The Vascular Disrupting Agent CA4P Improves the Antitumor Efficacy of CAR-T Cells in Preclinical Models of Solid Human Tumors.

Chimeric antigen receptor (CAR) T cell therapy remains relatively ineffective against solid tumors due to inadequate infiltration and in vivo expansion of CAR-T cells. Unlike hematological malignancies, solid tumors have vascular barriers that hinder CAR-T cells from reaching the tumor site. Here, we demonstrated that combretastatin A-4 phosphate (CA4P), a vascular disrupting agent (VDA), can significantly improve the infiltration ability of CAR-T cells in solid tumors as evidenced by elevated levels of IFN-γ. Moreover, combined treatment with CA4P and CAR-T cells greatly increased the therapeutic efficiency of the CAR-T cells in subcutaneous ovarian cancer mouse xenograft models and patient-derived xenograft (PDX) models of colon and ovarian carcinoma. Our findings highlight CA4P as an effective antitumor agent candidate for combination with CAR-T cells in clinical applications to treat solid tumors.

Chimeric Antigen Receptor-modified T Cells Repressed Solid Tumors and Their Relapse in an Established Patient-derived Colon Carcinoma Xenograft Model.

Adoptive transfer of T cells engineered with a chimeric antigen receptor (CAR) is deemed as the silver bullet to overcome the barriers of solid tumor treatment; however, the therapeutic application against solid tumors faces major challenges largely owing to the complex heterogeneity and immunosuppressive microenvironment of solid tumors. Preclinical development of CAR-T-cell products necessitates an appropriate animal model for the evaluation and improvement of their therapeutic capacities. Patient-derived xenograft (PDX) resembles real patients in several ways, and may serve as an attractive alternative to generate and evaluate the efficacy of CAR-T-cell products. In this study, we established and characterized a PDX mouse model implanted with colorectal cancer (CRC) xenograft. Human epidermal growth factor receptor 2 (HER2) expression in CRC specimens was detected by immunohistochemistry. The fragments of patient tumors were subcutaneously implanted into immunodeficient NOD-NPG mice after surgery. Furthermore, HER2-specific CAR-T cells were engineered and tested in our model to show their effectiveness in tumor clearance. Adoptive transfer of HER2-specific CAR-T cells resulted in the regression or even elimination of CRC xenograft and protection of relapse from rechallenged colon cancer tissue in PDX model. Significant survival advantage was achieved in these mice as compared with those transplanted with green fluorescent protein-T cells. Thus, this study showed that CAR-T-cell treatment may be a promising approach for solid tumor clearance and that the PDX model may be useful to evaluate the effects of CAR-T cells.

Transgenic chimera quail production by microinjecting lentiviral vector into the blood vessel of the early embryo.

In the past, several strategies have been used to generate transgenic birds. The most successful method has proven to be injection of lentiviral vector into the subgerminal cavity of the newly laid egg. In this study, we directly injected lentiviral vector into the blood vessel of HH13-15 quail embryos to produce transgenic chimeras. In the manipulated, hatched birds, the green fluorescent protein (GFP) gene driven by a cytomegalovirus (CMV) promoter was extensively expressed. All tissues analyzed were GFP-positive, and gonad cells from some of the manipulated embryos expressed GFP. The semen genome of 21.4% of mature male birds was determined to be GFP-positive by PCR, indicating these male birds were transgenic chimeras.

Contribution of blastoderm cells to Japanese quail (Coturnix coturnix japonica)-Peking duck (Anas platyrhynchos) chimeras.

The purpose of this study was to produce quail-duck chimeras by transferring stage X blastoderm cells and to detect the distribution of donor cells in heterogeneous embryos using PCR. Four experimental groups were made by transferring different amounts of quail blastoderm cells into duck recipients. In early embryonic stages, donor cells labeled with PKH26 fluorescent dye were observed in the head, neural tube and gonads by fluorescent microscopy. A total of 194 duck recipient embryos were injected and 93 survived to hatch. The average hatching rate was 48% (93/194); the hatching rate showed a significant difference among all the groups (P < 0.05). Sixteen somatic chimeras were obtained, 10 of which had black feathers derived from the donor quail. The PCR results showed that donor cells were distributed in various tissues and organs of the phenotypic chimeras. This is the first report on producing Japanese quail-Peking duck chimeras by transferring quail blastoderm cells into the subgerminal cavity of the duck. This technique will provide a basis for the investigation of fertilization barriers in interspecies germline chimeras and will aid conservation of endangered wild birds.

Ontogeny of ghrelin mRNA expression and identification of ghrelin-immunopositive cells in the gastrointestinal tract of the Peking duck, Anas platyrhynchos.

Ghrelin is an acylated peptide and an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), and stimulates growth hormone release and food intake in mammals. Peking duck is a very fast growing species of poultry. Although the sequence and structure of ghrelin have recently been determined, the expression of ghrelin in Peking duck has not been studied. Here, we investigated the tissue expression and distribution of ghrelin by RT-PCR and immunohistochemistry, respectively, in Peking duck at different stages of development. Ghrelin mRNA expression was mainly detected in the proventriculus and proventriculus-gizzard junction. It was first expressed, but weakly, on embryonic day 14 (E14); the expression increased by embryonic day 21 (E21), and was maintained at high levels between post-hatching-day 1 (P1) and post-hatching-day 60 (P60). Weak expression of ghrelin mRNA was also found in the gizzard and duodenum. In the gastrointestinal tract of growing Peking duck in P60, the largest number of ghrelin-ip cells was detected in the epithelium of the compound tubular glands in the proventriculus and the next largest number was in the proventriculus-gizzard junction. Very few ghrelin-ip cells were located in the epithelium of the simple tubular glands adjacent to the gizzard. No ghrelin-ip cells were observed elsewhere in the gastrointestinal tract. Ghrelin-ip cells were found in embryos as early as day E21; at the same time, the compound tubular glands in the proventriculus had formed. The numbers of ghrelin-ip cells on P1 were similar to those of E21 embryos. However, on P60, high numbers of strongly stained ghrelin-ip cells were found to be scattered in the epithelium of the compound tubular glands in the proventriculus. The density of ghrelin-ip cells (cells/mm(2)) in the proventriculus on P60 was significantly greater than those of P1 and E21 embryos. These results demonstrate that ghrelin is expressed in the Peking duck gastrointestinal tract, especially in the proventriculus, from mid-late-stage embryos to growing period and suggested an involvement of ghrelin in the development and biology of the gastrointestinal tract of the Peking duck.