A microfluidic device to study cancer metastasis under chronic and intermittent hypoxia

Metastatic cancer cells must traverse a microenvironment ranging from extremely hypoxic, within the tumor, to highly oxygenated, within the host''s vasculature. Tumor hypoxia can be further characterized by regions of both chronic and intermittent hypoxia. We present the design and characterization of a microfluidic device that can simultaneously mimic the oxygenation conditions observed within the tumor and model the cell migration and intravasation processes. This device can generate spatial oxygen gradients of chronic hypoxia and produce dynamically changing hypoxic microenvironments in long-term culture of cancer cells.     

Engineered three-dimensional microfluidic device for interrogating cell-cell interactions in the tumor microenvironment

Stromal cells in the tumor microenvironment play a key role in the metastatic properties of a tumor. It is recognized that cancer-associated fibroblasts (CAFs) and endothelial cells secrete factors capable of influencing tumor cell migration into the blood or lymphatic vessels. We developed a microfluidic device that can be used to image the interactions between stromal cells and tumor cell spheroids in a three dimensional (3D) microenvironment while enabling external control of interstitial flow at an interface, which supports endothelial cells. The apparatus couples a 200-μm channel with a semicircular well to mimic the interface of a blood vessel with the stroma, and the design allows for visualization of the interactions of interstitial flow, endothelial cells, leukocytes, and fibroblasts with the tumor cells. We observed that normal tissue-associated fibroblasts (NAFs) contribute to the “single file” pattern of migration of tumor cells from the spheroid in the 3D microenvironment. In contrast, CAFs induce a rapid dispersion of tumor cells out of the spheroid with migration into the 3D matrix. Moreover, treatment of tumor spheroid cultures with the chemokine CXCL12 mimics the effect of the CAFs, resulting in similar patterns of dispersal of the tumor cells from the spheroid. Conversely, addition of CXCL12 to co-cultures of NAFs with tumor spheroids did not mimic the effects observed with CAF co-cultures, suggesting that NAFs produce factors that stabilize the tumor spheroids to reduce their migration in response to CXCL12.     

An integrated microfluidic system for screening of phage-displayed peptides specific to colon cancer cells and colon cancer stem cells

Affinity reagents recognizing biomarkers specifically are essential components of clinical diagnostics and target therapeutics. However, conventional methods for screening of these reagents often have drawbacks such as large reagent consumption, the labor-intensive or time-consuming procedures, and the involvement of bulky or expensive equipment. Alternatively, microfluidic platforms could potentially automate the screening process within a shorter period of time and reduce reagent and sample consumption dramatically. It has been demonstrated recently that a subpopulation of tumor cells known as cancer stem cells possess high drug resistance and proliferation potential and are regarded as the main cause of metastasis. Therefore, a peptide that recognizes cancer stem cells and differentiates them from other cancer cells will be extremely useful in early diagnosis and target therapy. This study utilized M13 phage display technology to identify peptides that bind, respectively, to colon cancer cells and colon cancer stem cells using an integrated microfluidic system. In addition to positive selection, a negative selection process was integrated on the chip to achieve the selection of peptides of high affinity and specificity. We successfully screened three peptides specific to colon cancer cells and colon cancer stem cells, namely, HOLC-1, HOLC-2, and COLC-1, respectively, and their specificity was measured by the capture rate between target, control, and other cell lines. The capture rates are 43.40 ± 7.23%, 45.16 ± 7.12%, and 49.79 ± 5.34% for colon cancer cells and colon cancer stem cells, respectively, showing a higher specificity on target cells than on control and other cell lines. The developed technique may be promising for early diagnosis of cancer cells and target therapeutics.     

On-chip assay of the effect of topographical microenvironment on cell growth and cell-cell interactions during wound healing

Wound healing is an essential physiological process for tissue homeostasis, involving multiple types of cells, extracellular matrices, and growth factor/chemokine interactions. Many in vitro studies have investigated the interactions between cues mentioned above; however, most of them only focused on a single factor. In the present study, we design a wound healing device to recapitulate in vivo complex microenvironments and heterogeneous cell situations to investigate how three types of physiologically related cells interact with their microenvironments around and with each other during a wound healing process. Briefly, a microfluidic device with a micropillar substrate, where diameter and interspacing can be tuned to mimic the topographical features of the 3D extracellular matrix, was designed to perform positional cell loading on the micropillar substrate, co-culture of three types of physiologically related cells, keratinocytes, dermal fibroblasts, and human umbilical vein endothelial cells, as well as an investigation of their interactions during wound healing. The result showed that cell attachment, morphology, cytoskeleton distribution, and nucleus shape were strongly affected by the micropillars, and these cells showed collaborative response to heal the wound. Taken together, these findings highlight the dynamic relationship between cells and their microenvironments. Also, this reproducible device may facilitate the in vitro investigation of numerous physiological and pathological processes such as cancer metastasis, angiogenesis, and tissue engineering.     

On-chip three-dimensional tumor spheroid formation and pump-less perfusion culture using gravity-driven cell aggregation and balanced droplet dispensing

This paper presents a spheroid chip in which three-dimensional (3D) tumor spheroids are not only formed by gravity-driven cell aggregation but also cultured at the perfusion rates controlled by balanced droplet dispensing without fluidic pumps. The previous spheroid chips require additional off-chip processes of spheroid formation and extraction as well as bulky components of fluidic pumps. However, the present spheroid chip, where autonomous medium droplet dispensers are integrated on a well array, achieves the on-chip 3D tumor spheroid formation and perfusion culture using simple structure without bulky fluidic pumps. In the experimental study, we demonstrated that the spheroid chip successfully forms 3D tumor spheroids in the wide diameter range of 220 μm–3.2 mm (uniformity > 90%) using H358, H23, and A549 non-small cell lung cancer cells. At the pump-less perfusion culture (Q = 0.1–0.3 μl/min) of spheroids, the number of H358 cells in the spheroid increased up to 50% from the static culture (Q = 0 μl/min) and the viability of the cultured cells also increased about 10%. Therefore, we experimentally verified that the perfusion environment created by the spheroid chip offers a favourable condition to the spheroids with high increase rate and viability. The present chip achieves on-chip 3D tumor spheroid formation and pump-less perfusion culture with simple structure, thereby exhibiting potential for use in integrated in-vivo-like cell culture systems.     

CRISPR/Cas9-mediated activation of CDH1 suppresses metastasis of breast cancer in rats

BackgroundCancer is a life-threatening disease that affects approximately 18 million individuals worldwide. Breast cancer is the most common female neoplasm globally with more than 276,480 new cases of invasive breast cancer expected to be diagnosed in women in the U.S. alone in 2020. Genetic and epigenetic factors play role in the carcinogenesis and progression of this disease. In this study, MCF-7 adenocarcinoma cells were transfected with CRISPR/Cas9 plasmid to either knock out CDK11 or to activate CDH1. Treated cells were allografted into the mammary glands of female rats (150–190 g, 6–8 weeks) to evaluate the capability of these cells to control cancer progression and metastasis.ResultsqPCR data revealed a significant downregulation of CDK11 and upregulation of CDH1. Cell cycle analysis and apoptosis assays indicated the knockout of CDK11 and simultaneous activation of CDH1 resulted in cell cycle arrest at G2/M phase and accumulation of cells at G2. Meanwhile, the percentage of cells that underwent late apoptosis increased in both genome editing hits. Histopathological sectioning data indicated that untransfected MCF-7 cells were capable of developing tumors in the mammary gland and initiation g angiogenesis. Transfected cells significantly restricted cancer cell infiltration/invasion by minimally localizing tumors and inhibiting angiogenesis.ConclusionsAlthough further investigation is needed, the present data indicate the potentiality of using CRISPR/Cas9-based therapy as a promising approach to treat breast cancer. Impact: these data indicate targeting cancer-related genes via any genome editing tool might represent a novel approach to combat cancer.How to cite: Al-Mulhim F, Alqosaibi AI, Al-Muhnna A, et al. CRISPR/Cas9-mediated activation of CDH1 suppresses metastasis of breast cancer in rats. Electron J Biotechnol 2021;53. https://doi.org/10.1016/j.ejbt.2021.06.002     

Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip

Circulating tumor cells (CTCs) are found in the blood of patients with cancer. Although these cells are rare, they can provide useful information for chemotherapy. However, isolation of these rare cells from blood is technically challenging because they are small in numbers. An integrated microfluidic chip, dubbed CTC chip, was designed and fabricated for conducting tumor cell isolation. As CTCs usually show multidrug resistance (MDR), the effect of MDR inhibitors on chemotherapeutic drug accumulation in the isolated single tumor cell is measured. As a model of CTC isolation, human prostate cancer cells were mixed with mouse blood cells and the label-free isolation of the tumor cells was conducted based on cell size difference. The major advantages of the CTC chip are the ability for fast cell isolation, followed by multiple rounds of single-cell measurements, suggesting a potential assay for detecting the drug responses based on the liquid biopsy of cancer patients.     

Spheroid-on-chip microfluidic technology for the evaluation of the impact of continuous flow on metastatic potential in cancer models in vitro

The majority of cancer deaths are linked to tumor spread, or metastasis, but 3D in vitro metastasis models relevant to the tumor microenvironment (including interstitial fluid flow) remain an area of unmet need. Microfluidics allows us to introduce controlled flow to an in vitro cancer model to better understand the relationship between flow and metastasis. Here, we report new hybrid spheroid-on-chip in vitro models for the impact of interstitial fluid flow on cancer spread. We designed a series of reusable glass microfluidic devices to contain one spheroid in a microwell under continuous perfusion culture. Spheroids derived from established cancer cell lines were perfused with complete media at a flow rate relevant to tumor interstitial fluid flow. Spheroid viability and migratory/invasive capabilities were maintained on-chip when compared to off-chip static conditions. Importantly, using flow conditions modeled in vitro, we are the first to report flow-induced secretion of pro-metastatic factors, in this case cytokines vascular endothelial growth factor and interleukin 6. In summary, we have developed a new, streamlined spheroid-on-chip in vitro model that represents a feasible in vitro alternative to conventional murine in vivo metastasis assays, including complex tumor environmental factors, such as interstitial fluid flow, extracellular matrices, and using 3D models to model nutrient and oxygen gradients. Our device, therefore, constitutes a robust alternative to in vivo early-metastasis models for determination of novel metastasis biomarkers as well as evaluation of therapeutically relevant molecular targets not possible in in vivo murine models.     

In situ phase transitional polymeric vaccines for improved immunotherapy

Cancer vaccines have exhibited immense potential in cancer treatment. Through activating the host''s immune system, vaccines stimulate extensive functional T cells to eliminate cancer. However, the therapeutic efficacy of cancer vaccines is limited by their inferior lymph node delivery and inadequate uptake of dendritic cells. Herein, we propose an in situ phase transitional strategy on vaccine manufacturing to maximally enhance lymph node drainage while ensuring adequate dendritic cell uptake. The phase transitional vaccines, with dynamic size modulation property, retain a small size (24.4 ± 3.1 nm) during lymph node draining then transform into larger particles (483.0 ± 41.6 nm) on-site by external signal input. Results show that this strategy induced rapid and robust immune response in a mouse melanoma tumor model. Furthermore, a stronger humoral immune response was observed in mice when immunized with MHC-II restricted antigen, which demonstrated that lymph node-targeted cancer vaccine delivery could be effectively manipulated through dynamic size modulation.     

Spatio-temporal analysis of tamoxifen-induced bystander effects in breast cancer cells using microfluidics

The bystander effect in cancer therapy is the inhibition or killing of tumor cells that are adjacent to those directly affected by the agent used for treatment. In the case of chemotherapy, little is known as to how much and by which mechanisms bystander effects contribute to the elimination of tumor cells. This is mainly due to the difficulty to distinguish between targeted and bystander cells since both are exposed to the pharmaceutical compound. We here studied the interaction of tamoxifen-treated human breast cancer MCF-7 cells with their neighboring counterparts by exploiting laminar flow patterning in a microfluidic chip to ensure selective drug delivery. The spatio-temporal evolution of the bystander response in non-targeted cells was analyzed by measuring the mitochondrial membrane potential under conditions of free diffusion. Our data show that the bystander response is detectable as early as 1 hour after drug treatment and reached effective distances of at least 2.8 mm. Furthermore, the bystander effect was merely dependent on diffusible factors rather than cell contact-dependent signaling. Taken together, our study illustrates that this microfluidic approach is a promising tool for screening and optimization of putative chemotherapeutic drugs to maximize the bystander response in cancer therapy.     

One step antibody-mediated isolation and patterning of multiple cell types in microfluidic devices

Cell-cell interactions play a key role in regeneration, differentiation, and basic tissue function taking place under physiological shear forces. However, current solutions to mimic such interactions by micro-patterning cells within microfluidic devices have low resolution, high fabrication complexity, and are limited to one or two cell types. Here, we present a microfluidic platform capable of laminar patterning of any biotin-labeled peptide using streptavidin-based surface chemistry. The design permits the generation of arbitrary cell patterns from heterogeneous mixtures in microfluidic devices. We demonstrate the robust co-patterning of α-CD24, α-ASGPR-1, and α-Tie2 antibodies for rapid isolation and co-patterning of mixtures of hepatocytes and endothelial cells. In addition to one-step isolation and patterning, our design permits step-wise patterning of multiple cell types and empty spaces to create complex cellular geometries in vitro. In conclusion, we developed a microfluidic device that permits the generation of perfusable tissue-like patterns in microfluidic devices by directly injecting complex cell mixtures such as differentiated stem cells or tissue digests with minimal sample preparation.     

Quick chip assay using locked nucleic acid modified epithelial cell adhesion molecule and nucleolin aptamers for the capture of circulating tumor cells

The role of circulating tumor cells (CTCs) in disease diagnosis, prognosis, monitoring of the therapeutic efficacy, and clinical decision making is immense and has attracted tremendous focus in the last decade. We designed and fabricated simple, flat channel microfluidic devices polydimethylsiloxane (PDMS based) functionalized with locked nucleic acid (LNA) modified aptamers (targeting epithelial cell adhesion molecule (EpCAM) and nucleolin expression) for quick and efficient capture of CTCs and cancer cells. With optimized flow rates (10 μl/min), it was revealed that the aptamer modified devices offered reusability for up to six times while retaining optimal capture efficiency (>90%) and specificity. High capture sensitivity (92%) and specificity (100%) was observed in whole blood samples spiked with Caco-2 cells (10–100 cells/ml). Analysis of blood samples obtained from 25 head and neck cancer patients on the EpCAM LNA aptamer functionalized chip revealed that an average count of 5 ± 3 CTCs/ml of blood were captured from 22/25 samples (88%). EpCAM intracellular domain (EpICD) immunohistochemistry on 9 oral squamous cell carcinomas showed the EpICD positivity in the tumor cells, confirming the EpCAM expression in CTCs from head and neck cancers. These microfluidic devices also maintained viability for in vitro culture and characterization. Use of LNA modified aptamers provided added benefits in terms of cost effectiveness due to increased reusability and sustainability of the devices. Our results present a robust, quick, and efficient CTC capture platform with the use of simple PDMS based devices that are easy to fabricate at low cost and have an immense potential in cancer diagnosis, prognosis, and therapeutic planning.     

Quantitative impedimetric monitoring of cell migration under the stimulation of cytokine or anti-cancer drug in a microfluidic chip

Cell migration is a cellular response and results in various biological processes such as cancer metastasis, that is, the primary cause of death for cancer patients. Quantitative investigation of the correlation between cell migration and extracellular stimulation is essential for developing effective therapeutic strategies for controlling invasive cancer cells. The conventional method to determine cell migration rate based on comparison of successive images may not be an objective approach. In this work, a microfluidic chip embedded with measurement electrodes has been developed to quantitatively monitor the cell migration activity based on the impedimetric measurement technique. A no-damage wound was constructed by microfluidic phenomenon and cell migration activity under the stimulation of cytokine and an anti-cancer drug, i.e., interleukin-6 and doxorubicin, were, respectively, investigated. Impedance measurement was concurrently performed during the cell migration process. The impedance change was directly correlated to the cell migration activity; therefore, the migration rate could be calculated. In addition, a good match was found between impedance measurement and conventional imaging analysis. But the impedimetric measurement technique provides an objective and quantitative measurement. Based on our technique, cell migration rates were calculated to be 8.5, 19.1, and 34.9 μm/h under the stimulation of cytokine at concentrations of 0 (control), 5, and 10 ng/ml. This technique has high potential to be developed into a powerful analytical platform for cancer research.     

中医药调控肝癌血管生成的研究近况

肝细胞癌（HCC）的发生与发展与血管形成有关,肝癌血管新生为肿瘤细胞提供生长和转移的条件。而血管生成因子和血管抑制因子是肝癌血管生成与否的物质基础,鉴于此,抑制血管生成因子的表达或者让血管抑制因子表达占优势,即可抑制肿瘤血管生成。目前临床上已逐步开展抗肝癌血管生成治疗。研究表明,很多中药具有抗肿瘤血管新生的作用。     

Spatially gradated segregation and recovery of circulating tumor cells from peripheral blood of cancer patients

For cancer patients, the enumeration of rare circulating tumor cells (CTCs) in peripheral blood is a strong prognostic indicator of the severity of the cancer; for the general population, the capture of CTCs is needed for use as a clinical tool for cancer screening, early detection, and treatment assessment. Here, we present a fast, high-purity (∼90%) and high-efficiency (>90%) method for the segregation and undamaged recovery of CTCs using a spatially gradated microfluidic chip. Further, by lysing the red blood cells we achieved not only a significant reduction in the overall processing time but also mitigated the blood clogging problem commonly encountered in microfluidic-based CTC isolation systems. To clinically validate the chip, we employed it to detect and capture CTCs from 10 liver cancer patients. Positive CTC enumeration was observed in all the blood samples, and the readings ranged from a low of 1–2 CTCs (1 patient) to a high of >20 CTCs (2 patients) with the balance having 3–20 CTCs per 3-ml blood sample. The work here indicates that our system can be developed for use in cancer screening, metastatic assessment, and chemotherapeutic response and for pharmacological and genetic evaluation of single CTCs.     

Measurement of cell compressibility changes during epithelial–mesenchymal transition based on acoustofluidic microdevice

Epithelial–mesenchymal transition (EMT) confers migratory and invasiveness abilities on cancer cells, as well as leading to changes in biomechanical properties and cytoskeletal structure. Cell mechanical properties are considered to be promising label-free markers for diagnosis of cancer metastasis. In this work, cell compressibility, a novel and important parameter of cell mechanical properties, was measured directly and quickly using a specially designed acoustofluidic microdevice. The compressibilities of cells with different metastatic potentials were investigated. Based on a comparison of the measurement results, non-metastatic cells exhibited lower compressibility than metastatic cells. The correlation between cell compressibility and EMT status was further studied; the results showed that the acquisition of mesenchymal status was accompanied by an increase in cell compressibility. These findings imply strong correlations among cell compressibility, EMT status, and invasiveness. Therefore, cell compressibility represents a novel biomechanical marker for evaluating malignant transformation and metastasis of cancer.     

MicroRNAs: Potential biomarkers in cancer

microRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs, also known as micromanagers of gene expression. Polymorphisms in the miRNA pathway (miR-polymorphisms) are emerging as powerful tools to study the biology of a disease and have the potential to be used in disease prognosis and diagnosis. Advancements in the miRNA field also indicate a clear involvement of deregulated miRNA gene signatures in cancers, and several polymorphisms in pre-miRNA, miRNA binding sites or targets have been found to be associated with various cancers. The miRNA polymorphisms have also been reported to influence tumor aggressiveness as well as survival of cancer patients. miRNAs have a revolutionary impact on cancer research over recent years. They emerge as important players in tumorigenesis, leading to a paradigm shift in oncology. The extensive and comprehensive use of miRNA microarrays has enabled the identification of a number of miRNAs as potential biomarkers for cancer. Many miRNAs have been identified to act as oncogenes, tumor suppressors, or even modulators of cancer stem cells and metastasis. Some studies not only reported the identified miRNA biomarkers, but also deciphered their target genes and the underlying mechanisms. The rapid discovery of many miRNA targets and their relevant pathways has contributed to the development of miRNA-based therapeutics.     

Effect of vimentin on cell migration in collagen-coated microchannels: A mimetic physiological confined environment

Cancer cell migration through tissue pores and tracks into the bloodstream is a critical biological step for cancer metastasis. Although in vivo studies have shown that expression of vimentin can induce invasive cell lines, its role in cell cytoskeleton reorganization and cell motility under in vitro physical confinement remains unknown. Here, a microfluidic device with cell culture chamber and collagen-coated microchannels was developed as an in vitro model for physiological confinement environments. Using this microchannel assay, we demonstrated that the knockdown of vimentin decreases 3T3 fibroblast cell directional migration speed in confined microchannels. Additionally, as cells form dynamic membranes that define the leading edge of motile cells, different leading edge morphologies of 3T3 fibroblast and 3T3 vimentin knockdown cells were observed. The leading edge morphology change under confinement can be explained by the effect of vimentin on cytoskeletal organization and focal adhesion. The microfluidic device integrated with a time-lapse microscope provided a new approach to study the effect of vimentin on cell adhesion, migration, and invasiveness.     

ApoStream?, a new dielectrophoretic device for antibody independent isolation and recovery of viable cancer cells from blood

Isolation and enumeration of circulating tumor cells (CTCs) are used to monitor metastatic disease progression and guide cancer therapy. However, currently available technologies are limited to cells expressing specific cell surface markers, such as epithelial cell adhesion molecule (EpCAM) or have limited specificity because they are based on cell size alone. We developed a device, ApoStream^™ that overcomes these limitations by exploiting differences in the biophysical characteristics between cancer cells and normal, healthy blood cells to capture CTCs using dielectrophoretic technology in a microfluidic flow chamber. Further, the system overcomes throughput limitations by operating in continuous mode for efficient isolation and enrichment of CTCs from blood. The performance of the device was optimized using a design of experiment approach for key operating parameters such as frequency, voltage and flow rates, and buffer formulations. Cell spiking studies were conducted using SKOV3 or MDA-MB-231 cell lines that have a high and low expression level of EpCAM, respectively, to demonstrate linearity and precision of recovery independent of EpCAM receptor levels. The average recovery of SKOV3 and MDA-MB-231 cancer cells spiked into approximately 12 × 10⁶ peripheral blood mononuclear cells obtained from 7.5 ml normal human donor blood was 75.4% ± 3.1% (n = 12) and 71.2% ± 1.6% (n = 6), respectively. The intra-day and inter-day precision coefficients of variation of the device were both less than 3%. Linear regression analysis yielded a correlation coefficient (R²) of more than 0.99 for a spiking range of 4–2600 cells. The viability of MDA-MB-231 cancer cells captured with ApoStream was greater than 97.1% and there was no difference in cell growth up to 7 days in culture compared to controls. The ApoStream device demonstrated high precision and linearity of recovery of viable cancer cells independent of their EpCAM expression level. Isolation and enrichment of viable cancer cells from ApoStream enables molecular characterization of CTCs from a wide range of cancer types.     

High-efficiency rare cell identification on a high-density self-assembled cell arrangement chip

Detection of individual target cells among a large amount of blood cells is a major challenge in clinical diagnosis and laboratory protocols. Many researches show that two dimensional cells array technology can be incorporated into routine laboratory procedures for continuously and quantitatively measuring the dynamic behaviours of large number of living cells in parallel, while allowing other manipulations such as staining, rinsing, and even retrieval of targeted cells. In this study, we present a high-density cell self-assembly technology capable of quickly spreading over 300 000 cells to form a dense mono- to triple-layer cell arrangement in 5 min with minimal stacking of cells by the gentle incorporation of gravity and peripheral micro flow. With this self-assembled cell arrangement (SACA) chip technology, common fluorescent microscopy and immunofluorescence can be utilized for detecting and analyzing target cells after immuno-staining. Validated by experiments with real human peripheral blood samples, the SACA chip is suitable for detecting rare cells in blood samples with a ratio lower than 1/100 000. The identified cells can be isolated and further cultured in-situ on a chip for follow-on research and analysis. Furthermore, this technology does not require external mechanical devices, such as pump and valves, which simplifies operation and reduces system complexity and cost. The SACA chip offers a high-efficient, economical, yet simple scheme for identification and analysis of rare cells. Therefore, potentially SACA chip may provide a feasible and economical platform for rare cell detection in the clinic.