Fibroblasts Influence the Efficacy, Resistance, and Future Use of Vaccines and Immunotherapy in Cancer Treatment

Tumors are composed of not only epithelial cells but also many other cell types that contribute to the tumor microenvironment (TME). Within this space, cancer-associated fibroblasts (CAFs) are a prominent cell type, and these cells are connected to an increase in tumor progression as well as alteration of the immune landscape present in and around the tumor. This is accomplished in part by their ability to alter the presence of both innate and adaptive immune cells as well as the release of various chemokines and cytokines, together leading to a more immunosuppressive TME. Furthermore, new research implicates CAFs as players in immunotherapy response in many different tumor types, typically by blunting their efficacy. Fibroblast activation protein (FAP) and transforming growth factor β (TGF-β), two major CAF proteins, are associated with the outcome of different immunotherapies and, additionally, have become new targets themselves for immune-based strategies directed at CAFs. This review will focus on CAFs and how they alter the immune landscape within tumors, how this affects response to current immunotherapy treatments, and how immune-based treatments are currently being harnessed to target the CAF population itself

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

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

Example of flow cytometry gating for the caspase 3/7 activation assay.

Details of the experimental process can be found in the legend for Fig 4. (TIF)</p

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

Pancreatic cancer cell proliferation is decreased upon M344 treatment.

After treatment with M344 at various concentrations, the proliferation of (A) S2-013, (B) BxPC-3, (C) MIA PaCa-2, (D) T3M-4, and (E) CFPAC-1 cells was assessed by the MTT assay at 24, 48, and 72 hours. The graphs display the optical density (OD) at 570 nm relative to the OD for the 0.1% DMSO control. Each error bar represents the standard error of the mean. These results are representative of findings in 2–3 separate experiments for each cell line. 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<0.001, **** p<0.0001.</p

Immunoblotting of pancreatic cancer cell line HLA class I heavy chains following treatment of the cells with M344.

The immunoblot data displayed here correspond to Fig 9. The proteins were transferred after electrophoresis to a membrane that was then divided. The top portion was probed with anti-HSC 70 (loading control) antibody and the bottom portion was probed with the HC10 antibody (for HLA-B and–C heavy chains). The blots were imaged, and a long exposure and a short exposure are shown (on the left and right, respectively). (TIF)</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