Early neoplastic and metastatic mammary tumours of transgenic mice detected by 5-aminolevulinic acid-stimulated protoporphyrin IX accumulation.

A photodynamic technique for human breast cancer detection founded upon the ability of tumour cells to rapidly accumulate the fluorescent product protoporphyrin IX (PpIX) has been applied to transgenic mouse models of mammary tumorigenesis. A major goal of this investigation was to determine whether mouse mammary tumours are reliable models of human disease in terms of PpIX accumulation, for future mechanistic and therapeutic studies. The haeme substrate 5-aminolevulinic acid (5-ALA) (200 mg kg(-1)) was administered to mouse strains that develop mammary tumours of various histological subtypes upon expression of the transgenic oncogenes HRAS, Polyoma Virus middle T antigen, or Simian Virus 40 large T antigen in the mammary gland. Early neoplastic lesions, primary tumours and metastases showed consistent and rapid PpIX accumulation compared to the normal surrounding tissues, as evidenced by red fluorescence (635 nm) when the tumours were directly illuminated with blue light (380-440 nm). Detection of mouse mammary tumours at the stage of ductal carcinoma in situ by red fluorescence emissions suggests that enhanced PpIX synthesis is a good marker for early tumorigenic processes in the mammary gland. We propose the mouse models provide an ideal experimental system for further investigation of the early diagnostic and therapeutic potential of 5-ALA-stimulated PpIX accumulation in human breast cancer patients.

A genetically myeloablated MPS VII model detects the expansion and curative properties of as few as 100 enriched murine stem cells.

Causes of transplantation failures are often difficult to assess due to our inability to monitor hematopoietic stem cell (HSC) homing, distribution, and amplification in situ. We have developed a mouse model that permits histochemical localization of 1000-fold enriched HSC and quantification of their long-term expanded progeny in situ. The mice are genetically myeloablated (c-kit receptor mutated, W41/W41) and are beta-glucuronidase null (GUSB ; gus(mps)/gus(mps)). The GUSB- mice with mucopolysaccharidosis type VII (MPS VII), like a large number of human patients with similar diseases, have systemic lysosomal storage disease that leads to premature death. Congenic GUSB+, Lineage(lo), Sca-1(hi), c-Kit(hi), Hoechst(lo) HSC, at doses of 30, 100, 250, and 425 cells, implanted and amplified in adult W41/W41, gus(mps)/gus(mps) recipients in a dose-dependent manner. At autopsy, primary recipients of 100 and 425 donor cells had histologically identifiable donor GUSB+ cells in multiple sites and showed both myeloid and lymphoid expansion in bone marrow. Donor cells were rare in the liver and spleen of 100-cell recipients, but lysosomal storage was significantly reduced. The life span was significantly extended in engrafted recipients of 250 (36.7 +/- 3.84 weeks,p = 0.0316) and 425 (40.7 +/-1.53 weeks,p = 0.0033) cells compared to untreated mice (26.4 +/- 1.53 weeks). Secondary hosts of marrow from the recipients of 425 cells demonstrated continued expansion of the GUSB+ cells. Results indicate the genetically myeloablated MPS VII mice can be used to trace and enumerate donor cells long-term and to follow early engraftment events in situ.

beta2-microglobulin dependence of the lupus-like autoimmune syndrome of MRL-lpr mice.

MRL-lpr/lpr mice develop a distinctive immunologic disease characterized by accumulation of unusually large numbers of T cells in the peripheral lymphoid organs. Most of the accumulating T cells express an alpha beta-TCR but are peculiar in that they express neither CD4 nor CD8 co-ligands. Concurrent with lymphoaccumulation of such double negative (DN) T cells, MRL-lpr/lpr mice develop a lethal systemic lupus erythematosus-like autoimmune syndrome. This study focuses on the role of MHC class I molecules in this latter pathologic process. Highly backcrossed class I molecule-deficient MRL and MRL-lpr mice carrying a functionally defective allele of the gene beta 2-microglobulin (B2m) were produced. Class I deficient MRL-lpr/lpr mice demonstrated a substantial reduction in DN T cells, confirming other reports indicating that most DN T cells arise from progenitors positively selected on MHC class I molecules. Significantly, class I-deficient MRL-lpr/lpr mice also demonstrated a diminution of every autoimmune disease indicator analyzed including hypergammaglobulinemia; autoantibodies including anti-DNA, anti-Smith antigen, and rheumatoid factor; and glomerulonephritis. The results indicate that class I-dependent T cells are crucial not only for the development of DN T cells, but for multiple features of the MRL-lpr/lpr systemic lupus erythematosus syndrome. Moreover, the pattern of hypergammaglobulinemia suggests that the requirement for MHC class I proteins is restricted temporally to later stages of the disease.

The therapeutic efficacy of murine anti-tumor T cells: freshly isolated T cells are more therapeutic than T cells expanded in vitro.

Adoptive immunotherapy (AIT) involving transfer of tumor-sensitized T lymphocytes in combination with cyclophosphamide (CY)-injection results in the eradication of the C57BL/6J (B6) rhabdomyosarcoma, 76-9 and is associated with the accumulation of a large number of tumor-infiltrating lymphocytes (TIL). Using immune spleen cells (ISC) from B6 and congenic B6. PL. Thy-1a mice, it was shown that most (> or = 97%) of the TIL were donor-derived. This in situ increase in donor-derived T cells was confirmed by using positively-selected Thy- 1.1+ and Thy- 1.2+ TIL for AIT after isolating them from regressing tumors and expanding them in rIL-2. The extent of CD8+ TIL expansion in vivo correlated with the numbers of TIL adoptively transferred and this in turn determined the degree of anti-tumor effects. It was evident, however, that these in vitro-expanded TIL expressing mRNA for TNF alpha and IFN gamma were qualitatively different and therapeutically less efficacious than the T cells associated with ISC or with freshly-isolated TIL. Unlike freshly isolated TIL that expressed specific cytotoxicity towards the 76-9 targets in vitro, IL-2 expanded TIL killed 76-9 cells and unrelated tumor targets to the same extent. A cytotoxic CD8+ T cell line derived from ISC and selected for activity against the 76-9 tumor cells showed no therapeutic efficacy. The data suggest that, in this tumor model, expansion of CD8+ T cells in vitro selects against anti-tumor efficacy.

Qualitative and quantitative intratumoral changes in gene expression following cyclophosphamide injection and the adoptive transfer of T cells: the potential contribution of tumor-associated macrophages.

Adoptive immunotherapy (AIT) of mice bearing the MCA/76-9 rhabdomyosarcoma in combination with cyclophosphamide (CY) injection results in the permanent regression of tumors. This report is concerned with changes in the tumor-associated macrophage (TAM) population and the influence of both CY injection and CY/AIT on their potential functions. Sequential analyses of FcR, MAC-I and Class-II MHC antigen expressed by tumor-associated cells (TAC) showed that CY injection or CY/AIT induced marked increases in the proportions of all 3 parameters as compared with the relatively stable levels in progressing tumors. These changes were time- and treatment-related. The mean MAC-I fluorescence (antigen density per cell) increased nearly 2-fold by 48 hr after CY injection, regardless of subsequent AIT. In contrast, the density of Class-II antigen per cell declined by as much as 75% within 48 hr after CY injection and did not recover by 7 days. This initial decline was also seen after CY/AIT and was followed by a rapid recovery to near-normal values by day 7. Northern analysis of RNA isolated from whole tumor tissue indicated wide fluctuations in expression of the typical macrophage genes encoding the proteins MAC-I, IL-I alpha, IL-I beta, TNF alpha, IA beta and c-fms. However, with the exception of MAC-I and IL-1 alpha/IL-1 beta mRNA, the modifications appeared to be qualitative rather than representing changes in the proportions of TAM. The data suggest that the changes in membrane antigen and gene expression by TAM reflect a complex interaction between TAM and their environment, in particular tumor cells and tumor-infiltrating lymphocytes. In addition, it is evident that CY injection per se is responsible for defined fundamental changes that presumably influence the outcome of AIT.

Synergistic interaction of bacterial lipopolysaccharide and the monocyte-macrophage colony-stimulating factor: potential quantitative and qualitative changes in macrophage-produced cytokine bioactivity.

In this brief definitive report, we show that over a 6-h period and under serum-free conditions, recombinant monocyte-macrophage colony-stimulating factor 1 (rCSF-1) and lipopolysaccharide (LPS) synergize and induce macrophages to express higher levels of mRNA for interleukin 1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 and to release more bioactivity than macrophages treated with LPS alone. This synergy was regulated by the amount of LPS in the culture medium. Paraformaldehyde-fixed macrophages like-wise showed augmentation of IL-1 activity, but whereas all of the bioactivity associated with the fixed macrophages could be neutralized by anti-IL-1 alpha antibody only approximately 40% of the supernate activity could be attributed to IL-1 alpha. Preliminary data suggest that the augmenting effect induced by CSF-1 cannot be explained solely on a quantitative basis because the addition of rIL-1 alpha to supernates of macrophages treated with LPS alone or with the combination of LPS and CSF-1 resulted in an increase in thymocyte mitogenic activity to a level that could not be explained on an additive basis. Although the supernates contained TNF and IL-6, antibody neutralization assays made it unlikely that these were directly responsible for the augmenting effect. These results suggest that CSF-1 not only enhances basic genetic responses induced by LPS alone but also may induce a mechanism that amplifies cytokine bioactivity.

Differential in situ expansion and gene expression of CD4+ and CD8+ tumor-infiltrating lymphocytes following adoptive immunotherapy in a murine tumor model system.

In previous reports, we demonstrated that adoptively transferred T cells homed to the tumor site (among other sites) and that amplification of immune responses occurred in situ leading to the generation of cytotoxic CD8+ tumor-infiltrating lymphocytes (TIL) and macrophages. The present report extends these findings and shows that following adoptive immunotherapy (AIT) of mice bearing the immunogenic transplanted methylcholanthrene-induced rhabdomyosarcoma (MCA/76-9) there was a differential expansion of CD4+ and CD8+ TIL, the numbers peaking on days 6 and 8, respectively. At this time, CD8+ TIL accounted for the majority of Thy-1+ cells. Northern analyses of RNA extracted from positively selected (by panning) Thy-1+, CD8+ and CD4+ TIL isolated 8 days after AIT indicated the following: in five separate experiments, CD4+ cells expressed three- to sixfold more interleukin (IL)2 mRNA and six- to eightfold more IL6 mRNA than CD8+ cells, while CD8+ TIL expressed three- to sixfold more IL2 receptor (IL2R) mRNA and four- to sixfold more interferon-gamma mRNA than CD4+ cells. TIL cultured in 10% fetal bovine serum failed to release IL2 over a 24-h period, whereas both IL6 and interferon-gamma activities were demonstrable. The level of IL2R mRNA expression was reflected by a vigorous proliferative response of CD8+ TIL to exogenous recombinant IL2 and only a low response by CD4+ cells suggesting that most of the CD4+ TIL were in the resting stage. This was confirmed when it was shown that the incubation of panned CD4+ TIL with IL2 supplemented with irradiated spleen cells and "spent" 76-9 tumor culture supernatant (as a source of antigen) induced expansion of TIL resulting in a population consisting of greater than 90% CD4+ TIL. The overall data suggest that the relatively deactivated state of the CD4+ TIL at this particular time reflects the status of the rejection process in terms of the absence or low concentration of stimulating tumor-associated antigen.

Tumor-derived products induce Il-1 a, Il-1 b, Tnf a, and Il-6 gene expression in murine macrophages: distinctions between tumor- and bacterial endotoxin-induced gene expression.

The ability of progressing tumors to regulate host physiology is an important consideration in our understanding of tumor-host relationships. Previous data indicated that several lines of murine sarcoma cells produced one or more activities that were able to regulate both Il-1 a and Il-1 b gene transcription in macrophages (MO). We now describe an indepth analysis using Northern analysis and bioassays and show that two of these tumors produce one or more activities that when incubated with peritoneal MO result in the transcription of the Il-1a, Il-1 b, Tnf a, and Il-6 genes. Concordant with the Northern analyses was the finding that interleukin-1 (IL-1) and tumor necrosis factor (TNF) biological activities were detected in lysates of induced MO, fixed MO, and supernates of MO cultures. Induction of gene expression was shown to be distinct from that induced by bacterial endotoxin or lipopolysaccharide by a number of criteria. The data suggest that tumor cell products may play an important role in regulating several host physiological processes, particularly those involving Il-1a, Il-1 b, Tnf a, and Il-6 gene expression.

Regulation of systemic macrophage IL-1 gene transcription: the involvement of tumor-derived macrophage growth factor, CSF-1.

Previous data indicated that progressive growth of the C57BL/6J sarcoma, MCA/76-9, was associated with a systemic increase in the level of IL-1 activity in peritoneal and tumor-associated macrophages. In this paper, the hypothesis was tested that macrophage IL-1 alpha and beta gene expression was regulated by the specific macrophage growth factor, CSF-1, produced by tumor cells. The data indicated that all of the nine C57BL/6J, BALB/cJ, and C3H/HeJ sarcomas tested transcribed the CSF-1 gene. The tumor cell culture supernates induced proliferation of bone marrow cells that differentiated into macrophages. Moreover, proliferative activity of conditioned medium and the ability to induce macrophage differentiation were abrograted by neutralization with the 5A1, rat-anti-mouse monoclonal anti-CSF-1 antibody. Conditioned medium from tumor cell cultures, as well as recombinant CSF-1 and L cell-conditioned medium, induced transcription of the IL-1 alpha and beta genes, the latter being more strongly expressed. IL-1 gene transcription was not induced after neutralization of the culture supernates by the monoclonal antibody, 5A1. The overall data would support the hypothesis that the pathway in vivo leading to the increased IL-1 expression by macrophages is regulated by at least one tumor cell product, CSF-1.

Adoptive immunotherapy is suppressed in C57BL/6J and B6.C-H-2bm12 mice following recognition of congenic class II MHC antigen determinants.

In previous reports, we have shown that adoptive transfer of tumor-sensitized T (immune) cells to tumor-bearing mice that have received a prior injection of cyclophosphamide (CY) results in the induction of permanent tumor regression in syngeneic strains. It has also been shown that adoptive immunotherapy results in an increased expression of class II MHC antigens (Ia) by macrophages at the tumor site and in the peritoneal cavity and is associated with expansion of CD4+ and CD8+ T cells at the site of tumor regression. In this report, we use the Ia mutant strain, B6.C-H-2bm12, and congenic C57BL/6J (B6) mice to determine the relative importance of Ia expression in regulating amplification of immune responses following adoptive immunotherapy and to test the hypothesis that recognition of congenic Ia determinants will result in the induction of suppressor mechanisms that down-regulate active immunity. The data indicated that the adoptive transfer of immune congenic T cells (B6 immune cells into CY-treated tumor-bearing bm12 mice and vice-versa) down-regulated active immunity, while the transfer of syngeneic immune cells resulted in permanent tumor regression. By using radiation-chimeric mice, it was shown that down-regulation was associated with incompatibility of the transferred immune T cells and bone-marrow-derived cells (putatively expressing the Ia haplotype of donor-derived macrophages) and the appearance of long-lived splenic suppressor cells. Suppression per se was shown to be induced in response to the Ia difference between the two strains and not in response to the MCA/76-9 sarcoma, which appears to be one of the few tumors that can induce active immunity in both the syngeneic and congenic strains without obvious subsequent down-regulation by suppression.

Partial characterization of a low-molecular-weight, macrophage-derived inhibitor of DNA synthesis: a possible immunoregulatory molecule.

It has been previously reported that tumor-associated macrophages isolated after combination therapy contained an activity that inhibited thymocyte proliferation in the IL-1 comitogenic assay. The present report shows that this macrophage-derived inhibitory factor (MDIF) inhibits DNA synthesis in diverse adherent and nonadherent cells in vitro. The elution pattern seen after chromatography using Bio-Gel P10 or Sephadex G-25 columns indicated a molecular size of 3-6.5 kDa. However, full activity was retained when MDIF was passed through an Amicon filtration membrane having a cut off of 500 Da. It was also seen that other molecules less than 500 Da, such as thymidine, thimerosal, and PGE2, each of which was also shown to be growth inhibitory, eluted in a size range similar to that of MDIF after chromatographic separation. Ion exchange chromatography confirmed that MDIF was distinct from thymidine, and a radioimmunoassay indicated that PGE2 was not responsible for MDIF activity. Little or no inhibitory activity could be detected in macrophages isolated from progressing tumors or from tumors excised after cyclophosphamide treatment of tumor bearers. Given that this inhibitory activity was associated with macrophages derived from tumors induced to regress by combination therapy, the possibility is discussed that MDIF may play an important role during the regulation of immune reactions at the tumor site.

The immunological mouse mutants nude (nu) and rhino (hrrh) generate cytotoxic effector cells following adoptive immunotherapy but fail to reject a transplanted tumor.

Adoptive immunotherapy, consisting of cyclophosphamide injection and the i.v. transfer of tumor-sensitized T cells, resulted in rejection of the immunogenic fibrosarcoma, MCA/76-9, by syngeneic C57BL/6J (B6) mice. The same treatment of tumor-bearing congenic immunodeficient mice, homozygous for the deleterious mutations nude (nu) and rhino (hrrh), did not result in tumor rejection. Paradoxically, the intratumor and intrasplenic changes taking place in each of the three strains after therapy were indistinguishable. There was an increase in Thy-1+, Ly-2+, or L3T4+ cells at the tumor site 8 days after adoptive immunotherapy and a similar increase in Thy-1+ cells in the spleen. Moreover, the T cells isolated from the tumors or spleens from each genotype were shown to be specifically cytotoxic in vitro as well as in an in vivo Winn assay. Further evidence that immune amplification had occurred in the immunological mutant mice was provided by experiments showing (a) the ability of spleen cells from tumor-bearers and those tested after therapy to produce IL-2 in response to Con A stimulation and (b) an increase in class II-MHC antigen expression by tumor-associated macrophages. The data suggest that, although amplification of antitumor immune responses occurred in the immunological mutants, the absence of a critical host factor limited the potency of the antitumor response.

Activation of the IL-1 pathway during amplification of immune responses in tumor-bearing mice.

Previous work identified certain components of the immunological network that had been activated following the adoptive immunotherapy of tumor-bearing mice. The present report shows that part of the activation process involves the IL-1 pathway. Tumor-associated macrophages (TAM) from C57BL/6J mice bearing the immunogenic sarcoma, MCA/76-9, and tumor-bearers injected with cyclophosphamide (CY) or CY plus the intravenous transfer of tumor-sensitized lymphocytes showed relatively high levels of intracellular (IC) IL-1, as demonstrated in the mitogenic and comitogenic assays. Gel chromatography of IC IL-1 and extracellular (EC) IL-1 from TAM induced to secrete IL-1 by stimulation with lipopolysaccharide indicated a single peak of activity of similar molecular size. The active fractions of the EC IL-1 were found to increase in activity as they were diluted to a maximum of 1/64, beyond which IL-1 activity declined. Fractions of the IC IL-1 showed no increased activity on dilution. Filtrates (less than 10 kDa) obtained on concentration of the IC and EC IL-1 samples prior to fractionation were shown to contain an activity (3-5 kDa) that inhibited the uptake of [3H]TdR by thymocytes in the mitogenic and comitogenic assays. Membrane-bound IL-1 activity also was expressed by TAM and this coincided with the previously reported peak Ia expression by these cells. TAM were also shown to induce strong proliferative responses by allogeneic lymphocyte. Systemic amplification of antitumor responses was detected in mice bearing progressing tumors and in those that had received combination therapy as measured both by increases in free IL-1 in the peritoneal cavity and IL-1 within the peritoneal macrophages. These observations indicate that in addition to enhancement of Ia expression, the IL-1 pathway also is activated and amplified systemically in this model system of tumor progression and rejection.

The inability of adoptive immunotherapy to eradicate tumor cells in radiation-induced chimeric mice: the possible down-regulation of intratumor immune amplification.

Tumors were eradicated following adoptive immunotherapy of (C57BL/6J X DBA/2J) F1 (B6D2F1) hybrid mice bearing the C57B1/6J (B6) sarcoma, MCA/76-9, by treatment with cyclophosphamide (CY) and adoptively transferred tumor-sensitized B6D2F1 T cells. Identical treatment of B6----B6D2F1 or DBA----B6D2F1 chimeric tumor-bearing mice resulted in temporary tumor regression only. Immunotherapy in both systems resulted in the appearance at the tumor site of large numbers of T lymphocytes and macrophages, which were shown to be derived from the adoptively transferred donor immune B6D2F1 cells or from the original reconstituting bone marrow respectively. The TAC and TAL isolated from either B6D2F1 or chimeric mice expressed potent toxicity in vitro towards tumor target cells. In addition, TAC from both systems inhibited tumor growth in a Winn assay in an immunologically specific manner. Suppressor cells detected in spleens 8 days after CY injection of normal B6, DBA, B6D2F1 hybrid and chimeric mice and also after adoptive immunotherapy of tumor-bearing B6, B6D2F1 and chimeric mice and were shown to persist in the spleens of chimeric mice for at least 31 days after adoptive immunotherapy. In contrast, spleen cells taken 31 days after therapy of B6D2F1 mice did not contain detectable suppressor cells and were able to induce tumor eradication when adoptively transferred to CY-treated tumor-bearing B6D2F1 mice. The possibility is discussed that two forms of suppressor mechanisms are induced after adoptive immunotherapy of chimeric mice and that they may down-regulate donor T cell responses at the tumor site.

Amplification of immune T lymphocyte function in situ: the identification of active components of the immunologic network during tumor rejection.

Previous data had indicated that sarcoma-bearing mice receiving combination therapy consisting of a single i.p. injection of cytoxan (CY) and an i.v. injection of tumor-sensitized T cells (immune cells) rejected the neoplasm. The reaction was immunologically specific and dependent on donor T cells. This report is concerned with the hypothesis that the transfer of immune cells results in the amplification of T cell responses at the tumor site. Using C57BL/6J mice bearing the syngeneic rhabdomyosarcoma MCA/76-9, we show that 6 to 9 days after combination therapy those components usually associated with the immunologic network were present at the tumor site. Tumor-associated macrophages (TAM) and lymphocytes (TAL) were shown to produce IL 1 and IL 2, respectively. The TAM expressed Ir gene products (Ia) and were able to present the synthetic polymer GAT to specifically sensitized lymphocytes. In addition, it was demonstrated by in situ labeling with 3H-TdR that lymphocytes associated with the regressing tumors were proliferating. The peak incorporation occurred 7 days after therapy, 24 hr before a significant increase in the T cell content of the tumors. The data indicate that those facets of the immunologic network necessary for amplification were present at the site of rejection.

Distribution of tumor-sensitized cells during the induction of permanent tumor regression by chemoimmunotherapy: the use of glucose phosphate isomerase as a marker.

Using a previously described tumor model system, in which permanent tumor eradication was induced by treatment with cyclophosphamide (CY) and adoptively transferred tumor-sensitized (immune) spleen cells, we determined the distribution of the transferred cells in recipient mice. The experiments were carried out using the C57BL/6J (B6) sarcoma, MCA/76-9, and B6 and congeneic B6.CAST.Gpi-Ia strains, which are homozygous for the glucose phosphate isomerase (Gpi) alleles Ib and Ia respectively. Thus, tumor-bearing mice of the one strain were injected with CY (200 mg/kg) 10 days after implantation of tumor cells and 4 h later with an intravenous injection of 50 X 10(6) immune cells from presensitized mice of the other strain. It was observed that the donor-type isozyme was expressed in the tumor within 24 h of injection and continuously up to the time at which the tumor mass had totally regressed (11 days). The tumor-associated macrophage and neutrophil fractions were shown to express host-type isozyme, while the lymphocyte fraction expressed isozyme of both host and donor type. The donor-type isozyme did not appear in the blood until day 4 but persisted thereafter for long periods. The spleen and lymph nodes expressed donor-type isozyme by 1 day after chemoimmunotherapy and remained positive for the duration of the experiments. The adoptive transfer of normal (non-immune) spleen cells gave similar results, except that donor-type isozyme did not persist at the tumor site. The conclusion reached was that the injection of either normal or immune donor spleen cells after CY treatment of B6 mice gave rise to chimeric mice, in which the distribution of normal and immune donor cells was basically similar in terms of overall isozyme expression. However, only immune donor cells resulted in the appearance of both donor and host T cells at the tumor site, suggesting amplification of both donor and host lymphocyte function either at the tumor site or in the lymphoid tissues.