4 research outputs found
Nanostructure Embedded Microchips for Detection, Isolation, and Characterization of Circulating Tumor Cells
ConspectusCirculating
tumor cells (CTCs) are cancer cells that break away
from either a primary tumor or a metastatic site and circulate in
the peripheral blood as the cellular origin of metastasis. With their
role as a “tumor liquid biopsy”, CTCs provide convenient
access to all disease sites, including that of the primary tumor and
the site of fatal metastases. It is conceivable that detecting and
analyzing CTCs will provide insightful information in assessing the
disease status without the flaws and limitations encountered in performing
conventional tumor biopsies. However, identifying CTCs in patient
blood samples is technically challenging due to the extremely low
abundance of CTCs among a large number of hematologic cells. To address
this unmet need, there have been significant research endeavors, especially
in the fields of chemistry, materials science, and bioengineering,
devoted to developing CTC detection, isolation, and characterization
technologies.Inspired by the nanoscale interactions observed
in the tissue microenvironment,
our research team at UCLA pioneered a unique concept of “NanoVelcro”
cell-affinity substrates, in which CTC capture agent-coated nanostructured
substrates were utilized to immobilize CTCs with high efficiency.
The working mechanism of NanoVelcro cell-affinity substrates mimics
that of Velcro: when the two fabric strips of a Velcro fastener are
pressed together, tangling between the hairy surfaces on two strips
leads to strong binding. Through continuous evolution, three generations
(gens) of NanoVelcro CTC chips have been established to achieve different
clinical utilities. The first-gen NanoVelcro chip, composed of a silicon
nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer,
was created for CTC enumeration. Side-by-side analytical validation
studies using clinical blood samples suggested that the sensitivity
of first-gen NanoVelcro chip outperforms that of FDA-approved CellSearch.
In conjunction with the use of the laser microdissection (LMD) technique,
second-gen NanoVelcro chips (i.e., NanoVelcro-LMD), based on polymer
nanosubstrates, were developed for single-CTC isolation. The individually
isolated CTCs can be subjected to single-CTC genotyping (e.g., Sanger
sequencing and next-generation sequencing, NGS) to verify the CTC’s
role as tumor liquid biopsy. Created by grafting of thermoresponsive
polymer brushes onto SiNS, third-gen NanoVelcro chips (i.e., Thermoresponsive
NanoVelcro) have demonstrated the capture and release of CTCs at 37
and 4 °C, respectively. The temperature-dependent conformational
changes of polymer brushes can effectively alter the accessibility
of the capture agent on SiNS, allowing for rapid CTC purification
with desired viability and molecular integrity.This Account
summarizes the continuous evolution of NanoVelcro
CTC assays from the emergence of the original idea all the way to
their applications in cancer research. We envision that NanoVelcro
CTC assays will lead the way for powerful and cost-efficient diagnostic
platforms for researchers to better understand underlying disease
mechanisms and for physicians to monitor real-time disease progression
Reduction of Circulating Cancer Cells and Metastases in Breast-Cancer Models by a Potent EphA2-Agonistic Peptide–Drug Conjugate
EphA2
overexpression has been associated with metastasis in multiple
cancer types, including melanomas and ovarian, prostate, lung, and
breast cancers. We have recently proposed the development of peptide–drug
conjugates (PDCs) using agonistic EphA2-targeting agents, such as
the YSA peptide or its optimized version, 123B9. Although our studies
indicated that YSA– and 123B9–drug conjugates can selectively
deliver cytotoxic drugs to cancer cells in vivo, the relatively low
cellular agonistic activities (i.e., the high micromolar concentrations
required) of the agents toward the EphA2 receptor remained a limiting
factor to the further development of these PDCs in the clinic. Here,
we report that a dimeric version of 123B9 can induce receptor activation
at nanomolar concentrations. Furthermore, we demonstrated that the
conjugation of dimeric 123B9 with paclitaxel is very effective at
targeting circulating tumor cells and inhibiting lung metastasis in
breast-cancer models. These studies represent an important step toward
the development of effective EphA2-targeting PDCs
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Imprinted NanoVelcro Microchips for Isolation and Characterization of Circulating Fetal Trophoblasts: Toward Noninvasive Prenatal Diagnostics
Circulating
fetal nucleated cells (CFNCs) in maternal blood offer
an ideal source of fetal genomic DNA for noninvasive prenatal diagnostics
(NIPD). We developed a class of nanoVelcro microchips to effectively
enrich a subcategory of CFNCs, <i>i</i>.<i>e</i>., circulating trophoblasts (cTBs) from maternal blood, which can
then be isolated with single-cell resolution by a laser capture microdissection
(LCM) technique for downstream genetic testing. We first established
a nanoimprinting fabrication process to prepare the LCM-compatible
nanoVelcro substrates. Using an optimized cTB-capture condition and
an immunocytochemistry protocol, we were able to identify and isolate
single cTBs (Hoechst+/CK7+/HLA-G+/CD45–, 20 μm > sizes
> 12 ÎĽm) on the imprinted nanoVelcro microchips. Three cTBs
were polled to ensure reproducible whole genome amplification on the
cTB-derived DNA, paving the way for cTB-based array comparative genomic
hybridization (aCGH) and short tandem repeats analysis. Using maternal
blood samples collected from expectant mothers carrying a single fetus,
the cTB-derived aCGH data were able to detect fetal genders and chromosomal
aberrations, which had been confirmed by standard clinical practice.
Our results support the use of nanoVelcro microchips for cTB-based
noninvasive prenatal genetic testing, which holds potential for further
development toward future NIPD solution
Programming Thermoresponsiveness of NanoVelcro Substrates Enables Effective Purification of Circulating Tumor Cells in Lung Cancer Patients
Unlike tumor biopsies that can be constrained by problems such as sampling bias, circulating tumor cells (CTCs) are regarded as the “liquid biopsy” of the tumor, providing convenient access to all disease sites, including primary tumor and fatal metastases. Although enumerating CTCs is of prognostic significance in solid tumors, it is conceivable that performing molecular and functional analyses on CTCs will reveal much significant insight into tumor biology to guide proper therapeutic intervention. We developed the Thermoresponsive NanoVelcro CTC purification system that can be digitally programmed to achieve an optimal performance for purifying CTCs from non-small cell lung cancer (NSCLC) patients. The performance of this unique CTC purification system was optimized by systematically modulating surface chemistry, flow rates, and heating/cooling cycles. By applying a physiologically endurable stimulation (<i>i.e.</i>, temperature between 4 and 37 °C), the mild operational parameters allow minimum disruption to CTCs’ viability and molecular integrity. Subsequently, we were able to successfully demonstrate culture expansion and mutational analysis of the CTCs purified by this CTC purification system. Most excitingly, we adopted the combined use of the Thermoresponsive NanoVelcro system with downstream mutational analysis to monitor the disease evolution of an index NSCLC patient, highlighting its translational value in managing NSCLC