ROMAN MARTI DIAZ¹, MARIA FERNANDA MONTENEGRO ARCE², JUAN CABEZAS HERRERA³, LUIS SANCHEZ DEL CAMPO¹, JOSE NEPTUNO RODRIGUEZ LOPEZ³

(1) Department of Biochemistry and Molecular Biology A, Uniersidad de Murcia, 30100, España (Región de Murcia)
(2) NEUROPATOLOGÍAS DEL SISTEMA COLINÉRGICO, IMIB-Arrixaca, España
(3) TERAPIAS MOLECULARES Y BIOMARCADORES DE TUMORES SÓLIDOS, IMIB-Arrixaca, España

Cell surface expression of calreticulin (CRT) promotes the phagocytic uptake of dying, stressed, and cancer cells; however, the prophagocytic function of CRT is inhibited by CD47 (integrin-associated protein), which has previously been described as an antiphagocytic signal in several human cancers. Together, these studies indicate that the CRT–CD47 balance is an important determinant of cellular phagocytic uptake and manipulating this balance may be an important route to activate stronger antitumor immunity. Although translocation of CRT to the cell surface is a recognized hallmark of the immune modulation that suffer cancer cells after radiotherapy, the response of CD47 to ionizing radiation (IR) is still controversial. While some authors observed that radiation promoted loss of cell surface CD47 in human papillomavirus-positive cancer, others reported that CD47 is induced in radio-resistant breast cancer cells and irradiated tumors in a mechanism linked with cellular HER2 status. Therefore, to study the role of CD47 during radiotherapy, we generated a CD47 knockout breast cancer cell line using CRISPR-Cas9 technology, and were characterized for further immunological studies.

4T1-luc2-CD47-KO cells were generated using the CRISPR/Cas9 technique. Positives clones were validated by genomic DNA extraction followed by Sanger sequencing and western blot experiments. Cell proliferation was evaluated using colorimetric assays to analyze mitochondrial functions (MTT; Merck). For ionizing radiation assays, the cells were irradiated using an Andrex SMART 200E machine at a dose rate of 10 Gy. Apoptosis was quantified using a Cell Death Detection ELISA kit. For phagocytosis assays, breast cancer cells were labelled with the green fluorescence dye carboxyfluorescein succinimidyl ester (CFSE) and CFSE-labelled cells were added to cultures of mouse peripheral blood mononuclear cells (PBMC)-derived macrophages labelled with the red fluorescence dye PKH26.

To examine the potential role of CD47 in the immune response to breast cancer, we generated CD47 knockout cell lines (two clones derived from 4T1 mouse breast cancer cells) using CRISPR-Cas9. The mutated sequences were obtained and lack of CD47 expression was detected by Western blot (Fig. 1). Knockout of CD47 in 4T1-luc2 had not a strong consequence on cell growth, viability or cell morphology. Since we previously observed that IR showed a low impact on the expression and cell membrane exposition of CD47 in breast cancer cells, we hypothesized that depletion of this protein in these cells could improve the macrophage-dependent phagocytosis of irradiated cells. To prove this hypothesis, we performed experiments comparing the phagocytosis ratio of wild-type versus CD47-KO cells. Depletion of CD47 highly increased the macrophage-dependent phagocytosis of this murine cell line as determined by co-localization of both CFSE and PKH26 (Fig. 2).

Here, we generated a cell model that will be useful to study the response of the immune system toward breast cancer during radiotherapy. This model will be assayed in in vivo systems using immune competent BALB/c mice and an immunodeficient athymic nu/nu mouse model.