Advancements in 3D In Vitro Models for Colorectal Cancer

Abstract

The process of drug discovery and pre-clinical testing is currently inefficient, expensive, and time-consuming. Most importantly, the success rate is unsatisfactory, as only a small percentage of tested drugs are made available to oncological patients. This is largely due to the lack of reliable models that accurately predict drug efficacy and safety. Even animal models often fail to replicate human-specific pathologies and human body's complexity. These factors, along with ethical concerns regarding animal use, urge the development of suitable human-relevant, translational in vitro models.

Keywords: colorectal cancer; patient‐derived organoids; preclinical models for cancer; tumor microenvironment.

Figure 1

Crosstalk between CRC cells and different components of the TME. Schematic representation of the reciprocal communication network of colorectal cancer cells with the cell types that compose the tumor niche. Cells communicate mainly through soluble factors such as cytokines and chemokines, metabolites and extracellular vesicles, influencing tumor growth, immune responses, angiogenesis, and therapeutic outocomes in colorectal cancer. In each box are indicated the main soluble factors secreted by each cell type, that trigger pro‐tumoral pathways. Black arrows indicate direct communication, while dashed arrows signify indirect communication, not mediated by secreted factors. Among the principal components of the TME, cancer‐associated fibroblasts (CAFs) play a role in supporting colorectal cancer growth. CAFs and cancer cells exert a mutual influence by the indicated soluble cues (from CAFs to cancer cells,^{[ 20} , ²¹ , ²² , ²³ , ²⁴ , ²⁵ , ²⁶ , ²⁷ , ²⁸ , ²⁹ , ³⁰ , ³¹ , ³² , ³³ , ³⁴ , ³⁵ , ³⁶ , ³⁷ , ³⁸ , ^{39 ]} from cancer cells to CAFs.^{[ 21} , ²⁴ , ³⁶ , ⁴⁰ , ⁴¹ , ⁴² , ^{43 ]} Tumors require a blood supply for nutrients and oxygen. Blood vessels within the TME, known as tumor vasculature, can be abnormal, leaky, and disorganized. Cancer cells stimulate endothelial cells’ proliferation and neovascularization by releasing VEGF in the TME.^{[ 44 ]} The presence of cancer cells can trigger visceral adipose tissue to adopt an inflammatory pro‐tumoral phenotype,^{[ 46 ]} which in turn, produces molecules and metabolites that sustain tumor outgrowth.^{[ 45} , ⁴⁶ , ⁴⁷ , ⁴⁸ , ⁴⁹ , ⁵⁰ , ^{51 ]} The immune system, namely T cells, B cells, natural killer (NK) cells, macrophages, and dendritic cells, infiltrate the TME. They interact with cancer cells and can have both pro‐tumor and anti‐tumor effects. Here we reported secreted factors produced by immune cells that sustain pro‐tumoral adaptation of cancer cells,^{[ 52} , ⁵³ , ⁵⁴ , ⁵⁵ , ^{56 ]} and those secreted by cancer cells that induce a pro‐tumoral phenotype in immune cells.^{[ 55} , ⁵⁶ , ⁵⁷ , ⁵⁸ , ⁵⁹ , ⁶⁰ , ⁶¹ , ^{62 ]} The enteric nervous system has a pivotal role in physiological intestine functions. When properly stimulated by cancer cells,^{[ 63} , ^{66 ]} it can sustain colorectal cancer stem cells self‐renewal and tumorigenesis, proliferation and metastatic spread.^{[ 63} , ⁶⁴ , ⁶⁵ , ⁶⁶ , ⁶⁷ , ⁶⁸ , ^{69 ]} The intestinal microbiota can strongly impact tumor behaviour, by releasing in the TME metabolites and signaling molecules,^{[ 70} , ^{71 ]} causing local inflammation or genetic damage of colonic epithelial cells and subsequent oncogenic transformation. Cells resident in metastatic sites communicate with cancer cells and support their homing and growth by secreting specific factors within the niche. Here, we represented hepatocytes and their secreted factors as the more abundant cell type in the liver, the most frequent metastatic site for CRC.^{[ 72} , ^{73 ]}

Figure 2

Co‐evolution of colorectal cancer and its tumor microenvironment. Schematic of CRC development and progression illustrates the cellular composition and the ECM organization of healthy colon mucosa and its modifications during CRC evolution. The legend on the left provides a comprehensive list of the key cellular players involved in this process. a) HEALTHY COLON MUCOSA‐Intestinal crypt homeostasis relies on intestinal stem cells located at the bottom of the crypt, and differentiated cells, such as the enterocytes, goblet, tuft, enteroendocrine and Paneth cells (indicated as “intestinal epithelial cells”). In healthy colon tissue, the microenvironment comprises quiescent intestinal fibroblasts surrounded by a soft extracellular matrix (“soft ECM”), alongside a network of blood vessels, nerve cells from the mesenteric plexus, gut‐associated lymphoid tissue organized into Peyer's Plaques (depicted as grey structures on the right) and visceral adipose tissue. b) ADENOMA‐During the early phases of CRC progression, the immune system promotes immunosurveillance, an inflammatory response aimed to control tumor proliferation through both innate (neutrophil N1, macrophage M1, stimulatory dendritic cell – “sDC” ‐, natural killer NK1), and adaptive immune cells (B and T lymphocytes). Immune cells mainly implicated in this phase are shown in the left legend (middle panel, light blue). In this context, quiescent fibroblasts (light orange) become reversibly activated, proliferate and support the inflammatory response. Cancer cells and cancer stem cells are indicated in pink and dark pink, respectively. c) ADENOCARCINOMA‐Through adaptive mechanisms and the accumulation of genomic mutations, cancer cells gain the capability to evade immunosurveillance (immunoescape) and hijack the immune cells to promote their own proliferation. Cancer cells may inhibit the function of immune cells or promote the recruitment of immunosuppressive cells to the tumor. This step is characterized by an immunological switch toward a more tolerogenic and immunosuppressive phenotype. Immune cells mainly implicated in this phase are shown in the left legend (lower panel, dark blue), neutrophil N2, macrophage M2, regulatory/tolerogenic dendritic cell (rDC), natural killer NK2, myeloid‐derived suppressor cell (MDSC), lymphocytes and T regulatory cells (Treg). Moreover, fibroblasts activated during the inflammatory phase, acquire irreversible features and secrete cytokines that support tumor growth and extracellular matrix proteins. ECM surrounding the tumor is characterized by high stiffness due to the composition and spatial organization of collagen fibrils, linearized into tight bundles (“stiff ECM”), that substitute the random network of relaxed fibrils observed in the healthy ECM (“soft ECM”). Tumor cells also induce proliferation of endothelial cells, leading to neoangiogenesis. Adipose and nerve cells support tumor growth by secreting specific factors. As tumor progression continues, cancer cells may acquire migratory capabilities and enter the bloodstream to colonize distant organs.

Figure 3

Strategies to mimic CRC in vitro. 3D in vitro technologies to reproduce CRC TME and cell‐cell interactions are shown. For each system, relevance, versatility/applications (including advantages and limitations), technical issues, handling and related references are described in the boxes. The techniques are arranged from the least to the most complex (orange triangle below). a) Transwell is an indirect co‐culture technique that reproduces paracrine communication between cells that share the same medium but are physically separated by a permeable membrane. b) Spheroids and tumoroids are 3D cell aggregates that allow the self‐organization of different cell types (cancer cell and TME cells) and mimic some features of a solid tumor mass, such as detachment from a substrate, nutrients availability and gradients, cell‐cell interaction and paracrine signaling. c) Matrix droplets allow to reproduce the ECM complexity using different types of 3D matrices (hydrogel, Matrigel, collagen). This system can be combined with other technologies such as bioprinting or microfluidic devices (e; g). d) ALI is a cell culture system in which cells are seeded on a semi‐permeable membrane, either embedded within a matrix (as in c) or in liquid medium. Their basal membrane is in contact with culture medium and their apical surface is exposed to the air. e) 3D bioprinting is a technique that allows the fabrication of biological constructs through the precise deposition and localization of multiple layers of cells in supporting biopolymers. f) Decellularized scaffolds are natural ECMs that maintain the biochemical and ultrastructural properties of the tissue of origin. The process of decellularization enables complete cell removal while maintaining tissue architecture and ECM components. g) Cancer‐on‐a‐CHIP/Multi OoC are microfluidic devices designed to mimic in vitro the physiological conditions of tumors including cellular interactions, biochemical and niche factors gradients, tissue barriers, vascular perfusion and mechanical forces. h) Patient‐derived explants (PDEs) allow the culture of freshly resected tumor fragments or slices, while preserving the native 3D tissue architecture and cellular composition (tumor cells, stromal cell, TILs). Part of the figure is modified from Servier Medical Art (http://smart.servier.com/), licensed under a Creative Common Attribution 3.0 Generic License.

Figure 4

3D bioprinting. 3D bioprinting is an advanced technology that enables the creation of complex 3D biological structures that mimic the native tissue architecture, by precisely arranging multiple layers of cells and/or supporting biopolymers. The figure represents a schematic of a 3D bioprinting device (left upper panel) that was employed for the fabrication of a collagen‐based 3D scaffold, subsequently populated with tumor cells, cancer associated fibroblasts (CAFs) and tumor‐associated endothelial cells (TECs) (left lower panel). Adapted with permission.^{[ 107 ]} Copyright 2020, Ivyspring International Publisher. Transcriptomic analyses reveal that CRC avatars grown on 3D‐bioprinted scaffolds better reproduce gene expression of in vivo models, if compared to 2D cell‐cultures. This is shown in the right panel containing a clustered heatmap of 142 metabolic genes expressed in the 2D co‐cultured cells (2D‐co), 3D tumor tissue (3DT), and in vivo colorectal tumor (CT). Adapted with permission.^{[ 107 ]} Copyright 2020, Ivyspring International Publisher.

Figure 5

Preparation and Applications of dECM. The procedure to obtain dECM is described in the upper box, comprising the main steps of available protocols A–D). A) Following surgical resection, fresh colon tissues are promptly transferred to the collection medium and processed. B) In a petri dish, fat and blood clots are removed from tissues and rinsed in sterile PBS. C) Samples are cut and decellularization is achieved through chemical, enzymatic and/or physical treatments.^{[ 108} , ¹⁰⁹ , ^{112 ]} Upon completion of the process, tissue turns translucent and DNA content is significantly reduced. Immunofluorescence images in panel C show the loss of cellular content in both decellularized healthy mucosa and CRC samples (DAPI staining‐blue) while preserving the matrix structure (Laminin staining‐green). Adapted with permission.^{[ 111 ]} Copyright 2020, MDPI. D) Finally, the decellularized scaffold can be utilized for recellularization or pulverized into a fine powder and used as hydrogel in 3D co‐culture, microfluidic devices and bioprinting.^{[ 102} , ¹¹⁰ , ^{112 ]} The lower box shows the main applications of recellularized scaffolds. Recellularization of dECM derived from healthy mucosa, CRC or metastatic sites enables the creation of models for tumor progression and metastasis as well as in vitro platforms for drug screening or gene expression profiling.^{[ 109} , ¹¹⁰ , ^{111 ]} E) Example of histological characterization of CRC‐derived scaffold recellularized with HT‐29 cells, hematoxylin and eosin (H&E); periodic acid‐Shiff (PAS); collagen IV staining (Col IV); IF staining with epithelial marker E‐Cadherin (E‐Cad), basement membrane marker Laminin (Lam) and DAPI [adapted from reference 111]. G) Example of dECM application in chemotherapy response studies, IC50 values indicate higher resistance to 5FU in HT29 cultured within 3D scaffolds compared to 2D monolayers. Adapted with permission.^{[ 111 ]} Copyright 2020, MDPI. Part of the figure is modified from Servier Medical Art (http://smart.servier.com/), licensed under a Creative Common Attribution 3.0 Generic License.

Figure 6

Cancer on a chip (CoC). Schematic of multi‐organ on chip (Multi‐OoC) for CRC modelling. By combining multi‐organ systems and 3D cell culture, this technology enables the connection of CRC cells and TME models with multiple organs and tissues in a closed‐loop system. Moreover, this platform offers a powerful tool for mimicking cancer metastasis (metastasis‐on‐a‐chip). For example, it integrates CRC cells with liver, lung, and endothelial cells to reproduce the tropism of CRC cells during spreading to distant organs. Adapted with permission.^{[ 125 ]} Copyright 2018, Wiley Periodicals, Inc. Panel A) shows an example of a metastasis‐on‐a‐chip device consisting of a main microfluidic chamber seeded with CRC cells, connected to multiple downstream chambers in which endothelial (E), lung (Lu), liver (Li), and gel control constructs are housed B). Under recirculating fluid flow, the movement of cells can be tracked via fluorescent imaging (RFP+ cells) C) from the primary site to 4 downstream potential sites of metastasis, reproducing CRC cells spread and tropism for the liver and lungs. Adapted with permission.^{[ 125 ]} Copyright 2018, Wiley Periodicals, Inc. Part of the figure is modified from Servier Medical Art (http://smart.servier.com/), licensed under a Creative Common Attribution 3.0 Generic License.

Figure 7

Patient‐derived explants (PDE). Explants can be generated by fragmentation (PDE) or slicing (tumor slice) of fresh tumor tissue. In the left panels, representative H&E images of colorectal parental tumour tissue and derived explants (CRC‐PDE) over 4 weeks of culture; representative immunohistochemical images of T lymphocytes (CD3‐positive) and macrophages (CD68‐positive) detection on the CRC‐derived explants at weeks 2 of culture. Adapted with permission.^{[ 144 ]} Copyright 2021, MDPI. In the right panels, an example of PDE slice preparation, tumor tissue cores are collected from fresh specimens A) and placed on ice in a storage solution; then tissue cores are embedded in agarose individually B) or grouped C) and adhered to specimen disc D). The disc is placed on ice in the buffer tray with a cutting solution for slicing E). Vibratome cuts cores into tumor slices with desired thickness and placed them in a dish with medium for culture. Tumor slices can undergo evaluation for treatment response, viability, histology, imaging, and multi‐omics analyses. Adapted with permission.^{[ 131 ]} Copyright 2021, Elsevier.