Tumor-infiltrating lymphocytes and hepatocellular carcinoma: pathology and clinical management

The presence of tumor-infiltrating lymphocytes (TILs) in hepatocellular carcinoma (HCC) is relatively rare. The prognosis of patients with HCC and marked TILs is better than that of patients with HCC without TILs. TILs in HCC tissues are mainly T cells, and previous reports suggested that TILs might be important antitumor effector cells. TILs have been extensively analyzed, and subpopulations of CD3(+), CD4(+), and CD8(+) T cells are often present in HCC. Some studies have reported that the percentage of CD8(+) T cells, which might have cytotoxic activity, is decreased in tumors with TILs, as compared with noncancerous tissues. Although the antitumor effects of TILs seem to be impaired in HCCs, the underlying mechanism has remained unclear until quite recently. Pathological and in vitro studies have now shown that regulatory T cells play important roles in the deterioration of the antitumor effects of TILs. The aim of this review is to introduce recent pathological findings for TILs in HCC and to evaluate new therapeutic strategies in this field.     

乳腺癌微环境中T细胞与临床病理因素的相关性

目的:探讨乳腺癌中间质肿瘤浸润淋巴细胞（TILs）包括CD8+TILs、CD4+TILs、三维淋巴结构（TLS）与临床病理因素和分子亚型的相关性。方法:收集我院2015年至2017年乳腺癌标本200例,经常规石蜡包埋制片,判读HE染色切片间质TLS个数,采用免疫组织化学SP法检测200例病例中CD8+TILs和CD4+TILs浸润密度。结果:乳腺癌间质CD8+TILs、CD4+TILs、TLS与SBR分级、病理TNM分期、Ki67、p53呈正相关;CD8+TILs和CD4+TILs与淋巴结转移呈正相关;与患者年龄呈负相关（均P＜0.05）。CD8+TILs和CD4+TILs呈正相关（P＜0.05）。TLS以CD4+TILs为主,分别与CD8+TILs和CD4+TILs呈正相关（均P＜0.05）。在乳腺癌不同分子亚型中CD8+TILs、CD4+TILs和TLS存在显著差异。结论:乳腺癌中浸润的CD8+TILs、CD4+TILs、TLS反映局部肿瘤微环境的免疫状态,三者密切相关。乳腺癌组织中增多的CD4+TILs诱导CD8+TILs产生CD8+Treg细胞参与乳腺癌的免疫抑制,导致乳腺癌局部免疫功能下降,促进乳腺癌进展及转移。结合乳腺癌分子亚型和局部免疫状态的分析将更有利于预测乳腺癌进展的生物学潜能和对分子靶向性疗效和预后的评估。     

CD8+ lymphocyte infiltration and apoptosis in hepatocellular carcinoma.

AIMS: Tumour-infiltrating lymphocytes (TILs) play a role in local anti-tumour immunity. Tumour cells may escape from immune surveillance by expressing RCAS1, a receptor-binding cancer antigen expressed on SiSo cells, which inhibits T cell growth. In this study, the correlation between the density of CD8+ TILs, tumour cell apoptosis, and tumour RCAS1 expression was investigated in hepatocellular carcinoma (HCC). METHODS: We obtained tissues from 60 patients with surgically resected HCCs. CD8+ TILS, apoptotic cancer cells, and RCAS1 expressing cancer cells were identified by immunohistochemistry. RESULTS: The density of CD8+ T cells in tumours (mean: 9.5/HPF, HPF: high power field) was significantly less than in non-cancerous hepatic lobules (17.8/HPF, p<0.001) and in relation to the progression of tumour stage. The density of CD8+ T cells in tumours positively correlated with the occurrence of tumour cell apoptosis, but did not correlate with RCAS1 protein expression. CONCLUSIONS: CD8+ TILs may play a role in the occurrence of tumour cell apoptosis in HCC, but CD8+ TILs may not be controlled by RCAS1 in HCC.     

Suppression of hepatocellular carcinoma growth via oral immune regulation towards tumor-associated antigens is associated with increased NKT and CD8+ lymphocytes

BACKGROUND: Oral immune regulation towards viral proteins was previously shown to modulate the anti-HBV immune response. Adoptive transfer of orally immunomodulated lymphocytes suppressed the growth of hepatocellular carcinoma (HCC) expressing HBsAg in athymic mice. NKT lymphocytes play a role in the defense against tumor growth. AIM: To evaluate the effect of oral immune regulation towards HCC-associated antigens or HBV proteins on growth of HBsAg-expressing HCC, and to determine the role of NKT lymphocytes in immune modulation. METHODS: Sublethally irradiated athymic Balb/c mice were injected with 10(7) human hepatoma cells followed 10 days later by transplantation of 2 x 10(6) splenocytes from naive donor mice. Immune modulation was performed via feeding of HCC-extracted proteins or HBV antigens (HBsAg + Pre S1 + Pre S2). The control group was fed with bovine serum albumin (BSA). Mice were followed for survival, tumor volume, and serum alpha-fetoprotein levels. To determine the role of NKT cells in tumor suppression, cytokine expression and FACS analysis for CD4+, CD8+, and NK1.1+ T lymphocyte subsets were performed. RESULTS: Oral immune regulation towards HCC-extracted proteins induced complete tumor suppression in recipient mice. Mortality rates were 0% in HCC-immune-regulated mice, compared with an 80% mortality rate using HBV antigens and a 100% mortality rate in control mice. Oral immune regulation towards HCC prevented weight loss. No visible tumor mass was observed in orally immune-regulated mice as compared with 112 mm(3) in controls. Serum alphaFP levels were 0.9, 378 and 1,358 ng/ml in HCC, HBV immune-regulated and controls, respectively. Immune regulation towards HCC antigens significantly increased the NK1.1+ T lymphocytes/CD4+ and CD8+/CD4+ ratios. IFNgamma production increased two-fold. CONCLUSION: Oral immune regulation towards HCC antigens effectively enhanced the anti-tumor immune response, thus suppressing the growth of HCC in mice. This effect was associated with an increased NKT,CD8+/CD4+ lymphocyte ratio and may be mediated via enhancement of IFNgamma production.     

Inverse correlation between tumoral indoleamine 2,3-dioxygenase expression and tumor-infiltrating lymphocytes in endometrial cancer: its association with disease progression and survival.

PURPOSE: Tumor escape from host immune systems is a crucial mechanism for disease progression. We recently showed that the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) is a prognostic indicator for endometrial cancer. The purpose of the present study was to investigate the relationship between IDO expression and tumor-infiltrating lymphocytes (TIL) or natural killer (NK) cells and to clarify their prognostic effect in endometrial cancer. EXPERIMENTAL DESIGN: Immunohistochemical staining for IDO expression in endometrial cancer tissues (n = 65) was done. Tumor-infiltrating CD3+ and CD8+ lymphocytes, as well as CD57+ NK cells, were counted in serial tissue sections. RESULTS: High IDO expression in tumor cells was found in 32 of 65 cases and was positively correlated with myometrial invasion, nodal metastasis, and lymph-vascular space involvement. We also found a significant correlation between high IDO expression and reduced numbers of CD3+, CD8+, and CD57+ cells infiltrating into both the tumor epithelium and stroma. Patients with high IDO expression, a low number of stromal CD3 (<60), low intraepithelial CD8 (<25), or low stromal CD8 (<40) had significantly impaired progression-free survival. On multivariate analysis, IDO expression and the number of stromal CD3+ TILs were independent prognostic factors for impaired progression-free survival. CONCLUSIONS: Tumoral IDO expression correlated with a reduced number of TILs and NK cells in endometrial cancer, possibly contributing to disease progression and impaired clinical outcome. These findings suggest that targeting IDO to restore host antitumor immunity may be a therapeutic strategy for endometrial cancer.     

Expression of Fas antigen (CD95) in peripheral blood lymphocytes and in liver-infiltrating, cytotoxic lymphocytes in patients with hepatocellular carcinoma

BACKGROUND: Fas-expressing cytotoxic T lymphocytes (CTLs) are important antitumor immune effector cells in patients with hepatocellular carcinoma (HCC). The role of transforming growth factor beta 1 (TGF-beta1) in modulating the expression of Fas by CTLs is not known in HCC. The objectives of this study were to characterize the expression of Fas by CTLs and natural killer (NK) cells among peripheral blood lymphocytes (PBLs) and tumor-infiltrating lymphocytes (TILs) in patients with HCC and to correlate the association, if any, with serum TGF-beta1 levels. METHODS: PBLs from 18 patients with HCC and TILs from 5 HCC liver specimens were isolated, and Fas expression was analyzed by three-color flow cytometry. The results were compared with results from normal control volunteers (n = 19 individuals). Serum TGF-beta1 levels in patients with HCC were measured by enzyme-linked immunosorbent assay. RESULTS: The median percentage of Fas expression by CD3 positive T cells was significantly higher in patients with HCC compared with normal controls (54.37% vs. 32.03%, respectively; P = 0.0036), and this was attributable solely to Fas expression by CD4 positive PBLs (54.46% vs. 34.90%, respectively; P = 0.0234). In contrast, Fas expression was significantly higher in all the subtypes of TILs (CD3 positive, CD4 positive, CD8 positive, NK cells, and natural T cells) compared with controls (all P values were < 0.001). Tumor size was inversely proportional to the TGF-beta1 levels (correlation coefficient [r] = -0.725; P < 0.0001), which were correlated inversely with Fas expression by CD4 positive PBLs (r = -0.516; P = 0.01). CONCLUSIONS: In patients with HCC, TILs exhibit significantly increased expression of Fas compared with PBLs that may enhance their susceptibility to apoptotic mechanisms. Larger tumors were associated with lower serum TGFbeta1 levels, and this was correlated with greater Fas expression by CD4 positive PBLs.     

肝癌微环境中CD4+CD25+Treg细胞与肿瘤免疫细胞的关系

[目的]探讨肝癌微环境中Treg细胞的数量增多与HCC临床分期晚的可能原因。[方法]双重酶标免疫组织化学方法检测52例HCC组织中CD4^＋CD25＋Treg细胞以及CD4^＋CD25-T（CD4^＋T）细胞分布，免疫组化EnVision法检测20例HCC组织中CD8＋T细胞分布。[结果]正常肝脏组织中未发现Treg细胞。HCC组织中Treg细胞数量较癌旁组织明显增多（P=0．000），且肝癌组织中Treg细胞的数量与其浸润性CD4＋T淋巴细胞的数量以及CD4＋T/CD8^＋T比值呈显著负相关（r=-0．539，P=0．014；r=-0．545，P=0．000），而与浸润性CD8^＋T淋巴细胞的数量无明显相关性（f=-0．403，P=0．078）。[结论]Treg细胞在体内可能通过细胞接触的方式抑制CD8^＋T淋巴细胞的增殖来抑制肿瘤局部的免疫，去除或减少HCC微环境中浸润件Tree细胞数量可能提高肿瘤局部免疫治疗效果。     

肝癌小鼠脾脏免疫细胞的变化及其意义

目的:观察小鼠H22细胞原位种植性肝癌模型中,脾脏正性免疫细胞和负性免疫细胞比例的变化,并且观察脾脏切除后外周血和肿瘤组织免疫细胞比例的变化以及对肿瘤生长的影响。方法:建立小鼠H22细胞原位种植性肝癌模型,在荷瘤的不同时期,用流式细胞术检测脾脏中负性免疫细胞（MDSC、Treg）和正性免疫细胞（总CD3+T细胞、CD4+T细胞、CD8+T细胞、NK细胞、NKT细胞）比例的变化。荷瘤1 w后切除脾脏,用流式细胞术检测外周血及肿瘤组织中免疫细胞比例的变化,并比较切脾前后肿瘤重量、腹水量、荷瘤小鼠生存期的变化。 结果:荷瘤小鼠脾脏MDSC细胞的比例一直高于正常组;Treg细胞在荷瘤2 w时显著升高;总CD3+T细胞和CD4+T细胞在荷瘤1 w时显著升高,2 w时显著下降;CD8+T细胞在荷瘤2 w时明显下降;NK细胞在荷瘤3 w时明显下降;NKT细胞无显著变化。脾脏切除后外周血和肿瘤组织MDSC的比例下降,CD8+T细胞的比例升高;肿瘤重量和荷瘤小鼠生存期无显著变化,腹水量在荷瘤2 w时显著减少,腹水发生率明显降低。 结论:小鼠肝脏种植H22细胞株后,脾脏中正性免疫细胞的比例下降,负性免疫细胞的比例升高,脾脏的负性免疫状态逐渐占主导地位,从而促进了肝癌的发展。     

CD3+CD56+NKT细胞及CD3-CD56+NK细胞在恶性肿瘤患者的临床意义研究

背景与目的细胞毒性淋巴细胞在抗肿瘤免疫效应中发挥着重要作用,CD3 CD56 NKT细胞作为一类新的具有细胞毒性的效应细胞,目前关于其抗肿瘤意义的探讨主要集中于血液系统恶性疾病,而在实体肿瘤中的应用和临床价值研究尚少。本研究旨在初步探讨抗肿瘤细胞CD3 CD56 NKT细胞及CD3-CD56 NK细胞在恶性肿瘤患者外周血的表达状态及其临床意义。方法采用流式细胞术分析118例恶性肿瘤患者(55例肺癌患者和63例乳腺癌患者)及46例健康对照组外周血中的T细胞亚群及CD3 CD56 NKT细胞、CD3-CD56 NK细胞表达。结果恶性肿瘤患者组CD3 CD8 T细胞、CD3 CD56 NKT细胞以及CD3-CD56 NK细胞表达率均明显高于健康对照组(P<0.01)。肺癌患者中以CD3 CD8 T细胞和CD3 CD56 NKT细胞明显增加为主;乳腺癌患者中以CD3 CD56 NKT细胞和CD3-CD56 NK细胞明显增加为主。结论CD3 CD56 NKT细胞在肺癌和乳腺癌患者的抗肿瘤效应中占据重要地位,而CD3 CD8 CTL和CD3-CD56 NK细胞在不同类型肿瘤患者中具有不同的重要性。     

调节性T细胞与肿瘤

调节性T（regulatory T, Treg）细胞是一群具有抑制其他免疫细胞功能的负调控细胞，包括CD4+ Treg、CD8+ Treg、NKT Treg 和双阴性（double negative，DN）Treg细胞等四大类。研究显示，肿瘤微环境中Treg细胞数量升高，且这些升高的 Treg细胞能抑制抗肿瘤免疫、降低肿瘤免     

Peripheral blood and tumor infiltrating lymphocytes in non-small cell lung cancer: analysis at the population and clonal level

Tumor infiltrating (TIL) and peripheral blood lymphocytes (PBL) were isolated from 18 patients with non-small cell lung cancer undergoing radical surgery. Surface marker analysis revealed that TILs and PBLs mainly consisted of CD3+ T cells and that TILs generally displayed a lower CD4/CD8 ratio. Differences were found in the expression of CD25 (IL-2 receptor) and DR (MHC class II) antigens, which were increased in TILs, and in the percentage of CD16+ natural killer (NK) cells, which was reduced in TILs as compared to PBLs. Accordingly, the NK activity of TILs was lower than that of PBLs, whereas neither TILs nor PBLs expressed spontaneous cytolytic activity against fresh autologous tumor cells, melanoma cells and the "NK-resistant" A549 lung carcinoma cell line. After 4 days of culture in medium with recombinant-interleukin-2 (rIL-2), TILs and PBLs acquired cytolytic activity against all cell targets, but TILs expressed higher levels of cytotoxicity than autologous PBLs only in 3 patients out of 16 tested. More importantly, both TILs and PBLs displayed similar levels of cytotoxic activity against autologous tumor cells. TILs and PBLs from 8 patients were also analyzed by a limiting dilution microculture system. Cloning efficiency was remarkably lower in TILs, and surface marker analysis of T cell clones confirmed that an accumulation of CD8+ lymphocytes, which displayed cytolytic activity in a lectin-dependent assay, occurred at the tumor site. The non-MHC-restricted cytolytic activity of TIL- and PBL-derived T cell clones against K562, A549, and allogeneic melanoma cells and the cytolytic activity against autologous tumor cells showed no significant differences. Only 53% of TIL clones released IL-2 in response to PHA + TPA stimulation, whereas 68% of PBL-derived clones were IL-2 producers. Moreover, most PBL- and TIL-derived clones released tumor necrosis factor alpha in response to mitogen stimulation.     

肝细胞性肝癌组织浸润淋巴细胞表型与分布研究

目的:肝细胞性肝癌组织浸润淋巴细胞与外周血T 细胞表型可能与肿瘤进展及预后相关,本研究检测肝癌患者组织及外周血T 细胞表型与分布,分析淋巴细胞表型变化与预后的关系。方法:分析2007年10月至12月中山医院147 例肝癌及癌旁组织浸润淋巴细胞表型（T 细胞或B 细胞表面标志物:CD3、CD8、CD4、CD20、CD19、Foxp 3）,表型与临床病理特征及预后的关系;检测26例肝癌外周血CD3、CD8、CD4 +T细胞数量并其比例变化。结果:癌巢内肿瘤浸润细胞明显少于癌周组织（P < 0.01）,癌周淋巴细胞主要分布于癌旁正常肝组织、汇管区,其与患者肝炎病史及肝硬化相关,表型以CD3 +T细胞为主,其中又以CD8 + 细胞毒性T 细胞为主;CD4 染色在多数病例为阴性,Foxp 3 仅在个别病例（15/ 109）呈阳性。肿瘤浸润淋巴细胞B 细胞标志CD20、CD19均为阴性。肿瘤组织内CD8 +T细胞浸润数量与预后正相关,而癌周浸润淋巴细胞数目与患者转移及复发无显著关系。结论:肝癌肿瘤浸润细胞在癌巢内明显少于癌周组织,肿瘤及癌周浸润细胞以CD8 + 细胞毒性T 细胞为主。肿瘤组织内CD8 +T细胞浸润数量与预后相关,而癌周浸润淋巴细胞数量与患者转移及复发无显著关系。      

原发性肝癌患者外周血和癌组织中调节性T细胞数量及其临床意义

目的 探讨原发性肝癌患者外周血中调节性T细胞(Treg)占CD4+T细胞(Treg/CID4+)比值和肝癌组织中Treg数量及其临床意义.方法 63例原发性肝癌患者均行根治性手术切除,采用流式细胞术检测患者手术前外周血中Treg/CD4+比值,用免疫组织化学方法检测肿瘤组织中Treg的表达.结果 原发性肝癌患者外周血中Treg/CD4+比值显著高于乙型肝炎患者(P<0.01)及正常对照组(P<0.01).癌组织中Treg数量为(15.69±13.29)个/mm2,癌旁肝组织、10例乙型肝炎肝组织、10例正常肝组织中未见或极少见Treg.外周血中Treg/CD4+比值与癌组织中Treg数量呈正相关(P=0.024).外周血和癌组织中Treg数量高者,术后5年生存率低(P值分别为0.042、0.019);癌组织中Treg数量高者,术后5年无瘤生存率低(P=0.001).结论 原发性肝癌患者外周血和组织中调节性T细胞水平升高;外周血中Treg/CD4+比值及癌组织中Treg数量可作为预测患者根治术后预后的免疫学指标.     

髓系来源的抑制性细胞在小鼠H22原位种植性肝癌中的表达及其意义

目的:比较小鼠H22原位种植性肝癌模型中髓系来源的抑制性细胞（MDSCs）和调节性T细胞（Tregs）的表达，确定主要的免疫抑制性细胞。并探讨主要免疫抑制性细胞在小鼠H22原位种植性肝癌模型中的意义。方法:建立小鼠H22原位种植性肝癌模型，用流式细胞术的方法比较外周血、脾脏、肿瘤组织中MDSCs、Tregs的比例。分析MDSCs与CD4+T细胞、CD8+T细胞、NK细胞、NKT细胞的相关性。通过脾脏切除的方法下调MDSCs的表达，观察对肿瘤存活的影响。结果:MDSCs的比例无论在外周血、脾脏还是肿瘤组织中都显著高于Tregs。MDSCs的比例与CD4+T细胞、CD8+T细胞、NK细胞、NKT细胞呈负相关。下调MDSCs的比例可以显著延长小鼠存活期。结论:小鼠H22原位种植性肝癌中主要的免疫抑制性细胞是MDSCs。MDSCs的高表达预示着不良的小鼠存活期。     

CD8+ Foxp3+ regulatory T cells mediate immunosuppression in prostate cancer.

PURPOSE: Although elevated proportions of CD4(+)CD25(+) regulatory T (Treg) cells have been shown in several types of cancers, very little is known about the existence and function of CD8(+) Treg cells in prostate cancer. In this study, we investigated prostate tumor-derived CD8(+) Treg cells and their function. EXPERIMENTAL DESIGN: Tumor-infiltrating lymphocytes (TIL) from fresh tumor specimens of patients with prostate cancer were generated and subjected to phenotypic and suppressive function analyses. In particular, we investigated the role and function CD8(+) Treg cells in prostate cancer. RESULTS: We show that high percentages of CD4(+)CD25(+) T cells are probably present in the majority (70%) of prostate TILs. Remarkably, both CD4(+) and CD8(+) T-cell subpopulations possessed potent suppressive activity. T-cell cloning and fluorescence-activated cell sorting analyses showed the presence of CD8(+)CD25(+) Treg cell clones that expressed FoxP3 and suppressed na?ve T-cell proliferation, in addition to the previously known CD4(+)CD25(+) Treg cells. These CD8(+) Treg cells suppressed na?ve T-cell proliferation mainly through a cell contact-dependent mechanism. Importantly, the suppressive function of CD8(+) Treg cells could be reversed by human Toll-like receptor 8 (TLR8) signaling. CONCLUSION: Our study shows that like CD4(+)CD25(+) Treg cells, CD8(+) Foxp3(+) Treg cells present in prostate tumor-derived TILs suppress immune responses and that their suppressive function can be regulated by TLR8 ligands, raising the possibility that the manipulation of Treg cell function by TLR8 ligands could improve the efficacy of immunotherapy for prostate cancer patients.     

Reversed CD4/CD8 ratios of tumor-infiltrating lymphocytes are correlated with the progression of human cervical carcinoma.

BACKGROUND: To investigate the clinical significance of tumor-infiltrating lymphocytes (TILs) within the tumor milieu of human cervical carcinoma, the authors quantitatively measured and compared the subpopulations of lymphocytes infiltrating the neoplastic cervix. METHODS: A total of 30 patients with Stage Ia-IIa cervical carcinoma were enrolled. TILs were isolated from tissue specimens by means of a mechanical dispersal technique, and the immunocyte subsets were quantified with dual-color flow cytometry. Bulky tumor was defined as tumor size >4 cm in greatest dimension according to the 1995 staging of the International Federation of Gynecology and Obstetrics. RESULTS: The CD4/CD8 ratios of TILs were reversed in both cervical squamous cell carcinoma (n = 20) and cervical adenocarcinoma (n = 10). The proportion of CD4(+) T cells was significantly lower in tumors from patients with lymph node metastasis (n = 8) than in those from patients without lymph node metastasis (n = 22) (24.5 vs. 32.7, P = 0.001), as was the reversed CD4/CD8 ratio (0.50 vs. 0.81, P = 0.001). The proportion of CD4(+) T cells was much lower in bulky tumors (n = 5) than in nonbulky tumors (n = 25) (21.4 vs. 32.5, P < 0.001), reflecting in a more strongly reversed CD4/CD8 ratio (0.41 vs. 0.81, P = 0.001). CONCLUSIONS: Decreased proportions of tumor-infiltrating CD4+ T cells with reversed CD4/CD8 ratios are highly correlated with rapid tumor growth and lymph node metastasis in cervical carcinoma. The regional immune escape is of prognostic importance with regard to cancer progression.     

食管癌患者外周血中自然杀伤性T细胞含量的检测及其临床意义

 　目的　研究食管癌患者外周血中自然杀伤性（NK）T细胞在手术前后的表达情况。方法　采用流式细胞术（FCM）分析59例食管癌患者手术前后外周血中CD3、CD56、CD4、CD8抗体的表达,研究NKT细胞及其亚群的表达情况及所占比例。结果　随着CD+3 CD+56 CD+8／CD+3 CD+56 CD+4的比值逐渐升高,Ⅲ～Ⅳ期食管癌患者的比例逐渐降低,ANOVA示组间差异有统计学意义（P＜0.05）;CD+3 CD+56 CD+8／CD+3 CD+56 CD+4的比值与CD+3 CD+56 CD+4 NKT细胞非线性相关;CD+3 CD+56 CD+8 NKT细胞与NK细胞正相关,与CD+3 T细胞负相关。结论　CD+3 CD+56 CD+8／CD+3 CD+56 CD+4 的比值可能与肿瘤负荷有关,并且有助于判断食管癌患者的疾病程度及预后。     

Antitumor activity of alpha-galactosylceramide, KRN7000, in mice with the melanoma B16 hepatic metastasis and immunohistological study of tumor infiltrating cells

Liver metastasis of primary tumors is clinically a major problem. We examined the antitumor activity of KRN7000, an alpha-galactosylceramide, in mice with liver metastasis of the B16 melanoma. KRN7000 significantly inhibited tumor growth in the liver, and its potency was similar to that of interleukin-12. The KRN7000 administration resulted in a high percentage of cured mice, which acquired tumor-specific immunity. To study what kinds of antitumor effector cells participated in killing tumor cells, we then performed immunohistological analysis of tumor-infiltrating cells, and found that KRN7000 induced marked invasion of NK1.1+ cells, CD8+ cells, and F4/80+ cells (macrophages) into B16 tumor nodules. In addition, it appeared that KRN7000-treated, liver-associated macrophages possessed strong lytic activity against tumor cells. These results suggest that NK cells, NK1.1+ T (NKT) cells, cytotoxic T lymphocytes, and macrophages play an important role in killing tumor cells in the liver, and that KRN7000 may be useful for the treatment of cancer liver metastasis.