Carfilzomib modulates tumor microenvironment to potentiate immune checkpoint therapy for cancer (2021)
Figure 5

Figure 5. Carfilzomib shrinks tumor in vivo through promoting M2 macrophage polarization into M1-like macrophage

A. Macrophages play a role in mediating shrinkage of lung cancer by Carfilzomib. TetO‐EGFR (T790M/Del19)/CC10-rtTA (TD) mice were fed with doxycycline for 3 months to induce lung cancer. Tumor burdens were then documented through computed tomography (CT) imaging before treatment (PreRx). After two-week treatment with saline (iv), Clodrosome (iv), Carfilzomib (iv), or Carfilzomib plus Clodrosome, mice were CT-scanned again (PstRx). Treatment effect was determined by comparing tumor-areas of PreRx and PstRx through ImagJ analysis.

B. Statistics of the tumor burden in lung tissue. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test).

C. Representative images of Hematoxylin and eosin (H&E) staining of the lung tissue obtained from different treatment groups. Expression of Ki-67 in lung tissues was detected by immunohistochemistry method.

D. Statistics of the tumor area and the number of Ki67 positive cells in sections of lung tissue. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).

E. Carfilzomib treatment promotes the infiltration of M1 macrophages. Tumor bearing TD mice were treated as indicated in (A) and euthanized. Lung tissues were dissected for evaluating M1/M2 populations through analyze the expression of CD80 and CD206 by flow cytometry.

F. Statistics represent the proportion of CD80 or CD206 positive cells in lung tissue. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).

G. Carfilzomib treatment promotes the infiltration and activation of CD8+ T cells. Lung tissues of (E) were used for evaluating the infiltration of CD8+ T cells and their expression of CD69 by flow cytometry.

H. Statistics about the proportion of CD8+ or CD69+ positive cells in lung tissue. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).

I. Genetic confirmation of role played by macrophages in mediating carfilzomib's tumor shrinking effect through immune reconstitution with bone marrow from CD11B-DTR mice. TD mice were irradiated with X-ray (8 Gy for 20 min), transplanted with 5×106 of bone marrows from CD11B-DTR mice. Immune reconstituted mice induced to develop lung cancer by doxycycline-diet. Mice were treated for 2 weeks with saline (iv), DT (ip), Carfilzomib (iv), or Carfilzomib plus DT. Tumor burdens were documented through CT imaging before and after treatment.

J. Statistics of the tumor burden in lung tissues. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test).

K, L. Carfilzomib shrinks tumor through modulating tumor microenvironment. EG7 cells were inoculated in wild-type or LyzM-cre;lsl-dTA to allow tumor xenograft to reach a volume of around 80 mm3. Mice were randomized for treatment (n=5) by saline or Carfilzomib for two weeks. The xenografts were dissected to photograph (K) or weigh (L) after treatment. Data are shown as mean ±SD and n indicates the number of biological replicates. *p < 0.05, ***p < 0.001 (student's t-test).