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Cancer Technologies

Cancer Technologies - Poster Session B

Poster G10 - Co-culture between ER+ breast cancer and bacteria using a 3D printed platform to recreate the tumor microbiome.

Thursday, October 24, 2024
2:45 PM - 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  The human body contains many different microbiomes, with the most studied one being the gut microbiome. The gut microbiome is made up microbiota that are naturally found in the body and contribute to metabolism and infection prevention. Recent studies have shown that microbiomes, particularly the gut microbiome, can play a role in cancer prevention through the adjustment of patient diet or manipulation of the microbiome itself. Recent efforts have also identified that tissue-specific microbiomes can potentially assist in cancer resistance to chemotherapies or enhance tumor proliferation and metastasis. It has been suggested that the presence of a breast tissue-resident microbiome may have the same capabilities of advancing cancer proliferation and increasing drug resistance to endocrine therapies in estrogen receptor positive (ER+) breast cancer. One significant challenge in studying the bacteria-cancer axis is the lack of preclinical technologies that allow for the direct co-culture of these two dramatically different cell types. There have been extensive studies on the effects of the gut microbiome using a variety of techniques; however, the limited technologies capable of studying cell-to-cell communication between cancer cells and bacteria have made it difficult to identify the extent of the relationship between cancer and the breast microbiome. The goal of this work is to develop and optimize a novel 3D-printed technique to enable the successful co-culture of ER+ breast cancer cells and bacteria to study how cell-to-cell communication affects endocrine resistance.

Materials and

Methods:  To chemically connect yet physically separate two species, a 3D-printed device (herein called a 3D-printed insert) was designed featuring a grooved base and a three-footed insert with a wide lip, printed using acrylonitrile butadiene styrene (ABS) filament. The insert is designed to fit into a standard 6-well plate and hold an agar membrane that allows for the culture of cancer cells inside the well and bacteria on the agar (Fig. 1A). After printing, a multi-step process was used to decontaminate the ABS, minimizing contamination in the cancer cell chamber. The inserts and bases were soaked in a 70% ethanol solution for a minimum of two hours, transferred to a 1% antibiotic solution for an hour, then transferred back into a 70% ethanol solution for another hour. The 6-well plate was pre-seeded with the model ER+ breast cancer cell line (MCF-7) below the insert while the bacteria (E. coli 29522) was plated on the agar membrane. To ensure proper seeding, the E. coli was inoculated in 5 mL of LB broth and allowed to grow for 24 hours at 37°C before plating. The suspension was diluted the day of experimentation and seeded on the surface of the agar hydrogel. The co-culture 6-well plates were incubated for 24 or 48 h followed by terminal evaluation of cellular morphology using light microscopy, and AlamarBlue using a fluorometric plate reader. To evaluate endocrine resistance, the co-cultured plates were incubated in the presence or absence of 4-OHT (4-hydroxytamoxifen), a metabolite of the endocrine therapy tamoxifen.

Results, Conclusions, and Discussions: Prior work has fabricated the 3D-printed insert that acts as a physical barrier between the cancer cells and bacteria, while still allowing communication to pass between the two cell types. The insert was modeled off the Transwell Assay to allow for the passive diffusion of secreted factors between the cell types; however, the semi-permeable membrane used in the Transwell results in bacterial infiltration into the cancer cell culture chamber. The use of an agar hydrogel was implemented to prevent bacterial contamination in the bottom cancer cell chamber and provide nutrients for the bacteria. We have optimized the sterilization procedure of the ABS to prevent contamination in the bottom chamber, identified a sufficient cell density of bacteria to seed onto the agar, and successfully co-cultured MCF-7 cells with E. coli strain 29522 with minimal contamination and no changes in cancer cell morphology (Fig. 2) One challenge we faced was the use of the ABS filament, which extrudes more tightly than PLA (polylactic acid) filament. This served to prevent bacterial escape; however, ABS is difficult to print, leading to multiple failed prints/inserts. To further prevent contamination, the commercially available Flex Seal was applied on the inside and outside of inserts printed using PLA, yielding a success rate of about 66%. Additional work identified an optimal strain of E. coli for co-culture studies, as certain strains struggle to grow in the co-culture system. We identified two bacterial strains, E. coli 29522 and E. coli 10798, that were successfully co-cultured with the MCF-7 cells. We have demonstrated that cancer cells and bacteria can successfully grow using the insert system and that the presence of the bacteria itself does not alter cancer cell growth . Ongoing experiments work to perform co-culture experiments between the two cell lines in the presence or absence of 4-OHT to determine if bacteria can modulate the cancer cell response to endocrine therapies. Additionally, inconsistent growth rates were observed in the cancer cells between different time points. More trials and optimization are in progress due to unreliable results with varying data points, to be completed in the coming months.

Acknowledgements (Optional):