Functional Tumor Targeting Nano‐Systems for Reprogramming Circulating Tumor Cells with In Situ Evaluation on Therapeutic Efficiency at the Single‐Cell Level

Abstract

Tumor heterogeneity is primarily responsible for treatment resistance and cancer relapses. Being critically important to address this issue, the timely evaluation of the appropriateness of therapeutic actions at the single-cell level is still facing challenges. By using multi-functionalized nano-systems with the delivery vector composed of histone for plasmids loading, hyaluronic acid for tumor targeting, and a fusion peptide for C-X-C motif chemokine receptor 4 (CXCR4) targeting as well as nuclear localization, the reprogramming of circulating tumor cells (CTCs) with in situ detection on biomarkers at the single-cell level is realized. By efficient co-delivery of the genome editing plasmid for CXCR4 knockout and molecular beacons for detection of upregulated mRNA biomarkers into CTCs in unprocessed whole blood, the therapeutic outcomes of genome editing at the single-cell level can be in situ evaluated. The single-cell analysis shows that CXCR4 in CTCs of cancer patients is efficiently downregulated, resulting in upregulated anticancer biomarkers such as p53 and p21. The study provides a facile strategy for in-depth profiling of cancer cell responses to therapeutic actions at single-cell resolution to evaluate the outcomes of treatments timely and conveniently.

Recent progress of nanostructure-based enrichment of circulating tumor cells and downstream analysis

“…Based on the multi-functional nano-delivery system, Ren et al realized genome editing and in situ evaluation of therapeutic response to in vitro CTCs at the single-cell level, providing accurate information for personalized cancer treatment. 157 In this study, C-X-C motif chemokine receptor 4 (CXCR4), a typical target protein in cancer therapy, was selected as the representative target for genome editing in vitro . Single cell analysis showed that CXCR4 in CTCs of cancer patients was effectively downregulated, anti-cancer biomarkers such as p53 and p21 were upregulated, and the edited cancer cells showed inhibition of proliferation and inhibition of migration and invasion.…”

Engineered Extracellular Vesicle-Delivered CRISPR/Cas9 for Radiotherapy Sensitization of Glioblastoma

“…Permanent disruption of oncogene by CRISPR-Cas9 gene editing offers the ability to circumvent the repetitive dosage restrictions of conventional cancer therapies, improve treatment effectiveness, and reduce therapeutic costs. , However, an essential missing link in the therapeutic translation of CRISPR editing is the creation of effective and safe delivery systems for cancer. CRISPR/Cas9 therapeutic delivery using viral and nonviral-based delivery vehicles is rapidly expanding. , However, therapeutic delivery of Cas9 protein is currently hampered by the large size of Streptococcus pyogenes , Cas9 exceeding the carrying capacity of viral and nonviral vectors . In addition, long exposure time to the Cas9 nuclease and repeated doses may raise the risk for Cas9-related immunogenicity and off-target effects. , Therefore, to reduce this risk, it is necessary to develop a delivery system that may accomplish therapeutically relevant genome editing with reduced doses and a short Cas9 exposure time.…”

Detection of mRNAs of Ribosomal Protein L15 and E-Cadherin in Living Circulating Tumor Cells at Single Cell Resolution To Study Tumor Heterogeneity

“…By using delivery vectors to deliver RNA or DNA probes into living cells, detection of nucleic acids in living cells can be realized. The vectors for RNA or DNA probes explored cover a wide range of nano-materials based on inorganic materials, − organic materials, , metals, − and hybrid materials. − To efficiently deliver the probes into targeted cancer cells, various ligands, such as aptamers, − peptides, , polysaccharides, and small molecules, were incorporated to the delivery vectors. Nevertheless, these studies are mainly focused on detection of tumor cell lines and tumor tissues of tumor bearing mice ex vivo.…”

“…In our previous work, we designed biomacromolecule-based vectors for targeting delivery molecular beacons for detection of miRNA (e.g., miR-21) 26 and mRNA (e.g., CXCR4 mRNA, p53 mRNA, and p21 mRNA) 28 to CTCs. The biomacromolecule-based vectors with good biocompatibility minimize the unfavorable effects, such as perturbing RNA levels on treated cells, to ensure the accurate detection.…”