Acute hypoxia enhances spontaneous lymph node metastasis in an orthotopic murine model of human cervical carcinoma

An orthotopic mouse model of cervical carcinoma has been used to investigate the relationship between acute (cyclic) hypoxia and spontaneous lymph node metastasis in vivo. The human cervical carcinoma cell line ME-180 was stably transfected to express the fluorescent protein DsRed2, which allowed the in vivo optical monitoring of tumor growth and metastasis by fluorescent microscopy. The surgically implanted primary tumors metastasize initially to local lymph nodes and later to lung, a pattern consistent with the clinical course of the disease. The effect of acute hypoxia on the growth and spread of these tumors was examined by exposing tumor-bearing mice to treatment consisting of exposure to 12 cycles of 10 min 7% O(2) followed by 10 min air (total 4 h) daily during tumor growth. After 21 days, the tumors were excised, lymph node and lung metastases were quantified, and the hypoxic fraction and relative vascular area of the primary tumors were assessed by immunohistochemical staining for the hypoxic marker drug EF5 [2-(2-nitro-1H-imidazole-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] and the vascular marker CD31, respectively. In untreated mice, the primary tumor size was directly correlated with lymph node metastatic burden. The acute hypoxia treatment resulted in a significant decrease in the size of the primary tumors at the time of excision. However, the mice in the acute hypoxia group had an increased number of positive lymph nodes (2-4) as compared with control mice (1-3). Lung metastasis was not affected. The acute hypoxia treatment also decreased the relative vascular area in the primary tumors but did not affect the hypoxic fraction. These results suggest that fluctuating oxygenation in cervical carcinoma tumors may reduce tumor growth rate, but it may also enhance the ability of tumor cells to metastasize to local lymph nodes.     

Iron enhances tumor growth. Observation on spontaneous mammary tumors in mice.

Iron is essential for the growth of all cells, including tumor cells. The authors previously reported that a variety of transplantable tumors grew faster and larger in mice that were on an iron-rich diet compared with those on an iron-deficient diet. In this study the authors examined the relationship between iron in the diet and development of tumors in mice that are known to develop spontaneous tumors--C3H/HeN-MTV+(C3H-MTV+) mice that were congenitally infected with mammary tumor virus. These mice have a greater than 96% chance of developing mammary tumors between the ages of 7.2 and 9.2 months. Fifteen C3H-MTV+ weanlings were given a low-iron diet (5 mg iron/kg diet), and 15 were given diets with normal amounts of iron (180 mg Fe/kg diet). Thirteen of the 15 mice from the low-iron group and all 15 mice from the normal-iron group developed tumors. The average tumor growth rate in the normal-iron group was 112%/wk, compared with 62%/wk for the low-iron group. The difference in tumor growth rate between the two groups was significant (P = 0.02 by Student's t test). In this study, low iron intake did not prevent tumor development, but the results confirm the authors' previous report that iron nutrition of the host affects tumor growth; tumors grow better in an iron-rich environment. High levels of iron in the diet may enhance tumor growth, and this should be considered when treating patients with cancer.     

N-methyl-N-nitrosourea-induced rat mammary tumors. Hormone responsiveness but lack of spontaneous metastasis

N-Methyl-N-nitrosourea (MNU) given iv to rats 50-55 days old induced mammary tumors in 70% of F344/N and 91% W/ICRF inbred females with mean latency periods of 149 and 93 days, respectively. Reduction of the MNU dose did not affect tumor incidence in W/ICRF rats. Of the mammary tumors, 98% were classified histologically as adenocarcinomas, which grew progressively. Primary tumors of nonmammary origin were detected at low incidence. Upon histologic examination, no evidence was found for metastases of either the mammary or other primary tumors. No evidence for tumor-induced hypercalcemia was found. Oophorectomy at the time of MNU administration prevented tumor development; oophorectomy when at least 1 tumor/animal was palpable caused growth delay or regression. All MNU-induced and 7,12-dimethylbenz[a]anthracene-induced mammary tumors tested contained cytoplasmic estrogen receptor (ER) at similar concentrations and were indistinguishable histologically. MNU-induced tumors in F344 rats were transplantable and retained ER through three transplantations.     

Relationship of hypoxia to metallothionein expression in murine tumors

Purpose: To investigate if metallothionein, an endogenous chemo- and radioprotectant, is expressed in hypoxic cells in mouse C3H mammary carcinomas and if that expression responds to acute changes in tumor hypoxia.Methods and Materials: C3H mammary tumors were established in the hind legs of female CDF₁ mice. The mice were then subjected to air breathing (chronic hypoxia), carbogen breathing (acute decrease in hypoxia), or hydralazine injection (acute increase in hypoxia). Ninety minutes after the start of the experiment, tumors were excised, fixed in formalin, and sectioned. Hypoxic cells and metallothionein-containing cells were quantitated by image analysis. Pimonidazole hydrochloride and an IgG₁ mouse monoclonal antibody were used to detect hypoxia, and a mouse antimetallothionein monoclonal antibody (DAKO) was used to detect Type I and II metallothionein in sets of contiguous tissue sections.Results: The distribution of immunostaining intensity for metallothionein was the same in all three groups—heavy in hypoxic cells and light in other regions of the tumors. The acute increase in hypoxia caused by hydralazine injection was accompanied by an increase in metallothionein expression (p = 0.04). Carbogen breathing largely eliminated pimonidazole binding, but metallothionein expression persisted in the tumors of carbogen-breathing mice.Conclusions: Hypoxic cells in C3H mammary carcinomas strongly express metallothionein. Metallothionein expression is responsive to acute increases in hypoxia brought about by hydralazine injection. The effectiveness of hydralazine in enhancing the activation of bioreductive cytotoxins might be offset by the increased expression of metallothionein. The persistence of metallothionein in tumors of carbogen-breathing mice might contribute to a residual radioresistance in the tumors.     

Protective effects of amphotericin B against spontaneous and transplantable murine tumors.

Two tumor systems were used to test prophylactic effects of amphotericin B (AmB). When 0.5 mg AmB was given ip every 2 weeks to AKR mice beginning at 8 weeks of age, the 50% tumor incidence for spontaneous lymphoma development was delayed 2-3 months. In the second tumor system, BALB/c mice received injections of either 20 or 50 mug AmB before receiving MOPC-315-C cells sc. The mice given the low dose of AmB demonstrated a decreased tumor incidence and a reduced tumor growth rate, when compared with controls. Opposite effects were found for the group administered the high dose; tumor incidence and rate of growth were increased.     

Partial characterization of an antigen in spontaneous murine mammary tumors

Mammary carcinomas of three strains of mouse (Swiss Webster, A, and C3H/He) were investigated for the presence of a tumor-specific antigen. The antigen could be detected only in the outbred Swiss Webster mice. When rabbit antisera to pools of mammary tumor and normal tissues were absorbed three or more times with normal tissues (heart, lungs, spleen and liver) and examined by gel immunodiffusion, the results indicated that the antigen was present in tumor tissue but not in normal tissue. The antigen could not be demonstrated in other organs of tumor-bearing mice. Moreover, the precipitin band indicative of tumor antigen was easily removed by absorbing antitumor sera with pooled mammary tumor tissues. The results of lipid extraction procedures suggested that the antigen was not a lipid. The fact that it was not inactivated at 56°C for at least 30 minutes suggested that the antigen was fairly heat-stable. Trypsinization destroyed the ability of the antigen to react with absorbed anti-tumor sera, suggesting that it was probably protein in nature. With the cellular fractionation procedures employed, the antigen remained in the supernatant of the 105,000 × g fraction. This suggested that it was a cytoplasmic constituent. The results of immunofluorescent studies substantiated the impression that the antigen was in the cytoplasm of the tumor cells. No membrane or nuclear fluorescence could be detected.     

Direct evidence that hydralazine can induce hypoxia in both transplanted and spontaneous murine tumours.

Hydralazine can substantially decrease blood flow and increase hypoxia in transplanted tumours. Previous indirect studies have suggested that hydralazine does not induce such effects in spontaneous tumours. We have now directly investigated the ability of hydralazine to increase hypoxia in both transplanted and spontaneous murine tumours by measuring tumour oxygen partial pressure (pO2) distributions using an Eppendorf oxygen electrode. Spontaneous tumours arose at different sites in CDF1 mice, while transplanted tumours were produced by implanting a C3H mouse mammary carcinoma on the backs of the same mouse strain. Measurements of pO2 were made in anaesthetised mice immediately before and 45 min after an intravenous injection of 5 mg kg-1 hydralazine. In the transplanted tumours hydralazine significantly decreased tumour oxygenation, such that the percentage of pO2 values < or = 5 mmHg increased from 45% to 87%, and median pO2 decreased from 5 to 3 mmHg. Similar significant changes were induced by hydralazine in the spontaneous tumours, the percentage of pO2 values < or = 5 mmHg increasing from 60% to 94% while the median pO2 values decreased from 8 to 2 mmHg. These results clearly show that there is no difference in the response of transplanted and spontaneous mouse tumours to hydralazine.     

Characterization of a murine tumor of spontaneous origin with selective hepatic metastasis

The primary objective of this study was to establish the identity and characteristics of a murine uterine tumor of spontaneous origin in a C57Bl/6 Ros mouse that selectively metastasized to the liver. The primary tumor contained a minimum of two different cell types. One cell type was elongated and spindly and readily adhered to a plastic coated surface. From it, a permanent cell line, termed RCS-1 was established. The RCS-1 cell line was poorly tumorigenic in normal C57Bl/6 Ros mice. The other cell type, designated RCS-2, was more rounded in structure. It was maintained in vivo, enzymatically dissociated and used after short-term in vitro cell culture. The RCS-2 cells had phagocytic vacuoles, Fc, and C3 receptors. These cells phagocytosed antibody coated SRBCs and opsonized zymosan. Furthermore, these cells generated the superoxide anion. Thus, the RCS-2 cells are of macrophage origin. The spontaneous and experimental pattern of metastasis of the RCS-2 tumor cells was established. The RCS-2 tumor cells selectively metastasized to the liver by the hematogenous route. Metastatic RCS-2 tumor cells obtained from the liver (RCS-2M) retained their macrophage characteristics. These studies indicate that this murine uterine tumor of spontaneous origin consists of two cell types. One cell type is a reticulum cell sarcoma.     

A spontaneous murine melanoma lung metastasis comprised of host x tumor hybrids

Cells from a lung metastasis, arising from Cloudman S91 melanoma cells implanted s.c. in the tail of a BALB/c nu/nu mouse, were comprised chiefly of host x tumor hybrids. These lung metastasis cells showed: (a) 30-40% increased DNA content; (b) resistance to 10(-4) M hypoxanthine, 4 x 10(-7) M aminopterin, and 1.6 x 10(-5) M thymidine (HAT) + G418; and (c) the presence in genomic DNA of genes for both wt and albino tyrosinase, reflecting the DBA/2J (Cloudman S91) and BALB/c mouse genotypes, respectively. Individual clones of lung metastasis cells expressed enhanced pigmentation, motility, and responsiveness to MSH/IBMX, a behavior similar to that recently reported for artificially generated melanoma x macrophage fusion hybrids. These similarities suggested that the host fusion partner generating the lung metastasis hybrids might have been a macrophage, although formal proof for this was not possible. The results provide the first direct evidence that host x tumor hybridization could serve as an initiating mechanism for melanoma metastasis.     

Suppression of murine mammary carcinoma metastasis by the murine ortholog of breast cancer metastasis suppressor 1 (Brms1)

The murine ortholog (Brms1) of human breast cancer metastasis suppressor 1 shares 95% identity to the human metastasis suppressor, BRMS1, in amino acid structure. We tested Brms1 for suppression of metastasis of mouse mammary carcinoma cell line 4T1 in syngenic BALB/c mice, using orthotopic (mammary fat pad) injection as well as intravenous injection. As observed for BRMS1, transfection with Brms1 did not inhibit 4T1 primary tumor formation, but significantly suppressed lung colonization. We also show that Brms1 protein interacts with histone deacetylases, indicating involvement of Brms1 in murine Sin3-HDAC complex, like its human counterpart. Thus, because of similarities with its human ortholog, the results suggest that Brms1 will be useful as a model for studying mechanism of action of BRMS1.     

Evidence by cellular mosaicism for monoclonal metastasis of spontaneous mouse mammary tumors

Female (SHN X C3H/He)F1 mice carrying spontaneous mammary tumors with cellular mosaicism with respect to isozymes of phosphoglycerate kinase (PGK) showed a high incidence of lung metastases. On resection of successively appearing mammary tumors, mice survived a maximum of 8 tumors. In these mice with multiple primary mammary tumors and multiple metastases, not only each individual metastasis but also all the metastases in the same lung had the same pattern of PGK isozymes and the same histologic type in the PGK-mosaic background of the host, indicating the monoclonal origin of these metastases. In several cases, this single pattern of metastatic colonies coincided with that of only one of several primary tumors, indicating that this tumor had metastasized. In these cases, these tumors were not necessarily the earliest of the primary tumors, and they were not located in any particular site.     

Acute hypoxia in tumors: implications for modifiers of radiation effects

Radioresistant hypoxic cells have been shown to occur in nearly all the animal tumors studied to date. Furthermore, there is a large amount of evidence, albeit indirect, that hypoxic cells exist and impair the effectiveness of radiation therapy in some human cancers. Surprisingly little is known, however, about the natural history of such hypoxic cells. Recently in our laboratories, we have developed methods which enable us to select and analyse cells from tumors as a function of their distance from the tumor blood supply. Utilizing this technique, we have been able to demonstrate using SCCVII tumors greater than or equal to 500 mg that even cells close to the blood supply may become hypoxic at any particular time. This information provides direct evidence that, at least for that tumor, hypoxia can result from transient fluctuations in blood perfusion. The existence of acutely, as well as, chronically hypoxic cells within tumors has several implications for treatment strategies. Treatments designed to increase oxygen content in the blood may not be particularly effective in sensitizing acutely hypoxic cells. However small, freely diffusable radiosensitizers would distribute throughout the tumor, and should be equally effective in sensitizing both acutely and chronically hypoxic cells to radiation. Acute hypoxia may thus be the best possible indicator for the use of chemical radiosensitizers in radiation therapy.     

Rapid growth and spontaneous metastasis of human germinal tumors ectopically transplanted into scid (severe combined immunodeficiency) and scid-nudestreaker mice

Xenograft acceptance, growth and spontaneous metastasis of ectopically transplanted human germinal tumors were compared among scid mice, athymic nude mice and F2 hybrids constructed from scid and nude mice, in relation to the impairments of T and B cell functions in these mice. In scid mice which are deficient in T and B cell functions, human yolk sac tumor (YST-2) that originated from the ovary grew to enormous sizes in 100% of the animals after both subcutaneous and intraperitoneal transplantation, while only half (59.1% and 51.9%) of the subcutaneous and none of the intraperitoneal transplants were accepted in usual athymic nude mice (BALB/c-nu/nu and CD1-nu/nu). The YST-2 grew rapidly in scid mice, developing 3 to 10 times larger tumors compared to nude-streaker (AKR/J-nustr/nustr) and usual nude mice, respectively. Furthermore, ectopically transplanted tumors spontaneously metastasized to distant organs (mostly to the lung) in scid mice (but less frequently in leaky scid mice), while metastases have never been found in nude mice. Although a xenograft of human classic (typical or pure) seminoma of the testis has never been established in nude mice, it grows slowly in one-third (36.4%) of scid mice and very rapidly in all of scid-nustr (scid/scid; nustr/nustr) double mutant mice. Spontaneous metastases of xenografted seminomas were also observed in distant organs (lymph node, lung, liver, spleen, and kidney). The metastastic distribution of the two human germinal tumors in scid and scid-nustr mice mimics that found in human. These results (xenograft acceptance, growth of transplanted tumors and degree of metastatic spread) were compatible with the level of T and B cell impairments indicated by FACS analysis, as well as mitogen responses, serum IgG and morphological features of the thymus.     

Differential effect of protein-bound polysaccharide (PSK) on survival of experimental murine tumors.

The effect of protein-bound polysaccharide (PSK) on the survival of BALB/c and C57BL/6 mice after intravenous injections of syngeneic murine sarcomas (GR9.B9 and Meth-A), LSTRA lymphoma and B16 melanoma cells was studied. Pretreatment of mice with PSK significantly increased survival after the injection of either type of sarcoma cells, although the effect was attenuated when high numbers of cells were injected. Survival was not modified significantly in LSTRA lymphoma or B16 melanoma. Mice pretreated with anti-asialo GMI serum showed significantly decreased survival from all tumors in comparison with untreated mice injected with tumors, regardless of cell dose used. We observed an inverse correlation between H-2 antigen expression and in vitro NK sensitivity of tumor cells from all lines except B16 melanoma cells. These results clearly suggest that pretreatment of mice with PSK prolongs survival and inhibits metastasis formation in mice injected with sarcoma cells, being this effect highly selective, since survival was not improved in mice injected with LSTRA lymphoma or B16 melanoma.     

Inhibition of experimental and spontaneous pulmonary metastasis of murine RCT (+) sarcoma by beta-cyclodextrin-benzaldehyde

The effect of beta-cyclodextrin-benzaldehyde (CDBA) on pulmonary metastasis in C3H/He mice was examined. In experimental metastasis that was induced by iv injection of 1 X 10(6) RCT (+) cells, the highest inhibition was observed in the mice that were treated daily with CDBA (5 mg/day) for 1 week before tumor cell inoculation and further treated for 3 weeks after inoculation, when compared with those in other experimental groups that were given only pretreatment or posttreatment. The inhibitory effect was dose-dependent. In spontaneous metastasis that was induced by sc injection of 3 X 10(6) RCT (+) cells, the inhibition of metastasis was also observed in the mice treated with CDBA (5 mg/day) in the same manner as described above. However, the development of the primary tumor was not inhibited. CDBA-treated tumor-bearing mice showed almost as much NK activity as normal mice. Furthermore, although injection of 5-fluorouracil suppressed this activity to about 50% of that in normal mice, the combined treatment with CDBA could maintain the NK cell activity at the normal level. The results suggested that the inhibition of pulmonary metastasis might be induced by a combined effect of CDBA; that is, the direct inhibition of tumors and the maintenance of NK cell activity.     

Chemotherapeutic evaluation using clinical criteria in spontaneous, autochthonous murine breast tumors

Six drugs, three of which are considered to be active against human breast cancer [melphalan (PAM), cyclophosphamide (CTX), and 5-fluorouracil (FUra)] and three of which have failed to demonstrate activity against human breast cancer [N-phosphonacetyl-L-aspartate (PALA), cytarabine (ara-C), and 6-thioguanine (TG)], were tested at optimal weekly doses in (BALB/-cfC3H X DBA/8)F1 (CD8F1) mice bearing spontaneous, autochthonous breast tumors averaging 300 mg. When treatment was evaluated by laboratory criteria (i.e., tumor growth inhibition in comparison to vehicle-treated, size-matched controls), all six of the drugs tested were judged to be active. However, when the criteria for positive drug activity consisted of the attainment of tumor regressions of greater than or equal to 50% in greater than or equal to 20% of the treated individuals (i.e., analogous to clinical criteria), only the three drugs that are known to be active against human breast cancer (PAM, CTX, and FUra) were judged active against the spontaneous murine breast tumors. PALA, ara-C, and TG failed to demonstrate regressing activity against the spontaneous murine breast tumors. With a caveat concerning the limited spectrum of drugs evaluated in this study, it can be concluded that the CD8F1 breast tumor model demonstrated 100% correlation with human breast cancer in terms of both positive and negative drug sensitivity when the criteria for evaluation were parallel.     

The effect of OK-432 on modifying a spontaneous lung metastasis from murine fibrosarcomas

The authors have investigated the effect of OK-432 on the spontaneous metastasis from a murine fibrosarcoma, either an NFSA or a SANH, that had been transplanted into the right thigh of C3H mice. When the NFSA or the SANH tumors grew to 7 mm in diameter, they were irradiated with a single dose of 30 Gy and 20 Gy, respectively. A local administration of OK-432 (2.5 KE) around each of the tumors was initiated immediately after irradiation and continued twice a week. It was found that regrowth in both tumors was not modified by the OK-432, though the mean number of metastatic lung nodules per mouse from either tumor was significantly decreased. The incidence of a lung metastasis also was reduced by the OK-432 but not significantly. Further, mice that had received a SANH tumor transplant developed a lymph node metastasis in the abdominal cavity that was depressed by the OK-432.     

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.     

Lysis of spontaneous murine breast tumors by human interleukin 2-stimulated syngeneic T-lymphocytes

In vitro and in vivo cytotoxic assay systems show that primary explants of spontaneous mammary tumors from CD8F1 mice are susceptible to lysis by interleukin 2-stimulated, syngeneic T-lymphocytes.