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).
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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.
While the fabrication of a self-assembled network of capillaries and an engineered macro-network is not trivial but possible, the connection between the two is a real challenge. “The macro-to-micro connection proposed by B2B is truly innovative and, to the best of our knowledge, it’s the first time that it would be implemented” explains Andrea Banfi, leader of the related work package in B2B. The distinguishing factor of B2B, and the one that brings most of the complexity, is the size of the involved tissues, which require an extensive network of capillaries to penetrate the whole mass (range of cm3). In other techniques, such as the so-called “organ-on-a-chip” only a few thousand cells of each kind communicate with each other – so, the connecting system is greatly simplified.
“An extended network of capillaries is harder to reproduce because its development and final arrangement is linked to conditions evolving in the different micro-environments of the organoid, which cannot be precisely anticipated”, says Banfi, “To reproduce the real organ-level-complexity we should let this randomness take place.”
That’s why in B2B the functional connection between the micro- and macro-networks is built by spontaneous processes that rely on the normal biological behavior of endothelial cells and fluid flow. The endothelial cells that cover the engineered vase and the ones that form the capillaries should first recognize each other, then stick together and finally create a junction with a lumen. For this to happen, “we include in the micro-environment the right biological signals, e.g. growth factors and morphogens, to trigger the cascade of events leading to vessel assembly. Similarly, once connections are established, the fluid flow should naturally regulate and remodel the shape and size of the vascular network.” Overall, this spontaneous connection should not take more than a few days.
After the 1st part of the project, everything is ready to test this critical kiss: the set-up for the micro- and macro-networks have been identified and it’s time to place them together and let the system evolve. The first tests will provide useful feedback to fine-tune the two networks and ensure their compatibility.
In B2B, both the breast tumoroid and the ossicle have their self-assembled networks of capillaries.
In the first case, the tumoroid and its vascular network are generated in parallel. “We include cancer cells together with endothelial cells in a matrix of fibrin that contains growth factors for both tissues.” explains Andrea Banfi. Thanks to the biological signals, the system evolves by creating two ordered networks of cells of the same type: endothelial with endothelial and cancer with cancer. However, the two systems are in close contact, meaning that the tumoroid is fully vascularized.
Thanks to the large dimensions of the tumoroid, B2B reproduces well the stochastic growth of the cancer tissue. In some cases, it might exceed the flow capacity of the system, thus leading to the formation of necrotic and hypoxic areas. It is the presence of these areas that increases the metastatic predisposition of cells, as demonstrated by B2B partner Nicola Aceto, as if the lack of resources would trigger the need to migrate to new, richer areas. Only thanks to the peculiarities of the B2B system, based on spontaneous processes, it is possible to recreate such heterogeneity so vital for the selection of cells capable to metastasize (read more here).
Different is the approach for the ossicle, which is generated in vivo – by placing chondrogenic cells subcutaneously in a mouse. The resulting ossicle is then vascularized directly by the mouse system. “When we remove the ossicle, the major blood vessels are cut and we need to re-establish a connection, this time with the macro system“. This is quite hard as the vessels are placed randomly around the tissue, therefore it would be impossible to engineer something ad hoc.
The solution proposed by the team of Andrea Banfi is to generate a pervasive micro-network (of human origin) that extends over the whole surface around the tissue, touching and connecting, on one side, with the ossicle capillaries and, on the other side, with the macro network. When the connection happens, then the flow, and the physical laws governing it, remodel the dimensions of the used vessels and dismiss the unused ones.
As stated in the name of the project (from Breast to Bone), B2B relies on the connection between two tissues. Indeed, the two chambers, one with the breast tumoroid and the other with the ossicle, are connected with a system that resembles the physiological blood circulation, a set of vessels that brings the blood with oxygen and nutrients to the organs. As in the cardiovascular system, the B2B connecting network is made of two parts: the micro-network for the exchange of material from the tissue to the circulatory system and the macro network for its fast transportation from one side to another.
“As part of the micro-network it was crucial to include the capillaries” – explains Andrea Banfi, head of the Cell and Gene Therapy group at Basel University Hospital and the B2B partner responsible for the micro-network – “Two key events happen only there: the intra- and extravasation of the metastatic cells, the process by which cancer cells move, respectively, from the main tumor to the blood circulation and from the circulation to the target tissue”.
The two processes happen only in the smallest vessels (10-20 micrometers of caliber) because here the blood flow is slow enough to allow metastatic cells to roll and adhere to the blood vessels’ surface, the endothelium, and leak out. Instead, in larger vessels, the higher velocity of the flow hinders cell movement through the vessel wall. Also, structurally the capillaries encourage the exchange of cells and substances: they are made of a thin layer of mainly endothelial cells – so, to exit, a metastatic cell just needs to squeeze through the gaps between them. However, the small dimensions of the capillaries make them non-engineerable: therefore, they need to self-assemble under the guidance of provided biological signals and molecules (read more here).
The organoid chambers are connected by large macro-fluidic tubing, made of silicone, whose function is equivalent to that of large arteries and veins in the body, whose flux quickly transports substances from one side to the other. To bring flow from this tubing system to the self-assembled micro-vessels, an actual vascular network of decreasing size and increasing branching is required. This is the macro-vascular network, which is built by a set of engineered vessels bio-printed into and around the micro-vascularized organoids, whose diameters gradually range from large to small. This system follows the same laws that regulate the relationship between flow and dimension in the cardiovascular tree. But, unlike the self-assembled capillaries, at the moment the bio-printed vessels lack part of the structural features found in the physiological counterpart, like the smooth muscle surrounding arteries and responsible for their elasticity and contraction. This doesn’t impair the scope of the B2B device, as the global flow can be regulated by an external pump.
The integration between the macro- and micro-networks is one of the most innovative points in B2B. The full vascular network is realized in collaboration by two groups: the micro-network by Dr. Andrea Banfi’s lab at the Basel University Hospital and the macro-network by Lorenzo Moroni’s group at MERLN. Their joint efforts will result in an innovative vascular system with a smooth transition from the macro- to the micro-scale (read more here).
The B2B partner Andrea Banfi directs the Cell and Gene Therapy group at Basel University Hospital, in the Departments of Biomedicine and of Surgery. His research focus is the understanding of the basic principles governing the growth of blood vessels and translating this knowledge into the development of novel therapies. We asked him to introduce us to the concept of therapeutic angiogenesis.
“Therapeutic angiogenesis is the generation of blood vessels for therapeutic applications. Today, besides the application in tissue-engineering in vitro, like the one in B2B, there is also an increasing interest in the vascularization of ischemic tissues, in which the blood supply is reduced and needs to be restored for the normal organ function.
There are no pharmacological cures for this disease today. Only surgical interventions can substitute blocked arteries (e.g. by-pass surgery), but results are unsatisfactory, both because not every patient can be operated, and because the opened vessels re-close with time. By understanding how angiogenesis is regulated in nature, we might exploit similar signals to trigger new vascular growth directly in the tissue to generate a sort of long-lasting “biological by-pass”.
Common signals are molecules like the growth factor VEGF, but the body tightly regulates their production and avoids exceeding potentially harmful thresholds. To induce therapeutic blood vessels formation, it is necessary to exceed, under limited circumstances, the dose-limit that the body imposes. In our lab, we are investigating the optimal mix of stimuli, doses and duration of treatment to trigger efficient and long-lasting blood vessels formation.
During the very first tests, back in the early 2000s, therapies with VEGF failed to show efficacy in patients at safe doses. Subsequent retrospective analyses identified several issues underlying the discrepancy between the obvious biological function of VEGF and its difficulty as a drug. An important aspect relates to the fact that VEGF binds tightly to extracellular matrix and remains localized in the micro-environment around each producing cell. Therefore, it is important to ensure a homogeneous distribution of production levels in the tissue, otherwise a few hot spots – in which the local dose is toxic – will compromise safety, while the areas that don’t reach an effective dose compromise efficacy.
In our lab, we are currently working to overcome this issue. Homogeneous distributions of VEGF are rather hard to get, so we are exploring ways to stop the onset of the toxic behavior only in the hot spots. By administrating specific drugs during the critical first weeks after the therapy, we can block the aberrant angiogenesis while keeping the desired one. It’s a long way before reaching the clinical application, but I’m confident that we will finally find a way to apply angiogenesis in the fight against ischemia. “
The B2B Consortium collects the excellence of several sectors, each one contributing to a specific component of the device. Only the synergy between all the parts gives life to the B2B device.
The realization of the breast cancer model from a patient-derived primary lesion is led by Nicola Aceto at the Cancer Metastasis lab of the University of Basel, winner of an ERC starting grant. The lab has worked for many years with breast cancer to dissect the metastatic process.
The group of Eric Farrell at the Erasmus MC University Medical Center is in charge to develop the ossicle model. His lab, the Bone Tissue Engineering Research lab, studies the bone tissue development and in B2B it will build a bone-model with multiple tissues, including the mineralised matrix and the bone marrow.
The vascular network is realized in collaboration by two groups: Dr. Andrea Banfi’s Cell and Gene Therapy lab at the Basel University Hospital that studies the formation and self-assembling growth of blood vessels and Lorenzo Moroni’s Complex Tissue Regeneration group at MERLN that develops biofabrication technologies. Together they will develop an innovative vascular system allowing the transition from the macro- to the micro-scale of blood vessel branching and that can be attached directly to the self-assembled capillaries grown by the tumour tissues.
Finally, the Engineering for Health and Wellbeing Group at the CNR-IEIIT, headed by Silvia Scaglione, is responsible for the integration of these components into the final device. Her group is in charge to design and develop the full B2B platform which should ensure the right connection between the parts developed by the other groups. The first platform should be ready by the end of the first year, but the design will be constantly improved based on the collected findings, together with the company REACT4LIFE which will promote European market acceptance.
Once the system is set up, high-resolution imaging by the company BIOEMTECH will complement the work by assessing the correct development of the microvascular network, monitoring the circulation of the cancer cells and evaluating the metastasis formation in the bone-like structure.
Other two SMEs are involved in supporting the project: CITC assesses the degree of maturity reached by the technology, while IN supports the projects management, communication, dissemination of B2B results.
All the partners have already started to work since the very beginning of the project (July 2018) and they will constantly exchange information throughout the entire project lifetime to ensure the perfect synergy in the final device.
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STUDY CANCER METASTASIS IN CLINICALLY RELEVANT MODELS
Unable to validate some of their hypothesis, scientists often remain with many pending questions about cancer, especially regarding the metastatic process and the failure of preclinical studies: how come that some cancer cells exit the site of origin, enter the blood flow and attack another tissue? Why certain drugs do not have the expected effect in patients compared to pre-clinical studies in animal models? Such questions are hard to tackle with the cancer models available today.
Animal models, on the one hand, offer a unique venue to insert a tumour in a complex system made of connected organs. But the human-derived cancer becomes surrounded by a non-human physiological system. Therefore, the growth, development and response to drugs might differ, resulting in false positives that waste researchers’ time and money. The human metastatic process is even harder to reproduce in an animal model as it requires multiple connected organs.
On the other hand, the available in vitro approaches are generally bi-dimensional and lack the 3D complexity of a living organ. For example, standard cell cultures on a monolayer are an isolated system in which cells don’t behave differently according to the position and exposure, a behaviour far from the heterogeneity typical of cancer cells. The recent developed organs-on-a-chip (OOC) technology has upgraded the cell cultures to a 3D microfluidic chip where several 3D tissue constructs are connected with a network of sub-millimetre vases, that transport and distribute nutrients and soluble cues. However, the quantities involved are low – microliters and thousands of cells – making OOC suitable for automation and high-throughput screening but not for in-depth analysis.
To not jeopardize the reliability of the results and to understand the mechanism of metastasis, in vitro models should include all the factors that affect the process and better resemble the human physiology. The device developed within B2B will become the first cancer model that brings in vitro the 3D upgrade in clinically-relevant dimensions (macro-size tumour tissues), all in a connected system entirely based on human physiology.
The new technology should overcome the drawbacks of today’s in vitro and in vivo models by mimicking the human physiology as a system of connected organs. The connection via a fluidic system is particularly critical in B2B, as it will use macro-to-micro bioprinted vases that should reproduce the different sizes, branching and features of the blood vessels and at the same time be directly connected to the capillaries from the tumour tissues.
B2B has selected the metastatic process of breast cancer to the bone as its first application, since it represents a major hurdle in the fight of breast cancer. Breast cancer is the most common in women worldwide (25.4% of the total number of new cases diagnosed in 2018) and its most common metastatic site is indeed the bone (70% of the cases). In the B2B device, a patient-derived breast cancer lesion will be connected to an in vitro reconstructed bone, a marrow-containing ossicle. However, the technology developed in B2B is versatile and the same system might be applied in the future to study other types of cancers with similar features.
Research, in order to advance and fuel innovation, needs technological innovation itself. The EU-funded project B2B is doing research for researchers, to bring recent advances in fluidic systems and 3D printing to the biomedical sector.
“When a new technology reaches the biomedical field, all eyes are on the technology and its advantages – but to have a successful uptake, the technology should solve problems that matter to the end-users, namely the researchers.” explains Silvia Scaglione, the B2B coordinator. Silvia is well familiar with the problem as, during her PhD in Bioengineering and Bioelectronics, she worked side by side with cell biologists, biotechnologists and medical doctors. “In such a multidisciplinary environment, I got to know the frustrations and difficulties that the biomed researchers have to face day by day, and, as a bioengineer, I wanted to develop a technology that responded to their needs. That’s the idea that inspired B2B”.
Indeed, in cancer research, scientists are missing reliable cancer models to advance research. The general discontent is related to the faults of available models, unable to capture the complexity of the human disease (more here). B2B is developing a breakthrough in vitro alternative that is more clinically relevant than tumour spheroids and closer to the human physiology than animal models. In the B2B multidisciplinary team, scientists involved in cancer research work side by side with engineers and material scientists to develop an ad hoc technology that will simplify and enhance their research (more here).
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