The mechanism of tumor immune escape and progression in colorectal cancer (CRC) is widely investigated in-vitro to help understand and identify agents that might play a crucial role in response to treatment and improve the overall survival of CRC patients. Several mechanisms of immune escape and tumor progression, including expression of stemness markers, inactivation of immunoregulatory genes by methylation, and epigenetic silencing, have been reported in CRC, indicating the potential of demethylating agents as anti-cancer drugs. Of these, a chemotherapeutic demethylating agent, Decitabine (DAC), has been reported to induce a dual effect on both DNA demethylation and histone changes leading to an increased expression of target biomarkers, thus making it an attractive anti-tumorigenic drug.
We compared the effect of DAC in primary 1076 Col and metastatic 1872 Col cell lines isolated and generated from patients' tumor tissues. Both cell lines were treated with DAC, and the expression of the NY-ESO-1 cancer-testis antigen, the PD-L1 immunoinhibitory marker, and the CD44, Nanog, KLF-4, CD133, MSI-1 stemness markers were analyzed using different molecular and immunological assays.
DAC treatment significantly upregulated stemness markers in both primary 1076 Col and meta-static 1872 Col cell lines, although a lower effect occurred on the latter: CD44 (7.85 fold; ***p = 0.0001 vs. (4.19 fold; *p = 0.0120), Nanog (4.1 fold; ***p < 0.0001 vs.1.69 fold; ***p = 0.0008), KLF-4 (4.33 fold; ***p < 0.0001 vs.2.48 fold; ***p = 0.0005), CD133 (16.77 fold; ***p = 0.0003 vs.6.36 fold; *p = 0.0166), and MSI-1 (2.33 fold; ***p = 0.0003 vs.2.3 fold; ***p = 0.0004), respectively. Interestingly, in the metastatic 1872 Col cells treated with DAC, the expression of both PD-L1 and NY-ESO-1 was increased tenfold (*p = 0.0128) and fivefold (***p < 0.0001), respectively.
We conclude that the upregulation of both stemness and immune checkpoint markers by DAC treatment on CRC cells might represent a mechanism of immune evasion. In addition, induction of NY-ESO-1 may represent an immuno-therapeutic option in metastatic CRC patients. Finally, the combination of DAC and anti-PD-1/anti-PD-L1 antibodies treatment should represent a potential therapeutic intervention for this group of patients.
© 2023. The Author(s).

Teratogens are compounds that can induce birth defects upon exposure of the developing fetus. To date, most teratogen studies utilize pregnant rodents to determine compound teratogenicity in vivo. However, this is a low throughput approach that cannot easily meet the need for comprehensive high-volume teratogen assessment, a goal of the US Environmental Protection Agency. In addition, rodent and human development differ substantially, and therefore the use of assays using relevant human cells has utility. For these reasons, interest has recently focused on the use of human embryonic stem cells for teratogen assessment. Here we present a highly standardized and quantitative system for the detection and analysis of teratogens that utilizes well-characterized and purified highly pluripotent stem cells. We have devised strategies to mass-produce thousands of uniformly sized spheroids of human ESCs (hESCs) that can be caused to undergo synchronous differentiation to yield embryoid bodies (EBs) in the presence and absence of suspected teratogens. The system uses all human cells and rigorously controlled and standardized EB culture conditions. Furthermore, the approach has been made quantitative by using high-content imaging approaches. Our system offers distinct advantages over earlier EB systems that rely heavily on the use on mouse ESCs and EB aggregates of stochastic sizes. Together, our results show that thousands of suspected teratogens could be assessed using human EB-based approaches.

The in vivo osteogenesis potential of mesenchymal-like cells derived from human embryonic stem cells (hESC-MCs) was evaluated in vivo by implantation on collagen/hydroxyapatite scaffolds into calvarial defects in immunodeficient mice. This study is novel because no osteogenic or chondrogenic differentiation protocols were applied to the cells prior to implantation. After 6 weeks, X-ray, microCT, and histological analysis showed that the hESC-MCs had consistently formed a highly vascularized new bone that bridged the bone defect and seamlessly integrated with host bone. The implanted hESC-MCs differentiated in situ to functional hypertrophic chondrocytes, osteoblasts, and osteocytes forming new bone tissue via an endochondral ossification pathway. Evidence for the direct participation of the human cells in bone morphogenesis was verified by two separate assays: with Alu and by human mitochondrial antigen positive staining in conjunction with co-localized expression of human bone sialoprotein in histologically verified regions of new bone. The large volume of new bone in a calvarial defect and the direct participation of the hESC-MCs far exceeds that of previous studies and that of the control adult hMSCs. This study represents a key step forward for bone tissue engineering because of the large volume, vascularity, and reproducibility of new bone formation and the discovery that it is advantageous to not over-commit these progenitor cells to a particular lineage prior to implantation. The hESC-MCs were able to recapitulate the mesenchymal developmental pathway and were able to repair the bone defect semi-autonomously without preimplantation differentiation to osteo- or chondroprogenitors.