Chronic bacterial osteomyelitis suppression of tumor growth requires innate immune responses

Clinical studies over the past several years have reported that metastasis-free survival times in humans and dogs with osteosarcoma are significantly increased in patients that develop chronic bacterial osteomyelitis at their surgical site. However, the immunological mechanism by which osteomyelitis may suppress tumor growth has not been investigated. Therefore, we used a mouse model of osteomyelitis to assess the effects of bone infection on innate immunity and tumor growth. A chronic Staphylococcal osteomyelitis model was established in C3H mice and the effects of infection on tumor growth of syngeneic DLM8 osteosarcoma were assessed. The effects of infection on tumor angiogenesis and innate immunity, including NK cell and monocyte responses, were assessed. We found that osteomyelitis significantly inhibited the growth of tumors in mice, and that the effect was independent of the infecting bacterial type, tumor type, or mouse strain. Depletion of NK cells or monocytes reversed the antitumor activity elicited by infection. Moreover, infected mice had a significant increase in circulating monocytes and numbers of tumor associated macrophages. Infection suppressed tumor angiogenesis but did not affect the numbers of circulating endothelial cells. Therefore, we concluded that chronic localized bacterial infection could elicit significant systemic antitumor activity dependent on NK cells and macrophages.     

Cyclophosphamide induces the development of early myeloid cells suppressing tumor cell growth by a nitric oxide-dependent mechanism

Adoptive immunotherapy with cyclophosphamide (Cy) increases the host resistance against tumor growth. The precise mechanism(s) by which this therapy enhances tumor suppression is unclear. Cy induces the development of early myeloid cells that may be strongly antiproliferative through NO production. These cells are similar to the natural suppressor cells found in normal bone marrow with a potential antitumor effect. Here we have addressed whether the development of NO-producing cells may be involved in this tumor resistance in Cy-treated mice. The results show a synergism between Cy treatment and tumor-specific lymphocytes transferred systemically (i.v.) or locally (Winn's assay) that results in a strong tumor suppression. Inhibition of NO production by N(G)-monomethyl-L-arginine at the site of tumor inoculation results in a loss of the protection achieved by the combined therapy. Cy-treated mice develop splenic early myeloid (CD11b, Gr-1, CD31 (ER-MP12), ER-MP20, ER-MP54) cells producing large amounts of NO upon T cell-derived signals (IFN-gamma plus CD40 ligation) able to inhibit tumor cell growth in vitro. Early myeloid cells (ER-MP54(+)) and cells expressing inducible NO synthase are increased at the site of tumor challenge in mice treated with the combined therapy, but not in those treated with Cy or immune cell transfer alone. Thus, Cy induces the expansion of early myeloid cells, inhibiting tumor cell growth by a mechanism involving NO. Both the recruitment and the activation of these myeloid cells at the site of tumor challenge appear to be dependent on the presence of tumor-specific lymphocytes.     

Suppression of growth of a mouse plasmacytoma by the IgG2 fraction of syngeneic antitumor globulin

Summary The effects of syngeneic antitumor antibody on transplanted plasmacytoma cells have been examined. Globulin was prepared from ascites fluids produced in Balb/c mice injected with MOPC 315 tumor cells and bearing the solid tumor. Normal Balb/c mice were given inoculations of tumor cells that had been incubated with the antitumor globulin obtained at various intervals after immunization, or with portions of such globulins. These materials were prepared to express the IgG2 class antibody by three procedures: precipitation with heterologous anti-mouse IgG1, passage through columns of Sepharose anti-IgG1, or adsorption to and elution from heat- and formalin-killed protein A-bearing staphylococci. The original antitumor globulins showed differences with time relative to the second injection of the immunizing tumor, in that a number of the earlier pools led to some suppression of tumor growth, and a number of the later pools led to some enhancement. Of the globulins obtained later, preparations expressing the IgG2 class of antibody by precipitation with anti-IgG1 serum caused some suppression of tumor growth. Pronounced suppression of growth was consistently obtained with anti-MOPC 315 globulin freed of IgG1 by passing it through an anti-IgG1 immunoadsorbent, and with IgG2a preparations obtained by elution from the protein A-bearing staphylococci. Of the anti-IgG1 column-treated preparations, the suppressive effect was maximal in globulin obtained 15–26 days after the second immunization. The suppressive effect of these preparations could be removed by absorption with MOPC 315 cells, but not by cells of another Balb/c tumor nor by two other plasmacytomas.     

MAP1S Controls Breast Cancer Cell TLR5 Signaling Pathway and Promotes TLR5 Signaling-based Tumor Suppression

Targeting TLR5 signaling in breast cancer represents a novel strategy in cancer immunotherapy. However, the underlying mechanism by which TLR5 signaling inhibits cancer cell proliferation and tumor growth has not been elucidated. In this study, we found TLR5 agonist flagellin inhibited the cell state of activation and induced autophagy, and reported that autophagy protein MAP1S regulated the flagellin/TLR5 signaling pathway in breast cancer cells through enhancement of NF-κB activity and cytokine secretion. Remarkably, MAP1S played a critical role in tumor suppression induced by flagellin, and knockdown of MAP1S almost completely abrogated the suppression of tumor growth and migration by flagellin treatment. In addition, elevated expression of MAP1S in response to flagellin feed-back regulated tumor inflammatory microenvironment in the late stages of TLR5 signaling through degradation of MyD88 in autophagy process. These results indicate a mechanism of antitumor activity that involves MAP1S-controlled TLR5 signaling in breast cancer.     

Combination of IL-12 gene therapy and CTX chemotherapy inhibits growth of primary B16(F10) melanoma tumors in mice

We investigated suppression of murine B16(F10) melanoma tumor growth following a therapy which involved concomitant administration of cyclophosphamide and plasmid DNA bearing interleukin-12 gene. Since both therapeutic factors display antiangiogenic capabilities, we assumed that their use in blocking the formation of new blood vessels would result in augmented inhibition of tumor growth. This combined therapy regimen indeed resulted in a considerable suppression of tumor growth. We observed a statistically significant extension of treated animals' lifespan. Interestingly, the therapeutic effect was also obtained using a plasmid without an interleukin gene insert. This observation suggests that plasmid DNA, which has been widely applied for treating neoplastic tumors, contains element(s) that elicit immune response in mice.     

Antitumor resistance induced by zinostatin stimalamer (ZSS), a polymer-conjugated neocarzinostatin (NCS) derivative

Zinostatin stimalamer (ZSS) is a new anticancer agent derived from neocarzinostatin (NCS), which is synthesized by conjugation of one molecule of NCS and two molecules of poly(styrene-co-maleic acid). ZSS exhibited potent in vitro and in vivo antitumor activity in preclinical experiments, and a clinical trial of the intra-arterial administration of ZSS with iodized oil on hepatocellular carcinoma showed potent antitumor activity. We investigated the effect of ZSS and NCS on antitumor resistance and found that pretreatment with either drug suppressed the growth of MethA tumors in Balb/c mice and induced tumor eradication when given separately by single administration at therapeutic doses between 1 day and 4 weeks before tumor transplantation. The findings that the cytocidal activity of these drugs was not detected in vivo at the time of tumor transplantation and that tumor regression was preceded by a period of transient growth suggested that tumor regression was due to host-mediated antitumor activity induced by these drugs. Pretreatment with ZSS or NCS also suppressed the growth of Colon 26 carcinoma and Sarcoma 180. The finding that NCS showed the same effect as ZSS suggests that poly(styrene-comaleic acid) is not essential for the induction of hostmediated antitumor activity. Furthermore, apo-ZSS, which lacks cytocidal activity, did not induce antitumor activity. From this, it is suggested that the cytocidal effect of ZSS involves the induction of hostmediated antitumor resistance. In athymic Balb/cnu/nu mice, pretreatment with ZSS or NCS did not induce tumor eradication, suggesting that mature T lymphocytes play an important role in tumor eradication. Challenging MethA was rejected withot transient growth in mice that had been cured of MethA, but challenging Colon 26 was not, showing that anti-MethA resistance was augmented selectively in the MethaA-eradicated mice. Splenocytes from MethA-bearing mice pretreated with the drug showed tumorneutralizing activity beginning 14 days after tumor transplantation. Tumor-neutralizing activity was only induced after MethA transplantation. The effector cells of this tumor-neutralizing activity were Thy1.2⁺ T lymphocytes that had been passed through a nylonwool column, but no significant augmentation of cell-mediated cytotoxic activity of splenocytes from MethA-eradicated mice was observed in vitro.     

CD40 ligation in vivo can induce T cell independent antitumor effects even against immunogenic tumors

Antitumor effects of CD40 ligation appear to involve distinct antitumor effector cells in different experimental models. In this study, we tested whether T cells were required for antitumor effects of agonistic anti-CD40 mAb (alphaCD40) against immunogenic versus poorly immunogenic tumors. Treatment of mice bearing poorly immunogenic B16 melanoma and its more immunogenic variant, B16-hsp72.1, with alphaCD40 resulted in a similar level of tumor growth suppression. Depletion of T cells did not reduce the antitumor effects in these 2 tumor models. To generate antitumor T cell responses, C57BL/6 mice were immunized with irradiated B16-hsp72.1. Treatment of these vaccinated mice challenged with a high dose of B16-hsp72.1 tumor cells with alphaCD40 induced tumor growth suppression, which was reduced by T-cell depletion, demonstrating that T cells were involved in the antitumor effect of alphaCD40. However, immunized mice depleted of T cells and treated with alphaCD40 were still able to suppress tumor growth as compared to tumor growth in immunized, T cell-depleted mice not treated with alphaCD40, suggesting that T cells were not required for the antitumor effect of alphaCD40. To confirm a lack of correlation between tumor immunogenicity and T-cell requirement in antitumor effects of CD40 ligation, we found that alphaCD40 induced tumor growth suppression in nude and SCID/beige mice bearing highly immunogenic tumors such as Meth A sarcoma, suggesting that macrophages may play a role. Indeed, both poorly immunogenic and highly immunogenic tumors were sensitive to in vitro growth inhibition by macrophages from alphaCD40-treated mice. Taken together, our results indicate that antitumor effects induced by alphaCD40, even against immunogenic tumors, can be observed in the absence of T cells and may involve macrophages.     

Infection and cancer: the common vein

The role of infectious agents in the development of cancer is well documented. The pathogenesis of various human neoplasms ranging from non-Hodgkin lymphoma (NHL) to cervical carcinoma frequently involves a chronic, most often viral, infection. At the same time, there is compelling evidence that certain acute infections result in the inhibition of neoplastic growth. The basis for this phenomenon is often thought to be concomitant anti-tumor immunity. Yet, experimental data supporting this hypothesis are scarce, and other non-immune anti-tumor factors could be involved. For instance, since virtually all aggressive tumors outstrip their blood supply, development of new vessels, or angiogenesis, is a limiting factor during neoplastic growth. In this review, we will discuss recent studies that implicate anti-angiogenesis in infection-mediated tumor suppression and suggest that this mechanism could also complement cytotoxic immunity arising from the use of cancer vaccines.     

Immune reactivity in SL2 lymphoma-bearing mice compared with SL2-immunized mice

Summary We have studied the rather paradoxical phenomenon of the growth of an antigenic tumor in an immunocomponent host. This phenomenon was studied by comparing (a) the lymphocyte reactivity and (b) the macrophage cytotoxicity, during SL2 growth in DBA/2 mice (SL2-bearing mice) and in DBA/2 mice immunized against SL2 tumor cells (SL2-immune mice). Immune mice rejected a challenge of tumor cells. The immune T-lymphocytes rendered macrophages cytotoxic (arming) and were able to transfer tumor resistance to naive animals. Nonimmunized mice did not reject a challenge of SL2 cells. In these tumor-bearing mice various forms of immune reactivity were tested. Lymphocytes with the capacity to arm macrophages could not be found in the lymphoid organs. However, lymphocytes isolated from the tissue directly surrounding the subcutaneous SL2 tumor could arm macrophages in vitro.Shortly after subcutaneous tumor grafting cytotoxic macrophages were found in the peritoneal cavity. In the serum macrophage arming factors were detected that rendered macrophages cytotoxic in vitro. This cytotoxicity of the peritoneal macrophages and the presence of macrophage arming factors in the serum showed a similar biphasic pattern. The first phase of cytotoxicity between day 3 and 8 after tumor grafting was tumor (SL2) specific. The second phase from day 12 and onwards was not tumor specific. During the first 4 days after SL2 grafting the DBA/2 mice expressed a specific concomitant immunity to a second tumor graft. Then 7 or more days after grafting the first SL2 tumor, the concomitant immunity was nonspecific as the growth of a second SL2 tumor graft and a L5178Y (DBA/2) tumor graft were inhibited. In addition, the immune suppressive activity of serum and lymphocytes was tested. Neither serum nor lymphocytes from SL2-bearing mice suppressed the macrophage arming capacity of SL2 immune lymphocytes. Lymphocytes from tumor-bearing mice did not inhibit the capacity of SL2-immune lymphocytes to transfer resistance to naive animals. On the contrary, lymphocytes obtained from SL2-bearing mice 14 days after SL2 grafting transfered tumor resistance in a Winn-type assay. These data suggest that the growth of an antigenic tumor is due to the inability of the immune system to mount an effective antitumor effector cell population during tumor growth, rather than an immune suppression of the antitumor reactivity, as a limited immune reactivity could be detected in tumor-bearing mice, whereas immune suppression could not be detected.     

Functional activity of the lymphocytes of mouse lymph nodes in a system of viral carcinogenesis]

A study was made of the PHA-induced lymphocyte blastransformation (BT), and lymphocyte antitumor activity, using a model of progressive and regressive neoplastic process induced by the Moloney virus in mice BALB/c. The antitumor acitvity of lymphocytes was estimated by their ability to inhibit spheroid production by tumor cells. A lymphocyte BT and their antitumor activity was registered during a progressive growth of the tumor and its restoration as the tumor is regressing.     

Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation

Introduction of an exogenous retinoblastoma (RB) gene in RB-deficient retinoblastoma or osteosarcoma cells has been shown to suppress their neoplastic phenotype. In experiments designed to explore the potential mechanism of RB tumor suppression, we report here that the phosphorylation state of RB protein is modulated during normal cellular events. In resting cells, RB protein is present in its least phosphorylated form; in rapidly proliferating cells, RB protein is highly phosphorylated. Maximal phosphorylation is associated with S phase of the cell cycle. Induction of differentiation in several human leukemia cell lines by treatment with phorbol ester or retinoic acid leads to dephosphorylation of RB. Time course studies indicate that RB dephosphorylation precedes the total arrest of cell growth during differentiation. These observations strongly suggest that the function of RB protein is modulated by a phosphorylation/dephosphorylation mechanism during cell proliferation and differentiation.     

中药抗肿瘤作用的研究进展

应用中草药抗肿瘤的研究很早就已是医学科技工作者的重要研究领域;但是,近年来中草药抗肿瘤的研究变的越来越火热,越来越深入,许多研究工作已经不单纯局限于抗肿瘤的基本活性,更多的研究已经深入到抗肿瘤作用机理的层面;本文从中药有效成分对诱导肿瘤细胞凋亡、抑制肿瘤血管的生长、诱导肿瘤细胞分化、逆转肿瘤细胞多药耐药性、提高机体免疫力等方面对中药抗肿瘤研究的当前状况进行了综述,具体情况如下：     

Prostaglandin E and cancer Growth

Summary This paper reviews the evidence linking prostaglandin E (PGE) with the growth of neoplastic tissue. PGE ₂ is present in high concentrations in many natural and experimentally produced cancers. The immunosuppressive effect of some tumors in mice is due at least in part to a prostaglandin mechanism. The growth of these same tumors can be slowed and in some cases the tumor eliminated by administration of PG synthetase inhibitors. It is not yet clear whether the antitumor properties of these PG synthetase inhibitors are due to their releasing the hostimmune system from the chronic prostaglandin-mediated suppression of the tumor, resulting in an effective immune response to the tumor, or whether another mechanism is responsible. In humans overproduction of PGE ₂ by macrophages is partly responsible for the depressed PHA response in patients with Hodgkin's disease.     

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor

We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.     

Autophagy differentially controls plant basal immunity to biotrophic and necrotrophic pathogens

In plants, autophagy has been assigned 'pro-death' and 'pro-survival' roles in controlling programmed cell death associated with microbial effector-triggered immunity. The role of autophagy in basal immunity to virulent pathogens has not been addressed systematically, however. Using several autophagy-deficient (atg) genotypes, we determined the function of autophagy in basal plant immunity. Arabidopsis mutants lacking ATG5, ATG10 and ATG18a develop spreading necrosis upon infection with the necrotrophic fungal pathogen, Alternaria brassicicola, which is accompanied by the production of reactive oxygen intermediates and by enhanced hyphal growth. Likewise, treatment with the fungal toxin fumonisin B1 causes spreading lesion formation in atg mutant genotypes. We suggest that autophagy constitutes a 'pro-survival' mechanism that controls the containment of host tissue-destructive microbial infections. In contrast, atg plants do not show spreading necrosis, but exhibit marked resistance against the virulent biotrophic phytopathogen, Pseudomonas syringae pv. tomato. Inducible defenses associated with basal plant immunity, such as callose production or mitogen-activated protein kinase activation, were unaltered in atg genotypes. However, phytohormone analysis revealed that salicylic acid (SA) levels in non-infected and bacteria-infected atg plants were slightly higher than those in Col-0 plants, and were accompanied by elevated SA-dependent gene expression and camalexin production. This suggests that previously undetected moderate infection-induced rises in SA result in measurably enhanced bacterial resistance, and that autophagy negatively controls SA-dependent defenses and basal immunity to bacterial infection. We infer that the way in which autophagy contributes to plant immunity to different pathogens is mechanistically diverse, and thus resembles the complex role of this process in animal innate immunity.     

Cutting edge: differential effect of apoptotic versus necrotic tumor cells on macrophage antitumor activities.

Macrophages (Mphi) play essential roles both in tumor defense and normal tissue homeostasis by removal of transformed as well as damaged and disintegrating cells. Whereas tissue necrosis is known to provoke inflammatory responses, removal of apoptotic cells has been assumed to be immunologically inert. We now show that while Mphi exposure to necrotized tumor cells causes pronounced stimulation of Mphi antitumor activity, exposure of Mphi to apoptotic tumor cells in contrast results in impairment of Mphi-mediated tumor defense and even support of tumor cell growth. Given the fact that apoptosis is a consequence of various cancer treatment modalities, this may lead to a suppression of local antitumor reactions and thus actually counteract endogenous immune-mediated tumor defense mechanisms.     

肿瘤相关巨噬细胞：在肿瘤演进中的作用及潜在抗肿瘤靶点

炎症是公认的肿瘤十大特征之一,而肿瘤相关巨噬细胞是肿瘤微环境的重要组成部分,它影响肿瘤的生长、血管生成、免疫抑制、 转移和药物抗性。最新研究表明,肿瘤相关巨噬细胞还会影响抗肿瘤治疗的临床疗效。鉴于肿瘤相关巨噬细胞在肿瘤演进中起重要作用, 其作为潜在抗肿瘤靶点备受关注。基于最新的研究,对人类癌症中肿瘤相关巨噬细胞的主要功能、作用和特征以及用作新兴肿瘤治疗干预 靶点作一综述。     

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor

Summary We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.This paper was presented in part at the annual meeting of the American Association of Immunologists, Chicago, Illinois, 10–15 April 1983     

Chemoimmunotherapy with doxorubicin and caffeine combination enhanced ICD induction and T-cell infiltration in B16F10 melanoma tumors

Majority of chemotherapeutic agents can elicit antitumor immunity and modulate the composition, density, function, and distribution of tumor infiltrating lymphocytes (TILs), to influence differential therapeutic responses and prognosis in cancer patients. The clinical success of these agents, particularly anthracyclines like doxorubicin, not only depends on their cytotoxic activity but also by the enhancement of pre-existing immunity primarily through induction of immunogenic cell death (ICD). However, resistance for the induction of ICD either intrinsic or acquired is a major hurdle for most of these drugs. To enhance ICD by these agents, it has become clear that blockade of adenosine production or its signaling need to be specifically targeted as they represent highly resistant mechanisms. Given the prominent role of adenosine mediated immunosuppression and resistance to ICD induction in tumor microenvironment, combination strategies that involve ICD induction and adenosine signaling blockade are further warranted. In the present study, we investigated the antitumor effect of caffeine and doxorubicin combination therapy against 3-MCA-induced and cell-line induced tumors in mice. Our results demonstrated significant tumor growth inhibition by the combination therapy of doxorubicin and caffeine against both carcinogen-induced and cell-line induced tumor models. In addition, significant T-cell infiltration and enhanced ICD induction evidenced by increased intratumoral calreticulin and HMGB1 levels, was observed in B16F10 melanoma mice. The possible mechanism behind the observed antitumor activity might be due to the enhanced ICD induction and subsequent T-cell infiltration by the combination therapy. To prevent the development of resistance and to enhance the antitumor activity of ICD inducing drugs like doxorubicin, combination with adenosine-A2A receptor pathway inhibitors like caffeine might be a potential strategy.