Under the Influence of Tumor-adjacent Tissue: A 3D Co-culture Model of iPSC-derived Organoids and Patient-derived Microtumors

Abstract

Tumor diseases appear to be as individual as the patients themselves - whether solid or leukemic, the “enemy within” exhibits remarkable heterogeneities. As our understanding of the complex dynamics between tumors and their environment continues to grow, there is an urgent need to develop models that can most accurately recapitulate the patient situation. Such models would allow to study the interactions between tumors and their environment in a controlled setting, enabling the discovery of new treatment strategies, particularly with respect to tumor progression and metastasis, and thus of high relevance to patient outcomes. Tumor-adjacent tissue is likely to be the first point of contact with the growing tumor and thus may be a critical determinant of tumor progression. Despite this important fact, it has received little attention. Therefore, the present work aimed to establish an autologous and allogeneic co-culture system that allows for the investigation of how tumor-adjacent tissue influences tumor growth and invasion with a particular focus on breast cancer, the most common cancer type worldwide. Specifically, we introduce a novel 3D floating matrix co-culture model consisting of two parts: First, iPSC-derived mammary-like organoids (representing the tumor-adjacent mammary epithelium) and second, either breast cancer patient-derived microtumors or breast cancer cell line-derived spheroids. In a first step, CD34+ hematopoietic stem and progenitor cells were isolated from the peripheral blood of breast cancer patients. A comprehensive characterization of the newly established iPSC lines confirmed chromosomal stability, expression of endogenous stem cell markers, the loss of exogenous reprogramming factors and their potential to differentiate into the three germ layers. Next, the iPSCs were differentiated into mammary-like organoids, which were shown to display important features of the human mammary gland, including the expression of mammary luminal and basal epithelial markers as well as acinar-like structures. Expression of the milk protein, β-casein, was observed under prolactogenic culture conditions. For the subsequent co-cultivation with the organoids, microtumors were isolated from fresh primary tumor tissue. Microtumors are small fractions of the native tumor and therefore heterogenous in composition, containing not only cancer cells but also immune cell infiltrates, cancer-associated fibroblasts and components of the extracellular matrix. Using histological and protein-based approaches, we confirmed that the isolated microtumors closely resembled the corresponding primary tumor with respect to histological and molecular composition and preserve signaling pathway signatures of their native counterpart. Co-culture data revealed a significant increase in growth and invasiveness of microtumors derived from invasive ductal carcinoma of no special type in the presence of mammary-like organoids. Furthermore, a significant increase in the levels of the soluble markers fibronectin and metalloproteinase-2, both associated with tumor aggressiveness, invasion and metastasis, was detected in the co-cultures of mammary-like organoids and microtumors/spheroids, compared to the corresponding monocultures. We conclude that tumor-adjacent tissue may “fuel” tumor-progression, probably depending on the stage and the composition of the tumor.