The subsets of dendritic cells and memory T cells correspond to indoleamine 2,3-dioxygenase in stomach tumor microenvironment

The abnormal distributions of memory T cells (Tm) and dendritic cells (DC) in stomach cancer are not well understood. Indoleamine 2,3-dioxygenase (IDO), produced by DC, may be an important enzyme affecting function and proliferation of Tm. In this study, IDO expression was examined by immunohistochemical staining. The subsets of Tm and DC were counted by flow cytometry. The percentages of CD4?+?Tm and CD4?+?central Tm (Tcm) were lower in tumor tissues than in normal tissues (P?P?P?=?0.009). The high expression of IDO was more frequently observed in tumor tissues (P?=?0.001). The percentages of CD4?+?Tm and CD8?+?Tm were positively associated with DC1 percentage and ratio of DC1/DC2 (P?P?=?0.025). The patients with high IDO expression had significantly lower CD4?+?Tm (P?=?0.012) and CD8?+?Tm percentages (P?=?0.033), but higher CD8?+?Tem percentage (P?P?=?0.019). The CD4?+?Tm and CD8?+?Tem percentages were significantly associated with clinical stage and lymph node metastasis; the high IDO expressions were significantly associated with deeper tumor invasion (P?=?0.016) and lymph node metastasis (P?=?0.038). Thus, DC subsets, Tm subsets, and IDO expression were correlated with each other. They were associated with the established clinicopathologic features, such as tumor size, depth of invasion, lymph node metastasis, and clinical stage.     

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.

     

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.     

The significant increase and dynamic changes of the myeloid-derived suppressor cells percentage with chemotherapy in advanced NSCLC patients

Purpose

To investigate the correlations between myeloid-derived suppressor cells (MDSCs) in the peripheral blood and cancer stage, immune function, and chemotherapy.

Methods

Percentages of MDSCs (CD11b⁺CD14^?CD33⁺ cells) and lymphocyte subsets in peripheral blood mononuclear cells (PBMCs) of 94 patients with Non-small cell lung cancer (NSCLC) who were treated naïve and 30 healthy individuals were measured. Changes of the MDSCs percentage were further detected in patients with advanced NSCLC treated with systemic chemotherapy. Finally, coculture with CD8⁺ cells was developed to determine effect of MDSCs on IFN-γ secretion of T lymphocytes.

Results

MDSCs percentage of 94 patients with NSCLC was significantly higher than that of 30 healthy subjects (P < 0.05), the percentages were increased with tumor progression, in patients with stage III and IV percentages were significantly higher than those in stage I and II patients (P = 0.013). The MDSCs percentage was negatively related to percentage of CD8⁺ cells in the peripheral blood (r = ?0.354, n = 38, P = 0.029), and when they were cocultured, IFN-γ secretion of CD8⁺ cells was significantly decreased (P < 0.05). In 20 patients with advanced NSCLC who received systemic chemotherapy, nine partial remission (PR) cases got MDSCs percentage significantly decreased (P < 0.001), three stable disease (SD) cases remained invariable (P = 0.307) and eight progressive disease (PD) cases got significantly increased (P = 0.024).

Conclusion

The percentage of MDSCs in the patients was significantly higher than that of the healthy control subjects and it increased with tumor progression partially by inhibiting the CD8⁺ cell function. The dynamic changes of MDSCs percentage reflected the efficacy of systemic chemotherapy.     

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.

     

Activation of central/effector memory T cells and T‐helper 1 polarization in malignant melanoma patients treated with anti‐programmed death‐1 antibody

Human anti‐programmed death‐1 (PD‐1) antibody possesses the capability to revitalize host T cells and has been an effective therapy for metastatic malignant melanoma (MM). The precise subsets of T cells predominantly activated by anti‐PD‐1, however, have not yet been clarified. In this study, peripheral blood mononuclear cells obtained from MM patients scheduled to receive anti‐PD‐1 (nivolumab) therapy, and healthy subjects (HS), were systematically examined on flow cytometry to identify changes in the proportion of immune cell subsets. Compared with HS, MM patients prior to therapy had an increased proportion of activated CD8+ T cells with effector memory phenotypes (Tem), and PD‐1 positive subsets of CD4+ central memory T cells (Tcm) and T‐helper (Th)17 cells. After a single course of anti‐PD‐1 therapy, MM patients had an increase in activated Tem and Tcm subsets of CD4+ and CD8+ T cells, and activated Th1 plus T‐helper follicular 1 cells. There was no consistent change in the proportion of Tfh cells, B cells, natural killer cells, or dendritic cells. The observed activated phenotypes were attenuated during the course of therapy, but regulatory T cells belonging to the CD3+CD4+CD45RO+CD25high fraction increased at disease progression. Taken together, anti‐PD‐1 therapy modulates systemic immune reactions and exerts anti‐tumor effects, not only by revitalizing Tem and Tcm of CD4+ and CD8+ T cells, but also via a shift to a Th1 phenotype.     

Skewed immunological balance between Th17 (CD4+IL17A+) and Treg (CD4+CD25+FOXP3+) cells in human oral squamous cell carcinoma

Background

Several studies have documented modulation of Th17 and T regulatory (Treg) cells in various human malignancies which may vary with the type and extent of the disease. However, such data in patients with oral cancer is scarce and hence the current study was designed to elaborate the immunological balance between these two T cell subsets in oral cancer.

Methods and results

We analyzed various T cell subsets in the peripheral blood of 45 oral squamous cell carcinoma (OSCC) patients and 40 healthy volunteers. We found that, compared with the healthy controls, patients had a significantly (p?<?0.0001) higher proportion of both Th17 (CD4⁺IL17A⁺) and Treg (CD4⁺CD25⁺FOXP3⁺) cells, which further showed a reciprocal balance in relation to clinico-pathological parameters in patients. We also detected a circulating CD8⁺ subset of these cells in both patients and healthy controls, although the difference between the two groups was statistically insignificant. Higher frequencies of Th17 cells were found in patients with early stages and without lymph node involvement, while an increased prevalence of Tregs was associated with higher clinical stages and lymph node involvement. Moreover, Th17 cells were quantitatively and positively correlated to CD4⁺T and CD8⁺T cells and inversely correlated with Tregs. Contrarily, Tregs showed a negative association with CD4⁺T and CD8⁺T cells.

Conclusions

Our results suggest an increase in Th17/Tregs ratio in early stages and a decrease in this ratio in higher stages of oral cancer. Such counter regulation of Th17 and Tregs may be a significant prognostic factor in oral cancer patients.     

Clinical significance of regulatory T cells and CD8+ effector populations in patients with human endometrial carcinoma

BACKGROUND:

A study was carried out to determine the functional attributes of CD4⁺ CD25⁺ regulatory T cells in cancer progression by suppressing antitumor immunity.

METHODS:

Triple‐color flow cytometry was used to study the phenotype expression of CD4⁺ CD25⁺ regulatory T cells and CD8⁺ T cells in the peripheral blood lymphocytes (PBLs) and tumor‐infiltrating lymphocytes (TILs) of 57 cases of stage I to IV endometrial carcinoma. The expression of T cell subsets was correlated with clinical prognostic parameters.

RESULTS:

The prevalence of CD4⁺ CD25⁺ T cells was significantly higher in the TILs than PBLs. The expression of CD4⁺ CD25⁺ regulatory T cells in cancer milieu correlated with the tumor grade, stage, and myometrium invasion. The expression of FOXP3 and GITR in CD4⁺ CD25⁺ regulatory T cells was lower in PBLs than TILs. Most tumor‐infiltrating CD8⁺ T cells were CD28^? CD45RA^? CD45RO⁺ CCR7^?, suggesting good terminal differentiation. Most of them had an activated role with CD69⁺ CD103⁺ CD152⁺. Functionally, both granzyme B and perforin were scarcely expressed in peripheral regulatory T cells but were highly expressed in peripheral regulatory T cells in the tumor microenvironment. In contrast, CD8⁺ cytotoxic T cells derived from PBLs expressed both granzyme B and perforin, and at significantly higher levels than in TILs. Further functional assays demonstrated that Th1 cytokines and cytotoxic molecules can be synchronously up‐regulated in CD8⁺ cytotoxic T cells.

CONCLUSIONS:

Regulatory T cells in the tumor microenvironment may abrogate CD8⁺ T cell cytotoxicity in a granzyme B‐ and perforin‐dependent conduit. Decreases in both Th1 cytokines and cytotoxic enzymes are relevant for regulatory T cell‐mediated restraint of tumor clearance in vivo. Of clinical significance, the expression of regulatory T cells in TILs may mediate T cell immune repression within cancer milieu and thus greatly correlate with cancer progression. Cancer 2010. © 2010 American Cancer Society.     

Changes of immunological parameters with administration of Japanese Kampo medicine (Juzen-Taihoto/TJ-48) in patients with advanced pancreatic cancer

Background

The prognosis of pancreatic cancer is extremely poor regardless of various combination therapies. Immunoaugumentation against tumor cells was recently A focus. We reported that the population of Foxp3⁺CD25⁺CD4⁺ regulatory T cells (Foxp3⁺Treg) was the new parameter for the estimation of host immunity and had correlation with tumor aggressiveness. Here we show the immunoaugumentation effects of Japanese Kampo medicine, Juzen-Taihoto/TJ-48, empirically considered as an immunoaugumentation drug, with investigation of Treg and other immunological parameters.

Patients and method

Peripheral Foxp3⁺ Treg populations, CD4/CD8 ratio, and CD57⁺ cells (NK cells) populations in advanced pancreatic cancer patients (n = 30, stage VI A and B according to TNM classification) were estimated after TJ-48 administration for 14 days before the anti-cancer therapy.

Results

Treg populations were significantly increased compared to healthy donors (Mann–Whitney U test, P < 0.001). Administration of Juzen-Taihoto/TJ-48 significantly decreased Treg populations (Mann–Whitney U test, P < 0.001) and increased the CD4/CD8 ratio (Mann–Whitney U test, P < 0.01), even though CD57⁺ cell populations did not change significantly.

Conclusions

Juzen-Taihoto/TJ-48 increased regulatory activities in T cells through decreasing Foxp3⁺ Treg populations in advanced pancreatic cancer patients. This effect can lead to immunoaugumentation for various combination therapies.     

Targeting of the non-mutated tumor antigen HER2/neu to mature dendritic cells induces an integrated immune response that protects against breast cancer in mice

Introduction

Given their relative simplicity of manufacture and ability to be injected repeatedly, vaccines in a protein format are attractive for breast and other cancers. However, soluble human epidermal growth factor receptor (HER2)/neu protein as a vaccine has not been immunogenic. When protein is directly targeted to antigen uptake receptors, such as DEC205 (DEC), efficient processing and presentation of antigen take place. The aim of this study was to determine the immunogenicity of a HER2 protein vaccine that directly targets to DEC⁺ dendritic cells (DCs) in a mouse breast cancer model.

Methods

We genetically engineered the HER2 extracellular domain into a monoclonal antibody specific for DEC (DEC-HER2). Mice of various genetic backgrounds were immunized with DEC-HER2 in combination with DC maturation stimuli (poly IC ± CD40 Ab). Vaccine-induced T cell immunity was determined by analyzing the ability of CD4⁺/CD8⁺ T cell to produce interferon (IFN)-gamma and proliferate upon antigen rechallenge. Sera were assessed for the presence of antigen specific antibody (Ab). For vaccine efficacy, FVB/N mice were immunized with DEC-HER2 in combination with poly IC and protection against neu-expressing mammary tumors was assessed. Protection mechanisms and tumor-specific T cell responses were also evaluated.

Results

We demonstrate that DEC-HER2 fusion mAb, but not Ctrl Ig-HER2, elicits strong, broad and multifunctional CD4⁺ T cell immunity, CD8⁺ T cell responses, and humoral immunity specific for HER2 antigen. Cross-reactivity to rat neu protein was also observed. Importantly, mice xeno-primed with DEC-HER2 were protected from a neu-expressing mammary tumor challenge. Both CD4⁺ and CD8⁺ T cells mediated the tumor protection. Robust anti-tumor T cell immunity was detected in tumor protected mice.

Conclusions

Immunization of mice with HER2 protein vaccine targeting DEC⁺ DCs in vivo induced high levels of T- and B-cell immunity. Non-targeted HER2 protein was poorly immunogenic for CD4⁺ and CD8⁺ T cells. This vaccination approach provided long-term survival benefit for mice challenged with neu-expressing tumor following as little as 2.7 ??g of HER2 protein incorporated in the vaccine. Vaccine-induced CD4⁺ and CD8⁺ T cells were both essential for tumor protection. This immunization strategy demonstrates great potential towards the development of vaccines for breast cancer patients.     

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.

     

A different immunologic profile characterizes patients with HER-2-overexpressing and HER-2-negative locally advanced breast cancer: implications for immune-based therapies

Introduction

The clinical efficacy of trastuzumab and taxanes is at least partly related to their ability to mediate or promote antitumor immune responses. On these grounds, a careful analysis of basal immune profile may be capital to dissect the heterogeneity of clinical responses to these drugs in patients with locally advanced breast cancer undergoing neoadjuvant chemotherapy.

Methods

Blood samples were collected from 61 locally advanced breast cancers (36 HER2^- and 25 HER2⁺) at diagnosis and from 23 healthy women. Immunophenotypic profiling of circulating and intratumor immune cells, including regulatory T (Treg) cells, was assessed by flow cytometry and immunohistochemistry, respectively. Serum levels of 10 different cytokines were assessed by multiplex immunoassays. CD8⁺ T cell responses to multiple tumor-associated antigens (TAA) were evaluated by IFN-γ-enzyme-linked immunosorbent spot (ELISPOT). The Student's t test for two tailed distributions and the Wilcoxon two-sample test were used for the statistical analysis of the data.

Results

The proportion of circulating immune effectors was similar in HER2⁺ patients and healthy donors, whereas higher percentages of natural killer and Treg cells and a lower CD4⁺/CD8⁺ T cell ratio (with a prevalence of naïve and central memory CD8⁺ T cells) were observed in HER2^- cases. Higher numbers of circulating CD8⁺ T cells specific for several HLA-A*0201-restricted TAA-derived peptides were observed in HER2⁺ cases, together with a higher prevalence of intratumor CD8⁺ T cells. Serum cytokine profile of HER2⁺ patients was similar to that of controls, whereas HER2^- cases showed significantly lower cytokine amounts compared to healthy women (IL-2, IL-8, IL-6) and HER2⁺ cases (IL-2, IL-1β, IL-8, IL-6, IL-10).

Conclusions

Compared to HER2- cases, patients with HER2-overexpressing locally advanced breast cancer show a more limited tumor-related immune suppression. This may account for the clinical benefit achieved in this subset of patients with the use of drugs acting through, but also promoting, immune-mediated effects.     

Immune Analysis of Radium-223 in Patients With Metastatic Prostate Cancer

Background

Radium223 (Ra223) delivers high-energy radiation to osteoblastic metastasis of prostate cancer, resulting in irreparable double-stranded DNA damage. The effects of Ra223 on CD8+ T cell subsets in patients with prostate cancer is unknown.

High frequencies of less differentiated and more proliferative WT1‐specific CD8+ T cells in bone marrow in tumor‐bearing patients: An important role of bone marrow as a secondary lymphoid organ

In tumor‐bearing patients, tumor‐associated antigen (TAA)‐specific CTLs are spontaneously induced as a result of immune response to TAAs and play an important role in anti‐tumor immunity. Wilms’ tumor gene 1 (WT1) is overexpressed in various types of tumor and WT1 protein is a promising pan‐TAA because of its high immunogenicity. In this study, to clarify the immune response to the WT1 antigen, WT1‐specific CD8⁺ T cells that were spontaneously induced in patients with solid tumor were comparatively analyzed in both bone marrow (BM) and peripheral blood (PB). WT1‐specific CD8⁺ T cells more frequently existed in BM than in PB, whereas frequencies of naïve (CCR7⁺ CD45RA⁺), central memory (CCR7⁺ CD45RA?), effector‐memory (CCR7? CD45RA^?), and effector (CCR7? CD45RA⁺) subsets were not significantly different between BM and PB. However, analysis of these subsets for the expression of CD57 and CD28, which were associated with differentiation, revealed that effector‐memory and effector subsets of the WT1‐specific CD8⁺ T cells in BM had less differentiated phenotypes and more proliferative potential than those in PB. Furthermore, CD107a/b functional assay for WT1 peptide‐specific cytotoxic potential and carboxyfluorescein diacetate succinimidyl ester dilution assay for WT1 peptide‐specific proliferation also showed that WT1‐specific CD8⁺ T cells in BM were less cytotoxic and more proliferative in response to WT1 peptide than those in PB. These results implied that BM played an important role as a secondary lymphoid organ in tumor‐bearing patients. Preferential residence of WT1‐specific CD8⁺ T cells in BM could be, at least in part, explained by higher expression of chemokine receptor CCR5, whose ligand was expressed on BM fibroblasts on the WT1‐specific CD8⁺ T cells in BM, compared to those in PB. These results should provide us with an insight into WT1‐specific immune response in tumor‐bearing patients and give us an idea of enhancement of clinical response in WT1 protein‐targeted immunotherapy. (Cancer Sci 2010; 101: 848–854)     

Myeloid cells positive for CD10 at invasion front can predict poor outcome in stage II colorectal cancer

Background

Prediction of poor patient outcome as a effect of adjuvant therapy in stage II colorectal cancer (CRC) remains a matter of controversy. Tumor expression of CD10 and CD15 is reportedly related to poor outcome in CRC. In this study, we investigated whether the expression of CD10 and CD15 is a predictor of outcome and the effect of adjuvant therapy in stage II CRC.

Materials and methods

Immunohistochemical analyses for CD10 and CD15 and some additional markers (CD11b, CD14, CD163, CD3, and CD20) were performed using paraffin sections of CRC specimens from 57 patients who had undergone curative surgical treatments between 1998 and 2004. Forty of these patients received postoperative adjuvant therapy. We distinguished between expression in tumor cells (tCD10 and tCD15), in stromal cells (sCD10), and infiltrating immune cells (iCD10 and iCD15).

Results

Expression of iCD10 was observed in 21.1% (12/57) of the specimens examined. Of all expression patterns, only iCD10 expression at the cancer invasion front was a useful predictor of a disease-free period and overall survival in stage II CRC. Adjuvant therapy improved the outcome of iCD10⁺ patients. CD10⁺ immune cells were heterogeneous in origin and partially overlapped the cells positive for myeloid lineage markers, including CD11b and CD15.

Conclusions

iCD10 expression at the tumor invasion front is a useful marker for predicting a high risk of recurrence and mortality in stage II CRCs. CD10⁺ immune cells appear to be of myeloid origin.     

Decreased number and reduced NKG2D expression of Vδ1 γδ T cells are involved in the impaired function of Vδ1 γδ T cells in the tissue of gastric cancer

Background

In cancer patients, impaired function of immune cells—such as CD8⁺ T cells, NK cells, and dendritic cells—reportedly results in tumor progression. Although γδ T cells also play a critical role in tumor defense, their function remains unclear in cancer patients.

Methods

The frequency and function of γδ T cells in peripheral blood, normal gastric mucosa, and cancer tissue were evaluated by multicolor flow cytometry. We also determined NKG2D expression on γδ T cells in gastric cancer patients.

Results

The frequency of Vδ1 γδ T cells in gastric cancer tissue is significantly lower than in normal gastric mucosa; however, differences in the frequencies of Vδ2 and Vγ9 γδ T cells between normal gastric mucosa and gastric cancer tissue were not statistically significant. The Vδ1 γδ T cells from gastric cancer tissue produce significantly less IFN-γ than those from normal gastric mucosa do. Expression of NKG2D on Vδ1 γδ T cells from gastric cancer tissue was significantly lower than in normal gastric mucosa. We also found a significant correlation between NKG2D expression and IFN-γ production of Vδ1 γδ T cells in gastric cancer tissue.

Conclusion

Vδ1 γδ T cells show decreased frequency and impaired function in gastric cancer tissue, for which decreased NKG2D expression might be one of the mechanisms. Modalities specifically targeting NKG2D in Vδ1 γδ T cells may provide a breakthrough treatment for gastric cancer patients.     

CD44+/CD24− breast cancer cells isolated from MCF-7 cultures exhibit enhanced angiogenic properties

Background

Recent studies suggest that the relationship between cancer stem cells (CSCs) and the vascular niche may be bidirectional; the niche can support the growth and renewal of CSCs, and CSCs may contribute to the maintenance of the niche. There is little knowledge concerning the role of breast cancer stem cells in promoting tumor angiogenesis.

Aim

For human breast cancers, CSCs have been shown to be associated with a CD44⁺/CD24 ^? phenotype. We investigated the potential activities of CD44⁺/CD24 ^? breast cancer stem cells in promoting tumor angiogenesis.

Methods

The expression of pro-angiogenic genes was determined by quantitative real-time RT-PCR. Endothelial cell migration assays were employed to evaluate effects of conditioned media from CD44⁺/CD24 ^? on human umbilical vein endothelial cells. A chorioallantoic membrane (CAM) assay was used to study the potential of CD44⁺/CD24 ^? cells to promote angiogenesis.

Results

In our study, CD44⁺/CD24 ^? cells expressed elevated levels of pro-angiogenic factors compared with CD44⁺/CD24⁺ cells. CD44⁺/CD24 ^? cell-conditioned media significantly increased endothelial cell migration. Breast cancer cell lines enriched with CD44⁺/CD24 ^? cells were more pro-angiogenic in the CAM assay than those lacking a CD44⁺/CD24 ^? subpopulation. CD44⁺/CD24 ^? cells sorted from MCF-7 cell lines were more pro-angiogenic in a CAM assay than CD44⁺/CD24⁺ cells. Furthermore, the VEGF concentration was significantly higher in CD44⁺/CD24 ^? cell-conditioned media than in CD44⁺/CD24⁺ cell-conditioned media. The pro-angiogenic effect of CD44⁺/CD24 ^? cells on endothelial cells was abolished by bevacizumab.

Conclusion

Our findings demonstrate that CD44⁺/CD24 ^? breast cancer stem cells have substantial pro-angiogenic potential and activity. This provides new insights to explore in the development of targeted therapies.     

Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy

Introduction

The phenotypic and functional differences between cells that initiate human breast tumors (cancer stem cells) and those that comprise the tumor bulk are difficult to study using only primary tumor tissue. We embarked on this study hypothesizing that breast cancer cell lines would contain analogous hierarchical differentiation programs to those found in primary breast tumors.

Methods

Eight human breast cell lines (human mammary epithelial cells, and MCF10A, MCF7, SUM149, SUM159, SUM1315 and MDA.MB.231 cells) were analyzed using flow cytometry for CD44, CD24, and epithelial-specific antigen (ESA) expression. Limiting dilution orthotopic injections were used to evaluate tumor initiation, while serial colony-forming unit, reconstitution and tumorsphere assays were performed to assess self-renewal and differentiation. Pulse-chase bromodeoxyuridine (5-bromo-2-deoxyuridine [BrdU]) labeling was used to examine cell cycle and label-retention of cancer stem cells. Cells were treated with paclitaxol and 5-fluorouracil to test selective resistance to chemotherapy, and gene expression profile after chemotherapy were examined.

Results

The percentage of CD44⁺/CD24^- cells within cell lines does not correlate with tumorigenicity, but as few as 100 cells can form tumors when sorted for CD44⁺/CD24^-/low/ESA⁺. Furthermore, CD44⁺/CD24^-/ESA⁺ cells can self-renew, reconstitute the parental cell line, retain BrdU label, and preferentially survive chemotherapy.

Conclusion

These data validate the use of cancer cell lines as models for the development and testing of novel therapeutics aimed at eradicating cancer stem cells.     

ICOS+ Foxp3+ TILs in gastric cancer are prognostic markers and effector regulatory T cells associated with Helicobacter pylori

Regulatory T cells (Tregs) have an immunosuppressive role in the tumor microenvironment. Since effector Tregs (eTregs), which have highly suppressive functions, are located in a subpopulation of Foxp3⁺ CD4⁺ Tregs, the TCR‐inducible costimulatory receptor (ICOS) was applied as a marker of eTregs that infiltrated gastric cancer tissue and the induction pathway of ICOS⁺ Foxp3⁺ cells was analyzed by flow cytometry and immunohistochemistry. In tumor‐infiltrating lymphocytes (TILs), ICOS⁺ Foxp3⁺ CD4⁺ T cells were abundantly observed in the late stages of gastric cancer. ICOS⁺ CD4⁺ TILs exhibited the ability to produce IL‐10, but not IFN‐γ, TNF, or IL‐17 and also to suppress the proliferation of CFSE‐labeled responder CD8⁺ T cells. With the agonistic ICOS‐L protein (rICOS‐L Ig), ICOS⁺ Foxp3⁺ cells were efficiently induced from naive CD4⁺ T cells under a stimulation with TGF‐β and CD3/CD28 mAbs. Furthermore, when A*0201 PBMCs were cultured with the CMV or Melan‐A antigenic peptide and rICOS‐L Ig, the induction of CMV or Melan‐A tetramer‐binding CD8⁺ T cells, respectively, was inhibited. The expression of ICOS in Foxp3⁺ cells was closely related to plasmacytoid dendritic cells (pDCs) and their expression of ICOS‐L and TLR9 as well as Helicobacter pylori infection. Collectively, our results demonstrate the potential of ICOS as a promising target for direct Treg‐targeting therapeutic agents for gastric cancer, and that of eradicating therapy for H. pylori as an indirect immune therapy for gastric 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.