Genetic diversity of spontaneously developed primary and metastatic mammary tumor cells in mice

Metastatic cells are often considered to be clonal derivatives of one of the primary tumor cell subpopulations. To determine if the cells of spontaneously developed lung nodules in a mammary tumor-bearing mouse represent the major or minor population of the cells in the primary tumor, comparisons were made of the pattern of their mouse mammary tumor virus (MMTV) proviral integrations, and their insertional mutations of the mouse mammary tumor proto-oncogenes, int-1 and int-2. Of the 78 tumor-bearing C3H/He mice sacrificed, seven mice showed metastatic lung nodules, but only four mice had nodule tissues adequate for the present analysis. Examination of the primary tumors and the corresponding lung nodules of these four mice revealed that the number of newly integrated MMTV proviruses and their sites of integration in the genomic DNAs of primary tumors and tumor nodules were variable, and that the int-1 gene was disrupted in three of the primary tumors but not in any of the metastasized tumor tissue. These results support the notion that, at least in some mice, the majority of cells constituting the primary mammary tumors and the corresponding metastases are of different genotypes and raise the possibility that the activation of different int-proto-oncogenes may be involved in the genesis of different cell subpopulations including metastatic cells in the same mouse.     

Comparison of Expression Profiles of Metastatic versus Primary Mammary Tumors in MMTV-Wnt-1 and MMTV-Neu Transgenic Mice

Distant metastases of human breast cancers have been suggested to be more different from each other than from their respective primary tumors, based on expression profiling. The mechanism behind this lack of similarity between individual metastases is not known. We used cDNA microarrays to determine the expression profiles of pulmonary metastases and primary mammary tumors in two distinct transgenic models expressing either the Neu or the Wnt-1 oncogene from the mouse mammary tumor virus long terminal repeat (MMTV LTR). We found that pulmonary metastases are similar to each other and to their primary tumors within the same line. However, metastases arising in one transgenic mouse line are very different from either metastases or primary tumors arising in the other line. In addition, we found that, like their primary tumors, lung metastases in Wnt-1 transgenic mice harbor both epithelial and myoepithelial tumor cells and cells that express the putative progenitor cell marker keratin 6. Our data suggest that both gene expression profiles and cellular heterogeneity are preserved after breast cancer has spread to distant sites, and that metastases are similar to each other when their primary tumors were induced by the same oncogene and from the same subset of mammary cells.     

Clonal development of lymphomas induced by Rauscher leukemia virus

The number of cells from which tumors induced by neonatal injection of Rauscher Leukemia virus (RLV) develop was investigated using F₁ (C57BL/6 X Feral) female mice heterozygous at the X-linked phosphoglycerate kinase (PGK) locus. Because of inactivation of one of the two X chromosomes in each somatic cell which occurs during embryogenesis in female mice, only one of the two PGK genes is active in each somatic cell. Thus, tumors that develop clonally in these mice exhibit one enzyme type, B or A, whereas those with a multicellular origin may exhibit both enzymes. Seventeen of 22 PGK heterozygous mice injected with RLV developed lymphosarcomas after an average latency of 38 weeks. Twelve of these lymphomas exhibit a single-enzyme type, A or B. In three of five tumors with double-enzyme phenotypes, the minor enzyme component comprised 20% or less of the total PGK activity, and histological examination revealed that these tumors contained a mixture of malignant and normal-appearing cells, suggesting that the minor PGK component was not contributed by the malignant cells. In the two remaining lymphomas, the ratio of A:B PGK activity was close to l:l. However, lymphomas which developed in recipients after transplantation of cells from these two tumors into newborn mice showed only single-enzyme PGK phenotypes, suggesting that the neoplastic cells in the primary tumors had single-enzyme phenotypes. Thus, these results indicate that lymphosarcomas induced by RLV in mice develop clonally.     

Conditional activation of Neu in the mammary epithelium of transgenic mice results in reversible pulmonary metastasis

To determine the impact of tumor progression on the reversibility of Neu-induced tumorigenesis, we have used the tetracycline regulatory system to conditionally express activated Neu in the mammary epithelium of transgenic mice. When induced with doxycycline, bitransgenic MMTV-rtTA/TetO-NeuNT mice develop multiple invasive mammary carcinomas, essentially all of which regress to a clinically undetectable state following transgene deinduction. This demonstrates that Neu-initiated tumorigenesis is reversible. Strikingly, extensive lung metastases arising from Neu-induced mammary tumors also rapidly and fully regress following the abrogation of Neu expression. However, despite the near universal dependence of both primary tumors and metastases on Neu transgene expression, most animals bearing fully regressed Neu-induced tumors ultimately develop recurrent tumors that have progressed to a Neu-independent state.     

Lack of concordant p53 mutations in some paired primary and metastatic mouse squamous cell carcinomas induced by chemical carcinogenesis

We studied the frequency and pattern of p53 mutations in 16 mouse skin primary squamous carcinomas induced by chemical carcinogens and their 19 matched metastases. The molecular changes were analyzed by polymerase chain reaction-single-strand conformation polymorphism and subsequent direct sequencing analysis. Eleven mutations of the p53 gene were detected in a total of eight primary tumors, and 10 mutations were detected in nine metastases. Only four pairs had identical mutations in primary and paired metastatic tumors. Eight mutations in six pairs were detected in primary tumors but not in their metastases, and four mutations from three matched pairs were detected in metastases but not in primary tumors. The four pairs that contained the same mutations in both the primary and secondary tumors had lymph-node metastases, and all mutations occurred in exon 8. Conversely, five of six pairs with p53 mutations only in primary tumors had lung metastases and only one of the mutations occurred in exon 8. None of the mutations found only in metastases were located in exon 8. These data indicate that p53 mutations are prevalent in lymph-node metastases and infrequent in lung metastases of mouse skin tumors and that primary tumors with exon 8 mutations may be more likely to metastasize to the lymph nodes. © 1995 Wiley-Liss Inc.     

Flow cytometric analysis of primary lung carcinomas and their lymph node metastases

Specimens of primary lung carcinomas and lymph node metastases from the same 18 patients were investigated by means of flow cytometry. The number of DNA stemlines, DNA indices, the proportion of diploid cells in the tumors and the distribution of the cell cycle phases were compared. In 10 patients DNA stemlines and DNA indices were identical in primary tumors and metastases. In two cases the DNA indices were doubled in metastases. In 6 cases the primary tumors contained two abnormal DNA stemlines and their metastases contained only one aneuploid stemline. Gross differences between primary tumors and lymph node metastases with regard to the proportion of cell cycle phases could not be found. The large variation between primary tumors and lymph node metastases with regard to DNA stemlines indicates that flow cytometric analysis of lymph nodes gives only limited information about the primary tumors.     

Coexpression of cytokeratins characteristic for myoepithelial and luminal cell lineages in rat 13762NF mammary adenocarcinoma tumors and their spontaneous metastases.

We used immunohistochemical procedures to study the cellular expression and distribution of cytokeratins (CKs) in rat 13762NF mammary adenocarcinoma cells growing at mammary fat pad sites and at spontaneous lymph node and lung sites. In order to establish CK distribution in normal rat mammary epithelia, immature, resting, and lactating rat mammary glands were probed with a panel of monospecific antibodies that recognize individual CKs. Basal/myepithelial cells were distinguished by expression of CKs 5 and 14 and coexpression of vimentin from luminal cells, which expressed CKs 8, 18, and 19. Antibody to CK 7 recognized luminal epithelium of immature and resting, but not lactating, mammary glands. Myoepithelial cells of lactating mammary gland were weakly recognized by antibodies to CKs 7 and 19. Tumors formed by cell lines and clones derived from parental 13762NF tumor (MTPa, MTC, MTA, and MTF7) were not recognized by any of the anti-CK antibodies. Only vimentin was expressed in these tumors and their metastases. In tumors and metastases generated from cell lines and clones derived from lymph node (MTLY) and lung metastases (MTLn2 and MTLn3) of the 13762NF tumor we observed heterogeneous CK phenotypes. Expression of CKs 5 and 18 was greatly reduced or lacking, while CK 14 was coexpressed with CKs 7, 8, and 19 with or without vimentin. Tumors from the highly metastatic clone MTLn3 had a dominant cellular phenotype, expressing CKs 7, 8, 14, and 19 and vimentin, a pattern that did not match normal mammary epithelia, whether luminal, basal/myoepithelial, or the dual-phenotype stem cell, in which CKs 5, 8, 14, and 18 were coexpressed. MTLn3 lymph node and lung metastases expressed the same cellular phenotype as the s.c. growing MTLn3 tumor. The results appear to contradict the belief that malignant mammary tumors may be distinguished from benign tumors or hyperplastic growths by the lack of basal/myoepithelial markers.     

Lung colonization and metastasis of murine mammary tumors: relationship to various characteristics of the primary tumors

The ability to metastasize via the bloodstream of mammary tumors occurring in Balb/cfC3H and Balb/cfRIII mice (two substrains of identical Balb/c genotype carrying milk-transmitted C3H or RIII murine mammary tumor virus (MuMTV) infection, respectively) has been compared in MuMTV-free Balb/c virgin female recipients given intravenous tumor cell suspensions or subcutaneous solid tumor transplants from mammary tumor-bearing Balb/cfC3H and Balb/cfRIII breeding female donors. Tumor cell suspensions different for MuMTV inducing variant, growth rate, tumor size, and clinical duration, injected intravenously to Balb/c virgin female recipients, have been compared with respect to the foci of lung colonization induced in recipient hosts. The results obtained indicate that MuMTV variant, growth rate and clinical duration of the primary mammary tumor, but not the size of the primary tumor, significantly influence the lung colonization. Similar results were obtained with solid subcutaneous transplants of the same mammary tumors. The significance of these results for the understanding of the general mechanisms of tumor metastases is discussed.     

Effects of chronic indomethacin therapy on the development and progression of spontaneous mammary tumors in C3H/HEJ mice

The present study was designed to test the hypothesis that endogenous prostaglandin E (PGE) promotes the development, growth and metastasis of spontaneous mammary tumors in C3H/HeJ female retired breeder mice. The effect of chronic oral indomethacin (indo) therapy starting at 6 months of age was tested on these parameters as well as on animal survival, in comparison with control mice placed on 0.2% ethanol in drinking water for up to 25 months of age. Indo treatment delayed the initial (up to 27 weeks) development of primary tumors by 11–12 weeks; however, the subsequent rate of tumor appearance was unaffected (totaling 82% in indo-treated vs. 90% in controls by 25 months of age). Spontaneous regression of primary tumors (26% in controls) increased 2-fold (53%) with indo therapy. While the apparent reduction in the growth rate of primary tumors and the overall prolongation of animal survival were not significant, the lifespan of mice bearing multiple tumors was significantly prolonged by therapy. There was also a 2-fold reduction in the incidence of lung metastases in mice bearing detectable primary tumors, and this was more pronounced during the earlier phase of tumor development. Positive immunostaining for cyclooxygenase-2 enzyme (indicative of the cellular source of PGE) was exhibited by tumor cells, stromal cells and macrophages within the primary tumors. Tumors in indo-treated mice exhibited histological evidence of increased differentiation (acinar architecture), significant tumor cell death, mononuclear cell infiltration and reduction in vascularity, indicating that the beneficial effects of indo were due to multiple mechanisms, including improved immune response and reduced angiogenesis. Int. J. Cancer 73:371–380, 1997. © 1997 Wiley-Liss, Inc.     

CLONAL NATURE OF SPONTANEOUS AKR LEUKEMIA: STUDIES UTILIZING THE X-LINKED ENZYME PHOSPHOGLYCERATE KINASE

AKR mice heterozygous at the X-linked phosphoglycerate kinase (PGK) locus were used in experiments to determine the number of cells from which spontaneous thymic leukemias (thymomas) develop. Because only one of the two X-chromosomes is active in XX somatic cells, thymic leukemias that are clonal should display either type A or type B PGK, but not both, while those that are multicellular in origin may exhibit both enzymes. Spontaneous thymomas from 19 PGK heterozygous animals expressed exclusively (I I tumors) or predominantly (8 tumors) a single enzyme in contrast to non-malignant tissue from these animals which expressed both enzyme types in approximately equal ratios. When primary tumors expressing a single or predominant enzyme type were transplanted, the transplanted tumors invariably displayed the PGK phenotype that predominated in the initial tumor indicating that the minor PGK component was not contributed by malignant cells. These results indicate that spontaneous AKR leukemias are clonal.     

Clonal origin of lymph node metastases in bladder carcinoma

BACKGROUND: Evidence of genetic heterogeneity within urothelial carcinomas of the bladder has raised questions about the clonal origin of urothelial carcinoma and its metastases. High-grade urothelial carcinoma of the bladder frequently metastasizes to multiple regional lymph nodes in the pelvis. Whether or not these multiple lymph node metastases originate from the same tumor clone is uncertain. Molecular analysis of microsatellite alterations and X-chromosome inactivation status of distinct tumor cell populations from the same patient may further our understanding of the genetic basis of carcinoma progression and metastasis. METHODS: The authors examined 24 patients who underwent radical cystectomy for urothelial carcinoma. All patients had multiple (from two to four) lymph node metastases. Genomic DNA samples were prepared from formalin fixed, paraffin embedded tissue sections using laser-assisted microdissection. Loss of heterozygosity (LOH) assays for 3 microsatellite polymorphic markers on chromosome 9p21 (D9S171, region of putative tumor suppressor gene p16), 9q32 (D9S177, putative tumor suppressor gene involved in urothelial carcinoma tumorigenesis), and 17p13 (TP53, the p53 locus) were performed. In addition, X-chromosome inactivation analysis was performed in primary tumors and metastases from 10 female patients. RESULTS: In total, 79 tumors were analyzed. The overall frequency of allelic loss was 67% (16 of 24 tumors) in the primary urothelial carcinomas and 79% (19 of 24 tumors) in the metastatic carcinomas. The primary urothelial carcinoma showed LOH at the D9S171, D9S177, and TP53 loci in 39% (9 of 23 tumors), 30% (6 of 20 tumors), and 30% (7 of 23 tumors) of informative samples, respectively. LOH in > or = 1 lymph node metastases was seen at the D9S171, D9S177, and TP53 loci in 35% (8 of 23 tumors), 45% (9 of 20 tumors), and 48% (11 of 23 tumors) of informative samples, respectively. Eleven tumors demonstrated identical allelic loss patterns at all DNA loci both in the primary carcinoma and in all corresponding lymph node metastases. Three tumors showed allelic loss in the metastatic carcinoma but not in its matched primary carcinoma. Six tumors demonstrated a different LOH pattern in each of its lymph node metastases. Clonality analysis showed the same pattern of nonrandom X-chromosome inactivation both in the primary urothelial carcinoma and in all of the lymph node metastases in five of nine informative tumors studied. Four tumors showed a random pattern of X-chromosome inactivation in both the primary carcinoma and in the metastases. CONCLUSIONS: LOH and X-chromosome inactivation assays showed that multiple lymph node metastases and matched primary urothelial carcinomas of the bladder had the same clonal origin, suggesting that the capability for metastasis often arises in only a single clonal population in the primary tumor. The variable LOH patterns observed in some of the tumors likely reflect genetic divergence during the clonal evolution of urothelial carcinoma.     

Test of recurrence after experimental radiation therapy of chemically induced autochthonous tumors in mosaic mice

True recurrence was distinguished from induction of new second tumors after experimental radiation therapy using monoclonal tumors produced in the mosaic cell background of mice. The mice were C3H/He females heterozygous at the X-chromosome-linked locus of phosphoglycerate kinase (PGK) and consisted of two types (A and B) of somatic cells by inactivation of one of X-chromosomes. Sarcomas and carcinomas with a single PGK phenotype were produced by subcutaneous injection of 3-methylcholanthrene (MCA) into the groin of the mice, and locally and singly irradiated with 4-6.5 Krad of X rays generated by a 6 Mev linear accelerator, when they were 8-10 mm in diameter. Of 69 mice irradiated, 17 were available for comparison of the PGK and histological types of primary and recurrent tumors. Of these, 10 recurrent tumors with A-type PGK and 3 with B-type PGK exhibited the same PGK type as that of primary tumors, while one was distinguishable histologically. Only one recurrent tumor was of the opposite PGK and a different histological type from the primary tumor. Considering the probability of new tumor formation among A----A recurrent tumors, it was calculated that 79% (11/14) of the tumors that reappeared in the irradiated area were actually true recurrent tumors. Autochthonous tumors may be important in testing therapeutic methods.     

High frequency of clonally related tumors in cases of multiple synchronous lung cancers as revealed by molecular diagnosis.

In patients with multiple synchronous lung tumors, discrimination of multicentric lung cancers from intrapulmonary metastasis is important for treatment decision, but this is sometimes difficult. The aim of this study was to retrospectively distinguish multicentric lung cancers from intrapulmonary metastases in 14 such cases by loss of heterozygosity (LOH) and p53 mutational status. DNA was extracted from microdissected tumor cells in paraffin-embedded archival tissue, and 3p14.2, 3p21, 3p25, 9p21, and 18q21.1 were investigated for LOH. Exons 5-8 of the p53 gene were examined for mutations by the PCR, followed by single-strand conformation polymorphism analysis and DNA sequencing. For cases with the same LOH pattern, we calculated a clonality index, the probability of the given LOH pattern when these tumors were hypothesized to be independent in origin. Eleven of 14 cases (79%) were thus diagnosed as having pulmonary metastasis and only one case as having genuinely multicentric lung cancers. Two cases presented difficulty in diagnosis. In several cases, the LOH patterns conflicted with p53 mutation patterns, suggesting that clonal evolution is directly affected by certain genetic changes. The combination of p53 with LOH helped increase both the sensitivity and specificity of the assay.     

Lectin-binding patterns in transplantable mouse mammary tumors and their metastases.

Lectin binding was assessed in a transplantable pregnancy-dependent mouse mammary tumor line (TPDMT-4), its autonomous sublines (T4-0196 and T4-01165) and their artificial metastases (lung colonies), using the avidin-biotin-peroxidase technique. Soybean agglutinin (SBA) and peanut agglitinin (PNA) bound to the luminal surfaces of TPDMT-4 tumor cells, while dolicos biflorus agglutinin (DBA) showed no binding. In T4-0196 and T4-01165 tumors as well as their lung metastases, SBA and PNA binding was mixed and both positive and negative cells were detected, indicating that these lectins were not associated with the metastatic phenotype. Although the T4-0196 and T4-01165 sublines had a mixture of DBA-positive and DBA-negative cells, all the metastatic T4-0196 subclones contained only DBA-positive cells and all the metastatic T4-01165 subclones had DBA-negative cells. Thus DBA-positive, and DBA-negative subclones had respectively metastasized to the lungs from these autonomous sublines, implying that the carbohydrate moieties detected by DBA were not associated with metastatic potential but that the lung metastases were clonal in origin.     

DNA index and cell cycle analysis of primary breast cancer and synchronous axillary lymph node metastases

The DNA Index (DI) and the percentage of cells in S-phase (S-phase fraction, SPF) were measured by flow cytometry in 80 primary breast carcinomas and in 80 accompanying axillary lymph node metastases. The DI in primary tumors and metastases agreed in 61 cases (76%). Cases with diploid primary tumors revealed more constancy of the DI in comparison to the metastases than the cases with aneuploid primary tumors (91% and 70% respectively). The mean values of the SPF were in close agreement in the primary tumors and in the lymph node metastases (6.1% and 6.0% respectively). Differences between the SPF of the two groups could be detected only by the consideration of case-related data pairs. In 50 cases (62%), the percentage of SPF agreed approximately in primary tumors and in the correspondent metastases. The cases with diploid primary tumors revealed more agreement of the SPF in the primary site and the metastases than did cases with aneuploid primary tumors (78% and 56% respectively). In conclusion, diploid carcinomas and their metastases revealed more constancy of the DI and the percentage of SPF than aneuploid carcinomas. These findings agree well with a better prognosis of diploid mammary carcinomas, as reported in the literature. Comparisons between the DI and the SPF in primary tumors and the corresponding metastases could be a source of valuable information on the biological behaviour and the aggressiveness of mammary carcinomas.     

CXCR4 regulates growth of both primary and metastatic breast cancer

The chemokine receptor CXCR4 and its cognate ligand CXCL12 recently have been proposed to regulate the directional trafficking and invasion of breast cancer cells to sites of metastases. However, effects of CXCR4 on the growth of primary breast cancer tumors and established metastases and survival have not been determined. We used stable RNAi to reduce expression of CXCR4 in murine 4T1 cells, a highly metastatic mammary cancer cell line that is a model for stage IV human breast cancer. Using noninvasive bioluminescence and magnetic resonance imaging, we showed that knockdown of CXCR4 significantly limited the growth of orthotopically transplanted breast cancer cells. Mice in which parental 4T1 cells were implanted had progressively enlarging tumors that spontaneously metastasized, and these animals all died from metastatic disease. Remarkably, RNAi of CXCR4 prevented primary tumor formation in some mice, and all mice transplanted with CXCR RNAi cells survived without developing macroscopic metastases. To analyze effects of CXCR4 on metastases to the lung, an organ commonly affected by metastatic breast cancer, we injected tumor cells intravenously and monitored cell growth with bioluminescence imaging. Inhibiting CXCR4 with RNAi, or the specific antagonist AMD3100, substantially delayed the growth of 4T1 cells in the lung, although neither RNAi nor AMD3100 prolonged overall survival in mice with experimental lung metastases. These data indicate that CXCR4 is required to initiate proliferation and/or promote survival of breast cancer cells in vivo and suggest that CXCR4 inhibitors will improve treatment of patients with primary and metastatic breast cancer.     

Characterization of spontaneous metastases from autochthonous 3-methylcholanthrene-induced tumors

In mice bearing autochthonous 3-methylcholanthrene-induced tumors metastasis was rare, with only 2 out of 47 (4%) animals showing lung secondaries and 1 showing kidney lesions. Surgical excision of autochthonous growing tumors brought only a slight increase in incidence of metastasis (5 out of 42 mice, 12%). Cell lines were established by in vivo and/or in vitro passage from two kidney metastasis found in the same host (0.13-K1 and 0.13-K3) and from a spontaneous lung metastasis found in 2 mice (mR80/43 and mR80/17) and compared to lines from the respective primary tumors (0.13; R80/17; R80/43). Cell lines from metastases and primary tumors were heterogeneous in tumorigenicity, growth rate, metastatic potential (spontaneous), and colonizing capacity (i.v. inoculation). In particular, the mR80-43 line was more metastatic to lungs upon intravenous injection than the parent R80-43 primary tumor. Similarly the 013-K1 line from a kidney secondary caused more lung nodules when inoculated intravenously than the parent 0.13 line, but this was not the case with the 013-K3 line derived from another kidney secondary in the same host. The R80-17 and mR80-17 lines had similar lung-colonizing capacity. Lung colonizing ability was not strictly correlated to the capacity to form spontaneous metastases. Changes in lung-colonizing capacity occurred in part of the lines (013, 013-K1, R80-17, mR80-17) upon in vitro or in vivo passage. These findings with lines from spontaneous metastases from three autochthonous sarcomas extend previous observations on the heterogenous behavior of transplanted metastatic neoplasms.     

Discrimination of double primary lung cancer from intrapulmonary metastasis by p53 gene mutation

When multiple synchronous lung tumours are identified, discrimination of multicentric lung cancers from intrapulmonary metastases by clinical findings is often difficult. We used genetic alterations in p53 gene as a discrimination marker of double primary lung cancers from single lung cancer with intrapulmonary metastasis. Twenty of 861 patients with primary lung cancer who underwent lung resection were selected as subjects because they showed synchronous double solid tumours of the same histological type in the unilateral lung without distant metastases. In addition, they had been diagnosed as lung carcinoma with intrapulmonary metastasis by clinical and histological findings. DNAs were extracted from paraffin-embedded tissue of paired tumours from these 20 patients. Exons 5-9 of the p53 gene were examined for genetic alterations in the tumours by polymerase chain reaction, single-strand conformation polymorphism analysis and subsequent DNA sequencing analysis. Three different patterns in the distribution of p53 mutations in double lung tumours were observed: [A] mutation in only one of the tumours (four cases), [B] different mutations in the tumours (two cases), and [C] same mutation in both tumours (one case). The cases of [A] or [B] patterns could be classified as double primary lung cancers, while the case of the [C] pattern was suggested to be lung cancer with intrapulmonary metastasis. These results suggested that the multicentric cancers were more frequent than the intrapulmonary metastatic cancers in double cancer cases.     

Mediastinal lymph node metastases from gastrointestinal carcinoma

Mediastinal and hilar lymphadenopathy secondary to gastrointestinal metastases rarely have been reported and have been considered to occur only in conjunction with lymphangitic lung spread. The current report is of 15 cases of hilar and mediastinal metastases secondary to pancreatic, gastric, and colonic primary tumors. In only three of these cases was lymphangitic lung involvement noted. Possible pathways of spread of these tumors to the mediastinal lymph nodes are discussed.     

Spontaneous metastasis from primary C3H mouse mammary tumors

The normal incidence of metastasis was determined in 207 C3H/He and 42 C3Hf/He mice with spontaneous mammary tumors. The effects of early versus delayed surgical removal of the tumors on the incidence of metastasis were studied in the C3H/He mice. The presence of metastases was determined by histological examination, primarily of the lungs. The incidence of metastasis was proportional to the size the primary tumors were allowed to reach before surgery, with the highest incidence in mice not surgically cured. Tumors that developed early in the life of the mice had the greater tendency to metastasize. Immunogenic and non-immunogenic tumors occurred with similar frequency among 16 metastasizing tumors tested. Primary tumors and their metastases were equally immunogenic. All of 95 metastasizing adenocarcinomas grew extensively within pulmonary vessels with no tendency for active extravasation. In contrast, each of six metastasizing mammary sarcomas extravasated actively and probably extravasated early because intravascular growth was never observed.