New knowledge for me:
- sialic acids: protect cancer cells against innate immune cells killing.
2.CD20 exists in 95% lymphoma cells, 利妥昔单抗第一个单抗类药物。
CD47 in cancer cell: DON'T EAT ME!
methods
-
ADCP assay: use pHrodo red dye to mark cancer cell, and the color is green, once it has been eaten by macrophage, it would be digest in lysosome, and the pH condition of lysosome is low, the dye would become red, by detect the signal of red color, we can know the level of phagocytosis.
ADCP assay
Methods and results
1. cellular-intrinsic regulators of antibody-dependent cellular phagocytosis(ADCP) in cancer cell
methods:
Ramos lymphoma cells genome-wide CRISPR knockdout library.
( with anti-CD20)
results:
Found some gene which was already known as anti-phagocytic factor, like CD47, and some gene which was not linked to phagocytosis regulation, like APMAP.
2.to find the additional regulators of ADCP that are not endogenously expressed at high levels.
methods:
Ramos lymphoma cells genome-wide CRISPR activation library.
( with anti-CD20 and anti-CD47,cause CD47 is the key regulator of anti-phagocytic, so blocking CD47 can help find other phagocytic factors.)
results:
CD20 is a strong pro-phagocytic gene.
Reveal many regulators, and validated 9 of those genes by ADCP assay.
Aim3 4 :Does their screen platform have more application?
3.Do CRISPRa screen results in Ramon cell could used in other type of cancer?
methods:
SMAGP(from CRISPRa screen results) is high expresses in colon cancer cells.
results:
deletation of SMAGP in RKO cell sensetized to phagocytosis in the present of anti-CD47 antibody.
4.Do the overexpression against ADCP genes can affect the stage of ADCP--is the result of CRISPRa correct?
methods:
target cell-macrophage adhesion assay.
result:
most of those gene overexpression can against ADCP, but some of thoes have no influence.
Aim 5 6 7 8: choose a gene to further research.
5. APMAP loss sensitizes lymphoma cells to ADCP
methods:
genome-wide ADCP acreen in another lymphoma cell line: Karpas 299.
results:
APMAP is the highly anti-phagocytosis gene in two screen results, APMAP is not essential for growth in cell line and mouse.
APMAP-KO cell is sensetized to phagocytosis in the present of anti-CD20/47 antibody, but have no influence when ther is no anti-CD20/47 antibody. And APMAP-KO synergizes with cancer antigen-targeting antibody.
6. funtion domain of APMAP.
methods:
a series of flag-tagged APMAP alleles.
rescue expriment: KO cell line and add different APMAP.
E103 site: predict calcium-binding residue, homology to PON1.
results:
E103A exhibited the similar function of wt APMAP.
7.APMAP's function is conserved in other cancer cell line.
methods:
eight additional cancer cell lines.
results:
APMAP-targeting sgRNAs greatly sensitized cells to phagocytosis driven by anti-CD47.
8. APMAP's function verification in mouse model.
methods:
flanks of NSG mice, which lack adaptive immune cells but contain functional macrophages.
9.regulators in macrophage.
methods:
CRISPR KO screen to uptake WT or APMAP KO cell.
results:
GPR84 and GNB2 are specitial to uptake APMAP KO cell.
GNB2 and PREX1 are the downstream signal of GPR84.
The interesting thing is GPR84 agonists only enhance the phagocytosis of safe KO cell, but not APMAP KO cell. High cencentration of GPR84 could decrease the phagocytosis of both cell, this may reflect the desensitized.
