Novel Mouse Model for Analysis of Macrophage Function in Neuroblastoma

Background: Neuroblastoma is the third most common childhood cancer and accounts for 12% of cancer-associated deaths in children under the age of 15. Patients with high risk neuroblastoma have a poor 5-year survival rate of less than 50%. Neuroblastoma tumors treated with the histone deacetylase inhibitor (HDACi) vorinostat have increased infiltration of macrophages with upregulated immune cell-surface receptors. Neuroblastoma cells release VEGF and M-CSF, which may alter intratumoral macrophage populations. VEGF has also been implicated in alteration of amyloid precursor protein family processing. Our lab demonstrated that amyloid precursor protein 2 (APLP2), a member of the amyloid precursor protein family, plays an important role in the migration of tumor cells. APLP2 is known to be expressed by macrophages, but no studies have previously examined macrophage functions that are impacted by APLP2 in the context of neuroblastoma disease and its treatment by HDACi drugs. Significance of Problem: Because of the high morbidity and mortality associated with neuroblastoma, studies such as this one that are designed to comprehend the interaction of immunity and treatment in neuroblastoma are clinically significant. The results from this study are also expected to expand our comprehension of macrophage function and regulation, and thus will be of broad value in the immunology and oncology fields. Experimental Design and Results: We have treated neuroblastoma tumor cells in vitro with M344, an HDACi with structural similarity to vorinostat, and showed that M344 decreases neuroblastoma cell growth. In addition, we have generated mice that lack APLP2 expression in cells expressing the Csf-1 receptor (a protein characteristically expressed by macrophages and dendritic cells). We discovered that following polarization, macrophages collected from the bone marrow of these mice have an altered distribution of M1 and M2 sub-populations, which are macrophage sub-populations known to differ in their migratory capabilities. Furthermore, we have shown that M1 and M2 subpopulations of bone marrow-derived macrophages from normal mice differ in their expression of APLP2. Thus, APLP2 is influential in macrophage biology, and we have created a novel mouse model for defining its specific contributions in mice treated with HDACi that influence macrophage biology. Conclusions: Based on the data that we have acquired, we are well positioned to fully explore both the impact of HDACi drugs on macrophage/dendritic cell populations in a syngeneic neuroblastoma mouse model, and to define the role of APLP2 in the function of these cell populations in the context of neuroblastoma.https://digitalcommons.unmc.edu/chri_forum/1000/thumbnail.jp

Gemcitabine Modulates HLA-I Regulation to Improve Tumor Antigen Presentation by Pancreatic Cancer Cells

Pancreatic cancer is a lethal disease, harboring a five-year overall survival rate of only 13%. Current treatment approaches thus require modulation, with attention shifting towards liberating the stalled efficacy of immunotherapies. Select chemotherapy drugs which possess inherent immune-modifying behaviors could revitalize immune activity against pancreatic tumors and potentiate immunotherapeutic success. In this study, we characterized the influence of gemcitabine, a chemotherapy drug approved for the treatment of pancreatic cancer, on tumor antigen presentation by human leukocyte antigen class I (HLA-I). Gemcitabine increased pancreatic cancer cells’ HLA-I mRNA transcripts, total protein, surface expression, and surface stability. Temperature-dependent assay results indicated that the increased HLA-I stability may be due to reduced binding of low affinity peptides. Mass spectrometry analysis confirmed changes in the HLA-I-presented peptide pool post-treatment, and computational predictions suggested improved affinity and immunogenicity of peptides displayed solely by gemcitabine-treated cells. Most of the gemcitabine-exclusive peptides were derived from unique source proteins, with a notable overrepresentation of translation-related proteins. Gemcitabine also increased expression of select immunoproteasome subunits, providing a plausible mechanism for its modulation of the HLA-I-bound peptidome. Our work supports continued investigation of immunotherapies, including peptide-based vaccines, to be used with gemcitabine as new combination treatment modalities for pancreatic cancer

The histone deacetylase inhibitor M344 as a multifaceted therapy for pancreatic cancer.

The histone deacetylase (HDAC) inhibitor vorinostat, used with gemcitabine and other therapies, has been effective in treatment of experimental models of pancreatic cancer. In this study, we demonstrated that M344, an HDAC inhibitor, is efficacious against pancreatic cancer in vitro and in vivo, alone or with gemcitabine. By 24 hours post-treatment, M344 augments the population of pancreatic cancer cells in G1, and at a later time point (48 hours) it increases apoptosis. M344 inhibits histone H3 deacetylation and slows pancreatic cancer cell proliferation better than vorinostat, and it does not decrease the viability of a non-malignant cell line more than vorinostat. M344 also elevates pancreatic cancer cell major histocompatibility complex (MHC) class I molecule expression, potentially increasing the susceptibility of pancreatic cancer cells to T cell lysis. Taken together, our findings support further investigation of M344 as a pancreatic cancer treatment

M344 impairs viability in combination with gemcitabine.

S2-013 pancreatic cancer cells were treated with 0.1% DMSO, 10 μM M344, 100 nM gemcitabine, or 10 μM M344 + 100 nM gemcitabine. Viability was assessed by the trypan blue exclusion assay at 24, 48, and 72 hours and graphed as the number of live cells/total cells x 100. Each error bar represents the standard error of the mean. The results from 0.1% DMSO control treatment versus treatment with each M344 concentration were compared using Ordinary One-way ANOVA with Dunnett’s Multiple Comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the p values: * p<0.05, ** p< 0.01, **** p<0.0001.</p

In pancreatic cancer cells, M344 causes cell cycle arrest in G₁.

Treatment of S2-013 cells with 1 or 10 μM M344 resulted in large increases in the populations accumulated in G1 at 24 hours (A), 48 hours (B), and 72 hours (C), as shown by propidium iodide staining and flow cytometry. Statistical comparisons were made using a Two-way ANOVA with Tukey’s Multiple Comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the following p values: * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001.</p

M344-induced apoptosis is apparent by 48 hours and necrosis peaks at 72 hours.

S2-013 cells were treated with 0.1% DMSO control or M344 (1 μM or 10 μM) for 24, 48, or 72 hours. Caspase-3 and caspase-7 cleavage was simultaneously analyzed by using the CellEventTM Caspase-3/7 Green Flow Cytometry Assay Kit. The SYTOXTM AADvancedTM Dead Cell Stain included in the kit identified necrotic cells. Each error bar represents the standard error of the mean. Statistical comparisons of the results were done using a Two-way ANOVA with Tukey’s multiple comparisons test. The asterisks indicate the following p values: * <0.05, ** <0.01, *** <0.001, **** <0.0001.</p

M344 decreases orthotopic pancreatic tumor growth when used as a treatment alone or in combination with gemcitabine.

(A) S2-013 cells were orthotopically implanted into the pancreas of female NU/J mice. After 8 days, the tumor volume for each mouse was monitored twice weekly with the VisualSonic Vevo 3100 Imaging System. At 15 days post-implantation of tumor cells, the mice were randomized into control or treatment groups with matched average tumor volumes. M344 was administered intraperitoneally at 10 mg/kg for 5 days per week (5 days on, 2 days off). Gemcitabine was given every 3 days intraperitoneally at 50 mg/kg. On Day 25 post-tumor implantation, the mice were euthanized and the tumors were resected and weighed. The changes in tumor volume over time are shown in (B) and representative images of tumors at 25 days post implantation are shown in (C). For statistical analysis, ordinary One-way ANOVA with Dunnett’s Multiple Comparisons test in GraphPad Prism Version 8.4.2 was used. The asterisks indicate the following p values: * p<0.05, ** p< 0.01, *** p<0.001.</p

Compared to vorinostat, M344 decreases pancreatic cancer cell proliferation more effectively for a longer duration.

The proliferation of S2-013 cells was assessed by the MTT assay following treatment with 0.1% DMSO control or with 1 μM, 5 μM, 10 μM, or 25 μM M344 or vorinostat for 48 hours or 72 hours. The effects of M344 versus vorinostat at 48 hours (A) and at 72 hours (B) are shown. The same data are displayed for M344 (C) and vorinostat (D) at 48 hours versus 72 hours. The error bars represent the standard error of the mean. The results were compared using a Two-way ANOVA with Tukey’s multiple comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the following p values: ** p< 0.01, *** p<0.001, **** p<0.0001.</p

MHC class I expression on S2-013 pancreatic cancer cells is increased following M344 treatment.

After 24- or 48-hour treatments with 0.1% DMSO (vehicle control) or with M344 at 5 or 10 μM concentrations, cell surface expression was monitored using flow cytometry with the BB7.2 antibody for peptide-occupied HLA-A2 and the B1.23.2 antibody that detects HLA-B/C. The bar graphs depict the median fluorescence intensity (MFI) fold change relative to the 0.1% DMSO control treatment for (A) 24-hour M344 treatment and (B) 48-hour M344 treatment. Triplicate wells for each concentration and time point were analyzed. Each error bar represents the standard error of the mean. Statistical comparison of the results from the 0.1% DMSO control treatment versus treatment with each M344 concentration was performed using Ordinary One-way ANOVA with Dunnett’s Multiple Comparisons Test in GraphPad Prism Version 8.4.2. The asterisks indicate the p values: * pC) HLA-A2 expression at 24 hours post-treatment with M344, (D) HLA-A2 at 48 hours post-treatment with M344, (E) HLA-B/C at 24 hours post-treatment with M344, and (F) HLA-B/C at 48 hours post-treatment with M344. In the histograms, solid lines represent 0.1% DMSO treatment, dashed lines represent 5 μM M344 treatment, and solid gray areas represent 10 μM M344 treatment.</p

M344 treatment of pancreatic cancer cells increases global histone H3 acetylation more than vorinostat treatment.

The inhibition of histone H3 deacetylation in S2-013 cells by M344 versus vorinostat was compared by monitoring the percentage of acetylated histone H3 following 48-hour treatment with M344 or vorinostat (at 1 μM or 10 μM) or with the 0.1% DMSO vehicle alone. Global H3 acetylation was assessed using the EpiQuikTM Global Histone H3 Acetylation Assay Kit. The data are displayed in (A) for 1 μM M344 and vorinostat (and 0.1% DMSO control), and in (B) for 10 μM M344 and vorinostat (and 0.1% DMSO control). These data were compiled from two biological replicates with triplicate samples in the first assay and quintuplet samples in the second assay. The graphs display the percentage global histone H3 acetylation, calculated by this formula: OD (treated sample–blank) / OD (untreated control–blank) X 100%. Error bars represent the standard error of the mean. Statistical comparisons were made with One-Way ANOVA with Tukey’s multiple comparisons tests. The asterisks indicate the p values: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.</p