Introduction
Late-stage colorectal cancer (CRC) treatment options are limited due to efficacy, cost, and adverse events. The increasing prevalence of colorectal cancer and the advent of late detection have burdened oncologists, forcing them to choose from limited treatment modalities, most of which are not particularly beneficial for patients. To address this disparity and to find a suitable therapeutic intervention, the present study is performed employing in silico, in vitro and in vivo approaches.
 
Method
For the in silico analysis, the Connectivity Map (CMap) database was employed to identify gene expression signatures of capecitabine, cyclophosphamide, and mirtazapine in HT29 CRC cells. Signature sequences exhibiting substantial expression alterations (z-score±2) were screened to pinpoint genes modulated by the drug combinations. Furthermore, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, followed by Molecular Signature Database (MSigDB) analysis, to understand the impact of the significantly modulated genes on different pathways. In vitro studies were conducted utilising HCT116 CRC cell lines to assess cytotoxicity employing the MTT assay. IC50 values were determined for individual drugs, doublet, and triplet combinations. Regression analysis and ANCOVA were used to compare the triplet regimen with the standard drug, 5-fluorouracil (5-FU). Further, we have performed in vivo studies on 1,2-dimethyl hydrazine (DMH) Dihydrochloride induced male Swiss albino mice using multiple parameters. Animals were administered drugs for 21 days (5days/week), using metronomic doses for cytotoxic drugs: Cape (100mg/kg/day), Cyc (20mg/kg/day), and Mirt (7.5mg/kg/day). Animals were monitored weekly, followed by posthumous analysis for blood parameters, gross & histological analysis, and ELISA tests for VEGF and CEA biomarkers. Survival probabilities were also determined.
 
Result
CMap analysis showed that the triplet combination modulated key genes involved in CRC pathogenesis. KEGG analysis revealed that significantly modulated genes have high fold enrichments in nucleotide excision repair, proteoglycans and apoptosis pathways. Hallmark gene analysis in MSigDB database suggests that mitotic spindle and G2M checkpoint pathways were significantly affected by the modulated genes. The MTT assay showed that Cape (3.11µg), Cyc (4.12µg), and Mirt (6.06µg) had significantly reduced IC50 values in doublet and triplet combinations, with the triplet (1.09µg) being as potent as 5-FU (1.03µg). ANCOVA analysis reveals that higher doses of the triplet regimen may offer superior efficacy over 5-FU. Another interesting finding from the in vitro study is that the addition of Mirt to both individual cytotoxic drugs yields a lower IC50 compared to the combined IC50 of both cytotoxic drugs (Cape+Cyc). In vivo analysis using weekly parameters evaluation reveals improved QoL, while hematological parameters, gross colonoscopy, and vital organs histological analysis reveal inflammation. Further CEA and VEGF biomarker analysis shows reduced levels, survival analysis shows highest survival probabilities with Mirt, among the treatment groups.
 
Discussion/Conclusion
This study presents evidence suggesting that the metronomic combination of capecitabine, cyclophosphamide, and mirtazapine can be an effective therapeutic strategy for metastatic mCRC, showing antiangiogenic effect with improved tumor microenvironment, and also showing survival benefits. But, at the same time, inflammations and organ toxicity were reported, indicating further preclinical studies with dose modifications, possibly with reduced doses, are required.

Different in silico, in vitro, and in vivo approaches explored to study the metronomics combination of Capecitabine, Cyclophosphamide, and Mirtazapine in advanced colorectal cancer