Harm for Good: Lamprey's immune system in the fight against human brain cancer

Our brain is our everything. Violation of the work of this important organ leads to terrible, and sometimes fatal consequences. The complexity of the brain and its neural organization is colossal, which greatly complicates the process of treating a particular disease. As a rule, when we treat something, we try to get rid of the defects that the disease causes. But, what if these defects are used to combat what creates them? This is exactly what the authors of the study we are considering today decided to do. How did scientists apply the disruption of the blood-brain barrier, why do we need access to the extracellular matrix of the brain, and what role did lamprey parasitic on fish play in this? The report of the research group will tell us about this. Go.

Bit of theory

First of all, it is worth sorting out the characters in this laboratory play.


Blood-brain barrier

One of the main roles is played by the blood-brain barrier (BBB) ​​- a physiological barrier between the central nervous system (CNS) and the circulatory system. This barrier prevents the contact of nerve tissues with various components of the circulating blood, among which there may be toxins, microorganisms, cellular / humoral factors of the immune system that can respond to brain cells as foreign. The BBB can be compared to a bouncer in a very expensive club, letting in the central nervous system exclusively nutrients. But this bouncer is not very picky, he often does not miss the medications needed to treat the central nervous system. It turns out that a system aimed at the benefit of our health can be an obstacle to its treatment. Here is the irony in physiology.

However, the blood-brain barrier does not always work like a Swiss watch. In cases of stroke, tumors, various head injuries, chronic diseases, the BBB begins to fail, that is, to let in the central nervous system what it would have previously eliminated. In addition to the natural causes of the failure, there is also anthropogenic: focused high-intensity ultrasound and osmotic agents that disrupt the BBB. Why break something that ensures the normal functioning of the brain, you ask. Then, to deliver drugs that a full-fledged BBB will filter out. Anyway, the bouncer will not let the doctor into the club for a visitor who has fainted, because he does not have a club card.

The main and common for all cases, the result of disruption of the BBB is a pathological exposure of the extracellular matrix (ECM) of the brain, which is isolated under normal conditions.

The extracellular matrix is ​​the basis of connective tissue that provides mechanical support for cells and the transport of chemicals.

Therefore, scientists believe that by targeting certain parts of the brain with a pathologically disturbed BBB, it is possible to deliver drugs to those parts of the damaged central nervous system that were previously inaccessible precisely because of the BBB.

Thus, it is possible to create a ligand aimed at ECM, which will be effective in combating various diseases of the central nervous system, and not with one specific one, as previously developed methods.

To test the theory in practice, scientists decided to apply their method of drug delivery to incurable glioblastoma (brain cancer). This type of disease is quite rare, but it is extremely difficult to defeat. Even after chemotherapy, radiation therapy and surgery, survival is about 1-2 years.

Recent studies have shown that the use of immunotherapy through interleukin-13 * -based chimeric antigen receptors * is a promising treatment for glioblastoma.

Interleukins * are peptide information molecules produced by leukocytes, to a lesser extent by phagocytes and other tissues. Interleukins are part of the immune system.

Interleukin-13 * (IL13) is the main mediator of physiological changes caused by allergic inflammation in many tissues.

Chimeric antigen receptor * is a recombinant fusion protein that connects an antibody fragment that can selectively bind to specific antigens and signaling domains that activate T cells.

The use of chimeric antigenic receptors targeting the CSPG4 protein can also be a rather effective method of combating glioblastoma.

In addition, MRI analysis showed a violation of the blood-brain barrier inside glioblastoma. Therefore, treatments related to the extracellular matrix must be effective.

And here the immense imagination and creativity of scientists enters the scene. The fact is that you can use standard peptides and antibodies as ECM-targeted reagents, but this is not so much fun. Therefore, scientists decided to use variable lymphocyte receptors (VLR), that is, lampreys antigen receptors.


The lamprey class has about 40 species, most of which are parasites that feed on the blood of fish, to which they suck.

VLRs are sickle-rich, leucine-rich proteins that recognize antigenic targets with specificity and affinity * comparable to immunoglobulin-based antibodies.

Affinity * is the thermodynamic characteristic of the strength of the interaction of substances such as antigen and antibody.

Why precisely lampreys and their VLR? The fact is that between mammals and lampreys there is an evolutionary abyss of 500 million years. Thus, lamprey VLRs are more likely to recognize conserved proteins and glycans in ECM than mammalian antibodies.

To identify the VLRs that bind the brain ECM, scientists performed panning, a method for selecting specific bio-elements (proteins, petites, etc.) from VLR libraries of biomolecules. The result was a pool of ECM-binding clones * .

Clone * - a group of identical cells that share a common ancestor (primary source), that is, come from the same cell.

The resulting clone demonstrated preferential accumulation both in destroyed areas of the blood-brain barrier in animals with an osmotic disorder of the BBB or with glioblastoma. In addition, the clone was well aimed at liposomes * loaded with doxorubicin (an antibiotic).

Liposomes * - spherical intracellular organelles used to deliver drugs to certain tissues.

Study preparation

As we already learned earlier, ECM-binding VLRs were identified by panning VLR libraries. The library itself was obtained from a collection of lamprey VLRs immunized with mechanically isolated preparations of the plasma membrane of the microvasculature of the mouse brain, which contained the associated ECM of the brain.

The library was first enriched with ECM binders using two panning cycles on a decellularized * ECM generated by cultured mouse endothelial cells (bEnd.3 cell line).

Decellularization * is a method of purifying allografts from the cellular component to obtain a non-immunogenic, effective and safe construct based on a natural extracellular matrix.

Further, it was necessary to identify precisely those ECM-binding clones that predominantly bend bEnd.3 ECM, and not with the control group of mouse fibroblast ECMs (cell line 3T3).

Next, individual clones were placed in 96-well plates, after which they were expanded and induced to display VLR. After removing extra clones (so that there was no subcloning), scientists were able to conduct a comparative assessment of the binding of VLR to bEnd.3 and 3T3 ECM using ELISA screening ( 1a ).


Image No. 1 A

total of 285 clones were analyzed. As a result, it can be seen that the communication signals with bEnd.3 ECM are approximately 5 times stronger than the communication signals with 3T3 ECM ( 1b ).

Next, the ELISA results were checked by comparing images of bright field microscopy of clones associated with bEnd.3 and 3T3 ECM ( 1C ).

As can be seen in image 1c , clones P1C10 and P2C7 bind exclusively to bEnd.3 ECM, and the non-binding clone P1E9 practically does not show any connection with any type of ECM.

Next, scientists conducted a comparative analysis of a more practical method - on a section of the mouse brain. Eight of the 10 VLR clones that showed the best binding result in previous observations also showed binding ( 1d ) in this assay .

All binding clones showed a diffuse parenchymal EMC scheme without any additional enrichment (vascular or cellular).

Scientists identified 2 leaders according to the results of all the above observations - P1C10 and P3A8. It is these clones that will be considered in the future.

Research results

P1C10 and P3A8 were functionalized with Cy5 fluorescent dye. Direct immunostaining of murine tissues using VLR-Cy5 conjugates showed that P1C10-Cy5 has significant selectivity towards the ECM of the brain compared with tissues of the kidneys, heart, and liver ( 2a ).


Image No. 2a

Immunostaining * - a process that allows you to identify and localize an antigen in a specific area of ​​a cell, tissue or organ.

But P3A8-Cy5 binds to the brain and liver ECMs with the same intensity, but like P1C10-Cy5 does not show interest in kidney and heart ECMs.

Next, scientists tested the cross-reactivity of P1C10 and the ECM of the human brain (cryosections were used). The binding of P1C10-Cy5 to the ECM of the human brain resembles a picture of the same process, but involving the mouse brain ( 2b ). P1C10-Cy5 also successfully bound to ECM in cryosections of a sample of human glioblastoma ( 2c ).


Image No. 2b-e

Given these observations, scientists measured the affinity * P1C10.

Affinity * - the ability of a cell to capture and bind certain chemicals.

As a result, the dissociation constant (Kd) for binding to bEnd.3 ECM was 48.38 ± 6.05 nM ( 2d ).

Following this, scientists decided to check whether P1C10 will accumulate in places of destruction of the BBB in the brain of the mouse.

Modified by dye near infrared IR800 clones P1C10 or RBC36 were introduced into healthy laboratory mice in a volume of 1 mg / kg RBC36 is a VLR that recognizes the trisaccharide of the human H antigen, which is why it was used as an isotype control.

Next, mice were injected intravenously with mannitol (hexahydric alcohol) to temporarily open the BBB. After that, brain images were taken of mice to identify IR800 signals (image below).


Image No. 3

A comparative analysis showed that the accumulation of fluorescence in the brain (concentration of the test substance marked with IR800 dye) when using P1C10-IR800 is 3.3 times higher than with RBC36-IR800, and 7.6 times higher than when using saline. Consequently, P1C10 selectively accumulates in the brain after a malfunction of the blood-brain barrier.

Next, the scientists decided to check whether the VLR would target the naked extracellular matrix. For this, two models were created using murine GL261 and human glioblastoma U87 cells, which were introduced into the brain of experimental mice. As a result, tumors were formed with a chaotic vasculature and point disorders of the blood-brain barrier.

P1C10 or RBC36 in a volume of 1 mg / kg was administered intravenously to mice with embedded GB261 glioblastoma. After 30 minutes, brain samples were taken to analyze and visualize the IR800 signal (dye for P1C10 or RBC36).


Image No. 4

The average fluorescence intensity in the tumor region of GL261 in mice injected with P1C10-IR800 was 112 times higher than in the contralateral (opposite) region of the brain ( 4a ). But when using RBC36-IR800, the fluorescence intensity of the tumor region was only 9 times higher than the intensity in the opposite region.

In addition, it was found that the accumulation of P1C10-IR800 in the tumor itself is 13 times higher than the accumulation of RBC36-IR800.

These observations confirmed the ability of P1C10 to selectively target ECM in mouse tumors. Now it was necessary to test this talent on human brain tumors.

VLR was administered to mice with U87 (human brain tumor). Scientists tested not only P1C10, but also 192, which showed selective binding to the basolateral side of the vasculature of the brain in addition to the vessels of the kidneys and liver, as well as ECM.

P1C10, 192 or RBC36 in a volume of 3 mg / kg was administered intravenously and allowed to circulate freely for 30 minutes. After that, samples of organs of interest were taken to visualize the results.

Both VLR clones (P1C10 and 192) showed accumulation at the tumor borders, with P1C10 being distributed throughout the tumor ECM ( 4b) But 192 for the most part was concentrated outside the large tumor vessels.

None of the VLR clones accumulated in the healthy contralateral hemisphere of the mouse brain. And RBC36 was completely absent both in the healthy and in the tumor-containing parts of the brain.

Quantitative analysis showed that P1C10 accumulates in the U87 tumor 21.2 times more than in the contralateral region of the brain, 21.2 times the kidneys, 15.9 times the liver and 29.6 times the heart.

The accumulation of P1C10 and 192 in the tumor regions was 25.4 and 11.9 times greater than the accumulation of RBC36. At the same time, both VLR clones mainly accumulated in the areas of vascular defects of the tumor.

These observations confirm the effectiveness of P1C10 and its selective focus on brain ECM. It remains to be seen whether P1C10 can efficiently deliver drugs to where they need to.

Scientists have used VLR in combination with doxorubicin-loaded liposomes, which is highly visualized due to their own fluorescence, which greatly simplifies the process of analyzing the effectiveness of VLR application. Liposomes were obtained with an average diameter of 94.2 nm and a doxorubicin content of 1 to 2 mg / ml. Next, VLR clones were attached to the liposomes, which retained their binding activity after combining (image No. 5).


Image No. 5

To demonstrate the feasibility of treatment with doxorubicin-loaded liposomes targeting VLRs, tumor U87 cells (cultured on bEnd.3 ECM) were incubated with doxorubicin-loaded liposomes targeting P1C10 or RBC36.

Observations showed significant growth in the destroyed cells by liposomes targeting P1C10. The half-maximum effective concentration was 199.0 ± 1.7 nM for P1C10 and 3312.0 ± - 2.6 for RBC36.


Image # 6

And finally, scientists tested the potential therapeutic utility of targeting a pathologically damaged extracellular matrix of the brain. For this, doxorubicin-loaded liposomes in combination with P1C10, 192 or RBC36 were used in experimental mice with U87 cancer cells.

As seen in pictures 6aand 6b , the signal from doxorubicin is significantly stronger when using P1C10. It can be seen that this signal is sufficiently localized within the tumor and does not affect the contralateral (healthy) region of the brain.

Pictures 6c and 6d demonstrate that the accumulation of P1C10 in the tumor region is 7.6 times higher than in the healthy region. If we compare the degree of accumulation of P1C10 with RBC36, then the difference is 7.9 times in favor of P1C10.

For 4 weeks (on days 7, 14, 21, and 28), 12 mg / kg of doxorubicin was administered to experimental mice with U87 using P1C10, 192, or RBC36.

After the introduction of the tumor, the median survival was: 43 days for P1C10, 30 days for 192, and 28 days for RBC36.

A group of subjects with P1C10 showed a significant increase in survival compared to the other two groups. At the same time, indicators 192 and RBC36 do not differ significantly from indicators of control groups that did not receive treatment at all.

For a more detailed acquaintance with the nuances of the study, I recommend that you look into the report of scientists .

Epilogue

There would be no happiness, but misfortune helped. With this phrase you can quite accurately describe this study. Scientists used pathological disorders of the blood-brain barrier as a tool for delivering drugs to certain areas of the brain affected by a tumor, while avoiding healthy areas. And the scientists received help in this complex and noble endeavor, as they say, from where they did not wait. Variable receptor lymphocytes (VLR) lampreys became the basis of this work. Now it is safe to say that even parasites can be useful.

The treatment of brain tumors is fraught with a huge list of difficulties, and the success of this process is not as great as we would like. The creation of new methods and means to combat such serious diseases is really what science exists for. Knowing the world around and inside ourselves, we do not go against the will of nature, we only begin to understand it better, find new knowledge and apply it for good.


Thank you for staying with us. Do you like our articles? Want to see more interesting materials? Support us by placing an order or recommending it to your friends, a 30% discount for Habr users on a unique analogue of entry-level servers that we invented for you: The whole truth about VPS (KVM) E5-2650 v4 (6 Cores) 10GB DDR4 240GB SSD 1Gbps from $ 20 or how to divide the server? (options are available with RAID1 and RAID10, up to 24 cores and up to 40GB DDR4).

VPS (KVM) E5-2650 v4 (6 Cores) 10GB DDR4 240GB SSD 1Gbps until the summer for free when paying for a period of six months, you can order here .

Dell R730xd 2 times cheaper? Only we have 2 x Intel TetraDeca-Core Xeon 2x E5-2697v3 2.6GHz 14C 64GB DDR4 4x960GB SSD 1Gbps 100 TV from $ 199in the Netherlands! Dell R420 - 2x E5-2430 2.2Ghz 6C 128GB DDR3 2x960GB SSD 1Gbps 100TB - from $ 99! Read about How to Build Infrastructure Bldg. class using Dell R730xd E5-2650 v4 servers costing 9,000 euros for a penny?