In vitro and in vivo effects of an anti-mouse endoglin (CD105)–immunotoxin on the early stages of mouse B16MEL4A5 melanoma tumours

TGF-beta superfamily co-receptors are emerging as targets for cancer therapy, acting both directly on cells and indirectly on the tumour neovasculature. Endoglin (CD105), an accessory component of the TGF-beta receptor complex, is expressed in certain melanoma cell lines and the endothelial cells of tumour neovessels. Targeting endoglin with immunotoxins is an attractive approach for actively suppressing the blood supply to tumours. Here, we report evidence indicating that endoglin is expressed in mouse melanoma B16MEL4A5 and mouse fibroblast L929 cell lines. We prepared an immunotoxin to target endoglin by coupling the rat anti-mouse MJ7/18 (IgG2a) monoclonal antibody (mAb) to the non-toxic type 2 ribosome-inactivating protein nigrin b (Ngb) with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as a linker with a molar nigrin b at a MJ7/18 stoichiometry of 2:1. The MJ7-Ngb immunotoxin generated killed both cell lines, with IC₅₀ values of 4.2 × 10^?9 M for B16MEL4A5 and 7.7 × 10^?11 M for L929 cells. For in vivo assays of the immunotoxin, B16MEL4A5 cells were injected subcutaneously into the right flanks of 6-week-old C57BL/6 J mice. When the animals developed palpable solid tumours, they were subjected to treatment with the immunotoxin. While treatment with either MJ7/18 mAb or Ngb did not affect tumour development, treatment with the immunotoxin completely and steadily blocked tumour growth up to 7 days, after which some tumours re-grew. Thus, vascular-targeting therapy with this anti-vascular immunotoxin could promote the destruction of newly created tumour vessels at early stages of B16MEL4A5 tumour development and readily accessible CD105+ B16MEL4A5 melanoma cells.     

Gene transfer of a hybrid interleukin-1β gene to B16 mouse melanoma recruits leucocyte subsets and reduces tumour growth in vivo

 Interleukin(IL)-1 differs from most other cytokines in its lack of a signal sequence. This results in intracellular retention of the immature proform. The release of IL-1 has been shown to be restricted predominantly to activated monocytes and macrophages and to be associated with apoptosis of the producer cell. These features have limited the investigation of IL-1 in early immune responses. In order to study the biological effects of local IL-1β release during an antitumour immune response, we used B16 mouse melanoma cells transduced with mature human IL-1β cDNA constructs. To obtain a released form of human IL-1β (ssIL-1β), the signal sequence from the related IL-1 receptor antagonist was ligated to the cDNA that encoded the mature form of IL-1β. When cells of the poorly immunogenic B16 melanoma cell line were transduced with IL-1β by retroviral infection, high levels of the protein were detected intracellularly, whereas cells transduced with IL-1β containing the signal sequence secreted most of their protein. The in vitro growth of the melanoma cells was unaffected by the IL-1β or ssIL-1β gene transfer. In contrast, the in vivo subcutaneous tumour growth of the ssIL-1β-transduced B16 cells in syngeneic C57BL/6 mice was significantly reduced compared with the IL-1β- and the mock-transduced controls. Immunohistochemical analysis revealed the infiltration of macrophages to be strong in B16/ssIL-1β, moderate in B16/IL-1β and minimal in control tumours. Furthermore, a moderate infiltration of CD4⁺ cells and of scattered dendritic cells was detected in B16/ssIL-1β tumours whereas very few or no CD4⁺ cells and dendritic cells were seen in the B16/IL-1β or control tumours. Following in vivo growth, all the tumours up-regulated ICAM-1 on their cell surfaces. However, the percentage of ICAM-1-expressing cells was two- to fourfold higher in B16/ssIL-1β tumours compared to the control. The data suggest that IL-1β acts in vivo, either directly or indirectly, as a chemotactic factor for monocytes, T helper cells and dendritic cells. This supports IL-1β having a regulatory effect on tumour growth when locally released in the tumour area. Received: 12 November 1996 / Accepted: 6 May 1997     

Recombinant angioarrestin secreted from mouse melanoma cells inhibits growth of primary tumours

Angioarrestin is a recently described anti-angiogenic protein whose expression is down-regulated in solid tumours of various origins. It has a sequence identical to angiopoietin related protein-1. In this study we investigated anti-tumour properties of angioarrestin in B16 (F10) melanoma tumour model. We constructed an expression vector encoding human angioarrestin under the control of EF-1alpha promoter. This vector was transferred to B16 (F10) cells and recombinant angioarrestin secreted from the transfected cells was tested for anti-angiogenic activity using endothelial cell proliferation assay. Finally, mice were injected subcutaneously with cells that had been transfected with either angioarrestin-encoding vector or empty vector and tumor growth was compared. The obtained recombinant angioarrestin inhibited proliferation of bovine aortic endothelial cells. Tumours derived from an angioarrestin-secreting B16 (F10) cell clone grew in vivo more slowly than tumours derived from a cell clone transfected with empty vector. These data show, to our knowledge for the first time, that angioarrestin can inhibit primary melanoma tumour growth.     

Proteomic studies of B16 lines: involvement of annexin A1 in melanoma dissemination

To identify proteins involved in melanoma metastasis mechanisms, comparative proteomic studies were undertaken on B16F10 and B16Bl6 melanoma cell lines and their subsequent syngenic primary tumours as pulmonary metastases were present only in the mice bearing a B16Bl6 tumour. 2DE analyses followed by MALDI-TOF identification showed variations of 6 proteins in vitro and 13 proteins in vivo. Differential expressed proteins in tumours were related to energy production and storage. Two differentially expressed proteins which had not been previously associated to melanoma progression, annexin A1 (ANXA1) and creatine kinase B (CKB), were found both in cells and in tumours. To characterize ANXA1 involvement in melanoma B16 dissemination, we reduced ANXA1 protein level by siRNA and observed a significant decrease of B16Bl6 cell invasion through Matrigel coated chambers. We further demonstrated that the presence of several formyl peptide receptors (FPR1, FPRrs1 and 2) revealed by qRT-PCR, played a role in B16 invasion: incubation of B16Bl6 cells with the FPR agonist (fMLP) or antagonist (tBOC) enhanced or decreased Matrigel coated chamber invasion respectively, with a correlation of ANXA1 levels in both treatments. As ANXA1 could bind to FPRs, this should amplify invasion and enhance melanoma dissemination.     

Treatment with HPMA copolymer-based doxorubicin conjugate containing human immunoglobulin induces long-lasting systemic anti-tumour immunity in mice

Linkage of doxorubicin (Dox) to a water-soluble synthetic N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA) eliminates most of the systemic toxicity of the free drug. In EL-4 lymphoma-bearing C57BL/6 mice, a complete regression of pre-established tumours has been achieved upon treatment with Dox–PHPMA–HuIg conjugate. The treatment was effective using a range of regimens and dosages, ranging from 62.5 to 100% cured mice treated with a single dose of 10–20 mg of Dox eq./kg, respectively. Fractionated dosages producing lower levels of the conjugate for a prolonged time period had substantial curative capacity as well. The cured mice developed anti-tumour protection as they rejected subsequently re-transplanted original tumour. The proportion of tumour-protected mice inversely reflected the effectiveness of the primary treatment. The treatment protocol leading to 50% of cured mice produced only protected mice, while no mice treated with early treatment regimen (i.e. starting on day 1 after tumour transplantation) rejected the re-transplanted tumour. Exposure of the host to the cancer cells was a prerequisite for developing protection. The anti-tumour memory was long lasting and specific against the original tumour, as the cured mice did not reject another syngeneic tumour, melanoma B16-F10. The immunity was transferable to naïve recipients in in vivo neutralization assay by spleen cells or CD8⁺ lymphocytes derived from cured animals. We propose an effective treatment strategy which eradicates tumours without harming the protective immune anti-cancer responses.     

Effect of local interleukin-2 treatment on spontaneous tumours of different immunogenic strength

Transplantable tumour lines established from spontaneous tumours of BALB/c, CBA, and DBA/2 mice displayed different immunogenic strength. This report describes tumour susceptibility to interleukin-2 (IL-2) therapy in relation to tumour immunogenicity. The following tumour lines were used: X5, X6, and X9 mammary tumours of DBA/2, BALB/c, and CBA origin respectively, X7 carcinoma of BALB/c and X18 papilloma of CBA mice. Two spontaneous tumours of long transplantation history, SL2 lymphoma (SL2) of DBA/2 and Madison lung carcinoma M109 (M109) of BALB/c origin, were used as control systems. Experimental mice were transplanted with different inocula of tumour cells at day 0; treatment with IL-2 was initiated on days 1–3 or delayed until day 10 and consisted of daily injections of low doses of 5000 or 20 000 U/mouse given five times a week for a period of 3 weeks. Treatment of SL2 (2 × 10⁴ cells injected i.p.) consisted of i.p. injections of 5000 or 20 000 U IL-2/mouse given on days 10–14 after tumour transplantation. IL-2 therapy of SL2-bearing DBA/2JIco mice resulted in a significant proportion of cures; however, no response to IL-2 treatment was achieved in SL2-bearing DBA/2CrIiw mice. BALB/c mice with the i.p. transplant of M109 responded to IL-2 treatment with 40% increase in lifespan. The low-dose IL-2 therapy of the five spontaneous tumours resulted, in general, in transient growth inhibition of the i.m. transplants of lines X5, X6, and X7 provided that IL-2 was administered locally. The therapeutic effect depended on the number of transplanted tumour cells, the best results being achieved at cell numbers close to the dose-inducing tumour growth in 50% of animals. We found that the spontaneously arising tumours responding to IL-2 treatment were all slowly growing and immunogenic (X6 and X7) or might have viral association (X5) and, as such, might express foreign antigens. The data suggest a correlation between tumour immunogenicity and the therapeutic effect. However, IL-2 can still exert some effect against tumours with negligible immunogenicity. Received: 16 July 1998 / Accepted: 5 October 1998     

Effect of monoclonal antibodies to early pregnancy factor (EPF) on the in vivo growth of transplantable murine tumours

Summary Neutralisation studies with monoclonal antibodies (mAbs) specific for early pregnancy factor (EPF) have shown it to be essential for the continuation of pregnancy in mice and the growth of some tumour cells in vitro. These studies report that the mAbs are also able to limit the growth of two murine tumour lines transplanted s. c. The development of MCA-2 tumours in CBA mice was unaffected by the injection of 1 mg anti-EPF IgM at the time of tumour cell inoculation. However, four doses of 500 µg anti-EPF, injected one dose per day for 4 days after tumour cell inoculation, significantly retarded tumour development such that no tumours were palpable on day 13. A similar dose regimen of control IgM had no effect on tumour size. Dose/response studies revealed that lower doses of anti-EPF administered after tumour cell inoculation were effective in retarding the growth of the MCA-2 tumours. The effect of anti-EPF mAb administration on the growth rate of palpable B16 tumours established s. c. in C57BL/6 mice was also determined. Tumours injected with 6 mg anti-EPF 5/341 or anti-EPF 5/333 mAbs showed significant decrease in the uptake of [³H]thymidine into tumour tissue, measured 16 h after injection. Furthermore, titration of sera for active EPF showed that a significant reduction in the EPF titre was associated with a significant inhibition of tumour DNA synthesis. Thus it appears that neutralisation of EPF retards tumour growth both in vitro and in vivo. In vitro the effects must be due to anti-EPF mAb interfering with a direct mechanism that contributes to the maintenance of cells in the active growing phase. However, in vivo host immunological mechanism that are modified to allow tumour survival may also be affected. The presence of EPF-induced suppressor factors curculating in the serum of tumour-bearing mice has been confirmed and the contribution of such factors to tumour progression must now be investigated.     

In situ vaccination against a non-immunogenic tumour using intratumoural injections of liposomal interleukin 2

Cancers appear to escape surveillance by the immune system at least in part because they fail to induce a protective immune response. Therapeutic vaccines based on specific tumour antigens and tumour cells modified ex vivo by genetic techniques are but two strategies being used to circumvent this problem. In this report, we describe a simple, yet effective alternative in which tumour-specific responses are induced by in situ administration of a well-characterized liposomal formulation of the cytokine interleukin 2 (IL-2). Using the non-immunogenic B16 melanoma model, intratumoural injections of liposomal IL-2 L(IL2), were shown to induce a long-lived immune response specific for the injected tumour. In conjunction with subsequent removal of the primary tumours by surgery, the injections increased mean survival to 57 days from a control value of 32 days and partially protected surviving mice against re-challenge with B16. L(IL2) induced an early infiltration of inflammatory cells within the tumours which was followed several days later by an influx of CD3+ T cells. The cellular influx and a coincident decrease in tumour growth were noted in both injected tumours and a second non-injected tumour on the same animal, thereby demonstrating the systemic nature of the immune response. Intratumoural injections of soluble IL-2 at the same dose failed to induce B16-specific cellular immunity or to prolong survival of the mice. Thus, liposomal formulation of the cytokine was fundamental to successful induction of immunity by this in situ vaccination regimen.     

1,25-Dihydroxyvitamin D3 enhances the growth of tumors in athymic mice inoculated with receptor rich osteosarcoma cells

We tested the influence of daily subcutaneous injections of 12.5 and 25 pmol of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the growth of tumors arising from intracutaneous inoculations of athymic nude mice with rat osteogenic sarcoma cells (ROS) and human melanoma cells. Both doses of 1,25(OH)2D3 increased plasma calcium levels after 3 weeks and produced a striking enhancement in tumor weight when the mice received 1,25(OH)2D3 receptor-rich ROS17/2.8 cells. In contrast, 1,25(OH)2D3 caused no consistent effect on tumor weight in mice given G-361 melanoma cells with low receptor copy number or receptor deficient ROS 24/1 cells. Thus, 1,25(OH)2D3 stimulated tumor growth in a receptor dependent fashion, in vivo, instead of inhibiting it as predicted from the reduction of proliferation of cultured cells in the presence of 1,25(OH)2D3.     

Immunosuppressive effect of bone marrow-derived mesenchymal stem cells in inflammatory microenvironment favours the growth of B16 melanoma cells

Mesenchymal stem cells (MSCs) are studied for their potential clinical use in regenerative medicine, tissue engineering and tumour therapy. However, the therapeutic application of MSCs in tumour therapy still remains limited unless the immunosuppressive role of MSCs for tumour growth in vivo is better understood. In this study, we investigated the mechanism of MSCs favouring tumour escape from immunologic surveillance in inflammatory microenvironment. We first compared the promotive capacity of bone marrow-derived MSCs on B16 melanoma cells growth in vivo, pre-incubated or not with the inflammatory cytokines interferon (IFN)-γ and tumour necrosis factor (TNF)-α. We showed that the development of B16 melanoma cells is faster when co-injected with MSCs pre-incubated with IFN-γ and TNF-α compared with control groups. Moreover, tumour incidence increases obviously in allogeneic recipients when B16 melanoma cells were co-injected with MSCs pre-incubated with IFN-γ and TNF-α. We then demonstrated that the immunosuppressive function of MSCs was elicited by IFN-γ and TNF-α. These cytokine combinations provoke the expression of inducible nitric oxide synthase (iNOS) by MSCs. The impulsive effect of MSCs treated with inflammatory cytokines on B16 melanoma cells in vivo can be reversed by inhibitor or short interfering RNA of iNOS. Our results suggest that the MSCs in tumour inflammatory microenvironment may be elicited of immunosuppressive function, which will help tumour to escape from the immunity surveillance.     

Immunogenicity of 2 murine B16 melanoma variants with high metastatic potential

The expression of tumor-associated transplantation antigens (TATA) by two metastatic variants, isolated from B16 melanoma in vivo, was examined. The first, YB16 melanoma (amelanotic), was selectioned after a successive s. c. transplantations of B16 melanoma cells on the coisogenic Yellow AY/a mutant mice of C57BL/6J mice. The second, MB16 melanoma, characterized by a variable pigmentation, was obtained from a s. c. transplantation of YB16 melanoma cells on C57BL/6J mice. The comparison of TATA expressed by the two variants and the B16 melanoma, made between different modes of inducing tumor-rejection activity, revealed that i) these two variants failed to induce an autologous antitumor response, ii) they were resistant to crossed immunization with an immunogenic preparations of B16 melanoma and iii) only MB16 melanoma preparations reduced significantly the tumoral incidence of B16 melanoma cells. These data leads us to suggest i) that the s. c. transplantation of B16 melanoma cells on Yellow AY/a mice resulted in the selection of nonimmunogenic, amelanotic and metastatic cell population of YB16 melanoma and ii) the existence of an epigenetic regulation of melanogenesis and expression of TATA in MB16 melanoma cells carried on C57BL/6J mice.     

Paclitaxel treatment enhances lymphatic metastasis of B16F10 melanoma cells via CCL21/CCR7 axis

Chemotherapeutic drugs have been successfully used to treat several cancers, including melanoma. However, metastasis occasionally occurs after chemotherapy. Here, we reported that paclitaxel (PTX) treatment for B16F10 tumour in mice led to an enhanced lymphatic metastasis of the melanoma cells, although a significant inhibition of tumour growth at the injection site was observed. Further study demonstrated that PTX upregulated the expression of C-C chemokine receptor type 7 (CCR7) in B16F10 cells, enhancing their migration through the activation of JNK and p38 signalling pathways. Loss of CCR7 or blockade of C-C motif chemokine ligand 21 (CCL21)/CCR7 axis abolished the pro-migration effect of PTX on B16F10 melanoma cells. Importantly, combination of PTX and CCR7 mAb could simultaneously delay the tumour growth and reduce the lymphatic metastasis in B16F10 melanoma. The blockade of CCL21/CCR7 axis may collectively serve as a strategy for lymphatic metastasis in some melanoma after chemotherapy.     

Antimetastatic activity of Sulforaphane

The effect of Sulforaphane on the inhibition of lung metastasis induced by B16F-10 melanoma cells was studied in C57BL/6 mice by three different modalities of administration-simultaneous, prophylactic and after tumour developed. Of this simultaneous mode of Sulforaphane administration was found to be most effective. There was 95.5% inhibition of lung tumour nodule formation and 94.06% increase in the life span of metastatic tumour bearing animals. Highly elevated levels of lung hydroxyproline, lung uronic acid, lung hexosamine, serum sialic acid and serum gamma-glutamyl transpeptidase (GGT) in the metastatic control animals was found to be significantly lowered in the Sulforaphane treated animals. Histopathological analysis of lung tissues also correlated with these results. In the in vitro system Sulforaphane showed a significant inhibition in the invasion of B16F-10 melanoma cells across the collagen matrix. (3)H-thymidine proliferation assay showed that Sulforaphane could inhibit the proliferation of B16F-10 melanoma cells in vitro. Gelatin zymographic analysis showed that Sulforaphane could inhibit the activation of matrix metalloproteinases. These findings suggest that Sulforaphane reduced the invasion of B16F-10 melanoma cells by the inhibition of activation of matrix metalloproteinases, thereby inhibiting lung metastasis.     

In vitro and in vivo melanogenesis inhibition by biochanin A from Trifolium pratense

Our previous study showed that a methanol extract from Trifolium pratense exerted potent inhibitory activity on melanogenesis in mouse B16 melanoma cells. In the present study, the active compound in this Chinese herb extract was isolated and identified as biochanin A by mass spectrum, (1)H-NMR, and (13)C-NMR analysis. The inhibitory effects of biochanin A on melanogenesis were investigated in vitro in cultured melanoma cells and in vivo in zebrafish and mice. Biochanin A dose-dependently inhibited both melanogenesis and cellular tyrosinase activity in B16 cells and in zebrafish embryos. Application of a cream containing 2% biochanin A twice daily to the skin of mice also increased the skin-whitening index value after 1 week of treatment, and the increase continued for another 2 weeks. Biochanin A was confirmed as a good candidate for use as a skin-whitening agent in the treatment of skin hyperpigmentation disorders.     

Radiosensitization of a mouse melanoma by withaferin A: in vivo studies

The radiosensitizing effect of a plant withanolide, withaferin A, on the B16F1 mouse melanoma was studied in vivo. Treatment of 100 mm3 tumours with 10 to 60 mg/kg withaferin A intraperitoneally produced a dose dependent increase in growth delay and volume doubling time. Injection of 30-50 mg/kg withaferin A, followed by 30 Gy local gamma irradiation, significantly enhanced the tumour response. No systemic or local adverse reactions were noted in these groups. The drug was most effective when injected intraperitoneally 1 h before irradiation. However, neither the individual agents nor their combination could produce any complete response (tumour cure). Melanoma is a relatively radioresistant tumour. The present results indicate that the radiation response of this tumour can be significantly enhanced by pretreatment with withaferin A.     

Immunization of mice with melanoma cells transfected to secrete the superantigen, staphylococcal enterotoxin A

Immunization of mice with a melanoma vaccine coupled with staphylococcal enterotoxin A (SEA) inhibits the growth of primary melanoma tumors in mice. We have now successfully transfected B16 cells with the sea gene and have immunized C57BL/6 mice subcutaneously once per week for 4 weeks prior to tumor challenge with vaccines of irradiated B16 cells or, 4 weeks following tumor challenge of naïve mice with B16 cells, with irradiated B16 cells transfected with the sea gene. Primary tumor growth following both types of treatments was inhibited significantly. To characterize immune responses to these immunogens, we examined the production of antibodies to the B700 melanoma antigen, the stimulation of endogenous IL-2 production, the expression of CD4, CD8, Vβ and CD25 T cell markers, and the induction of NK activity. At 4 weeks following immunization of mice, there was a significant increase (P<0.05) in levels of interleukin-2 production by splenocytes from mice immunized with SEA-secreting B16 cells or with the parental B16 cells, compared to controls. Levels of antibodies to the B700 melanoma antigen were also significantly higher in mice immunized with the SEA-secreting B16 cells, as was expression of CD4, CD8, CD25 and Vβ T cell antigens, particularly CD4. Natural killer cell activity (at various E:T ratios) was tenfold higher in splenocytes of mice immunized with SEA-secreting B16 cells, and fivefold higher in mice immunized with the parental B16 cells, compared to controls.?These data confirm the possibility of using irradiated murine melanoma cells transfected to secrete SEA in vaccines targeted at preventing the development and growth of melanoma.     

Monoclonal antibodies NORM-1 and NORM-2 induce more normal behavior of tumor cells in vitro and reduce tumor growth in vivo

In this study, large numbers of hybridomas (produced by syngeneic immunization with B16 mouse melanoma and fusion with NS-1 myeloma cells) were screened for the production of antibodies that affected morphology and growth of animal and human tumor cells in vitro. Two such antibodies, NORM-1 and NORM-2 (both IgG2a), inhibited the growth of B16 melanoma cells in soft agar and increased the serum requirements of tumor cells in tissue culture. Antibody NORM-2 also inhibited the growth of SV40-transformed 3T3 cells in agar and caused them to deposit more fibronectin into extracellular matrix. These antibodies thus seem to induce a more normal behavior of tumor cells in vitro. In vivo both antibodies reduced the number of growing lung tumors of B16 melanoma in C57BL/6 mice by 70%-90% when injected 3 days after the tumor cells. By immunoprecipitation of 35S-methionine-labeled cell extracts, NORM-2 antibody recognized a 59 kd protein in B16 mouse and in A375 human melanoma cells but not in 3T3 fibroblasts.     

Anti-tumor activity of class II MHC antigen-restricted cloned autoreactive T cells. II. Novel immunotherapy of B16 melanomas by local and systemic adoptive transfer

Lyt-1+, L3T4a+ autoreactive cloned T cells, producing lymphotoxin (LT) and interferon-gamma (IFN-gamma) in response to self-class II major histocompatibility complex antigen in vitro were examined for their anti-tumor effect in vivo against B16 melanomas. Without the aid of exogenous interleukin 2, the autoreactive T cells, when injected immediately and at an equal cell number into the site of s.c. inoculated B16 melanoma cells inhibited tumor growth in sublethally irradiated and nonirradiated syngeneic mice. The autoreactive T cells also induced regression of tumors established 3 days earlier. Normal spleen cells or class II-restricted cloned T cells specific for chicken gamma-globulin (CGG) had no inhibitory effect on tumor growth. A single injection of autoreactive T cells delayed tumor growth and prolonged the survival of mice that had received a lethal dose of B16 melanoma cells. The autoreactive T cells caused extensive necrosis at the injection site. A treatment regime consisting of two successive injections of anti-I-Ab monoclonal antibody 3JP prevented the inhibition of tumor growth, supporting the hypothesis that the autoreactive T cells inhibited the growth of melanomas by releasing LT and IFN-gamma upon recognition of I-A antigen-bearing cells at the injection site. The CGG-specific control T cells did not cause necrosis and survived within the nests of uninhibited tumor cells. Autoreactive T cells administered i.v. immediately after i.v. injection of B16 melanoma cells markedly reduced pulmonary metastases, whereas CGG-specific T cells did not. These results indicate that autoreactive T cells can function in vivo as inhibitors of tumor growth.     

Growth of human pheochromocytomas in the anterior eye chamber of the rat. A histochemical study on amine and peptide content of pheochromocytoma tumour cells

Tissue pieces from seven benign human pheochromocytomas have been successfully transplanted to the anterior eye chamber of cyclosporin-treated rats. In vivo observations showed that 74-99% of the tumour transplants were vascularized within one to two days after transplantation. No increase in the size of the transplants was noted during the observation period (1-4 weeks). Tumour transplants grown in non-immunosuppressed rats were initially vascularized but rejection started to occur one week after transplantation. Histochemical analysis of tumour transplants grown in immunosuppressed rats demonstrated numerous tumour cells with strong catecholamine fluorescence, some of which formed long cell processes on the host iris. Immunocytochemical analysis of tumour transplants demonstrated positively labelled tumour cells after incubation with antisera against neuropeptide Y, enkephalin, vasoactive intestinal polypeptide, somatostatin, substance P, dopamine-beta-hydroxylase, tyrosine hydroxylase and serotonin. A similar histochemical and immunocytochemical pattern was observed in primary tumours but tumour cells sending out cell processes were observed less frequently. Human pheochromocytomas may thus be successfully grown in oculo in cyclosporin-treated rats. This may prove to be a suitable model for the study of storage and release of catecholamines and neuropeptides from pheochromocytoma tumour cells.