Abdal, Ebrahim Mohammed; (2023) Interactions between prostate cancer and monocytes in a 3D immunocompetent tumouroid model. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Monocytes (U937) are innate immune cells having important roles in regulating progression in prostate cancer (PCA). Utilisation of three-dimensional (3D) culture models provides more realistic representations of tumour microenvironment (TME) compared to two-dimensional (2D) culture models. The aim of this study was to incorporate monocytes in a 3D tumouroid prostate cancer (PCA) model under different biophysical environments, to ultimately create a simple immunocompetent in vitro cancer model. The work built on the existing in-house model of a tumouroid, which is a 3D collagen construct containing cancer cells and compressed to physiological stiffness to mimic native tissues. Investigations focused on monocyte behaviour, cytokine secretion and effect on PCA spheroid growth. U937 were added and their behaviour examined by microscopy over 7 days in three types of 3D constructs, under different biophysical conditions: acellular 3D collagen gels, either non compressed hydrogels or compressed (RAFT method); and tumouroids, which consisted of PCA (LNCaP and PC3) cells within the compressed gel. Confocal microscopy determined fluorescently-tagged U937 remained viable and penetration depth was similar in all conditions, with that in non-compressed gels and immunocompetent tumouroids (i.e., gels already containing cancer cells) slightly higher (95µm and 114μm respectively, v. 61.6µm in compressed, NS). Interestingly, it appeared that monocyte numbers (i.e., fluorescence levels) were greater in non-compressed than compressed acellular gels, (day 7 85 vs. 56 AU, p<0.05). Spheroid growth of LNCaP PCA cells over time (14 days), determined via fluorescent microscopy, increased more in immunocompetent tumouroids (4609µm2 ) than control tumouroids (4366µm2 ) (day 14, p<0.05). Further comparisons of experiments utilising PMA stimulated U937 monocytes (more differentiated) showed an increase in cancer spheroid diameter within tumouroids compared to un-stimulated monocytes (3114µm2 v. 2568µm2 , day 10, p<0.05). U937 cells cytokine production in gels was investigated via Luminex followed by ELISA. The Luminex analyses showed in compressed gels upregulated cytokines including: VEGF (20,015.3AU, vs. uncompressed 4,990AU, p<0.05); and to a lesser extent IL-10 (368 AU vs. uncompressed 343.3AU, p>0.05) and TNF-α (436 AU vs. uncompressed 386AU, p>0.05). Subsequent ELISA analyses showed that for LNCaP PCA tumouroids, VEGF and TNF-α levels were similar in simple and immunocompetent cultures (i.e., with added U937 monocytes), (Day 14: 0.52 and 0.23AU for simple; v. 0.52 and 0.21AU for immunocompetent respectively). Hypoxia and cytokine expression in immunocompetent PCA tumouroids, constructed with two different cancer cell lines, was analysed. Under hypoxia, VEGF increased more in LNCaP than PC3 immunocompetent tumouroids (Day 14, 0.47 v. 0.25AU, p<0.05) . Conversely, IL-10 peaked in PC3 than LNCaP immunocompetent tumouroids (Day 14, 0.26 v. 0.22 AU, p>0.05). TNF-α levels increased in LNCaP compared to PC3 immunocompetent tumouroids (Day 14, 0.30 v. 0.21AU, p>0.05) respectively. Additionally, in normoxia, PMA stimulated U937 cells in PCA (LNCaP) immunocompetent tumouroids compared to previous experiments on day 10 had elevated; VEGF (0.36 AU, p<0.05), IL-10 (0.24 AU, p<0.05) and TNF-α (0.28 AU, p<0.05). TNF-α secretion was higher than all previous experiments. In conclusion, this is the first report of tumouroids successfully incorporating monocytes, a key immune cell in cancer progression. The work demonstrates monocytes promoting cancer growth and altered expression of key cytokines especially increase in protumourigenic VEFG. This is the basis for developing an immunocompetent tumouroid platform to eventually use as a drug testing platform. Furthermore, tumouroids have the real potential to reduce animal experimentation and provide insight into complex tumour interactions in a biomimetic environment.

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