Addition of HER2 and CD44 to 18F-FDG PET-based clinico-radiomic models enhances prediction of neoadjuvant chemoradiotherapy response in esophageal cancer.

OBJECTIVES: To assess the complementary value of human epidermal growth factor receptor 2 (HER2)-related biological tumor markers to clinico-radiomic models in predicting complete response to neoadjuvant chemoradiotherapy (NCRT) in esophageal cancer patients. METHODS: Expression of HER2 was assessed by immunohistochemistry in pre-treatment tumor biopsies of 96 patients with locally advanced esophageal cancer. Five other potentially active HER2-related biological tumor markers in esophageal cancer were examined in a sub-analysis on 43 patients. Patients received at least four of the five cycles of chemotherapy and full radiotherapy regimen followed by esophagectomy. Three reference clinico-radiomic models based on 18F-FDG PET were constructed to predict pathologic response, which was categorized into complete versus incomplete (Mandard tumor regression grade 1 vs. 2-5). The complementary value of the biological tumor markers was evaluated by internal validation through bootstrapping. RESULTS: Pathologic examination revealed 21 (22%) complete and 75 (78%) incomplete responders. HER2 and cluster of differentiation 44 (CD44), analyzed in the sub-analysis, were univariably associated with pathologic response. Incorporation of HER2 and CD44 into the reference models improved the overall performance (R2s of 0.221, 0.270, and 0.225) and discrimination AUCs of 0.759, 0.857, and 0.816. All models exhibited moderate to good calibration. The remaining studied biological tumor markers did not yield model improvement. CONCLUSIONS: Incorporation of HER2 and CD44 into clinico-radiomic prediction models improved NCRT response prediction in esophageal cancer. These biological tumor markers are promising in initial response evaluation. KEY POINTS: • A multimodality approach, integrating independent genomic and radiomic information, is promising to improve prediction of γpCR in patients with esophageal cancer. • HER2 and CD44 are potential biological tumor markers in the initial work-up of patients with esophageal cancer. • Prediction models combining 18F-FDG PET radiomic features with HER2 and CD44 may be useful in the decision to omit surgery after neoadjuvant chemoradiotherapy in patients with esophageal cancer.

An Electrochemical Immunosensor Based on Gold-Graphene Oxide Nanocomposites with Ionic Liquid for Detecting the Breast Cancer CD44 Biomarker.

We develop a highly sensitive electrochemical immunosensor for the detection of a cluster of differentiation-44 (CD44) antigen, a breast cancer biomarker. The hybrid nanocomposite consists of graphene oxide, ionic liquid, and gold nanoparticles (GO-IL-AuNPs) immobilized on a glassy carbon electrode. GO favors the immobilization of antibodies because of the availability of oxygen functionalities. However, 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM.BF4) and AuNPs facilitate electron transfer and increase the effective surface area, which enhances the performance of the immunosensor. Furthermore, UV-visible, fourier transform infrared and Raman spectroscopy, X-ray diffraction, field emission scanning electron microscopy, transmission electron microscopy, voltammetry, and electrochemical impedance spectroscopy characterization techniques have been employed to investigate the structural and chemical properties of the nanomaterials. The quantitative detection of CD44 antigen has been accomplished via differential pulse voltammetry and EIS detection techniques. It has been quantified that the proposed immunosensor offers excellent detection ability in both phosphate-buffered saline (PBS) and serum samples. Under optimum conditions, the linear detection range of the immunosensor for CD44 antigen is 5.0 fg mL-1 to 50.0 µg mL-1 and the limit of detection is 2.0 and 1.90 fg mL-1 as observed via DPV and EIS, respectively, in PBS. Additionally, the immunosensor has high sensitivity and specificity and can be successfully applied for the detection of CD44 antigen in clinical samples.

ABCG2, CD44 and SOX9 are increased with the acquisition of drug resistance and involved in cancer stem cell activities in head and neck squamous cell carcinoma cells.

Cancer stem cells are a sub-population of cancer cells with self-renewal activity that play key roles in tumor resistance to chemotherapy and radiation. Several cancer stem cell markers have been identified to correlate with clinical prognosis. However, which marker is associated with which cancer stem cell characteristic is unclear. The present study aimed to clarify the relationship between cancer stem cell markers associated with drug resistance acquisition and the characteristics of cancer stem cells. We generated cisplatin-resistant head and neck squamous cell carcinoma cells by culturing cells in increasing concentrations of cisplatin. The cisplatin-resistant head and neck squamous cell carcinoma cells also acquired multidrug resistance and were named resistant HSC-3 (R HSC-3) cells. R HSC-3 showed no differences in cell proliferation or cell cycle distributions compared with parental cells. R HSC-3 cells showed increased drug excretion ability and elevated expression of ATP-binding cassette subfamily G member 2 (ABCG2), a drug excretion pump. R HSC-3 cells also highly expressed CD44, a cancer stem cell marker, and exhibited enhanced cell invasion and spheroid formation abilities. Furthermore, the stem cell-related factor SRY-box transcription factor 9 (SOX9) was identified as increased in R HSC-3 cells by microarray analysis. Knockdown experiments showed that SOX9 and ABCG2 were involved in the drug excretion ability of R HSC3 cells and ABCG2 was involved in the spheroid formation ability of R HSC-3 cells. These results indicate that CD44, SOX9 and ABCG2 expression levels were enhanced in head and neck squamous cell carcinoma cells that acquired multidrug resistance and that these molecules are important for maintaining cancer stem cell characteristics. Overall, regulating CD44, SOX9 and ABCG2 may be a strategy to inhibit cancer stem cells.

Correlation of Soluble CD44 Expression in Saliva and CD44 Protein in Oral Leukoplakia Tissues.

The aim of this study was to determine whether and how pan-CD44 protein expression in leukoplakia tissues correlates with positive SolCD44 test presence and their role in oral leukoplakia. SolCD44 and total protein expression in saliva were determined using an OncAlert® Oral Cancer Rapid test. Comparison of paired associations of total protein, SolCD44, mean number of CD44 expressed epithelial layers in leukoplakia tissue, and macrophages below the basement membrane between control group and patients with leukoplakia showed statistically significant results (p < 0.0001). It is shown that the total protein indicates low or elevated risk of possible malignant transformation processes in leukoplakia. Statistically significant differences between higher total protein level and clinical forms of oral leukoplakia (p < 0.0001), as well as CD44-labeled epithelial cell layer decrease (p < 0.0001), were found. This possibly points to the onset of the stemness loss in leukoplakia tissue. CD9 antigen expression in the exosomes of the oral epithelium explained the intercellular flow of SolCD44 and other fluids in the leukoplakia area. We conclude that the OncAlert® Oral Cancer Rapid test is a valuable screening method in daily clinical practice, in terms of complementing clinical diagnostics methods and to assess the potential for early malignancy.

Effect of CD44 on differentiation of human amniotic mesenchymal stem cells into chondrocytes via Smad and ERK signaling pathways.

CD44 antigen (CD44) is a transmembrane protein found in cell adhesion molecules and is involved in the regulation of various physiological processes in cells. It was hypothesized that CD44 directly affected the chondrogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs). In the present study, the expression of chondrocyte­associated factors was detected in the absence and presence of the antibody blocker anti­CD44 antibody during the chondrogenic differentiation of hAMSCs. Following inhibition of CD44 expression, the transcriptional levels of chondrocyte­associated genes SRY­box transcription factor 9, aggrecan and collagen type II α 1 chain, as well as the production of chondrocyte markers type II collagen and aggrecan were significantly decreased in hAMSCs. Further investigation indicated that there was no significant change in total ERK1/2 expression following inhibition of CD44 expression; however, phosphorylated (p)­ERK1/2 expression was decreased. The expression of p­Smad2/3 was also upregulated following CD44 inhibition. These data indicated that CD44 may affect the differentiation of hAMSCs into chondrocytes by regulating the Smad2/3 and ERK1/2 signaling pathway.

LXRα Promotes the Differentiation of Human Gastric Cancer Cells through Inactivation of Wnt/ß-catenin Signaling.

LXRα is a subtype of the liver X receptors (LXRs). There is accumulating evidence to support the involvement of LXRα in a variety of malignancies. However, the function and specific mechanism of LXRα in gastric cancer (GC) remain unclear. In this study, the expression of LXRα was significantly lower in poorly differentiated and undifferentiated GC tissues compared with well- and moderately differentiated GC tissues by immunohistochemistry analysis. The activation of LXRα leads to the decreased expression of ß-catenin, CD44, and Cyclin D1, whereas the inhibition of LXRα has opposite effect. The same results were obtained in animal experiments. Furthermore, results showed that CD44 and Cyclin D1 expression significantly decreased when Wnt/ß-catenin signaling was blocked in LXRα silent GC cells, whereas it was significantly increased when Wnt/ß-catenin signaling was activated in LXRα over-expressed GC cells. CD44 and Cyclin D1, downstream targets of Wnt/ß-catenin signaling, are specific markers for cell differentiation. Therefore, we conclude that LXRα may promote the differentiation of human GC cells through inactivation of Wnt/ß-catenin signaling.

Isolation, Characterization and Proliferation of Cancer Cells from Breast Cancer Patients.

INTRODUCTION: The most cases of cancer death, which are in the first rank among cancers suffered by women is breast cancer. The breast cancer therapy for patients has been done, but still not optimal, so it is necessary to understand the mechanism of therapy in model cell of breast cancer. AIM: This study aim to develop an isolation technique of breast cancer cell from patients as a cancer cell model. MATERIAL AND METHODS: Breast cancer cell isolation is performed by enzymatic methods using the collagen I and hyaluronidase. Then, breast cancer cells were characterized using flow cytometry based on the CD44/CD24 expression where MDA-MB468 and MCF-7 breast cancer cell lines were used as positive controls. Estrogen receptor (ER), progesterone receptor (PR), p53, HER2, and Ki67 expression were assessed using an immunohistochemistry assay. RESULT AND DISCUSSION: The morphology of cancer cells was fibroblast like cells on the day 7th after isolation. Isolated breast cancer cells expressed 95.33±0.47% of CD44+/CD24+ and human epidermal growth factor receptor 2 (HER2) low expressions. Isolation of breast cancer cells can use In-house enzymatic protocol. Isolated breast cancer showed the same expression as MDA-MB468 (CD44+/CD24+) and HER2- compared to MCF-7 cell lines (CD44-/CD24+). CONCLUSION: These cells belonged to a basal type of breast carcinoma and expressed CD44+/CD24+, then isolated BCCs can be used as model cancer cells for further research.

CXCL12 modulates the radiosensitivity of cervical cancer by regulating CD44.

The aim of the present study was to investigate the regulation of stromal cell­derived factor 1 (CXCL12) in the radioresistance of cervical cancer, which was upregulated in tumors in our previous study. A CCK­8 assay was used to detect cell viability. Flow cytometry was used to measure cell apoptosis and the expression levels of CD44 and CXCR4. ELISA was performed to measure the expression level of CXCL12 protein and CXCL12 mRNA was detected by reverse transcription­quantitative polymerase chain reaction assays. Cell viability and apoptosis were determined with or without treatment with CXCL12 small interfering (si)RNA to examine the function of CXCL12 in Hela cells. The expression level of CD44 antigen (CD44) and C­X­C chemokine receptor type 4 (CXCR4) were measured using flow cytometry in the presence of CXCL12 and irradiation. In the present study, it was demonstrated that inhibition of CXCL12 reduced cell viability and increased cellular apoptosis in Hela cells treated with irradiation. Following treatment with CXCL12 siRNA, the expression level of CD44 was downregulated and the expression level of CXCR4 was upregulated. This effect of regulation additionally occurred in the presence of irradiation. In conclusion, the present data demonstrated that CXCL12 served an important role in the radioresistance of cervical cancer, suggestinh a novel therapeutic target.

TNFAIP3 promotes survival of CD4 T cells by restricting MTOR and promoting autophagy.

Autophagy plays important roles in metabolism, differentiation, and survival in T cells. TNFAIP3/A20 is a ubiquitin-editing enzyme that is thought to be a negative regulator of autophagy in cell lines. However, the role of TNFAIP3 in autophagy remains unclear. To determine whether TNFAIP3 regulates autophagy in CD4 T cells, we first analyzed Tnfaip3-deficient naïve CD4 T cells in vitro. We demonstrated that Tnfaip3-deficient CD4 T cells exhibited reduced MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) puncta formation, increased mitochondrial content, and exaggerated reactive oxygen species (ROS) production. These results indicate that TNFAIP3 promotes autophagy after T cell receptor (TCR) stimulation in CD4 T cells. We then investigated the mechanism by which TNFAIP3 promotes autophagy signaling. We found that TNFAIP3 bound to the MTOR (mechanistic target of rapamycin) complex and that Tnfaip3-deficient cells displayed enhanced ubiquitination of the MTOR complex and MTOR activity. To confirm the effects of enhanced MTOR activity in Tnfaip3-deficient cells, we analyzed cell survival following treatment with Torin1, an MTOR inhibitor. Tnfaip3-deficient CD4 T cells exhibited fewer cell numbers than the control cells in vitro and in vivo. In addition, the impaired survival of Tnfaip3-deficient cells was ameliorated with Torin1 treatment in vitro and in vivo. The effect of Torin1 was abolished by Atg5 deficiency. Thus, enhanced MTOR activity regulates the survival of Tnfaip3-deficient CD4 T cells. Taken together, our findings illustrate that TNFAIP3 restricts MTOR signaling and promotes autophagy, providing new insight into the manner in which MTOR and autophagy regulate survival in CD4 T cells.

Osteopontin increases hepatocellular carcinoma cell growth in a CD44 dependant manner.

AIM: To investigate the role of osteopontin (OPN) and its splice variants in the proliferation of hepatocellular carcinoma (HCC). METHODS: The expression of OPN variants in HCC cell lines as well as HCC tissue samples and non-tumour tissue was studied using polymerase chain reaction. OPN variant cDNAs were cloned into a mammalian expression vector allowing both transient expression and the production of stable OPN expressing cell lines. OPN expression was studied in these cells using Western blotting, immunofluoresnce and enzyme linked immunosorbent assay. A CD44 blocking antibody and siRNA targeting of CD44 were used to examine the role of this receptor in the OPN stimulated cell growth observed in culture. Huh-7 cells stably expressing either OPN-A, -B or -C were injected subcutaneously into the flanks of nude mice to observe in vivo tumour growth. Expression of OPN mRNA and protein in these tumours was examined using reverse transcription-polymerase chain reaction and immunohistochemistry. RESULTS: OPN is expressed in HCC in 3 forms, the full length OPN-A and 2 splice variants OPN-B and -C. OPN variant expression was noted in HCC tissue as well as cognate surrounding cirrhotic liver tissue. Expression of these OPN variants in the HCC derived cell line Huh-7 resulted in secretion of OPN into the culture medium. Transfer of OPN conditioned media to naïve Huh-7 and HepG2 cells resulted in significant cell growth suggesting that all OPN variants can modulate cell proliferation in a paracrine manner. Furthermore the OPN mediated increase in cellular proliferation was dependent on CD44 as only CD44 positive cell lines responded to OPN conditioned media while siRNA knockdown of CD44 blocked the proliferative effect. OPN expression also increased the proliferation of Huh-7 cells in a subcutaneous nude mouse tumour model, with Huh-7 cells expressing OPN-A showing the greatest proliferative effect. CONCLUSION: This study demonstrates that OPN plays a significant role in the proliferation of HCC through interaction with the cell surface receptor CD44. Modulation of this interaction could represent a novel strategy for the control of HCC.