Ole Thastrup

Signal Transduction via the Mitogen-activated Protein Kinase Pathway Induced by Binding of Coagulation Factor VIIa to Tissue Factor

The putative role of tissue factor (TF) as a receptor involved in signal transduction is indicated by its sequence homology to cytokine receptors (Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6934–6938). Signal transduction induced by binding of FVIIa to cells expressing TF was studied with baby hamster kidney (BHK) cells stably transfected with TF and with a reporter gene construct encoding a luciferase gene under transcriptional control of tandem cassettes of signal transducer and activator of transcription (STAT) elements and one serum response element (SRE). FVIIa induced a significant luciferase response in cells expressing TF, BHK(+TF), but not in cells without TF. The BHK(+TF) cells responded to the addition of FVIIa in a dose-dependent manner, whereas no response was observed with active site-inhibited FVIIa, which also worked as an antagonist to FVIIa-induced signaling. Activation of the p44/42 MAPK pathway upon binding of FVIIa to TF was demonstrated by suppression of signaling with the specific kinase inhibitor PD98059 and demonstration of a transient p44/42 MAPK phosphorylation. No stimulation of p44/42 MAPK phosphorylation was observed with catalytically inactive FVIIa derivatives suggesting that the catalytic activity of FVIIa was obligatory for activation of the MAPK pathway. Signal transduction caused by a putative generation of FXa activity was excluded by experiments showing that FVIIa/TF-induced signaling was not quenched by tick anticoagulant protein, just as addition of FXa could not induce phosphorylation of p44/42 MAPK in BHK(+TF) cells. These results suggest a specific mechanism by which binding of FVIIa to cell surface TF independent of coagulation can modulate cellular functions and possibly play a role in angiogenesis and tumor metastasis as indicated by several recent observations.

Excitation Wave Propagation as a Possible Mechanism for Signal Transmission in Pancreatic Islets of Langerhans

In response to glucose application, beta-cells forming pancreatic islets of Langerhans start bursting oscillations of the membrane potential and intracellular calcium concentration, inducing insulin secretion by the cells. Until recently, it has been assumed that the bursting activity of beta-cells in a single islet of Langerhans is synchronized across the whole islet due to coupling between the cells. However, time delays of several seconds in the activity of distant cells are usually observed in the islets of Langerhans, indicating that electrical/calcium wave propagation through the islets can occur. This work presents both experimental and theoretical evidence for wave propagation in the islets of Langerhans. Experiments with Fura-2 fluorescence monitoring of spatiotemporal calcium dynamics in the islets have clearly shown such wave propagation. Furthermore, numerical simulations of the model describing a cluster of electrically coupled beta-cells have supported our view that the experimentally observed calcium waves are due to electric pulses propagating through the cluster. This point of view is also supported by independent experimental results. Based on the model equations, an approximate analytical expression for the wave velocity is introduced, indicating which parameters can alter the velocity. We point to the possible role of the observed waves as signals controlling the insulin secretion inside the islets of Langerhans, in particular, in the regions that cannot be reached by any external stimuli such as high glucose concentration outside the islets.

Characteristics of stably expressed human dopamine D1a and D1b receptors: Atypical behavior of the dopamine D1b receptor

Human dopamine D1a and D1b receptors were stably expressed in Baby Hamster Kidney (BHK) or Chinese Hamster Ovary (CHO) cells. [3H]SCH23390 saturation experiments indicated the presence of only a single binding site in the D1a expressing cell line with a Kd of 0.5 nM. In D1b expressing cell lines, two binding sites were observed with Kd values of 0.5 and 5 nM in CHO cells and 0.05 and 1.6 nM in BHK cells, respectively. Neither of the receptors affected Ca2+ metabolism whereas they both were coupled in a stimulatory fashion to adenylyl cyclase. The pharmacological profile of both the D1a and D1b receptors as assessed from inhibition of specific [3H]SCH 23390 binding was classical D1-like. Thus, benzazepine derivatives as well as the atypical neuroleptics, clozapine and fluperlapine, exhibited high affinity whereas D2 selective compounds like sulpiride and spiperone had low affinity for these receptors. Besides SCH 23390, only NNC 112, fluphenazine and bulbocapnine were able to discriminate between the two states of the D1b receptor. In case of the D1a receptor, the Ki values obtained in binding experiments were very similar to Ki values obtained from inhibition of dopamine stimulated adenylyl cyclase. In the D1b expressing cell line, the Ki values obtained from inhibition of the dopamine stimulated adenylyl cyclase indicated a significantly better correlation with the state of the D1b receptor showing high affinity for antagonists. In agreement with observations from binding experiments, dopamine was around 20 fold more potent in stimulating adenylyl cyclase via the D1b receptor as compared to the D1a receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of activated Ca2+ entry in the rat pancreatoma cell line, AR4-2J

The characteristics of Ca2+ entry activated by surface receptor agonists and membrane depolarization were studied in the rat pancreatoma cell line, AR4-2J. Ca2+ mobilization activated by substance P, bombesin, or muscarinic receptor stimulation was found to involve both Ca2+ release and entry. In addition, depolarization of the surface membrane of AR4-2J cells with elevated concentrations of K+ activated Ca2+ entry. Ca2+ entry induced by membrane depolarization was inhibited by the L-channel antagonist, nimodipine, while that due to surface receptor agonists was not inhibited by this agent. The microsomal Ca(2+)-ATPase inhibitor, thapsigargin, caused both depletion of the agonist-sensitive intracellular Ca2+ pool and sustained Ca2+ influx indistinguishable from that produced by bombesin or methacholine. These results confirm that, unlike the pancreatic acinar cells from which they are presumably derived, AR4-2J cells express voltage-sensitive, dihydropyridine-inhibitable Ca2+ channels. However, in contrast to previous reports with this cell line, in the AR4-2J cells in use in our laboratory, and under our experimental conditions, surface receptor agonists (including substance P) do not cause Ca2+ influx through voltage-sensitive Ca2+ channels. Instead, we conclude that agonist-activated Ca2+ mobilization is initiated by (1,4,5)IP3-mediated intracellular Ca2+ release and that Ca2+ influx is regulated primarily, if not exclusively, by the state of depletion of the (1,4,5)IP3-sensitive intracellular Ca2+ pool.

Pancreatic islets cultured on extracellular matrix: An excellent preparation for microfluorometry

We have examined the use of extracellular matrix (ECM) as a substratum for mouse pancreatic islet cultures with special reference to microfluorometry work. We find the techniques for obtaining and maintaining ECM-producing cell cultures and coating of glass straightforward. The islets attached readily to ECM-coated glass and with time spread out. Cultures were probed for glucose responses after one day, one week and two weeks. Upon stimulation with 10 mM glucose, the well-established initial changes in [Ca2+]i, (monitored with Fura-2), as well as oscillations upon prolonged exposure, were observed. At 20 mM glucose only an elevated level without oscillations was seen. Similar responses were observed during the two weeks in culture. After one week in culture or more, single cell measurements of qualitative changes in [Ca2+]i and cell membranepotential (monitored with the dye bis-(1,3-dibutyl-barbituric acid) trimethine oxonol (DiBAC4(3)) could be performed concurrently on single cells at the islet periphery with ordinary fluorescence microscopy equipment. Simultaneous measurements of NAD(P)H and flavine adenine dinucleotide (FAD) fluorescence as well as measurements of rhodamine 123 fluorescence for mitochondrial membrane potential showed stable metabolic responses to glucose over the entire culture period. The islets had clearly elevated insulin secretion at 10 and 20 mM glucose compared to the secretion at 3 mM. In conclusion, mouse pancreatic islets cultured for up to two weeks on ECM-coated glass coverslips provide a functionally well- preserved and reproducible preparation for microfluorometry. The largest benefit of the method is that whole islet responses as well as single cell responses at the islet periphery can be monitored, using the same preparation.

Flow injection microscopy: a novel tool for the study of cellular response and drug discovery

Studying responses of live cells to agonists, antagonists and other physical stimuli offers insight into their complex membrane and internal biochemistry. An experimental technique has been developed in which responses of living cells in an inverted radial flow chamber are continuously monitored while being repeatedly stimulated using controlled pulses of a biologically active ligand. Precisely defined flow conditions result in reproducible peaks which can be numerically analysed by comparison with a tracer curve obtained by substituting a dye for the stimulus. Exploratory studies have demonstrated that the flow injection technique can provide a novel method for kinetics of receptor binding and cellular responses. Flow injection microscopy (FIM) allows identification of biologically active ligands and their ranking based on measurement of the cellular responses in a short time frame. The use of FIM for rapid drug screening, through monitoring of the initial kinetics of cellular responses, is demonstrated on a model system.

Short-term spheroid culture of primary colorectal cancer cells as an in vitro model for personalizing cancer medicine

Chemotherapy treatment of cancer remains a challenge due to the molecular and functional heterogeneity displayed by tumours originating from the same cell type. The pronounced heterogeneity makes it difficult for oncologists to devise an effective therapeutic strategy for the patient. One approach for increasing treatment efficacy is to test the chemosensitivity of cancer cells obtained from the patient’s tumour. 3D culture represents a promising method for modelling patient tumours in vitro. The aim of this study was therefore to evaluate how closely short-term spheroid cultures of primary colorectal cancer cells resemble the original tumour. Colorectal cancer cells were isolated from human tumour tissue and cultured as spheroids. Spheroid cultures were established with a high success rate and remained viable for at least 10 days. The spheroids exhibited significant growth over a period of 7 days and no difference in growth rate was observed for spheroids of different sizes. Comparison of spheroids with the original tumour revealed that spheroid culture generally preserved adenocarcinoma histology and expression patterns of cytokeratin 20 and carcinoembryonic antigen. Interestingly, spheroids had a tendency to resemble tumour protein expression more closely after 10 days of culture compared to 3 days. Chemosensitivity screening using spheroids from five patients demonstrated individual response profiles. This indicates that the spheroids maintained patient-to-patient differences in sensitivity towards the drugs and combinations most commonly used for treatment of colorectal cancer. In summary, short-term spheroid culture of primary colorectal adenocarcinoma cells represents a promising in vitro model for use in personalized medicine.

Abstract 2906: Characterization of genetic intratumor heterogeneity of colorectal cancer and matching organoids

Background: In the recent years it has become evident that intratumor heterogeneity of solid tumors, such as colorectal cancer (CRC), complicates development of efficient therapy strategies. A minor clone of the tumor might have the ability for treatment resistance and hence the ability for re-establishing the tumor. Organoid cultures derived from the patients’ tumors can be used for ex vivo drug screening prior to treatment of the patient. However this approach is only efficient if the organoids represent the heterogeneity of the tumor. In this study we aim to characterize the genetic intratumor heterogeneity of CRC by performing whole exome sequencing (WES) on multiple biopsies per tumor, and to see how well the heterogeneity is reflected in matching organoid cultures. Methods: From five CRC patients, three biopsies were collected from separate areas of each tumor. Each biopsy was divided in two: one half was fresh frozen for DNA purification, and the other half was grown in vitro as organoid culture. When available, lymph node metastases (LNMs) were included. WES was performed using SeqCap EZ Exome v3.0, and Illumina NextSeq500. Matched germline DNA was used as reference to identify somatic mutations and copy number variations (CNVs) using Mutect2 and FACETS, respectively. Results: Each tumor contained multiple mutations that were present in all biopsies and in the organoids as well, representing a common ancestral branch. However all tumor biopsies and organoids also contained private mutations. These constituted on average 10% of the mutations (range 3-18%) indicating spatial genetic heterogeneity in all tumors. Each organoid had private mutations not seen in the tumor area of origin and vice versa, indicating that each area contained multiple clones and only a subset was represented in the organoids. The extent of mutational differences between organoids and their area of origin was similar to the mutational differences between tumor areas. Implying that organoids do not reflect their local origin better than a single biopsy reflects the whole tumor. In one patient with LNMs it was observed that the mutational profile of the metastases resembled only one of the examined tumor areas. Surprisingly, not all the ancestral mutations found in the tumor biopsies were observed in the LNMs, suggesting that at least two clones co-existed in the area of origin and just one of these formed the metastases. Conclusion: In the five patients studied; spatial genetic heterogeneity was observed, meaning that multiple biopsies are needed to picture the whole tumor. Genetic heterogeneity was also observed between primary tumor and metastases and our data support that the metastases were formed from a single cancer clone that did not dominate the primary tumor. From a genetic point of view, organoids do not seem to fully reflect the tumor area of origin and less so the whole tumor, indicating that care should be taken when using organoids as models of the primary tumor. Citation Format: Sigrid S. Arnadottir, Maria Jeppesen, Philippe Lamy, Iver Nordentoft, Michael Knudsen, Soren Vang, Mogens R. Madsen, Jacob Thastrup, Ole Thastrup, Claus L. Andersen. Characterization of genetic intratumor heterogeneity of colorectal cancer and matching organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2906. doi:10.1158/1538-7445.AM2017-2906

Abstract 2020: Spheroid culture of primary colorectal cancer cells from liver metastases as an in vitro model of patient tumors

More than 65 % of patients with colorectal cancer require chemotherapeutic treatment during the course of their disease. However, colorectal tumors demonstrate pronounced heterogeneity, leading to significant individual variation in sensitivity to standard chemotherapeutic drugs. Tools for assessing chemosensitivity and tailoring therapeutic regimens to the individual patient are therefore urgently needed. A promising approach is to isolate viable cancer cells from patient tissue and to test the chemosensitivity of the individual tumor in vitro. The aim of the current study was to establish primary colorectal cancer cell cultures from patient liver metastases and investigate if the cultured cells retained important characteristics of the original tumor. Small cell clusters were isolated from metastatic tissue and allowed to form cell spheroids in suspension culture. The cell composition of patient spheroids was characterized by immunostaining with cell type specific antibodies. Cancer stem cells were identified by staining for the markers CD44, CD166 and Lgr5. Spheroid size was monitored over time and growth curves established for each patient. The distribution of proliferating and apoptotic cells in the spheroids was assessed by staining for specific markers. Overall, the 3-D culture system was verified as a tumor model by comparing the results obtained for the spheroids to paraffin-embedded sections of the original tumors. Successful spheroid culture was obtained from more than 80 % of tissue samples. Immunostaining confirmed that the primary cultures were composed of epithelial cells with minimal contamination by other cell types. Cell morphology and expression of intestinal markers confirmed their malignant nature and colorectal origin. The dominating cell populations of the original tumor could generally be identified in the derived spheroids as well. The spheroids demonstrated significant growth in vitro with no decrease in proliferation or increase in apoptosis during short-term culture. In summary, spheroid cultures of colorectal cancer cells can be established from liver metastases of individual patients with a high success rate and the spheroids retain important cellular characteristics of the original tumor. Spheroid culture therefore represents a promising technique for establishing primary cultures for chemotherapeutic screening of the individual patient. Citation Format: Maria Jeppesen, Grith Hagel, Ben Vainer, Henrik Harling, Ole Thastrup, Lars Nannestad Jorgensen, Jacob Thastrup. Spheroid culture of primary colorectal cancer cells from liver metastases as an in vitro model of patient tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2020. doi:10.1158/1538-7445.AM2014-2020

Abstract 2718: Spheroid culture of colorectal cancer cells from patient tumor tissue preserves important cellular characteristics of the parental tumor.

At the time of diagnosis more than 65% of patients with colorectal cancer have advanced disease and require chemotherapeutic treatment. However, colorectal tumors demonstrate pronounced heterogeneity, leading to significant individual variation in sensitivity to standard chemotherapeutic drugs. Tools for assessing chemosensitivity and tailoring therapeutic regimens to the individual patient are therefore urgently needed. A promising approach is to isolate viable cancer cells from patient tissue and to test the chemosensitivity of the individual tumor in vitro. The aim of the current study was to establish primary colorectal cancer cell cultures from patient tissue and investigate if the cultured cells retain important characteristics of the original tumor. Small cell clusters were isolated from colorectal tumor tissue and allowed to form cell spheroids in suspension culture. The cell type composition of patient spheroids was characterized by immunostaining with cell type specific antibodies. Spheroid size was monitored over time and growth curves established for each patient. The distribution of proliferating and apoptotic cells in the spheroids was assessed by staining for specific markers. The 3D culture system was verified as a tumor model by comparing the results obtained for the spheroids to paraffin-embedded sections of the original tumors. Successful spheroid culture was obtained from more than 80% of tissue samples. Immunostaining confirmed that the primary cultures were composed of colorectal cancer cells, without significant contaminations by other cell types. Growth could be observed as an increase in spheroid size. The dominating cell populations of the original tumor could generally be identified in the derived spheroids as well. In summary, spheroid cultures of colorectal cancer cells can be established from tumor tissue of individual patients with a high success rate and the formed spheroids retain important cellular characteristics of the original tumor. Culture of colorectal cancer cells as spheroids represents a promising method for establishing primary cultures for chemotherapeutic screening of the individual patient. Citation Format: Maria Jeppesen, Grith Hagel, Anders Glenthoj, Ole Thastrup, Lars Nannestad Jorgensen, Jacob Thastrup. Spheroid culture of colorectal cancer cells from patient tumor tissue preserves important cellular characteristics of the parental tumor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2718. doi:10.1158/1538-7445.AM2013-2718