Proliferation of CD4+CD25high+Foxp3+ regulatory T lymphocytes in ex vivo expanded ascitic fluid from primary and recurrent ovarian carcinoma

Objective

Regulatory T lymphocytes evoke the immune tolerance by suppressing and inactivating cytotoxic T lymphocytes. The objective of this study was to compare the proportion of regulatory T lymphocytes, precisely defined as CD4⁺CD25^high+Foxp3⁺ T lymphocytes, in primary and recurrent ovarian carcinoma before and after ex vivo expansion of ascites with interleukin-2 (IL-2).

Methods

Ascitic fluid samples were obtained from 26 patients with ovarian carcinoma. Lymphocytes were isolated from ascites and cell markers were analyzed by flow cytometry using anti-CD3/CD4/CD8/CD16/CD56/CD25 and anti-Foxp3 antibodies. Lymphocytes were incubated for 2 to 3 weeks and expanded ex vivo by IL-2 stimulation and their phenotypes were analyzed by flow cytometry.

Results

Following ex vivo expansion, ascitic fluid lymphocytes increased by a greater extent in the recurrent group than in the primary group. The proportion of ex vivo-expanded lymphocytes changed as follows; CD4⁺ T lymphocytes increased, CD8⁺ T lymphocytes decreased, and the proportion of CD3^-CD16⁺56⁺ NK cells was unchanged. The proportion of CD4⁺CD25^high+Foxp3⁺ regulatory T lymphocytes in CD4⁺ T lymphocytes increased after ex vivo expansion in both groups, but to a greater degree in the recurrent group.

Conclusion

This study showed that regulatory T lymphocytes, neither cytotoxic T lymphocytes nor NK cells, were extensively increased after ex vivo expansion, especially in recurrent ovarian carcinoma. These results may provide information that helps to guide the future development of adoptive immunotherapy against ovarian carcinoma.     

PD-1+ CD8+ T cells are exhausted in tumours and functional in draining lymph nodes of colorectal cancer patients

Background:

The blockade of PD-1–PD-L1 pathway is emerging as an effective therapeutic strategy for several advanced cancers. But the immune regulatory role of PD-1–PD-L1 pathway is not clear in colorectal cancer (CRC) patients. This study aims to evaluate the role of PD-1–PD-L1 pathway in CD8⁺ T-cell functions in tumour-draining lymph nodes (TDLNs) and tumours of CRC patients.

Methods:

PD-1 expression on CD8⁺ T cells was examined by flow cytometry, and PD-L1 expression in TDLNs and tumour tissues were examined by immunohistochemistry. Production of IFN-γ, IL-2 and expression of granzyme B, perforin in CD8⁺ T cells were detected by intracellular staining.

Results:

PD-1 expression is markedly upregulated on CD8⁺ T cells in TDLNs and tumours compared with that in peripheral blood. PD-1-expressing CD8⁺ T cells are competent for production of cytokine (IL-2 and IFN-γ) and perforin in the tumour-free lymph nodes (TFLNs), but exhibit exhausted phenotypes in tumours. In addition, PD-L1 is highly expressed in tumours rather than TFLNs, which is closely correlated with the impairment of IFN-γ production of tumour-infiltrating PD-1⁺ CD8⁺ T cells.

Conclusions:

Our findings suggest a suppressive effect of PD-1 on CD8⁺ T-cell function in tumours, but not in TFLNs.     

CD44 targets Na+/H+ exchanger 1 to mediate MDA-MB-231 cells' metastasis via the regulation of ERK1/2

Background:

CD44, a transmembrane glycoprotein expressed in a variety of cells and tissues, has been implicated in tumour metastasis. But the molecular mechanisms of CD44-mediated tumour cell metastasis remain to be elucidated.

Methods:

The downregulation of CD44 was determined by immunofluorescence. Moreover, the motility of breast cancer cells was detected by wound-healing and transwell experiments. Then the spontaneous metastasis of CD44-silenced MDA-MB-231 cells was tested by histology with BALB/c nude mice.

Results:

A positive correlation between CD44 and Na⁺/H⁺ exchanger isoform 1 (NHE1) was found in two breast cancer cells. CD44 downregulation could inhibit the metastasis of MDA-MB-231 cells and the expressions of Na⁺/H⁺ exchanger 1. Moreover, CD44 overexpression upregulated the metastasis of MCF-7 cells, but the elevated metastatic ability was then inhibited by Cariporide. Interestingly, during these processes only the p-ERK1/2 was suppressed by CD44 downregulation and the expression of matrix metalloproteinases and metastatic capacity of MDA-MB-231 cells were greatly inhibited by the MEK1 inhibitor PD98059, which even had a synergistic effect with Cariporide. Furthermore, CD44 downregulation inhibits breast tumour outgrowth and spontaneous lung metastasis.

Conclusions:

Taken together, this work indicates that CD44 regulates the metastasis of breast cancer cells through regulating NHE1 expression, which could be used as a novel strategy for breast cancer therapy.     

Synergy between the ectoenzymes CD39 and CD73 contributes to adenosinergic immunosuppression in human malignant gliomas

Background

The importance of ectoenzymes CD39 and CD73 in mediating adenosinergic immunosuppression has been recognized, but their roles in human malignant glioma–associated immunosuppression remain largely unknown.

Methods

In this study, the ectoenzyme characteristics of malignant glioma cells and infiltrating CD4⁺ T lymphocytes isolated from newly diagnosed malignant glioma patients were investigated. The ectoenzyme activities of both cell populations were determined by nucleotide hydrolysis assay. The immunosuppressive property of the CD39-CD73 synergic effect was evaluated via responder T-cell proliferation assay.

Results

We observed that CD39⁻CD73⁺ glioma cells and infiltrating CD4⁺CD39^highCD73^low T lymphocytes exhibited 2 distinct but complementary ectoenzyme phenotypes, which were further verified by enzyme activity assay. The nucleotide hydrolysis cascade was incomplete unless CD39 derived from T lymphocytes and CD73 collaborated synergistically. We demonstrated that increased suppression of responder CD4⁺ T-cell proliferation suppression was induced by CD4⁺CD39⁺ T cells in the presence of CD73⁺ glioma cells, which could be alleviated by the CD39 inhibitor {"type":"entrez-protein","attrs":{"text":"ARL67156","term_id":"1186396857","term_text":"ARL67156"}}ARL67156, the CD73 inhibitor APCP, or the adenosine receptor A_2aR antagonist {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261. In addition, survival analysis suggested that CD73 downregulation was a positive prognostic factor related to the extended disease-free survival of glioblastoma patients.

Conclusions

Our data indicate that glioma-derived CD73 contributes to local adenosine-mediated immunosuppression in synergy with CD39 from infiltrating CD4⁺CD39⁺ T lymphocytes, which could become a potential therapeutic target for treatment of malignant glioma and other immunosuppressive diseases.     

Effects of Recombinant Erythropoietin on Breast Cancer-Initiating Cells

Background

Cancer anemia causes fatigue and correlates with poor treatment outcome. Erythropoietin has been introduced in an attempt to correct these defects. However, five recent clinical trials reported a negative impact of erythropoietin on survival and/or tumor control, indicating that experimental evaluation of a possible direct effect of erythropoietin on cancer cells is required. Cancer recurrence is thought to rely on the proliferation of cancer initiating cells (CICs). In breast cancer, CICs can be identified by phenotypic markers and their fate is controlled by the Notch pathway.

Methods

In this study, we investigated the effect of erythropoietin on CICs in breast cancer cell lines. Levels of erythropoietin receptor (EpoR), CD24, CD44, Jagged-1 expression, and activation of Notch-1 were assessed by flow cytometry. Self-renewing capacity of CICs was investigated in sphere formation assays.

Results

EpoR expression was found on the surface of CICs. Recombinant human Epo (rhEpo) increased the numbers of CICs and self-renewing capacity in a Notch-dependent fashion by induction of Jagged-1. Inhibitors of the Notch pathway and PI3-kinase blocked both effects.

Conclusions

Erythropoietin functionally affects CICs directly. Our observation may explain the negative impact of recombinant Epo on local control and survival of cancer patients with EpoR-positive tumors.     

Numbers and cytotoxicities of CD3+CD56+ T lymphocytes in peripheral blood of patients with acute myeloid leukemia and acute lymphocytic leukemia

Recent reports have highlighted the role of cellular immunity in anti-tumor defenses. T lymphocytes are known to play important part in anti-cancer immunity. The number and function of T lymphocytes are altered in chronic leukemia patients. CD3⁺CD56⁺ T lymphocytes have also been found to be abnormal in cancer patients. We therefore investigated changes in the number and cytotoxicity of CD3⁺CD56⁺ T lymphocytes in the peripheral blood of acute leukemia (AL) patients (excluding acute promyelocytic leukemia), to improve our understanding of the role of this T lymphocyte subset. We analyzed CD3⁺CD56⁺ T lymphocyte numbers and cytotoxicities in healthy controls, AL patients, and AL patients with complete remission. Lymphocyte counts were performed in peripheral blood and flow cytometry was used to determine cell numbers and cytotoxicities. The absolute number of CD3⁺CD56⁺ T lymphocytes was increased in AL patients (including acute myeloid [AML] and acute lymphocytic leukemia [ALL]) compared with healthy controls (P < 0.05), but their functioning was significantly reduced (P < 0.05). The number of CD3⁺CD56⁺ T lymphocytes in AML and ALL patients who achieved remission following chemotherapy was close to healthy controls (P > 0.05), but their functioning was still significantly reduced (P < 0.05). In addition, the number of CD3⁺CD56⁺ T lymphocytes increased significantly in AML patients with increased peripheral blood white blood cell (WBC) counts, and in ALL patients without increased WBCs. These results suggest that cellular immunity may respond to AML and ALL, but that lymphocyte cytotoxicity remains impaired. Dysfunction of CD3⁺CD56⁺ T lymphocytes in AML and ALL patients may contribute to the failure of the host immune response against leukemic blasts.     

The intratumoural subsite and relation of CD8+ and FOXP3+ T lymphocytes in colorectal cancer provide important prognostic clues

Background:

To find improved tools for prognostic evaluation in patients with colorectal cancer (CRC), we have analysed how infiltration of cytotoxic T lymphocytes (CD8⁺) and regulatory T lymphocytes (FoxP3⁺) correlates to prognosis, not only according to quantity and relation, but also to subsite within tumours of different molecular characteristics (microsatellite instability and CpG island methylator phenotype status).

Methods:

CD8 and FOXP3 expression was evaluated by immunohistochemistry in 426 archival tumour tissue samples from patients surgically resected for CRC. The average infiltration of CD8⁺ and FOXP3⁺ cells was assessed along the tumour invasive front, in the tumour centre and within the tumour epithelium (intraepithelial).

Results:

We found that infiltration of CD8⁺ T lymphocytes within the tumour epithelium provided the strongest prognostic information (P<0.001). At the tumour invasive front and tumour centre, FOXP3 expression withheld the strongest association to prognosis (P<0.001), suggesting FOXP3⁺ T-lymphocyte infiltration to be a better prognostic tool than CD8⁺ T lymphocytes at these intratumoural subsites. We further analysed the possible prognostic impact of the relation between these T-cell subsets, finding that a high intraepithelial CD8 expression was associated with a better patient outcome, independent of FOXP3 infiltration. In groups of low intraepithelial CD8 expression, however, a high infiltration rate of FOXP3⁺ cells at the tumour invasive front, significantly improved prognosis.

Conclusions:

Analyses of intraepithelial infiltration of CD8⁺ T lymphocytes, infiltration of FOXP3⁺ T lymphocytes at the tumour front or centre, and the relation between these subsets, may be a valuable tool for predicting prognosis in colon cancer.     

Mobilized CD34+ cells as a biomarker candidate for the efficacy of combined maximal tolerance dose and continuous infusional chemotherapy and G-CSF surge in gastric cancer

We investigated whether the level of bone marrow-derived progenitor cells and mature endothelial cells could be used as predictors of clinical outcome in patients receiving taxotere-based chemotherapy for advanced gastric cancer. Peripheral blood mononuclear cells were obtained from 49 gastric cancer patients who received taxotere combined with 5-FU and leucovorin and prophylactic G-CSF treatment. To categorize the cells, the cell markers CD34, vWF, P1H12, and CD31 were stained. Changes in these cells were examined before and after chemotherapy, and the clinical significance of these changes to response prediction and prognosis were investigated. Before the second cycle of chemotherapy, the number of CD34⁺/vWF⁺ and CD34⁺ cells was higher in non-responders as compared to the responders. Patients with 6.2 CD34⁺/vWF⁺ cells/ml had a shorter progression free survival (3.7 months) as against patients with <6.2 CD34⁺/vWF⁺/ml (6.0 months, p = 0.076). Patients with 5.8 CD34⁺ cells/ml had shorter progression free survival (4.0 months) than patients with <5.8 CD34⁺ cells/ml (6.1 months, p = 0.046). In an ex vivo pharmacokinetic study, the maximum inhibition (I_max) for HUVEC and YCC3 cells was 13.0 ± 6.6% and 74.0 ± 2.0%, respectively. The time to reach I_max (T_max) was 72 h in all HUVEC cells and 0.5 hours in YCC3 cells. We suggested that CD34⁺/vWF⁺ and CD34⁺ cells can be used as a biomarker for prediction and CD34⁺ cells for prognosis.     

CD4+ and CD8+ T cells have opposing roles in breast cancer progression and outcome

The Cancer Immunoediting concept has provided critical insights suggesting dual functions of immune system during the cancer initiation and development. However, the dynamics and roles of CD4⁺ and CD8⁺ T cells in the pathogenesis of breast cancer remain unclear. Here we utilized two murine breast cancer models (4T1 and E0771) and demonstrated that both CD4⁺ and CD8⁺ T cells were increased and involved in immune responses, but with distinct dynamic trends in breast cancer development. In addition to cell number increases, CD4⁺ T cells changed their dominant subsets from Th1 in the early stages to Treg and Th17 cells in the late stages of the cancer progression. We also analyzed CD4⁺ and CD8⁺ T cell infiltration in primary breast cancer tissues from cancer patients. We observed that CD8⁺ T cells are the key effector cell population mediating effective anti-tumor immunity resulting in better clinical outcomes. In contrast, intra-tumoral CD4⁺ T cells have negative prognostic effects on breast cancer patient outcomes. These studies indicate that CD4⁺ and CD8⁺ T cells have opposing roles in breast cancer progression and outcomes, which provides new insights relevant for the development of effective cancer immunotherapeutic approaches.     

CD45RA-Foxp3high but not CD45RA+Foxp3low suppressive T regulatory cells increased in the peripheral circulation of patients with head and neck squamous cell carcinoma and correlated with tumor progression

Background

T regulatory cells (Tregs) contribute to the progression of head and neck squamous cell carcinoma (HNSCC) by suppressing antitumor immunity. However, little is known regarding the functional heterogeneity of Tregs in HNSCC patients.

Methods

Using multicolor flow cytometry, the frequency of three Treg subsets, separated on the basis of CD45RA and Foxp3, from the peripheral circulation of newly-presenting HNSCC patients (19 oral cavity squamous cell carcinoma, 20 hypopharyngeal squamous cell carcinoma, 18 nasopharyngeal squamous cell carcinoma, 19 oropharyngeal squamous cell carcinoma, and 36 laryngeal squamous cell carcinoma) were assessed with regard to 31 healthy donors and clinicopathological features. Moreover, the functional capacity of each Treg subsets was evaluated based on CD45RA and CD25 expression.

Results

The frequency of Tregs in the peripheral circulation of HNSCC patients as a whole cohort was higher than in healthy donors (P < 0.0001). However, the frequency of Tregs was similar between patients with oral cavity squamous cell carcinoma and healthy donors (P = 0.269). Further dividing Tregs into three subsets based on Foxp3 and CD45RA expression revealed that the frequency of CD45RA^-Foxp3^high Tregs and CD45RA^-Foxp3^lowCD4⁺ T cells in patients with HNSCC developing from different subsites was higher than in healthy donors (P < 0.0001, P < 0.0001), whereas the frequency of CD45RA⁺Foxp3^low Tregs was lower than in healthy donors (P < 0.0001). Functionally study revealed that CD45RA^-CD25⁺⁺⁺ Tregs significantly inhibit the proliferation of CD4⁺CD25^- T cells (P < 0.001) and secrete lower levels of cytokines (P < 0.01) compared with CD45RA^-CD25⁺⁺CD4⁺ T cells. Importantly, the frequency of CD45RA^-Foxp3^high Tregs positively correlate with tumor stage (P < 0.0001) and nodal status (P < 0.0001).

Conclusions

CD45RA^-Foxp3^high Tregs increase in the peripheral circulation of HNSCC patients, and correlate with tumor stage and nodal status; suggesting a role in tumor progression which may be manipulated by future immunotherapy.     

Lecithin: Cholesterol Acyltransferase and Na+-K+-ATPase Activity in Patients with Breast Cancer

Purpose

The aim of this study was to determine whether plasma lecithin:cholesterol acyltransferase (pLCAT) and erythrocyte membrane Na⁺-K⁺-ATPase ase (emNaKATPs) activity have a correlation in breast cancer. This study compared these parameters at time points before and after treatment with radiotherapy.

Methods

The levels of pLCAT and emNaKATPs were assessed in 30 patients with breast carcinoma and 20 control subjects. While emNaKATPs was measured with spectrophotometric method, pLCAT levels was measured using a specific enzyme-linked immunosorbent assay.

Results

pLCAT levels, both before and after radiotherapy, were found to be decreased in breast cancer patients than in the controls groups (p<0.001 and p<0.001, respectively). Also, pLCAT levels after radiotherapy were found to be decreased in breast cancer patients than the pLCAT levels before radiotherapy (p<0.001). The emNaKATPs activity were higher in the control group than in the breast cancer patients before/after radiotherapy (RT) (p<0.001 and p<0.001, respectively). At the same time, emNaKATPs activity before RT was higher in the breast cancer patients than emNaKATPs activity after RT (p<0.001). There was a significant correlation between pLCAT and emNaKATPs activity in breast cancer patients receiving radiotherapy (r=0.63, p<0.001), but no correlation between in breast cancer patients before RT and control group (r=0.023, p>0.05).

Conclusion

The results of the present study demonstrated that decreased pLCAT and emNaKATPs activity levels in breast cancer patients after/before RT than control group. In addition, decreased emNaKATPs activity in breast cancer patients receiving radiotherapy may be due to decreased pLCAT concentrations and RT beam. In our opinion, altered activities of pLCAT and emNaKATPs are linked to the treatment effect of radiotherapy. These data may clarify the development of cell membrane dysfunction and lipid metabolism in breast cancer patients receiving radiotherapy.     

miR-548c-5p inhibits proliferation and migration and promotes apoptosis in CD90+ HepG2 cells

Background

Since the introduction of the theory of tumour stem cells (TSCs), the liver cancer stem cell (LCSC)-like cells have become one of the focuses in the research on liver cancer.

Materials and methods.

In this study, CD90⁺ cells were applied as the possible LCSC-like cells, and the miRNA and gene expression were analyzed in the CD90⁺ HepG2 cells. The pilot study showed miR-548c-5p exerted potential effect on the CD90⁺ HepG2 cells and was thereafter applied for the further study. CD90⁺ HepG2 cells were assigned to miR-548c-5p mimic transfection group and control group. MTT assay was performed to detect the proliferation of CD90⁺ HepG2 cells. The migration and invasion abilities were examined by wound healing assay and transwell migration assay, respectively. A detection of apoptosis was performed by fluorescence microscopy.

Results

Our results showed that caspase-3 and bcl-2 were down-regulated while caspase-8 was up-regulated in the CD90⁺ HepG2 cells. Moreover, the miR-548c-5p transfection could down-regulate the expression of β-catenin, Tg737, bcl-2, bcl-XL, and caspase-3, inhibit the proliferation, migration and invasion and promote the apoptosis of the CD90⁺ HepG2 cells.

Conclusions

Our findings indicate the imbalance between apoptosis and anti-apoptosis in the LCSC-like cells, which influence the biological features of LCSC-like cells. miRNA plays a regulatory role in the LCSC-like cells among which miR-548c-5p might be a suppressor.     

Expansion of CD3+CD8+PD1+ T lymphocytes and TCR repertoire diversity predict clinical responses to adoptive cell therapy in advanced gastric cancer

The adoptive cell therapy (ACT) and delivery of ex vivo activated cellular products, such as dendritic cells (DCs), NK cells, and T cells, have shown promise for the treatment of gastric cancer (GC). However, it is unknown which cells can improve patient survival. This study was focused on the antitumour activity of a subset of these cellular products and their relationships with clinical outcomes. Nineteen patients were enrolled at the Capital Medical University Cancer Center, Beijing Shijitan Hospital, from June 1, 2013, to May 30, 2016. CD8⁺PD1⁺ T-cell sorting was carried out using flow cytometry, and the T-cell receptor (TCR) repertoire during ex vivo expansion for 15 days was analyzed by next-generation sequencing. After 15 days of culture, the number of CD8⁺ T cells had increased significantly, and the number of CD4⁺ T cells had increased correspondingly. After ex vivo expansion, CD8⁺ T cells exhibited significantly enhanced expression of PD-1, LAG-3, and TIM-3 but not 4-1BB. Survival analysis showed that patients with a pro/pre value of CD8⁺PD-1⁺ T cells >2.4 had significantly favorable overall survival (OS) (median OS time, 248 days versus 96 days, P=0.02) and progression-free survival (PFS) (median PFS time, 183 days vs. 77 days, P=0.002). The sorted CD8⁺PD-1⁺ T cells displayed enhanced antitumor activity and increased IFN-γ secretion after coculture with autologous tumor cell lines. TCR repertoire diversity was decreased after ex vivo expansion, which decreased the Shannon index and increased the clonality value. The prognosis of patients was significantly improved and was associated with the extent of CD8⁺PD-1⁺ T-cell expansion. In summary, this study showed that after ex vivo expansion for 15 days, CD8⁺PD-1⁺ T cells could be identified as tumor-reactive cells in patients treated for GC. Changing TCR species can predict the extent of CD3⁺CD8⁺PD1⁺ T-cell growth and the effect of ACT treatment.     

Prognostic and predictive value of immunological parameters for chemoradioimmunotherapy in patients with pancreatic adenocarcinoma

Background:

Chemoradioimmunotherapy of patients with pancreatic adenocarcinoma from the CapRI trial did not show any benefit of interferon-α in addition to a 5-fluorouracil (5FU)-based treatment. The aim of this study was to identify immunological parameters in patients from this trial to be used for predictive and/or prognostic purposes.

Methods:

The following methods were used: tumour immunohistology, FACS analyses, cytokine measurement, as well as cytotoxicity and ELIspot. Immunological parameters were correlated with patients'' survival using the Kaplan–Meier method.

Results:

Irrespective of therapy type, high lymphocyte accumulation in tumours and frequencies of NK cells and effector (eff) CD8⁺ T cells in peripheral blood of the patients were associated with patients'' survival. Amount of CD3⁺ and effector-memory CD8⁺ blood lymphocytes, expression of CD152 and interleukin (IL)-2 serum level showed a predictive value for chemoradioimmunotherapy. Tumoural accumulation of CD3⁺ and CD8⁺ cells was predictive for outcome of chemotherapy alone. Besides, we identified the frequencies of CD3⁺ lymphocytes, effCD8⁺ T cells and NK cells in the peripheral blood of the patients, and IL-10 amount in serum, to be predictive values for 5FU-based chemotherapy.

Conclusions:

Immunological parameters, identified in this trial as possible markers, may be of interest in personalized medicine towards the improvement of the treatment and prognosis of pancreatic carcinoma patients.     

Na+/K+-ATPase β2-subunit (AMOG) expression abrogates invasion of glioblastoma-derived brain tumor-initiating cells

Background

Mechanisms of glioma invasion remain to be fully elucidated. Glioma cells within glioblastoma multiforme (GBM) range from well-differentiated tumor cells to less-differentiated brain tumor-initiating cells (BTICs). The β2-subunit of Na⁺/K⁺-ATPase, called the adhesion molecule on glia (AMOG), is highly expressed in normal glia but is thought to be universally downregulated in GBM. To test our hypothesis that expression of AMOG is heterogeneous in GBM and confers a less invasive phenotype, we compared it between BTICs and differentiated cells from patient-matched GBM and then tested GBM invasion in vitro after AMOG overexpression.

Methods

Immunohistochemistry, immunoblotting, and real-time PCR were used to characterize AMOG protein and mRNA expression in tumor samples, BTICs, and differentiated cells. Matrigel invasion assay, scratch assay, and direct cell counting were used for testing in vitro invasion, migration, and proliferation, respectively.

Results

While AMOG expression is heterogeneous in astrocytomas of grades II–IV, it is lost in most GBM. BTICs express higher levels of AMOG mRNA and protein compared with patient-matched differentiated tumor cells. Overexpression of AMOG decreased GBM cell and BTIC invasion without affecting migration or proliferation. Knockdown of AMOG expression in normal human astrocytes increased invasion.

Conclusions

AMOG expression inhibits GBM invasion. Its downregulation increases invasion in glial cells and may also represent an important step in BTIC differentiation. These data provide compelling evidence implicating the role of AMOG in glioma invasion and provide impetus for further investigation.     

Biological significance of fluorine-18-α-methyltyrosine (FAMT) uptake on PET in patients with oesophageal cancer

Purpose:

¹⁸F-FAMT as an amino-acid tracer for positron emission tomography (PET) is useful for detecting human neoplasms. ¹⁸F-FAMT is accumulated in tumour cells solely via L-type amino-acid transporter 1 (LAT1). This study was conducted to investigate the biological significance of ¹⁸F-FAMT uptake in patients with oesophageal cancer.

Methods:

From April 2008 to December 2011, 42 patients with oesophageal cancer underwent both ¹⁸F-FAMT PET/CT and ¹⁸F-FDG PET/CT before surgical treatment. The immunohistochemical analysis of LAT1, CD98, Ki-67, CD34, p53, p-Akt and p-mTOR was performed on the primary lesions. In vitro experiments were performed to examine the mechanism of ¹⁸F-FAMT uptake.

Results:

High uptake of ¹⁸F-FAMT was significantly associated with advanced stage, lymph node metastasis and the expression of LAT1, CD98, Ki-67 and CD34. LAT1 expression yielded a statistically significant correlation with CD98 expression, cell proliferation, angiogenesis and glucose metabolism. In vitro experiments revealed that ¹⁸F-FAMT was specifically transported by LAT1.

Conclusions:

The uptake of ¹⁸F-FAMT within tumour cells is determined by the LAT1 expression and correlated with cell proliferation and angiogenesis in oesophageal cancer. The present experiments also confirmed the presence of LAT1 as an underlying mechanism of ¹⁸F-FAMT accumulation.     

Head and neck cancer relapse after chemoradiotherapy correlates with CD163+ macrophages in primary tumour and CD11b+ myeloid cells in recurrences

Background:

We investigated the prognostic role of tumour-associated macrophages (TAMs) in patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive chemoradiotherapy (CRT).

Methods:

The expression of CD68+, CD163+ and CD11b+ cells was assessed using immunohistochemistry in n=106 pre-treatment tumour biopsy samples and was correlated with clinicopathological characteristics, including T-stage, N-stage, grading, tumour localisation, age and sex as well as local failure-free survival (LFFS), distant metastases-free survival (DMFS), progression-free (PFS), and overall survival (OS). Finally, TAMs expression and vessel density (CD31) were examined in n=12 available early local recurrence samples and compared with their matched primary tumours . The diagnostic images and radiotherapy plans of these 12 patients were also analysed. All local recurrences occurred in the high radiation dose region (⩾70 Gy).

Results:

With a median follow-up of 40 months, OS at 2 years was 60.5%. High CD163 expression in primary tumours was associated with decreased OS (P=0.010), PFS (P=0.033), LFFS (P=0.036) and DMFS (P=0.038) in multivariate analysis. CD163 demonstrated a strong prognostic value only in human papillomavirus (p16^INK4)-negative patients. Early local recurrence specimens demonstrated a significantly increased infiltration of CD11b+ myeloid cells (P=0.0097) but decreased CD31-positive vessel density (P=0.0004) compared with their matched primary samples.

Conclusions:

Altogether, baseline CD163 expression predicts for an unfavourable clinical outcome in HNSCC after definitive CRT. Early local recurrences showed increased infiltration by CD11b+ cells. These data provide important insight on the role of TAMs in mediating response to CRT in patients with HNSCC.     

Specific recognition and inhibition of Ewing tumour growth by antigen-specific allo-restricted cytotoxic T cells

Background:

The development of a successful immunotherapy is hampered by an ineffective T-cell repertoire against tumour antigens and the inability of the patient''s immune system to overcome tolerance-inducing mechanisms. Here, we test the specific recognition and lytical potential of allo-restricted CD8⁺ T cells against Ewing tumour (ET) associated antigens Enhancer of Zeste, Drosophila Homolog 2 (EZH2), and Chondromodulin-I (CHM1) identified through previous microarray analysis.

Methods:

Following repetitive CHM1³¹⁹ (VIMPCSWWV) and EZH2⁶⁶⁶ (YMCSFLFNL) peptide-driven stimulations with HLA-A^*0201⁺ dendritic cells (DC), allo-restricted HLA-A^*0201⁻ CD8⁺ T cells were stained with HLA-A^*0201/peptide multimers, sorted and expanded by limiting dilution.

Results:

Expanded T cells specifically recognised peptide-pulsed target cells or antigen-transfected cells in the context of HLA-A^*0201 and killed HLA-A^*0201⁺ ET lines expressing the antigen while HLA-A^*0201^– ET lines were not affected. Furthermore, adoptively transferred T cells caused significant ET growth delay in Rag2^−/−γ_C^−/− mice. Within this context, we identified the CHM1³¹⁹ peptide as a new candidate target antigen for ET immunotherapy.

Conclusion:

These results clearly identify the ET-derived antigens, EZH2⁶⁶⁶ and CHM1³¹⁹, as suitable targets for protective allo-restricted human CD8⁺ T-cell responses against non-immunogenic ET and may benefit new therapeutic strategies in ET patients treated with allogeneic stem cell transplantation.     

鼻咽癌组织与外周血CD4+CD25+调节性T细胞检测及临床意义

 目的 通过检测鼻咽癌患者肿瘤组织及外周血中CD4⁺T、CD8⁺T、CD4⁺CD25^-T、CD4⁺CD25⁺T细胞的频数,寻找客观、全面评价鼻咽癌患者免疫状态的临床指标。方法 采用流式细胞术检测40例初诊鼻咽癌患者及10例正常对照鼻咽部组织和外周血CD4⁺T、CD8⁺T、CD4⁺CD25^-T、CD4⁺CD25⁺T细胞比例。结果 鼻咽癌患者CD4⁺T细胞比例及CD4⁺/ CD8⁺T比值均低于对照组（P<0.05）,而CD8⁺T细胞两组间差异无统计学意义（P>0.05）,但是CD4⁺/ CD8⁺T比值在鼻咽癌组织与外周血间差异无统计学意义（P>0.05）。鼻咽癌组织及外周血中CD4⁺CD25⁺T细胞比例都高于对照组（P<0.05）,同时癌组织中该细胞比例远远高于外周血（P<0.05）。在鼻咽癌组织中CD4⁺CD25⁺T细胞与CD8+ T细胞、CD4⁺CD25^-T细胞呈负相关（r分别为-0.70、-0.675,P<0.05）,而在外周血中没有相关关系（P>0.05）。在不同T(原发肿瘤大小)组间,T4组的鼻咽癌组织中CD4⁺CD25⁺T细胞分别高于T1、T2、T3各组（P<0.05）,而在T1、T2、T3各组间差异无统计学意义（P>0.05）;鼻咽癌中CD4⁺CD25⁺T细胞比例与患者有无淋巴结转移并无关系（P>0.05）;鼻咽癌组织中Ⅲ+Ⅳ期组CD4⁺CD25⁺T细胞比例高于Ⅰ+Ⅱ期组（P<0.05）,而在外周血中两组间差异无统计学意义（P>0.05）。结论 CD4⁺CD25⁺T细胞与鼻咽癌病程进展无相关性,但是联合检测患者肿瘤组织及外周血中CD4⁺CD25⁺T细胞的频数并结合既往CD4⁺/ CD8⁺T比值会全面反应患者免疫状态,为临床治疗提供依据。     

Intratumoral regulatory T cells upregulate immunosuppressive molecules in head and neck cancer patients

Background:

Although regulatory T cells (Treg) are highly enriched in human tumours compared with peripheral blood, expression of the immune-checkpoint receptors, immunosuppressive molecules and function of Treg in these two sites remains undefined.

Methods:

Tumour-infiltrating lymphocytes and peripheral blood lymphocytes were isolated from a cohort of head and neck squamous cell carcinoma (HNSCC) patients. The immunosuppressive phenotypes and function of intratumoral Treg were compared with those of peripheral blood Treg.

Results:

The frequency of immune-checkpoint receptor-positive cells was higher on intratumoral FOXP3⁺CD25^hi Treg compared with circulating Treg (CTLA-4, P=0.002; TIM-3, P=0.002 and PD-1, P=0.002). Immunosuppressive effector molecules, LAP and ectonucleotidase CD39 were also upregulated on intratumoral FOXP3⁺ Treg (P=0.002 and P=0.004, respectively). CTLA-4 and CD39 were co-expressed on the majority of intratumoral FOXP3⁺CD4⁺ Treg, suggesting that these molecules have a key role in regulatory functions of these cells in situ. Notably, intratumoral Treg exhibited more potently immunosuppressive activity than circulating Treg.

Conclusion:

These results indicate that intratumoral Treg are more immunosuppressive than circulating Treg and CTLA-4 and CD39 expressed can be potential target molecules to inhibit suppressive activities of intratumoral Treg in situ.