Human tumor antigens inducing in vivo delayed hypersensitivity and in vitro mitogenic activity

Significant immunogenic properties were observed with an antigen extracted from tumor-liberated particles (TLP). Several different tumors were used to obtain antigens by such cell analysis and purification procedures as: gel filtration on Sephadex G-100, ion-exchange chromatography, affinity chromatography, and gel filtration on Sephadex G-200. The molecular weight determined on SDS-PAGE was about 214,000 daltons. In vivo, TLP yields delayed hypersensitivity reactions both in autologous and homologous hosts. TLP does not seem to have any nonspecific mitogenic activity, but it does have specific mitogenic activity, since lymphocyte blastogenesis (TLP-induced in presensitized patients) could mean its ability when intradermally inoculated to sensitize a lymphocytic clone.     

Modulation of murine tumor major histocompatibility antigens by cytokines in vivo and in vitro

The capacity of different cytokines to upregulate major histocompatibility complex (MHC) expression on murine tumor cells in vitro, and on s.c. tumors or pulmonary metastases in vivo has been examined. Interleukins-1, -2, and -4 (IL-1, -2, -4), tumor necrosis factor-alpha (TNF-alpha), alpha-interferon (IFN-alpha), and gamma-interferon (IFN-gamma), were incubated with tissue culture lines of murine tumor cells displaying low (MCA-101), intermediate (MCA-102, -106), or high (MCA-105) Class I expression. IFN-alpha and IFN-gamma significantly increased Class I but not Class II antigens on all lines. TNF-alpha, IL-1, -2, and -4 had no significant effect on Class I or II expression in vitro. Mice bearing pulmonary metastases or s.c. lesions generated by MCA-101 and -102 were treated with IFN-alpha or IFN-gamma i.p. or i.v., or with a single dose of TNF-alpha i.v. Immunoperoxidase staining of lung metastases or subcutaneous tumors showed an increase in Class I but not Class II expression on MCA-102 tumors treated with IFN-alpha or IFN-gamma. IL-1, -2, or TNF-alpha had no effect on MHC Class I or II expression in vivo. None of the cytokines tested could upregulate MHC Class I or II expression on MCA-101 tumors in vivo. Flow cytometry analysis demonstrated an increase in Class I but not Class II expression on MCA-102 and MCA-106 tumor cells from s.c. tumor treated with IFN-alpha or IFN-gamma. A kinetic analysis of the flow cytometry data revealed that augmented MCA-102 Class I levels persisted for several days after cessation of in vivo therapy with IFN-alpha. Our data suggest one possible mechanism for the synergistic antitumor effects of IL-2 and IFN-alpha.     

Selective depletion of nk cell activity in vivo and its effect on the growth of NK-sensitive and nk-resistant tumor cell variants

Intravenous injection of rabbit anti-asialo-GM₁ serum, an antiserum previously shown to eliminate splenic natural killer (NK) activity in vitro, profoundly depressed NK activity in CBA, DBA/2 and BALB/c nu/nu mice. The effect on NK activity was selective, as treatment of mice with anti-asialo-GM₁ serum did not affect the development of other cytotoxic cells including cytotoxic macrophages following injection of poly I:C, or cytotoxic T cells in response to allogeneic cells. The role of NK cells in controlling tumor cell growth was investigated using an NK-sensitive (cl 27v-IC2) and an NK-resistant (cl 27av) subline of the murine lymphoma L5178Y. Initial studies showed that cl 27v-IC2 cells were at least 100 times less tumorigenic than were cl 27av cells in both syngeneic DBA/2 mice and BALB/c nu/nu mice. In addition, treatment of DBA/2 mice with poly I:C, which boosted NK activity, markedly depressed the growth of cl 27v-IC2 cells, but not of cl 27av cells. On the other hand, treatment of DBA/2 mice and BALB/c nu/nu mice with antiasialo-GM1₁ serum led to a marked increase in tumorigenicity of cl 27v-IC2 cells, but had no effect on the tumorigenicity of cl 27av cells. In addition, the protection against cl 27v-IC2 growth afforded by poly-I:C treatment was abrogated by injection of anti-asialo-GM₁ serum. The possibility that the effects observed were caused by binding of the injected antibodies to the tumor cells was minimized by: (1) using a clone of tumor cells (cl 27v-IC2) that lacks chemically detectable asialo-GM₁, and (2) pre treating animals with anti-asialo-GM₁ rather than administering antiserum and tumor cells concurrently. These studies provided compelling evidence that NK cells could play an active role in controlling tumor growth. Selective depletion of NK activity by injection of antiasialo-GM₁ serum is a method which would be generally applicable to studying the role of NK cells in disease processes.     

Lipoic acid inhibits cell proliferation of tumor cells in vitro and in vivo

Cancer cells convert glucose preferentially to lactate even in the presence of oxygen (aerobic glycolysis–Warburg effect). New concepts in cancer treatment aim at inhibition of aerobic glycolysis. Pyruvate dehydrogenase converts pyruvate to acetylCoA thus preventing lactate formation. Therefore, the aim of this study was to evaluate compounds that could activate pyruvate dehydrogenase in cancer cells. We investigated the effects of (R)-(+)-α-lipoic acid (LPA) and dichloroacetate (DCA), possible activators of pyruvate dehydrogenase, on suppression of aerobic glycolysis and induction of cell death. The neuroblastoma cell lines Kelly, SK-N-SH, Neuro-2a and the breast cancer cell line SkBr3 were incubated with different concentrations (0.1–30 mM) of LPA and DCA. The effects of both compounds on cell viability/proliferation (WST-1 assay), [18F]-FDG uptake, lactate production and induction of apoptosis (flow cytometric detection of caspase-3) were evaluated. Furthermore, NMRI nu/nu mice that had been inoculated s.c. with SkBr3 cells were treated daily for four weeks with LPA (i.p, 18.5 mg/kg) starting at day 7 p.i.. Tumor development was measured with a sliding calliper and monitored via [18F]-FDG-PET. Residual tumors after therapy were examined histopathologically. These data suggests that LPA can reduce (1) cell viability/proliferation, (2) uptake of [18F]-FDG and (3) lactate production and increase apoptosis in all investigated cell lines. In contrast, DCA was almost ineffective. In the mouse xenograft model with s.c. SkBr3 cells, daily treatment with LPA retarded tumor progression. Therefore, LPA seems to be a promising compound for cancer treatment.     

Enhancement of in vitro and in vivo tumor cell radiosensitivity by valproic acid

Valproic acid (VA) is a well-tolerated drug used to treat seizure disorders and has recently been shown to inhibit histone deacetylase (HDAC). Because HDAC modulates chromatin structure and gene expression, parameters considered to influence radioresponse, we investigated the effects of VA on the radiosensitivity of human brain tumor cells grown in vitro and in vivo. The human brain tumor cell lines SF539 and U251 were used in our study. Histone hyperacetylation served as an indicator of HDAC inhibition. The effects of VA on tumor cell radiosensitivity in vitro were assessed using a clonogenic survival assay and gammaH2AX expression was determined as a measure of radiation-induced DNA double strand breaks. The effect of VA on the in vivo radioresponse of brain tumor cells was evaluated according to tumor growth delay analysis carried out on U251 xenografts. Irradiation at the time of maximum VA-induced histone hyperacetylation resulted in significant increases in the radiosensitivity of both SF539 and U251 cells. The radiosensitization was accompanied by a prolonged expression of gammaH2AX. VA administration to mice resulted in a clearly detectable level of histone hyperacetylation in U251 xenografts. Irradiation of U251 tumors in mice treated with VA resulted in an increase in radiation-induced tumor growth delay. Valproic acid enhanced the radiosensitivity of both SF539 and U251 cell lines in vitro and U251 xenografts in vivo, which correlated with the induction of histone hyperacetylation. Moreover, the VA-mediated increase in radiation-induced cell killing seemed to involve the inhibition of DNA DSB repair.     

Specificity of adhesion between murine tumor cells and capillary endothelium: an in vitro correlate of preferential metastasis in vivo

We have compared the rate and extent of adhesion of various types of mouse tumor cells to endothelial cells derived from different organ sources. Our panel of tumors has included sarcoma, bladder carcinoma, glioma, teratoma, hepatoma, endothelioma, mammary adenocarcinoma, and lymphoma cells. Endothelial cell monolayers have included murine microvascular endothelial cells from ovary, brain, lung, and liver as well as large vessel endothelium from thoracic duct and dorsal aorta. Tumor cells differ both in the adhesive propensity and adhesive preference for different endothelial cells. Some, but not all, of the adhesive preferences correlate with the known in vivo metastatic behavior of these tumors. Our results support the hypothesis that endothelial cell surface-associated specificities may play a significant role in determining the pattern of metastasis.     

In vivo and in vitro production and detection of monoclonal antibodies to surface components on metastatic variants of murine tumor cells

Mouse-mouse and rat-mouse hybridoma cell lines secreting complement-dependent cytotoxic or cell-binding monoclonal antibodies have been produced against cell surface components of two murine metastatic tumor systems: B16 melanoma and RAW117 lymphosarcoma. We have used both in vivo and in vitro spleen cell culture methods for immunization and have made a number of methodologic alterations to increase the yield and survival of hybridomas including media osmolality, pH, serum type, macrophage feeder layers and supplemented amino acids, vitamins and metabolites. Using complement-dependent cytotoxicity or lectin immobilization of tumor cells to polystyrene via a water-soluble carbodiimide for an enzyme-linked immunosorbant assay we were able to rapidly screen hybridoma cultures for specific antibody secretion.     

In vivo and in vitro reactivity of rat spleen cells against regressor and progressor colon-cancer cell variants

Previous reports demonstrated that progressor and regressor tumor-cell variants isolated from the same colon carcinoma chemically induced in a BD-IX rat differed in their capacity to induce an immune response. The present study was aimed at analyzing the characteristics of the responses to the regressor REGb and progressor PROb clones. Spleen cells from rats bearing early REGb tumors neutralized PROb cell tumorigenicity in a Winn-type local transfer assay, but responded occasionally to REGb and PROb cells in vitro. However, spleen cells from rats immunized by several injections of REGb and PROb cells strongly proliferated when cultured with PROb or REGb cells. This response was selective for the cell lines generated from the individual tumor at the origin of PROb and REGb lines, was dependent on CD4⁺ spleen cells, and was partially inhibited by an antibody against major histocompatibility complex class-II molecules. Although PROb cells shared tumor-rejection antigen(s) with REGb cells, splenocytes from PROb tumor-bearing rats did not neutralize PROb-cell tumorigenicity in vivo, nor did they proliferate when cultured with PROb or REGb cells in vitro. The unresponsiveness of spleen cells from PROb tumor-bearing rats was not due to the presence of immune suppressive cells, and a defect of antigen-presenting cells was shown to be unlikely. This unresponsiveness was limited to a lymphocyte subpopulation, since spleen cells from tumor-bearing rats responded normally to stimulation by PHA or allogeneic lymphocytes. These results strongly suggest that unresponsiveness of spleen cells from tumor-bearing rats is due to a tumor-specific anergy of the lymphocyte clones able to respond to tumorassociated antigens.     

Analysis of tumor cell surface antigens

The cell surface analysis is a very useful tool to diagnose tumor cells, especially hematological disorders such as leukemia and lymphoma. Particularly owing to the recent spread of flow cytometry and development of monoclonal antibodies, a single color analysis has become to be used as routine clinical series at many institutions. Recently a two color analysis with fluorescein isothiocyanate (FITC) and phycoerythrin (PE) has become to be easily applied to a cell surface staining and a more detail analysis has become to be available in studying cell surface antigens. By using a two color technic, it has been found that adult T cell leukemia cells, which are positive for CD4 (helper/inducer T lymphocytes) in a single color analysis, are derived from suppressor inducer T cells (CD4+--Leu 8+). The increased cells in thymoma have also been identified to be positive for CD4 and CD8 simultaneously (CD4+--CD8+). We conclude that a two color analysis is a very useful tool to study more detail and precise cell surface markers. A flow chart to diagnose hematological disorders with cell surface markers is also proposed in this article.     

灵芝精粉和孢子粉混合物抑制肿瘤细胞生长的实验研究

背景与目的：灵芝是一种具有滋补强身作用的传统中草药。研究表明灵芝对小鼠移植性肿瘤具有生长抑制作用,这种作用一般认为是通过机体免疫系统介导的。我们实验室近来发现灵芝主要成分灵芝精粉和孢子粉混合物（lucid garoderma and lucid garoderma spore,MLGLGS）能抑制拓扑异构酶ⅠⅡ活性,因此,本研究欲进一步观察MLGLGS在体内外对人肿瘤细胞生长的抑制作用。方法：运用MTT法、SRB法研究MLGLGS体外对小鼠白血病P388细胞和10种不同类型人肿瘤细胞株的生长抑制作用,并利用裸小鼠移植瘤模型研究MLGLGS体内对人肺腺癌LAX-83细胞和人胃腺癌SGC-7901细胞的生长抑制作用。结果：MLGLGS对P388、U-937和HL-60细胞的IC50值都小于2mg/ml,对2株人胃癌和2株人肺癌细胞的IC50都小于4mg/ml。体内试验结果显示：当MLGLGS为1g/kg时,对人肺腺癌移植瘤的生长抑制率为49.47％;当MLGLGS为2g/kg时,对人胃腺癌移植瘤的生长抑制率为43.09％。结论：高浓度、高剂量MLGLGS有抑制肿瘤细胞生长的作用。     

MAZ51, an indolinone that inhibits endothelial cell and tumor cell growth in vitro, suppresses tumor growth in vivo

We have recently described MAZ51, an indolinone that blocks the ligand-induced autophosphorylation of VEGFR-3, a receptor tyrosine kinase that plays a central role in the regulation of lymphangiogenesis. Here we show that MAZ51 is able to block the proliferation of VEGFR-3-expressing human endothelial cells and is less potently able to induce their apoptosis. MAZ51 also inhibits the proliferation and induces the apoptosis of a variety of non-VEGFR-3-expressing tumor cell lines. These data suggest that MAZ51 blocks the activity of tyrosine kinases in addition to VEGFR-3. In vivo, MAZ51 significantly inhibits the growth of rat mammary carcinomas. These data establish MAZ51 as a compound with antitumor properties that inhibits tumor growth directly and also indirectly by interfering with tumor-host interactions.     

An exponential-Gompertzian description of LoVo cell tumor growth from in vivo and in vitro data

The data of nine monolayer cultures, 48 multicellular spheroids, and 19 s.c. xenografts of LoVo cells were fitted, on an individual basis, by exponential and Gompertzian equations, respectively. The mean growth parameters alpha 0 (initial growth rate) and beta (retardation factor) of the three experimental systems presented a strong linear correlation alpha 0 = 6.88 beta + 0.56, r = 0.9843. This implies that, at the particular tumor size of 155 microns in diameter (mean +/- SD = 50-310 microns), the tumor growths described by Gompertzian curves (from spheroids as well as from s.c. xenografts) have the same growth rate as monolayer cultures. This occurrence strongly supports an exponential-Gompertzian growth model, where an exponential monolayer-like phase changes to a Gompertzian growth, controlled by environmental conditions, when the tumor size has reached a critical value.     

Tumor-specific and forssman antigens of guinea-pig hepatoma cells: comparison of tumor cells grown in vivo and in vitro.

Cloned tumor-cell lines were derived from two antigenically distinct ascites variants of diethylnitrosamine-induced guinea-pig hepatomas (designated line 1 and line 10). Cell-surface antigens on the ascites and in vitro-grown tumor cells were analyzed with immunofluorescence, C1 fixation and transfer, and antibody-complement-mediated cytotoxity tests. Tumor-specific and Forssman antigens continued to be expressed during 3-6 months in vitro cultivation. Differences between ascites and cultured cells were noted in the degree of antigen expression and sensitivity to antibody-complement-mediated cytotoxicity. Cells grown in vitro exhibited a greater number of Forssman and tumor-specific antigen sites than in vivo grown cells as determined by the quantitative C1 fixation and transfer test. Immunofluorescent staining indicated that some cloned lines were considerably more homogeneous in terms of antigen expression than were the cultured, non-cloned parent cells or ascites-grown cells. Cloned lines were frequently more sensitive to the cytotoxic action of antibody and complement than were the in vivo grown and cultured non-cloned parent tumor cells. Sensitivity to cytotoxicity did not necessarily correlate, however, with the degree of antigen expression. These results suggest that; (1) the expression of Forssman and tumor-specific antigens does not diminish on cells cultivated in vitro and (2) ascites hepatoma cells in vivo are a heterogeneous population of cells differing in their degree of antigen expression and sensitivity to antibody-complement-mediated cytotoxicity.     

Glycosaminoglycan production by murine melanoma variants in vivo and in vitro

Glycosaminoglycan (GAG) synthesis of B-16 melanoma metastatic variants was examined in vivo and in vitro to begin to assess the relationship between the presence of these polymers and the process of primary invasion and metastasis. The variants that were examined for GAG production included the F-1 line that exhibits low metastatic potential, the F-10 line selected for high metastatic potential, and the BL6 line selected for high invasiveness. The F-1 cell line was routinely less invasive than the F-10 and BL6 lines when injected s.c. into the legs of irradiated Swiss Webster mice. All cell lines formed palpable tumors after s.c. injection, but histological sections revealed early and extensive invasion in only F-10 and BL6 tumors. The F-1 tumors were surrounded by a connective tissue capsule and did not begin to invade into host tissue until this structure disappeared approximately 16 days after injection of tumor cells. Some consistent alterations in GAG synthesis, particularly the release of hyaluronic acid and heparan sulfate, were observed among the cell lines in vivo and in vitro, although differences observed in vitro were small and variable. In vivo all tumors were surrounded by a hyaluronic acid-rich zone that was concentrated at the tumor-stromal interface and was transitory. Hyaluronate occurred as a diffuse band around BL6 and F-10 tumors but was confined to a capsule surrounding the less aggressive F-1 tumor. In vitro the BL6 and F-10 cell lines released larger amounts of heparan sulfate and hyaluronic acid than did the F-1 cell line. Differences in release of chondroitin sulfate by the cell lines were not observed. Differences in trypsin-releasable GAG, presumably associated with the glycocalyx, were also not apparent. These results link the release in vitro and organization in vivo of hyaluronic acid and heparan sulfate to invasion and metastasis.     

UV radiation-induced tumors in haired mice: identification as squamous cell carcinomas

The most common tumor induced by UV radiation in haired mice is considered to be a fibrosarcoma on the basis of its presentation as a nodule in the skin and on the basis of a spindled appearance upon light microscopic examination. A squamous cell carcinoma is thought to be a much less common tumor. In the present report this concept was reevaluated in mammary tumor virus-free C3H/HeNCr (C3H-) mice. From first appearance, almost all lesions upon gross morphologic examination have an epidermal component and initially are similar to solar keratoses in humans. The lesions then become nodular and eventually develop central ulceration, often with a rolled border characteristic of squamous cell carcinomas. The morphology upon light microscopic examination ranged from well-differentiated squamous cell carcinoma to a poorly differentiated spindle cell neoplasm. Occasionally, variable patterns of squamous differentiation were seen in the same lesion. Immunoperoxidase examination with a polyclonal antikeratin serum demonstrated the presence of keratin in 84 of 87 tumors. Frequent, poorly formed desmosomes were found on ultrastructural examination. These tumors usually had a regressor phenotype upon transplantation into recipients. In conclusion, almost all UV radiation-induced tumors in C3H- mice are squamous cell carcinomas, and these tumors are usually antigenic.     

Thymoglobulin targets multiple plasma cell antigens and has in vitro and in vivo activity in multiple myeloma.

Multiple myeloma is characterized by the proliferation of clonal plasma cells that have a heterogeneous expression of various cell surface markers, precluding successful use of monoclonal antibodies for therapeutic targeting of the tumor cell. Thymoglobulin (rabbit-derived polyclonal anti-thymocyte globulin), by virtue of its method of preparation, contains antibodies against several B-cell and plasma cell antigens and offers an attractive option for immunotherapy of myeloma. Here, we demonstrate potent anti-myeloma activity of the rabbit anti-thymocyte globulin preparation Thymoglobulin in vitro and in vivo in an animal model of myeloma. Thymoglobulin was able to induce dose- and time-dependent apoptosis of several myeloma cell lines, including those resistant to conventional anti-myeloma agents. Importantly, the anti-myeloma activity was preserved even when myeloma cells were grown with different cytokines demonstrating the ability to overcome microenvironment-mediated resistance. Thymoglobulin induced apoptosis of freshly isolated primary myeloma cells from patients. Using a competitive flow cytometric analysis, we were able to identify the potential antigen targets for Thymoglobulin preparation. Finally, in a plasmacytoma mouse model of myeloma, Thymoglobulin delayed the tumor growth in a dose-dependent manner providing convincing evidence for continued evaluation of this agent in the clinic in patients with myeloma, either alone or in combination with other agents.     

Gonadotropin-releasing hormone receptor levels and cell context affect tumor cell responses to agonist in vitro and in vivo

Activation of gonadotropin-releasing hormone (GnRH) receptors inhibits proliferation of transformed cells derived from reproductive tissues and in transfected cell lines. Hence, GnRH receptors represent a therapeutic target for direct action of GnRH analogues on certain proliferating cells. However, more cell biological data are required to develop this particular application of GnRH analogues. Therefore, we compared the effects of GnRH receptor activation in transfected HEK293 cells (HEK293([SCL60])) with transfected human ovarian cancer cell lines SKOV3 and EFO21, human hepatoblastoma HepG2 cells, and rat neuroblastoma B35 cells. Marked differences in receptor levels, magnitude of inositol phosphate generation, and dynamics of inositol phosphate turnover occurred in the different cells. Activation of GnRH receptors, expressed at high or moderate levels, inhibited the growth of HEK293([SCL60]) and B35 cells, respectively. Western blotting detected markers of apoptosis [cleaved poly(ADP-ribose) polymerase, caspase-9] in HEK293([SCL60]) and B35 following treatment with 100 nmol/L d-Trp(6)-GnRH-I. Cell growth inhibition was partially or completely rescued with inhibitor Q-VD-OPh or Ro32-0432. Low levels of GnRH receptor expression in transfected SKOV3, EFO21, or HepG2 activated intracellular signaling but did not induce apoptosis or significantly affect cell proliferation. Tumor xenografts prepared from HEK293([SCL60]) regressed during treatment with d-Trp(6)-GnRH-I and growth of xenografts derived from transfected B35 was slowed. SKOV3 xenografts were not growth inhibited. Therefore, differences in levels of GnRH receptor and signaling differentially affect the apoptotic machinery within cell lines and contribute to the cell type-specific effects of GnRH on growth. Further studies should exploit the growth-inhibitory potential of GnRH receptor activation in abnormal cells in diseased human tissues.     

Characterization of in vitro immunoselected variants from a highly metastatic murine tumor for alterations in malignant behavior in vivo

A new Ly-6.2- antigen-loss variant (called L61 -M1) of the highly metastatic DBA/2 mouse (Ly-6.2+) MDAY-D2 tumor has been obtained by means of a monoclonal anti-Ly-6.2 antibody in an in vitro immunoselection technique. Whereas L61 -M1 grew poorly when inoculated subcutaneously into the syngeneic host, it grew and metastasized in a similar way to the parental MDAY-D2 tumor when inoculated into immunosuppressed, athymic nude mice. L61 -M1 as well as another Ly-6.2- variant of the same MDAY-D2 tumor (called L61 ) which is poorly metastatic in the syngeneic host salvaged exogenous fucose into glycoproteins and glycolipids at rates 5.5 and 7.8 times that of the parental MDAY-D2 line. In contrast, the Ly-6.2- variants exhibited a 50-70% decrease in the incorporation of exogenous mannose into glycoproteins and glycolipids. L61 -M1 and L61 also exhibited alterations in the structures of the oligosaccharide moieties linked to the cell surface glycoproteins and/or glycolipids. Thus, the in vitro immunoselection technique can be used to obtain a panel of variants with stable phenotypic alterations in their growth and metastatic capacities. Such mutants may, like previously described lectin-resistant mutants, be useful in studying the contribution of cell surface glycoproteins and glycolipids to tumorigenicity and metastasis.