Precision medicine research progress based on colorectal cancer organoids

Abstract

Organoids are cultures that use stem cells to induce differentiation or clinical samples as raw materials to form three-dimensional spatial structures through specific culture systems, thereby simulating the structure and function of tissues in vivo. Organoids are characterized by their ease of construction, long-term stable passage, clinical authenticity, and application in high-throughput drug screening. Patient-derived organoids have great application value in colorectal cancer research and individualized treatment. In this review, recent advances in colorectal cancer organoid culture technology, clinical individualized drug sensitivity assessment, new drug screening, drug mechanism research, nanomedicine-based applications, and imaging technology are summarized. These advancements will provide support for the precise diagnosis and treatment of colorectal cancer and the development of new treatment strategies, which will have significant scientific value and clinical transformation value.

Organoids are the intersection of clinical and basic research

Malignant tumor is a major disease that seriously endangers human health. As tumor treatment enters the era of precision diagnosis and treatment, the comprehensive application of surgery, chemoradiotherapy, targeted therapy, immunotherapy and other means has improved the treatment effect and prolonged the survival time of tumor patients [1]. Long-term survival with tumor makes tumor gradually become a chronic disease. However, it is difficult to overcome the clinical problems such as recurrence, drug resistance and metastasis caused by the malignant biological behavior of tumors. Accurate monitoring and evaluation of the dynamic changes in tumor tissues during comprehensive treatment is an important basis for the realization of individualized treatment, the development of anti-tumor new drugs, the screening of tumor markers, and the exploration of the mechanism of tumor occurrence and development. The construction of biological models suitable for reflecting the real state in vivo is the premise of accurate monitoring and evaluation.

Samples generated in clinical work are not only an important tool for disease diagnosis and treatment, but also a valuable resource for medical research. The characteristics and quality of samples directly determine their clinical diagnosis and treatment and scientific research value. The introduction of formalin-fixed tissue samples in the late 19th century promoted the rapid development of pathology. Formalin fixation preserves histological morphology and denatures proteins and DNA without degradation, but it does not block RNA degradation [2]. Since the 20th century, the use of ultra-low temperature preservation of tissue samples has preserved the activity of biological macromolecules such as protein, DNA, RNA, etc., which makes the construction of large-scale biobank a reality and greatly promotes the progress of biomedicines [3]. Cryopreservation technology can also realize the freezing and recovery of cell lines and some tissues [4]. However, in general, for most clinical tissue specimens, formalin and cryopreservation fix the tissues in the “in vitro state”, and the test results obtained on these tissue samples reflect the “in vitro state of the body”, and the continuous and dynamic evolution of the body can only be indirectly speculated, but cannot be directly confirmed. Therefore, from the clinical perspective, the development of in vitro tissue culture technology and the realization of the qualitative change of clinical samples from dead to alive will provide a strong support for the exploration of the mechanism of the occurrence and development of diseases and the research and development of treatment strategies.

The technology of ex vivo tissue culture of primary cells originated in the early 20th century and has been widely used in the research of various diseases [5]. Cell culture has the advantages of convenient observation, controllable conditions, and diverse detection methods. Since the establishment of the human tumor cell line Hela in 1951, scientists have used a variety of immortalized cell lines to study the malignant biological behavior and development mechanism of tumors, covering the structure and function of biological macromolecules, signal transduction networks, metabolic remodeling and therapeutic strategies, which has greatly promoted people’s understanding of tumors and improved the ability of diagnosis and treatment [6]. However, long-term cultured cell lines lose their original three-dimensional structure, are isolated and lack of cell interaction, which cannot effectively reflect the cell function in the complex environment in vivo. More importantly, with the deepening of the concept of precision medicine, there is a growing demand for therapeutic strategies based on the characteristics of individual tissues and cells. Long-term cultured cell lines derived from a few patients decades ago cannot effectively match the individualized treatment needs. Therefore, from the perspective of research, it is necessary to break through the limitations of traditional two-dimensional cell culture technology and develop new cultures with complex structures and similar in vivo functions to further understand the mechanism of disease occurrence and development.

The common needs of clinical and basic research drive technological progress, resulting in organoids, a new culture model. Organoids are cultures that use stem cells to induce differentiation or clinically derived samples as raw materials to form three-dimensional spatial structures through specific culture systems, so as to simulate the structure and function of tissues in vivo [7, 8]. A large number of studies and reviews on organoids have shown that, compared with two-dimensional cell lines, organoids can better simulate the complex structure of body tissues, and compared with model animals, organoids are easy to construct, long-term stable passage, and suitable for high-throughput drug screening [9, 10], which can provide an important platform for the study of disease mechanisms, drug screening and individualized treatment. With the continuous progress of technology, the application range of organoids continues to expand, from cancer research [11, 12] to regenerative medicine [13, 14], showing a good prospect, can be said to be the intersection of clinical and basic research needs, but also a new starting point of clinical and basic research [15, 16]. In order to reflect the latest research progress of colorectal cancer organoids, we reviewed the research progress of colorectal cancer organoids in the past three years.

Progress of colorectal cancer organoid culture technology

With the development of theory and culture technology, and the control of microbial contamination, the success rate of organoid culture is constantly improving, Organoid systemic solutions were generated [17]. Microbial contamination is a major problem in organoid culture of colorectal cancer. Aiming at the problem of microbial contamination in organoid culture, although the protocol for generating organoids from colorectal tissue has been extensively optimized, there is still a lack of standardization of tissue processing, resulting in microbial contamination of organoid cultures. Marinucci et al. tested the efficacy of phosphate buffered saline(PBS), penicillin/streptomycin (P/S), and primocin alone or in combination to prevent organoid culture contamination during the washing step prior to tissue processing. They found that prior to tissue processing, adding primocin to the tissue washing solution is able to eliminate the risk of microbial contamination in organoids cultures, and that the use of P/S negatively impacts organoids growth [18]. This protocol is helpful to improve the success rate of organoid culture in colorectal cancer.

In terms of organoid culture technology, cell embedment in solid matrices is the current mainstay for the growth and propagation of patient-derived colorectal organoids and tumoroids [19, 20]. But cell embedment in solid matrices is inadequate for scalable experimental and high-throughput screening applications. Due to solid stress accumulation, oxygen and nutrient transport [21], organoid growth in the solid matrix is limited and frequent passage is required to maintain and expand the culture. Solid matrix must be removed by mechanical or enzymatic methods to separate organoids and tumor-like cells for proliferation or experimental applications [8]. In order to improve the culture efficiency of organoids, Tan et al. developed a protocol for growing organoids in low-viscosity matrix (LVM) suspension culture, which can be rapidly expanded and easily adapted to high-throughput screening. LVM suspension cultures could be easily harvested, with floating live organoids readily separated from tissue or cell debris precipitating at well bottoms, and could be dispensed using liquid-handling systems without additional cooling requirements to keep Matrigel in a liquid state. This technology can effectively reduce the manpower and cost of organoid establishment [22].

Organoids embedded in solid matrices are difficult to access the apical surface, which to some extent hampers drug penetration [23]. In order to improve the culture efficiency and drug penetration of organoids, Zhao et al. established an innovative two-dimensional (2D) and three-dimensional (3D) model for patient-derived cancer cells (PDCCs) culture and air-liquid interface (ALI) organotypic culture. In the ALI organotypic culture system, Matrigel is replaced by 3T3-J2 feeder cells, a type of stromal cell designed to support the physiological microenvironment. The 3D organotypic culture produced in the ALI system is a multilayered tumor cell that is physiologically closer to the histological features of its parent tumor and has an open “lumen” that maintains the apic-basal polarity of the parent tumor. Both 2D and 3D models maintained the transcriptomic characteristics of the respective primary tumors and showed the same trend in response to 5-fluorouracil [24]. With its superior gas exchange ability and nutrient supply, Air-liquid interface provides a good microenvironment for the growth and development of organoids, and has great advantages in the culture of organoids such as lung and gastrointestinal tract [25, 26]. This technology not only simulates the in vivo environment, but also improves cell survival and functional performance, thus providing strong support for disease mechanism research, drug screening, and personalized therapy [27].

With the development of microfluidic technology, the development of OrganoidChip has become an important direction in the study of colorectal cancer. OrganoidChip combines organoid and microfluidic technology to simulate the physiological microenvironment of the intestine on a single platform, thereby improving the efficiency and accuracy of drug screening [28–31]. This integrated technology not only enables real-time monitoring of drug response, but also enables multiple experiments under different biological conditions, promoting the development of personalized medicine. Pinho et al. combined microfluidic technology with 3D tumor organoid models to develop a low-cost microfluidic device for growing and expanding organoids - OrganoidChip for reproducing tumor tissue and in vivo functions. The viability and proliferative activity of colorectal cancer organoids were significantly improved when they were cultured in OrganoidChip. There were significant morphological differences between colorectal cancer organoids cultured in OrganoidChip and those cultured on plates. The colorectal cancer organoids cultured in organoid chips showed late morphology, which was more in line with the needs of organoid culture. There was no significant difference in response to 5-fluorouracil (5-FU) treatment between organoids on chip and those on the plate [32]. Jung et al. demonstrated a scalable organoid production platform with 8 pore strips, each with a total of 72 micropores, and single cells self-organizing into organoids in a medium containing 2% matrix glue. It is functionally compatible with existing 96-well plates and traditional organoid culture methods based on Matrigel, but the consistency, uniformity and reproducibility of organoids are significantly improved, showing better consistency in drug screening [33].

Although organoid culture techniques have undergone multiple innovations, their ability to fully demonstrate tumor heterogeneity remains limited. Due to the heterogeneity of tumors, tumor organoids from multiple cells may contain different cell subpopulations, and the throughput of conventional organoid culture platforms is generally insufficient to analyze rare phenotypes [34]. This puts forward higher requirements for the resolution and throughput of tumor organoid technology. In a recent study, Zihe Chen’s team developed a microfluidic chip with 30,000 micropores that is specifically designed for single-cell culture methods for selective expansion and differential induction of cancer stem cells. This method can generate a large number of single-cell-derived tumor organoids (STOS) and is particularly suitable for high-throughput tumor heterogeneity analysis. A comprehensive analysis of histometry, immunohistochemistry, drug response assessment, cell invasion assessment, and biomarker detection revealed heterogeneity among STOS. Compared with previous organoids-on-a-chip technologies, this method has advantages in cell recovery, prevent cell loss, convenience compatibility of stem cell expansion and compatibility of on-chip detection [34].

The methods of organoid culture are constantly innovated, but the growth of organoids always depends on the induction of medium. Although most colorectal cancer organoid culture systems successfully construct organoids and preserve tumor heterogeneity, they often neglect the protection of tumor heterogeneity and purity. The purity of tumor organoids is also a concern in organoid culture system [35]. Therefore, it is important to establish an optimal culture system of colorectal cancer organoids that can better retain the characteristics of the original tumor. Wang et al. conducted a comprehensive assessment of the relationship between culture conditions and the biological characteristics of organoids in patients with colorectal cancer and found that conditioned medium was superior to chemically defined medium in long-term culture of tumor epithelial cells [36]. Tan et al. explored organoid culture formulations and found that activation of Wnt and EGF signals and suppression of BMP signals were not necessary for the survival of most colorectal cancer organoids. They designed a growth factor reduction medium containing FGF10, A83-01 (TGF-β type I receptor inhibitor), SB202190 (p38 MAPK inhibitor), gastrin, and niacinamide. The use of this medium can maintain tumor characteristics in long-term colorectal cancer organoid culture, as demonstrated by histopathology, genetic stability, tumorigenicity, and clinical therapeutic response [37]. Integrating genomic, transcriptomic, and proteomic data as well as correlation analysis of thawing, time, and culture conditions can be used for detailed characterization of organoid molecular stability.

In terms of organoid cell sources, in addition to traditional surgical specimens, other specimens from patients can also be used for organoid culture. Bergin et al. systematically described a protocol for establishing a serial tumor organoid platform using primary colorectal cancer tissues, and introduced in detail the isolation, organoid embedding, serial passage, and cancer stem cell (CSC) related analysis techniques of primary colorectal cancer stem cells [38]. Engel et al. generated organoids from surgical samples of primary and metastatic colorectal cancer. These organoids retained key histopathological features similar to the primary tumors and exhibited a high degree of concordance in critical driver mutations [39]. Angelis et al. isolated circulating tumor cells (CTCS) from the in-situ xenograft model of colorectal cancer and used CTCS to generate organoids. CTC-derived organoids (CTCDO) were characterized by proteomic analysis, immunohistochemistry, immunofluorescence, flow cytometry, tumor-forming capacity, and drug screening analyses, and a number of intracellular and extracellular markers were identified and validated on CTCS isolated from peripheral blood of CRC patients [40]. Roberto Coppo et al. investigated the preparation and culture of organoids using the spheroid method derived from cancer tissue. They also developed a protocol for measuring spheroid formation and growth from single cells, allowing for the tracking of growth fate and the isolation of slow-growing cells in human colorectal cancer organoids [41].Yurong Song generated a model of organoid derived from intestinal tumors in MSH2-deficient mice that were of epithelial origin, had stem-cell characteristics, and had a high frequency of frame-shift mutations with MSI-H and chromosomal instability and intratumor and intertumor heterogeneity. An in-situ model in the cecum showed progressive tumor growth leading to the development of adenocarcinoma with mucinous features and distant metastasis to the liver and lymph nodes. Organoids exhibiting MSI-H cancer characteristics can be utilized to study the biology of mismatch repair-deficient (MMRd) cancers [42].

Progress of colorectal cancer organoids as a platform for individualized treatment drug screening

Traditional drug screening methods primarily use two-dimensional cell cultures and animal models, which have notable limitations in mimicking human physiological and pathological conditions. The introduction of organoid technology offers a promising new approach to drug screening. Organoids can effectively represent patients’ responses to drugs, particularly in the context of personalized medicine. Conducting drug response tests on patient-derived organoids enables researchers to predict individual patient reactions to specific drugs, forming a foundation for clinical treatment [43]. Furthermore, organoids exhibit a high success rate in culture and expansion, enhancing the efficiency and reliability of their use in drug screening [44].

A study published in Science demonstrated that the drug sensitivity test results of organoids from metastatic gastrointestinal cancer closely align with clinical outcomes, and these organoids can show the dynamic changes of internal molecules in tumors during treatment [45]. Zeng et al. created an organoid biobank from 22 colorectal cancer patients. They used this biobank to assess the impact of hyperthermia alongside chemotherapy. Their study revealed that hyperthermia significantly boosted the effectiveness of raltitrexed [46]. Yang et al. combined liquid biopsy with organoid-model drug testing, and the combination of biomarker-based drug prediction with organoid-based functional drug sensitivity determination may lead to more effective cancer therapy [47]. Yi et al. evaluated the drug sensitivity of four agents—5-fluorouracil (5-FU), cisplatin, oxaliplatin, and irinotecan—using organoids derived from colorectal cancer patients. The median lethal dose of 5-FU was found to be the lowest when used to treat the organoids. The effectiveness of chemotherapy depends on specific patient characteristics, such as age, tumor location, disease stage, and tumor type. Patient organoids responded to chemotherapy in a way that matched the responses of actual patients who experienced cancer recurrence after surgery [48]. Thng et al. established three pairs of matched metastatic colorectal cancer (metCRC) PDOs which were derived from the primary sites (ptCRC) and metastatic lesions (mCRC). They screened the drug in vitro in metCRC PDO and found that epigenomic variations between primary tumours and metastases may have contributed to the differential sensitivity profiles of primary and metastatic tumours [49]. Martini et al. created a biobank of organoids from both primary and metastatic colorectal cancer. They utilized in vitro chemosensitivity data to demonstrate how organoids could predict chemotherapy responses and clinical outcomes in patients with metastatic colorectal cancer (mCRC) [50]. Mauri et al. presented a case of oligometastatic colorectal cancer in a patient who underwent organoid-based drug testing. The results showed resistance to oxaliplatin but sensitivity to 5-FU, SN-38, and panitumumab. This information guided the clinical decision to use FOLFIRI and panitumumab as perioperative treatment options, leading to a significant reduction in lesions. This allows for a second liver metastasectomy and improves the chance of cure [51]. Ramzy et al. designed a personalized treatment regimen for patients with colorectal cancer (CRC) by combining ex vivo organoid efficacy testing with mathematical modeling, with four low-dose co-optimized drug combinations (ODCs). The cell viability was inhibited by up to 88%, which was significantly better than the clinical dose of FOLFOXIRI [52]. Jensen et al. recruited a cohort of 34 patients with colorectal cancer to perform organoid susceptibility testing to guide clinical administration. Seventeen of these patients met the primary endpoint, 2-month progression-free survival (PFS), beyond the predefined level. The median PFS was 67 days, and the median overall survival was 189 days [53]. Tang et al. performed drug testing on 277 organoid samples from 242 CRC patients receiving FOLFOX or XELOX chemotherapy and defined an IC50 cutoff for the PDTO drug trial to effectively distinguish chemotherapy-sensitive from chemotherapy-insensitive CRC patients and predict a survival benefit [54]. Zhu et al. developed an organoid based pharmacokinetic/pharmacodynamic (PK/PD) model that can reflect the PK profile of patients with colorectal cancer after oxaliplatin or irinotecan treatment, which provides an important tool for predicting the clinical response to different drugs [55]. Zhang et al. used a cancer tissue sphere of origin (CTOS) approach to generate organoids from heterogeneous population specimens of cancer and evaluated the ability of CTOS to predict clinical drug response. By analyzing the relationship between the activity of drug-treated CTOS, drug targets and target-related pathways, the effective target-related pathways inherent in tumors were determined. These pathways were highly matched to the aberrant pathways revealed by whole-exome sequencing. The results of drug susceptibility testing were highly matched with the clinical response [56]. Smabers et al. optimized organoid drug screening methods for relevance to patient response and explored the potential to predict response to standard chemotherapy. After optimization, the correlation coefficient between organoid and patient response was 0.58 for 5-FU, 0.61 for irinotecan, and 0.60 for oxaliplatin-based chemotherapy. This study highlights the critical impact of screening methods in determining the correlation between organoid drug screening and patient outcomes in metastatic colorectal cancer [57].

Organoids can be used not only for sensitivity testing of existing clinical drugs, but also for screening of new drugs. In 2022, Professor Toshiro Sato, one of the pioneers of organoid technology, established a screening platform for organoid drugs derived from colorectal cancer patients in Japan to promote the understanding of the biology and clinical authenticity of colorectal cancer drugs [58]. Herpers et al. performed a large-scale functional screening of dual-targeted bispecific antibodies (bAbs) on colorectal cancer organoids. Functional evaluation of more than 500 therapeutic bAbs targeting Wnt and RTK pathways, using high-content imaging, identified a bAb-MCLA-158 that specifically triggers EGFR degradation in LGR5 + cancer stem cells but has minimal toxicity to healthy LGR5 + col on stem cells. MCLA-158 has shown therapeutic properties in preclinical models of several epithelial cancer types, such as growth inhibition in KRAS-mutant colorectal cancer, blockade of metastasis initiation, and inhibition of tumor growth [59]. Mao et al. developed an organoid-based drug screening system, which combined drug library with computational drug prediction to identify 34 drugs with anti-colorectal cancer effects, and divided drug response characteristics into five representative models based on transcriptome analysis: differentiation induction, growth inhibition, metabolic inhibition, immune response promotion, and cell cycle inhibition. This approach provides a valuable resource for developing new clinical therapies for CRC with representative transcriptome characteristics of drug responses [12]. Cioce et al. screened drugs approved by the US Food and Drug Administration (FDA) through colorectal cancer organoids and found that pentoxifylline and 5-fluorouracil (5-FU) synergistically inhibited organoid growth by interfering with the IL-6-STAT3 axis [60]. Luo et al. established a patient-derived high-risk colorectal adenoma organoid (HRCA-PDO) biobanking and used a customized library of 139 compounds for drug screening. Four drugs were selected, including metformin, BMS754807, panostat and AT9283, and their inhibition effects on HRCA PDO were generally consistent. As a representative, metformin was found to hinder the growth of HRCA-PDO in vitro and in vivo by limiting stemness maintenance [61]. Lee et al. found that the combination of biguanides and chemotherapy agents effectively reduced organoid growth in colorectal cancer, and significant therapeutic effect would be obtained by combining AMPK activators and cancer metabolic inhibitors [62].

The research on colorectal cancer organoids as a screening platform for personalized therapy has shown extensive application potential and importance. Advances in this technology allow us to more precisely evaluate the efficacy of different drugs against a patient’s specific tumor, which in turn advances precision medicine. However, how to balance the perspectives and findings of different studies remains an urgent challenge. Different researchers may adopt different methods and standards in organoid model construction, drug sensitivity testing and clinical relevance assessment, which affects the comparability and consistency of research results. Therefore, future research needs to be further discussed in the aspects of standardizing experimental conditions, optimizing organoid culture techniques and building a more comprehensive drug evaluation system.

Progress of drug mechanism and detection technology based on colorectal cancer organoids

As an alternative model of two-dimensional cells and animals, organoids are increasingly used in the study of drug mechanism and detection methods. In terms of mechanism of drug action, Tung et al. used ATAC-Seq and RNA-Seq integrated transcriptome and chromatin accessibility analysis in colorectal cancer organoids and found significant chromatin changes in oxaliplatin-resistant tumor cells. A set of genes associated with increased chromatin accessibility were found to be up-regulated in one organoid. Knockdown of FGFR1 and XTR helped to overcome oxaliplatin resistance, and oxaliplatin in combination with FGFR1 inhibitor (PD166866) or OXTR antagonist (L-368,899) inhibited resistant organoids. However, oxaliplatin treatment did not activate FGFR1 or OXTR expression in the other resistant organoid, indicating that the changes in chromatin accessibility are patient-specific [63]. Chen generated organoids from oxaliplatin-resistant and treatment-sensitive colorectal cancer patients and performed single-cell RNA sequencing (scRNA-Seq) to identify resistance-related signaling pathways (e.g., oxidative phosphorylation and ATP metabolism), resistance driver genes (STMN1, VEGFA and NDRG1) and transcription factors (E2F1, BRCA1, MYBL2, CDX2 and CDX1) [64]. Li found that lnc-RP11-536 K7.3 could promote proliferation, glycolysis, angiogenesis and chemoresistance of colorectal cancer through the SOX2/USP7/HIF-1ɑ signaling axis in resistant organoids [65]. Shen et al. found that KLF5 inhibitor ML264 increased the sensitivity of colorectal cancer organoids to oxaliplatin by inhibiting the KLF5/Bcl-2/capase3 signaling pathway [66]. Papaccio et al. combined the drug response with baseline proteome and transcriptome characteristics by SWATH-MS and RNA-seq analysis, and found that oxaliplatin unresponsive organoids showed enrichment of t-RNA aminoacylation process and showed a shift to oxidative phosphorylation pathway dependence. Protein-transcriptomic data fusion confirmed these results in a highly integrated network of functional processes involved in drug differential responses [67]. Sever et al. found that β-hydroxybutyrate (BOHB) produced synergistic anti-tumor effects with oxaliplatin in CRC organoids by altering energy metabolism and increasing reactive oxygen species (ROS) levels [68].Boos et al. found that the adaptation of colorectal cancer organoids to combination chemotherapy was accompanied by transcriptomic changes rather than genetic mutations. Drug-resistant cells escape apoptosis and up-regulate the expression of MYC/E2F1 and/or interferon-α related genes. The introduction of KRASG12D mutations further increases the resistance of CRC organoids to combination therapy. AURKA inhibition restored the apoptotic response of drug-resistant KRAS wild-type organoids. AURKA expression is increased in primary tumors and derived liver metastases [69].

In addition to chemotherapy drugs, colorectal cancer organoids are also used to study the mechanism of action of natural products. Our team established organoids from patients with colorectal cancer and tested the response of organoids to curcumin by metabolomics technology. It was found that curcumin regulates phenylalanine, tyrosine and tryptophan biosynthesis, niacin and niacinamide metabolism, and purine metabolism [70]. Kim et al. found that the natural plant flavonoid Fisetin significantly reduced organoid activity in A dose-dependent manner, significantly delayed the growth of PDOX tumors, and significantly increased the level of A kinase anchoring protein 12 (AKAP12) after treatment with Fisetin. Inhibition of vascular endothelial growth factor (VEGF) and epithelial cell adhesion molecule (EpCAM), thereby inhibiting angiogenesis [71].

Organoids, as models of clinical authenticity, have been increasingly used in the study of tumor-related genes and signaling pathways. Laoukili et al. found that glutamate-cysteine ligase (GCL) was highly expressed in BRAFV600E colorectal cancer tissues and organoids, and GCL knockout in organoids inhibited distant liver and lung metastasis. Mechanism studies showed that GCL-driven glutathione synthesis reduced oxidative stress [72]. Dijkstra et al. revealed the relationship between SMAD4 and colorectal cancer migratory in colorectal cancer organoids through the multi-omics method. The loss of SMAD4 leads to abnormalities in signaling pathways such as TGF-β, WNT and VEGF, and at the same time, leads to increased secretion of proteins known to be involved in various colorectal cancer pro-migratory processes, thereby activating processes involved in regulating cell motility and proliferation. SMAD4 mutant organoids can also secrete DKK3 which reduces the anti-tumor effect of natural killer cells [73]. Tian et al. studied the organoids and animal models of colorectal cancer and found that pharmacological inhibition of NOTUM prevented the growth of primary adenocarcinoma and inhibited metastasis and colonization of distal organs through the target glypian [74].

Organoids have played a role in the field of nanorelated medicine. Targeted diagnostic nanoparticles (NPs) can generate heat for ablation and visualize tumors through their fluorescence, providing advantages for the detection and treatment of tumor disseminated nodules. A major obstacle to the clinical transformation of nanoparticles is their interaction with the three-dimensional tumor microenvironment. The ablation potential of targeted cd44 polymer nanoparticles using hyaluronic acid (HA) as a targeting agent for colorectal cancer organoids was reported. It was found that the diffusion of non-targeted nanoparticles was uniform, while the diffusion of targeted nanoparticles was weakened due to nanoparticle - matrix interaction. This study demonstrates that organoid models can be used to evaluate how nanoparticles spread within tumors [75]. Deng et al. studied the inhibitory effect of nano-carriers on colorectal cancer by using organoid models. Polydopamine (PDA)-mediated hydroxyapatite nanoclusters (c-HAP) were used to load DOX (c-HAP/DOX), and c-HAP/DOX could release enough DOX to produce cytotoxicity in a slightly acidic environment. It showed excellent antitumor effect in organoids [76].

Based on the above studies, colorectal cancer organoids can more truly reflect the biological characteristics and drug response of colorectal cancer. This research strategy not only contributes to the in-depth understanding of the pathogenesis of colorectal cancer, but also provides a new perspective for the screening of new drugs and the analysis of the mechanism of action. In organoid research, the key role of detection technology cannot be ignored. Precise detection techniques allow researchers to efficiently assess the effects of drugs on colorectal cancer organoids and reveal their molecular mechanisms. Advances in these technologies, especially in the fields of genomics, transcriptomics and metabolomics, have injected new vitality into organoid research, allowing us to analyze the complexity of drug action from multiple dimensions and layers. However, there are still many challenges in this field, such as the establishment of standardization of organoids, the stability of long-term culture, and how to effectively translate research results into clinical applications. Although there may be differences in experimental design and results, comparative analysis, data integration and multi-center collaboration can more objectively evaluate the applicability and limitations of organoids in the study of drug mechanism of action. Future research should continue to explore the diversity and complexity of colorectal cancer organoids to drive advances in personalized therapy and ultimately achieve the goal of precision medicine.

Progress in the application of colorectal cancer organoids in the study of tumor microenvironment

Tumor tissue is composed of tumor cells, functional microenvironment cells, and the extracellular matrix. While organoids are believed to replicate the original tumor’s three-dimensional structure, genetics, and cellular diversity, they lack a complete tumor microenvironment. Reproducing complete tumor tissue and addressing the interaction between organoids and other cells is essential [77]. Tumor-associated fibroblasts (CAFs) are vital components of the tumor microenvironment. They perform multiple functions, such as matrix deposition and remodeling, signaling interactions with cancer cells, and engaging with infiltrating white blood cells. Luo et al. developed the technology of co-culture of colorectal cancer organoids and patient-derived CAFs using three-dimensional hyaluronic acid-gelatin hydrogel. CAFs could maintain the proliferation of colorectal cancer organoids in the hydrogel without adding growth factors to the co-culture. The CRC-DO-CAFs model was proved to be suitable for evaluating the efficacy of standard drugs [78]. Atanasova et al. developed a colorectal cancer organoid model containing matched epithelial-derived tumor cells and stromal fibroblasts, and organoids growing with fibroblasts showed greater tumor heterogeneity and were very similar to in vivo tumor morphology [79]. Farin et al. established a cancer organoid biobank of organoids and cancer-associated fibroblasts (CAF) from 30 colorectal cancer patients. Coculture with CAFs improved transcriptome fidelity and directed the expression of subtype specific matrix genes [80]. Naruse et al. evaluated the interaction between tumor cells and CAFs through co-culture of colorectal cancer organoids and paired cancer-associated fibroblasts. They identified several genes that were highly expressed in original colorectal cancer tissues but down-regulated in organoids. However, co-culturing with CAFs re-expressed these genes, including those related to immune response and external stimulation, like the REG family and dual oxidase enzymes, which are associated with malignant functions. Resulting in tumor cell proliferation and/or an anti-apoptotic state and drug-resistant phenotype [81]. Kobayashi et al. reproduced the fibroblast-rich histology of human CRC liver metastasis through portal vein injection of colorectal cancer organoids to produce a desmoplasia response. Compared with the intrasplenal injection model, this model is tissue-specific and has a higher liver tumor burden, simplifying the analysis of mouse survival. By injecting tumor organoids expressing luciferase, tumor growth dynamics can be monitored through in vivo imaging [82]. Wallisch et al. proposed a co-culture system that enables drug testing of colorectal cancer organoids and fibroblasts without additional matrix components [83].

In addition to fibroblasts, there are many types of immune cells in the tumor microenvironment, such as macrophages. Co-culture of immune cells and organoids can not only reveal the interaction between the two, but also guide immunotherapy. Subtil et al. established a model of dendritic cell (DC) and metastatic colorectal cancer (mCRC) organoid co-culture that reveals how mCRC organoids regulate and shape monocyte-derived DC (MoDC) behavior, phenotype, and function in the collagen matrix [84]. Li et al. predicted the cancer-tumor microenvironment interaction from the single cell transcriptome of colorectal cancer, and co-cultured human monocyte-derived macrophages with organoids for the study of the relationship between macrophages and colorectal cancer [85]. Teijeira et al. co-cultured colorectal cancer organoids expressing different levels of CEA with T lymphocytes, and found that the killing of tumor cells in colon cancer cultures depended on the level of CEA expression on the surface. Therefore, low affinity reagents (CEA-TCB) would not mediate the killing of human pre-activated T cells when the CEA expression threshold was lower. The high-affinity construct (CEACAM5-TCB) remained active on organoids with low CEA expression. The heterogeneity of CEA expression levels was simulated by co-culture of high and low CEA organoids, showing a weak bystander killing effect. At the same time, they co-cultured tumor organoids, autologous fibroblasts, and T cells to test the co-stimulatory effects of fibroblast Activating Protein (FAP) targeting 4-1BBL bispecific antibody fusion protein, which is currently undergoing clinical trials. Anti-FAP-4-1BBL co-stimulation was observed in the co-culture model, which can release IFN-γ and kill tumor cells more effectively [86].

Progress of imaging methods for organoids of colorectal cancer

Organoid imaging technology is an important tool in organoid research, which can simulate the structure and function of real organs in vitro, and is widely used in the fields of disease simulation, drug screening and cell therapy. In order to obtain the necessary information such as morphology, structure, cell function and dynamic signals of organoids, it is particularly important to directly monitor the culture process of organoids. The development of organoid imaging technology provides a new perspective and tool for organoid research [87].

In the past, it was difficult to capture the organoids distributed in the different layers of Matrigel in a single layer image due to limited imaging technology, resulting in researchers taking a lot of time to understand the status of all the organoids in the 3D culture model. With the development of 3D organoid culture model and the progress of 3D imaging system, Z-stack technology has been widely used in the field of drug screening [88, 89]. With Z-stack imaging, researchers can capture multiple facets of organoids in the same image, allowing for a more complete analysis of survival after drug treatment. Li et al. used fluorescent dye CalcIN-AM to identify living cells in organoids and Z-stack image processing to capture all organoids cultured in Matrigel, thus proposing a new method for high-throughput imaging and analysis of organoids [90].

The development of organoid high-throughput imaging analysis technology has promoted the efficiency of drug efficacy evaluation in organoids. Daniel et al. developed a tool for rapidly and nondestructively imaging organoid treatment responses, monitoring metabolic and morphological changes at the level of individual organoids. Wide-field one-photon redox imaging was used to segment and track individual organoids to extract morphological and metabolic variables of treatment response [91]. Edoardo D’Imprima et al. proposed a multi-scale imaging method to track organoids’ growth, detect their morphology with fluorescent markers, identify areas of interest, and analyze their 3D ultrastructure. They used automatic image segmentation to annotate and quantitatively analyze subcellular structures in colorectal cancer organoid, identifying local tissues in dense and polarized epithelium with diffractive limited cell connections [92].

The development of organoid imaging technology provides a new perspective and tool for organoid research. Traditional imaging techniques often face challenges in dealing with three-dimensional structures, especially in observing the dynamic changes between cells and the internal structure of tissues. The researchers used immunofluorescence staining and confocal microscopy to image the internal structure of colorectal cancer [93]. In recent years, with advances in tissue transparency and three-dimensional imaging technology, researchers have been able to obtain three-dimensional images of organoids at higher resolution and analyze their internal structure and cell composition [94]. For example, the use of low-coherence holography allows researchers to image organoids in real time over long periods of time without labeling, capturing the dynamic activity and growth of cells. According to a recent study, Mahn Jae Lee et al. used low-coherence Holotomography (HT) to continuously, marker-free monitor mouse intestinal organoids to reveal their developmental trajectoras and pharmacological responses. This technique has a unique advantage in classifying living and non-living cells within organs and provides unmatched detail in describing 3D morphological changes following drug exposure. The technique further enables quantitative measurements of organoid volume, protein concentration and dry mass, setting a new standard for comprehensive and rigorous statistical evaluation of organoid biological research [95].

These technological advances not only improve the quality and efficiency of imaging, but also provide a solid foundation for the application of organoids in drug screening and disease models. With the continuous progress of imaging technology, we are expected to achieve dynamic monitoring and real-time analysis of organoids in the future, which will bring a new turning point for personalized medicine. Although organoid imaging technology shows great potential, there are still some differences in views and findings between different studies. For example, some studies highlight the advantages of organoids in drug metabolism and toxicity testing, while others point to their limitations in simulating complex physiological environments. This situation requires us to consider the results of various studies when evaluating and applying organoid imaging techniques. Only through interdisciplinary cooperation, integrating knowledge from different fields, can the application potential of organoids be more fully understood. Through continuous exploration and innovation, we expect organoid imaging technology to open new horizons for the development of future medicine.

Summary

In view of the above studies, in the rapid development of colorectal cancer organoid research, we can see its far-reaching impact on regenerative medicine and personalized medicine. Organoids, as miniature organs cultured in vitro, can not only simulate the structure and function of real organs, but also provide more accurate models for studying disease mechanisms and drug screening. Making full use of the abundant clinical tumor samples in various medical institutions, the establishment of tumor organoid biobank plays an important supporting role in promoting the development of tumor medicine. By establishing colorectal cancer organoids, conducting mechanism research and multi-omics analysis, analyzing the regularity and characteristics of the occurrence and development of colorectal cancer, building a platform for drug screening and clinical drug sensitivity, and finally realizing individualized treatment are the greatest value of conducting organoid research. This work will provide support for the accurate diagnosis and treatment of tumors and the development of new treatment strategies, which not only has important scientific value, but also has good social value.

Author contributions

Pang, Hu and Dai wrote the main manuscript text. Lou , Xu and Chen participated in writing and revising the article. All authors reviewed the manuscript.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing Interests

The authors declare no competing interests.