# Applications of Tumor Cells in an _In Vitro_ 3D Environment
## 7 Applications of 3D Models of Cancer Cells  
### Colorectal Cancer  
CRC is the third most common cause of cancer-related deaths worldwide [20; 47]. Due to the anatomical proximity, CRC most often metastasizes in the liver. Up to 25% of patients present with CRC liver metastases at the time of diagnosis, and 50-60% of CRC patients will develop liver metastases at some point later on [5]. Unfortunately, all patients are not eligible for hepatectomy at the time of diagnosis and will, therefore, suffer from progressive metastatic CRC. The incidents of CRC have been increasing, especially in younger patients [16]. Despite significant improvements in treatment, the prognosis for patients with metastatic CRC (mCRC) is still poor [48]. Molecular profiling consists of a pathologic assessment of the tumor tissue to test for microsatellite instability-high (MSI-H) and mismatch repair-deficient (MMR-D), as well as for sequential variants in KRAS, NRAS, and BRAF genes that have been applied for the prognosis of mCRC and the prediction of response to adjuvant therapy in some local clinical practices [49; 50]. Novel and more effective therapeutic treatments are, therefore, necessary. Two-dimensional (2D) cell cultures are not accurately representing the interactions between the CRC cells and other components of the TME, including immune cells, cancer-associated fibroblasts (CAFs), and stromal cells, _in vivo_. It has been shown that the stromal compartment of CRC cells can be involved in alterations in disease progression and therapeutic response [51]. In addition, cytokines and growth factors that are present can affect CRC initiation and progression [16]. The CRC HCT116 cell line is frequently used for culturing 3D spheroids. The cell line has a mutation in codon 13 of the ras proto-oncogene [52]. [PERSON] et al. used HCT116 cells to detect over 1350 proteins with very little variation between replicate spheroids [53]. Moreover, cellular populations of the CRC cell line HT29 showed differences between the outer proliferating layer, the senescent middle layer, and the necrotic core, which is in line with comparable _in vivo_ models [54]. The HCT116 and HT29 cancer cell lines belong to the most often utilized CRC model cells in 2D cultures and have, therefore, frequently been investigated in 3D spheroid cultures [55; 56]. [PERSON] et al. published one of the first devices that allowed the building of tissue microarrays of spheroids cultured under various conditions or from different cell lines straight from the culture plate, suited to spheroids grown in high-throughput plate-based formats [57]. Eleven different cell lines were cultured and analyzed, including HCT116. The HCT116 cell line did not require addition or Cultrex basement membrane extract and formed compact spheroids. Moreover, [PERSON] et al. used immunohistochemistry to detect protein markers, but the method could equally be applied to detect RNA or DNA markers, e.g., using _in situ_ hybridization methods [57]. [PERSON] et al. described the time line for the first organoid studies on CRC cells performed less than fifteen years ago [16]. Indeed, the patient material of tumor tissue was more comparable with CRC cells than spheroids based on cell lines [58]. Organoids are being used to examine the molecular and genetic bases of CRC initiation and progression [59]. Patient-derived organoids (PDOs) are robust 3D _in vitro_ models that retain the histopathological features of _in vivo_ tumors, including patient-specific drug responses [60]. [PERSON] et al. analyzed the interplay between EGF signaling and the activation of MAPK/ERK using PDOs from KRAS and BRAF mutant CRCs. Without activity through the EGF receptor (EGF-R), signaling by these two oncogenes is insufficient to sustain the full cell proliferative potential. The study provided data that revealed that EGFR signaling actively amplifies the signaling output of oncogenic MAPK effectors. Individual treatment of CRC is preferred since the disease is very heterogeneous.