MHC-UNRESTRICTED MUC1-SPECIFIC T CELL RECEPTOR FOR CANCER IMMUNOTHERAPY/GENE THERAPY

MUC1 glycoprotein is overexpressed on the surface of a variety of epithelial tumors and has been under investigation as a target for immunotherapy. A number of cytotoxic lymphocyte clones were generated in our laboratory from breast and pancreatic cancer patients that recognized MUC1 on the surface of tumor cells in a TCR-mediated MHC-unrestricted manner. The purpose of this study was to test the feasibility, efficacy and safety of using MHC-unrestricted MUC1-specific T cell receptor (TCR) gene transfer as a tool for cancer immunotherapy. The TCR £ and £] chains were cloned from one MHC-unrestricted MUC1-specific CTL clone (MA). Various configurations of chimeric TCRs were constructed and were expressed on the surface of a variety of cell lines in vitro. The TCR-deficient T cell line, Jurkat JRT3.5, transfected with the TCR £ and £] chains from MA CTL clone fluxed calcium in response to stimulation by a MUC1+ pancreatic human tumor, HPAF. BWZ murine thymoma cells transfected with a single-chain TCR (scTCR) consisting of the TCR extracellular domain and the CD3 ƒê signaling domain) were triggered to secrete IL-2 in response to stimulation with different MUC1+ tumor cells. The tumor recognition and rejection functions of this scTCR were tested in vivo when SCID mice were reconstituted with bone marrow (BM) cells transduced with scTCR-MFG retroviral supernatant and challenged with HPAF tumor cells. Tumor growth in mice reconstituted with scTCR-transduced BM cells was significantly slower (P<0.05) than that seen in the control group. Tumor sections from TCR-reconstituted mice were infiltrated by neutrophils and macrophages, and to lesser extent, by NK cells. FACS analyses showed that BM cells transduced with scTCR-MFG could differentiate in vivo into multiple immune lineages including T cells, B cells, granulocytes, monocytes and NK cells that express the scTCR. The scTCR was expressed on higher percentages of cells of the innate immune system when compared to T and B cells. Human MUC1 transgenic (Tg.) mice reconstituted with BM cells transduced with this MUC1-specific TCR did not show any signs of autoimmunity, abnormal cellular infiltration or destruction of MUC1-expressing tissues. Transduction of BM with tumor-specific TCR represents a potentially efficacious gene therapy/immunotherapy approach. MUC1-specific MHC-unrestricted TCR will make this treatment applicable to all cancer patients with MUC1+ tumors, regardless of their HLA type

Cloning and Expression of Human Membrane-Bound and Soluble Engineered T Cell Receptors for Immunotherapy

We report here the design and construction of several gene vectors for expression in mammalian cells of membrane-bound and soluble human T cell receptors (TR). We designed a vector (TR-ALPHA-IRES-TR-BETA pEF4) that encodes high-level expression of the full-length TR on the surface of T cells. Furthermore, we engineered TR that does not require the presence of endogenous CD3 molecules for surface expression and thus expression is not limited to T cells. We also constructed a vector encoding a single-chain TR (scTR) as a fusion protein of V-ALPHA-V-BETA-C-BETA with CD3Z. Since it is encoded and expressed as a single molecule, this scTR is well suited for gene therapy. Lastly, we successfully used a mammalian expression vector for generation of soluble human TR. The approaches we used here for manipulation of a human tumor-specific TR can be useful for other investigators interested in TR-based immunotherapy

CDH1 and IL1-beta expression dictates FAK and MAPKK-dependent cross-talk between cancer cells and human mesenchymal stem cells

Is Figure S1 showing transfer of cellular components between hMSCs and cancer cells. (DOCX 214 kb

In vitro differentiation of human skin-derived multipotent stromal cells into putative endothelial-like cells

<p>Abstract</p> <p>Background</p> <p>Multipotent stem cells have been successfully isolated from various tissues and are currently utilized for tissue-engineering and cell-based therapies. Among the many sources, skin has recently emerged as an attractive source for multipotent cells because of its abundance. Recent literature showed that skin stromal cells (SSCs) possess mesoderm lineage differentiation potential; however, the endothelial differentiation and angiogenic potential of SSC remains elusive. In our study, SSCs were isolated from human neonatal foreskin (hNFSSCs) and adult dermal skin (hADSSCs) using explants cultures and were compared with bone marrow (hMSC-TERT) and adipose tissue-derived mesenchymal stem cells (hADMSCs) for their potential differentiation into osteoblasts, adipocytes, and endothelial cells.</p> <p>Results</p> <p>Concordant with previous studies, both MSCs and SSCs showed similar morphology, surface protein expression, and were able to differentiate into osteoblasts and adipocytes. Using an endothelial induction culture system combined with an in vitro matrigel angiogenesis assay, hNFSSCs and hADSSCs exhibited the highest tube-forming capability, which was similar to those formed by human umbilical vein endothelial cells (HUVEC), with hNFSSCs forming the most tightly packed, longest, and largest diameter tubules among the three cell types. CD146 was highly expressed on hNFSSCs and HUVEC followed by hADSSCs, and hMSC-TERT, while its expression was almost absent on hADMSCs. Similarly, higher vascular density (based on the expression of CD31, CD34, vWF, CD146 and SMA) was observed in neonatal skin, followed by adult dermal skin and adipose tissue. Thus, our preliminary data indicated a plausible relationship between vascular densities, and the expression of CD146 on multipotent cells derived from those tissues.</p> <p>Conclusions</p> <p>Our data is the first to demonstrate that human dermal skin stromal cells can be differentiated into endothelial lineage. Hence, SSCs represents a novel source of stem/stromal cells for tissue regeneration and the vascularization of engineered tissues. Moreover, the CD146 investigations suggested that the microenvironmental niche might contribute to direct stromal cells multipotency toward certain lineages, which warrants further investigation.</p

Bone morphogenetic protein 2 (BMP2) induces growth suppression and enhances chemosensitivity of human colon cancer cells

Additional file 5: Figure S4. BMP2 expression in CRC cell lines from the Cancer Cell Line Encyclopedia. Data are presented as mRNA expression (Log2)

Gene expression data analysis identifies multiple deregulated pathways in patients with asthma

©2018 The Author(s).Peer reviewedPublisher PD

CXCR7 signaling promotes breast cancer survival in response to mesenchymal stromal stem cell-derived factors

Abstract The interaction between cancer cells and molecular cues provided by tumor stromal cells plays a crucial role in cancer growth and progression. We have recently reported that the outcome of interaction between tumor cells and stromal cells is dependent on the gene expression signature of tumor cells. In the current study, we observed that several cancer cell lines, e.g., MCF7 breast cancer line, exhibited growth advantage when cultured in the presence of conditioned media (CM) derived from human bone marrow stromal stem cells (hBMSCs). Regarding the underlying molecular mechanism, we have identified CXCR7 as highly expressed by MCF7 cells and that it mediated the enhanced growth in response to hBMSC CM. Regarding the clinical relevance, we found an inverse correlation between the level of tumor gene expression of CXCR7 in bladder, breast, cervical, kidney, liver, lung, pancreatic, stomach, and uterine cancers, and patients’ overall survival. Interestingly, significant positive correlation between CXCR7 and CXCL12 gene expression (Pearson = 0.3, p = 2.0 × 10–16) was observed in breast cancer patients, suggesting a biological role for the CXCR7/CXCL12 genetic circuit in breast cancer biology. Our data provide insight into the molecular mechanisms by which stromal-derived microenvironmental cues mediate CXCR7 signaling and growth enhancement of breast cancer cells. Therapeutic targeting of this circuit might provide novel therapeutic opportunity for breast cancer

Genome-wide mRNA and miRNA expression profiling reveal multiple regulatory networks in colorectal cancer

Despite recent advances in cancer management, colorectal cancer (CRC) remains the third most common cancer and a major health-care problem worldwide. MicroRNAs have recently emerged as key regulators of cancer development and progression by targeting multiple cancer-related genes; however, such regulatory networks are not well characterized in CRC. Thus, the aim of this study was to perform global messenger RNA (mRNA) and microRNA expression profiling in the same CRC samples and adjacent normal tissues and to identify potential miRNA-mRNA regulatory networks. Our data revealed 1273 significantly upregulated and 1902 downregulated genes in CRC. Pathway analysis revealed significant enrichment in cell cycle, integrated cancer, Wnt (wingless-type MMTV integration site family member), matrix metalloproteinase, and TGF-β pathways in CRC. Pharmacological inhibition of Wnt (using XAV939 or IWP-2) or TGF-β (using SB-431542) pathways led to dose- and time-dependent inhibition of CRC cell growth. Similarly, our data revealed up- (42) and downregulated (61) microRNAs in the same matched samples. Using target prediction and bioinformatics, ~77% of the upregulated genes were predicted to be targeted by microRNAs found to be downregulated in CRC. We subsequently focused on EZH2 (enhancer of zeste homolog 2 ), which was found to be regulated by hsa-miR-26a-5p and several members of the let-7 (lethal-7) family in CRC. Significant inverse correlation between EZH2 and hsa-miR-26a-5p (R(2)=0.56, P=0.0001) and hsa-let-7b-5p (R(2)=0.19, P=0.02) expression was observed in the same samples, corroborating the belief of EZH2 being a bona fide target for these two miRNAs in CRC. Pharmacological inhibition of EZH2 led to significant reduction in trimethylated histone H3 on lysine 27 (H3K27) methylation, marked reduction in cell proliferation, and migration in vitro. Concordantly, small interfering RNA-mediated knockdown of EZH2 led to similar effects on CRC cell growth in vitro. Therefore, our data have revealed several hundred potential miRNA-mRNA regulatory networks in CRC and suggest targeting relevant networks as potential therapeutic strategy for CRC