Prostaglandin E2 receptor heterogeneity and dysfunction in mammary tumor cells

We have reported previously that murine mammary tumor cell subpopulations isolated from one spontaneous adenocarcinoma are heterogenous in terms of prostaglandin E2 (PGE2) synthetic capacity. We have also shown that tumor-PGE2 contributes to the ability of these cells to grow and metastasize in vivo (Fulton and Heppner: Cancer Research 45:4779-4784, 1985). In the present study, we have asked whether exogenous PGE2 has direct effects on the proliferation of these cells in vitro and if such responses can be attributed to the capacity of these cells to 1) bind PGE2 and 2) activate adenylate cyclase via the PGE2 receptor. We report that PGE2, at concentrations below 1 x 10(-5) M, does not affect the proliferation rate of these cells. This unresponsiveness is not due to the absence of receptors for PGE2. However, marked heterogeneity in receptor binding and function was detected in these closely related cell lines. Two metastatic lines (66 and 410.4) have high-affinity receptors for PGE2 (average Kd = 4.3 x 10(-9) M/L and 4.2 x 10(-9) M/L, respectively) and similar binding capacities (4.1 x 10(-4) and 2.9 x 10(4) binding sites, respectively). Two nonmetastatic lines, 410 and 67, have receptors with lower affinity (Kd = 8.3 x 10(-9) M/L and 1.6 x 10(-7) M/L, respectively) and binding capacities of 2.8 x 10(5)/410 cell or 7.3 x 10(4)/67 cell. A third nonmetastatic line (168) exhibits no specific binding. PGE2 receptor stimulation leads to elevated intracellular cAMP in lines 66, 410, and 67. Line 410.4 cells appear to have a functional lesion in the PGE2 receptor resulting in a failure to elevate cAMP in response to receptor occupancy. Adenylate cyclase can, however, be activated in these cells by cholera toxin, NaF, or forskolin. In comparison to the other cell lines, line 168 cells respond poorly to all cAMP-stimulating agents. Thus, we have found that PGE2 binding is a heterogenous property for these cells, and, in addition, we have identified an apparent uncoupling of PGE2 receptor to the adenylate cyclase system in one cell line.     

Inhibition of experimental metastasis with indomethacin: role of macrophages and natural killer cells

The metastatic capacity of murine mammary tumor line 410.4 is greatly increased by treatment of the host with asialo-GM1 antiserum (5-fold), 2-chloroadenosine (4-fold) or k-carrageenan (2.5-fold). This suggests that both NK cells and macrophages contribute to control of metastatic dissemination. The metastatic potential of these cells is associated with the synthesis of high levels of prostaglandin E2 (PGE2) (1). When line 410.4 cells are cultured in vitro in the presence of the prostaglandin synthesis inhibitor indomethacin (INDO) 1 microM) their subsequent lung colonization ability (experimental metastasis) is reduced by 50-90% as compared to solvent-treated cells. The inhibitory effect of INDO is highly dependent on the presence of asGM1 positive cells, and is compromised to a lesser extent by treatments directed towards macrophages. The INDO-mediated inhibition is neither due to differential arrest of tumor cells in the lung nor does it appear to be due to shifts in the replication cycle.     

Effect of ouabain resistance on human diploid fibroblasts carrying other genetic variants.

Human skin fibroblasts from a Lesch-Nyhan male with the G6PD A variant and those from a female with the cri-du-chat deletion have been treated with ethyl methane sulfonate and subjected to ouabain selection. Sixteen ouabain-resistant clones were obtained from 1.2 × l0⁸ cells plated in 10^?6 M ouabain. Some variation in degree of ouabain resistance was noted among subclones. The ouabain-resistant phenotype was stable and was associated with G6PD and HGPRT phenotypes and karyotypes similar to the parent strains except that tetraploidy characterized 2 of 5 of the clones karyotyped. The ouabain-resistant cells were used to study the influence of the mutation leading to ouabain resistance on the transfer of nucleotides from cell to cell. Results indicate that contact-feeding of nucleotides is not dependent upon the ouabain-sensitive Na⁺K⁺ transport system. It is suggested that ouabain resistance may provide a simple assay to detect agents mutagenic for human cells, and that cells carrying both HGPRT as well as ouabain-resistant markers are ideal for cell hybridization with strains having no selectable markers.     

A low-level expression of human MUC1 mucin enhances lethality of murine tumor cells

We report here the development of a mouse mammary adenocarcinoma cell line containing full-length human MUC1 cDNA that can be more lethal than the parental cell line. The metastatic murine mammary adenocarcinoma cell line 410.4 was transfected with cDNA coding for a 42-tandem-repeat version of human MUC1. Two cell lines were selected, one for stable, high expression in vitro of cell-surface MUC1 (GZHi) and one for stable, low expression in vitro of cell-surface MUC1 (GZLo). Following subcutaneous challenge of CB6F1 mice with various doses of tumor cells, GZHi tumors showed loss of MUC1 expression; negligible amounts of serum MUC1 mucin were detected and the mice survived longer than mice challenged with GZLo or wild-type (410.4) tumor cells. Mice challenged with GZLo tumor cells had shorter survival times than mice challenged with either GZHi or 410.4 tumor cells. GZLo-challenged mice that showed rapidly increasing serum MUC1 mucin levels several weeks prior to death had a shorter survival than mice without detectable rising MUC1 serum levels. Surprisingly, SCID-BEIGE mice challenged with GZLo cells also survived for a shorter time than those challenged with either GZHi or 410.4 cells. This suggests that MUC1 mucin may also enhance the aggressiveness of GZLo tumors by non-immune mechanisms. Received: 30 November 1999 / Accepted: 13 March 2000     

Prostaglandin E2 receptor modulation affects tumor cell adhesion to laminin.

We have shown previously that murine mammary adenocarcinoma cells both synthesize prostaglandin E2 (PGE2) and have a high affinity receptor for this ligand. Modulation of either PGE synthesis or PGE receptor function changes the metastatic potential of these cells. Because of the importance of laminin and laminin receptors to the metastatic process, we asked whether or not the PGE receptor participates in tumor cell-laminin interactions. As has been reported for many other tumor cells, laminin and the laminin-derived peptide PA22-2, containing the sequence IKVAV, mediate attachment of line 410.4 mammary tumor cells in vitro. We now demonstrate that the attachment of 410.4 cells to laminin or peptide PA22-2 was significantly inhibited by three PGE receptor antagonists, LE0101, SC19220, and sodium meclofenamate. LE0101 was most active, inhibiting tumor cell adhesion in a dose-dependent manner in the absence of nonspecific toxicity. These receptor antagonists had no effect on the PA22-2-mediated attachment of a PGE receptor negative tumor cell line, except at the highest concentration of LE0101 tested. No inhibition of adhesion to Type I collagen was seen. These results indicate that the PGE2 receptor modulates tumor cell adhesion to laminin which may subsequently affect the in vivo process of metastasis.     

Phenotypic characterization of ouabain-resistant Aedes albopictus cells.

The phenotype of a ouabain-resistant Aedes albopictus cell line has been partially characterized. Treatment of ouabain-sensitive cells with 0.005-1.0 mM ouabain resulted in an 80% reduction in the uptake of 86rubidium (86Rb+), an ion with an affinity for the K+ pump binding site; ouabain-resistant cells showed only a 40% reduction with 1.0 mM ouabain. When ouabain-sensitive cells were incubated in the presence of ouabain (0.1 mM) for one and one-half to three hours, the molar ratio of intracellular Na+/K+ rose from 0.2 to 4.2. In ouabain-resistant cells, a similar treatment had very little effect. Based on [3H] ouabain-binding studies, ouabain-resistant cells were estimated to have 60% fewer binding sites per cell than ouabain-sensitive cells. The spontaneous mutation rate from ouabain sensitivity to ouabain resistance was calculated to be 1-6 x 10(-8) mutations/cell/generation, a value similar to that reported for mammalian cells at the analogous locus.     

Erythroid differentiation and the Na+,K+-pump in ouabain-sensitive and ouabain-resistant Friend erythroleukemia cell lines

The selection and biochemical characterization of ouabain-resistant erythroleukemia cell lines are described. Treatment of ouabain-resistant Friend erythroleukemia cell (FLC) lines with 1 mM ouabain demonstrated a reduced ouabain-sensitive 86Rb+-uptake after Na+-preloading in comparison with ouabain-sensitive cells. The ouabain- and diuretic (piretanide)-insensitive component of the 86Rb+-uptake (residual influx) was significantly enhanced in the ouabain-resistant FLC clones. Measurements of the Na+,K+-ATPase activity (E.C. 3.6.1.3) in plasma membrane preparations of the ouabain-resistant FLC clone B6/2 indicated that a ouabain-resistant Na+,K+-ATPase activity of about 20% of the total enzyme activity existed in the presence of 1 mM ouabain. Further experiments showed that the Na+,K+-ion-gradient in ouabain-resistant B6/2 cells was unaffected by ouabain exposure whereas the gradient collapsed in wild type 12 N cells. Another property of the ouabain-resistant cell lines was a decrease of the 86Rb+-uptake due to the Na+,K+, 2Cl(-)-cotransport system measured as piretanide-sensitive 86Rb+-uptake. The data on ion transport mechanisms in QuaR and QuaS FLC are discussed with respect to mutagen-induced and spontaneous cellular ouabain resistance. In addition, the role of altered ion transport mechanisms is considered for induced erythroid differentiation.     

Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix

A clinically relevant model of spontaneous breast cancer metastasis to multiple sites, including bone, was characterized and used to identify genes involved in metastatic progression. The metastatic potential of several genetically related tumor lines was assayed using a novel real-time quantitative RT-PCR assay of tumor burden. Based on this assay, the tumor lines were categorized as nonmetastatic (67NR), weakly metastatic to lymph node (168FARN) or lung (66cl4), or highly metastatic to lymph node, lung, and bone (4T1.2 and 4T1.13). In vitro assays that mimic stages of metastasis showed that highly metastatic tumors lines were more adhesive, invasive, and migratory than the less metastatic lines. To identify metastasis-related genes in this model, each metastatic tumor was array profiled against the nonmetastatic 67NR using 15,000 mouse cDNA arrays. A significant proportion of genes relating to the extracellular matrix had elevated expression in highly metastatic tumors. The role of one of these genes, POEM, was further investigated in the model. In situ hybridization showed that POEM expression was specific to the tumor epithelium of highly metastatic tumors. Decreased POEM expression in 4T1.2 tumors significantly inhibited spontaneous metastasis to the lung, bone, and kidney. Taken together, our data support a role for the extracellular matrix in metastatic progression and describe, for the first time, a role for POEM in this process.     

Low incidence of tumourigenicity in agarose colonies from spontaneous murine mammary tumours

In these experiments individual colonies growing in agarose seeded with monocellular suspensions from freshly disaggregated naturally-occurring mouse mammary tumours, induced by the murine mammary tumour virus (MMTV), were reimplanted into mammary fat pads of virus-free mice. It was found that only a small proportion of these colonies generated tumours and that the implantation of multiple colonies in each site did not result in disproportionate, synergistic, increase in tumour takes. It was also observed that the proportion of colonies which were tumourigenic on reimplantation differed for each donor tumour and represented only a small fraction of the total cell population (0.001%-0.1%). However, this value was significantly higher in tumours which produced large numbers of deposits in lung colony assays following i.v. injections, than in tumours of low pulmonary colonisation potential. A point of particular interest was that tumours derived from agarose colonies of spontaneously metastatic donor tumours were substantially more spontaneously metastatic themselves than those from nonmetastatic donors, indicating that this property is heritable through numerous cell divisions, manipulations in vitro and transplantation procedures. From these results it is concluded that measurement of clonogenicity in agar is useful as an index of the capability of a tumour to propagate itself and to colonise new sites, but that individual agarose colonies are not all the progeny of potentially immortal stem cells.     

Association of hst gene expression with metastatic phenotype in mouse mammary tumors

From the pregnancy-dependent mouse mammary tumor TPDMT-4, four autonomous sublines were established after its independent progression under different conditions. Despite their similar growth rates in inguinal fat pads, three sublines formed lung metastases, and one did not when they were injected i.v. into mice as a single cell suspension. The TPDMT-4 tumor and the nonmetastatic subline expressed mRNA for the orf gene of mouse mammary tumor virus, whereas all metastatic sublines did not. This suggested that the loss of its expression may have been a prerequisite for the progression toward metastatic ability. To identify the gene(s) participating in the generation and the progression of TPDMT-4, the expression of 23 different oncogenes was analyzed. The expression of int-2 was detected in TPDMT-4 and in all sublines, indicating that TPDMT-4 was generated by activation of this gene, whereas hst expression occurred only in the metastatic sublines. These results demonstrated that the hst gene may contribute to tumor progression from a nonmetastatic to a metastatic phenotype in the mouse mammary tumor system.     

Spontaneous fusion in vivo between normal host and tumor cells: possible contribution to tumor progression and metastasis studied with a lectin-resistant mutant tumor.

Previous studies demonstrated that growth in DBA/2 mice of MDW4, a wheat germ agglutinin-resistant (WGAr) mutant of the highly metastatic MDAY-D2 DBA/2 mouse tumor, led to the emergence of WGA-sensitive (WGAs) revertants having higher ploidy levels at the site of inoculation as well as at distant visceral metastases. The results implied that MDW4 was nonmetastatic but progressed to become metastatic in vivo only after a cellular change took place which was accompanied by extinction of the WGAr phenotype and acquisition of a higher number of chromosomes. Results presented here provide strong and direct evidence for the underlying mechanism being spontaneous cell fusion in vivo between the MDW4 (WGAr) tumor cells and normal host cells, at least some of which are of bone marrow origin. Thus, growth of the H-2d MDW4 tumor cells in (C3H X DBA/2)F1 (H-2k X H-2d) or (C57BL/6 X DBA/2)F1 (H-2b X H-2d) mice led to the appearance of WGAs revertants bearing the H-2k or H-2b major histocompatibility complex antigens associated with the C3H or C57BL/6 parental strains, respectively. Similarly, WGAs revertants of MDW4 were found to express H-2k antigens after growth in CBA/HT6T6 (H-2k) leads to DBA/2 bone marrow radiation chimeras. Attempts to mimic the in vivo hybridization process were successful in that in vitro somatic cell fusion between an ouabain-resistant (OuaR), 6-thioguanine-resistant (Thgr) derivative of the MDW4 mutant and either normal bone marrow or spleen cells resulted in loss of the WGAr phenotype in the hybrids (thus showing its recessive character) and increased malignant properties in vivo. An analysis of spontaneous frequencies of re-expression of various drug resistance genetic markers in several hybrid metastatic cells was also consistent with chromosome segregation of the sensitive alleles. The results show that tumor progression and the emergence of metastatic cell variants could arise as a consequence of tumor X host cell fusion followed by chromosome segregation. We also discuss the possibility that this type of event may normally be a very rare one during the growth of tumors, the frequency of which can be artificially amplified by the use of certain classes of lectin-resistant mutants carrying particular cell surface alterations.     

HH2A,an immortalized bovine mammary epithelial cell line,expresses the gene encoding mammary derived growth inhibitor (MDGI)

Summary We have established and partially characterized a spontaneously immortalized bovine mammary epithelial cell line, designated HH2a. The cells express the gene encoding for mammary derived growth inhibitor (MDGI) when grown on released collagen gels in the presence of lactogenic hormones. This is the first report of a cell line that expresses MDGI. Immunohistochemical studies showed that HH2a cells contain keratin intermediate filaments and desmosomes. When plated on confluent monolayer of live fibroblasts, HH2a cells extensively contacted with fibroblasts. When embedded in the collagen gels, they rearranged themselves to produce three-dimensional duct-like outgrowths extending into the matrix. The HH2a cell line should be useful in investigations of the roles of cell-cell and cell-extracellular interactions in regulation of breast epithelial cell proliferation, and of the hormonal regulation of MDGI gene expression.     

Ouabain-resistant mechanism of volume control and the ultrastructural organization of liver slices recovering from swellingin vitro

Summary We have studied the net extrusion of water by liver slices recovering from swelling at 1°C and have attempted to relate this to ultrastructural alterations. Special attention was paid to the ouabain-resistant extrusion of water. The restoration of many details of intracellular architecture was dissociated from the net loss of water, since an osmotic stimulus (produced by 5% inulin) caused a passive withdrawal of water with little recovery of structure. Also, a similar recovery of structure was produced during active extrusion of water in the absence and presence of ouabain, even though ouabain reduced the water extrusion by 50%. The time-course of water extrusion in the presence of ouabain was correlated with the formation of cytoplasmic vesicles. Incubation without K⁺ in the medium had similar effects to those caused by ouabain. Colchicine had little effect on water extrusion in presence or absence of ouabain except at concentrations which reduced tissue ATP levels and caused much necrosis. Cytochalasin B alone had little effect on water extrusion, but led to the accumulation of many vesicles in the cytoplasm and appeared to abolish the access of such vesicles to the canaliculi. In the presence of ouabain, cytochalasin B had a similar effect on ultrastructure, and totally prevented the ouabain-resistant water extrusion. Ni²⁺ had rather similar effects to cytochalasin B both in the presence and absence of ouabain, although to a smaller degree. The results support our previous suggestion that the ouabain-resistant water extrusion proceeds by secretion of water into cytoplasmic vesicles, followed by the exocytotic expulsion of the vesicular contents into the bile canaliculi. Microfilaments appear to play an important role in this process.     

Ouabain-resistant transfectants of the murine ouabain resistance gene contain mutations in the alpha-subunit of the Na,K-ATPase.

A 6.5-kilobase murine genomic DNA fragment isolated by Levenson et al. (Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1489-1493) (called the ouabain resistance gene) has been shown to produce ouabain resistance in primate cells. Preliminary sequence information has revealed no homology with the coding sequence of the Na,K-ATPase. We have introduced this murine sequence into monkey and murine cells in an attempt to characterize its mechanism of action. In our experiments, transfection of this DNA fragment is associated with the low frequency (1 in 8 x 10(5) cells) appearance of ouabain-resistant clones of CV1, COS, and NIH 3T3 cells, an event not seen in control transfections. Characterization of a new clone of ouabain-resistant CV1 cells (called OR8 cells) revealed a 5-fold increase in the IC50 for ouabain inhibition of rubidium uptake and a 10-fold increase in cell survival on ouabain. Although the murine sequence was detectable in Southern blots of ouabain-resistant cells soon after transfection, this exogenous DNA was rapidly lost despite continued exposure to ouabain. Furthermore, we were unable to detect message expression by this genomic sequence in any of the three cell types tested. Instead, we found that all three ouabain-resistant cell lines exhibited point mutations in a domain of the alpha-subunit that has been implicated in ouabain sensitivity (H1-H2). One of these mutations (Asp121-Asn121 in OR8 cells) has been previously reported to cause ouabain resistance (Price, E.M., Rice, D.A., and Lingrel, J.B. (1989) J. Biol. Chem. 264, 21902-21906). Other novel mutations in the H2 transmembrane domain were also detected. We postulate that the "ouabain resistance gene" is important in the early selection process on ouabain but that the permanent ouabain-resistant phenotype is due to a stable mutation in one allele of the alpha-subunit of the Na,K-ATPase.     

Ouabain-Sensitive and Ouabain-Resistant Net Uptake of Potassium into Astrocytes and Neurons in Primary Cultures

Inhibition of net uptake of 42K by different concentrations of ouabain was studied in primary cultures of astrocytes and in primary cultures of neurons in order to investigate whether there is a pronounced difference between ouabain sensitivity in the two cell types and to determine the genuine magnitudes of the ouabain-sensitive and the ouabain-resistant potassium uptakes. In morphologically differentiated astrocytes, obtained after treatment with dibutyryl cyclic AMP (dBcAMP), the sensitivity to ouabain was slightly lower than in neurons, but astrocytes which had not been treated with dBcAMP showed sensitivity similar to the neurons (which likewise were not treated). In the presence of elevated potassium concentrations (12 and 24 mM) ouabain sensitivity was decreased, although only by a factor of 2-3. Accordingly, maximum inhibition of the uptake required under all conditions studied, at most, 1.0 mM ouabain. Like total uptake, this ouabain-sensitive uptake was several times less intense in neurons than in astrocytes, where it reached its maximum value at an external potassium concentration of 12 mM. Subtraction of the ouabain-sensitive uptake from the total uptake revealed a considerable ouabain-resistant uptake. This ouabain-resistant uptake was studied in detail in the astrocytes, where it was found to increase with increasing potassium concentration over the whole concentration range 3-24 mM and to exceed substantially the maximum amount that can be accumulated by diffusion.     

Reduced ouabain inhibition of Na,K-activated adenosine triphosphatase in cultured cell recipients of the ouabain-resistance gene

Using sequential DNA-mediated gene transfer, a mouse DNA sequence (ouaR) that confers resistance to the cytotoxic effects of ouabain in cultured cells was recently isolated. To determine the basis of this resistance, we examined the ouabain-resistant phenotype of cells from each stage of the transfers that led to isolation of the ouaR gene, as well as the recipient of the isolated gene. Membranes prepared from the original DNA donor, the two intermediate stages of the sequential gene transfer, and the final isolated gene recipient contained at least two functionally distinct forms of the Na,K-ATPase. In addition to a form indistinguishable in ouabain affinity from that in the parental lines, these ouabain-resistant cells contained a form characterized by a low affinity for the glycoside. That the low-affinity form contributed to ouabain resistance was suggested by the correlation between its relative abundance and the ability to limit the increase in intracellular Na+ content when exposed to ouabain. Maintenance of the final recipient of the isolated ouaR gene for 24 h in 10 microM ouabain had no effect on the specific activity of the resistant form of the Na,K-ATPase, implying that the low-affinity form was not uniquely inducible by ouabain. These results suggest that the ouabain-resistant phenotype conferred by ouaR is attributable to expression of a Na,K-ATPase with a low affinity for the glycoside.     

A 6-thioguanine-resistant variant of the rat mammary adenocarcinoma 13762 that is more immunogenic

Summary A 6-thioguanine-resistant (TgR) variant of the metastatic mammary tumor 13762 was found to be very immunogenic. This TgR variant was nontumorigenic and nonmetastatic, whereas the parent 13762 cell line is very tumorigenic and metastatic in normal syngeneic animals. The TgR variant was tumorigenic in irradiated animals. The mechanism of the hosts' immune rejection of this TgR variant was investigated. A ⁵¹Cr-release cytotoxic cell assay was used to assess lymphocyte cell-mediated cytotoxicity (CMC) of tumor-draining lymph nodes and spleens from animals injected with tumor cells. In a secondary CMC response of splenic T cells from animals injected with TgR cells, there was a much stronger response as compared to animals injected with 13762 cells. This strong cytotoxic T cell response was short-term and correlated to the host rejection of TgR cells. Previously, we selected revertant cell lines (TgRrev, TgRrevM) from the TgR variant line that were more metastatic and tumorigenic. The revertant cell lines induced a lower CMC response than the TgR line, but a higher response compared to the parent 13762 line. The poor CMC response from 13762 tumorbearing animals was investigated and appeared to be due to a suppressor T cell response.     

Behavior of metastatic and nonmetastatic breast tumors in old mice

Breast cancer incidence and mortality increase with age. A better understanding of the biological behavior of metastatic and nonmetastatic breast tumors in older subjects may help to develop improved breast cancer therapies. In this study, we used syngeneic metastatic (4TO7cg) and nonmetastatic (64pT) mouse breast tumor models at three age levels to evaluate various characteristics that are considered to be important for effective anti-breast cancer immunotherapy. These included tumor size and growth, metastases, vascularization, gene expression levels of the tumor-associated antigen (TAA) Mage-b (homologous to human MAGE-B) in primary breast tumors and metastases, and the presence of CD4(+) and CD8(+) T cells in the inguinal lymph nodes at the site of the tumor. The primary breast tumors and metastases were generated by injection of mouse mammary tumor cell lines 4TO7cg or 64pT into a mammary fat pad of normal 3-, 9-, or 21/24-month old BALB/c mice. In the nonmetastatic breast tumor model, significantly smaller tumors were observed in old compared with young mice. This was associated with a significant increase in the percentage of CD8(+) T cells in inguinal lymph nodes and significantly higher Mage-b expression levels in the primary tumors at old age. In the metastatic (4TO7cg) breast tumor model, a less pronounced, not statistically significant, smaller tumor size was found in the old mice, without a difference in the percentage of CD8(+) T cells or Mage-b expression levels. However, in this mouse model almost all metastases showed high levels of Mage-b expression (2- to 3-fold higher than the primary tumors in the same animals) regardless of age. These results indicate that the metastatic and nonmetastatic breast tumor models could be useful model systems to analyze how breast cancer vaccines for humans can be tailored to old age.     

Concomitant tumor immunity in the mammary gland

Summary The mouse mammary fatpad is immunologically privileged relative to the subcutis for the transplantation of nonmalignant tissues. Mouse mammary tumors, however, induce tumor-specific immunity in the mammary fatpad. Immunizing tumor cells were injected into the fatpad or subcutis at several time periods before challenge tumor cells were injected into the subcutis on the contralateral side and the time of onset of concomitant immunity was determined. Mice immunized by transplantion of tumor cells into the subcutis became resistant to challenge tumor transplants before mice immunized with tumor cells transplanted into the fatpad. By utilizing a metastatic tumor line which is resistant to thioguanine and ouabain, it was directly demonstrated that tumor cells from a s.c. implant reached the regional inguinal lymph node before tumor cells from a mammary fatpad tumor implant.     

Characterization of tumor-associated lymphocytes in a series of mouse mammary tumor lines with differing biological properties

Tumor-associated lymphocytes were isolated by isokinetic gradient separation from five related mouse mammary tumor lines with different immunological and growth characteristics. Although considerable variation in recovery rates was seen from experiment to experiment, the five tumor types were found to have reproducible and characteristic patterns of T lymphocyte subpopulations, as detected by cytotoxicity assay using monoclonal antisera to Thy-1, Lyt-1, and Lyt-2 antigens. Tumors of line 168, which are weakly immunogenic at best, had the lowest numbers of recovered ALS+, Thy1+ lymphocytes (12% and 9%, respectively), in contrast to immunogenic lines (mean 38% and 26%, respectively). Line 68H tumors, which grow after prolonged latency periods and also produce tumor cell variants in vivo, were unique in that the numbers of recovered Lyt 1+ lymphocytes exceeded the number of Lyt 2+ lymphocytes, whereas these two T cell subpopulations were either equal or Lyt 2+ cells predominated in the other faster growing, non-variant-producing tumors. No differences in T lymphocyte distribution were associated with the presence or absence of metastatic behavior. These results indicate that distinctive lymphocyte infiltrates may be characteristic of tumors with distinct biological differences.