Poster BB5 - Sirtuin-5 shRNA Knockdown Decreases Spheroid Invasion of Triple Negative Breast Cancer Cells

Introduction: In the U.S, 12% of women will be diagnosed with breast cancer within their lifetime. This highly heterogeneous disease has many different subtypes with triple-negative breast cancer (TNBC) recognized as having the worst prognosis. TNBC is aptly named for its lack of detectable estrogen, progesterone, and human growth factor 2 (HER2) receptors. For this reason, traditional hormone and HER2-targeted therapies are ineffective for TNBC patients. Diversifying chemotherapies beyond these treatments, like through the targeting of cancer cell metabolism, may address these shortcomings. Sirtuins are proteins that protect metabolic enzymes from degradation through deacetylation. One such protein, SIRT5, is more highly overexpressed in TNBC compared to other breast cancer subtypes. Elevated SIRT5 levels support proliferation and anchorage-independent growth of breast cancer cells, allowing for malignant transformation and homing into distal metastatic sites. Genetic disruption of SIRT5 has been shown to impair proliferation of breast cancer cells in 2D cell culture and delay metastasis in SIRT5-deficient mice. However, how SIRT5 disruption affects invasion, the first step of the metastatic cascade, remains unknown. Understanding these connections is critical as tumor invasion is an intrinsically energy-consuming process and thus, dependent on metabolic changes. To investigate how SIRT5 knockdown (KD) affects tumor invasion, spheroids were generated from genetically modified TNBC MDA-MB-231 cells, embedded into fibrillar type I collagen hydrogels, and monitored for tumor cell invasion. Utilizing this model along with various imaging modalities and image analysis of the spheroids will contribute to a better understanding of SIRT5-based therapies on breast cancer progression.

Materials and

Methods: Genetically modified MDA-MB-231 cells were engineered via shRNA lentiviral knockdown of SIRT5. Three different cell lines were produced: shRNA control, SIRT5 shRNA KD1, and SIRT5 shRNA KD2. Spheroids were made via the hanging droplet method using a solution of cell suspension, methylcellulose, and collagen type I (Figure 1A). To study SIRT5-disruption and its effect on breast cancer cell invasion, spheroids were embedded in fibrillar type I collagen hydrogels formed inside a polydimethylsiloxane (PDMS) ring mold (Figure 1B). Spheroids were monitored utilizing brightfield microscopy for two days. They were subsequently fixed with paraformaldehyde, stained with DAPI and Phalloidin, and imaged with confocal microscopy. ImageJ was used to analyze the spheroid invasion area. GraphPad was used to determine statistical significance of the data.

Results, Conclusions, and Discussions: Bright-field imaging showed that Day 0 spheroids (directly after embedding) featured distinct, smooth boundaries (Figure 1C). On day 1 spheroids begin invading into the surrounding collagen matrix. By Day 2, it was qualitatively observed that many cells were invading from all directions of the spheroid. Compared to MDA-MB-231 shRNA control spheroids, the SIRT5 shRNA knockdown spheroids showed a significant decrease in invasion, as measured by relative change in spheroid area (Figure 1D). Confocal images capture the spheroid and the invading cells on the periphery (Figure 1E). The data shown here suggests that SIRT5 disruption hinders the invasion of TNBC cell lines. While the current study focuses primarily on MDA-MB-231 cells, previous 2D in vitro studies have shown promise of SIRT5 disruption in other breast cancer cell lines. Other works have also shown that SIRT5-deficient mice have mild phenotypes, which suggest low toxicity. Traditional cancer drug development utilizing cell-in-well 2D in vitro models often lack physiologically relevant cell and matrix interactions, which contributes to the finding that 95% of drugs that are effective in preclinical trials fails to translate in clinic. Our spheroid-in-collagen hydrogel is a highly tunable model that captures the cell-cell and cell-matrix interactions found natively in the breast microenvironment. Additionally, patient-derived breast cancer cells can be utilized to make spheroids, providing a model that can be used for better precision medicine. Thus, future studies can utilize this model platform to explore the use of SIRT5 disruption for both non-TNBC cell lines and patient-derived cells. Altogether, our work highlights the potential of using this model to screen for the efficacy of SIRT5-targeted inhibition therapies for TNBC.

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