Poster T2 - The Impact of Vitamin B12 on Tumor Innervation and Cancer Viability in a 3D Platform Mimicking Tumor Environment

Introduction: In 2021, around 38 million people (11.6% of US population) had been diagnosed with diabetes. One of the most common treatments for this condition is a drug that reduces blood glucose levels such as metformin. It is widely known that long-term metformin administration leads to vitamin B12 deficiency causing unwanted side effects. General practice for this condition is supplementing vitamin B12 to uplift the serum levels in patients with diabetes treated with metformin. Vitamin B12 such as cobalamin is known to play a crucial role in nerve injury recovery as it enhances survival of neurons, promotes nerve growth, and remyelinates axons. However, there exists a correlation between vitamin B12 serum level and 1-year risk of cancers such as head/neck, leukemia/lymphoma, liver, lung, and pancreatic cancer. Since it is well-studied that nerve growth in tumor site could promote cancer proliferation, migration, and survival, we hypothesize that the supplement of vitamin B12 after metformin treatment could lead to undesirable innervation in tumor site that aggravates the cancer condition. To verify the hypothesis, we employ our lab-developed 3D platform which mimics native tumors to investigate the effects of different concentrations of vitamin B12, specifically methylcobalamin, on tumor nerve growth.

Materials and

Methods: Cancer cell lines with increasing levels of malignancy as follows MCF-10A, MCF-10AneoT, and MCF-10CA1a provided by Karmanos Cancer Institute were used in this study. In addition, to imitate the presence of fibroblasts in native breast tumors, Adipose-derived stem cells (ADSCs) were added to co-culture samples. Collagen I hydrogel from isolated rat tail tendons was used to create our 3D model. Both cancer cells and ADSCs were mixed in the 5mg/mL collagen solution before being extruded into a PDMS ring mold. Collagen arrangement and cell morphology after being embedded in the hydrogel were imaged using Olympus IX83 confocal microscope. Gelation kinetic and Alamar blue assays were employed to test the quality of our collagen I gel and decide suitable concentrations of methylcobalamin used in our study, respectively. After 5 days of treatment, culture media was replaced with serum-free media, and after 24 hours, media was collected for Luminex assay for neurotrophic factors NT-3, b-NGF, GDNF, and BDNF. Dorsal root ganglia (DRG) were isolated from 3- to 4-week-old male BALB/c mice spinal cords and positioned on top of the MCF-containing gels, samples were later treated with multiple concentrations of methylcobalamin, the axonal sprouts originating from DRG were stained with neurofilament for imaging and measured using Sholl analysis.

Results, Conclusions, and Discussions: The gelation kinetic result shows that the solidifying time of our 5mg/mL collagen I gel is similar to that of commercial collagen I from Corning Inc. Reflectance images show that the lab-made gel has decent collagen distribution and preserves the morphology of embedded cells after 7 days of culture. Alamar blue results show that the lab’s collagen I gel maintains cell proliferation and metabolic activities for at least 7 days of culture. A significant drop in viability of MCF-10A (Fig. 1a), MCF-10A-neoT, MCF-10Ca1a, and ADSCs (Fig. 1b) treated with 4nM of methylcobalamin was observed on days 4 and 7 compared to 0nM, 0.5nM, 1nM, and 2nM. Therefore, 0nM of methylcobalamin was decided to represent vitamin B12 deficiency state, 0.5nM as normal state, and 2nM as excess state. Administration of methylcobalamin at 2nM shows a significant increase in BDNF (Fig. 2), a slight uplift in NT-3 with MCF-10CA1a, and an increase in GDNF with MCF-10A compared to normal state. ADSCs treated with 0.5nM of methylcobalamin have the highest production of GDNF, BDNF (Fig. 3), and b-NGF. Luminex results of our cocultures clearly reveal that ADSCs produce significantly larger amounts of neurotrophic factors compared to MCF cells (Fig. 3) which is rational as fibroblasts tend to produce more of those factors than epithelial cells. Among epithelial cell lines, MCF-10CA1a produced the largest amount of BDNF, b-NGF, and NT-3 while MCF-10A had the highest yield of GDNF. This shift in neurotrophic factor production suggests cell preferences change as they progress to malignancy. In healthy epithelial cells, MCF-10A, excess vitamin B12 led to an increase in GDNF production known to have function in development of multiple organs in the body. On the other hand, in more cancerous state, MCF-10CA1a, excess vitamin B12 resulted in elevated yield of the other three which play significant roles in nerve survival and growth, suggesting that supplement of vitamin B12 could promote tumor innervation, especially in a malignant stage of cancer. It is interesting to know that excess vitamin B12 somewhat decreases production of neurotrophic factors in fibroblasts, suggesting dissimilar effects of vitamin B12 on different cell types.

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