Analysis of the Heterogeneity of CD4+CD25+ T Cell TCR β CDR3 Repertoires in Breast Tumor Tissues, Lung Metastatic Tissues, and Spleens from 4T1 Tumor-Bearing BALB/c Mice

To study the homogeneity and heterogeneity of CD4+CD25+ T cells receptor β-chain complementarity determining region 3 (TCR β CDR3) repertoires in breast tumor tissues, lung metastatic tissues, and spleens from 4T1 tumor-bearing BALB/c mice. We used high-throughput sequencing to analyze the characteristics and changes of CD4+CD25+ TCR β CDR3 repertoires among tumor tissues, lung metastatic tissues, and spleens. The diversity of the CD4+CD25+ TCR β CDR3 repertoires in breast tumor tissue was similar to that of lung metastatic tissues and less pronounced than that of spleen tissues. Breast tumor tissues and lung metastatic tissues had a greater number of high-frequency CDR3 sequences and intermediate-frequency CDR3 sequences than those of spleens. The proportion of unique productive CDR3 sequences in breast tumor tissues and lung metastatic tissues was significantly greater than that in the spleens. The diversity and frequency of the CDR3 repertoires remained homogeneous in breast tumors and lung metastatic tissues and showed great heterogeneity in the spleens, which suggested that the breast tissues and lung metastatic tissues have characteristics of CD4+CD25+ T cells that relate to the tumor microenvironment. However, the number and characteristics of overlapping CDR3 sequences suggested that there were some different CD4+CD25+ T cells in tumors and in the circulatory immune system. The study may be used to further explore the characteristics of the CDR3 repertoires and determine the source of the CD4+CD25+ T cells in the breast cancer microenvironment.

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CCL2 and IL18 expressions may associate with the anti-proliferative effect of noncontact electro capacitive cancer therapy in vivo

F1000Research ◽

10.12688/f1000research.20727.1 ◽

2019 ◽

Vol 8 ◽

pp. 1770 ◽

Cited By ~ 1

Author(s):

Rarastoeti Pratiwi ◽

Nyoman Yudi Antara ◽

Lalu Gunawan Fadliansyah ◽

Syamsul Arif Ardiansyah ◽

Luthfi Nurhidayat ◽

...

Keyword(s):

Cancer Therapy ◽

Breast Tumor ◽

Metastatic Breast ◽

Intermediate Frequency ◽

Necrosis Factor Alpha ◽

Breast Tumor Tissue ◽

Proliferative Effect ◽

Tumor Tissues ◽

Tnf Α

Background: Noncontact Electro Capacitive Cancer Therapy (ECCT) is a novel treatment modality in cancer. Chemokine (C-C motif) ligand 2 (CCL2) has a major role in the outgrowth of metastatic breast cancer. Interleukin 18 (IL18) plays a role in macrophage alteration, which leads to excessive angiogenesis. This study aims to elaborate on the association of CCL2, IL18, IL23α, and TNF-α (tumor necrosis factor-alpha) expression with the anti-proliferative effect of ECCT in rat breast tumor tissue. Methods: Low intensity (18 Vpp) and intermediate frequency (150 kHz) alternating current-electric field (AC-EF) between two capacitive electrodes were exposed as external EF to a rat cage. Twenty-four rats were divided into four groups of six replicates. Breast tumor tissues were collected from 7, 12-dimethylbenz[a]anthracene (DMBA)-induced rats. Two groups were none DMBA-induced rats without ECCT exposure (NINT) and with (NIT). The other two groups were DMBA-induced rats without ECCT exposure (INT) and with (IT). Mammary glands and breast tumor tissues were collected from each group and preserved. Hematoxylin-eosin and immunohistochemistry staining were performed on paraffin sections of tissues using anti-PCNA, anti-ErbB2, anti-Caspase3, and anti-CD68. CCL2, IL18, IL23α, and TNF-α mRNA relative expressions were analyzed using qRT-PCR. Results: ECCT exposure may cause the reduction of PCNA protein expression as well as ErbB2 on breast tumor tissues, but it causes the increase of Caspase3 and macrophage CD68 protein. In rat breast tumor tissues of IT groups, the mRNA expression of CCL2 and IL18 are significantly down-regulated, in contrast with the up-regulated expression of these cytokines in tumor tissues of the INT group. IL23α and TNF- α expression remained similar in both groups. Conclusion: CCL2 and IL18 expressions have an association with the inhibition of breast tumor cell proliferation affected by ECCT exposure

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CCL2 and IL18 expressions may associate with the anti-proliferative effect of noncontact electro capacitive cancer therapy in vivo

F1000Research ◽

10.12688/f1000research.20727.2 ◽

2020 ◽

Vol 8 ◽

pp. 1770

Author(s):

Rarastoeti Pratiwi ◽

Nyoman Yudi Antara ◽

Lalu Gunawan Fadliansyah ◽

Syamsul Arif Ardiansyah ◽

Luthfi Nurhidayat ◽

...

Keyword(s):

Cancer Therapy ◽

Breast Tumor ◽

Metastatic Breast ◽

Intermediate Frequency ◽

Necrosis Factor Alpha ◽

Breast Tumor Tissue ◽

Proliferative Effect ◽

Tumor Tissues ◽

Tnf Α

Background: Noncontact Electro Capacitive Cancer Therapy (ECCT) is a novel treatment modality in cancer. Chemokine (C-C motif) ligand 2 (CCL2) has a major role in the outgrowth of metastatic breast cancer. Interleukin 18 (IL18) plays a role in macrophage alteration, which leads to excessive angiogenesis. This study aims to elaborate on the association of CCL2, IL18, IL23α, and TNF-α (tumor necrosis factor-alpha) expression with the anti-proliferative effect of ECCT in rat breast tumor tissue. Methods: Low intensity (18 Vpp) and intermediate frequency (150 kHz) alternating current-electric field (AC-EF) between two capacitive electrodes were exposed as external EF to a rat cage. Twenty-four rats were divided into four groups of six replicates. Breast tumor tissues were collected from 7, 12-dimethylbenz[a]anthracene (DMBA)-induced rats. Two groups were non DMBA-induced rats without ECCT exposure (NINT) and with (NIT). The other two groups were DMBA-induced rats without ECCT exposure (INT) and with (IT). Mammary glands and breast tumor tissues were collected from each group and preserved. Hematoxylin-eosin and immunohistochemistry staining were performed on paraffin sections of tissues using anti-PCNA, anti-ErbB2, anti-Caspase3, and anti-CD68. CCL2, IL18, IL23α, and TNF-α mRNA relative expressions were analyzed using qRT-PCR. Results: ECCT exposure may cause the reduction of PCNA protein expression as well as ErbB2 on breast tumor tissues, but it causes the increase of Caspase3 and macrophage CD68 protein. In rat breast tumor tissues of IT groups, the mRNA expression of CCL2 and IL18 are significantly down-regulated, in contrast with the up-regulated expression of these cytokines in tumor tissues of the INT group. IL23α and TNF- α expression remained similar in both groups. Conclusion: CCL2 and IL18 expressions have an association with the inhibition of breast tumor cell proliferation affected by ECCT exposure

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Systemic infection with Candida albicans in breast tumor bearing mice: Cytokines dysregulation and induction of regulatory T cells

Journal de Mycologie Médicale ◽

10.1016/j.mycmed.2018.10.006 ◽

2019 ◽

Vol 29 (1) ◽

pp. 49-55 ◽

Cited By ~ 4

Author(s):

N. Ahmadi ◽

A. Ahmadi ◽

E. Kheirali ◽

M. Hossein Yadegari ◽

M. Bayat ◽

...

Keyword(s):

T Cells ◽

Candida Albicans ◽

Regulatory T Cells ◽

Breast Tumor ◽

Systemic Infection ◽

Tumor Bearing

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Long non-coding RNA UCA1 promotes breast cancer by upregulating PTP1B expression via inhibiting miR-206

Cancer Cell International ◽

10.1186/s12935-019-0958-z ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 5

Author(s):

Yi Li ◽

Qingan Zeng ◽

Jiliang Qiu ◽

Ting Pang ◽

Jianzhong Xian ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Tumor ◽

Breast Cancer Cells ◽

Transcriptional Level ◽

Normal Tissues ◽

Non Coding Rna ◽

Tumor Tissues ◽

Breast Tissues ◽

Long Non Coding Rna

Abstract Background The long non-coding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) is involved in various cancers and often functions through microRNAs. The pro-survival protein PTP1B is known to play important roles in cancer development. However, the connection between UCA1 and PTP1B in breast cancer is not well studied. Methods In this study, we first evaluated the correlation between UCA1 level and PTP1B expression in breast tissues, which showed the expression of PTP1B were much higher in the breast tumor tissues than in the peritumor normal tissues. The UCA1 level was positively associated with PTP1B expression in breast tumor tissues. Results We observed that UCA1 could up-regulate PTP1B expression in breast cancer cells. We also found that miR-206 could inhibit the expression of PTP1B by directly binding to the 3′-UTR of its mRNA. Interestingly, UCA1 could increase the expression of PTP1B through sequestering miR-206 at post-transcriptional level. The results also suggested that UCA1-induced PTP1B expression facilitated the proliferation of breast cancer cells. Conclusions We conclude that UCA1 can up-regulates PTP1B to enhance cell proliferation through sequestering miR-206 in breast cancer. Our finding provides new insights into the mechanism of breast cancer regulation by UCA1, which could be a potential target for breast cancer treatment. Trial registration 2012N5hSYSU48573. Registered at Oct 12, 2012

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HCA587 protein vaccine induces specific antitumor immunity mediated by CD4+ T-cells expressing granzyme B in a mouse model of melanoma

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200728131951 ◽

2020 ◽

Vol 20 ◽

Author(s):

Weiming Yang ◽

Weiheng Zhang ◽

Xiaozhong Wang ◽

Liming Tan ◽

Hua Li ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Antitumor Effect ◽

Granzyme B ◽

Cell Subset ◽

Cell Mediated Immunity ◽

Protein Vaccine ◽

Tumor Tissues ◽

Wide Range ◽

Ifn Γ

Background: The antigen HCA587 (also known as MAGE-C2), which is considered a cancer-testis antigen, exhibits upregulated expression in a wide range of malignant tumors with unique immunological properties, and may thus serve as a promising target for tumor immunotherapy. Objective: To explore the antitumor effect of the HCA587 protein vaccine and the response of humoral and cell-mediated immunity. Methods: The HCA587 protein vaccine was formulated with adjuvants CpG and and ISCOM. B16 melanoma cells were subcutaneously inoculated to C57BL/6 mice, followed by treatment with HCA587 protein vaccine subcutaneously. Mouse survival was monitored daily, and tumor volume was measured every 2 to 3 days. The tumor sizes, survival time and immune cells in tumor tissues were detected. And the vital immune cell subset and effector molecules were explored. Results: After treatment with HCA587 protein vaccine, the vaccination generated elicited significant immune responses, which delayed tumor growth and improved animal survival. The vaccination increased the proportion of CD4+ T cells expressing IFN-γ and granzyme B in tumor tissues. Depletion of CD4+T cells resulted in an almost complete abrogation of the antitumor effect of the vaccination, suggesting that the antitumor efficacy was mediated by CD4+ T cells. In addition, knockout of IFN-γ resulted in a decrease in granzyme B levels which were secreted by CD4+ T cells, and the antitumor effect was also significantly attenuated. Conclusion: The HCA587 protein vaccine may increase the levels of granzyme B expressed by CD4+ T cells, and this increase is dependent on IFN-γ, and the vaccine resulted in a specific tumor immune response and subsequent eradication of the tumor.

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678 Addition of a single bacteria facilitates anti-tumor immunity and long-term survival in colorectal cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0678 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A717-A717

Author(s):

Abigail Overacre-Delgoffe ◽

Anthony Cillo ◽

Hannah Bumgarner ◽

Ansen Burr ◽

Justin Tometich ◽

...

Keyword(s):

Colorectal Cancer ◽

T Cells ◽

Tumor Immunity ◽

Tumor Burden ◽

Intestinal Microbiome ◽

Tumor Control ◽

Mesenteric Lymph Nodes ◽

Term Survival ◽

16S Sequencing ◽

Tumor Bearing

BackgroundColorectal cancer remains one of the most common and deadliest cancers worldwide and effective therapies are lacking. While immunotherapy has revolutionized treatment for many cancers, the overwhelming majority of colorectal cancer patients are non-responsive and the 5-year survival rate for advanced disease is <20%. Immunotherapeutic response has been associated with select members of the microbiome in melanoma; however, the potential benefit in colorectal cancer and the underlying mechanisms remain unclear. We sought to determine how specific members of the intestinal microbiome affect anti-tumor immunity in colorectal cancer (CRC) in hopes of discovering novel treatments and revealing potential hurdles to current therapeutic response in CRC patients.MethodsWe utilized a carcinogen-induced mouse model of CRC and colonized half of the tumor-bearing mice with Helicobacter hepaticus (Hhep) 7 weeks post AOM. Tumor number was assessed 12 weeks post AOM. We isolated lymphocytes from the lamina propria, colonic epithelium, mesenteric lymph nodes, and tumor(s) to track the spatial and transcriptional Hhep-specific and endogenous immune responses during tumor progression through 5’ single cell RNAseq, flow cytometry, and immunofluorescence. In addition, we utilized 16S sequencing and FISH to track Hhep colonization, location within the colon, and its impact on the surrounding microbiome.ResultsWe have found that rational modification of the microbiome of colon tumor-bearing mice through addition of a single bacteria, Hhep, led to tumor control or clearance and a significant survival advantage. Colonization led to the expansion of the lymphatic network and development of numerous peri- or intra-tumoral tertiary lymphoid structures (TLS) composed of Hhep-specific CD4 T follicular helper cells (TFH) as well as the bacteria itself. This led to an overall ‘heating’ of the tumor, wherein we saw an increase of CD4 T cell infiltration to the tumor core as well as an increase in CD103+ type 1 DC (cDC1) recruitment through increased chemokines such as CCL5 and XCL1. Hhep-specific TFH were both necessary and sufficient to drive TLS formation, increased immune invasion, and anti-tumor immunity.ConclusionsWe have shown that addition of a single bacteria, Hhep, leads to a reduction in CRC tumor burden or clearance through lymphatic expansion, TLS formation, and remodeling of the tumor microenvironment, and that Hhep-specific T cells are required for tumor control. These studies suggest that rational modification of the microbiome and microbiome-specific T cells can positively impact anti-tumor immunity and may represent a unique immunotherapeutic target to turn resistant tumors into responsive tumors.

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49 In situ phenotypic analysis of T cells in the tumor microenvironment of a pre-clinical model of non-small cell lung cancer (NSCLC) by tissue sectioning and whole-mount immunofluorescence imaging

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0049 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A52-A52

Author(s):

Elen Torres ◽

Stefani Spranger

Keyword(s):

T Cells ◽

Spatial Distribution ◽

T Cell ◽

Spatial Location ◽

Cell Infiltration ◽

Cell Populations ◽

Tumor Bearing ◽

T Cell Infiltration ◽

Volume Imaging ◽

Whole Mount

BackgroundUnderstanding the interactions between tumor and immune cells is critical for improving current immunotherapies. Pre-clinical and clinical evidence has shown that failed T cell infiltration into lung cancer lesions might be associated with low responsiveness towards checkpoint blockade.1 For this reason, it is necessary to characterize not only the phenotype of T cells in tumor-bearing lungs but also their spatial location in the tumor microenvironment (TME). Multiplex immunofluorescence staining allows the simultaneous use of several cell markers to study the state and the spatial location of cell populations in the tissue of interest. Although this technique is usually applied to thin tissue sections (5 to 12 µm), the analysis of large tissue volumes may provide a better understanding of the spatial distribution of cells in relation to the TME. Here, we analyzed the number and spatial distribution of cytotoxic T cells and other immune cells in the TME of tumor-bearing lungs, using both 12 µm sections and whole-mount preparations imaged by confocal microscopy.MethodsLung tumors were induced in C57BL/6 mice by tail vein injection of a cancer cell line derived from KrasG12D/+ and Tp53-/- mice. Lung tissue with a diverse degree of T cell infiltration was collected after 21 days post tumor induction. Tissue was fixed in 4% PFA, followed by snap-frozen for sectioning. Whole-mount preparations were processed according to Weizhe Li et al. (2019) 2 for tissue clearing and multiplex volume imaging. T cells were labeled with CD8 and FOXP3 antibodies to identify cytotoxic or regulatory T cells, respectively. Tumor cells were labeled with a pan-Keratin antibody. Images were acquired using a Leica SP8 confocal microscope. FIJI3 and IMARIS were used for image processing.ResultsWe identified both cytotoxic and regulatory T cell populations in the TME using thin sections and whole-mount. However, using whole-mount after tissue clearing allowed us to better evaluate the spatial distribution of the T cell populations in relation to the tumor structure. Furthermore, tissue clearance facilitates the imaging of larger volumes using multiplex immunofluorescence.ConclusionsAnalysis of large lung tissue volumes provides a better understanding of the location of immune cell populations in relation to the TME and allows to study heterogeneous immune infiltration on a per-lesion base. This valuable information will improve the characterization of the TME and the definition of cancer-immune phenotypes in NSCLC.ReferencesTeng MW, et al., Classifying cancers based on T-cell infiltration and PD-L1. Cancer Res 2015;75(11): p. 2139–45.Li W, Germain RN, and Gerner MY. High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging. Nat Protoc 2019;14(6): p. 1708–1733.Schindelin J, et al, Fiji: an open-source platform for biological-image analysis. Nat Methods 2012;9(7): p. 676–82.

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Early Detection and Dynamic Changes of Circulating Tumor Cells in Transgenic NeuN Transgenic (NTTg) Mice with Spontaneous Breast Tumor Development

Cancers ◽

10.3390/cancers13133294 ◽

2021 ◽

Vol 13 (13) ◽

pp. 3294

Author(s):

Wen-Sy Tsai ◽

Tsung-Fu Hung ◽

Jia-Yang Chen ◽

Shu-Huan Huang ◽

Ying-Chih Chang

Keyword(s):

Tumor Cells ◽

Circulating Tumor Cells ◽

Breast Tumor ◽

Early Stage ◽

Tumor Development ◽

P Value ◽

Vascular Density ◽

Dynamic Changes ◽

Tumor Bearing ◽

Development Methods

Background: This study used NeuN transgenic (NTTg) mice with spontaneous breast tumor development to evaluate the dynamic changes of circulating tumor cells (CTCs) prior to and during tumor development. Methods: In this longitudinal, clinically uninterrupted study, we collected 75 μL of peripheral blood at the age of 8, 12, 16, and 20 weeks in the first group of five mice, and at the age of 32 weeks, the time of tumor palpability, and one week after tumor palpability in the second group of four mice. Diluted blood samples were run through a modified mouse-CMx chip to isolate the CTCs. Results: The CTC counts of the first group of mice were low (1 ± 1.6) initially. The average CTC counts were 16 ± 9.5, 29.0 ± 18.2, and 70.0 ± 30.3 cells per 75 μL blood at the age of 32 weeks, the time of tumor palpability, and one week after tumor palpability, respectively. There was a significant positive correlation between an increase in CTC levels and tumor vascular density (p-value < 0.01). This correlation was stronger than that between CTC levels and tumor size (p-value = 0.076). The captured CTCs were implanted into a non-tumor-bearing NTTg mouse for xenografting, confirming their viability and tumorigenesis. Conclusion: Serial CTCs during an early stage of tumor progression were quantified and found to be positively correlated with the later tumor vascular density and size. Furthermore, the successful generation of CTC-derived xenografts indicates the tumorigenicity of this early onset CTC population.

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