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

Amyloid Precursor-like Protein 2 Expression Increases during Pancreatic Cancer Development and Shortens the Survival of a Spontaneous Mouse Model of Pancreatic Cancer.

In the United States, pancreatic cancer is a major cause of cancer-related deaths. Although substantial efforts have been made to understand pancreatic cancer biology and improve therapeutic efficacy, patients still face a bleak chance of survival. A greater understanding of pancreatic cancer development and the identification of novel treatment targets are desperately needed. Our analysis of gene expression data from patient samples showed an increase in amyloid precursor-like protein 2 (APLP2) expression within primary tumor epithelium relative to pancreatic intraepithelial neoplasia (PanIN) epithelial cells. Augmented expression of APLP2 in primary tumors compared to adjacent stroma was also observed. Genetically engineered mouse models of spontaneous pancreatic ductal adenocarcinoma were used to investigate APLP2\u27s role in cancer development. We found that APLP2 expression intensifies significantly during pancreatic cancer initiation and progression in the LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model, as shown by immunohistochemistry analysis. In studies utilizing pancreas-specific heterozygous and homozygous knockout of APLP2 in the KPC mouse model background, we observed significantly prolonged survival and reduced metastatic progression of pancreatic cancer. These results demonstrate the importance of APLP2 in pancreatic cancer initiation and metastasis and indicate that APLP2 should be considered a potential therapeutic target for this disease

Evaluating Targets and Therapeutics for the Treatment of Pancreatic Cancer

Pancreatic cancer has a dismally low survival rate, due to inadequate understanding of the processes that are involved in disease development and progression. Despite the identification of oncogenic drivers such as KRAS and p53, there is a need for the identification of molecular targets to improve and develop novel therapeutic approaches for the treatment of pancreatic cancer. Studies from our laboratory have identified and evaluated targets and therapeutic approaches that can aid in our understanding of pancreatic cancer disease progression and improve patient outcomes. Through the use of epidermal growth factor receptor (EGFR) ligands (EGF and TGF-α) and small molecule inhibitor erlotinib, we evaluated the effects that EGFR activation and inhibition have on MHC class I expression. Activation of EGFR by EGF and TGF-α led to decreased expression of MHC class I at the cell surface. EGFR and MHC class I were also found to associate with one another. Inhibition of EGFR by erlotinib led to significant increases in MHC class I expression at the cell surface. We further investigated the ability of an histone deacetylase (HDAC) inhibitor (M344) to enhance MHC class I expression on pancreatic cancer cells, and found M344 increased it, while also inhibiting cell viability, proliferation, migration and having longer lasting anti-proliferative effects than the FDA-approved vorinostat. In combination with gemcitabine, M344 was found to significantly slow tumor growth in an orthotopic mouse model. Utilizing our novel KPC mouse model with pancreas-specific loss of amyloid precursor-like protein 2 (APLP2) (KPCA), which has been shown to prolong survival and inhibit metastasis, we investigated the role APLP2 has on collagen deposition during tumor development and the ability of APLP2 to alter pancreatic cancer cells to sense the tumor microenvironment. Loss of APLP2 led to a biphasic shift in collagen deposition, decreased integrin expression, and increased actin expression. Overall, we have shown that APLP2 and its downstream targets provide novel therapeutic strategies, while the use of erlotinib and M344 could potentially improve efficacy for the treatment of pancreatic cancer when used in a combinatorial approach with immunotherapy

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

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

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

Migration of pancreatic cancer cells is reduced upon treatment with M344.

Transwell assays were performed to assess the migration of (A) S2-013, (B) BxPC-3, (C) MIA PaCa-2, (D) T3M-4, and (E) CFPAC-1 cells following treatment with 1 μM or 10 μM of M344 (or with 0.1% DMSO, as the vehicle control) for 24 h. For the transwell assays, after treatment the cells were plated into 3 separate 8-μm inserts (1x105 cells/insert) in the presence of 1 μM M344, 10 μM M344, or 0.1% DMSO and incubated for 24 h. Cells were fixed and stained, and photographs of 3 random fields were taken for each of the inserts. The results of counts of the stained, migrated cells were averaged. The graph shows the average numbers of cells that migrated, and each error bar represents the standard error of the mean. The presented graph for the S2-013 cells is representative of the results from 3 separate experiments; the full experimental set of counts for each of the other cell lines was done once. The results of 0.1% DMSO control treatment versus the 1 μM or the 10 μM M344 treatment were compared using Student’s t test. The asterisks indicate the following p values: * p<0.05, ** p< 0.01, *** p<0.001, and **** p<0.0001.</p