Mesenchymal Stem Cells Increase Drug Tolerance of A431 Cells Only in 3D Spheroids, Not in 2D Co-Cultures

Abstract

Mesenchymal stem cells (MSCs) are an integral part of the tumor microenvironment (TME); however, their role is somewhat controversial: conflicting reports suggest that, depending on the stage of tumor development, MSCs can either support or suppress tumor growth and spread. Additionally, the influence of MSCs on drug resistance is also ambiguous. Previously, we showed that, despite MSCs proliferating significantly more slowly than cancer cells, there are chemotherapeutic drugs which proved to be similarly toxic to both cell types. Here we established 2D co-cultures and 3D co-culture spheroids from different ratios of GFP-expressing, adipose tissue-derived MSCs and A431 epidermoid carcinoma cells tagged with mCherry to investigate the effect of MSCs on cancer cell growth, survival, and drug sensitivity. We examined the cytokine secretion profile of mono- and co-cultures, explored the inner structure of the spheroids, applied MSC-(nutlin-3) and cancer cell-targeting (cisplatin) treatments separately, monitored the response with live-cell imaging and identified a new, double-fluorescent cell type emerging from these cultures. In 2D co-cultures, no effect on proliferation or drug sensitivity was observed, regardless of the changes in cytokine secretion induced by the co-culture. Conversely, 3D spheroids developed a unique internal structure consisting of MSCs, which significantly improved cancer cell survival and resilience to treatment, suggesting that physical proximity and cell-cell connections are required for MSCs to considerably affect nearby cancer cells. Our results shed light on MSC-cancer cell interactions and could help design new, better treatment options for tumors.

Keywords: cancer cells; drug resistance; mesenchymal stem cells; spheroids; tumor microenvironment.

Figure 1

Representative image of cytokine secretion profile of 2D mono- and co-cultures of MSCs and A431 cancer cells. (A) representative images of mono- and co-cultures at day 0 and day 5. The number of MSCs and A431 cells were counted using Burker chambers under a fluorescence microscope. Scale bar on the left column is 200 µm and 100 µm on the right. The black arrow represents the change from brightfield to fluorescent imaging on the same field of view. (B) Cytokine secretion profile of MSCs, A431 cells, and co-cultures on the human cytokine profile membrane. (C) Relative cytokine secretion (normalized to cell numbers) of mono- and co-cultures.

Figure 2

Cisplatin and nutlin-3 sensitivity of 2D mono- and co-cultures of MSCs and A431 cells. (A) Endpoint images of mono- and co-cultures of A431-mch and Ad-MSC-GFP 3 cells mixed in different ratios, following treatment with with 2 μM, 10 μM, and 20 μM cisplatin and 8 μM and 30 μM nutlin-3 for 5 days. Scale bar is 250 μm. Scale bar represents 250 µm. (B) Growth curves based on live cell imaging of mono- and co-cultures of MSCs (green) and cancer cells (red), imaged every 5 h for 120 h. p values and related significance levels were: p > 0.05 ns; p ≤ 0.05 *; p ≤ 0.01 **.

Figure 3

(A) Schematic experimental workflow of spheroid formation. (B,C) Confocal images showing the inner structure of 3D spheroids established from Ad-MSC-GFP 3 and A431-mCh cells. Cells mixed in different ratios were grown to allow for the formation of spheroids. Despite varying initial MSC:cancer cell ratios, the structure of the 3D co-cultures always shows the same pattern. Ad-MSC-GFP 3 cells create an inner scaffold for A431-mCh cells. Spheroids from A431-mCh cells die and collapse on their own, but if they are co-cultured with MSCs, they can survive on the surface of MSCs. X, Y and Z axis are the same size on all images. (D) Two-photon microscopy images of spheroids established using different cell ratios. In the first 3 columns Z-projection of the 3 replicates are shown. Scale bar 50 µm. The last column shows 3D reconstructed images of a single spheroid with the given cell ratios.

Figure 4

Analysis of GFP+/mCh+ double-positive cells. (A) Flow cytometry analysis of the composition of MSC, cancer cell and co-cultures, revealing that 0.8–1.1% of the co-culture shows double positivity. Green and red dots represent GFP- and mCh-positive cells, respectively. (B) Visualization of GFP+/mCh+ cells by fluorescent microscopy. White arrows point to double positive cells. Scale bar represent 70 μm. (C) F-actin staining with phalloidin (purple) of GFP- (green) and mCh-expressing (red) co-cultures and GFP+/mCh+ cells. Nuclei were visualized using DAPI (blue). Scale bars represent 200 μm. Double positive cells were magnified in the upper right corner of the merged images on both panel (B,C).

Figure 5

Cisplatin and nutlin-3 sensitivity of 3D Ad-MSC-GFP 3, A431-mCh and co-cultures. (A) Endpoint images of 3D co-cultured A431-mch and Ad-MSC-GFP 3 cells mixed in different ratios, treated with 2 μM, 10 μM, 20 μM cisplatin and 8 μM, 30 μM nutlin-3. Scale bars represent 250 μm. Cells were imaged every 5 h, creating a 120h record, and (B) growth curves were plotted by intensity analysis of GFP and mCherry fluorescence at each time point. (C) FACS analysis of mono- and co-cultured spheroids showing the number of GFP (Y axis, green)- and mCh (X axis, red)-expressing cells. Dead cells were stained with TO-PRO 3 (purple). (D) Quadrant statistics as determined by flow cytometry. P values and related significance levels were: p ≤ 0.05 *; p ≤ 0.01 **; p ≤ 0.001 ***.