Cetuximab strongly enhances immune cell infiltration into liver metastatic sites in colorectal cancer

Cetuximab has activity against colorectal cancers. Recent studies demonstrated that cetuximab induces antibody‐dependent cell‐mediated cytotoxicity via immune cells, and a new immune‐related mechanism of inducing immunogenic cell death. This study aimed to evaluate the immune responses induced by cetuximab in tumor microenvironments at liver metastasis sites of metastatic colorectal cancer patients. We assessed immune cell infiltration in the liver metastatic sites of 53 colorectal cancer patients. These patients were divided into three groups according to the treatment before operation: chemotherapy with cetuximab, chemotherapy without cetuximab, and no chemotherapy. The inflammatory cells in the liver metastatic sites were assessed by hematoxylin–eosin staining, focusing on the invasive margin. The overall inflammatory reaction and number of lymphoid cells were assessed with a four‐point scoring system. We then assessed immune cell infiltration (CD3, CD8 and CD56) in 15 liver metastatic sites. Hematoxylin–eosin staining demonstrated more inflammatory cells in the chemotherapy with cetuximab group than in the other groups (P < 0.001). Of note, inflammatory cells were found in intratumoral areas, and the destruction of cancer cell foci was observed in the chemotherapy with cetuximab group. Moreover, a higher infiltration of CD3+ (P = 0.003), CD8+ (P = 0.003) and CD56+ (P = 0.001) cells was observed in the chemotherapy with cetuximab group than in the other groups. These results suggest that cetuximab might have an immune‐enhancing effect. As such, the immune‐related mechanism of action of cetuximab may enhance the efficacy of combination therapy, such as chemotherapy and immunotherapy using therapeutic peptides.     

The immune system as anti-tumor sentinel: molecular requirements for an anti-tumor immune response.

The concept behind immune surveillance against cancer is that tumor cells continuously develop, but that there may not be clinical evidence of their presence because the immune system recognizes the cells as foreign and destroys them. A clear role for the immune system in preventing and/or eliminating tumors is emerging as insights into the molecular requirements for the induction and effector function of cytolytic T lymphocytes (CTL) have been gained. Using murine tumor rejection models, the role of particular molecular components of the immune system in controlling tumor growth has been defined. However, tumor rejection does not always occur spontaneously in vivo, indicating that defects in the generation or execution of an anti-tumor immune response may be common. Understanding defects when they arise should allow for development of new therapeutic approaches in tumor-bearing individuals. Many clinical studies are underway to test strategies to induce or heighten an antitumor immune response in cancer patients.     

HLA-G modulates immune responses by diverse receptor interactions

HLA-G regulates immune responses as it binds different receptors expressed on natural killer (NK) cells, T cells and myeloid cells. HLA-G1 can inhibit NK- and T-cell-mediated lysis of target cells by its interaction with the inhibitory receptors ILT2 and ILT4. Engaging KIR2DL4 triggers different reactions depending on the activation state of the effector cells. The indirect recognition of HLA-G as peptide presented by HLA-E and recognized by the CD94/NKG2 receptor family might further power the battle between the immune system and tumor cells. Secreted HLA-G5 can also bind CD8 and induces Fas/Fas ligand-mediated apoptosis in activated CD8+ lymphocytes.     

射频消融术联合经皮椎体后凸成形术对脊柱转移性肿瘤患者疼痛及疗效的影响研究

目的探讨经皮椎体后凸成形术(PKP)联合射频消融术(RFA)对脊柱转移性肿瘤患者疼痛及疗效的影响。方法回顾性分析227例(共405节椎体)脊柱转移性肿瘤患者的病历资料,其中联合组(采用PKP联合RFA治疗)患者164例,245节椎体,PKP组(单独采用PKP治疗)患者63例,160节椎体。比较两组患者疼痛缓解情况、Oswestry功能障碍指数、视觉模拟评分及影像学改变情况。结果两组患者术前视觉模拟评分比较,差异无统计学意义(P﹥0.05);术后1天、术后5个月,联合组患者视觉模拟评分均低于PKP组,差异均有统计学意义(P﹤0.05)。术后1周、术后2个月,联合组患者疼痛缓解有效率均高于PKP组,差异均有统计学意义(P﹤0.05);术后5个月,联合组患者疼痛缓解有效率明显高于PKP组,差异有统计学意义(P﹤0.01)。两组患者术前Oswes-try功能障碍指数比较,差异无统计学意义(P﹥0.05);术后3天、1周、2个月、5个月,联合组患者Oswestry功能障碍指数均明显低于PKP组,差异均有统计学意义(P﹤0.01)。术前两组患者椎体前缘、椎体中部高度比较,差异均无统计学意义(P﹥0.05);术后,联合组患者椎体前缘、椎体中部高度均明显高于PKP组,差异均有统计学意义(P﹤0.01)。结论 PKP联合RFA治疗脊柱转移性肿瘤可缓解患者疼痛,改善患者日常活动功能,更好地提高病变椎体高度,增强脊柱的稳定性,效果显著。     

癸酸能够活化CD8+ T细胞并提高其抗肿瘤免疫反应能力

目的：探究中链脂肪酸癸酸对CD8+ T细胞活化的影响,及其对CD8+ T细胞介导的抗肿瘤免疫反应的作用和机制。方法：建立C57BL/6小鼠黑色素瘤B16F10 皮下荷瘤模型,随机分为癸酸组（10 mg/kg 癸酸灌胃）和对照组（等量溶剂灌胃）,观察癸酸对小鼠肿瘤生长以及生存率的影响,采用流式细胞术检测肿瘤微环境中浸润CD8+ T细胞的活化水平。建立B16F10-OVA和OT-I T细胞共培养体系,采用流式细胞术检测癸酸对CD8+ T细胞的肿瘤细胞杀伤能力的影响。采用α-CD8抗体清除B16F10 荷瘤小鼠体内CD8+ T细胞,观察对小鼠肿瘤体积的影响。小鼠原代CD8+ T细胞经癸酸处理后,采用WB、ELISA及qPCR、流式细胞术检测T细胞受体（TCR）活化、效应细胞因子产生以及增殖和代谢水平。在B16F10荷瘤小鼠模型中,观察α-PD-1抗体联合癸酸给药对小鼠肿瘤生长以及生存率的影响。结果：在小鼠黑色素瘤荷瘤模型中,与对照组相比,癸酸组小鼠移植瘤体积显著降低且生存率显著提高（均P<0.05）,肿瘤浸润CD8+ T细胞IFN-γ和TNF-α的表达水平显著升高（P<0.01）。经癸酸处理的OT-I T细胞对B16F10-OVA细胞的杀伤水平显著升高（P<0.01）。在荷瘤小鼠模型中用α-CD8 抗体清除CD8+ T 细胞后,癸酸对移植瘤的抑制作用显著降低（P<0.000 1）。小鼠原代CD8+ T细胞经癸酸处理后,TCR活化水平显著升高、细胞因子IL-2和IFN-γ的产生增多、线粒体代谢水平显著上调（均P<0.05）。在黑色素瘤荷瘤小鼠模型中,癸酸与α-PD-1抗体联用,能够显著抑制小鼠移植瘤生长并提高其生存率（均P<0.05）。结论：癸酸能够促进CD8+ T细胞活化、增强其抗肿瘤免疫反应能力。     

Results after simultaneous surgery and RFA liver ablation for patients with colorectal carcinoma and synchronous liver metastases

BackgroundApproximately 20% of patients with colorectal cancer present with synchronous liver metastases (sCRLM). These patients can be treated with a “one-step procedure” or staged resection, with or without radiofrequency ablation (RFA). Colorectal surgery in combination with intraoperative RFA leads to concerns regarding postoperative complications and survival. The purpose was to evaluate the one-step procedure with or without RFA in patients with sCRLM.Materials and methodsBetween January 2000 and September 2018, patients with sCRLM were selected in two tertiary referral centers and retrospectively analyzed. Postoperative morbidity and survival were analyzed.ResultsFrom a total of 410 patients presenting with sCRLM, 329 patients underwent a staged resection and 81 a one-step procedure. The 3-year overall survival (OS) was respectively 66% and 69% for one-step procedure and staged resection (P = 0.24). A total of 18 patients underwent RFA during the one step procedure. No significant differences were shown in postoperative complications whether intraoperative RFA was used in patients with sCRLM. In the one-step procedure, the 3-year OS was respectively 43% and 72% wheter patients did or did not receive RFA (P = 0.19).ConclusionOS for patients with sCRLM was similar for both one-step procedure and staged resection. Intraoperative RFA for sCRLM is technically safe.     

Vector-based delivery of tumor-associated antigens and T-cell co-stimulatory molecules in the induction of immune responses and anti-tumor immunity

It has now been demonstrated in both experimental models and recent clinical trials that certain "self" antigens, which are functionally non-immunogenic in the host, can become immunogenic if presented to the immune system in a certain way. Here, we describe recombinant vaccines and vaccine strategies that have been developed to induce and potentiate T-cell responses of the host to such self-antigens. These strategies include: (a) the use of recombinant poxvirus vectors in which the tumor-associated antigen (TAA) is inserted as a transgene. Recombinant vaccinia vaccines and recombinant avipox (replication-defective) vaccines have been employed to break tolerance to a self-antigen; (b) the use of diversified prime and boost strategies using different vaccines; and (c) the insertion of multiple T-cell co-stimulatory molecules into recombinant poxvirus vectors, along with the TAA gene, to enhance T-cell immune responses to the TAA and induce anti-tumor immunity.     

肿瘤射频消融治疗后的免疫反应及抗肿瘤效应

射频消融治疗不仅造成局部肿瘤细胞的坏死,产生免疫原性的肿瘤相关抗原,还可产生大量炎性细胞因子和多种免疫原性介质,促进免疫细胞的局部浸润和活化,刺激免疫系统产生一定的抗肿瘤效应,但是这种抗肿瘤作用的强度较弱,不足以抵抗肿瘤生长。将射频消融治疗联合特定的免疫治疗如免疫刺激剂、过继免疫细胞治疗、树突状细胞疫苗、单克隆抗体等,可以取得最大临床获益,是免疫治疗有机融人肿瘤综合治疗的一个很好的模式。     

Human-human hybridomas for the study of anti-tumor immune response in patients with colorectal cancer

Human-human hybridoma technology was evaluated for the study of humoral immune reactions of colorectal cancer patients against their own tumors. Six fusions were carried out with lymphocytes from mesenteric lymph nodes from patients with colorectal cancer, using the human B-lymphoma cell line LICR-LON-HMy-2 as fusion partner. A total of 294 wells with cell growth were obtained. Supernatants from 26 of these reacted in enzyme-linked immuno-sorbent assay (ELISA) with one or more colon cancer cell lines. Cells from only one of these wells (D 4213) could be cloned. The clone was shown to produce antibody which by immunocytochemical analysis reacted with a panel of colon cancer cell lines and melanoma cell lines but not with several other cancer cell lines or normal human leukocytes. By immunohistochemical analysis on formalin-fixed paraffin-embedded tissue this antibody reacted strongly with antigen expressed by autologous and allogeneic colorectal cancers. Faint staining could occasionally be observed on normal colon epithelium. D4213 is a hybrid cell line since it is tetraploid and produces kappa and lambda light chains as well as gamma and mu chains, whereas HMy-2 produces only kappa and gamma chains. The study produces only kappa and gamma chains. The study suggests that patients may possess B cells producing antibody reactive with their own malignant cells.     

Combined liver surgery and RFA for patients with gastroenteropancreatic endocrine tumors presenting with more than 15 metastases to the liver

Aim

The aim of this study was to report the feasibility and early survival results of liver metastases (LM) resection combining cytoreductive surgery and radiofrequency ablation (RFA) during a one-step procedure, in patients presenting more than 15 bilateral LM from well-differentiated endocrine carcinoma. It is an extensive application of the current guidelines.

Methods

In this retrospective review of a prospectively collected database, we used a combination of hepatectomy to treat large or contiguous LM, and extensively used multiple RFA to treat the remaining LM which were smaller than 2.5 cm. Patients were selected based on a low natural tumor burden slope, and the technical feasibility of treating all the detectable LM.

Results

From January 2002 to May 2007, 16 patients with a median of 23 LM per patient (mean number: 25.7 ± 12; range16–89) underwent this procedure. A mean of 15 ± 9 LM per patient were surgically removed and a mean of 12 ± 8 (median of 10) LM per patient were RF ablated. No mortality occurred. Morbidity was observed in 11 patients (69%). The 3-year overall survival and disease-free survival rates were similar to those observed in our preliminary series of 47 hepatectomized patients with a median of 7 LM per patient.

Conclusion

This new one-step combined technique allowed us to apply an “upgraded” therapeutic approach to a selection of patients presenting a median of 23 LM per patient and to improve their prognosis, putting it on par with that obtained by conventional hepatectomy.     

Effective induction of anti-tumor immune responses with oligomannose-coated liposome targeting to intraperitoneal phagocytic cells

We recently established a novel drug delivery system (DDS) using oligomannose-coated liposomes (OMLs) which are probably taken up by macrophages (M) to carry anti-cancer drugs to milky spots known as preferential metastatic sites of gastric cancers [Y. Ikehara, T. Niwa, L. Biao, S.K. Ikehara, N. Ohashi, T. Kobayashi, Y. Shimizu, N. Kojima, H. Nakanishi, A carbohydrate recognition-based drug delivery and controlled release system using intraperitoneal macrophages as a cellular vehicle, Cancer Res. 66 (2006) 8740–8748]. In the present study, we applied this intraperitoneal DDS for systemic cancer immunotherapy employing ovalbumin (OVA) as a model antigen. The cells taking up the OMLs containing FITC-OVA injected into the peritoneal cavity were predominantly M, as they showed adhesive characteristics and expressed F4/80 and CD11b almost exclusively. The phagocytic cells also took up bare OVA directly to the same extent as OML-enclosed OVA (OML-OVA), as it is a highly mannosilated protein. The phagocytic cells taking up OML-OVA, however, could activate OVA-specific CD8⁺ (from OT-I: H-2K^b/OVA_257–264-specific) and CD4⁺ (from OT-II: H-2A^b/OVA_323–339-specific) T cells much more effectively in vitro than those taking up bare OVA. Furthermore, only the mice pre-immunized with OML-OVA rejected E.G7-OVA (OVA-transfected EL4) but not EL4. These results indicate that the OMLs can also be used as an effective antigen delivery system for cancer immunotherapy activating both CTL and Th subsets.     

Recombinant fowlpox viruses encoding the anchor-modified gp100 melanoma antigen can generate antitumor immune responses in patients with metastatic melanoma.

PURPOSE: The purpose of this study was to evaluate the immunological responses and therapeutic effectiveness of immunization with fowlpox vaccines encoding the gp100 melanoma antigen in patients with metastatic melanoma. Experimental Design: In three consecutive clinical trials, patients were immunized with recombinant fowlpox viruses encoding three different forms of the melanoma/melanocyte-associated antigen gp100: (a) the native, full-length gp100 molecule; (b) the gp100 molecule with two amino acids modified to increase binding to HLA-A*0201 molecules; and (c) a "minigene" construct encoding a single, modified epitope gp100:209-217(210M) targeted to the endoplasmic reticulum. The immunogenicity of these constructs was studied using peripheral blood mononuclear cells to measure epitope-specific release of IFN-gamma. RESULTS: Reactivity against gp100 was not seen in any patient before receiving fowlpox immunization. Whereas just one of seven patients developed reactivity after receiving fowlpox encoding native gp100, 10 of 14 patients who received fowlpox encoding the anchor modified full-length gp100 exhibited reactivity against the native gp100 molecule, and 12 of 16 patients were successfully immunized after inoculation with the modified minigene construct (p2 = 0.02). There was no difference in the latter group between those randomized to vaccination by i.v. or i.m. routes. There was one partial cancer regression in the group of 46 patients receiving virus in the absence of interleukin (IL)-2. Once patients showed evidence of progressive disease, they were eligible for "cross-over" treatment to IL-2 alone or with the fowlpox virus. None of the 13 patients receiving the full-length or modified full-length forms of gp100 responded when receiving IL-2, whereas 6 of 12 patients who received the fowlpox containing the minigene construct and then received IL-2 showed objective cancer regressions, including three patients with complete regression. CONCLUSIONS: These data underscore the importance of modifying anchor residues of nonmutated self-antigen peptides to generate cellular immune responses after immunization and support the further investigation of recombinant fowlpox viruses encoding modified epitopes administered in combination with IL-2.     

An autopsy case of sepsis following radiofrequency ablation (RFA) for metastatic liver carcinoma after bile duct reconstruction

An autopsy case of sepsis following radiofrequency ablation (RFA) for metastatic liver carcinoma after bile duct reconstructive operation is reported. A 72-year-old man underwent pylorus-preserving pancreaticoduodenectomy and reconstruction with the Suzuki-method (PD-III) for extrahepatic bile duct cancer in October 1998. A metastatic lesion was recognized in the liver (S3) in November 2001. Percutaneous RFA was performed for a recurrent lesion. A metastatic lesion was recognized again in the same segment in February 2002. Percutaneous RFA was performed again on February 26 and March 12. The patient was discharged without hemorrhage, infection, or hepatic failure on March 22. He complained of general fatigue on March 26. He was diagnosed with liver abscess, sepsis, acute renal failure, and disseminated intravascular coagulation, and received intensive care, but died on April 1. The autopsy revealed liver necrotic abscess at the RFA locus and multiple microabscesses of the liver, heart, and kidney.     

Effects of recombinant leukocyte interferon (rIFN-alpha A) on tumour growth and immune responses in patients with metastatic melanoma.

Studies were initiated to assess the response of patients with disseminated melanoma to recombinant alpha interferon (rIFN-alpha A) and to monitor effects of rIFN-alpha A on several tests of immune function. Twenty patients were treated with rIFN-alpha A given by i.m. injection in escalating doses from 15 to 50 X 10(6) um-2. The responses of two patients were considered unevaluable. Of the remainder there was complete remission of tumour in two and stable disease in two. Subsequent progression of tumour in one of the latter patients coincided with development of antibodies to IFN. Side effects (usually fatigue) were dose rate limiting in 11 patients. Laboratory tests on samples taken 6 hours after rIFN-alpha A indicated a marked lymphopenia and a reduction in natural killer (NK) cell activity particularly against K562 target cells. Longer term changes measured in samples taken 2 days after the previous rIFN-alpha A injections consisted of neutropenia and an increase in the T4/T8 ratio due mainly to a relative increase in OKT4 positive T cells compared to OKT8 positive T cells. NK activity against the K562 target cell increased in most patients during the first week of treatment and then returned to below or near pretreatment levels thereafter against the K562 target cell. This contrasted with NK activity against the melanoma target cell which showed a more gradual increase over the duration of the treatment in 6 patients. The latter correlated with an increase in mitogen stimulated IL 2 production from their blood lymphocytes and may indicate that the cytotoxic activity resulted from lymphokine-activated killer (LAK) cells. These results confirm the activity of rIFN-alpha A against melanoma in certain patients. They suggest that further studies are needed to select patients who may respond to rIFN-alpha A and to optimize treatment regimens. Tests of IL 2 production and LAK activity may assisted in achieving these objectives.     

苓甲抗癌复方提取物在荷瘤小鼠中抗肿瘤免疫应答机制

目的:初步探究苓甲抗癌复方提取物(LAM)在荷瘤小鼠中抗肿瘤免疫应答机制.方法:LAM治疗荷瘤小鼠,检测小鼠生长、瘤块体积与重量的变化;流式细胞术和ELISA分析小鼠脾脏中Treg细胞、NK细胞和各种细胞因子;Western blot分析LAM对Treg细胞相关蛋白作用.结果:LAM抑制荷瘤小鼠肿瘤的生长,引发肿瘤坏死,抑制肿瘤促增殖蛋白Ki67的表达,降低血液中CA153的含量;降低脾脏中Treg细胞比例和血清中TGF-β与IL-10的含量,提高IL-2、IFN-γ、穿孔素、颗粒酶B的含量和NK细胞的比例.结论:LAM抑制肿瘤的生长,并抑制Treg细胞的数量与功能,提高机体的免疫功能.     

Specific anti-tumor responses by cultured immune spleen cells. II. Culture supernatants induce specific anti-tumor cytotoxicity by non-immune lymphoid cells in vitro.

Sera from tumor-bearing mice induce specific cytotoxicity to tumor cells by non-immune lymphoid cells (antiserum-dependent cytotoxicity or ADC). When spleen cells from BALB/c mice bearing autochthonous or syngeneic sarcomas were cultured in vitro, culture supernatants were obtained which specifically sensitized sarcoma cells to injury in vitro by normal lymph-node cells (LNC). Culture supernatants of spleen cells from mice whose transplanted sarcomas had been excised also induced ADC. The ADC activity resided in the mouse immunoglobulin fraction of the culture supernatants and its synthesis did not depend on the presence of theta-positive cells. Following a brief in vivo exposure to culture supernatant with known ADC activity, LNC from non-immune mice specifically destroyed tumor cells in an in vitro assay.     

Dissociation of anti-tumor immune responses in rats immunized with solubilized tumor-associated antigens from a methylcholanthrene-induced fibrosarcoma.

Soluble tumor antigens were prepared from chemically-induced rat fibrosarcoma KMT-17 cells by various methods [Na-deoxycholate (DOC), 3 M-KCI extraction, and crude membrane preparations by mechanical disruption]. Soluble tumor antigens prepared by DOC extraction (DOC-STA) could be detected by a radioisotopic footpad assay (FPA) and they showed the strongest antigenic activity in KMT-17 immune rats. Anti-tumor immune responses in rats previously immunized with DOC-STA were measured by FPA, Winn assay, and transplantation resistance. Significant responses detected by the FPA and Winn assay were demonstrated in rats immunized with DOC-STA. However, rats previously immunized with DOC-STA showed a significant enhancement of tumor growth when challenged with KMT-17 cells. This enhancement was specific for the tumor line used. Normal rats which received adoptive transfer of thymus and spleen cells from rats immunized with DOC-STA produced specific enhancement of tumor growth as compared with non-treated rats. Administration of cyclophosphamide before immunization with DOC-STA abrogated the enhanced tumor growth in the host. These results suggest that immunization with soluble tumor antigens specifically enhanced tumor grwoth by the induction of immunosuppressor cells. Dissociation between the anti-tumor immunity detected by the FPA and Winn assay and the enhanced tumor growth detected by transplantation resistance in rats immunized with DOC-STA is discussed.     

MHC independent anti-tumor immune responses induced by Hsp70-enriched exosomes generate tumor regression in murine models

The ideal cancer vaccine should work regardless of MHC types but currently the barrier generated by MHC specificity hampers the development of human cancer vaccines, requesting to identify strong immunogenic molecules that can induce anti-cancer immune responses without being affected by MHC polymorphism. Tumor-derived exosomes are small membrane vesicles containing tumor antigens as well as other immunologically important molecules such as MHC molecules and heat shock proteins (HSPs). Because of their potential immunogenicity, the plausible utility of tumor-derived exosomes as an MHC independent cancer vaccine was proposed. Here, we investigated whether Hsp70-enriched tumor exosomes can induce stronger immunogenicity as compared to normal tumor-derived exosomes in autologous as well as allogeneic murine models in vitro and in vivo. Western blotting showed that the exosomes of heat-treated tumor cells (HS Exo) contained higher amounts of Hsp70 than the exosomes of untreated cells (CNTL Exo). In both MHC type-identical and -irrelevant antigen-presenting cell models in vitro, HS Exo triggered the increased expressions of MHC class II molecules. Crucially, HS Exo performed greater therapeutic capability in regressing pre-established MHC type-identical and -irrelevant tumors than CNTL Exo in vivo. The analyses of anti-tumor function in allogeneic mouse model demonstrated that HS Exo elicited Th1-polarized immune responses defined by the increased productions of IgG2a and IFN-γ. In summary, the Hsp70-enriched exosomes extracted from heat-treated tumors induced strong Th1 immune responses, resulting in eliminating cancer cells in allogeneic hosts in vivo. These results indicate that HS Exo is a potent MHC independent cell-free cancer therapeutic agent that can be developed for clinical trials.