Cancer cells regulate lymphocyte recruitment and leukocyte-endothelium interactions in the tumor-draining lymph node

The physiologic function of the secondary lymphoid organs to recruit large numbers of na?ve lymphocytes increases the probability that antigens encounter their rare, sometimes unique, specific T lymphocytes and initiate a specific immune response. In peripheral lymph nodes (LNs), this recruitment is a multistep process, initiated predominantly within the high endothelial venules (HEVs), beginning with rolling and chemokine-dependent firm adhesion of the lymphocytes on the venular endothelium surface. We report here that, in C57BL/6 mice, the recruitment of na?ve lymphocytes is impaired in LNs draining a B16 melanoma tumor. Intravital microscopy analysis of the tumor-draining LNs revealed that this effect is associated with an important defect in lymphocyte adhesion in the HEVs and a progressive decrease in the expression of the LN chemokine CCL21. In parallel with these effects, the tumor up-regulated, essentially through a P-selectin-dependent mechanism, the rolling and sticking of circulating polymorphonuclear cells within the LN low-order venules where few rolling and sticking events are usually observed. These effects of the tumor were independent of the presence of metastasis into the LN and occurred as long as the tumor developed. Together, these results indicate that the tumor proximity disturbs the LN physiology by modifying the molecular, spatial, and cellular rules that usually control leukocyte-endothelium interactions into the peripheral LNs. In addition, they emphasize a new role for the low-order venules of the peripheral LNs, which compared with the HEVs, seem to be the preferential port of entry for cells linked to inflammatory processes.     

Effect of anti-CD3 antibody on the generation of interleukin-2-activated lymphocytes from tumor tissues of gastrointestinal cancer.

BACKGROUND. The efficiency of anti-CD3 antibody (OKT3) for adoptive immunotherapy using lymphokine-activated killer (LAK) cells generated from tumor-infiltrating lymphocytes (TIL), regional lymph node lymphocytes (RLNL), and peripheral blood lymphocytes (PBL) was investigated. METHODS. TIL, RLNL, and PBL derived from 39 patients with gastrointestinal cancers (16 gastric cancers, 17 colorectal cancers, and 6 esophageal cancers) were cultured for 4 weeks with 200 U/ml of recombinant interleukin-2. To one group, solid-phase 10 micrograms/ml OKT3 was added during the initial culture period (day 2 or 4). Cytotoxicity against K562 cells (NK-like activity) and Daudi cells (LAK activity) and the phenotypes of effector cells generated after culturing for 2-3 weeks were studied. RESULTS. Proliferative responses were significantly increased by OKT3 in each type of effector cell (P less than 0.01); in particular, TIL expanded more by OKT3 than PBL and RLNL (P less than 0.01). The population of CD8+ CD11b- cytotoxic T-cells in OKT3-stimulated groups was significantly larger than that in unstimulated groups (P less than 0.01), whereas no differences were observed with CD4+ cells (helper/inducer T-cells) and CD8+ CD11b+ cells (suppressor T-cells). OKT3 enhanced the NK-like activity of TIL and PBL but did not affect their LAK activity. OKT3 suppressed the NK and LAK activity of RLNL. CONCLUSIONS. OKT3 stimulation did not significantly enhance the LAK activity, but the authors propose that OKT3 could be an effective addition to adoptive immunotherapy using TIL due to an increased proliferation and generation of a large cytotoxic T-cell population.     

Susceptibility of chemoresistant murine and human tumor cells to lysis by interleukin 2-activated lymphocytes

The sensitivity of three different human and murine doxorubicin (Dx)-sensitive or -resistant pairs of tumor cells to recombinant interleukin 2 (rIL2)-activated lymphocytes was studied. In two pairs of these sublines (LoVo human colon carcinoma and B16 mouse melanoma sublines), resistance to Dx was induced in vitro, while in the third pair (9229 human metastatic melanoma clones), Dx resistance was spontaneously present in clone 9229.24. Dx-resistant cells were efficiently lysed by rIL2-activated lymphocytes in a short-term 51Cr release assay; in some experiments a trend toward higher lysis of Dx-resistant cells was present. We then tested the tumor cell growth-inhibitory activity of rIL2-activated lymphocytes in the human tumor clonogenic assay after lymphocyte-tumor coculture. Complete inhibition of tumor cell growth was obtained with five of six sublines or clones (both Dx sensitive and resistant) after 3 to 6 days of coculture at effector lymphocyte/target tumor cell ratios of 5 to 50/1; a maximum 99% inhibition was observed with the melanoma clone 9229.4 even after coculture for 6 days at an effector lymphocyte/target tumor cell ratio of 50/1. By using lower effector lymphocyte/target tumor cell coculture ratios (1, 5, 25/1), it was shown that all the three Dx-resistant cell types were significantly more affected by activated lymphocytes than their Dx-sensitive counterparts. The LoVo/DX subline was also more lysed than its Dx-sensitive counterpart LoVo/H subline by an antitumor monoclonal antibody in a complement-mediated cytotoxicity assay, despite the fact that both sublines expressed a similar amount of antigen on the cell surface. These data indicate that Dx-resistant cancer cells are more susceptible to the lysis by rIL2-activated lymphocytes than their Dx-sensitive counterparts and that a complete inhibition of their clonogenic potential can be obtained in vitro.     

Susceptibility of multidrug-resistant human leukemia cell lines to human interleukin 2-activated killer cells.

Considering the possibility to overcome drug resistance by other treatment strategies than chemotherapy we investigated the susceptibility of three independently selected multidrug-resistant sublines of the T-lymphoblastoid leukemic cell line CCRF-CEM to lymphokine-activated killer (LAK) cells. We found that two of the multidrug-resistant sublines were significantly less susceptible targets to LAK cells. A third one, however, was as susceptible as the parental CCRF-CEM cell line. Moreover, a multidrug-resistant subline that reverted to an almost drug-sensitive phenotype was observed to be also revertant for resistance against LAK cells. We found an inverse relationship between the expression of the mdr1 gene (P-glycoprotein) and the susceptibility to LAK cells. Verapamil, a calcium channel blocker, while increasing the drug sensitivity of a multidrug-resistant subline, did not induce a reversal of the suppression of LAK susceptibility. The possibility of enhanced resistance to LAK cells of multidrug-resistant cells should be taken into account when one is looking for therapy strategies to overcome multidrug resistance.     

Phase II trial of autologous tumor vaccination, anti-CD3-activated vaccine-primed lymphocytes, and interleukin-2 in stage IV renal cell cancer.

PURPOSE: Previous preclinical and clinical studies have demonstrated that autologous tumor vaccines can induce relatively specific tumor-reactive T cells in draining lymph nodes. The adoptive transfer of these cells can result in tumor regression. PATIENTS AND METHODS: Patients with stage IV renal cell cancer (RCC) were vaccinated with irradiated autologous tumor cells admixed with Calmette-Guérin bacillus. Approximately 7 days later, vaccine-primed lymph nodes (VPLNs) were harvested and the lymphoid cells secondarily activated with anti-CD3 monoclonal antibody and expanded in interleukin 2 (IL-2). The activated cells were subsequently infused intravenously along with the concomitant administration of bolus IL-2 (360,000 U/kg intravenously x 15 doses). RESULTS: Thirty-nine patients were entered onto the study, of whom 34 completed an initial course of cell therapy consisting of a mean (SEM) number of 4.3 (2.2) x 10(10) VPLN cells. Among subjects who received cell therapy, there were nine responses (four complete responses [CRs] and five partial responses [PRs]), for an overall response rate of 27%. The durations of the CRs were > 48, 45, > 35, and 12 months, and the durations of the PRs were > 63, 48, 15, 12, and 4 months. Cultured tumor cells were available to assess in vitro cytokine release of VPLN cells in 24 subjects. The median cytokine release ratio of interferon gamma (IFNgamma) to IL-10 for responders and nonresponders was 992 and 5, respectively, which was significantly different (P =.047). CONCLUSION: The treatment protocol resulted in durable tumor responses in patients with advanced RCC. The ratio of IFNgamma and IL-10 cytokines released in response to tumor by the VPLN cells was a significant correlate with tumor response.     

Lymphokine-activated killer activity in long-term cultures with anti-CD3 plus interleukin 2: identification and isolation of effector subsets

Peripheral blood lymphocytes cultured in recombinant interleukin 2 during 3 to 5 days (short-term cultures) develop the ability to lyse natural killer-resistant tumor lines and fresh tumor cells, i.e., express lymphokine-activated killer (LAK) function. Phenotypic analysis has shown these cells to be natural killer cells, i.e., CD16+ and/or Leu 19+ cells. CD3+,CD16- T-cells, instead, develop very low LAK function in these cultures. We recently reported the development of long-term (up to 21 days) cultured cells with LAK activity by stimulation with OKT3 + interleukin 2(IL2). These culture conditions repeatedly resulted in a several hundred-fold expansion in cell number. Specific LAK activity on Day 14 of culture was comparable to that of 3-day LAK cultures and could be further enhanced by the addition of interleukin 1 beta, beta-, or gamma-interferon. Total LAK activity was greatly increased in OKT3 + IL2 cultures over that found in short-term cultures. Isolation of effectors mediating LAK function in long-term cultures stimulated with OKT3 + IL2 showed that both CD3+,CD16- cells and CD16+,CD3- cells tested on Day 14 of culture expressed equivalent levels of LAK activity as shown by lysis of natural killer-resistant targets, HL60 and Daudi. Further dissection of the subpopulations developing LAK activity demonstrated that, in addition to CD16+,CD3- cells, CD3+, CD4-,CD8- cells and Leu 19+,CD3-,CD16- cells also developed high LAK activity in long-term cultures with OKT3 + IL2. Further, long-term culture with OKT3 + IL2 induced increases in the numbers not only of CD3+,CD4-,CD8- cells but also of CD16+,CD3- and Leu 19+,CD3-,CD16- cells. Although there is a significant increase in the number of CD3+,CD8+ cells, neither these, nor the CD3+,CD4+ cells, mediate LAK activity to the same extent as the populations mentioned above.     

Nitric oxide is an effector molecule in inhibition of tumor cell growth by rIFN-γ-activated rat neutrophils

This study was designed to determine the effector molecule responsible for the tumor-inhibitory activity of rat neutrophils treated with rat recombinant interferon gamma (rIFN-γ) in vitro. The results show that nitric oxide (NO) production by neutrophils is dependent on rIFN-γ concentration, and that neutrophil-mediated tumor cytostasis is in turn dependent on the amount of NO. NO production and tumor cytostatis by rIFN-γ-activated neutrophils were inhibited completely by N^G monomethyl-L-arginine (N^GMMA), a specific competitive NO production inhibitor. Tumor cytostasis was also inhibited by oxyhemoglobin (HbO₂), an NO scavenger. An extracellular oxygen radical scavenger, superoxide dismutase (SOD), was found to increase tumor cell inhibition by rIFN-γ-activated neutrophils by a factor of 4. This SOD-enhanced cytostasis was not even inhibited by catalase. Tumor cytostasis was slightly increased by a hydroxyl radical-(-OH) scavanger, dimethylthiourea (DMTU), which did not affect NO production by rIFN-γ-activated neutrophils. Our findings suggest that tumor cytostasis of neutrophils activated by rIFN-γ is mediated by L-arginine-derived nitrogen oxidation products, and that O₂⁻ produced by these neutrophils reduces NO-mediated tumor cytostasis at low NO concentrations. Int. J. Cancer 71:223-230, 1997. © 1997 Wiley-Liss, Inc.     

淋巴结转移瘤细胞与原发灶瘤细胞的药物敏感性相关性分析

目的：研究淋巴结转移的肿瘤细胞与原发灶的肿瘤细胞是否具有一致的化疗药敏感谱。方法：比较20例非小细胞肺癌患者的原发灶与淋巴结转移灶的肿瘤细胞对5-氟尿嘧啶,丝裂霉素,顺铂,氨甲喋呤,紫杉醇等五种化疗药物的敏感性。结果：患者对各化疗药的敏感性差异很大。但是同一患者的淋巴结转移瘤细胞与原发灶瘤细胞的化疗药敏感谱相似,淋巴结转移瘤细胞与原发灶瘤细胞对顺铂的敏感性高度相关（r=0.73）。结论：淋巴结转移瘤细胞可以替代原发灶瘤细胞进行药敏测试,这对于原发灶不明或者取样困难的病例具有重要意义。     

淋巴结转移瘤细胞与原发灶瘤细胞的药物敏感性相关性分析

目的:研究淋巴结转移的肿瘤细胞与原发灶的肿瘤细胞是否具有一致的化疗药敏感谱。方法:比较20例非小细胞肺癌患者的原发灶与淋巴结转移灶的肿瘤细胞对5-氟尿嘧啶,丝裂霉素,顺铂,氨甲喋呤,紫杉醇等五种化疗药物的敏感性。结果:患者对各化疗药的敏感性差异很大。但是同一患者的淋巴结转移瘤细胞与原发灶瘤细胞的化疗药敏感谱相似,淋巴结转移瘤细胞与原发灶瘤细胞对顺铂的敏感性高度相关(r=0.73)。结论:淋巴结转移瘤细胞可以替代原发灶瘤细胞进行药敏测试,这对于原发灶不明或者取样困难的病例具有重要意义。     

Cyclooxygenase-2 promotes tumor lymphangiogenesis and lymph node metastasis in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer and frequently metastasizes to the cervical lymph nodes, leading to poor survival of patients with OSCC. However, the mechanism of lymph node metastasis is not fully understood. To clarify the molecular mechanism underlying OSCC metastasis to regional lymph nodes, the highly metastatic fluorescent labeled OSCC cell line SAS-LM3 was successfully established allowing us to monitor the progression of lymph node metastases in a non-invasive manner. SAS-LM3 tumors showed increased lymphangiogenesis and elevated expression of VEGF-C, a potent stimulator of lymphangiogenesis, compared to parental SAS tumors. SAS-LM3 showed high expression of cyclooxygenase-2 (COX-2) compared to parental SAS cells and immunohistochemical analysis demonstrated intense COX-2 expression at the primary site. Inactivation of COX-2 by knockdown or the COX-2 inhibitor NS-398 decreased VEGF-C expression. Administration of COX-2 inhibitor NS-398 in SAS-LM3 tumor-bearing mice suppressed tumor lymphangiogenesis and lymphatic metastases. Collectively, our results indicate that COX-2 promotes tumor lymphangiogenesis and lymph node metastasis of OSCC. COX-2 ablation holds promise as a potential therapeutic approach for lymph node metastasis in OSCC.     

Studies concerning the regional lymph node in cancer. VIII. Effect of two asynchronous tumor foci on lymph node cell cytotoxicity.

Results suggest that cytotoxicity by cells from nodes regional to a primary tumor is unique. While a primary tumor was present, A) cytotoxicity was displayed by cells from lymph nodes (RLNs) to that tumor, B) cells from nonregional lymph nodes regional (NRLNs) possessed lesser cytotoxicity which failed to increase in response to a second tumor focus in an area drained by those nodes, and C) the second focus attenuated cytotoxicity of cells from LNs regional to the primary tumor. Following removal of the primary tumor, cells from RLNs rapidly lost cytotoxicity and with passage of time were unable to regain that function in response to a second tumor focus. In contrast, cells from NRLNs demonstrated increased cytotoxicity at any time following removal of the primary tumor when exposed to a second focus. These observations suggest that nodes regional to a distant metastatic focus may be unable to react to it and thus contribute little to the host response generated by the primary tumor. In addition, since nodes regional to a primary tumor manifest diminished cytotoxicity in the presence of a distant tumor focus, tumor cells gaining access and lodging in those nodes subsequent to the development of other metastatic foci are likely to proliferate, resulting in the "positive" lymph node. The findings are in keeping with our contention that host factors responsible for metastases, and perhaps metastases themselves, are at least in part responsible for growth of tumor in RLNs. They also have relevance to the site of administration of specific immunotherapeutic agents and to the significance of the removal of RLNs with a primary tumor.     

Bispecific antibody-directed antitumor activity of human CD4+ helper/killer T cells induced by anti-CD3 monoclonal antibody plus interleukin 2.

Freshly isolated human CD4+ T cells can not respond to recombinant interleukin 2 (rIL-2) because of their lack of p75 IL-2 receptor expression. However, we succeeded in inducing a marked proliferation of purified CD4+ T cells by activation with rIL-2 plus anti-CD3 monoclonal antibody (mAb) cross-linked to a plastic plate. The proliferated CD4+ T cells produced a significant amount of IL-2 upon stimulation with phorbol ester plus A23187. Interestingly, CD4+ T cells activated with anti-CD3 mAb plus rIL-2 revealed a strong cytotoxic activity against Fc receptor (FcR)-positive tumor cells in the presence of anti-CD3 mAb. Moreover, the CD4+ T cells could lyse FcR-negative glioma cells by targeting with bispecific mAb containing anti-CD3 mAb and anti-glioma mAb. Thus, we demonstrated that rIL-2 and immobilized anti-CD3 mAb allowed the rapid generation of human CD4+ helper/killer T cells, which may be useful for the development of a new adoptive tumor immunotherapy.     

Paraaortic lymph node micrometastasis and tumor cell microinvolvement in advanced gastric carcinoma

Background. Paraaortic lymph node dissection in advanced gastric carcinoma is controversial. The purpose of this study was to investigate the incidence and significance of micrometastasis (MM) or tumor cell microinvolvement (TCM) in these critical lymph nodes. Methods. A total of 2339 lymph nodes, including 390 paraaortic nodes, obtained from 47 patients with advanced gastric carcinoma were examined immunohistochemically, using cytokeratin antibody. Results. Lymph node metastasis was found in 95 of the 390 paraaortic nodes of 14 patients by routine histological examination. MM or TCM was immunohistochemically detected in 45 of the 295 negative paraaortic lymph nodes from 15 of 33 patients (MM, n = 5; TCM, n = 10). The 5-year-survival rate in the paraaortic node-negative group and cytokeratin-positive group was significantly higher that that of the hematoxilin and eosin-positive group. The total number of lymph node metastases by hematoxylin and eosin staining and the pathological lymph node compartments, by cytokeratin-positive nodes, were prognostic factors by multivariate analysis. Conclusions. We demonstrated a high rate of MM or TCM in the paraaortic lymph nodes and suggest that such harbored metastases are related to the prognosis of patients with advanced gastric carcinoma. On the basis of this study, a multi-institutional study should be considered. Received for publication on June 7, 1999; accepted on Sept. 30, 1999     

Spontaneous regression of cervical lymph node metastasis in a patient with mesopharyngeal squamous cell carcinoma of the tongue: possible association between apoptosis and tumor regression

Background We report a case of mesopharyngeal squamous cell carcinoma with spontaneous regression of lymph node metastasis. Spontaneous regression of lymph node metastasis of head and neck carcinoma has not been reported previously. Possible causes of the regression of lymph node metastasis include regression of lymphocytic division transiently inflated by an immunological stimulus, and en-bloc tumor necrosis due to degradation of vascularity, such as thromboembolism and intranodal hemorrhage. However, the patient's history and repeated imaging analyses suggested that these factors were not responsible for the regression. To clarify the etiology of this rare phenomenon, we investigated the cause of spontaneous regression with analyses of paraffin-embedded sections. Methods The frequency of cystic lesions, en-bloc necrotic lesions, and apoptosis of carcinoma were investigated with immunohistochemical analysis, and these features were compared with those in specimens from five other patients with head and neck squamous cell carcinoma. Results The present case revealed no tendency towards microscopically confirmed cystic formation or necrosis, but the frequency of apoptosis was significantly higher than that in the other five cases. The apoptotic tendency was not restricted to the lymph node in which spontaneous regression was confirmed clinically, but was also consistently observed in other lymph nodes and in the primary lesion that was detected and radically ablated 2 months after complete neck regional dissection had been done. Conclusion Our case may be the first case of squamous cell carcinoma undergoing spontaneous regression in which enhanced apoptosis was demonstrated quantitatively. The findings were considered to contribute to evidence of spontaneous regression in squamous cell carcinoma of the head and neck resulting from enhanced apoptosis.     

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。