Influence of ovarian cancer type I and type II microenvironment on the phenotype and function of monocyte-derived dendritic cells

PURPOSE

The aim of this study was to evaluate the influence of ovarian cancer cell lysates isolated from type I or type II ovarian cancer (OC) on the phenotype of monocyte-derived dendritic cells (Mo-DCs) and the cytokine profile. We also determined whether the Mo-DCs and tumor microenvironment, reflected by peritoneal fluid (PF) from type I or II ovarian cancer, could promote regulatory T cell (Tregs) differentiation from naive CD4⁺ lymphocytes in vitro.

RESULTS

Our results show a significant role of the ovarian cancer microenvironment reflected by PF from type I or II OC in the inhibition of the DC differentiation process. Interestingly, the percentage of cells co-expressing CD45 and CD14 antigens in the cultures stimulated with PF from both type I and type II OC was higher than in the control. Furthermore, the percentage of cells expressing CD1a, i.e., a marker of immature DCs, was significantly reduced in the cultures stimulated with PF from type I and type II OC. The results obtained show that ovarian cancer type II lysates induce differentiation of monocytes into macrophage-like cells with a CD1a⁺/HLA-DR⁺/CD83^? phenotype and significantly higher CD86/HLA-DR expression. We show that ovarian cancer type II Mo-DCs are able to prevent an immune response by release of IL-10, whereas OC type I Mo-DCs can promote the generation of Tregs.

CONCLUSIONS

We demonstrate that each type of ovarian cancer can induce a unique phenotype of DCs and differentiation of Tregs, both associated with immune-suppressive function, which may be an obstacle while developing effective anticancer dendritic cell vaccination.

     

The correlation of CD19 + CD24 + CD38 + B cells and other clinicopathological variables with the proportion of circulating Tregs in breast cancer patients

Background

T regulatory cells (Tregs) are known to negatively control immune response. The frequency of these cells was inversely correlated with clinical outcomes of breast cancer. CD19⁺CD24^hiCD38^hi cells also play a critical role in inflammation and autoimmune disease. However, their function in tumor immune response is less studied. In this study we aimed to determine the role of CD19⁺CD24^hiCD38^hi cells and some other clinicopathological variables in increasing the proportion of Tregs in breast cancer patients.

Methods

We selected 47 patients with invasive ductal breast carcinoma and 50 healthy controls and obtained their blood samples.

Results

The proportion of circulating CD4⁺CD25⁺Foxp3⁺ Tregs and CD19⁺CD24^hiCD38^hi cells was significantly increased in breast cancer patients. We also found that increased proportion of Tregs in breast cancer is correlated with HER2 amplification, advanced clinical stages, serum TGF-β1 and increased CD19⁺CD24^hiCD38^hi cells in the peripheral blood.

Conclusion

Altogether, our data suggest that as much as Tregs, CD19⁺CD24^hiCD38^hi B cells could also have a part in the suppression of immune response in breast cancer.

     

Tumor-infiltrating CD45RO<Superscript>+</Superscript> memory cells are associated with a favorable prognosis breast cancer

Background

CD45RO is a marker for memory lymphocytes. Whether CD45RO⁺ tumor-infiltrating lymphocytes (TILs) prevent breast cancer recurrence is unclear.

Methods

In the present study, we evaluated CD45RO expression in TILs as a predictor of prognosis in 98 patients with breast cancer who underwent radical surgery without neoadjuvant chemotherapy. Patients were classified as CD45RO⁺/TILs^High or CD45RO⁺/TILs^Low based on median immunohistochemistry levels.

Results

CD45RO⁺/TILs^High were associated with smaller tumor size. The CD45RO⁺/TILs^High group also had significantly fewer metastatic lymph nodes (P = 0.0082) and fewer peritumoral lymphatic invasions (P = 0.0284). The CD45RO⁺/TILs^High group enjoyed longer recurrence-free survival (P = 0.0453) but not cancer-specific survival (P = 0.0640) in univariate analysis.

Conclusions

These results suggested that CD45RO⁺ effector cells may both help eradicate local tumors and prevent metastases to the lymphatic systems in breast cancer patients. High ratio of CD45RO expressing TILs was associated with recurrence-free survival improvement and a trend toward cancer-specific survival improvement in breast cancer patients.

     

Cross-priming of cyclin B1, MUC-1 and survivin-specific CD8+ T cells by dendritic cells loaded with killed allogeneic breast cancer cells

Introduction

The ability of dendritic cells (DCs) to take up whole tumor cells and process their antigens for presentation to T cells ('cross-priming') is an important mechanism for induction of tumor specific immunity.

Methods

In vitro generated DCs were loaded with killed allogeneic breast cancer cells and offered to autologous naïve CD8⁺ T cells in 2-week and/or 3-week cultures. CD8⁺ T cell differentiation was measured by their capacity to secrete effector cytokines (interferon-γ) and kill breast cancer cells. Specificity was measured using peptides derived from defined breast cancer antigens.

Results

We found that DCs loaded with killed breast cancer cells can prime naïve CD8⁺ T cells to differentiate into effector cytotoxic T lymphocytes (CTLs). Importantly, these CTLs primed by DCs loaded with killed HLA-A*0201^- breast cancer cells can kill HLA-A*0201⁺ breast cancer cells. Among the tumor specific CTLs, we found that CTLs specific for HLA-A2 restricted peptides derived from three well known shared breast tumor antigens, namely cyclin B1, MUC-1 and survivin.

Conclusion

This ability of DCs loaded with killed allogeneic breast cancer cells to elicit multiantigen specific immunity supports their use as vaccines in patients with breast cancer.     

Higher densities of Foxp3<Superscript>+</Superscript> regulatory T cells are associated with better prognosis in triple-negative breast cancer

Purpose

The role of Forkhead Box Protein 3 (Foxp3) expressing regulatory T cells (Tregs) in breast cancer remains unclear. We examined the abundance and localisation of total T cells, B cells and Tregs within samples from triple-negative breast cancers (TNBCs) and asked whether these parameters were associated with clinicopathological features of the cancer or clinical outcomes.

Methods

Samples from TNBCs diagnosed between 2003 and 2010 in Singapore were divided into “high” and “low” intra-tumoural or stromal groups, based on whether they had higher or lower than median densities of specific tumour-infiltrating lymphocyte populations (CD3⁺ total T cells, Foxp3⁺CD3⁺ Tregs, or CD20⁺ B cells) in the intra-tumoural space or stroma.

Results

Of the 164 samples, patients bearing tumours with high Tregs within their intra-tumoural, but not stromal, areas experienced significantly longer overall and disease-free survival compared to individuals with low Treg densities. These “high intra-tumoural Treg” tumours were also characterised by relatively higher frequencies of CD8⁺ T cells and CD20⁺ B cells, and expressed significantly higher levels of some genes associated with inflammation, immune cell functions and trafficking, altogether consistent with a more “immune-activated” tumour microenvironment, in contrast to tumours bearing lower densities of Tregs.

Conclusions

In summary, the combination of high densities of intra-tumoural Tregs, CD8⁺ T cells and CD20⁺ B cells represents a favourable prognostic panel in TNBCs. These data also indicate new avenues for further investigation on the interaction between immune cell types within the tumour microenvironment and highlight the potential of Treg density and localisation within tumours to affect clinical outcome.

     

Functional heterogeneity of circulating T regulatory cell subsets in breast cancer patients

Background

Regulatory T cells (Tregs) play a major role in tumor escape from immunosurveillance by suppressing effector cells. The number of Tregs is increased in tumor sites and peripheral blood of breast cancer patients. However, the data regarding phenotypic and functional heterogeneity of Treg subpopulations in breast cancer are limited. The present study aimed to investigate the number and suppressive potential of Tregs that possess natural naïve-(N nTregs), effector/memory-like (EM nTregs), and Tr1-like phenotypes in breast cancer patients and healthy women.

Methods

The study included 10 HW and 17 primary breast cancer patients. Numbers of CD4⁺CD25⁺FoxP3⁺CD45RA⁺ N nTregs, CD4⁺CD25⁺FoxP3⁺CD45RA^? EM nTregs, and CD4⁺IL-4^?IL-10⁺ Tr1 subsets and the expression of CTLA-4, CD39, GITR, LAP, and IL-35 by these Treg subsets were measured in freshly obtained peripheral blood by flow cytometry.

Results

Herein, we demonstrate that the percentages of N nTregs, EM nTregs, CD25⁺ and FoxP3⁺ Tr1 cells are elevated in the peripheral blood of breast cancer patients, but do not correlate with cancer stages. Nevertheless, the frequency of CD25⁺ Tr1 cells was associated with nodal involvement, while the number of EM nTregs correlated with clinical outcome. The expression of CTLA-4 and IL-35 by all assessed Treg subsets was increased throughout all tumor stages (I–III).

Conclusions

Collectively, the current study shows phenotypic alterations in suppressive receptors of Treg subsets, suggesting that breast cancer patients have increased activity of N nTregs, EM nTregs and Tr1 cells; and EM nTregs and CD25⁺ Tr1 cells represent prospective markers for assessing disease prognosis.

     

Breast cancer stem cells characterized by CD70 expression preferentially metastasize to the lungs

Background

Cancer stem cells (CSCs) are believed to form metastases. We sought to determine whether CD70?+?subpopulation in human breast cancers represents the CSCs accounting for distant metastasis.

Methods

We measured the expression levels of CD70 in breast cancer cell lines and 122 primary breast cancer samples. We characterized the functional roles of CD70?+?subpopulation in distant metastasis of breast cancers.

Results

We observed a distinct pattern of CD70 expression in a panel of primary breast carcinoma samples, indicating that CD70 serves as a biomarker of lung-specific metastasis. CD70? and CD70+?cell populations isolated from breast cancer cell lines exhibited epithelial and mesenchymal phenotypes, respectively. CD70+?cells, but not CD70? cells, possessed self-renewal and differentiation potentials. Tumorsphere formation in suspension cultures and in vivo tumorigenicity were significantly greater in CD70+?cells than in CD70? cells. Furthermore, the development of lung metastases induced by orthotopic injection was markedly increased in mice inoculated with CD70?+?cells. CD70 contributed to the promotion of lung metastases by enhancing self-renewal potential of CD70?+?cells.

Conclusions

We isolated CSCs from primary human breast cancers and found that CD70?+?subpopulations mediate lung-specific metastasis. These findings might be used to aid in selection of patients for postoperative adjuvant therapy.

     

Simultaneous detection of circulating immunological parameters and tumor biomarkers in early stage breast cancer patients during adjuvant chemotherapy

Background

Chemotherapy-induced immune suppression has mainly been studied in patients with advanced cancer, but the influence of chemotherapy on the immune system in early stage cancer patients has so far not been studied systematically. The aim of the present study was to monitor the immune system during anthracycline- and taxane-based adjuvant chemotherapy in early stage breast cancer patients, to assess the impact of circulating tumor cells on selected immune parameters and to reveal putative angiogenic effects of circulating endothelial cells.

Methods

Peripheral blood samples from 20 early stage breast cancer patients were analyzed using a flow cytometric multi-color of antibodies to enumerate lymphocyte and dendritic cell subsets, as well as endothelial and tumor cells. An enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of various serological factors.

Results

During chemotherapy, all immunological parameters and angiogenesis surrogate biomarkers showed significant decreases. The numbers of circulating tumor cells showed significant inverse correlations with the numbers of T helper cells, a lymphocyte subset directly related to effective anti-tumor responses. Reduced T helper cell numbers may contribute to systemic immunosuppression and, as such, the activation of dormant tumor cells.

Conclusions

From our results we conclude that adjuvant chemotherapy suppresses immune function in early stage breast cancer patients. In addition, we conclude that the presence of circulating tumor cells, defined as pan-cytokeratin⁺, CD326⁺, CD45^? cells, may serve as an important indicator of a patient’s immune status. Further investigations are needed to firmly define circulating tumor cells as a predictor for the success of breast cancer adjuvant chemotherapy.

     

Oncotype DX breast cancer recurrence score can be predicted with a novel nomogram using clinicopathologic data

Purpose

Breast cancer is a leading cause of cancer deaths in women, but despite steady improvements in therapies, treatment is still suboptimal. Immunotherapy holds promise as a more effective therapy for breast cancer; supporting this, our prior study showed that patients possessing HER2-reactive CD8+ T cells in blood experience survival superior to patients without these cells. Here, we define a composite set of biomarkers that identify patients with T cell responses to tumour antigens.

Methods

We assessed T cell responses following in vitro stimulation with the HER2, MUC1 and SUR tumour-associated antigens (TAA) by flow cytometry and intracellular cytokine staining in 50 breast cancer patients. We also measured HLA type, serum cytokines, tumour-infiltrating leukocytes and blood leukocyte populations.

Results

We found few correlations between TAA-reactive T cells and HLA type, serum cytokines and tumour-infiltrating leukocytes, whereas blood leukocyte phenotypes broadly correlated with TAA responses. This showed monocytes, natural killer cells, dendritic cells and T cells to be inversely associated with both CD4+ and CD8+ T cells reactive to tumour antigens. Moreover, combining multiple parameters improved the accuracy in predicting patients with TAA-responsive T cells.

Conclusion

This study therefore defines composite immune profiles that identify patients responding to TAAs which may allow better personalisation of cancer therapies.

     

Influence of comorbidity on chemotherapy use for early breast cancer: systematic review and meta-analysis

Purpose

The recent increase in the incidence of ductal carcinoma in situ (DCIS) has sparked debate over the classification and treatment of this disease. Although DCIS is considered a precursor lesion to invasive breast cancer, some DCIS may have more or less risk than is realized. In this study, we characterized the immune microenvironment in DCIS to determine if immune infiltrates are predictive of recurrence.

Methods

Fifty-two cases of high-grade DCIS (HG-DCIS), enriched for large lesions and a history of recurrence, were age matched with 65 cases of non-high-grade DCIS (nHG-DCIS). Immune infiltrates were characterized by single- or dual-color staining of FFPE sections for the following antigens: CD4, CD8, CD20, FoxP3, CD68, CD115, Mac387, MRC1, HLA-DR, and PCNA. Nuance multispectral imaging software was used for image acquisition. Protocols for automated image analysis were developed using CellProfiler. Immune cell populations associated with risk of recurrence were identified using classification and regression tree analysis.

Results

HG-DCIS had significantly higher percentages of FoxP3⁺ cells, CD68⁺ and CD68⁺PCNA⁺ macrophages, HLA-DR⁺ cells, CD4⁺ T cells, CD20⁺ B cells, and total tumor infiltrating lymphocytes compared to nHG-DCIS. A classification tree, generated from 16 immune cell populations and 8 clinical parameters, identified three immune cell populations associated with risk of recurrence: CD8⁺HLADR⁺ T cells, CD8⁺HLADR^? T cells, and CD115⁺ cells.

Conclusion

These findings suggest that the tumor immune microenvironment is an important factor in identifying DCIS cases with the highest risk for recurrence and that manipulating the immune microenvironment may be an efficacious strategy to alter or prevent disease progression.

     

Implications of NOVA1 suppression within the microenvironment of gastric cancer: association with immune cell dysregulation

Background

The neuronal splicing factor neuro-oncological ventral antigen 1 (NOVA1) is enriched in normal fibroblasts. Stromal spindle cells such as fibroblasts are major components of tissue inflammation and tertiary lymphoid structures within the microenvironment that contribute to the survival and growth of cancer cells. In the present study, we investigated changes of NOVA1 expression in tertiary lymphoid structures in early and advanced gastric cancer microenvironments in terms of tumor progression and immune regulation.

Methods

Using immunohistochemistry, we analyzed NOVA1 expression in tumor cells, T cells, and stromal spindle cells as well as infiltrating densities of CD3⁺ T cells, forkhead box P3 positive (FOXP3⁺) regulatory T cells, CD68⁺ macrophages, CD163⁺ M2 macrophages, and myeloperoxidase-positive neutrophils in 396 surgically resected gastric cancer tissues.

Results

Suppressed NOVA1 expression in tumor cells, T cells, and stromal spindle cells was closely related to decreased infiltration of FOXP3⁺ regulatory T cells, increased infiltration of CD68⁺ macrophages and CD163⁺ M2 macrophages, more advanced tumor stage, and inferior overall survival rate. In addition, low infiltration of CD3⁺ T cells and FOXP3⁺ regulatory T cells and high infiltration of CD68⁺ macrophages were associated with inferior overall survival. Specifically, weak NOVA1 expression in tumor cells was independently related to more advanced tumor stage and inferior overall survival.

Conclusions

NOVA1 suppression was frequently noted in the gastric cancer microenvironment, and attenuated NOVA1 expression in tumor cells was associated with tumor progression and poor prognosis. This finding seems to be related to immune dysfunction through changes in the immune cell composition of T cells and macrophages.

     

Tumor-associated macrophages and crown-like structures in adipose tissue in breast cancer

Purpose

We aimed to evaluate macrophage infiltration and to identify the status of crown-like structures (CLSs) in mammary adipose tissue of human breast tissue in cases with and without breast cancer.

Methods

Breast adipose tissue was obtained from reduction mammoplasty (N = 56, Group 1), non-neoplastic breast tissue of breast cancer patients (N = 84, Group 2), and breast cancer with adipose stroma (N = 140, Group 3). Immunohistochemical staining of CD68 and CD163 was performed, and the infiltrating macrophages and CLSs within breast adipose tissue were evaluated.

Results

Group 3 had the largest number of CD68-positive (CD68⁺) and CD163-positive (CD163⁺) macrophages and CLSs within adipose tissue (P < 0.001). Among Group 3, cases with high levels of CD68⁺ and CD163⁺ macrophages commonly had a higher histologic grade (P = 0.016 and P = 0.045), and cases with CD163⁺ CLSs were correlated with old age (P = 0.042), estrogen receptor negativity (P = 0.013), human epidermal growth factor receptor-2 positivity (P = 0.043), and non-luminal A type (P = 0.039). Upon univariate analysis, high levels of CD163⁺ macrophages were associated with shorter disease-free survival in node-negative breast cancer patients (P = 0.033), and CD68⁺ CLSs were associated with shorter overall survival in node-positive breast cancer patients (P = 0.015).

Conclusions

CD68⁺ and/or CD163⁺ tumor-associated macrophage infiltration as well as CLSs are present in adipose tissue nearby the breast cancer lesion, and are associated with various clinicopathologic parameters of breast cancer.

     

Systemic inflammation is associated with the density of immune cells in the tumor microenvironment of gastric cancer

Background

The neutrophil–lymphocyte ratio (NLR) and the prognostic nutritional index (PNI) are markers of systemic inflammation known to be useful prognostic indicators of malignancy. However, little evidence has defined the influence of inflammation on the tumor microenvironment.

Methods

Two hundred eighty-eight patients who underwent curative surgery for gastric cancer were included. Preoperative peripheral blood samples were used to analyze the NLR and PNI. The optimal cutoff levels for the NLR and PNI were defined by receiver operating characteristic curve analysis for survival (NLR = 2.7, PNI = 47.7). The densities of specific immune cells (CD3⁺, CD4⁺, CD8⁺) within the tumor microenvironment were measured in tumor microarrays by immunohistochemical analysis.

Results

Two hundred thirty-five patients (81.6 %) had a low NLR and 53 patients (18.4 %) had a high NLR. One hundred seventeen patients (40.6 %) had a low PNI and 171 patients (59.4 %) had a high PNI. CD3⁺ and CD8⁺ immune cell density were not associated with the NLR and PNI. However, in the high-NLR group compared with the low-NLR group, CD4⁺ immune cell density was significantly decreased (P < 0.001). Similarly, the density of CD4⁺ immune cells was also significantly decreased in the low-PNI group compared with the high-PNI group (P = 0.007). A high NLR and a low PNI were correlated with worse overall survival in multivariate analysis (P = 0.028 and P = 0.002 respectively).

Conclusions

The NLR and PNI are associated with the density of CD4⁺ immune cells in the tumor microenvironment, which leads to prognostic values of systemic inflammation in gastric cancer.

     

HIV-1 matrix protein p17 and its variants promote human triple negative breast cancer cell aggressiveness

Background

The introduction of cART has changed the morbidity and mortality patterns affecting HIV-infected (HIV⁺) individuals. The risk of breast cancer in HIV⁺ patients has now approached the general population risk. However, breast cancer has a more aggressive clinical course and poorer outcome in HIV⁺ patients than in general population, without correlation with the CD4 or virus particles count. These findings suggest a likely influence of HIV-1 proteins on breast cancer aggressiveness and progression. The HIV-1 matrix protein (p17) is expressed in different tissues and organs of successfully cART-treated patients and promotes migration of different cells. Variants of p17 (vp17s), characterized by mutations and amino acid insertions, differently from the prototype p17 (refp17), also promote B-cell proliferation and transformation.

Methods

Wound-healing assay, matrigel-based invasion assay, and anchorage-independent proliferation assay were employed to compare the biological activity exerted by refp17 and three different vp17s on the triple-negative human breast cancer cell line MDA-MB 231. Intracellular signaling was investigated by western blot analysis.

Results

Motility and invasiveness increased in cells treated with both refp17 and vp17s compared to untreated cells. The effects of the viral proteins were mediated by binding to the chemokine receptor CXCR2 and activation of the ERK1/2 signaling pathway. However, vp17s promoted MDA-MB 231 cell growth and proliferation in contrast to refp17-treated or not treated cells.

Conclusions

In the context of the emerging role of the microenvironment in promoting and supporting cancer cell growth and metastatic spreading, here we provide the first evidence that exogenous p17 may play a crucial role in sustaining breast cancer cell migration and invasiveness, whereas some p17 variants may also be involved in cancer cell growth and proliferation.

     

The prognostic value of androgen receptors in breast cancer subtypes

Introduction

We hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype.

Materials and methods

Normal breast parenchyma?≥?5 cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44⁺CD49f⁺CD133/2⁺ mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples.

Results

8 of 8 IBC samples expressed isolated CD44⁺CD49f⁺CD133/2⁺ stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p?=?0.001). Similarly, the median number of CD44⁺CD49f⁺CD133/2⁺ cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p?=?0.007). 7 of 8 IBC samples expressed CD68?+?histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p?=?0.001). In the validation cohort, the median number of CD68?+?cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p?=?0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p?=?0.02) and with a 79-gene IBC signature (p?<?0.001).

Conclusions

Normal tissue from IBC patients is enriched for both mammary stem cells and macrophages and has higher association with both a tumorigenic stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.

     

The DC-SIGN-CD56 interaction inhibits the anti-dendritic cell cytotoxicity of CD56 expressing cells

Background

This study aimed to clarify interactions of the pattern-recognition receptor DC-SIGN with cells from the HIV-infected peripheral blood lymphocyte cultures.

Methods

Cells from control and HIV-infected peripheral blood lymphocyte cultures were tested for the surface expression of DC-SIGN ligands. The DC-SIGN ligand expressing cells were analyzed for the role of DC-SIGN-ligand interaction in their functionality.

Results

In the vast majority of experiments HIV-infected lymphocytes did not express detectable DC-SIGN ligands on their cell surfaces. In contrast, non-infected cells, carrying NK-specific marker CD56, expressed cell surface DC-SIGN ligands. The weakly polysialylated CD56 was identified as a novel DC-SIGN ligand. The treatment of DC-SIGN expressing dendritic cells with anti-DC-SIGN antibodies increased the anti-dendritic cell cytotoxicity of CD56^pos cells. The treatment of CD56^pos cells with a peptide, blocking the weakly polysialylated CD56-specifc trans-homophilic interactions, inhibited their anti-dendritic cells cytotoxicity.

Conclusions

The interaction between DC-SIGN and CD56 inhibits homotypic intercellular interactions of CD56^pos cells and protects DC-SIGN expressing dendritic cells against CD56^pos cell-mediated cytotoxicity. This finding can have an impact on the development of approaches to HIV infection and cancer therapy as well as in transplantation medicine.

     

Phenotype plasticity rather than repopulation from CD90/CK14+ cancer stem cells leads to cisplatin resistance of urothelial carcinoma cell lines

Background

Tumour heterogeneity and resistance to systemic treatment in urothelial carcinoma (UC) may arise from cancer stem cells (CSC). A recent model describes cellular differentiation states within UC based on corresponding expression of surface markers (CD) and cytokeratins (CK) with CD90 and CK14 positive cells representing the least differentiated and most tumourigenic population. Based on the fact that this population is postulated to constitute CSCs and the origin of cisplatin resistance, we enriched urothelial carcinoma cell lines (UCCs) for CD90 and studied the tumour-initiating potential of these separated cells in vitro.

Methods

Magnetic- and fluorescence-activated- cell sorting were used for separation of CD90⁺ and CD90^? UCCs. Distribution of cell surface markers CD90, CD44, and CD49f and cytokeratins CK14, CK5, and CK20 as well as the effects of short- and long-term treatment with cisplatin were assessed in vitro and measured by qRT-PCR, immunocytochemistry, reporter assay and flow cytometry in 11 UCCs.

Results

We observed cell populations with surface markers according to those reported in tumour xenografts. However, expression of cytokeratins did not concord regularly with that of the surface markers. In particular, expression of CD90 and CK14 diverged during enrichment of CD90⁺ cells by immunomagnetic sorting or following cisplatin treatment. Enriched CD90⁺ cells did not exhibit CSC-like characteristics like enhanced clonogenicity and cisplatin resistance. Moreover, selection of cisplatin-resistant sublines by long-term drug treatment did not result in enrichment of CD90⁺ cells. Rather, these sublines displayed significant phenotypic plasticity expressing EMT markers, an altered pattern of CKs, and WNT-pathway target genes.

Conclusions

Our findings indicate that the correspondence between CD surface markers and cytokeratins reported in xenografts is not maintained in commonly used UCCs and that CD90 may not be a stable marker of CSC in UC. Moreover, UCCs cells are capable of substantial phenotypic plasticity that may significantly contribute to the emergence of cisplatin resistance.

     

Memory CD4<Superscript>+</Superscript> T cell subsets in tumor draining lymph nodes of breast cancer patients: A focus on T stem cell memory cells

Background

The compartments of memory T cells play a fundamental role in the immune system by substantiating specific and acquired immunity. A new subset of memory cells, T stem cell memory (T_SCM) cells, with stem cell-like properties, a high capacity to proliferate, a long survival, and an ability to differentiate into all effector and memory cells has recently been introduced. In the present study, we aimed to determine the frequency of CD4⁺ T_SCM and other T memory cell subsets in tumor draining lymph nodes of breast cancer patients.

Materials and methods

Mononuclear cells were obtained from axillary lymph nodes of 52 untreated patients with breast cancer (BC) and stained with fluorochrome conjugated anti-CD4, ?CCR7, ?CD45RO and -CD95 antibodies to detect different subtypes of memory cells in CD4⁺ lymphocyte populations. Data were acquired using a four-color FACSCalibur flow cytometer and analyzed using CellQuest Pro software.

Results

We found that >70% of CD4⁺ lymphocytes in draining lymph nodes of BC patients exhibited a memory phenotype of which 7.04 ± 1.04% had a T_SCM phenotype (CD4⁺CCR7⁺CD45RO^?CD95⁺). The frequency of T_SCM cells was significantly higher in tumor positive lymph nodes compared to tumor negative lymph nodes (p = 0.026) as well as among those patients who had at least one affected lymph node (p = 0.012). Moreover, we found that the total frequency of central memory T cells (T_CM) with a low expression of CD45RO was significantly higher among these patients. The percentage of CD45RO^Low T_CM cells was also found to increase with tumor progression from stage I to stage III (p = 0.020). On the other hand, we found that the percentage of CD95^Hi effector memory T cells (T_EM) was significantly decreased in involved lymph nodes (p = 0.009).

Conclusion

Our data suggest that following long-term exposure to putative tumor antigens, T_SCM cells proliferate to generate a pool of committed memory and effector T cells. As the tumor progresses, the immunosuppressive milieu induced by tumor cells may slow down the differentiation of CD45RO^Low T_CM cells to more functional sub-populations.

     

Nuclear β-catenin and CD44 upregulation characterize invasive cell populations in non-aggressive MCF-7 breast cancer cells

Background

In breast cancer cells, the metastatic cell state is strongly correlated to epithelial-to-mesenchymal transition (EMT) and the CD44⁺/CD24^- stem cell phenotype. However, the MCF-7 cell line, which has a luminal epithelial-like phenotype and lacks a CD44⁺/CD24^- subpopulation, has rare cell populations with higher Matrigel invasive ability. Thus, what are the potentially important differences between invasive and non-invasive breast cancer cells, and are the differences related to EMT or CD44/CD24 expression?

Methods

Throughout the sequential selection process using Matrigel, we obtained MCF-7-14 cells of opposite migratory and invasive capabilities from MCF-7 cells. Comparative analysis of epithelial and mesenchymal marker expression was performed between parental MCF-7, selected MCF-7-14, and aggressive mesenchymal MDA-MB-231 cells. Furthermore, using microarray expression profiles of these cells, we selected differentially expressed genes for their invasive potential, and performed pathway and network analysis to identify a set of interesting genes, which were evaluated by RT-PCR, flow cytometry or function-blocking antibody treatment.

Results

MCF-7-14 cells had enhanced migratory and invasive ability compared with MCF-7 cells. Although MCF-7-14 cells, similar to MCF-7 cells, expressed E-cadherin but neither vimentin nor fibronectin, β-catenin was expressed not only on the cell membrane but also in the nucleus. Furthermore, using gene expression profiles of MCF-7, MCF-7-14 and MDA-MB-231 cells, we demonstrated that MCF-7-14 cells have alterations in signaling pathways regulating cell migration and identified a set of genes (PIK3R1, SOCS2, BMP7, CD44 and CD24). Interestingly, MCF-7-14 and its invasive clone CL6 cells displayed increased CD44 expression and downregulated CD24 expression compared with MCF-7 cells. Anti-CD44 antibody treatment significantly decreased cell migration and invasion in both MCF-7-14 and MCF-7-14 CL6 cells as well as MDA-MB-231 cells.

Conclusions

MCF-7-14 cells are a novel model for breast cancer metastasis without requiring constitutive EMT and are categorized as a "metastable phenotype", which can be distinguished from both epithelial and mesenchymal cells. The alterations and characteristics of MCF-7-14 cells, especially nuclear β-catenin and CD44 upregulation, may characterize invasive cell populations in breast cancer.