Guadecitabine, in combination with Cyclophosphamide, promotes anti- cancer immunity in BALB/c mice bearing 4T1 mouse mammary carcinoma

Background: The extremely high mortality rate of patients diagnosed with triple negative breast cancer makes it one of the deadliest forms of cancer. Due to the heterogenous nature of tumors, complete clearance is not achieved and clonal selection occurs resulting in tumor cells evading the immune system. I aim to design a therapeutic intervention that is able to elicit an effective immune response against the tumor and instill immunological memory to eradicate primary and metastatic lesions. I hypothesize that the combination of Guad and Cyp will synergize and promote anticancer immunity via increased expression of neo-tumor antigens and depletion of MDSCs and T-regs. Methods: Guadecitabine (Guad), is a second-generation DNA methyltransferase inhibitor (DMNTi) that has been reported to increase antigenicity and deplete myeloid-derived suppressor cells (MDSC’s). Cyclophosphamide (Cyp) is a chemotherapy that has been shown to deplete regulatory T-cells (T-regs). Both MDSD’s and T-regs suppress antitumor immunity. BALB/c mice were challenged with 4T1 tumor cells subcutaneously in the mammary fat pad region. 4T1-bearing mice were administered low-dose Guad and Cyp for ten consecutive days. Tumor growth curves, tumor-infiltrating lymphocytes (TILs) were measured and MDSC’s and T- regs levels were assessed by flow cytometry. Results: Results from this experiment showed significant synergy between Guad and Cyp with both drugs reducing the tumor size over monotherapy. Conclusions: Further analysis of the data along with future experiments will elucidate if this synergy is driven by the depletion of MDSC’s and T-regs alone or the increase in tumor antigenicity inducing increased numbers of TILs.https://scholarscompass.vcu.edu/gradposters/1078/thumbnail.jp

Determining Effective Treatment Regimens for Breast Cancer Using Combined Immunotherapy and Chemotherapy in Vivo

Breast cancer has the highest incidence rate of all cancers globally in women, and those of African descent, especially West African females, face higher rates of triple-negative breast cancer (TNBC), a more aggressive form of breast cancer. Immunotherapy for breast cancer is a relatively new treatment option, and research is ongoing to identify the best combination treatments for increasing survival of those diagnosed with TNBC. Eganelisib (IPI-549: a PI3K-gamma inhibitor that works to shift M2 macrophages to M1 to augment T cell function) with other combinatory treatments has shown promising results in reducing tumor growth and increasing survival in mice. We have been conducting experiments to determine the most effective treatment regimen that will reduce growth of 4T1 mammary cancers, a murine TNBC model in syngeneic BalbC female mice. Combinations of eganelisib, cyclophosphamide, and anti-PD-1 or anti-PD-L1 have been tested to determine how immunotherapy and chemotherapy can induce a strong immune response, resulting in better responses to chemotherapy. Our current data indicates that a treatment regimen combining eganelisib, cyclophosphamide, and anti-PD-1 was most effective at suppressing tumor growth, compared to other treatments that only included one or two of these treatments. Mean tumor sizes of mice treated with a combination of eganelisib and other treatments were 187±70 mm3, 232±71 mm3, and 227±59 mm3 at the end of the experiments, compared to control data of 576±137 mm3, 414±31 mm3, and 591±200 mm3, respectively. These results could lead to further research on effective immunotherapy treatment combinations for TNBC.https://scholarscompass.vcu.edu/uresposters/1417/thumbnail.jp

Identification of molecular signatures specific for distinct cranial sensory ganglia in the developing chick

Background The cranial sensory ganglia represent populations of neurons with distinct functions, or sensory modalities. The production of individual ganglia from distinct neurogenic placodes with different developmental pathways provides a powerful model to investigate the acquisition of specific sensory modalities. To date there is a limited range of gene markers available to examine the molecular pathways underlying this process. Results Transcriptional profiles were generated for populations of differentiated neurons purified from distinct cranial sensory ganglia using microdissection in embryonic chicken followed by FAC-sorting and RNAseq. Whole transcriptome analysis confirmed the division into somato- versus viscerosensory neurons, with additional evidence for subdivision of the somatic class into general and special somatosensory neurons. Cross-comparison of distinct ganglia transcriptomes identified a total of 134 markers, 113 of which are novel, which can be used to distinguish trigeminal, vestibulo-acoustic and epibranchial neuronal populations. In situ hybridisation analysis provided validation for 20/26 tested markers, and showed related expression in the target region of the hindbrain in many cases. Results One hundred thirty-four high-confidence markers have been identified for placode-derived cranial sensory ganglia which can now be used to address the acquisition of specific cranial sensory modalities.</p

Ex vivo expansion of tumor-reactive T cells by means of bryostatin 1/ionomycin and the common gamma chain cytokines formulation.

It was reported that breast cancer patients have pre-existing immune responses against their tumors(1,2). However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses(3). However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively(4). This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7+IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined

Activated NKT cells and NK cells render T cells resistant to myeloid-derived suppressor cells and result in an effective adoptive cellular therapy against breast cancer in the FVBN202 transgenic mouse.

Attempts to cure breast cancer by adoptive cellular therapy (ACT) have not been successful. This is primarily due to the presence of tumor-induced immune-suppressive mechanisms as well as the failure of tumor-reactive T cells to provide long-term memory responses in vivo. To address these clinically important challenges, we developed an ex vivo protocol for the expansion of tumor-reactive immune cells obtained from tumor-bearing animals prior to or after local radiation therapy. We used an Ag-free protocol that included bryostatin 1/ionomycin and sequential common γ-chain cytokines (IL-7/IL-15 + IL-2). The proposed protocol expanded tumor-reactive T cells as well as activated non-T cells, including NKT cells, NK cells, and IFN-γ-producing killer dendritic cells. Antitumor efficacy of T cells depended on the presence of non-T cells. The effector non-T cells also rendered T cells resistant to myeloid-derived suppressor cells. Radiation therapy altered phenotypic distribution and differentiation of T cells as well as their ability to generate central memory T cells. ACT by means of the expanded cells protected animals from tumor challenge and generated long-term memory responses against the tumor, provided that leukocytes were derived from tumor-bearing animals prior to radiation therapy. The ex vivo protocol was also able to expand HER-2/neu-specific T cells derived from the PBMC of a single patient with breast carcinoma. These data suggest that the proposed ACT protocol should be studied further in breast cancer patients

Tumor-reactive immune cells protect against metastatic tumor and induce immunoediting of indolent but not quiescent tumor cells.

Two major barriers to cancer immunotherapy include tumor-induced immune suppression mediated by myeloid-derived suppressor cells and poor immunogenicity of the tumor-expressing self-antigens. To overcome these barriers, we reprogrammed tumor-immune cell cross-talk by combined use of decitabine and adoptive immunotherapy, containing tumor-sensitized T cells and CD25(+) NKT cells. Decitabine functioned to induce the expression of highly immunogenic cancer testis antigens in the tumor, while also reducing the frequency of myeloid-derived suppressor cells and the presence of CD25(+) NKT cells rendered T cells, resistant to remaining myeloid-derived suppressor cells. This combinatorial therapy significantly prolonged survival of animals bearing metastatic tumor cells. Adoptive immunotherapy also induced tumor immunoediting, resulting in tumor escape and associated disease-related mortality. To identify a tumor target that is incapable of escape from the immune response, we used dormant tumor cells. We used Adriamycin chemotherapy or radiation therapy, which simultaneously induce tumor cell death and tumor dormancy. Resultant dormant cells became refractory to additional doses of Adriamycin or radiation therapy, but they remained sensitive to tumor-reactive immune cells. Importantly, we discovered that dormant tumor cells contained indolent cells that expressed low levels of Ki67 and quiescent cells that were Ki67 negative. Whereas the former were prone to tumor immunoediting and escape, the latter did not demonstrate immunoediting. Our results suggest that immunotherapy could be highly effective against quiescent dormant tumor cells. The challenge is to develop combinatorial therapies that could establish a quiescent type of tumor dormancy, which would be the best target for immunotherapy