Experimental study on immunological effect of splenectomy on tumor-bearers

In view of popularity of splenectomy in combination with gastrectomy for gastric cancer, immunological effect of splenectomy was studied experimentally, using SD rats and 3-methylcholanthrene-induced mammary cancer, MRMT -1. Splenectomy was performed on day -14, 2, 7, 14 or 21 of sc inoculation of 200 mg (about 4 X 10(6) cells) of MRMT -1 at the back of 4-week-old female rats. Tumor growth and immunocompetence of peripheral lymphocytes, spleen cells and thymus cells were examined. Tumor growth on day 35 tended to be inhibited in rats splenectomized on day -14, 2, or 14, while it tended to be promoted in rats splenectomized on day 7. PHA-induced blastogenesis and NK activity of spleen cells were reduced on day 2, rose on day 7 and gradually decreased on days 14 and 21. Winn's neutralization assay revealed that spleen cells on days 2, 14 and 21 of tumor inoculation had suppressor activities. In conclusion, spleen cells of tumor-bearing rats showed a reduced immunocompetence on day 2 (the early tumor-bearing stage) and on day 14 and later (the late tumor-bearing stage), while they showed a raised immunocompetence on day 7 (the middle tumor-bearing period).     

Experimental study on preventive effects of lung metastases using LAK cells induced from various lymphocytes--special references to enhancement of lung metastasis after laparotomy stress

In 5 week-old female SD rats, ether anesthesia or laparotomy for 30 minutes was done, followed by iv inoculation of 1 x 10(6) MRMT-1 cells. On day 14, number of lung metastatic nodules was examined. Preventive effect of LAK cells on lung metastases was also observed. LAK cells were induced from regional lymph node lymphocytes (LN), thymus cells (TC) and spleen cells (SC) of the rats on day 7 after tumor inoculation by incubation with 1 micrograms/ml of R-IL2 (TGP-3) for 4 days. 1) Lung metastases were noted in 100% after MRMT-1 intravenous inoculation, while number of lung metastatic nodules was reduced after iv administration of LAK cells. 2) When MRMT-1 cells were inoculated intravenously after laparotomy stress for 30 minutes, number of lung metastatic nodules was significantly increased and LAK cells reduced it significantly. 3) When R-IL2 sc administration for 4 days was accompanied, number of lung metastatic nodules was less than that of without R-IL2 substitution. 4) The antitumor effect of LAK cells was the strongest in LN-LAK, while it was the weakest in SC-LAK. In conclusion, usefulness of LAK cells for preventing lung metastases by surgical stress was indicated. This fact suggested that possibility of adoptive immunotherapy using LAK cells might be useful for prevention of lung metastases after laparotomy.     

In vivo antitumor effect of LAK cells induced from various lymphocytes--experimental study in rats

A MRMT-1 was inoculated in the thigh of SD rats. On day 7, regional lymph node lymphocytes (TB-LN), thymus cells (TB-TC) and spleen cells (TB-SC) or non-tumor bearing spleen cells (NB-SC), were obtained. LAK were induced with lug/ml of R-IL2 (TGP-3) using above lymphocytes, and were examined as for their in vivo and in vitro cytotoxic activities. In vitro: Against MRMT-1, cytotoxic activity of LAK increased with lapse of culturing time. Thus on day 4 of culture, it was the highest (46.5%) in TB-SC-LAK, while that of TB-LN-LAK was the lowest (5.8%). Against YAC-1, they also increased except TB-LN-LAK. In vivo: When the mixture of MRMT-1 and LAK was sc inoculation, diameter of the tumor were the smallest in TB-LN-LAK and the largest in TB-SC-LAK. When it was injected intraperitoneally, in non-irradiated rats, the survival prolongation was the best in TB-LN-LAK and the worst in TB-SC-LAK. In pre-irradiated rats; the cytotoxicity was almost the same as the results of in vitro. When it was injected intravenously, number of lung metastatic nodes were the smallest in TB-LN-LAK and the largest in TB-SC-LAK. R-IL2 substitution therapy was also effective. In conclusion, the effectiveness of LAK-AIT was estimated more accurately by in vivo cytotoxic assay. TB-LN was most useful as a source of LAK cells.     

Immunological evaluation of splenectomy in tumor-bearing mice

The effect of splenectomy upon neoplastic outgrowth was examined after inoculation of methylcholanthrene-induced C3H/He murine tumors. Three days or 20 days after tumor inoculation, splenectomy resulted in significant retardation of tumor growth when compared with sham operation, while splenectomy 6, 9, 15 days after tumor inoculation did not alter the tumor outgrowth. These results suggest that spleen might have immunologically negative element in early or late stage of tumor burden. In fact, spleen cells from mice bearing MCA-F tumors for 3 days or 30 days nonspecifically facilitated the tumor outgrowth in Winn assay. The non-specific tumor-enhancing cells were radioresistant (700 rads), capable of phagocytizing carbonyl-iron and adherent to plastic dish suggesting those were tumor enhancing macrophages. On the other hand, spleen cells from tumor-bearing mice for 9 to 15 days specifically reduced the tumor growth in Winn assay, and those cytotoxic cells were radio-sensitive (700 rads) T cell population.     

Effect of regional or distant lymph node removal and of splenectomy on immunologic responses in Ehrlich tumor-bearing mice

To study the role of regional (RLN) and distant (DLN) lymph nodes and of the spleen in the regulation of tumor immunity, we monitored tumor growth in mice subjected to lymph node removal or splenectomy. We found that tumor growth was facilitated when RLN were removed before or soon after tumor inoculation and that RLN removal after extirpation of the immunizing tumor decreased the host resistance to a subsequent challenge. When splenectomy preceded tumor inoculation, tumor growth was not affected, however, marked growth inhibition was observed when splenectomy was performed 5 days after tumor inoculation. Furthermore, splenectomy combined with extirpation of the immunizing tumor increased the host resistance to a subsequent challenge. We conclude from these studies that RLN or the spleen may be of immunologic importance in the host’s tumor resistance.     

Antitumor immunity in the relatively early period after cryosurgery. Experimental study using rat's metastasizing mammary tumor

Six-week-old female Sprague-Dawley rats were inoculated with a mammary tumor, MRMT-1. At 2 weeks after inoculation, one of the following 4 treatments was done: surgical tumor excision (SE), cryosurgery (CR), surgical excision plus re-inoculation of freezing-thawing-produced vaccine (FT), and surgical excision plus fasting for 72 hours (FA). In FT and FA groups, incidence of metastatic death were higher than in SE group, while that in CR group was similar as in SE group. Specific footpad reactivity at 2 and 3 weeks after treatment was lower in CR than in SE group. Winn's neutralization assay revealed that antitumor activity of spleen cells at 1 and 3 week(s) after treatment was lower in CR than in SE groups. In vivo observation on effect of inactivated serum at 1 week after treatment showed tumor enhancement in SE group and tumor inhibition in CR group. In conclusion, the observed mild reduction in antitumor immunity in the relatively early period after cryosurgery might not be due to blocking action of superfluous tumor antigens, but probably due to activation of suppressor cells consequent on cryosurgical stress or on slow absorption of tumor antigens.     

脾肠联合移植对大鼠小肠移植免疫耐受的影响

目的　探讨脾肠联合移植对小肠移植免疫耐受的诱导作用。方法　选用SD大鼠为供体、Wistar大鼠为受体 ,进行异位全小肠和脾脏联合移植。实验分 3组 ,每组 6只。A组 :小肠移植非免疫干预组 ;B组 :受体脾切除 ,脾肠联合移植组 ;C组 :小肠移植环孢霉素A(CsA)治疗组。术后 3、5、7、10d取移植小肠回肠段 0 .5～ 1.0cm进行病理学检查 ,用病理图像分析系统测量黏膜厚度 ,绒毛高度和隐窝深度。采用TdT介导的脱氧核苷酸原位末端标记法 (TUNEL)检测 3、5、7d移植小肠黏膜细胞凋亡 ,评价移植小肠急性排斥反应损伤程度。结果　A、B两组均有不同程度的急性排斥反应损伤 ,但A组高于B组 ,移植后 3、5、7d分别属于轻、中、重度排斥 ,10d黏膜基本完全脱落。B组移植后 3、5d符合轻度排斥 ,7d中度以下排斥 4只 ,重度排斥 2只 ,10d中度排斥 2只 ,重度 4只 ,但仍可见黏膜层。C组移植后 10d 1只出现轻度排斥其余未见明显损伤。黏膜厚度、绒毛高度和隐窝深度B组明显高于A组 ,黏膜上皮细胞凋亡数目较A组低。结论　受体脾切除 ,脾肠联合移植可减轻移植小肠急性排斥反应损伤 ,诱导一定程度的小肠移植免疫耐受     

Bidirectional effects of splenectomy on the growth of syngeneic tumor in mice

The effects of splenectomy on tumor growth following inoculation with a relatively large number of cells (1 X 10(7) ) and a smaller number of cells (5 X 10(5) ) of Meth I tumor were studied. When 1 X 10(7) tumor cells were inoculated, tumor growth in splenectomized mice was depressed, while tumor in sham-operated mice grew progressively. On the contrary, when 5 X 10(5) tumor cells were inoculated, the tumor take was lower in sham-operated than in splenectomized mice. The spleen cells from mice inoculated with either a large or small number of tumor cells, showed an equally potent cytotoxic activity, but no detectable suppressor cell activity. On the other hand, the activity of immunosuppressive factor was detected in sera from mice inoculated with 1 X 10(7) tumor cells, but not in those given 5 X 10(5) cells. The effect of splenectomy on tumor growth is, thus, bidirectional, depending on the dose of tumor cells inoculated.     

Experimental study on the absorption mechanism of cryo-necrotized tumor antigens

On day 7 of MRMT-1 tumor inoculation at the thigh in 5 week-old female SD rats, the tumor was treated cryosurgically by contact method at -170 degrees C and 2 cycle freezing. Postoperative changes in local blood circulation were observed by colloidal carbon perfusion and hydrogen-clearance methods, and pathway and time course of tumor antigen absorption were observed by measuring 3H-thymidine (injected intra-tumorally) uptake in various organs. The observations were done at 0.5, 1, 2, 3, 6, 9, 12, 24, 48, 72, 120 and 168 hour(s) after cryosurgery. The following results were obtained. At 0.5 to 1 hour after cryosurgery: vascular stasis, dilatation and tortuosity were observed. There were no inflow of colloidal carbon and almost no uptake of 3H-thymidine. At 6 hours: vascular stasis and sludging became more marked. There was no inflow of carbon, while uptake of 3H-thymidine was markedly increased. At 24 hours: some carbon inflows in fine vessels and partial recovery of blood circulation in the peritumoral region were observed. Rate of 3H-thymidine uptake was more increased. At 72 hours: carbon inflow and blood circulation were further increased, while the cryo-necrotized tumor showed hyaline degeneration. At 168 hours: increase of newly formed vessels and recovery of blood circulation were remarkable, but uptake rate of 3H-thymidine was decreased according to increased demarcation of the cryo-necrotized tumor. From the above results, it was suggested that the absorption of cryo-necrotized tumor antigens was starting through lymphatic channels in the early period, then also through newly formed capillaries surrounding the tumor at 24 hours after cryosurgery and continued until 72 hours after cryosurgery.     

Laparoscopic splenectomy and nephrectomy in a rat model

Background: In experimental studies on the effects of laparoscopic procedures on tumor biology, a localized tumor model is desirable. The spleen and the kidney are preferable, because these organs are amenable to tumor placement and subsequent removal. This study describes the technique of laparoscopic splenectomy and nephrectomy in the rat model. Methods: Pneumoperitoneum was established by CO₂ insufflation. Laparoscopic splenectomy involved two-handed dissection, intracorporeal ligation, and division of gastrosplenic attachments and hilar and short gastric vessels. Laparoscopic nephrectomy was done by intracorporeal ligation and division of the renal vessels and the ureter after mobilization of the kidney. Results: Laparoscopic splenectomy was performed in six rats; laparoscopic nephrectomy was done in six rats. Operative time ranged from 45 to 90 min for splenectomy and from 40 to 65 min for nephrectomy. Postoperatively, two rats died from hemorrhage. Necropsy of the rats after 10 days revealed adhesion in three rats after splenectomy and in four rats after nephrectomy. Inflammatory processes were found around the silk ligatures in all rats after splenectomy; in two rats wound infections occurred at the port sites. Conclusions: Laparoscopic splenectomy and nephrectomy in the rat proved technically feasible and may provide new localized tumor models suitable to be used in further studies on the oncological effects of laparoscopic surgery.     

肝癌合并肝硬化时脾脏对机体免疫状态影响的实验研究

目的 建立大鼠肝硬化肝癌模型，并行脾脏切除，探讨脾脏在肿瘤免疫中的作用。方法 将健康雄性SD大鼠55只背部皮下注射40％四氯化碳（CCl4)花生油溶液建立肝硬化模型，将Walker-256癌肉瘤接种于肝脏内并行脾切除术，2周后观察肿瘤的生长、转移、腹水、NK细胞活性及CD25。结果 切脾组腹水量、肿瘤转移较对照组明显，肿瘤体积明显大于对照组（P＜0.01)，荷瘤后2周两组NK细胞活性均较荷瘤前降低（P＜0.01)，而荷瘤前与荷瘤后切脾组与模拟切脾组之间比较无统计学差异（P＞0.05)。荷瘤2周后两组动物CD25较荷瘤前升高（P＜0.01)，而荷瘤前后切脾组与对照组之间比较无变化（P＞0.05)。结论 荷瘤早期切除脾脏加速肿瘤的生长、转移，而对NK细胞活性及CD25的表达影响不及肿瘤对其的影响。     

IL-12 cDNA direct injection: antimetastatic effect from a single injection in a murine hepatic metastases model

BACKGROUND: Interleukin 12 (IL-12) gene therapy is an effective antitumor agent in local and metastatic murine tumor models. We sought to evaluate the antimetastatic effect of IL-12 cDNA in a liver metastases model. MATERIALS AND METHODS: A liver metastases model was induced by creating a "primary" splenic tumor through inoculation of 1 x 10(5) TS/A adenocarcinoma cells directly into the inferior pole of the spleen in female BALB/c mice. On day 4, 50 microg of IL-12 cDNA or control plasmid DNA was injected into splenic tumor, followed by splenectomy on day 8. Mice were sacrificed on day 25 to assess liver tumor burden. IL-12 mRNA and mIL-12 and IFN-gamma protein levels were assessed after IL-12 injection. Peripheral blood CD4+, CD8+, and NK cells were quantified on day 14 using FACS. To determine the significance of site of cytokine DNA injection, IL-12 cDNA was injected on day 4 into splenic tumor or into the non-involved spleen after isolation of the inferior and superior portions of the spleen, respectively, with surgical clips. Splenectomy was performed on day 8 and sacrifice was performed on day 25. RESULTS: IL-12 mRNA was detected in the liver 8 h after injection, with a peak at 24 h. After splenic injection, protein levels of IL-12 and IFN-gamma were detectable in the liver and spleen 24 h after treatment. IL-12 and IFN-gamma were not detectable in control animals. In the peripheral blood, there was a marked increase in NK cells (13% of total lymphocytes versus 4%, control) and in the CD4+/CD8+ ratio (5.5 versus 1.9). At day 25, there was a marked antimetastatic effect after IL-12 injection into either splenic tumor [liver:body weight, 6.2 versus 10.9 (control), P = 0.007] or non-involved spleen (6.8 g versus 10.7 g, P = 0.005). There was no difference in the antimetastatic effect between animals injected into splenic tumor or non-involved spleen (P = 0.3). CONCLUSION: Injection with a single dose of IL-12 cDNA into splenic tumor or non-involved spleen resulted in a profound antimetastatic effect. Splenic IL-12 injection results in mRNA expression in the liver, protein expression in the liver and spleen, and a marked increase in NK cells and the CD4+/CD8+ ratio in peripheral blood.     

The role of the spleen in tumor bearing host: II. The influence of splenectomy in mice

The effect of splenectomy on neoplastic outgrowth was examined prior to and after implantation of methylcholanthrene-induced C₃H/He murine tumors. Splenectomy performed 12 days before tumor inoculation did not affect the tumor outgrowth, however, both splenectomy and sham operation performed shortly before tumor inoculation resulted in significant tumor facilitation compared with the non-operated group, suggesting that this accelerated tumor was not related to the presence or absence of splenic tissue, but rather to systemically-induced immunosuppression. While splenectomy performed 6, 9, 12 days after tumor inoculation did not alter the tumor growth, splenectomy performed early (3 days) or late stage (20 days) after tumor cell challenge revealed a retarded neoplastic outgrowth, compared with the sham operated group. These results suggest that splenectomy in very early and late stages of tumor-bearing host may be effective for tumor treatment.     

Bidirectional effects of splenectomy on the growth of syngeneic tumor in mice

The effects of splenectomy on tumor growth following inoculation with a relatively large number of cells (1×10⁷) and a smaller number of cells (5×10⁵) of Meth I tumor were studied. When 1×10⁷ tumor cells were inoculated, tumor growth in splenectomized mice was depressed, while tumor in sham-operated mice grew progressively. On the contrary, when 5×10⁵ tumor cells were inoculated, the tumor take was lower in sham-operated than in splenectomized mice. The spleen cells from mice inoculated with either a large or small number of tumor cells, showed an equally potent cytotoxic activity, but no detectable suppressor cell activity. On the other hand, the activity of immunosuppressive factor was detected in sera from mice inoculated with 1×10⁷ tumor cells, but not in those given 5×10⁵ cells. The effect of splenectomy on tumor growth is, thus, bidirectional, depending on the dose of tumor cells inoculated.     

Adverse effect of splenectomy on growth and survival with murine lymphosarcoma

Recent reports suggest that splenectomy may improve host resistance and inhibit solid tumor growth. The effect of splenectomy on lymphoid tumors in less clear. This study evaluates and compares the effect of splenectomy on tumor growth, therapy and survival in murine lymphosarcoma and mammary tumor. Gardner lymphosarcoma (5 x 10(5) cells) was implanted subcutaneously into 400 6C3HED mice (20 g). Two hundred mice underwent splenectomy 10 days previously. Animals were randomly placed into four groups. Group I (tumor alone) and Group II (splenectomy and tumor) received no further therapy. Group III (tumor) and Group IV (splenectomy and tumor) received cyclophosphamide (50 mg/kg/day x 3 days) beginning 10 days after implantation. The rate of implantation was similar in all groups (greater than 90%). Tumor growth was localized in controls, but was widespread in splenectomized mice. Survival analysis at 30 days showed an increased mortality in untreated mice (Group II) following splenectomy (p less than .02). Survival was (13/102) 12.75% Group I versus (8/103) 7.77% Group II. Survival was similar in mice receiving chemotherapy (36%) and was (p less than .001) greater than the untreated groups (12%). A similar protocol in mice with mammary tumor showed no differences between groups in tumor localization or survival postsplenectomy. These data suggest that splenectomy adversely affects localization and survival in murine lymphosarcoma but not in solid tumor. The variable effect of this operation on the natural history of lymphoid versus solid neoplasia questions the advisability of splenectomy in staging of patients with lymphosarcoma.     

Antitumor activity of interleukin-12 against murine bladder cancer

PURPOSE: We investigated the antitumor activity of interleukin-12 (IL-12) against MBT-2, a murine bladder carcinoma, to clarify whether or not IL-12 is effective against urothelial tumors. MATERIALS AND METHODS: MBT-2, a murine carcinogen-induced, poorly differentiated transitional cell carcinoma of C3H/He origin, was used. Three or 10 days after the subcutaneous administration of MBT-2 cells, C3H/He mice were injected intraperitoneally with IL-12 five times per wk. for 2 wk. Tumor growth was measured twice weekly. Spleen cells from the C3H/He mice that had rejected MBT-2 after the IL-12 treatment were examined for MBT-2-specific cytolytic T lymphocytes (CTL) activity and cytokine production. RESULTS: Tumor growth and acceptance was obviously suppressed when C3H/He mice were treated with IL-12 from 3 days after the tumor inoculation. In the spleen cells from the C3H/He mice that had rejected MBT-2, MBT-2-specific CTL activity and secretion of IL-2 and interferon (IFN)-gamma were clearly detected. However, the established MBT-2 tumor cells were not rejected when C3H/He mice were given IL-12 from 10 days after the tumor inoculation, although the tumor growth was transiently suppressed during the IL-12 treatment. CONCLUSION: These data demonstrate that IL-12 is considerably effective against murine bladder cancer and suggest the clinical application of IL-12 against human bladder cancer.     

Experimental model of liver metastases of the mouse]

3 syngeneic tumor-mouse systems were used to establish experimental model of liver metastasis. Only one system, SC42-DS, was suitable for the purpose. When 2 x 10(5) SC42 tumor cells was injected into the spleen of DS mouse and splenectomy was done after 24 hours preventing of spleen tumor due to implantation, multiple nodular liver metastasis were observed in all mice after 14 days. The numbers of nodulus were well correlated with the liver weight.     

脾切除对心脏移植大鼠外周血淋巴细胞凋亡及调节性T淋巴细胞的影响

目的 探讨脾切除对同种异体心脏移植大鼠外周血淋巴细胞凋亡及调节性T淋巴细胞的影响.方法 以Wistar大鼠为供者、SD大鼠为受者,进行腹部异位心脏移植,同时切除受者的脾脏(心脏移植切脾组),并以不切脾者为对照(心脏移植对照组),另设不行任何处理的对照组和单纯切脾的单纯切脾组.术后第1、3、5、7天.取各组受者的移植心脏和外周血,观察移植心脏的组织学变化和细胞超微结构改变情况,以流式细胞仪检测外周血淋巴细胞的凋亡率及CD4+ CD25+ T淋巴细胞的变化,逆转录聚合酶链反应检测CD4+ CD25+ T淋巴细胞上Foxp3 mRNA的表达情况,记录移植心脏的存活时间.结果 心脏移植对照组移植心脏存活时间为(7.47±2.24)d,心脏移植切脾组移植心脏存活时间为(17.63±4.54)d,二者间的差异有统计学意义(P<0.05).心脏移植对照组的移植心脏肿胀,质硬,色暗,间质水肿、出血,弥漫性炎症细胞浸润,大量心肌细胞坏死、溶解,横纹不清;心脏移植切脾组的移植心脏质软,色红,局部灰白,外膜下以及细胞间局灶性水肿,炎症细胞浸润,心肌细胞结构完整,横纹清晰;心脏移植切脾组的细胞超微结构改变轻于心脏移植对照组.心脏移植切脾组术后第5天和第7天的淋巴细胞凋亡率分别为(7.62±2.15)%和(9.41±3.82)%,明显高于心脏移植对照组(P<0.05,P<0.05).心脏移植切脾组术后第3、5、7天时的CD4+ CD25+ T淋巴细胞明显多于心脏移植对照组(P<0.01,P<0.01,P<0.01),其Foxp3 mRNA的表达也较心脏移植对照组明显上调.结论 脾切除使心脏移植大鼠外周血淋巴细胞凋亡率增加,调节性T淋巴细胞增多,其Foxp3 mRNA表达上调,这些变化与移植心脏病理改变呈负相关.     

Transition from T cell protection to T cell enhancement during tumor growth in an allogeneic host.

The cell-mediated immune response of mice toward a lethal allogeneic tumor was investigated during tumor development. The activity of spleen cells from the tumor-bearing mice was studied by transferring them together with 3LL tumor cells into normal C3H/eb recipient mice. The activity depended upon the time interval between inoculation of the tumor and transfer. Spleen cells taken relatively early, 1 week after tumor inoculation, mediated protection against tumor growth. In contrast, spleen cells taken 4 weeks after tumor inoculation markedly enhanced tumor growth. The tumor-enhancing cells, like the tumor-protecting cells, appeared to be T lymphocytes. The enhancing activity could be transferred by extra cellular medium prepared by incubating the enhancing T cells. Protecting activity could not be transferred by cell-free medium prepared from the protecting T cells. Both activities were found to exist to a relatively slight degree in populations of spleen cells from normal mice. The transition from T cell protection to T cell enhancement might be a determining factor in the outcome of the host-tumor relationship.     

Significance of splenectomy in the treatment of cancer

With inoculation of a large amount of tumor cells, the tumor growth of splenectomized mice was depressed compared to sham operated mice. On the contrary, with inoculation of a small amount of tumor cells the occurrence of tumor was lower in sham-operated mice. The effect of splenectomy on tumor growth was bidirectional depending on the dose of the inoculate. The effect was due to the production of the immunosuppressive factor in sera obtained from mice inoculated with a large but not small amount of tumor cells. Studies for the late survivals of 113 patients who had received curative total gastrectomy with or without splenectomy revealed that the non-splenectomized group showed a significantly better late survival rate than the splenectomized group when the splenic hilar lymph nodes were not involved with cancer metastasis.