Enhanced plant-derived vesicles for nucleotide delivery for cancer therapy.

Small RNAs (microRNAs [miRNAs] or small interfering RNAs [siRNAs]) are effective tools for cancer therapy, but many of the existing carriers for their delivery are limited by low bioavailability, insufficient loading, impaired transport across biological barriers, and low delivery into the tumor microenvironment. Extracellular vesicle (EV)-based communication in mammalian and plant systems is important for many physiological and pathological processes, and EVs show promise as carriers for RNA interference molecules. However, some fundamental issues limit their use, such as insufficient cargo loading and low potential for scaling production. Plant-derived vesicles (PDVs) are membrane-coated vesicles released in the apoplastic fluid of plants that contain biomolecules that play a role in several biological mechanisms. Here, we developed an alternative approach to deliver miRNA for cancer therapy using PDVs. We isolated vesicles from watermelon and formulated a hybrid, exosomal, polymeric system in which PDVs were combined with a dendrimer bound to miRNA146 mimic. Third generation PAMAM was chosen due to its high branching structure and versatility for loading molecules of interest. We performed several in vivo experiments to demonstrate the therapeutic efficacy of our compound and explored in vitro biological mechanisms underlying the anti-tumor effects of miRNA146, which are mostly related to its anti-angiogenic activity.

EphA2- and HDAC-Targeted Combination Therapy in Endometrial Cancer.

Endometrial cancer is the most frequent malignant tumor of the female reproductive tract but lacks effective therapy. EphA2, a receptor tyrosine kinase, is overexpressed by various cancers including endometrial cancer and is associated with poor clinical outcomes. In preclinical models, EphA2-targeted drugs had modest efficacy. To discover potential synergistic partners for EphA2-targeted drugs, we performed a high-throughput drug screen and identified panobinostat, a histone deacetylase inhibitor, as a candidate. We hypothesized that combination therapy with an EphA2 inhibitor and panobinostat leads to synergistic cell death. Indeed, we found that the combination enhanced DNA damage, increased apoptosis, and decreased clonogenic survival in Ishikawa and Hec1A endometrial cancer cells and significantly reduced tumor burden in mouse models of endometrial carcinoma. Upon RNA sequencing, the combination was associated with downregulation of cell survival pathways, including senescence, cyclins, and cell cycle regulators. The Axl-PI3K-Akt-mTOR pathway was also decreased by combination therapy. Together, our results highlight EphA2 and histone deacetylase as promising therapeutic targets for endometrial cancer.

Enhancing oral delivery of plant-derived vesicles for colitis.

Plant-derived vesicles (PDVs) are attractive for therapeutic applications, including as potential nanocarriers. However, a concern with oral delivery of PDVs is whether they would remain intact in the gastrointestinal tract. We found that 82% of cabbage PDVs were destroyed under conditions mimicking the upper digestive tract. To overcome this limitation, we developed a delivery method whereby lyophilized Eudragit S100-coated cabbage PDVs were packaged into a capsule (Cap-cPDVs). Lyophilization and suspension of PDVs did not have an appreciable impact on PDV structure, number, or therapeutic effect. Additionally, packaging the lyophilized Eudragit S100-coated PDVs into capsules allowed them to pass through the upper gastrointestinal tract for delivery into the colon better than did suspension of PDVs in phosphate-buffered saline. Cap-cPDVs showed robust therapeutic effect in a dextran sulfate sodium-induced colitis mouse model. These findings could have broad implications for the use of PDVs as orally delivered nanocarriers of natural therapeutic plant compounds or other therapeutics.

Dynamic Contrast-Enhanced Magnetic Resonance Imaging as Imaging Biomarker for Vascular Normalization Effect of Infigratinib in High-FGFR-Expressing Hepatocellular Carcinoma Xenografts.

PURPOSE: Overexpression of fibroblast growth factor receptor (FGFR) contributes to tumorigenesis, metastasis, and poor prognosis of hepatocellular carcinoma (HCC). Infigratinib-a pan-FGFR inhibitor-potently suppresses the growth of high-FGFR-expressing HCCs in part via alteration of the tumor microenvironment and vessel normalization. In this study, we aim to assess the utility of dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) as a non-invasive imaging technique to detect microenvironment changes associated with infigratinib and sorafenib treatment in high-FGFR-expressing HCC xenografts. PROCEDURES: Serial DCE-MRIs were performed on 12 nude mice bearing high-FGFR-expressing patient-derived HCC xenografts to quantify tumor microenvironment pre- (day 0) and post-treatment (days 3, 6, 9, and 15) of vehicle, sorafenib, and infigratinib. DCE-MRI data were analyzed using extended generalized kinetic model and two-compartment distributed parameter model. After treatment, immunohistochemistry stains were performed on the harvested tumors to confirm DCE-MRI findings. RESULTS: By treatment day 15, infigratinib induced tumor regression (70 % volume reduction from baseline) while sorafenib induced relative growth arrest (185 % volume increase from baseline versus 694 % volume increase from baseline of control). DCE-MRI analysis revealed different changes in microcirculatory parameters upon exposure to sorafenib versus infigratinib. While sorafenib induced microenvironment changes similar to those of rapidly growing tumors, such as a decrease in blood flow (F), fractional intravascular volume (vp), and permeability surface area product (PS), infigratinib induced the exact opposite changes as early as day 3 after treatment: increase in F, vp, and PS. CONCLUSIONS: Our study demonstrated that DCE-MRI is a reliable non-invasive imaging technique to monitor tumor microcirculatory response to FGFR inhibition and VEGF inhibition in high-FGFR-expressing HCC xenografts. Furthermore, the microcirculatory changes from FGFR inhibition manifested early upon treatment initiation and were reliably detected by DCE-MRI, creating possibilities of combinatorial therapy for synergistic effect.

Ribociclib enhances infigratinib-induced cancer cell differentiation and delays resistance in FGFR-driven hepatocellular carcinoma.

BACKGROUND & AIMS: Infigratinib is a pan-FGFR (fibroblast growth factor receptor) inhibitor that has shown encouraging activity in FGFR-dependent hepatocellular carcinoma (HCC) models. However, long-term treatment results in the emergence of resistant colonies. We sought to understand the mechanisms behind infigratinib-induced tumour cell differentiation and resistance and to explore the potential of adding the CDK4/6 inhibitor ribociclib to prolong cell differentiation. METHODS: Nine high and three low FGFR1-3-expressing HCC patient-derived xenograft (PDX) tumours were subcutaneously implanted into SCID mice and subsequently treated with either infigratinib alone or in combination with ribociclib. Tumour tissues were then subjected to immunohistochemistry to assess cell differentiation, as indicated by the cytoplasmic-to-nuclear ratio and markers such as CYP3A4, HNF4α and albumin. Western blot analyses were performed to investigate the signalling pathways involved. RESULTS: Infigratinib induced cell differentiation in FGFR1-3-dependent HCC PDX models, as indicated by an increase in the cytoplasmic/nuclear ratio and an increase in CYP3A4, HNF4α and albumin. Resistant colonies emerged in long-term treatment, characterised by a reversal of differentiated cell morphology, a reduction in the cytoplasmic-to-nuclear ratio and a loss of differentiation markers. Western blot analyses identified an increase in the CDK4/Cdc2/Rb pathway. The addition of ribociclib effectively blocked this pathway and reversed resistance to infigratinib, resulting in prolonged cell differentiation and growth inhibition. CONCLUSIONS: Our findings demonstrate that the combined inhibition of FGFR/CDK4/6 pathways is highly effective in providing long-lasting tumour growth inhibition and cell differentiation and reducing drug resistance. Therefore, further clinical investigations in patients with FGFR1-3-dependant HCC are warranted.

Bevacizumab Augments the Antitumor Efficacy of Infigratinib in Hepatocellular Carcinoma.

The fibroblast growth factor (FGF) signaling cascade is one of the key signaling pathways in hepatocellular carcinoma (HCC). FGF has been shown to augment vascular endothelial growth factor (VEGF)-mediated HCC development and angiogenesis, as well as to potentially lead to resistance to VEGF/VEGF receptor (VEGFR)-targeted agents. Thus, novel agents targeting FGF/FGF receptor (FGFR) signaling may enhance and/or overcome de novo or acquired resistance to VEGF-targeted agents in HCC. Mice bearing high- and low-FGFR tumors were treated with Infigratinib (i.e., a pan-FGFR kinase inhibitor) and/or Bevacizumab (i.e., an angiogenesis inhibitor). The antitumor activity of both agents was assessed individually or in combination. Tumor vasculature, intratumoral hypoxia, and downstream targets of FGFR signaling pathways were also investigated. Infigratinib, when combined with Bevacizumab, exerted a synergistic inhibitory effect on tumor growth, invasion, and lung metastasis, and it significantly improved the overall survival of mice bearing FGFR-dependent HCC. Infigratinib/Bevacizumab promoted apoptosis, inhibited cell proliferation concomitant with upregulation of p27, and reduction in the expression of FGFR2-4, p-FRS-2, p-ERK1/2, p-p70S6K/4EBP1, Cdc25C, survivin, p-Cdc2, and p-Rb. Combining Infigratinib/Bevacizumab may provide therapeutic benefits for a subpopulation of HCC patients with FGFR-dependent tumors. A high level of FGFR-2/3 may serve as a potential biomarker for patient selection to Infigratinib/Bevacizumab.

FGF401 and vinorelbine synergistically mediate antitumor activity and vascular normalization in FGF19-dependent hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a lethal cancer with limited therapeutic options, and standard therapy with sorafenib provides only modest survival benefits. Fibroblast growth factor 19 (FGF19) has been proposed as a driver oncogene, and targeting its receptor, FGFR-4, may provide a better alternative to standard therapy for patients with FGF19-driven tumors. Sixty-three HCC patient-derived xenograft (PDX) models were screened for FGF19 expression. Mice bearing high and low FGF19-expressing tumors were treated with FGF401 and/or vinorelbine, and the antitumor activity of both agents was assessed individually and in combination. Tumor vasculature and intratumoral hypoxia were also examined. High FGF19 expression was detected in 14.3% (9 of 63) of the HCC models tested and may represent a good target for HCC treatment. FGF401 potently inhibited the growth of high FGF19-expressing HCC models regardless of FGF19 gene amplification. Furthermore, FGF401 inhibited the FGF19/FGFR-4 signaling pathway, cell proliferation, and hypoxia, induced apoptosis and blood vessel normalization and prolonged the overall survival (OS) of mice bearing high FGF19 tumors. FGF401 synergistically acted with the microtubule-depolymerizing drug vinorelbine to further suppress tumor growth, promote apoptosis, and prolong the OS of mice bearing high FGF19 tumors, with no evidence of increased toxicity. Our study suggests that a subset of patients with high FGF19-expressing HCC tumors could benefit from FGF401 or FGF401/vinorelbine treatment. A high level of FGF19 in a tumor may serve as a potential biomarker for patient selection.

Vinorelbine Augments Radiotherapy in Hepatocellular Carcinoma.

There is a need to improve the effectiveness of radiotherapy (RT) in hepatocellular carcinoma (HCC). Therefore, the purpose of this study was to explore the efficacy and toxicity of the anti-microtubule agent Vinorelbine as a radiosensitizer in HCC. The radio sensitivity of 16 HCC patient-derived xenograft (PDX) models was determined by quantifying the survival fraction following irradiation in vitro, and Vinorelbine radio sensitization was determined by clonogenic assay. Ectopic HCC xenografts were treated with a single dose of 8 Gy irradiation and twice-weekly 3 mg/kg Vinorelbine. Tumor growth and changes in the proteins involved in DNA repair, angiogenesis, tumor cell proliferation, and survival were assessed, and the 3/16 (18.75%), 7/16 (43.75%), and 6/16 (37.5%) HCC lines were classified as sensitive, moderately sensitive, and resistant, respectively. The combination of RT and Vinorelbine significantly inhibited tumor growth, DNA repair proteins, angiogenesis, and cell proliferation, and promoted more apoptosis compared with RT or Vinorelbine treatment alone. Vinorelbine improved HCC tumor response to standard irradiation with no increase in toxicity. HCC is prevalent in less developed parts of the world and is mostly unresectable on presentation. Vinorelbine and conventional radiotherapy are cost-effective, well-established modalities of cancer treatment that are readily available. Therefore, this strategy can potentially address an unmet clinical need, warranting further investigation in early-phase clinical trials.