(F-230) Microengineering of Interstitial Fluid Pressure in Solid Tumor Microtissue Models

Introduction:: Current in vitro tumor models lack the appropriate vasculature that surrounds the tumor microenvironment (TME) [1] when encapsulating tumor cells in a biomimetic extracellular matrix (ECM), such as Matrigel [2]. Such models are still simplistic, and more efforts are expected to improve our ability to regulate TME factors. In this work, we present a breast solid tumor model using hydrogel engineering and freeform reversible embedding of suspended hydrogels (FRESH) method [3] in a collagen-based ECM. We study the effects of interstitial fluid pressure (IFP) on tumor cells embedded in cell spheroids. Solid tumors show increased IFP (10 to 100 mmHg) compared to normal tissue, resulting in smaller pressure gradients, that lead to a reduced or inefficient uptake of therapeutic agents from the transcapillary flow [4]. With an increased IFP in the solid tumor, the surrounding blood vessels shift and become ‘leaky’ for cancer cells to infiltrate through its layers [5]. By controlling the flow in the blood vessels, we can manipulate the IFP of tumor cells, to improve the uptake of therapeutic drugs, and reduce blood vessel infiltration of cancer cells, while also hindering the solid tumor-to-stroma signaling, which is also seen in terms of a reduction in the formation of cancer-associated fibroblasts (CAFs) [6].

Materials and Methods:: Rat-tail collagen (type I) was extracted following standard methods [7]. The collagen spheroids were formed using the freeze-drying method [8], and vapor crosslinking was done by a novel technique in a vacuum chamber [9]. These ECM spheroids were used to encapsulate the tumor cells (MD-231, ATCC) at a density of 5 M/mL in the microporous scaffold, enabling the cells to adhere, proliferate, and migrate. The collagen solution at 4 mg/mL (acetic acid) was extruded in a gelatin support bath (4.5% Gelatin in 0.125% CaCL₂) as described in the FRESH method to create vessels ranging from 200 µm to 500 µm. The perfusability of the collagen-based vessels was then modified by optimizing their composition (i.e., 4-8 mg/ml), porosity (i.e., 80-97%), and stiffness (i.e., 10 kPa-1 MPa). The hollow of the blood vessels was seeded with mesenchymal stem cells (MSCs) that differentiated into endothelial cells (ECs) in the presence of VEGF (50 ng/ml) and sheer flow (10 µl/min), creating a lumen within 48 hours [10]. The MD-231 cells were encapsulated in collagen to form the spheroids, which were embedded on the surface of the blood vessels. These were then placed onto a microfluidics setup with perfusion to stimulate blood flow to the TME [11]. We measured the levels of the biomarker HIF1-A, CD44, and MMP1, along with epithelial-to-mesenchymal (EMT) biomarkers such as SNAIL-1, Vimentin, and E-Cadherin [12]. Increased IFP acts as a barrier to therapeutics from the capillary network and also causes the differentiation of Stromal fibroblasts to CAFs [13].

Results, Conclusions, and Discussions:: We will study the role of IFP in tumor spheroids and how this can be modulated to improve drug uptake and thereby improve treatment efficacy. Solid tumors show elevated IFP that acts as a barrier to therapeutics from the transcapillary network. There is an elevation of hypoxia and EMT biomarkers, and a marked increase in stromal fibroblasts to CAFs differentiation. Regulating the flow to the tumor mass will provide a means of controlling the tumor IFP, which can reduce tumor proliferation and metastasis, and improve drug efficacy. Such a model will provide key insights into the role of IFP and the mechanisms underlying tumor-stromal interactions, which can be used to develop novel therapeutic strategies to target the TME. In addition, the FRESH method will be an efficient approach to creating vascular models [14]. The next steps will be to include other cell types and biomaterials to study the effects of IFP on cancer metastasis and cancer cell deposition in other organs. We will also acquire more clinical data for understanding the role of IFP in other solid tumors such as prostate cancer.

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References (Optional): :

1. Germain, N., et al., Current Advances in 3D Bioprinting for Cancer Modeling and Personalized Medicine. Int J Mol Sci, 2022. 23(7).

2. Tevis, K.M., Y.L. Colson, and M.W. Grinstaff, Embedded Spheroids as Models of the Cancer Microenvironment. Adv Biosyst, 2017. 1(10).

3. Hinton, T.J., et al., Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels. Sci Adv, 2015. 1(9): p. e1500758.

4. Shang, M., et al., Microfluidic studies of hydrostatic pressure-enhanced doxorubicin resistance in human breast cancer cells. Lab Chip, 2021. 21(4): p. 746-754.

5. Ferretti, S., et al., Tumor interstitial fluid pressure as an early-response marker for anticancer therapeutics. Neoplasia, 2009. 11(9): p. 874-81.

6. Kharaishvili, G., Simkova, D., Bouchalova, K. et al. The role of cancer-associated fibroblasts, solid stress and other microenvironmental factors in tumor progression and therapy resistance. Cancer Cell Int 14, 41 (2014).

7. Rajan, N., et al., Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nat Protoc, 2006. 1(6): p. 2753-8.

8. Lowe, C.J., et al., Production of Highly Aligned Collagen Scaffolds by Freeze-drying of Self-assembled, Fibrillar Collagen Gels. ACS Biomater Sci Eng, 2016. 2(4): p. 643-651.

9. Nair, M., et al., Tunable bioactivity and mechanics of collagen-based tissue engineering constructs: A comparison of EDC-NHS, genipin and TG2 crosslinkers. Biomaterials, 2020. 254: p. 120109.

10. Huang, Y., et al., Effects of shear stress on differentiation of stem cells into endothelial cells. World J Stem Cells, 2021. 13(7): p. 894-913.

11. Cao, X., et al., A Tumor-on-a-Chip System with Bioprinted Blood and Lymphatic Vessel Pair. Adv Funct Mater, 2019. 29(31).

12. Piotrowski-Daspit, A.S., J. Tien, and C.M. Nelson, Interstitial fluid pressure regulates collective invasion in engineered human breast tumors via Snail, vimentin, and E-cadherin. Integr Biol (Camb), 2016. 8(3): p. 319-31.

13. Libutti SK, Tamarkin L, Nilubol N. Targeting the invincible barrier for drug delivery in solid cancers: interstitial fluid pressure. Oncotarget. 2018 Nov 6;9(87):35723-35725.

14. Shieh, A.C. and M.A. Swartz, Regulation of tumor invasion by interstitial fluid flow. Phys Biol, 2011. 8(1): p. 015012.