In Flight miRNA Isolation and Recovery on the ISS

Due to advancements in RNA research, mi (micro) RNAs and other small nucleotide RNAs have become a major research field in biology including spaceflight research. The regulation of RNA transcription and processing by miRNAs makes miRNAs an appealing topic for genetics and molecular research. It has been estimated that over 60% of human gene transcripts are targets of miRNA regulation. In fact, this is true for all organisms, including plants and insects. Small nucleotide RNAs can also play a role in regulating gene expression, meaning that gene expression alone is not a complete picture of the potential genetic changes that occur in an organism during spaceflight. The goal of the WetLab-2 project is to isolate and recover miRNAs from various tissue sources on the International Space Station (ISS). No system currently exists that can isolate and recover small nucleotide RNA in space. However, the WetLab-2 system that was validated on the ISS in 2016 can be adapted to fit this purpose. We are currently testing the new modified protocols by running plant and mouse blood experiments in parallel, allowing us to demonstrate the effectiveness of the procedure on different sample types. We expect to be able to optimize and implement the modified miRNA protocols for use on future ISS flights

In Flight MiRNA Isolation and Recovery on the ISS Using the Wetlab-2 System

Due to advancements in RNA research, mi (micro) RNAs and other small nucleotide RNAs have become a major research field in biology including spaceflight research. The regulation of RNA transcription and processing by miRNAs makes miRNAs an appealing topic for genetics and molecular research. It has been estimated that over 60% of human gene transcripts are targets of miRNA regulation. In fact, this is true for all organisms, including plants and insects. Small nucleotide RNAs can also play a role in regulating gene expression, meaning that gene expression alone is not a complete picture of the potential genetic changes that occur in an organism during spaceflight. The goal of the WetLab-2 project is to isolate and recover miRNAs from various tissue sources on the International Space Station (ISS). No system currently exists that can isolate and recover small nucleotide RNA in space. However, the WetLab-2 system that was validated on the ISS in 2016 can be adapted to fit this purpose. We are currently testing the new modified protocols by running plant and mouse blood experiments in parallel, allowing us to demonstrate the effectiveness of the procedure on different sample types. We expect to be able to optimize and implement the modified miRNA protocols for use on future ISS flights

Optimizing Protocols for the Derivation of Primary Cultures from Mammalian Tissue

The isolation of primary cell cultures from fresh mammalian tissue is a major interest of our laboratory. Thus, the ability to generate cancer cell lines from cancer tissue, or to generate non-malignant cultures form the host stroma surrounding a tumor, allows us to develop new laboratory models to study the behavior of cancer cells and its microenvironment. We report our proof of principle study on optimizing the derivation of primary culture, using rat tissues as a model. Thus, rat kidney and rat skin samples were isolated, minced with surgical blades, and then incubated with an enzyme digestion cocktail (Collagenase III- 4mg/mL, Collagenase IV-2mg/mL, Hyaluronidase-2mg/mL, in phosphate buffered saline, pH 7.4). Thereafter, the digested tissues were plated in tissue culture dishes in serum (20%) containing media. Three weeks after plating, very few cells were visible in any of the preparations. However, four weeks after plating we noted the appearance of colonies in rat kidney preparations. These colonies have since rapidly grown into larger colonies of fibroblast-like cells. These results demonstrate that we have an effective protocol for the derivation of fresh primary cultures from fresh tissues. Our plans for further optimization of the isolation procedure, for the eventual derivation of primary cultures from human and dog tumors (and from their benign stromal components such as fibroblasts and endothelial cells), will be discussed

Protein Expression Analysis of Cell Lines Derived from Drug Resistant Tumors

There are a number of effective treatments for breast cancer, including chemotherapy and targeted strategies such as the use of the Her-2 targeting drugs lapatinib and trastuzumab. However, in a number of cases, tumors that initially respond to therapy eventually develop drug resistance, leading to the relapse of the disease. By studying protein levels in different drug resistant variants, we have observed two mechanisms by which drug resistance may develop. Thus, some Her-2 human breast cancer cells (e.g., MDA-MB-231H2N) treated with the clinically used trastuzumab agent, can escape therapy by shedding, or losing, the target Her-2 protein. Similarly, EMT-6 mouse mammary cancer models that are resistant to alkylating agents (such as cisplatin, thiotepa, or cyclophosphamide) lose or reduce (i.e., between 3- to 5-fold) the expression of proteins such as MLH1 and PMS2, both of which are important for the sensitivity of cancer cells to alkylating agents. Thus, taken together our results suggest that tumors can escape therapy by losing those proteins that are either the target of the therapy or that modulate sensitivity to a given treatment. We will report our findings, and describe our ongoing efforts to optimize the analysis of protein levels in tumor cells that show resistance to currently available anti-cancer strategies

Messenger mRNA Analysis as a Means to Identify Mechanisms of Cancer Drug Resistance

On principal approach to identify the mechanisms by which cancers escape from anti-cancer therapies is the analysis of messenger RNAs (mRNAs) in those tumor cells that survive treatment. Thus, by studying Her-2 positive human breast tumor cells that survived anti-Her-2 based therapies (i.e., using anti-Her-2 antibody) we observed the 3-fold or greater overexpression of Vascular Endothelial Growth Factor (VEGF) mRNA. Similarly, by studying the mRNAs of tumor cell cultures resistant to chemotherapy (such as cisplatin), we found that low levels of mRNAs for DNA mismatch repair genes correlates with drug resistance. These results suggest that two possible strategies of sensitizing drug resistant cancer cells may be to inhibit VEGF overexpression, or to prevent the suppression of DNA mismatch repair gene expression, observed in drug resistant cells. Furthermore, we are also optimizing our RNA extraction and analysis protocol to facilitate future analysis of drug resistant variants, including the analysis of tumor cells resistant to the clinically relevant therapies that combine anti-Her-2 targeting strategies with conventional chemotherapy

In Vitro Evaluation of Human Her2-Positive Breast Cancer Cells

One in four cases of breast cancer shows over expression of the Her-2 protein, and there is an urgent need to improve therapies for this disease. We have evaluated two Her-2 positive human breast cancer cell lines, MDA-231H2N and HCC1419. MDA-231 human breast cancer cell, which are Her-2 negative, were used as a control. The cell lines were evaluated in vitro for their relative growth rate, their growth under reduced (i.e. 3% serum) conditions, or as spheroid cultures, and for their sensitivity to drugs such as ceramide analogs and tyrosine kinase inhibitors (i.e. CLM3, CLM29, and CLM94) that are being evaluated in our laboratory. Her-2 transduced variants (MDA-231-H2N) showed a 50% increase in doubling rate and enhanced growth in low-serum media compared to MDA-231 cells. Only HCC1419 grew as compact spheroids, even 96 hours after plating on solidified agar. All three cancer cell lines showed sensitivity to ceramide analogs and tyrosine kinase inhibitors tested. Following 48 hour treatment, the greatest cytotoxicity was observed with 25mM ceramide analog C6 (N-Hexanoyl-D-erythro-sphingosine), and 25mM of the tyrosine kinase inhibitor CLM29 (pyrazolo[3,4-d]pyrimidine). The activity of these agents was unaffected by Her-2 transduction or over expression. Our results provide additional characterization of available models of human Her-2 positive breast cancer cell lines, and suggest that the agents C6 and CLM29 merit further evaluation as potential anti-breast cancer agents

Evaluation of CTLA-4 Blockage with Metronomic Chemotherapy for the Treatment of Preclinical Breast Cancer

The targeting of the CTLA-4 protein with the antibody ipilimumab has been a success in terms of producing an increase in the survival of patients with unresectable melanoma, and clinical trials are ongoing to evaluate this strategy in other tumor types. Our aim in this study was to evaluate the combination of CTLA-4 blocking with metronomic chemotherapy regimens. To that end, we subcutaneously implanted murine EMT-6 breast tumor cells into syngeneic Balb/c mice (n=6-8/group) and evaluated therapies on the established tumors. Murine CTLA-4 blocking was achieved using anti-mouse CD152 (CTLA-4), clone 9H10, injected on day 1 (100ug/mouse) and on day 6 (35ug/mouse) of therapy. Anti-CTLA-4 therapy was administered on its own or combined with metronomic regimens. These included; a) Bolus (150mg/kg, i.p.) cyclophosphamide (CTX) followed by metronomic CTX (20mg/kg/day, p.o.), b) metronomic CTX, and c) sequential gemcitabine therapy (160mg/kg every 3 days, i.p.) given to the tumors relapsing after the anti-CTLA-4 therapy. We observed that control (saline) treated tumors, or tumors treated with Bolus CTX plus metronomic CTX, grew rapidly and had to be sacrificed 4 weeks after tumor implantation. Anti-CTLA-4 monotherapy produced an initial tumor regression followed by tumor relapses, 2-3 weeks later, in 5/6 mice. Surprisingly, the Bolus CTX plus metronomic CTX hindered the effective CTLA-4 therapy, failed to produce tumor regression, and resulted in rapidly growing tumors. The combination of anti-CTLA-4 plus metronomic CTX also produced tumor regression and resulted in a longer delay in the appearance of relapsing tumors (p \u3c 0.05 compared to anti-CTLA-4 alone), which also eventually appeared in 5/6 mice. A Kaplan Meier plot showed that the anti-CTLA-4 plus metronomic CTX regimen significantly improved survival compared to the anti-CTLA-4 monotherapy (p \u3c 0.05). The regimen involving first line anti-CTLA4 therapy followed by a second line gemcitabine therapy, produced a sustained tumor regression that continued for over 100 days. In this group, 5/7 mice did not show a tumor regrowth; 1 mouse showed a tumor regrowth under continuous gemcitabine therapy with concomitant development of lung metastasis. Tumor cells lines were derived from the relapsing tumor and from the lung metastasis. Collectively our data shows that Bolus plus metronomic CTX may compromise anti-CTLA-4 therapy. Furthermore, anti-CTLA-4 therapy may be effectively combined with metronomic CTX, or with a sequential gemcitabine therapy, in a preclinical model of breast cancer

Development of New Models to Study Human Her-2 Positive Breast Cancer

To study the evolution of human Her-2 positive breast cancer, we evaluated a model of three dimensional spheroid co-culture using H2N human breast cancer cells that express high amounts of Her-2 protein, with the Her-2 negative MDA-231 cells. Human HCC1419, which can form compact spheroids, were used as controls. In tissue culture plates, H2N cells were found to have a doubling rate of about 48 hours, compared to around 144 hours for MDA231. Since H2N cells were engineered to express a fluorescent protein, we could note that these cells did not immediately overgrow the non-fluorescent MDA231 cells in a mixed three dimensional mass – suggesting that this model could allow for the study of long term evolution of specific subpopulation of tumor cells within a heterogeneous tumor mass. When the breast cancer cells were grown on tissue culture plastic, we evaluated a drug screen of newly synthesized potential anticancer compounds. One compound, CLM29 was observed to inhibit the growth of all three breast cancer cell lines. Furthermore, 25 micromolar treatment of H2N or of MDA231, but not of HCC1419 cells, lead over 4 days to the formation of cellular neurite-like extensive protrusions, suggesting the activation of a differentiation of breast cancer cells. Our results show the effective use of three different human breast cancer cell lines to develop models to follow the evolution of specific subtypes of breast cancer within a tumor mass, and to test new compounds with potential anti-breast cancer activity

Abstract 3261: Impact of CTLA-4 blockade in conjunction with metronomic chemotherapy on preclinical breast cancer growth

Background:Although there are reports that metronomic cyclophosphamide (CTX) can be immune stimulating, the impact of its combination with anti-CTLA-4 immunotherapy for the treatment of cancer remains to be evaluated.Methods:Murine EMT-6/P breast cancer, or its cisplatin or CTX-resistant variants, or CT-26 colon, were implanted into Balb/c mice. Established tumours were monitored for relative growth following treatment with anti-CTLA-4 antibody alone or in combination with; (a) metronomic CTX (ldCTX; 20 mg kg-1 day-1), b) bolus (150 mg kg-1) plus ldCTX, or (c) sequential treatment with gemcitabine (160 mg kg-1 every 3 days).Results:EMT-6/P tumours responded to anti-CTLA-4 therapy, but this response was less effective when combined with bolus plus ldCTX. Anti-CTLA-4 could be effectively combined with either ldCTX (without a bolus), or with regimens of either sequential or concomitant gemcitabine, including in orthotopic EMT-6 tumours, and independently of the schedule of drug administration. Tumour responses were confirmed with CT-26 tumours but were less pronounced in drug-resistant EMT-6/CTX or EMT-6/DDP tumour models than in the parent tumour. A number of tumour bearing mice developed spontaneous metastases under continuous therapy. The majority of cured mice rejected tumour re-challenges.Conclusions:Metronomic CTX can be combined with anti-CTLA-4 therapy, but this therapy is impaired by concomitant bolus CTX. Sequential therapy of anti-CTLA-4 followed by gemcitabine is effective in chemotherapy-naive tumours, although tumour relapses can occur, in some cases accompanied by the development of spontaneous metastases.British Journal of Cancer advance online publication, 5 January 2017; doi:10.1038/bjc.2016.429 www.bjcancer.com