CD44 In Cancer Cells Research Articles

Remodelling of the bone marrow microenvironment by stromal hyaluronan modulates the malignancy of breast cancer cells

BackgroundHyaluronan (HA) is an abundant component of the bone marrow (BM) extracellular matrix. Here, we investigated the abnormal deposition of HA in the BM microenvironment and its remodelling in mediating the malignancy of breast cancer cells (BCCs).MethodsBCCs were transplanted into nude mice by intracardiac injection. The BCCs were cocultured with BM-derived stromal HS5 cells. Then, the abnormal metabolism of HA and its correlation with the malignant growth and the intracellular signalling pathways of the BCCs were investigated. After knockdown/out of the HA receptor CD44 in cancer cells by shRNA and CRISPR/Cas9, the mechanism was investigated in vivo through intratibial inoculation and in vitro by coculture with HS5 cells.ResultsThe malignancy of cancer cells was highly related to the degree of accumulation of HA in the BM. Further, stromal cell-derived HA, especially the mixed complex, significantly promoted the growth of BCCs and osteolysis by binding to the CD44 receptor. Additionally, the investigation of the underlying mechanism revealed that the PI3K, Cyclin D1, and CDK4 pathways were involved in the effect of bone stromal cell-derived HA on the BCC activities.ConclusionThese data suggested that HA in abnormal BM stroma might be a therapeutic candidate for bone metastasis of breast cancer.19kbyyRgNofaNy4kV-u_efVideo Graphical abstract

Correlation of PKM2 and CD44 Protein Expression with Poor Prognosis in Platinum-Treated Epithelial Ovarian Cancer: A Retrospective Study.

CD44, a surface marker for cancer stem cells, interacts with PKM2, a key regulator of aerobic glycolysis, and enhances the glycolytic phenotype of cancer cells leading to antioxidant protection and macromolecules’ synthesis. To clarify the clinical importance of this “cross-talk” as a mechanism of drug resistance, we assessed the expression both of PKM2 and of CD44 in cancer cells of patients with epithelial ovarian cancer (EOC) treated with platinum-based treatment. One hundred and seventy-one patients with EOC were assessed for PKM2mRNA expression and PKM2 and CD44 proteins detection. Associations with progression-free survival (PFS) and overall survival (OS) were assessed with Kaplan–Meier and adjusted Cox regression models. PKM2mRNA and protein as well as CD44 protein were detectable in the majority of patients. Positive correlation between PKM2 and CD44 protein expression was observed (Spearman rho = 0.2, p = 0.015). When we used the median to group patients into high versus low expression, high PKM2mRNA and protein levels were significantly associated with lower progression-free survival (PFS; p = 0.003 and p = 0.002, respectively) and shorter overall survival (OS; p ≤ 0.001 and p = 0.001, respectively). However, high CD44 protein expression was significantly correlated only with shorter OS (p = 0.004). Moreover, patients with both high PKM2 and CD44 protein levels experienced shorter PFS and OS (p = 0.007 and p = 0.003, respectively) compared to patients with low expression of both proteins. Finally, higher PKM2mRNA and protein expression as well as CD44 protein expression (HR: 2.16; HR: 1.82; HR: 1.01, respectively) were independent prognostic factors for decreased median OS (mOS), whereas only PKM2 protein expression (HR: 1.95) was an independent prognostic factor for decreased median PFS (mPFS). In conclusion, PKM2 expression is a negative prognostic factor in EOC patients, but the interaction between CD44 and PKM2 that may be implicated in EOC platinum-resistance needs further investigation.

Fluorescent vesicular nanointerface: construction and ratiometric sensing behavior toward CD44 protein

A fluorescent vesicular nanointerface for CD44 protein sensing has been achieved by using amphiphilic Eu3+ complexes and derivatives of a pair of fluorophores, fluorescein and coumarin. Both Eu3+ complex and coumarin were designed to have hydrophobic tails, which facilitated their co-assembly to form vesicles (EuL3+/CA). The negatively charged fluorescein-labeled hyaluronic acid (HA-FA) could bond onto EuL3+/CA surface via coulombic interaction. The vesicles usually emit in the blue region, but they exhibited green fluorescence after HA-FA was bonded to the vesicle surface. This is due to the fluorescence resonance energy transfer (FRET) process between coumarin and fluorescein. As the CD44 protein was introduced to the system, intensity of the green emission of the assembly decreased, while the blue emission enhanced. This phenomenon is accounted for the specific binding between CD44 and hyaluronic acid thereby prohibiting the FRET process between HA-FA and EuL3+/CA. The vesicular nanointerface was found to be sensitive and selective to CD44 sensing (DL=0.179 μg/mL) while the amino acids and proteins tested did not affect the fluorescent properties of the assembly. What is more, the assembly could not only be used to detect CD44 in buffer solution, but also be applied to detect CD44 in cancer cells.

Development and mechanistic insight into enhanced cytotoxic potential of hyaluronic acid conjugated nanoparticles in CD44 overexpressing cancer cells

The overexpression of CD44 in cancer cells reroutes number of oncogenic pathways including the central Pi3K/Akt/NF-kB pathway leading to cancer progression and malignancy. Herein, we developed hyaluronic acid-modified poly(dl-lactic-co-glycolic acid)-poly (ethylene glycol) nanoparticles (PLGA-PEG-HA NPs) for targeted delivery of TTQ (thio-tetrazolyl analog of a clinical candidate, IC87114) to CD44 overexpressing cancer cells. The PLGA-PEG co-polymer was synthesized and characterized by NMR and FTIR. The co-polymer based nanoparticles were prepared by solvent evaporation method and hyaluronic acid (HA) was conjugated on to the nanoparticle surface via EDC/NHS chemistry. The PLGA-PEG–HA NPs had a desirable particle size (<200nm) with reduced polydispersibility and exhibited spherical shape under atomic force microscope (AFM). In vitro cytotoxicity and cellular uptake studies demonstrated higher cytotoxicity and enhanced intracellular accumulation of PLGA-PEG-HA NPs compared to PLGA-PEG NPs in high CD44 expressing MiaPaca-2 cells compared to MDA-MB-231 and MCF7 cells. At the molecular level, the PLGA-PEG-HA NPs were found to be inducing premature senescence with increase in senescence associated β-galactosidase activity and senescence specific marker p21 expression through modulation of Pi3K/Akt/NF-kB signaling pathway in MiaPaca-2 cells. These findings collectively indicated that HA-modified nanoparticles might serve as a promising nanocarrier for site-specific drug delivery, and can be explored further to increase the therapeutic efficacy of anticancer drugs via targeting to CD44 over-expressing cancer cells.

Modulation of CD44 Activity by A6-Peptide.

Hyaluronan (HA) is a non-sulfated glycosaminoglycan distributed throughout the extracellular matrix that plays a major role in cell adhesion, migration, and proliferation. CD44, a multifunctional cell surface glycoprotein, is a receptor for HA. In addition, CD44 is known to interact with other receptors and ligands, and to mediate a number of cellular functions as well as disease progression. Studies have shown that binding of HA to CD44 in cancer cells activates survival pathways resulting in cancer cell survival. This effect can be blocked by anti-CD44 monoclonal antibodies. A6 is a capped, eight l-amino acid peptide (Ac-KPSSPPEE-NH2) derived from the biologically active connecting peptide domain of the serine protease, human urokinase plasminogen activator (uPA). A6 neither binds to the uPA receptor (uPAR) nor interferes with uPA/uPAR binding. A6 binds to CD44 resulting in the inhibition of migration, invasion, and metastasis of tumor cells, and the modulation of CD44-mediated cell signaling. A6 has been shown to have no dose-limiting toxicity in animal studies. A6 has demonstrated efficacy and an excellent safety profile in Phase 1a, 1b, and 2 clinical trials. In animal models, A6 has also exhibited promising results for the treatment of diabetic retinopathy and wet age-related macular degeneration through the reduction of retinal vascular permeability and inhibition of choroidal neovascularization, respectively. Recently, A6 has been shown to be directly cytotoxic for B-lymphocytes obtained from patients with chronic lymphocytic leukemia expressing the kinase, ZAP-70. This review will discuss the activity of A6, A6 modulation of HA and CD44, and a novel strategy for therapeutic intervention in disease.

CD44 as a potential diagnostic tumor marker.

CD44 is a cellular protein that has been intensively studied in relation to carcinogenesis over the last decade. It is altered during inflammatory responses and cellular malfunctioning during tumor progression. Tumors of epithelial origin express CD44 in multiple isoforms called variants; some isoforms are related to specific cancer cells. An increase of CD44 specific isoforms is detected in certain leukemic proliferations. Most published data indicates a partial involvement of CD44 in cancer cells, either in invasiveness or self-renewability. However, there is still uncertainty regarding the exact mechanism by which CD44 participates in growth of cancer or the inflammatory response. This review focuses on CD44 prevalence in cancer cell. It considers tumorigenic behavior of cells that highly express CD44 as an early marker for neoplastic stem cell proliferation. We will discuss multiple examples of tumor in this paper, with an emphasis of 2 solid tumors; namely, breast and colon cancer.

Expression of stem cell marker CD44 in prostate cancer biopsies predicts cancer grade in radical prostatectomy specimens.

Cancer stem cells play an important role in development and progression of many cancer types including prostate adenocarcinoma. We used a stem cell marker CD44 to evaluate the prevalence of prostate cancer stem cells in prostate biopsies and in matched radical prostatectomy specimens. We tested both types of specimen for the existence of a correlation between the immunohistochemical expression of CD44 and Gleason grade, pathological stage (pT) according to TNM, patient age and preoperative plasma PSA levels in 52 patients. We found a positive correlation between the expression of CD44 in cancer cells from prostate biopsies and in matched radical prostatectomy specimens. We also observed that higher level of CD44 expression in cancer cells correlated with lower Gleason score, both in prostate biopsies and in radical prostatectomies. To the best of our knowledge we showed for the first time, that the level of CD44 expression in prostate biopsies correlates with that observed in matched radical prostatectomy specimens. Since the level of CD44 expression was shown to predict a response to anti cancer therapy in several types of human tumors, CD44 assessment might support a clinical decision making process in prostate cancer patients.

Macrophage-derived reactive oxygen species suppress miR-328 targeting CD44 in cancer cells and promote redox adaptation

CD44 is frequently overexpressed in a wide variety of epithelial malignancies including gastrointestinal cancer and causes resistance to currently available treatments. MicroRNAs (miRNAs) are non-coding RNAs that regulate molecular pathways in cancer by targeting various genes. The aim of this study was to investigate the regulation of CD44 expression by miRNAs and to develop new molecular targets in gastrointestinal cancer. We performed miRNA screening in six human gastrointestinal cancer cell lines and identified three candidate miRNAs that could regulate CD44 expression in gastrointestinal cancer. Among these, we focused on miR-328 and examined its functional relevance using growth assays and cytotoxicity assays. CD44 expression was reduced in gastrointestinal cancer cell lines forced to express miR-328, leading to inhibition of cancer cell growth in vitro and in vivo, and impaired resistance to chemotherapeutic drugs and reactive oxygen species (ROS). In contrast, induction of CD44 expression by miR-328 inhibitor led to promotion of cancer cell growth. Furthermore, we revealed that ROS produced by macrophages triggered CD44 expression through suppression of miR-328 in gastric cancer cells. Finally, tumor-infiltrating macrophages (CD68 and CD163) were closely related to both miR-328 downregulation and CD44 upregulation in 63 patients with surgically resected gastric cancer. These findings suggest that macrophages in the tumor microenvironment may cause increased CD44 expression through miR-328 suppression, resulting in tumor progression by enhancing ROS defense. miR-328-CD44 signaling mediated by macrophages may thus represent a potential target for the treatment of gastrointestinal cancer.

Abstract 254: Expression of the putative stem cell marker CD44 in prostate biopsies correlates with Gleason score in radical prostatectomies in patients with prostate cancer.

Abstract AIM: Prostate cancer is one of the leading causes of death among male oncology patients. There is growing evidence to support the hypothesis that cancer cells in the prostate are of stem cell origin. In this study we used CD44, as one of the most promising putative stem cell markers, to evaluate the prevalence of prostate cancer cells with stem cell like features in prostate biopsies and in radical prostatectomy specimens. We tested both types of specimen for the existence of a correlation between the expression of CD44 and commonly used prostate cancer prognostic factors such as Gleason grade, pathological stage (pT) according to TNM, patient age and preoperative plasma PSA levels. M&amp;M: Formalin fixed paraffin embedded tissue samples from fifty two prostate biopsies and matched radical prostatectomy specimens were retrieved from the archives of the Pathology Department at the City Hospital, Poznan, Poland or from a collection of cases sent for consultation in the private laboratory of one of the authors (JB). Immunohistochemistry was performed on sections from all paraffin blocks using an anti-CD44 primary antibody (Abcam) according to the manufacturer's protocol. Using an OLYMPUS BX41 microscope, we counted at least 100 cancer cells from core biopsies and 500 cancer cells from radical prostatectomy specimens. Only cells with clearly visible membranous staining at 10x magnification were called positive for the expression of CD44. The percentage of cancer cells expressing CD44 was used for further analysis. The statistical analysis was based on Spearman and Person correlations performed in Microsoft Office Excel 2007. RESULTS: CD44 positive cancer cells were identified in forty three prostate biopsies (82%) and forty seven radical prostatectomy specimens (90%). There was a positive correlation between the expression of CD44 in cancer cells from prostate biopsies and in radical prostatectomy specimens. We also observed a negative correlation between CD44 expression in cancer cells and Gleason score, both in prostate biopsies and in radical prostatectomy material. When other clinical data were analysed, such as patient age, preoperative PSA serum levels or tumour stage (pT), only the first parameter was found to correlate positively with CD44 expression in prostate biopsies. CONCLUSION: To the best of our knowledge this study showed for the first time, that the level of CD44 expression in prostate biopsies correlates with that observed in matched radical prostatectomy specimens. Additionally, we confirmed the results of other authors which show that the percentage of CD44 expressing cancer cells in prostate biopsies negatively correlates with Gleason grade of tumours, both in prostate biopsies and in radical prostatectomy material. Citation Format: Konstanty Korski, Anna Malicka, Jan Breborowicz. Expression of the putative stem cell marker CD44 in prostate biopsies correlates with Gleason score in radical prostatectomies in patients with prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 254. doi:10.1158/1538-7445.AM2013-254

Abstract 5207: FGFR inhibitor regulates CD133 expression in FGFR2-amplified gastric cancer cells

Abstract The underlying mechanism regulating the expression of the cancer stem cell / tumor-initiating cell marker CD133/prominin-1 in cancer cells remains largely unclear, although knowledge of this mechanism would likely provide important biological information regarding cancer stem cells. We recently have reported that the expression of CD133 in cancer cells could be regulated though the inhibition of signal transduction by molecular-targeted drugs (Matsumoto K. et al. Cancer Res. 2009). Here, we screened the expression changes of CD133 using wide variety of molecular-targeted drugs in FGFR2-amplified and CD133-overexpressing gastric cancer cell lines. CD133 expression was remarkably upregulated by FGFR inhibitor PD173074 exposure in these cells. Further analysis revealed that PD173074 upregulated the CD133 expression both mRNA and protein levels in dose- and time-dependent manners. In addition, the expression change was correlated with the mRNA expression levels of stem cell factors including OCT3/4, SOX2 and NANOG. As expected, PD173074 exhibited a potent growth inhibitory effect on FGFR2-amplified gastric cancer cells in vitro. These results suggest that the mechanism regulating the expression of CD133 is involved in FGFR2 signaling and also in addicted signaling pathway for cancer cells in FGFR2-amplified gastric cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5207. doi:10.1158/1538-7445.AM2011-5207

CD133 suppresses neuroblastoma cell differentiation via signal pathway modification

CD133 (prominin-1) is a transmembrane glycoprotein expressed on the surface of normal and cancer stem cells (tumor-initiating cells), progenitor cells, rod photoreceptor cells and a variety of epithelial cells. Although CD133 is widely used as a marker of various somatic and putative cancer stem cells, its contribution to the fundamental properties of cancer cells, such as tumorigenesis and differentiation, remains to be elucidated. In the present report, we found that CD133 was expressed in several neuroblastoma (NB) cell lines/tumor samples. Intriguingly, CD133 repressed NB cell differentiation, for example neurite extension and the expression of differentiation marker proteins, and was decreased by several differentiation stimuli, but accelerated cell proliferation, anchorage-independent colony formation and in vivo tumor formation of NB cells. NB cell line and primary tumor-sphere experiments indicated that the molecular mechanism of CD133-related differentiation suppression in NB was in part dependent on neurotrophic receptor RET tyrosine kinase regulation. RET transcription was suppressed by CD133 in NB cells and glial cell line-derived neurotrophic factor treatment failed to induce RET in CD133-expressing cells; RET overexpression rescued CD133-related inhibition of neurite elongation. Of note, CD133-related NB cell differentiation and RET repression were mainly dependent on p38MAPK and PI3K/Akt pathways. Furthermore, CD133 has a function in growth and RET expression in NB cell line- and primary tumor cell-derived tumor spheres. To the best of our knowledge, this is the first report of the function of CD133 in cancer cells and our findings may be applied to improve differentiation induction therapy for NB patients.

Abstract 4302: Human endometrial cancer cell CD133+ cell fractions are regulated by methylation

Abstract Like other solid tumors, endometrial tumors have been shown to contain a subset of tumor initiating cells although little is known about how these rare sub-fractions are regulated. Our primary objective was to analyze potential epigenetic regulation of such a tumor initiating cell population in human endometrial cancer cells. To accomplish this, we demonstrated by flow cytometry that primary endometrial tumors contain CD133+ cells. To assess their tumor initiating capacity, serially transplanted endometrial human tumor explants generated in NOD/SCID mice were harvested, enzymatically dissociated, depleted of H-2Kd+ mouse cells and sorted via flow cytometry to generate relatively pure (&amp;gt; 98.8 %) CD133+ and CD133− fractions. These positive and negative fractions were serially diluted and subcutaneously injected into immunocompromised mice. The CD133+ fractions had a significantly increased capacity for tumor formation relative to the CD133− fractions and this difference was more pronounced as the number of injected cells decreased. Interestingly, the level of CD133+ tumor cells appeared to be enriched following serial transplantation as evidenced by flow cytometric and immunohistochemical analyses. It has been proposed that methylation may play a role in regulation of tumor initiating cells. To investigate this possibility in endometrial cancer, we isolated DNA from serially transplanted tumors and analyzed the methylation status of CpG islands located upstream of the CD133 transcription start site. This region was shown to be a target of methylation, which led us to determine whether changing the methylation status would alter CD133 expression in endometrial cancer cells. We treated 4 individual human endometrial cancer cell lines with either vehicle or 5 μM 5-aza-2′-deoxyctidine (5-aza-DC), for 72 hours, and evaluated post-treatment levels of CD133 expression by RT-PCR and flow cytometry. In the tested cell lines, CD133 mRNA levels, as measured by RT-PCR, were increased following treatment with 5-aza-DC suggesting that methylation of the CD133 promoter was suppressing its expression. To extend this finding, we analyzed the percentage of CD133 expressing cells in either vehicle treated or 5-aza-DC by flow cytometry. Consistent with the RT-PCR results, the frequency of CD133-expressing cells was increased in 3 of the 4 cell lines following treatment with 5-aza-DC. It is not clear however, if the 5-aza-DC mediated demethylation and subsequent shift in the percentage of CD133+ cells correlates with a shift in the frequency of cells that have increased tumor initiating capacity. Nevertheless, CD133 expression in human endometrial cancer cells is regulated at least in part by methylation and altering this may cause these tumor cells to become more sensitive to standard chemotherapy regimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4302.

MTOR Signal and Hypoxia-Inducible Factor-1α Regulate CD133 Expression in Cancer Cells

The underlying mechanism regulating the expression of the cancer stem cell/tumor-initiating cell marker CD133/prominin-1 in cancer cells remains largely unclear, although knowledge of this mechanism would likely provide important biological information regarding cancer stem cells. Here, we found that the inhibition of mTOR signaling up-regulated CD133 expression at both the mRNA and protein levels in a CD133-overexpressing cancer cell line. This effect was canceled by a rapamycin-competitor, tacrolimus, and was not modified by conventional cytotoxic drugs. We hypothesized that hypoxia-inducible factor-1 alpha (HIF-1 alpha), a downstream molecule in the mTOR signaling pathway, might regulate CD133 expression; we therefore investigated the relation between CD133 and HIF-1 alpha. Hypoxic conditions up-regulated HIF-1 alpha expression and inversely down-regulated CD133 expression at both the mRNA and protein levels. Similarly, the HIF-1 alpha activator deferoxamine mesylate dose-dependently down-regulated CD133 expression, consistent with the effects of hypoxic conditions. Finally, the correlations between CD133 and the expressions of HIF-1 alpha and HIF-1 beta were examined using clinical gastric cancer samples. A strong inverse correlation (r = -0.68) was observed between CD133 and HIF-1 alpha, but not between CD133 and HIF-1 beta. In conclusion, these results indicate that HIF-1 alpha down-regulates CD133 expression and suggest that mTOR signaling is involved in the expression of CD133 in cancer cells. Our findings provide a novel insight into the regulatory mechanisms of CD133 expression via mTOR signaling and HIF-1 alpha in cancer cells and might lead to insights into the involvement of the mTOR signal and oxygen-sensitive intracellular pathways in the maintenance of stemness in cancer stem cells.

High stromal hyaluronan level is associated with poor differentiation and metastasis in prostate cancer

Several epithelial tumours accumulate hyaluronan (HA) which promotes cancer cell invasion and metastasis. We analysed the expression of HA and its receptor CD44 and their prognostic value in 166 prostate cancer patients followed up for a mean of 13 years; standard deviation (S.D.) 2.7; range 8.7–21.4 years. HA was detected with a specific biotinylated probe prepared from cartilage aggrecan and link protein, and CD44 with an antibody recognising all forms of CD44. The peri- and intratumoral stroma from half of the patients strongly expressed immunohistochemically detectable HA in ⩽15% of the stromal area; the tumours in the remaining half expressed HA in >15% of the area. The staining of cancer cells for HA was scored positive or negative, and for CD44 the median value of 80% of positive tumour cells was used as a cut-off point. The expression of HA in cancer cells was weakly associated with perineural infiltration of the tumour (P=0.03) and high Gleason score (P=0.002). There was also a significant inverse relationship between the expression of HA and CD44 in cancer cells (P<0.001). The high level of HA in the peri-and intratumoral stroma was related to metastasis, high T-category, high Gleason score, perineural infiltration and high mitotic activity of the tumour (for all P<0.001). There was a significant inverse relationship between the expression of CD44 in cancer cells and high level of strong expression of HA in the tumour stroma (P<0.001). A low fraction of CD44-positive cells was related to a high TM-category, high Gleason score and rapid cell proliferation (for all P<0.0001; M/V P value=0.0013). In the univariate survival analysis, the high level of strong expression of HA in tumour stroma predicted an unfavourable outcome in the entire series (P=0.003) and also in the M0 tumours (P=0.07), while in T1–2 M0 tumours the prognostic value did not reach the level of statistical significance (P=0.1). A low fraction of CD44-positive cells predicted a poor outcome in the entire series (P<0.001) and also in M0 tumours (P=0.003). Cancer cell-associated HA expression had no prognostic value in any tumour categories. In the multivariate analysis of prognostic factors, HA expression in the cancer cells or in the tumour stroma had no additional value to the standard prognostic factors TM-classification, Gleason score and CD44 expression. Our results show that stromal HA accumulation is related to several malignant features and adverse clinical outcome in prostate cancer. However, further studies based on uniformly treated patient cohorts are needed to establish the clinical significance of these findings in current clinical practice.

Loss of standard type of CD44 expression in invaded area as a good indicator of lymph-node metastasis in colorectal carcinoma.

Recent advances have made possible the treatment of small invasive colorectal cancer by means of polypectomy or endoscopic mucosal resection. CD44 expression in cancer cells was identified as an indicator of lymph-node metastasis, which could be evaluated in specimens removed by colonoscopy. The correlation between lymph-node metastasis and the expression of standard-type CD44 in cancer cells was examined immunohistologically using the invaded cancer cells of 61 tissue samples of superficially invasive colorectal cancer. We defined the above as invasive cancer restricted within the colorectal wall. Of the 61 samples, 31 had submucosal invasion and 30 had muscular invasion. Standard-type CD44 expression in the area of invasion in cases with lymph-node metastasis was remarkably down-regulated. In 43 cases with no lymph-node metastasis, 36 (83.7 percent) of patients had CD44 expression in invaded cells, whereas only two of 18 cases (11.1 percent) with lymph-node metastasis had expression of standard-type CD44 in the same area (P < 0.0001). A total of 69.6 percent (16/23) of patients with loss of standard-type CD44 expression in invaded sites were found to have positive metastasis in the lymph nodes. These results suggest that standard-type CD44 in invasive colon cancer cells could suppress metastasis to the regional lymph nodes. In cases of invasive colorectal cancer, the loss of standard-type CD44 expression in the invaded area is a sensitive marker for metastasis to the lymph nodes. Further investigation with larger patient groups is required to clarify the reliability of loss of standard-type CD44 expression as an indicator for additional surgery after endoscopic resection of submucosal invasive colorectal carcinoma.