Combined treatment with radioestradiol-lucanthone in mouse C3HBA mammary adenocarcinoma and with estradiol-lucanthone in an estrogen bioassay

C3H female mice bearing transplantable estrogen-nondependent adenocarcinomas were treated subcutaneously with either estradiol, tritiated (³H)estradiol, lucanthone, ³H-estradiol followed by lucanthone, or lucanthone followed by ³H-estradiol. Tumor growth and host survival were ascertained. As single agents, lucanthone or ³H-estradiol had slight tutor-inhibiting effects with small tumors. Combined treatment with radioestrogen followed by htcatlthone 4 hrs later blocked early tumor growth for several days and permanently reduced the growth rate thereafter; it also lengthened survival by some 67%. On the other hand, combined treatment with lucanthone followed 1 hr later by ³H-estradiol did not have the same effect. The increase in adrenal gland weight that occurs as this tumor grows was significantly reduced by treatment with lucanthone, and more so by ³HE2…..+Lu; both of these treatmelnts also depressed the lymphatic system. It is suggested that the antitumor effects observed may be mediated-systemically via the adrenal. None of the treatments were effective against advanced tumors. Lucantbone was also administered subcutaneously to w female Swiss mice in conjunction with estradiol. Evidence showed that lucanthone interferes with the immature mouse uterine weight response to estrogen, especially what given 1 hr before the hormone. Although an estrogen dose-response relationship was demonstrated for all groups, the hormone and the DNA intercalating agent interfered with each other in a way that suggests possiblle steric or competitive inhibition. Thus lucanthone modulates two estrogen effects, namely, the effect of estrogen out an estrogen-response tissue (uterine weight, especially when given prior to the hormone), and the effect of radioestrogen on an estrogen-nondependent antonomous tumor (when given after the hormone). A novel antitumor strategy, here called radiocytochemotherapy, is described, and possible applications to human tumors are discussed.     

Enhanced growth of an estrogen receptor-negative endometrial adenocarcinoma by estradiol in athymic mice

The aim of this study was to investigate the effects of estradiol and tamoxifen (TAM) on the growth of human endometrial carcinomas in athymic mice. Tissues from primary tumors were implanted into estradiol-treated mice. In passage 2, animals were treated with (a) placebo, (b) estradiol, (c) estradiol plus TAM, and (d) TAM alone. The size of the tumors was measured weekly. Estrogen receptors (ER) were determined with the dextran-coated charcoal method and/or ER enzyme-linked immunoassay. Progesterone receptors were measured with the dextran-coated charcoal technique. Of 16 primary tumors, 2 grew in the athymic mice and were studied further. Tumor EL was positive for ER (145 fmol/mg protein) and progesterone receptors (993 fmol/mg protein). Tumor EL in passage 2 was not significantly stimulated by estradiol, but was stimulated by a combination of estradiol and TAM. Treatments (estradiol, estradiol plus TAM, or TAM) all increased tumor growth in passage 3. Tumor BR and a metastasis BR-MET were ER and progesterone receptor negative, applying dextran-coated charcoal, ER enzyme-linked immunoassay, and immunocytochemistry. The BR and BR-MET cells contain the complete ER gene but do not express any measurable amounts of ER mRNA as quantitated by Northern blot analysis, using a complete ER complementary DNA probe. In all animal passages the growth rate was significantly higher in estradiol-treated mice compared with the control. TAM alone had some growth stimulatory effect, but much smaller than observed in the estradiol group. TAM inhibited estradiol-stimulated growth. These results suggest that estradiol and possibly TAM are capable of stimulating tumor growth in the athymic mice independently from ER, potentially through a host-mediated mechanism.     

Application of bioluminescence imaging to therapeutic intervention of herpes simplex virus type I – Thymidine kinase/ganciclovir in glioma

Lentiviral vector containing the HSV1-tk and firefly luciferase (fLuc) gene was infected into C6 and C6-TL expressing HSV1-tk and fLuc gene was generated. C6-TL showed higher [¹²⁵I]IVDU uptake than C6. The survival rate of C6-TL decreased more rapidly with increasing GCV dose and was well correlated with fLuc activity. The images of microPET clearly demonstrated higher uptake of [¹⁸F]FHBG into the C6-TL tumor. Inhibition of tumor growth was observed in C6-TL tumor-bearing mice treated with GCV through tumor size measurement and bioluminescence imaging. The therapeutic effect of HSV1-tk/GCV system can be monitored using bioluminescent imaging and tumor size measurement.     

Immunological studies on mouse mammary tumors. I. Induction of resistance to tumor isograft in C3H/He mice

An isografted tumor MM2, originating from a spontaneous mammary tumor in a C3H/He/mouse, killed 4- to 5-week-old mice within 30 days through intraperitoneal injection of 2 × 10⁴ cells per mouse. It was shown that resistance to the isograft was induced by injection of 1.5 × 10⁵ or 2.0 × 10⁵ tumor cells sensitized with serum (5 μg antibody N) of rabbits immunized with the tumor. Four weeks after injection of the sensitized MM2 cells, 173 C3H/He mice were challenged intraperitoneally with fresh unsensitized MM2 in doses of 5 × 10⁵ to 1 × 10⁶ cells per mouse. Of these, 119 mice survived without any sign of tumor growth while all untreated mice died. The mean survival time for untreated mice was 18.1 days (± 1.7) when inoculated with 5 × 10⁵ cells and 16.2 days (± 1.8) when inoculated with 1 × 10⁶ cells. After the second challenge with the same number of fresh unsensitized cells, 117 out of 119 mice survived without any sign of tumor growth. All of 15 mice randomly selected from these resistant mice were tested for acceptance of skin grafts from normal C3H/He mice. The grafts showed no rejection even 150 days after the second graft. After hyperimmunization of these resistant mice, the serum of the spleen extract taken from the mice inhibited growth of tumors when the tumors were premixed in vitro and injected intraperitoneally into C3H/He mice.     

Estrogen responsive proliferation of clonal human breast carcinoma cells in athymic mice

A clone of MCF-7 (MCF-7ED), a cell in continuous in vitro cultivation derived from an estrogen-responsive human breast carcinoma, requires estrogen supplementation for progressive exponential (doubling time: 50–85 h) tumor growth in the mammary fat of athymic mice. The plasma concentration of estradiol which stimulated exponential growth of MCF-7ED corresponded to physiologic premenopausal levels in women. The tumors were carcinomas with murine and MCF-7ED cells intermixed. MCF-7ED cells could be repurified in subcultures of mixed tumors. Comparative studies of breast and non-breast cell lines showed concordance between the presence of estradiol receptor, sensitivity to the anti-estrogen tamoxifen for growth in vitro, and estradiol dependence for tumorigenic growth in athymic mice. Progesterone alone did not stimulate MCF-7ED growth, but acted synergistically with estrogen. Progesterone's action was to decrease tumor latent period, not to increase final tumor incidence or growth rate. Under estrogen-deficient conditions, conditions approximating postmeno-pausal status in women, (10^?10 M in plasma), a dormant state was established between MCF-7ED cells and murine mammary stroma which could be maintained several months. The dormant state could be broken by introduction of estradiol, but not progesterone. This system should be useful for defining host and cancer cell determinants in estrogen-responsive breast cancer growth.     

Relationship between stages of mammary development and sensitivity to gamma-ray irradiation in mammary tumorigenesis in rats

Mature Wistar-MS rats were ovariectomized and treated with estradiol benzoate and/or progesterone. Control animals were treated with olive oil. The rats were then exposed to γ-rays and implanted with a pellet of diethylstilbestrol. The incidence of mammary tumors in rats treated with estradiol benzoate or with progesterone was significantly higher than in rats in the non-treated control group, whereas, in rats treated with both estradiol benzoate and progesterone, the incidence was not significantly different from that in the controls. Histological examination of the mammary tumors showed 2 types of neoplasm: adenocarcinoma and fibroadenoma. Interestingly, over half of all the tumors in the rats treated with estradiol benzoate were adenocarcinomas, while fibroadenomas were mainly induced in the rats treated with progesterone or with both estradiol benzoate and progesterone. The expression of estrogen and progesterone receptors in the tumor tissues showed some differences according to whether the groups were treated with estradiol benzoate or with progesterone. Morphologically, mammary glands at irradiation showed well-developed lobuloalveoli in both the estradiol-benzoate-treated rats and in those rats treated with both estradiol benzoate and progesterone. This was consistent with the higher incorporation of [³H]thymidine into the DNA in the mammary glands of rats in both of these groups. Our findings suggest that a more advanced developmental stage of the mammary glands, dependent upon ovarian hormones, is related to a higher incidence of mammary tumors induced by irradiation. © 1995 Wiley-Liss, Inc.     

5-氮杂-2’-脱氧胞苷对人前列腺癌裸鼠移植瘤生长及雄激素受体表达影响的研究

目的探讨5-氮杂-2’-脱氧胞苷(5-Aza-2’deoxycytidine,5-Aza-CdR)对LNCaP荷瘤裸鼠的肿瘤生长及雄激素受体(androgen receptor,AR)表达的影响。方法裸鼠皮下接种LNCaP细胞,建立人前列腺癌裸鼠移植瘤模型,待肿瘤长至300 mm3大小时,荷瘤裸鼠腹腔内注射5-Aza-CdR,0.25 mg/kg,隔天1次。对照组给予生理盐水注射。每3天测量肿瘤体积1次,共观察24天。实验结束时处死裸鼠,取出肿块称重后,肿瘤组织抽提RNA和蛋白质,分别应用实时定量PCR和western blot方法,检测AR在mRNA和蛋白质水平上的表达。结果 LNCaP荷瘤裸鼠经5-Aza-CdR治疗后,与对照组相比,肿瘤生长缓慢。第24天观察结束时,5-Aza-CdR处理组肿瘤体积[(982±83)mm3]显著低于对照组[(1 867±195)mm3](P<0.01)。移植瘤重量与对照组相比明显下降[(796±178)mg vs(1 267±252)mg],抑瘤率为37.2%,差异有统计学意义(P<0.01)。5-Aza-CdR组游离PSA(FPSA)浓度相对对照组显著下降,分别为(40.25±13.3)ng/mL和(79.7±13.2)ng/mL,差异有统计学意义(P<0.01)。荷瘤裸鼠经5-Aza-CdR处理后,AR在mRNA水平上的表达下降为对照组的0.5倍,在蛋白质水平上的表达也显著降低(P<0.01)。结论 5-Aza-CdR能够有效抑制LN-CaP荷瘤裸鼠移植瘤的生长,并显著降低AR基因表达,为前列腺癌的去甲基化治疗提供了理论依据。     

Early detection of the CH1 murine lymphoma by tumor-associated idiotype in the serum.

In this report, we describe a sensitive C-dependent^{2 51}Cr release inhibition assay using anti-idiotype antiserum which allows early detection of the CH1 murine B-cell lymphoma by the presence of 19S tumor-associated immunoglobulin in the serum. The assay is based on the finding that sera from mice bearing the CH1 lymphoma specifically inhibit C-dependent ⁵¹Cr release from labelled CH1 cells in the presence of A-Id serum. Groups of mice were injected ip with 10², 10³, or 10⁴ CH1 cells and serum samples were collected every other day until host death. Tumor-specific idiotype was detectable as early as 8 days after a 10⁴ CH1 tumor cell challenge and by 12–14 days in mice inoculated with 10³ or 10² CH1 cells. The amount of idiotype increased in parallel with tumor burden and reached a peak titer just before host death In all cases, tumor-associated idiotype was present in the serum time significantly prior to observation of a visible tumor mass (ca. day 20–28). In subsequent experiments, mice were inoculated with 10⁴ CH1 cells and treated 16 days later with a single dose of melphalan (10 mg/kg), vincristine sulfate (0.75 mg/kg), or cyclophosphamide (100 mg/kg). Serum samples were taken from these animals and the level of serum idiotype was monitored. After M or VS drug therapy, the level of idiotype decreased in all mice, however host survival was not significantly prolonged. By contrast, mice treated with Cy survived significantly longer than untreated controls, and the serum idiotype titer in these mice decreased to undetectable levels from day 20–28. By day 37, idiotype was detectable again in the serum and all mice died of ascites tumor by day 44. The results of these experiments suggest that the presence of idiotype in the serum of a lymphoma-bearing host may provide a marker which will allow early detection of tumor growth during periods of tumor remission induced by conventional chemotherapy.     

Influence of route of administration on [3H]-camptothecin distribution and tumor uptake in CASE-bearing nude mice: whole-body autoradiographic studies

 Camptothecin (CPT) inhibits the growth of a wide variety of experimental tumors. As a part of our exploration of this drug for use as a cancer chemotherapeutic agent, we studied the effect of route of administration on the absorption, distribution and tumor uptake of [³H]-CPT. The rate of disappearance of [³H]-CPT-derived radioactivity from blood during the first 48 h was highest following oral than following intravenous (i.v.) administration. Thereafter blood levels were low irrespective of route of administration. Considerable [³H]-CPT-derived radioactivity was detected in urine and feces up to 48 h after dosing. Distribution studies were conducted using quantitative whole-body autoradiography (WBA). These studies revealed that independent of the route of administration, [³H]-CPT was rapidly excreted in the bile (gallbladder) followed by elimination into the small and large intestinal tract. Levels of CPT-derived radioactivity in the kidneys were minimal and mostly localized in the renal pelvis. Hepatic concentrations of CPT were low and were almost equal to those of the tumor. The lungs of animals treated i.v. showed higher uptake of radioactivity than those treated intramuscularly or orally. Tumor/blood ratios were slightly higher following oral administration than following administration by other routes. This study indicates that CPT is primarily eliminated via the bile. The gastrointestinal tract is the major site of accumulation and excretion of CPT. Received: 13 May1994/Accepted: 18 March 1996     

Characterization of organ-specific immunoliposomes for delivery of 3′,5′-O-dipalmitoyl-5-fluoro-2′-deoxyuridine in a mouse lung-metastasis model

A previous study has shown that lipophilic prodrugs can be delivered efficiently to normal lung endothelium by incorporation into liposomes covalently conjugated to monoclonal antibody (mAb) 34A against the lung endothelial anticoagulant protein thrombomodulin. In the present study, the potential use of these lung-targeted immunoliposomes (34A-liposomes) for delivery of a lipophilic prodrug, 3',5'- O-dipalmitoyl-5-fluoro-2'-deoxyuridine (dpFUdR), to the tumor-bearing lung was examined using BALB/c mice bearing experimental lung metastasis induced by i.v. injection of EMT-6 mouse mammary tumor cells. Immunohistochemical examination of the tumor-bearing lung showed specificity of mAb 34A to lung endothelium. Tumor cells appeared to localize just outside of the normal blood vessels and were within a small diffusion distance from the mAb 34A-binding sites. ¹¹¹In-labeled 34A-liposomes containing monosialoganglioside (G _M1) were prepared that included [ ³H]-dpFUdR at 3.0 mol% in the lipid mixture. In vitro cell binding studies further demonstrated that 34A-liposomes bound specifically to normal mouse lung cells that expressed thrombomodulin but not to EMT-6 cells. Biodistribution study showed efficient and immunospecific accumulation of [ ³H]-dpFUdR incorporated into 34A-liposomes in the lung at a level parallel with that of ¹¹¹In-labeled 34A-liposomes, indicating that the drug is delivered to the target organ in intact liposomes. Liposomal dpFUdR appeared to be metabolized in the lung to the parent drug FUdR at a rate slower than in the liver and spleen. Furthermore, treatment of lung-metastasis-bearing mice with dpFUdR incorporated into 34A-liposomes on days 1 and 3 after tumor cell injection resulted in a significant increase in the median survival time of treated mice as compared with control mice (%T/C value, 165%). dpFUdR either dispersed in emulsion or incorporated into antibody-free liposomes was ineffective in prolonging the survival of mice. These results indicate the potential effectiveness of organ-specific immunoliposomes containing a lipophilic prodrug for the targeted therapy of metastatic tumors.     

Effects of host hormonal status on binding of activated estrogen receptor to nuclei from R3230AC and 7,12-dimethylbenz[a]anthracene-induced mammary tumors

The effects of various hormonal perturbations that alter growth of two different rat mammary tumors in vivo were investigated by study of the interactions of [3H]estradiol-charged estrogen receptors ([3H]ER) with tumor nuclei in vitro. Nuclei from the transplantable R3230AC adenocarcinoma were isolated after ovariectomy, estrogen treatment, or progesterone treatment. Saturable specific binding of [3H]ER to nuclei was assayed in this in vivo-like system. Scatchard analysis of [3H]ER-nuclear binding data indicated that these perturbations did not affect affinity, which ranged from Kd 1.0 to 2.4 nM. However, the number of [3H]ER-binding sites/nucleus was altered according to the treatment: intact rats, 94,500 +/- 4,200; ovariectomy, 70,400 +/- 3,200; ovariectomy plus estradiol, 82,100 +/- 5,800; and ovariectomy plus progesterone, 73,900 +/- 2,500. Nuclei from primary tumors induced by 7,12-dimethylbenz(a)anthracene displayed similar affinities for [3H]ER, although these tumors had fewer binding sites per nucleus. Animals bearing 7,12-dimethylbenz(a)-anthracene-induced tumors were either ovariectomized or made diabetic by administration of streptozotocin, perturbations that cause regression of the majority of tumors. The number of [3H]ER binding sites per nucleus, in tumors classified according to growth characteristics in host animals subsequent to hormonal perturbation, was: intact growing 36,300 +/- 3,400; ovex regressing, 15,400 +/- 3,400; ovex, estrogen-treated growing, 28,100 +/- 2,700; diabetic regressing, 19,500 +/- 2,400; diabetic static, 32,100 and diabetic growing, 42,000 +/- 7,100. These results indicate that (a) the number of nuclear ER-binding sites can be reduced by hormonal interventions that cause tumor regression and (b) endogenous ovarian hormones may play a role in regulating nuclear ER binding.     

Biodistribution of paclitaxel and poly(l-glutamic acid)-paclitaxel conjugate in mice with ovarian OCa-1 tumor

Purpose: Poly(l-glutamic acid)-paclitaxel (PG-TXL) is a water-soluble paclitaxel (TXL) conjugate made by conjugating TXL to poly(l-glutamic acid) via ester bonds. In preclinical studies, PG-TXL has shown significant antitumor activity against a variety of solid tumors. To elucidate the relationship between tissue distribution and antitumor efficacy of PG-TXL, we studied and compared the biodistribution of PG-TXL and TXL. Methods: Female C3Hf/Kam mice bearing syngeneic ovarian OCa-1 tumors were injected with either [³H]TXL or PG-[³H]TXL at an equivalent TXL dose of 20 mg/kg. Mice were killed at various times after drug injection, and samples of blood, spleen, liver, kidney, lung, heart, muscle, brain, fat, and tumor were removed and the radioactivity counted. In addition, concentrations of free [³H]TXL released from PG-[³H]TXL in the spleen, liver, kidney, and tumor were analyzed by using high-performance liquid chromatography (HPLC). Whole-body autoradiographs of mice killed 1 day and 6 days after administration of PG-[³H]TXL were obtained to study the intratumoral distribution of PG-TXL. Results: When [³H]TXL was conjugated to polymer, the biodistribution pattern of PG-[³H]TXL differed from that of [³H]TXL. Based on area under the tissue concentration-time curve (AUC) values, tumor exposure to [³H]TXL was five times greater when administered as PG-TXL than as TXL formulated in Cremophor EL/alcohol vehicle. Furthermore, concentrations of free paclitaxel released from PG-[³H]TXL remained relatively constant in tumor tissue, being 489, 949 and 552 ng/g tumor tissue at 5, 48 and 144 h after dosing, respectively. Autoradiographic images of mice injected with PG-[³H]TXL revealed that radioactivity was primarily located in the periphery of the tumor on day 1 after drug administration and was homogeneously diffused into the center of the tumor by day 6. Over the 144-h study period, [³H]TXL concentrations, predominantly as the inactive conjugate, were higher in tissues with a more abundant reticular endothelial system (i.e. liver, kidney, spleen, lung) than in tissues with less abundant or lacking RE systems (i.e. muscle, fat, brain). Both [³H]TXL and PG-[³H]TXL were excreted primarily through the hepatobiliary route, with a small fraction of each drug (5% and 8.7%, respectively) excreted into the urine within 48 h. Conclusions: This study indicates that the distribution to tumor tissue was enhanced when [³H]TXL was administered as a macromolecular conjugate, and that free TXL was released and maintained within the tumor for a prolonged period. Thus, the antitumor activity of PG-TXL observed in preclinical studies may be attributed in part to enhanced tumor uptake of PG-TXL. Received: 22 December 1999 / Accepted: 24 May 2000     

Stimulation of tumor growth in adult rats in vivo during an acute fast

These experiments investigate an increase in tumor growth that occurs in adult rats in vivo during an acute fast. The effects of feeding, fasting, and underfeeding on the growth of Morris hepatomas 5123C and 7288CTC in Buffalo rats and of Walker carcinoma 256 and Jensen sarcoma in Sprague-Dawley rats were studied. Animals were matched for tumor size and growth during a period of ad libitum feeding preceding the fasting or underfeeding. Tumor growth was documented by increased size and incorporation of [methyl-3H]thymidine into tumor DNA. Fasting increased the rate of growth of the tumors 3 to 4 times over that measured in fed rats. This effect began during the first day of fasting and ended abruptly on refeeding. After refeeding tumor growth slowed to the rate in fed rats. Tumors from fed or fasted rats were not different in cellularity or dry weight/g wet weight. A positive growth response in the tumor required lipolysis and ketosis in the host. No stimulation was observed during an acute fast in either immature rats or in mature rats whose weights had been reduced by underfeeding. These animals have small fat stores and show no increase in arterial blood free fatty acid or ketone body concentrations during an acute fast. Finally, underfeeding of adult rats raised the blood concentrations of these nutrients to values that were intermediate between those in fasted and fed rats. Tumor growth rates in these rats were intermediate between those in fasted and fed rats. The results support the proposal that an increase in availability of free fatty acids and/or ketone bodies is the stimulus that increases the rate of tumor growth during an acute fast.     

A new triphenylethylene derivative,TAT-59; hormone receptors; insulin-like growth factor 1; and growth suppression of hormone-dependent MCF-7 tumors in athymic mice

TAT-59 {(E)-4-[1-[4-[2-(dimethylamino)ethoxy]-phenyl]-2-(4-isopropyl)phenyl-1-butenyl]-phenyl-monophosphate} treatment was performed on hormone-dependent MCF-7 tumors in athymic mice. TAT-59 given at 1, 5, and 20 mg/kg inhibited the estrogen-stimulated growth of MCF-7 tumors in athymic mice in a dose-dependent fashion. The most clear decrease in tumor growth was shown in the TAT-59 alone group, although it was not dramatic. Average serum concentrations of DP-TAT-59 {(Z)-[1-[4-[2-(dimethylamino)-ethoxy]phenyl]-2-(4-isopropyl)phenyl-1-butenyl]-4-hydroxybenzene} and DM-DP-TAT-59(desmethyl-DP-TAT-manner. Much higher levels of DP-TAT-59 and DM-DP-TAT-59 wer shown in tumors (target tissues of estrogen) as compared with muscles (nontarget tissues of estrogen) or serum. A serum concentration of DP-TAT-59 or DM-DP-TAT-59 corresponding to the physiologic levels of serum estradiol in premenopausal women was sufficient to inhibit the estrogen-stimulated growth of MCF-7 tumors in mice. TAT-59 induced a dose-dependent increase in estrogen receptor levels in the MCF-7 tumors. In contrast, it prevented the estradiol (E₂)-induced increase in progesterone receptor levels in a dose-dependent manner. Insulin-like growth factor 1 levels measured in the MCF-7 tumors significantly decreased in the TAT-59 alone group and in the no treatment group as compared with the E₂ alone group. These results show the pronounced antiestrogenic action of TAT-59 on hormone-dependent MCF-7 tumors in athymic mice.     

Tumor growth suppression by the combination of nanobubbles and ultrasound

We previously developed novel liposomal nanobubbles (Bubble liposomes [BL]) that oscillate and collapse in an ultrasound field, generating heat and shock waves. We aimed to investigate the feasibility of cancer therapy using the combination of BL and ultrasound. In addition, we investigated the anti‐tumor mechanism of this cancer therapy. Colon‐26 cells were inoculated into the flank of BALB/c mice to induce tumors. After 8 days, BL or saline was intratumorally injected, followed by transdermal ultrasound exposure of tumor tissue (1 MHz, 0–4 W/cm², 2 min). The anti‐tumor effects were evaluated by histology (necrosis) and tumor growth. In vivo cell depletion assays were performed to identify the immune cells responsible for anti‐tumor effects. Tumor temperatures were significantly higher when treated with BL + ultrasound than ultrasound alone. Intratumoral BL caused extensive tissue necrosis at 3–4 W/cm² of ultrasound exposure. In addition, BL + ultrasound significantly suppressed tumor growth at 2–4 W/cm². In vivo depletion of CD8⁺ T cells (not NK or CD4⁺ T cells) completely blocked the effect of BL + ultrasound on tumor growth. These data suggest that CD8⁺ T cells play a critical role in tumor growth suppression. Finally, we concluded that BL + ultrasound, which can prime the anti‐tumor cellular immune system, may be an effective hyperthermia strategy for cancer treatment.     

Effect of dietary fat saturation on survival of mice with L1210 leukemia.

We examined L1210 murine leukemia growth rate and survival of host male DBA/2J mice fed a diet rich in either polyunsaturated fat (16% sunflower oil) or saturated fat (16% coconut oil). The survival of mice that received transplants of L1210 leukemia cells was longer among the animals that had ingested a diet rich in the saturated fat as compared to those fed the more unsaturated fat. In duplicate experiments, the mean survivals of mice fed coconut oil were 200.9 +/- 1.6 and 202.5 +/- 3.4 hours compared to 188.7 +/- 5.3 and 187.6 +/- 3.5 hours for those fed sunflower oil. Tumor growth rate or the rate of DNA synthesis by the leukemia cells did not differ between the two experimental groups. Therefore, the alteration in survival was apparently due to an effect of the diets on the responses of the hosts rather than their effect on tumor size or growth rate.     

Modulation of immune response and tumor development in tumor-bearing mice treated by the thymic factor thymostimulin

Thymostimulin (TS), a partially purified thymic factor, has a significant impact on tumor development in C57B1/6 mice inoculated with Lewis lung carcinoma (3LL) cells, as judged by its effect on time of tumor appearance after tumor cell transplantation. In a previous study, we determined the conditions under which survival rate of the tumor-bearing mice can be significantly increased by TS treatment. In the study communicated here we analyzed host defense mechanisms that are modified by TS treatment in the tumor-bearing mice. In general, immune parameters that were increased or stimulated by the presence of the tumor were further increased in the TS-treated animals (number of lymphoid spleen cells, their response in mixed lymphocyte tumor cultures, their natural killer cell activity, and their ability to produce colony-stimulating factor), or reached earlier maximum levels (spontaneous [3H]thymidine incorporation, a reflection of in vivo spleen cell activation). Responses which reflect tumor-induced immunosuppression (proliferative response induced by phytohemagglutinin or concanavalin A stimulation) were restored to normal level by TS. Specific tumor-related reactions (specific cell-mediated cytotoxicity) were preserved in the TS-treated animals. The wide spectrum of TS effects had, nevertheless, certain elements of selectivity; e.g. colony-stimulating factor, but no interferon production is enhanced by TS in the tumor-bearing mice in diametric contrast to TS effect in Mengo virus-infected mice. The spectrum of TS effects was also dependent on the type of tumor cell used. The results indicate that the significant effect of TS on 3LL tumor development in mice is associated with a strong, multifaceted effect of TS on the immune system.     

Kinetics of inflammation and sarcoma cell development in primary moloney sarcoma-virus-induced tumors

The kinetics of ‘sarcoma’ cell appearance and inflammatory cell infiltration into primary Moloney sarcoma virus (MSV)-induced tumors has been studied in normal and partially immunosuppressed anti-lymphocyte-globulin (ALG)-treated A/WySn mice. The ALG treatment facilitated an earlier appearance of gp70-positive ‘sarcoma’ cells accompanied by an intense inflammatory response. Time course pulse-labelling studies showed that a decreasing proportion of gp70-positive ‘sarcoma’ cells were incorporating [³H]-thymidine in vivo in both normal and immunosuppressed mice as the lesion progressed to maximum size. ALG-treated mice inoculated with MSV were given a single injection of [³H]-thymidine immediately prior to extensive appearance of gp70-positive ‘sarcoma’ cells (day 6) and were studied for the presence of labelled gp70-positive cells 2 h, 2 days and 6 days post labelling. The data indicate that few cells divided during the time in which an 8- to 10-fold increase in virus-positive ‘sarcoma’ cell number occurred. These experiments provide evidence that recruitment rather than proliferation of virus-positive cells occurred. The results also suggest that, within the tumor, defense mechanisms develop which restrict sarcomacell proliferation also in ALG-treated mice. The composition of inflammatory cells during the course of tumor progression and regression in the uncompromised mice revealed that the proportion of lymphocytes was highest early and late during tumor development, dropping to a minimum at the peak size of the tumor. Macrophages and granulocytes constituted 70–80% of the total cells recovered at peak tumor size and the onset of regression. In the ALG-treated mice, the proportions of macrophages, granulocytes, and infrequent lymphocytes remained constant throughout the duration of the tumor progression. A decrease in total cells, including a decrease in gp70-positive cells, was noticed before a change in the proportion of infiltrating lymphocytes occurred due to recovery of the immune system. The composition of the inflammatory cells at onset of regression suggests that macrophages and granulocytes are of major importance in the defense against MSV infection.     

Irrigation does not dislodge or destroy tumor cells adherent to the tumor bed

Local recurrences in the surgical bed after tumor resection may be due to residual tumor cells “dropping” into the wound. Irrigation with water is often used to remove these cells. We designed experiments to determine whether irrigation would prevent tumor recurrence. Surgical wounds of uniform size in C57BL/6 mice were seeded with 5 × 10², 5 × 10³, 5 × 10⁴, 5 × 10⁵, or 5 × 10⁶ viable syngeneic B16-F10 melanoma cells to test the hypothesis that irrigation with water would decrease local tumor recurrence. The tumor-contaminated wounds were irrigated with distilled water or with saline (0.9% NaCl) immediately or 5, 30, 60, 120, or 240 min after seeding. Control wounds were seeded but not irrigated. The technique of irrigation was altered in a second group of experiments such that the amount of time the tumor cells were exposed to the water or saline was 5, 10, or 15 min. To determine the rapidity and durability of tumor cell attachment to host tissue, 1 × 10⁴ viable B16-F10 tumor cells were seeded in vitro onto freshly cut disks of syngeneic mouse dermis. The tissue was irrigated with saline or distilled water 0, 2, 5, 10, 15, 30, 60, 120, or 240 min later. Tumor growth was observed in all the mice and neither the mechanical action of irrigation nor the hypotonic effect of distilled water changed the rate of growth. Scanning electron microscopy (SEM) demonstrated stable and firm attachment to mouse tissue within seconds of seeding with no noticeable dislodgement or cytotoxicity by either saline or water irrigation. The data suggest that the commonly used technique of irrigating the bed of the resected tumor may not be of value in preventing local recurrences. © 1993 Wiley-Liss, Inc.     

Radioisotope assay for evaluation of in vivo natural cell-mediated resistance of mice to local transplantation of tumor cells

A radioisotopic assay based on the rate of elimination of [¹²⁵I]dUrd-labelled tumor cells from the local site of inoculation was used to study anti-tumor natural cellmediated immunity in vivo. [¹²⁵I]dUrd-labelled YAC-I, RL♂ I, and M109 tumor cells were inoculated into the footpads of mice and the radioactivity remaining at the site was measured at various times. This method allowed quantitation, in individual mice, of the actual number of tumor cells inoculated and their local clearance. It was found that 90–99% of inoculated YAC-I or RL♂ I cells were eliminated in the first 24–48 h following transplantation. Participation of NK cells in the local elimination of the transplanted tumor cells was supported by the following findings: (1) The rate of elimination of NK-sensitive tumor cells was higher than that of the NK-resistant M 109 tumor cells. (2) There was a positive correlation between the level of in vitro spleen-cell NK activity of the different strains of mice and the rate of in vivo elimination of YAC-I. (3) Inoculation of poly I:C or cyclophosphamide increased or decreased, respectively, the clearance of tumor cells in vivo. (4) Local adoptive transfer of normal spleen cells accelerated the rate of tumor cell elimination in vivo. (5) This adoptive transfer of the cytotoxicity was mediated by non-adherent, non-T cells that expressed asialo GMI. Despite these correlations between NK activity and the in vivo assay, two major discrepancies were found: YAC-I cells were eliminated at the same rate in young and old mice, and beige mice were as effective for in vivo clearance of tumor cells as were normal C57BL/6 mice. These data suggested that other natural effector cells may participate in the in vivo destruction of the transplanted tumor cells or that different regulatory processes may operate in vivo. Our findings provide further evidence that natural cell-mediated immunity can play an important role in the in vivo elimination of the transplanted tumor cells and indicate that this can occur at the local site of tumor inoculation as well as in the lungs or other organs.