Colon cancer is the fourth most common malignant tumor worldwide and is one of the leading causes of cancer death in developed western countries [1]. The latest International Agency for Research on Cancer (IARC) statistics estimate that there are approximately 1.14 million new cases and 538,000 deaths between 2022 and February 8, 2024 [2]. Globally, age-standardized incidence rates (ASR) were high in Oceania (ASR 19.77), Europe (ASR 18.64) and North America (ASR 17.08), which exceeded double or even more in comparison to the other continents. Advances in the development of treatment modalities such as surgical approaches and radiotherapy techniques have improved the survival rate of colon cancer patients. Also, chemotherapy or immunotherapy will be provided as an adjuvant option. Different from chemotherapy, which kills cancer via extensive cytotoxic properties, immunotherapy uses the host immune system to targeted kill tumor cells [3]. However, only a few colon cancer patients who are microsatellite instability (MSI) types can benefit from immunotherapy. The majority of colon cancer patients are microsatellite stable (MSS) types and do not respond to immunotherapy [4]. Besides, the prognosis and survival rate of colon cancer patients is still unsatisfactory. Therefore, it is urgent to find adjuvant treatment to improve the survival rate. The occurrence, development, and metastasis of tumors are closely related to diet and nutrition, especially in colon cancer. Therefore, from the perspective of precise nutrition, adjuvant treatment in daily food can inhibit or even block the occurrence and development of colon cancer [5].

Based on the reduction of exploitable land resources, more attention is being paid to marine resources. Marine algae are the most abundant resources in the ocean. Marine algae are the most abundant edible non-animal resource in the oceans. In Asian countries, seaweed-rich diets have been associated with lower rates of chronic diseases such as cancer, cardiovascular and heart disease [6]. Caulerpa lentillifera is a common edible economic green algae in Southeast Asian countries, also known as sea grapes or green caviar [7]. It naturally grows in several Southeast Asian countries, including China, Japan, Philippines, and Vietnam, with wide commercial cultivation [8]. A study showed that the polysaccharide contents of C. lentillifera from different regions were mostly >40 %, which was the main component of it [9]. Previous studies have found that its polysaccharide has immunomodulatory [10,11], anti-cancer [12], obesity prevention [13], antiviral [14], neuroprotective and restorative [15], and gut microbiota regulation [16] effects. The anti-cancer activity of C. lentillifera polysaccharides was only demonstrated by Nurkolis et al. [12] which were found to show a wide activity against the cell lines of colon cancer HCT-8, hepatocellular cancer KAIMRC1, breast cancers MDA-MB-231 and MCF-7, and leukemia HL-60. As an edible algae, exploring the role of its polysaccharides on colon cancer can better develop the application from a dietary therapy perspective. However, more in-depth studies of C. lentillifera polysaccharide on anti-colon cancer activity and mechanism are still needed.

The model of tumor research has an important advancement with the application of three-dimensional (3D) cell cultures, especially organoids [17]. Typically, we perform tumor studies with two-dimensional (2D) monolayer cell or animal models. The 2D monolayer cells are a basic model that offers insight into diseases with less time, low cost, and high reproducibility but does not show the complexity of the in vivo microenvironment [18]. Animal models provide the most realistic representation of human function and cellular interactions but are complicated to implement due to limitations in maintenance costs, space, and experimental cycle time [19]. As a better alternative to tumor research models, the advantages of the 3D cell culture models lie in the middle ground between them. It has been proven to be closer to in vivo natural systems than 2D models with a shorter cycle time than animal models and can be used for precursor experiments performed before animal experimental validation [20]. In particular, the establishment of patient-derived organoids (PDOs) led us to a more complex physiological environment than the 3D culture of monotypic cell lines, containing undifferentiated stem cells while allowing stem cell differentiation [21]. Moreover, this kind of in vitro multicellular model with high similarity to the phenotypic and genetic characteristics of the original tissue from the patient, has a great contribution to drug screening and mechanism studies for human disease [22].

Based on the absence of more in-depth studies, we investigated the activity and mechanism of C. lentillifera polysaccharide against colon cancer. In this study, we prepared a C. lentillifera polysaccharide (CLP) from Ningbo, China, and demonstrated its physicochemical properties. Aiming at a relatively comprehensive manner, the anti-colon cancer activity of CLP was evaluated by a variety of models with 3D models as the main focus. First of all, the HT29 cell (MSS phenotype) was used for the evaluation of anti-colon cancer activity in 2D monolayer and 3D spheroid cells. Further, transcriptomics and metabolomics in HT29 spheroid cells were performed to investigate the potential molecular mechanism. At last, the activity on other colon cancer models was analyzed, including 2D monolayer and 3D spheroid cells of HCT116 and LoVo (both MSI phenotype), patient-derived organoids, and CT26 (MSS phenotype) bearing BALB/c mice. Further, transcriptomics and metabolomics were performed to investigate the potential molecular mechanism. This study can clarify the anti-colon cancer activity and mechanism of CLP in multiple dimensions, provide a research basis for the development of adjuvant therapeutic food, and enrich the research on the biological activity of C. lentillifera.