c-abl function in normal and chronic myelogenous leukemia hematopoiesis: in vitro studies with antisense oligomers.

By using antisense oligomers the functional role of the c-abl proto-oncogene in the in vitro growth of bone marrow hematopoietic progenitors from normal subjects and patients with chronic myelogenous leukemia (CML) has been evaluated. Light density bone marrow cells (LDBMs) were depleted of adherent cells, pre-incubated for 15 h with the appropriate oligomer at a concentration of 14 microns, and then plated in methylcellulose for the evaluation of colony formation. Both anti-exon Ia and anti-exon Ib antisense oligomers produced a significant inhibition of normal day 14 CFU-GM growth in vitro (n = 5, 41 +/- 11%, and 36 +/- 7%, respectively; p less than 0.01). In contrast, normal BFU-E growth was not significantly influenced by antisense oligomers (n = 5, 14 +/- 21% and 7 +/- 19%, respectively; p less than 0.05). These findings were confirmed by plating CD34 positive progenitors. When interleukin 3 (IL-3) (100 ng/ml) was added to the culture medium during the preincubation of LDBMCs, the inhibitory effects of antisense oligomers on normal CFU-GM growth were abolished. Seven patients with CML were also studied, all of whom had cytogenetic evidence of 100% clonal hematopoiesis. In five patients in the chronic phase, antisense oligomers were inhibitory on in vitro growth of both day 14 CFU-GM (37 +/- 20% and 37 +/- 15%, p less than 0.05) and BFU-E (45 +/- 15% and 41 +/- 11%, p less than 0.05), and this inhibition was not removed by pre-incubation with IL-3. No significant effect was observed on cluster or colony formation in two patients with CML in accelerated or blastic phase, and on in vitro growth of clonogenic cells from the Ph1-positive K-562 cell line. These findings (i) confirm previous observations showing a lineage specific requirement of c-abl function in normal hematopoiesis, and (ii) suggest that the residual c-abl expression has a role in chronic phase CML hematopoiesis, as its inhibition impairs both myeloid and erythroid colony formation in vitro.     

Slowing growth and histology changes in Lewis lung carcinoma implanted in a partly denervated muscle

The purpose of this study is to test the effect of partial muscle denervation on a tumor inocculated on the same muscle. Experiments were performed on male C57BL mice divided into two numerically equal A and B groups, each of which was subdivided into three subgroups AA, AB, Ac and BA, BB, BC, respectively. The right sciatic nerve of each mice of the A group was incised and partly dissected (day 0). Then Lewis lung carcinoma (LLC) cells were implanted on the right gastrocnemia of each mice of both groups on day 0 for subgroups AA and BA, on day 1 for subgroups AB and BB, and on day 4 for subgroups AC and BC. The incision and dissection of the sciatic nerve caused a significant reduction in the size of the tumor in all three A subgroups of mice. Fourteen days after implantation, the size of the primary tumors implanted on the right hind leg was 2.6 +/- 0.32 cm3, 3.5 +/- 0.21 cm3, and 4.7 +/- 0.31 cm3 for the three BA, BB, BC subgroups of the control group compared to 1.4 +/- 0.19 cm3, 2.0 +/- 0.28 cm3, and 3.0 +/- 0.27 cm3 for the three subgroups AA, AB, Ac, in which sciatic nerve was incised and dissected, indicating inhibition of the tumor growth of 46.2%, 43.0% and 36.2%, respectively. Although a smaller part of gastrocnemius is innervated by the anterior femoral nerve, which was left intact, denervation of the sciatic nerve caused inhibition of the tumor growth. This may be due to worse nutrition and/or blood supply of the LLC tumor. Mitochondria function and the degree of apoptosis of the tumor cells are further studied by us.     

B7-H3对小鼠Lewis肺癌细胞生长的影响

目的观察B7-H3对Lewis肺癌(LLC)细胞生长的影响并探讨其作用机制。方法采用40只C57BL/6小鼠,右腋下接种Lewis肺癌细胞悬液,建立皮下种植瘤模型,随机分为4组,在瘤体附近皮下围绕瘤体分别注射多西他赛、B7-H3及多西他赛+B7-H3,以注射空质粒作为对照。注射后第1、3、7、14天测量肿瘤的大小,计算肿瘤的体积;采用免疫组织化学及Western blot检测瘤组织中B7-H3的表达;采用ELISA检测小鼠血清IL-12的水平。结果多西他赛和B7-H3均能分别抑制肿瘤的生长,两者联合则抑制作用增强,多西他赛组、B7-H3组及多西他赛+B7-H3组的抑瘤率分别为32.98%、28.59%和4595%,上述处理后肿瘤细胞凋亡率均比对照组明显提高(P<0.01);B7-H3组及多西他赛+B7-H3组肿瘤组织中B7-H3的表达下调,但差异无统计学意义(P>0.05);小鼠血清中IL-12的表达在B7-H3组及多西他赛+B7-H3组均较对照组水平升高(P<0.05)。结论 B7-H3能抑制小鼠Lewis肺癌细胞的生长,其机制可能与通过IL-12诱导激活细胞毒性T细胞而产生抗肿瘤免疫应答有关。     

Ionizing radiation and busulfan induce premature senescence in murine bone marrow hematopoietic cells

Exposure of murine bone marrow (BM) cells to ionizing radiation (IR; 4 Gy) resulted in >95% inhibition of the frequency of various day types of cobblestone area-forming cells in association with the induction of apoptosis in hematopoietic stem cell alike cells (Lin(-) ScaI(+) c-kit(+) cells; IR: 64.8 +/- 0.4% versus control: 20.4 +/- 0.5%; P < 0.001) and progenitors (Lin(-) ScaI(-) c-kit(+) cells; IR: 46.2 +/- 1.4% versus control: 7.8 +/- 0.5%; P < 0.001). Incubation of murine BM cells with busulfan (BU; 30 micro M) for 6 h also inhibited the cobblestone area-forming cell frequency but failed to cause a significant increase in apoptosis in these two types of hematopoietic cells. After 5 weeks of long-term BM cell culture, 33% and 72% of hematopoietic cells survived IR- and BU-induced damage, respectively, as compared with control cells, but they could not form colony forming units-granulocyte macrophages. Moreover, these surviving cells expressed an increased level of senescence-associated beta-galactosidase, p16(Ink4a), and p19(Arf). These findings suggest that IR inhibits the function of hematopoietic stem cell alike cells and progenitors primarily by inducing apoptosis, whereas BU does so mainly by inducing premature senescence. In addition, induction of premature senescence in BM hematopoietic cells also contributes to IR-induced inhibition of their hematopoietic function. Interestingly, the induction of hematopoietic cell senescence by IR, but not by BU, was associated with an elevation in p53 and p21(Cip1/Waf1) expression. This suggests that IR induces hematopoietic cell senescence in a p53-p21(Cip1/Waf1)-dependent manner, whereas the induction of senescence by BU bypasses the p53-p21(Cip1/Waf1) pathway.     

Imaging of human tumor xenografts with an indium-111-labeled anti-epidermal growth factor receptor monoclonal antibody

The mouse monoclonal antibody (mAb) 225 IgG1 against the epidermal growth factor (EGF) receptor has been investigated for its capacity to localize in human tumor xenografts. The EGF receptor is the product of the c-erb-B proto-oncogene (also known as EGFR). Elevated expression of EGF receptors has been demonstrated in many human tumors and tumor cell lines. We studied A431 human vulvar squamous cell carcinoma cells, with 2 X 10(6) receptors per cell; MDA-MB-468 (MDA 468) human breast adenocarcinoma cells, with 3 X 10(5) receptors per cell; and MCF-7 human breast adenocarcinoma cells, with 5 X 10(3) receptors per cell. The 111In-labeled pentetic acid (DTPA), derivative of mAb 225 (111In-DTPA-225) was injected intraperitoneally into nude mice bearing subcutaneous tumor xenografts. We measured uptake by quantifying radioactivity in tumor and normal tissues and by obtaining gamma camera images. Uptake in A431 xenografts was 28% +/- 2.4% of the injected dose per gram of tumor on day 3 and 12.4% +/- 3.0% on day 7. Distribution ratios comparing uptake in the tumor with that in normal tissues were consistently greater than 4. In contrast, there was far less uptake of the control mAb KS1/4S-1 labeled with 111In. This conjugate, 111In-DTPA-KS1/4S-1, has an IgG1 isotype but does not bind to human or murine cells. Imaging of the tumor with mAb 225 was excellent, especially on days 3-7. MDA 468 xenografts exhibited reduced localization of mAb 225 in the tumor. For MCF-7 xenografts, the tumor uptake of mAb 225 after 7 days was only 0.70% +/- 0.10% of the injected dose per gram of tumor, which was comparable to the uptake of the KS1/4S-1 control mAb. The ratio of the concentration of radioactivity in the tumor to that in normal tissue (distribution ratio) showed poor selectivity of uptake, and imaging was not obtained. These observations suggest that labeled mAb can target the product of a proto-oncogene, the EGF receptor, when it is expressed at high levels in human tumor xenografts.     

Identification of a protein factor secreted by 3T3-L1 preadipocytes inhibitory for the human MCF-7 breast cancer cell line.

The 3T3-L1 cell line is a preadipocyte cell line derived from the Swiss 3T3 mouse fibroblast cell line. We have compared the effect of 3T3-L1 conditioned medium (3T3-L1 CM) and Swiss 3T3 conditioned medium (3T3 CM) on the growth of normal mouse mammary cells (NMMG) and the human MCF-7 breast carcinoma cell line. 3T3 CM increased the growth of both NMMG and MCF-7 cells by 19 +/- 2% (SD) and 24 +/- 3%, respectively, and increased thymidine incorporation by 74 +/- 4% and 104 +/- 8%, respectively. Conditioned medium from 3T3-L1 cells stimulated the growth of NMMG cells by 64 +/- 2%; in contrast, 3T3-L1 CM inhibited the growth of MCF-7 cells by 36 +/- 1%. In parallel with these growth studies, thymidine incorporation increased by 20 +/- 4% in NMMG cells and decreased by 72 +/- 5% in the MCF-7 cells. Moreover, a similar effect was also noted in NCI H630 colon cancer cells, where 3T3-L1 CM produced a 58 +/- 4% decrease in growth and a 82 +/- 6% decrease in thymidine incorporation. Heating the 3T3-L1 CM at 100 degrees C for 30 min destroyed all inhibitory activity. Several known inhibitory growth factors (fibroblast growth factor, 20 ng/ml; interleukin 6, 1000 units/ml; tumor necrosis factor alpha, 15 ng/ml; transforming growth factor beta, 1 ng/ml) were tested for activity in the MCF-7 cells. Tumor necrosis factor alpha and transforming growth factor beta produced a 97% and 67% inhibition of thymidine uptake, respectively, whereas interleukin 6 and fibroblast growth factor had no effect. Neither transforming growth factor beta nor tumor necrosis factor alpha activity was detectable in 3T3-L1 CM using an enzyme-linked immunosorbent assay. High-performance liquid chromatography fractionation of the 3T3-L1 CM revealed that the inhibitory activity eluted at a molecular weight of 67,000; moreover, silver staining of these eluates on a denaturing polyacrylamide gel revealed that M(r) 69,000 peptide was the predominant protein band in the inhibitory fractions. Thus 3T3-L1 CM stimulates the growth of normal breast epithelial cells and inhibits the growth of MCF-7 breast cancer cells. This inhibitory activity appears to be due to a protein secreted by 3T3-L1 preadipocytes.     

Combination of Fasl and GM-CSF confers synergistic antitumor immunity in an in vivo model of the murine Lewis lung carcinoma

Gene transfer of Fas ligand (FasL) to tumor cells has been demonstrated to inhibit tumor growth in vivo, and neutrophils are primarily responsible for this immunoprotection. The granulocyte-macrophage colony stimulating factor (GM-CSF) secreted by tumor vaccine can recruit dendritic cells (DCs) for efficient antigen presentation to T cells that generate the tumor-specific response. To investigate whether the combination of FasL and GM-CSF can efficiently suppress tumor growth, we have established Lewis lung carcinoma (LLC-1) cells that are transduced with GM-CSF (LLC/GM-CSF), FasL (LLC/FasL) or both genes (LLC/FasL/GM-CSF) to test their tumorigenic potential in vivo. Mice inoculated with LLC/GM-CSF display high survival rates along with reduction of tumor growth. In contrast, none of the mice injected with LLC/FasL or LLC/FasL/GM-CSF develop tumors. Specific memory immune response and delayed LLC-1 tumor growth are found in mice immunized with LLC-1/FasL or LLC-1/FasL/GM-CSF. Furthermore, therapeutic effects are observed only when LLC-1/FasL/GM-CSF tumor vaccine is employed to retard growth of preexisting LLC-1 tumors. Tumor growth is also completely suppressed in mice injected with a mixture of LLC-1 and LLC-1/FasL/GM-CSF. In addition, IL-12 production, cytotoxic T-cell activity and IgG against LLC-1 are manifested in mice injected with LLC/FasL/GM-CSF. Our data show that FasL-induced pathway triggers expression of proinflammatory cytokines, including IL-1 beta, IL-6, MIP-2 and MCP-1, while GM-CSF-dependent pathway promotes functional maturation and activation of DCs. Taken together, the results indicate that dual gene-based delivery with FasL and GM-CSF may serve as a more effective tumor vaccine to suppress lung cancer cell growth in vivo.     

Treatment of hamster pancreatic cancer with alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis

Polyamines are essential for cell division and growth. Inhibition of polyamine biosynthesis by alpha-difluoromethylornithine (DFMO) on the growth of hamster H2T pancreatic cancer was investigated both in vitro and in vivo. Cell-doubling time (TD) and survival fraction were determined after a single treatment with DFMO (5 mM). We examined the ability of putrescine to reverse the growth-inhibitory effect of DFMO. The TD for cells treated with DFMO in vitro was 49.6 +/- 5.7 versus 25.4 +/- 2.6 hours for control. The addition of putrescine to DFMO-treated H2T cells showed a reversal of the growth-inhibitory effect of DFMO. Cytotoxicity in vitro increased with prolonged treatment; the survival fraction after 24 hours of treatment was 32%; after 48 hours, 19%; after 72 hours, 13%; and after 92 hours, 8%. We performed two separate animal experiments. In experiment I, H2T cells were injected into the cheek pouch of male Syrian golden hamsters; controls did not receive DFMO. Continuous treatment with 3% DFMO in the drinking water was begun 7 days before, on the day of, or 7 days after tumor cell injection. In experiment II, 4 groups were treated identically to those in experiment I. An additional group of 10 hamsters received 3% DFMO and no tumor, and another additional group of 10 hamsters were housed individually with 3% DFMO begun 7 days after tumor cell injection. Tumor size, body weight, water, and food intake were measured. DFMO treatment in vivo significantly inhibited tumor size and inhibited growth of pancreatic cancer by as much as 50% of control. Our results demonstrate a significant antiproliferative effect of DFMO on the growth of pancreatic adenocarcinoma both in vitro and in vivo.     

Endothelial Rac1 is essential for hematogenous metastasis to the lung

A variety of vasoactive stimuli induce endothelial permeability through Rac1, a membrane of Rho small GTPases. Here, we determine whether tumor-secreted vasoactive stimulant through Rac1 inducing permeability contributes to hematogenous metastasis. Activation of Rac1 was assayed in human umbilical vein endothelial cells (HUVEC), transendothelial passages were measured by Transwell chambers, and hematogenously metastatic mouse model was generated by intravenous injection with Lewis lung carcinoma cells (LLC). LLC secreted abundant vascular endothelial growth factor (VEGF) in the culture media and sera of mice bearing LLC xenografts or metastatic LLC, and VEGF activated Rac1 through VEGF receptors/PI3Kβ signaling cascade, resulting in hyperoxidative stress and consequent hyperpermeability in HUVEC. Moreover, in co-culture of LLC and HUVEC, significant increases in endothelial permeability and transendothelial migration of LLC were robustly attenuated by either anti-VEGF neutralizing antibody or Rac1 knockdown in HUVEC. Finally, in metastatic mouse model, deletion of one copy of Rac1 in endothelium not only significantly attenuated LLC-induced vascular permeability, but robustly reduced the metastasis of LLC to lungs. This study supports that tumor-secreted vasoactive stimuli activate Rac1 to induce permeability and consequent transendothelial migration of tumor cells, and that loss of Rac1 function in endothelium is an effective therapeutic intervention for hematogenous metastasis.     

Secreted protein acidic and rich in cysteine promotes glioma invasion and delays tumor growth in vivo

Secreted protein acidic and rich in cysteine (SPARC) is highly expressed in human astrocytomas, grades II-IV. We demonstrated previously that SPARC promotes invasion in vitro using the U87MG-derived clone U87T2 and U87T2-derived SPARC-transfected clones, A2b2, A2bi, and C2a4, in the spheroid confrontation assay. Additional in vitro studies demonstrated that SPARC delays growth, increases attachment, and modulates migration of tumor cells in extracellular matrix-specific and concentration-dependent manners. Therefore, we propose that SPARC functionally contributes to brain tumor invasion and delays tumor growth in vivo, and that the effects of SPARC are related to the level of SPARC secreted into the extracellular matrix. To test these hypotheses, we stereotactically injected these clones into nude rat brains (six animals were injected per clone). Animals were sacrificed on day 7 to assess growth and invasion for all clones at the same time in tumor development. To determine whether SPARC delayed but did not inhibit growth, rats were injected with U87T2 or clone A2b2, and the animals were sacrificed on days 9 (U87T2) and 20 (A2b2), when the animals demonstrated neurological deficit. Brains were removed, fixed, photographed, paraffin embedded, and sectioned. Sections were then serially stained with H&E for morphological assessment of invasion and to measure tumor volume, immunohistochemically stained to visualize SPARC, subjected to in situ hybridization with the human AluII DNA-binding probe to identify human cells, and immunohistochemically stained with MIB-1 to measure proliferation index. The results demonstrate that SPARC promotes invasion in vivo at day 7. Both the low (A2bi) and the high (A2b2) SPARC-secreting clones produced invasive tumors, invading with fingerlike projections and satellite masses into adjacent brain, as well as along the corpus collosum. The intermediate SPARC secreting clone (C2a4) primarily migrated as a bulk tumor along the corpus collosum. SPARC significantly decreased tumor growth at day 7, as measured both by adjusted MIB-1 proliferation indices (U87T2 = 95.3 +/- 1.4 versus A2bi = 73.4 +/- 4.0, A2b2 = 30.8 +/- 6.7 and C2a4 = 15.7 +/- 13.0) and tumor volumes (U87T2 = 13.4 +/- 0.6 mm(3) versus A2bi = 4.5 +/- 0.6 mm(3), A2b2 = 1.1 +/- 0.1 mm(3), and C2a4 = 0.4 +/- 0.1 mm(3)). Furthermore, SPARC delayed but did not inhibit tumor growth. The patterns of invasion and the extent of growth delay correlated with the level of SPARC expression. We propose that the ability of SPARC to promote invasion depends on the level of its secretion and the resultant modulation of the level of adherence and motility induced. This demonstration that SPARC functionally contributes to brain tumor invasion in vivo suggests that SPARC is a candidate therapeutic target for the design of therapies directed toward inhibition of the invasive phenotype.     

Treatment of primary radiogenic C57BL mouse cell leukemia/lymphoma by 1,3-bis(2-chloroethyl)-1-nitrosourea chemotherapy and adjuvant cellular therapy

Primary radiation-induced or radiation leukemia virus (RadLV)-induced T-leukemias/lymphomas were treated in vivo in an early to advanced state by using 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU was given at various times after the tumor induction procedure. Death from RadLV lymphomas which had been initiated in 33 +/- 3 day old C57BL mice by intrathymic injection of RadLV was scored in untreated, BCNU-treated or BCNU and cellular adjuvant treated mice. Intrathymic RadLV injection in 33 +/- 3 day old mice produced tumors in 98% of injected mice. Median survival time (MST) was increased by BCNU and by BCNU plus bone marrow cell therapy whether done 33 or 47 days after RadLV. There was increased in MST from 108 days to 171 days by BCNU and bone marrow cell therapy given 33 days after tumor initiation and to 195 days when therapy was given 47 days after initiation. In radiation-induced lymphomas produced by 190 rad every week X 4 of 33 +/- 3 day old mice, spleen cell (X 1) therapy or BCNU treatment increased the MST of treated mice from 142 days to 177 days after iv spleen cells or to 195 days after iv-ip spleen cells, and this protocol produced 31% long-term cures. Cellular adjuvant therapy combined with BCNU chemotherapy was effective for curing the lymphomas but cellular adjuvant therapy alone was also highly effective for therapy.     

All-trans-retinoic acid effects the growth, differentiation and apoptosis of normal human myeloid progenitors derived from purified CD34+ bone marrow cells.

We have previously shown that all-trans retinoic acid (ATRA) increases the number of CFU-GM colonies grown from unseparated human bone marrow cells with crude sources of colony stimulating factors. In this study, we further characterized the effect of ATRA on the growth of CFU-GM stimulated by individual cytokines from multiple samples of CD34+ enriched or purified human bone marrow cells. The number of IL-3- or GM-CSF-induced CFU-GM with 3 x 10(-7) M ATRA was 3.25+/-1.13, and 2.17+/-0.8-fold greater respectively, compared to controls without ATRA, while G-CSF had no effect and the ratio of colony-induced with or without ATRA was 1.06+/-0.17 (P = 0.00012). No colonies grew with ATRA + IL-6 or ATRA without a cytokine. Maximum enhancing effect on IL-3-induced CFU-GM occurred when ATRA was added on day 2, gradually diminished when delaying ATRA, and in cultures of day 9 or older adding ATRA had no effect. In 14 days liquid cultures of purified CD34+ cells with IL-3, ATRA increased the number of myeloid differentiated cells to 91-95%, compared to 37-70% with IL-3 alone. In addition, the number of apoptotic cells using the annexin V method increased after 14 days from 5.1% with IL-3 to 17.1% with IL-3 + ATRA and by the TUNEL in situ method from 10-26% to 60-95%, respectively. This study demonstrates that ATRA consistently enhances the growth of myeloid progenitors from CD34+ cells. This effect is dependent on the stimulating cytokine, suggesting the myeloid cells responding to ATRA are the less mature CFU-GMs that are targets of IL-3 and GM-CSF and not the G-CSF-responding mature progenitors. The growth stimulation by ATRA and IL-3 is also associated with granulocyte differentiation and increased apoptosis. These studies further suggest a potential role of pharmacological doses of ATRA on the development of normal human hematopoietic cells.     

Significant antitumor effect from bone-seeking,alpha-particle-emitting (223)Ra demonstrated in an experimental skeletal metastases model

The therapeutic efficacy of the alpha-particle-emitting radionuclide (223)Ra (t(1/2) = 11.4 days) in the treatment against experimental skeletal metastases in rats was addressed. Biodistribution studies, involving measurement of (223)Ra in bone marrow samples, were performed in rats after i.v. injection. To study the therapeutic effect of (223)Ra, an experimental skeletal metastases model in nude rats was used. Animals that had received 10(6) MT-1 human breast cancer cells were treated with (223)Ra doses in the range of 6-30 kBq after 7 days. The biodistribution experiment demonstrated that (223)Ra was selectively concentrated in bone as compared with soft tissues. The femur content of (223)Ra was 800 +/- 56% of injected dose per gram tissue times gram body weight (b.w.; mean +/- SD) 1 day after the injection and 413 +/- 23% of injected dose per gram tissue times gram b.w. at 14 days. The femur:kidney ratio increased from (5.9 +/- 2.0).10(2) at 1 day to (7.2 +/- 3.0).10(2) at 14 days, whereas the femur:liver ratio increased from (6.2 +/- 0.2).10(2) to (9.1 +/- 6.6).10(2). Femur:spleen ratio increased from (8.1 +/- 0.3).10(2) at 1 day to (6.4 2.2).10(3) at 14 days. The femoral bone:marrow ratio was 6.5 +/- 2.1 after day 1 and larger than 15 at day 14. All of the tumor-bearing control animals had to be sacrificed because of tumor-induced paralysis 20-30 days after injection with tumor cells, whereas the rats treated with > or =10 kBq of (223)Ra had a significantly increased symptom-free survival (P < 0.05). Also 36% (5 of 14) of rats treated with 11 kBq and 40% (2 of 5) of rats treated with 10 kBq were alive beyond the 67-day follow-up period. No signs of bone marrow toxicity or b.w. loss were observed in the groups of treated animals. The significant antitumor effect of (223)Ra at doses that are tolerated by the bone marrow is most likely linked to the intense and highly localized radiation dose from alpha-particles at the bone surfaces. The results of this study indicate that (223)Ra should be additionally studied as a potential bone marrow-sparing treatment of cancers involving the skeleton.     

18F]3'-deoxy-3'-fluorothymidine-PET in NHL patients: whole-body biodistribution and imaging of lymphoma manifestations--a pilot study

OBJECTIVE: The nucleoside 3'-deoxy-3'-fluorothymidine (alovudine) is an antiviral agent accumulating in proliferating cells. We prospectively evaluated the biodistribution of the PET tracer [18F]3'-deoxy-3'-fluorothymidine (FLT) and its value in detecting manifestations of non-Hodgkin's lymphoma (NHL). METHODS: In this pilot study, 7 patients (6 male, 1 female) with indolent NHL (2), NHL in transformation (2) or aggressive NHL (3) were examined. Patients received initial staging or restaging with an interval of at least 10 weeks between therapy and positron emission tomography (PET). Mean doses of 324 +/- 165 MBq FLT were injected intravenously. Static PET scans were performed 50-70 minutes after application. Maximum standardized uptake values (SUV) of organs and NHL manifestations were calculated. FLT-positive lesions were verified by biopsies (3 lesions) or aspiration smears (5 lesions in 4 patients). RESULTS: Biodistribution: The tracer accumulated physiologically in hematopoietic marrow and in the liver. It was renally excreted. SUV of organs 1 hour after injection were: bones with hematopoietic marrow, 9.9 +/- 4.7; liver, 5.2 +/- 1.0; kidneys, 4.0 +/- 1.7; spleen, 3.0 +/- 1.2; bones without hematopoietic marrow, 1.9 +/- 1.1; lungs, 0.8 +/- 0.3. NHL manifestations: In 7 patients, diagnosis was verified as true positive by histology/cytology. Maximum lymphoma SUV of FLT positive lesions were 6 for the indolent group and 8-11 for lymphoma in transformation. In the aggressive group, SUV were 6, 14, and 17. The low SUV in this group was found in a highly proliferate large-cell anaplastic lymphoma combined with marked sclerosis of 30%. CONCLUSIONS: In this pilot study, FLT-PET was suitable in the imaging of NHL manifestations. In NHL with normal cellularity, FLT accumulated more intensely in aggressive NHL and NHL in transformation than in indolent NHL. Bones with hematopoietic marrow and the liver were the organs with the highest physiological uptake.     

125I粒子植入联合抗PD-1治疗对小鼠Lewis肺癌的抑制作用研究

目的:研究125I粒子植入对免疫微环境的影响,以及125I粒子植入联合抗程序性死亡受体-1(programmed cell death receptor-1,PD-1)治疗的抗肿瘤疗效.方法:在小鼠右后肢皮下注射Lewis肺癌(LLC)细胞构建肿瘤模型,利用流式细胞术分析125I粒子植入后PD-1、程序性死亡配体1(p...     

Prostate cancer radiosensitization in vivo with adenovirus-mediated p53 gene therapy.

An adenovirus 5 vector containing wild-type p53 cDNA (Ad5-p53) and a cytomegalovirus promoter was used to generate p53 transgene expression. Control vector (Ad5-pA) contained the poly-adenosine sequence. PC3 cells (2 x 10(6)) were injected s.c. into the legs of nude mice. Treatment with Ad5-p53 was initiated at a tumor volume of 200 mm3. Three intratumoral injections (days 1, 4, and 7) were given with 3 x 10(8) plaque-forming units, followed by 5 Gy pelvic irradiation (day 8) in one fraction using a cobalt-60 source. Tumor volume measurements were obtained every 2 days. LNCaP cells (2 x 10(6)) were injected orthotopically into the prostates of nude mice, and tumor weight was approximated using serum prostate-specific antigen (PSA) obtained from weekly tail vein bleedings. The target PSA for the start of the studies was 5 ng/ml. The intraprostatic injections of Ad5-p53 were done twice (days 1 and 2) and followed by 5 Gy pelvic irradiation on day 3. The PC3 tumor volume growth curves were log transformed and fitted using linear regression. The times (in days) for the tumors to reach 500 mm3 were calculated as 10.7 +/- 0.7 (+/- SE) for the saline control (no virus), 9.8 +/- 2.1 for Ad5-pA, 15.6 +/- 1.6 for Ad5-p53, 14.6 +/- 1.5 radiation therapy (RT; 5 Gy), 14.6 +/- 1.5 for Ad5-pA plus RT, and 31.4 +/- 5.3 for Ad5-p53 plus RT. The Ad5-p53 plus RT times were significantly different from the other groups. An enhancement factor of 3.4 was calculated, indicating supra-additivity. LNCaP tumor growth was determined via weekly serum PSA measurements. Treatment failure was determined using two PSA-based methods; a serum PSA of > 1.5 ng/ml or two rises in PSA during 6 weeks posttreatment. The results were similar using either end point. Treatment with Ad5-p53 plus 5 Gy resulted in significantly fewer PSA failures (<30%), as compared with Ad5-p53 alone (64-73%) and the other controls (approximately 80-100%) These results are also consistent with a supra-additive inhibition of tumor growth. Tumor growth in vivo was inhibited supra-additively when p53null and p53wildtype prostate tumors were treated with Ad5-p53 and 5 Gy radiation.     

Surgery promotes implantation of disseminated tumor cells, but does not increase growth of tumor cell clusters

INTRODUCTION: Local recurrence and peritoneal dissemination is common after intentionally curative resection of colorectal carcinoma. It is not yet clear which mechanisms stimulate post-operative intra-abdominal tumor development. Enhanced adhesion or growth of tumor cells and/or post-operative immuno suppression may influence tumor recurrence. AIMS OF THE STUDY: In the present study, we evaluated effects of local and remote surgery on intra-abdominal tumor development. MATERIALS AND METHODS: A standardized intra-abdominal trauma was inflicted by rubbing both uterus horns in laparotomy groups, while a dorsolateral thoracotomy was performed in thoracotomy groups (on day -1, 0, or +3). To induce tumor development rats were injected intra-peritoneally with the coloncarcinoma cell line CC531s on day 0 and evaluated after 21 days. RESULTS: Rats undergoing laparotomy and injection on day 0 showed significantly higher tumorload than control rats (195 +/- 20 vs. 47 +/- 29, P < 0.001). When a laparotomy was performed, the day before tumor inoculation even higher tumorload was seen (245 +/- 37 vs. 195 +/- 20, P < 0.01). Strikingly, performing a thoracotomy on the day before or on the same day as tumor inoculation resulted in enhanced tumorload compared to controls as well (135 +/- 84 vs. 47 +/- 29; P < 0.001 and 88 +/- 38 vs. 47 +/- 29; P < 0.02, respectively). Either laparotomy or thoracotomy 3 days after tumor cell inoculation did not affect growth of pre-existing tumor cell clusters. CONCLUSIONS: The (post) surgical intra-peritoneal microenvironment enhances successful implantation of spilled tumor cells, whereas growth of adhered tumor cell clusters is not affected. The inflammatory response as a result of remote surgery promotes successful tumor development as well.     

Reduction in carboplatin hematopoietic toxicity in tumor bearing mice: comparative mechanisms and effects of interleukin-1 beta and corticosteroids

Increasing clinical evidence suggests that treatment of certain cancers is more effective with high dose chemotherapy compared to standard dose chemotherapy. Efforts at reduction of high dose chemotherapy hematotoxicity have focused on post-chemotherapy administration of hematopoietic growth factors or stem cells. A pretreatment strategy to induce hematopoietic resistance has not been extensively examined experimentally or clinically. Pretreatment with agents can provide an alternative or additional method to reduce high-dose chemotherapy hematotoxicity. We have previously described a murine model in which normal mice were injected with high dose carboplatin (CB, 600 mg/m2, intravenously). Within 7-12 days post-therapy, severe granulocytopenia and thrombocytopenia were induced and resulted in a granulocytopenic mortality of 70-80%. Utilizing this model, we observed that pretreatment of mice with interleukin (IL)-1 beta and/or a corticosteroid effectively reduced CB hematotoxicity. In the current studies, we demonstrated that C3H/HeJ mice bearing 0.25-0.5 cm RIF-1 tumors, exhibited a tumor associated decrease in hematopoietic tolerance to CB compared to normal mice. However, IL-1 beta (1 ug/mouse/day for 7 days), cortisone acetate (CA 0.5 mg/mouse/day for 7 days) or both CA and IL-1 beta, decreased CB induced mortality rates (control = 73%, IL-1 beta = 46%, CA = 30%, IL-1 beta + CA = 5%). Pretreatment with IL-1 beta, CA, or both ameliorated CB-induced absolute granulocyte, lymphocyte and platelet count nadirs. Bone marrow granulocyte-macrophage colony forming units (CFU-GM) from mice treated with IL-1 beta demonstrated increased ex-vivo resistance to cisplatin, without altering its sensitivity to high specific activity 3H-thymidine (a measure of cell fraction in S-phase). Bone marrow CFU-GM from mice treated with CA exhibited increased resistance to both cisplatin and to high specific activity 3H-thymidine. Pretreatment with IL-1 beta, CA or both did not alter tumor sensitivity to CB in vivo. These data suggest that IL-1 beta, CA or the combination may be clinically useful in reducing the hematotoxicity of CB.     

Effects of non-motorized voluntary running on experimental and spontaneous metastasis in mice

The present study investigated the effects of non-motorized voluntary running on experimental metastasis of B16BL/6 melanoma and spontaneous metastasis of Lewis lung carcinoma (LLC) in male C57BL/6 mice. After 9 weeks of running, mice (n=30 per group) received an intravenous injection of B16BL/6 cells or a subcutaneous injection of LLC cells, and then they were continued with their running activities. Experiments were terminated 2 weeks after the intravenous injection of B16BL/6 cells or 2 weeks after surgical removal of the primary tumor from mice subcutaneously injected with LLC cells. Mice in the running group ran an average of 4-6 km/day for the duration of the experiment. Voluntary running reduced body weight compared with the sedentary controls, but there were no differences in the number and size of lung metastases between groups with either model. Voluntary running significantly reduced plasma insulin and leptin levels and increased adiponectin level in mice with and without LLC compared with the sedentary controls. Having LLC significantly increased plasma concentrations of vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB), PDGF-AB and monocyte chemotactic protein-1 (MCP-1) in mice. Voluntary running significantly increased plasma PDGF-BB and PDGFAB, but not VEGF and MCP-1, in mice with LLC compared to their sedentary counterparts. In conclusion, non-motorized voluntary running was favorable to body weight and the expression of related adipokines, but at 4-6 km/day it did not affect either experimental or spontaneous metastasis in mice.     

Utilization of bone marrow-derived endothelial cell precursors in spontaneous prostate tumors varies with tumor grade

Id1 and Id3 genes are required for vascularization, growth, and metastasis of xenograft tumors. In Id-deficient mice, tumor transplantation and proangiogenic factors fail to mobilize and recruit circulating endothelial precursor cells (CEPs) and hematopoietic cells, leading to defective tumor angiogenesis in various models. To investigate the requirement of Id genes and bone marrow incorporation in spontaneous prostate tumors, we crossbred Id mutant mice with the transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Id1-/- Id3+/- TRAMP mice display delayed tumor growth at 24 weeks compared with wild-type TRAMP mice. Id1 and Id3 were strongly expressed in the endothelial cells of poorly differentiated prostate adenocarcinoma but not in the vasculature of well-differentiated tumors, a finding that is corroborated in human prostate tumor samples. In Id-deficient TRAMP mice, the poorly differentiated tumors show extensive hemorrhage, whereas well-differentiated tumors exhibit none. Transplantation with Id wild-type bone marrow significantly reduced the hemorrhage in poorly differentiated prostate adenocarcinomas with bone marrow-derived endothelial cells contributing to 14% of the tumor blood vessels. However, in well-differentiated prostate adenocarcinomas, there was little evidence of bone marrow-derived endothelial cell incorporation. These differences in the expression of Id genes, the effects of Id loss, and the recruitment of bone marrow-derived endothelial precursor cells in tumor vasculature between well-differentiated and poorly differentiated prostate adenocarcinoma suggest that tumor angiogenesis varies depending on the tumor grade.