3D Cell Printing of Metastatic Melanoma Model with Blood and Lymphatic Vessel

Abstract

Metastatic melanoma, an extremely deadly form of skin cancer, presents the highest mortality risk. The primary cause of death in melanoma cases stems from its ability to spread to distant organs via the bloodstream and lymphatic system. Effectively managing metastatic melanoma is challenging due to the rapid emergence of tumor resistance to standard chemotherapy and its persistent carcinogenic nature. Pathological observations from melanoma patients indicate that melanoma is a complex tissue comprised of cancer cells and a dynamic combination of stromal cells. As a result, researchers theorize that the interactions between melanoma cells and stromal cells play a critical role in the disease's progression and metastasis. Particularly, the recognition that the majority of tumors present as detectable, fibrous masses has led researchers to hypothesize that cancer-associated fibroblasts (CAFs) play a significant role in the tumor stroma. Co-culture experiments have shown that CAFs facilitate the movement of cancer cells by creating microscopic paths for their invasion. Moreover, by secreting growth factors that stimulate tumor growth and the extracellular matrix (ECM), CAFs establish supportive environments that protect cancer cells from chemotherapy-induced cell death. Within this nurturing microenvironment, cancer cells can continuously multiply and infiltrate the neighboring blood vessels (BVs). Notably, in the progression of melanoma, lymphatic vessels (LVs) act as the primary route for cancer cells to metastasize through the lymphatic system before disseminating throughout the body via BVs. Clinically, the presence of metastasis in regional lymph nodes is a critical indicator of distant metastasis and mortality in melanoma cases. Thus, cancer models that can replicate the melanoma microenvironment, including both blood and lymphatic vasculature, could prove invaluable for investigating the mechanisms underlying melanoma metastasis and assessing the cellular response to anticancer medications. Preclinical models using animals have traditionally served as the benchmark for studying cancer progression. However, their ability to faithfully replicate the clinical environment of human cancer is questionable due to inherent physiological differences. In recent times, three-dimensional (3D) cancer models, specifically spheroids, have emerged as vital tools in cancer research, faithfully mimicking the structural intricacies of solid tumors observed in vivo. Organ-on-a-chip systems based on microfluidics, which simulate vascularized cancer models, have also provided valuable insights into the interplay between tumors and blood vessels, as well as the process of metastasis. Nonetheless, the spontaneous formation of vascular networks in organ-on-a-chip systems poses challenges in achieving precise spatial control between tumors and vasculature, potentially affecting cell-to-cell interactions and the metastatic behavior of cancer cells. Moreover, issues related to scaling in microfluidic systems make it difficult to generate and position spheroids larger than 400 µm. These challenges may hinder the accurate and consistent reproduction of tumor interactions with the vasculature, including angiogenesis and the metastasis of melanoma. The advancement of 3D cell printing technology has enabled precise and programmable deposition of bioinks, which are printable hydrogels containing cells. This technology allows for the fabrication of complex tissue constructs in vitro. When it comes to melanoma cells, an ideal bioink should closely mimic the structural and biochemical properties of the ECM. In an effort to replicate the intricate microenvironment of metastatic melanoma, Schmid et al. developed a printable bioink. However, the study of metastasis was limited in murine models, relying on the arteriovenous loop and lacking the ability to fully recapitulate tumor-vascular interactions in human melanoma. Recent advancements in bioprinting vascularized tissues have provided new opportunities for the development of advanced tumor-vascular models. Meng et al. designed a metastatic cancer model that investigated key aspects of metastatic dissemination, including invasion, intravasation, and angiogenesis. By utilizing bioprinting, they were able to precisely position capsules that released growth factors, inducing cancer migration and angiogenesis. While these models have made significant progress in replicating tumor-vascular interactions and the metastatic cascade, they have overlooked the inclusion of the lymphatic system, an important component in the development of in vitro cancer models. In this context, Cao et al. proposed a tumor-on-chip device that featured a bioprinted pair of blood vessels BV and LV. This innovative platform successfully replicated the transport kinetics of drugs within the TME. However, the engineered acellular vessels fell short in faithfully reproducing the melanoma microenvironment, and inadequate attention was given to studying tumor-stroma interactions. These limitations may impede comprehensive investigations into the progression and metastasis of melanoma in future studies. In this study, we introduce a novel method for constructing an integrated cancer platform that combines blood and lymphatic vessels using an in-bath cell printing technique. Our initial step involved creating a bioink derived from decellularized extracellular matrix (SdECM) obtained from biomimetic skin tissue. This bioink enabled us to directly print multiple cellular components at precise locations. Importantly, the SdECM bioink retained the microstructure of the extracellular matrix and various growth factors, thus providing a biologically and physically representative microenvironment similar to native skin. Consequently, we were able to print melanoma spheroids with different geometries within the SdECM bioink bath, accurately reproducing the essential characteristics of the tumor stroma. The biomimetic bioink bath included a set of biomimetic BV and LV. Using this platform, we successfully replicated several critical steps in metastatic dissemination. As the melanoma spheroids matured, they developed resistance to BRAF inhibitors by creating tumor stromal niches. Moreover, the melanoma cells displayed relentless intravasation, disrupting the surrounding vasculature. As a proof of concept, we evaluated the effectiveness of combination therapy targeting both melanoma cells and their stroma. Different combinations of inhibitors elicited distinct responses in melanoma progression and endothelial dysfunction within the platform. This advanced 3D melanoma model in vitro, which more accurately emulates the complex TME, serves as a valuable tool for investigating the interaction between cancer cells and their microenvironment, as well as for screening potential anticancer drugs. The dissertation is organized as follows: 1) Development of an in-bath 3D cell printing technique for fabricating perfusable melanoma spheroids with metastatic properties; 2) Recreation of a complex TME in vitro by incorporating tumor stroma with a pair of blood and lymphatic vessels; 3) Application of the system to study the anticancer effects of combination targeted therapy.