The role of immune effector cells in flavone acetic acid-induced injury to tumor cells in EMT6 spheroids.

Inhibition of tumor blood flow appears to a major antitumor mechanism of flavone acetic acid (FAA), although non-ischemic processes may also be a significant role. To distinguish between direct and immune effector cell-mediated cytotoxicity as the basis for non-ischemic killing, effects of FAA were compared in EMT6 spheroids grown entirely in vitro and spheroids recovered from the peritoneal cavities of mice after six days of in vivo growth (ex vivo spheroids). Approximately 50% of the cells in the latter case were of host origin (macrophages and lymphocytes). Ex vivo spheroids showed specific histological changes when exposed to FAA, including tumor cell rounding, apoptosis, depression of mitotic activity and dissolution of necrotic debris in the spheroid core. Quantitation of histological changes indicated these effects to be significantly greater in ex vivo than in vitro spheroids. The histological changes in FAA treated ex vivo spheroids were partially inhibited by dexamethasone. Oxygen tension did not influence the response of spheroids to FAA. The results suggest that immune effector cells, probably macrophages, mediate blood flow-independent antitumor effects of FAA.     

Adjuvant chemoimmunotherapy with LMF + BCG in node-negative and node-positive breast cancer patients: 10 year results

A total of 254 patients with stages T1–3a/N0–1/M0 operable breast cancer were randomized to either surgery alone or surgery plus adjuvant chemoimmunotherapy (LMF + BCG). Ten-year results are presented for RFS (relapse-free survival) and OAS (overall survival) in the whole patient population as well as in the most important menopausal and nodal subgroups.LMF + BCG significantly increased RFS in the whole patient population as well as in node-positive women. The earlier impressive RFS and OAS gains for node-negative patients were fading after 5 and 8 years respectively, leaving marginal trends in favour of the LMF + BCG treated women. Node-positive patients treated with LMF + BCG continue to demonstrate a marginal gain in RFS up to 10 years. This gain is nearly exclusively expressed in postmenopausal node-positive women, an observation which can be made in the node-negative patient group as well. Despite the still continuing increase in RFS, no OAS benefit was observed for node-positive women with LMF + BCG at any time of the study.Dose still remains a critical factor in cancer therapy. However, at 10 years of follow-up, a full dose of LMF (⩾90%) during the six cycles no longer affects OAS favourably.There was no indication of any adverse long-term toxicity of LMF + BCG in our study after a median follow-up of 10 years, especially no increase of second tumours.In the node-negative patient population, the presence or absence of intramammary lymphatic infiltration seems to be a significant prognostic factor within this nodal subgroup.     

Doxorubicin plus interleukin-2 chemoimmunotherapy against breast cancer in mice

As recently characterized, following s.c. implantation into syngeneic C57BL/6 mice, E0771 tumor invades locally into dermal layers and peritoneum, metastasizes to the lung, and induces a nonspecific immunosuppression in the host. Using this breast medullar adenocarcinoma model, a therapy consisting of a single moderate dose of doxorubicin followed by twice daily moderate doses of interleukin-2 for 30 days was examined for efficacy and mechanism of action when given to animals with established disease. This combination treatment, but not combinants alone, resulted in tumor-free long-term survival of 40% of the mice without significant toxicity and 83% of survivors had immune memory specific for E0771 cells. Treatment also decreased immune suppression induced by E0771 tumor. Full response to treatment required functional CD8(+) T cells, whereas depletion of natural killer cells caused only a reduction in response rate. A serum "biomarker" profile that correlated with, and seemed predictive of, response to treatment was identified by nuclear magnetic resonance-based metabonomic analysis. The efficacy of this nontoxic treatment and the potential to be able to predict which individual is responding to treatment are characteristics that make this chemoimmunotherapy attractive for clinical testing.     

Mechanisms of in vivo generation of cytotoxic effector cells against tumor in tumor-bearing mice

Our previous study showed that spleen cells from BALB/c mice bearing RL male 1 lymphoma inhibited the growth of RL male 1 lymphoma by the Winn-type adoptive transfer assay. Although this antitumor activity was mediated by the T-cell subset manifesting the surface phenotype of cytotoxic T-lymphocytes (CTLs), this antitumor activity of spleen cells was not detected by the in vitro cell-mediated cytotoxicity assay (4-h 51Cr release assay). The present study is concerned with the hypothesis that the maturation of the CTLs directed against RL male 1 may be arrested in spleens of the RL male 1-bearing mice and the differentiation into mature CTLs may occur at the tumor site; i.e., the immature CTLs (activated precytotoxic T-cells) may acquire the killing activity when they contact tumor cells at the tumor site. This report shows that BALB/c mice bearing the progressive RL male 1 lymphoma were able to generate CTLs against RL male 1 in the peritoneal cavities when the mice were inoculated i.p. with the irradiated RL male 1 cells. The cytotoxic activity of the peritoneal exudate cells of the RL male 1-bearing mice appeared 3 to 5 days after i.p. inoculation of the irradiated RL male 1 cells and rapidly decreased on day 7 after inoculation. In addition, spleen cells from the RL male 1-bearing mice after i.p. inoculation of the irradiated RL male 1 cells were not cytotoxic, suggesting a highly localized response. The cytotoxic effector cells induced in the peritoneal cavities consisted of T-lymphocytes and natural killer cells. Both cell types were simultaneously induced in the peritoneal cavities of the RL male 1-bearing mice. The T-cell subset mediating cytolytic activity against RL male 1 was shown to consist of Lyt-1+2+ T-cells which were defined by cytolysis with anti-Lyt-1 or anti-Lyt-2 antibody and complement. On the other hand, the normal BALB/c mice inoculated i.p. with the irradiated RL male 1 cells generated natural killer cells in the peritoneal cavities and spleens but the CTLs were not induced. The results from the present and previous studies suggest that precytotoxic or immature cytotoxic T-cells in spleens of the tumor-bearing mice migrate into the circulation and then mature CTLs develop at the tumor site.     

Breast cancer cell CD200 expression regulates immune response to EMT6 tumor cells in mice

CD200 has been characterized as an important immunoregulatory molecule, increased expression of which can lead to decreased transplant rejection, autoimmunity, and allergic disease. Elevated CD200 expression has been reported to be associated with poor prognosis in a number of human malignancies. We have found that cells of the transplantable EMT6 mouse breast cancer line growing in vitro express low levels of CD200, but levels increase markedly during growth in immunocompetent mice. Similar increased in vivo expression does not occur in NOD-SCID.IL-2^γr−/− mice or mice with generalized over-expression of a CD200 transgene. In both mice, tumor growth occurs faster. Altered CD200 expression in control versus transgenic mice is accompanied by reproducible changes in tumor-infiltrating host cells, and altered cell composition in lymph nodes draining the tumor (DLN). Neutralization of expressed CD200 by anti-CD200mAbs leads to decreased tumor growth in immunocompetent mice, with improved detection of cytotoxic anti-tumor immune cells in DLN. Finally, we report that tumor growth in vivo can be monitored by levels of soluble CD200 (sCD200) in serum of tumor-bearing animals.     

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.     

Equilibrium between host and cancer caused by effector T cells killing tumor stroma

The growth of solid tumors depends on tumor stroma. A single adoptive transfer of CD8(+) CTLs that recognize tumor antigen-loaded stromal cells, but not the cancer cells because of MHC restriction, caused long-term inhibition of tumor growth. T cells persisted and continuously destroyed CD11b(+) myeloid-derived, F4/80(+) or Gr1(+) stromal cells during homeostasis between host and cancer. Using high-affinity T-cell receptor tetramers, we found that both subpopulations of stromal cells captured tumor antigen from surrounding cancer cells. Epitopes on the captured antigen made these cells targets for antigen-specific T cells. These myeloid stromal cells are immunosuppressive, proangiogenic, and phagocytic. Elimination of these myeloid cells allowed T cells to remain active, prevented neovascularization, and prevented tumor resorption so that tumor size remained stationary. These findings show the effectiveness of adoptive CTL therapy directed against tumor stroma and open a new avenue for cancer treatments.     

Adjuvant chemo-endocrine therapy for androgen-dependent mammary tumor in mice

Summary About 500 male DS mice grafted with androgen-dependent Shionogi carcinoma 115 (SC115) were used. When the tumor diameter reached about 20 mm (approximately 25 days after transplantation), excision of the tumor and/or castration were carried out. The injection of cyclophosphamide (80 mg/kg body weight × 3 at 7-day intervals) was started from the day after excision.In mice with excised tumor, adjuvant chemo-endocrine therapy was the most effective treatment examined; cumulative 120-day mortalities after transplantation of tumors in non-treated, adjuvant endocrine therapy, adjuvant chemotherapy and adjuvant chemo-endocrine therapy groups were 91%, 29%, 21% and 0%, respectively. Castration induced development of clusters of androgen-independent cancer cells in androgendependent SC115 tumor. In mice without tumor excision, the chemo-endocrine therapy was again the most effective treatment, though 86% of mice died by the 120th day after tumor transplantation. These findings suggest the usefulness of adjuvant chemo-endocrine therapy for achieving complete remission in hormonedependent tumors. Address for reprints: K. Matsumoto, M.D., Dept. of Pathology, Osaka University, Medical School, Kita-ku, Osaka 430, Japan.     

Generation of tumor-reactive effector lymphocytes using tumor RNA-introduced dendritic cells in gastric cancer patients

Anti-tumor effector cells were generated by stimulating peripheral blood lymphocytes with cultured dendritic cells (DCs) and mRNA extracted from the gastric cancer cell line MKN45 or ascites tumor cells of gastric cancer patients. DCs were generated from an adherent fraction of peripheral blood mononuclear cells (PBMCs) in the presence of GM-CSF and IL-4. mRNA was extracted from tumor cells and subjected to T7-amplification. The DCs were electroporated (150 V/150 microF) with amplified mRNA and used after maturation with TNF-alpha to stimulate PBMCs to generate tumor RNA-introduced DC-activated killer (TRiDAK) cells. It was found that tumor RNA could efficiently be introduced into cultured DCs by electroporation (55% efficiency, 78% viability), and tumor RNA-introduced DCs could reproducibly stimulate lymphocytes to be tumor-reactive TRiDAK cells. The TRiDAK cells expressed an IFN-gamma response specific for tumor cells, but not for normal cells. Mock DCs or normal cell RNA-introduced DCs did not induce any killer cells. RNA-specific recognition of the effector cells generated was demonstrated using an amplified EGFP-mRNA system. The tumor killing activity of TRiDAK cells was inhibited not only with the anti-HLA class I antibody but also with the anti-HLA class II antibody as well as the anti-TCR antibody. TRiDAK cells reactive with autologous tumor cells could be generated in a CEA-positive gastric cancer patient with malignant ascites, in whom effector cell generation using DCs and CEA peptides had failed. These results suggest that TRiDAK cell generation is safe, feasible, and active in gastric cancer patients with malignant ascites, and is superior to other effector cell generation systems using DCs and epitope peptides. The adoptive immunotherapy of cancer using TRiDAK cells may be warranted in a clinical setting. This is the first study investigating anti-tumor effector cell generation using cultured DCs and tumor mRNA from gastric cancer cells.     

Cellular basis for the immune response to methylcholanthrene-induced tumors in mice. Heterogeneity of effector cells

Immune resistance to methylcholanthrene-induced tumors has two phases, an early specific and a late non-specific phase. Both phases were found to be T-cell-dependent in vivo. Thus, adult thymectomized, irradiated, bone-marrow-protected mice bearing H-1 tumor isografts showed impaired resistance to challenge with homologous (H-I) and heterologous (H-3) tumor cells. In each case resistance was restored by injection of thymus cells. In vitro analysis of the cellular basis of resistance revealed that different mechanisms were involved in the two phases. The cytotoxic effect of immune spleen cells taken during the early specific phase was inhibited by pretreatment with anti-θ serum and complement and by removal of macrophages. Neither procedure, however, interfered with the cytotoxic potential of immune spleen cells taken during the late non-specific phase of immunity. Passage of immune spleen cells through rabbit-IgG anti-mouse immunoglobulin-coated columns (which yielded a T-cell-enriched, B-cell-depleted population) resulted in abrogation of cytotoxicity whether the cells were obtained during the early or the late phase of resistance. The inability of late-phase spleen cells to kill was explicable in terms of B-cell removal since T-cells and macrophages had been shown to be ineffective at that time. In contrast, the failure of column-treated cells from the early phase to kill was found to be due to removal of adherent cells rather than B-cells since cytotoxicity (1) was abrogated by passage through control columns coated with rabbit-IgG anti-sheep red blood cell antibody which did not retain B-cells and (2) could be restored by addition of immune macrophages (from anti-θ serumtreated spleens). Taken together, these results indicate that the cellular basis of immune resistance to methylcholanthrene-induced tumors is heterogeneous. The early specific phase seems to be mediated by an interaction between T-cells and macrophages; the late non-specific phase, although T-cell dependent in its induction, depends on a different effector mechanism, possibly involving a cell or its products of the B lineage.     

抗人P185 erbB2 scFv-Fc-IL-2调变肿瘤细胞表面分子和激活免疫效应细胞

将抗人P185erbB2scFvFcIL2融合蛋白（HFI）作用于人卵巢癌细胞株SKOV3细胞和人外周血单个核细胞（PBMC）,通过体外实验阐明HFI调变肿瘤细胞表面分子和激活免疫效应细胞的抗肿瘤机制,为HFI临床应用提供实验依据。〖HT5W〗方法： 〖HT5"SS〗MTT法检测细胞增殖、杀伤活性;流式细胞术观察细胞表面分子的表达水平;生物活性法检测细胞膜相关TNF杀伤活性;RTPCR检测细胞穿孔素表达水平。〖HT5W〗结果： 〖HT5"SS〗HFI处理后,未观察到对SKOV3细胞增殖活性的直接抑制作用;SKOV3细胞表面杀伤相关分子ICAM1、Fas表达率分别由24.85%、0.53%增高到85.36%、59.19%（P<0.01);人PBMC的增殖活性增强,CD3+CD8+T细胞和CD3CD16+CD56+NK细胞分别由24.37%、6.90%提高到38.80%、13.45%（P<0.01);CD25、LFA1、FasL表达水平分别由399%、86.52%、5.02%提高到12.96%、99.06% 16.19%（P<0.01);穿孔素基因、膜相关TNF均表达增强,LAK样、NK样杀伤活性在各效靶比时均明显增高（P<0.01)。〖HT5W〗结论： 〖HT5"SS〗HFI提高SKOV3细胞杀伤相关分子ICAM1、Fas表达水平,并且对人PBMC有明显的增殖活化作用,通过激活LFA1/ICAM1、Fas/FasL途径提高杀伤介质穿孔素和膜相关TNF的释放,增强LAK样、NK样杀伤活性。     

Prognostic role of circulating tumor cells and disseminated tumor cells in patients with prostate cancer: a systematic review and meta-analysis

Circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) displayed their roles in prognosis prediction in prostate cancer. The objective of the present study was to conduct a systematic review and meta-analysis of published literature while investigating the correlation between survival outcome and CTCs or DTCs counts in patients with prostate cancer. Relevant literature was searched in Pubmed and Embase. Survival data of included study were extracted. Forrest plots were used to estimate the effect of CTCs/DTCs on the survival of patients. Publication bias was evaluated using Begg’s test. The estimated HRs and 95 % confidence interval for the effect of CTCs/DTCs on overall survival (OS) and biochemical relapse-free survival (bRFS) or disease-free survival (DFS) were 2.43 [2.07, 2.86] (p?<?0.00001) and 2.15 [1.69, 2.73] (p?<?0.00001), respectively. Subgroup analysis revealed that CTCs were also relevant to poor prognosis (hazard ratio (HR) 2.43 [2.05, 2.89] for OS, HR 2.46 [2.08, 2.90] for bRFS/DFS). A similar result was yielded in DTCs (1.47 [1.21, 1.80] for DFS). CTCs/DTCs could also predict poor OS in metastatic prostate cancer (2.37 [1.99, 2.82], p?<?0.00001) and in localized stage (HR 1.84 [1.47, 2.28], p?<?0.00001). In addition, CTCs/DTCs detected by different methods, especially by CellSearch system (HR for OS 2.36 [1.95, 2.85] and HR for bRFS/DFS 2.53 [1.66, 3.85]), were relevant to poor prognosis. Available evidence supported the notion of the strong prognostic value of CTCs. CTCs are promising biomarkers that are clinically implemented in the therapeutic decision-making process in patients with prostate cancer.     

Adjuvant chemoimmunotherapy following regional therapy for isolated recurrences of breast cancer (stage IV NED).

A combination of 5-fluorouracil, Adriamycin, cyclophosphamide, and BCG (FAC-BCG) was evaluated as adjuvant treatment in breast cancer patients following surgical excision and/or radiation of first site of recurrent disease. In a group of 68 patients treated with FAC-BCG, the estimated proportion remaining free of disease at 2 years from first recurrence was 69%, compared to 24% in 60 historical control patients (P less than 0.01). Estimated 2-year survival rate from first recurrence was 85% for the FAC-BCG patients and 78% for the controls (P = 0.07). This regimen has significantly prolonged the disease-free interval from the first recurrence, but additional follow-up is needed to determine its effect on long-term survival.