Patel, Tushar

Cancer stem cells (CSC) within non–small cell lung carcinoma (NSCLC) tumors drive NSCLC progression, metastasis, relapse, and intrinsic chemoresistance. Understanding the mechanisms that support the malignant phenotypes of NSCLC CSCs may provide insights for improved NSCLC therapeutic interventions. Here, we report that expression of RAB27B, a small GTPase, is significantly upregulated in NSCLC CSCs when compared with bulk cancer cells (BCC). Short hairpin RNA–mediated knockdown of RAB27B leads to a loss of stem cell marker gene expression and reduced NSCLC spheroid growth, clonal expansion, transformed growth, invasion, and tumorigenicity. We find that NSCLC CSCs secrete significantly more extracellular vesicles (EV) than BCCs, and that this is RAB27B-dependent. Furthermore, CSC-derived EVs, but not BCC-derived EVs, induce spheroid growth, clonal expansion, and invasion in BCCs. Finally, RAB27B is required for CSC-derived EV-induced stemness in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating, invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Our findings further suggest that inhibition of RAB27B-dependent EV secretion may be a potential therapeutic strategy for NSCLC. Significance: Expression of RAB27B in CSCs leads to elevated levels of EVs that mediate communication between CSCs and BCCs that maintains a stem-like phenotype in NSCLC cells.

Non-alcoholic fatty liver disease (NAFLD) is a fast-growing public health problem and predisposes to hepatocellular carcinoma (HCC) in a significant proportion of patients. Metabolic alterations might underlie the progression of NAFLD to HCC, but the magnitudes of risk and population-attributable risk fractions (PAFs) for various metabolic conditions that are associated with HCC risk in patients with NAFLD are unknown. We investigated the associations between metabolic conditions and HCC development in individuals with a prior history of NAFLD. The study included 11,245 participants in the SEER-Medicare database, comprising 1310 NAFLD-related HCC cases and 9835 NAFLD controls. We excluded individuals with competing liver diseases (e.g., alcoholic liver disease and chronic viral hepatitis). Baseline pre-existing diabetes mellitus, dyslipidemia, obesity, hypertension, hypothyroidism, and metabolic syndrome were assessed. Multivariable-adjusted logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). PAFs were also calculated for each metabolic condition. The results show that diabetes (OR = 2.39, 95% CI: 2.04–2.79), metabolic syndrome (OR = 1.73, 95% CI: 1.49–2.01), and obesity (OR = 1.62, 95% CI: 1.43–1.85) were associated with a higher HCC risk in individuals with NAFLD. The highest PAF for HCC was observed for pre-existing diabetes (42.1%, 95% CI: 35.7–48.5), followed by metabolic syndrome (28.8%, 95% CI: 21.7–35.9) and obesity (13.2%, 95% CI: 9.6–16.8). The major predisposing factors for HCC in individuals with NAFLD are diabetes mellitus, metabolic syndrome, and obesity, and their control would be critically important in mitigating the rising incidence of NAFLD-related HCC.

Tunneling nanotubes (TNTs) are thin, F-actin-based membranous protrusions that connect distant cells and can provide e a novel mechanism for intercellular communication. By establishing cytoplasmic continuity between interconnected cells, TNTs enable the bidirectional transfer of nuclear and cytoplasmic cargo, including organelles, nucleic acids, drugs, and pathogenic molecules. TNT-mediated nucleic acid transfer provides a unique opportunity for donor cells to directly alter the genome, transcriptome, and metabolome of recipient cells. TNTs have been reported to transport DNA, mitochondrial DNA, mRNA, viral RNA, and non-coding RNAs, such as miRNA and siRNA. This mechanism of transfer is observed in physiological as well as pathological conditions, and has been implicated in the progression of disease. Herein, we provide a concise overview of TNTs’ structure, mechanisms of biogenesis, and the functional effects of TNT-mediated intercellular transfer of nucleic acid cargo. Furthermore, we highlight the potential translational applications of TNT-mediated nucleic acid transfer in cancer, immunity, and neurological diseases.

Extracellular vesicles (EV) within the cellular secretome are emerging as modulators of pathological processes involved in tumor growth through their ability to transfer donor‐derived RNA into recipient cells. While the effects of tumor and stromal cell EVs within the tumor microenvironment have been studied, less is known about the contributions of normal, nontransformed cells. We examined the impact of EVs within the cellular secretome from nonmalignant cells on transformed cell growth and behavior in cholangiocarcinoma cells. These effects were enhanced in the presence of the pro‐fibrogenic mediator TGF‐β. We identified miR‐195 as a TGF‐β responsive miRNA in normal cells that can be transferred via EV to tumor cells and regulate cell growth, invasion, and migration. The effects of miR‐195 involve modulation of the epithelial–mesenchymal transition through direct effects on the transcription factor Snail. These studies provide in vitro and in vivo evidence for the impact of normal cellular secretome on transformed cell growth, show the importance of EV RNA transfer, and identify mechanisms of EV‐mediated transfer of miRNA as a contributor to tumor development, which may provide new therapeutic opportunities for targeting human cholangiocarcinoma.

Background and Aims Intrahepatic cholangiocarcinoma (iCCA) and hepatocellular carcinoma (HCC) are the most common primary liver cancers (PLCs). Differences in their clinical features and outcomes are open for investigation in a large‐scale study. We aim to investigate the differences in clinical features and outcomes between iCCA and HCC. Approach and Results The Surveillance, Epidemiology, and End Results Program 18 Database (2000‐2017) was used to extract demographic and clinical features of HCC and iCCA patients. Logistic regression analysis was performed to identify factors associated with iCCA diagnosis versus HCC. Cox regression analysis was used to assess factors affecting overall survival (OS). There were 13,611 iCCA and 96,151 HCC patients. Half of iCCA (50.7%) and three quarters of HCC (76.3%) patients were male. Diagnosis in recent year, age (<50 or ≥65), female sex, non‐Hispanic White race, higher income, rural area, and higher tumor burden were independently associated with iCCA diagnosis versus HCC. Patients with iCCA had worse OS than those with HCC (9 vs. 13 months; P < 0.001). However, OS was comparable between iCCA and HCC in multivariable analysis (adjusted hazard ratio [aHR] = 1.02; 95% CI = 0.99‐1.05). In subgroup analyses, iCCA was associated with better OS than HCC in patients with tumor ≥5 cm (aHR = 0.83; 95% CI = 0.80‐0.86), lymph node involvement (aHR = 0.76; 95% CI = 0.72‐0.81), distant metastasis (aHR = 0.76; 95% CI = 0.73‐0.79), poorly/undifferentiated tumors (aHR = 0.88; 95% CI = 0.83‐0.94), and those receiving noncurative treatment (aHR = 0.96; 95% CI = 0.93‐0.98). Conclusions We identified the demographic, socioeconomic, and clinical features associated with iCCA diagnosis over HCC among patients with PLC. Although iCCA patients presented at an advanced stage, OS was similar between iCCA and HCC in multivariable analysis. iCCA was associated with longer OS for subgroups with poor prognostic features.

Extracellular vesicles (EVs) are a heterogeneous group of biological nano‐sized vesicles that are released from cells and contribute to intercellular communication. Emerging knowledge about their biogenesis, composition, release, and uptake has resulted in broad interest in elucidating their potential roles in disease pathophysiology. The distinct biological properties of these biological nanoparticles emphasize several appealing advantages for potential therapeutic applications compared with the use of synthetic nanoparticles. When administered systemically, EVs are taken up and sequestered within the liver, further emphasizing opportunities for therapeutic use. Consequently, there is growing interest in their use for liver diseases. EVs can be used directly as therapeutics, and several studies have highlighted the intrinsic therapeutic properties of mesenchymal stem cell–derived EVs for chronic and acute liver diseases. Alternatively, EVs can be modified to facilitate their use for the delivery of therapeutic cargo. In this review, we discuss the cellular sources of EV, provide a concise overview of their potential use in diverse processes, and outline several promising applications for the use of EV‐based therapeutics for liver diseases. The use of EV‐based therapeutics provides a viable approach to target hepatic pathophysiology.

By exploiting their biological functions, the use of biological nanoparticles such as extracellular vesicles can provide an efficient and effective approach for hepatic delivery of RNA‐based therapeutics for the treatment of liver cancers such as hepatocellular cancer (HCC). Targeting liver cancer stem cells (LCSC) within HCC provide an untapped opportunity to improve outcomes by enhancing therapeutic responses. Cells with tumor‐initiating capabilities such as LCSC can be identified by expression of markers such as epithelial cell adhesion molecule (EpCAM) on their cell surface. EpCAM is a target of Wnt/β‐catenin signaling, a fundamental pathway in stem‐cell growth. Moreover, mutations in the β‐catenin gene are frequently observed in HCC and can be associated with constitutive activation of the Wnt/β‐catenin pathway. However, targeting these pathways for the treatment of HCC has been challenging. Using RNA nanotechnology, we developed engineered biological nanoparticles capable of specific and effective delivery of RNA therapeutics targeting β‐catenin to LCSC. Extracellular vesicles isolated from milk were loaded with small interfering RNA to β‐catenin and decorated with RNA scaffolds to incorporate RNA aptamers capable of binding to EpCAM. Cellular uptake of these EpCAM‐targeting therapeutic milk‐derived nanovesicles in vitro resulted in loss of β‐catenin expression and decreased proliferation. The uptake and therapeutic efficacy of these engineered biological nanotherapeutics was demonstrated in vivo using tumor xenograft mouse models. Conclusion: β‐catenin can be targeted directly to control the proliferation of hepatic cancer stem cells using small interfering RNA delivered using target‐specific biological nanoparticles. Application of this RNA nanotechnology–based approach to engineer biological nanotherapeutics provides a platform for developing cell‐surface molecule–directed targeted therapeutics.

AbstractExtracellular vesicles are endogenous biological nanoparticles that have potential for use as therapeutic nanoparticles or as delivery vehicles for therapeutic agents. Milk nanovesicles (MNV) are extracellular vesicles isolated from bovine milk that have been explored for use as delivery vehicles for RNA therapeutics such as small interfering RNA (siRNA). We performed in vivo toxicological studies of MNV or therapeutic MNV (tMNV) loaded with siRNA as a prelude to their clinical use. Development toxicity was assessed in zebrafish embryos. Acute toxicity was assessed in both mice and zebrafish whereas safety, biochemical, histological and immune effects after multiple dosing were assessed in mice. Zebrafish embryo hatching was accelerated with MNV and tMNV. While acute toxicity or effects on mortality were not observed in zebrafish, developmental effects were observed at high concentrations of MNV. There was a lack of discernable toxicity, mortality and systemic inflammatory or immunological responses in mice following administration of either MNVs or tMNVs. The tolerability and lack of discernable developmental or systemic in vivo toxicity support their use as biological nano‐therapeutics. Adoption of a standardized protocol for systematic analysis of in vivo safety and toxicity will facilitate preclinical assessment of EV based formulations for therapeutic use.

Hepatocellular carcinoma (HCC) is a leading cause of cancer‐related death worldwide. Although HCC can respond to immune checkpoint inhibitors, such as monoclonal antibodies against programmed death 1 (PD‐1), many patients fail to respond or develop secondary resistance. Activation of Wnt/β‐catenin signaling can contribute to immune evasion. Mutations in β‐catenin are among the most frequent mutations associated with HCC. Thus, our aim was to directly target β‐catenin to enhance the therapeutic response to immune checkpoint inhibition. A synthetic transgenic mouse model of experimental HCC induced by tyrosine‐protein kinase Met/β‐catenin expression and extracellular vesicles (EVs) as a therapeutic delivery agent was used to evaluate the efficacy of directly targeting β‐catenin on the response to anti‐PD‐1. These studies showed that (1) oncogenic β‐catenin could be therapeutically targeted using a biological nanoparticle‐based delivery approach, (2) targeting β‐catenin using small interfering RNA (siRNA) delivered within EVs can reduce tumor growth, and (3) the therapeutic response to anti‐PD‐1 can be enhanced by concomitantly targeting β‐catenin using therapeutic EVs. These preclinical studies establish the efficacy of the use of biological nanoparticles as an endogenous delivery vehicle for therapeutic RNA delivery and support the use of therapeutic strategies targeting tumor‐intrinsic β‐catenin as an adjunct to anti‐PD‐1‐based therapy.Conclusion:Combination therapy with anti‐PD‐1 and β‐catenin siRNA delivered using biological nanoparticles provides an effective strategy for the treatment of HCC. This strategy could be further exploited into targeted approaches for immune potentiation by countering oncogene‐mediated resistance to immunotherapies.