Introduction

Liver metastasis is one of the main reasons for colorectal cancer (CRC)-related deaths due to the lack of effective therapeutic. CRC cell line-derived xenograft (CDX) mouse models developed by orthotopic injection in the cecum, ectopic injection in the spleen or the portal vein are essential invivo tools for exploring the mechanism of liver metastasis and testing potential chemotherapeutic agents. We did this study to evaluate and optimize the establishment of these models, and emphasize characteristics of liver metastatic foci derived from different CRC cell lines.

Aims & Methods

Luciferin-expressing HCT116 cells, SW480 cells and SW620 cells were used to induce mouse models of CRC with liver metastases by the cecum injection, intrasplenic injection and intraportal injection. The metastatic characteristics of tumors were confirmed and evaluated by bioluminescence imaging, macroscopic and microscopic appearance. Furthermore, we improved and evaluated the procedures of these CRC CDX mouse models, and comprehensively summarized their performances in the aspect of liver metastasis with three popular cell lines.

Results

Models established by cecal injection and intrasplenic injection displayed similarly in the operation difficulty, cell number to need and frequency of metastasis. The bioluminescence imaging usually had a strong and concentrated signal in cecum, but a weak or moderate signal in the spleen respectively, and accompanied with a diffuse signal or no signal in the liver. The model for HCT116-induced liver metastasis could be well established by intrasplenic injection, showing granular lesions with tumor pleomorphic variation, invasion, erosion and necrosis. SW480 and SW620-derived models by cecal or intrasplenic injection characterized macroscopically with nodular, white grey metastatic foci throughout the liver tissue, and identified with scattered, sub-rotund, uniform-sized and infiltrative pathological changes. However, the metastatic lesions were more and larger in the interior of liver tissues in the cecum injection model within 6 weeks, suggesting its superiority. Intraportal injection had technique difficulty in the discovery and puncture of the portal vein, probably leading to post-operated hemostasis and mouse deaths. The metastatic foci developed from intraportal injection were hardly displayed obvious findings in macroscopic appearance, and few or small-sized sporadic lesions under microscopy could be detected.

TABLE 1: The opitimization and evaluation of the establishment of three CDX models.

Conclusion

The study provides not only useful information for the establishment of CRC CDX models for liver metastasis, but also novel insights for identifying characteristics of metastatic foci induced by different cell lines.

Disclosure

No conflict of interest

Introduction

Colorectal cancer (CRC)-driven liver metastasis formation is one of the main drivers of cancer-related deaths. As CRC development has formerly been identified as a disease of elderly people, recent epidemiological data shows an increasing incidence of CRC in people younger than 50 years of age (early-onset colorectal cancer, EOCRC) while less people are being diagnosed with CRC after the age of 50 years (late-onset CRC, LOCRC). Recent studies have identified disparities between these two forms, with early-onset cases often diagnosed at more advanced stages, including elevated risks for recurrence and metastasis formation, including liver metastasis.

Aims & Methods

Here, we have studied how CRC and CRC-dependent liver metastasis develops in an age-dependent manner by using 8-weeks old and 78-weeks old C57BL/6 mice from the same vendor under the same housing conditions. To this end, we used an orthotopic mouse model of CRC-related liver tumor formation, in which colorectal tumor organoids were engrafted in the liver via injection into the portal circulation, solely leading to liver metastasis formation without any primary colon tumor growth. To study age-dependency of primary CRC growth, we injected CRC organoids into the mucosa of the rectum. We further analyzed the molecular mechanisms of disease development, using antibody depletion, genetic mouse and ex vivo tumor models, as well as spatial transcriptomics approaches.

Results

Interestingly, we could demonstrate that young mice exhibit more and larger liver metastases compared to older counterparts, while the formation of primary CRC was unaffected by the age of the host. Liver metastases in old mice display increased necrosis formation which is accompanied by an elevated abundance of different immune cells such as CD8⁺ T cells. Spatial transcriptomics analysis further revealed a distinct anaplerotic metabolic phenotype in liver metastases of young hosts, showing that tumor-surrounding metabolic factors differ between young and old mice, which highlights important metastatic niche dependencies to control tumor growth. Current investigations therefore are directed towards elucidating the factors driving enhanced metastatic development in young hosts and their interactions within the tumor microenvironment, focusing on spatial metabolomics approaches and human cohorts for early-onset CRC.

Conclusion

Our findings demonstrate more aggressive liver metastasis formation in young hosts, as similarly observed in humans, and offer valuable insights into the pathogenesis of early-onset colorectal cancer.

Introduction

Circular RNAs (circRNAs) are a class of singlestranded, covalently closed RNA molecules that are formed by precursor mRNA back-splicing or skipping events of thousands of genes in eukaryotes. CircRNAs play an essential role in the initiation and progression of human cancers by participating in the regulation of one or several cancer hallmarks.

Aims & Methods

In our study, we aimed to reveal the functions and mechanisms of circRNAs in colorectal cancer (CRC) progression. High-throughput RNA sequencing (RNA-seq) was performed to assess RNA expression profiles in 10 paires of CRC tumor tissues and matched adjacent peritumor tissues, then the expression level of top five dysregulated circRNAs were tested in CRC cancer cell lines. The circLRBA with concordant alteration in RNA-seq data and cell lines was validated in tumor and corresponding adjacent tissues (85 pairs) from a CRC cohort by qRT-PCR analysis followed by correlation and prognosis analysis between circLRBA and clinical characteristics. Sanger sequencing, RNase R and Actinomycin D assays were used to verify the ring structure of circLRBA. Nuclear-cytoplasmic separation experiment and the fluorescence in situ hybridization (FISH) were performed to show the cellular location of circLRBA. Functional experiments, including CCK8 assay, colony formation assay, transwell assay, 3D multicellular tumor spheroid invasion assay and orthotopic-injected CRC cell line-derived xenograft (CDX) mouse models, were performed by using circLRBA overexpression and knockdown cell lines to investigate the effects of circLRBA on CRC progression in vitro and in vivo. Mechanistically, RNA-sequencing was used to reveal the dysregulated genes followed by circLRBA overexpression. Bioinformatic analysis was used to predict the possible downstream pathways. Furthermore, Biotin‑labeled RNA pulldown and RNA immunoprecipitation (RIP) assays were carried out to identify the proteins directly interacting with circLRBA.

Results

Totally 853 differentially expressed circRNAs were identified, including 778 down-regulated and 75 up-regulated circRNAs. We identified a circular RNA generated from the LRBA gene, designated circLRBA, that was significantly down-regulated in CRC tissues and associated with unfavorable clinicopathological parameters. CircLRBA was dominantly distributed in the cytoplasm in several CRC cell lines. Gain‑ and loss‑of‑function experiments suggested that circLRBA could inhibit CRC cell proliferation, metastasis and apoptosis in vitro and in vivo. RNA-seq of circLRBA-overexpressing cells and the controls revealed that these dysregulated genes were significantly enriched in pathways of malignant neoplasm of colon stage IV (DisGeNET database), protein localization to endoplastic reticulum and lysosomal lumen (Gene Ontology analyse). Furthermore, RNA pulldown and RIP assays validated that circLRBA directly binds to the HSPA8 protein, which is an essential chaperone for mediating cell autophagy by translocating proteins containing the KFERQ-like motif in their amino acid sequence to undergo lysosomal degradation.

Conclusion

Our study uncovered a novel LRBA gene-generated circRNA with a tumor suppression function. CircLRBA probably inhibited CRC progression by chaperone-mediated autophagy (CMA) through interacting with HSPA8. However, further exploration is needed to reveal the substrate protein of HSPA8 and its effects on CRC. CircLRBA is expected to be a new therapeutic target for CRC patients in the future.

Disclosure

No conflict of interest.

Introduction

Over the last four decades, metal-based antitumor medications, notably cisplatin, have been extensively utilized in chemotherapy, despite notable side effects. Colorectal cancer, a significant contributor to cancer-related mortality worldwide, has prompted extensive research into innovative treatment methods. Thiazoles and their derivatives constitute integral components of certain cytostatic agents, garnering considerable attention in studies exploring their biological and pharmacological properties.

Aims & Methods

The primary objective of this research is to examine the in vitro cytotoxic effect of newly synthesized palladium(II) complexes with 2-aminothiazole derivatives on colorectal carcinoma cell lines. The cytotoxic effects of newly synthesized palladium(II) complexes (C1, C2) and corresponding ligands (L1, L2) were determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay against both mouse (CT26) and human (HCT116) colorectal carcinoma cell lines. Non-tumor cells, specifically mouse mesenchymal stem cells (mMSCs), were employed as controls.

Results

The tested complexes and corresponding ligands reduce the viability of tumor cells in a dose-dependent manner. The C1 complex demonstrated strong cytotoxic activity against all tested tumor cell lines, with the HCT116 cell line being more sensitive to its toxic effects. All tested complexes and corresponding ligands reduced the viability of non-tumor mMSC cells, with mMSC cells being particularly sensitive to the cytotoxic effect of the C2 complex. The IC50 (half maximal inhibitory concentration) value obtained for the C1 complex was higher compared to the gold standard, cisplatin. The C1 complex exhibits the highest cytotoxic capacity towards tumor cells, followed by the highest IC50 values for control.

Conclusion

Palladium(II) complexes with 2-aminothiazole derivatives demonstrate direct antitumor activity in vitro. The C1 complex exhibits a high level of cytotoxicity. Further research is necessary to indicate the potential use of these compounds in experimental tumor therapy.

Disclosure

yes

Introduction

​The actual screening of colorectal cancer (CRC) includes people of 45 years old and patients with alcoholic liver cirrhosis (LC) have an elevated risk of developing colon cancer. Curative treatment of CRC in patients with an existing diagnosis of LC can be a challenge, requiring major surgical resection and may include liver-toxic chemotherapy. Postoperative morbidity and mortality are elevated in patients with LC. For improved survival, optimization of liver function is more important than adjuvant chemotherapy.

Aims & Methods

Our study included 149 hospitalized LC patients Classes Child- Pugh A, B and C, meanage 56 years old, during April 2023-March 2024, of which 13 cases presented lower bleeding, iron deficiency anemia, intestinal transit disorders, abdominal pain, and familial history of CRC. Clinical and paraclinical diagnostic of CRC in LC patients were made by digital rectal examination, hematological samples, rectosigmoidoscopy, total colonoscopy, abdominal ultrasound, computed tomography and magnetic resonance imaging.

Results

Biological tests indicated anemia, trombocitopenia, increased ESR, biochemical liver tests increased, occult bleeding (FIT), and the increase of the tumor markers CA19-9, AFP, CEA that is particularly useful for monitoring treated CRC. Flexible rectosigmoidoscopy detected six cases of rectal cancer, three cases of rectosigmoidian tumors, and total colonoscopy was the "gold standard" imaging investigation that detected two cases of descending colon cancer, one case of hepatic flexure tumor and one patient with ascending colon cancer. Of patients with LC Class Child Pugh A, 6 cases were operated by laparotomy, being referred to Oncology Institute with favorable outcomes, and the rest of seven patients with LC Class Child Pugh B and C had advanced CRC, being treated by chemotherapy. Abdominal ultrasound visualizes liver metastases, abdominal lymph nodes and CT, MRI were the main methods used for staging CRC before surgical and oncological treatment.

Conclusion

Colonoscopy is the exploration of choice in CRC detection with 97% sensitivity, providing histopathological examination by tumor biopsy and also diagnosis of synchronous cancer. Early diagnosis of CRC is essential to prevent complications and increase the survival rate in LC. Men with LC are predominantly affected of CRC. Abdominal ultrasound, CT or MRI are the main imaging investigations recommended in CRC staging before the surgical and oncological treatment. Laparotomy should be preferred in patients with LC. Survival rate is strongly associated with the improvements in liver function. Echoendoscopy, immunoscintigraphy with monoclonal antibodies can be useful in CRC and genetic testing of tumor DNA markers in stool can be recommended in families with genetic syndromes (Turcot, FAP).

Introduction

Metastasis is the major cause of mortality among colorectal cancer (CRC) patients and represents a crucial cancer research challenge. Metastasis is the results of an intimate crosstalk between cancer cells and the surrounding tumor microenvironment, where neoplastic cells exploit the inefficient and leaky tumor vessels to disseminate to secondary organs¹. In this context, the extracellular matrix protein Multimerin-2, specifically deposited by endothelial cells (EC), is instrumental for the maintenance of vascular homeostasis, ensuring the proper formation of cell-cell junctions via the VEGFR2 signaling².

Aims & Methods

We observed that Multimerin-2 is down modulated in tumors both at transcriptional and post-translational levels. Thus we hypothesized that Multimerin-2-devoid vessels may enhance cancer cell dissemination, being characterized by a compromised endothelial barrier.
Multimerin-2 expression was analyzed in human CRC samples through IHC, ELISA and in silico by the tnmplot.com tool. The effects of Multimerin-2 loss were assessed in vitro through transmigration assays upon Multimerin-2 down-regulation in EC cells and the molecular mechanism verified by WB and qRT-PCR. The putative Multimerin-2-affected metastatic dissemination was assessed through intravenous injection of MC38 colon cancer cells in wt and Multimerin-2^-/-mice.

Results

Multimerin-2 expression is significantly reduced within the tumor tissue respect to the normal counterpart among CRC patients, despite at variable degree. Importantly, in vitro, we found an increased number of CRC cells transmigrating through a Multimerin-2-depleted endothelium. This phenomenon was not VEGFR2-dependent since the use of a VEGFA blocking agents did not affect the extravasation of tumor cells. Indeed, under tumor-mimicking conditions, the knock-down of Multimerin-2 in ECs induces the upregulation of the vascular adhesion molecule VCAM1, a molecule exploited also by cancer cells to transpass the endothelium³. Accordingly, the intravenous injection of B16F10 syngeneic melanoma cells, as well as MC38 colon cancer cells, in Multimerin-2^-/-mice led to the formation of a higher number of metastatic lesions in the lungs.

Conclusion

Overall, these data indicate that Multimerin-2 expression is required to maintain vascular stability and halt the escape of cancer cells, suggesting that the Multimerin-2-devoid tumor vessels may represent a preferential route for metastatic dissemination, during CRC progression.

References

¹ Lugano R. et al, Tumor angiogenesis: causes, consequences, challenges and opportunities, Cell Mol Life Sci., 2020
² Pellicani R. et al, Multimerin-2 Maintains Vascular Stability and Permeability, Matrix Biol., 2020
³ Wieland E. et al, Endothelial Notch1 Activity Facilitates Metastasis, Cancer Cell 2017