Murine Mammary Adenocarcinoma Research Articles

Nitric Oxide Synthase Inhibition by NG-Nitro- l-Arginine Methyl Ester Inhibits Tumor-Induced Angiogenesis in Mammary Tumors

Using a murine breast cancer model, we earlier found a positive correlation between the expression of nitric oxide synthase (NOS) and tumor progression; treatment with inhibitors of NOS, N(G)-methyl-L-arginine (NMMA) and N(G)-nitro-L-arginine methyl ester (L-NAME), had antitumor and antimetastatic effects that were partly attributed to reduced tumor cell invasiveness. In the present study, we used a novel in vivo model of tumor angiogenesis using subcutaneous implants of tumor cells suspended in growth factor-reduced Matrigel to examine the angiogenic role of NO in a highly metastatic murine mammary adenocarcinoma cell line. This cell line, C3L5, expresses endothelial (e) NOS in vitro and in vivo, and inducible (i) NOS in vitro on stimulation with lipopolysaccharide and interferon-gamma. Female C3H/HeJ mice received subcutaneous implants of growth factor-reduced Matrigel inclusive of C3L5 cells on one side, and on the contralateral side, Matrigel alone; L-NAME and D-NAME (inactive enantiomer) were subsequently administered for 14 days using osmotic minipumps. Immediately after sacrifice, implants were removed and processed for immunolocalization of eNOS and iNOS proteins, and measurement of angiogenesis. Neovascularization was quantified in sections stained with Masson's trichrome or immunostained for the endothelial cell specific CD31 antigen. While most tumor cells and endothelial cells expressed immunoreactive eNOS protein, iNOS was localized in endothelial cells and some macrophages within the tumor-inclusive implants. Measurable angiogenesis occurred only in implants containing tumor cells. Irrespective of the method of quantification used, tumor-induced neovascularization was significantly reduced in L-NAME-treated mice relative to those treated with D-NAME. The quantity of stromal tissue was lower, but the quantity of necrotic tissue higher in L-NAME relative to D-NAME-treated animals. The total mass of viable tissue (ie, stroma and tumor cells) was lower in L-NAME relative to D-NAME-treated animals. These data suggest that NO is a key mediator of C3L5 tumor-induced angiogenesis, and that the antitumor effects of L-NAME are partly mediated by reduced tumor angiogenesis.

Induction of thymidine phosphorylase expression and enhancement of efficacy of capecitabine or 5'-deoxy-5-fluorouridine by cyclophosphamide in mammary tumor models.

Thymidine phosphorylase (dThdPase) is an essential enzyme for the activation of the oral cytostatic drugs capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine, Xeloda(trade mark)) and its intermediate metabolite doxifluridine [5'-deoxy-5-fluorouridine (5'-dFUrd, Furtulon((R)))] to 5-fluorouracil (5-FUra) in tumors. In a previous study, we found that several cytostatics were able to up-regulate tumor levels of dThdPase in a human colon cancer xenograft model. In the present study, we confirmed that the administration of cytostatics used for breast cancer treatment, such as taxanes and cyclophosphamide (CPA), up-regulated the tumor level of dThdPase in mammary tumor models as well. Because the dThdPase up-regulation was observed even when CPA was given orally, we investigated further the usefulness of combination therapy with the 2 oral drugs, 5'-dFUrd/capecitabine and CPA in mammary tumor models. Daily oral administration of CPA up-regulated human dThdPase levels in the tumor tissue of mice bearing a human mammary tumor xenograft, MX-1, whereas in the small intestine and liver, it did not affect levels of pyrimidine nucleoside phosphorylases (PyNPase) including dThdPase and uridine phosphorylase. The preferential up-regulation of PyNPase activity in the tumor by CPA administration was also confirmed in mice bearing a syngeneic murine mammary adenocarcinoma, A755. In both models, combination therapy of 5'-dFUrd/capecitabine with CPA showed synergistic antitumor activity, without significant potentiation of toxicity. In contrast, treatment with CPA and either 5-FUra or UFT (a mixture of tegafur and uracil) in combination showed only additive activity. Our results suggest that CPA and capecitabine/5'-dFUrd, both available for oral administration, would be good partners, and that clinical trials with this drug combination against breast cancer are warranted.

Soluble factors from the target organ enhance tumor cell angiogenesis: role of laminin SIKVAV sequence.

The ability of tumor cells to respond to microenvironmental factors present in the target organ determines in part the successful development of a metastasis. In a previous work it was demonstrated that the conditioned medium (CM) from lungs of normal mice stimulates in vitro migration, proliferation and uPA activity of cells from a murine mammary adenocarcinoma moderately metastatic to lung. This CM also enhanced local and metastatic tumor growth. Here, we show that lung CM enhanced neovascularization when inoculated together with LM3 tumor cells into the skin of syngeneic mice. A similar tumor-induced angiogenesis response was obtained when lung CM was injected systemically. Western blot analysis of lung CM revealed the presence of some laminin fragments containing the sequence SIKVAV. To determine whether those molecules were responsible for the observed angiogenic effects, the CM was depleted of the peptides containing the SIKVAV sequence. We observed that the SIKVAV-depleted lung CM lost its ability to induce an enhancement of the tumor neovascular response. Our results suggest a role for the target organ in facilitating the neovascularization of tumor cells, probably through the participation of active peptides derived from the proteolytic degradation of the basement membrane component laminin.

Nitric-oxide production by murine mammary adenocarcinoma cells promotes tumor-cell invasiveness.

The role of nitric oxide (NO) in tumor biology remains controversial and poorly understood. While a few reports indicate that the presence of NO in tumor cells or their micro-environment is detrimental for tumor-cell survival, and consequently their metastatic ability, a large body of data suggests that NO promotes tumor progression. The purpose of this study was to identify the source of NO in the spontaneously metastasizing C3-L5 murine mammary-adenocarcinoma model, the role of tumor-derived NO in tumor-cell invasiveness, and the mechanisms underlying the invasion-stimulating effects of tumor-derived NO. The source of NO was established by immunocytochemical localization of NO synthase (NOS) enzymes in C3-L5 cells in vitro and transplanted tumors in vivo. An in vitro transwell Matrigel invasion assay was used to test the invasiveness of C3-L5 cells in the presence or the absence of NO blocking agents or iNOS inducers (IFN-gamma and LPS). The mechanisms underlying the invasion-stimulating effects of tumor-derived NO were examined by measuring mRNA expression of matrix metalloproteinases (MMP)-2 and -9, and tissue inhibitors of metalloproteinases (TIMP) 1, 2 and 3 in C3-L5 cells in various experimental conditions. Results showed that C3-L5 cells expressed high level of eNOS protein in vitro, and in vivo, both in primary and in metastatic tumors. C3-L5 cells also expressed iNOS mRNA and protein when cultured in the presence of IFN-gamma and LPS. Constitutively produced NO promoted tumor-cell invasiveness in vitro by down-regulating TIMP 2 and TIMP 3. In addition, there was up-regulation of MMP-2, when extra NO was induced by IFN-gamma and LPS. In conclusion, NO produced by C3-L5 cells promoted tumor-cell invasiveness by altering the balance between MMP-2 and its inhibitors TIMP-2 and 3. Thus, our earlier observations of anti-tumor and anti-metastatic effects of NO inhibitors in vivo in this tumor model can be explained, at least in part, by reduced tumor-cell invasiveness.

Topical and intratumoral photodynamic therapy with 5-aminolevulinic acid in a subcutaneous murine mammary adenocarcinoma

One of the most promising substances used in photodynamic therapy (PDT) is 5-aminolevulinic acid (ALA), which induces endogenous synthesis and accumulation of porphyrins in malignant cells. In this paper we have shown that both topical and intratumoral administration of ALA in a subcutaneously implanted mammary carcinoma produced a significant synthesis of porphyrins and subsequent sensitization to laser light. Porphyrin accumulation was greater when ALA was administered intratumorally and tumour/normal skin porphyrin concentration ratios were higher compared with topical application. Irradiation was optimal between 2 and 3 h after topical application of 50 mg of a 20% ALA cream and 2–4 h after intratumoral administration of 30 mg ALA/cm 3. The pattern of tumour response evaluated as the delay of tumour growth was similar following either route of drug administration. Applications of PDT were performed once, twice or three times in the study. The response to successive applications was constant for the same tumour, indicating that no resistance was acquired. Microscopic analysis showed both induction of foci of necrosis and haemorrhage, morphological features of apoptotic cells and total absence of cellular immune response. This paper reports on PDT with topical ALA in a subcutaneous carcinoma leading to tumour growth delay. These findings may have great relevance in the treatment of cutaneous metastasis of mammary carcinomas.

MCP-1 expression as a potential contributor to the high malignancy phenotype of murine mammary adenocarcinoma cells

The search for mechanisms that regulate tumor progression has motivated the authors’ laboratory to establish a unique murine model system, consisting of two lines of DA3 mammary adenocarcinoma cells that were derived originally from a common ancestor but differed in their malignant potential. Studies indicated that the highly malignant phenotype manifested by one of the cell lines (termed Ly-6 hi DA3 cells) was associated with high expression of the Ly-6E.1 antigen. To characterize the mechanisms controlling the high malignancy phenotype expressed by Ly-6 hi DA3 cells, the study was focussed on the potential contribution of tumor-derived factors to the high malignancy phenotype expressed by these cells. To this end, the expression of CC chemokines, major chemoattractants of monocytes and T cells, by the highly malignant Ly-6 hi DA3 cells as compared to the low malignancy Ly-6 lo DA3 cells was evaluated. The results indicate that the highly malignant cells express higher levels of factors that induce monocyte migration than the low malignancy cells. Two CC chemokines were shown to be highly produced by Ly-6 hi DA3 cells, MIP-1α and MCP-1, of which only the latter was shown to contribute to the high migratory activity expressed by the high malignancy Ly-6 hi DA3 cells. Since MCP-1 may attract monocytes to tumor sites, these findings suggest that monocyte-derived mediators, such as growth factors or angiogenic cytokines, have pro-malignancy effects that contribute to the high malignancy phenotype expressed by Ly-6 hi DA3 cells.

A synthetic, nitrogenated antimetastatic and anti-angiogenic compound with low toxicity in vivo.

The design of more effective therapies for metastatic disease involves development of new compounds able to specifically block the malignant process. We demonstrated previously that a new synthetic nitrogenated compound 3'-1-chloroethyl-2,3-dihydro-1H-imidazo-(2, 1-i)-purine-4-ium-7-yl-3'-deoxy-1',5', 6'-tri-O-(methylsulfonyl)-muco-inositol chloride (DIC) had an anti-proliferative activity on tumor cells in vitro. In the present work we demonstrate that DIC induces apoptosis on the LM3 murine mammary adenocarcinoma cell line in vitro and has anti-angiogenic activity in vivo. We also evaluated toxicity, biodistribution and anti-neoplastic properties of DIC in vivo. Toxicity studies allowed us to establish the LD50 (750 mg/kg body weight). Administration of 250 mg/kg/day (LD10) for 6 days did not cause overt toxic effects. Biodistribution assays revealed that DIC was rapidly eliminated (60% at t=10 min), although it accumulated in tumor tissue at higher concentrations than in other tissues. Daily s.c. treatment with DIC (LD10) for 24 days significantly reduced the number of spontaneous lung metastases. These results suggest that DIC has the ability of impairing the metastatic development by inhibiting angiogenesis and inducing apoptosis on tumor cells.

Impact of the antimetastatic drug Batimastat on tumor growth and PO2 measured by EPR oximetry in a murine mammary adenocarcinoma.

Patients treated for early breast cancer have only a 10–15% probability of local relapse (Fowble, 1991), but the likelihood of eventual recurrence with distant metastases can be much higher depending on the original stage of the cancer. It is from this systemic disease that patients die (Willner et al., 1997). Treatment of malignant tumors has relied heavily on the use of cytotoxic agents, targeted at the increased metabolic and proliferative activity of the cancer cell. An alternative strategy has been to develop low toxicity therapies that can be administered continuously over a period of several years to stabilize malignant disease, and it is in this category that treatment with matrix metallopro-teinase inhibitors (MMPIs) lie.

Progesterone receptor involvement in independent tumor growth in MPA-induced murine mammary adenocarcinomas

We have developed a model of hormonal carcinogenesis in BALB/c female mice, in which MPA induced ductal mammary adenocarcinomas, expressing high levels of estrogen and progesterone receptors (ER and PR). A series of tumor lines, retaining both PR and ER expression, were obtained from selected tumors, which are maintained by syngeneic passages. In this model progesterone behaves as the growth-stimulating hormone (progesterone-dependent or PD tumors), whereas estrogens induce tumor regression. Through selective treatments we were able to derive a series of progesterone-independent (PI) variants. These lines do not require progesterone treatment to grow in ovariectomized female BALB/c mice, but retain, however, the expression of ER and PR. The aim of this paper is to investigate a possible regulatory role of the progesterone receptor (PR) on PI tumor growth. ER and PR were detected by immunocytochemistry in all lines studied. They were also characterized using biochemical assays and Scatchard plots. No differences in K d of PR or ER were detected in PI variants. AR or GR were not detected in tumor samples using biochemical assays. Estradiol (5 mg silastic pellet) induced complete tumor regression in all tumors tested. We also evaluated the effects of different antiprogestins on tumor growth. Onapristone (10 mg/kg/day) and mifepristone (4.5 mg/kg/day) were able to induce complete tumor regression. The antiandrogen flutamide (5 mg silastic pellet) had no effect on tumor growth in agreement with the lack of androgen receptors. We used an in vitro approach to corroborate that the antiprogestin-induced inhibition was not attributable to an intrinsic effect. Cultures of a selected PI line were treated with PR antisense oligodeoxynucleotides (ASPR) to inhibit in vitro cell proliferation. A significant decrease of 3H-thymidine uptake was observed in cells of a PI line growing in the presence of 2.5% charcoalized fetal calf serum and 0.8–20 μg/ml ASPR. It can be concluded that the PR pathway is an essential path in the growth stimulation of PI tumors.

Downregulation of fibronectin transcription in highly metastatic adenocarcinoma cells

Silencing of fibronectin (FN) expression seems to be one of the key mechanisms underlying metastatic behaviour. An inverse correlation exists between FN expression levels and the metastatic potential of two related murine mammary adenocarcinomas, M3 and MM3. Primary cultures of M3 tumour, which is moderately metastatic to lung (40% incidence), show a conspicuous FN extracellular matrix (ECM) and high levels of FN mRNA, while primary cultures of the highly metastatic MM3 tumour (95% lung incidence) are negative for FN in immunofluorescence and show at least 40-fold lower levels of FN mRNA, only detectable by RT-PCR, with a different pattern of alternatively spliced EDI isoforms compared to M3 cells. We show that the FN promoter sequence is not altered in MM3 cells. Transfection experiments with CAT constructs indicate that silencing occurs at the transcriptional level, involving the 220-bp proximal promoter region.

Canarypox virus-mediated interleukin 12 gene transfer into murine mammary adenocarcinoma induces tumor suppression and long-term antitumoral immunity.

The antitumoral activity of recombinant canarypox virus vectors (ALVAC) expressing murine interleukin 12 (IL-12) was evaluated in the syngeneic, nonimmunogenic murine mammary adenocarcinoma model (TS/A). Seven-day preestablished subcutaneous tumors (5- to 6-mm mean diameters) were injected on days 7, 10, 14, 17, 21, and 24 with the vector ALVAC-IL12 at 2.5 x 10(5) TCID50 (50% tissue culture infective dose). Total tumor regression occurred in 40 to 50% of the treated mice. Furthermore, 100% of the cured mice were protected against a contralateral subsequent challenge with the TS/A parental cells on day 28. The ALVAC-IL12 treatment is not effective in nude mice, suggesting the critical role of T cells. CD4 and CD8 T cells infiltrated the tumors treated with ALVAC-IL12 in the BALB/c model. Furthermore, in vivo depletion of CD4+ T cells totally abrogated the induction of the long-term antitumoral immune response by ALVAC-IL12. Interestingly, some tumor growth inhibition was also observed with ALVAC-betaGal treatment and a vaccinal effect was found in 33% of the treated animals, suggesting an adjuvant effect of the vector itself. Other ALVAC vectors expressing murine cytokines (IL-2, GM-CSF, IFN-gamma) were evaluated in the same model. Major antitumoral activity was observed with ALVAC-GM-CSF. However, a combination of ALVAC-GM-CSF and ALVAC-IL12 had no synergistic effect. These results suggest that in vivo gene transfer with canarypox virus expressing IL-12 may provide an effective and safe strategy for the treatment of human cancers.

Canarypox Virus-Mediated Interleukin 12 Gene Transfer into Murine Mammary Adenocarcinoma Induces Tumor Suppression and Long-Term Antitumoral Immunity

Canarypox Virus-Mediated Interleukin 12 Gene Transfer into Murine Mammary Adenocarcinoma Induces Tumor Suppression and Long-Term Antitumoral Immunity

Greater antitumor efficacy of paclitaxel administered before epirubicin in a mouse mammary carcinoma.

Combined treatment with paclitaxel and anthracyclines is increasingly being tested in clinical practice. Epirubicin is in general administered before paclitaxel. We have investigated, using a murine mammary adenocarcinoma, whether the efficacy and toxicity of this combination is influenced by treatment sequence, different time intervals and dose intensity. The tumor was transplanted into the right hind foot of C3D2F1 female mice. Paclitaxel was administered i.p. in doses ranging from 15 mg/kg to 75 mg/kg and epirubicin (i.p. or i.v.) in doses from 9 mg/kg to 30 mg/kg. The hepatic and peritoneal toxicity observed with epirubicin administration increased in combined treatments (stronger with i.p. than i.v. epirubicin administrations) and was dose-dependent. When paclitaxel and epirubicin were administered simultaneously or paclitaxel was given 24 h before epirubicin, the same tumor growth delays were obtained in all groups. A smaller effect was observed when paclitaxel was administered 24 h after epirubicin. Increasing the epirubicin or paclitaxel dose led to higher tumor growth delays but also an increased toxicity. In conclusion, in this experimental model, the administration of 45 mg/kg paclitaxel before 15 mg/kg epirubicin was very effective and the increased toxicity can be limited by introducing an interval of 24 h between drug administrations. These results should be considered when designing clinical trials.

Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo.

Suicide genes such as cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) encode products that convert nontoxic substances (prodrugs) into toxic metabolites. Suicide gene transfer is currently being used in cancer therapy or can be used as a safety modality. To analyze the reliability of suicide genes as a safety modality for a vaccination study with viable cytokine/B7 gene-modified tumor cells, the individual and combined efficacy of the two suicide genes was compared for in vitro and in vivo cell killing of a murine mammary adenocarcinoma cell line (TS/A). To adapt the system to an in vivo gene delivery situation, bulk cultures cotransfected with the CD and TK gene were used instead of selected clones. In vitro, both CD and TK conferred sensitivity to the respective prodrug but the combined cytotoxic effects of both gene products were always superior. For in vivo analysis BALB/c mice were injected subcutaneously with CD- and TK-modified TS/A cells, treated with prodrugs, and tumor size was evaluated for a period of 100 days. In the in vivo situation the combination of both enzyme/prodrug systems was again most effective. The highest single concentration of 5-FC (500 mg/kg) or GCV (100 mg/kg) was not able to fully protect the animals from developing tumors, whereas a combination of 5-FC (250 mg/kg) and GCV (50 mg/kg) resulted in complete tumor eradication. In nude mice treated in the same way, most CD/TK tumors could not be eliminated. Furthermore, BALB/c mice cured of TS/A-CD/TK tumors developed a systemic tumor immunity against challenge with parental TS/A cells. These findings indicate that reliable tumor elimination by the suicide genes depends on T cells. The cooperative effect of both suicide genes was confirmed in vitro with the human renal cell carcinoma line RCC26. We conclude that TK and CD together, but neither gene alone, act as a safety mechanism for the elimination of tumor cells in a reliable fashion and suggest that a rapid and quantitative antigen release by effective TK- and CD-mediated tumor destruction is necessary for T cell immunity to develop.

Differential modulation by dietary n-6 or n-9 unsaturated fatty acids on the development of two murine mammary gland tumors having different metastatic capabilities

It is widely known that dietary lipids can modify the ability of different cancers to grow up and metastasize, especially mammary gland tumors. However, it is still unclear whether n-6 fatty acids behave as tumor promoters in this gland cell population. The effect of different nutritional polyunsaturated fatty acids (PUFAs) on tumor growth parameters of two transplantable murine mammary gland adenocarcinomas of low and high metastatic ability was tested on hosts fed diets with corn oil (CO) rich in 18:2 n-6, evening primrose oil (EPO) containing 18:3 n-6 (GLA) and a third formula supplemented with olein (O) 18:1 n-9, which induces an essential fatty acid deficiency (EFAD). Tumor growth parameters were not adversely affected in the corn oil group with respect to stock-fed controls. Furthermore, metastatic spreading diminished in this group. EPO showed a moderate antitumor activity whereas the n-9-enriched diet showed no clear-cut effects. In both mammary gland tumors, n-6 fatty acid-rich lipids formulae, containing GLA and linoleic acid, were not tumor promoters. On the contrary, both exhibited anticancer activity.

Combination of cisplatin and radiation in cell culture: effect of duration of exposure to drug and timing of irradiation.

Responses to the combination of cisplatin (CDDP) and radiation in experimental and clinical studies have been reported to vary from high radiosensitization to clear sub-additivity. We examined the combined effect of CDDP with ionizing radiation in both murine mammary adenocarcinoma (EMT-6) and human ovarian carcinoma (OV-1063) cells with special reference to the duration of CDDP exposure and timing of irradiation. Cell survival was measured with a colorimetric assay of cell density. The nature of interaction of cisplatin and radiation was evaluated using isobolograms and a combination index (CI). Exposure of both cell lines to CDDP for 24 hr before irradiation yielded an additive or slightly sub-additive response only if the exposure was extended for a few more hours after irradiation. In EMT-6 cells, the combination of radiation with subsequent continuous as well as short-term (4 to 6 hr) CDDP treatment was found to have a clear sub-additive effect; dose escalation of each modality reduced the additional effect of the other. The sub-additive effect may be explained by a radiation-induced arrest of cells in late S phase, which was dose- and time-dependent. Post-radiation exposure to CDDP further increased the S-phase arrest. In contrast, a 2 hr post-radiation drug exposure resulted in a supra-additive combined effect. Our results stress the crucial role of the timing and the doses of both modalities as well as the duration of post-radiation drug exposure on their combined effect.

Combination of cisplatin and radiation in cell culture: Effect of duration of exposure to drug and timing of irradiation

Responses to the combination of cisplatin (CDDP) and radiation in experimental and clinical studies have been reported to vary from high radiosensitization to clear sub-additivity. We examined the combined effect of CDDP with ionizing radiation in both murine mammary adenocarcinoma (EMT-6) and human ovarian carcinoma (OV-1063) cells with special reference to the duration of CDDP exposure and timing of irradiation. Cell survival was measured with a colorimetric assay of cell density. The nature of interaction of cisplatin and radiation was evaluated using isobolograms and a combination index (CI). Exposure of both cell lines to CDDP for 24 hr before irradiation yielded an additive or slightly sub-additive response only if the exposure was extended for a few more hours after irradiation. In EMT-6 cells, the combination of radiation with subsequent continuous as well as short-term (4 to 6 hr) CDDP treatment was found to have a clear sub-additive effect; dose escalation of each modality reduced the additional effect of the other. The sub-additive effect may be explained by a radiation-induced arrest of cells in late S phase, which was dose- and time-dependent. Post-radiation exposure to CDDP further increased the S-phase arrest. In contrast, a 2 hr post-radiation drug exposure resulted in a supra-additive combined effect. Our results stress the crucial role of the timing and the doses of both modalities as well as the duration of post-radiation drug exposure on their combined effect. Int. J. Cancer 75:635–642, 1998. © 1998 Wiley-Liss, Inc.

The immune response elicited by mammary adenocarcinoma cells transduced with interferon-gamma and cytosine deaminase genes cures lung metastases by parental cells.

The parental cells of the TSA murine mammary adenocarcinoma (TSA-pc) were transfected with both the interferon-gamma (IFN-y) gene and the cytosine deaminase (CD) suicide gene to obtain a therapeutic vaccine active against TSA-pc lung metastases. Even in the absence of treatment with the prodrug 5-fluorocytosine (5-FC), the local growth of double transfectants (CD-y clones) was inhibited by a marked recruitment of granulocytes and macrophages. In mice harboring TSA-pc micrometastases, therapeutic vaccination with either IFN-gamma or CD single transfectants reduced the number of lung nodules, whereas CD-gamma double transfectants abrogated metastasis growth in up to 80% of mice. Treatment of mice with 5-FC did not alter the curative efficacy of CD-gamma double-transfectant cells. By contrast, in mice vaccinated with CD single-transfectant cells, 5-FC treatment caused a significant loss of their curative activity. Host T cells played an active role in the cure of lung metastases, because vaccination of nude mice with CD-gamma cells was uneffective.

Characterization of the specific response to serotonin of mouse tumour-feeding arterioles

Purpose: To investigate the role of tumour versus non-tumour factors in the specific response to serotonin (5-HT) of tumour-feeding arterioles (TFA). Materials and methods: Using mouse models of intra-vital microscopy, the response to topical administration of 5-HT was studied in arterioles feeding tumours: fibrosarcoma (Meth A), murine mammary adenocarcinoma (EMT6) and human colo-rectal carcinoma (HRT18) intra-cutaneously implanted. Results: For all types of tumour, 5-HT induced a far more pronounced constriction of TFA than of control arterioles. The presence of a tumour implanted in the connective tissue between the skin and the cremaster muscle also affected the reactivity of muscle arterioles. Conversely, the response to serotonin by neovessels grown after implantation of an exogenous element under the skin did not differ from that of control arterioles. Conclusions: Changes in reactivity to serotonin were not dependent on the type of tumour implanted in the skin and were not present for a non-tumour implant. The presence of the tumour can alter the reactivity of vessels from tissue in contact with the tumour even if these vessels did not feed the tumour. This phenomenon is local and was not found in the vessels at a distance from the tumour.

Inhibition of lung colonisation of a mouse mammary carcinoma by therapeutic vaccination with interferon-alpha gene-transduced tumor cells.

A spontaneously metastatic murine mammary adenocarcinoma, TSA, has been transduced with the gene for interferon alpha1 (IFN-alpha). Transfectants were used for the immunotherapy of mice bearing lung colonies induced by the intravenous inoculation of non-transduced parental cells. A significant reduction in the number of tumor colonies was obtained when repeated subcutaneous administrations of mitomycin C-blocked transfectant cells were given, commencing 3 days after an intravenous challenge with TSA cells. Intraperitoneal vaccination induced a stronger anti-tumor response than subcutaneous vaccination, and the proportion of tumor-free mice reached 50%. The potency of IFN-alpha transfectants was similar to that of IFN-gamma transfectants previously obtained from TSA. Admixture of IFN-alpha and IFN-gamma transfectant cells in the same vaccine did not increase the curative effect over that of single vaccines. In nude mice vaccination with IFN-alpha or IFN-gamma transfectants did not lead to a reduction in the number of lung colonies, indicating that an intact T cell response was required for the therapeutic effect observed in immunocompetent mice.