Lactic Acidosis Interferes With Toxicity of Perifosine to Colorectal Cancer Spheroids: Multimodal Imaging Analysis

Abstract

Colorectal cancer (CRC) is a disease with constantly increasing incidence and high mortality. The treatment efficacy could be curtailed by drug resistance resulting from poor drug penetration into tumor tissue and the tumor-specific microenvironment, such as hypoxia and acidosis. Furthermore, CRC tumors can be exposed to different pH depending on the position in the intestinal tract. CRC tumors often share upregulation of the Akt signaling pathway. In this study, we investigated the role of external pH in control of cytotoxicity of perifosine, the Akt signaling pathway inhibitor, to CRC cells using 2D and 3D tumor models. In 3D settings, we employed an innovative strategy for simultaneous detection of spatial drug distribution and biological markers of proliferation/apoptosis using a combination of mass spectrometry imaging and immunohistochemistry. In 3D conditions, low and heterogeneous penetration of perifosine into the inner parts of the spheroids was observed. The depth of penetration depended on the treatment duration but not on the external pH. However, pH alteration in the tumor microenvironment affected the distribution of proliferation- and apoptosis-specific markers in the perifosine-treated spheroid. Accurate co-registration of perifosine distribution and biological response in the same spheroid section revealed dynamic changes in apoptotic and proliferative markers occurring not only in the perifosine-exposed cells, but also in the perifosine-free regions. Cytotoxicity of perifosine to both 2D and 3D cultures decreased in an acidic environment below pH 6.7. External pH affects cytotoxicity of the other Akt inhibitor, MK-2206, in a similar way. Our innovative approach for accurate determination of drug efficiency in 3D tumor tissue revealed that cytotoxicity of Akt inhibitors to CRC cells is strongly dependent on pH of the tumor microenvironment. Therefore, the effect of pH should be considered during the design and pre-clinical/clinical testing of the Akt-targeted cancer therapy.

Infrared Laser Desorption of Intact Nanoparticles for Digital Tissue Imaging

“…To distinguish the outer layer with proliferating cells from the other zones, a nuclear protein Ki-67 with characteristic spatial distribution was chosen. This protein is commonly used as a marker of proliferation, since its expression is bound to phases of cell cycle G1, S, G2, and mitosis; it is found almost exclusively in the outer layers of spheroid sections …”

Caspase-8 Deficient Osteoblastic Cells Display Alterations in Non-Apoptotic Pathways

“…Cell proliferation was determined by using the MTT assay as described previously ( Pavlatovská et al, 2020 ). Briefly, 5 × 10 3 of MC3T3-E1 control and Casp8 −/− cells were seeded in a 24-well plate.…”

Synergistic cytotoxicity of perifosine and ABT‐737 to colon cancer cells

“…Cells (10 5 cells/ml) were seeded in control and acidic media in normoxia or hypoxia for 72 h then, the depleted media were replaced with fresh media and the cells were treated with perifosine in combination with ABT‐737 (final concentrations of both: 2.5, 5, 10, 20 and 40 μM) for 48 h. Control cells were treated with 50% ethanol as solvent. The 3D models were treated with perifosine and/or ABT‐737 (final concentrations: 5–20 μM) for 48 h under normal conditions, as acidosis and hypoxia naturally occur in the 3D tumour models 10,33 . MTT assay was performed for both monolayers and spheroids as described 10 …”

“…Spheroids were treated with a 20 μM final concentration of perifosine and/or 20 μM ABT‐737 for 48 h under normal conditions. Cell viability up to 100 μm from the spheroid surface was determined using calcein‐AM/propidium iodide live/dead cell staining and emitted fluorescence was detected using laser scanning confocal microscopy (LSCM) (TCS SP8, Leica Microsystems, Wetzlar, Germany) as described 10 . The final ratio of dead to live cells was examined using TissueQuest software (TissueGnostics, Vienna, Austria).…”

“… 7 , 8 However, the activity of Akt inhibitors is highly pH‐dependent, as they lose efficacy under acidic conditions. 9 , 10 Unfortunately, an acidic environment and low tumour oxygen levels are hallmarks of cancer and contribute to cell resistance. 11 …”