A CRITICAL KISS: FROM MACRO TO MICRO

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.

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TO EACH TISSUE, ITS MICRO-NETWORK

Example of micro-network (capillaries) with endothelial cells in green. The cell nuclei (5 microns on average) are visible in blue or pink (if proliferating). Courtesy of Andrea Banfi.

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.

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MACRO & MICRO in B2B

3D rendering of a micro-network: endothelial capillaries in red, supporting cells in green. Courtesy of Andrea Banfi.

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 branching of an artery-vein pair. Courtesy of Andrea Banfi.

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).

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Therapeutic Angiogenesis

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.  

New blood vessels forming from pre-existing vessels.
www.scientificanimations.com – http://www.scientificanimations.com/wiki-images/

“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. “

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THE PROJECT SYNERGY: THE CONSORTIUM

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.

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RESEARCH TO INNOVATE CANCER RESEARCH

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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AN INTRINSICALLY FET PROJECT

B2B it’s a Future & Emerging Technologies (FET) project. This EU-funded Programme is all about transforming advanced scientific ideas into radically new technologies for the future. As for the B2B platform, the breakthrough technology should not be incremental but truly revolutionary, changing the today paradigms. We asked our project coordinator Silvia Scaglione to describe the innovation behind B2B and why it fits well in the FET Programme.

“B2B has several strengths that make it a very innovative project. First, it’s a bottom-up project that responds to the scientists’ request for a reliable model to study cancer. The success is further ensured by the multidisciplinary Consortium, a fertile environment in which different expertise come together and complement each other. Having myself a technical background but working with biologists and medical doctors, I know very well how hard is to cross-link activities between these fields and thus deliver technologies that can offer effective solutions to the problems of the biomedical sector.

As every cutting-edge project, B2B has several connected risks and for each, we have foreseen a mitigation action to ensure that we have a functioning platform at the end of the project. Each component of the device has its critical point; for example, bioprinted branching vessels should connect well with both the external fluidic system and the self-assembled capillaries from the tumour tissues. The inclusion of the bone marrow in the ossicle model is also challenging, but we have planned two alternatives for its development (i.e. directly in vitro or first in vivo and subsequently transplanted in vitro). Lastly, the breast cancer model should support the migration of the tumour cells, which imply that the cancer cells exit the site of origin and penetrate into the blood system. Only after facing and overcoming these difficulties, we will be able to create a breakthrough device that brings an innovation leap into cancer research.

The new technology should first bring tangible benefits to researchers, enabling them to finally validate hypotheses that never saw the light of day. Nevertheless, on the long-term we expect a great impact in the society as a whole, since achieved results might reduce failures during preclinical studies, resulting in new therapies that will reach the market in a faster and more cost-competitive way.

From the moment it was conceived, the project fitted extremely well within the FET programme, having a strong technological side and a visionary approach far beyond the state-of-the-art. We were very proud to get funds from the FET funding scheme, as the programme rewards excellence and tackles critical points of doing research. Part of the funds is promptly available from the very beginning of the project, thus allowing the partners to acquire resources and start immediately with their work. We are strongly committed to making the most of our 4-year funding.”

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B2B project has started

B2B project has officially started on July 1, 2018. During the next 4 years, the Consortium will be busy developing a novel hybrid device able to model the spontaneous breast cancer metastasis to the bone. The project has been funded for a total of 3.8M€ under the FET OPEN call of the European Union’s Horizon 2020 Framework Programme. The full official information about the project is available in CORDIS.

 

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