Anti-KRT6A monoclonal antibody, clone MM131

Introduction: Reconstruction of respiratory epithelium is critical for the fabrication of bioengineered airway implants. Epithelial differentiation is typically achieved using bovine pituitary extract (BPE). Due to the xenogenic nature and undefined composition of BPE, an alternative for human clinical applications, devoid of BPE, must be developed. The goal of this study was to develop two different BPE-free media, with and without select pituitary hormone (PH), which could initiate epithelial differentiation for use in human implantation. Methods: The ability of the two BPE-free media to initiate epithelial differentiation of adherent, non-expanded stromal-vascular cells grown on porcine small intestinal submucosa was compared to traditional BPE-containing media (M1). Nanostring (R) was used to measure differences in gene expression of sternness (MSC), basal cell (basal), and ciliated markers (muco-cil), and staining was performed support the gene data. Results: Compared to baseline, both BPE-free media upregulated epithelial and sternness genes, however this was to a lower degree than BPE-containing media. In general, the expression of basal cell markers (COL17A1, DSG3, ITGA6, KRT6A, LOXL2) and secreted mucous proteins (PLUNC, MUC5B, SCGB2A1) was upregulated. The gene expression of ciliated markers C9orf24, TUBA3 and DNCI2B but not of the key transcription factor for cilagenesis FOXJ1 were upregulated, indicating that mucus-secreting cell differentiation occurs more rapidly than ciliogenesis. The ability of the adherent stromal vascular cells to upregulate gene expression of both epithelial and sternness markers suggests maintenance of the self-renewal capacity of undifferentiated and/or basal cell-like cells contributing to proliferation and ensuring a persisting source of cells for regenerative medicine applications. Conclusion: This study provides the initial step to defining a BPE-free epithelial differentiation medium for clinical translation. Thus, either of the proposed BPE-free medium are viable alternatives to BPE-containing medium for partial epithelial differentiation for human translational applications. (C) 2018 Published by Elsevier Inc.

Background: Compelling evidence shows that progestins regulate breast cancer growth. Using preclinical models, we demonstrated that antiprogestins are inhibitory when the level of progesterone receptor isoform A (PR-A) is higher than that of isoform B (PR-B) and that they might stimulate growth when PR-B is predominant. The aims of this study were to investigate ex vivo responses to mifepristone (MFP) in breast carcinomas with different PR isoform ratios and to examine their clinical and molecular characteristics. Methods: We performed human breast cancer tissue culture assays (n=36) to evaluate the effect of MFP on cell proliferation. PR isoform expression was determined by immunoblotting (n=282). Tumors were categorized as PRA-H (PR-A/PR-B >= 1.2) or PRB-H (PR-A/PR-B <= 0.83). RNA was extracted for Ribo-Zero-Seq sequencing to evaluate differentially expressed genes. Subtypes and risk scores were predicted using the PAM50 gene set, the data analyzed using The Cancer Genome Atlas RNA-seq gene analysis and other publicly available gene expression data. Tissue microarrays were performed using paraffin-embedded tissues (PRA-H n=53, PRB-H n=24), and protein expression analyzed by immunohistochemistry. All statistical tests were two-sided. Results: One hundred sixteen out of 222 (52.3%) PR+ tumors were PRA-H, and 64 (28.8%) PRB-H. Cell proliferation was inhibited by MFP in 19 of 19 tissue cultures from PRA-H tumors. A total of 139 transcripts related to proliferative pathways were differentially expressed in nine PRA-H and seven PRB-H tumors. PRB-H and PRA-H tumors were either luminal B or A phenotypes, respectively (P=.03). PRB-H cases were associated with shorter relapse-free survival (hazard ratio [HR]=2.70, 95% confidence interval [CI]=1.71 to 6.20, P=.02) and distant metastasis-free survival (HR=4.17, 95% CI=2.18 to 7.97, P<.001). PRB-H tumors showed increased tumor size (P<.001), Ki-67 levels (P<.001), human epidermal growth factor receptor 2 expression (P=.04), high grades (P=.03), and decreased total PR (P=.004) compared with PRA-H tumors. MUC-2 (P<.001) and KRT6A (P=.02) were also overexpressed in PRB-H tumors. Conclusion: The PRA/PRB ratio is a prognostic and predictive factor for antiprogestin responsiveness in breast cancer.