Elimination of Cancer Cells in Co-Culture: Role of Different Nanocarriers in Regulation of CD47 and Calreticulin-Induced Phagocytosis

Abstract

Under healthy conditions, pro-and anti-phagocytic signals are balanced. Cluster of Differentiation 47 (CD47) is believed to act as an anti-phagocytic marker that is highly expressed on multiple types of human cancer cells including acute myeloid leukemia (AML) and lung and liver carcinomas, allowing them to escape phagocytosis by macrophages. Downregulating CD47 on cancer cells discloses calreticulin (CRT) to macrophages and recovers their phagocytic activity. Herein, we postulate that using a modified graphene oxide (GO) carrier to deliver small interfering RNA (siRNA) CD47 (CD47_siRNA) in AML, A549 lung, and HepG2 liver cancer cells in co-culture in vitro will silence CD47 and flag cancer cells for CRT-mediated phagocytosis. Results showed a high knockdown efficiency of CD47 and a significant increase in CRT levels simultaneously by using GO formulation as carriers in all used cancer cell lines. The presence of CRT on cancer cells was significantly higher than levels before knockdown of CD47 and was required to achieve phagocytosis in co-culture with human macrophages. Lipid nanoparticles (LNPs) and modified boron nitride nanotubes (BNPs) were used to carry CD47_siRNA, and the knockdown efficiency values of CD47 were compared in three cancer cells in co-culture, with an achieved knockdown efficiency of >95% using LNPs as carriers. Interestingly, the high efficiency of CD47 knockdown was obtained by using the LNPs and BNP carriers; however, an increase in CRT levels on cancer cells was not required for phagocytosis to happen in co-culture with human macrophages, indicating other pathways' involvement in the phagocytosis process. These findings highlight the roles of 2D (graphene oxide), 1D (boron nitride nanotube), and "0D" (lipid nanoparticle) carriers for the delivery of siRNA to eliminate cancer cells in co-culture, likely through different phagocytosis pathways in multiple types of human cancer cells. Moreover, these results provide an explanation of immune therapies that target CD47 and the potential use of these carriers in screening drugs for such therapies in vitro.

Harnessing graphene oxide nanocarriers for siRNA delivery in a 3D spheroid model of lung cancer

“…17 We and others have tested the siRNA targeting CD47 (CD47_siRNA) to effectively knock down CD47 protein expression in several cancer cell lines (Scheme 1), inhibiting the anti-phagocytosis signal of such cancer cells and demonstrating their susceptibility to macrophages following treatment. 11,[17][18][19] In order to achieve transfection, the siRNA must enter the body, pass through the circulation and reach the tumour site and penetrate the cell membrane to bind the mRNA inside the cancerous cells. During this journey, several difficulties and barriers can arise which limit the efficacy and use of siRNA.…”

Attenuation of Chronic Inflammation in Intestinal Organoids with Graphene Oxide-Mediated Tumor Necrosis Factor-α_Small Interfering RNA Delivery

“…GO and its derivatives, obtained by covalent modification of surface functional groups with polymers such as polyethylene glycol (PEG), chitosan, dextran, and dendrimers (e.g., polyamidoamine, PAMAM), have been used to achieve gene silencing in various cancer cells. ,− We have previously shown that GO-mediated delivery of siRNA targeting CD47 in different cancer cell lines downregulated CD47 protein expression, which can inhibit the antiphagocytosis “do not eat me” signal. This increases their susceptibility to phagocytic clearance by macrophages. ,, Additionally, when CD47 was knocked down, there was a significant increase in the production of IL-6, TNF-α, IL-1β, and IL-8 in the culture media compared to the control (IL-8, IL-6, and TNF-α showed a remarkable increase of over 65 times, and IL-1β increased >40 times, compared to the control: cancer cells in coculture without transfection), strongly implying that their main function is to target and eliminate cancer cells . Furthermore, GO size, surface modifications (PEG and PAMAM), and cargo-to-GO ratios play important roles in transfection efficiency of CD47_siRNA in both two-dimensional (2D) and three-dimensional (3D) cultures of lung cancer cell lines.…”

“…GO has a large surface area characterized by the presence of many functional groups such as hydroxyl, carboxyl, and carbonyl groups. , These functional groups can be further modified to achieve enhanced biocompatibility, aqueous solubility, and high cargo loading capacity. ,− The large surface area to volume ratio of GO reduces cytotoxicity and supports effective endocytosis due to its increased interactions with cells . GO and its derivatives, obtained by covalent modification of surface functional groups with polymers such as polyethylene glycol (PEG), chitosan, dextran, and dendrimers (e.g., polyamidoamine, PAMAM), have been used to achieve gene silencing in various cancer cells. ,− We have previously shown that GO-mediated delivery of siRNA targeting CD47 in different cancer cell lines downregulated CD47 protein expression, which can inhibit the antiphagocytosis “do not eat me” signal. This increases their susceptibility to phagocytic clearance by macrophages. ,, Additionally, when CD47 was knocked down, there was a significant increase in the production of IL-6, TNF-α, IL-1β, and IL-8 in the culture media compared to the control (IL-8, IL-6, and TNF-α showed a remarkable increase of over 65 times, and IL-1β increased >40 times, compared to the control: cancer cells in coculture without transfection), strongly implying that their main function is to target and eliminate cancer cells .…”

“…This increases their susceptibility to phagocytic clearance by macrophages. 35 , 36 , 40 Additionally, when CD47 was knocked down, there was a significant increase in the production of IL-6, TNF-α, IL-1β, and IL-8 in the culture media compared to the control (IL-8, IL-6, and TNF-α showed a remarkable increase of over 65 times, and IL-1β increased >40 times, compared to the control: cancer cells in coculture without transfection), strongly implying that their main function is to target and eliminate cancer cells. 35 Furthermore, GO size, surface modifications (PEG and PAMAM), and cargo-to-GO ratios play important roles in transfection efficiency of CD47_siRNA in both two-dimensional (2D) and three-dimensional (3D) cultures of lung cancer cell lines.…”

Nanosystem Delivers Senescence Activators and Immunomodulators to Combat Liver Cancer

“…[25][26][27] Because the EPR effect is limited, to improve the active targeting of NPs to tumors, we prepared NPs modified with a CD47 antibody (AbCD47). Because CD47 is highly expressed on HCC, [5] AbCD47 can target and block CD47 on the surface of tumor cells to disable the "Don't eat me" signal. However, tumor heterogeneity causes the amount of CD47 to vary from cell to cell, [28] leading to targeting failure.…”