Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most frequent of the keratinocyte-derived malignancies with actinic keratosis (AK) as a precancerous lesion. To comprehensively delineate the underlying mechanisms for the whole progression from normal skin to AK to invasive cSCC, we performed single-cell RNA sequencing (scRNA-seq) to acquire the transcriptomes of 138,982 cells from 13 samples of six patients including AK, squamous cell carcinoma in situ (SCCIS), cSCC, and their matched normal tissues, covering comprehensive clinical courses of cSCC. We identified diverse cell types, including important subtypes with different gene expression profiles and functions in major keratinocytes. In SCCIS, we discovered the malignant subtypes of basal cells with differential proliferative and migration potential. Differentially expressed genes (DEGs) analysis screened out multiple key driver genes including transcription factors along AK to cSCC progression. Immunohistochemistry (IHC)/immunofluorescence (IF) experiments and single-cell ATAC sequencing (scATAC-seq) data verified the expression changes of these genes. The functional experiments confirmed the important roles of these genes in regulating cell proliferation, apoptosis, migration, and invasion in cSCC tumor. Furthermore, we comprehensively described the tumor microenvironment (TME) landscape and potential keratinocyte-TME crosstalk in cSCC providing theoretical basis for immunotherapy. Together, our findings provide a valuable resource for deciphering the progression from AK to cSCC and identifying potential targets for anticancer treatment of cSCC.

Identification of key genes in cutaneous squamous cell carcinoma: a transcriptome sequencing and bioinformatics profiling study.

BACKGROUND: Long-term exposure to ultraviolet (UV) radiation can cause cutaneous squamous cell carcinoma (cSCC), which is one of the most common malignant cancers worldwide. Actinic keratosis (AK) is generally considered a precancerous lesion of cSCC. However, the pathogenesis and oncogenic processes of AK and cSCC remain elusive, especially in the context of photodamage. METHODS: In this study, transcriptome sequencing was performed on AK, cSCC, normal sun-exposed skin (NES) tissues, and normal non-sun-exposed skin (NNS) from 24 individuals. Bioinformatics analysis to identify the differentially expressed genes (DEGs) of 4 groups, and potential key genes of cSCC were validated by real-time quantitative reverse transcription PCR (qRT-PCR). RESULTS: A total of 46,930 genes were differentially expressed in the 4 groups, including 127 genes that were differentially expressed between NES and NNS, 420 DEGs in AK compared to NES, 1,658 DEGs in cSCC compared to NES, and 1,389 DEGs in cSCC compared to AK. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis suggested that the DEGs are involved in multiple pathways, including extracellular matrix (ECM)-receptor interaction, immune, inflammatory, microbial infection, and other related pathways. Finally, 5 new genes (HEPHL1, FBN2, SULF1, SULF2, and TCN1) were confirmed significantly upregulated in cSCC. CONCLUSIONS: Using transcriptome sequencing and integrated bioinformatical analysis, we have identified key DEGs and pathways in cSCC, which could improve our understanding of the cause and underlying molecular events of AK and cSCC. HEPHL1, FBN2, SULF1, SULF2, and TCN1 may be novel potential biomarkers and therapeutic targets of cSCC.

[Clinical Analysis of 153 Cases of Refractory/Relapsed Diffuse Large B-cell Lymphoma].

OBJECTIVE: To analyze the clinical features and treatment methods of refractory/relapsed diffuse large B cell lymphoma (DLBCL) patients, and to explore the curative effect and the main factors affecting prognosis. METHODS: Clinical data of 1043 cases of DLBCL in our hospital from January 2008 to April 2016 were retrospectively analyzed, then the clinical data of 153 patients with refractory/relapsed lymphoma were selected and analyzed for determing the relationship of the related factors with therapeutic effect and prognosis. Treatment methods include chemotherapy alone and chemotherapy combined with radio-therapy, the first line regimen was CHOP or R-CHOP program, the salvage regimens are ICE, Hyper CVAD or EPOCH, etc. The median age of these 153 patients was 50 years old, the ratio of male and female was 1.59:1. RESULTS: 4 cases were not treated in 153 patients, 6 cases (4.03%) of 149 patients have been treated and achieved complete remission(CR), 18 cases (12.08%) achieved partial remission(PR), and the total response rate was 16.1%. Single factor analysis showed that the patients' serum LDH values, IPI score, bulky, extra-node involvement, bone marrow infiltration and the salvage regimen all could influence the survival rate, with statistically significantce (P<0.05). CONCLUSION: Refractory/relapsed DLBCL mainly occurs in the middle-aged and male, the serum LDH value, IPI score, bulky and scope of lesions are mainly factors influencing the prognosis of refractory/relapsed DLBCL patients.

The application of mRNA-based gene transfer in mesenchymal stem cell-mediated cytotoxicity of glioma cells.

Since the tumor-oriented homing capacity of mesenchymal stem cells (MSCs) was discovered, MSCs have attracted great interest in the research field of cancer therapy mainly focused on their use as carries for anticancer agents. Differing from DNA-based vectors, the use of mRNA-based antituor gene delivery benefits from readily transfection and mutagenesis-free. However, it is essential to verify if mRNA transfection interferes with MSCs' tropism and their antitumor properties. TRAIL- and PTEN-mRNAs were synthesized and studied in an in vitro model of MSC-mediated indirect co-culture with DBTRG human glioma cells. The expression of TRAIL and PTEN in transfected MSCs was verified by immunoblotting analysis, and the migration ability of MSCs after anticancer gene transfection was demonstrated using transwell co-cultures. The viability of DBTRG cells was determined with bioluminescence, live/dead staining and real time cell analyzer. An in vivo model of DBTRG cell-derived xenografted tumors was used to verify the antitumor effects of TRAIL- and PTEN-engineered MSCs. With regard to the effect of mRNA transfection on MSCs' migration toward glioma cells, an enhanced migration rate was observed with MSCs transfected with all tested mRNAs compared to non-transfected MSCs (p<0.05). TRAIL- and PTEN-mRNA-induced cytotoxicity of DBTRG glioma cells was proportionally correlated with the ratio of conditioned medium from transfected MSCs. A synergistic action of TRAIL and PTEN was demonstrated in the current co-culture model. The immunoblotting analysis revealed the apoptotic nature of the cells death in the present study. The growth of the xenografted tumors was significantly inhibited by the application of MSCPTEN or MSCTRAIL/PTEN on day 14 and MSCTRAIL on day 28 (p<0.05). The results suggested that anticancer gene-bearing mRNAs synthesized in vitro are capable of being applied for MSC-mediated anticancer modality. This study provides an experimental base for further clinical anticancer studies using synthesized mRNAs.

PTEN-mRNA engineered mesenchymal stem cell-mediated cytotoxic effects on U251 glioma cells.

Mesenchymal stem cells (MSCs) have been considered to have potential as ideal carriers for the delivery of anticancer agents since the capacity for tumor-oriented migration and integration was identified. In contrast to DNA-based vectors, mRNA synthesized in vitro may be readily transfected and is mutagenesis-free. The present study was performed in order to investigate the effects of phosphatase and tensin homolog (PTEN) mRNA-engineered MSCs on human glioma U251 cells under indirect co-culture conditions. PTEN-bearing mRNA was generated by in vitro transcription and was transfected into MSCs. The expression of PTEN in transfected MSCs was detected by immunoblotting, and the migration ability of MSCs following PTEN-bearing mRNA transfection was verified using Transwell co-cultures. The indirect co-culture was used to determine the effects of PTEN-engineered MSCs on the viability of U251 glioma cells by luminescence and fluorescence microscopy. The synthesized PTEN mRNA was expressed in MSCs, and the expression was highest at 24 h subsequent to transfection. An enhanced migration rate was observed in MSCs transfected with PTEN mRNA compared with non-transfected MSCs (P<0.05). A significant inhibition of U251 cells was observed when the cells were cultured with conditioned medium from PTEN mRNA-engineered MSCs (P<0.05). The results suggested that anticancer gene-bearing mRNA synthesized in vitro is capable of being applied to a MSC-mediated anticancer strategy for the treatment of glioblastoma patients.

[Cytogenetic Abnormalities and Outcomes of 117 Patients with Multiple Myeloma Detected by FISH].

OBJECTIVE: To analyze the cytogenetic abnormalities and prognostic outcomes of patients with multiple myeloma (MM) detected by fluorescence in situ hybridization (FISH). METHODS: The clinical record of 117 newly-diagnosed patients with MM treated in department of hematology and geriatric hematology of our hospital for 7 years were collected, and their molecular cytogenetic abnormalities detected by FISH and the clinical outcome were analyzed retrospectively. RESULTS: The detected rate of cytogenetic abnormality was 76.9%(90/117), the most common abnormality deteted by FISH was 1q21+ (71.1%), followed by 13q- (56.6%). The cross comparison method showed that 13q- and 17p13-, t(11;14) and t(4;14) were related respectively. All the patients with cytogenetic abnormalities showed no significant difference in the overall survival from cytogenetic normal patients. CONCLUSION: The positive rate of molecular cytogenetic abnormalities detected by FISH in MM patients is high, but data from larger and longer studies are needed to evaluate the prognostic outcomes.

Therapeutic potential of CAR-T cell-derived exosomes: a cell-free modality for targeted cancer therapy.

Chimeric antigen receptor (CAR)-based T-cell adoptive immunotherapy is a distinctively promising therapy for cancer. The engineering of CARs into T cells provides T cells with tumor-targeting capabilities and intensifies their cytotoxic activity through stimulated cell expansion and enhanced cytokine production. As a novel and potent therapeutic modality, there exists some uncontrollable processes which are the potential sources of adverse events. As an extension of this impactful modality, CAR-T cell-derived exosomes may substitute CAR-T cells to act as ultimate attackers, thereby overcoming some limitations. Exosomes retain most characteristics of parent cells and play an essential role in intercellular communications via transmitting their cargo to recipient cells. The application of CAR-T cell-derived exosomes will make this cell-based therapy more clinically controllable as it also provides a cell-free platform to diversify anticancer mediators, which responds effectively to the complexity and volatility of cancer. It is believed that the appropriate application of both cellular and exosomal platforms will make this effective treatment more practicable.

PDX-1 mRNA-induced reprogramming of mouse pancreas-derived mesenchymal stem cells into insulin-producing cells in vitro.

Pancreatic islet transplantation has remained an effective therapy for type 1 diabetes since 2000. Its widespread use has been prohibited by the shortage of suitable donors. It is critical to explore an applicable alternative for ß-cell replacement. This study was performed to generate insulin-producing cells (IPCs) from pancreas-derived mesenchymal stem cells (pMSCs). pMSCs were isolated from discarded pancreatic tissue in the filter liquor during islet isolation procedure in mice and ex vivo expanded in culture. IPCs were induced by transfection of pancreas and duodenal transcription factor 1 (PDX-1) mRNA in vitro. Some islet characteristics were identified on pMSC-derived IPCs in mRNA and protein levels. Our results demonstrated that mouse pMSCs can be transdifferentiated into effective glucose-responsive insulin-producing cells through transfecting synthetic modified PDX-1 mRNA in vitro. The study of PDX-1 mRNA-induced pMSC reprogramming may pave the way toward the development of a novel ß-cell source for the treatment of diabetes.

Cancer cell-oriented migration of mesenchymal stem cells engineered with an anticancer gene (PTEN): an imaging demonstration.

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered to hold great potential as ideal carriers for the delivery of anticancer agents since the discovery of their tumor tropism. This study was performed to demonstrate the effects of phosphatase and tensin homolog (PTEN) engineering on MSCs' capacity for cancer cell-oriented migration. METHODS: MSCs were engineered with a PTEN-bearing plasmid and the expression was confirmed with Western blotting. A human glioma cell line (DBTRG) was used as the target cell; DBTRG cell-oriented migration of MSCs was monitored with a micro speed photographic system. RESULTS: The expression of transfected PTEN in MSCs was identified by immunoblotting analysis and confirmed with cell viability assessment of target cells. The DBTRG cell-oriented migration of PTEN-engineered MSCs was demonstrated by a real-time dynamic monitoring system, and a phagocytosis-like action of MSCs was also observed. CONCLUSION: MSCs maintained their capacity for cancer cell-directed migration after they were engineered with anticancer genes. This study provides the first direct evidence of MSCs' tropism post-anticancer gene engineering.