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Supplemental materials from experiments testing the migration potential and non-specific phagocytic activity of SP LAM subsets as well as the T-ALL model in CCR2-/- mice.

Expression of chemokines in WT and CCR2-/- T-ALL mice.

Cell cycle analysis of SP LAM subsets and phenotypic evolution of MOMF in the liver of T-ALL mice.

Macrophages play important roles in both physiologic and pathologic processes and arise from successive waves of embryonic and adult hematopoiesis. Monocyte-derived macrophages (MOMF) exert distinct functions under pathologic conditions, and leukemia-associated macrophages (LAM) show considerable diversities in activation and functional phenotype. However, their origin and pathologic roles have not been well elucidated. Here we used wild-type and CCR2−/− mice to study the pathologic roles of monocyte-derived LAM in extramedullary tissues in models of Notch1-induced T-cell acute lymphoblastic leukemia (T-ALL). MOMF existed in the resting liver and spleen. In the spleen, Ly6C+ monocytes gave rise to the Ly6C+ macrophage subset. Furthermore, an increase of monocyte-derived LAM, including the Ly6C+ subset, was detected in the extramedullary tissues in leukemic mice. More monocyte-derived LAM, including Ly6C+ LAM, was detected in the spleens of leukemic mice transplanted with exogeneous mononuclear cells. Moreover, Ly6C+ LAM exhibited increased M1-related characteristics and contributed to sterile inflammation. In CCR2−/− leukemic mice, reduced Ly6C+ LAM, relieved sterile inflammation, and reduced distribution of leukemia cells were detected in extramedullary tissues. In addition, monocyte-derived Ly6C+ LAM expressed high levels of CCL8 and CCL9/10. Blocking CCR1 and CCR2 relieved hepatosplenomegaly and inhibited the extramedullary distribution of leukemia cells in T-ALL mice. Collectively, our findings reveal the multifaceted pathologic roles of monocyte-derived LAM in T-ALL progression.Significance:This study links monocyte-derived leukemia-associated macrophages with noninfectious inflammation and extramedullary distribution of leukemia cells during leukemia progression, providing new insight into macrophage-based immunotherapy in leukemia.

Including Figure S1-S12 and Table S1-S4

Supplemental materials to study the role of the CCL8-CCR1/CCR2 and CCL9/10-CCR1 axes.

Triple-negative breast cancer (TNBC) exhibits a high mortality rate and is the most aggressive subtype of breast cancer. As previous studies have shown that histone deacetylases (HDAC) may represent molecular targets for TNBC treatment, we screened a small library of synthetic molecules and identified a potent HDAC inhibitor (HDACi), YF438, which exerts effective anti-TNBC activity both in vitro and in vivo. Proteomic and biochemical studies revealed that YF438 significantly downregulated mouse double minute 2 homolog (MDM2) expression. In parallel, loss of MDM2 expression or blocking MDM2 E3 ligase activity rendered TNBC cells less sensitive to YF438 treatment, revealing an essential role of MDM2 E3 ligase activity in YF438-induced inhibition of TNBC. Mechanistically, YF438 disturbed the interaction between HDAC1 and MDM2, induced the dissociation of MDM2-MDMX, and subsequently increased MDM2 self-ubiquitination to accelerate its degradation, which ultimately inhibited growth and metastasis of TNBC cells. In addition, analysis of clinical tissue samples demonstrated high expression levels of MDM2 in TNBC, and MDM2 protein levels closely correlated with TNBC progression and metastasis. Collectively, these findings show that MDM2 plays an essential role in TNBC progression and targeting the HDAC1–MDM2–MDMX signaling axis with YF438 may provide a promising therapeutic option for TNBC. Furthermore, this novel underlying mechanism of a hydroxamate-based HDACi in altering MDM2 highlights the need for further development of HDACi for TNBC treatment.Significance:This study uncovers the essential role of MDM2 in TNBC progression and suggests that targeting the HDAC1–MDM2–MDMX axis with a hydroxamate-based HDACi could be a promising therapeutic strategy for TNBC.

RNA-seq analysis of SP normal macrophage and LAM subsets.

Gating strategy for macrophages in the liver and SP as well as monocytes/ macrophages in the transplantation experiments at the steady state.

Primers used in real-time RT-PCR

Triple-negative breast cancer (TNBC) exhibits a high mortality rate and is the most aggressive subtype of breast cancer. As previous studies have shown that histone deacetylases (HDAC) may represent molecular targets for TNBC treatment, we screened a small library of synthetic molecules and identified a potent HDAC inhibitor (HDACi), YF438, which exerts effective anti-TNBC activity both in vitro and in vivo. Proteomic and biochemical studies revealed that YF438 significantly downregulated mouse double minute 2 homolog (MDM2) expression. In parallel, loss of MDM2 expression or blocking MDM2 E3 ligase activity rendered TNBC cells less sensitive to YF438 treatment, revealing an essential role of MDM2 E3 ligase activity in YF438-induced inhibition of TNBC. Mechanistically, YF438 disturbed the interaction between HDAC1 and MDM2, induced the dissociation of MDM2-MDMX, and subsequently increased MDM2 self-ubiquitination to accelerate its degradation, which ultimately inhibited growth and metastasis of TNBC cells. In addition, analysis of clinical tissue samples demonstrated high expression levels of MDM2 in TNBC, and MDM2 protein levels closely correlated with TNBC progression and metastasis. Collectively, these findings show that MDM2 plays an essential role in TNBC progression and targeting the HDAC1–MDM2–MDMX signaling axis with YF438 may provide a promising therapeutic option for TNBC. Furthermore, this novel underlying mechanism of a hydroxamate-based HDACi in altering MDM2 highlights the need for further development of HDACi for TNBC treatment. Significance: This study uncovers the essential role of MDM2 in TNBC progression and suggests that targeting the HDAC1–MDM2–MDMX axis with a hydroxamate-based HDACi could be a promising therapeutic strategy for TNBC.

Macrophages play important roles in both physiologic and pathologic processes and arise from successive waves of embryonic and adult hematopoiesis. Monocyte-derived macrophages (MOMF) exert distinct functions under pathologic conditions, and leukemia-associated macrophages (LAM) show considerable diversities in activation and functional phenotype. However, their origin and pathologic roles have not been well elucidated. Here we used wild-type and CCR2−/− mice to study the pathologic roles of monocyte-derived LAM in extramedullary tissues in models of Notch1-induced T-cell acute lymphoblastic leukemia (T-ALL). MOMF existed in the resting liver and spleen. In the spleen, Ly6C+ monocytes gave rise to the Ly6C+ macrophage subset. Furthermore, an increase of monocyte-derived LAM, including the Ly6C+ subset, was detected in the extramedullary tissues in leukemic mice. More monocyte-derived LAM, including Ly6C+ LAM, was detected in the spleens of leukemic mice transplanted with exogeneous mononuclear cells. Moreover, Ly6C+ LAM exhibited increased M1-related characteristics and contributed to sterile inflammation. In CCR2−/− leukemic mice, reduced Ly6C+ LAM, relieved sterile inflammation, and reduced distribution of leukemia cells were detected in extramedullary tissues. In addition, monocyte-derived Ly6C+ LAM expressed high levels of CCL8 and CCL9/10. Blocking CCR1 and CCR2 relieved hepatosplenomegaly and inhibited the extramedullary distribution of leukemia cells in T-ALL mice. Collectively, our findings reveal the multifaceted pathologic roles of monocyte-derived LAM in T-ALL progression. Significance: This study links monocyte-derived leukemia-associated macrophages with noninfectious inflammation and extramedullary distribution of leukemia cells during leukemia progression, providing new insight into macrophage-based immunotherapy in leukemia.

Background: One of the major concerns in cancer treatment and clinical translation of many anti-cancer compounds has been their potential toxicities, especially hepatotoxicity. Nanoparticle (NP) holds great potential to solve the problem, as many clinical studies have not shown increased hepatotoxicity for nanotherapeutics with higher accumulation in the liver. However, there are few studies investigating how nanoparticle assists in reducing hepatotoxicity. Herein, we demonstrated that nanoformulation of known hepatotoxic anti-cancer compounds showed lower hepatotoxicity than their small molecule counterparts. Importantly, we demonstrated that slower drug release is associated with reduced hepatotoxicity. We also showed that that Kupffer cell uptake in the liver reduce nanotherapeutics’ hepatotoxicity. Methods: Two different antineoplastic drugs with high hepatotoxicity, SN-38 and wortmannin (Wtmn), were encapsulated into lipid shell-PLGA core nanoparticles separately. NP release kinetics were controlled by adjusting lipids/polymer ratio for fast (Fast-NPs), medium (Medium-NPs) and slow drug release profile (Slow-NPs), respectively. Hepatotoxicity of NPs or free drugs was analyzed by assessing serum ALT and AST levels post intravenous injection at ½ MTD in CD-1 mice. IHC staining of HO-1 and Mn-SOD was also used to show liver damage. In vitro hepatotoxicity of primary hepatocytes after Kupffer cell uptake was assessed by MTS assay and LDH assay. In vivo macrophage depletion was achieved using clodrosome. Results: Nanoformulations of SN-38 and Wtmn showed lower ALT and AST levels than their small molecule counterparts 12 h and 24 h post treatment. The liver damage was further confirmed with IHC staining of Mn-SOD and HO-1. To address the effect of drug release kinetics on hepatotoxicity, we showed that over 90% of drugs were released within 24 h in Fast-NP. While the release rate of Slow-NPs was 66.8% for SN-38 and 67.5% for Wtmn. We showed that Slow-NPs of Wtmn resulted in minimal increase of ALT and AST levels. Compared to the baseline in untreated mice, the ALT level was 2.3-fold for Slow-NPs, 4.4-fold for Medium-NPs and 6-fold for fast-NPs; meanwhile, the AST level was 1.6-fold, 2.5-fold and 3.6-fold, respectively. To assess the effect of Kupffer cell uptake in hepatotoxicity, we found that the toxicity of the nanotherapeutics for primary hepatocytes significantly reduced after Kupffer cell uptake in vitro. We further confirmed it in vivo by depleting Kupffer cells in CD-1 mice. We demonstrated that ALT and AST levels of nanotherapeutics significantly increased to the levels comparable to free drugs after Kupffer cell depletion. Conclusions: We demonstrate that nanoparticle reduces hepatotoxicity of cancer treatment by controlled release and Kupffer cell uptake. Our work bridges an important knowledge in nanoparticle drug delivery and clinical translation of nanomedicine. Citation Format: Yu Mi, Feifei Yang, Andrew Z. Wang. Nanoparticle reduces hepatotoxicity of cancer treatment by controlled release and Kupffer cell uptake [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3899.

Background: Cancer immunotherapy is an exciting new approach to cancer treatment and there is strong interest in strategies to improve the long-term durable response rates of cancer immunotherapy. One approach is to combine checkpoint inhibitors such as aPD-1 with T cell activator such as OX40 agonist to further increase immune activation. We hypothesized that we can improve the therapeutic efficacy of this approach by temporally control the activation of OX40 and inhibition of PD-1 pathways. To accomplish this, we utilized nanoparticles that can deliver anti-OX40 and anti-PD1 antibodies simultaneously to T cells. Methods: Agonist antibody (anti-OX40) and antagonist antibody (anti-PD1) were conjugated to PLGA-PEG-Maleimide nanoparticles (AANPs) with precise ratio control and quantified by ELISA. Their specific binding to the target proteins was shown in vitro by flow cytometry. The tumor inhibition efficiency was assessed on mice bearing different tumor models. Two tumors were inoculated subcutaneously (105 B16F10 cells) or on fourth mammary fat pad (105 4T1 cells) on both flanks of mice. One side of tumor was irradiated once and AANPs were injected twice every 3 days. In vivo depletion experiments were tested on C56Bl6 mice and CD11b/c mice. Different populations of T cells in tumor and spleen were analyzed by flow cytometry and by fluorescent IHC staining. T cell killing assay and IFN-γ ELISpot were studied. Co-localization was demonstrated with fluorescent labeled antibodies and the corresponding AANPs. Results: AANPs showed a 30% cure rate, compared to 10% of free antibodies, 0% of anti-PD1 conjugated NPs, and 0% of anti-OX40 conjugated NPs in B16F10 melanoma model. We then re-challenged the cured mice with 2×105 B16F10 cells and none of the mice developed another tumor. In 4T1 breast cancer model, the survival rate on day 39 was 50% with AANPs treatment, compared to 22% in the mixture of anti-PD1 conjugated nanoparticles and anti-OX40 conjugated nanoparticles, and 0% of free antibodies. We demonstrated that AANPs led to a higher medium TCD8+/Treg ratio in tumors. The therapeutic effect was mediated by CD8+ T cells as elimination of these cells abrogated the therapeutic effects. In vitro study confirmed that AANPs were able to improve T cell stimulation compared to free antibodies by increasing IFN-γ excretion (2x). We further confirmed co-localization of antibodies with AANPs on tumor infiltration T cells in vivo. Our data demonstrated that spatial-temporal delivery of agonist and antagonist could improve T cell activation and cancer immunotherapy. Conclusions: Our data demonstrates that spatial-temporal delivery of agonist and antagonist can improve T cell activation and cancer immunotherapy. Citation Format: Yu Mi, Christof C. Smith, Feifei Yang, Jonathan Serody, Benjamin Vincent, Andrew Z. Wang. Spatial-temporal delivery of OX40 agonist and PD-1 inhibitor using nanoparticles improves therapeutic efficacy of cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 978. doi:10.1158/1538-7445.AM2017-978