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.

Combined Targeting of AKT and mTOR Synergistically Inhibits Formation of Primary Colorectal Carcinoma Tumouroids In Vitro: A 3D Tumour Model for Pre-therapeutic Drug Screening.

BACKGROUND: Pre-therapeutic analysis of three-dimensional spheroid cultures of primary tumour samples is a promising approach of assessing susceptibility to potential treatment. The phosphatidylinositol-3-kinase/AKT serine/threonine kinase/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway is frequently activated in colorectal cancer (CRC). In previous work, we showed combined inhibition of AKT and mTOR to be highly synergistic in cell lines from patients with hepatocellular carcinoma and cholangiocarcinoma in vitro as well as in vivo in murine xenograft tumour models. MATERIALS AND METHODS: Patient-derived xenograft colorectal carcinoma cell lines HROC80 T1 M1, HROC147 T0 M1, HROC147Met, HROC277 T0 M1 and HROC277Met2 were treated with AKT inhibitor MK2206, mTOR inhibitor RAD001 or the combination of both drugs. The sensitivity of these cell lines to inhibition was evaluated by calculation of combinatory indices after bromodeoxyuridine assays and analysis of the respective pathways by western blotting. Furthermore, the dual inhibition of AKT and mTOR was confirmed in vivo in a xenograft mouse model. Additionally, primary CRC samples of four patients were embedded in a three-dimensional matrix and the sensitivity of these samples was analyzed by measurement of the spheroid area. RESULTS: In this study, we demonstrate that combined treatment with MK2206 and RAD001 resulted in strong synergistic effects on growth of several primary CRC cell lines and reduced the growth of a patient-derived CRC xenograft in a xenotransplantation mouse model in vivo. Interestingly, the response to treatment varied between cell lines derived from the primary lesion and a liver metastasis of the same patient. In addition, combined treatment with AKT and mTOR inhibitors resulted in a synergistic inhibition of tumouroid growth in all four of the primary patient samples, analyzed in a three-dimensional spheroid model in vitro. CONCLUSION: Our data demonstrate that combined treatment with AKT and mTOR inhibitors exhibits synergistic effects on proliferation of cell lines and primary tumour cells from patients with CRC and may be a promising approach for the treatment of CRC.

Characterization of genetic intratumor heterogeneity in colorectal cancer and matching patient-derived spheroid cultures.

Patient-derived in vitro cultures of colorectal cancer (CRC) may help guide treatment strategies prior to patient treatment. However, most previous studies have been performed on a single biopsy per tumor. The purpose of this study was to analyze multiple spatially distinct biopsies from CRCs and see how well intratumor heterogeneity (ITH) was recapitulated in matching patient-derived spheroids. Three to five biopsies were collected from six CRC tumors. Each biopsy was split in two; one half was used for spheroid culturing, while the other half was used for DNA and RNA purification. For two patients, lymph node metastases were analyzed. Somatic mutations were called from whole exome sequencing data. Each tumor contained mutations shared across all biopsies and spheroids, including major CRC drivers such as APC, KRAS, and TP53. At the same time, all tumors exhibited ITH on both mutation and copy number level. The concordance between biopsies and spheroids ranged between 40 and 70% for coding mutations. For three patients, the biopsy and spheroid from matching areas clustered together, meaning that the spheroid resembled the area of origin more than the other areas. However, all biopsies and spheroids contained private mutations. Therefore, multiple cultures from spatially distinct sites of the tumor increase the insight into the genetic profile of the entire tumor. Molecular subtypes were called from RNA sequencing data. When based on transcripts from both cancer and noncancerous cells, the subtypes were largely independent of sampling site. In contrast, subtyping based on cancer cell transcripts alone was dependent on sample site and genetic ITH. In conclusion, all examined CRC tumors showed genetic ITH. Spheroid cultures partly reflected this ITH, and having multiple cultures from distinct tumor sites improved the representation of the genetic tumor subclones. This should be taken into account when establishing patient-derived models for drug screening.

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.

Chemical synthesis on SU-8.

In this paper we describe a highly effective surface modification of SU-8 microparticles, the attachment of appropriate linkers for solid-supported synthesis, and the successful chemical modification of these particles via controlled multi-step organic synthesis leading to molecules attached in an unambiguous manner to the support surface.

Changes in intracellular cAMP reported by a Redistribution assay using a cAMP-dependent protein kinase-green fluorescent protein chimera.

We report on a novel method to monitor changes in intracellular cAMP concentration ([cAMP]i) within intact living cells using a chimeric fusion of the catalytic subunit of cAMP-dependent protein kinase to green fluorescent protein (PKAcat-GFP). In stably transfected unstimulated fibroblasts, fusion protein fluorescence is highly concentrated in aggregates throughout the cytoplasm and absent in the nucleus. Elevation of [cAMP]i disperses GFP fluorescence from the cytoplasmic aggregates within minutes. Spot-photobleach measurements show that the rate of exchange of GFP-labeled catalytic subunits at these aggregates increases in proportion to [cAMP]i. For any given stimulus, the response curve for dispersal of GFP fluorescence from aggregates agrees closely with the increase in total [cAMP]i as measured by standard in vitro methods (SPA). The redistribution of fluorescence is completely reversible: reduction of [cAMP]i results in return of fluorescence to the cytoplasmic aggregates. Consistent behaviour of PKAcat-GFP is seen in different cell backgrounds. We demonstrate that PKA Redistribution assays are suitable for measurement of changes in [cAMP]i brought about by both Gs- and Gi-protein-coupled receptor stimulation as well as by inhibition of cAMP phosphodiesterases.