The extracellular matrix (ECM) is highly dynamic as it is constantly deposited, remodeled and degraded to maintain tissue homeostasis. ECM is a major structural component of the tumor microenvironment, and cancer development and progression require its extensive reorganization. Cancerized ECM is biochemically different in its composition and is stiffer compared to normal ECM. The abnormal ECM affects cancer progression by directly promoting cell proliferation, survival, migration and differentiation. The restructured extracellular matrix and its degradation fragments (matrikines) also modulate the signaling cascades mediated by the interaction with cell-surface receptors, deregulate the stromal cell behavior and lead to emergence of an oncogenic microenvironment. Here, we summarize the current state of understanding how the composition and structure of ECM changes during cancer progression. We also describe the functional role of key proteins, especially tenascin C and fibronectin, and signaling molecules involved in the formation of the tumor microenvironment, as well as the signaling pathways that they activate in cancer cells. 

/pdf/the-functional-role-of-extracellular-matrix-proteins-in-2zdlu8a6.pdf

The Functional Role of Extracellular Matrix Proteins in Cancer

The tumour microenvironment plays a critical role in tumour progression and drug resistance processes. Non-malignant cell players, such as fibroblasts, endothelial cells, immune cells and others, interact with each other and with the tumour cells, shaping the disease. Though the role of each cell type and cell communication mechanisms have been progressively studied, the complexity of this cellular network and its role in disease mechanism and therapeutic response are still being unveiled. Animal models have been mainly used, as they can represent systemic interactions and conditions, though they face recognized limitations in translational potential due to interspecies differences. In vitro 3D cancer models can surpass these limitations, by incorporating human cells, including patient-derived ones, and allowing a range of experimental designs with precise control of each tumour microenvironment element. We summarize the role of each tumour microenvironment component and review studies proposing 3D co-culture strategies of tumour cells and non-malignant cell components. Moreover, we discuss the potential of these modelling approaches to uncover potential therapeutic targets in the tumour microenvironment and assess therapeutic efficacy, current bottlenecks and perspectives.

3D Cancer Models: Depicting Cellular Crosstalk within the Tumour Microenvironment.

Cancer still ranks as a leading cause of mortality worldwide. Although considerable efforts have been dedicated to anticancer therapeutics, progress is still slow, partially due to the absence of robust prediction models. Multicellular tumor spheroids, as a major three-dimensional (3D) culture model exhibiting features of avascular tumors, gained great popularity in pathophysiological studies and high throughput drug screening. However, limited control over cellular and structural organization is still the key challenge in achieving in vivo like tissue microenvironment. 3D bioprinting has made great strides toward tissue/organ mimicry, due to its outstanding spatial control through combining both cells and materials, scalability, and reproducibility. Prospectively, harnessing the power from both 3D bioprinting and multicellular spheroids would likely generate more faithful tumor models and advance our understanding on the mechanism of tumor progression. In this review, the emerging concept on using spheroids as a building block in 3D bioprinting for tumor modeling is illustrated. We begin by describing the context of the tumor microenvironment, followed by an introduction of various methodologies for tumor spheroid formation, with their specific merits and drawbacks. Thereafter, we present an overview of existing 3D printed tumor models using spheroids as a focus. We provide a compilation of the contemporary literature sources and summarize the overall advancements in technology and possibilities of using spheroids as building blocks in 3D printed tissue modeling, with a particular emphasis on tumor models. Future outlooks about the wonderous advancements of integrated 3D spheroidal printing conclude this review.

https://ijb.whioce.com/index.php/int-j-bioprinting/article/viewFile/444/pdf

Using Spheroids as Building Blocks Towards 3D Bioprinting of Tumor Microenvironment.

Immunotherapy discovery on tumor organoid-on-a-chip platforms that recapitulate the tumor microenvironment.

Cancer, one of the most dangerous diseases with a high mortality rate, has attracted much attention and interest. Advances in tissue engineering and microfluidics have led to the development of tumor-on-chips (TOCs) capable of in-vitro modeling the dynamic microenvironment, growth, and inter-cellular interactions of tumor tissues, providing a promising platform for oncology research. The search for powerful analytical tools for TOCs has facilitated the integration of electrochemical biosensors due to their high sensitivity and selectivity, low cost, portability, ease of fabrication, and low sample consumption. Recent reports have demonstrated promising applications of integrated electrochemical biosensors in cancer research at cellular and molecular levels in TOCs. In this review, we will introduce TOCs that model different stages of the tumor and highlight the use of electrochemical biosensors for the analysis of tumor cells and biomarkers. Future work may focus on the development of flexible and robust electrochemical biosensors with high mechanical compliance with tumor tissues for long-term monitoring of the tumor microenvironment. 

Emerging Tumor-on-Chips with Electrochemical Biosensors

Endothelial cells (ECs) are an important component of the tumor microenvironment, playing key roles in tumor development and progression that span from angiogenesis to immune regulation and drug resistance. Heterotypic tumor spheroids are one of the most widely used in vitro tumor microenvironment models, presenting improved recapitulation of tumor microenvironments compared to 2D cultures, in a simple and low-cost setup. Heterotypic tumor spheroid models incorporating endothelial cells have been proposed but present multiple limitations, such as the short culture duration typically obtained, the use of animal-derived matrices, and poor reproducibility; the diversity of culture conditions employed hinders comparison between studies and standardization of relevant culture parameters. Herein, we developed long-term cultures of triple heterotypic spheroids composed of the HCC1954 tumor cell line, human fibroblasts, and ECs. We explored culture parameters potentially relevant for EC maintenance, such as tumor cell line, seeding cell number, cell ratio, and agitation vs. static culture. In HCC1954-based spheroids, we observed maintenance of viable EC for up to 1 month of culture in agitation, with retention of the identity markers CD31 and von Willebrand factor. At the optimized tumor cell:fibroblast:EC ratio of 1:3:10, HCC1954-based spheroids had a higher EC area/total spheroid area at 1 month of culture than the other cell ratios tested. EC maintenance was tumor cell line-dependent, and in HCC1954-based spheroids it was also dependent on the presence of fibroblasts and agitation. Moreover, vascular endothelial growth factor (VEGF) supplementation was not required for maintenance of EC, as the factor was endogenously produced. ECs co-localized with fibroblasts, which accumulated preferentially in the core of the spheroids and secreted EC-relevant extracellular matrix proteins, such as collagen I and IV. This simple model setup does not rely on artificial or animal-derived scaffolds and can serve as a useful tool to explore the culture parameters influencing heterotypic spheroids, contributing to model standardization, as well as to explore molecular cross talk of ECs within the tumor microenvironment, and potentially its effects on drug response.

/pdf/heterotypic-tumor-spheroids-in-agitation-based-cultures-a-94796z2r1z.pdf

Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial Cells

Colorectal cancer (CRC) is one of the most common cancers worldwide. Although short-term cultures of tumour sections and xenotransplants have been used to determine drug efficacy, the results frequently fail to confer clinically useful information. Biomarker discovery has changed the paradigm for advanced CRC, though the presence of a biomarker does not necessarily translate into therapeutic success. To improve clinical outcomes, translational models predictive of drug response are needed. We describe a simple method for the fast establishment of CRC patient-derived explant (CRC-PDE) cultures from different carcinogenesis pathways, employing agitation-based platforms. A total of 26 CRC-PDE were established and a subset was evaluated for viability (n = 23), morphology and genetic key alterations (n = 21). CRC-PDE retained partial tumor glandular architecture and microenvironment features were partially lost over 4 weeks of culture. Key proteins (p53 and Mismatch repair) and oncogenic driver mutations of the original tumours were sustained throughout the culture. Drug challenge (n = 5) revealed differential drug response from distinct CRC-PDE cases. These findings suggest an adequate representation of the original tumour and highlight the importance of detailed model characterisation. The preservation of key aspects of the CRC microenvironment and genetics supports CRC-PDE potential applicability in pre- and co-clinical settings, as long as temporal dynamics are considered.

/pdf/patient-derived-explants-of-colorectal-cancer-2kkd1ifo8k.pdf

Teresa Franchi-Mendes

Papers

3D Cancer Models: Depicting Cellular Crosstalk within the Tumour Microenvironment.

Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial Cells

Patient-Derived Explants of Colorectal Cancer: Histopathological and Molecular Analysis of Long-Term Cultures