Xenograft System Research Articles

Protein Kinase Cζ Represses the Interleukin-6 Promoter and Impairs Tumorigenesis In Vivo

Gene alterations in tumor cells that confer the ability to grow under nutrient- and mitogen-deficient conditions constitute a competitive advantage that leads to more-aggressive forms of cancer. The atypical protein kinase C (PKC) isoform, PKCzeta, has been shown to interact with the signaling adapter p62, which is important for Ras-induced lung carcinogenesis. Here we show that PKCzeta-deficient mice display increased Ras-induced lung carcinogenesis, suggesting a new role for this kinase as a tumor suppressor in vivo. We also show that Ras-transformed PKCzeta-deficient lungs and embryo fibroblasts produced more interleukin-6 (IL-6), which we demonstrate here plays an essential role in the ability of Ras-transformed cells to grow under nutrient-deprived conditions in vitro and in a mouse xenograft system in vivo. We also show that PKCzeta represses histone acetylation at the C/EBPbeta element in the IL-6 promoter. Therefore, PKCzeta, by controlling the production of IL-6, is a critical signaling molecule in tumorigenesis.

Genetically Engineered Mouse Models of Brain Cancer and the Promise of Preclinical Testing

Recent improvements in the understanding of brain tumor biology have opened the door to a number of rational therapeutic strategies targeting distinct oncogenic pathways. The successful translation of such “designer drugs” to clinical application depends heavily on effective and expeditious screening methods in relevant disease models. By recapitulating both the underlying genetics and the characteristic tumor‐stroma microenvironment of brain cancer, genetically engineered mouse models (GEMMs) may offer distinct advantages over cell culture and xenograft systems in the preclinical testing of promising therapies. This review focuses on recently developed GEMMs for both glioma and medulloblastoma, and discusses their potential use in preclinical trials. Examples showcasing the use of GEMMs in the testing of molecularly targeted therapeutics are given, and relevant topics, such as stem cell biology, in vivo imaging technology and radiotherapy, are also addressed.

Pre-clinical evidence for nab-paclitaxel efficacy in the treatment of adrenocortical cancer

22070 Background: Adrenocortical carcinoma (ACC) is a rare and lethal cancer with an average incidence of 0.7 in 1 million. Response to current standard therapy including mitotane is approximately 22%. Expression profiling analysis of human tumors has identified potential therapeutic targets which were then tested in in vitro and in vivo systems. Methods: RNA samples from 25 ACC tumors and 4 normal adrenal glands were profiled on both the Affymetrix Human Genome U133 Plus 2 and Agilent 22K element expression arrays. Novel therapeutic targets identified were tested in comparison with mitotane in two ACC cell lines (H295R and SW-13) in vitro and in vivo in a Scid mouse xenograft system. Protein levels of selected genes were subsequently assayed by IHC of a xenograft tumor TMA. Results: Expression of SPARC (secreted protein acidic rich in cysteine), an albumin binding matrix protein involved in cell structure and cell cycle progression, is upregulated within the ACC tumor population 1.50 + 0.81 (μ+SD) and 3.54+ 2.37 (μ+SD) fold on the Affymetrix and Agilent platforms, respectively. The anti-microtubular drugs paclitaxel and nanoparticle albumin bound paclitaxel (ABRAXANE®) exhibited in vitro inhibition of H295R and SW-13 cells at IC50 concentrations of 0.33 μM and 0.0078 μM for paclitaxel and 0.35 μM and 0.0087 μM for nab-paclitaxel compared to mitotane concentrations of 15.9 μM and 46.4 μM. IHC staining of treated xenograft tumors shows no induction of MDR1 with nab-paclitaxel treatment. Conclusions: Biological insight garnered through expression profiling of ACC tumors supports the clinical investigation of nab-paclitaxel use in the treatment of ACC in which there is over-expression of SPARC. No significant financial relationships to disclose.

Detection of zona pellucida proteins during human folliculogenesis

The stage of folliculogenesis at which the human zona pellucida (ZP) is initiated and the cells responsible for the origin of the ZP continue to be controversial. This study characterizes the development of the ZP during human folliculogenesis using ovarian samples donated from patients requesting ovarian storage. Follicles (from n = 18 patients, 14-40 years old) within fresh tissue and following development in a xenograft system were stained, using immunohistochemical techniques, for the presence of the three human ZP proteins, ZP1, ZP2 and ZP3. Over 500 primordial follicles and >20 follicles at each developmental stage were examined. All three ZP proteins were detected within the oocyte of the primordial follicle. Presence of ZP1 and ZP3 was observed in the majority of primordial oocytes (93% and 95%, respectively), whereas ZP2 was detected in only 32% of these follicles. The three ZP proteins were detected in the cytoplasm of cuboidal granulosa cells and their distribution correlates with developmental stages throughout folliculogenesis. ZP proteins were detected in both the oocyte and the granulosa cells as early as the primordial follicle stage in the human. The detection of ZP proteins in the quiescent primordial follicle suggests that these proteins have been present since oogenesis.

Adult Human Stem Cells Exert Therapeutic Effects To Repair Damaged Tissues in Xenograft Systems through Secretion of Trophic Factors Rather Than Direct Incorporation and Expansion.

Human stem cells from adult sources have been shown, in our laboratory and others, to promote tissue repair. Different populations of stem cells have been shown to contribute to the regeneration of muscle, liver, heart, and vasculature, although the mechanisms by which they accomplish this are still not well understood. Stem cells are known, however, to secrete a variety of factors that have both paracrine and autocrine activities. One theory of tissue repair and regeneration by adult stem cells is that they home to hypoxic and/or inflamed areas, and release trophic factors that hasten endogenous repair. These secreted bioactive factors suppress the local immune system, enhance angiogenesis, inhibit fibrosis and apoptosis, and stimulate recruitment, retention, mitosis and differentiation of tissue-residing stem cells. These effects, which are referred to as trophic effects, are distinct from the direct differentiation of stem cells into the tissue to be regenerated. We tested human umbilical cord blood (UCB) derived CD34+, ALDHhiLin− and ALDHloLin− cells following transplantation to NOD/SCID or NOD/SCID/B2M null mice with experimentally induced acute myocardial infarction (AMI). We used combined nanoparticle labeling and whole organ fluorescent imaging to detect homing of the cells to multiple organs 48 hours post transplantation. Long term engraftment and the regenerative effect of cell treatment was assessed four weeks post transplant. There was superior homing of ALDHhiLin− cells to the site of injury, as compared to CD34+ or ALDHloLin− cells at 48 hours post transplantation. At four weeks post transplantation, ALDHhiLin− cells engrafted multiple organs, including the heart, liver and kidney, at higher frequencies than ALDHloLin− cells. We found no donor derived cardiomyocytes and only few endothelial cells of donor origin. However, there was a significant increase in the density of large caliber vessels in the central infarct zone of ALDHhiLin− cell-transplanted mice, as compared to PBS and ALDHhiLin− cell treated groups. Tissue staining in the damaged regions revealed that the transplanted human cells had undergone very few cell divisions after homing to the area of hypoxia or inflammation. It is important to note that, with over 300 mice now analyzed in tissue repair experiments in our group, no adult human stem cell-derived tumors have ever been observed, even though the mouse strains used have no capacity to reject the human cells. The paucity of human cells remaining in the tissue after repair suggest that the tissue improvements that were observed were not the result of generation of transplanted cell-derived endothelial cells or cardiac tissue, but suggest that cytokines secreted from transplanted cells potentiated angiogenic activity and tissue repair by the endogenous murine cells. Our data indicate that adult human stem cells do not become a significant part of the damaged tissue, but rapidly home to and persist only temporarily at a site of hypoxia or inflammation to exert significant trophic effects on tissue repair, and to enhance recovery of the vasculature.

Human FLT3/FLK2 Targets Hematopoietic Stem Cells and Granulocyte/Macrophage Progenitors.

FLT3/FLK2, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis, and the constitutively active form of the FLT3 mutation is one of the most common genetic abnormalities in acute myelogenous leukemia. In murine hematopoiesis, Flt3 is not expressed in self-renewing long-term hematopoietic stem cells (LT-HSCs), but its expression is restricted to the multipotent and the lymphoid progenitor stages at which cells are incapable of self-renewal. In order to test whether Flt3 expression can delineate such a developmental pathway also in human hematopoiesis, we have analyzed the expression of human Flt3 (hFlt3) in prospectively-purified human stem and progenitors (PNAS 2002) by utilizing 7-color FACS and a highly efficient xenograft systems. We have found that Flt3 expression in early hematopoiesis is completely different between human and mice: hCD34+hCD38-hCD90+Lin-LT-HSCs capable of long-term reconstitution in xenogeneic hosts uniformly express hFlt3, and its expression is upregulated through hCD34+hCD38+hCD45RA-hCD123+Lin-common myeloid progenitors (CMPs) to hCD34+hCD38+hCD45RA+hCD123+Lin-granulocyte/macrophage progenitors (GMPs), but hCD34+hCD38+hCD45RA-hCD123-Lin- megakaryocyte/erythrocyte progenitors (MEPs) shut off its expression in human. Furthermore, we have also demonstrated that hFlt3 signaling can prevent stem and progenitors from apoptotic cell death in vitro without any effects on lineage fate decision. Next, we tried to find key molecules for Flt3-Flt3 ligand (FL)-mediating anti-apoptotic effect. First, we tested expression pattern of anti-apoptotic Bcl-2 family genes in HSCs, CMPs, GMPs, MEPs and common lymphoid progenitors (CLPs) in human hematopoiesis. Mcl-1, an indispensable survival factor for murine hematopoiesis (Science, 2005), was also expressed at the highest level in human HSCs, whereas Bcl-2 and Bcl-xL was highly expressed in GMPs and MEPs, respectively. Next, we examined whether FL stimulation can upregulate the expression of Bcl-2 family genes in human purified HSCs and progenitors. FL significantly upregulated the expression of Mcl-1, but not of Bcl-2 or Bcl-xL in HSCs as well as CMPs and GMPs. In conclusion, our data show that the distribution of Flt3 is quite different in mouse and human hematopoeisis. Human Flt3 targets LT-HSCs and myeloid progenitors except for MEPs. Flt3 signaling might support cell survival in early hematopoiesis including the HSC and the myeloid progenitor stages through upregulation of Mcl-1. This is a striking example that the expression pattern of key molecules could be significantly different between human and mouse.

Bortezomib Sensitizes Non–Hodgkin's Lymphoma Cells to Apoptosis Induced by Antibodies to Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (TRAIL) Receptors TRAIL-R1 and TRAIL-R2

Non-Hodgkin's lymphoma (NHL) is an increasingly common disease that, despite advances in antibody-targeted therapy, still requires novel therapeutic approaches. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates a major nonmitochondrial pathway for tumor cell killing through binding to a receptor family, some activating and some decoy. Agonistic antibodies to the receptors TRAIL-R1 and TRAIL-R2 can mimic many of the effects of TRAIL. We are investigating the effects of such agonistic antibodies, mapatumumab directed at TRAIL-R1 and lexatumumab directed at TRAIL-R2, on NHL cell lines. These antibodies induce apoptosis through caspase-8 but also activate BID to involve the mitochondrial pathway and activate caspase-9. In addition, we find signaling through both the nuclear factor-kappaB and c-Jun NH2-terminal kinase pathways. Because the proteasome inhibitor bortezomib also affects these pathways, we have investigated the combination of TRAIL-R antibodies and bortezomib and show enhanced apoptosis and signaling as well as enhanced killing of NHL cells in a severe combined immunodeficient mouse/human NHL cell line xenograft system. The combination of bortezomib and TRAIL signaling warrants further investigation as a therapeutic regimen. Understanding the multiple intracellular pathways of TRAIL activation may lead to rationally designed therapeutic trials.

Targeting the mitotic spindle in the treatment of adrenocortical cancer

10572 Background: Adrenocortical carcinoma (ACC) is a rare and lethal cancer with an average incidence of 1 in 1.4 million. Response to current standard therapy including mitotane is approximately 22%. Herein we use gene expression profiling to identify novel targets for therapeutic intervention. Methods: The transcriptomes of 29 ACC tumors and 4 normal adrenal glands were profiled on Affymetrix Human Genome and Agilent 22K element expression arrays in order to identify genes that could represent potential therapeutic targets. Agents known to inhibit the selected targets were tested in comparison with mitotane in two ACC cell lines (H295R and SW-13) in vitro and are currently being tested in a Scid mouse xenograft system. Results: Expression of numerous kinesin family members is dysregulated within the ACC tumor population. Of interest in the ACC cells EG5 (KIF11, KSP), a kinesin important for mitotic spindle formation, is up-regulated 5.81+ 4.42 (μ+SD) and 6.48 + 0.99 fold on the Agilent and Affymetrix platforms respectively. In addition, SPARC, an albumin binding matrix associated protein is up-regulated in ACC tumors by 3.69 + 2.37 (μ+SD) and 1.56 + 0.44 on the two platforms. The anti-microtubular drugs paclitaxel and albumin bound paclitaxel (abraxane) exhibited in vitro inhibition of H295R and SW-13 cells at IC50 concentrations of 0.33 μM and 0.0078 μM for paclitaxel and 0.35 μM and 0.0087 μM for Abraxane compared to mitotane concentrations of 15.9 μM and 46.4 μM. Treatment effects of monastrol, paclitaxel, and Abraxane are currently being tested in xenograft murine models. Testing of the EG5 inhibitor S-trityl-L- cysteine is ongoing in the in vitro system. Conclusions: KIF11 inhibitors have been proposed in the treatment of cancer. These compounds may offer better anti-mitotic activity than other tubulin interacting agents with less neurotoxic side effects. Based on biological insight garnered through expression profiling of ACC tumors, the current ongoing research is investigating the use of abraxane and EG5 inhibitors in the in vitro and in vivo treatment of ACC. No significant financial relationships to disclose.

Human amniotic fluid-derived stem cells are rejected after transplantation in the myocardium of normal, ischemic, immuno-suppressed or immuno-deficient rat

Human amniotic fluid-derived stem (AFS) cells, similarly to embryonic stem cells, could possess privileged immunological characteristics suitable for a successful transplantation even in a discordant xenograft system. We investigated whether AFS cells could be fruitfully used in a rat model of myocardial infarction. c-kit immunomagnetic-sorted AFS cells were characterized by flow cytometric analysis and cytospins as well as reverse-transcription polymerase chain reaction, Western blotting and immunocytochemistry for cardiovascular differentiation markers. In vitro, AFS cell phenotypic conversion was assayed by cardiovascular-specific induction media or co-cultured with rat neonatal cardiomyocytes. AFS cells showed mRNAs and/or protein for endothelial (angiopoietin, CD146) and smooth muscle (smoothelin) cells, and cardiomyocyte (Nkx2.5, MLC-2v, GATA-4, β-MyHC) markers. Acquisition of a cardiomyocyte-like phenotype in rare AFS cells could be seen only in co-cultures with rat neonatal cells. In vivo, AFS cells xenotransplantated in a rat model of myocardial infarction, with or without cyclosporine treatment, or in intact heart from immuno-competent or immuno-deficient animals were acutely rejected due to the different recruitment of recipient CD4 +, CD8 + T and B lymphocytes, NK cells and macrophages. This reaction is most likely to be linked to expression of B7 co-stimulatory molecules CD80 and CD86 as well as macrophage marker CD68 on AFS cells. Xenotransplanted AFS cells gave also rise in some animals to cell masses in the subendocardium and myocardium suggestive of a process of chondro-osteogenic differentiation. Despite AFS cells in vitro can differentiate to some extent to cells of cardiovascular lineages, their in vivo use in xenotransplantation for cell therapy of myocardial infarction is hampered by their peculiar immunogenic properties and phenotypic instability.

HIV-1 infection and CD4 T cell depletion in humanized Rag2-/-yc-/- mice

Previously well established mouse-human xenograft systems, namely, the SCID-hu and hu-PBL-SCID mouse models have been instrumental for several advances in HIV research. However, as these models lacked the capacity for generating primary immune responses, viral pathogenic effects on the human cells could only be evaluated in the context of the absence of an active immune response. Intra-hepatic injection of human CD34 hematopoietic stem cells into immunodeficient Rag2 common gamma chain knock out (Rag2-/-γc-/-) mice results in multilineage human hematopoiesis with de novo production of T cells, B cells, monocytes and dendritic cells, and the generation of a functional immune system. Therefore this in vivo model shows great potential to study HIV infection and evaluation of therapeutic approaches. As a first step towards developing this model, humanized Rag mice (Rag-Hu mice) were infected intraperitonially with either X4 tropic or R5 tropic HIV-1. Prolonged viremia (up to 12 weeks so far) could be demonstrated in the infected mice. CD4 T cell depletion together with lymphadenopathy was also seen in viremic mice. Further characterizations are currently underway. Several new experimental approaches are now possible in these humanized mice that include vaccine testing, viral latency and evaluation of novel therapeutics.

HFlt3 Expression Marks Long-Term Hematopoietic Stem Cells, but Not the Pathway Common to Granulocyte/Monocyte and Lymphoid Lineages in Human Hematopoiesis.

Flt3 is a tyrosine kinase receptor, whose signals support proliferation and differentiation of early hematopoietic progenitors in cooperation with other cytokine signals. The concept of myeloid vs. lymphoid commitment in murine hematopoiesis has been challenged based on the analysis of Flt3 expression (Adolfsson et al, Cell, 2006): Long-term murine hematopoietic stem cells (HSCs) with self-renewal activity do not express murine Flt3 (mFlt3), but mFlt3 is upregulated in a fraction of progenitors common to granulocyte/monocyte and lymphoid lineages, suggesting the existence of the 3rd pathway of murine hematolymphoid development. In order to test whether Flt3 expression can delineate such a developmental pathway also in human hematopoiesis, we have analyzed the expression of human Flt3 (hFlt3) in prospectively-purified human stem and progenitors (PNAS 99, 2002) by utilizing 7-color FACS and a highly efficient xenograft systems. Surprisingly, all single human hCD34+hCD38-Thy-1+Lin-long-term HSCs expressed Flt3 at a low level. Purified Flt3lo HSCs can reconstitute NOD-scid/IL2Rγnull newborns (Blood 106, 2005) for a long-term after adoptive transfer of only 100 cells, and generated all hematopoietic cells including GM, megakaryocyte/erythrocyte (MegE), T, B and NK cells, providing formal proof that hFlt3 is expressed in long-term multipotent human HSCs. In the myeloid pathway, hCD34+hCD38+hCD45RA-IL-3RαloLin-CMPs expressed low to negative levels of hFlt3, and hCD34+hCD38+CD45RA-IL-3Rα-Lin-MEPs did not express Flt3. However, in marked contrast to murine progenitors, hCD34+hCD38+hCD45RA+IL-3RαloLin-GMPs uniformly expressed hFlt3 at the highest level. We then separated CMPs into 2 fractions with negative and low level expression of Flt3. In vitro culture in the presence of cytokine cocktail revealed that hFlt3lo CMPs differentiated into hFlt3-CMPs, hFlt3-MEPs and hFlt3hi GMPs, while hFlt3-CMPs mainly generated hFlt3-MEPs in vitro, suggesting that hFlt3lo CMPs is located upstream of the hFlt3-CMP stage that has skewed differentiation potential into the MegE lineage. In vitro differentiation activity of these progenitors was not affected with or without hFlt3 ligand in the culture. hFlt3 ligand, however, appeared to be able to maintain survival of hFlt3-expressing stem and progenitor cells at least in vitro. Thus, in striking contrast to mice, hFlt3 expression is already initiated at the most primitive long-term HSC stage, and is progressively upregulated in accordance with GM lineage differentiation. These data collectively suggest that the role of Flt3 signaling in hematopoietic development might be quite different between human and mouse, and that hFlt3 signaling may especially play a critical role in maintenance of long-term HSCs and in development of GM cells in human hematopoiesis.

Suppression effects of human recombinant tissue inhibitor of metalloproteinases-1(TIMP-1) on tumor proliferation using in vivo treatment model of well-differentiated colon cancer cell line, HT29.

To investigate the suppressive effect of human recombinant TIMP-1 (rh-TIMP-1) on tumor proliferation using an in vivo xenograft system, HT29 was suspended in 0.1 ml phosphate buffered saline (PBS) and then subcutaneously injected in the back of female mice (BALB/C nu/nu). The mice were divided into 2 groups an and the tumor diameter was measured after rh-TIMP-1 (2 mg/kg) (rh-TIMP-1 group) or PBS (control group) was administered injections according to the following schedules. Schedule 1 : Beginning 2 weeks after the subcutaneous injection of HT29, an intraperitoneal injection of rh-TIMP-1 or PBS were performed twice a day (every 12 h) for 14 consecutive days. Schedule 2 : Beginning 1 week after the subcutaneous injection of HT29, an intraperitoneal injection was performed twice a day for 14 consecutive days. Schedule 3 : Intraperitoneal injections were started simultaneously with the subcutaneous injection of HT29, and then performed twice a day for 21 consecutive days. The mice were sacrificed and the tumors extirpated for immunohistochemical investigation. In addition, gelatin zymography and a cell proliferation assay were performed. With Schedule 1, the changes in the tumor diameter in the rh-TIMP-1 group followed the same course as those in the control group, and no suppressive effect on tumor proliferation was observed. However, with Schedule 3, a remarkable suppressive effect was observed throughout the treatment period. In immunostaining, more cases negative for MMP-9 were observed in the rh-TIMP-1 group than in the control group. Cases negative for CD34 were significantly more observed in the rh-TIMP-1 group than in the control group with Schedule 3. All of the results were obtained through the suppressive effect of rh-TIMP-1 on angiogenesis.

Establishment and characterization of human urothelial cancer xenografts in severe combined immunodeficient mice

To establish and characterize a murine xenograft model of human urothelial cancer in severe combined immunodeficient (SCID) mice for therapeutic simulation. Pieces of 30 freshly resected urothelial tumors (24 obtained from bladder and 6 from ureter or pelvis) were implanted subcutaneously into SCID mice, and xenograft tumors were passed in tumorigenic cases. At each passage, histopathology, TP53 mutational status assessed by yeast p53 functional assay, and the Ki-67 labeling index (LI) were examined to evaluate the preservation of original features. A growth delay assay after single-dose irradiation was performed in four representative xenografts. Tumor growth was observed in 18 mice (60%, 18/30). Histologically, 15 of the 18 were epithelial carcinomas similar to the original tumors, whereas the other 3 were Epstein-Barr virus-associated lymphoproliferative disease, resulting in a 50% (15/30) take rate. No correlation was found between the tumor take rate and the clinicopathologic features, TP53 mutational status, or Ki-67 LI of the patients' tumors. Of these 15 xenografts, 11 xenografts were passed from 3 to 10 generations. TP53 mutational status remained stable during the passages, and the Ki-67 LI of eight xenografts was within a range of 50% of the LI of the original tumors, although the other three xenografts increased by over 50%. Specific growth delay after irradiation, independent of the original tumor growth speed and Ki-67 LI, was observed in four xenografts. SCID mice are useful recipients for investigations of human urothelial cancer with a wide biological range. This easy-to-handle xenograft system can help to develop a better in vivo preclinical evaluation system for therapeutic agents as well as the investigation of tumor pathophysiology.

Effects of cryotherapy or chemotherapy on apoptosis in a non-small-cell lung cancer xenografted into SCID mice

Lung cancers are among the most frequent and the most lethal tumours. They are mainly treated by surgery or by chemotherapy, but in the most advanced stages a local cryotherapy can be proposed as a palliative option for bronchial clearance. This therapy, based on the cytotoxic effects of low temperatures, acts by mechanisms which are not yet totally understood. The aim of this work was to investigate in vivo the biological effects of cryotherapy in a model of human non-small-cell lung cancer. We used a xenograft system: cells from the A549 cell line (adenocarcinoma) were injected subcutaneously into SCID mice. Cryotherapy was performed (three cycles, nitrous oxide cryoprobe). Chemotherapy (intravenous injection of Vinorelbine (Navelbine), 4.8 mg/kg) was used as a control treatment. Tumour nodes were excised at variable time points and studied morphologically. The induction of apoptosis was analysed by immunohistochemical staining of cleaved caspase-3 and by TUNEL. Results showed that cryotherapy was an efficient technique to induce cell death either by necrosis or by apoptosis. Necrosis was found near the cryoprobe impact site and was maximal 2 h after treatment (65%); a second peak was observed after 4 days (77%). Around this central necrotic area, apoptotic cells were found. Apoptosis was maximal after 8 h (47%). Chemotherapy induced apoptosis in a fewer number of cells and this effect was not time-dependent. Taken together, these results demonstrate the differential effects of cryotherapy and chemotherapy in vivo, suggesting different modes of action and the potential benefit to combine them.

Mise au point d’un modèle d’étude des effets d’une cryothérapie sur des tumeurs pulmonaires

Adenocarcinomas are today the most frequent lung cancers. They are mainly treated by surgery or by chemotherapy, but for the most advanced stages a local cryotherapy can be proposed as a palliative option for bronchial desobstruction. Aim of the study. – The aim of this work was to establish an experimental model to study in vivo the biological effects of this technique to propose it as a neoadjuvant treatment. Materials and methods. – A xenograft system was used: cells from the A549 cell line were injected subcutaneously into SCID mice. Tumour nodes could be treated after seven weeks. The histological study showed that these tumours faithfully reproduced the morphological features of adenocarcinoma, and developed an intratumoral neovascularization. Two protocols of cryotherapy (1 vs 3 cycles of freezing) were performed and the induction of apoptosis was analyzed by immunohistochemical staining of cleaved caspase-3. Results. – The basal expression of cleaved caspase-3 in untreated tumours (23%) increased after cryotherapy. The increase was maximal eight hours after treatment (up to 47% of positive cells) and was less important with the first protocol, suggesting a lesser efficiency in the induction of apoptosis. Conclusion. – The establishment of this model, which is faithful to physiological features, allowed us to demonstrate in vivo time and dose-dependent effects of cryotherapy.

Quantifying transient hypoxia in human tumor xenografts by flow cytometry.

Transient hypoxia is a poorly understood and potentially important factor that may limit tumor response to various forms of therapy. We assessed transient hypoxia on a global scale in two different human tumor xenografts by sequentially administering two hypoxia markers followed by quantification of hypoxic cells using flow cytometry. High levels of the first hypoxia marker (pimonidazole) were maintained in the circulation over an 8-hour period by multiple hourly injections, providing a "time-integrated" hypoxia measure showing an asymptotic increase in the total number of hypoxic cells. Subsequent administration of a second hypoxia marker (CCI-103F) showed that substantial numbers of the previously pimonidazole-labeled cells were no longer hypoxic during the circulation lifetime of the second marker. The overall fraction of tumor cells that demonstrated changes in hypoxic status with time increased with different kinetics and by different magnitudes in the two xenograft systems. Specifically, up to 20% of the cells in SiHa (human cervical squamous cell carcinoma) tumors and up to 8% of the cells in WiDr (human colon adenocarcinoma) tumors were intermittently hypoxic over an 8-hour period. Also, the tumor cells that demonstrated transient hypoxia were typically not adjacent to functional tumor blood vessels. Similar approaches could be used in the clinic to provide information on the duration of intermittent hypoxia episodes and the fraction of transiently hypoxic tumor cells, which would, in turn, have important implications for the strategic improvement of cancer therapy.

Immune-deficient mouse models for analysis of human stem cells.

The field of murine models of xenotransplantation has grown immensely over the past two decades. The explosive growth in this field is in part due to the fact that good in vitro methods do not exist yet to allow examination of human stem cell homing into the bone marrow compartment versus other tissues, long-term survival of human stem cells, or differentiation into tissues outside of the hematopoietic system. Since these important aspects of human stem cell biology can be examined in vivo using immune-deficient mice, the number of different strains and models is constantly increasing. The current review discusses the merits and drawbacks of each immune-deficient mouse xenograft system as it stands to date and reviews how each immune-deficient mouse model has been used to further our knowledge of human hematopoietic stem cell biology.

Pathogenesis of X-Linked Charcot-Marie-Tooth Disease: Differential Effects of Two Mutations in Connexin 32

X-linked Charcot-Marie-Tooth disease is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding connexin 32 (Cx32). Cx32 is expressed at the paranodes and Schmidt-Lantermann incisures of myelinating Schwann cells in which it is believed to form a reflexive pathway between the abaxonal and adaxonal cytoplasmic domains. Patients with the Val181Ala (V181A) mutation have a severe peripheral neuropathy. Experiments using a nude mouse xenograft system show that Schwann cells expressing only this mutant form of Cx32 are profoundly impaired in their ability to support the earliest stages of regeneration of myelinated fibers. Coupling between paired Xenopus oocytes expressing V181A is reduced compared with the coupling between oocytes expressing wild-type human Cx32 (32WT), and protein levels assayed by Western blot are substantially lower. Immunocytochemisty shows that Neuro2a cells expressing the V181A mutant have very few gap junction plaques compared with cells expressing 32WT; Cx32 protein levels are lower in these cells than in those expressing 32WT. Because failure of normal regeneration is evident before formation of myelin, loss of function of Cx32 may impact on the function of precursors of the myelinating Schwann cell before the formation of the hypothesized reflexive pathway. The Glu102Gly (E102G) mutation leads to a milder phenotype. Early regeneration is normal in grafts with Schwann cells expressing the E102G mutant. The only abnormality detected in the behavior of its channel is increased sensitivity to acidification-induced closure, a property that may lead to reduced gap junction coupling during periods of metabolic stress. This restricted functional abnormality may explain the relatively mild phenotype seen in the xenograft model and in E102G patients.

Antitumor effect of arsenic trioxide in murine xenograft model.

Arsenic trioxide, As(2)O(3) (ATO), has been established to be an effective agent for treating acute promyelocytic leukemia, but its effect on solid tumors has not been fully explored. In the present study in a murine xenograft system, we found that ATO significantly inhibited tumor growth of the inoculated human hepatocellular carcinoma cell line HuH7 when administered either intravenously or intratumorally. Pathological examination revealed that ATO induced extensive cell death in the tumor. Some of the dead cells in intratumorally ATO-treated mice showed characteristic features of apoptosis, such as nuclear condensation and fragmentation, and were TUNEL-positive. The measurement of arsenic by using particle induced X-ray emission revealed that arsenic was accumulated more in the tumor than in brain, kidney or liver after the intravenous injection of ATO, which is consistent with the hemorrhagic cell death observed in ATO-treated tumor tissues. Thus, ATO appears to have potential for the treatment of solid tumors, as well as hematopoietic malignancies.

Human papillomavirus type 59 immortalized keratinocytes express late viral proteins and infectious virus after calcium stimulation

Human papillomavirus type 59 (HPV 59) is an oncogenic type related to HPV 18. HPV 59 was recently propagated in the athymic mouse xenograft system. A continuous keratinocyte cell line infected with HPV 59 was created from a foreskin xenograft grown in an athymic mouse. Cells were cultured beyond passage 50. The cells were highly pleomorphic, containing numerous abnormally shaped nuclei and mitotic figures. HPV 59 sequences were detected in the cells by DNA in situ hybridization in a diffuse nuclear distribution. Southern blots were consistent with an episomal state of HPV 59 DNA at approximately 50 copies per cell. Analysis of the cells using a PCR/reverse blot strip assay, which amplifies a portion of the L1 open reading frame, was strongly positive. Differentiation of cells in monolayers was induced by growth in F medium containing 2 mM calcium chloride for 10 days. Cells were harvested as a single tissue-like sheet, and histologic analysis revealed a four-to-six cell-thick layer. Transcripts encoding involucrin, a cornified envelope protein, and the E1^E4 and E1^E4^L1 viral transcripts were detected after several days of growth in F medium containing 2 mM calcium chloride. The E1^E4 and L1 proteins were detected by immunohistochemical analysis, and virus particles were seen in electron micrographs in a subset of differentiated cells. An extract of differentiated cells was prepared by vigorous sonication and was used to infect foreskin fragments. These fragments were implanted into athymic mice. HPV 59 was detected in the foreskin xenografts removed 4 months later by DNA in situ hybridization and PCR/reverse blot assay. Thus, the complete viral growth cycle, including production on infectious virus, was demonstrated in the HPV 59 immortalized cells grown in a simple culture system.