Fibroblast growth factors (FGFs) have mitogenic activity toward a wide variety of cells of mesenchymal, neuronal, and epithelial origin and regulate events in normal embryonic development, angiogenesis, wound repair, and neoplasia. FGF-2 is expressed in many normal adult tissues and can regulate migration and replication of intestinal epithelial cells in culture. However, little is known about the effects of FGF-2 on intestinal epithelial stem cells during either normal epithelial renewal or regeneration of a functional epithelium after injury. In this study, we investigated the expression of FGF-2 in the mouse small intestine after irradiation and determined the effect of exogenous FGF-2 on crypt stem cell survival after radiation injury. Expression of FGF-2 mRNA and protein began to increase at 12 h after γ-irradiation, and peak levels were observed from 48 to 120 h after irradiation. At all times after irradiation, the higher molecular mass isoform (∼24 kDa) of FGF-2 was the predominant form expressed in the small intestine. Immunohistochemical analysis of FGF-2 expression after radiation injury demonstrated that FGF-2 was predominantly found in the mesenchyme surrounding regenerating crypts. Exogenous recombinant human FGF-2 (rhFGF-2) markedly enhanced crypt stem cell survival when given before irradiation. We conclude that expression of FGF-2 is induced by radiation injury and that rhFGF-2 can enhance crypt stem cell survival after subsequent injury.

In that salmonellac have been iplicted in an unprecedented array of diseases, sequences found to be specific to this species are often thought to be involved in the virulence attributes not seen in other enteric bacteria. To identify the molecular, genetic, and phenotypic characteristics that differentiate bacterial species, we analyzed five cloned DNA agments that were originally described as being confined to Salnonella.

The anti-apoptotic effect of PGE(2) was examined in Jurkat cells (human T-cell leukemia) by incubation with PGE(2) (5 nM) prior to treatment with the cancer chemotherapeutic agent camptothecin. Apoptosis was evaluated by caspase-3 activity in cell extracts and flow cytometry of propidium iodide-labeled cells. Pre-incubation with PGE(2) reduced camptothecin-induced caspase activity by 30% and apoptosis by 35%, respectively. Pharmacological data demonstrate that the EP4 receptor is responsible for mediating the protection from camptothecin-induced apoptosis. Pre-treatment of the cells with the EP4 antagonist (EP4A) prior to PGE(2) and camptothecin abolished the increased survival effect of PGE(2). Specific inhibition of the downstream of PI3 kinase or AKT/protein kinase but not protein kinase A prevents the observed increase in cell survival elicited by PGE(2). These findings have critical implications regarding the mechanism and potential application of PGE(2) receptor specific inhibition in cancer therapy.

The biological activities of PGE2 are mediated through EP receptors (EP1–EP4), plasma membrane G protein-coupled receptors that differ in ligand binding and signal-transduction pathways. We investigated gastrointestinal EP2 receptor expression in adult mice before and after radiation injury and evaluated intestinal stem cell survival and crypt epithelial apoptosis after radiation injury in EP2 null mice. EP2 was expressed throughout the gut. Intestinal EP2 mRNA increased fivefold after γ-irradiation. Crypt survival was diminished in EP2 −/− mice (4.06 crypts/cross section) compared with wild-type littermates (8.15 crypts/cross section). Radiation-induced apoptosis was significantly increased in EP2 −/− mice compared with wild-type littermates. Apoptosis was 1.6-fold higher in EP2 −/− mice (5.9 apoptotic cells/crypt) than in wild-type mice (3.5 apoptotic cells/crypt). The EP2receptor is expressed in mouse gastrointestinal epithelial cells and is upregulated following radiation injury. The effects of PGE2on both crypt epithelial apoptosis and intestinal crypt stem cell survival are mediated through the EP2 receptor.

Next-generation sequencing technologies have provided insights into the biology and mutational landscape of cancer. Here we evaluate the relevance of cancer neoantigens in human breast cancers. Using patient-derived xenografts from three patients with advanced breast cancer (xenografts were designated as WHIM30, WHIM35, and WHIM37), we sequenced exomes of tumor and patient-matched normal cells. We identified 2091 (WHIM30), 354 (WHIM35), and 235 (WHIM37) nonsynonymous somatic mutations. A computational analysis identified and prioritized HLA class I–restricted candidate neoantigens expressed in the dominant tumor clone. Each candidate neoantigen was evaluated using peptide-binding assays, T-cell cultures that measure the ability of CD8+ T cells to recognize candidate neoantigens, and preclinical models in which we measured antitumor immunity. Our results demonstrate that breast cancer neoantigens can be recognized by the immune system, and that human CD8+ T cells enriched for prioritized breast cancer neoantigens were able to protect mice from tumor challenge with autologous patient-derived xenografts. We conclude that next-generation sequencing and epitope-prediction strategies can identify and prioritize candidate neoantigens for immune targeting in breast cancer.

Purpose Mammaglobin-A (MAM-A) is overexpressed in 40–80% of primary breast cancers. We initiated a phase 1 clinical trial of a MAM-A DNA vaccine to evaluate its safety and biological efficacy. Experimental Design Breast cancer patients with stable metastatic disease were eligible for enrollment. Safety was monitored with clinical and laboratory assessments. The CD8 T cell response was measured by ELISPOT, flow cytometry, and cytotoxicity assays. Progression-free survival was described using the Kaplan-Meier product limit estimator. Results Fourteen subjects have been treated with the MAM-A DNA vaccine and no significant adverse events have been observed. Eight of fourteen subjects were HLA-A2+, and the CD8 T cell response to vaccination was studied in detail. Flow cytometry demonstrated a significant increase in the frequency of MAM-A-specific CD8 T cells following vaccination (0.9 ± 0.5% vs. 3.8 ± 1.2%, p < 0.001), and ELISPOT analysis demonstrated an increase in the number of MAM-A-specific IFN-γ-secreting T cells (41 ± 32 vs. 215 ± 67 spm, p < 0.001). Although this study was not powered to evaluate progression-free survival, preliminary evidence suggests that subjects treated with the MAM-A DNA vaccine had improved progression-free survival compared to subjects who met all eligibility criteria, were enrolled in the trial, but were not vaccinated because of HLA phenotype. Conclusion The MAM-A DNA vaccine is safe, capable of eliciting MAM-A-specific CD8 T cell responses, and preliminary evidence suggests improved progression-free survival. Additional studies are required to define the potential of the MAM-A DNA vaccine for breast cancer prevention and/or therapy.

Background Preclinical studies and early clinical trials have shown that targeting cancer neoantigens is a promising approach towards the development of personalized cancer immunotherapies. DNA vaccines can be rapidly and efficiently manufactured and can integrate multiple neoantigens simultaneously. We therefore sought to optimize the design of polyepitope DNA vaccines and test optimized polyepitope neoantigen DNA vaccines in preclinical models and in clinical translation. Methods We developed and optimized a DNA vaccine platform to target multiple neoantigens. The polyepitope DNA vaccine platform was first optimized using model antigens in vitro and in vivo. We then identified neoantigens in preclinical breast cancer models through genome sequencing and in silico neoantigen prediction pipelines. Optimized polyepitope neoantigen DNA vaccines specific for the murine breast tumor E0771 and 4T1 were designed and their immunogenicity was tested in vivo. We also tested an optimized polyepitope neoantigen DNA vaccine in a patient with metastatic pancreatic neuroendocrine tumor. Results Our data support an optimized polyepitope neoantigen DNA vaccine design encoding long (≥20-mer) epitopes with a mutant form of ubiquitin (Ubmut) fused to the N-terminus for antigen processing and presentation. Optimized polyepitope neoantigen DNA vaccines were immunogenic and generated robust neoantigen-specific immune responses in mice. The magnitude of immune responses generated by optimized polyepitope neoantigen DNA vaccines was similar to that of synthetic long peptide vaccines specific for the same neoantigens. When combined with immune checkpoint blockade therapy, optimized polyepitope neoantigen DNA vaccines were capable of inducing antitumor immunity in preclinical models. Immune monitoring data suggest that optimized polyepitope neoantigen DNA vaccines are capable of inducing neoantigen-specific T cell responses in a patient with metastatic pancreatic neuroendocrine tumor. Conclusions We have developed and optimized a novel polyepitope neoantigen DNA vaccine platform that can target multiple neoantigens and induce antitumor immune responses in preclinical models and neoantigen-specific responses in clinical translation.

The microcolony assay following gamma irradiation (IR) is a functional assay of intestinal stem cell fate. The cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1/Sdi1) (p21) regulates cell cycle arrest following DNA damage. To explore the role of p21 on stem cell fate, we examined the effects of p21 deletion on intestinal crypt survival following IR and expression of the stem/progenitor cell marker Musashi-1 (Msi-1) and the antiapoptotic gene survivin. Intestinal stem cell survival in adult wild-type (WT) and p21(-/-) mice was measured using the microcolony assay. Msi-1, p21, and survivin mRNA were measured using real-time PCR and immunohistochemical analysis. Laser capture microdissection (LCM) was used to isolate mRNA from the crypt stem cell zone. No differences in radiation-induced apoptosis were observed between WT and p21(-/-) mice. However, increased crypt survival (3.0-fold) was observed in p21(-/-) compared with WT mice 3.5 days after 13 Gy. Msi-1 and survivin mRNA were elevated 12- and 7.5-fold, respectively, in LCM-dissected crypts of p21(-/-) compared with WT mice. In conclusion, deletion of p21 results in protection of crypt stem/progenitor cells from IR-induced cell death. Furthermore, the increase in crypt survival is associated with increased numbers of Msi-1- and survivin-expressing cells in regenerative crypts.