The ultimate goal of the B2B device is to follow the metastasis that travels from the cancer chamber to the bone. Metastatic cells substantially differ from any other cell in the ossicle; for example, they express specific markers on the surface which allow easy identification. However, the method used to detect cancer cells within the ossicle might impair the fate of the sample.
“Some methods are very invasive and destructive for the tissue, so we perform them only at the end of the experiment” points out Dr. Farrell, “For example, we can precisely localize the metastasis by dissecting the pellet and looking at the slices with the microscope. Another harsh method implies to crush the pellet, generate a cell suspension and quantify the cancer cells by means of flow cytometry. But in both cases, the ossicle is not recoverable and thus the experiment comes to an end.”
A less invasive option is to visualize the cells directly within the tissue, without the need to destroy the sample. With this purpose in mind, the breast cancer cells used in B2B have been modified to express a luciferase, a bioluminescent protein (see link to Aceto news). As such the cancer cells can be seen with light-sensitive apparatus even when buried inside the tissue. Light penetration might be an issue in the most hidden and internal part of the sample, but if the number of emitting cells is high enough, the signal can be detected. In B2B, we will employ all these methods, as each of them helps to answer different questions. The destruction of the ossicle remains a bottleneck for now, as the generation of a new one is not trivial. That’s why an in vitro alternative is much needed.
In B2B two approaches have been planned in order to generate the ossicle for the bone chamber. The first, and more standardized, consists of the generation of the ossicle in vivo (in murine models) and only later to transfer it into the dedicated chamber. This approach starts with the insertion of patient-derived chondrocytes, the cell forming the cartilage, in mice with a weakened immune system, to reduce the chances of rejection. There, the human cartilage is slowly converted into an ossicle thanks to the action of the animal blood flow: nutrients and bone-forming cells are brought in and they trigger the remodeling of the cellular mass into an ossicle – including the cavity to accommodate the bone marrow.
Besides this reliable and relatively standard approach, the team of Eric Farrell, Associate Professor at the Department of Oral and Maxillofacial Surgery at Erasmus University Medical Center (The Netherlands) and partner in B2B, has set the challenge to generate the ossicle without the support of animal models. “We hope in the future to generate as much as bone-like structure directly in vitro. But to achieve this, we first need to understand step by step how the transformation happens in animals and then mimic it in a test tube,” explains Dr. Farrell. Besides avoiding animal experimentation, the in vitro approach brings other benefits: the system is more reproducible thus reducing the intrinsic variability associated with animal models; it’s easily scalable, enabling also high-throughput experiments, and, once the protocol is set-up, the process should take less time and effort to be completed compared to the animal counterpart.
“The level of complexity that we try to reach within the B2B project is quite new. In vitro scientists usually focus on the very early stages of the bone formation, for example, the mineralization step; our challenge is to include the blood vessels and other types of cells during this process, thus adding an extra layer of complexity.” At this stage of the project, the team at Erasmus MC is able to generate cartilage in vitro and is currently working on the mineralization step and the introduction of other types of cells e.g. immune cells, osteoclasts, blood vessels, etc. To ease this part, the initial idea was to host an expert on angiogenesis from Prof. Banfi’s group in Basel (link to the news about it), to support the co-culture of endothelial cells with the cartilage system; but the exchange had to be postponed due to the COVID-19 emergency. “In the meantime, we are dissecting the events that occur in vivo, step by step,” says Prof.Farrell. This approach will still set an important base for bone-generation in tissue engineering.
The B2B device is a radically new future technology that opens the way to a new type of in vitro modelling. “The device falls right in between two types of in vitro models: the macro 3D models and the organ on a chip (OOC), which instead are on the micron scale.” says Prof. Moroni “On one hand the OOC devices create a fully controlled environment but they are not yet able to mimic the complexity of the tissue and organs that one would wish to test. On the other hand, 3D macro models have much more complexity but much less control of the operational settings – sometimes they are more like a black box as you do not fully grasp what’s happening inside.”
With B2B, we aim to improve the complexity of the tissue model from the dimensionality point of view yet maintaining the control typical of the OOC platforms. A key point of the B2B device is that it doesn’t just reproduce the macro and the micro, but it bridges them. “That’s something very challenging because it needs to integrate processes that are happening at the tissue level with those happening at the cellular level – to synthetize and complement these two aspects is really hard” explains Prof. Moroni.
If successful, the device will be applicable beyond the breast-to-bone metastasis: “There are several mechanisms in which both the cellular and the tissue level need to be taken into account; like the crosstalk between organs.” For example, the thyroid gland releases certain hormones that influence the reproductive system or our brain – so with a B2B-like device, we might be able to model and understand fundamental physiological mechanisms, not just a pathological condition. Today there is no in vitro model able to capture similar crosstalks – That’s why B2B would be a breakthrough technology, not just incremental but truly revolutionary.
R4L among the top 23 EIC-funded SMEs to meet ROCHE
For the third straight year, the European Innovation Council has organized the EIC Corporate Days, gathering innovative European SMEs and large corporates to take full advantage of partnering opportunities.
This year, the pharmaceutical giant ROCHE was also invited to meet EIC-funded SMEs and participate in the EIC Corporate Day initiative. From 22 till 25 June 2020, a limited hand-picked number of EIC companies had the opportunity to pitch and present their innovative solutions and to engage in One-to-One Business meetings with Roche representatives. The selected EIC-funded SMEs had also the opportunity to attend the Roche Partnering for Innovation Summit Summit, with workshops, a marketplace, discussions and keynote speeches.
Our partner R4L was selected as one of the 23 EIC top innovators that had the chance to pitch their product MIVO and the B2B project to Roche. According to Maurizio Aiello, CEO of R4L and partner in B2B: “Less than the 8% of the application (23 out of 302) got selected as promising companies for potential future collaboration with Roche; this great result confirms the high potential of our technology, which is tightly connected to the B2B research line. We are glad for this opportunity and looking forward to expanding our objectives.”
Congratulation to our partner R4L and for a more extensive view on the innovations by R4L check out this interview to CEO Maurizio Aiello.
In order to build the macro-network, the initial method used by the group of Lorenzo Moroni, Professor of Biofabrication for regenerative medicine at the University of Maastricht and partner in B2B, it’s a hybrid process between bioprinting and additive manufacturing. As he explains “We first create fibers that correspond to the dimensions of the vessels that we want to make. These fibers are made of a material that can be washed out by a water solution; so, once they are embedded in the gels that comprise the beast or the bone compartments, the fibers can be leached away, leaving their trace as a template.” This template is later covered by endothelial cells, thus forming a network of vessels that is expected to behave as a biological unit.
“While the technique in itself is not new, the innovation relies on the level of branching that we wish to obtain in the same construct.” Indeed, the laboratory of Prof. Moroni at MERLN is currently working to build the full network in one manufacturing session, which imply to fine-tune and control of several processing parameters to recreate the required change of dimensions and number of branching.
The design of the branching within the network needs to be carefully planned too. That’s why the Moroni’s lab takes advantage of computational modelling to figure out how to reproduce a physiological vascular tree. “Classical principle of microfluidics not always mimic very well the natural vessels in our body. We have developed an improved model to better understand the physiological level of branching and the architecture of a branched network”.
While computational power might help, it also brings another challenge: translate the physiological features into machine code and extract from a computational model a real prototype.
The network in B2B mimics as much as possible the physiological vascular network found in the real tissues, both in the breast and the bone side. One of the strengths and most innovative aspects is the fact that its vessels branchout and change the diameter (see news on mesoscale).
Another feature that brings the device closer to its physiological counterpart is the endothelialisation of the vessels: endothelial cells cover their surfaces making such vessels fully biological. The cells are actually included only after the manufacturing process, once the template is removed (see news on additive manufacturing).
The flow supported by the B2B network will be calibrated as close as possible to the physiological one, with a similar flow rate and shear stress on the covering cell. Otherwise, several problems might arise: turbulence effects and blood clots might cause the formation of micro-thrombi, or a higher flow rate might lead to bulged vessels thus provoking a synthetic-like form of an aneurism. Special care is needed at the level of the bifurcations, as the blood that flows through these regions has a more turbulent flow and the impact on these areas is more vigorous.
Other types of complexity instead fall outside the scope of the B2B project. For the time being in the project, the circulating fluid in the network will be a mix of culture media: the ones typically used in the breast and bone compartments and a classical media that maintains the endothelial cells alive and functional. The resulting media should be able to transport possible metastasis from breast to bone. As a possible future improvement, the cultural media could be substituted with blood, which would increase the physiological biomimicry of the B2B device, but would also bring more challenges related to studying the possible occurring of clotting. For as much as this is an important challenge to tackle in implantable synthetic vascular grafts, it remains of less importance for 3D in vitro models.
The macrovascular part of the B2B device is a network aimed at replicating the key features of vascular network found in our body – therefore it includes a smooth transition from relatively large vessel down to micro-vessels.
“To cover the mesoscale that lies in between the macro and the micro scales, that’s where the real challenge is. In B2B, we will try to cover this gap and bridge vessels of few mm with those in the sub-mm scale”, explains Prof. Moroni, head of the Complex Tissue Regeneration department at MERLN and responsible for the development of the macro-network in the B2B device.
Indeed, the current technology allows us to build wonderful scaffolds above the mm scale, and, thanks to the biological angiogenesis, we can well reproduce micro-vessels or capillaries that are below the tens of microns scale. In B2B instead, the network will start from vessels in the order of a few mm of diameter and it will go down to reach a scale of tens of microns. According to Prof. Moroni, “This is a challenge not only from the technological point of view but also from the physiological point of view, as we need to ensure that the bio-fabricated network maintains a correct flow in all the sub-branches of the network.”
He continues: “It’s already quite difficult to have a single vessel which is physiologically functional in the order of few mm.” For example, an unmet clinical need is the creation of an artificial coronary artery (5 mm), for which the solutions developed so far still present some shortcomings as they tend to form blood clots. “We start from this challenge, but in B2B we expand it to embrace multiscale complexity by aiming to manufacture an array of branching vessels of different size.”
Another challenging aspect the B2B technology is the connection between the macro-network and the micro-vessel, the latter created by the group of A. Banfi at University of Basel (read the news here). “It might sound logical but it’s harder than one can imagine, because the two parts have a different nature: one is biological (the capillaries) and the other one comes from a bioengineering approach – therefore, their compatibility and communication is not granted.” The two systems need to merge and create a continuous lumen that runs from the macro to the micro.
The B2B device is a unique environment to study a complex process in a controlled system. In an in vivo study, for example, several factors come into play e.g. the diet of the animal, its immune system, etc., and it’s much harder to define the impact of each one of these variables.
A simplified system like the B2B device helps instead to clear up our view of the process and to focus on key contributing factors. For example, in a controlled situation, it is easier to link a perturbation, as the addition of a drug, to a response without worrying about the influence of other factors. This is essential for the first application that we have envisioned for the B2B device, namely, to test compounds that might inhibit the formation of metastasis.
“In this regard, the B2B device is truly unique because it lets us model the whole process of cancer development, including both the growth of the mass at the primary site and the metastasis formation” as pointed out by Prof. Aceto “Therefore, we can look for anticancer treatments that are not only reducing the mass of the primary tumor but also the number of metastasis found downstream.”
The B2B device might have even wider applications. “It is essentially a chamber that hosts the spontaneous formation of a vascularized tissue – and it doesn’t need to be a cancerogenic tissue.” Indeed, the device can host other tissues and model their pathological or healthy behavior in a 3D setting. The potential is definitely very high.
The main purpose of the B2B device is to follow and dissect the steps of the metastatic process. Therefore, once some cancer cells have left the primary site, we aim to isolate and analyze their features. The main problem during this phase is that within the samples the metastatic cells are rare. “We are talking probably about less than a hundred cells – therefore, we need tools capable to detect and isolate such a small amount.”
The solution is to label the cancer cells before their insertion into the chamber. The labeling can be done with a fluorescent protein (GFP, RFP, etc.) or a luciferase – a protein found in fireflies that is responsible for their ability to emit light. According to Prof. Aceto “Such method allows us to isolate the tumor cells found in samples taken from the circulation of the B2B device. The situation is harder within the bone tissue due to the complexity of the environment. But even then, using microscopic imaging, we can recognize the metastatic cells among the bone ones thanks to their fluorescence or luminescence.”
In the isolated cancer cells, we plan to study the expression levels of some protein markers. In this way we can address specific questions; e.g. in order to understand if the cells are still proliferating while they go through the circulation, we can stain the samples with antibodies that detect proliferation markers. The limitation of this approach is that it relies on a group of pre-selected markers. To obtain a more general characterization of the metastatic cells we plan to perform a whole transcriptome analysis. The sequencing of the RNAs provides a snapshot about which genes are expressed by a cell in a particular moment and their level of expression. By comparing these expression profiles with the ones found in non-metastatic cells (the ones that remain in the primary tumor), we can identify molecular signals that might be responsible for the metastatic behavior. The same information can also be used to infer something about the microenvironment or stresses that have generated such behavior. This knowledge will guide us in the selection of the type of drugs to test as metastatic inhibitors (link to 3rd news – see below).
The breast cancer chamber: as close as possible to reality
In the B2B device, just as in real life, the metastatic process starts within a primary tumor. Indeed, the device hosts a patient-derived breast cancer lesion in a dedicated chamber, which is developed by the Cancer Metastasis group led by Nicola Aceto at the University of Basel.
“We designed the breast cancer chamber so that it is as close to reality as possible”, explains Prof. Aceto. “First, it hosts a 3Dgrowingtumor mass, as opposed to more static systems which usually enable the proliferation only in two dimensions.” In B2B instead, the tumor mass can grow autonomously in three dimensions, mimicking the spontaneous behavior of a tumor. “To increase the resemblance,” continues Nicola, “we opted for patient-derived circulating tumor cells. The classic cancerogenic cell lines have been propagated for so many years that they have adapted over time. Our patient-derived cells are instead grown in the lab for the minimum amount of time and then placed directly into the device, where they continue to grow.”
The B2B breast cancer lesion has also a network of self-assembled capillaries that connect the tissue to a network of larger artificial vessels (more here). To develop these natural capillaries, “we will include within the tumor mass some endothelial cells –cells that naturally form blood vessels – and as the tumor grows, they will grow too and form a microcapillary network.” This network is then put in contact with the macro network, developed by the University of Maastricht. The smooth transition from small natural vessels to big artificial ones is one of the most critical and innovative features of the B2B device (more here).
Finally, thanks to the B2B concept the extravasation of cells is spontaneous and not forced by the system. “We expect that some cancer cells will recognize the presence of blood vessels and they will spontaneously migrate there and join the systemic circulation” says Nicola “While we are fairly positive that this phenomenon will happen, its frequency might depend on several factors e.g. how invasive the primary cells are, how many capillaries there are in the system, what is the micro-environment, etc.” These parameters might be used later on to fine-tune the system and modulate the metastasis frequency.