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Background: MicroRNAs (miRNAs) regulate expression of many cancer-related genes through posttranscriptional repression of their mRNAs. In this study we investigate the proto-oncogene MYCN as a target for miRNA regulation. Methods: A luciferase reporter assay was used to investigate software-predicted miRNA target sites in the 3′-untranslated region (3′UTR) of MYCN . The miRNAs were overexpressed in cell lines by transfection of miRNA mimics or miRNA-expressing plasmids. Mutation of the target sites was used to validate MYCN 3′UTR as a direct target of several miRNAs. To measure miRNA-mediated suppression of endogenous N-myc protein, inhibition of proliferation and inhibition of clonogenic growth, miRNAs were overexpressed in a MYCN -amplified neuroblastoma cell line. Results: The results from this study show that MYCN is targeted by several miRNAs. In addition to the previously shown mir-34a/c , we experimentally validate mir-449 , mir-19a/b , mir-29a/b/c, mir-101 and let-7e/mir-202 as direct MYCN -targeting miRNAs. These miRNAs were able to suppress endogenous N-myc protein in a MYCN -amplified neuroblastoma cell line. The let-7e and mir-202 were strong negative regulators of MYCN expression. The mir-101 and the let-7 family miRNAs let-7e and mir-202 inhibited proliferation and clonogenic growth when overexpressed in Kelly cells. Conclusion: The tumour-suppressor miRNAs let-7 and mir-101 target MYCN and inhibit proliferation and clonogenic growth of MYCN -amplified neuroblastoma cells.

Traditional cancer treatments, such as chemotherapy, are often incapable of achieving complete responses as standalone therapies. Hence, current treatment strategies typically rely on a combination of several approaches. Nanoparticle-based photothermal therapy (PTT) is a technique used to kill cancer cells through localized, severe hyperthermia that has shown promise as an add-on treatment to multiple cancer therapies. Here, we evaluated whether the combination of gold nanoshell (NS)-based PTT and liposomal doxorubicin could improve outcome in a mouse model of colorectal cancer. First, NS-based PTT was performed on tumor-bearing mice. Radiolabeled liposomes were then injected at different timepoints to follow their accumulation in the tumor and determine the ideal injection time after PTT. In addition, fluorescent liposomes were used to observe the liposomal distribution in the tumor after PTT. Finally, we combined PTT and doxorubicin-loaded liposomes and studied the effect of the treatment strategy on the mice by following tumor growth and survival. PTT significantly improved liposomal accumulation in the tumor, but only when the liposomes were injected immediately after the therapy. The liposomes accumulated mostly in regions adjacent to the ablated areas. When PTT was combined with liposomal doxorubicin, the mice experienced a slowdown in tumor growth and an improvement in survival. According to our preclinical study, NS-based PTT seems promising as an add-on treatment for liposomal chemotherapy and potentially other systemic therapies, and could be relevant for future application in a clinical setting.

Introduction/Background*The primary objective of this multicentre, phase II study was to evaluate the preliminary efficacy of O (3000mg, IV q2W) in combination with D (1500mg, IV, q4W) in relapsed OC patients with the CD73 expression on intraepithelial inflammatory cells in the TME, defined by disease-control rate (DCR) at 16 weeks. Key eligibility criteria include: histologically confirmed OC with CD73 expression; relapsed disease; measurable disease; no prior immunotherapy; mandatory paired biopsy. 25 patients have been enrolled. Treatment was continued until disease progression, unacceptable toxicity, or withdrawal of consent. Primary endpoint is DCR; secondary endpoints include ORR, DoR, PFS, OS, safety and tolerability. Responses are also corelated to CD8 and PD-L1 positive expression in tumours. Assessment of PD-L1 (≥5%) and CD8 (≥5%) were performed by immunohistochemistry on archival tumour specimens. Clinical trial information: NCT03267589MethodologyThe primary objective of this multicentre, phase II study was to evaluate the preliminary efficacy of O (3000mg, IV q2W) in combination with D (1500mg, IV, q4W) in relapsed OC patients with the CD73 expression on intraepithelial inflammatory cells in the TME, defined by disease-control rate (DCR) at 16 weeks. Key eligibility criteria include: histologically confirmed OC with CD73 expression; relapsed disease; measurable disease; no prior immunotherapy; mandatory paired biopsy. 25 patients have been enrolled. Treatment was continued until disease progression, unacceptable toxicity, or withdrawal of consent. Primary endpoint is DCR; secondary endpoints include ORR, DoR, PFS, OS, safety and tolerability. Responses are also corelated to CD8 and PD-L1 positive expression in tumours. Assessment of PD-L1 (≥5%) and CD8 (≥5%) were performed by immunohistochemistry on archival tumour specimens. Clinical trial information: NCT03267589Result(s)*80% of patients had received ≥2 lines of therapy for relapse. DCR was 27%, median PFS was 2.7months and median OS was 8.4 months. 74% of patients were CD8-positive, 42% were PD-L1-positive, while 37% were both CD8 & PD-L1-positive. Biomarker positivity was not significantly associated with better DCR (p=0.584). TEAE: 1 thromboembolic event (grade 3), one neutropenia (grade 4), one cardiac arrest (grade5).Conclusion*Combination of Oleclumab-durvalumab is feasible and demonstrate modest preliminary activity in relapsed OC. Further biomarker analysis research to predict response is ongoing.

ObjectivesPlatinum-resistant ovarian cancer (OC) remains a challenge with few or no treatment options. Methylation of the HOXA9 gene has been found in plasma of patients with OC. It does not, however, occur in blood from healthy individuals. The aim of this study was to evaluate if HOXA9 methylation could predict outcome and identify patients who can benefit from palliative chemotherapy.MethodsPlasma from 27 patients with platinum-resistant OC was analyzed by digital PCR with a HOXA9 methylation-specific assay at baseline and before cycle two. The fractional abundance of methylated HOXA9 was calculated and the patients with values increasing above the 95% confidence interval of baseline values was compared with patients having stable or decreasing values. The primary endpoint was progression free survival (PFS).ResultsAt baseline 22 patients (81.5%) had measurable HOXA9 methylation in plasma. Patients (N=4) with a significant increase in HOXA9 methylation after the first cycle had a median PFS of 1.4 months compared to 5.4 months in patients (N=23) with stable or decreasing HOXA9 (p=0.0019). Nine patients were HOXA9 negative before cycle two. The median PFS in this group was 9 months compared to 2.6 months for patients (N=18) with measurable HOXA9 at second cycle (p=0.001) (figure 1).Abstract 335 Figure 1PFS HOXA9 status at 2nd treatment cycleConclusionsThe study demonstrated that an increase in HOXA9 methylated DNA could be used as an early marker to predict poor outcome in platinum-resistant OC. Furthermore, absence of HOXA9 methylation was prognostic favorable indicating the potential to identify patients who can benefit from palliative chemotherapy.

Polycations such as polyethylenimine (PEI) are used in many novel nonviral vector designs and there are continuous efforts to increase our mechanistic understanding of their interactions with cells. Even so, the mechanism of polyplex escape from the endosomal/lysosomal pathway after internalization is still elusive. The “proton sponge ” hypothesis remains the most generally accepted mechanism, although it is heavily debated. This hypothesis is associated with the large buffering capacity of PEI and other polycations, which has been interpreted to cause an increase in lysosomal pH even though no conclusive proof has been provided. In the present study, we have used a nanoparticle pH sensor that was developed for pH measurements in the endosomal/lysosomal pathway. We have carried out quantitative measurements of lysosomal pH as a function of PEI content and correlate the results to the “proton sponge ” hypothesis. Our measurements show that PEI does not induce change in lysosomal pH as previously suggested and quantification of PEI concentrations in lysosomes makes it uncertain that the “proton sponge ” effect is the dominant mechanism of polyplex escape.

Analyzing rare DNA and RNA molecules in limited sample sizes, such as liquid biopsies and single cells, often requires preamplification, which makes downstream analyses particularly sensitive to polymerase chain reaction (PCR) generated contamination. Herein, we assessed the feasibility of performing Cod uracil-DNA N-glycosylase (Cod UNG) treatment in combination with targeted preamplification, using deoxyuridine triphosphate (dUTP) to eliminate carry-over DNA. Cod UNG can be completely and irreversibly heat inactivated, a prerequisite in preamplification methods, where any loss of amplicons is detrimental to subsequent quantification. Using 96 target assays and quantitative real-time PCR, we show that replacement of deoxythymidine triphosphate (dTTP) with dUTP in the preamplification reaction mix results in comparable dynamic range, reproducibility, and sensitivity. Moreover, Cod UNG essentially removes all uracil-containing template of most assays, regardless of initial concentration, without affecting downstream analyses. Finally, we demonstrate that the use of Cod UNG and dUTP in targeted preamplification can easily be included in the workflow for single-cell gene expression profiling. In summary, Cod UNG treatment in combination with targeted preamplification using dUTP provides a simple and efficient solution to eliminate carry-over contamination and the generation of false positives and inaccurate quantification.

Methanogenic archaea are genotypically and phenotypically diverse organisms that are integral to carbon cycling in anaerobic environments. Owing to their genetic tractability and ability to be readily cultivated, Methanosarcina spp. have become a powerful model system for understanding methanogen biology at the cellular systems level. However, relatively little is known of how genotypic and phenotypic variation is partitioned in Methanosarcina populations inhabiting natural environments and the possible ecological and evolutionary implications of such variation. Here, we have identified how genomic and phenotypic diversity is partitioned within and between Methanosarcina mazei populations obtained from two different sediment environments in the Columbia River Estuary (Oregon, USA). Population genomic analysis of 56 M. mazei isolates averaging <1% nucleotide divergence revealed two distinct clades, which we refer to as ‘mazei-T’ and ‘mazei-WC’. Genomic analyses showed that these clades differed in gene content and fixation of allelic variants, which point to potential differences in primary metabolism and also interactions with foreign genetic elements. This hypothesis of niche partitioning was supported by laboratory growth experiments that revealed significant differences in trimethylamine utilization. These findings improve our understanding of the ecologically relevant scales of genomic variation in natural systems and demonstrate interactions between genetic and ecological diversity in these easily cultivable and genetically tractable model methanogens.

Background: The accumulation of liposome encapsulated chemotherapy in solid cancers is dependent on the presence of the enhanced permeability and retention (EPR) effect. Positron emission tomography (PET) imaging with a liposome encapsulated radioisotope, such as liposome encapsulated Cu-64 (64Cu-liposome) may help to identify tumors with high liposome accumulation, and thereby stratify patients based on expected benefit from liposomal chemotherapy. However, intravenous administration of liposomes without a cytotoxic content is complicated by the accelerated blood clearance (ABC) phenomenon for succeeding therapeutic liposome dosing. Alternative markers for assessing the tumor’s EPR level are therefore warranted. Materials and Methods: To increase our understanding of EPR variations and to ultimately identify an alternative marker for the EPR effect, we investigated the correlation between 64Cu-liposome PET/CT (EPR effect) and 68Ga-RGD PET/CT (neoangiogenesis), 18F-FDG PET/CT (glycolysis), diffusion-weighted MRI (diffusivity) and interstitial fluid pressure in two experimental cancer models (CT26 and COLO 205). Results: 64Cu-liposome and 68Ga-RGD SUVmax displayed a significant moderate correlation, however, none of the other parameters evaluated displayed significant correlations. These results indicate that differences in neoangiogenesis may explain some EPR variability, however, as correlations were only moderate and not observed for SUVmean, 68Ga-RGD is probably insufficient to serve as a stand-alone surrogate marker for quantifying the EPR effect and stratifying patients.

Active, ligand-mediated, targeting of functionalized liposomes to folate receptors (FRs) overexpressed on cancer cells could potentially improve drug delivery and specificity. Studies on folate-targeting liposomes (FTLs) have, however, yielded varying results and generally fail to display a clear benefit of FR targeting. Tumor accumulating potential of FTLs and NTLs were investigated in a FR overex-pressing xenograft model by positron emission tomography/computed tomography imaging. Tumors displayed significantly lower activity of FTLs than NTLs. Furthermore, FTLs displayed worse circulating properties and increased liver-accumulation than NTLs. This study underlines that long-circulating properties of liposomes must be achieved to take advantage of EPR-dependent tumor accumulation which may be lost by functionalization. FR-functionalization negatively affected both tumor accumulation and circulation properties.