Modelling the complex nature of the tumor microenvironment: 3D tumor spheroids as an evolving tool

Cancer remains a serious burden in society and while the pace in the development of novel and more effective therapeutics is increasing, testing platforms that faithfully mimic the tumor microenvironment are lacking. With a clear shift from animal models to more complex in vitro 3D systems, spheroids emerge as strong options in this regard. Years of development have allowed spheroid-based models to better reproduce the biomechanical cues that are observed in the tumor-associated extracellular matrix (ECM) and cellular interactions that occur in both a cellâ cell and cell-ECM manner. Here, we summarize some of the key cellular interactions that drive tumor development, progression and invasion, and how successfully are these interactions recapitulated in 3D spheroid models currently in use in the field. We finish by speculating on future advancements in the field and on how these can shape the relevance of spherical 3D models for tumor modelling.Authors would like to acknowledge the fnancial support from the European Research Council through the Starting Grant “CapBed” (ERC-2018-STG-805411) and FCT/MCTES (Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia, e Ensino Superior) through the grant SFRH/BD/119756/2016 (D.B.R.). The authors would additionally like to thank the contributions to this work from the project “TERM RES Hub—Scientifc Infrastructure for Tissue Engineering and Regenerative Medicine”, reference PINFRA/22190/2016 (Norte-01-0145-FEDER-022190), funded by the Portuguese National Science Foundation (FCT) in cooperation with the Northern Portugal Regional Coordination and Development Commission (CCDR-N). Ultimately, we would like to equally acknowledge fnancial support fromhttps://doi.org/10.54499/ UIDB/50026/2020);https://doi.org/10.54499/UIDP/50026/2020) andhttps://doi. org/10.54499/LA/P/0050/2020)

Hind limb ischemia in type 1 diabetic mice as useful tool to evaluate the neovascularization of tissue engineering constructs

Hind-limb ischemia has been used in type 1 diabetic mice to evaluate treatments for peripheral arterial disease or mechanisms of vascular impairment in diabetes [1]. Vascular deficiency is not only a pathophysiological condition, but also an obvious circumstance in tissue regeneration and in tissue engineering and regenerative medicine (TERM) strategies. We performed a pilot experiment of hind-limb ischemia in streptozotocin(STZ)-induced type 1 diabetic mice to hypothesise whether diabetes influences neovascularization induced by biomaterials. The dependent variables included blood flow and markers of arteriogenesis and angiogenesis. Type 1 diabetes was induced in 8-week-old C57BL/6 mice by an i.p. injection of STZ (50 mg/kg daily for 5 days). Hind-limb ischemia was created under deep anaesthesia and the left femoral artery and vein were isolated, ligated, and excised. The contralateral hind limb served as an internal control within each mouse. Non-diabetic ischaemic mice were used as experiment controls. At the hind-limb ischemia surgical procedure, different types of biomaterials were placed in the blood vessels gap. Blood flow was estimated by Laser Doppler perfusion imager, right after surgery and then weekly. After 28 days of implantation, surrounding muscle was excised and evaluated by histological analysis for arteriogenesis and angiogenesis. The results showed that implanted biomaterials were promote faster restoration of blood flow in the ischemic limbs and improved neovascularization in the diabetic mice. Therefore, we herein demonstrate that the combined model of hind-limb ischemia in type 1 diabetes mice is suitable to evaluate the neovascularization potential of biomaterials and eventually tissue engineering constructs.  Acknowledgments: TCS and RPP acknowledge RL3-TECT-NORTE-01-0124-FEDER-000020, co-financed by ON.2-O Novo Norte; and FCT grants: SFRH/BPD/101952/2014 and SFRH/BPD/101886/2014, respectively. LPS acknowledges FCT grant SFRH/BD/78025/2011

Stromal vascular fraction from adipose tissue and cell sheet engineering to build vascularization units for tissue engineering and regenerative medicine

Vascularization holds the gold key for the effective survival and engraftment of complex engineered tissues and organs for Tissue Engineering and Regenerative Medicine. The lack of adequate vascularization post-transplantation often results in cell necrosis and ultimate failure and rejection of the engineered construct. Herein, we propose a strategy capable of surpassing this obstacle. Harnessing easy accessible adipose tissue stromal vascular fraction (SVF) as a source for cells with intrinsic angiogenic potential, and cell sheet technology we were able to engineer cell sheets with high angiogenic potential. SVF was isolated from the adipose tissue of healthy human subjects after enzymatic digestion and 2x105 nucleated cells/well were seeded on 24 well plates for cell sheet formation. To further boost cells’ angiogenic potential, hypoxic conditions of 5% of oxygen were provided to some of the cells while the rest was cultured in typical normoxia, for up to 8 days of culture, in basal medium. Flow cytometry analysis demonstrated the presence of a heterogeneous population of mesenchymal progenitors, endothelial and hematopoietic cells. Furthermore, the proliferation of SVF cells was evaluated through dsDNA quantification, which showed higher numbers for cells in hypoxic conditions, at earlier time points. Immunocytochemistry against CD31 and CD146 revealed the presence of an interconnected and highly branched network of vessel-like structures, more prominent for cells in hypoxia after 5 days of culture and quite similar for both conditions after 8 days, in the absence of any specific media supplementation. In vivo testing using the cell sheets detached from the wells and HIF expression analysis are currently underway. Taken together, the great potentiality of cell sheet technology with SVF cells cultured in hypoxia opens new exciting perspectives and may represent tremendously valuable vascularization units for tissue engineering strategies

Stem cell-containing hyaluronic acid-based spongy hydrogels for integrated diabetic wound healing

The detailed pathophysiology of diabetic foot ulcers is yet to be established and improved treatments are still required. We propose a strategy that directs inflammation, neovascularization, and neoinnervation of diabetic wounds. Aiming to potentiate a relevant secretome for nerve regeneration, stem cells were precultured in hyaluronic acid-based spongy hydrogels under neurogenic/standard media before transplantation into diabetic mice full-thickness wounds. Acellular spongy hydrogels and empty wounds were used as controls. Reepithelialization was attained 4 weeks after transplantation independently of the test groups, whereas a thicker and more differentiated epidermis was observed for the cellular spongy hydrogels. A switch from the inflammatory to the proliferative phase of wound healing was revealed for all the experimental groups 2 weeks after injury, but a significantly higher M2(CD163 þ )/M1(CD86 þ ) subtype ratio was observed in the neurogenic preconditioned group that also failed to promote neoinnervation. A higher number of intraepidermal nerve fibers were observed for the unconditioned group probably due to a more controlled transition from the inflammatory to the proliferative phase. Overall, stem cell-containing spongy hydrogels represent a promising approach to enhance diabetic wound healing by positively impacting re-epithelialization and by modulating the inflammatory response to promote a successful neoinnervation.The authors would like to acknowledge Gene2Skin Project (H2020-TWINN2015-692221) and Fundac¸a˜o para a Cieˆncia e Tecnologia for SFRH/BD/ 78025/2011 (LPdS), SFRH/BPD/96611/2013 (MTC), SFRH/BPD/101886/2014 (RPP), SFRH/BPD/101952/2014 (TCS) grants. Moreover, the authors would also like to acknowledge Teresa Oliveira for histology support, Andreia Carvalho for hASCs supply, Luca Gasperini for cell profiler analysis, and Manuela E. L. Lago and Carla M. Abreu for intraepidermal nerve fiber quantification.info:eu-repo/semantics/publishedVersio

Performance of Beef Heifers of Various Genetic Groups, Supplemented or Not, in Coastcross Pastures

The objective of this study was to determine whether the performance of beef heifers of different genetic groups was affected by breed x nutritional environment interactions. Sixty four weaned heifers, 16 per genetic group: ½ Angus + ½ Nellore (AN), ½ Canchim + ½ Nellore (CN), ½ Simmental + ½ Nellore (SN) and pure Nellore (NE), were used with or without 3.0 kg of concentrate.animal-1 .day-1 in a fertilized coastcross pasture under rotational grazing system. There were effects of genetic group and supplementation (P\u3c 0.05) on the weight and age at first estrus, but there was no interaction between them. In a rotational grazing system with 4000 kg of available dry matter per hectare with 13% of crude protein, the crossbred AN, supplemented or not, was more precocious (111 days) than Nellore heifers, showing the first estrus at 356 days of age and 324 kg of live weight

Integrin-specific hydrogels for growth factor-free vasculogenesis

Integrin-binding biomaterials have been extensively evaluated for their capacity to enable de novo formation of capillary-like structures/vessels, ultimately supporting neovascularization in vivo. Yet, the role of integrins as vascular initiators in engineered materials is still not well understood. Here, we show that αvβ3 integrin-specific 3D matrices were able to retain PECAM1+ cells from the stromal vascular fraction (SVF) of adipose tissue, triggering vasculogenesis in vitro in the absence of extrinsic growth factors. Our results suggest that αvβ3-RGD-driven signaling in the formation of capillary-like structures prevents the activation of the caspase 8 pathway and activates the FAK/paxillin pathway, both responsible for endothelial cells (ECs) survival and migration. We also show that prevascularized αvβ3 integrin-specific constructs inosculate with the host vascular system fostering in vivo neovascularization. Overall, this work demonstrates the ability of the biomaterial to trigger vasculogenesis in an integrin-specific manner, by activating essential pathways for EC survival and migration within a self-regulatory growth factor microenvironment. This strategy represents an improvement to current vascularization routes for Tissue Engineering constructs, potentially enhancing their clinical applicability.The authors would like to acknowledge the financial support from the Consolidator Grant “ECM_INK” (ERC-2016-COG-726061) and the Starting Grant “CapBed” (ERC2018-STG-805411), to the FSE/POCH (Fundo Social Europeu através do Programa Operacional do Capital Humano) under the scope of the PD/169/2013, NORTE-08- 5369-FSE-000037 (H.R.M.), and to FCT/MCTES (Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia, e Ensino Superior) through the grants SFRH/BD/119756/2016 (D.B.R.), Ph.D. grant PD/BD/135252/2017 (S.F.R.) and IF/00347/ 2015 (R.P.P.)

Spongy-like hydrogels prevascularization with the adipose tissue vascular fraction delays cutaneous wound healing by sustaining inflammatory cell influx

In vitro prevascularization is one of the most explored approaches to foster engineered tissue vascularization. We previously demonstrated a benefit in tissue neovascularization by using integrin-specific biomaterials prevascularized by stromal vascular fraction (SVF) cells, which triggered vasculogenesis in the absence of extrinsic growth factors. SVF cells are also associated to biological processes important in cutaneous wound healing. Thus, we aimed to investigate whether in vitro construct prevascularization with SVF accelerates the healing cascade by fostering early vascularization vis-à-vis SVF seeding prior to implantation. Prevascularized constructs delayed re-epithelization of full-thickness mice wounds compared to both non-prevascularized and control (no SVF) groups. Our results suggest this delay is due to a persistent inflammation as indicated by a significantly lower M2(CD163+)/M1(CD86+) macrophage subtype ratio. Moreover, a slower transition from the inflammatory to the proliferative phase of the healing was confirmed by reduced extracellular matrix deposition and increased presence of thick collagen fibers from early time-points, suggesting the prevalence of fiber crosslinking in relation to neodeposition. Overall, while prevascularization potentiates inflammatory cell influx, which negatively impacts the cutaneous wound healing cascade, an effective wound healing was guaranteed in non-prevascularized SVF cell-containing spongy-like hydrogels confirming that the SVF can have enhanced efficacy.Authors would like to acknowledge the financial support from the Consolidator Grant “ECM_INK” (ERC-2016-COG-726061) and the Starting Grant “CapBed” (ERC-2018-STG-805411), to the FSE/POCH (Fundo Social Europeu através do Programa Operacional do Capital Humano) under the scope of the PD/169/2013, NORTE-08-5369-FSE-000037 (H.R.M.), and to FCT/MCTES (Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia, e Ensino Superior) through the grants SFRH/BD/119756/2016 (D.B.R.), PhD grant PD/BD/135252/2017 (S.F.R.) and IF/00347/2015 (R.P.P.). Authors would also like to acknowledge BioRender.com as a platform for image creation