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Unige iGEM 2019

Results

Co-culture coverslip dose-response

As a first proof of concept, we wanted to prove that a co-culture method has a higher physiological relevance than a single cell type culture method. For this aim, we chose a well known model: the response of breast cancer (BC) cells to Tamoxifen (Tam).

Tam is a pro-drug that needs to be metabolized to 4-hydroxytamoxifen (4-OHT) by the liver to inhibit the estrogen receptor (ERα) in breast tumors, meaning that this response depends on a physiological interaction between two distant tissues, liver and breast tumor.

Our first hypothesis was that a co-culture of BC cells with liver cancer (LC) cells would improve the action of Tamoxifen when compared to a monoculture of BC cells. We monitored the activity of ERα by using an ERα-dependent luciferase reporter construct in BC cells cultured with or without LC cells. A dose-response was performed with increasing concentration of Tam.

We showed that the co-culture BC + LC increased the efficiency of Tam by almost 20-fold (IC50 : 20nM) when compared to the culture of BC cells alone (IC50 : 337 nM) (Figure 1). This result demonstrates that the co-culture recapitulates at least a part of the physiological interaction between liver and breast tumors for the action of Tam on ERα activity. In addition, it suggests that a co-culture method would allow the discovery of different pro-drugs such as Tam, which could not be detected with the current drug screening method.

Figure 1: Pharmacological inhibition of ERα in BC cells co-cultured with LC cells. BC cells stably expressing luciferase under the control of ERα were cultured with or without LC cells and they were treated with increasing concentrations of Tam or 4-OHT for 48 hours. BC cells were lysed and bioluminescence was measured to determine ERα activity. Graph shows Relative Luminescence Units (RLU) as a function of Log[inhibitor concentration].

Cloning and test of fluorescent reporter results

Cloning of the construct zipGFP-Casp3 into a lentiviral vector

To generate stable cell lines reporting apoptotic activity, we decided to use the zipGFP-Casp3 construct from Addgene (#81241). In this construct, GFP domains are zipped with coiled coils that are cut by executioner Caspase-3 when caspase-dependent apoptosis is triggered in the cells. Caspase-3 activation leads to the cleavage of the coiled coils and increases the fluorescence intensity of GFP by several fold.

Figure 1: β-sheets 1-10 of GFP (above) and β-sheet 11 (below) are zipped at the N- and C-terminus. A Caspase-3 proteolytic sequence is shown in pink. After Caspase-3 cleavage, both parts of GFP assemble to form a fluorescent GFP (green).

To amplify the zipGFP-Casp3 insert by PCR and to integrate it into a pHAGE vector, we designed primers that are specific to the 5’- and 3’-ends of the zipGFP-Casp3 sequence. We flanked 5-ends of the primers with 20bp that are homologous to the 5’ and 3’ insertion site of the pHAGE vector (after it's cleavage by SpeI (5’) and ClaI (3’) restrictions enzymes), for the forward and reverse primer, respectively. After amplification of the insert, homologous sequences will allow end-joining with the pHAGE vector by the Gibson assembly method.

In parallel, we cleaved the pHAGE vector with the restriction enzymes SpeI and ClaI. After agarose gel electrophoresis, we cut the bands corresponding to the cleaved pHAGE vector (6kb), and the band of the zipGFP-Casp3 insert (3kb).

Figure 2: Agarose gel showing the cleaved pHAGE vector and the zipGFP-Casp3 insert used for the cloning experiment.

Gibson assembly cloning

Gibson assembly was used to assemble several DNA fragments together in the proper sequence and orientation.

Figure 3: Gibson assembly scheme.

Addgene reference

After cloning of zipGFP-Casp3 insert into pHAGE lentiviral vector, we transformed stellar competent cells, selecting them on Ampicilin-agar plates and we screened ampicillin-resistant clones by using PCR on a colony with a couple of primers overlapping the junction between the pHAGE vector and the insert, to check both the presence of the insert and its orientation.

Figure 4: Agarose gel showing colony PCR of 14 colonies after Gibson assembly on zipGFP-Casp3 insert and pHAGE vector.

A positive colony (band size: 600bp) was amplified, DNA was extracted, purified and sequenced. This construct can then be used to prepare lentiviruses to generate stable cell lines.

Figure 5: Map of the lentiviral pHAGE-zipGFP-Casp3 construct.

Test of the lentiviral zipGFP-Casp3 reporter construct

We wanted to ensure that after cloning the zipGFP-Casp3 reporter construct into the pHAGE lentiviral vector, it was still working as a reporter of apoptotic activity. To do so, we transiently transfected the original zipGFP-Casp3 construct from Addgene, and in parallel we transfected our lentiviral zipGFP-Casp3 construct in HEK293T cells. Then, we induced apoptosis by treating cells overnight with Staurosporine 1µM, an inducer of caspase-dependent apoptosis.

By normalizing the GFP intensities by the mCherry intensities, we determined the proportion of cells that underwent caspase-dependent apoptosis. We could detect that treating the cells with Staurosporin increased apoptosis, with both the construct from Addgene and our lentiviral construct, thus validating the use of the our lentiviral zipGFP-Casp3 construct to monitor apoptosis.

Figure 6: Test of the zipGFP-Casp3 reporter on HEK293T cells treated with or without Staurosporin 1µM for 18 hours.

Creation of a new part: cloning of CTCF-ERE-SYFP2

In order to monitor the activity of Estrogen Receptor (ERα), we decided to generate a fluorescent reporter under the control of ERα. Indeed, in our experiment of monolayer cell co-culture, we chose the pharmacological inhibition of ERα by Tamoxifene as a model to assess a physiological interaction. However, the reporter that we used is a bioluminescent reporter (Luciferase), that creates a signal that is too bright to be compatible with our fluorescence acquisition method. We designed our construct by fusing an Estrogen Response Element (ERE) followed by a mini-Thymidine Kinase Promoter (mini-TK), to the gene coding the Super Yellow Fluorescent Protein 2 (SYFP2). This gene was followed by a polyadenylation site (pA) to stop the transcription of SYFP2 in mammalian cells. To help in the cloning of this construct, we obtained the generous help provided by Twist Bioscience in the context of the iGEM competition, and we asked them to synthesize the full insert of the reporter construct: ERE-miniTK-SYFP2-pA. This insert can be seen as an assembly of two different BioBricks: a regulatory BioBrick with the ERE, and a reporter BioBrick with SYFP2.

Because we want to generate stable reporting cells to monitor the activity of ERα, we thought about the genomic context of our reporter construct. In mammals, gene expression depends on the 3D organization of the genome, and promoter regulation can be influenced by distant response elements like our ERE. To avoid misregulations of our reporter by the genomic regions surrounding the insertion site, we decided to flank our reporter construct with CTCF sites. CTCT is an insulator protein that creates DNA loops and control the 3D structure of mammalian genomes. By making a loop, CTCF insulates a specific genomic region and avoids unspecific regulations by distant regulatory sequences.

After we received the synthetic DNA construct from Twist Bioscience, we did a PCR with specific primers flanked with CTCF sites. This allowed us to have the following construct: CTCF-ERE-mini-TK-SYFP2-pA-CTCF.

Figure 7: Agarose gel showing the CTCF-ERE-SYFP2 inserts used for the cloning of the basic part. Band size: 1.3kb.

We then used the cleaved pHAGE vector from the cloning of the zipGFP-Casp3 construct and we cloned the CTCF-ERE-SYFP2 insert with the Gibson assembly method. We screened positive clone by colony PCR and we expected a band at a size of 600bp for positive clones:

Figure 8: Agarose gel showing colony PCR of 12 colonies after Gibson assembly on CTCF-ERE-SYFP2 insert and pHAGE vector.

A positive colony (band size: 600bp) was amplified, DNA was extracted, purified and sequenced. This construct can then be used to prepare lentiviruses to generate stable cell lines.

Sequence obtained for our insert:

Figure 9: Map of the lentiviral pHAGE-CTCF-ERE-SYFP2 construct.

Test of the new iGEM basic part: CTCF-ERE-SYFP2.

After cloning the CTCF-ERE-SYFP2, we wanted to investigate whether this construct is inducible by estrogen (E2) treatment. We transiently transfected the pHAGE-CTCF-ERE-SYFP2 construct with or without co-transfection with an ERα-coding plasmid in HEK293T cells. As a positive control for SYFP2 expression, we also performed a transfection of HEK293T cells with a plasmid coding for SYFP2. An mCherry-coding plasmid was co-transfected in all conditions to normalize SYFP2 fluorescence intensities. Then, we treated cells overnight with E2 100nM to activate ERα.

After normalization of SYFP2 signal by mCherry signal, we showed that only the condition where the ERE-SYFP2construct and ERα-coding plasmid were co-transfected is inducible by E2 treatment. This validate the estrogen-inducibility of our contruct.

The new iGEM basic part that we designed, the CTCF-ERE-SYFP2, can thus be used as a estrogen receptor activity reporter using SYFP2 fluorescence as a readout.

Figure 10: Test of the zipGFP-Casp3 reporter on HEK293T cells treated with or without Staurosporin 1µM for 18 hours.

Creation of a new part: cloning of CTCF-ERE-SCFP3A

For the cloning of the CTCF-ERE-SCFP3A construct, we followed exactly the same strategy as for the cloning of the CTCF-ERE-SYFP2 construct. Instead of using SYFP2 as the fluorescent reporter gene, we choose Super Cyan Fluorescent Protein 3A (SCFP3A) to increase the number of fluorescent combinations we can do with other reporters.

After we received the synthetic DNA construct from Twist Bioscience, we conducted a PCR with specific primers flanked with CTCF sites. This allowed us to have the following construct: CTCF-ERE-mini-TK-SCFP3A-pA-CTCF.

Figure 11: Agarose gel showing the CTCF-ERE-SCFP3A insert used for the cloning of the basic part. Band size: 1.3kb.

We then used the cleaved pHAGE vector from the cloning of the zipGFP-Casp3 construct and we cloned the CTCF-ERE-SCFP3A insert with the Gibson assembly method. We screened positive clone by colony PCR and we expected a band at a size of 600bp for positive clones:

Figure 12: Agarose gel showing colony PCR of 12 colonies after Gibson assembly on CTCF-ERE-SCFP3A insert and pHAGE vector.

A positive colony (band size: 600bp) was amplified, DNA was extracted, purified and sequenced. This construct can then be used to prepare lentiviruses to generate stable cell lines.

Sequence obtained for our insert:

Figure 13: Map of the lentiviral pHAGE-CTCF-ERE-SCFP3A construct

Test of the new iGEM basic part: CTCF-ERE-SCFP3A

We wanted to validate that the CTCF-ERE-SCFP3A worked and we did exactly the same experiment as for CTCF-ERE-SYFP2, except that we replaced it by CTCF-ERE-SCFP3A. Unfortunately, we observed that there was no fluorescence emission for the wavelength corresponding to SCFP3a. This construct is not validated and we need to change our design strategy to use SCFP3A as a reporter of estrogen receptor activity. An additional solution could be to use another blue fluorescent protein with a different excitation-emission spectra, or alternatively try to strengthen the Estrogen Receptor Element by adding more sites bound by ERα.

References:

Gibson, D.G., et al., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods, 2009. 6(5): p. 343-5.

Addgene Gibson assembly

T-L, Schepis A, Ruiz-González R, Zhang Q, Yu D, Dong Z, et al. Rational Design of a GFP-Based Fluorogenic Caspase Reporter for Imaging Apoptosis In Vivo. Cell Chemical Biology. 21 juill 2016;23(7):875‑82.

Test of the FUCCI dual-reporter system

As a first biological activity readout, we decided to use the FUCCI dual-reporter because it allows to monitor two different proliferative states: cells in the G1 phase of the cell cycle are red while cells in the remaining phases of the cell cycle (S, G2 and M) are green. This reporter system is based on the cell cycle-dependent degradation of two fusion proteins, mAG-Geminin which is degraded in the G1 phase of the cell cycle, and mKO2-Cdt1 which is degraded in S, G2 and M phase.

Figure 1: Schematics of the FUCCI dual-reporter system on cells: Cells fluoresce red during the G1 phase, and green for all the other phases of the cell cycle.

We wanted to use this reporter system to test our concept of measuring multiple biological readouts.Indeed, even if the FUCCI reporter can only measure proliferation, it gives information about two different states of proliferation with two different fluorescent proteins. Being able to measure both fluorescence intensities in two different cell types would show that our concept is viable.

We used the lentiviral FUCCI construct from Addgene (#86849) to generate stable reporter cell lines expressing the FUCCI dual-reporter system. First, we wanted to validate the FUCCI construct in a cell reporter assay. We transiently transfected HEK293T cells with the FUCCI plasmid or with an empty vector as a control. Then, we deprived cells without serum or we growed them in 15% Fetal Bovine Serum (FBS) and after 24 hours we measured the fluorescence intensities with a multiplate reader.

By normalizing the mAG intensities by the mKO2 intensities, we determined the proportion of cells that were actively proliferating by entering into the cell cycle. We observed that with the FUCCI reporter, we demonstrated that increasing the concentration of FBS to 15% stimulated the proliferation of cells, thus validating the use of the FUCCI reporter for our project.

Figure 2: Test of the FUCCI reporter on HEK293T cells with or without 15% FBS for 24 hours.

References:

Merten, O.W., M. Hebben, and C. Bovolenta, Production of lentiviral vectors. Mol Ther Methods Clin Dev, 2016. 3: p. 16017.

SpectraMax MiniMax cytometer

Encapsulation results

To test the technology of cell encapsulation, we first encapsulated human Liver Cancer (LC) cells (HepG2) and human Breast Cancer (BC) cells (MDA-MB134) in separated alginate capsules (Figure1). Right after encapsulation, cells are in suspension within the capsule and we could not distinguish LC from BC cells. After seven days in culture, cells formed spheroids and we could observe two morphologically different kind of spheroids. However, because both LC and BC spheroids were mixed in a co-culture, we could not identify them individually. This is why in the Design section, we contemplated labeling alginate with fluorophores to make color-encoded capsules to facilitate the identification of different types of spheroids in co-culture.

Figure 1: Light microscope pictures showing encapsulated cells right after encapsulation (left) and 7 days after encapsulation (right).

Labeling the alginate capsule

Alginate is a molecule with a carboxylic acid function that can react with amines after its activation with N-Hydroxysulfosuccinimide (sulfo-NHS). We choose an amine-containing fluorophore that emits in the far-red: ATTO 647N. After covalent modification of alginate with ATTO647N (Alginate-ATTO 647N), we assessed whether we can make capsules stably labeled with this fluorophore, and we could obtain far-red fluorescent capsules (Figure 2). It means that fluorescently-labeled alginate can be use to make color-encoded capsules.

Figure 2: Picture showing far-red fluorescent alginate capsules without cells.

Encapsulation of fluorescent cells

The next step was to evaluate whether we can encapsulate fluorescent cells and see the emission of the reporter's fluorescent light through the capsule wall. We transiently transfect HEK293T human cells with mCherry-encoding plasmid, and 24 hours later we encapsulated those cells. We showed in Figure 3 that red fluorescent cells can be clearly seen through the alginate capsules, validating the fact that we can encapsulate fluorescent reporter cells and measure the fluorescence from cells.

Figure 3: Capsules with mCherry-overexpressing cells.

Measurement of two cellular states in two cell types in co-culture

After validating the use of fluorescently-labeled capsule for the identification of encapsulated cell types, and the detection of fluorescent cells through the capsule to measure reported biological activities, we combined both together to assess whether we can measure two different biological activities in two different cell types in encapsulated 3D co-culture. We used the FUCCI dual-reporter system because it reports two different cellular states with two different fluorescent colors: cells in the G1 phase of the cell cycle (latent) are red while cells in all the other phase of the cell cycle (active) are green.

BC cells that stably express the FUCCI reporter were encapsulated in far-red alginate (BC capsules). BC cells are green or red because of the FUCCI reporter, so we artificially change the color of the capsules with the fluorescence analysis software from far-red to blue to make the visualization more convenient. As shown in Figure 4, we can clearly discriminate the fluorescence coming from the capsules and from each individual encapsulated BC cells.

Figure 4: FUCCI-expressing BC cells encapsulated in alginate-ATTO 647N.

In parallel, LC cancer cells that stably express the FUCCI reporter were encapsulated in unlabeled alginate (LC capsules) to discriminate them from BC capsules. We can observe green and red LC cells inside a non-fluorescent alginate capsule (Figure 5).

Figure 5: FUCCI-expressing LC cells encapsulated in unlabeled alginate.

Then, we did a co-culture by mixing BC capsules with LC capsules together in the same well. Because of the different labeling of the capsules, we successfully discriminated between BC capsules (blue) and LC capsules (non-fluorescent) (Figure 6). Identification of the capsules according to their fluorescence profile allows the analyze of images with segmentation processes leading to the individual measurement of each cellular state in each capsule type.

Figure 6: Co-culture of FUCCI-BC cells in far-red capsules (blue) with FUCCI-LC cells in unlabeled capsules (non-fluorescent) in FBS 0%. Cell segmentation profile used to quantify the different fluorescent intensities following different combinations is shown at the right.

In this experiment, we grow the cells in three different serum (FBS) concentrations (FBS 0%(Figure 6), FBS 5% (Figure 7), FBS 15% (Figure 8)) for 4 days to modulate the proliferation rates of the different cell populations in co-culture.

Figure 7: Co-culture of FUCCI-BC cells in far-red capsules (blue) with FUCCI-LC cells in unlabeled capsules (non-fluorescent) in FBS 5%. Cell segmentation profile was used to quantify the different fluorescent intensities following different combinations, which are shown to the right.

Figure 8: Co-culture of FUCCI-BC cells in far-red capsules (blue) with FUCCI-LC cells in unlabeled capsules (non-fluorescent) in FBS 15%. Cell segmentation profile was used to quantify the different fluorescent intensities following different combinations, which is shown to the right.

Figure 9 demonstrates that two different cellular states reported by two different fluorescent reporters (green fluorescent reporter gene for proliferating cells; red fluorescent reporter gene for non-proliferating cells, both included in the FUCCI reporter) can be quantified simultaneously in a 3D co-culture of two different cell types encapsulated in two different alginate capsules (far-red-labeled alginate capsules for BC cells; unlabelled alginate capsules for LC cells). Results from figure 9 also show that the fluorescence of reporters can be discriminated between them and with the fluorescence of the labelled alginate capsules by microscopy acquisition followed by image processing and analysis with the software MetaXpress (Molecular Devices).

Figure 9: Quantification of two cellular proliferation states in BC cells and LC cells in co-culture.

From this experiment, we showed that BC cells proliferate more with FBS 15% but also that the overall number of cells, even not-proliferating cells, is increased. The overall increase of proliferation during the previous days is probably responsible for the large number of total cells. For LC cells, surprisingly we observed a decrease of the total number of cells with FBS 15%. Because LC cells were already proliferating far more than BC cells with low serum concentration, this result suggests that with FBS 15%, LC cells proliferated until reaching the maximal size that can be contained inside the capsule. Then, many cells probably entered into a quiescent state, explaining the decrease of the expression of the reporter.

We conclude from this experiment that our drug testing concept works as expected. We can consider that the proof of our concept is validated by the results of this experiment: measuring multiple biological activities or cellular states simultaneously in different co-culture cell types.

Additional combinations with labeling of alginate with AMCA?

In order to make additional combinations, we wanted to label alginate with another fluorophore. We chose AMCA (aminomethylcoumarin) which is a blue fluorophore that we can use in combination with alginate-ATTO 647N (far-red) and unlabeled alginate, to have a total of three different color-encoded capsules.

After covalent modification of alginate with AMCA, we encapsulated two different fluorescent reporter BC cells: FUCCI-BC cells in alginate-ATTO647N capsules, and zipGFP-BC cells in alginate-AMCA capsules. zipGFP-Casp3 (zipGFP) is a fluorescent reporter expressing constitutively mCherry to check for the cells having the reporter. The activity of GFP of this reporter is increased by the cleavage by executioner Caspase-3 of inhibitory sequences impairing the maturation of GFP. More the apoptotic activity is high, more the fluorescence of GFP is bright. By putting FUCCI and zipGFP together, we aimed at measuring two different biological activities at the same time but because these two reporters use the same fluorescent colors, we had to encapsulate the cells expressing these two reporters in different capsules.

Unfortunately, results from Figure 10 show that the capsules containing the zipGFP reporter are unlabeled, indicating that AMCA was not properly linked to the alginate, leading to unlabeled alginate.

Figure 10: Co-culture of FUCCI-BC cells in far-red capsules (blue) with zipGFP-BC cells encapsulated in alginate-AMCA (not-fluorescent).

Labeling of alginate with Fluoresceinamine

Because we failed to label alginate with AMCA, we decided to use Fluoresceinamine which is a green fluorophore that can be used with alginate-ATTO 647N and unlabeled alginate.

As shown in Figure 11, labeling of alginate with fluoresceinamine was successful. It allowed the identification of FUCCI-BC cells (green capsules), and of zipGFP-BC cells (blue capsules). It means also that a third type of cells that are encapsulated in unlabeled alginate can be added in co-culture, and it would lead to the discrimination of three different cell types.

Figure 11: Co-culture of zipGFP-BC cells in far-red capsules (blue) with FUCCI-BC cells encapsulated in alginate-Fluoresceinamine (green).

Multiple biological activities and interactions between cell types

Our very first motivation to design this new cell culture method was to reproduce physiologically relevant interactions between tissues. We have tested a part of this hypothesis on monolayer cell co-culture with BC cells together with LC cells and we showed that it increased the ability of Tamoxifen to inhibit its target, the estrogen receptor, by 17-fold when compared to a culture of BC cells alone.

At high concentrations, Tamoxifen induces apoptosis. We decided to check whether the toxicity of Tamoxifen is increased in BC cells when they are co-cultured with LC cells. zipGFP-BC cells were encapsulated in alginate-ATTO 647N, LC cells in alginate-fluoresceinamine, and FUCCI-BC cells in unlabeled capsules (Figures 12 and 13). We treated cells with concentrations of Tamoxifen above 1µM to assess its toxicity. At these concentrations, inhibition of proliferation is maximal and we cannot expect more cytostatic activity. FUCCI-BC cells were used here as a control to assess whether we can finely discriminate different types of pharmacological activities (cytostatic vs cytotoxic) according to different ranges of drug concentration, where a high range of Tamoxifen concentrations induced cytotoxicity.

Figure 12: Co-culture of zipGFP-BC cells in far-red capsules (blue) with FUCCI-BC cells in unlabeled capsules (non-fluorescent).

Figure 13: Co-culture of zipGFP-BC cells in far-red capsules (blue) with FUCCI-BC cells in unlabeled capsules (non-fluorescent) with LC cells in green capsules (green).

Figure 14: Caspase-dependent apoptotic activity in BC cells treated with Tamoxifen and co-cultured with or without LC cells.

Figure 15: Proliferation states of BC cells treated with Tamoxifen and co-cultured with or without LC cells.

Results from Figure 14 showed that the co-culture of BC cells with LC cells increased by 3-fold the cytotoxicity of Tamoxifen for concentrations above 5µM, when compared to BC cells alone. However, the cytostatic activity of Tamoxifen is unchanged for this concentration range, either for BC cells alone or in co-culture with LC cells (Figure 15). These results show that our cell culture method allows the discrimination of different pharmacological activities for specific ranges of drug concentration, and that it integrates the physiological interactions between the treated tissue and other physiologically relevant tissues that can modify the activity of a compound.