MULTI-MODAL IMAGING OF TUMOR VASCULATURE AND TREATMENT RESPONSE IN PRECLINICAL CANCER MODELS

Multimodal avbildning av tumorvaskulatur og behandlingsrespons i prekliniske modeller av kreft Kreft, en sykdom forårsaket av ukontrollert celledeling, forårsaker betydelig sykelighet og dødelighet i alle folkegrupper. Noen kreftpasienter har meget god prognose etter behandling med kirurgi, stråleterapi eller kjemoterapi, som utgjør bærebjelkene i dagens kreftbehandling. For andre pasienter er det dessverre vanskelig å oppnå et godt behandlingsresultat. Dette skyldes ulike faktorer som dårlige diagnostiske verktøy, inoperabilitet på grunn av tumorens størrelse og lokalisering, metastaserende kreftceller, behandlingsresistens eller dosebegrensende bivirkninger. Det er derfor et stort behov for nye behandlingstilbud for kreftpasienter. Mange nye legemidler rettet mot deregulerte signalveier i kreftceller er under klinisk utprøvning, og det blir ofte hevdet at denne typen legemidler kan bidra til effektiv, persontilpasset kreftbehandling. For å oppnå dette, trengs det nye metoder for å identifisere hvilke pasienter som vil ha utbytte av de ulike legemidler. Bedre metoder for å predikere behandlingsrespons krever at det utvikles og valideres biomarkører som kan brukes til å monitorere kreftutvikling og behandlingsrespons. Medisinsk avbildning kan potensielt brukes til å identifisere både responderende og behandlingsresistente pasienter tidlig i behandlingen, og dermed hjelpe til å skreddersy behandlingsopplegg for den enkelte kreftpasient. For å oppnå dette trengs det mer kunnskap om hvordan disse nye legemidlene påvirker fysiologiske egenskaper i tumorer. Angiogenese – dannelse av nye blodkar – er essensielt for tumorvekst og progresjon. Hemming av angiogenesen er derfor ansett som et attraktivt prinsipp for behandling av kreft. I tillegg har det vist seg at legemidler som hemmer signaloverføring i kreftceller, og dermed begrenser celledeling og tumorvekst, kan påvirke blodårenes funksjon. Økt forståelse av angiogenesen og hvordan nye legemidler kan påvirke vaskulær funksjon i tumorer kan derfor bidra til å øke behandlingseffekten i persontilpasset kreftbehandling. Hovedmålet i dette doktorgradsarbeidet var å evaluere hvordan ulike avbildningsmodaliteter kan brukes til å vurdere morfologi og funksjon av blodkar i tumorer, og å karakterisere cellulær og vaskulær respons på behandling med et legemiddel som hemmer kreftcellers signalveier. Avhandlingen består av tre artikler, hvor multimodal avbildning ble brukt i studier i xenograft-tumorer i mus. I den første artikkelen ble mikro-CT, mikro-MRI og in vivo MRI brukt til å studere vaskulatur i tumorer med ulik romlig oppløsning. Dette gjorde det mulig å sammenligne ulike teknikker for å estimere blodvolum i tumorer, og dermed bekrefte nøyaktigheten av blodvolumestimater basert på in vivo MRI. I den andre artikkelen ble effekten av PI3K-hemmeren BEZ235 evaluert i to forskjellige xenograft-modeller av eggstokkreft ved hjelp av in vivo MRI, mikro-CT og histopatologiske metoder. Vi fant at en krefttype med høy aktivitet i PI3K-signalveien responderte godt på behandlingen mens en krefttype med lav aktivitet responderte dårlig. I tillegg fant vi at MRI-markører for celletetthet og histopatologiske markører for proliferasjon hadde bedre prediktive egenskaper enn MRI- og CT-baserte markører for vaskulære endringer. I den tredje artikkelen kombinerte vi dynamisk in vivo MRI og intravital mikroskopi for å kunne studere opptaket av nanopartikler i xenograftede eggstokkreft-tumorer. Her fant vi at denne fremgangsmåten var godt egnet til å studere dynamikken i nanopartikkel-opptaket i tumorene in vivo, noe som kan være av betydning i forståelsen av hvordan nanopartikler kan brukes til målrettet levering av legemidler til en tumor. Samlet utgjør disse tre artiklene en helhet, der en rekke forskjellige bruksområder for multimodal avbildning i prekliniske kreftmodeller blir presentert og evaluert. Resultatene i avhandlingen bidrar til utvikling innenfor preklinisk kreftforskning gjennom økt forståelse av hvordan medisinske avbildningsteknikker gjenspeiler fysiologiske forhold i tumorer. Funnene i avhandlingen kan også bidra til mer rasjonell bruk av MR-avbildning i klinisk, persontilpasset kreftbehandling

Anti-angiogenic therapy affects the relationship between tumor vascular structure and function: a correlation study between micro-CT angiography and DCE-MRI

Purpose: To compare the effects of two anti-angiogenic drugs, bevacizumab and a cytosolic phospholipase A2-α inhibitor (AVX235), on the relationship between vascular structure and dynamic contrast enhanced (DCE)-MRI measurements in a patient-derived breast cancer xenograft model. Methods: Mice bearing MAS98.12 tumors were randomized into three groups: bevacizumab-treated (n=9), AVX235-treated (n=9), and control (n=8). DCE-MRI was performed pre-and post-treatment. The median initial area under the concentration-time curve (IAUC60) and volume transfer constant (Ktrans) were computed for each tumor. The tumors were excised for ex vivo micro-CT angiography, from which the vascular surface area (VSA) and fractional blood volume (FBV) were computed. Spearman correlation coefficients (ρ) were computed to evaluate the associations between the DCE-MRI and micro-CT parameters. Results: With the groups pooled, IAUC60 and Ktrans correlated significantly with VSA (ρ=0.475 and 0.527, P=0.019 and 0.008). There were no significant correlations within the control group. There were various significant correlations within the treatment groups but the correlations in the bevacizumab group were of opposite sign, e.g., Ktrans vs. FBV: AVX235 ρ=0.800 (P=0.014), bevacizumab ρ=-0.786 (P=0.023). Conclusion: DCE-MRI measurements can highly depend on vascular structure. The relationship between vascular structure and function changed markedly after antiangiogenic treatment

Anti-vascular effects of the cytosolic phospholipase A2 inhibitor AVX235 in a patient-derived basal-like breast cancer model

Background Group IVA cytosolic phospholipase A2 (cPLA2α) plays an important role in tumorigenesis and angiogenesis. It is overexpressed in basal-like breast cancer (BLBC), which is aggressive and usually triple-negative, making it unresponsive to current targeted therapies. Here, we evaluated the anti-angiogenic effects of a specific cPLA2α inhibitor, AVX235, in a patient-derived triple-negative BLBC model. Methods Mice bearing orthotopic xenografts received i.p. injections of AVX235 or DMSO vehicle daily for 1 week and then every other day for up to 19 days. Six treated and six control mice were terminated after 2 days of treatment, and the tumors excised for high resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS) and prostaglandin E2 (PGE2) enzyme immunoassay (EIA) analysis. A 1-week imaging study was performed on a separate cohort of mice. Longitudinal dynamic contrast enhanced (DCE)-MRI was performed before, after 4 days, and after 1 week of treatment. The mice were then perfused with a radiopaque vascular casting agent, and the tumors excised for micro-CT angiography. Subsequently, tumors were sectioned and stained with lectin and for Ki67 or α-smooth muscle actin to quantify endothelial cell proliferation and vessel maturity, respectively. Partial least squares discriminant analysis was performed on the multivariate HR MAS MRS data, and non-parametric univariate analyses using Mann–Whitney U tests (α = 0.05) were performed on all other data. Results Glycerophosphocholine and PGE2 levels, measured by HR MAS MRS and EIA, respectively, were lower in treated tumors after 2 days of treatment. These molecular changes are expected downstream effects of cPLA2α inhibition and were followed by significant tumor growth inhibition after 8 days of treatment. DCE-MRI revealed that AVX235 treatment caused a decrease in tumor perfusion. Concordantly, micro-CT angiography showed that vessel volume fraction, density, and caliber were reduced in treated tumors. Moreover, histology showed decreased endothelial cell proliferation and fewer immature vessels in treated tumors. Conclusions These results demonstrate that cPLA2α inhibition with AVX235 resulted in decreased vascularization and perfusion and subsequent inhibition of tumor growth. Thus, cPLA2α inhibition may be a potential new therapeutic option for triple-negative basal-like breast cancer

Autocrine activin A signaling in cancer cells controls secretion of IL-6 and autophagy in cancer cachexia

Background - The majority of patients with advanced cancer develop cachexia, a weight loss syndrome that severely reduces quality of life and limits survival. Our understanding of the underlying mechanisms that cause the condition is limited, and there are currently no treatment options that can completely reverse cachexia. Several tumor-derived factors and inflammatory mediators have been suggested to contribute to weight loss in cachectic patients. However, inconsistencies between studies are recurrent. Activin A and IL-6 are among the best studied factors that seem to be important, and several studies support their individual role in cachexia development. Methods – We investigated the interplay between activin A and IL-6 in the cachexia inducing TOV21G cell line both in culture and in tumours in mice. We previously found that the human TOV21G cells secrete IL-6 that induce autophagy in reporter cells and cachexia in mice. Using this established cachexia cell model, we targeted autocrine Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation activin A by genetic, chemical and biological approaches. The secretion of IL-6 from the cancer cells was determined both in culture and tumor-bearing mice by a species specific ELISA. Autophagy reporter cells were used to monitor the culture medium for autophagy inducing activities and muscle mass changes were evaluated in tumor bearing mice. Results – We show that activin A acts in an autocrine manner to promote the synthesis and secretion of IL-6 from cancer cells. We find that this is important for the ability of the cancer cells to accelerate autophagy in non-cancerous cells. Consistently, interfering with activin A signaling in cachectic tumor-bearing mice reduces serum levels of cancer cell-derived IL-6 and reverses cachexia. Thus, our data support a functional link between activin and IL-6 signaling pathways, and indicate that interference with activin A-induced IL-6 secretion from the tumor has therapeutic potential for cancer-induced cachexia

MRI Reveals the in Vivo Cellular and Vascular Response to BEZ235 in Ovarian Cancer Xenografts with Different PI3-Kinase Pathway Activity

Background: The phosphoinositide-3 kinase (PI3K) pathway is an attractive therapeutic target. However, difficulty in predicting therapeutic response limits the clinical implementation of PI3K inhibitors. This study evaluates the utility of clinically relevant magnetic resonance imaging (MRI) biomarkers for noninvasively assessing the in vivo response to the dual PI3K/mTOR inhibitor BEZ235 in two ovarian cancer models with differential PI3K pathway activity. Methods: The PI3K signalling activity of TOV-21G and TOV-112D human ovarian cancer cells was investigated in vitro. Cellular and vascular response of the xenografts to BEZ235 treatment (65 mg kg−1, 3 days) was assessed in vivo using diffusion-weighted (DW) and dynamic contrast-enhanced (DCE)-MRI. Micro-computed tomography was performed to investigate changes in vascular morphology. Results: The TOV-21G cells showed higher PI3K signalling activity than TOV-112D cells in vitro and in vivo. Treated TOV-21G xenografts decreased in volume and DW-MRI revealed an increased water diffusivity that was not found in TOV-112D xenografts. Treatment-induced improvement in vascular functionality was detected with DCE-MRI in both models. Changes in vascular morphology were not found. Conclusions: Our results suggest that DW- and DCE-MRI can detect cellular and vascular response to PI3K/mTOR inhibition in vivo. However, only DW-MRI could discriminate between a strong and weak response to BEZ235