUC = 0.944) and RAI group (2.23 vs. 0.37, AUC = 0.979) at the cutoff >0.86 (92.6% accuracy, 88.6% sensitivity, 97% specificity) in the whole cohorts (C-statistics = 0.943/Brier = 0.083) and RAI subgroup (C-statistic = 0.978/Brier = 0.049). The high-score group had a three-fold increased progression risk (hazard ratio = 2.71, 95%CI = 1.86-3.96, p < 0.001) and shorter survival times (17.3 vs. 70.79 months, p < 0.001). Our prognostic microRNA signature and nomogram showed excellent predictive accuracy for progression-free survival in DTC.

Image-Guided Intensity-Modulated Radiation Therapy for IgG4-Related Ophthalmic Disease.

BACKGROUND: IgG4-related ophthalmic disease is a rare, newly recognized entity with high failure rates on first-line therapy of systemic corticosteroids and no other proven management options. Case Presentation. Here, we present the clinical course of a patient with IgG4 ophthalmic disease who achieved a favorable response from radiotherapy. Our patient initially presented with a history of recurrent painful flares of orbital inflammation, a pathologic diagnosis follicular lymphoid hyperplasia from a right lacrimal gland biopsy, and MRI imaging noting expansion of the lateral rectus muscle of the right eye. Initial treatment with dacryoadenectomy and multiple courses of corticosteroids failed to keep his symptoms at bay. Further evaluation revealed florid IgG4 staining. In this context, he was evaluated for image-guided intensity-modulated radiotherapy (IG-IMRT) to the orbit to 20 Gy in 10 fractions. His ophthalmic symptoms resolved. CONCLUSIONS: This treatment experience suggests radiotherapy may be a favorable option for symptom relief in patients with IgG4-related ophthalmic disease not controlled by corticosteroids.

CD4+ T helper 17 cell response of aged mice promotes prostate cancer cell migration and invasion.

BACKGROUND: Aging is the most important risk factor for prostate cancer (PCa), but how age contributes to PCa is poorly understood. Aging is characterized by low-grade systemic inflammation (i.e., inflammaging) that is often attributed to the progressive activation of immune cells over time, which may play an important role in prostate carcinogenesis. Th17 response is elevated in aging humans and mice, but it remains unknown whether it is increased in prostate tissue or contributes to prostate carcinogenesis during aging. In this study, we aimed to determine the role of age-related Th17 response in PCa cell growth, migration, and invasion. METHODS: C57BL/6J (B6) mouse was used as an aging animal model and the prostate histopathology during aging was analyzed. Splenic CD4+ T cells were isolated from young (16-20 weeks old) and aged (96-104 weeks old) mice, and cultured in the presence of plate-bound anti-CD3/anti-CD28, with or without Th17 differentiation conditions. The cells were collected and used for subsequent flow cytometry or quantitative reverse transcription polymerase chain reaction. The supernatant was collected and used to treat PCa cell lines. The treated PCa cells were analyzed for cell viability, migration, invasion, and nuclear factor kappa B (NF-κB) signaling. RESULTS: Aged mice had enlarged prostate glands and increased morphological alterations, with not only increased inflammatory cell infiltration but also increased Th17 cytokines in prostate tissue, compared to young mice. Naïve CD4+ T cells from aged mice differentiated increased interleukin (IL)-17-expressing cells. CD4+ T cells from aged mice spleen had increased Th17 cells, Th17 cytokines and Th17/Treg ratio compared to young mice. Factors secreted from aged CD4+ T cells, especially from ex vivo differentiated Th17 cells, not only promoted PCa cell viability, migration, and invasion but also activated the NF-κB signaling in PCa cells compared to young mice. CONCLUSIONS: These results indicate that age-related CD4+ T cells, especially Th17 cells-secreted factors have the potential to contribute to prostate carcinogenesis. Our work could prompt further research using autochthonous PCa mouse models at different ages to elucidate the functional role of Th17 response in prostate carcinogenesis during aging.

Somatic mutations in the DNA repairome in prostate cancers in African Americans and Caucasians.

Most hereditary tumors show aberrations in DNA repair genes or their regulators. In contrast, only a minority of sporadic tumors show alterations in these genes. As a result, genomic instability is currently considered an enhancer of tumorigenesis rather than an obligatory event in this process. However, tumor heterogeneity presents a significant technical challenge for most cancer genomics studies performed at less than 100× mean resolution depth. To address the importance of genomic instability in prostate carcinogenesis and tumor progression, we performed ultrahigh depth exome sequencing of 124 DNA damage repair/response (repairome) genes in 63 tumors and matched normal tissue samples in African Americans and Caucasians. The average sequence depth was 712-fold for DNA isolated from normal tissue and 368-fold for FFPE tumors. We identified 671 somatic mutations in tumors from African Americans and 762 somatic mutations in tumors in Caucasians. The most frequently mutated DNA repairome genes were EXO1, ATR, POLQ, NEIL3, ERCC6, BRCA2, BRCA1, XPC, JAG1, RPA1, POLE, ATM, and LIG1 in African American men, and POLQ, NEIL3, POLB, BRCA2, EXO1, ERCC6, ATR, RBBP8, BRCA1, ATM, JAG1, XPC, and POLE in Caucasians. We found that 89% of tumors had at least one mutation in nucleotide excision repair pathway genes in African Americans, whereas >40% of tumors had mutations in base excision repair pathway genes in Caucasians. We further identified a marginal increase in mutation rate in tumors in African Americans with increasing age. Tumors in Caucasians did not show a correlation with age, but a progressive increase in the mutation rate was observed at higher Gleason scores. Our data reveal significant differences in the molecular signatures in the DNA repairome in prostate cancer between African Americans and Caucasians. These data also have substantial implications regarding the well-known health disparities in prostate cancer, such as the higher mortality in African Americans than Caucasians.

Patient-Derived Xenografts as an Innovative Surrogate Tumor Model for the Investigation of Health Disparities in Triple Negative Breast Cancer.

Despite a decline in overall incidence rates for cancer in the past decade, due in part to impressive advancements in both diagnosis and treatment, breast cancer (BC) remains the leading cause of cancer-related deaths in women. BC alone accounts for ∼30% of all new cancer diagnoses in women worldwide. Triple-negative BC (TNBC), defined as having no expression of the estrogen or progesterone receptors and no amplification of the HER2 receptor, is a subtype of BC that does not benefit from the use of estrogen receptor-targeting or HER2-targeting therapies. Differences in socioeconomic factors and cell intrinsic and extrinsic characteristics have been demonstrated in Black and White TNBC patient tumors. The emergence of patient-derived xenograft (PDX) models as a surrogate, translational, and functional representation of the patient with TNBC has led to the advances in drug discovery and testing of novel targeted approaches and combination therapies. However, current established TNBC PDX models fail to represent the diverse patient population and, most importantly, the specific ethnic patient populations that have higher rates of incidence and mortality. The primary aim of this review is to emphasize the importance of using clinically relevant translatable tumor models that reflect TNBC human tumor biology and heterogeneity in high-risk patient populations. The focus is to highlight the complexity of BC as it specifically relates to the management of TNBC in Black women. We discuss the importance of utilizing PDX models to study the extracellular matrix (ECM), and the distinct differences in ECM composition and biophysical properties in Black and White women. Finally, we demonstrate the crucial importance of PDX models toward novel drug discovery in this patient population.

Chaperone-Mediated Autophagy in the Liver: Good or Bad?

Hepatitis C virus (HCV) infection triggers autophagy processes, which help clear out the dysfunctional viral and cellular components that would otherwise inhibit the virus replication. Increased cellular autophagy may kill the infected cell and terminate the infection without proper regulation. The mechanism of autophagy regulation during liver disease progression in HCV infection is unclear. The autophagy research has gained a lot of attention recently since autophagy impairment is associated with the development of hepatocellular carcinoma (HCC). Macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) are three autophagy processes involved in the lysosomal degradation and extracellular release of cytosolic cargoes under excessive stress. Autophagy processes compensate for each other during extreme endoplasmic reticulum (ER) stress to promote host and microbe survival as well as HCC development in the highly stressed microenvironment of the cirrhotic liver. This review describes the molecular details of how excessive cellular stress generated during HCV infection activates CMA to improve cell survival. The pathological implications of stress-related CMA activation resulting in the loss of hepatic innate immunity and tumor suppressors, which are most often observed among cirrhotic patients with HCC, are discussed. The oncogenic cell programming through autophagy regulation initiated by a cytoplasmic virus may facilitate our understanding of HCC mechanisms related to non-viral etiologies and metabolic conditions such as uncontrolled type II diabetes. We propose that a better understanding of how excessive cellular stress leads to cancer through autophagy modulation may allow therapeutic development and early detection of HCC.

Drug resistance profiling of a new triple negative breast cancer patient-derived xenograft model.

BACKGROUND: Triple-negative breast cancer (TNBC) represents an aggressive subtype with limited therapeutic options. Experimental preclinical models that recapitulate their tumors of origin can accelerate target identification, thereby potentially improving therapeutic efficacy. Patient-derived xenografts (PDXs), due to their genomic and transcriptomic fidelity to the tumors from which they are derived, are poised to improve the preclinical testing of drug-target combinations in translational models. Despite the previous development of breast and TNBC PDX models, those derived from patients with demonstrated health-disparities are lacking. METHODS: We use an aggressive TNBC PDX model propagated in SCID/Beige mice that was established from an African-American woman, TU-BcX-2 K1, and assess its metastatic potential and drug sensitivities under distinct in vitro conditions. Cellular derivatives of the primary tumor or the PDX were grown in 2D culture conditions or grown in mammospheres 3D culture. Flow cytometry and fluorescence staining was used to quantify cancer stem cell-like populations. qRT-PCR was used to describe the mesenchymal gene signature of the tumor. The sensitivity of TU-BcX-2 K1-derived cells to anti-neoplastic oncology drugs was compared in adherent cells and mammospheres. Drug response was evaluated using a live/dead staining kit and crystal violet staining. RESULTS: TU-BcX-2 K1 has a low propensity for metastasis, reflects a mesenchymal state, and contains a large burden of cancer stem cells. We show that TU-BcX-2 K1 cells have differential responses to cytotoxic and targeted therapies in 2D compared to 3D culture conditions insofar as several drug classes conferred sensitivity in 2D but not in 3D culture, or cells grown as mammospheres. CONCLUSIONS: Here we introduce a new TNBC PDX model and demonstrate the differences in evaluating drug sensitivity in adherent cells compared to mammosphere, or suspension, culture.

A novel patient-derived xenograft model for claudin-low triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) subtypes are clinically aggressive and cannot be treated with targeted therapeutics commonly used in other breast cancer subtypes. The claudin-low (CL) molecular subtype of TNBC has high rates of metastases, chemoresistance and recurrence. There exists an urgent need to identify novel therapeutic targets in TNBC; however, existing models utilized in target discovery research are limited. Patient-derived xenograft (PDX) models have emerged as superior models for target discovery experiments because they recapitulate features of patient tumors that are limited by cell-line derived xenograft methods. METHODS: We utilize immunohistochemistry, qRT-PCR and Western Blot to visualize tumor architecture, cellular composition, genomic and protein expressions of a new CL-TNBC PDX model (TU-BcX-2O0). We utilize tissue decellularization techniques to examine extracellular matrix composition of TU-BcX-2O0. RESULTS: Our laboratory successfully established a TNBC PDX tumor, TU-BCX-2O0, which represents a CL-TNBC subtype and maintains this phenotype throughout subsequent passaging. We dissected TU-BCx-2O0 to examine aspects of this complex tumor that can be targeted by developing therapeutics, including the whole and intact breast tumor, specific cell populations within the tumor, and the extracellular matrix. CONCLUSIONS: Here, we characterize a claudin-low TNBC patient-derived xenograft model that can be utilized for therapeutic research studies.

PRL­3 increases the aggressive phenotype of prostate cancer cells in vitro and its expression correlates with high-grade prostate tumors in patients.

The increased expression of phosphatase of regenerating liver-3 (PRL­3) has been shown to be associated with the aggressive and metastatic phenotype of different solid tumors. However, it is not known whether PRL­3 plays a similar role in the progression of prostate cancer (PCa). In this study, immunoblot analysis of androgen receptor (AR)-positive PCa lines (LNCaP and LNCaP­SF) revealed the constitutive cytoplasmic expression of PRL­3, and stimulation with R1881 (AR agonist) rapidly increased the nuclear translocation of PRL­3. The AR-negative cell lines exhibited negligible PRL­3 expression, and the ectopic overexpression of PRL­3 increased both the proliferative and invasive potential of PC3 and DU145 cells. In addition, we measured PRL­3 protein expression in human prostate tumor sections. A high-density prostate tumor microarray (TMA) was immunostained to assess whether PRL­3 expression and its subcellular localization (cytoplasmic and nuclear levels) is associated with the Gleason score (GS), Gleason grade (GG) and tumor stage (T-stage). Digital image analysis (DIA) revealed that PRL­3 expression was significantly higher in the malignant cores, as compared to the non­malignant areas. Increases in both total and nuclear PRL­3 levels were also associated with a higher GS and GG. Metastatic tumors (T4­stage) had lower cytoplasmic, but higher nuclear PRL­3 levels. Furthermore, the nuclear/cytoplasmic ratio for PRL­3 in the tumors graded as GS7 could effectively distinguish between indolent (3+4) and aggressive (4+3) disease. Thus, our experiments using PCa lines suggested that PRL­3 is an AR-regulated gene and its androgen-induced nuclear localization may increase the aggressive behavior of PCa cells. Furthermore, the digital analysis of immunostained tumor sections suggested that PRL­3 may be an effective biomarker of high-grade PCa, and its nuclear/cytoplasmic ratio may be used to distinguish between indolent vs. aggressive tumors.