FAQ2023-12-08T13:44:13+01:00

Shipment of Your Compounds

What is the shipping address for compounds?2022-03-29T11:17:07+02:00

Oncolines B.V.
Attn. Mr. J. de Roos
Building OP
Kloosterstraat 9
5349 AB Oss
The Netherlands
Tel.: +31 412 700 501
Mail: services@oncolines.com

Do we need to include a shipment declaration for customs?2020-10-13T14:55:19+02:00

Your shipper should be able to advise. Description of the materials should be on the Airway Bill.
Description of the materials: “Novel research compound for NON-human research”.
Please add MSDS (Safety Sheet).

Are there timeslots for shipment of compounds?2021-09-10T21:42:43+02:00

You can ship your compound at any time. The turnaround time starts at arrival of the compounds at Oncolines.

Deliverables

What is the status of IP that is generated during the studies?2021-09-29T11:22:26+02:00

The study results are owned by the Client. Oncolines destroys study related material after a pre-defined period.

Quality

What is the maximum concentration of DMSO in the wells?2019-10-03T13:11:29+02:00

The maximum concentration of DMSO in the wells is 0.4%. All wells contain the same DMSO concentration, including the control.

Is the 3 day assay format optimal for all of the cell lines, in regards to doubling time/assay window?2019-10-03T13:07:46+02:00

We have selected cell lines for the panel that show sufficient window in a 3 day proliferation assay. We have also validated the assays for five day incubation time.

How many passages are your cell stocks?2019-10-17T11:37:02+02:00

Our assay stocks have undergone 10 passages at maximum.

How do you monitor the quality of cell growth?2019-10-10T15:31:10+02:00

The doubling time of each cell line is monitored in each study. When the doubling time is beyond the quality criteria, the cell stock will be replaced.

Have you optimized the assay for each cell line?2019-10-03T13:15:44+02:00

Yes, we have optimized the cell number for each cell line. This is to ensure that they are in exponential growth phase when we expose them to compounds.

Have you observed differences in drug responses with longer incubation times?2019-10-03T13:10:43+02:00

3 versus 5 days incubation time can indeed make a difference for the effect of compounds. In particular, this was shown for the efficacy (% effect), which is related to number of cell cycles. See for instance Maia et al. (2015).

Have you looked at 3D cultures or spheroid assays with real time analysis in comparison to your proliferation assays?2019-10-03T15:08:53+02:00

We have done real time image analysis to look at chromosome mis-segregation induced by TTK inhibitors. This is illustrated by Libouban et al. (2017).

Does the presence of DMSO interfere with the readout of the proliferation assays?2019-10-03T15:03:21+02:00

The level of DMSO (0.4% in the wells) does not interfere with the readout and interpretation of the results.

Do you include control samples?2019-10-03T15:04:22+02:00

We always include untreated wells in the studies, as well as the reference compound doxorubicin.

Can you work with compounds that are unstable in solution at ambient?2019-10-03T13:19:02+02:00

We can solve the compound at the day of the experiment. For frozen solutions, the total time between thawing and aliquoting on cells can be decreased to approximately 1 hour.

Can you work with compounds that are dissolved in buffers instead of DMSO?2023-01-26T16:06:28+01:00

Yes, we have worked with antibodies, antibody-drug conjugates, peptides, … etc.
Water soluble compounds are dissolved in PBS, as water is not isotonic.

Proliferation Assays

How did you select cell lines for the Oncolines® panel?2022-10-17T10:59:18+02:00

The selection of cell lines was based on a good representation of:

  1. Cancer gene mutations – according to occurrence in patients
  2. Tissue types – currently 25

Other selection criteria are the growth characteristics of the cell lines and the absence of restrictions for use. We have a commercial licence from the ATCC for all cell lines in the Oncolines® panel.

Can we choose the dilution steps for the proliferation assays?2020-04-28T14:28:05+02:00

You are free to choose your preferred dilution steps. By default we use √10 dilution steps. With 9 dilution points, the √10 dilution steps cover 4 log decades. With a maximum test concentration of 31.6 µM, the mimimum test concentration is 3.16 nM.

Are Oncolines® studies restricted to the 102 cancer cell lines?2022-10-17T11:00:32+02:00

Based on your request we can add cell lines to a panel study. We have validated proliferation assays for more than 200 cell lines.

Bioinformatics Analysis – Gene Mutation Analysis

Which mutations are the ‘clinically actionable’ mutations?2019-10-03T15:15:40+02:00

These are mutations which are very frequently found on specific gene positions in cancer patients; so called ‘hotspot’ mutations (e.g. BRAF[V600E]). For completeness, copy number variations in the genes with hotspot mutations are included. The gene alterations included in this analysis are all well-known to be relevant in cancer.

What is the proprietary selection of cancer genes?2019-10-03T15:13:36+02:00

The ‘proprietary selection’ of cancer genes refers to the list of 98 genes, after filtering of the genes from the public domain. The aim of the filtering was to generate a list of genes with reported relevance in the cancer field, so to exclude genes that may not be relevant for cancer after all. Non-coding mutations have been filtered out (e.g. silent mutations) and only mutations that have been reported in cancer patients were kept. In addition, relevant copy number variations are included in the analysis.

The subset of 38 ‘commonly occurring and well known genes’ are a subset of the 98 genes. It contains the 35 most frequently mutated, amplified or deleted genes (TP53, CDKN2A, KRAS etc.) + 3 well known genes that everybody would miss if they are not included (BRCA2, SMAD4, and the BCR-ABL translocation). The ANOVA analysis is restricted to 38 genes for statistical reasons (due to the number of cell lines).

Can you run the analyses for various growth parameters, for instance GI50, AUC?2019-10-17T11:41:24+02:00

Yes – based on your specific needs we can adapt the analysis.

By what criteria were the oncogenes selected for the genomic analysis?2019-10-03T15:11:08+02:00

The criteria for selection of oncogenes are a.o.:

  1. Single nucleotide polymorphisms removed
  2. Only mutations in protein coding sequences retained
  3. Mutations filtered for occurrence in cancer patients

The mutant status of relevant oncogenes and tumor suppressors in the cell lines, e.g. B-RAF, CDKN2A, CTNNB1, EGFR, H-RAS, K-RAS, N-RAS, PIK3CA, TP53 etc. was confirmed by in-house DNA sequencing.

Bioinformatics Analysis – Tissue Sensitivity Analysis

What is the annotation of tissues based on?2019-10-03T15:25:14+02:00

Annotation of disease indication conforms to the Cellosaurus database. The associated tissue types are hierarchically organized according to the Oncotree classification.

Many of the previous profiling studies also report a significant difference in compound sensitivity for cell lines grown in suspension vs other cancer types. Are such trends observed here?2019-10-03T15:22:11+02:00

Indeed we observe that cell lines from hematological origins, which are all grown in suspension, are more sensitive to some cytotoxic therapies. Some of this is also discussed in our earlier work (Uitdehaag et al. (2014)). We do not know if this originates from the way of growing, from the hematological lineage, or from the historic focus on leukemias for drug development.

Bioinformatics Analysis – Comparative Analysis

Where can I find the list of anti-cancer agents for comparative profiling?2021-09-07T16:06:59+02:00

The Anti-Cancer Drugs Database can be requested via services@oncolines.com, or you can call us at +31 412 700 501.

Is there a color legend for the network tree?2019-10-03T15:26:56+02:00

The colors are indicative for clusters of compounds.

In the network tree, are the compounds listed closely to my study compound more similar than the ones listed farther out?2019-10-03T15:26:08+02:00

The distance between compounds is not related to similarity. The relative similarity ranking of your compound with other compounds, is shown in a separate correlation table.

Bioinformatics Analysis – Gene Expression Analysis

What is the value annotation for the gene expression? Is this fold change in relation to something?2019-10-03T15:33:45+02:00

It is log2-transformed ‘RMA-normalized data’ based on microarray. Since it is log2, a difference of 1 unit between genes and/or cell lines is similar to a 2-fold difference. For instance, Gene X has an expression value of 3 in Cell line A and an expression value of 4 in Cell line B. The difference is 1 unit (= factor 2), so the expression of Gene X is 2x higher in Cell line B than in Cell line A. A difference of 2 units means a 22 = 4-fold difference in expression; a difference of 3 means a 23 = 8-fold difference in expression.

As you see, the values are relative values, meaning that the values can be used to compare genes and cell lines. The values cannot be used in an absolute manner, for instance ‘1 ug of my sample contains 10,000 mRNA transcripts of Gene X’.

Is the gene expression data based on microarray or RNA seq?2022-12-15T14:26:27+01:00

RNAseq data. Both methods are reproducible and have a high correlation between gene expression profiles generated by the two.

SynergyFinder™

Why do you test 3 ratios of the mixture compounds in SynergyFinder™; 1:4, 1:1, and 4:1?2020-02-07T14:40:06+01:00

We test 3 ratios to confirm that a synergy in a specific mixture is observed in different settings. Generally, you would expect that a synergy is ratio independent. NB. when a synergy is observed, we always re-test it in an independent replicate experiment.

What are the differences between Loewe, Bliss, and HAS, and which model are you using?2023-01-26T16:08:15+01:00

In general there are two synergy ‘Schools of Thought’: Bliss additivity and Loewe additivity. The first uses %-effects and the other focuses on dosage. This is explained well by Foucquier and Guedj (2015). ‘HAS’, highest-single-agent, is also a Bliss-based model, using %-effect, but very simply taking the most potent compound as a reference. We do not use this. We base our calculations on Loewe additivity and use the isobologram and the Chou-Talalay CI Index as ways to visualize and quantify synergistic effects.

 

I heard about SynergyFinder™ and SynergyScreen™ as separate approaches, could you explain?2023-01-26T16:08:49+01:00

SynergyFinder™ is generally used for small-scale combination studies. The advantage is the determination of synergy according to curve shift analysis, CI Index and isobolograms; it is based on full dose response curves of both single agents and 3 mixtures per combination (ratios 1:4; 1:1; 4:1). Literature references are: Uitdehaag et al. (2014); Straetemans et al. (2005) and Chou (2010).

SynergyScreen™ is generally used for broad combination screening. Advantage: less laborious and therefore quicker and lower price. It concerns determination of synergy according to curve shift analysis; based on full dose response curve of 1 single compound and mixture curve of that compound with a fixed concentration of the 2nd compound.

How do you mix compounds to achieve equipotent mixtures?2023-01-26T16:09:59+01:00

Let’s say we need 50 µL of the mixture, IC50 of compound A is 1 nM and IC50 of compound B is 10 nM. In that case we take 25 µL of compound A and 25 µL of compound B. The total of 50 µL is subsequently diluted to generate the dose response curve. Since the compounds are equally effective, 25 µL of compound A can be added to 25 µL of compound B without a ‘dilution effect’.

Could you tell us in what case you will conclude that the combination showed synergy?2019-10-07T12:11:02+02:00

The combination is synergistic when the CI is lower than 1. In those cases, the experiment will be replicated before submission of the final report. Just to confirm that the observed synergistic effect is reproducible.

Could you tell us in what case we can expect synergy effect in vivo?2019-10-03T15:44:29+02:00

We have several cases of CI of around 0.7 in our cell-based studies, which are confirmed synergies in vivo and have been approved for clinical use.

For instance, the combination of dabrafenib and trametinib for patients with BRAF mutation and the combination of olaparib and cisplatin for ovarian cancer patients.

Mechanistic Cell Biology

What is the workflow for custom-based studies in Mechanistic Cell Biology?2021-09-10T21:40:58+02:00

Based on initial discussions about your interests in proof of concept studies, we prepare a workplan and a quotation. Using your input we’ll provide a proposal that is focused on your specific goals. After agreement, we will start the work. Study results and scientific insights are discussed during regular update meetings. It is work in progress, meaning that the workplan can be adapted based on new results if desired.

Can we collaborate on shared scientific publications?2019-10-03T15:59:59+02:00

The study results are owned by the Client. Of course we can support in the preparation of new scientific papers if desired.

Other Services

I cannot find ResidenceTimer™, for kinetic binding experiments, on your website. Is this still offered?2021-09-10T21:39:59+02:00

Yes, ResidenceTimer™ is part of Oncolines’ Precision Medicine Services portfolio. Uitdehaag et al. (2017) and Willemsen-Seegers et al. (2017) have described the technology.

For more info, check www.residencetimer.com/residencetimer/, contact us via services@oncolines.com or give us a call at +31 412 700 501.

I cannot find QuickScout™, for kinase activity assays, on your website. Is this still offered?2021-09-10T21:39:15+02:00

Yes, Oncolines is representing Carna Bioscience’s QuickScout™ in Europe. The biochemical assays are described by Uitdehaag et al. (2019) and Uitdehaag et al. (2014).

For more info, check www.residencetimer.com/quickscout/, contact us via services@oncolines.com or give us a call at +31 412 700 501.

I cannot find NFK GreenScreen™, enzyme activity assays for tryptophan catalyzing enzymes, on your website. Is this still offered?2021-09-16T16:25:10+02:00

Yes, NFK GreenScreen™ is supplied by Oncolines to customers worldwide. The technology is described by Seegers et al. (2014).

For more info, check our website on biochemical assays: www.residencetimer.com. Of course you can also contact us via services@oncolines.com or give us a call at +31 412 700 501.

I cannot find Arginase Gold™, activity assays for Arginase inhibitors, on your website. Is it still offered?2023-01-07T17:08:47+01:00

Yes, Arginase Gold™ is supplied by Oncolines to customers worldwide. The technology is described by Grobben et al. (2020).

For more info, check our website on biochemical assays: www.residencetimer.com. Of course, you can also contact us via services@oncolines.com or give us a call at +31 412 700 501.

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