Weeks Of Tumor Growth Research Articles

Accelerated Pancreatic Cancer Growth: High Fat Diet Or Obesity?

Introduction: Obesity is a well accepted risk factor for development and accelerated growth of pancreatic cancer. We have reported increased pancreatic cancer growth in two discrete murine models of obesity: congenital obesity and obesity induced by high-fat diet (HFD). In the HFD model, not all mice on HFD become obese; nevertheless, pancreatic cancer size correlates directly with body weight. The current study was undertaken to evaluate the hypothesis that obesity, rather than high-fat diet is responsible for accelerated pancreatic cancer growth. Methods: Thirty male C57BL/6J mice were studied. At 5 weeks of age, 20 mice were fed high fat diet (60% fat; HFD) and 10 were fed 10% fat (low fat) diet. At 19 weeks of age, all mice were inoculated in the flank with Pan02 murine pancreatic cancer cells. After 5 weeks of tumor growth, sera and tumors were collected. Apoptosis was determined by apoptag assay. Serum was analyzed by ELISA and colorimetric assay for insulin, glucose, leptin, and adiponectin. Student's t-test, Wilcoxian Rank-Sum, and Pearson's correlation were applied where appropriate. Two way ANOVA was used to determine whether differences were related to diet or weight. P<0.05 was accepted as statistically significant. Results: Two way ANOVA showed that diet did not influence tumor weight (p=0.58); however, tumor weight had a strong positive correlation with body weight (p=0.005). As we have previously described, mice were segregated into overweight (OW - heavier than the mean body weight of HFD mice–35.5g, n=15) and lean (less than 35.5g, n=14). Two thirds of animals in each group had been fed the HFD. Tumors in OW mice were twice as large as those in lean animals (1.23±0.2g vs 0.6±0.1g; p=0.001). Tumors in OW mice had significantly fewer apoptotic cells than those in lean mice (0.8±0.4 vs 2.4±0.5; p<0.05). No changes in serum insulin, glucose, or leptin were observed between groups. Serum adiponectin had a strong negative correlation with tumor size (R=0.45; p<0.05) Conclusions: These data show that: 1) high fat diet did not influence the growth of murine pancreatic cancer; 2) Tumor size correlated positively with body weight; 3) Tumors were significantly larger in overweight compared to lean mice; 4) Tumors in overweight mice had significantly fewer apoptotic cells than those in lean mice; and 5) Serum adiponectin had a significant negative correlation with tumor size. We conclude that obesity, rather than diet influences the accelerated pancreatic cancer growth observed in obesity.

Transforming Growth Factor-β–Stimulated Clone-22 Is an Androgen-Regulated Gene That Enhances Apoptosis in Prostate Cancer following Insulin-Like Growth Factor-I Receptor Inhibition

PURPOSE: Inhibition of insulin-like growth factor (IGF) signaling using the human IGF-I receptor monoclonal antibody A12 is most effective at inducing apoptosis in prostate cancer xenografts in the presence of androgen. We undertook this study to determine mechanisms for increased apoptosis by A12 in the presence of androgens. Experimental Methods: The castrate-resistant human xenograft LuCaP 35 V was implanted into intact or castrate severe combined immunodeficient mice and treated with A12 weekly. After 6 weeks of tumor growth, animals were sacrificed and tumors were removed and analyzed for cell cycle distribution/apoptosis and cDNA arrays were done. RESULTS: In castrate mice, the tumors were delayed in G(2) with no apoptosis; in contrast, tumors from intact mice underwent apoptosis with either G(1) or G(2) delay. Transforming growth factor-beta-stimulated clone-22 (TSC-22) was significantly elevated in tumors from the intact mice compared with castrate mice, especially in those tumors with the highest levels of apoptosis. To further determine the function of TSC-22, we transfected various human prostate cancer cell lines with a plasmid expressing TSC-22. Cell lines overexpressing TSC-22 showed an increase in apoptosis and a delay in G(1). When these cell lines were placed subcutaneously in athymic nude mice, a decreased number of animals formed tumors and the rate of tumor growth was decreased compared with control tumors. CONCLUSIONS: These data indicate that IGF-I receptor inhibition in the presence of androgen has an enhanced effect on decreasing tumor growth, in part, through increased expression of the tumor suppressor gene TSC-22. (Clin Cancer Res 2009;15(24):7634-41).

Elevated mPer1 gene expression in tumor stroma imaged through bioluminescence

The tumor stroma has significant effects on cancer cell growth and metastasis. Interactions between cancer and stromal cells shape tumor progression through poorly understood mechanisms. One factor regulating tumor growth is the circadian timing system that generates daily physiological rhythms throughout the body. Clock genes such as mPer1 serve in molecular timing events of circadian oscillators and when mutated can disrupt circadian rhythms and accelerate tumor growth. Stimulation of mPer1 by cytokines suggests that the timing of circadian oscillators may be altered by these tumor-derived signals. To explore tumor and stromal interactions, the pattern of mPer1 expression was imaged in tumors generated through subcutaneous injection of Lewis lung carcinoma (LLC) cells. Several imaging studies have used bioluminescent cancer cell lines expressing firefly luciferase to image tumor growth in live mice. In contrast, this study used non-bioluminescent cancer cells to produce tumors within transgenic mice expressing luciferase controlled by the mPer1 gene promoter. Bioluminescence originated only in host cells and was significantly elevated throughout the tumor stroma. It was detected through the skin of live mice or by imaging the tumor directly. No effects on the circadian timing system were detected during three weeks of tumor growth according to wheel-running rhythms. Similarly, no effects on mPer1 expression outside the tumor were found. These results suggest that mPer1 activity may play a localized role in the interactions between cancer and stromal cells. The effects might be exploited clinically by targeting the circadian clock genes of stromal cells.

Obesity potentiates the growth and dissemination of pancreatic cancer

Obesity is an independent risk factor for pancreatic cancer development and progression, although the mechanisms underlying this association are completely unknown. The aim of the current study was to investigate the influence of obesity on pancreatic cancer growth using a novel in vivo model. Lean (C57BL/6 J) and obese (Lep(Db) and Lep(Ob)) mice were inoculated with murine pancreatic cancer cells (PAN02), and studied after 5 weeks of tumor growth. Tumor histology was evaluated by hematoxylin and eosin staining, cellular proliferation was assessed by 5-bromodeoxyuridine, and apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. Serum adiponectin, leptin, and insulin levels were assayed. Obese mice developed larger tumors, and a significantly greater number of mice developed metastases; mortality was also greater in obese mice. Tumor apoptosis did not differ among strains, but tumors from both obese strains had greater proliferation relative to those growing in lean animals. Serum adiponectin concentration correlated negatively and serum insulin concentration correlated positively with tumor proliferation. Intratumoral adipocyte mass in tumors from both obese strains was significantly greater than that in tumors of lean mice. Data from this novel in vivo model suggest that the altered adipokine milieu and insulin resistance observed in obesity may lead directly to changes in tumor microenvironment, thereby promoting pancreatic cancer growth and dissemination.

5.02] Preliminary results: In vitro and in vivo evaluation of photodynamic techniques for the experimental treatment of human hepatoblastoma and neuroblastoma

Objective Hepatoblastoma and neuroblastoma are cancers which occur in early childhood. Intraoperative fluorescence guidance or post-surgical photodynamic therapy (PDT) might improve treatment outcome. In this study, human cell lines derived from these cancers were studied in vitro and in an animal model for their responsiveness to photodynamic methods using 5-aminolevulinic acid (5-ALA) as a precursor of the fluorescent photosensitizer protoporphyrin IX (PpIX). Material and methods Cell cultures of human hepatoblastoma (HuH6) and neuroblastoma (MHH-NB-11) were incubated in 96-well plates with 5-ALA (medac, Wedel, Germany) at increasing concentrations and incubation times to determine optimal incubation parameters by measurement of PpIX-fluorescence. Human fibroblast cells (N1) served as controls. Cell survival following PDT with increasing light doses was tested with the CTB assay (Promega, Madison, WI, USA) at 150 μg/ml 5-ALA for 4 h. HuH6-cells were also studied in vivo after laparoscopic implantation of 3×106 cells between peritoneum and abdominal wall of immunoincompetent nude rats. After 7 weeks of tumor growth, fluorescence kinetics (tumor, adjacent peritoneum, liver, colon) was assessed by fiber-based measurements following i.p. injection of 5-ALA at 500 mg/kg bodyweight (10 animals) and on further 24 animals, laser irradiation was performed with a light dose of 50 J/cm2 at 25 or 100 mW/cm2. Two days later, rats were again injected with 5-ALA and irradiated tissues were excised and studied by conventional and fluorescence microscopy. Results In vitro, PpIX fluorescence approached saturation at 150 μg/ml 5-ALA-concentration, whereas there was no saturation effect observed with respect to incubation time, 4 h was set as practically feasible. HuH6 and NB-11-cells showed comparable fluorescence intensities, whereas N1 fluorescence was low. After PDT, cell survival was lowest for HuH6-cells (LD50 at 1.5 J/cm2) compared to MHH-NB-11 (LD50 at 6 J/cm2) and N1-cells (LD50>20 J/cm2). In vivo, tumor fluorescence was highest at around 200 min post injection with a maximum contrast to the surrounding peritoneum at approx. 150 min. The mean contrast of tumor fluorescence versus fluorescence measured in peritoneum and liver was 22 at this time (not corrected for optical parameters), whereas versus colon it was 5. PDT response was visible in irradiated tumor and liver tissue. The depth of necrosis could be assessed by observation of fluorescence on cryosections of excised tissue. While peritoneum exposed to PDT remained unaffected, irradiation of the liver led to superficial necrosis and sensitized tumor showed deep necrosis. Conclusion The human hepatoblastoma and neuroblastoma cells were susceptible to selective PpIX accumulation and PDT effect. In vivo, HuH6-tumors showed high and selective PpIX accumulation and extended necrosis after PDT. The possibility of a PDT-induced necrosis of normal liver tissue must be considered for clinical trials.

Murine mammary tumor growth is blunted in dystrophin‐deficient mdx mice

Breast cancer (BC) is the second leading cause of cancer death in women in the US. Changes to the dystrophin glycoprotein complex (DGC), a multi‐protein structure that likely plays mechanical and signaling roles, have been reported in BC cells. For example, expression of α‐ and β‐dystroglycan appears to be inversely related to tumor stage. However, systemic absence of the DGC, as seen in the mdx mouse model of Duchenne muscular dystrophy, does not appear to induce spontaneous mammary tumor formation, indicating that presence of the DGC may be necessary for breast cancer initiation. We therefore hypothesized that breast tumor growth would be altered in mdx mice compared to wild type (C57BL/6) mice. We injected E0771 murine mammary adenocarcinoma cells into mdx and C57BL/6 mice. After 3 weeks of tumor growth, blood, skeletal muscles and tumors were collected and analyzed. Growth of E0771 tumors and serum content of migration and invasion chemokine markers, RANTES and MCP‐1, was dramatically blunted in mdx mice. The dystrophin protein was not detectable in E0771 cells or tumors, suggesting that its expression, like that of other DGC components, may be suppressed or altered in this cancer cell line. The exact mechanism(s) of tumor inhibition in mdx mice is not presently known; however, our results suggest a mechanism for suppressing BC growth and progression may depend on the absence of one or more proteins in the DGC.

190. Obesity Potentiates the Growth and Dissemination of Pancreatic Cancer

Background. Obesity is an independent risk factor for pancreatic cancer development and progression, although the mechanisms underlying this association are completely unknown. The aim of the current study was to investigate the influence of obesity on pancreatic cancer growth using a novel in vivo model. Methods. Lean (C57BL/6J) and obese (Lep Db and Lep Ob ) mice were inoculated with murine pancreatic cancer cells (PAN02), and studied after 5 weeks of tumor growth. Tumor histology was evaluated by hematoxylin and eosin staining, cellular proliferation was assessed by 5-bromodeoxyuridine, and apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. Serum adiponectin, leptin, and insulin levels were assayed. Results. Obese mice developed larger tumors, and a significantly greater number of mice developed metastases; mortality was also greater in obese mice. Tumor apoptosis did not differ among strains, but tumors from both obese strains had greater proliferation relative to those growing in lean animals. Serum adiponectin concentration correlated negatively and serum insulin concentration correlated positively with tumor proliferation. Intratumoral adipocyte mass in tumors from both obese strains was significantly greater than that in tumors of lean mice. Conclusion. Data from this novel in vivo model suggest that the altered adipokine milieu and insulin resistance observed in obesity may lead directly to changes in tumor microenvironment, thereby promoting pancreatic cancer growth and dissemination.

μ Opioid Receptor Stimulates a Growth Promoting and Pro-Angiogenic Tumor Microenvironment by Transactivating VEGF Receptor-2/Flk-1.

Like VEGF, morphine stimulates MAPK/ERK and Akt, leading to the promotion of angiogenesis via NO dependent signaling (Cancer Res 62: 4491, 2002). Morphine acts via pertussis toxin (PT)-dependent G-protein coupled receptors (GPCRS), while VEGF acts via receptor tyrosine kinases (RTKs). We showed that PT-dependent GPCRs transactivate VEGF receptor-2/Flk1 via small GTPase RhoA (JBC 277: 4679, 2002; JBC 278:20738, 2003). Therefore, we hypothesized that morphine via the mu opioid receptor (MOR) transactivates Flk1 and promotes a pro-angiogenic microenvironment. Morphine-induced proliferation of human umbilical vein endothelial cells (HUVEC) was completely abrogated by Y-27632 (100 μM), a highly selective and potent inhibitor of Rho-associated protein kinases, suggesting the activation of Rho signaling by morphine. Addition of 1 μM morphine potentiated VEGF-induced (10 ng/ml) proliferation of HUVEC by 25%. We observed a 30% increase in intracellular calcium release after VEGF stimulation of HUVEC pre-incubated with morphine as compared to HUVEC pre-incubated with PBS, detected by a change in the fluorescence ratio of the Fura-2 AM dye. These findings show that morphine, via MOR and Rho signaling, transactivates Flk1 leading to the stimulation of calcium signaling and endothelial cell proliferation. To functionally corroborate our hypothesis, we used MOR knockout (MOR-KO) mice and injected them with MOR-replete T241 fibrosarcoma cells. T241 fibrosarcoma tumor growth in vivo showed appearance of palpable and measurable tumors 2 days earlier in wild type (wt) as compared to MOR-KO mice. Tumor growth and angiogenesis were decreased by 20–35% in MOR-KO mice as compared to wt littermates during 3 weeks of tumor growth. None of the MOR-KO showed signs of lung metastasis versus 40% wt mice with metastasis. Morphine (1.42 for the first 2 wks and 2.14 mg/Kg/day later, respectively) stimulated 20–35% tumor growth in wt, but not in MOR-KO mice. Western immunoblotting showed a 10-fold increase in the expression of phospho-Flk1 in morphine treated wt tumors as compared to PBS-treated wt mice. Morphine did not stimulate phospho-Flk1 expression in MOR-KO mice. Western analysis of immunoprecipitates obtained with α-MOR antibody showed the expression of Flk1 and phospho-Flk1 in wt, but were not expressed in MOR-KO tumors. Thus, MOR stimulates the transactivation of Flk1 in wt mice but not in MOR-KO. These in vitro and in vivo data using MOR-KO mice and the MOR agonist, morphine, show that MOR stimulates endothelial proliferation, angiogenesis and promotes tumor growth and metastasis directly as well as by transactivating Flk1 phosphorylation. We speculate that MOR is a critical component of the ‘angiogenic switch', which regulates the pro-angiogenic and growth promoting tumor microenvironment. Thus, MOR provides a novel target for developing anti-angiogenic therapies.

Functional interactions between tumor and peripheral nerve: changes in excitability and morphology of primary afferent fibers in a murine model of cancer pain

We used a murine model to investigate functional interactions between tumors and peripheral nerves that may contribute to pain associated with cancer. Implantation of fibrosarcoma cells in and around the calcaneus bone produced mechanical hyperalgesia of the ipsilateral paw. Electrophysiological recordings from primary afferent fibers in control and hyperalgesic mice with tumor revealed the development of spontaneous activity (0.2‐3.4 Hz) in 34% of cutaneous C‐fibers adjacent to the tumor (9‐17 d after implantation). C‐fibers in tumor‐bearing mice exhibited a mean decrease in heat threshold of 3.5 +/− 0.10 °C. We also examined innervation of the skin overlying the tumor. Epidermal nerve fibers (ENFs) were immunostained for protein gene product 9.5, imaged using confocal microscopy, and analyzed in terms of number of fibers per millimeter of epidermal length and branching (number of nodes per fiber). Divergent morphological changes were linked to tumor progression. Although branching of ENFs increased significantly relative to control values, in later stages (16‐24 d after implantation) of tumor growth a sharp decrease in the number of ENFs was observed. This decay of epidermal innervation of skin over the tumor coincided temporally with gradual loss of electrophysiological activity in tumor‐bearing mice. The development of spontaneous activity and sensitization to heat in C‐fibers and increased innervation of cutaneous structures within the first 2 weeks of tumor growth suggest activation and sensitization of a proportion of C‐fibers. The decrease in the number of ENFs observed in later stages of tumor development implicates neuropathic involvement in this model of cancer pain.

Functional Interactions between Tumor and Peripheral Nerve: Changes in Excitability and Morphology of Primary Afferent Fibers in a Murine Model of Cancer Pain

We used a murine model to investigate functional interactions between tumors and peripheral nerves that may contribute to pain associated with cancer. Implantation of fibrosarcoma cells in and around the calcaneus bone produced mechanical hyperalgesia of the ipsilateral paw. Electrophysiological recordings from primary afferent fibers in control and hyperalgesic mice with tumor revealed the development of spontaneous activity (0.2-3.4 Hz) in 34% of cutaneous C-fibers adjacent to the tumor (9-17 d after implantation). C-fibers in tumor-bearing mice exhibited a mean decrease in heat threshold of 3.5 +/- 0.10 degrees C. We also examined innervation of the skin overlying the tumor. Epidermal nerve fibers (ENFs) were immunostained for protein gene product 9.5, imaged using confocal microscopy, and analyzed in terms of number of fibers per millimeter of epidermal length and branching (number of nodes per fiber). Divergent morphological changes were linked to tumor progression. Although branching of ENFs increased significantly relative to control values, in later stages (16-24 d after implantation) of tumor growth a sharp decrease in the number of ENFs was observed. This decay of epidermal innervation of skin over the tumor coincided temporally with gradual loss of electrophysiological activity in tumor-bearing mice. The development of spontaneous activity and sensitization to heat in C-fibers and increased innervation of cutaneous structures within the first 2 weeks of tumor growth suggest activation and sensitization of a proportion of C-fibers. The decrease in the number of ENFs observed in later stages of tumor development implicates neuropathic involvement in this model of cancer pain.

Pathological angiogenesis in a murine model of human Wilms' tumor

Background/Purpose: Pathological vascular architecture is a feature of neoangiogenic processes such as diseases of the retina and tumor growth. The authors hypothesized that experimental human Wilm's tumors would display a vascular architecture similar to retinal diseases that are driven by vascular endothelial growth factor (VEGF).Methods: Human Wilms' tumors were established in the right kidneys of nude mice. After 4.5 weeks of tumor growth, fluorescein angiograms were performed before death. Representative sections of tumors and contralateral, control kidneys were evaluated by fluorescent microscopy.Results: Fluorescein angiograms demonstrated a characteristic pathological architecture. Vascular tortuosity, capillary tufting, and hemorrhage were noted. These features were not present in normal kidneys.Conclusions: Vascular architecture of Wilms' tumor displays the specific features previously described in diseases of the retina, which have been shown to be driven by VEGF, suggesting that neoangiogenesis in this model is also VEGF driven.

-Glutamyl Transpeptidase Mediation of Tumor Glutathione Utilization In Vivo

Glutathione is a tripeptide used by cells to protect against oxidative and free radical damage. It may also be involved in biochemical mechanisms that cause some tumors to become resistant to anticancer drugs. gamma-Glutamyl transpeptidase (GGTP) is a membrane-bound enzyme that cleaves extracellular glutathione, providing cells with amino acids necessary for intracellular synthesis of this compound. Increased expression of GGTP has been found in a number of human tumors; however, few studies have examined the contribution of GGTP to tumor glutathione metabolism in vivo. Our goals were to study the utilization of host glutathione by 3-methylcholanthrene (MCA)-induced sarcomas grown in rats and to evaluate the involvement of tumor GGTP in this process. The left ovaries of 21 female Fischer 344 rats were isolated by laparotomy and placed in subcutaneous positions through stab wounds in the abdominal wall. A 3-mm cube of MCA sarcoma was then sutured to each of the isolated ovaries. The MCA implants obliterated the ovarian tissue, yielding isolated tumors with one arterial supply (the ovarian artery) and one draining vein (the ovarian vein, referred to as the tumor vein). After 2 weeks of tumor growth, blood was drawn from the tumor vein, the inferior vena cava (IVC), and the aorta of 16 animals. Glutathione and cysteine concentrations in plasma samples from this blood were determined by high-performance liquid chromatography and used to calculate glutathione and cysteine utilization ratios for the tumor and the systemic circulations ([(concentration aorta-concentration tumor vein)/concentration aorta] x 100 and [(concentration aorta-concentration IVC)/concentration aorta ] x 100, respectively). The utilization ratios from these control animals were compared with those from acivicin (AT-125; an irreversible GGTP inhibitor)-treated rats (the remaining five animals). Data are presented as mean +/- standard deviation; reported P values are from two-tailed tests of statistical significance. In the control animals, glutathione and cysteine concentrations were significantly lower in the tumor vein (3.55 +/- 1.9 and 5.69 +/- 2.8 microM, respectively) and in the IVC (5.65 +/- 2.3 and 7.17 +/- 2.4 microM, respectively) than in the artery (12.48 +/- 5.7 and 12.33 +/- 5.9 microM, respectively; all P values < .05). In addition, the glutathione utilization ratio was significantly higher for the tumor circulation than for the systemic circulation (69% +/- 14% versus 52% +/- 14%; P < .003). The combined glutathione and cysteine utilization ratio was also significantly higher for the tumor circulation than for the systemic circulation (116% +/- 35% versus 88% +/- 28%; P < .02). Treatment with AT-125 lowered the tumor glutathione utilization ratio significantly (45% +/- 12% for treated animals versus 69% +/- 14% for control animals; P < .005). Our results show that glutathione and cysteine in the host circulation are used by MCA sarcomas. The significant reduction in tumor utilization of serum glutathione after treatment with AT-125, a GGTP inhibitor, indicates that GGTP is important in tumor glutathione metabolism.

Anticatabolic Effect of the β2-Agonist Cimaterol in Vivo in Tumor-Bearing Animals

Loss of lean body mass occurs in cancer and may adversely affect outcome. The β2-agonist cimaterol increases muscle mass and protein content in tumor-bearing animals, in part by decreasing protein degradation, but the effect of the drug on protein synthesis remains uncertain. To determine the influence of cimaterol on protein synthesis, a methylcholanthrene sarcoma was transplanted sc into the dorsum of male Fischer-344 rats. After 3 weeks of tumor growth, tumor-bearing and control animals received daily sc injections of the β2-agonist cimaterol (0.15 mg/kg) for 5 days. Rate of protein synthesis was measured using iv [3H]phenylalanine (25 μCi/100 g body wt) and cold phenylalanine (150 μmole/100 g body wt) in a flooding dose. Extensor digitorum longus muscles were harvested 10 min later, homogenized, and assayed for [3H]phenylalanine uptake (bound) (dpm/mg muscle) and tissue-specific (free) radioactivity to determine protein synthesis rate (Ks: %/24 hr). There was a significant increase in protein synthesis rate in control and tumor-bearing animals receiving cimaterol compared to that in freely feeding, food-deprived, or matched-carcass-weight nontumor-bearing controls, as well as compared to that in tumor-bearing controls. We conclude that the anabolic effects of cimaterol are due to both decreased protein degradation and increased muscle protein synthesis. Therefore, β2-agonists may prove useful in prevention and/or treatment of cancer cachexia.

Dominated and resistant subpopulation causes regrowth after response to 1,3-Bis(2-chloroethyl)-1-nitrosourea treatment of a heterogeneous small cell lung cancer xenograft in nude mice: Aabo K, Roed H, Vindelov LL, Spang-Thomsen M. Univ. Inst. of Pathological Anatomy, University of Copenhagen, Frederik d. V's Vej 11, DK-2100 Copenhagen Ø. Cancer Res 1994;54:3295–3299

In order to address the question of the influence of a primarily chemoresistant tumor cell subpopulation on the progression of a heterogeneous tumor after cytotoxic therapy, in vitro established human small cell lung cancer cell lines of a 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-sensitive (592) and a resistant (NYH) tumor were used to produce mixed solid tumors in nude mice. Mixtures of 592/NYH (9:1 and 1:1) were inoculated s.c. After 3-4 weeks of tumor growth, the mice were stratified according to tumor size and randomized to treatment with BCNU 40 mg/kg i.p. (10% of lethal dose) or no treatment. Tumor growth curves were used to calculate the effect of the treatment, and changes in the relative proportions of 592 and NYH in the mixed tumors were monitored by flow cytometric DNA analysis by which the two cell lines were distinguishable due to differences in DNA content. A significant response was demonstrated in the 9:1 mixed tumors in which only 592 cells were detectable at the start of the treatment. The response was short and less pronounced compared with tumors containing only 592. In the regrowing tumors after treatment, only NYH was detected. In untreated 9:1 mixed control tumors, only 592 cells were detectable throughout the entire observation period. It is substantiated that the 592 cells were able to inhibit the growth of the NYH cells completely when grown together in 9:1 mixed tumors. This was not the case in the 1:1 mixed tumors. The 1:1 mixed tumors did not respond to BCNU, although 592 was eradicated. These results indicate that resistant and undetectable (dominated) subpopulations in heterogeneous tumors may be responsible for relapse and that the fractional size and the growth characteristics of the resistant subpopulation may determine the magnitude of the clinical response to cytotoxic treatment.

Low-dose chemotherapy delays relapse of a dominated and resistant sub-population in a heterogeneous human SCLC xenograft in nude mice.

We investigated the influence of cellular heterogeneity on the response to low-dose BCNU chemotherapy of an artificially mixed human small-cell lung cancer (SCLC) xenograft in nude mice containing a BCNU-sensitive and dominating sub-population and a BCNU-resistant and undetectable (dominated) sub-population. The cell lines differed in DNA content, making them distinguishable by DNA flow cytometry (FCM). After 3 weeks of tumor growth, the mice were stratified according to tumor size and randomized to 2 different low-dose treatments with BCNU or no treatment. After a further 3 to 4 weeks, a high-dose treatment (LD10) was given to both groups of treated tumors. Changes in the relative proportions of and cell lines in the tumors were measured by FCM on fine-needle tumor aspirates. At the time of low-dose treatment, all the tumors were totally dominated by the sensitive cells. A temporary response was seen after low-dose treatment. After the high-dose treatment, a similar short response was seen. In the non-treated group, the sensitive cells continued to dominate. At the time of tumor regrowth after the low-dose treatment, most of the tumors continued to be dominated by the sensitive population. At the time of progression after the response to the high-dose treatment, the resistant cell line was the predominant population. If compared with a single high-dose BCNU treatment, the response of tumors treated with a low dose was superior, indicating that the presence of a dominating and slower growing sub-population influenced the outcome of the treatment.

CONCENTRATION AND DISTRIBUTION OF ELEMENTS IN NORMAL AND TUMOR TISSUE OF THE RAT ANALYZED BY THE HAMBURG PROTON MICROPROBE

Concentrations of chemical elements in different organs of healthy, of tumor (rhabdomyosarcoma R1H) bearing and of cis-diamminodichloroplatinum (cisDDP) treated rats were analyzed with a 2 MeV proton microprobe. Characteristic concentrations were found in spleen, liver, kidney, muscle and tumor. Bromine concentrations were elevated after three weeks of tumor growth. As a result of cisDDP application, they dropped considerably. These alterations were found not only in tumor tissue but also in organs not directly involved in tumor growth (e.g. liver and spleen). Platinum as a marker for the drug could be detected in all tissues except muscle. The highest concentrations were found in the kidney. With the scanning microprobe, elemental maps in several tissues were investigated. These could be correlated to histological details (e.g. in spleen and tumor).

Inhibition of pancreatic glucagon gene expression in mice bearing a subcutaneous glucagon-producing GLUTag transplantable tumor.

Transgenic mice that express a glucagon gene-simian virus-40 large T-antigen (GLUTag) fusion gene develop neuroendocrine carcinoma of the large bowel. This glucagon-producing tumor was implanted sc and reproducibly formed tumors in nude mice. The transplanted GLUTag tumor expressed large amounts of proglucagon mRNA transcripts, and the levels of proglucagon mRNA transcripts remained constant during 2-8 weeks of tumor growth. The posttranslational processing of proglucagon in the transplantable tumor resembled that detected in the original transgenic tumor, with the liberation of glicentin, oxyntomodulin, glucagon, glucagon-like peptide (1-37) [GLP-1-(1-37)] and GLP-1-(7-37). Tumor-bearing mice demonstrated progressive elevations in the plasma levels of proglucagon-derived peptides. Elevated plasma levels of glucagon-like immunoreactive peptides and immunoreactive glucagon were associated with a marked reduction in the levels of pancreatic glucagon mRNA transcripts by 4 weeks, and after 8 weeks of tumor growth, the levels of glucagon mRNA transcripts in the pancreas were not detectable by Northern blot analysis. Synthesis of the proglucagon-derived peptides was also significantly suppressed at 4-8 weeks in the pancreas of tumor-bearing animals. Histological examination of the endocrine pancreas in mice carrying the GLUTag tumor for 6-8 weeks demonstrated a marked reduction in the number and size of the islets of Langerhans and a disproportionately greater decrease in the number of cells exhibiting glucagon immunoreactivity. By electron microscopy, the residual A-cells were small, compressed at the periphery of the islets, and had poorly developed cytoplasmic organelles. In contrast, no changes in mouse glucagon gene expression or islet morphology were detected in control animals without tumors or mice carrying a sc v-jun-induced fibrosarcoma. The suppression of pancreatic A-cell function and islet size in mice with elevated plasma levels of the proglucagon-derived peptides raises the possibility that a proglucagon-derived peptide may participate in a negative feedback loop, inhibiting expression of the glucagon gene in the A-cells of the endocrine pancreas.

In vivo NMR T2 relaxation of experimental brain tumors in the cat: A multiparameter tissue characterization

Experimental gliomas (F98) were inoculated in cat brain for the systematic study of their in vivo T 2 relaxation time behavior. With a CPMG multi-echo imaging sequence, a train of 16 echoes was evaluated to obtain the transverse relaxation time and the magnetization M(0) at time t = 0. The magnetization decay curves were analyzed for biexponentiality. All tissues showed monoexponential T 2, only that of the ventricular fluid and part of the vital tumor tissue were biexponential. Based on these NMR relaxation parameters the tissues were characterized, their correct assignment being assured by comparison with histological slices. T 2 of normal grey and white matter was 74 ± 6 and 72 ± 6 msec, respectively. These two tissue types were distinguished through M(0) which for white matter was only 0.88 of the intensity of grey matter in full agreement with water content, determined from tissue specimens. At the time of maximal tumor growth and edema spread a tissue differentiation was possible in NMR relaxation parameter images. Separation of the three tissue groups of normal tissue, tumor and edema was based on T 2 with T 2 (normal) < T 2 (tumor) < T 2 (edema). Using M(0) as a second parameter the differentiation was supported, in particular between white matter and tumor or edema. Animals were studied at 1–4 wk after tumor implantation to study tumor development. The magnetization M(0) of both tumor and peritumoral edema went through a maximum between the second and third week of tumor growth. T 2 of edema was maximal at the same time with 133 ± 4 msec, while the relaxation time of tumor continued to increase during the whole growth period, reaching values of 114 ± 12 msec at the fourth week. Thus, a complete characterization of pathological tissues with NMR relaxometry must include a detailed study of the developmental changes of these tissues to assure correct experimental conditions for the goal of optimal contrast between normal and pathological regions in the NMR images.

Macrophage-mediated suppression of natural killer cell activity in mice bearing Lewis lung carcinoma.

The natural killer (NK) cell cytotoxic capacity of C57BL/6 mice with implantation of Lewis lung carcinoma (LLC) was quantitated during tumor growth. The NK activity became suppressed at 1 week of tumor growth and remained suppressed. The mechanisms for the suppression during the first 3 weeks of tumor growth included secretion of prostaglandin E2 (PGE2) by both the LLC tumor and host macrophages. With tumor growth, plasma PGE2 concentrations progressively increased. Oral administration of indomethacin to tumor-bearing mice prevented the rise in serum PGE2 concentrations and the suppression of NK activity. Cultured LLC cells and splenic macrophages isolated from mice during the first 3 weeks of tumor growth secreted increased amounts of PGE2. Macrophages from tumor-bearer spleen cells were shown to suppress NK activity. Depletion of these macrophages restored the NK activity, and addition of these macrophages to normal spleen cells resulted in an indomethacin-sensitive suppression of the NK response. The mechanisms of suppression in mice bearing large tumors were different than those observed with smaller tumors. With a large tumor burden, the plasma PGE2 concentrations declined. Indomethacin treatment did not prevent the suppression of NK activity, and depletion of splenic macrophages did not restore NK cytotoxicity.

A granulocytosis-inducing tumor inhibits the production of B lymphocytes in murine bone marrow.

Mice bearing a transplantable CE mammary carcinoma have been shown to have greatly augmented rates of neutrophil production coupled with a marked diminution of bone marrow lymphocytes. The objective of the present study was to test whether the loss of lymphocytes, and especially of B cells, from the bone marrow and spleen of tumor-bearing animals was due to a reduced rate of cell production and if so, at what level this response was regulated. A modified 3H-TdR pulse and chase analysis was used to assess the rates of production of small lymphocytes and B cells (stained for c mu and s mu) at weekly intervals after CE tumor transplantation. 3H-TdR was infused continuously for 24 hr, and radioautographs were prepared of bone marrow and spleen cells 0, 24, and 48 hr after termination of the infusion. Pre-B cells (c mu+s mu-) essentially disappeared from the femoral bone marrow by the end of 1 wk of tumor growth, followed by a great reduction in the number of c mu+s mu+ cells in the marrow and s mu + cells in the spleen. Although pre-B cells appeared in the peripheral marrow (caudal vertebrae, metatarsal bones) and spleen of tumor-bearing mice, these cells could not compensate for the continued decrease in the numbers of more mature B cells. In normal mice, during the 48-hr chase period, newly formed, 3H-TdR-labeled, small lymphocytes and s mu+ cells continued to emerge from the prelabeled precursor compartment at a steady rate, but after 1 wk of tumor growth, the number of small lymphocytes and s mu+ cells emerging from the precursor compartment fell steadily during the 48-hr chase period. During the second and third weeks of tumor growth, a steady state appears to have been reached in B cell production, which was at a level approximately 10 times below that of normal. Because pre-B cells are normally maintained by a less mature precursor population (2), the initial disappearance of c mu+s mu- cells suggests that the CE mammary carcinoma exerts its modulatory influence on primary B cell production by inhibiting or eliminating the cells that eventually feed into the pre-B compartment. The nature of the regulatory factors apparently secreted by the tumor and the more precise identity of the target cells are under investigation.