Size-Dependent Penetration of Nanoparticles in Tumor Spheroids: A Multidimensional and Quantitative Study of Transcellular and Paracellular Pathways.

Tumor penetration of nanoparticles is crucial in nanomedicine, but the mechanisms of tumor penetration are poorly understood. This work presents a multidimensional, quantitative approach to investigate the tissue penetration behavior of nanoparticles, with focuses on the particle size effect on penetration pathways, in an MDA-MB-231 tumor spheroid model using a combination of spectrometry, microscopy, and synchrotron beamline techniques. Quasi-spherical gold nanoparticles of different sizes are synthesized and incubated with 2D and 3D MDA-MB-231 cells and spheroids with or without an energy-dependent cell uptake inhibitor. The distribution and penetration pathways of nanoparticles in spheroids are visualized and quantified by inductively coupled plasma mass spectrometry, two-photon microscopy, and synchrotron X-ray fluorescence microscopy. The results reveal that 15 nm nanoparticles penetrate spheroids mainly through an energy-independent transcellular pathway, while 60 nm nanoparticles penetrate primarily through an energy-dependent transcellular pathway. Meanwhile, 22 nm nanoparticles penetrate through both transcellular and paracellular pathways and they demonstrate the greatest penetration ability in comparison to other two sizes. The multidimensional analytical methodology developed through this work offers a generalizable approach to quantitatively study the tissue penetration of nanoparticles, and the results provide important insights into the designs of nanoparticles with high accumulation at a target site.

Engineered Cancer-Derived Small Extracellular Vesicle-Liposome Hybrid Delivery System for Targeted Treatment of Breast Cancer.

Cancer-derived small extracellular vesicles (sEVs) may be a promising drug delivery system that targets cancer cells due to their unique features, such as native homing ability, biological barrier crossing capability, and low immune response. However, the oncogenic cargos within them pose safety concerns, hence limiting their application thus far. We proposed using an electroporation-based strategy to extract the endogenous cargos from cancer-derived sEVs and demonstrated that their homing ability was still retained. A membrane fusion technique was used to fuse these sEVs with liposomes to form hybrid particles, which possessed both benefits of sEVs and liposomes. Anti-EGFR monoclonal antibodies were modified on the hybrid particles to improve their targeting ability further. The engineered hybrid particles showed higher drug loading ability that is 33.75 and 43.88% higher than that of liposomes and sEVs, respectively, and improved targeting ability by 52.23% higher than hybrid particles without modification. This delivery system showed >90% cell viability and enhanced treatment efficiency with 91.58 and 79.26% cell migration inhibition rates for the miR-21 inhibitor and gemcitabine, respectively.

Ropporin-1 and 1B Are Widely Expressed in Human Melanoma and Evoke Strong Humoral Immune Responses.

Antibodies that block immune regulatory checkpoints (programmed cell death 1, PD-1 and cytotoxic T-lymphocyte-associated antigen 4, CTLA-4) to mobilise immunity have shown unprecedented clinical efficacy against cancer, demonstrating the importance of antigen-specific tumour recognition. Despite this, many patients still fail to benefit from these treatments and additional approaches are being sought. These include mechanisms that boost antigen-specific immunity either by vaccination or adoptive transfer of effector cells. Other than neoantigens, epigenetically regulated and shared antigens such as NY-ESO-1 are attractive targets; however, tissue expression is often heterogeneous and weak. Therefore, peptide-specific therapies combining multiple antigens rationally selected to give additive anti-cancer benefits are necessary to achieve optimal outcomes. Here, we show that Ropporin-1 (ROPN1) and 1B (ROPN1B), cancer restricted antigens, are highly expressed and immunogenic, inducing humoral immunity in patients with advanced metastatic melanoma. By multispectral immunohistochemistry, 88.5% of melanoma patients tested (n = 54/61) showed ROPN1B expression in at least 1 of 2/3 tumour cores in tissue microarrays. Antibody responses against ROPN1A and ROPN1B were detected in 71.2% of melanoma patients tested (n = 74/104), with increased reactivity seen with more advanced disease stages. Thus, ROPN1A and ROPN1B may indeed be viable targets for cancer immunotherapy, alone or in combination with other cancer antigens, and could be combined with additional therapies such as immune checkpoint blockade.

Progressive silicone lymphadenopathy post mastectomy and implant reconstruction for breast cancer.

A 56-year-old woman with a 12-year history of recurrent triple-negative invasive carcinoma of the breast presented with progressive enlargement of lymph nodes in the setting of established rupture of the ipsilateral silicone breast implant. Although this was proven to be benign on cytology, its progressive nature led to repeated core biopsies for histology, which were necessary given the high-risk nature of triple-negative breast cancer and the multiple proven previous recurrences. The histology demonstrated features of silicone deposits without evidence of malignancy. This case demonstrates the dilemma in surveillance of high-risk patients with breast cancer who have had previous silicone lymphadenopathy.

Characterising the phenotypic evolution of circulating tumour cells during treatment.

Real-time monitoring of cancer cells' phenotypic evolution during therapy can provide vital tumour biology information for treatment management. Circulating tumour cell (CTC) analysis has emerged as a useful monitoring tool, but its routine usage is restricted by either limited multiplexing capability or sensitivity. Here, we demonstrate the use of antibody-conjugated and Raman reporter-coated gold nanoparticles for simultaneous labelling and monitoring of multiple CTC surface markers (named as "cell signature"), without the need for isolating individual CTCs. We observe cell heterogeneity and phenotypic changes of melanoma cell lines during molecular targeted treatment. Furthermore, we follow the CTC signature changes of 10 stage-IV melanoma patients receiving immunological or molecular targeted therapies. Our technique maps the phenotypic evolution of patient CTCs sensitively and rapidly, and shows drug-resistant clones having different CTC signatures of potential clinical value. We believe our proposed method is of general interest in the CTC relevant research and translation fields.

Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags.

Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research.

Capture and On-chip analysis of Melanoma Cells Using Tunable Surface Shear forces.

With new systemic therapies becoming available for metastatic melanoma such as BRAF and PD-1 inhibitors, there is an increasing demand for methods to assist with treatment selection and response monitoring. Quantification and characterisation of circulating melanoma cells (CMCs) has been regarded as an excellent non-invasive candidate but a sensitive and efficient tool to do these is lacking. Herein we demonstrate a microfluidic approach for melanoma cell capture and subsequent on-chip evaluation of BRAF mutation status. Our approach utilizes a recently discovered alternating current electrohydrodynamic (AC-EHD)-induced surface shear forces, referred to as nanoshearing. A key feature of nanoshearing is the ability to agitate fluid to encourage contact with surface-bound antibody for the cell capture whilst removing nonspecific cells from the surface. By adjusting the AC-EHD force to match the binding affinity of antibodies against the melanoma-associated chondroitin sulphate proteoglycan (MCSP), a commonly expressed melanoma antigen, this platform achieved an average recovery of 84.7% from biological samples. Subsequent staining with anti-BRAF(V600E) specific antibody enabled on-chip evaluation of BRAF(V600E) mutation status in melanoma cells. We believe that the ability of nanoshearing-based capture to enumerate melanoma cells and subsequent on-chip characterisation has the potential as a rapid screening tool while making treatment decisions.

Monitoring response to therapy in melanoma by quantifying circulating tumour DNA with droplet digital PCR for BRAF and NRAS mutations.

We assessed the utility of droplet digital PCR (ddPCR) to evaluate the potential of using circulating tumour DNA (ctDNA) as a post therapy monitoring tool in melanoma by comparing it to serum LDH levels and RECIST scores. ddPCR was shown to be reliable in distinguishing mutant from wild type alleles with no false positives. Subsequently, we quantified ctDNA ((V600E)BRAF,(V600K)BRAF or (Q61H)NRAS) in 6 stage IV melanoma patients across several time points during their treatment course. All tested patients had detectable ctDNA, which exhibited dynamic changes corresponding to the changes in their disease status. The ctDNA levels fell upon treatment response and rose with detectable disease progression. In our group of patients, ctDNA was more consistent and informative than LDH as a blood-based biomarker. In addition, BRAF mutant ctDNA as detected by ddPCR could be used diagnostically where the tumour block was unavailable. In conclusion, this study demonstrates the applicability of using ddPCR to detect and quantify ctDNA in the plasma of melanoma patients.