Sijie Hao

Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78–83%), high retention of cell viability (71–74%), high tumour cell enrichment against leukocytes (1.7–2 × 103), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4–0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

Mitochondria-Targeting Polydopamine Nanoparticles To Deliver Doxorubicin for Overcoming Drug Resistance

Mitochondria play a critical role in diverse cellular processes, such as energy production and apoptosis regulation. The mitochondria-targeted drug delivery is becoming a potential novel strategy for overcoming drug resistance in cancer therapy. Herein, we synthesize nature-inspired dopamine-derived polydopamine (PDA) nanoparticles. Using triphenylphosphonium (TPP) as the mitochondrial penetration molecule to improve the target efficiency, we synthesize poly(ethylene glycol) (PEG)-modified PDA (PDA-PEG) and TPP-functionalized PEG-modified PDA (PDA-PEG-TPP) nanoparticles. Then anticancer drug doxorubicin (DOX) was loaded on PDA-PEG and PDA-PEG-TPP (PDA-PEG-DOX and PDA-PEG-TPP-DOX) nanoparticles, which are apt to deliver DOX to cell nuclei and mitochondria, respectively. To mimic the repeated anticancer drug treatment in clinical cases, we repeatedly treated the MDA-MD-231 cancer cells for a long time using DOX-loaded nanoparticles and find that the mitochondria targeting PDA-PEG-TPP-DOX has higher potential to overcome the drug resistance than the regular delivery nanoparticles PDA-PEG-DOX. These results indicate the promising potential of applying PDA-PEG-TPP-DOX nanoparticles in mitochondria-targeted drug delivery to overcome the drug resistance in long-time anticancer chemotherapy.

A combinatory strategy for detection of live CTCs using microfiltration and a new telomerase-selective adenovirus

Circulating tumor cells (CTC) have become an important biomarker for early cancer diagnosis, prognosis, and treatment monitoring. Recently, a replication-competent recombinant adenovirus driven by a human telomerase gene (hTERT) promoter was shown to detect live CTCs in blood samples of patients with cancer. Here, we report a new class of adenoviruses containing regulatory elements that repress the hTERT gene in normal cells. Compared with the virus with only the hTERT core promoter, the new viruses showed better selectivity for replication in cancer cells than in normal cells. In particular, Ad5GTSe, containing three extra copies of a repressor element, displayed a superior tropism for cancer cells among leukocytes and was thus selected for CTC detection in blood samples. To further improve the efficiency and specificity of CTC identification, we tested a combinatory strategy of microfiltration enrichment using flexible micro spring arrays and Ad5GTSe imaging. Our experiments showed that this method efficiently detected both cancer cells spiked into healthy blood and potential CTCs in blood samples of patients with breast and pancreatic cancer, demonstrating its potential as a highly sensitive and reliable system for detection and capture of CTCs of different tumor types. Mol Cancer Ther; 14(3); 835–43. ©2015 AACR.

A Spontaneous 3D Bone-On-a-Chip for Bone Metastasis Study of Breast Cancer Cells

Bone metastasis occurs at ≈70% frequency in metastatic breast cancer. The mechanisms used by tumors to hijack the skeleton, promote bone metastases, and confer therapeutic resistance are poorly understood. This has led to the development of various bone models to investigate the interactions between cancer cells and host bone marrow cells and related physiological changes. However, it is challenging to perform bone studies due to the difficulty in periodic sampling. Herein, a bone-on-a-chip (BC) is reported for spontaneous growth of a 3D, mineralized, collagenous bone tissue. Mature osteoblastic tissue of up to 85 µm thickness containing heavily mineralized collagen fibers naturally formed in 720 h without the aid of differentiation agents. Moreover, co-culture of metastatic breast cancer cells is examined with osteoblastic tissues. The new bone-on-a-chip design not only increases experimental throughput by miniaturization, but also maximizes the chances of cancer cell interaction with bone matrix of a concentrated surface area and facilitates easy, frequent observation. As a result, unique hallmarks of breast cancer bone colonization, previously confirmed only in vivo, are observed. The spontaneous 3D BC keeps the promise as a physiologically relevant model for the in vitro study of breast cancer bone metastasis.

Size-based separation methods of circulating tumor cells

Abstract Circulating tumor cells (CTCs) originate from the primary tumor mass and enter into the peripheral bloodstream. Compared to other “liquid biopsy” portfolios such as exosome, circulating tumor DNA/RNA (ctDNA/RNA), CTCs have incomparable advantages in analyses of transcriptomics, proteomics, and signal colocalization. Hence, CTCs hold the key to understanding the biology of metastasis and play a vital role in cancer diagnosis, treatment monitoring, and prognosis. Size‐based enrichment features are prominent in CTC isolation. It is a label‐free, simple and fast method. Enriched CTCs remain unmodified and viable for a wide range of subsequent analyses. In this review, we comprehensively summarize the differences of size and deformability between CTCs and blood cells, which would facilitate the development of technologies of size‐based CTC isolation. Then we review representative size‐/deformability‐based technologies available for CTC isolation and highlight the recent achievements in molecular analysis of isolated CTCs. To wrap up, we discuss the substantial challenges facing the field, and elaborate on prospects. Graphical abstract Figure. No Caption available.

Preoccupation of Empty Carriers Decreases Endo-/Lysosome Escape and Reduces the Protein Delivery Efficiency of Mesoporous Silica Nanoparticles

Endo-/lysosome escape is a major challenge in nanoparticle-based protein delivery for cancer therapy. To enhance the endo-/lysosomal escape and increase the efficacy of protein delivery, current strategies mainly focus on destroying endo-/lysosomes by employing modified nanoparticles, such as pH-sensitive polyplexes, cell-penetrating peptides, and photosensitive molecules. Herein, we hypothesize that pretreatment with empty nanocarriers might make endo-/lysosomes occupied and affect the endo/lysosomal escape of protein subsequently delivery by nanocarriers. We first treated breast carcinoma MDA-MB-231 cells with a high concentration of empty nanocarriers, mesoporous silica nanoparticles (MSN), to occupy the endo-/lysosome. After 2 h, we treated the cells with a lower concentration of fluorescein isothiocyanate-labeled MSN (MSN-FITC) and investigated the intracellular spatial and temporal distribution of MSN-FITC and their colocalization with endo-/lysosomes. We discovered the preoccupation of endo-/lysosomes by the empty nanocarriers did exist, mainly through changing the spatial distribution of the subsequently introduced nanocarriers. Furthermore, for the protein delivery, we observed reduced MSN-saporin delivery after endo-/lysosome preoccupation by MSN empty carriers. A similar result is observed for the delivery of cytochrome C by MSN but not for the small-molecule anticancer drug doxorubicin. The results show that the empty nanocarriers inhibit the endo-/lysosome intracellular trafficking process and decrease the endo-/lysosome escape of proteins subsequently delivered by the nanocarriers. This new discovered phenomenon of declined endo-/lysosome escape after endo-/lysosome preoccupation indicates that repeated treatment by nanomaterials with low protein-loading capacity may not yield a good cancer therapeutic effect. Therefore, it provides a new insightful perspective on the role of nanomaterial carriers in intracellular protein delivery.

Nucleus of Circulating Tumor Cell Determines Its Translocation Through Biomimetic Microconstrictions and Its Physical Enrichment by Microfiltration

The mechanism of cells passing through microconstrictions, such as capillaries and endothelial junctions, influences metastasis of circulating tumor cells (CTCs) in vivo, as well as size-based enrichment of CTCs in vitro. However, very few studies observe such translocation of microconstrictions in real time, and thus the inherent biophysical mechanism is poorly understood. In this study, a multiplexed microfluidic device is fabricated for real-time tracking of cell translocation under physiological pressure and recording deformation of the whole cell and nucleus, respectively. It is found that the deformability and size of the nucleus instead of the whole cell dominate cellular translocation through microconstrictions under a normal physiological pressure range. More specifically, cells with a large and stiff nucleus are prone to be blocked by relatively small constrictions. The same phenomenon is also observed in the size-based enrichment of CTCs from peripheral blood of metastatic cancer patients. These findings are different from a popular viewpoint that the size and deformability of a whole cell mainly determine cell translation through microconstrictions, and thus may elucidate interactions between CTCs and capillaries from a new perspective and guide the rational design of size-based microfilters for rare cell enrichment.

Self-Assembly of Extracellular Vesicle-like Metal–Organic Framework Nanoparticles for Protection and Intracellular Delivery of Biofunctional Proteins

The intracellular delivery of biofunctional enzymes or therapeutic proteins through systemic administration is of great importance in therapeutic intervention of various diseases. However, current strategies face substantial challenges owing to various biological barriers, including susceptibility to protein degradation and denaturation, poor cellular uptake, and low transduction efficiency into the cytosol. Here, we developed a biomimetic nanoparticle platform for systemic and intracellular delivery of proteins. Through a biocompatible strategy, guest proteins are caged in the matrix of metal-organic frameworks (MOFs) with high efficiency (up to ∼94%) and high loading content up to ∼50 times those achieved by surface conjunction, and the nanoparticles were further decorated with the extracellular vesicle (EV) membrane with an efficiency as high as ∼97%. In vitro and in vivo study manifests that the EV-like nanoparticles can not only protect proteins against protease digestion and evade the immune system clearance but also selectively target homotypic tumor sites and promote tumor cell uptake and autonomous release of the guest protein after internalization. Assisted by biomimetic nanoparticles, intracellular delivery of the bioactive therapeutic protein gelonin significantly inhibits the tumor growth in vivo and increased 14-fold the therapeutic efficacy. Together, our work not only proposes a new concept to construct a biomimetic nanoplatform but also provides a new solution for systemic and intracellular delivery of protein.

In Situ Caging of Biomolecules in Graphene Hybrids for Light Modulated Bioactivity

Remote and noninvasive modulation of protein activity is essential for applications in biotechnology and medicine. Optical control has emerged as the most attractive approach owing to its high spatial and temporal resolutions; however, it is challenging to engineer light responsive proteins. In this work, a near-infrared (NIR) light-responsive graphene-silica-trypsin (GST) nanoreactor is developed for modulating the bioactivity of trypsin molecules. Biomolecules are spatially confined and protected in the rationally designed compartment architecture, which not only reduces the possible interference but also boosts the bioreaction efficiency. Upon NIR irradiation, the photothermal effect of the GST nanoreactor enables the ultrafast in situ heating for remote activation and tuning of the bioactivity. We apply the GST nanoreactor for remote and ultrafast proteolysis of proteins, which remarkably enhances the proteolysis efficiency and reduces the bioreaction time from the overnight of using free trypsin to seconds. We envision that this work not only provides a promising tool of ultrafast and remotely controllable proteolysis for in vivo proteomics in study of tissue microenvironment and other biomedical applications but also paves the way for exploring smart artificial nanoreactors in biomolecular modulation to gain insight in dynamic biological transformation.

A bone-on-a-chip microdevice for long-term spontaneous 3D bone tissue formation and cancer bone metastasis

Bone is one of the preferred places for cancer metastasis, especially for breast, prostate, lung and melanoma cancers. In general, the prognosis for bone metastasis is abysmal with few treatment options available. Some colonies of cancer cells can reside in bone tissue as dormant cells for years before they become aggressive and grow into macrometastases. The interaction between cancer cells and bone tissue during this long period of time is currently not well understood. One technical barrier to study this in vitro is the lack of realistic bone tissue model suit for long-term study. Herein, we reported a bone-on-a-chip microdevice that can spontaneously form of a 3D, mineralized, collagenous bone tissue from an inoculum of isolated osteoblastic cell line without any artificial scaffold materials. Based on the principle of simultaneous-growth-dialysis, phenotypically mature osteoblastic tissue of up to 85 μm thickness containing heavily mineralized collagen fibers naturally formed in 720 hours without the aid of differentiation agents. Moreover, we have examined co-culture of metastatic human breast cancer cells MDA-MB-231 with osteoblastic tissues and observed important hallmarks of breast cancer colonization previously confirmed in vivo. With simple manipulation and ultra-low unit cost, the spontaneous 3D bone-on-a-chip shows promise as a physiologically-relevant model for the in vitro study of cancer bone metastasis.