Establishment and Validation of a New Co-Culture for the Evaluation of the Permeability through the Blood–Brain Barrier in Patients with Glioblastoma

Round 1

Reviewer 1 Report (Previous Reviewer 2)

The revise manuscript submitted by the authors is understandable.

However, the authors' conclusions are still unclear. Please provide a clearly stated description of which vitro BBB model you would recommend if researchers wish to deliver drugs with what characteristics for brain tumor treatment.

It is not clear what difference in usefulness the authors found between the two models (MDCK and MDCK-MDR1).

This is related to the authors' response to an initial review, but please include it in the body of the text.

Author Response

Please provide a clearly stated description of which vitro BBB model you would recommend if researchers wish to deliver drugs with what characteristics for brain tumor treatment.

Thanks for your comment. We have included a small protocol with our recommendations in the conclusions section:

With the available knowledge, the protocol that these authors recommend to follow when trying to develop a new tool to treat glioblastoma would be:

1) Checking the access of the molecule in the MDCK-MDR1 BBB model at different concentrations (one of them, the one selected as effective to kill glioma cells) to evaluate if it is or it is not substrate of the Pgp transporter.

2) If the new molecule is substrate of Pgp, then the access in glioblastoma illness, should be evaluated combining MDCK-MDR1 and U87-MG cells. If the molecule, does not bind the Pgp, the permeability should be tested with MDCK and U87-MG cell lines.

It is not clear what difference in usefulness the authors found between the two models (MDCK and MDCK-MDR1).This is related to the authors' response to an initial review, but please include it in the body of the text.

When using molecules that access to the CNS by passive routes there is not any difference in usefulness between the MDCK and the MDCK-MDR1 as the only difference between them is tat the second cell lie has an extra expression of the efflux transporter Pgp. This has been specified when talking about the fluorescein experiments.

Fluorescein uses a passive access route to cross the BBB and, that’s why, no statistically significant differences can be observed when comparing the results from the MDCK and the MDCK-MDR1 cells, whose only variance is the extra Pgp transporter in the second ones.

When the drugs tested are substrate of the Pgp transporter, then, using the MDCK-MDR1 cell line is more useful. Although, in this experiment, differences are not observed as it has been explained in the text.

For letrozole and gemcitabine, drugs which are exclusively substrate of the Pgp efflux transporter (table 2), it would be expected to have a lower access to the basolateral chamber when using the healthy MDCK-MDR1 cell line, due to its extra level of Pgp, but this lower access is not observed (figure 3). This fact can be explained by the concentrations used to carry out the tests. As said before, the concentration selected for the in vitro experiments was the equivalent to the maximum plasma concentration reported in rats and, at that concentration, the transporter may be saturated and no differences are observed between the transfected and the non-transfected cell lines.

Reviewer 2 Report (New Reviewer)

The experimental design uses an in vitro model based on coculture of epithelial cells and only tumoral cells, without a control of normal astrocytes in coculture, and the drugs used in the experiments are not specific for the treatment of glioblastoma. 

 In the conclusions, the authors should make a clear description of their suggested BBB model consistent with appropriate drug delivery for brain tumor treatment. 

 

The authors should clarify these aspects.

Author Response

The experimental design uses an in vitro model based on coculture of epithelial cells and only tumoral cells, without a control of normal astrocytes in coculture, and the drugs used in the experiments are not specific for the treatment of glioblastoma. 

Thanks for your comments. We will try to answer them.

On the one hand, normal astrocytes were not tested in coculture as a control, because the three monolayers healthy BBB models, have been previously evaluated to check the access of substances to the CNS with no need of extra cells to simulate the BBB.

On the other hand, just the drugs for which we had in vivo information in healthy animals and animals with glioblastoma were tested as said in the text:

Four compounds were used for carrying out this study as, when looking at the bibliography, they were the only ones whose biodistribution had been tested both in healthy animals and in animals with glioblastoma.

We would have preferred to use drugs for treating glioblastoma in our study, but no biodistribution studies in healthy animals were available in bibliography.

We say in the conclusion section that more studies with more drugs are needed to improve the robustness of our observations, but, at least, we now have the first step to contribute to the development of drugs for the treatment of glioblastoma reducing the number of animals used in research.

In the conclusions, the authors should make a clear description of their suggested BBB model consistent with appropriate drug delivery for brain tumor treatment.

We have included a small protocol with our recommendations in the conclusions section:

With the available knowledge, the protocol that these authors recommend to follow when trying to develop a new tool to treat glioblastoma would be:

1) Checking the access of the molecule in the MDCK-MDR1 BBB model at different concentrations (one of them, the one selected as effective to kill glioma cells) to evaluate if it is or it is not substrate of the Pgp transporter.

2) If the new molecule is substrate of Pgp, then the access in glioblastoma illness, should be evaluated combining MDCK-MDR1 and U87-MG cells. If the molecule, does not bind the Pgp, the permeability should be tested with MDCK and U87-MG cell lines.

Reviewer 3 Report (New Reviewer)

In this work the authors aim to the creation and validation of a new in vitro model capable of predicting the in vivo permeability across blood-brain barrier in the presence of glioblastoma testing several drugs

In my opinion, the paper is quite interesting for the scientific community working in this field and I suggest the publication.

Anyway, here are some minor revision:

-         In line 14, pag 1, please check the sentence “The in vitro method selected was a cell co-culture model of epithelial cell lines ……”.

-         In line 45, pag 2, please replace “;” with “,”.

Author Response

In this work the authors aim to the creation and validation of a new in vitro model capable of predicting the in vivo permeability across blood-brain barrier in the presence of glioblastoma testing several drugs

In my opinion, the paper is quite interesting for the scientific community working in this field and I suggest the publication.

Anyway, here are some minor revision:

-         In line 14, pag 1, please check the sentence “The in vitro method selected was a cell co-culture model of epithelial cell lines ……”.

-         In line 45, pag 2, please replace “;” with “,”

Thank you very much for your comments. We have corrected both sentences.

- Line 14, pag 1: The selected in vitro method was a cell co-culture model of epithelial cell lines (MDCK and MDCK-MDR1) with a glioblastoma cell line (U87-MG).

- Line 45, pag 2: In light of the origin of the cells (primary or immortalised cell lines), its source (rodent, porcine, bovine, and human) and the transporters-expressed, numerous cellular models can be proposed, facilitating the creation of detailed tools for assessing the BBB physiology, pathophysiology and streamlining the drug development process [2,3].

Round 2

Reviewer 1 Report (Previous Reviewer 2)

I am pleased that the authors submitted the revised manuscript in response to my points of view.

The authors have clarified the message that this manuscript provides to readers and the problems that may arise when conducting research based on this manuscript.

I hope that many researchers will use these methods reported in this manuscript to investigate promising new therapeutic strategies for glioma.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The present article title " Establishment and validation of a new co-culture for the evaluation of the permeability through the Blood−brain barrier in patients with glioblastoma " aims to propose an improved method for mimicking BBB in vitro to test drug permeability. Authors made the conclusion that  MDCK and MDCK-MDR1 culture obtain a great predictability for the in vivo situation. The ideas and results are interesting, but I have serious concerns about the writing style and the experimental design. I suggest the author resubmit after correction. 

 

1. The writing needs intensity English editing. Several one sentence paragraphs in the introduction, and in a discontinuous order. Several sentences are too long and not clear, such as line 54-55,  61-63, 87-89 is very difficult to read, 

 

2. The result is writing like figure legend, which is not appreciated in formal scientific writing style. The “Result part” should descript experiment rationale, data comparison, interpretation, but not let the readers look into figures and find out by themself. 

 

3. The in vivo and in vitro correlation is done by others research data, and only four points (four compounds) were used. This is not a good research design, which should be done parallel and more compounds and different concentrations should be used, or it should not make the conclusion that  MDCK and MDCK-MDR1 culture obtain a great predictability for the in vivo situation.

 

Author Response

REVIEWER 1

The present article title " Establishment and validation of a new co-culture for the evaluation of the permeability through the Blood−brain barrier in patients with glioblastoma " aims to propose an improved method for mimicking BBB in vitro to test drug permeability. Authors made the conclusion that MDCK and MDCK-MDR1 culture obtain a great predictability for the in vivo situation. The ideas and results are interesting, but I have serious concerns about the writing style and the experimental design. I suggest the author resubmit after correction.

Thank you very much for your kind suggestions. We have tried to explain better the experimental design to be clear for other researchers.

The experimental design has been modified:

In 2015, Mendes et al. developed a new in vitro model with the hCMEC/D3 and the U87-MG cell lines which was able to simulate the disruption of the BBB which is known that glioblastoma provokes in vivo.

In 2013, our group developed Innovative in vitro method to predict rate and extent of drug delivery to the brain across the blood-brain barrier using MDCK and MDCK-MDR1 cells lines PMID:23977999

In 2021, Sanchez-Dengra et all establish the correlation between hCMEC/D3, MDCK and MDCK-MRD1 to simulate BBB PMID:33838261

In this work a correlation capable of predicting the in vivo permeability behaviour of different active compounds in patients with glioblastoma using an in vitro method (culture cells). This group is always working according to 3Rs principles, it would be better to have an "animal-free" alternative technique. For this purpose, the experimental design was: 

MDCK, MDCK-MDR1 and hCMEC/D3 cells lines has been chosen to add U87-MG cell line and creating a new model capable of predicting the in vivo permeability behaviour of different active compounds in patients with glioblastoma. All cells were defrosted and maintained in 75 cm² flasks at 37.5 ºC, with a 90% humidity and 5% CO₂, changing the culture media every other day and sub culturing them two times a week with a cell density of 6.5·10⁵ cells/cm2, for the MDCK, MDCK-MDR1 and hCMEC/D3 cells lines, and 7,5·10⁵ cells/cm², for the U87-MG cell line. This maintenance was done during 2 months.

The media used for each cell line is different, according to characteristic of the cells. The MDCK were preserved with DMEM, MDCK-MDR1 cell lines and the glioblastoma tumor line U87-MG were grown with DMEM+pyruvate media. Both medias enriched with 1% glutamine, 1% non-essential amino acids, 1%HEPES to maintain the pH of the media, 1%penicillin-streptomycin to avoid the contamination and 10% Fetal Bovine Serum (FBS) to growing of the cells. The hCMEC/D3 cell line was maintained in EBM-2 medium enriched with the same products (FBS, penicillin-streptomycin, HEPES) and CD-LP, hydrocortisone solution and ascorbic acid solution, both filtered with a 0.2µm filter, in addition, each flask of hCMEC/D3, bFGF was added at a concentration of 1 ng/mL.

Permeability experiments are carried out on cell monolayers grown on a polycarbonate membrane, 0.4 µm pore size, and with a surface area of 4.2 cm2. For this study, the MDCK, MDCK-MDR1 and hCMEC/D3 cells were seeded (6.5*10⁵ cells/cm² in the apical side of 6-well transwells plates and they were let to grow until confluence. The confluence was reached in more or less 6-8 days. When the glioblastoma illness wanted to be simulated, the second day after seeding the MDCK, MDCK-MDR1 and hCMEC/D3 cells, the U87-MG cell line was added to the basolateral compartment as previously done by Mendes et all [18]. After a week, the confluence of the monolayers was checked by means of measuring the TEER values of each well. If the resistance is good the experiments were carried out under agitation and at 37 ºC, adding the drug solution in the apical chamber and taking samples from the basolateral chamber after 15, 30, 60, 90 minutes. With the aim of reproducing the in vivo conditions, the concentrations of the drugs tested were equivalent to the maximum plasma concentration reported in rats, that is, 50 μM for ganciclovir (46.9 μM - [19]), 5 μM for letrozole (3.5 μM - [20]), 50 μM for methotrexate (44 μM - [21]) and 75 μM for gemcitabine (76 μM - [22]). After the last sampling time, cells were washed with HBSS, the TEER value was measured again to test the membrane were not broken during the experiment due to the drug or the experimental process. After that, the monolayers were disrupted with methanol to recover the drug from inside the cells. Finally, samples were centrifuged and analyze by HPLC.

 

1. The writing needs intensity English editing. Several one sentence paragraphs in the introduction, and in a discontinuous order. Several sentences are too long and not clear, such as line 54-55,  61-63, 87-89 is very difficult to read, 

English has been reviewed and several sentences have been modified.

Later on, researchers developed immortalised cell-lines, such as the hCMEC/D3 [6,7] and the Madin-Darby Canine Kidney Cell line (MDCK). The hCMEC/D3 cell line has a human origin and expresses most of the ABCB, ABCC and ABCG transporters. The MDCK cell line has a non-cerebral origin, but it is widely used for their robustness (high TEER values) and their capability of transfectation with specific gene transporters, such as the MDR1 gene which expresses the P-gp/ABCB1 transporter. As for today, the models proposed with MDCK cells surpass those with cerebral cells in the in vitro – in vivo correlation of passive compounds, with R2 = 0.64 [8] or 0.72 [9] de-pending on the works consulted. Nevertheless, when evaluating active-transported com-pounds [8] a non-significant correlation (R2 = 0.40) is obtained. So, the histological differ-ences between MDCK and cerebral lines cannot be overlooked.

In vitro studies have shown the impact of the glioblastoma in healthy, intact blood-brain barrier models. Mendes et al. [18] proposes a novel in vitro coculture cell BBB-pathological model using U87 cells (glioma cell line) and immortalised human brain capillary endothelial cells (hCMEC/D3 cell line). In their work, Mendes et al., carried out numerous measurements so as to assess the glioma impact in the barrier. Among them, the TEER measurements and the permeability assays, which employed fluorescein isothiocy-anate (FD) of different molecular weights, were the most useful to compare the alterations induced by the glioma cells in the BBB. Mendes et al. noted that, in the presence of the glioma cells, the coculture expressed lower TEER values and an increased paracellular permeability. Cell morphology alterations were also detected.

When the same study is repeated but combining the MDCK and the MDCK-MDR1 cells with the U87-MG cell line, then, it is observed that there is a clear increment in the permeation for the fluorescein with the worst access to the CNS when the BBB is intact (the 70 kDa one) (figure 2).

 

2. The result is writing like figure legend, which is not appreciated in formal scientific writing style. The “Result part” should descript experiment rationale, data comparison, interpretation, but not let the readers look into figures and find out by themself. 

We have added several paragraphs to the results section to improve the discussion.

It can be observed that there is a clear increase in the permeation of all drugs when the U87-MG cells are added to the MDCK and the MDCK-MDR1 models, with the excep-tion of letrozole. Nonetheless, when the hCMEC/D3 experiments are checked, it can be seen that there is not such a clear increment for any of drugs. Moreover, the “healthy” permeability value is always greater when using the hCMEC/D3 monolayer than with the MDCK and the MDCK-MDR1 cell lines.

The lower tightness in the hCMEC/D3 cells makes the drugs to cross more easily the BBB and, thus, there is not a relevant increment when adding the glioblastoma cells to the culture. In the other cells, the extra access obtained due to the lack of robustness provoked by the glioblastoma cells can be clearly observed, because drugs move from being not ac-cessible to enter without difficulties to the CNS.

Table 3 shows the ratios obtained after dividing the apparent permeability of the BBB+glioblastoma model over the healthy permeability value. The calculation of the ratios shows in a simplest way the increment in permeability observed after adding glioblasto-ma cells to the basolateral chamber of a BBB model. These increases can also be observed in figure 3, comparing the blue (monolayer) and the yellow (coculture) lines.

Having such type of high‐throughput screening tool would save both money and time to pharmaceutical industries when developing new drugs to the treatment of brain cancer. It is because only the drugs with a good access to the CNS in in vitro illness condi-tions would move to more expensive in vivo studies.

From an ethical point of view, the co-culture with glioblastoma cells will also have advantages. For instance, the use of this in vitro model would contribute to the accom-plishment of the Rs principles of the use of animals in research (reduction, refinement and replacement). Moreover, less human studies would be needed as the number of failures would be lower.

Figure 4 shows a comparison of the initial TEER values for the three cell lines tested with and without the glioblastoma co-culture. In all cases, the initial TEER value is signif-icantly lower when the U87-MG cells are used. So, the glioblastoma cells are able to alter the tightness of the BBB.

The correlations obtained after representing together the in vitro and the in vivo ratios are represented in figure 5. From these data, it can be extracted that the simplest cell lines (MDCK and MDCK-MDR1) co-cultured with U87-MG cells reflect in a more reliable way what happens in vivo than the co-culture with the most complex one (hCMEC/D3). The coefficient of determination (r2) for the correlation with the hCMEC/D3 cells is 0.271, while this coefficient for the other cell lines is greater than 0.800.

 

3. The in vivo and in vitro correlation is done by others research data, and only four points (four compounds) were used. This is not a good research design, which should be done parallel and more compounds and different concentrations should be used, or it should not make the conclusion that  MDCK and MDCK-MDR1 culture obtain a great predictability for the in vivo situation.

Thanks for your comment. I totally agree but in this study we need reported in vivo data in healthy rats and in rats with glioblastoma with different compounds. So, only 4 compounds have been reported.

We clarified in the manuscript:

Four compounds were used for carrying out this study as, when looking at the bibliography, they were the only ones whose biodistribution had been tested both in healthy animals and in animals with glioblastoma.

We have clarified our conclusions as well:

The objective of this work was the establish the correlation between in vivo behaviour and a cell co-culture model of epithelial cell lines (MDCK and MDCK-MDR1) with a glioblastoma cell line (U87-MG) able to predict the in vivo behaviour of compounds. The differences in the TEER values between the monolayer and co-culture models have ratified the damage to the in-tegrity of the cell barrier due to damage to the tumor line. It has been seen that use of the U87-MG cell line with the MDCK and MDCK-MDR1 cells gives higher permeabilities than monolayers models. In addition, the MDCK and MDCK-MDR1 cell lines are valid for pre-dicting the access of drugs to the CNS in the presence of glioblastoma, and have a higher predictive value than the co-culture model with the hCMEC/D3 cell line proposed by other authors. However, to improve the robustness of these conclusions more studies should be done, in vitro and in vivo, to increase the number of compounds included in the correlations.

Author Response File: Author Response.docx

Reviewer 2 Report

I read with great interest the manuscript entitled "Establishment and validation of a new co-culture for the evaluation of the permeability through the Blood-brain barrier in patients with glioblastoma.  As the authors explain, drugs that are expected to have an effect on the central nervous system have a blood-brain barrier (BBB) problem, and there is a great deal of work involved in the drug development process.  There are various limitations to in vitro studies conducted during the drug screening phase of drug discovery research and the formulation of new protocols, and research continues on methods to accurately characterize the BBB in vitro.

The authors identify various questions about in vitro BBB methods reported previously and compare them to methods capable of resolving these questions.  The authors' main focus is to evaluate the passage of BBB through brain tumors.  The authors examine how the BBB is a drug transport system in the presence of tumor cells. Although the BBB is completely disrupted in the central part of primary brain tumors, there is a functioning barrier system, also known as the blood-tumor barrier (BTB), in the surrounding area.  To simulate this, the authors investigated drug transport in co-culture with the glioma cell line U87.

Major comments.

The difference between MDCK and MDCK-MDR1 is thought to be the degree of the ABCB1 activation, which would be expected to allow easier passage of drugs delivered through it. However, in this study the differences between MDCK and MDCK-MDR1 are not clear for both Letrozole and Gemcitabin.  Please discuss about this.

The difference between MDCK and MDCK-MDR1 and hCMEC is the completeness of the simulated BBB, which is based on letrozole, which does not pass, and methotrexate, which does.  Both Ganciclovir and Gemcitabin, which are difficult to pass in hCMEC, have similarly improved passage.  What are the authors' thoughts on the transport mechanism involved in this?

Please specify for what purpose co-culture with glioma cell lines is highly useful, as there is insufficient discussion to reach the conclusion the authors were aiming for.

Author Response

REVIEWER 2

I read with great interest the manuscript entitled "Establishment and validation of a new co-culture for the evaluation of the permeability through the Blood-brain barrier in patients with glioblastoma.  As the authors explain, drugs that are expected to have an effect on the central nervous system have a blood-brain barrier (BBB) problem, and there is a great deal of work involved in the drug development process.  There are various limitations to in vitro studies conducted during the drug screening phase of drug discovery research and the formulation of new protocols, and research continues on methods to accurately characterize the BBB in vitro.

The authors identify various questions about in vitro BBB methods reported previously and compare them to methods capable of resolving these questions.  The authors' main focus is to evaluate the passage of BBB through brain tumors.  The authors examine how the BBB is a drug transport system in the presence of tumor cells. Although the BBB is completely disrupted in the central part of primary brain tumors, there is a functioning barrier system, also known as the blood-tumor barrier (BTB), in the surrounding area.  To simulate this, the authors investigated drug transport in co-culture with the glioma cell line U87.

Major comments.

Thank you for your fantastic summary and we have tried to answer to your comments and improve the paper.

 

The difference between MDCK and MDCK-MDR1 is thought to be the degree of the ABCB1 activation, which would be expected to allow easier passage of drugs delivered through it. However, in this study the differences between MDCK and MDCK-MDR1 are not clear for both Letrozole and Gemcitabin.  Please discuss about this.

ABCB1 (P-gp1) is an efflux transporter. So, the extra expression of ABCB1 in the MDCK-MDR1 cell line should hinder the passage through the BBB of those drugs which are substrate of it (in this case, letrozole, methotrexate and gemcitabine). Nonetheless, in this study, the differences in permeability between the MDCK and MDCK-MDR1 cell lines were not significant, fact that can be explained by a couple of reasons:

1. A) In the healthy BBB model (the monoculture), the absence of difference may be due to the use of just a drug concentration, the maximum detected in plasma in the in vivo studies. Depending on the affinity of the drug for the P-gp1 a different concentration will saturate the transporter, making it not useful anymore. This is what it may have happened in this study.
2. B) In the non-healthy BBB model (coculture), as the BBB is disrupted, TEER values are significantly lower, the action of the efflux transporter may be non-important, as the drug crosses the barrier faster than the returning speed to the bloodstream.

 

The difference between MDCK and MDCK-MDR1 and hCMEC is the completeness of the simulated BBB, which is based on letrozole, which does not pass, and methotrexate, which does.  Both Ganciclovir and Gemcitabin, which are difficult to pass in hCMEC, have similarly improved passage.  What are the authors' thoughts on the transport mechanism involved in this?

In hCMEC/D3 monolayers, the permeability values of ganciclovir, methotrexate and gemcitabine are greater than the permeability values obtained in the other monolayers, as it can be observed in the following table (Peff x 10⁶, cm/s):

	MDCK	MDCK-MDR1	hCMEC/D3
Ganciclovir	80.71	104.05	221.93
Methotrexate	44.73	35.63	95.37
Gemcitabin	55.85	89.25	179.38

So, we consider that there is not increase in the passage of the drugs when tested in the hCMEC/D3 with glioblastoma, because they can already cross the BBB when it is healthy.

 

Please specify for what purpose co-culture with glioma cell lines is highly useful, as there is insufficient discussion to reach the conclusion the authors were aiming for.

Thanks for your comment. We have improved the discussion about this aspect.

Knowing that the U87-MG cell line is able to alter the behaviour of different BBB monolayers is interesting, but is not really useful, unless this alteration can be correlated with what happens in vivo. Only in that case, the in vitro studies could be used as a high‐throughput screening tool in the development of treatments against glioblastoma.

Having such type of high‐throughput screening tool would save both money and time to pharmaceutical industries when developing new drugs to the treatment of brain cancer. It is because only the drugs with a good access to the CNS in in vitro illness conditions would move to more expensive in vivo studies.

From an ethical point of view, the co-culture with glioblastoma cells will also have advantages. For instance, the use of this in vitro model would contribute to the accomplishment of the Rs principles of the use of animals in research (reduction, refinement and replacement). Moreover, less human studies would be needed as the number of failures would be lower.

 

Author Response File: Author Response.docx