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Objective(s) Breast tumors show heterogeneity containing cancer stem cells as a small subpopulation of a tumor mass. CD44 as a cancer stem cells antigen is abnormally expressed by carcinomas of epithelial origin. Also, overexpression of CD44 variable isoforms (CD44v) is associated with malignancy in breast cancer. In the present research, our objective was to evaluate the immunogenicity of prepared nanoparticles containing a novel recombinant CD44v (rCD44v) protein in the mouse model. Materials and methods CD44 gene was expressed in E. coli BL21 DE3 using the pET28a-CD44 vector. The expressed rCD44v protein was purified, encapsulated into the chitosan nanoparticles, and administered to BALB/c mice. ELISA was used to evaluate the immunoglobulin levels of immunized animals. For challenge experiment, 2 × 10 6 4T1-CD44 tumor cells were injected subcutaneously in mice, and tumor size, necrosis, and metastases were measured. Finally, cell proliferation assay, cytokines assay, and neutralization assay of the mouse anti-rCD44v on the human breast cancer cell line were examined. Results The measured size of chitosan-rCD44v nanoparticles was 146.5 nm. Recombinant CD44v encapsulated by chitosan nanoparticles increases immunological responses via the adjuvant nature of chitosan nanoparticles. In the immunized mice, IgG and IgA titers were significantly increased. Tumor growth in injection and nano-injection test groups compared with the mice control groups displayed a significant reduction ( P  < 0.05). A high amount of splenocytes secreting IFNγ and IL-17 was seen in immunized mice with rCD44v ( P  < 0.05). Furthermore, a smaller size of lung metastases compared to the control mice groups was detected. Conclusion The encapsulated rCD44v within the chitosan nanoparticles induced a significant immune response in mice and can establish significant protection against breast cancer. Therefore, it can be considered a vaccine candidate for breast cancer therapeutic modalities. Highlights rCD44v encapsulated by chitosan nanoparticles increases immunological responses. rCD44v increased IgG and IgA titers and some cytokines in the immunized mice. The rCD44v protein has potential features to be used as a vaccine for breast cancer.

GSH biosynthesis is enhanced in cancer cells that express the variant isoform of the surface antigen CD44 (CD44v), which is overexpressed in certain types of cancer. The GSH-responsive prodrug Ns-Dox was prepared by modifying the GSH-responsive group 2-nitrobenzene sulfonyl (Ns) with the model drug doxorubicin (Dox). Its function was evaluated based on its molecular interaction with model DNA in terms of its binding constant (Ka). The association constant of Ns-Dox was lower, and its interaction with model DNA was weaker compared to that of Dox, suggesting that Ns-Dox may act as a less toxic prodrug. HCT116 cells with high CD44v expression and GSH levels and BT474 cells with low CD44v expression and GSH levels were used. The addition of Ns-Dox to HCT116 cells produced cytotoxic effects similar to those of Dox. In contrast, a significant difference in viability was observed between Ns-Dox- and Dox-treated BT474 cells at low concentrations. These findings suggest that Ns-Dox functions as a prodrug with low environmental toxicity and a lower GSH concentration in cancer cells. It is efficiently activated to Dox in cells with high GSH production, demonstrating its cell-killing effects.

Spliced variant isoforms of CD44 (CD44v) are a marker of cancer stem cells in solid tumors. They stabilize the xCT subunit of the transporter system xc(–) and thereby promote synthesis of the antioxidant glutathione. Salazosulfapyridine (SASP) is an inhibitor of xCT and suppresses the proliferation of CD44v‐positive cancer cells. Chemotherapy‐naïve patients with advanced non‐squamous non‐small‐cell lung cancer were enrolled in a dose‐escalation study (standard 3 + 3 design) of SASP in combination with cisplatin and pemetrexed. The primary end‐point was the percentage of patients who experience dose‐limiting toxicity. Fifteen patients were enrolled in the study. Dose‐limiting toxicity was observed in one of six patients at a SASP dose of 1.5 g/day (elevation of aspartate and alanine aminotransferase levels, each of grade 3), two of five patients at 3 g/day (hypotension or pneumonitis, each of grade 3), and two of three patients at 4.5 g/day (anorexia of grade 3). The maximum tolerated dose was thus 3 g/day, and the recommended dose was 1.5 g/day. The overall response rate was 26.7% and median progression‐free survival was 11.7 months, much longer than that for cisplatin–pemetrexed alone in previous studies. Exposure to SASP varied markedly among individuals according to ABCG2 and NAT2 genotypes. The serum concentration of free CD44v protein was increased after the first cycle of treatment, possibly reflecting death of cancer stem cells. Salazosulfapyridine was thus given safely in combination with cisplatin–pemetrexed, with the addition of SASP tending to prolong progression‐free survival. This trial is registered in the UMIN Clinical Trials Registry as UMIN000017854. Salazosulfapyridine (SASP) has been reported to suppress the proliferation of CD44v‐positive cancer stem cells. In this phase I study, SASP was administered safely in combination with cisplatin‐pemetrexed, with the addition of SASP tending to prolong progression‐free survival.

Expression of CD44, especially the variant isoforms (CD44v) of this major cancer stem cell marker, contributes to reactive oxygen species (ROS) defense through stabilizing xCT (a cystine–glutamate transporter) and promoting glutathione synthesis. This enhances cancer development and increases chemotherapy resistance. We investigate the role of CD44v in the regulation of the ROS defense system in cholangiocarcinoma (CCA). Immunohistochemical staining of CD44v and p38MAPK (a major ROS target) expression in Opisthorchis viverrini‐induced hamster CCA tissues (at 60, 90, 120, and 180 days) reveals a decreased phospho‐p38MAPK signal, whereas the CD44v signal was increased during bile duct transformation. Patients with CCA showed CD44v overexpression and negative‐phospho‐p38MAPK patients a significantly shorter survival rate than the low CD44v signal and positive‐phospho‐p38MAPK patients (P = 0.030). Knockdown of CD44 showed that xCT and glutathione levels were decreased, leading to a high level of ROS. We examined xCT‐targeted CD44v cancer stem cell therapy using sulfasalazine. Glutathione decreased and ROS increased after the treatment, leading to inhibition of cell proliferation and induction of cell death. Thus, the accumulation of CD44v leads to the suppression of p38MAPK in transforming bile duct cells. The redox status regulation of CCA cells depends on the expression of CD44v to contribute the xCT function and is a link to the poor prognosis of patients. Thus, an xCT inhibitor could inhibit cell growth and activate cell death. This suggests that an xCT‐targeting drug may improve CCA therapy by sensitization to the available drug (e.g. gemcitabine) by blocking the mechanism of the cell's ROS defensive system. The redox status regulation of CCA cells depends on the expression of CD44v to contribute the xCT function and is a link to the carcinogenesis and poor prognosis of patients.

Cancer stem cells (CSCs) are thought to play important roles in cancer malignancy. Previously, we successfully induced sphere cancer stem‐like cells (CSLCs) from several cell lines and observed the property of chemoresistance. In the present study, we examined the metastatic potential of these induced CSLCs. Sphere cancer stem‐like cells were induced from a human hepatoma cell line (SK‐HEP‐1) in a unique medium containing neural survival factor‐1. Splenic injection of cells into immune‐deficient mice was used to assess hematogenous liver metastasis. Transcriptomic strand‐specific RNA‐sequencing analysis, quantitative real‐time PCR, and flow cytometry were carried out to examine the expression of epithelial‐mesenchymal transition (EMT)‐related genes. Splenic injection of CSLCs resulted in a significantly increased frequency of liver metastasis compared to parental cancer cells (P < .05). In CSLCs, a mesenchymal marker, Vimentin, and EMT‐promoting transcription factors, Snail and Twist1, were upregulated compared to parental cells. Correspondingly, significant enrichment of the molecular signature of the EMT in CSLCs relative to parental cancer cells was shown (q < 0.01) by RNA‐sequencing analysis. This analysis also revealed differential expression of CD44 isoforms between CSLCs and parental cancer cells. Increasing CD44 isoforms containing an extra exon were observed, and the standard CD44 isoform decreased in CSLCs compared to parental cells. Interestingly, another CD44 variant isoform encoding a short cytoplasmic tail was also upregulated in CSLCs (11.7‐fold). Our induced CSLCs possess an increased liver metastatic potential in which promotion of the EMT and upregulation of CD44 variant isoforms, especially short‐tail, were observed. Previously, we successfully induced sphere cancer stem‐like cells (CSLCs) from several cell lines and observed the property of chemoresistance. In this study, our induced CSLC possess an increased liver metastatic potential in which promotion of the EMT and up‐regulation of CD44 variant isoforms, especially short‐tail, were observed.

A previous dose-escalation study of sulfasalazine (SSZ), an inhibitor of cystine-glutamate exchange transporter xc (–), in the variant form of CD44 (CD44v)-positive cancer stem cells (CSCs) suggested that administration of SSZ induces the reduction of CD44v-positive cells and intracellular reduced glutathione (GSH) levels in patients with advanced gastric cancer (AGC). Here we report a study to evaluate SSZ in combination with cisplatin in patients with CD44v-expressing AGC refractory to cisplatin. SSZ was given by oral administration four times daily with 2 weeks on and 1 week off. Cisplatin at 60 mg/m 2 was administered every 3 weeks. Of the 15 patients who underwent prescreening of CD44v expression, 8 patients were positive, and 7 patients were treated with the dose level of SSZ at 6 g/day. One patient experienced dose-limiting toxicity (DLT) as grade 3 anorexia. Although no other patients experienced DLT, 4 patients required dose interruption or reduction of SSZ; thus, we terminated further dose escalation. No patient achieved objective response, but 1 patient completed six cycles with stable disease for more than 4 months as well as reduction of intratumoral GSH level. The combination of SSZ plus cisplatin was manageable, although dose modification was frequently required during a short observational period.

Effective therapies have yet to be established for pancreatic ductal adenocarcinomas (PDAC) even though it is the most aggressive cancer. In the present study, PDAC was analyzed using novel rat mAbs against membrane proteins in conjunction with flow cytometry and immunohistochemistry. Human epidermal growth receptor (HER)1–4, mesenchymal to epithelial transition factor (MET), sphingosine‐1‐phospahate receptor 1 (S1PR1), l‐type amino acid transporter 1 (LAT1), system x−c transporter (xCT), alanine‐serine‐cysteine transporter (ASCT2), cationic amino acid transporter 1 (CAT1) and variant CD44 (CD44v) were expressed at high frequencies in both in vitro and in vivo PDAC. Internalization of membrane proteins by mAbs and growth inhibition by toxin‐linked mAbs were demonstrated in many PDAC cell lines, and mAbs against S1PR1, ASCT2, HER3 and CD44v inhibited the growth of xenografted MIA PaCa‐2 PDAC cells. Furthermore, CD44v‐high PDAC showed high mRNA expression of HER1–3, MET and CD44v, and was correlated with poor prognosis. Taken together, our results suggest that CD44v, S1PR1, HER3, MET and the above‐mentioned cancer‐associated amino acid transporters might be promising targets for the diagnosis and treatment of PDAC. Novel mAbs could detect cancer‐associated membrane proteins on various type of pancreatic ductal adenocarcinomas (PDAC) by flow cytometry with viable PDAC cells and by immunohistochemistry with PDAC tissues. Results of protein expression were substantiated by mRNA expression by The Cancer Genome Atlas. PDAC will be handled by targeting mAb‐defined single or combinations of multiple molecules such as variant CD44 (CD44v), sphingosine‐1‐phospahate receptor 1 (S1PR1), human epidermal growth receptor (HER)1–4, mesenchymal to epithelial transition factor (MET), l‐type amino acid transporter 1 (LAT1), system x−c transporter (xCT), alanine‐serine‐cysteine transporter (ASCT2) and cationic amino acid transporter 1 (CAT1).

Background and purpose CD44 variants have been associated with tumor invasion and metastasis, but CD44 expression patterns have not been systematically investigated in pancreatic carcinoma. This study systematically investigated whether CD44 expression patterns are involved in pancreatic carcinoma metastasis and prognosis. Methods We applied primers specific for all CD44 variants and CD44s to analyze the expression patterns of CD44 (CD44v2-CD44v10 and CD44s) using quantitative real-time PCR (qRT-PCR). We then further evaluated their roles in pancreatic carcinoma metastasis and prognosis using clinical survival analysis. Results Increased CD44v expression and decreased CD44s expression were found in metastatic pancreatic carcinoma in three different cell lines and in human tumor tissue. Clinical analysis showed that CD44v6 + and CD44v9 + were correlated with lymph node metastasis, liver metastasis and TNM stage. However, CD44s − was associated with liver metastasis, tumor differentiation and TNM stage. Survival analysis showed that patients with CD44v6 + /CD44s − or CD44v6 + /CD44s − had lower overall survival (OS) rates, although the individual expression of CD44v6, CD44v9 and CD44s was also related to decreased OS rates. Univariate analysis showed that lymph node metastasis; vessel invasion; hepatic metastases; TNM stage; and individual or co-expression of CD44v6, CD44v9 and CD44s were risk factors affecting survival. Multivariate analysis showed that CD44v6 + /CD44s − was an independent predictor of survival. Conclusions We found that CD44v6 + , CD44v9 + and CD44s − were associated with pancreatic carcinoma metastasis and progression and that CD44v6 + /CD44s − was an independent risk factor affecting survival in pancreatic carcinoma. Therefore, the different expression patterns of CD44v/CD44s may determine pancreatic carcinoma prognosis. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1579257224116287 .

Background CD44, as a tumor‐associated marker, can be used to detect stem cells in breast cancer. While CD44 is expressed in normal epithelial cells, carcinoma cells overexpress CD44. Aims In the current study, we designed a recombinant protein that included the variable component of the CD44 (CD44v) extracellular domain to apply in clinical diagnosis of breast cancer. Methods A total of 100 CD44v amino‐acid residues were determined, and the structure was examined using bioinformatics tools. The construct was inserted into the PET28a vector and transformed in E. coli BL21(DE3). A nearly 12 kDa fusion protein was obtained by Ni‐NTA affinity metal chromatography. Recombinant CD44v was examined by Western blotting, ELISA, and immunohistochemistry (IHC) assays. Results The findings revealed that the structure of rCD44v was stable, and its antigenic domain was exposed. The recombinant CD44v was confirmed by western blotting, and the presence of antibodies against recombinant CD44v protein in the patient's serum was detected by the ELISA. Our data demonstrated a link between CD44v serum levels and the prevalence of breast cancer. Conclusion Assessments of antiCD44v antibodies with rCD44v could be a useful tool for identifying breast cancer in its early stages, which can lead to better outcomes.

Podoplanin and CD44 are transmembrane glycoproteins involved in inflammation and cancer. In this paper, we report that podoplanin is coordinately expressed with the CD44 standard (CD44s) and variant (CD44v) isoforms in vivo—in hyperplastic skin after a pro-inflammatory stimulus with 12-O-tetradecanoylphorbol-13-acetate (TPA)—and in vitro—in cell lines representative of different stages of mouse-skin chemical carcinogenesis, as well as in human squamous carcinoma cell (SCC) lines. Moreover, we identify CD44v10 in the mouse-skin carcinogenesis model as the only CD44 variant isoform expressed in highly aggressive spindle carcinoma cell lines together with CD44s and podoplanin. We also characterized CD44v3-10, CD44v6-10 and CD44v8-10 as the major variant isoforms co-expressed with CD44s and podoplanin in human SCC cell lines. Immunofluorescence confocal microscopy experiments show that these CD44v isoforms colocalize with podoplanin at plasma membrane protrusions and cell–cell contacts of SCC cells, as previously reported for CD44s. Furthermore, CD44v isoforms colocalize with podoplanin in chemically induced mouse-skin SCCs in vivo. Co-immunoprecipitation experiments indicate that podoplanin physically binds to CD44v3-10, CD44v6-10 and CD44v8-10 isoforms, as well as to CD44s. Podoplanin–CD44 interaction is mediated by the transmembrane and cytosolic regions and is negatively modulated by glycosylation of the extracellular domain. These results point to a functional interplay of podoplanin with both CD44v and CD44s isoforms in SCCs and give insight into the regulation of the podoplanin–CD44 association.