Blockade of p38 kinase impedes the mobilization of protumorigenic myeloid populations to impact breast cancer metastasis

Abstract

Patients with metastatic breast cancer (MBC) have limited therapeutic options and novel treatments are critically needed. Prior research implicates tumor-induced mobilization of myeloid cell populations in metastatic progression, as well as being an unfavorable outcome in MBC; however, the underlying mechanisms for these relationships remain unknown. Here, we provide evidence for a novel mechanism by which p38 promotes metastasis. Using triple-negative breast cancer models, we showed that a selective inhibitor of p38 (p38i) significantly reduced tumor growth, angiogenesis, and lung metastasis. Importantly, p38i decreased the accumulation of myeloid populations, namely, myeloid-derived suppressor cells (MDSCs) and CD163⁺ tumor-associated macrophages (TAMs). p38 controlled the expression of tumor-derived chemokines/cytokines that facilitated the recruitment of protumor myeloid populations. Depletion of MDSCs was accompanied by reduced TAM infiltration and phenocopied the antimetastatic effects of p38i. Reciprocally, p38i increased tumor infiltration by cytotoxic CD8⁺ T cells. Furthermore, the CD163⁺ /CD8⁺ expression ratio inversely correlated with metastasis-free survival in breast cancer, suggesting that targeting p38 may improve clinical outcomes. Overall, our study highlights a previously unknown p38-driven pathway as a therapeutic target in MBC.

Keywords: breast cancer; macrophages; metastasis; myeloid-derived suppressor cells; p38 kinase; tumor microenvironment.

Figure 1.. Tumor growth and metastasis are reduced by systemic treatment with a p38 inhibitor.

(A) 4T1-Luc mammary carcinoma cells (50,000) were orthotopically implanted into female BALB/c female 6-week old mice (9 mice per group). Starting on day 14 post-tumor implantation, when the tumor size reached ~100mm³, mice were treated by daily oral gavage with vehicle-control (saline) or p38i (Ralimetinib). (B) Quantification of bioluminescence in live animals at day 34. (C) Tumor weight at day 34. (D) Survival was evaluated using Kaplan-Meier estimator based on time-to-arrive at 1cm3 of tumor size measured in 4T1 tumor-bearing mice in non-treated (4T1) and Ralimetinib-treated groups, (n=9). Comparison was made using the Log-rank test (P<0.01). (E) Tumor blood vessels were visualized by anti-CD31 staining, bar=100μm. (F) The microvessel density was evaluated on comparable size tumors using CD31-stained sections in six fields for each case (3 tumors/group) and presented as mean area per field (0.2 mm²). Statistical significance was determined using the two-tailed unpaired t-test (P<0.05). (G) Detection of metastases in H&E-stained lung sections. (H) Luminescence activity in whole-cell extracts from the lungs of control or p38i-treated comparable size groups, 3 mice per group. (I) Immunoblots of whole tumor extracts from mice bearing 4T1 tumors from the vehicle control and p38i-treated groups.

Figure 2.. Blockade of p38 kinase reduces angiogenesis at the early tumor growth stage.

(A) The study outline: 4T1-Luc mammary carcinoma cells (200,000) were orthotopically implanted into BALB/c female 6-week old mice (9 mice per group). Treatment with p38i/Ralimetinib at 30mg/kg was initiated on day 4 by daily oral gavage. (B) Bioluminescence images of tumor-free (naïve), tumor-bearing (tumor), or tumor-bearing and p38i-treated mice. Note the absence of luminescence at the lungs. (C) Quantification of bioluminescence in live animals on day 14. (D) Tumor weight (median) at day 14 of the study. (E) Images of CD31-stained tumor sections from untreated or p38i-treated tumor-bearing mice, bar=100μm. (F) Quantification of the microvessel density in CD31-stained sections of comparable-size tumors (G), a mean area per field (0.2 mm²), 6 fields per tumor section, 3 tumors per group. (G) Comparison of tumor volumes from untreated and p38i-treated groups. Statistical significance was determined using the two-tailed unpaired t-test (P<0.05; ns, not significant, P>0.05).

Figure 3.. Blockade of p38 reduces accumulation of MDSCs in the lungs and the expansion of PMN-MDSCs and megakaryocytes in the spleen of tumor-bearing mice.

(A) Lung tissues from tumor-free (naïve), 4T1-Luc tumor-bearing (Tu) and tumor-bearing mice treated with p38i were stained for Ly6G, a marker of PMN-MDSCs, bar=200μm. (B) Quantification of Ly6G⁺ cells in the lung sections, 3 fields per section, 3 mice per group. (C) Flow cytometry analysis of CD11b⁺Ly6C^loLy6G⁺ cells in whole-lung tissues, 3 mice per group. (D) H&E-stained spleen tissues at 20x magnification (top row, bar=1mm), bottom at 200x, bar=100μm. Yellow arrows show megakaryocytes; WP, white pulp; RP, red pulp. Note an increase in the RP and spleen size in tumor-bearing mice compared to tumor-free mice, and their reduction by p38i/Ralimetinib. (E) Spleen weights were measured at day 14 of the study, n=5. (F) Evaluation of megakaryocytes (MK) in the spleen of tumor-free (naïve), tumor-bearing (Tu), and tumor-bearing treated with p38i (Tu+p38i). (G) Spleen sections stained for Ly6G, bar=100μm. (H) Flow cytometry data for CD11b⁺Ly6C^loLy6G⁺ cells in whole-spleen tissue from 3 mice per group. (I) Quantification of granulocytes in peripheral blood using complete blood count (CBC) assays. Comparisons were done with material from naïve mice and mice with comparably sized tumors, and statistical significance was determined using the two-tailed unpaired t-test (*P<0.05; **P<0.01; ***P<0.001).

Figure 4.. Evaluation of CD163⁺ macrophages in tumor tissues.

(A) Quantification of monocytes in peripheral of tumor-free mice (naïve), tumor-bearing untreated (Tu) or p38i-treated mice by CBC assays at day 14 of the study. (B) Images of CD163-stained tumor sections, bar=100μm. (C) Quantification of CD163⁺ cells in tumor tissues, 3 fields/section, 3 sections/group. (D) Images of CD163 and F4/80-stained sections of tumor xenografts of MDA-MB-231 human breast cancer cells (empty-vector control and dnp38) in SCID mice, bar=200μm. (E) Images of CD8A-stained 4T1-Luc tumor sections, bar=100μm. (F) Quantification of CD8A⁺ cells in tumor tissues, 3 fields/section, 3 sections/group. (G) The Kaplan–Meier estimator of metastasis-free survival in patients with BC, n=3,951.

Figure 5.. Depletion of PMN-MDSCs by anti-Ly6G antibody impedes the pre-metastatic niche.

(A) Panels show bioluminescence images and their quantification in live animals at day 14. 4T1-Luc mammary carcinoma cells (200,000) were orthotopically implanted in female BALB/c 6-week old mice, 7 mice/group. The next day, mice were administrated i.p. injections of vehicle-control (saline) or antibody to Ly6G scheduled on every other day. (B) Tumor volume at day 14. Statistical significance was determined using the two-tailed unpaired t-test (*P<0.05). (C) Blood vessels (CD31), tumor-associated macrophages (CD163) and tumor-infiltrating T cells (CD8A) were visualized in tumor sections from comparable-size tumors (day 14) treated with anti-Ly6G or isotype-control (CTR) antibody, bar=100μm. (D) Panels show Ly6G stained sections of the lungs and liver of mice treated with anti-Ly6G or isotype-control (CTR) antibody, bar=100μm.

Figure 6.. The p38 kinase controls the tumor-immune crosstalk that mediates the expansion of pro-tumor myeloid populations.

(A) Expression of myeloid cell markers and cytokine/chemokine receptors in the lungs from tumor-free (naïve) and 4T1 tumor-bearing mice (tumor). (B) Levels of secreted factors were measured in serum-free media conditioned for 48 hours by human MDA-MB-231 breast cancer cells, expressing the empty-vector (CTR) or dnp38 (dn-p38) constructs. Two biologically independent replicates were assayed using the Luminex 200 System. (C) Quantitative RT-PCR of CCL2/MCP-1 in total RNA from MDA-MB-231 and WI-38 mesenchymal stromal cells which were treated with vehicle (DMSO) or p38i (Ralimetinib) for 48 hours. (D) Expression profiles of breast cancer TCGA dataset. (E) Kaplan-Meier estimation of recurrence-free survival for CCR1 and S100A8 in BC patients (n=3,951). (F) The working model: the p38 kinase in tumor and mesenchymal stromal cells in the TME mediates expansion and mobilization of pro-tumor myeloid cells by controlling the tumor-immune crosstalk.