Hepatocellular carcinoma (HCC) is a common and deadly cancer. Most cases of HCC arise in a cirrhotic/fibrotic liver, indicating that environment may play a paramount role in cancer genesis. Previous ...studies from our group and others have shown that, in desmoplastic cancers, there is a rich intercellular communication between activated, cancer‐associated fibroblasts and cancer cells. Moreover, extracellular vesicles (EVs), or exosomes, have been identified as an important arm of this intercellular communication platform. Finally, these studies have shown that EVs can carry microRNA (miR) species in vivo and deliver them to desmoplastic cancers. The precise role played by activated liver fibroblasts/stellate cells in HCC development is insufficiently known. Based on previous studies, it appears plausible that activated fibroblasts produce signals carried by EVs that promote HCC genesis. In the current study, we first hypothesized and then demonstrated that stellate cell‐derived EVs 1) can be loaded with an miR species of choice (miR‐335‐5p); 2) are taken up by HCC cells in vitro and more importantly in vivo; 3) can supply the miR‐335‐5p cargo to recipient HCC cells in vitro as well as in vivo; and 4) inhibit HCC cell proliferation and invasion in vitro as well as induce HCC tumor shrinkage in vivo. Finally, we identified messenger RNA targets for miR‐335 that are down‐regulated after treatment with EV‐miR‐335‐5p. This study informs potential therapeutic strategies in HCC, whereby stellate cell‐derived EVs are loaded with therapeutic nucleic acids and delivered in vivo. (Hepatology 2018;67:940–954)
We report on a therapeutic approach using thermo‐responsive multi‐fingered drug eluting devices. These therapeutic grippers referred to as theragrippers are shaped using photolithographic patterning ...and are composed of rigid poly(propylene fumarate) segments and stimuli‐responsive poly(N‐isopropylacrylamide‐co‐acrylic acid) hinges. They close above 32 °C allowing them to spontaneously grip onto tissue when introduced from a cold state into the body. Due to porosity in the grippers, theragrippers could also be loaded with fluorescent dyes and commercial drugs such as mesalamine and doxorubicin, which eluted from the grippers for up to seven days with first order release kinetics. In an in vitro model, theragrippers enhanced delivery of doxorubicin as compared to a control patch. We also released theragrippers into a live pig and visualized release of dye in the stomach. The design of such tissue gripping drug delivery devices offers an effective strategy for sustained release of drugs with immediate applicability in the gastrointestinal tract.
Thermoresponsive polymeric grippers for controlled drug release (“theragrippers”) close spontaneously above 32 °C and grip onto tissue. They were loaded with drugs mesalamine and doxorubicin, which eluted for up to 7 days. Theragrippers show improved site‐specific delivery and offer a novel strategy for sustained release with immediate applicability in the gastrointestinal tract.
The cancer microenvironment plays a central role in cancer development, growth, and homeostasis. This paradigm suggests that cancer fibroblasts support cancers, probably in response to stimuli ...received from the cancer cells. We aimed at investigating whether extracellular vesicles (EVs) can shuttle microRNA (miR) species between cancer‐associated fibroblasts (CAFs) and cancer cells. To this end, we extracted EVs according to published protocols. EVs were studied for their miR content by quantitative reverse‐transcription polymerase chain reaction. EVs were transfected with select miR species and utilized in vitro as well as in vivo in a rat model of cholangiocarcinoma (CCA). We found that miR‐195 is down‐regulated in CCA cells, as well as in adjoining fibroblasts. Furthermore, we report that EVs shuttle miR‐195 from fibroblasts to cancer cells. Last, we show that fibroblast‐derived EVs, loaded with miR‐195, can be administered in a rat model of CCA, concentrate within the tumor, decrease the size of cancers, and improve survival of treated rats. Conclusion: EVs play a salient role in trafficking miR species between cancer cells and CAFs in human CCA. Understanding of these mechanisms may allow devising of novel therapeutics. (Hepatology 2017;65:501‐514).
Ever since their discovery, microRNAs (miRNAs) have been the subject of intense investigation of their roles in cells and tissues, both normal and disease state. Although some of the precise ...mechanisms of biogenesis and actions of miRNAs remain debatable, the fact that miRNAs are dysregulated in diseases such as cancer is undisputed. For many miRNA species, computational databases predict often numerous targets; however, experimental verification in vitro and in vivo is still lacking. For some miRNAs, species-specific targets have been validated; nevertheless, the precise mechanisms those targets act in and whether they are the only truly important ones remain to be discovered. The authors take a closer look at the current status of the role of miRNAs in the diagnosis and biology of cholangiocarcinomas where the perhaps biggest impact in the short term comes from the use of biomarkers in the early diagnosis.
The biliary system is routinely accessed for clinical purposes via endoscopic retrograde cholangiopancreatography (ERCP). We previously pioneered ERCP-mediated hydrodynamic injection in large animal ...models as an innovative gene delivery approach for monogenic liver diseases. However, the procedure poses potential safety concerns related mainly to liver or biliary tree injury. Here, we sought to further define biliary hydrodynamic injection parameters that are well-tolerated in a human-sized animal model. ERCP was performed in pigs, and hydrodynamic injection carried out using a novel protocol to reduce duct wall stress. Each pig was subjected to multiple repeated injections to expedite testing and judge tolerability. Different injection parameters (volume, flow rate) and injection port diameters were tested. Vital signs were monitored throughout the procedure, and liver enzyme panels were collected pre- and post-procedure. Pigs tolerated repeated biliary hydrodynamic injections with only occasional, mild, isolated elevation in aspartate aminotransferase (AST), which returned to normal levels within one day post-injection. All other liver tests remained unchanged. No upper limit of volume tolerance was reached, which suggests the biliary tree can readily transmit fluid into the vascular space. Flow rates up to 10 mL/sec were also tolerated with minimal disturbance to vital signs and no anatomic rupture of bile ducts. Measured intrabiliary pressure was up to 150 mmHg, and fluid-filled vesicles were induced in liver histology at high flow rates, mimicking the changes in histology observed in mouse liver after hydrodynamic tail vein injection. Overall, our investigations in a human-sized pig liver using standard clinical equipment suggest that ERCP-guided hydrodynamic injection will be safely tolerated in patients. Future investigations will interrogate if higher flow rates and pressure mediate higher DNA delivery efficiencies.
Liver cancer is the fourth leading cause of cancer-related mortality and is distinguished by a relative paucity of chemotherapy options. It has been hypothesized that intratumor genetic heterogeneity ...may contribute to the high failure rate of chemotherapy. Here, we evaluated functional heterogeneity in a cohort of primary human liver cancer organoid lines. Each primary human liver cancer surgical specimen was used to generate multiple cancer organoid lines, obtained from distinct regions of the tumor. A total of 27 liver cancer lines were established and tested with 129 cancer drugs, generating 3,483 cell survival data points. We found a rich intratumor, functional (drug response) heterogeneity in our liver cancer patients. Furthermore, we established that the majority of drugs were either ineffective, or effective only in select organoid lines. In contrast, we found that a subset of drugs appeared pan-effective, displaying at least moderate activity in the majority of these cancer organoid lines. These drugs, which are FDA approved for indications other than liver cancers, deserve further consideration as either systemic or local therapeutics. Of note, molecular profiles, obtained for a reduced sample set, did not correlate with the drug response heterogeneity of liver cancer organoid lines. Taken together, these findings lay the foundation for in-depth studies of pan-effective drugs, as well as for functional personalized oncology approaches. Lastly, these functional studies demonstrate the utility of cancer organoid drug testing as part of a drug discovery pipeline.
Extracellular vesicles (EVs) play critical roles in intercellular communication by transporting bioactive cargo to recipient cells. EVs have been implicated in a range of physiological and ...pathological processes, including tumor progression, metastasis, immune modulation, and drug resistance. The objective of this review is to present a thorough overview of recent studies focusing on EVs in hepatocellular carcinoma (HCC), with an emphasis on their potential utility as diagnostic biomarkers as well as therapeutic agents. Initially, we explore the utility of EVs as diagnostic biomarkers for HCC, followed by a discussion of their potential as carriers of therapeutic payloads. Additionally, we delve into the emerging field of therapeutic EVs for modulating tumor immune responses. Through this review, our ultimate aim is to provide a comprehensive understanding of the opportunities and challenges in the clinical translation of EV research in the domain of HCC.
Reliable establishment of tumor organoids is paramount to advance applications of organoid technology for personalized medicine. Here, we share our multi-center experience on initiation and ...tumorigenic confirmation of hepatobiliary cancer organoids. We discuss current concerns, propose potential solutions, and provide future perspectives for improvements in hepatobiliary cancer organoid establishment.
Reliable establishment of tumor organoids is paramount to advance applications of organoid technology for personalized medicine. Here, we share our multi-center experience on initiation and tumorigenic confirmation of hepatobiliary cancer organoids. We discuss current concerns, propose potential solutions, and provide future perspectives for improvements in hepatobiliary cancer organoid establishment.
Thermally activated, untethered microgrippers can reach narrow conduits in the body and be used to excise tissue for diagnostic analyses. As depicted in the figure, the feasibility of an in vivo ...biopsy of the porcine bile duct using untethered microgrippers is demonstrated.
Background & Aims A long duration of inflammatory bowel disease (IBD) increases the risk for colorectal cancer. Mutation analysis of limited numbers of genes has indicated that colorectal tumors that ...develop in patients with IBD differ from those of patients without IBD. We performed whole-exome sequencing analyses to characterize the genetic landscape of these tumors. Methods We collected colorectal tumor and non-neoplastic tissues from 31 patients with IBD and colorectal cancer (15 with ulcerative colitis, 14 with Crohn’s disease, and 2 with indeterminate colitis) and performed whole-exome sequencing analyses of the microdissected tumor and matched nontumor tissues. We identified somatic alterations by comparing matched specimens. The prevalence of mutations in sporadic colorectal tumors was obtained from previously published exome-sequencing studies. Results Two specimens had somatic mutations in the DNA proofreading or mismatch repair genes POLE , MLH1 , and MSH6 and the tumor cells had a hypermutable phenotype. The remaining tumors had, on average, 71 alterations per sample. TP53 was the most commonly mutated gene, with prevalence similar to that of sporadic colorectal tumors (63% of cases). However, tumors from the patients with IBD had a different mutation spectrum. APC and KRAS were mutated at significantly lower rates in tumors from patients with IBD than in sporadic colorectal tumors (13% and 20% of cases, respectively). Several genes were mutated more frequently or uniquely in tumors from patients with IBD, including SOX9 and EP300 (which encode proteins in the WNT pathway), NRG1 (which encodes an ERBB ligand), and IL16 (which encodes a cytokine). Our study also revealed recurrent mutations in components of the Rho and Rac GTPase network, indicating a role for noncanonical WNT signaling in development of colorectal tumors in patients with IBD. Conclusions Colorectal tumors that develop in patients with IBD have distinct genetic features from sporadic colorectal tumors. These findings could be used to develop disease-specific markers for diagnosis and treatment of patients with IBD and colorectal cancer.
PDF
