Breast cancer metastasis: immune profiling of lymph nodes reveals exhaustion of effector T cells and immunosuppression

Sentinel lymph nodes are the first nodes draining the lymph from a breast and could reveal early changes in the host immune system upon dissemination of breast cancer cells. To investigate this, we performed single‐cell immune profiling of lymph nodes with and without metastatic cells. Whereas no significant changes were observed for B‐cell and natural killer (NK)‐cell subsets, metastatic lymph nodes had a significantly increased frequency of CD8 T cells and a skewing toward an effector/memory phenotype of CD4 and CD8 T cells, suggesting an ongoing immune response. Additionally, metastatic lymph nodes had an increased frequency of TIGIT (T‐cell immunoreceptor with Ig and ITIM domains)‐positive T cells with suppressed TCR signaling compared with non‐metastatic nodes, indicating exhaustion of effector T cells, and an increased frequency of regulatory T cells (Tregs) with an activated phenotype. T‐cell alterations correlated with the percentage of metastatic tumor cells, reflecting the presence of metastatic tumor cells driving T effector cells toward exhaustion and promoting immunosuppression by recruitment or increased differentiation toward Tregs. These results show that immune suppression occurs already in early stages of tumor progression.     

A histomorphological study of regional lymphnodes in carcinomas of breast, stomach and colon.

INTRODUCTION: Immune response plays an important role in the interaction between malignant neoplasms and their host, which is reflected as histologic changes in the lymph nodes draining tumours. MATERIAL: 641 regional lymph nodes from 64 primary carcinomas of breast, stomach and colon were examined to assess such a response. METHODS: The lymphnodes were classified into one of the following histological patterns--lymphocyte predominance (LP), germinal centre predominance (GCP), lymphocyte depletion (LD) and unstimulated (U). RESULTS: LP (T Cell response) was the frequently observed pattern in the lymph nodes of breast and stomach. A similar pattern was observed among the survivors irrespective of the involvement of lymph node with tumour. There was a significant association between the histologic pattern of lymph node and stage & grade of the disease in breast carcinoma. CONCLUSION: The correlation of lymph node histology with grade and extent of the disease & survival indicate that the immune system is important in regulating the growth of malignant neoplasms. Such information may help as prognostic indicator and as therapeutic guide for immunotherapy.     

Interferon production in lymph nodes draining breast carcinoma and its augmentation by OK-432

In breast cancer patients on whom modified radical mastectomy is performed, relatively more of the regional lymph nodes draining the breast carcinoma remain in comparison with standard radical mastectomy. Therefore, investigation of the functions of lymph nodes draining breast carcinoma has become important. Lymphocyte subsets of 33 axillary lymph nodes from 19 breast cancer patients were analysed using flow cytometry. In axillary lymph nodes, both OKT-3(+) cells and OKT-8(+) cells were decreased in comparison with those in peripheral blood. However, the OKT 4/8 ratio was increased in axillary lymph nodes. These findings suggest that axillary lymph nodes are immunologically more functional against cancer spread than peripheral blood. OK-M1(+) cells, Leu-7(+) cells and Leu-11a(+) cells were decreased in axillary lymph nodes in comparison with peripheral blood. The ability of IFN production in axillary lymph nodes and peripheral blood was analysed using the cytopathic effect of VSV-sindbis virus. After 72 hours incubation, IFN production of axillary lymph nodes showed maximum titer. When lymph nodes were co-cultured with OK-432, IFN production of axillary lymph nodes was strongly augmented. IFN production of axillary lymph nodes draining breast carcinoma were increased in comparison with peripheral blood. Axillary lymph nodes draining breast carcinoma would thus seem to be important as cytokine-producing organs. IFN has been found to be an activator of NK cells, cytotoxic T cells and IL-2 production. Axillary lymph nodes may therefore play an important role against the spread of breast cancer.     

Altered local and systemic immune profiles underlie lymph node metastasis in breast cancer patients

Cancer‐mediated immune dysfunction contributes to tumor progression and correlates with patient outcome. Metastasis to tumor draining lymph nodes (TDLNs) is an important step in breast cancer progression and is used to predict patient outcome and survival. Although lymph nodes are important immune organs, the role of immune cells in TDLNs has not been thoroughly investigated. We hypothesized that the host immune response in node negative (NN) patients is more intact and thereby can resist tumor invasion compared to node positive (NP) patients. As such, lymph node metastasis requires breakdown of the host immune response in addition to escape of cancer cells from the tumor. To investigate the immunological differences between NN and NP breast cancer patients, we purified and profiled immune cells from the three major compartments where cancer and immune cells interact: tumor, TDLNs and peripheral blood. Significant down‐regulation of genes associated with immune‐related pathways and up‐regulation of genes associated with tumor‐promoting pathways was consistently observed in NP patients' TDLNs compared to NN patients. Importantly, these signatures were seen even in NP patients' tumor‐free TDLNs, suggesting that such immune changes are not driven solely by local tumor invasion. Furthermore, similar patterns were also observed in NP patients' tumor and blood immune cells, suggesting that immunological differences between NN and NP patients are systemic. Together, these findings suggest that alterations in overall immune function may underlie risk for LN metastasis in breast cancer patients.     

Spontaneous mammary carcinomas fail to induce an immune response in syngeneic FVBN202 neu transgenic mice

FVBN202 mice, which are transgenic for the rat neu gene, spontaneously develop mammary carcinomas between 6 and 7 months of age. We investigated whether these spontaneous tumors (spontaneous breast carcinoma cells, SBCC) could elicit an immune response in naive 6- to 8-week-old FVBN202 transgenic and FVBN nontransgenic mice. After s.c. injection of SBCC, the recently activated T cells, which were identified by their reduced expression of CD62L (L-selectin), were isolated from the draining lymph nodes, expanded with anti-CD3 and IL-2, and their cytokine response to tumor cells in vitro was analyzed. Tumor-vaccine draining lymph node lymphocytes (TVDLN) from transgenic mice failed to make IFN-gamma in response to the tumor cells. However, TVDLN from the nontransgenic mice exhibited a tumor-specific IFN-gamma response against the SBCC. This indicates that the SBCC are immunogenic. The lack of response in transgenic mice could not be attributed to cytokine immune deviation or T-cell signaling defects. Although transgenic mice were tolerant to their own tumors, their immune competence was established by their ability to respond in an allogeneic mixed lymphocyte reaction, to reject an allogeneic breast carcinoma cell line, and to produce a tumor-specific IFN-gamma response against a syngeneic cancer cell line. This transgenic mouse model provides the opportunity to investigate the immune response against a primary tumor cell culture rather than cell lines or clones and should prove useful for developing immunotherapies that overcome tolerance to self-tumor antigens.     

Mage-b vaccine delivered by recombinant Listeria monocytogenes is highly effective against breast cancer metastases

New therapies are needed that target breast cancer metastases. In previous studies, we have shown that vaccination with pcDNA3.1-Mage-b DNA vaccine is effective against breast cancer metastases. In the study presented here, we have further enhanced the efficacy of Mage-b vaccination through the improved delivery of the vaccine using recombinant Listeria monocytogenes (LM). Three overlapping fragments of Mage-b as well as the complete protein-encoding region of Mage-b have been expressed as a fusion protein with a truncated non-cytolytic form of listeriolysin O (LLO) in recombinant LM. These different Mage-b vaccine strains were preventively tested for their efficacy against breast cancer metastases in a syngeneic mouse tumour model 4T1. The LM-LLO-Mage-b/2nd, expressing position 311-660 of the cDNA of Mage-b, was the most effective vaccine strain against metastases in the 4T1 mouse breast tumour model. Vaccination with LM-LLO-Mage-b/2nd dramatically reduced the number of metastases by 96% compared with the saline group and by 88% compared with the vector control group (LM-LLO), and this correlated with strong Mage-b-specific CD8 T-cell responses in the spleen, after restimulation with Mage-b. However, no effect of LM-LLO-Mage-b/2nd was observed on 4T1 primary tumours, which may be the result of a complete absence of Mage-b-specific immune responses in the draining lymph nodes. Vaccination with LM-LLO-Mage-b/2nd could be an excellent follow-up after removal of the primary tumour, to eliminate metastases and residual tumour cells.     

T cells sensitized with breast tumor progenitor cell vaccine have therapeutic activity against spontaneous HER2/neu tumors

Cancer progenitor cells are critical for tumor initiation and recurrence so they are an important therapeutic target. We tested whether T cells could recognize tumor antigens expressed by breast cancer progenitor cells and acquire therapeutic activity against established metastases or delay onset of spontaneous tumors. Breast tumors were derived from HER2/neu transgenic mice and propagated in vitro under conditions that selected progenitor cells which were then used as an irradiated whole cell vaccine. A minor subset of recently sensitized T cells was isolated from vaccine-draining lymph nodes then activated in vitro to achieve numerical expansion. We show that the tumor progenitor cell vaccines reversed tolerance to a known HER2/neu epitope, otherwise inhibited by Treg cells. Additional shared tumor antigens were recognized because a Neuneg subclone also induced a Th1 type immune response against breast tumors. Adoptive transfer of in vitro activated lymph node T cells-mediated regression of established metastases from multiple independently derived breast tumor lines. Moreover, adoptive transfer of effector T cells into Neu-tolerant mice, months before the onset of spontaneous tumors, significantly postponed tumor development. Interestingly, T-cell-mediated lysis of metastases stimulated an IgG response to HER2/neu as well as other shared antigens. In summary, tumor progenitor cells contain shared antigens which can lead to a cross-protective T-cell response. Moreover, antigens acquired during immune-mediated tumor destruction are presented in a manner conducive to reversal of tolerance and Ig class switching. These complementary effector mechanisms might augment therapy by eliminating refractory breast cancer stem cells.     

Activated T lymphocytes in infiltrates and draining lymph nodes of nasopharyngeal carcinoma

Lymphocytes isolated from the tumors and draining lymph nodes of nasopharyngeal carcinoma (NPC) patients exhibit the following characteristics of immune activation: (1) stable E rosette formation, (2) natural attachment to various human cells, (3) sensitivity in vitro to the lytic effect of glucocorticoids. Although the NPC T cells attach in vitro to various cells they kill only EBV-genome-carrying targets. These findings suggest the occurrence of a local cellular immune response in NPC, possibly directed to EBV-determined antigens.     

Immunological characterization of mononuclear cells in peripheral blood and regional lymph nodes of breast cancer patients.

Mononuclear cells from peripheral blood and draining lymph nodes of 40 patients with invasive locoregional breast cancer were examined for immunological cell surface markers (E, EAhuman, EAox, EAC, SIg pos.). Concomitantly, blood lymphocytes from 36 healthy women and axillary and mesenteric lymph-nodes from patients without malignant diseases were tested as controls. In peripheral blood of tumor patients E rosette-forming cells were slightly diminished as compared to the control group, whereas EAox and EAC rosette-forming cells were increased. These differences may be age-dependent rather than tumor-related. In the draining lymph nodes of breast cancer patients as well as in the control lymph nodes, the percentages of EAC rosette-forming cells and SIg positive lymphocytes were significantly increased compared to peripheral blood, whereas E and EAhuman rosette values remained unchanged. Percentages of EAox rosettes on the other hand were strongly diminished in the draining lymph nodes, suggesting that the EAhuman and EAox rosetting techniques detect 2 types of Fc-receptor bearing cells. No significant differences were found between the cell surface marker analysis of tumor-free and metastatic lymph nodes of breast cancer patients and the control lymph nodes.     

In vivo detection and partial characterization of effector and suppressor cell populations in spleens of mice with large metastatic fibrosarcomas

The MC-2 fibrosarcoma, which is a transplantable tumour syngeneic for BALB/c mice, metastasizes to lymph nodes draining subcutaneous inoculation sites, and also to the lungs. T cell-mediated immunity was detected in Winn assays using spleens from excision immunized mice. T cell-mediated anti-tumour immunity was also detected in spleens from mice with small tumours but disappeared as the tumour burden increased. Protective immune spleen cell activity in the Winn assay was inhibited by prior addition of spleen cells from mice with large tumours, causing increased tumour incidence. Splenic metastases occasionally occurred in the MC-2 model, but were not demonstrable by bioassay in any of the experiments detecting splenic suppressor cell activity. In vivo protective activity was restored to advanced-stage tumour-bearer spleens by whole-body ionizing irradiation (0.5 and 2.5 Gy) of donor mice 15 h before sampling. Spleen cells from mice with small tumours remained protective after 1.5, 2.5 and 4.0 Gy of irradiation in vivo. These results are consistent with the properties of radiosensitive suppressor T cells. In contrast to reports in other tumour models, suppressor cells were not detected until late in the course of MC-2 development. This is surprising in view of the aggressively metastatic nature of MC-2. It is postulated that modulation of the anti-tumour immune response by the suppressor cells is associated with metastasis in this tumour model. The late appearance of both suppressor cells and metastatic cells in the spleen may reflect similar processes occurring earlier in regional lymph nodes.     

乳腺癌患者淋巴结CD4＋CD25＋T细胞和相关细胞因子的检测及相关性分析

目的探讨CD4＋CD25＋T细胞在乳腺癌发生、发展中的作用。方法将取自35例乳腺癌患者的105个肿大淋巴结,制备成单细胞悬液,应用流式细胞仪检测CD4＋CD25＋T细胞比例及CD4＋CD25-、CD8＋T细胞、NK细胞的相对水平。采用定量RT-PCR法,检测IL-2、IL-10、TGF-β1和IFN-γ的细胞因子水平。结果乳腺癌患者淋巴结中的CD4＋CD25＋T细胞水平（在CD4＋T细胞中的百分含量）与淋巴结转移相关,转移淋巴结中其水平明显高于未转移淋巴结。乳腺癌患者淋巴结中CD4＋CD25＋T细胞与CD4＋CD25-、CD8＋T细胞和NK细胞的水平呈负相关关系。乳腺癌患者淋巴结中CD4＋CD25＋T细胞水平与TGF-β1呈正相关,与IL-2、IL-10、IFN-γ无相关性。乳腺癌患者淋巴结中TGF-β1、IL-10、IFN-γ水平与淋巴结转移相关,转移淋巴结中TGF-β1、IL-10含量高而IFN-γ含量较低。IL-2与淋巴结转移无相关性。结论乳腺癌患者转移淋巴结中的CD4＋CD25＋T细胞水平高于未转移淋巴结。     

Regulatory B cells producing IL-10 are increased in human tumor draining lymph nodes

The contribution of different immune cell subsets, especially T cells, in anti-tumor immune response is well established. In contrast to T cells, the anti-tumor contribution of B cells has been scarcely investigated. B-cells are often overlooked, even though they are important players in a fully integrated immune response and constitute a substantial fraction of tumor draining lymph nodes (TDLNs) known also as Sentinel Nodes. In this project, samples including TDLNs, non-TDLNs (nTDLNs) and metastatic lymph nodes from 21 patients with oral squamous cell carcinoma were analyzed by flow cytometry. TDLNs were characterized by a significantly higher proportion of B cells compared with nTDLNs (P = .0127). TDLNs-associated B cells contained high percentages of naïve B cells, in contrary to nTDLNs which contained significantly higher percentages of memory B cells. Patients having metastases in TDLNs showed a significantly higher presence of immunosuppressive B regulatory cells compared with metastasis-free patients (P = .0008). Elevated levels of regulatory B cells in TDLNs were associated with the advancement of the disease. B cells in TDLNs were characterized by significantly higher expression of an immunosuppressive cytokine—IL-10 compared with nTDLNs (P = .0077). Our data indicate that B cells in human TDLNs differ from B cells in nTDLNs and exhibit more naïve and immunosuppressive phenotypes. We identified a high accumulation of regulatory B cells within TDLNs which may be a potential obstacle in achieving response to novel cancer immunotherapies (ICIs) in head and neck cancer.     

Outcome of treatment for breast cancer patients with chest wall recurrence according to initial stage: implications for post-mastectomy radiation therapy

PURPOSE: The impact of postmastectomy radiation therapy (PMRT) on overall survival (OS) for patients with Stage II breast cancer with 1-3 positive lymph nodes is controversial. We sought to compare the outcome of salvage treatment for patients with chest wall recurrence (CWR) according to initial disease stage to shed light on the potential benefit of PMRT in specific subgroups of patients. METHODS: We retrospectively reviewed information concerning 96 patients with CWR who were not previously treated with PMRT. The patients were divided according to their initial extent of disease: T1-T2N0 (Group 1), T1-T2 with 1-3 positive lymph nodes (Group 2), and T3-T4 or > or =4 positive lymph nodes (Group 3). The OS and distant metastasis-free survival (DMFS) from the time of CWR were compared using the method of Kaplan and Meier, and a Cox regression model was used for a multivariate analysis. RESULTS: Group 1 had an improved OS and DMFS compared with Group 2 and Group 3 (p < 0.001), but there were no differences in OS or DMFS between Group 2 and Group 3 (p = 0.250 and p = 0.492, respectively). The respective 5-year rates for the three groups were as follows: OS 79.9% vs. 41.9% vs. 29.1%; DMFS 75.2% vs. 33.6% vs. 25.9%. CONCLUSIONS: Breast cancer patients with T1-T2N0 breast cancer who develop a CWR have a significantly better outcome than those with lymph node-positive disease. Patients with T1-T2 tumors and one to three positive lymph nodes have a similar outcome after CWR as those with larger tumors or more than four positive lymph nodes. These data should be considered when weighing the risks and benefits of PMRT for patients with Stage II breast cancer with one to three positive lymph nodes.     

Response of T and B lymphocytes in the spleen, lymph nodes and mammary tumors in rats treated with human soluble tumor-associated antigens and commercial human albumin

We showed previously that soluble tumor-associated antigens (sTAA) isolated from breast cancer patients could suppress chemically-induced tumorigenesis in rats in comparison to the effect of commercial human albumin (CHA). Herein we analyze the possible mechanism of those findings. The following groups of mammary tumor-bearing rats were used in the studies: i) control rats treated with saline; ii) rats treated with CHA; and iii) rats treated with human sTAA. Different zones of the spleen, regional lymph nodes and tumors and their cellular content (B and T cells) were analyzed using the methods of morphometry and immunohistochemistry. Treatment of tumor-bearing rats with CHA resulted in a significant decrease in the size of the germinal center of the follicles. The number of B lymphocytes in the mantle layer of the follicles, the marginal zone and red pulp decreased significantly. The number of CD8+ T cells also decreased in the marginal zone and red pulp, whereas the number of CD4+ T cells increased in the periarterial lymph sheath (PALS) and the red pulp. Reaction of the spleen to vaccination with sTAA manifested in a significant increase in the size of most areas of the white pulp and in the number of B lymphocytes. In lymph nodes from control rats or those treated with CHA, CD8+ lymphocytes mainly accumulated in the paracortical zone. In rats treated with sTAA, CD8+ lymphocytes accumulated also in the medulla. The number of CD4+ T cells in these rats sharply increased and accumulated mainly in the medulla around the vessels. The total number of lymphocytes was changed differently in different areas of tumors (peripheral vs. at depth). The number of CD8+ cells significantly increased at depth of tumors, and also the ratio in the number of these cells at depth of tumors compared to a periphery increased. No difference was found in response of lymph cells to different types of treatment. All findings indicated a strict antitumor effect of vaccination with the sTAA, which prevents the development of insufficiency of the immune system when an intensive immune reaction takes place.     

Administration of 6-gingerol greatly enhances the number of tumor-infiltrating lymphocytes in murine tumors

Tumor-infiltrating lymphocytes (TILs) play critical roles in host antitumor immune responses. It is known that cancer patients with tumor-reactive lymphocyte infiltration in their tumors have better prognoses, while patients with tumors infiltrated by immunosuppressive cells have worse prognoses. We found that administration of 6-gingerol, which is a component of ginger, inhibited tumor growth in several types of murine tumors, such as B16F1 melanomas, Renca renal cell carcinomas and CT26 colon carcinomas, which were established by inoculating tumor cells on the flanks of mice. However, administration of 6-gingerol did not lead to complete eradication of the tumors. 6-Gingerol treatment of tumor-bearing mice caused massive infiltration of CD4 and CD8 T-cells and B220(+) B-cells, but reduced the number of CD4(+) Foxp3(+) regulatory T-cells. The CD8 tumor-infiltrating T lymphocytes in 6-gingerol-treated mice strongly expressed IFN-γ, a marker of activation of cytotoxic T lymphocytes (CTL) CD107a and chemokine receptors that are expressed on T(H) 1 cells, such as CXCR3 and CCR5. To test whether 6-gingerol could promote infiltration of tumor antigen-specific CD8 T-cells into tumors, we adoptively transferred CFSE-labeled OT-1 CD8 T-cells into EG7 tumor-bearing mice. We found that CD8 T cells isolated from 6-gingerol pretreated OT-1 mice, but not from control OT-1 mice, massively infiltrated tumors and tumor draining lymph nodes and divided several times. Our results strongly suggest that 6-gingerol can be used in tumor immunotherapy to increase the number of TILs.     

Proliferative patterns of lymphocytes in lymph nodes during tumour development: involvement of T and B cell areas

DNA-synthetizing lymphocytes were identified in the lymph nodes regional and more distal to the site of developing P-815 tumours by incorporation of [3H]-thymidine followed by autoradiography of lymph node sections. It appeared that not only T but also B cell areas of draining and to a lesser extent of distal lymph nodes were stimulated by the growing tumour. This result was unexpected since neither humoral nor tumour cell-bound antibody could be identified so far as a functional correlate of B cell stimulation. In general the proliferative response of lymphocytes followed a biphasic pattern with an early peak of reactivity on days 2-3 and a second peak around day 12-15 after tumour cell inoculation. In the draining (axillary) lymph node the second peak of reactivity was suppressed, possibly as a consequence of metastatic tumour cells in this node when tumour cells were inoculated in the flank. The pattern of lymphocyte stimulation revealed larger individual variations after tumour cell inoculation in the flank than the foot pad. These results were associated with a slower and less regular drainage of carbon particles from the flank to the axillary and exceptionally the brachial lymph node than from the foot pad to the popliteal node after injection of India ink.     

The histogenesis of tumours induced in golden hamsters by murine sarcoma virus-Harvey (MSV-H)

Newborn golden hamsters were inoculated subcutaneously with a cell-free filtrate of cultured mouse 3T3 cells infected with murine sarcoma virus-Harvey (MSV-H). An electron microscopical study was made of the lesions that subsequently developed in these hamsters. Two weeks after inoculation, cysts developed eccentrically in the hila of lymph nodes draining the inoculation site. Type H (radial) virus-like particles were seen in the endothelial cells that lined the cysts and in the serous fluid that filled the cysts. During the 3–6 weeks after inoculation, nodules appeared in lymph nodes, subcutaneous and muscle fascia. They were composed of pleomorphic cells that interlocked with each other and resembled the fixed phagocytic histiocytes of normal lymph nodes. The pleomorphic interlocking cells infiltrated and replaced lymph nodes and perinodal tissues. Some lymph nodes were replaced by abnormal mast cells. During the 12–24 weeks after inoculation tumours were found that involved the paws or limb muscles. The predominant cells in these tumours were similar to the pleomorphic interlocking cells seen in early developing lesions. Similar cells were also found in lymph nodes draining the tumours. It is concluded that tumours induced in golden hamsters by MSV-H are mesenchymal tumours of histiocytic origin.     

The tumour associated antigen CA15.3 in primary breast cancer. Evaluation of 667 cases

CA15.3 preoperatory serum levels have been determined in 667 patients with primary untreated breast cancer and in 193 controls. The relationships between CA15.3 and several clinical and pathological parameters were evaluated. CA15.3 levels showed a highly significant direct relationship with stage, T, pT, N and the number of positive lymph nodes. The close relationship between CA15.3 and the number of positive lymph nodes was also demonstrated in a subgroup of 406 patients in which more than ten lymph nodes had been examined. CA15.3 levels were correlated with tumour size in patients without axillary metastasis as well as with the number of positive lymph nodes in pT1 tumours. CA15.3 was significantly higher in medullary than in ductal carcinoma. No relationships were found between serum CA15.3 and receptor status. We conclude from the present findings that CA15.3 in primary untreated breast cancer is a marker of tumour burden as well as of the tendency of local invasiveness (relationship between CA15.3 and nodal status in pT1 tumours).