CD44 variant ABs & kit for characterising & isolating cancer stem cells

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CD44v positive cancer stem cells are reported to be resistant to chemo and radiation therapy, in part due to elevated resistance to ROS and slow growth. Serial adoptive transfer studies have shown that these cells are highly enriched in tumorigenic potential. Thus, these cells are thought to survive therapeutic tumor mass reduction and upon epithelial mesenchymal transition seed distant metastases, ultimately leading to the lethality of both solid and hematopoietic cancers.

The transmembrane signaling protein CD44 binds through its extracellular domain to multiple extracellular matrix components including hyaluronan and fibronectin. It is encoded by 20 exons, 10 of which are variably included in the mature mRNA via alternative splicing controlled in part by the splicing factor ESRP1. These 10 exons contribute to variation in the membrane proximal extracellular domain of the protein. Certain variant isoforms have been shown to confer binding to growth factor receptors and importantly to a subunit of the cystine-glutamate antiporter responsible for replenishing intracellular GSH levels, thereby contributing to ROS resistance in some cancer stem cells.

Combinatorial splicing of the variant exons can theoretically yield 1024 different CD44 isoforms. Which of these potential isoforms are actually produced and physiologically relevant in different normal and tumorigenic contexts remains to be explored. Apart from the paucity of cancer stem cells in tumor explants and difficulty in their purification, the lack of a comprehensive set of validated variant isoform-specific antibodies has limited progress in understanding the role of CD44v in cancer stem cell biology.

Our rat anti-human CD44v9 monoclonal antibody recognizes CD44v8-10 and has been cited in numerous publications including the landmark paper mentioned above describing the association of CD44v8-10 with the cystine-glutamate transporter. It has been used in multiple applications, including immunoblot, IHC, IF, ELISA, flow cytometry, IP, MACS and photo-immunotherapy. In addition, we hope you will find useful our new RV3-conjugated magnetic bead kit for easy isolation of CD44V9 positive cancer stem cells. Finally, we carry a rat anti-mouse CD44v10-e16 that has been validated in flow cytometry and IHC.

Name	Host	Reactivity	Clone	Applications
Anti CD44 v10-e16 (Cat No. CAC-LKG-M002)	RT	MS	RM1	FC IHC
Anti CD44 v9 (Cat No. CAC-LKG-M001)	RT	HU	RV3	FC WB IHC(p) IF ELISA IP MACS Photo-immunotherapy
Anti CD44ICD (Cat No. KAL-KO601)	Rabbit	HU	-	IP ICC IF IHC WB
Magnetic Cell separation Kit for Human CD44v9+ Cancer Stem Cell	RT	HU	RV3	Cancer stem cell isolation

Year	Publications citing anti-CD44 splice variant clones RM1 and RV3 or CD44 ICD rabbit polyclonal
2019	Characterization of CD44 intracellular domain interaction with RUNX2 in PC3 human prostate cancer cells. 2019. Cell Communication and Signaling 17:80. Anti-human CD44-ICD antibody (Rabbit polyclonal available from Cosmo Bio) was used in western blot analysis of prostate cancer cell lines (PC3, LnCaP and PCa2b). A previous study of breast cancer cells showed that a CD44-ICD/RUNX2 complex occurred on the metastasis-related MMP-9 gene promoter region. This study shows a functional relationship between CD44-ICD and RUNX2 in the prostate cancer cell line PC3. Elevated RUNX2 expression in PC3 cells was associated with increased nuclear CD44-ICD/RUNX2 interaction and expression of MMP-9 as well as increased migration and tumorsphere formation.
2019	Sulfasalazine could modulate the CD44v9-xCT system and enhance cisplatin-induced cytotoxic effects in metastatic bladder cancer. 2019. Cancer Science. 110:1431–1441. Anti-human CD44v9 antibody (clone RV3 available from Cosmo Bio) was used in immunohistochemical analyses of radical cystectomies from muscle invasive bladder cancer (MIBC) patients. CD44v9 expression was independently associated with disease recurrence and cancer-specific death in MIBC. Cytotoxic effects, glutathione levels, and reactive oxygen species production by sulfasalazine (SSZ) and CD44v9 and phospho-p38 MAPK protein expression by SSZ with or without cisplatin (CDDP) were assessed in MBT-2V cells with highly metastatic potential. SSZ exerted cytotoxic effects against MBT-2V cells by inhibiting glutathione levels and inducing the production of reactive oxygen species. SSZ in combination with CDDP appeared to exert strong cytotoxic effects against MBT-2V cells by inhibiting CD44v9 expression and upregulating phospho-p38 MAPK expression. The inhibitory effects of SSZ with or without CDDP were also investigated using an MBT-2V lung metastatic model. In the murine lung metastatic BC model, SSZ significantly prolonged animal survival. Furthermore, the combination of SSZ with CDDP exerted stronger inhibitory effects on the establishment of lung tumor nodules than SSZ or CDDP alone.
2019	Cancer stem-cell marker CD44v9-positive cells arise from Helicobacter pylori-infected CAPZA1-overexpressing cells. 2019. Cellular and Molecular Gastroenterology and Hepatology. 8(3):319 - 334. Anti-human CD44v9 antibody (clone RV3 available from Cosmo Bio) was used in immunohistochemical and western blot analyses of Mongolian gerbil gastric mucosa, human gastric adenocarcinomas and the gastric cancer cell lines (MKN28 and AGS). This paper demonstrates the role of CAPZA1 in CD44v9 expression and shows that the presence of CAPZA1-overexpressing cells induces expansion of CD44v9-positive cells. The induction of CD44v9 expression in CAPZA1-overexpressing cells infected with H pylori is caused by aberrant beta-catenin signaling via intracellularly accumulated CagA and by alternative splicing of CD44 total via overexpression of ESRP1. Accumulation of CagA in CAPZA1-overexpressing cells also aberrantly induced SALL4 and KLF5 expression. These findings show that over-expression of CAPZA1, in combination with accumulation of CagA, predisposes cells to develop into CD44v9-positive cells in H pylori–infected gastric mucosa.
2018	Redox regulation in stem-like cancer cells by CD44 variant isoforms. Oncogene. 2013 32(44):5191-8. This review paper describes the discovery of a critical association between a variant form of CD44 (CD44v8-10; recognized by clone RV3 available from Cosmo Bio) and xCT, a subunit of the cystine-glutamate antiporter required for generation of intracellular antioxidant GSH. CD44v8-10 is thought to be a cancer stem cell marker that acts in part by promoting resistance to ROS, thereby accounting for enhanced resistance to chemo and radiotherapy.
2018	Functional loss of p53 cooperates with the in vivo microenvironment to promote malignant progression of gastric cancers. Sci Rep. 2018 8(1):2291. Anti-mouse CD44v monoclonal Ab (Cosmo Bio) was used in immunohistochemical analysis of gastric epithelia and gastric epithelial tumor cells. By 20 weeks of age mice whose gastric mucosa overexpressed Wnt1, Cox2 and microsomal prostaglandin E synthase-1 (Gan mice) developed dysplastic gastric tumors of low malignancy. In this paper the authors crossed Gan mice with p53 KO mice to produce p53 deficient Gan mice. Gastric epithelial cells from p53 KO Gan mice were tumorigenic when transplanted into immunocompetent recipients and serial transfers gradually gave rise to malignant gastric cancer. CD44v expression was noted to increase in p53 KO Gan mouse gastric tumors but to diminish in undifferentiated portions of highly malignant serially transferred tumors.
2018	Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli. Cell Mol Gastroenterol Hepatol. 2018 5(4):523-538. Anti-mouse CD44v10-e16 rat monoclonal Ab (clone RM1 available from Cosmo Bio) was used in immunohistochemical staining of mouse gastric epithelium. Evidence is presented that impaired BMP signaling coupled with Helicobacter felis infection leads to loss of parietal cells and to activation and expansion of a subset of Lgr5 positive chief cells that express CD44v9, a cancer stem cell marker of SPEM known to give rise to gastric carcinogenesis.
2018	Variant isoforms of CD44 involves acquisition of chemoresistance to cisplatin and has potential as a novel indicator for identifying a cisplatin-resistant population in urothelial cancer. Cancer. 2018 18(1):113. Anti-human CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) was used in immunohistochemistry of human urothelial cancer tissue and in immunofluorescence staining of human bladder cancer cell lines. Positivity for CD44v9 (CD44v8-10) was shown to be associated with a significantly higher incidence of high grade urothelial tumors. Further, positivity for CD44v9 (CD44v8-10) was also shown to be a strong prognostic indicator of poor cancer specific survival in UC patients treated with CDDP-based chemotherapy and also found to be a significant risk factor for cancer specific mortality.
2018	Prominent role of RAB39A-RXRB axis in cancer development and stemness. Oncotarget. 2018 9(11): 9852–9866. Using global expression profiling of human osteosarcoma cells and normal fibroblast cell lines, RAB39A was discovered to be a functional cancer stem cell marker whose knockdown and overexpression correlated with loss and gain of in vitro sphere-forming and in vivo xenograft tumor forming ability. Anti-human CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) was used in flow cytometry of sarcoma cell lines to show that knockdown of RAB39A led to a decrease in the expression of CD44v9. Differential global expression profiling of RAB39A knockdown spheres led to the identification of RXRB as a major downstream effector of RAB39A, suggesting both molecules as therapeutic targets in cancer cells.
2017	Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma. J Cancer. 2017 8(10):1774-1785. Anti-human CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) was used in immunohistochemistry and flow cytometry to screen multiple lung cancer cell lines for properties associated with cancer stem cells (CSCs). Staining for aldehyde dehydrogenase (ALDH) was also combined with CD44v9 with the hope of improving the ability to detect CSCs. The results suggest that CD44v9 is a good marker for lung CSCs in human lung adenocarcinoma cell lines. Use of ALDH as second marker provided evidence that lung CSCs may be heterogeneous with different CSC properties distributed over distinct cell subsets.
2017	CD44v8-10 as a potential theranostic biomarker for targeting disseminated cancer cells in advanced gastric cancer. Sci Rep. 2017 7(1):4930. Anti-human CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) was used to demonstrate predominant expression of CD44v8-10 in gastric cancer cell lines and advanced gastric tumors. CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) was conjugated with near infrared red (NIR) fluors ATTO680 for immunohistochemical detection of CD44v8-10-expressing cells in gastric tumor tissues; and Methylene Blue (MB) for use in in vitro photoimmunotherapy. MB causes not only NIR fluorescence, but also the highly efficient generation of singlet oxygen, thereby selectively killing cancer cells. In mixtures of cell lines either expressing CD44v8-10 or not and stained with MB-conjugated CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) only the CD44v8-10-expressors were subject to photolysis. Together, the results suggest the applicability of CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) for NIR fluorescence detection and photoimmunotherapy of both primary gastric cancer cells and disseminated cancer stem cell-like CD44v8-10(+) cells in the peritoneal cavity in advanced gastric cancer.
2017	Isoform switch of CD44 induces different chemotactic and tumorigenic ability in gallbladder cancer. Int J Oncol. 2017 51(3):771-780. Anti-human CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) was used in flow cytometry to sort and analyze the human gall bladder cancer cell line NOZ. The NOZ cell line is shown to comprise two populations that express different CD44 isoforms: CD44s and CD44v9, accounting for 70% and 30% of the CD44+ cells, respectively. Interestingly, the CD44s cells exhibited elevated migration and invasion phenotypes and expressed mesenchymal markers ZEB1, ZEB2 and Vimentin while the CD44v9 cells did not. Instead, they exhibited enhanced tumorigenicity in a mouse xenograft model and expressed high levels of the epithelial marker E-cadherin and the splicing factor ESRP1. Also, subculture of CD44v9 cells gave rise to both CD44v9 and CD44s cells, while subculture of CD44s cells only gave rise to CD44s cells.
2017	SIRT1 Regulates the Chemoresistance and Invasiveness of Ovarian Carcinoma Cells. Transl Oncol. 2017 10(4): 621–631. Anti-human CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) was used in immunoblot assays to show that SIRT1 upregulates CD44v9 in ovarian carcinoma cells, correlating with acquisition of enhanced resistance to oxidative stress. Genetic and pharmacological knockdown of SIRT1 downregulated CD44v9 while upregulation led to enhanced CD44v9. The cancer stem cell marker CD44v9 is known to be involved in stabilizing the xCT glutamate-cystine antiporter system that promotes GSH production and protection from ROS.
2017	Phase I study of salazosulfapyridine in combination with cisplatin and pemetrexed for advanced non-small-cell lung cancer. Cancer Sci. 2017 108(9):1843-1849. Anti-human CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) was used to show that effectiveness of salazosulfapyridine treatment in combination with cisplatin and pemetrexed for advanced non-small-cell lung cancer was associated with an increase in serum CD44v9, likely due to apoptotic death of CD44v9+ CSC. Serum samples collected from 14 patients before and after 21 days of salazosulfapyridine treatment were tested by ELISA for human CD44v9 (clone RV3 available from Cosmo Bio).
2017	cRGD peptide installation on cisplatin-loaded nanomedicines enhances efficacy against locally advanced head and neck squamous cell carcinoma bearing cancer stem-like cells. J Control Release. 2017 Sep 10;261:275-286. Anti-human CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) was used in immunohistochemical and flow cytometric analyses to confirm the expression of αvβ5 integrins on the CD44v-positive fractions of two human oral carcinoma cells (HSC-2 and SAS-L1). By installing the αvβ5 integrin-targeting cyclic Arg-Gly-Asp (cRGD) peptide on the surface of cisplatin (CDDP)-containing micelles (CDDP/m), therapeutic strategies were tested for improved efficacy against head and neck squamous cell cancer (HNSCC)-bearing cancer stem cells (CSCs). cRGD-installed CDDP/m (cRGD-CDDP/m) exhibited significant antitumor activity against both orthotopic HSC-2 and SAS-L1-Luc tumors, and rapidly and selectively accumulated in lymph node metastases of the SAS-L1-Luc tumor and reduced their growth rate.
2017	Inflammatory mediator ultra-low-molecular-weight hyaluronan triggers necrosis of B-precursor leukemia cells with high surface CD44 expression. Cell Death Dis. 2017 8(6): e2857. Ultra low molecular weight hyaluronan is shown to induce features consistent with necrotic cell death in B-precursor leukemia cells that express high levels of CD44. Anti-human CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) was used in flow cytometry to show that this property did not correlate with the magnitude of ultra-low molecular weight hyaluronan-mediated inhibition of leukemic cells.
2017	Increased Expression of CD44v9, A Cancer Stem Cell Marker, in Head And Neck Squamous Cell Carcinoma Cells after Irradiation. 2017 Int J Cancer Oncol. 4(2): 1-5. FITC-conjugated anti-human CD44s clone SFF-2, APC-conjugated anti-human CD44v9 (clone RV3 available from Cosmo Bio), APC-conjugated anti-human CD44v3 clone 3G5 and APC-conjugated anti-human CD44v6 clone 2F10 were used in flow cytometry on 5 different human squamous cell carcinoma cell lines to show that after exposure to 60 Gy of irradiation expression of CD44s decreased while variant CD44 expression increased, especially CD44v9. These changes correlated with increased sphere forming ability.
2017	Distinct metaplastic and inflammatory phenotypes in autoimmune and adenocarcinoma-associated chronic atrophic gastritis. United European Gastroenterol J. 2017 5(1):37-44. Anti-human CD44v9 rat monoclonal Ab (clone RV3 from Cosmo Bio) was used in immunohistochemistry to show that patients suffering from either autoimmune or adenocarcinoma-associated chronic atrophic gastritis exhibited elevated CD44v9 expression in spasmolytic polypeptide-expressing metaplastic (SPEM) tissue.
2017	CD44 variant 9 expression as a predictor for gastric cancer recurrence: immunohistochemical and metabolomic analysis of surgically resected tissues. Biomed Res. 2017 38(1):41-52. Anti-human CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) was used in immunohistochemistry to analyze 103 gastric cancer patient specimens for CD44v9 expression and correlation with five-year recurrence-free survival rate and metabolomic analysis. The results show that high level expression of CD44v9 correlated with significantly lower five-year recurrence-free survival rate and reduced glutathione disulfide levels.
2017	A signalling cascade of IL-33 to IL-13 regulates metaplasia in the mouse stomach. Gut. 2018 67(5):805-817. Anti-human CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) was used in immunocytochemical analyses to identify spasmolytic polypeptide-expressing metaplasia (SPEM) in the stomach by quantitating the number of cells triple-positive for GSII-lectin, CD44v9 and gastric intrinsic factor (GIF). The authors conclude: Metaplasia induction and macrophage polarization after parietal cell loss is coordinated through a cytokine signaling network of IL-33 and IL-13, linking a combined response to injury by both intrinsic mucosal mechanisms and infiltrating M2 macrophages.
2017	Targeted Apoptosis of Parietal Cells Is Insufficient to Induce Metaplasia in Stomach. Gastroenterology. 2017 152(4):762-766.e7. Anti-mouse CD44v10-e16 (clone RM1 available from Cosmo Bio), ortholog of human v9, was used in IHC(p) to characterize mouse stomach. To test whether parietal cell atrophy causes metaplasia in the stomach, mice that expressed the diphtheria toxin receptor specifically in parietal cells to induce their death, were found to exhibit elevated proliferation in the normal stem cell zone and neck but not metaplastic reprogramming of chief cells.
2016	Comparable roles of CD44v8-10 and CD44s in the development of bone metastases in a mouse model. Oncol Lett. 2016 12(4):2962-2969. Anti-human CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) was used in immunoblots to confirm the overexpression of CD44v8-10 in lung and breast cancer cell lines. No significant difference was detected in the phenotype of cell lines transfected to overexpress CD44s versus CD44v, in terms of in vitro invasiveness, migration, and tumor sphere formation or on the development of bone metastases.
2016	Clinical significance of CD44 variant 9 expression as a prognostic indicator in bladder cancer. Oncol Rep. 2016 36(5):2852-2860. Anti-human CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) was used in immunohistochemistry to assess CD44v9 expression in 98 pathologically confirmed bladder tumor specimens and in two human bladder cancer cell lines. Expression was then correlated with clinical outcome. Elevated CD44v9 expression was correlated with significantly lower progression free and cancer-specific survival rates. In vitro siRNA knockdown of CD44v9 led to lower invasion ability and shorter migration distance, increased expression of the epithelial marker E-cadherin and decreased expression of EMT markers N-cadherin, Snail and Slug.
2016	Calreticulin is highly expressed in pancreatic cancer stem-like cells. Cancer Sci. 2016 107(11):1599-1609. CD44v9-expressing cells from pancreatic cancer cell lines were magnetically enriched using anti-human CD44v9 rat monoclonal Ab (clone RV3 available from Cosmo Bio) with MACS (Miltenyi) and sorted/analyzed by flow cytometry. Resected pancreatic tumor samples were analyzed by immunohistochemistry for CD44v9-expression. Interestingly, CD44v9 level showed no correlation with clinical outcome.
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CD44 variant ABs & kit for characterising & isolating cancer stem cells

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