CAR T Cell Therapy: Remedies of Current Challenges in Design, Injection, Infiltration and Working.

Chimeric antigen receptor (CAR) T cell therapy, as an innovative immunotherapy, plays a huge role in current cancer therapy. Although CAR T cell therapy has demonstrated therapeutic effects in some subtypes of B cell leukemia or lymphoma, there are many challenges that limit the therapeutic efficacy of CAR T cells in solid tumors. And how to efficiently transport CAR T cells to tumor tissues is a continuing concern for us. In this review, experiments have been extensively studied and compared. We finally compared the influence of different injection methods on therapeutic efficacy. We also carefully explored the difficulties of designing, homing, and working of CAR T cells, and ultimately came up with better solutions for each process to help CAR T cells reach tumor tissue more efficiently and quickly. These results will have significant implications for guiding CAR T cell therapy in cancer treatment.

PKIB involved in the metastasis and survival of osteosarcoma.

Osteosarcoma is frequently metastasized at the time of diagnosis in patients. However, the underlying mechanism of osteosarcoma metastasis remains poorly understood. In this study, we evaluated DNA methylation profiles combined with gene expression profiles of 21 patients with metastatic osteosarcoma and 64 patients with non-metastatic osteosarcoma from TARGET database and identified PKIB and AIM2 as hub genes related to the metastasis of osteosarcoma. To verify the effects of PKIB on migration and invasion of osteosarcoma, we performed wound-healing assay and transwell assay. The results showed that PKIB significantly inhibited the migration and invasion of osteosarcoma cells, and the Western blot experiments showed that the protein level of E-cad was upregulated and of VIM was downregulated in 143-B cell recombinant expression PKIB. These results indicate that PKIB inhibit the metastasis of osteosarcoma. CCK-8 assay results showed that PKIB promote the proliferation of osteosarcoma. In addition, the Western blot results showed that the phosphorylation level of Akt was upregulated in 143-B cells overexpressing PKIB, indicating that PKIB promotes the proliferation of osteosarcoma probably through signaling pathway that Akt involved in. These results give us clues that PKIB was a potential target for osteosarcoma therapy. Furthermore, combined clinical profiles analysis showed that the expression of AIM2- and PKIB- related risk scores was significantly related to the overall survival of patients with osteosarcoma. Thus, we constructed a nomogram based on AIM2 and PKIB expression-related risk scores for osteosarcoma prognostic assessment to predict the 1-, 2-, 3-, and 5-year overall survival rate of patients with metastatic osteosarcoma, assisting clinicians in the diagnosis and treatment of metastatic osteosarcoma.

Clinical Strategies for Enhancing the Efficacy of CAR T-Cell Therapy for Hematological Malignancies.

Chimeric antigen receptor (CAR) T cells have been successfully used for hematological malignancies, especially for relapsed/refractory B-cell acute lymphoblastic leukemia and non-Hodgkin's lymphoma. Patients who have undergone conventional chemo-immunotherapy and have relapsed can achieve complete remission for several months with the infusion of CAR T-cells. However, side effects and short duration of response are still major barriers to further CAR T-cell therapy. To improve the efficacy, multiple targets, the discovery of new target antigens, and CAR T-cell optimization have been extensively studied. Nevertheless, the fact that the determination of the efficacy of CAR T-cell therapy is inseparable from the discussion of clinical application strategies has rarely been discussed. In this review, we will discuss some clinical application strategies, including lymphodepletion regimens, dosing strategies, combination treatment, and side effect management, which are closely related to augmenting and maximizing the efficacy of CAR T-cell therapy.

CAR T targets and microenvironmental barriers of osteosarcoma.

Osteosarcoma (OS) is one of the most common malignancies in children and adolescents. Multimodal chemotherapy and aggressive surgical resection have improved the prognosis of patients with osteosarcoma. However, the prognosis of OS patients with unresectable advanced tumors, distant metastasis or chemotherapy is still poor. Chimeric antigen receptor (CAR) T cells have achieved remarkable success in the treatment of hematologic malignancies, injecting new vitality into the field of adoptive cell therapy. However, the efficacy in solid tumors has been largely limited. The reason for the poor curative effect of solid tumors is mainly the heterogeneity of solid tumor antigen, immune escape, tumor microenvironment barrier, resistance of immunosuppressive cells and inhibitory factors, which lead to the obstruction of CAR T cell infiltration and the aggravation of failure. Potential antigenic targets for osteosarcoma CAR T cell therapy are under continuous exploration. Some of the antigenic targets, such as anti-HER2-CAR T cells, have achieved good results in preclinical studies, and some of them have entered clinical studies and achieved certain clinical effects. In this review, we discuss the research progress of potential antigen targets and osteosarcoma microenvironment of CAR T cells in the treatment of osteosarcoma.

The role of non­coding RNAs in the regulation, diagnosis, prognosis and treatment of osteosarcoma (Review).

Osteosarcoma (OS) is the most common primary bone tumor worldwide. OS exhibits a range of aggressive behaviors, including early metastasis potential, rapid progression, poor clinical prognosis and insensitivity to chemoradiotherapy. Non­coding RNAs are transcripts that do not encode proteins. A significant number of studies published on OS have been focused on the aberrant expression of non­coding RNAs and their involvement in tumor initiation and progression. It has been confirmed that non­coding RNAs exert their regulatory functions at both the transcriptional and post­transcriptional level, which leads to tumor initiation or progression in OS. According to present knowledge, this review provides a state­of­the­art overview of the functions and mechanisms of microRNAs, long non­coding RNAs and circular RNAs in terms of their involvement with OS. The review also covers their potential clinical application in the diagnosis, prognosis and treatment of OS. It is hoped that the information presented in this review on the involvement of non­coding RNAs in OS will lead to a more comprehensive understanding of OS and provide a useful perspective on the potential diagnostic and therapeutic applications of non­coding RNAs for patients with OS.

Consequences of Mutations and Abnormal Expression of SMAD4 in Tumors and T Cells.

SMAD4 is a typical tumor suppressor in the TGF-ß signaling pathway. In human cancers, SMAD4 is frequently mutated and inactivated. In recent years, the consequences of mutations and inactivation of SMAD4 are gradually becoming clearer. Most of the mutations have negative consequences and reduce the chances of survival of their carriers. Loss of SMAD4 functions due to mutations or abnormal expression can suppress the inhibition of tumor growth and support the tumor progression. Functions of SMAD4 and its variants in T cells are being studied extensively, to better understand the SMAD4 functions in T cells. In this review, we mainly discuss the recently reported consequences of mutations and abnormal expression of SMAD4 in tumors, and the effects of loss, deficiency or mutation of SMAD4 and its T cells, to show the use of SMAD4 mutations in cancer diagnosis and therapeutic strategies.

Mechanisms linking mitochondrial mechanotransduction and chondrocyte biology in the pathogenesis of osteoarthritis.

Mechanical loading is essential for chondrocyte health. Chondrocytes can sense and respond to various extracellular mechanical signals through an integrated set of mechanisms. Recently, it has been found that mitochondria, acting as critical mechanotransducers, are at the intersection between extracellular mechanical signals and chondrocyte biology. Much attention has been focused on identifying how mechanical loading-induced mitochondrial dysfunction contributes to the pathogenesis of osteoarthritis. In contrast, little is known regarding the mechanisms underlying functional alterations in mitochondria induced by mechanical stimulation. In this review, we describe how chondrocytes perceive environmental mechanical signals. We discuss how mechanical load induces mitochondrial functional alterations and highlight the major unanswered questions in this field. We speculate that AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis, may play an important role in coupling force transmission to mitochondrial health and intracellular biological responses.

Novel Alternatively Spliced Variants of Smad4 Expressed in TGF-ß-Induced EMT Regulating Proliferation and Migration of A549 Cells.

INTRODUCTION: Non-small cell lung cancer (NSCLC) is a worldwide malignance threatening human life. TGF-ß/Smad signaling is known to regulate cell proliferation, differentiation, migration and growth. As the only co-Smad playing crucial roles in TGF-ß signaling, Smad4 is reported to be frequently mutated or to occur as alternatively spliced in tumor cells. Smad4 was reported to be involved in the TGF-ß-induced EMT process. However, whether the alternative splicing occurs in the TGF-ß-induced EMT process in NSCLC was not clear. METHODS: In our current study, we explored the alternative splicing of Smad4 during the process of TGF-ß-induced EMT in A549 cells. 10 ng/mL TGF-ß was used to induce EMT. Then, nest-PCR and agarose electrophoresis were performed to detect the expression of Smad4 variants and sequencing to get the variant DNA sequences. For recombinant expression of variants of Smad4 in A549 cells, we used lentiviral variants to infect cells. In order to explore the effects of variants on the proliferation and migration of A549 cells, the MTT assay, colony formation assay and wound-healing assay were done. The effects of variants on E-cad and VIM protein expression were explored through Western blot. RESULTS: There were several novel gene fragments expressed in TGF-ß-induced A549 cells, and the sequencing results showed that they were indeed the Smad4 variants that were not reported. For recombinant expression of Smad4 variants in A549 cells, we found that they have significant effects on the proliferation and migration of cells, and also regulated the E-cad and VIM protein expression. CONCLUSION: Our results indicated that novel Smad4 variants were expressed in TGF-ß-induced EMT process. The functional study showed that these novel variants regulate cell proliferation and migration and affect E-cad and VIM protein expression, showing the potential as targets for cancer therapy.

Biophysical basis underlying dynamic Lck activation visualized by ZapLck FRET biosensor.

Lck plays crucial roles in TCR signaling. We developed a new and sensitive FRET biosensor (ZapLck) to visualize Lck kinase activity with high spatiotemporal resolutions in live cells. ZapLck revealed that 62% of Lck signal was preactivated in T-cells. In Lck-deficient JCam T-cells, Lck preactivation was abolished, which can be restored to 51% by reconstitution with wild-type Lck (LckWT) but not a putatively inactive mutant LckY394F. LckWT also showed a stronger basal Lck-Lck interaction and a slower diffusion rate than LckY394F. Interestingly, aggregation of TCR receptors by antibodies in JCam cells led to a strong activation of reconstituted LckY394F similar to LckWT. Both activated LckY394F and LckWT diffused more slowly and displayed increased Lck-Lck interaction at a similar level. Therefore, these results suggest that a phosphorylatable Y394 is necessary for the basal-level interaction and preactivation of LckWT, while antibody-induced TCR aggregation can trigger the full activation of LckY394F.

Sensitive FRET Biosensor Reveals Fyn Kinase Regulation by Submembrane Localization.

Fyn kinase plays crucial roles in hematology and T cell signaling; however, there are currently limited tools to visualize the dynamic Fyn activity in live cells. Here we developed and characterized a highly sensitive Fyn biosensor based on fluorescence resonance energy transfer (FRET) to monitor Fyn kinase activity in live cells. Our results show that Fyn kinase activity can be induced in both mouse embryonic fibroblasts (MEFs) and T cells by ligand engagement. Two different motifs were further introduced to target the biosensor at the cellular membrane microdomains in MEFs, revealing that the Fyn-tagged biosensor had 70% greater response to growth factor stimulation than the Lyn-tagged version. This suggests that the plasma membrane microdomains can be categorized into different functional subdomains. Further experiments show that while the membrane accessibility is necessary for Fyn activation, the localization of Fyn outside of its microdomains causes its hyperactivity, indicating that membrane microdomains provide a suppressive microenvironment for Fyn regulation in MEFs. Interestingly, a relatively high Fyn activity can be observed at perinuclear regions, further supporting the notion that the membrane microenvironment has a significant impact on the local molecular functions. Our work hence highlights a novel Fyn FRET biosensor for live cell imaging and its application in revealing an intricate submembrane regulation of Fyn in live MEFs.

Alternative Splicing of SMAD4 and Its Function in HaCaT Cells in Response to UVB Irradiation.

Alternative splicing is one of the most common mechanisms of human gene regulation and plays a crucial role in increasing the diversity of functional proteins. Many diseases are linked to alternative splicing, especially cancer. SMAD4 is a member of the SMAD family and plays a critical role in mediating of TGF-ß signal transduction and gene regulatory events. Smad4 is a tumour suppressor and acts as a shuttling protein between nucleus and cytoplasm. The splicing variants of Smad4 have been found in many cancers. The present study performed nested PCR to detect alternative splicing of Smad4 in HaCaT cells lines in response to UVB irradiation. The UVB induced a novel Smad4B isoform that led to decrease the Smad4 expression. The hnRNPA1 splicing factor is responsible for Smad4 alternative splicing in response to UVB. The UVB increased the expression of SF2 and hnRNPA1 Splicing factors. The hnRNPA1 overexpression induced Smad4B by regulating Smad4 alternative splicing. The Smad4B isoform supported the function of Smad4 full length in UVB resistance with certain limitation. The western blot analyses showed that the overexpressed Smad4 full length significantly increased N-cadherin expression while Smad4B overexpression decreased the expression the N-cadherin (P<0.05). Furthermore, overexpression of the isoform in HaCaT cells decreased cell invasion as compared to Smad4 full-length overexpression. These results will be helpful to understand the importance of Smad4 alternative splicing in skin tumorigenesis.

The structure-function relationships of insulin-like growth factor 1 Ec in C2C12 cells.

Insulin-like growth factor 1 (IGF1) is a crucial growth factor, that regulates skeletal muscles development during cell growth and repair. Recently, its alternative splicing variant, named IGF1Ec, also named mechano-growth factor (MGF), has gained attentions as a new damage repair factor. However, the structure-function relationships of IGF1Ec have not been fully clarified due to contradictory reports. In this study, we systematically investigated physiologic responses of C2C12 muscle cells to IGF1Ec, IGF1 and MGF E peptide. Our data indicate that while the N-terminal sequence of IGF1Ec, which is homolog in part with IGF1, promotes proliferation; the C-terminal sequence of IGF1Ec, which is identical to MGF E, promotes differentiation and migration of C2C12 cells. Our results suggest that MGF E cannot completely replace all the functions of IGF1Ec on muscle repair and regeneration, and elucidate the relationships between structure and function of IGF1Ec.

CRIM1, a newfound cancer-related player, regulates the adhesion and migration of lung cancer cells.

CRIM1 is a member of the bone morphogenetic protein (BMP) antagonists; however, the role of CRIM1 in controlling cancer cell behavior remains unknown. This study investigated its function in the A549 cell line in vitro. The results show that treating cells with CRIM1 peptide could increase the migration and adhesion of A549. Consistently, silencing the CRIM1 expression decreased the migration and adhesion of A549. Furthermore, the CRIM1 protein expression was increased in A549 which were treated with transforming growth factor beta 1 to induced EMT. However, CRIM1 peptide treatment could increase the expression of N-CAD and E-CAD expression. Finally, overexpression of the oncogene YAP1 resulted in an up-regulation of the CRIM1 expression in A549, suggesting that CRIM1 was a target of the Hippo pathway. These observations provide evidence for the first time that CRIM1 plays a role in cancer cells by enhancing the migration and adhesion and increasing the expression of N-CAD and E-CAD.

Examination of alternate codon bias solutions for expression and purification of recombinant mechano-growth factor in Escherichia coli.

Mechano-growth factor (MGF), an alternative splicing variant of insulin-like growth factor-1 (IGF-1) gene, promotes cell proliferation and inhibits cell differentiation. It also plays an important role in tumor development. It is important to optimize the production process and achieve MGF protein because there is no commercial MGF protein available. In this study, the human MGF gene is cloned into pGEX-4T-1 and the recombinant human MGF (rhMGF) protein could be expressed in Rosetta (DE3) by isopropyl ß-D-1-thiogalactopyranoside induction but not in BL21 (DE3). Mutation from rare codons to Escherichia coli preferred ones is performed. We obtain MGF(Mut54-56) and MGF(Mut-total) fragments through site-directed mutagenesis and overlapping PCR. Both pGEX-4T-1/MGF(Mut54-56)- and pGEX-4T-1/MGF(Mut-total)-transformed BL21 (DE3) can be induced to express rhMGF protein. To optimize the production technology, expression and purification of rhMGF are analyzed and compared in Rosetta (DE3) and BL21 (DE3). Results indicate that rhMGF expression in BL21 (DE3) is significantly higher than that in Rosetta (DE3). The protein yield of pGEX-4T-1/MGF(Mut-total) in BL21 (DE3) is higher than that of pGEX-4T-1/MGF(Mut54-56). We test the biological activity of MGF protein purified by affinity chromatography in C2C12 cell line and find that rhMGF promotes cell proliferation significantly. In conclusion, we establish a method to produce rhMGF economically with high biological activity in BL21 (DE3).