Figure 3: Cyclophosphamide Promotes Lymphoma Phagocytosis by Macrophages.

(A) Tumor cells from the spleen and BM of mice (n=3 per condition) engrafted with DFBL-20954 were isolated by mouse cell depletion 16 hours after treatment with PBS, doxorubicin (Dox), or cyclophosphamide (CTX). Dead cells were removed and the remaining live tumor cells were incubated ex vivo with bone marrow-derived macrophages in the presence and absence of Alem (Alem). Two-sided Welch’s t-test, *p<0.05, **p<0.01, ***p<0.001

(B) Surface expression by mean fluorescence intensity (MFI) compared to isotype control or total protein level by ELISA for the indicated proteins in the bone marrow and spleen of DFBL-20954-engrafted mice (n=3-6 per condition). Paired two-sided t-test.

(C) DFBL-20954 cells (N=3 mice) were isolated from BM of untreated mice, labeled with CFSE and co-cultured with bone-marrow derived macrophages in the presence or absence of Alem and Prostaglandin E2 (PGE2, 2ng/ml). Phagocytosis was quantified as percent of CD11b⁺ cells that were CFSE⁺. The experiment was performed three times and a representative example is shown.

(D) As in (C) but with recombinant human galectin-1 (Gal-1, 500 ng/ml) and lactose (20 mM).

(E) DFBL-20954 engrafted mice (n=3-6 per condition) were treated with the indicated agents and assayed for surface expression of the indicated markers on viable tumor cells. CD47 and VISTA were quantified at 16 hours post-treatment; CD52 and Calreticulin at 48 hours post-treatment. PGE2 and Gal-1 levels were quantified by ELISA 48 hours post-treatment.