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Monitoring cell behaviour in hydrogel-based 3D models is critical for assessing their growth and response to cytotoxic treatment. Resazurin-based PrestoBlue and AlamarBlue reagents are frequently used metabolic activity assays when determining cell responses. However, both assays are largely applied to cell monolayer cultures but yet to have a defined protocol for use in hydrogel-based 3D models. The assays' performance depends on the cell type, culture condition and measurement sensitivity. To better understand how both assays perform, we grew pancreatic cancer cells in gelatin methacryloyl and collagen hydrogels and evaluated their metabolic activity using different concentrations and incubation times of the PrestoBlue and AlamarBlue reagents. We tested reagent concentrations of 4 % and 10 % and incubation times of 45 min, 2 h and 4 h. In addition, we co-cultured cancer cells together with cancer-associated fibroblasts and peripheral blood mononuclear cells in gelatin methacryloyl hydrogels and subjected them to gemcitabine and nab-paclitaxel to evaluate how both assays perform when characterising cell responses upon drug treatment. CyQuant assays were conducted on the same samples and compared to data from the metabolic activity assays. In cancer monocultures, higher reagent concentration and incubation time increased fluorescent intensity. We found a reagent concentration of 10 % and an incubation time of 2 h suitable for all cell lines and both hydrogels. In multicellular 3D cultures, PrestoBlue and AlamarBlue assays detected similar cell responses upon drug treatment but overestimated cell growth. We recommend to assess cell viability and growth in conjunction with CyQuant assays that directly measure cell functions., Competing Interests: Declaration of competing interest The authors do not have any conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

CMS4 colorectal cancer (CRC), based on the consensus molecular subtype (CMS), stratifies patients with the poorest disease-free survival rates. It is characterized by a strong mesenchymal stromal cell (MSC) signature, wound healing-like inflammation and therapy resistance. We utilized 2D and 3D in vitro, in vivo , and ex vivo models to assess the impact of inflammation and stromal cells on immunosuppression in CMS4 CRC. RNA sequencing data from untreated stage II/III CRC patients showed enriched TNF-α signatures in CMS1 and CMS4 tumors. Secretome from TNF-α treated cancer cells induced an immunomodulatory and chemotactic phenotype in MSC and cancer-associated fibroblasts (CAFs). Macrophages in CRC tumours migrate and preferentially localise in stromal compartment. Inflammatory CRC secretome enhances expression of PD-L1 and CD47 on both human and murine stromal cells. We demonstrate that TNF-α-induced inflammation in CRC suppresses macrophage phagocytosis via stromal cells. We show that stromal cell-mediated suppression of macrophage phagocytosis is mediated in part through PD-1 signaling. These data suggest that re-stratification of CRC by CMS may reveal patient subsets with microsatellite stable tumors, particularly CMS4-like tumors, that may respond to immunotherapies., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s).)

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense and stiff extracellular matrix (ECM) associated with tumor progression and therapy resistance. To further the understanding of how stiffening of the tumor microenvironment (TME) contributes to aggressiveness, a three-dimensional (3D) self-assembling hydrogel disease model is developed based on peptide amphiphiles (PAs, PA-E3Y) designed to tailor stiffness. The model displays nanofibrous architectures reminiscent of native TME and enables the study of the invasive behavior of PDAC cells. Enhanced tuneability of stiffness is demonstrated by interacting thermally annealed aqueous solutions of PA-E3Y (PA-E3Y h ) with divalent cations to create hydrogels with mechanical properties and ultrastructure similar to native tumor ECM. It is shown that stiffening of PA-E3Y h hydrogels to levels found in PDAC induces ECM deposition, promotes epithelial-to-mesenchymal transition (EMT), enriches CD133 + /CXCR4 + cancer stem cells (CSCs), and subsequently enhances drug resistance. The findings reveal how a stiff 3D environment renders PDAC cells more aggressive and therefore more faithfully recapitulates in vivo tumors., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Tumours are heterogeneous tissues containing diverse populations of cells and an abundant extracellular matrix (ECM). This tumour microenvironment prompts cancer cells to adapt their metabolism to survive and grow. Besides epigenetic factors, the metabolism of cancer cells is shaped by crosstalk with stromal cells and extracellular components. To date, most experimental models neglect the complexity of the tumour microenvironment and its relevance in regulating the dynamics of the metabolism in cancer. We discuss emerging strategies to model cellular and extracellular aspects of cancer metabolism. We highlight cancer models based on bioengineering, animal, and mathematical approaches to recreate cell-cell and cell-matrix interactions and patient-specific metabolism. Combining these approaches will improve our understanding of cancer metabolism and support the development of metabolism-targeting therapies., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Pancreatic cancer is a devastating malignancy with minimal treatment options. Standard-of-care therapy, including surgery and chemotherapy, is unsatisfactory, and therapies harnessing the immune system have been unsuccessful in clinical trials. Resistance to therapy and disease progression are mediated by the tumor microenvironment, which contains excessive amounts of extracellular matrix and stromal cells, acting as a barrier to drug delivery. There is a lack of preclinical pancreatic cancer models that reconstruct the extracellular, cellular, and biomechanical elements of tumor tissues to assess responses toward immunotherapy. To address this limitation and explore the effects of immunotherapy in combination with chemotherapy, a multicellular 3D cancer model using a star-shaped poly(ethylene glycol)-heparin hydrogel matrix is developed. Human pancreatic cancer cells, cancer-associated fibroblasts, and myeloid cells are grown encapsulated in hydrogels to mimic key components of tumor tissues, and cell responses toward treatment are assessed. Combining the CD11b agonist ADH-503 with anti-PD-1 immunotherapy and chemotherapy leads to a significant reduction in tumor cell viability, proliferation, metabolic activity, immunomodulation, and secretion of immunosuppressive and tumor growth-promoting cytokines., (© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Background: The small GTP-binding protein Rab31 plays an important role in the modulation of tumor biological-relevant processes, including cell proliferation, adhesion, and invasion. As an underlying mechanism, Rab31 is presumed to act as a molecular switch between a more proliferative and an invasive phenotype. This prompted us to analyze whether Rab31 overexpression in breast cancer cells affects expression of genes involved in epithelial-to-mesenchymal transition (EMT)-like processes when compared to Rab31 low-expressing cells., Methods: Commercially available profiler PCR arrays were applied to search for differentially expressed genes in Rab31 high- and low-expressing CAMA-1 breast cancer cells. Differential expression of selected candidate genes in response to Rab31 overexpression in CAMA-1 cells was validated by independent qPCR and protein assays., Results: Gene expression profiling of key genes involved in EMT, or its reciprocal process MET, identified 9 genes being significantly up- or down-regulated in Rab31 overexpressing CAMA-1 cells, with the strongest effects seen for TGFB1, encoding TGF-ß1 (> 25-fold down-regulation in Rab31 overexpressing cells). Subsequent validation analyses by qPCR revealed a strong down-regulation of TGFB1 mRNA levels in response to increased Rab31 expression not only in CAMA-1 cells, but also in another breast cancer cell line, MDA-MB-231. Using ELISA and Western blot analysis, a considerable reduction of both intracellular and secreted TGF-ß1 antigen levels was determined in Rab31 overexpressing cells compared to vector control cells. Furthermore, reduced TGF-ß activity was observed upon Rab31 overexpression in CAMA-1 cells using a sensitive TGF-ß bioassay. Finally, the relationship between Rab31 expression and the TGF-ß axis was analyzed by another profiler PCR array focusing on genes involved in TGF-ß signaling. We found 12 out of 84 mRNAs significantly reduced and 7 mRNAs significantly increased upon Rab31 overexpression., Conclusions: Our results demonstrate that Rab31 is a potent modulator of the expression of TGF-ß and other components of the TGF-ß signaling pathway in breast cancer cells., (© 2021. The Author(s).)

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. CRC develops in a complex tumour microenvironment (TME) with both mesenchymal stromal cells (MSCs) and immune infiltrate, shown to alter disease progression and treatment response. We hypothesised that an accessible, affordable model of CRC that combines multiple cell types will improve research translation to the clinic and enable the identification of novel therapeutic targets. A viable gelatine-methacrloyl-based hydrogel culture system that incorporates CRC cells with MSCs and a monocyte cell line was developed. Gels were analysed on day 10 by PCR, cytokine array, microscopy and flow cytometry. The addition of stromal cells increased transcription of matrix remodelling proteins FN1 and MMP9, induced release of tumour-promoting immune molecules MIF, Serpin E1, CXCL1, IL-8 and CXCL12 and altered cancer cell expression of immunotherapeutic targets EGFR, CD47 and PD-L1. Treatment with PD153035, an EGFR inhibitor, revealed altered CRC expression of PD-L1 but only in gels lacking MSCs. We established a viable 3D model of CRC that combined cancer cells, MSCs and monocytic cells that can be used to research the role the stroma plays in the TME, identify novel therapeutic targets and improve the transitional efficacy of therapies.

Ovarian cancer (OvCa) is one of the leading causes of gynecologic malignancies. Despite treatment with surgery and chemotherapy, OvCa disseminates and recurs frequently, reducing the survival rate for patients. There is an urgent need to develop more effective treatment options for women diagnosed with OvCa. The tumor microenvironment (TME) is a key driver of disease progression, metastasis and resistance to treatment. For this reason, 3D models have been designed to represent this specific niche and allow more realistic cell behaviors compared to conventional 2D approaches. In particular, self-assembling peptides represent a promising biomaterial platform to study tumor biology. They form nanofiber networks that resemble the architecture of the extracellular matrix and can be designed to display mechanical properties and biochemical motifs representative of the TME. In this review, we highlight the properties and benefits of emerging 3D platforms used to model the ovarian TME. We also outline the challenges associated with using these 3D systems and provide suggestions for future studies and developments. We conclude that our understanding of OvCa and advances in materials science will progress the engineering of novel 3D approaches, which will enable the development of more effective therapies.

The tumor microenvironment evolves during malignant progression, with major changes in nonmalignant cells, cytokine networks, and the extracellular matrix (ECM). In this study, we aimed to understand how the ECM changes during neoplastic transformation of serous tubal intraepithelial carcinoma lesions (STIC) into high-grade serous ovarian cancers (HGSOC). Analysis of the mechanical properties of human fallopian tubes (FT) and ovaries revealed that normal FT and fimbria had a lower tissue modulus, a measure of stiffness, than normal or diseased ovaries. Proteomic analysis of the matrisome fraction between FT, fimbria, and ovaries showed significant differences in the ECM protein TGF beta induced (TGFBI, also known as βig-h3). STIC lesions in the fimbria expressed high levels of TGFBI, which was predominantly produced by CD163-positive macrophages proximal to STIC epithelial cells. In vitro stimulation of macrophages with TGFβ and IL4 induced secretion of TGFBI, whereas IFNγ/LPS downregulated macrophage TGFBI expression. Immortalized FT secretory epithelial cells carrying clinically relevant TP53 mutations stimulated macrophages to secrete TGFBI and upregulated integrin αvβ3, a putative TGFBI receptor. Transcriptomic HGSOC datasets showed a significant correlation between TGFBI expression and alternatively activated macrophage signatures. Fibroblasts in HGSOC metastases expressed TGFBI and stimulated macrophage TGFBI production in vitro . Treatment of orthotopic mouse HGSOC tumors with an anti-TGFBI antibody reduced peritoneal tumor size, increased tumor monocytes, and activated β3-expressing unconventional T cells. In conclusion, TGFBI may favor an immunosuppressive microenvironment in STICs that persists in advanced HGSOC. Furthermore, TGFBI may be an effector of the tumor-promoting actions of TGFβ and a potential therapeutic target. SIGNIFICANCE: Analysis of ECM changes during neoplastic transformation reveals a role for TGFBI secreted by macrophages in immunosuppression in early ovarian cancer., (©2021 The Authors; Published by the American Association for Cancer Research.)

Patient-derived in vivo models of human cancer have become a reality, yet their turnaround time is inadequate for clinical applications. Therefore, tailored ex vivo models that faithfully recapitulate in vivo tumour biology are urgently needed. These may especially benefit the management of pancreatic ductal adenocarcinoma (PDAC), where therapy failure has been ascribed to its high cancer stem cell (CSC) content and high density of stromal cells and extracellular matrix (ECM). To date, these features are only partially reproduced ex vivo using organoid and sphere cultures. We have now developed a more comprehensive and highly tuneable ex vivo model of PDAC based on the 3D co-assembly of peptide amphiphiles (PAs) with custom ECM components (PA-ECM). These cultures maintain patient-specific transcriptional profiles and exhibit CSC functionality, including strong in vivo tumourigenicity. User-defined modification of the system enables control over niche-dependent phenotypes such as epithelial-to-mesenchymal transition and matrix deposition. Indeed, proteomic analysis of these cultures reveals improved matrisome recapitulation compared to organoids. Most importantly, patient-specific in vivo drug responses are better reproduced in self-assembled cultures than in other models. These findings support the use of tuneable self-assembling platforms in cancer research and pave the way for future precision medicine approaches., (© 2021. The Author(s).)

Prostate cancer (PCa) is the second most commonly diagnosed cancer in men, and bone is the most frequent site of metastasis. The tumor microenvironment (TME) impacts tumor growth and metastasis, yet the role of the TME in PCa metastasis to bone is not fully understood. We used a tissue-engineered xenograft approach in NOD-scid IL2Rγ null (NSG) mice to incorporate two levels of humanization; the primary tumor and TME, and the secondary metastatic bone organ. Bioluminescent imaging, histology, and immunohistochemistry were used to study metastasis of human PC-3 and LNCaP PCa cells from the prostate to tissue-engineered bone. Here we show pre-seeding scaffolds with human osteoblasts increases the human cellular and extracellular matrix content of bone constructs, compared to unseeded scaffolds. The humanized prostate TME showed a trend to decrease metastasis of PC-3 PCa cells to the tissue-engineered bone, but did not affect the metastatic potential of PCa cells to the endogenous murine bones or organs. On the other hand, the humanized TME enhanced LNCaP tumor growth and metastasis to humanized and murine bone. Together this demonstrates the importance of the TME in PCa bone tropism, although further investigations are needed to delineate specific roles of the TME components in this context., (© 2021. Crown.)

As cancer-associated factors, kallikrein-related peptidases (KLKs) are components of the tumour microenvironment, which represents a rich substrate repertoire, and considered attractive targets for the development of novel treatments. Standard-of-care therapy of pancreatic cancer shows unsatisfactory results, indicating the need for alternative therapeutic approaches. We aimed to investigate the expression of KLKs in pancreatic cancer and to inhibit the function of KLK6 in pancreatic cancer cells. KLK6, KLK7, KLK8, KLK10 and KLK11 were coexpressed and upregulated in tissues from pancreatic cancer patients compared to normal pancreas. Their high expression levels correlated with each other and were linked to shorter survival compared to low KLK levels. We then validated KLK6 mRNA and protein expression in patient-derived tissues and pancreatic cancer cells. Coexpression of KLK6 with KRT19, αSMA or CD68 was independent of tumour stage, while KLK6 was coexpressed with KRT19 and CD68 in the invasive tumour area. High KLK6 levels in tumour and CD68+ cells were linked to shorter survival. KLK6 inhibition reduced KLK6 mRNA expression, cell metabolic activity and KLK6 secretion and increased the secretion of other serine and aspartic lysosomal proteases. The association of high KLK levels and poor prognosis suggests that inhibiting KLKs may be a therapeutic strategy for precision medicine.

Three-dimensional (3D) cancer models are invaluable tools designed to study tumour biology and new treatments. Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest types of cancer, has been progressively explored with bioengineered 3D approaches by deconstructing elements of its tumour microenvironment. Here, we investigated the suitability of collagen-nanocellulose hydrogels to mimic the extracellular matrix of PDAC and to promote the formation of tumour spheroids and multicellular 3D cultures with stromal cells. Blending of type I collagen fibrils and cellulose nanofibres formed a matrix of controllable stiffness, which resembled the lower profile of pancreatic tumour tissues. Collagen-nanocellulose hydrogels supported the growth of tumour spheroids and multicellular 3D cultures, with increased metabolic activity and matrix stiffness. To validate our 3D cancer model, we tested the individual and combined effects of the anti-cancer compound triptolide and the chemotherapeutics gemcitabine and paclitaxel, resulting in differential cell responses. Our blended 3D matrices with tuneable mechanical properties consistently maintain the growth of PDAC cells and its cellular microenvironment and allow the screening of anti-cancer treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Curvello, Kast, Abuwarwar, Fletcher, Garnier and Loessner.)

The tumour microenvironment (TME) comprises not only malignant and non-malignant cells, but also the extracellular matrix (ECM), secreted factors, and regulators of cellular functions. In addition to genetic alterations, changes of the biochemical/biophysical properties or cellular composition of the TME have been implicated in drug resistance. Here, we review the composition of the ECM and different elements of the TME contributing to drug resistance, including soluble factors, hypoxia, extracellular acidity, and cell adhesion properties. We discuss selected approaches for modelling the TME, current progress, and their use in low-and high-throughput assays for preclinical studies. Lastly, we summarise the status quo of advanced 3D cancer models compatible with high-throughput screening (HTS), the technical practicalities and challenges., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Neuroblastoma is a paediatric malignancy of the developing sympathetic nervous system. About half of the patients have metastatic disease at the time of diagnosis and a survival rate of less than 50%. Our understanding of the cellular processes promoting neuroblastoma metastases will be facilitated by the development of appropriate experimental models. In this study, we aimed to explore the invasion of neuroblastoma cells and organoids from patient-derived xenografts (PDXs) grown embedded in 3D extracellular matrix (ECM) hydrogels by time-lapse microscopy and quantitative image analysis. We found that the ECM composition influenced the growth, viability and local invasion of organoids. The ECM compositions induced distinct cell behaviours, with Matrigel being the preferred substratum for local organoid invasion. Organoid invasion was cell line- and PDX-dependent. We identified six distinct phenotypes in PDX-derived organoids. In contrast, NB cell lines were more phenotypically restricted in their invasion strategies, as organoids isolated from cell line-derived xenografts displayed a broader range of phenotypes compared to clonal cell line clusters. The addition of FBS and bFGF induced more aggressive cell behaviour and a broader range of phenotypes. In contrast, the repression of the prognostic neuroblastoma marker, MYCN , resulted in less aggressive cell behaviour. The combination of PDX organoids, real-time imaging and the novel 3D culture assays developed herein will enable rapid progress in elucidating the molecular mechanisms that control neuroblastoma invasion.

The main reasons for the slow progress in improving survival outcomes for ovarian cancer are the 'one-size-fits-all' therapy and lack of clinically relevant experimental models that represent the advanced stages of the human disease. The interaction of tumour cells with their surrounding niche, the tumour microenvironment, influences the spread of ovarian cancer cells within the peritoneum and their responses to therapeutics. Scientists are increasingly using 3D cell culture models to dissect the role of the tumour microenvironment in cancer development and progression and the treatment of this disease. In this chapter, we will briefly describe the tumour microenvironment of ovarian cancer. Then, we will review some of the clinically relevant experimental approaches, such as spheroid, organoid and organotypic models, that have been developed for the 3D culture of ovarian cancer cells using different tools, including hydrogels, scaffolds and cancer-on-a-chip devices, to mimic selected components of the tumour microenvironment., (© 2021. Springer Nature Switzerland AG.)

Bioengineered three-dimensional (3D) matrices expand our experimental repertoire to study tumor growth and progression in a biologically relevant, yet controlled, manner. Here, we used peptide amphiphiles (PAs) to coassemble with and organize extracellular matrix (ECM) proteins producing tunable 3D models of the tumor microenvironment. The matrix was designed to mimic physical and biomolecular features of tumors present in patients. We included specific epitopes, PA nanofibers, and ECM macromolecules for the 3D culture of human ovarian cancer, endothelial, and mesenchymal stem cells. The multicellular constructs supported the formation of tumor spheroids with extensive F-actin networks surrounding the spheroids, enabling cell-cell communication, and comparative cell-matrix interactions and encapsulation response to those observed in Matrigel. We conducted a proof-of-concept study with clinically used chemotherapeutics to validate the functionality of the multicellular constructs. Our study demonstrates that peptide-protein coassembling matrices serve as a defined model of the multicellular tumor microenvironment of primary ovarian tumors., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

In tumor engineering, 3D approaches are used to model components of the tumor microenvironment and to test new treatments. Pancreatic cancers are a cancer of substantial unmet need and survival rates are lower compared to any other cancer. Bioengineering techniques are increasingly applied to understand the unique biology of pancreatic tumors and to design patient-specific models. Here we summarize how extracellular and cellular elements of the pancreatic tumor microenvironment and their interactions have been studied in 3D cell cultures. We review selected clinical trials, assess the benefits of therapies interfering with the tumor microenvironment and address their limitations and future perspectives., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The quest for predictive tumor markers for osteosarcoma (OS) has not well progressed over the last two decades due to a lack of preclinical models. The aim of this study was to investigate if microenvironmental modifications in an original humanized in vivo model alter the expression of OS tumor markers. Human bone micro-chips and bone marrow, harvested during hip arthroplasty, were implanted at the flanks of NOD/scid mice. We administered recombinant human bone morphogenetic protein 7 (rhBMP-7) in human bone micro-chips/bone marrow group I in order to modulate bone matrix and bone marrow humanization. Ten weeks post-implantation, human Luc-SAOS-2 OS cells were injected into the humanized tissue-engineered bone organs (hTEBOs). Tumors were harvested 5 weeks post-implantation to determine the expression of the previously described OS markers ezrin, periostin, VEGF, HIF1α and HIF2α. Representation of these proteins was analyzed in two different OS patient cohorts. Ezrin was downregulated in OS in hTEBOs with rhBMP-7, whereas HIF2α was significantly upregulated in comparison to hTEBOs without rhBMP-7. The expression of periostin, VEGF and HIF1α did not differ significantly between both groups. HIF2α was consistently present in OS patients and dependent on tumor site and clinical stage. OS patients post-chemotherapy had suppressed levels of HIF2α. In conclusion, we demonstrated the overall expression of OS-related factors in a preclinical model, which is based on a humanized bone organ. Our preclinical research results and analysis of two comprehensive patient cohorts imply that HIF2α is a potential prognostic marker and/or therapeutic target. STATEMENT OF SIGNIFICANCE: This study demonstrates the clinical relevance of the humanized organ bone microenvironment in osteosarcoma research and validates the expression of tumor markers, especially HIF2α. The convergence of clinically proven bone engineering concepts for the development of humanized mice models is a new starting point for investigations of OS-related marker expression. The validation and first data set in such a model let one conclude that further clinical studies on the role of HIF2α as a prognostic marker and its potential as therapeutic target is a condition sine qua non., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Peritoneal invasion through the mesothelial cell layer is a hallmark of ovarian cancer metastasis. Using tissue engineering technologies, we recreated an ovarian tumor microenvironment replicating this aspect of disease progression. Ovarian cancer cell-laden hydrogels were combined with mesothelial cell-layered melt electrospun written scaffolds and characterized with proliferation and transcriptomic analyses and used as intraperitoneal xenografts. Here we show increased cancer cell proliferation in these 3D co-cultures, which we validated using patient-derived cells and linked to peritoneal tumor growth in vivo. Transcriptome-wide expression analysis identified IGFBP7, PTGS2, VEGFC and FGF2 as bidirectional factors deregulated in 3D co-cultures compared to 3D mono-cultures, which we confirmed by immunohistochemistry of xenograft and patient-derived tumor tissues and correlated with overall and progression-free survival. These factors were further increased upon expression of kallikrein-related proteases. This clinically predictive model allows us to mimic the complexity and processes of the metastatic disease that may lead to therapies that protect from peritoneal invasion or delay the development of metastasis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Tissue engineering technologies have produced controllable and reproducible three-dimensional (3D) models that mimic the architecture and complexity of native tissues. In particular cell biology-based research is driven by the development of cell culture platforms and techniques that allow the analysis of cells cultured in 3D. Here we summarize several easy-to-follow methods for the characterization of cells that have been encapsulated and grown in hydrogels to measure their cell viability, metabolic activity, and mechanical properties of cell-containing hydrogels. We also describe an enzymatic approach for the digestion of cell-containing hydrogels and cell recovery thereby maintaining high cell viability for subsequent analysis.

The primary tumor microenvironment is inherently important in prostate cancer (PCa) initiation, growth and metastasis. However, most current PCa animal models are based on the injection of cancer cells into the blood circulation and bypass the first steps of the metastatic cascade, hence failing to investigate the influence of the primary tumor microenvironment on PCa metastasis. Here, we investigated the spontaneous metastasis of PC3 human PCa cells from humanized prostate tissue, containing cancer-associated fibroblasts (CAFs) and prostate lymphatic and blood vessel endothelial cells (BVEC), to humanized tissue-engineered bone constructs (hTEBC) in NOD-SCID IL2Rγ null (NSG) mice. The hTEBC formed a physiologically mature organ bone which allowed homing of metastatic PCa cells. Humanization of prostate tissue had no significant effect on the tumor burden at the primary site over the 4 weeks following intraprostatic injection, yet reduced the incidence and burden of metastases in the hTEBC. Spontaneous PCa metastases were detected in the lungs and spleen with no significant differences between the humanized and non-humanized prostate groups. A significantly greater metastatic tumor burden was observed in the liver when metastasis occurred from the humanized prostate. Together, our data suggests that the presence of human-derived CAFs and BVECs in the primary PCa microenvironment influences selectively the metastatic and homing behavior of PC3 cells in this model. Our orthotopic and humanized prostate cancer model developed via convergence of cancer research and tissue engineering concepts provides an important platform to study species-specific PCa bone metastasis and to develop and test therapeutic strategies., Competing Interests: C.M. is a founder, shareholder and director of GELOMICS PTY Ltd, a start-up company developing and distributing hydrogels for 3D cell culture applications. D.W.H. is a founder and shareholder of GELOMICS PTY Ltd. All other authors declare no competing interests.

Background: Tissue kallikrein-related peptidases 4, 5, 6 and 7 (KLK4-7) strongly increase the malignancy of ovarian cancer cells. Deciphering their downstream effectors, we aimed at finding new potential prognostic biomarkers and treatment targets for ovarian cancer patients. KLK4-7-transfected (OV-KLK4-7) and vector-control OV-MZ-6 (OV-VC) ovarian cancer cells were established to select differentially regulated factors., Methods: With three independent approaches, PCR arrays, genome-wide microarray and proteome analyses, we identified 10 candidates (MSN, KRT19, COL5A2, COL1A2, BMP5, F10, KRT7, JUNB, BMP4, MMP1). To determine differential protein expression, we performed western blot analyses, immunofluorescence and immunohistochemistry for four candidates (MSN, KRT19, KRT7, JUNB) in cells, tumour xenograft and patient-derived tissues., Results: We demonstrated that KLK4-7 clearly regulates expression of MSN, KRT19, KRT7 and JUNB at the mRNA and protein levels in ovarian cancer cells and tissues. Protein expression of the top-upregulated effectors, MSN and KRT19, was investigated by immunohistochemistry in patients afflicted with serous ovarian cancer and related to KLK4-7 immunoexpression. Significant positive associations were found for KRT19/KLK4, KRT19/KLK5 and MSN/KLK7., Conclusion: These findings imply that KLK4-7 exert key modulatory effects on other cancer-related genes and proteins in ovarian cancer. These downstream effectors of KLK4-7, MSN and KRT19 may represent important therapeutic targets in serous ovarian cancer.

Introduction: Aberrant levels of kallikrein-related peptidases (KLK1-15) have been linked to cancer cell proliferation, invasion and metastasis. In ovarian cancer, the KLK proteolytic network has a crucial role in the tissue and tumor microenvironment. Publically available ovarian cancer genome and expression data from multiple patient cohorts show an upregulation of most KLKs. Areas covered: Here, we review the expression levels of all 15 members of this family in normal and ovarian cancer tissues, categorizing them into highly and moderately or weakly expressed KLKs, and their association with patient prognosis and survival. We summarize their tumor-biological functions determined in cell-based assays and xenograft models, further highlighting their suitability as cancer biomarkers and attractive candidates for drug development. Finally, we discuss some different pharmaceutical approaches, including peptide-based and small molecule inhibitors, cyclic peptides, depsipeptides, engineered natural inhibitors, antibodies, RNA/DNA-based aptamers, prodrugs, miRNA and siRNA. Expert opinion: In light of the results from clinical and tumor-biological studies, together with the available pharmaceutical tools, we suggest KLK4, KLK5, KLK6 and possibly KLK7 as preferred targets for inhibition in ovarian cancer.

Background: Existing preclinical murine models often fail to predict effects of anti-cancer drugs. In order to minimize interspecies-differences between murine hosts and human bone tumors of in vivo xenograft platforms, we tissue-engineered a novel orthotopic humanized bone model., Methods: Orthotopic humanized tissue engineered bone constructs (ohTEBC) were fabricated by 3D printing of medical-grade polycaprolactone scaffolds, which were seeded with human osteoblasts and embedded within polyethylene glycol-based hydrogels containing human umbilical vein endothelial cells (HUVECs). Constructs were then implanted at the femur of NOD-scid and NSG mice. NSG mice were then bone marrow transplanted with human CD34  +  cells. Human osteosarcoma (OS) growth was induced within the ohTEBCs by direct injection of Luc-SAOS-2 cells. Tissues were harvested for bone matrix and marrow morphology analysis as well as tumor biology investigations. Tumor marker expression was analyzed in the humanized OS and correlated with the expression in 68 OS patients utilizing tissue micro arrays (TMA)., Results: After harvesting the femurs micro computed tomography and immunohistochemical staining showed an organ, which had all features of human bone. Around the original mouse femur new bone trabeculae have formed surrounded by a bone cortex. Staining for human specific (hs) collagen type-I (hs Col-I) showed human extracellular bone matrix production. The presence of nuclei staining positive for human nuclear mitotic apparatus protein 1 (hs NuMa) proved the osteocytes residing within the bone matrix were of human origin. Flow cytometry verified the presence of human hematopoietic cells. After injection of Luc-SAOS-2 cells a primary tumor and lung metastasis developed. After euthanization histological analysis showed pathognomic features of osteoblastic OS. Furthermore, the tumor utilized the previously implanted HUVECS for angiogenesis. Tumor marker expression was similar to human patients. Moreover, the recently discovered musculoskeletal gene C12orf29 was expressed in the most common subtypes of OS patient samples., Conclusion: OhTEBCs represent a suitable orthotopic microenvironment for humanized OS growth and offers a new translational direction, as the femur is the most common location of OS. The newly developed and validated preclinical model allows controlled and predictive marker studies of primary bone tumors and other bone malignancies., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Modern tissue engineering technologies have delivered tools to recreate a cell's naturally occurring niche in vitro and to investigate normal and pathological cell-cell and cell-niche interactions. Hydrogel biomaterials mimic crucial properties of native extracellular matrices, including mechanical support, cell adhesion sites and proteolytic degradability. As such, they are applied as 3D cell culture platforms to replicate tissue-like architectures observed in vivo, allowing physiologically relevant cell behaviors. Here we review bioengineered 3D approaches used for prostate and breast cancer. Furthermore, we describe the synthesis and use of gelatin methacryloyl-based hydrogels as in vitro 3D cancer model. This platform is used to engineer the microenvironments for prostate and breast cancer cells to study processes regulating spheroid formation, cell functions and responses to therapeutic compounds. Collectively, these bioengineered 3D approaches provide cell biologists with innovative pre-clinical tools that integrate the complexity of the disease seen in patients to advance our knowledge of cancer cell physiology and the contribution of a tumor's surrounding milieu.

Mutagens like oxidants cause lesions in the DNA of ovarian and fallopian tube epithelial cells, resulting in neoplastic transformation. Reduced exposure of surface epithelia to oxidative stress may prevent the onset or reduce the growth of ovarian cancer. Lycopene is well-known for its excellent antioxidant properties. In this study, the potential of lycopene in the prevention and treatment of ovarian cancer was investigated using an intraperitoneal animal model. Lycopene prevention significantly reduced the metastatic load of ovarian cancer-bearing mice, whereas treatment of already established ovarian tumors with lycopene significantly diminished the tumor burden. Lycopene treatment synergistically enhanced anti-tumorigenic effects of paclitaxel and carboplatin. Immunostaining of tumor and metastatic tissues for Ki67 revealed that lycopene reduced the number of proliferating cancer cells. Lycopene decreased the expression of the ovarian cancer biomarker, CA125. The anti-metastatic and anti-proliferative effects were accompanied by down-regulated expression of ITGA5, ITGB1, MMP9, FAK, ILK and EMT markers, decreased protein expression of integrin α5 and reduced activation of MAPK. These findings indicate that lycopene interferes with mechanisms involved in the development and progression of ovarian cancer and that its preventive and therapeutic use, combined with chemotherapeutics, reduces the tumor and metastatic burden of ovarian cancer in vivo ., Competing Interests: None.

Tissue engineering technology platforms constitute a unique opportunity to integrate cells and extracellular matrix (ECM) proteins into scaffolds and matrices that mimic the natural microenvironment in vitro. The development of tissue-engineered 3D models that mimic the endosteal microenvironment enables researchers to discover the causes and improve treatments for blood and immune-related diseases. The aim of this study was to establish a physiologically relevant in vitro model using 3D printed scaffolds to assess the contribution of human cells to the formation of a construct that mimics human endosteum. Melt electrospun written scaffolds were used to compare the suitability of primary human osteoblasts (hOBs) and placenta-derived mesenchymal stem cells (plMSCs) in (non-)osteogenic conditions and with different surface treatments. Using osteogenic conditions, hOBs secreted a dense ECM with enhanced deposition of endosteal proteins, such as fibronectin and vitronectin, and osteogenic markers, such as osteopontin and alkaline phosphatase, compared to plMSCs. The expression patterns of these proteins were reproducibly identified in hOBs derived from three individual donors. Calcium phosphate-coated scaffolds induced the expression of osteocalcin by hOBs when maintained in osteogenic conditions. The tissue-engineered endosteal microenvironment supported the growth and migration of primary human haematopoietic stem cells (HSCs) when compared to HSCs maintained using tissue culture plastic. This 3D testing platform represents an endosteal bone-like tissue and warrants future investigation for the maintenance and expansion of human HSCs., Statement of Significance: This work is motivated by the recent interest in melt electrospinning writing, a 3D printing technique used to produce porous scaffolds for biomedical applications in regenerative medicine. Our team has been among the pioneers in building a new class of melt electrospinning devices for scaffold-based tissue engineering. These scaffolds allow structural support for various cell types to invade and deposit their own ECM, mimicking a characteristic 3D microenvironment for experimental studies. We used melt electrospun written polycaprolactone scaffolds to develop an endosteal bone-like tissue that promotes the growth of HSCs. We combine tissue engineering concepts with cell biology and stem cell research to design a physiologically relevant niche that is of prime interest to the scientific community., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)