Primary Tumor Cell Cultures Research Articles

Gastrointestinal stromal tumors (GISTs) express somatostatin receptors and bind radiolabeled somatostatin analogs

Background. Gastrointestinal stromal tumors (GISTs) can be effectively treated with tyrosine kinase inhibitors (TKIs). However, some patients with GIST develop drug resistance, and alternative treatment strategies are therefore needed. The aim of this study was to analyze the expression of somatostatin receptors (SSTR) in GIST as a target for peptide receptor-mediated radiotherapy (PRRT). Material and methods. Expression profiling of SSTR1–5 was performed on biopsies from 34 GISTs (16 gastric tumors, 15 small intestinal tumors, and three rectal tumors). SSTR scintigraphy (111In-octreotide) and measurement of 111In activity in tumor specimens was performed in seven patients. Uptake and internalization of 177Lu- octreotate was studied in primary cell cultures from two patients. Results. Quantitative PCR analysis showed expression of SSTR1 and SSTR2 in the majority of tumors, while SSTR3–5 were expressed at low levels. Immunohistochemical analysis confirmed the presence of SSTR1 and SSTR2 proteins in all GISTs, and SSTR3–5 in a subset of tumors. Diagnostic imaging by SSTR scintigraphy, using 111In-octreotide, demonstrated tumor uptake of 111In in three of six GIST patients. Measurement of 111In activity in excised tumor specimens from five patients gave tumor-to-blood (T/B) activity ratios of between eight and 96. Tumor cells in primary culture (gastric and small intestinal GIST) specifically bound and internalized 177Lu when incubated with the therapeutic compound 177Lu-octreotate for 4–48 hours (p < 0.05). Conclusion. Peptide receptor-mediated radiotherapy via SSTR may provide a novel treatment strategy in carefully selected GIST patients with TKI-resistant tumors.

Chemokine 25–induced signaling suppresses colon cancer invasion and metastasis

Chemotactic cytokines (chemokines) can help regulate tumor cell invasion and metastasis. Here, we show that chemokine 25 (CCL25) and its cognate receptor chemokine receptor 9 (CCR9) inhibit colorectal cancer (CRC) invasion and metastasis. We found that CCR9 protein expression levels were highest in colon adenomas and progressively decreased in invasive and metastatic CRCs. CCR9 was expressed in both primary tumor cell cultures and colon-cancer-initiating cell (CCIC) lines derived from early-stage CRCs but not from metastatic CRC. CCL25 stimulated cell proliferation by activating AKT signaling. In vivo, systemically injected CCR9+ early-stage CCICs led to the formation of orthotopic gastrointestinal xenograft tumors. Blocking CCR9 signaling inhibited CRC tumor formation in the native gastrointestinal CCL25+ microenvironment, while increasing extraintestinal tumor incidence. NOTCH signaling, which promotes CRC metastasis, increased extraintestinal tumor frequency by stimulating CCR9 proteasomal degradation. Overall, these data indicate that CCL25 and CCR9 regulate CRC progression and invasion and further demonstrate an appropriate in vivo experimental system to study CRC progression in the native colon microenvironment.

Specific Binding and Uptake of 131I-MIBG and 111In-Octreotide in Metastatic Paraganglioma - Tools for Choice of Radionuclide Therapy

Tumor-specific uptake of the radiolabeled nor-epinephrine analogue meta-iodobenzylguanidine via norepinephrine transporter or radiolabeled somatostatin analogues octreotide/octreotate via somatostatin receptors offers possibilities to diagnose and treat metastatic pheochromocytoma/paraganglioma. High uptake of 123I-meta-iodobenzylguanidine is dependent on high expression of vesicular monoamine transporters responsible for mediating uptake of biogenic amines into dense core granules. A patient with metastatic paraganglioma (liver and bone metastases) underwent surgical removal of the primary after injection of 131I-meta-iodobenzylguanidine and 111In-octreotide. Radioactivity was determined in biopsies from tumor and normal tissue biopsies. The tumor/blood concentration value was high: 180 for 131I-meta-iodobenzylguanidine 3 h after injection and 590 for 111In-octreotide 27 h after injection. Studies of primary tumor cell cultures demonstrated increased cell membrane binding and internalization over time for 131I-meta-iodobenzylguanidine. The vesicular monoamine transporter antagonist reserpine and the norepinephrine transporter inhibitor clomipramine reduced internalization by 90% and 70%, respectively, after 46 h of incubation. The results demonstrated increased cell membrane binding and internalization over time also for 111In-octreotide. Internalization was highest for a low concentration of 111In-octreotide. Excess of octreotide reduced internalization of 111In-octreotide with 75% after 46 h of incubation. In conclusion, uptake and tumor/blood concentration values of radiolabeled meta-iodobenzylguanidine and somatostatin analogues can be determined for metastatic pheochromocytoma/paraganglioma to evaluate the possibility to use one or both agents for therapy. For this patient, the high tumor/blood values clearly demonstrated that therapy using both radiopharmaceuticals would be most beneficial. In vitro studies verified specific cell-membrane binding and internalization in tumor cells of both radiopharmaceuticals.

Abstract 5207: The chemokine 25 and chemokine receptor 9 axis suppresses colon cancer invasion and metastasis

Abstract Chemokines help regulate tumor cell invasion and metastasis. Here, we show that the Chemokine 25/Chemokine Receptor 9 (CCL25/CCR9) axis suppresses colon cancer (CRC) invasion and metastasis. CCR9 protein expression levels are highest in colon adenomas, and progressively decrease in invasive and metastatic CRCs. CCR9 is expressed in both primary tumor cell cultures and colon cancer initiating cell lines (CCIC) derived from early stage (I/II) CRCs. CCL25 stimulates cell proliferation through a network of transcription factors downstream of AKT signaling. In vivo, systemically injected CCR9+ early stage CCIC spontaneously form orthotopic gastrointestinal xenograft tumors. Blocking CCR9 signaling prevents CRC tumor formation in the native gastrointestinal CCL25+ microenvironment, while increasing extra-intestinal tumor multiplicity. NOTCH signaling, which promotes CRC metastasis, increases extra-intestinal tumor multiplicity by stimulating CCR proteosomal degradation. These data provide insights into the mechanisms that regulate CRC progression, invasion and metastasis and describe a novel in vivo experimental system to study these processes. Our findings also raise a safety concern for ongoing Inflammatory Bowel Disease clinical trials using CCR9 antagonists. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5207. doi:1538-7445.AM2012-5207

Abstract 1355: Utility of primary cultured cells in personalized chemotherapy for angiosarcoma of the oral cavity

Abstract Current treatments for major malignant tumors are based on evidences accumulated over the world and clinical trials with molecular target drugs that are expected to improve their prognosis are ongoing. In contrast, rare malignant tumors have no standard therapies and few evidences for effective drugs. Moreover, epidemiology, etiology, pathogenesis and molecular mechanisms are unknown because published data are based on a few case series and experimental materials can be used in vitro do not exist. Thus we have to manage a treatment case by case when facing a rare tumor. One of the useful methods to evaluate drug efficacy is the collagen gel droplet embedded culture drug sensitivity test (CD-DST), but a resected tumor tissue as an examination materials is limited. Primary cultured cells are thought to be an alternative material for various analyses. Angiosarcoma is a rare soft-tissue sarcoma of endothelial cell origin showing a poor prognosis. A 72-year-old man who has large masses on upper and lower gingiva came to our department and a palliative excision was performed. He also had multiple nodules in both lungs and cervical lymph nodes. Histopathological diagnosis was angiosarcoma because the immunohistochemical stainings showed that vimentin, CD31 and vW factor were positive. We attempted to culture the excised tumor specimen in vitro and obtained primary cultured cells with stable proliferation, and confirmed their in vivo tumorigenicity in athymic nude mice. First, both primary tumor tissues and cultured cells were assessed their sensitivity against 8 anti-tumor drugs by CD-DST. We evaluated their growth inhibitory effects as the ratio of total volume of colonies in the treated group to that in the untreated group (T/C%). The most effective drug, gemcitabine, had 20.9 T/C% in tumor tissues and 35.4 T/C% in cultured cells, and the second, docetaxel, had 32.2 T/C% and 40.2 T/C%, respectively. The other drugs showed the similar sensitivity between both types of samples. Next, we determined the gene expression profiles of both samples and a normal gingival tissue by microarray analysis. The data showed that 2,498 genes in primary tumor tissues and 2,893 genes in primary cultured cells had more than 5-fold change compared to control. Of these, 1,627 genes were common, and then we found the overexpression of 170 cancer-related genes including Aurora kinase A (AURKA) among these genes. Finally, we investigated the effect of a selective AURKA inhibitor MLN8237 on the growth of primary cultured angiosarcoma cells in vitro, and then observed the growth inhibitory effect of MLN8237 against these cells. These results suggest that primary cultured tumor cells may be useful for predicting the efficacies of various anti-tumor drugs and developing novel therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1355. doi:1538-7445.AM2012-1355

Abstract 5270: Evaluation of Cell-able spheroid culture system for culturing patient derived primary tumor cells

Abstract Tumor cell microenvironment has significant impact on growth kinetics, cell signaling and response to drug treatments. 3D models are more biologically relevant models compared to 2D models and have gained preference among researchers and drug developers. It is well recognized that primary tumor cell cultures grown in 2D monolayer quickly lose the ability to proliferate while 3D culture conditions on extracellular matrix allows for spheroid formation and proliferation. Cell-able, a novel micropatterned plate has cell adhesion areas in the well bottom where the size and shape of these areas are tightly controlled. Micro-fabrication of the well bottom enables rapid cell attachment and spheroid formation. Cell-able plates are potentially an alternative to culturing cells in 3D without extracellular matrix. We have evaluated spheroid formation, morphology, duration of viability, histopathology and expression of markers in patient-derived primary tumor cells from different tumor types when cultured on Cell-able plates compared to Cultrex extracellular matrix (ECM) coated plates. In addition we have utilized Cell-able plates to grow patient-derived tumor cells and evaluate the anti-proliferative responses to cytotoxic and targeted agents using high content imaging coupled with immunofluorescence characterization enabling subpopulation analysis within the spheroids. Our results indicate that patient-derived tumor cells from multiple indications as well as tumor cell lines can be cultured on Cell-able plates as spheroids that resemble the micro architecture of tumors and are therefore a suitable 3D model. The results of high content imaging analysis suggest that Cell-able plates could be used as a valuable tool in in vitro predictive assay models in oncology drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5270. doi:1538-7445.AM2012-5270

Clinical Application of the Adenosine Triphosphate-based Response Assay in Intravesical Chemotherapy for Superficial Bladder Cancer

To investigate correlations between adenosine triphosphate chemotherapy response assay (ATP-CRA) and clinical outcomes after ATP-CRA-based chemotherapy for drug selection in patients receiving intravesical chemotherapy to prevent recurrence of superficial bladder cancer after surgery. The chemosensitivities of 12 anticancer drugs were evaluated, including 5-Fu ADM, and EPI, using ATP-CRA and primary tumor cell culture in 54 patients. In addition, a further 58 patients were treated according to clinical experience. Differences in post-chemotherapeutical effects between drug sensitivity assay and experience groups were compared. The evaluable rate of the test was 96.3%, the clinical effective rate was 80.8%, the sensitivity rate was 97.6% (41/42), the specificity was 20%, the total predicting accuracy was 74.3%, the positive predictive value was 83.7% (41/49), the negative predictive value was 66.7% (2/3); in the drug sensitivity test group, the clinical effective rate was 80.8%, the experience group response rate was 63.8%, with a significant difference in clinical effects between the ATP-based sensitivity and experience groups (χ2 =7.0153, P<0.01). ATP-CRA is a stable, accurate and potentially practical chemosensitivity test providing a predictor of chemotherapeutic response in patients with superficial bladder cancer.

18F‐FDG microPET imaging detects early transient response to an IGF1R inhibitor in genetically engineered rhabdomyosarcoma models

Alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS) are among the most common and most treatment resistant soft tissue sarcomas of childhood. Here, we evaluated the potential of (18)F-Fluorodeoxyglucose (FDG) as a marker of therapeutic response to picropodophyllin (PPP), an IGF1R inhibitor, in a conditional mouse model of ARMS and a conditional model of ERMS/undifferentiated pleomorphic sarcoma (UPS). Primary tumor cell cultures from Myf6Cre,Pax3:Fkhr,p53 and Pax7CreER,Ptch1,p53 conditional models of ARMS and ERMS/UPS were found to be highly sensitive to PPP (IC(50) values 150 and 200 nM, respectively). Animals of each model were then treated with 80 mg/kg/day PPP by intraperitoneal injection for 12 days and imaged by (18)F-FDG microPET. Tumor volumes on day 4 for PPP-treated ARMS and ERMS mice were lower than untreated control mouse tumor volumes, although treated tumors were larger than day 0. However, tumor FDG uptake was significantly reduced on day 4 for PPP-treated mice compared to pretreatment baseline or untreated control mice on day 4 (P < 0.05). Nevertheless, by day 12 tumor volumes and FDG uptake for treated mice had increased significantly, indicating rapidly evolving resistance to therapy. (18)F-FDG PET imaging is a potential imaging biomarker of molecular susceptibility to targeted agents early in treatment for this aggressive form of sarcoma, but may find best use serially for Phase I/II studies where chemotherapy and targeted agents are combined to cytoreduce tumors and abrogate Igf1r inhibitor resistance.

Activation of mTOR signaling in medullary and aggressive papillary thyroid carcinomas

Because mammalian target of rapamycin (mTOR) may be involved in thyroid carcinogenesis, we investigated the expression and activation patterns of mTOR signaling proteins in thyroid carcinoma cells and tumors and their association with tumor histology and aggressiveness. Tissue specimens from 50 patients with thyroid cancer were analyzed for eIF4E, a critical downstream target of the mTOR pathway, using immunohistochemistry. In addition, fresh-frozen samples from patients, and primary tumor cell cultures were analyzed for expression and activation of mTOR signaling proteins by Western blot. Moreover, pharmacologic studies with rapamycin were performed. High expression of eIF4E was observed in medullary thyroid carcinomas (MTC) and in aggressive variants of papillary thyroid carcinomas (PTC) as compared with conventional PTC and follicular thyroid carcinomas (P < .0001). The level of eIF4E expression also correlated with tumor stage (P = .002). Using Western blot analysis, p-rpS6, p-4EBP1, 4EBP1, and eIF4E were detected at higher levels in aggressive PTC and MTC cells. Treatment of MTC cells with increasing concentrations of rapamycin resulted in significant cell death and in decreased cell growth associated with deactivation of the mTOR pathway. mTOR signaling, which controls protein synthesis through regulation of translation initiation, is activated in aggressive PTC and MTC and represents a promising target for investigational therapies in these patients.

Prenyl Transferases Are Involved in the Regulation of CD20 Levels and Influence Anti-CD20 Monoclonal Antibody-Mediated Activation of Complement-Dependent Cytotoxicity,

Abstract 3722Anti-CD20 monoclonal antibodies (mAbs) (rituximab or ofatumumab) are being successfully used in the treatment of non-Hodgkin's lymphomas (NHL) and B-cell chronic lymphocytic leukemia (B-CLL). They exert antitumor effects by triggering indirect effector mechanisms of the immune system, such as activation of complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Moreover, upon crosslinking with secondary antibodies, anti-CD20 mAbs can induce cell death. It is frequently underscored that CD20 expression levels in various B-cell tumors is relatively constant. However, accumulating evidence indicates that CD20 can be modulated at transcriptional, posttranscriptional, and even posttranslational levels. Moreover, it has been clearly shown in vitro that CDC efficacy of anti-CD20 mAbs clearly depends on CD20 expression. We have previously observed that statins impair detection of CD20 in non-Hodgkin lymphoma cells and impair rituximab-mediated CDC and ADCC (Winiarska et al. PLoS Med 2008). Statins are inhibitors of cholesterol synthesis and decrease production of prenyl groups (farnesyl and geranylgeranyl PPi), which are necessary for posttranslational modification of approximately 1% of cellular proteins. In experiments aimed at elucidation of the molecular mechanisms of statin-mediated modulation of CD20 we observed that neither geranylgeranyl transferase (GGT) nor farnesyl transferase (FNT) inhibitors could mimic statins effects. On the contrary, prenyltransferase inhibitors improved rituximab-mediated CDC. Therefore, we decided to investigate in more detail the interaction of prenyltransferase inhibitors and anti-CD20 mAbs. In the initial experiments we evaluated the effects of three different farnesyl transferase inhibitors as well as three different geranylgeranyl transferase inhibitors. Among all FNT and GGT inhibitors, L-744,832 seemed to produce the most significant influence on both rituximab-mediated CDC and CD20 levels (Figure). Moreover, L-744,832 significantly increased rituximab-mediated CDC in 3 out of 5 primary tumor cell cultures isolated from patients with NHL or CLL. Therefore, L-744,832 was selected for further more systematic studies. Interestingly, in Raji cells L-744,832 did not improve rituximab-mediated ADCC and only at the highest non-toxic concentrations it sensitized to rituximab+crosslinking antibody-mediated cytotoxicity. In 10 out of 17 (58.8%) primary lymphoma/leukemia cells L-744,832 increased CD20 expression by at least 20% as measured with flow cytometry. Moreover, we observed that upon L-744,832 treatment CD20 is up-regulated in Raji cells at both mRNA as well as protein level. Experiments aimed at investigation of FTI influence on proteasome activity as well as CD20 endocytosis and shedding revealed that L-744,832 influences CD20 levels independently from its posttranslational regulation. To verify whether modulation of CD20 levels by L-744,832 results from specific inhibition of farnesyltransferase or is an off-target effect of this compound we performed FNT B subunit (FNTB) knock-down experiments that resulted in increased CD20 levels by almost 60%. Incubation of Raji cells with a transcription inhibitor cycloheximide completely prevented L-744,832-mediated increase of CD20 levels in WB. Therefore, a chromatin immunoprecipitation assay was performed to determine whether inhibition of FNT activity is associated with binding of transcription factors to the promoter of cd20 gene. These studies revealed that L-744,832 promotes binding of PU.1 and Oct2, but not TFE3 to target DNA sequences within cd20 promoter in Raji cells. To conclude, our studies indicate for the first time that CD20 expression can be modulated by prenyltransferase inhibitors. While inhibition of FNT activity significantly up-regulates expression of CD20, the influence of GGT inhibitors on this protein is more complex, and requires further studies. Furthermore, pre-incubation of NHL and CLL cells with L-744,832, a FNT inhibitor, potentiates anti-CD20 mAb-mediated activation of the complement-mediated cytotoxicity. Therefore, the combination seems to be promising and its efficacy should be determined in patients with NHL or CLL. [Display omitted] Disclosures:Winiarska:Genmab A/S: Research Funding. Golab:Genmab A/S: Research Funding.

Retinoic acid pathway activity in wilms tumors and characterization of biological responses in vitro

BackgroundWilms tumor (WT) is one of the most common malignancies in childhood. With current therapy protocols up to 90% of patients can be cured, but there is still a need to improve therapy for patients with aggressive WT and to reduce treatment intensity where possible. Prior data suggested a deregulation of the retinoic acid (RA) signaling pathway in high-risk WT, but its mode of action remained unclear.ResultsThe association of retinoid signaling and clinical parameters could be validated in a large independent tumor set, but its relevance in primary nephrectomy tumors from very young children may be different. Reduced RA pathway activity and MYCN overexpression were found in high risk tumors as opposed to tumors with low/intermediate risk, suggesting a beneficial impact of RA especially on advanced WT. To search for possible modes of action of retinoids as novel therapeutic options, primary tumor cell cultures were treated in vitro with all-trans-RA (ATRA), 9cis-RA, fenretinide and combinations of retinoids and a histone deacetylase (HDAC) inhibitor. Genes deregulated in high risk tumors showed opposite changes upon treatment suggesting a positive effect of retinoids. 6/7 primary cultures tested reduced proliferation, irrespective of prior RA signaling levels. The only variant culture was derived from mesoblastic nephroma, a distinct childhood kidney neoplasm. Retinoid/HDAC inhibitor combinations provided no synergistic effect. ATRA and 9cis-RA induced morphological changes suggestive of differentiation, while fenretinide induced apoptosis in several cultures tested. Microarray analysis of ATRA treated WT cells revealed differential expression of many genes involved in extracellular matrix formation and osteogenic, neuronal or muscle differentiation. The effects documented appear to be reversible upon drug withdrawal, however.ConclusionsAltered retinoic acid signaling has been validated especially in high risk Wilms tumors. In vitro testing of primary tumor cultures provided clear evidence of a potential utility of retinoids in Wilms tumor treatment based on the analysis of gene expression, proliferation, differentiation and apoptosis.

Novel activity of acriflavine against colorectal cancer tumor cells

A high-throughput screen of the cytotoxic activity of 2000 molecules from a commercial library in three human colon cancer cell lines and two normal cell types identified the acridine acriflavin to be a colorectal cancer (CRC) active drug. Acriflavine was active in cell spheroids, indicating good drug penetration and activity against hypoxic cells. In a validation step based on primary cultures of patient tumor cells, acriflavine was found to be more active against CRC than ovarian cancer and chronic lymphocytic leukemia. This contrasted to the activity pattern of the CRC active standard drugs 5-fluorouracil, irinotecan and oxaliplatin. Mechanistic studies indicated acriflavine to be a dual topoisomerase I and II inhibitor. In conclusion, the strategy used seems promising for identification of new diagnosis-specific cancer drugs.

Phenotype-based drug screening in primary ovarian carcinoma cultures identifies intracellular iron depletion as a promising strategy for cancer treatment

Primary cultures of patient tumor cells (PCPTC) have been used for prediction of diagnosis-specific activity and individual patient response to anticancer drugs, but have not been utilized as a model for identification of novel drugs in high throughput screening. In the present study, ovarian carcinoma cells from three patients were tested in response to a library of 3000 chemically diverse compounds. Eight hits were retrieved after counter screening using normal epithelial cells, and one of the two structurally related hit compounds was selected for further preclinical evaluation. This compound, designated VLX 50, demonstrated a broad spectrum of activity when tested in a panel of PCPTCs representing different forms of leukemia and solid tumors and displayed a high tumor to normal cell activity. VLX 50 induced delayed cell death with some features of classical apoptosis. Significant in vivo activity was confirmed on primary cultures of human ovarian carcinoma cells in mice using the hollow fiber model. Mechanistic exploration was performed using gene expression analysis of drug exposed tumor cells to generate a drug-specific signature. This query signature was analyzed using the Gene Set Enrichment Analysis and the Connectivity Map database. Strong connections to hypoxia inducible factor 1 and iron chelators were retrieved. The mechanistic hypothesis of intracellular iron depletion leading to hypoxia signaling was confirmed by a series of experiments. The results indicate the feasibility of using PCPTC for cancer drug screening and that intracellular iron depletion could be a potentially important strategy for cancer therapy.

Abstract 1316: Overactivation of AKT promotes hormone-independent mammary tumors

Abstract Using a mouse mammary tumor model induced by medroxyprogesterone acetate (MPA) that transits through different stages of hormone dependence, we have previously reported that the activation of the PI3K/AKT pathway is critical for the growth of hormone-independent (HI) mammary tumors, but not for the growth of a hormone-dependent (HD) tumor variant. The main aim of this work was to explore whether the activation of the PI3K/AKT pathway per se is responsible for the transition from a HD to a HI tumor phenotype. To answer this question we infected primary cultures of epithelial cells obtained from HD tumors with a retrovirus that directs the expression of a constitutively active form of AKT1 (myristoylated AKT1, myrAKT1), or with the empty vector as control. We inoculated 103 cells in nude mice (n = 6). MyrAKT1 expressing HD-derived cells were able to form mammary tumors even in the absence of MPA, that is, they behaved as HI cells. These tumors were highly differentiated and displayed a ductal phenotype with a pattern of expression of cytokeratin 8 and laminin-1 typical of HI tumors. Ligand-independent phosphorylation of progesterone receptor (PR) in Ser 190 was also increased in myrAKT1 tumors supporting our hypothesis that overactivation of AKT is directly involved in the acquisition of hormone independence. Furthermore, comparing myrAKT tumors with uninfected HD tumors revealed that overactivation of AKT did not increase the incidence of lung metastasis, indicating that AKT1 is involved specifically with tumor differentiation and growth rather than with tumor cell invasiveness. In vitro, primary cultures of mammary tumor cells modified to express myrAKT1 showed similar levels of proliferation determined by Ki67 and H3-tymidine incorporation as compared to control (cells infected with the empty vector), suggesting that the in vivo tumorigenic effect of myrAKT1 cells described above might require cell-cell or cell-matrix interactions absent in vitro. Interestingly, we found that conditioned medium from myrAKT1 tumor cells increase endogenous AKT1 activity when tested in untransfected cells. Moreover, conditioned medium from HI carcinoma associated fibroblasts (CAF-HI) increased the level of activation of the PI3K/AKT pathway in epithelial cells. Altogether, these results indicate that paracrine signals derived from the tumor cells and/or its microenvironment participate in the activation of the PI3K/AKT pathway, ultimately leading to tissue differentiation and ligand-independent mammary tumor growth. Thus, the PI3K/AKT pathway might be a key therapeutic target to prevent progression to autonomous and endocrine resistant breast tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1316. doi:10.1158/1538-7445.AM2011-1316

Abstract 1197: EphA3 kinase ablation induces glioblastoma differentiation and prevents tumor progression

Abstract Purpose: Eph receptors constitute the largest sub-family of receptor tyrosine kinases and interact with membrane-bound ligands termed ephrins. Eph and ephrins have many vital functions including cell adhesion, migration and axon guidance. Eph and ephrins have been found to be aberrantly expressed in many malignancies including brain tumor. The purpose of this study was to investigate EphA3 receptor function in the most common and aggressive form of brain tumor, Glioblastoma (GBM). Methods: Gene expression was investigated by Q-PCR, IHC and flow cytometry in high grade glioma (HGG) surgical specimens and primary derived serum free cell cultures. Targeted reduction of Eph expression was performed using both a constitutive and inducible shRNA system. Murine in-vivo studies were performed using both subcutaneous and orthotopic ‘intracranial’ xenografts in immuno-compromised animals. Signaling pathways were assessed by western blotting. Results: To establish whether the receptor tyrosine kinase EphA3 was over expressed in HGG we assessed 12 normal human brain specimens, 56 HGG specimens and 26 HGG primary cultures. EphA3 mRNA expression was negligible in normal brain while 30% of clinical specimens and 46% of primary cultured tumor cells expressed EphA3. EphA3 protein was also detected in HGG clinical specimens using IHC. To further investigate EphA3 function the receptor was down regulated using shRNA in an EphA3+ GBM neurosphere cell line. Constitutive and inducible down regulation of the EphA3 receptor resulted in initiation of neuronal and glial cell differentiation following activation of the ERK/MAPK pathway. A reduction in stem/progenitor cell proliferation was also observed following EphA3 knockdown by shRNA (46%) or by alternately inhibiting EphA3 function using soluble ephrin A5-Fc (33%). CFSE division tracking identified slower cell division in populations in which EphA3 signaling was attenuated. In-vivo studies were performed using a NOD/SCID mouse subcutaneous and intracranial xenograft model. Results highlighted a marked reduction in tumor formation in the EphA3 knockdown as opposed to control tumors. Subcutaneous control tumors formed with a median survival of 66 days while EphA3 knockdown animals survived beyond 100 days (p&amp;lt;0.05). Similar to the subcutaneous xenograft model a marked lack of intracranial tumor formation was observed when EphA3 was neutralized. Control mice formed large well vascularized invasive tumors with a median survival of 62 days. All EphA3 knockdown animals were free of tumor following autopsy at 145 days when the experiment was terminated (p&amp;lt;0.05). Importantly a mutant EphA3 rescue of the knockdown culture returned the tumorigenic potential of these cells. Conclusions: We propose EphA3, in part, regulates cancer stem cell self renewal and cell division rate in GBM and could prove a potential therapeutic target and marker of brain tumor initiating stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1197. doi:10.1158/1538-7445.AM2011-1197

Proximity ligation in situ assay for monitoring the global DNA methylation in cells

BackgroundDNA methylation has a central role in the epigenetic control of mammalian gene expression, and is required for X inactivation, genomics imprinting and silencing of retrotransposons and repetitive sequences. Thus, several technologies have been developed to measure the degree of DNA methylation.ResultsWe here present the development of the detection of protein-protein interactions via the adaptation of the proximity ligation in situ technology to evaluate the DNA methylation status in cells since the quantification of Dnmt1/PCNA interaction in cells reflects the degree of DNA methylation.ConclusionThis method being directly realizable on cells, it appears that it could suggest a wide range of applications in basic research and drug development. More particularly, this method is specially adapted for the investigations realized from a weak quantity of biologic materiel such as stem cells or primary cultured tumor cells for examples.

Primary culture of patient tumor cells in multicellular spheroid clusters

Primary culture of patient tumor cells in multicellular spheroid clusters

Testosterone regulates cell proliferation in aggressive fibromatosis (desmoid tumour)

Background:Aggressive fibromatosis (desmoid tumour) is a locally invasive tumour caused by mutations resulting in β-catenin protein stabilisation. Apc1638N mice are predisposed to developing aggressive fibromatosis tumours, and male mice develop greater numbers of tumours than female mice, suggesting a role for androgens in this tumour type.Methods:Human aggressive fibromatosis tumours were examined for the expression of the androgen receptor, and primary human tumour cell cultures were treated with testosterone. Orchidectomised Apc1638N mice were investigated for the development of tumours, and were treated with testosterone to study the effect of tumour formation and the level of β-catenin.Results:Androgen receptors are universally expressed in human aggressive fibromatosis tumours. Testosterone increased the proliferation rate and β-catenin protein level in a dose-dependent manner in human aggressive fibromatosis tumours. Orchiectomy reduced the number and size of tumours that formed in male Apc1638N mice to a similar level as observed in female mice. Testosterone treatment increased the number of tumours that formed in orchidectomised male mice, and resulted in a marked increase in β-catenin protein levels.Conclusion:Testosterone regulates β-catenin protein level and proliferation rate in this mesenchymal tumour. This work identifies the therapeutic use of testosterone blockade in aggressive fibromatosis as an area for further investigation.

Evasion Mechanisms to Igf1r Inhibition in Rhabdomyosarcoma

Inhibition of the insulin-like growth factor 1 receptor (Igf1r) is an approach being taken in clinical trials to overcome the dismal outcome for metastatic alveolar rhabdomyosarcoma (ARMS), an aggressive muscle cancer of children and young adults. In our study, we address the potential mechanism(s) of Igf1r inhibitor resistance that might be anticipated for patients. Using a genetically engineered mouse model of ARMS, validated for active Igf1r signaling, we show that the prototypic Igf1r inhibitor NVP-AEW541 can inhibit cell growth and induce apoptosis in vitro in association with decreased Akt and Mapk phosphorylation. However, drug resistance in vivo is more common and is accompanied by Igf1r overexpression, Mapk reactivation, and Her2 overexpression. Her2 is found to form heterodimers with Igf1r in resistant primary tumor cell cultures, and stimulation with Igf2 leads to Her2 phosphorylation. The Her2 inhibitor lapatinib cooperates with NVP-AEW541 to reduce Igf1r phosphorylation and to inhibit cell growth even though lapatinib alone has little effect on growth. These results point to the potential therapeutic importance of simultaneous targeting of Igf1r and Her2 to abrogate resistance.

Comparison of signal transduction drug response markers in immortalized cell lines and primary patient specimens

Abstract Background: The epidermal growth factor pathway is an important driver of cancer growth. Erlotinib is a small molecule protein-kinase inhibitor targeting the epidermal growth factor receptor (EGFR) that may benefit a select subtype of cancer patients. However, identification of the characteristics of patients who may benefit from the use of erlotinib is not easy. Previous publication explored the activation of signal transduction proteins in the EGFR pathway as a method of determining erlotinib response and demonstrated that lung cancer cell lines characterized by increased pEGFR/pAKT ratios are more resistant to erlotinib. The aim of this study was to compare the activation of signal transduction markers in the EGFR pathway between immortalized cell lines and primary cultures of patient-derived tumor cells. Predictive potential of pEGFR/pAKT ratios in both immortalized cell lines and primary cultures of lung cancer cells was also compared. Methods: Western blotting and In-Cell western analysis were used to determine the activation state of the EGFR signaling pathway. Signaling pathway components were compared in the presence and absence of erlotinib treatment in immortalized lung cancer cell lines and primary cultures of lung cancer specimens. Chemosensitivity of cell lines and specimens to erlotinib was determined using an in vitro drug response marker. Cells were treated with a 10 dose concentration range of erlotinib for 72 hours and the remaining post-treatment live cells were enumerated by nuclear staining. Results: The pEGFR/pAKT ratio was increased in erlotinib-responsive immortalized cell lines when compared to non-responsive cell lines (p&amp;lt;0.05). This difference in pEGFR/pAKT ratio was significant after treatment with erlotinib as well. The difference in pEGFR/pAKT ratios in response to erlotinib treatment between erlotinib-responsive and non-responsive primary cultures of lung cancer cells was not, however, statistically significant. Conclusion: These studies confirmed that pEGFR/pAKT ratios may be indicative of erlotinib response in immortalized lung cancer cell lines. However, when evaluating the signal transduction markers in patient-derived primary cultures, no correlation with in vitro erlotinib response was found. These results highlight the value of using in vitro drug response markers, which utilize primary patient tissue, in the development of predictive biomarkers.