Inhibition of Lewis lung carcinoma in mice by local microwave hyperthermia combined with immunomodulating Propionibacterium granulosum KP-45

C-57 BL/6 mice with Lewis lung carcinoma were treated 2 weeks after tumor implantation with local microwave hyperthermia (2450 MHz, tumor temperature 43.5 degrees C, 30 min) and/or intratumoral or intraperitoneal injection of 1 mg cell walls of Propionibacterium granulosum KP-45. Tumor growth, survival time of the animals, and the delayed skin hypersensitivity to oxazolone were followed up, as well as the 3H-thymidine uptake of tumorous tissues and the number of lung metastases. The combined treatment of microwave hyperthermia with immunomodulating P. granulosum KP-45 resulted in significantly stronger inhibition of tumor growth than with each of these methods alone. The number of lung metastases could be significantly lowered, and the skin reactivity to oxazolone remained enhanced during the whole observation period (over 70 days after tumor implantation). The implications of the test observation are discussed.     

Therapy of spontaneous pulmonary metastases of a murine mammary carcinoma with anaerobic corynebacteria

Summary Corynebacterium parvum given intravenously or into the tumor 7 or 2 days before surgical removal of primary tumors greatly reduced the number of spontaneous pulmonary metastases of a syngeneic mammary carcinoma of C3Hf/Bu mice. When a single dose of 6000 rads -rays was used to eliminate primary tumors the number of lung metastases significantly increased. Administration of C. parvum before irradiation not only prevented this metastasis — facilitating effect of radiation, but also reduced the number of lung nodules below that in amputated mice. Metastatic spread was not altered by postoperative treatment of mice with single doses of C. parvum or C. granulosum. However, significantly more mice had lung metastases if they were given 2 intraperitoneal injections of C. granulosum.     

Activation of natural resistance against lung metastasis of an adenocarcinoma in T-cell depressed spontaneously hypertensive rats by infection with Listeria monocytogenes

Summary We report here our study of the role of natural host defense mechanisms mediated by macrophages and natural killer (NK) cells in an experimental model of spontaneous pulmonary metastases of a mammary adenocarcinoma SST-2 in spontaneously hypertensive rats (SHR) with congenital T-cell depression. To activate macrophages and NK cells, Listeria monocytogenes (LM) was injected IV into SHR which had received a transplantation of SST-2. To assess the antimetastatic responses induced by LM, the number of lung nodules and the lung weight in SHR were evaluated 30 days after tumor inoculation. The growth of lung metastases, though not of primary tumors, was significantly reduced if 10⁷ LM were injected IV into SHR 2, 10 and 20 days after the SC transplantation of 5×10⁴ or 5×10⁵ SST-2. An inhibitory effect of LM on pulmonary metastases was also observed in tumor-excised rats, in which the number of lung metastases and the lung weight were enhanced as compared with those in tumor-bearing rats which had not undergone surgery. Peritoneal resident cells which were harvested from rats injected with LM showed a significant augmentation of tumoricidal activity against SST-2 cells as measured by in vitro cytotoxicity. Similarly, the NK activity of spleen cells of SHR injected with LM increased significantly when compared with untreated SHR. These data suggest that the inhibition of metastatic growth, though not of pirmary tumor growth, was accomplished by the, possibly T-cell independent, activation of macrophages and NK cells.     

Immunochemotherapy of breast cancer with Propionibacterium granulosum

Summary Eighty-two patients with metastatic breast cancer were treated with adriamycin, cyclophosphamide, and 5-fluorouracil (FAC). These patients were divided by randomization into two groups. One was receiving cytostatic drugs only, while another received the identical cytostatic medication plus additional therapy consisting of IV infusions of gamma-radiation-sterilized cells of Propionibacterium granulosum strain KP-45. Metastases were localized in six different sites and/or organs. Multiple parameters for monitoring changes in metastatic lesions and laboratory tests were performed serially in all patients. The addition of P. granulosum to standard (FAC) chemotherapy of metastatic breast cancer influenced metastates in the liver. It also resulted in increased survival time within the observation period. Possible mechanisms of immunomodulating activity of P. granulosum in this disease are discussed.     

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification. Download video file.^{(48M, mov)}     

Model for mediastinal lymph node metastasis produced by orthotopic intrapulmonary implantation of lung cancer cells in mice

This study is designed to establish a pulmonary tumor model to investigate the biology and therapy of lung cancer in mice. Current methods for forming a solitary intrapulmonary nodule and subsequent metastasis to mediastinal lymph nodes are not well defined. Lewis lung carcinoma cell (LLC) suspensions were orthotopically introduced into the lung parenchyma of C57/BL6 mice via a limited skin incision without thoracotomy followed by direct puncture through the intercostal space. The implantation process was performed within approximately 50 sec per mouse, and the operative mortality was less than 5%. Single pulmonary nodules developed at the implanted site in 93% of animals and subsequent mediastinal lymph nodes metastasis were observed in all mice that were succeeded to form a lung nodule after intrapulmonary implantation. The size of tumor nodule and the weight of mediastinal lymph node increased in a time-dependent manner. The mean survival time of mice implanted successfully with LLC cells was 21 +/- 2 days (range; 19-24 days). Histopathological analysis revealed that no metastatic tumor was detectable in the mediastinal lymph nodes on day 11, but metastatic foci at mediastinal lymph nodes were clearly observed on days 17 and 21 after implantation. Other metastases in distant organs or lymph nodes were not observed at 21 days after the implantation. Comparative studies with intrapleural and intravenous injections of LLC cells suggest that the mediastinal lymph node metastasis by intrapulmonary implantation is due to the release of tumor cells from the primary nodule, and not due to extrapulmonary leakage of cells. An intravenous administration of CDDP on day 1 after tumor implantation tended to suppress the primary tumor nodule and significantly inhibited the lymph node metastasis. Thus, a solitary pulmonary tumor nodule model with lymph node metastasis approximates clinical lung cancer, and may provide a useful basis for lung cancer research.     

Systemic treatment with interleukin-4 induces regression of pulmonary metastases in a murine renal cell carcinoma model

Advanced metastatic renal cell carcinoma has been shown to be responsive to immunotherapy but the response rate is still limited. We have investigated the therapeutic potential of systemic interleukin-4 (IL-4) administration for the treatment of pulmonary metastases in the murine Renca renal adenocarcinoma model. Renca cells were injected iv in Balb/c mice to induce multiple pulmonary tumor nodules. From Day 5, Renca-bearing mice were treated with two daily injections of recombinant murine IL-4 for 5 consecutive days. IL-4 treatment induced a significant reduction in the number of lung metastases in a dose-dependent manner and significantly augmented the survival of treated animals. Immunohistochemistry studies, performed on lung sections, showed macrophage and CD8⁺ T cell infiltration in the tumor nodules 1 day after the end of IL-4 treatment. The CD8 infiltration increased by Day 7 after IL-4 treatment. Granulocyte infiltration was not detectable. To clarify further the role of the immune system in IL-4 anti-tumor effect, mice were depleted of lymphocyte subpopulations by in vivo injections of specific antibodies prior to treatment with IL-4. Depletion of CD8⁺ T cells or AsGM1⁺ cells abrogated the effect of IL-4 on lung metastases, whereas depletion of CD4⁺ T cells had no impact. These data indicate that CD8⁺ T cells and AsGM1⁺ cells are involved in IL-4-induced regression of established renal cell carcinoma.     

Reciprocal effects of Th1 and Treg cell inducing pathogen-associated immunomodulatory molecules on anti-tumor immunity

We have addressed the hypothesis that pathogen-associated immunomodulatory molecules may influence anti-tumor immunity through their pro- and anti-inflammatory activities and abilities to induce effector and regulatory T (Treg) cells. We found that CpG oligonucleotides (CpG) and cholera toxin (CT), which promote Th1 or Th2/Treg cell biased responses, respectively, had differential effects on tumor growth. Therapeutic peritumoral administration of CpG significantly reduced subcutaneous tumor growth and prolonged survival, whereas CT enhanced tumor growth and reduced survival. Peritumoral administration of CpG enhanced the frequency of IFN-γ-secreting and reduced IL-10-secreting CD4⁺ and CD8⁺ T cells, in the tumor and in the draining lymph nodes, whereas, CT significantly enhanced the frequency of CD4⁺CD25⁺Foxp3⁺ Treg cells, but reduced IFN-γ-secreting T cells infiltrating the tumor. In contrast to the beneficial effect of CpG in mice with subcutaneous tumors, CpG or CT had no protective effect against tumor growth in the lungs when given therapeutically by the nasal route. However, prophylactic intranasal administration of CpG significantly reduced the number of lung metastases and this was associated with an enhanced frequency of IFN-γ-secreting CD8⁺ T cells in the draining lymph node and enhanced tumor-specific CTL responses. Our findings demonstrate that pathogen-associated molecules can either inhibit or enhance anti-tumor immunity by selectively promoting the induction of effector or regulatory T cells, and that the environment of the growing tumor influences the protective effect. Joanne Lysaght and Andrew G. Jarnicki contributed equally.     

Antimetastatic effect of recombinant human macrophage-colony-stimulating factor against lung and liver metastatic B16 melanoma

 We studied the effect of recombinant human macrophage-colony-stimulating factor (rhM-CSF) on the formation of lung and liver metastases following the i.v. injection of the B16 melanoma subline (B16 LiLu) into mice. When rhM-CSF was administered before the B16 inoculation, the number of tumor metastases decreased in the lung and liver. However, the administration of rhM-CSF after B16 inoculation did not produce an antimetastatic effect in the lung, but did in the liver. B16 cells labeled with 5-[¹²⁵I]-iodo-2′-deoxyuridine (¹²⁵I-dUrd) were injected and the arrest of tumor cell emboli was examined in the capillary beds of the lung and liver of mice treated with either vehicle or rhM-CSF. In both groups, there were the same numbers of B16 cells in both the lung and the liver 3 minutes after the B16 injection, and almost all tumor cells died within 24 h. However, the number of cells surviving in the lung was decreased in mice injected with rhM-CSF (37%). There was no difference in the number of cells in the livers of mice treated either with vehicle or rhM-CSF in the first 24 h after tumor cell injection. The administration of rhM-CSF increased NK 1.1⁺ cells in the mouse spleen and facilitated NK activity in vivo. At the same time, the administration of an anti-NK 1.1 antibody blocked the antimetastatic effect of rhM-CSF in the lung but not in the liver. The antibody was effective only when it was injected before the B16 inoculation. These results suggest that the antimetastatic effect of rhM-CSF in the lung was mediated by NK 1.1⁺ cells within 24 h of B16 injection. In contrast, the antimetastatic effect of rhM-CSF in the liver was mediated not only by NK 1.1⁺ cells but also by other antimetastatic systems such as macrophages. Received: 8 April 1996 / Accepted: 26 November 1996     

Comparison of Immunity in Mice Cured of Primary/Metastatic Growth of EMT6 or 4THM Breast Cancer by Chemotherapy or Immunotherapy

Purpose

We have compared cure from local/metastatic tumor growth in BALB/c mice receiving EMT6 or the poorly immunogenic, highly metastatic 4THM, breast cancer cells following manipulation of immunosuppressive CD200:CD200R interactions or conventional chemotherapy.

Methods

We reported previously that EMT6 tumors are cured in CD200R1KO mice following surgical resection and immunization with irradiated EMT6 cells and CpG oligodeoxynucleotide (CpG), while wild-type (WT) animals developed pulmonary and liver metastases within 30 days of surgery. We report growth and metastasis of both EMT6 and a highly metastatic 4THM tumor in WT mice receiving iv infusions of Fab anti-CD200R1 along with CpG/tumor cell immunization. Metastasis was followed both macroscopically (lung/liver nodules) and microscopically by cloning tumor cells at limiting dilution in vitro from draining lymph nodes (DLN) harvested at surgery. We compared these results with local/metastatic tumor growth in mice receiving 4 courses of combination treatment with anti-VEGF and paclitaxel.

Results

In WT mice receiving Fab anti-CD200R, no tumor cells are detectable following immunotherapy, and CD4+ cells produced increased TNFα/IL-2/IFNγ on stimulation with EMT6 in vitro. No long-term cure was seen following surgery/immunotherapy of 4THM, with both microscopic (tumors in DLN at limiting dilution) and macroscopic metastases present within 14 d of surgery. Chemotherapy attenuated growth/metastases in 4THM tumor-bearers and produced a decline in lung/liver metastases, with no detectable DLN metastases in EMT6 tumor-bearing mice-these latter mice nevertheless showed no significantly increased cytokine production after restimulation with EMT6 in vitro. EMT6 mice receiving immunotherapy were resistant to subsequent re-challenge with EMT6 tumor cells, but not those receiving curative chemotherapy. Anti-CD4 treatment caused tumor recurrence after immunotherapy, but produced no apparent effect in either EMT6 or 4THM tumor bearers after chemotherapy treatment.

Conclusion

Immunotherapy, but not chemotherapy, enhances CD4⁺ immunity and affords long-term control of breast cancer growth and resistance to new tumor foci.     

Anti-invasive and anti-metastatic effect of lysine oxidase from Trichoderma sp. in vitro and in vivo]

A new fungal strain, Trichoderma sp., discovered in Moscow, produces the antitumor enzyme, lysine-oxidase, which demonstrates an anti-invasive effect in vitro and anti-metastatic activity in vivo. Maximal inhibition of the in vitro invasion of MM1 clone cells was obtained when the tumor cells were pretreated with 2.5 mU/ml of lysine-oxidase; the pretreatment caused a 1.9-times reduction in cell growth and a 1.6-times reduction in the invasive capacity. We studied its anti-metastatic effect on the spreading Lewis lung carcinoma (3LL) in mice from which the primary tumor had been removed. The administration of the enzyme (50 U/kg, i.v.) significantly decreased not only the extent but the number of lung metastases, as compared with the untreated mice. In addition to that, the lysine-oxidase treatment considerably increases the life-span of mice from which the primary tumor had been removed (200 days after 3LL implantation, lysine-oxidase treatment caused surviving of 50% mice in experimental group).     

Immunologically nonspecific enhancement of artificial lung metastases in tumor-bearing mice

Summary Experiments designed to investigate concomitant enhancement of tumor growth in the lungs of tumor-bearing mice are reported. When a fibrosarcoma (NFSA)³ that had arisen spontaneously in a C₃Hf/Bu mouse was transplanted into the hind legs of syngeneic mice 2 weeks prior to IV tumor challenge, the tumor-bearing mice developed more lung colonies than did normal controls. Paradoxically, the same mice demonstrated concomitant resistance to an IM tumor challenge. Animals bearing the tumor for only 1 week and those from which the tumor had been excised after 2 weeks' growth showed neither enhancement nor resistance to lung colony growth. Enhancement in mice bearing the tumor for 2 weeks was shown not to be due to metastases seeded from the primary tumor. Tumor-bearing animals receiving whole-body irradiation (WBI) 1 day before or 12 days after tumor transplantation showed no enhancement compared with nontumor-bearing WBI controls. Mice receiving partial body radiation with the thorax shielded also failed to show concomitant enhancement. Adult thymectomy alone did not affect the enhancement, while thymectomy followed by whole-body irradiation and bone marrow-cell reconstitution abolished it. Although apparently dependent upon relatively long-lived T cells, enhancement was not tumor-specific; mice bearing another fibrosarcoma (FSA), which does not cross react immunologically with NFSA, also showed enhancement when challenged IV with NFSA. Treatment of tumor-bearing mice with C. parvum prior to IV challenge prevented the enhancement phenomenon.Animals used in this study were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care, and in accordance with current United States Department of Agriculture and Department of Health, Education, and Welfare, National Institutes of Health, regulations and standards.Abbreviations used here are: NFSA, spontaneously arisen fibrosarcoma; FSA, methylcholanthrene-induced fibrosarcoma; LCR, lung colony ratio (number of lung colonies in experimental group over those in control group); TG time, tumor growth time (the time required for a tumor to reach 500 mm³ after transplantation); TxRB mice, thymectomized whole body-radiated and bone marrow-reconstituted mice.     

Biodistribution and tumor localization of lymphokine-activated killer T cells following different routes of administration into tumor-bearing animals

The efficiency of adoptive cellular immunotherapy of cancer might depend on the number of effector cells that reach the malignant tissues. In the present study, the biodistribution and tumor localization of ex vivo lymphokine-activated T killer (T-LAK) cells was investigated. Methods: T-LAK cells were labeled with ¹²⁵I-dU or the fluorescent dye tetramethylrhodamine isothiocyanate (TRITC) and transferred by intravenous, -cardiac, -portal or -peritoneal injection into normal (C57BL/6) mice or mice with syngeneic day-7 to day-12 B16 melanoma metastases established in various organs. The overall biodistribution of the T-LAK cells was measured by gamma counting and their tumor localization by fluorescence microscopy. Results: At 16 h after intravenous injection, the organ distribution of ¹²⁵I-dU-labeled T-LAK cells was identical in normal and tumor-bearing animals. Fluorescence microscopy of lung tissue from animals receiving TRITC-labeled T-LAK cells revealed, however, a fivefold higher accumulation of T-LAK cells in lung metastases than in the surrounding normal lung tissue (1174 and 226 cells/mm² respectively). Some pulmonary metastases were, however, resistant to infiltration. Very few intravenously injected cells redistributed to other organs or to tumors in these, since only 60 and 30 T-LAK cells/mm² were found within metastases of the adrenal glands and the liver respectively. However, following injection of T-LAK cells via the left ventricle of the heart, a threefold increase (from 60 to 169 cells/mm²) in the number of transferred cells in metastases of the adrenal glands was observed. Moreover, following locoregional administration of T-LAK cells into the portal vein, tenfold higher numbers (from 30 to 400 cells/mm²) were found in hepatic metastases than were observed following intravenous or intracardiac injection. In the liver, a surprisingly large number of intraportally injected T-LAK cells (approx. 1300/mm²) were observed to accumulate in the perivascular spaces of the portal, but not the central veins. Even though some superficial ovarian and liver metastases were separated from the peritoneal cavity by only the peritoneal lining, no localization into these metastases was seen following intraperitoneal injection of the T-LAK cells. While treatment of tumor-bearing animals with T-LAK cells plus IL-2 reduced lung metastases by 76% as compared to treatment with IL-2 alone (P < 0.03), no significant reduction of liver metastases was seen. Conclusions: T-LAK cells are able to localize substantially into tumor metastases in various anatomical locations, but mainly following locoregional injection. This finding might have important implications for the design of future clinical protocols of adoptive immunotherapy based on T cells. Received: 29 April 1999 / Accepted: 5 August 1999     

Detection of Cancer Metastases with a Dual-labeled Near-Infrared/Positron Emission Tomography Imaging Agent

By dual labeling a targeting moiety with both nuclear and optical probes, the ability for noninvasive imaging and intraoperative guidance may be possible. Herein, the ability to detect metastasis in an immunocompetent animal model of human epidermal growth factor receptor 2 (HER-2)-positive cancer metastases using positron emission tomography (PET) and near-infrared (NIR) fluorescence imaging is demonstrated. METHODS: (⁶⁴Cu-DOTA)_n-trastuzumab-(IRDye800)_m was synthesized, characterized, and administered to female Balb/c mice subcutaneously inoculated with highly metastatic 4T1.2neu/R breast cancer cells. (⁶⁴Cu-DOTA)_n-trastuzumab-(IRDye800)_m (150 µg, 150 µCi, m = 2, n = 2) was administered through the tail vein at weeks 2 and 6 after implantation, and PET/computed tomography and NIR fluorescence imaging were performed 24 hours later. Results were compared with the detection capabilities of F-18 fluorodeoxyglucose (¹⁸FDG-PET). RESULTS: Primary tumors were visualized with ¹⁸FDG and (⁶⁴Cu-DOTA)_n-trastuzumab-(IRDye800)_m, but resulting metastases were identified only with the dual-labeled imaging agent. ⁶⁴Cu-PET imaging detected lung metastases, whereas ex vivo NIR fluorescence showed uptake in regions of lung, skin, skeletal muscle, and lymph nodes, which corresponded with the presence of cancer cells as confirmed by histologic hematoxylin and eosin stains. In addition to detecting the agent in lymph nodes, the high signal-to-noise ratio from NIR fluorescence imaging enabled visualization of channels between the primary tumor and the axillary lymph nodes, suggesting a lymphatic route for trafficking cancer cells. Because antibody clearance occurs through the liver, we could not distinguish between nonspecific uptake and liver metastases. CONCLUSION: (⁶⁴Cu-DOTA)_n-trastuzumab-(IRDye800)_m may be an effective diagnostic imaging agent for staging HER-2-positive breast cancer patients and intraoperative resection.     

Further studies of the therapeutic effectsof murine melanoma-specific monoclonal antibodies

The results presented here further characterize four murine monoclonal antibodies (mAb) that recognize melanoma-specific antigens (9B6, T97, 2-3-1 and 2-3-3). These melanoma-specific mAbs are of the IgG_2b isotype and are significantly therapeutic when administered systemically against established pulmonary melanoma metastases. Here we show a consistent and significantly inhibition of the growth of melanoma lung metastases by all four mAbs and the existence of a time ‘window’ at days 5–8 after tumor inoculation for optimal therapy. Since these mAbs were found not to be cytotoxic or cytolytic in vitro, we looked for host immune response regulation as being responsible for the therapeutic effects. Natural killer (NK) cells were implication as one arm of the host immune system involved in this response since depletion of NK cells in vivo by αasialoGM1 or αNK1.1 antibodies partially abrogated the inhibitory effect of the mAbs. The observed antimetastatic effects could also be partially abrogated using antibodies directed against the T-cell subset surface markers, CD4⁺ and CD8⁺. Intramuscular melanoma tumor growth was also found to be suppressed by mAb 2-3-1, but only if administered in the area of tumor growth and only if multiple inoculations are administered over a 13-day period. The beneficial effect of mAb antimetastatic therapy was found to be useful against several syngeneic melanomas, including JB/MS, B16 and several sublines of the B16 F10 melanoma.     

Active immunization with a Her-2/neu-targeting Multi-peptide B cell vaccine prevents lung metastases formation from Her-2/neu breast cancer in a mouse model

In pre-clinical and clinical settings, active immunization with a Her-2/neu vaccine (HerVaxx), comprising B-cell peptide from Trastuzumab binding site, has been shown to reduce primary tumor growth via induction of polyclonal anti-tumor immune responses and immunological memory. Here, we tested the combination of HerVaxx and the recently identified B-cell epitope/mimotope of Pertuzumab, i.e. a multi-peptide B-cell vaccine, for preventing Her-2/neu lung metastases formation in a mouse model. Active immunization with the multi-peptide vaccine was associated with decreased lung weights, and histological evaluation of the lungs showed that the significant reduction of lung metastases was associated with increased CD4⁺ and CD8⁺ T cell infiltration. Notably, along with the overall reduction of lungs weights and Her-2 positive metastases, a formation of Her-2/neu-negative tumors but with increased PD-L1 expression was observed. Our results might pave the way to a multi-peptide B-cell Her-2/neu vaccine serving as a secondary intervention in adjuvant settings to prevent tumor recurrence and spread. Moreover, combination therapy targeting PD-L1 may result in total remission of metastases. Such a therapy may be used clinically to alternately target Her-2/neu and PD-L1 in metastatic breast cancer.     

Brain metabolism of sarcoma 45-bearing rats undergoing radiation and the effect of hyperthermia on the tumor

Activity of hexokinase and acetylcholinesterase and pyridoxal co-enzyme content of brain subcellular fractions were studied in rats, bearing sarcoma 45, after local exposure of the tumor to 20 Gy X-radiation and microwave hyperthermia. The carbohydrate metabolism was sharply inhibited while the pyridoxal coenzyme content and acetylcholinesterase activity increased.     

Prognostic significance of tumor-infiltrating T-lymphocytes in primary and metastatic lesions of advanced stage ovarian cancer

Purpose  Ovarian cancer patients with intra-tumoral CD3⁺ T-lymphocytes in primary tumor tissue have a better prognosis. This study aims to analyze the presence and relative influence of three important T-lymphocyte subsets, tumor-infiltrating CD8⁺ cytotoxic T-lymphocytes (CTL), CD45R0⁺ memory T-lymphocytes, and FoxP3⁺ regulatory T-lymphocytes (Treg), in primary tumor tissue and omental metastases of patients with ovarian cancer. Experimental design  The number of CD8⁺, CD45R0⁺, and FoxP3⁺ T-lymphocytes was determined by immunohistochemistry on a tissue micro array containing ovarian tumor tissue and/or omental metastases obtained at primary debulking surgery from 306 FIGO stage I–IV ovarian cancer patients. Immunohistochemistry data were correlated to clinicopathological parameters and survival data. Results  High number of CD8⁺ CTL and a high CD8⁺/FoxP3⁺ ratio in ovarian-derived tumor tissue were associated with increased disease-specific survival and proved to be independent prognostic factors in multivariate analyses. In advanced stage patients, the presence of CD8⁺ CTL, CD45R0⁺ memory T-lymphocytes, FoxP3⁺ Treg or a high CD8⁺/FoxP3⁺ ratio in ovarian-derived tumor tissue was associated with an increased disease specific survival in univariate analysis, as was the presence of CD45R0⁺ memory T-lymphocytes and FoxP3⁺ Treg in omental metastases. Furthermore, in advanced stage patients CD8⁺ cytotoxic and FoxP3⁺ regulatory T-lymphocytes infiltrating ovarian-derived tumor tissue were independent predictors of increased prognosis. Conclusions  T-lymphocytes infiltrating primary and metastatic ovarian cancer sites are associated with improved prognosis. These associations are especially distinct in advanced stage patients, underlining the potential for immunotherapy as a broadly applicable therapeutic strategy. Ninke Leffers and Marloes J. M. Gooden contributed equally.     

放疗联合热疗治疗复发性卵巢癌患者的临床疗效及对患者免疫功能的影响

目的:探讨放疗联合热疗治疗复发性卵巢癌患者的临床疗效及对患者免疫功能的影响。方法:回顾性选取2011年1月-2014年5月在我院诊治的复发性卵巢癌患者99例,按治疗方法将患者分为对照组47例和观察组52例。对照组患者给予三维适行放射治疗,观察组在对照组的基础上给予盆腔部位热疗。治疗后,比较两组患者的临床疗效及免疫功能。结果:治疗后,对照组总缓解率为53.19%,观察组总缓解率为78.85%,显著高于对照组(P0.05);治疗后,观察组与对照组外周血中CD3+、CD4+及CD4+/CD8+含量均较治疗前明显升高(P0.05),而CD8+含量则显著降低(P0.05),且观察组患者CD3+、CD4+及CD4+/CD8+显著高于对照组(P0.05),CD8+水平则明显低于对照组(P0.05)。两组患者治疗后骨髓抑制、胃肠道反应及泌尿系统反应的发生率、1年及2年生存率比较均无显著性差异(P0.05),观察组治疗后3年生存率显著高于对照组(P0.05)。结论:与单纯三维适行放射治疗相比,放疗联合热疗能有效提高复发性卵巢癌的临床疗效,显著增强患者免疫功能,延长生存期,且不会增加不良反应。     

FTY720 suppresses liver tumor metastasis by reducing the population of circulating endothelial progenitor cells

Background

Surgical procedures such as liver resection and liver transplantation are the first-line treatments for hepatocellular carcinoma (HCC) patients. However, the high incidence of tumor recurrence and metastasis after liver surgery remains a major problem. Recent studies have shown that hepatic ischemia-reperfusion (I/R) injury and endothelial progenitor cells (EPCs) contribute to tumor growth and metastasis. We aim to investigate the mechanism of FTY720, which was originally applied as an immunomodulator, on suppression of liver tumor metastasis after liver resection and partial hepatic I/R injury.

Methodology/Principal Findings

An orthotopic liver tumor model in Buffalo rat was established using the hepatocellular carcinoma cell line McA-RH7777. Two weeks after orthotopic liver tumor implantation, the rats underwent liver resection for tumor-bearing lobe and partial hepatic I/R injury. FTY720 (2 mg/kg) was administered through the inferior caval vein before and after I/R injury. Blood samples were taken at days 0, 1, 3, 7, 14, 21 and 28 for detection of circulating EPCs (CD133+CD34+). Our results showed that intrahepatic and lung metastases were significantly inhibited together with less tumor angiogenesis by FTY720 treatment. The number of circulating EPCs was also significantly decreased by FTY720 treatment from day 7 to day 28. Hepatic gene expressions of CXCL10, VEGF, CXCR3, CXCR4 induced by hepatic I/R injury were down-regulated in the treatment group.

Conclusions/Significance

FTY720 suppressed liver tumor metastasis after liver resection marred by hepatic I/R injury in a rat liver tumor model by attenuating hepatic I/R injury and reducing circulating EPCs.