Flow cytometric immunophenotypic characteristics of 36 cases of plasma cell leukemia

Prospective flow cytometric analysis of antigens expression on bone marrow and peripheral blood plasma cells of 36 plasma cell leukemia (PCL) patients enabled to establish the following immunophenotype of leukemic plasma cell: CD38⁺⁺, CD138⁺, CD54⁺, CD49d⁺, CD29⁺, CD44⁺, CD126⁺, CD19⁻, CD45⁻. In one-third of patients PCL cells express CD56, CD71 and CD117. Expression of CD54 on plasma cells was higher as compared to expression of adhesion molecules CD11a, CD18 and CD11b (p < 0.01). Expression of CD18, CD11a, CD11b was lower on bone marrow and higher on peripheral blood cells. In conclusion, impaired expression of adhesion molecules such as CD11a/CD18 or CD56 may explain hematogenic dissemination characterizing PCL.     

Circulating tumor cells with a putative stem cell phenotype in peripheral blood of patients with breast cancer

The CD44⁺/CD24^−/low and ALDH1⁺ cell phenotypes are associated with stemness and enhanced tumorigenic potential in breast cancer. We assessed the expression of CD44, CD24 and ALDH1 on tumor cells circulating in the peripheral blood (CTCs) of patients with metastatic breast cancer using triple-marker immunofluorescence microscopy. Among a total of 1439 CTCs identified in 20 (66.7%) out of 30 patients, 35.2% had the stem-like/tumorigenic phenotype CD44⁺/CD24^−/low, whereas 17.7% of the CTCs analyzed in seven patients, were ALDH1^high/CD24^−/low. In conclusion, we report the existence of a subpopulation of CTCs with putative stem cell progenitor phenotypes in patients with metastatic breast cancer.     

Enriched CD44/CD24 population drives the aggressive phenotypes presented in triple-negative breast cancer (TNBC)

The mechanism underlying the aggressive behaviors of triple negative breast cancer (TNBC) is not well characterized yet. The association between cancer stem cell (CSC) population and the aggressive behaviors of TNBC has not been established. We found the CD44⁺/CD24⁻ cell population was enriched in TNBC tissues and cell lines, with a higher capacity of proliferation, migration, invasion and tumorigenicity as well as lower adhesion ability. The CD44⁺/CD24⁻ cell population with cancer stem cell-like properties may play an important role in the aggressive behaviors of TNBC. This discovery may lead to new therapeutic strategies targeting CD44⁺/CD24⁻ cell population in TNBC.     

Cancer stem cell marker expression in hepatocellular carcinoma and liver metastases is not sufficient as single prognostic parameter

Recent findings suggest that the presence of cancer stem cells could be linked with patients’ survival. We profiled suggested cancer stem cell markers in tissue specimens of hepatocellular carcinoma and colorectal carcinoma liver metastases. About 1% of cells co-expressed cancer stem cell antigens, but there was no correlation between the amount of CD133⁺, CD133⁺/CD44⁺ or CD133⁺/CD24⁻ cells and the patients’ clinical–pathological status or with the cancer stem cell marker-positive cells localization. CD133⁺ and CD133⁻ fractions of Huh7 cells did not differ in migratory properties. Therefore, presence of markers alone should be taken with caution as single prognostic parameters.     

Epithelial mesenchymal transition in early invasive breast cancer: an immunohistochemical and reverse phase protein array study

Epithelial mesenchymal transition (EMT), as defined by loss of epithelial characteristics and gain of a mesenchymal phenotype, has been reported in vivo although the occurrence of events remains unclear. This study aims at exploration of EMT portraits of breast cancer (BC) with relevance to different molecular pathways, especially potential EMT triggers and BC molecular subtypes. Immunohistochemical (IHC) expression of markers/triggers of EMT was studied on a well-defined cohort of invasive non-lobular BC (n = 1,035), prepared as tissue microarrays. IHC panel of biomarkers included cadherins (cad; E-cad and N-cad), TGFβ1, PIK3CA, pAkt, and others. Reverse phase protein array (RPPA) was performed for quantitative analysis of proteins extracted from formalin fixed paraffin embedded tissues of a subset of cases from this cohort. Four combinatorial phenotypic groups representing cadherin switch were defined, including E-cad⁺/N-cad^?, E-cad^?/N-cad^?, E-cad⁺/N-cad⁺, and E-cad^?/N-cad⁺. Statistically significant association was noticed between these phenotypes and histological tumour grade, tumour type and size and NPI staging classes. The E-cad/N-cad switch occurred more frequently in the triple negative molecular class, both basal and non-basal, and in the HER2⁺ subtype than in luminal BC. Significant outcome differences were observed between cadherin switch combinatorial groups regarding BCSS and DMFS (p < 0.001). Results of RPPA confirm those observed using IHC regarding differential expressions of EMT markers/triggers. EMT/cadherin switch programs in BC appear to occur in synergy with TGFβ1 and PIK3/Akt pathways activation. These data explain, at translational proteomic level, the molecular heterogeneity and in turn the varied clinical behaviour of BC molecular subtypes. RPPA is a promising high-throughput technique in monitoring subtle quantitative changes in protein expression in archival material.     

CD133 expression is not selective for tumor-initiating or radioresistant cell populations in the CRC cell lines HCT-116

Background and purpose

CD133 is controversially discussed as putative (surrogate) marker for cancer stem/tumor-initiating cell populations (CSC/TIC) in epithelial tumors including colorectal carcinomas (CRCs). We studied CD133 expression in established CRC cell lines and examined in vitro behavior, radioresponse and in vivo tumor formation of CD133^+/− subpopulations of one cell line of interest.

Materials and methods

Ten CRC cell lines were analyzed for CD133 expression using flow cytometry and Western blotting. CD133⁺ and CD133⁻ HCT-116 subpopulations were separated by FACS and studied in 2-D and 3-D culture and colony formation assays after irradiation. Subcutaneous xenograft formation was monitored in NMRI (nu/nu) mice.

Results and conclusions

CRC cell lines could be classified into three groups: (i) CD133⁻, (ii) CD133⁺ and (iii) those with two distinct CD133⁺ and CD133⁻ subpopulations. Isolated CD133^+/− HCT-116 subpopulations were studied relative to the original fraction. No difference was found in 2-D growth, spheroid formation or radioresponse in vitro. Also, tumor formation and growth rate did not differ for the sorted subpopulations. However, a subset of xenografts originated from CD133⁻ HCT-116 showed a striking enrichment in the CD133⁺ fraction. Our data show that CD133 expression is not selective for sphere forming, tumor-initiating or radioresistant subpopulations in the HCT-116 CRC cell lines. This implies that CD133 cannot be regarded as a CSC/TIC marker in all CRC cell lines and that functional measurements of tumor formation have to generally accompany CSC/TIC-directed mechanistic or therapeutic studies.     

Enrichment of N-Cadherin and Tie2-bearing CD34/CD38/CD123 leukemic stem cells by chemotherapy-resistance

Acute myeloid leukemia (AML) arises from genetic changes at the level of stem cell, various mutations have been elucidated, including AML1–ETO fusion gene has been shown as the representative target of cellular transformation for LSCs originating from hematopoietic stem cells (HSCs) compartment. LSCs resemble HSCs with respect to self-renewal capacity and chemotherapy-resistance. However, LSCs possess specific cell-surface markers, they are proposed to reside within the CD34⁺/CD38⁻/CD123⁺ compartment. And the interaction mediated by adhesion molecules between LSCs and niche played a role in chemoresistance of LSCs. Therefore, study on the LSCs surface makers related to niche is helpful for the potential target therapy in the future. In this study, the proportions of CD34⁺/CD38⁻/CD123⁺ LSCs compartment co-expressing the three adhesion molecules, N-Cadherin, Tie2 and CD44, respectively, from AML patients before and after chemotherapy were analyzed. We demonstrated N-Cadherin and Tie2 positive CD34⁺/CD38⁻/CD123⁺ LSCs populations could be enriched by chemotherapy. Furthermore, AML1/ETO fusion signals and MDR1 expression were detected on the CD34⁺/CD38⁻/CD123⁺ LSCs populations expressing N-Cadherin and Tie2. Therefore, N-Cadherin and Tie2 are probably the potential markers for identification of LSCs.     

CD133 expression is not selective for tumor-initiating or radioresistant cell populations in the CRC cell line HCT-116

Background and purpose

CD133 is controversially discussed as putative (surrogate) marker for cancer stem/tumor-initiating cell populations (CSC/TIC) in epithelial tumors including colorectal carcinomas (CRCs). We studied CD133 expression in established CRC cell lines and examined in vitro behavior, radioresponse and in vivo tumor formation of CD133^+/− subpopulations of one cell line of interest.

Materials and methods

Ten CRC cell lines were analyzed for CD133 expression using flow cytometry and Western blotting. CD133⁺ and CD133⁻ HCT-116 subpopulations were separated by FACS and studied in 2-D and 3-D culture and colony formation assays after irradiation. Subcutaneous xenograft formation was monitored in NMRI (nu/nu) mice.

Results and conclusions

CRC cell lines could be classified into three groups: (i) CD133⁻, (ii) CD133⁺ and (iii) those with two distinct CD133⁺ and CD133⁻ subpopulations. Isolated CD133^+/− HCT-116 subpopulations were studied relative to the original fraction. No difference was found in 2-D growth, spheroid formation or radioresponse in vitro. Also, tumor formation and growth rate did not differ for the sorted subpopulations. However, a subset of xenografts originated from CD133⁻ HCT-116 showed a striking enrichment in the CD133⁺ fraction. Our data show that CD133 expression is not selective for sphere forming, tumor-initiating or radioresistant subpopulations in the HCT-116 CRC cell line. This implies that CD133 cannot be regarded as a CSC/TIC marker in all CRC cell lines and that functional measurements of tumor formation have to generally accompany CSC/TIC-directed mechanistic or therapeutic studies.     

Y-box-binding protein 1 (YB1) in breast carcinomas: Relation to aggressive tumor phenotype and identification of patients at high risk for relapse

Aims

To investigate the expression pattern of Y-box-binding protein 1 (YB1) in breast carcinomas, its clinicopathological and prognostic value, and its association with the breast cancer stem cell phenotype [CD44⁺/CD24^−/low].

Methods and results

Immunohistochemistry was performed on 225 paraffin embedded specimens of invasive breast carcinomas to detect the expression of the proteins YB1, ER, PR, HER2, p53, Ki67, bcl-2, CD44 and CD24. YB1 protein was detected in the nuclei, the cytoplasm and the stroma of the tumor cells. Cytoplasmic YB1 was detected more often in carcinomas of ductal type (p = 0.002), of higher nuclear grade (p < 0.001), with lack of ER expression (p = 0.002), positive expression of p53 and Ki67 (p = 0.002 and p = 022, respectively), and with present CD44⁺/CD24^−/low breast cancer stem cells (p = 0.001), while its association with bcl-2 was found to be inverse (p = 0.042). Nuclear YB1 was found to exert unfavorable impact on the disease-free survival of the unselected patients (p = 0.05) and the patients having been subjected to adjuvant chemotherapy and radiotherapy (p = 0.036 and p = 0.05, respectively).

Conclusions

Cytoplasmic YB1 is associated with an aggressive and “stem cell-like” tumor phenotype, while nuclear localization discriminates patients at high risk for recurrence, especially those who are subjected to chemo- and radiotherapy.     

Distinct sensitivity of CD8CD4 and CD8CD4 leukemic cell subpopulations to cyclophosphamide and rapamycin in Notch1-induced T-ALL mouse model

The Notch1 signaling pathway plays an essential role in cell growth and differentiation. Over-expression of the intracellular Notch1 domain (ICN1) in murine hematopoietic cells is able to induce robust T-cell acute lymphoblastic leukemia (T-ALL) in mice. Here we explored the drug sensitivity of T-ALL cells in two subpopulations of CD8⁺CD4⁺ and CD8⁺CD4⁻ cells in Notch1-induced T-ALL mice. We found that Notch1 induced T-ALL cells could be decreased by chemotherapeutic drug cyclophosphamide (CTX). CD8⁺CD4⁻ T-ALL cells were more sensitive to CTX treatment than CD8⁺CD4⁺ T-ALL cells. The percentage of apoptotic cells induced by CTX treatment was higher in CD8⁺CD4⁻ T-ALL cells. T-ALL cells were also inhibited by inhibitor of mTORC1 rapamycin. CD8⁺CD4⁺ T-ALL cells were more susceptible to rapamycin treatment than CD8⁺CD4⁻ T-ALL cells. Rapamycin treatment selectively arrested more CD8⁺CD4⁺ T-ALL cells at G0 phase of cell cycle. A combination of the two drugs significantly improved overall survival of T-ALL bearing mice when compared with CTX or rapamycin alone. These results indicated that CD8⁺CD4⁺ and CD8⁺CD4⁻ leukemia cell populations had distinct drug sensitivity.     

Histological and prognostic importance of CD44+/CD24+/EpCAM+ expression in clinical pancreatic cancer

CD44⁺/CD24⁺/EpCAM⁺ cells have been reported to be cancer stem cells in pancreatic cancer; however, the histological and clinical importance of these cells has not yet been investigated. Here we clarified the characteristics of CD44⁺/CD24⁺/EpCAM⁺ cells in clinical specimens of pancreatic cancer using immunohistochemical assay. We used surgical specimens of pancreatic ductal adenocarcinoma from 101 patients. In view of tumor heterogeneity, we randomly selected 10 high‐power fields per case, and triple‐positive CD44⁺/CD24⁺/EpCAM⁺ expression was identified using our scoring system. The distribution, histological characteristics, and prognostic importance of CD44⁺/CD24⁺/EpCAM⁺ cells were then analyzed. As a result, the distribution of CD44⁺/CD24⁺/EpCAM⁺ cells varied widely among the 101 cases examined, and CD44⁺/CD24⁺/EpCAM⁺ expression was correlated with poor glandular differentiation and high proliferation. Survival analysis showed that CD44⁺/CD24⁺/EpCAM⁺ expression was not correlated with patient outcome; however, CD44⁺/CD24⁺ expression appeared to be correlated with poor prognosis. In conclusion, CD44⁺/CD24⁺/EpCAM⁺ expression overlapped with poorly differentiated cells and possessed high proliferative potential in clinical pancreatic cancer. In particular, the presence of double‐positive CD44⁺/CD24⁺ expression seemed to have clinical relevance, associating with poor prognosis.     

CD15<Superscript>+</Superscript>/CD16<Superscript>low</Superscript> human granulocytes from terminal cancer patients: granulocytic myeloid-derived suppressor cells that have suppressive function

Myeloid-derived suppressor cells (MDSCs) are a subpopulation of myeloid cells with immunosuppressive function whose numbers are increased in conditions such as chronic infection, trauma, and cancer. Unlike murine MDSCs defined as CD11b⁺/Gr-1⁺, there are no specific markers for human MDSCs. The goal of this study was to delineate a specific human MDSCs subpopulation in granulocytes from terminal cancer patients and investigate its clinical implications. Here, we show that the CD15⁺/CD16^low subset was increased in terminal cancer patients compared with healthy donors (P = 0.009). Phorbol 12-myristate 13-acetate-activated granulocytes (CD16^low/CD66b⁺⁺/CD15⁺) that have a phenotype similar to MDSCs from cancer patients, effectively suppressed both proliferation and cytotoxicity of normal T cells. Among cancer patients, T-cell proliferation was highly suppressed by granulocytes isolated from terminal cancer patients with a high proportion of CD15⁺/CD16^low cells. Patients with low peripheral blood levels of CD15⁺/CD16^low cells had significantly longer survival than those with high levels (P = 0.0011). Patients with higher levels of CD15⁺/CD16^low also tended to have poor performance status (P = 0.05). These data suggest that CD15⁺/CD16^low granulocytes found in terminal cancer patients may play a role in the progression of cancer by inhibiting tumor immunity.     

CD44 variant 9 is a potential biomarker of tumor initiating cells predicting survival outcome in hepatitis C virus‐positive patients with resected hepatocellular carcinoma

This study investigated whether the expression of CD44 variant 9 (CD44v9) might be a functional marker of tumor‐initiating stem‐like cells in primary hepatocellular carcinomas (HCCs) of hepatitis C virus (HCV)⁺ patients and provide an indicator of patient survival, as well as associated mechanisms. A total of 90 HCV⁺ HCC patients who underwent surgery from 2006 to 2011 were enrolled and monitored for 2–8 years. Expression of CD44v9 was validated immunohistochemically in all HCCs, followed by comparative proteome, survival, and clinicopathological analyses. CD44 variant 8–‐10 was further evaluated in diethylnitrosamine‐induced HCCs of C57Bl/6J mice. Focally localized CD44v⁺ cells with a membranous staining pattern were detected in human HCV⁺ and mouse HCCs. CD44v9⁺ cells of HCCs were predominantly negative for Ki67 and P‐p38, indicating decrease of cell proliferation in the CD44v9⁺ tumor cell population, likely to be related to suppression of intracellular oxidative stress due to activation of Nrf2‐mediated signaling, DNA repair, and inhibition of xenobiotic metabolism. CD44v9 IHC evaluation in 90 HCV⁺ HCC cases revealed that positive expression was significantly associated with poor overall and recurrence‐free survival, a younger age, poor histological differentiation of HCCs, and high alkaline phosphatase levels compared with patients with negative expression. CD44v9 is concluded to be a potential biomarker of tumor‐initiating stem‐like cells and a prognostic marker in HCV⁺ HCC patients associated with Nrf2‐mediated resistance to oxidative stress.     

Cancer Stemness,Immune Cells,and Epithelial–Mesenchymal Transition Cooperatively Predict Prognosis in Colorectal Carcinoma

Background

Tumor tissues consist of heterogeneous cancer cells and stroma cells, including cancer stem cells and immune cells. Epithelial–mesenchymal transition (EMT) programs closely associate with acquisition of stemness. We investigated for the first time the clinical significance of combining cancer stem cells, immune cells, and EMT traits.

Identification and analysis of CD133 melanoma stem-like cells conferring resistance to taxol: An insight into the mechanisms of their resistance and response

The presence and the involvement of cancer stem-like cells (CSCs) in tumor initiation and progression, and chemo-resistance are documented. Herein, we functionally analyzed melanoma stem-like cells (MSC)/CD133⁺ cells on their resistance and response to taxol-induced apoptosis. Besides being taxol resistant, the CD133⁺ cells demonstrated a growth advantage over the CD133⁻ subpopulation. Taxol induced apoptosis on CD133⁻ cells, but not on CD133⁺ cells. In the CD133⁻ subpopulation, the exposure to taxol induced the activation of apoptosis signal-regulating kinase1 (ASK1)/c-jun-N-terminal kinase (JNK), p38, extracellular signal regulated kinase (ERK) pathways and Bax expression, while in CD133⁺ cells taxol was able only to enhance the activity of the ERK pathway. In CD133⁺ cells, the direct gene transfer of Bax overcame the acquired resistance to taxol. Taken together, our data provide an insight into the mechanistic cascade of melanoma resistance to taxol and suggest Bax gene transfer as a complementary approach to chemotherapy.     

The CD44<Superscript>+</Superscript>/CD24<Superscript>-</Superscript>phenotype is enriched in basal-like breast tumors

Introduction  

Human breast tumors are heterogeneous and consist of phenotypically diverse cells. Breast cancer cells with a CD44⁺/CD24^- phenotype have been suggested to have tumor-initiating properties with stem cell-like and invasive features, although it is unclear whether their presence within a tumor has clinical implications. There is also a large heterogeneity between tumors, illustrated by reproducible stratification into various subtypes based on gene expression profiles or histopathological features. We have explored the prevalence of cells with different CD44/CD24 phenotypes within breast cancer subtypes.     

Effects of dexmedetomidine on immune response in patients undergoing radical and reconstructive surgery for oral cancer

Oral cancer is one of the most common malignancies in the world. The present study aimed to investigate the effects of dexmedetomidine on immune response in patients undergoing radical and reconstructive surgery for oral cancer. Patients were randomly divided into the dexmedetomidine and control groups. Within 15 min before anesthesia induction, dexmedetomidine was infused with a 0.5 µg·kg⁻¹ loading dose followed by a maintenance dose of 0.4 µg·kg⁻¹·h⁻¹ to the end of operation in the dexmedetomidine group, whereas the same volume of saline was administered in the control group. Blood samples were obtained at five time-points: 30 min Before induction (T₀), 1 h after induction (T₁), end of the operation (T₂) and 24 (T₃) and 48 h (T₄) after the operation. The T lymphocyte subsets (including CD3⁺, CD4⁺ and CD8⁺ cells) and CD4⁺/CD8⁺ ratio, B lymphocytes, dendritic cells and myeloid-derived suppressor cells (MDSCs) were analyzed by flow cytometry. All immunological indicators, except CD8⁺ cells, significantly decreased between the two groups at T_1–3 compared with T₀ (P<0.05). The percentages of CD3⁺, CD4⁺, dendritic cells and the CD4⁺/CD8⁺ ratios were significantly higher at T_2–4 and the percentages of MDSCs were significantly lower at T_2–4 in the dexmedetomidine group compared with the control group (all P<0.05). These findings suggested that dexmedetomidine can attenuate immunosuppression in patients undergoing radical and reconstructive surgery for oral cancer.     

CD44v3 and VEGF-C Expression and its Relationship with Lymph Node Metastasis in Squamous Cell Carcinomas of the Uterine Cervix

Background: To investigate the expression of CD44v3 and vascular endothelial growth factor-C (VEGF-C) and their relationship with lymph node metastasis in squamous cell carcinomas (SCC) of the uterine cervix. Materials and Methods: Expression of CD44v3 and VEGF-C was analyzed in 109 cases of cervical SCC by immunohistochemistry (IHC). The relationship was analyzed between expression and the patient age, histological differentiation, formation of tumor emboli in lymphoid vessels, lymph node metastasis, FIGO staging, and TNMclassification. Results: Expression rates for both CD44v3 and VEGF-C were 43.1% in cervical SCC. The cells with positive immunohistochemical staining of CD44v3 were distributed mainly around the keratin pearls in well differentiated carcinomas, but distributed diffusely in the moderately and poorly differentiated lesions. VEGF-C was found stained positively in most of the tumor cells. There were differences in expression between normal epithelium and atypical hyperplasia as well as carcinoma. Both CD44v3 and VEGF-C were found to beassociated positively with lymph node metastasis and TNM classification (both p=0.000). Neither CD44v3 nor VEGF-C was found to be associated with patient age, histological differentiation, formation of tumor emboli in lymphoid vessels and FIGO staging. CD44v3 was found to be associated with VEGF-C positively (p=0.000). Conclusions: Abnormal expression of CD44v3 and VEGF-C is associated closely with the lymph node metastasis in cervical SCC, and these agents may cooperate in carcinogenesis and development of metastatic lesions.     

CD44+/CD24- phenotype contributes to malignant relapse following surgical resection and chemotherapy in patients with invasive ductal carcinoma

Background

Invasive ductal carcinoma is the most common type of breast malignancy, with varying molecular features and resistance to treatment. Although CD44+/CD24- cells are believed to act as breast cancer stem cells and to be linked to poor prognosis in some patients, the association between these cells and tumor recurrence or metastasis in all or some types of invasive ductal carcinoma is unclear.

Methods

A total of 147 randomly selected primary and secondary invasive ductal carcinoma samples were assayed for expression of CD44, CD24, ER, PR, and Her2. The association between the proportions of CD44+/CD24- tumor cells and the clinico-pathological features of these patients was evaluated.

Results

CD44+/CD24- tumor cells were detected in 70.1% of the tumors, with a median proportion of 5.8%. The proportion of CD44+/CD24- tumor cells was significantly associated with lymph node involvement (P = 0.026) and PR status (P = 0.038), and was correlated with strong PR status in patients with recurrent or metastatic tumors (P = 0.046) and with basal-like features (p = 0.05). The median disease-free survival (DFS) of patients with and without CD44⁺/CD24^-/low tumor cells were 22.9 ± 2.2 months and 35.9 ± 3.8 months, and the median overall survival (OS) of patients with and without CD44⁺/CD24^-/low tumor cells were 39.3 ± 2.6 months and 54.0 ± 3.5 months, respectively, and with both univariate and multivariate analyses showing that the proportion of CD44⁺/CD24^-/low tumor cells was strongly correlated with DFS and OS.

Conclusion

The prevalence of CD44+/CD24- tumor cells varied greatly in invasive ductal carcinomas, with the occurrence of this phenotype high in primary tumors with high PR status and in secondary tumors. Moreover, this phenotype was significantly associated with shorter cumulative DFS and OS. Thus, the CD44⁺/CD24^- phenotype may be an important factor for malignant relapse following surgical resection and chemotherapy in patients with invasive ductal carcinoma.