• Origin: The MC38 cell line is derived from C57BL/6 murine colon adenocarcinoma cells. The cell line is a commonly used murine model for colorectal carcinoma.
• Background Information: The gene encodes for a receptor tyrosine kinase‑like orphan receptor that plays a role in regulating neurite outgrowth within the central nervous system. The resulting protein is a glycosylated type I transmembrane protein and belongs to the ROR subfamily of cell‑surface receptors. It acts as a pseudokinase, meaning it is catalytically inactive, and is believed to participate in the non‑canonical Wnt signalling pathway. Expression of this gene is high during early embryonic development, but drops to very low levels in adult tissues. Notably, upregulation of this gene is associated with B‑cell chronic lymphocytic leukaemia. Furthermore, alternative splicing produces multiple transcript variants that give rise to distinct protein isoforms.
• Gene targeting strategy: The exon3 to exon4 of mouse Ror1 gene were replaced by human ROR1 coding sequence in the B-hROR1 low MC38. Human ROR1 is expressed on the surface of B-hROR1 low MC38.
• Tumorigenicity: Confirmed in C57BL/6JNifdc mice.
• Application: The B-hROR1 low MC38 tumor model can be used for preclinical efficacy studies.
• Notes:

Inoculated cell lines can be suspended with RPMI-1640 stock solution.

Before implementing the project, it is recommended to perform tumor growth experiments. The recommended cell inoculation amount is between 5E5-1E6.

In the experiment, it is necessary to ensure that the number of animals inoculated subcutaneously is at least 1.6 times the actual grouping number.

• Human ROR1 was detected on the surface of both B-hROR1 low MC38 and B-hROR1 MC38, with the expression level on B-hROR1 low MC38 being lower than that on B-hROR1 MC38.

Human ROR1 expression analysis in B-hROR1 MC38 by flow cytometry. Single cell suspensions from wild-type MC38, B-hROR1 low MC38 #1-G05 and B-hROR1 MC38 #1-G06 cultures were stained with anti-human ROR1 antibody (Biolegend, 357804). Human ROR1 was detected on the surface of both B-hROR1 low MC38 and B-hROR1 MC38.

Subcutaneous tumor growth of B-hROR1 low MC38 cells. B-hROR1 low MC38 cells (5×10⁵) and wild-type MC38 cells (5×10⁵) were subcutaneously implanted into C57BL/6JNifdc mice (male, 8-week-old, n=6). Tumor volume and body weight were measured twice a week. (A) Average tumor volume. (B) Body weight. Volume was expressed in mm³ using the formula: V=0.5 × long diameter × short diameter². Results indicate that B-hROR1 low MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

• Human ROR1 was detected on the surface of B-hROR1 low MC38 tumors cells.

Human ROR1 expression were evaluated​​ on B-hROR1 low MC38 by flow cytometry. ​​These cells​​ were subcutaneously transplanted into C57BL/6JNifdc mice (male, 8-week-old, n=6). At the end of the experiment, tumor cells were harvested and ​​analyzed with species-specific anti-human ROR1 antibody (Biolegend, 357804) expression​​ by flow cytometry. ​​