Figure 2: Bone Marrow Resistance to Alemtuzumab Involves Microenvironmental Remodeling.
(A) 1×106 DFBL-20954 cells were injected into NSG mice and Alem treatment (daily x 2) was initiated on the indicated days 8, 12 and 16 (base of blue arrows). The tips of the blue arrows correspond to tumor burden of mice treated with Alem at 48 hours after the second treatment dose. N=3 mice for each condition at each time point.
(B) Assessment of DFBL-20954 lymphoma cells after in vivo treatment with APC-conjugated Alem. Representative flow cytometry plots shown.
(C) On day 10 after transplant of 1×106 DFBL-20954 cells, mice were treated with clodronate liposomes (clod, 200ul) or PBS. Alem (Alem) was given as a single dose of 10mg/kg i.v. on day 12. Tumor burden was assessed 48 hours after treatment. Two-sided Welch t-test, *p<0.05, **p<0.01, ***p<0.001.
(D) On day 19 after transplant of 106 DFBL-20954 cells, mice received clodronate or PBS (−). On day 21, mice received CTX, Alem (10mg/kg i.v. single dose), the combination or vehicle (PBS) and assessed for tumor burden on day 23. Mouse cells were depleted and human cell numbers and viability were assessed with trypan blue staining. Tumor burden is reported as the number of viable tumor cells. Two-sided Welch t-test.
(E) Primary, viable BM (7-AAD−hCD19−CD11b+F4/80+) monocytes/macrophages were sorted from unengrafted or lymphoma-engrafted NSG mice. Bone marrow tumor cells from corresponding engrafted mice or Raji cells were incubated in the presence or absence of Alem. All comparisons between unengrafted and vehicle under the same conditions (n=3 per condition) were non-significant.
(F) Primary viable splenic (7AAD−hCD19−CD11b−F4/80+) and BM (hCD19−CD11b+F4/80+) monocytes/macrophages were sorted from unengrafted or lymphoma-engrafted NSG mice and cultured with BM lymphoma cells from the corresponding xenograft that were treated ex vivo with Alem. All comparisons between splenic and BM macrophages under the same conditions (n=3 per condition) were non-significant.
(G) Ratio of lymphoma (hCD19+hCD45+) to macrophage (hCD19−CD11b−F4/80+) cells 48 hours after chemotherapy initiation of DFBL-20954-engrafted mice. Two-sided Welch t-test, *p<0.05, **p<0.01, ***p<0.001. Dox vs CTX BM Not significant
(H) Mice were engrafted with DFBL-20954 and given clodronate (25ul) or vehicle on day 19 followed by Alem or vehicle on day 21 and 22 and then sacrificed 48 hours after dosing. Spleen tumor burden shown. Two-sided Welch t-test, *p<0.05, **p<0.01, ***p<0.001.
(I) Macrophage BM (hCD19−CD11b+F4/80+) cellularity based on flow cytometry 8 days after initiation of the indicated agents. Two-sided Welch t-test, *p<0.05, **p<0.01, ***p<0.001. Comparison of PBS vs CTX shown for both xenografts. CTX vs Dox across all conditions significant (p<0.001 for DFBL-20954 and p<0.05 for DFBL-69487).
(J) Representative anti-F4/80 staining (brown) of fixed femurs from mice engrafted with DFBL-20954 or DFBL-69487 and harvested 40 hours after treatment with the indicated agents. Images are 1x and 20x magnified as indicated.