Mutation of KRAS in colorectal adenocarcinoma in Greenland.

KRAS mutation is one of the most frequent driver mutations in colorectal cancer (CRC) and is also a prognostic biomarker. The aim of the present study was to determine the frequency of KRAS mutations over time in the Greenlandic population diagnosed with CRC. In total, 578 patients with the diagnosis of adenocarcinoma between 1988 and 2017 were identified. The status of KRAS and the mutational subtypes of KRAS mutations were determined in 102 representative samples by the Idylla™ platform in the time periods 1988-1990, 2002-2004, and 2015-2017. The results showed that the frequency of the KRAS mutations increased significantly, from 27% in 1988-1990 to 43% in 2015-2017 (p < 0.001). Furthermore, the most frequent subtypes of KRAS mutations in Greenland were G12D (c.35G > A) with 14%, G12V (c.35G > T) with 7%, and G13D (c.38G > A) with 6%. In conclusion, this study showed that the frequency of KRAS mutations in CRC has been increasing in recent decades in the specific population of Greenland. The results of this study may be used in initiatives related to targeted therapy of CRC in specific ethnicities and in investigations focusing on the environmental factors of cancer-related somatic mutations.

Characterization of in vitro 3D cultures.

Over the past decade, 3D culture models of human and animal cells have found their way into tissue differentiation, drug development, personalized medicine and tumour behaviour studies. Embryoid bodies (EBs) are in vitro 3D cultures established from murine pluripotential stem cells, whereas tumoroids are patient-derived in vitro 3D cultures. This thesis aims to describe a new implication of an embryoid body model and to characterize the patient-specific microenvironment of the parental tumour in relation to tumoroid growth rate. In this thesis, we described a high-throughput monitoring method, where EBs are used as a dynamic angiogenesis model. In this model, digital image analysis (DIA) is implemented on immunohistochemistry (IHC) stained sections of the cultures over time. Furthermore, we have investigated the correlation between the genetic profile and inflammatory microenvironment of parental tumours on the in vitro growth rate of tumoroids. The EBs were cultured in spinner flasks. The samples were collected at days 4, 6, 9, 14, 18 and 21, dehydrated and embedded in paraffin. The histological sections were IHC stained for the endothelial marker CD31 and digitally scanned. The virtual whole-image slides were digitally analysed by Visiopharm® software. Histological evaluation showed vascular-like structures over time. The quantitative DIA was plausible to monitor significant increase in the total area of the EBs and an increase in endothelial differentiation. The tumoroids were established from 32 colorectal adenocarcinomas. The in vitro growth rate of the tumoroids was followed by automated microscopy over an 11-day period. The parental tumours were analysed by next-generation sequencing for KRAS, TP53, PIK3CA, SMAD4, MAP2K1, BRAF, FGFR3 and FBXW7 status. The tumoroids established from KRAS-mutated parental tumours showed a significantly higher growth rate compared to their wild-type counterparts. The density of CD3+ T lymphocytes and CD68+ macrophages was calculated in the centre of the tumours and at the invasive margin of the tumours. The high density of CD3+ cells and the low density of CD68+ cells showed a significant correlation with a higher growth rate of the tumoroids. In conclusion, a novel approach for histological monitoring of endothelial differentiation is presented in the stem cell-derived EBs. Furthermore, the KRAS status and density of CD3+ T cells and macrophages in the parental tumour influence the growth rate of the tumoroids. Our results indicate that these parameters should be included when tumoroids are to be implemented in personalized medicine.

The cancer angiogenesis co-culture assay: In vitro quantification of the angiogenic potential of tumoroids.

The treatment response to anti-angiogenic agents varies among cancer patients and predictive biomarkers are needed to identify patients with resistant cancer or guide the choice of anti-angiogenic treatment. We present "the Cancer Angiogenesis Co-Culture (CACC) assay", an in vitro Functional Precision Medicine assay which enables the study of tumouroid induced angiogenesis. This assay can quantify the ability of a patient-derived tumouroid to induce vascularization by measuring the induction of tube formation in a co-culture of vascular cells and tumoroids established from the primary colorectal tumour or a metastasis. Furthermore, the assay can quantify the sensitivity of patient-derived tumoroids to anti-angiogenic therapies. We observed that tube formation increased in a dose-dependent manner upon treatment with the pro-angiogenic factor vascular endothelial growth factor A (VEGF-A). When investigating the angiogenic potential of tumoroids from 12 patients we found that 9 tumoroid cultures induced a significant increase in tube formation compared to controls without tumoroids. In these 9 angiogenic tumoroid cultures the tube formation could be abolished by treatment with one or more of the investigated anti-angiogenic agents. The 3 non-angiogenic tumoroid cultures secreted VEGF-A but we observed no correlation between the amount of tube formation and tumoroid-secreted VEGF-A. Our data suggests that the CACC assay recapitulates the complexity of tumour angiogenesis, and when clinically verified, could prove a valuable tool to quantify sensitivity towards different anti-angiogenic agents.

Application of digital image analysis on histological images of a murine embryoid body model for monitoring endothelial differentiation.

The in vitro 3D model established from murine pluripotential stem cells (i.e., embryoid bodies (EBs)) is a dynamic model for endothelial differentiation. The aim of the present study was to investigate whether digital image analysis (DIA) can be applied on histological sections of EBs in order to quantify endothelial differentiation over time. The EBs were established in suspension cultures for 21 days in three independent replicate experiments. At day 4, 6, 9, 14, 18, and 21, the EBs were fixed in formaldehyde, embedded in paraffin and immunohistochemically (IHC) stained for CD31. The IHC-stained slides were digitally scanned and analysed using the Visiopharm® Quantitative Digital Pathology software Oncotopix™. The EBs developed CD31+ vascular-like structures during their differentiation. The quantitative DIA of the EBs showed that the log10 values of the relative CD31+ areas increased from -0.574 ± 0.470 (mean ± SD) at day 4 to 0.093 ± 0.688 (mean ± SD) at day 21 (p < 0.001). The approach presented in this study is a fast, quantitative and reproducible alternative method for an otherwise time-consuming and observer-dependent histological investigation. The future perspectives for such a system would be implementation of a modified version of the method on different 3D cultures and IHC markers.

KRAS mutations in the parental tumour accelerate in vitro growth of tumoroids established from colorectal adenocarcinoma.

The aim of the present study was to characterize a patient-derived in vitro 3D model (ie tumoroid) established from colorectal adenocarcinoma. This study investigated the growth rate of tumoroids and whether the Kirsten rat sarcoma (KRAS) mutations in the parental tumour accelerate this rate. The tumoroids were established from surgical resections of primary and metastatic colorectal adenocarcinoma from 26 patients. The in vitro growth rate of these tumoroids was monitored by automated imaging and recorded as relative growth rate. The KRAS hotspot mutations were investigated on the parental tumours by Ion Torrent™ next-generation sequencing. The KRAS mutations were detected in 58% of the parental tumours, and a significantly higher growth rate was observed for tumoroids established from the KRAS-mutated tumours compared to wild-type tumours (P < 0.0001). The average relative growth rate (log10) on day 10 was 0.360 ± 0.180 (mean ± SD) for the KRAS-mutated group and 0.098 ± 0.135 (mean ± SD) for the KRAS wild-type group. These results showed that the presence of KRAS mutations in parental tumours is associated with an acceleration of the growth rate of tumoroids. The future perspective for such a model could be the implementation of chemoassays for personalized medicine.

The density of infiltrating T cells and macrophages in the parental tumour correlates with growth rate of tumoroids established from colorectal adenocarcinoma.

The aim of the present study was to investigate the correlation between the density of infiltrating T cells and macrophages in the parental colorectal cancer (CRC) and the growth rate of tumoroids (i.e. a patient-derived in vitro 3D model). Tumoroids were established from fresh specimens of primary and metastatic CRC from 29 patients. The in vitro growth rate of tumoroids was monitored by automated imaging. The density of infiltrating T cells and macrophages was determined in the centre of the tumour (CT) and at the invasive margin (IM) of the parental tumours. This was performed by digital image analysis on the whole-slide scanned images using Visiopharm® software. Tumoroids with higher density of infiltrating CD3+ lymphocytes in the IM of their parental tumour showed a higher growth rate (P < .0005). The average relative growth rate (log10) during the period from day 1 to day 11 was 0.364 ± 0.006 (mean ± SD) for the CD3+ (IM)-high group and 0.273 ± 0.008 (mean ± SD) for the CD3+ (IM)-low group. In contrast, the density of CD68+ infiltrating macrophages in the parental tumours showed significant inverse effect on the growth rate of the tumoroids (P < .0005). The present study showed that the density of immune cells in the parental CRC correlates with the growth rate of the tumoroids. The future perspective for such a 3D model could be in vitro investigations of the tumour-associated inflammatory microenvironment as well as personalized cancer immunotherapy.

Reinforcement of the bactericidal effect of ciprofloxacin on Pseudomonas aeruginosa biofilm by hyperbaric oxygen treatment.

Chronic Pseudomonas aeruginosa lung infection is the most severe complication in cystic fibrosis patients. It is characterised by antibiotic-tolerant biofilms in the endobronchial mucus with zones of oxygen (O2) depletion mainly due to polymorphonuclear leucocyte activity. Whilst the exact mechanisms affecting antibiotic effectiveness on biofilms remain unclear, accumulating evidence suggests that the efficacy of several bactericidal antibiotics such as ciprofloxacin is enhanced by stimulation of the aerobic respiration of pathogens, and that lack of O2 increases their tolerance. Reoxygenation of O2-depleted biofilms may thus improve susceptibility to ciprofloxacin possibly by restoring aerobic respiration. We tested such a strategy using reoxygenation of O2-depleted P. aeruginosa strain PAO1 agarose-embedded biofilms by hyperbaric oxygen treatment (HBOT) (100% O2, 2.8bar), enhancing the diffusive supply for aerobic respiration during ciprofloxacin treatment. This proof-of-principle study demonstrates that biofilm reoxygenation by HBOT can significantly enhance the bactericidal activity of ciprofloxacin on P. aeruginosa. Combining ciprofloxacin treatment with HBOT thus clearly has potential to improve the treatment of P. aeruginosa biofilm infections.

Increased bactericidal activity of colistin on Pseudomonas aeruginosa biofilms in anaerobic conditions.

Tolerance towards antibiotics of Pseudomonas aeruginosa biofilms is recognized as a major cause of therapeutic failure of chronic lung infection in cystic fibrosis (CF) patients. This lung infection is characterized by antibiotic-tolerant biofilms in mucus with zones of O2 depletion mainly due to polymorphonuclear leukocytic activity. In contrast to the main types of bactericidal antibiotics, it has not been possible to establish an association between the bactericidal effects of colistin and the production of detectable levels of OH ˙ on several strains of planktonic P. aeruginosa. Therefore, we propose that production of OH ˙ may not contribute significantly to the bactericidal activity of colistin on P. aeruginosa biofilm. Thus, we investigated the effect of colistin treatment on biofilm of wild-type PAO1, a catalase-deficient mutant (ΔkatA) and a colistin-resistant CF isolate cultured in microtiter plates in normoxic- or anoxic atmosphere with 1 mM nitrate. The killing of bacteria during colistin treatment was measured by CFU counts, and the OH⋅ formation was measured by 3(')-(p-hydroxylphenyl fluorescein) fluorescein (HPF) fluorescence. Validation of the assay was done by hydrogen peroxide treatment. OH⋅ formation was undetectable in aerobic PAO1 biofilms during 3 h of colistin treatment. Interestingly, we demonstrate increased susceptibility of P. aeruginosa biofilms towards colistin during anaerobic conditions. In fact, the maximum enhancement of killing by anaerobic conditions exceeded 2 logs using 4 mg L(-1) of colistin compared to killing at aerobic conditions.