Searching for Synergy : Radiosensitization of 177Lu-DOTATATE

Cancers presents a major health challenge, and there is a pressing need to develop new therapeutic strategies. Surgery, chemotherapy and radiation are the most commonly used treatments for cancer today. Radiation can be given as targeted radionuclide therapy (TRT), i.e., systemic administration of a radiolabeled cancer-targeting molecule. This is especially suitable for inoperable and disseminated tumors. 177Lu-DOTATATE, a TRT directed against the somatostatin receptors (SSTRs), was recently approved for therapy of a subset of neuroendocrine tumors (NETs). Although it has prolonged the life of NET patients, complete remission is seldom achieved. Consequently, to increase the efficacy of the treatment, this thesis aimed to assess potential radiosensitizing strategies for 177Lu-DOTATATE. The two radiosensitization targets in focus were HSP90, a chaperone protein with numerous oncogenic client proteins, and p53, a central regulator of DNA damage. In papers I and II, we investigated the HSP90-inhibitor Onalespib, as a treatment for NETs, and as a potential radiosensitizer. The drugs were assessed in vitro and in vivo. We concluded that Onalespib reduced NET cell growth and acted synergistically with 177Lu-DOTATATE. Inhibition of EGFR, a HSP90 client protein, was suggested as a mediator of the observed synergy. Furthermore, the combination had a favorable toxicity profile.  In paper III, we assessed the novel stapled peptide VIP116, which inhibits the p53 repressors MDM2 and MDM4, as a potentiator of 177Lu-DOTATATE in wildtype p53 neuroblastoma cells. Combination therapy exhibited growth-inhibitory effects, with resulting additive or synergistic effects. The treatment-mediated effects on p53 signaling were characterized, revealing a possible involvement of V-myc myelocytomatosis viral oncogene homolog, neuroblastoma derived (MYCN), a prognostic marker for poor survival in neuroblastoma. In paper IV, we aimed to improve targeted delivery of VIP116, with the use of lipid bilayer disks (lipodisks). VIP116 was successfully loaded onto epidermal growth factor receptor (EGFR)-targeting lipodisks, leading to specific delivery and reduction of viability of EGFR expressing tumor cells. The study provided a proof-of-concept for utilizing lipodisks as a drug delivery system for p53-stabilizing peptides. In conclusion, we have investigated, and found, suitable candidates for potentiating 177Lu-DOTATATE therapy. We have addressed the feasibility of the treatments, toxicity and targeted delivery. Moreover, the work has explored the biology of TRT. This is an area in need of more attention, as more and more radionuclide-based therapies are entering clinicals trials and reaching approval

Radiosensitising tumour cells : Optimisations in vitro and in vivo

One in three people in Sweden will sometime during their life be diagnosed with cancer. The most important therapies used today to treat these patients are surgery, radiation and chemotherapy. Small stapled peptides targeting the inhibition of tumour suppressor protein p53 have great potential of becoming radiosensitising drugs and increase efficacy of radiation therapy. This project aimed to test two modifications of stapled peptides PM2 and PM3 to gain further knowledge about their properties and optimise specificity and delivery of them. Radioiodination was optimised for labelling yield, oxidative stress and purification. Final product was quality tested on tumour cells to assess whether the modification affected the biological efficacy of the peptides. Results showed no reduction of efficacy. The compound was injected in tumour-bearing nude mice, resulting in tumour tissue absorption and low risk for toxicity in critical organs. Lipid bilayer disks (lipodisks) were used as a potential delivery system. The structure has previously been tested as carrier for amphiphilic peptides and shows great potential. Furthermore, it is non-toxic and biocompatible. Association of PM2 and PM3 to disks was measured with the use of fluorescence emission shift and QCM-D. Results showed that PM3 has high affinity to the disks. The study conducted here show that a drug delivery system consisting of tumour-targeting lipodisks carrying radiosensitising peptides have great potential. By adding a therapeutic radionuclide, the system can also be used for targeted radionuclide therapy

Radiosensitising tumour cells : Optimisations in vitro and in vivo

One in three people in Sweden will sometime during their life be diagnosed with cancer. The most important therapies used today to treat these patients are surgery, radiation and chemotherapy. Small stapled peptides targeting the inhibition of tumour suppressor protein p53 have great potential of becoming radiosensitising drugs and increase efficacy of radiation therapy. This project aimed to test two modifications of stapled peptides PM2 and PM3 to gain further knowledge about their properties and optimise specificity and delivery of them. Radioiodination was optimised for labelling yield, oxidative stress and purification. Final product was quality tested on tumour cells to assess whether the modification affected the biological efficacy of the peptides. Results showed no reduction of efficacy. The compound was injected in tumour-bearing nude mice, resulting in tumour tissue absorption and low risk for toxicity in critical organs. Lipid bilayer disks (lipodisks) were used as a potential delivery system. The structure has previously been tested as carrier for amphiphilic peptides and shows great potential. Furthermore, it is non-toxic and biocompatible. Association of PM2 and PM3 to disks was measured with the use of fluorescence emission shift and QCM-D. Results showed that PM3 has high affinity to the disks. The study conducted here show that a drug delivery system consisting of tumour-targeting lipodisks carrying radiosensitising peptides have great potential. By adding a therapeutic radionuclide, the system can also be used for targeted radionuclide therapy

Searching for Synergy : Radiosensitization of 177Lu-DOTATATE

Cancers presents a major health challenge, and there is a pressing need to develop new therapeutic strategies. Surgery, chemotherapy and radiation are the most commonly used treatments for cancer today. Radiation can be given as targeted radionuclide therapy (TRT), i.e., systemic administration of a radiolabeled cancer-targeting molecule. This is especially suitable for inoperable and disseminated tumors. 177Lu-DOTATATE, a TRT directed against the somatostatin receptors (SSTRs), was recently approved for therapy of a subset of neuroendocrine tumors (NETs). Although it has prolonged the life of NET patients, complete remission is seldom achieved. Consequently, to increase the efficacy of the treatment, this thesis aimed to assess potential radiosensitizing strategies for 177Lu-DOTATATE. The two radiosensitization targets in focus were HSP90, a chaperone protein with numerous oncogenic client proteins, and p53, a central regulator of DNA damage. In papers I and II, we investigated the HSP90-inhibitor Onalespib, as a treatment for NETs, and as a potential radiosensitizer. The drugs were assessed in vitro and in vivo. We concluded that Onalespib reduced NET cell growth and acted synergistically with 177Lu-DOTATATE. Inhibition of EGFR, a HSP90 client protein, was suggested as a mediator of the observed synergy. Furthermore, the combination had a favorable toxicity profile.  In paper III, we assessed the novel stapled peptide VIP116, which inhibits the p53 repressors MDM2 and MDM4, as a potentiator of 177Lu-DOTATATE in wildtype p53 neuroblastoma cells. Combination therapy exhibited growth-inhibitory effects, with resulting additive or synergistic effects. The treatment-mediated effects on p53 signaling were characterized, revealing a possible involvement of V-myc myelocytomatosis viral oncogene homolog, neuroblastoma derived (MYCN), a prognostic marker for poor survival in neuroblastoma. In paper IV, we aimed to improve targeted delivery of VIP116, with the use of lipid bilayer disks (lipodisks). VIP116 was successfully loaded onto epidermal growth factor receptor (EGFR)-targeting lipodisks, leading to specific delivery and reduction of viability of EGFR expressing tumor cells. The study provided a proof-of-concept for utilizing lipodisks as a drug delivery system for p53-stabilizing peptides. In conclusion, we have investigated, and found, suitable candidates for potentiating 177Lu-DOTATATE therapy. We have addressed the feasibility of the treatments, toxicity and targeted delivery. Moreover, the work has explored the biology of TRT. This is an area in need of more attention, as more and more radionuclide-based therapies are entering clinicals trials and reaching approval

p53-Mediated Radiosensitization of 177Lu-DOTATATE in Neuroblastoma Tumor Spheroids

p53 is involved in DNA damage response and is an exciting target for radiosensitization in cancer. Targeted radionuclide therapy against somatostatin receptors with 177Lu-DOTATATE is currently being explored as a treatment for neuroblastoma. The aim of this study was to investigate the novel p53-stabilizing peptide VIP116 in neuroblastoma, both as monotherapy and together with 177Lu-DOTATATE. Five neuroblastoma cell lines, including two patient-derived xenograft (PDX) lines, were characterized in monolayer cultures. Four out of five were positive for 177Lu-DOTATATE uptake. IC50 values after VIP116 treatments correlated with p53 status, ranging between 2.8–238.2 μM. IMR-32 and PDX lines LU-NB-1 and LU-NB-2 were then cultured as multicellular tumor spheroids and treated with 177Lu-DOTATATE and/or VIP116. Spheroid growth was inhibited in all spheroid models for all treatment modalities. The most pronounced effects were observed for combination treatments, mediating synergistic effects in the IMR-32 model. VIP116 and combination treatment increased p53 levels with subsequent induction of p21, Bax and cleaved caspase 3. Combination treatment resulted in a 14-fold and 1.6-fold induction of MDM2 in LU-NB-2 and IMR-32 spheroids, respectively. This, together with differential MYCN signaling, may explain the varying degree of synergy. In conclusion, VIP116 inhibited neuroblastoma cell growth, potentiated 177Lu-DOTATATE treatment and could, therefore, be a feasible treatment option for neuroblastoma

Radiosensitising tumour cells : Optimisations in vitro and in vivo

One in three people in Sweden will sometime during their life be diagnosed with cancer. The most important therapies used today to treat these patients are surgery, radiation and chemotherapy. Small stapled peptides targeting the inhibition of tumour suppressor protein p53 have great potential of becoming radiosensitising drugs and increase efficacy of radiation therapy. This project aimed to test two modifications of stapled peptides PM2 and PM3 to gain further knowledge about their properties and optimise specificity and delivery of them. Radioiodination was optimised for labelling yield, oxidative stress and purification. Final product was quality tested on tumour cells to assess whether the modification affected the biological efficacy of the peptides. Results showed no reduction of efficacy. The compound was injected in tumour-bearing nude mice, resulting in tumour tissue absorption and low risk for toxicity in critical organs. Lipid bilayer disks (lipodisks) were used as a potential delivery system. The structure has previously been tested as carrier for amphiphilic peptides and shows great potential. Furthermore, it is non-toxic and biocompatible. Association of PM2 and PM3 to disks was measured with the use of fluorescence emission shift and QCM-D. Results showed that PM3 has high affinity to the disks. The study conducted here show that a drug delivery system consisting of tumour-targeting lipodisks carrying radiosensitising peptides have great potential. By adding a therapeutic radionuclide, the system can also be used for targeted radionuclide therapy

Patients experience of chronic pain after cardiac surgery – a two year follow up : Patienters erfarenhet av kronisk smärta - en tvåårig uppföljning

Background and objective: Approximately 6000 cardiac surgeries were performed in Sweden 2013 and a known complication after such procedure is chronic pain. One surgical technique in open cardiac surgery includes a sternal retraction and this creates sensitive areas of pain which could lead to nerve damage. Few studies have been looking at this in a qualitative way. The aim of this study was to illuminate the  experiences of chronic pain in former patients, two years after cardiac surgery. Methods: This study is of qualitative design. Four women and six men (age 43 to 87 years), assessed as having chronic pain after surgery, were interviewed two years after cardiac surgery. The data was analyzed using qualitative content analysis with an inductive approach. Findings: Three categories and nine sub-categories were formulated. The first category was ‘feeling limitation in daily life’, with the subcategories; ‘everyday activities become painful’, ‘losing control of the body’, ‘pain is unpredictable’and‘insecurity’. The second category was;‘being reminded of illness’, with the subcategories; ‘the scar is a visually memory’and ‘being grateful’.The last category was; ‘coping with pain’, with the subcategories; ‘adaption for handling pain’, ‘different shades of pain’ and ‘pain is transient’. Conclusion: Chronic pain is a personal experience and can be described in a both physical and emotional way. Findings showed that participants, in some way, almost daily were reminded about their experiences of cardiac surgery either by looking at their scar or that they were restricted in daily activities. Despite the limitation in life; could participants describe their experience of chronic pain as manageable.  Pain was described in several ways, and the participants had different coping strategies. Most of the participants also were assured that pain eventually would pass or get better. Bakgrund och syfte: Cirka 6000 öppna hjärtoperationer genomfördes i Sverige 2013 och en känd komplikation är kronisk smärta. En av de kirurgiska teknikerna är öppen hjärtkirurgi vilken inkluderar delning av sternum, vilket kan åstadkomma ett flertal smärtkänsliga områden, som i sin tur kan leda till nervskador. Få studier har belyst detta kvalitativt. Syftet med denna studie var att belysa patienters upplevelse av kronisk smärta, två år efter öppen hjärtkirurgi. Metod: Föreliggande studie har en kvalitativ design. Fyra kvinnor och sex män (43-87 år), intervjuades två år efter öppen hjärtkirurgi. Dataanalysen var kvalitativ innehållsanalys med en induktiv ansats. Resultat: Tre kategorier och nio underkategorier konstruerades. Den första kategorin var “att känna begränsningar i vardagligt liv”, med underkategorierna; ‘vardagsaktiviteter blir smärtsamma’, ‘förlora kontrollen över kroppen’, ‘smärtan är oförutsägbar’ och ‘osäkerhet’. Den andra kategorin var; ‘en påminnelse om sjukdom’, med underkategorierna; ‘ärret är en visuell påminnelse’ och ‘tacksamhet’. Den sista kategorin var: ‘hantera smärta’, med underkategorierna; ‘anpassning för att hantera smärtan’, ‘olika nyanser av smärta’ och ‘smärtan är övergående’. Slutsats: Smärta är en personlig upplevelse och kan beskrivas både fysiskt och känslomässigt. Resultatet visade att deltagarna, på något sätt, nästan dagligen påmindes om deras erfarenhet efter hjärtkirurgi, antingen genom att titta på ärret eller genom att de blev begränsad i sina vardagliga aktiviteter. Trots begränsning i livet, kunde deltagarna beskriva upplevelsen av smärta som hanterbar. Deltagarna beskrev smärtan på olika sätt och de hade olika copingstrategier, de flesta av deltagarna var övertygade om att smärtan skulle gå över eller bli bättre

The HSP90 inhibitor onalespib potentiates Lu-177-DOTATATE therapy in neuroendocrine tumor cells

Lu-177-DOTATATE was recently approved for the treatment of somatostatin receptor (SSTR)-positive neuroen-docrine tumors (NETs). However, despite impressive response rates, complete responses are rare. Heat shock protein 90 (HSP90) inhibitors have been suggested as suitable therapeutic agents for NETs, as well as a potential radiosensitizers. Consequently, the aim of this study was to investigate whether the HSP90-inhibitor onalespib could reduce NET cell growth and act as a radiosensitizer when used in combination with Lu-177-DOTATATE. The NET cell lines BON, NCI-H727 and NCI-H460, were first characterized with regards to Lu-177-DOTATATE uptake and sensitivity to onalespib treatment in monolayer cell assays. The growth inhibitory effects of the monotherapies and combination treatments were then examined in three-dimensional multicellular tumor spheroids. Lastly, the molecular effects of the treatments were assessed. Lu-177-DOTATATE uptake was observed in the BON and NCI-H727 cells, while the NCI-H460 cells exhibited no detectable uptake. Accordingly, Lu-177-DOTATATE reduced the growth of BON and NCI-H727 spheroids, while no effect was observed in the NCI-H460 spheroids. Onalespib reduced cell viability and spheroid growth in all three cell lines. Furthermore, the combination of onalespib and Lu-177-DOTATATE exerted synergistic therapeutic effects on the BON and NCI-H727 spheroids. Western blot analysis of BON spheroids revealed the downregulation of epidermal growth factor receptor (EGFR) and the upregulation of gamma H2A histone family member X (gamma H2AX) following combined treatment with onalespib and Lu-177-DOTATATE. Moreover, flow cytometric analyses revealed a two-fold increase in caspase 3/7 activity in the combination group. In conclusion, the findings of this study demonstrate that onalespib exerts antitumorigenic effects on NET cells and may thus be a feasible treatment option for NETs. Furthermore, onalespib was able to synergistically potentiate Lu-177-DOTATATE treatment in a SSTR-specific manner. The radiosensitizing mechanisms of onalespib involved the downregulation of EGFR expression and the induction of apoptosis. Consequently, the combination of onalespib and Lu-177-DOTATATE may prove to be a promising strategy with which to improve therapeutic responses in patients with NETs. Further studies investigating this strategy in vivo regarding the therapeutic effects and potential toxicities are warranted to expand these promising findings

The radiosensitizer Onalespib increases complete remission in Lu-177-DOTATATE-treated mice bearing neuroendocrine tumor xenografts

Purpose: Lu-177-DOTATATE targeting the somatostatin receptor (SSTR) is utilized for treatment of neuroendocrine tumors (NETs). Onalespib, a heat shock protein 90 (HSP90) inhibitor, has demonstrated radiosensitizing properties and may thus enhance the effect of Lu-177-DOTATATE. Consequently, the aim of this study was to assess the potential of Onalespib in combination with Lu-177-DOTATATE in vivo and to examine the toxicity profiles of the treatments. Methods: Lu-177-DOTATATE selectivity and distribution in NET xenografts were studied using biodistribution and autoradiography. Therapeutic effects of Onalespib in combination with Lu-177-DOTATATE were studied in NET xenografts. Histological analyses were used to assess molecular effects from treatment and to establish toxicity profiles. Results: Biodistribution and autoradiography confirmed the SSTR-selective tumor uptake of Lu-177-DOTATATE, which was unaffected by Onalespib treatment. Immunohistochemistry verified molecular responses to Onalespib therapy in the tumors. While Onalespib and Lu-177-DOTATATE monotherapies resulted in a 10% and 33% delay in tumor doubling time compared with control, the combination treatment resulted in a 73% delayed tumor doubling time. Moreover, combination treatment increased complete remissions threefold from Lu-177-DOTATATE monotherapy, resulting in 29% complete remissions. In addition, histological analyses demonstrated radiation-induced glomerular injury in the Lu-177-DOTATATE monotherapy group. The damage was decreased tenfold in the combination group, potentially due to Onalespib-induced HSP70 upregulation in the kidneys. Conclusion: Treatment with Onalespib potentiated Lu-177-DOTATATE therapy of NET xenografts with a favorable toxicity profile. Utilizing Onalespib's radiosensitizing properties with Lu-177-DOTATATE may lead to better therapeutic results in the future and may reduce unwanted side effects in dose-limiting organs.De två första författarna delar förstaförfattarskapet.</p

Ett nytt multiplext PCR-protokoll för identifiering och detektion av Shigella och enteroinvasiv E. coli (EIEC) från livsmedel

This report is the result of a project in the course Independent Projekt in Molecular Biotechnology at Uppsala University during the spring of 2014. The foremost purpose of the course is to give students the opportunity to carry through exstensive work in a project environment. This project was formed based on a comission from the biotechnology company SweTree Technologies, and the goal has been to compose a summary of the different techniques and methods that exist in the field of mass propagation of trees through the method of somatic embryogenesis. The project group has obtained information about the area mainly throgh reading patents, trying to find key components and bottlenecks in other companies’ somatic embryogenesis technologies. This paper is divided into different sections, containing the patents of the automation of different steps in the process. This is to make it easier for readers to find information about the area they are interested in, as well as to illustrate the main parts of the process as percieved by the project group. Currently, there are several automated solutions for almost every step in the process, some of which are already in use. All the information obtained shows that the cost and labour has decreased with the development of this technology. While there is still room for significant devolopment in order to produce a complete automated process, there is no doubt that this method is becoming an ever more important asset in the area of forestry. Our hope is that this report may be a useful tool for companies or laymen to geta grasp of the field of automated mass production of trees