High Drug Resistance in Feline Mammary Carcinoma Cell Line (FMCm) and Comparison with Human Breast Cancer Cell Line (MCF-7)

Drug repurposing and drug combination are important therapeutic approaches in cancer therapy. Drug repurposing aims to give new indications to drugs, rather than the original indication, whereas drug combination presupposes that the effect that is obtained should be more beneficial than the effect obtained by the individual drugs. Previously, drug repurposing and the combination of different drugs was evaluated in our research group against human breast cancer cells (MCF-7 cells). Our results demonstrated that the response obtained through the combination of drugs, when compared with the single drugs, led to more synergic responses. Therefore, using potential drugs for repurposing, combined with a reference drug in breast cancer (5-Fluorouracil), was the major aim of this project, but for the first time using the feline mammary carcinoma cell line, FMCm. Surprisingly, the feline neoplastic cells demonstrated considerable resistance to the drugs tested in isolation, and the combination was not effective, which contrasted with the obtained MCF-7 cells’ response.

Environmental control of mammary carcinoma cell expansion by acidification and spheroid formation in vitro

Breast cancer is the leading cause of cancer death among women worldwide. Like other cancers, mammary carcinoma progression involves acidification of the tumor microenvironment, which is an important factor for cancer detection and treatment strategies. However, the effects of acidity on mammary carcinoma cell morphology and phenotype have not been thoroughly characterized. Here, we evaluated fundamental effects of environmental acidification on mammary carcinoma cells in standard two-dimensional cultures and three-dimensional spheroids. Acidification decreased overall mammary carcinoma cell viability, while increasing their resistance to the anthracycline doxorubicin. Environmental acidification also increased extracellular vesicle production by mammary carcinoma cells. Conditioned media containing these vesicles appeared to increase fibroblast motility. Acidification also increased mammary carcinoma cell motility when cultured with fibroblasts in spheroids. Taken together, results from this study suggest that environmental acidification induces drug resistance and extracellular vesicle production by mammary carcinoma cells that promote tumor expansion.

In Vitro Effects of Polar Materials from Thermally Abused Frying Oil on Mammary Carcinoma Cell Migration (P05-004-19)

Objectives Widely consumed foods such as chicken, fish, and potatoes are regularly prepared by deep frying. The frying process involves temperatures exceeding 180 degrees Celsius and repeated frying cycles that result in thermally induced chemical changes of the oil's lipid structures. One such chemical change is an accumulation of polar compounds, including secondary lipid oxidation products, which are associated with several disease pathologies. Many European countries adhere to strict limits of less than 30% polar compounds within recycled fryer oils. There are no such regulations in the United States. Using a murine model of late-stage breast cancer (BC), we previously demonstrated an increased metastatic burden in mice consuming a diet of thermally abused frying oil (TAFO) compared with mice consuming fresh vegetable oil. To further understand this observation, we assessed 1) the amount of polar material in oil recycled for 300 minutes of deep frying, and 2) the effect the fractionated polar material from TAFO has on in vitro migration of 4T1 murine cancer cells. Methods We used silica column chromatography to separate the TAFO into polar and non-polar fractions. The polar fraction of TAFO (TAFO-PF) was retained from oil used to fry fish nuggets for a duration of 300 minutes. In vitro wound healing migration assays were conducted in the presence or absence of TAFO-PF in a concentration dependent manner. We assessed the wound closure rates (motility) of the highly metastatic 4T1 murine mammary carcinoma cell line. Live images were captured every hour for 24 hours to measure cell migration using brightfield microscopy. Results We found that after 300 minutes of frying, oil contained 74 ± 7.8 μg/mL of polar compound. 4T1 cells incubated over a period of 24 hours with diluted TAFO-PF achieved faster wound closure rates compared with cells incubated in growth media alone. Conclusions Our results suggest that TAFO-PF increases motility as an indicator of the metastatic potential of BC cells. Ongoing work is being focused on conducting in vitro invasion assays on both 4T1 and human BC MDA-MB-231 cell lines in order to further understand the potential mechanisms and effects TAFO-PF has on cancer metastatic progression. Funding Sources NIEHS Training (T32) Grant ES007326 Fellowship to JRH and ABO; UIUC Campus Research Board Beckman Grant and UIUC Hatch 1011659_ILLU-698-357.

Immunotherapeutic efficacy of a Lactobacillus casei lysate as an adjuvant combined with a heated-4T1 mammary carcinoma cell lysate in a murine model of breast cancer

Background Immunotherapy, during which the immune system of the patient is manipulated to act against tumors has been among the most successful methods in the treatment of breast cancer, a leading cause of mortality among women worldwide. Objectives To investigate the immunotherapeutic efficacy of Lactobacillus casei lysate as an adjuvant in combination with a heated-4T1 mammary carcinoma cell lysate in a model of breast cancer. Methods After ethics committee approval of all animal procedures, a murine model of breast cancer was induced in BALB/c mice using 4T1 cells. These mice were immunized with a combination of lysates of heated 4T1 cells and L. casei. Subsequent changes in tumor size and weight, and the production of TNF-α, IL-2, IL-12, IL-17, and IL13 were measured. Lung weights were measured as an indicator of metastasis to other organs. Results The tumor size and weight in mice immunized with the combined vaccine were significantly reduced compared with controls. The combined immunotherapy altered the pattern of cytokine production to the advantage of antitumor immunity, and was significantly more potent than immunization with heated-4T1-cell lysate or L. casei lysate alone. Conclusions Coadministration of L. casei lysate enhanced the immunotherapeutic efficacy of the heated-4T1-cell lysate as a source of tumor-associated antigens. L. casei can potentially be used as an adjuvant combined with sources of tumor antigens in the treatment of cancers, and as a safe alternative to the current adjuvants that cause greater irritation to hosts. Further studies are required to clarify the mechanisms underlying these effects.

Cytotoxicity and Apoptosis Induction of Ardisia crispa and Its Solvent Partitions against Mus musculus Mammary Carcinoma Cell Line (4T1)

This study was conducted to investigate the cytotoxicity and apoptosis effect of A. crispa extract and its solvent partition (ethyl acetate and aqueous extract) against Mus musculus mammary carcinoma cell line (4T1). The normal mouse fibroblast cell line (NIH3T3) was used as comparison for selective cytotoxicity properties. The cytotoxicity evaluation was assessed using MTT assay. AO/PI dual fluorescent staining assay and Annexin V-FITC were used for apoptosis analysis. Results showed that 80% methanol extract from leaves showed most promising antimammary cancer agent with IC50 value of 42.26±1.82 μg/mL and selective index (SI) value of 10.22. Ethyl acetate was cytotoxic for both cancer and normal cell while aqueous extract exhibited poor cytotoxic effect. 4T1 cells labelled with AO/PI and Annexin V-FITC and treated with 80% methanol extract demonstrated that the extract induces apoptosis to 4T1 mammary cancer cells. In conclusion, 80% methanol extract of A. crispa was selectively cytotoxic towards 4T1 cells but less cytotoxic towards NIH3T3 cells and induced the cancerous cells into apoptotic stage as early as 6 hours.