• 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 ROR1 encodes a receptor tyrosine kinase that has been implicated in nervous system development, specifically in the maintenance of neural progenitor cell fate, neurite extension and synapse formation. The encoded protein, likely a pseudokinase that lacks catalytic activity, may also regulate adipogenesis. PD-L1 is an immune checkpoint protein expressed on the surface of variouscells, including tumor cells and immune cells. It binds to PD-1 on T cells, leading to T cell exhaustion, reduced cytokine production, and impaired anti-tumor immunity. Combining anti-ROR1 therapies with immune checkpoint inhibitors has emerged as a promising strategy to enhance therapeuticoutcomes by simultaneously targeting tumor and immune evasion.
• Gene targeting strategy: The exogenous promoter, human PD-L1 and luciferase coding sequences were inserted to replace part of murine exon 3. The insertion disrupts the endogenous murine Pdl1 gene, resulting in a non-functional transcript. The exogenous promoter and human ROR1 coding sequence was inserted to replace part of murine exon 2 and all of exon 3. The insertion disrupts the endogenous murine Ror1 gene, resulting in a non-functional transcript. To increase the protein expression level of human ROR1, the coding sequence of human ROR1 CDS was randomly inserted into the genome of B-hPD-L1 plus/hROR1 MC38 plus. 
• Application: The B-hPD-L1 plus/hROR1 MC38 plus tumor models can be used for preclinical evaluation of monoclonal antibody drugs and bispecificantibody drugs targeting humanROR1 and PD-1/PD-L1.

B-hPD-L1 plus/hROR1 MC38 plus cells were subcutaneously transplanted into B-hROR1 mice (female, 8-week-old, n=6), and on 33 days post inoculation, tumor cells were harvested and assessed for human PD-L1 and human ROR1 expression with anti-mouse PD-L1 (Biolegend, 124312), anti-human PD-L1 (Biolegend, 329706) and anti-hROR1 (Biolegend, 357804) antibodies by flow cytometry. As shown, human PD-L1 and human ROR1 was highly expressed on the surface of tumor cells. Therefore, B-hPD-L1 plus/hROR1 MC38 plus cells can be used for in vivo efficacy studies of novel PD-L1 and ROR1 therapeutics.

PD-L1 and ROR1 expression analysis in B-hPD-L1 plus/hROR1 MC38 plus cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hPD-L1 plus/hROR1 MC38 plus cultures were stained with species-specific anti-mouse PD-L1 (Biolegend, 124312), anti-human PD-L1 (Biolegend, 329706) and anti-hROR1 (Biolegend, 357804) antibodies. Human PD-L1 and human ROR1 was detected on the surface of B-hPD-L1 plus/hROR1 MC38 plus cells but not on wild-type MC38 cells. The 1-C02 clone of B-hPD-L1 plus/hROR1 MC38 plus cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-hPD-L1 plus/hROR1 MC38 plus cells. B-hPD-L1 plus/hROR1 MC38 plus cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into B-hROR1 mice (female, 8-week-old, n=6). Tumor volume and body weight were measured twice a week. (A) Average tumor volume ± SEM. (B) Body weight (Mean± SEM). Volume was expressed in mm³ using the formula: V=0.5 X long diameter X short diameter2. As shown in panel A, B-hPD-L1 plus/hROR1 MC38 plus were able to establish tumors in vivo and can be used for efficacy studies.