Deciphering the Biology of Circulating Tumor Cells through Single-Cell RNA Sequencing: Implications for Precision Medicine in Cancer.

Circulating tumor cells (CTCs) hold unique biological characteristics that directly involve them in hematogenous dissemination. Studying CTCs systematically is technically challenging due to their extreme rarity and heterogeneity and the lack of specific markers to specify metastasis-initiating CTCs. With cutting-edge technology, single-cell RNA sequencing (scRNA-seq) provides insights into the biology of metastatic processes driven by CTCs. Transcriptomics analysis of single CTCs can decipher tumor heterogeneity and phenotypic plasticity for exploring promising novel therapeutic targets. The integrated approach provides a perspective on the mechanisms underlying tumor development and interrogates CTCs interactions with other blood cell types, particularly those of the immune system. This review aims to comprehensively describe the current study on CTC transcriptomic analysis through scRNA-seq technology. We emphasize the workflow for scRNA-seq analysis of CTCs, including enrichment, single cell isolation, and bioinformatic tools applied for this purpose. Furthermore, we elucidated the translational knowledge from the transcriptomic profile of individual CTCs and the biology of cancer metastasis for developing effective therapeutics through targeting key pathways in CTCs.

Alpha-mangostin inhibits dengue virus production and pro-inflammatory cytokine/chemokine expression in dendritic cells.

Dengue virus (DENV) is transmitted to humans via the bite of an Aedes mosquito, causing dengue fever, dengue hemorrhagic fever, or dengue shock syndrome. In the human skin, DENV first infects keratinocytes, dendritic cells, and macrophages. Monocytes that are recruited to the site of infection and differentiate into monocyte-derived dendritic cells (moDCs) are also infected by DENV. DENV-infected DCs secrete pro-inflammatory cytokines and chemokines to modulate the immune response. The viral load and massive pro-inflammatory cytokine/chemokine production, referred to as a 'cytokine storm', are associated with disease severity. We propose that an ideal drug for treatment of DENV infection should inhibit both virus production and the cytokine storm, and previously, we reported that alpha-mangostin (α-MG) inhibits both DENV replication and cytokine production in hepatocytes. However, the effect of α-MG on DENV-infected moDCs remains unknown. In this study, we investigated the effects of α-MG on DENV infection and pro-inflammatory cytokine/chemokine production in primary moDCs generated ex vivo from monocytes of healthy individuals. α-MG at the non-toxic concentrations of 20 and 25 µM reduced DENV production by more than 10-fold and 1,000-fold, respectively. Treatment with α-MG efficiently inhibited the infection of immature moDCs by all four serotypes of DENV. Time-of-addition studies suggested that α-MG (25 µM) inhibits DENV at the early stage of replication. In addition, α-MG markedly reduced cytokine/chemokine (TNF-α, CCL4, CCL5, CXCL10, IL6, IL1ß, IL10, and IFN-α) transcription in DENV-infected immature moDCs. These findings suggest the potential of α-MG to be developed as a novel anti-DENV drug.

Vivo-morpholino oligomers strongly inhibit dengue virus replication and production.

Dengue virus (DENV) infection is a worldwide public health problem, which can cause severe dengue hemorrhagic fever (DHF) and life-threatening dengue shock syndrome (DSS). There are currently no anti-DENV drugs available, and there has been an intensive search for effective anti-DENV agents that can inhibit all four DENV serotypes. In this study, we tested whether vivo-morpholino oligomers (vivo-MOs), whose effect on DENV infection has not previously been studied, can inhibit DENV infection. Vivo-MOs were designed to target the top of 3' stem-loop (3' SL) in the 3' UTR of the DENV genome and tested for inhibition of DENV infection in monkey kidney epithelial (Vero) cells and human lung epithelial carcinoma (A549) cells. The results showed that vivo-MOs could bind to a DENV RNA sequence and markedly reduce DENV-RNA, protein, and virus production in infected Vero and A549 cells. Vivo-MOs at a concentration of 4 µM could inhibit DENV production by more than 104-fold when compared to that of an untreated control. In addition, vivo-MOs also inhibited DENV production in U937 cells and primary human monocytes. Therefore, vivo-MOs targeting to the 3' SL in the 3' UTR of DENV genomes are effective and have the potential to be developed as anti-DENV agents.

Unlocking the tumor-immune microenvironment in osteosarcoma: insights into the immune landscape and mechanisms.

Osteosarcoma has a unique tumor microenvironment (TME), which is characterized as a complex microenvironment comprising of bone cells, immune cells, stromal cells, and heterogeneous vascular structures. These elements are intricately embedded in a mineralized extracellular matrix, setting it apart from other primary TMEs. In a state of normal physiological function, these cell types collaborate in a coordinated manner to maintain the homeostasis of the bone and hematopoietic systems. However, in the pathological condition, i.e., neoplastic malignancies, the tumor-immune microenvironment (TIME) has been shown to promote cancer cells proliferation, migration, apoptosis and drug resistance, as well as immune escape. The intricate and dynamic system of the TIME in osteosarcoma involves crucial roles played by various infiltrating cells, the complement system, and exosomes. This complexity is closely associated with tumor cells evading immune surveillance, experiencing uncontrolled proliferation, and facilitating metastasis. In this review, we elucidate the intricate interplay between diverse cell populations in the osteosarcoma TIME, each contributing uniquely to tumor progression. From chondroblastic and osteoblastic osteosarcoma cells to osteoclasts, stromal cells, and various myeloid and lymphoid cell subsets, the comprehensive single-cell analysis provides a detailed roadmap of the complex osteosarcoma ecosystem. Furthermore, we summarize the mutations, epigenetic mechanisms, and extracellular vesicles that dictate the immunologic landscape and modulate the TIME of osteosarcoma. The perspectives of the clinical implementation of immunotherapy and therapeutic approaches for targeting immune cells are also intensively discussed.

Establishment, characterization, and genetic profiling of patient-derived osteosarcoma cells from a patient with retinoblastoma.

Osteosarcoma is the most common malignant bone cancer in pediatric patients. Patients who respond poorly to chemotherapy experience worse clinical outcomes with a high mortality rate. The major challenge is the lack of effective drugs for these patients. To introduce new drugs for clinical approval, preclinical studies based on in vitro models must demonstrate the potency of the tested drugs, enabling the drugs to enter phase 1 clinical trials. Patient-derived cell culture is a promising testing platform for in vitro studies, as they more accurately recapitulate cancer states and genetic profiles compared to cell lines. In the present study, we established patient-derived osteosarcoma cells (PDC) from a patient who had previously been diagnosed with retinoblastoma. We identified a new variant of a germline mutation in the RB1 gene in the tissue of the patient. The biological effects of this PDC were studied to observe whether the cryopreserved PDC retained a feature of fresh PDC. The cryopreserved PDC preserved the key biological effects, including cell growth, invasive capability, migration, and mineralization, that define the conserved phenotypes compared to fresh PDC. From whole genome sequencing analysis of osteosarcoma tissue and patient-derived cells, we found that cryopreserved PDC was a minor population in the origin tissue and was selectively grown under the culture conditions. The cryopreserved PDC has a high resistance to conventional chemotherapy. This study demonstrated that the established cryopreserved PDC has the aggressive characteristics of osteosarcoma, in particular the chemoresistance phenotype that might be used for further investigation in the chemoresistant mechanism of osteosarcoma. In conclusion, the approach we applied for primary cell culture might be a promising method to generate in vitro models for functional testing of osteosarcoma.

Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains.

Second-generation anti-CD19-chimeric antigen receptor T cells (anti-CD19-CAR2 T cells) are effective for treating B-cell malignancies; however, anti-CD19-CAR2 T cells can induce human anti-mouse immune responses because anti-CD19 single-chain variable fragment (scFv) in the CAR molecules is derived from a murine FMC63 (mFMC63) monoclonal antibody. Consequently, the persistence of mFMC63-CAR2 T cells and their therapeutic efficiency in patients are decreased, which results in tumor relapse. In an attempt to remedy this shortcoming, we generated a new anti-CD19-CAR T cells containing fully human anti-CD19 scFv (Hu1E7-CAR4 T cells) to pre-clinically evaluate and compare with mFMC63-CAR4 T cells. The human anti-CD19 scFv (Hu1E7) was isolated from a human scFv phage display library and fused to the hinge region of CD8α, the transmembrane domain of CD28, three intracellular costimulatory domains (CD28, 4-1BB, and CD27), and a CD3ζ signaling domain (28BB27ζ). Compared to mFMC63-CAR2 T cells (BBζ) and mFMC63-CAR3 (BB27ζ), the mFMC63-CAR4 T cells (28BB27ζ) exerted superior anti-tumor activity against Raji (CD19+) target cell. The Hu1E7-CAR4 and mFMC63-CAR4 T cells demonstrated comparable cytotoxicity and proliferation. Interestingly, compared to mFMC63-CAR4 T cells, the Hu1E7-CAR4 T cells secreted lower levels of cytokines (IFN-γ and TNF-α), which may be due to the lower binding affinity of Hu1E7-CAR4 T cells. These findings demonstrated the successfulness in creation of a new CAR T cells containing a novel fully human-derived scFv specific to CD19+ cancer cells. In vivo studies are needed to further compare the anti-tumor efficacy and safety of Hu1E7-CAR4 T cells and mFMC63-CAR4 T cells.

Gemcitabine enhances cytotoxic activity of effector T-lymphocytes against chemo-resistant cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) can resist chemotherapy resulting in treatment failure. Gemcitabine, a chemotherapeutic drug, can sensitize cancer cells to become susceptible to cytotoxic T-lymphocytes (CTLs). We, therefore, hypothesized that a combination of gemcitabine and CTLs would be more effective for CCA treatment than individual therapy. To test this hypothesis, we conducted an in vitro study using gemcitabine combined with CTLs to treat gemcitabine-resistant CCA (KKU-213) cells. KKU-213 cells were pretreated with gemcitabine and tested for killing by CTLs activated by dendritic cells that were prepared by three different methods, including: (i) monocyte-derived dendritic cells pulsed with cancer cell lysate (Mo-DC + Lys), (ii) self-differentiated dendritic cells pulsed with cancer-cell lysate (SD-DC + Lys), and (iii) SD-DC presenting tumor-associated antigen PRKAR1A (SD-DC-PR). KKU-213 cells pretreated with gemcitabine were killed by CTLs activated by either SD-DC + Lys or SD-DC-PR more efficiently than those activated by Mo-DC + Lys. Furthermore, KKU-213 cells pretreated with a low dose (2 µM) of gemcitabine significantly enhanced the cytotoxic activity of CTLs activated by either SD-DC + Lys or SD-DC-PR at all evaluated effector (E) to target cell (T) ratios. At an E:T ratio of 5:1, KKU-213 cells pretreated with gemcitabine enhanced the cytotoxic activity of CLTs by approximately 2.5-fold (greater than 50% cell death) compared to untreated condition. The upregulation of HLA class I upon pretreatment of KKU-213 cells with gemcitabine may suggest a mechanism that leads to alteration of the antigen presentation process to promote CTL functions. These findings support the concept of combination therapy for overcoming chemo-resistant CCA.

Inhibition of dengue virus replication in monocyte-derived dendritic cells by vivo-morpholino oligomers.

Skin dendritic cells (DCs) are primary target cells of dengue virus (DENV) infection and they play an important role in its immunopathogenesis. Monocyte-derived dendritic cells (MDDCs) represent dermal and bloodstream DCs that serve as human primary cells for ex vivo studies of DENV infection. Improved understanding of the mechanisms that effectuate the inhibition of DENV replication in MDDCs will accelerate the development of antiviral drugs to treat DENV infection. In this study, we investigated whether or not vivo-morpholino oligomer (vivo-MO), which was designed to target the top of the 3' stem-loop (3' SL) at the 3' UTR of the DENV genome, could inhibit DENV infection and replication in MDDCs. The findings of this study revealed that vivo-MO-1 could inhibit DENV-2 infection in MDDCs, and that it could significantly reduce DENV RNA, protein, and viral production in a dose-dependent manner. Treatment of MDDCs with 4 µM of vivo-MO-1 decreased DENV production by more than 1,000-fold, when compared to that of the vivo-MO-NC control. Thus, vivo-MO-1 targeting of DENV RNA demonstrates potential for further development into an anti-DENV agent.

Novel bioactive peptides demonstrating anti-dengue virus activity isolated from the Asian medicinal plant Acacia Catechu.

The therapeutic activities of food-derived bioactive proteins and peptides are attracting increased attention within the research community. Medicinal plants used in traditional medicines are an excellent source of bioactive proteins and peptides, especially those traditionally prepared by water extraction for use as tea or food supplement. In this study, novel bioactive peptides were isolated from enzymatic digests of 33 Thai medicinal plants. The inhibitory activity of each against dengue virus (DENV) infection was investigated. Of 33 plants, peptides from Acacia catechu extract demonstrated the most pronounced anti-DENV activity. Half maximal inhibitory concentration of 0.18 µg/ml effectively inhibited DENV foci formation. Treatment with 1.25 µg/ml crude peptide extract could reduce virus production less than 100-fold with no observable cell toxicity. Peptide sequences were determined by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry. Two bioactive peptides isolated from Acacia catechu inhibited DENV foci formation >90% at the concentration of 50 µM; therefore, they are recommended for further investigation as antiviral peptides against DENV infection.