Interactions between leukemia and feeders in co-cultivation under hypoxia

Abstract

Background

Leukemia is driven by complex interactions within the inherently hypoxic bone marrow microenvironment, impacting both disease progression and therapeutic resistance. Co-cultivation of leukemic cells with feeder cells has emerged as a valuable tool to mimic the bone marrow niche. This study explores the interplay between human commercial SD-1 and patient-derived UPF26K leukemic cell lines with feeders - human fibroblasts (NHDF) and mesenchymal stem cells (hMSCs) under normoxic and hypoxic conditions.

Results

Co-cultivation with feeders significantly enhances proliferation and glycolytic activity in the SD-1 cells, improving their viability, while this interaction inhibits the growth and glucose metabolism of the feeders, particularly NHDF. In contrast, UPF26K cells show reduced proliferation when co-cultivated with the feeders while this interaction stimulates NHDF and hMSCs proliferation and glycolysis but reduce their mitochondrial metabolism with hypoxia amplifying these effects.

Conclusions

Cells that switch to glycolysis during co-cultivation, particularly under hypoxia, benefit most from these low oxygen conditions. Due to this leukemic cells’ response heterogeneity, targeting microenvironmental interactions and oxygen levels is crucial for personalized leukemia therapy. Advancing co-cultivation models, particularly through innovations like spheroids, can further enhance in vitro studies of primary leukemic cells and support the testing of novel therapies.

Background

The bone marrow (BM) microenvironment, characterized by lower oxygen levels (hypoxia - HX), varies in location-specific intensity, creating conditions that significantly affect cell survival [40]. The BM niche comprises a mix of multipotent mesenchymal stem cells (MSCs), vascular networks, nerve fibers, mature blood cells, hematopoietic stem cells and other components essential for hematopoiesis and stem cell differentiation [75]. These cells secrete proteins, cytokines and growth factors that maintain physiological homeostasis and contribute to immune evasion and drug resistance [12, 49]. However, replicating the BM microenvironment in vitro remains challenging due to the difficulty of mimicking physiological oxygen levels. The tumor microenvironment shares similarities with the BM niche, including the presence of stromal, immune and tumor cells that may invade surrounding tissues or metastasize. Both environments are characterized by HX, acidity, immunosuppression and nutrient deficiency, all of which support cancer progression [67]. In myeloid malignancies, disruptions in the BM microenvironment transform the normal hematopoietic niche into a leukemic one, influencing leukemia initiation and therapy response [51]. Leukemic cells, created from poorly differentiated blood cells, adapt to nutrient-poor environments by adjusting to varying levels of glucose, glutamine, pH and oxygen [63, 87]. As with other cancer cells, they bypass normal growth controls, often relying on glycolysis even in the presence of oxygen, a phenomenon known as the Warburg effect [81]. The optimization of the in vitro cultivation conditions is crucial to improving the understanding of leukemic cell behavior since primary leukemic cells are extremely sensitive to manipulation, which leads to rapid senescence and limited lifespans without external support, e.g. growth factors.

Recent studies highlight the role of stromal cells in promoting Acute Lymphoblastic Leukemia (ALL) cell survival and chemoresistance within the BM microenvironment. Various in vitro and in vivo models have been used to explore these interactions. Traditional two-dimensional (2D) co-cultivation models, where leukemic cells are grown with stromal cells like MSCs, are commonly employed to study cell-cell interactions and drug resistance [33, 54]. Many of these studies focus on signaling pathways, like Wnt or epithelial-mesenchymal transition, both of which enhance chemoresistance and ALL cell survival [62, 85]. However, these models may not capture the full complexity of the BM niche. More advanced three-dimensional (3D) models, such as spheroids and microfluidic systems, better simulate in vivo conditions by incorporating extracellular matrix components and oxygen gradients [9, 11]. Animal models like patient-derived xenografts (PDX) are also used to mimic the human BM niche, but they can be resource-intensive and lack experimental control [62].

In contrast to models that rely on activating complex signaling pathways or altering stromal cell phenotypes, our approach uses a straightforward co-cultivation of human fibroblasts (NHDF) and mesenchymal stem cell (hMSCs) feeders, which were not growth-arrested, to observe natural metabolic and viability changes in the leukemic and the feeder cells, without additional cellular manipulation. NHDFs, often considered to be more differentiated or aged forms of hMSCs, share significant similarities with hMSCs, thus rendering them a suitable preliminary model for the study of the more valuable hMSCs [71]. Given the dynamic interplay between leukemic cells and their microenvironments, human feeders from various sources (e.g. fetal muscle and skin, bone marrow, adipose tissue, etc.) offer a more natural in vitro environment for the testing of leukemic cells [86, 88].

After refining optimal in vitro conditions, leukemia research could further benefit from spheroid models, a 3D approach gaining traction for its ability to better mimic the tumor microenvironment. These models support primary leukemic cells under lab conditions more effectively than traditional 2D cultures. While recent studies published by Balandrán et al. and Haselanger et al. [4, 33] have successfully applied spheroids to leukemia research, this approach remains emerging and is not yet widely implemented. Unlike animal models, spheroids enable more controlled experimentation with reduced variability and ethical concerns, while offering faster and more precise insights into cell interactions, drug resistance and treatment responses, ultimately paving the way for more effective personalized therapies with greater predictive accuracy [58, 83]. Acknowledging the importance of HX in complex BM and tumor niches, our study employs both normoxic (NX, 20% O₂) and hypoxic (HX, 1% O₂) conditions to align in vitro studies with physiological realities to explore how various cultivation conditions (oxygen level and type of feeders) affect the viability of leukemic cells from the same diagnosis (Adult B Acute Lymphoblastic Leukemia– ALL). ALL, a malignancy arising from lymphoid precursors that deviate from normal T- or B-cell development [47], was modeled using both the commercial SD-1 cell line and patient-derived UPF26K cells, thereby allowing a direct comparison of their responses under various cultivation conditions. We hypothesize that leukemic cell survival and metabolism are not only influenced by HX but also by direct interactions with different feeder cells, which may either support or inhibit leukemic cell growth depending on their adaptations. By investigating metabolic and proliferative changes in both commercial and patient-derived leukemic cells under NX and HX co-cultivation conditions, we aim to uncover novel metabolic dependencies that could inform microenvironment-driven therapeutic strategies. These findings will enhance the predictive accuracy of in vitro leukemia models and contribute to the development of more personalized treatment approaches.

Results

The influence of the cultivation conditions on the proliferation and metabolism of the leukemia cells

The impact of the cultivation conditions on the proliferation of the commercial SD-1 cells and patient-derived UPF26K cells was identified by testing two selected types of media– IMDM (as their standard cultivation medium) and a mix of αMEM + IMDM (1:1) (a medium used for co-cultivation with feeders) under NX– 20% O₂ and HX– 1% O₂ over time.

The SD-1 cells (Fig. 1A) increased in number during short-term (24–72 h) cultivation to a similar extent in the various media and under varying oxygen conditions, as demonstrated by their similar doubling time rates (approx. 25 h) (Fig. 1C). Interestingly, the pre-incubation of SD-1 cells under HX for 7 days before the estimation of the doubling time almost tripled the doubling time (approx. 70 h). The UPF26K cells followed a similar proliferation trend in the various cultivation media and under varying oxygen conditions (Fig. 1B) and evinced similar doubling times (approx. 28 h) (Fig. 1C). Interestingly, the pre-incubation of the UPF26K cells under HX for 7 days led to a decrease in proliferation, but to a lower extent than for the SD-1 cells (approx. 38 h).

Fig. 1

Proliferation of SD-1 and UPF26K cells under different cultivation conditions in the individual cultivation. (A, B) Proliferation (cell number over time) of SD-1 (A) and UPF26K (B) cells grown in “IMDM” (standard control medium) and a “mix” media (mixture of αMEM and IMDM) under normoxia (NX – 20% O₂) and hypoxia (HX – 1% O₂). Data are shown as box and whisker plots: boxes represent the interquartile range (IQR), the median (horizontal line), minimum and maximum values (whiskers) and individual data points (dots) are plotted. (C) Doubling time of SD-1 and UPF26K cells after 2 days of cultivation (darker bars of appropriate cell type) and after 7 days of preincubation under HX (lighter bars – “HX 7d preincubation”). Bar graphs show individual data points (dots) and standard deviation (SD). The numbers above the boxplots and bars indicate the mean for each group. In (A) and (B), the control condition (“% of ctrl-NX IMDM 24 h”) set as 100%, represents the individual cultivation of particular cell type cultivated in a IMDM medium after 24 h under NX. Statistical analyses were performed using a nonparametric, two-tailed Mann–Whitney U test for two-sample comparisons. Statistical significance (p ≤ 0.05) is indicated in the graphs using symbols that denote significant differences between the following comparisons: * = all individual cultivation samples vs. NX IMDM individual control medium at 24 h, significance brackets = differences for the same sample (i.e., the same cell type and cultivation medium) grown under NX vs. HX

The metabolic activity of the tested cells was evaluated using MTS assay after their cultivation under differing conditions (media and oxygen level).

The metabolic (mitochondrial) activity of the SD-1 cell line did not vary across the different media. However, compared to NX, HX significantly induced metabolic activity at 24 h (Fig. 2A). The activity was comparable under both oxygen conditions after 48 and 72 h.

Fig. 2

Metabolism of SD-1 and UPF26K cells under different cultivation conditions in the individual cultivation. Metabolic activity in SD-1 (A) and UPF26K (B) cells, measured using the MTS assay in “IMDM” (standard control medium) and a “mix” media (mixture of αMEM and IMDM) under NX and HX. Data are shown as box and whisker plots: boxes represent the IQR, the median (horizontal line), minimum and maximum values (whiskers) and individual data points (dots) are plotted. The numbers above the boxplots indicate the mean for each group. (C) Extracellular flux analysis of SD-1 and UPF26K cells cultivated under varying oxygen conditions (2 days and 7 days) using the SeaHorse ATP rate assay. Data are shown as stacked bar graphs: each stacked bar reflects the sum of the two measured components (e.g., “% Glycolysis” in blue and “% Oxidative Phosphorylation” in orange). The bar height corresponds to the mean value, with error bars indicating the SD. Numbers within each segment denote the mean percentage contribution of that component. (D) Light microscopy images of SD-1 and UPF26K cells after 48 h of cultivation under different conditions (the scale bar in the images represents 100 μm). In (A) and (B), the control condition (“% of ctrl-NX IMDM 24 h”) set as 100%, represents the individual cultivation of particular cell type cultivated in a IMDM medium after 24 h under NX. Statistical analyses were performed using a nonparametric, two-tailed Mann–Whitney U test for two-sample comparisons. Statistical significance (p ≤ 0.05) is indicated in the graphs using symbols that denote significant differences between the following comparisons: * = all individual cultivation samples vs. NX IMDM individual control medium at 24 h, significance brackets = differences for the same sample (i.e., the same cell type and cultivation medium) grown under NX vs. HX

SeaHorse ATP rate assay was used after 2 and 7 days of cultivation (Fig. 2C) for the determination of the contribution of glycolysis and oxidative phosphorylation (OXPHOS) to the cell metabolism. Surprisingly, the SD-1 cells exhibited the same rate of glycolysis and OXPHOS (approx. 50% each) under both oxygen conditions. A slight increase in glycolysis (57%) was observed following long-term HX incubation (7 d), although this change was not statistically significant.

In contrast, the metabolic activity of the UPF26K cells was significantly influenced by the differing cultivation conditions (Fig. 2B). While the media type had no effect, HX notably stimulated the increase in the metabolic activity of the UPF26K cells under all the conditions tested, as determined by the MTS assay.

The SeaHorse ATP rate assay confirmed the findings (Fig. 2C); after 48 h of HX incubation, the cells exhibited a predominantly OXPHOS-driven profile (88% OXPHOS vs. 12% glycolysis), a difference that was statistically proven. Following long-term (7 d) exposure to HX conditions, a modest shift was observed with the increase of glycolysis (35%) while OXPHOS remained still dominant (65%), although without statistical significance.

Although the organization of the SD-1 cells within the samples was not influenced by the cultivation conditions (Fig. 2D), the organization of the UPF26K cells was strongly affected by the oxygen conditions after 48 h (Fig. 2D), with more clusters visibly formed under the NX than under the HX conditions in both media types; the cluster size, however, remained unchanged.

The results confirmed that mix media can be used in co-cultivation experiments without affecting the leukemic cells. Although short term HX (48 h) exerted no impact on the cell proliferation, it influenced their metabolism, whereas longer HX cultivation (7 d) lowered the leukemic cell proliferation level.

Influence of the cultivation conditions on the feeders’ proliferation and metabolism

Aimed at identifying the impact of the cultivation conditions on the proliferation of the non-growth-arrested NHDF and hMSCs feeders, we tested two media types– αMEM (their standard cultivation medium) and a mix of αMEM and IMDM (1:1) (used for co-cultivation with leukemic cells) under NX and HX over time.

The NHDF evinced only a minimal difference in their proliferative activity in the various media during short-term (48 h) incubation under the two oxygen conditions, as demonstrated by their doubling time (approx. 30 h) (Fig. 3A). However, long-term (14 d) cultivation under HX markedly reduced NHDF colony formation (Fig. 3B) and quality (Fig. 3C), especially in the standard αMEM medium.

Fig. 3

Proliferation of NHDF and hMSCs cells under different cultivation conditions in the individual cultivation. Doubling time (A) of NHDF and hMSCs after 48 h of cultivation. Colony formation assay (CFU-F) (B) assesses colony formation efficiency and proliferative potential after 14 days under different cultivation conditions. Bar graphs show individual data points (dots) and SD. The numbers above the bars indicate the mean for each group. Light microscopy images (C) showing size and morphology of NHDF and hMSCs´ colonies (the scale bar in the images represents 200 μm). In (A) and (B), statistical analyses were performed using a nonparametric, two-tailed Mann–Whitney U test for two-sample comparisons. Statistical significance (p ≤ 0.05) is indicated in the graphs using symbols that denote significant differences between the following comparisons: * = all individual cultivation samples vs. NX αMEM individual control medium (for each cell type separately), # = HX individual cultivation samples vs. HX αMEM individual control medium, significance brackets = differences for cells cultivated in the same cultivation medium grown under NX vs. HX

The hMSCs proliferated in a similar way in the various cultivation media under NX (approx. 50 h), but their proliferative activity increased significantly under HX during short-term (48 h) incubation, with a more pronounced effect in the mix media, as evinced by the decreased doubling time (approx. 38 h) (Fig. 3A). The cultivation media did not affect colony formation; however, HX reduced their colony-forming ability to less than half during long-term (14 d) incubation (Fig. 3B). Surprisingly, the colony quality (Fig. 3C) was comparable under all the conditions.

The metabolic activity of the NHDF cells decreased in the mix media compared to the standard medium and overtime; no significant difference was observed between the NX and HX conditions (Fig. 4A). The SeaHorse ATP rate assay (Fig. 4C) showed that most of the ATP (71%) in the NHDF came from OXPHOS after 48 h under NX. However, after 48 h under HX, the NHDF cell metabolic activity switched, i.e. most of the ATP came from glycolysis (77%), which further increased to 85% after 7 days under HX.

Fig. 4

Metabolism of NHDF and hMSCs cells under different cultivation conditions in the individual cultivation. Metabolic activity in NHDF (A) and hMSCs (B) cells, measured using the MTS assay in a control (αMEM) medium and a mixture of αMEM and IMDM (mix) media under NX and HX. Data are shown as box and whisker plots: boxes represent the IQR, the median (horizontal line), minimum and maximum values (whiskers) and individual data points (dots) are plotted. The numbers above the boxplots indicate the mean for each group. (C) Extracellular flux analysis of NHDF and hMSCs cells cultivated under varying oxygen conditions (2d and 7 d) using the SeaHorse ATP rate assay. Data are shown as stacked bar graphs: each stacked bar reflects the sum of the two measured components (e.g., “% Glycolysis” in blue and “% Oxidative Phosphorylation” in orange). The bar height corresponds to the mean value, with error bars indicating the SD. Numbers within each segment denote the mean percentage contribution of that component. In (A) and (B), the control condition (“% of ctrl-NX αMEM 24 h”) set as 100%, represents the individual cultivation of particular cell type cultivated in a αMEM medium after 24 h under NX. Statistical analyses were performed using a nonparametric, two-tailed Mann–Whitney U test for two-sample comparisons. Statistical significance (p ≤ 0.05) is indicated in the graphs using symbols that denote significant differences between the following comparisons: * = all individual cultivation samples vs. NX αMEM individual control medium at 24 h, # = HX individual cultivation samples vs. HX αMEM individual control medium at 24 h, significance brackets = differences for the same sample (i.e., the same cell type and cultivation medium) grown under NX vs. HX

In contrast, the metabolic activity of the hMSCs, as measured by the MTS assay, significantly and continuously increased under HX but without the impact of the media type. Interestingly, the SeaHorse ATP rate assay revealed a similar metabolic shift in the hMSCs as in the NHDF cells (Fig. 4C). After 48 h under NX, the majority of ATP was generated through OXPHOS (70%), but under HX, the hMSCs switched to glycolysis (73%), which continued for 7 days of HX exposure (75%).

The results confirmed that the mix medium can be used in co-cultivation experiments without affecting the proliferation and metabolism of the feeder cells. Short-term HX does not affect NHDF cell proliferation; HX decreases their colony-forming ability over time. In contrast, short-term HX enhances hMSCs´ proliferation, while long-term HX exerts a negative impact on their colony-forming ability.

Influence of co-cultivation and hypoxia on the leukemic and the feeder cells

Both types of leukemia cells (SD-1 and UPF26K) were co-cultivated with both types of feeders (NHDF and hMSCs) and compared to the cells cultivated individually under various oxygen conditions.

We observed a significant increase in the SD-1 cell number after co-cultivation with the NHDF feeders under both NX and HX conditions compared to the SD-1 cells cultivated alone (Fig. 5A). Moreover, the cell number under NX was significantly higher than under HX, despite there being no difference in the cell number for the SD-1 cells cultivated individually under both conditions.

Fig. 5

Viability of leukemic cells and feeders under individual cultivation and co-cultivation conditions. Analysis of cell number (A, D), metabolic activity (B, E) and 2-DG uptake (C, F) in leukemic cells (SD-1, UPF26K) (A, B, C) and feeders (NHDF, hMSCs) (D, E, F) after 48 h of co-cultivation under NX and HX conditions. Data are shown as box and whisker plots: boxes represent the IQR, the median (horizontal line), minimum and maximum values (whiskers) and individual data points (dots) are plotted. The numbers above the boxplots indicate the mean for each group. The control condition (NX individual cultivation in MIX = N × SD-1 or NX UPF26K or NX NHDF or NX hMSC) set as 100% represents the individual cultivation of particular cell type cultivated in a MIX medium under NX. Statistical analyses were performed using a nonparametric, two-tailed Mann–Whitney U test for two-sample comparisons. Statistical significance (p ≤ 0.05) is indicated in the graphs using symbols that denote significant differences between the following comparisons: * = all co-cultivation samples and HX individual samples vs. NX individual cultivation control, # = HX co-cultivation samples vs. HX individual cultivation control, + = different co-cultivation counterparts within the same oxygen level (e.g. SD-1 from NX co-cultivation with NHDF vs. SD-1 from NX co-cultivation with hMSCs), significance brackets = differences for the same sample (i.e., the same cell type and co-cultivation counterparts) grown under NX vs. HX

An increase was also observed in the SD-1 cell number after SD-1 cell and hMSCs co-cultivation (Fig. 5A); however, it was not significant and occurred only under NX. While the individually cultivated SD-1 cells evinced similar cell numbers under HX and NX, their metabolic activity was significantly higher under HX than NX (Fig. 5B). Conversely, the metabolic activity of the SD-1 cells co-cultivated with both feeders was comparable under both oxygen conditions and decreased in comparison to the individually cultivated cells.

Further analysis of the metabolism of the co-cultivated leukemic cells was performed applying a short incubation period with fluorescently labeled glucose analog 2-deoxyglucose (2-DG) (Fig. 5C), which indirectly indicated glycolysis involvement in the cell metabolism. The individually cultivated SD-1 cells exhibited a similar 2-DG uptake under HX and NX; however, the cells co-cultivated with NHDF under HX exhibited enhanced 2-DG uptake, which most likely indicated increased glycolysis. This increase was also noticeable after co-cultivation with the hMSCs; however, this was confirmed only via the more sensitive Glass’s Delta statistical analysis (0.6 with NHDF, 1.1 with hMSCs under HX compared to individual SD-1 cultivation under NX). In contrast, reduced 2-DG uptake by the SD-1 cells was apparent under NX when co-cultivated with both feeders compared to individual cultivation.

In summary, although the SD-1 cells co-cultivated with NHDF feeders increased in number under both oxygen conditions, their metabolic (mitochondrial) activity remained comparable; 2-DG uptake increased only under HX. No significant changes in the SD-1 proliferation or metabolism were detected for the co-cultivation with hMSCs.

Conversely, a significant decrease in the number of UPF26K cells co-cultivated with the two feeders was detected under both NX and HX conditions compared to the individually cultivated cells (Fig. 5A). However, a significantly higher cell number was detected under HX than NX after co-cultivation with both types of feeders; this difference was not observed in the individually cultivated UPF26K cells.

The metabolic activity of the UPF26K cells after co-cultivation with both feeder types was significantly elevated under NX and, in the case of NHDF, also under HX as compared to their individual cultivation, concerning which no significant differences between the conditions were observed (Fig. 5B). Nevertheless, a significant increase was observed in the metabolic activity under NX compared to HX in both co-cultivated UPF26K cells.

The UPF26K cells, when co-cultivated with NHDF, evinced an increased 2-DG uptake compared to individual cultivation (Glass´s Delta − 1.4 under NX; 1.2 under HX). However, no significant change was observed after 2-DG uptake for the hMSCs (both HX and NX conditions) (Fig. 5C).

In addition, the NHDF and hMSCs feeders were tested following co-cultivation with both types of leukemia cells (SD-1 and UPF26K) and compared to the respective individual cultivation controls.

A significantly reduced number of NHDF was detected after co-cultivation with the SD-1 cells under both NX and HX compared to the individually cultivated NHDF (Fig. 5D). Conversely, the number of NHDF after co-cultivation with the UPF26K cells significantly increased under both NX and HX. In a similar way to the individually cultivated NHDF cells, those co-cultivated with UPF26K evinced higher cell numbers under HX than NX, although the difference was not significant.

The metabolic activity of NHDF concerning individual cultivation under HX was significantly lower than under NX (Fig. 5E). This trend was also observed for co-cultivation with both the SD-1 and UPF26K cells. Moreover, a significant increase was apparent under NX with the SD-1 cells, whereas no change was observed for the UPF26K cells compared to individual cultivation under NX. A significant increase was evident for co-cultivation with the SD-1 cells under HX and a decrease for the UPF26K cells compared to the individually cultivated NHDF.

The determination of the 2-DG uptake revealed a logically reversed trend (Fig. 5F); decreased uptake was detected in the NHDFs from co-cultivation with the SD-1 cells, whereas an increased uptake was noted for the UPF26K cells. Comparable 2-DG uptakes were detected under both oxygen conditions.

HMSCs co-cultivation with both types of leukemic cells revealed their higher number under HX than for individual cultivation (Fig. 5D). However, no difference in the cell number was observed under NX. The difference between the hMSCs under NX and HX was not significant either for individual cultivation or for co-cultivation with the SD-1 cells. However, the number of hMSCs was significantly higher under HX than NX with the UPF26K cells.

Subsequently, a decrease in the metabolic activity of the hMSCs was observed after co-cultivation with both leukemic cells under HX (Fig. 5E). Such a decrease was apparent under NX only after co-cultivation with the SD-1 cells. A significant difference in the metabolic activity between the NX and HX conditions was proven only for the hMSCs co-cultivated with the UPF26K cells.

The co-cultivation of hMSCs with SD-1 appeared to lower the 2-DG uptake in hMSCs (0.9 (NX) and 1.1 (HX) according to Glass’s Delta) (Fig. 5F). In contrast, an increase in the 2-DG uptake compared to individual cultivation was observed with the UPF26K cells. No impact was detected of the oxygen level on the 2-DG uptake in the hMSCs.

Impact of co-cultivation and hypoxia on the leukemic cell distribution

The bond strength between the leukemic cells and the feeders and the distribution of the leukemic cells on the feeder monolayer were assessed after the washing procedure. The interaction between the SD-1 cells and the NHDF feeders seems to be strong and unaffected by the oxygen conditions (Fig. 6A). Conversely, the SD-1 cells look tightly bonded to the hMSCs under NX only (Fig. 6B). The UPF26K cells exhibited a notably weaker bond with both feeders than the SD-1 cells under all the cultivation conditions except of a stronger attachment to the hMSCs under NX (Fig. 6C, D).

Fig. 6

Effects of co-cultivation and hypoxia on bond strength and leukemic distribution on feeders. Visualization of bond strength, referring to the adhesion between leukemic cells and the feeder monolayer and distribution changes in leukemic cells following the separation and processing of cell suspensions from the feeder monolayer after 48 h of co-cultivation under NX and HX conditions (the scale bar in the images represents 100 μm). Presence of SD-1 and NHDF (A), SD-1 and hMSCs (B), UPF26K and NHDF (C) and UPF26K and hMSCs (D), after their co-cultivation and washing on the cultivation well

Impact of co-cultivation and hypoxia on the mitochondria morphology of the feeders

The next stage comprised the evaluation of the mitochondria of the feeders via fluorescent microscopy following co-cultivation with the leukemic cells under NX and HX. The mitochondrial network morphology varied between the NX and HX conditions in all the cultivation setups. The mitochondria were more fragmented under HX, whereas they were elongated under NX. Generally, mitochondrial fragmentation under HX suggests cellular stress or a metabolic shift, whereas elongation under NX indicates efficient energy production [24, 76].

The mitochondrial network of the NHDF after co-cultivation with SD-1 under NX (Fig. 7C) suggested higher degrees of density and connectivity between the mitochondria than for the individual cultivation of the NHDF, which was supported by a detected increase in their metabolic activity (Fig. 5E). In contrast, the NHDF mitochondria were strongly fragmented, large, rounded and localized in the perinuclear space under HX. The mitochondrial network of the NHDF after co-cultivation with the UPF26K cells under NX (Fig. 7E) was comparable to that from their individual cultivation in line with the non-significant difference in their metabolic activity (Fig. 5E); however, it was partially fragmented under HX. The significant decrease in the metabolic activity (Fig. 5E) suggested that the mitochondria were less functional than for individual cultivation under the same conditions.

Fig. 7

Impact of co-cultivation and hypoxia on feeder cells’ mitochondrial status. Changes in the mitochondrial network of feeder cells, indicated by staining with MitoTracker CMX/Ros dye, which accumulation is based on mitochondrial membrane potential under NX and HX conditions (the scale bar in the images represents 10 μm). Mitochondrial networks in individually cultivated NHDF (A) and hMSCs (B); NHDF (C) and hMSCs (D) co-cultivated with SD-1, and NHDF (E) and hMSCs (F) co-cultivated with UPF26K cells

In contrast, the hMSCs co-cultivated with SD-1 under NX (Fig. 7D) exhibited a more scattered mitochondrial network across the entire cell body than for individual cultivation. The significant change in the mitochondrial status was also supported by the determination of the metabolic activity (Fig. 5E). The mitochondrial network was even more fragmented and concentrated perinuclearly under HX. The hMSCs co-cultivated with UPF26K under NX (Fig. 7F) exhibited a slightly scattered mitochondrial network; however, compared to the hMSCs co-cultivated with SD-1 (Fig. 7D), the network was more similar to that for individual cultivation (Fig. 7B). Again, the mitochondria were more fragmented and densely localized around the cell nucleus under HX than for the individually cultivated hMSCs; in this case the metabolic activity was strongly reduced (Fig. 5E).

Gene expression profiles of the leukemic and feeder cells after co-cultivation under NX/HX

The gene expressions were examined of the selected metabolic-related genes in all the tested cells after co-cultivation at various oxygen levels. Glycolysis was represented by GLUT1 (glucose transporter), OXPHOS by SDHA (a mitochondrial complex II component), HX by HIF1α (master regulator of the cellular response to HX) and cell-cell interaction by PGE (which mediates interactions between different cell types).

In SD-1 cells, the expression of the glucose transporter GLUT1 was notably reduced after co-cultivation with both the NHDF and hMSCs (Fig. 8A). The expression of SDHA remained relatively stable across the various conditions, except for a significant decrease in the SD-1 cells co-cultivated with hMSCs under NX. HIF1α evinced a significant increase in SD-1 cells when co-cultivated with both feeder types; this was more pronounced under HX. In addition, the PGE expression was markedly upregulated in the SD-1 cells co-cultivated with NHDF. However, no change was detected in the PGE expression after co-cultivation with the hMSCs.

Fig. 8

Gene expression responses to co-cultivation and hypoxia of leukemic cells and feeders. The graphs show the gene expression profiles of leukemic cells SD-1 and UPF26K (A) and feeder cells NHDF and hMSCs (B), measured via SYBR Green based qRT-PCR, after 48 h of co-cultivation under NX and HX conditions. The bar graphs specifically illustrate the expression levels of selected genes associated with metabolism and HX. For all the graphs, expression data have been normalized to the expression levels observed under NX conditions in individual cultivation, which are designated as a baseline value of 1. GAPDH and Ubiquitin C were employed as reference genes for mRNA normalization. Data are shown as bar graph with the SD. Statistical analyses were performed using a nonparametric, two-tailed Mann–Whitney U test for two-sample comparisons. Statistical significance (p ≤ 0.05) is indicated in the graphs using symbols that denote significant differences between the following comparisons: * = all co-cultivation samples and HX individual samples vs. NX individual cultivation control, # = HX co-cultivation samples vs. HX individual cultivation control, + = different co-cultivation counterparts within the same oxygen level (e.g. SD-1 from NX co-cultivation with NHDF vs. SD-1 from NX co-cultivation with hMSCs)

.

For UPF26K cells, an increase in the GLUT1 expression was observed only after co-cultivation with both feeders under HX. Surprisingly, an increase in the GLUT1 expression was also detected in the UPF26K cells co-cultivated with hMSCs under NX. HIF1α exhibited a considerable increased expression in the UPF26K cells co-cultivated with NHDF under both NX and HX, but only under HX for the hMSCs. SDHA and PGE maintained stable expressions with no significant changes across all the conditions.

The NHDF feeders exhibited a decrease in the GLUT1 gene expression after co-cultivation with both the SD-1 and UPF26K leukemic cells under both conditions, as well as for their individual cultivation (Fig. 8B). The SDHA expression significantly increased after co-cultivation with the SD-1 cells but was notably reduced with the UPF26K cells under both oxygen conditions. Surprisingly, the HIF1α expression was significantly downregulated in the NHDF cells under HX; moreover, also under NX following co-cultivation with both leukemic cell types. In addition, the expression of PGE in the NHDF cells evinced a slight increase only after co-cultivation with the SD-1 cells, but a decrease (not statistically significant) with the UPF26K cells.

The hMSCs reacted to HX via the increased expression of GLUT1, SDHA and HIF1α; however, co-cultivation changed the situation significantly. The SD-1 cells increased the expression of SDHA but reduced the expression of GLUT1 and HIF1α under HX, while the UPF26K cells reduced the expression of all these genes. The PGE gene expression remained relatively stable and was in line with the control levels of the individual cells.

The results of the influence of co-cultivation and HX on the leukemic and feeder cells are shown in Table 1.

Table 1 Influence of the co-cultivation and hypoxia on the leukemic and the feeder cells

Discussion

Leukemia cell proliferation and metabolic activity in the individual cultivation

The consistent proliferation was determined for the commercial SD-1 and the patient-derived UPF26K cells under the various short-term (2-day) cultivation conditions (different media and oxygen levels). The comparable proliferation in the two tested media suggests that mix media are suitable for co-cultivation experiments. Even though short-term cultivation under HX did not impact cell proliferation, prolonged exposure to HX (7-day preincubation) retarded their proliferation, thus indicating the growth inhibition influence of HX, as addressed in Petit et al. [64], who proposed that ALL cells are resilient to short-term cultivation condition changes but sensitive to prolonged HX. Moreover, comparable results were observed using hematopoietic stem cells (HSCs) [23, 75], which are progenitors of ALL cells situated in bone marrow niches. Interestingly, the proliferation of the UPF26K cells slowed down only from 28 to 38 h compared to slowing down from 25 to 70 h for the SD-1 cells. This is in line with a proposed hypothesis regarding the inherent heterogeneity within leukemic cells of the same diagnosis [14, 45]. This observation, while preliminary, suggests a unique adaptation of these cells to environmental changes, aligning with findings by Notta et al. [59] on intra-tumor heterogeneity in leukemia. The patient-specific factors influencing leukemia cell behavior observed in our study complement also the work by Frismantas et al. [25] who demonstrated variable responses in patient-derived ALL samples. Further studies using a broader range of cell lines are warranted to substantiate these findings.

The comparable metabolic activity of the SD-1 cells in both the tested media suggests that mix media can be used for co-cultivation experiments. However, HX initially enhances the metabolic activity of these cells, which then declines to comparable levels under both conditions (Fig. 2A), thus indicating metabolic adjustment after longer-time exposure to HX [35, 56]. The metabolic activity was measured via a commercial test (MTS [20], which is generally used for the detection of mitochondrial activity. However, Xiao et al. [84] noted that the detected activity of dehydrogenases occurs not only in mitochondria but also in cytoplasm, thus also supporting other cellular functions (glycolysis, biosynthesis, detoxification); therefore, the interpretation of our results is ambiguous. Comparable metabolisms regardless of the oxygen levels were confirmed for the SD-1 cells via the extracellular flux analysis, which suggested that SD-1 cells rely more on glycolysis than OXPHOS over time (Fig. 2C), as supported by Suganuma et al. and Vander Heiden et al. [74, 77].

Over the short-term, the UPF26K cells exhibited comparable metabolic activities in both media; however, the increased metabolic (mitochondrial) activity under HX was supported by the results of two distinct methods (MTS (Fig. 2B) and SeaHorse (Fig. 2C)). Compared to the SD-1 cells, where this increase was only transient and at the outset, it persisted over a longer time for the UPF26K cells, which demonstrated distinct metabolic responses to the oxygen levels. This finding is particularly noteworthy as it differs from the typical Warburg phenotype observed in many cancer cells, however, aligns with recent studies by Lagadinou et al. and Kuntz et al. [41, 42] suggesting that some leukemic cells maintain or upregulate oxidative metabolism as a survival mechanism. Increased OXPHOS potentially indicates enhanced malignancy or a shift to a more energy-efficient state [2, 27] moreover, it supports efficient ATP production and aids immune evasion and treatment resistance [89]. However, the long-term incubation of UPF26K under HX (7 days) induced the induction of glycolysis (12% after 2 days vs. 35% after 7 days) (Fig. 2C).

The results suggest that SD-1 cells remain unchanged under changing oxygen conditions due to reliance on glycolysis under all circumstances, whereas UPF26K cells evince a more dynamic metabolic response to HX due to the primary OXPHOS-dependent metabolism, thus highlighting their unique metabolic profiles and potential therapeutic relevance. Different oxygen levels also exert impacts on cell organization, the reason for which is unknown.

Feeder cell proliferation and metabolic activity in the individual cultivation

The minimal differences in the NHDF proliferation following short-term cultivation in the various media and the oxygen levels are shown in Fig. 3. However, long-term HX significantly impaired colony formation and quality (Fig. 3B) by altering the multifunctional proteins, thereby impacting the intercellular communication in healthy and disease fibroblasts [6]. Similarly, the hMSCs were unaffected by the type of the cultivation medium (Fig. 3A) under NX. However, short-term HX increased their proliferation, while long-term HX significantly reduced their colony-forming ability (Fig. 3B) without altering their quality (Fig. 3C). This indicates a time-dependent response to oxygen deprivation that involves initial adaptation followed by growth limitations, similar to observations by Boyette et al. [8] and Holzwarth et al. [34]. Previous studies [28, 29, 69] showed that HX enhances hMSCs expansion and preserves differentiation, thus emphasizing the crucial role of oxygen in stem cell behavior and tissue development. These findings suggest that oxygen levels potentially impact the roles of NHDF and hMSCs in their further co-cultivation with leukemia cells.

The metabolic responses of NHDF and hMSCs to the various cultivation conditions were distinct. Despite the absence of the impact of the medium type, the NHDF cells exhibited a decrease in the metabolic (mitochondrial) activity as detected by the MTS assay over time (Fig. 4A) and shifted towards glycolysis, which they maintained even after 7 days of HX exposure (Fig. 4C). Similarly increased glycolysis under HX in fibroblasts was reported byJena et al. [37] and Mordhorst et al. [52].

Conversely, the hMSCs exhibited a continuous increase in their metabolic (mitochondrial) activity, particularly under HX (Fig. 4B). In a similar way to NHDF, the extracellular flux analysis (Fig. 4C) detected a switch in the hMSCs from OXPHOS under NX to glycolysis under HX. Both principally distinct methods suggested a simultaneous increase in the mitochondrial activity and glycolysis in the hMSCs under HX, which highlights their metabolic flexibility and adaptation ability, as observed by Buizer et al. and Grayson et al. [10, 28].

The distinct metabolic responses between NHDF and hMSCs may arise from their differing functions and adaptability. The NHDF cells shifted their ATP production predominantly to glycolysis under HX. In contrast, the hMSCs produced ATP mostly via glycolysis, as presented in Nuschke et al. [60], while simultaneously maintaining active mitochondria, thus indicating their regenerative properties and plasticity to oxygen fluctuations.

Impact of co-cultivation on the behavior of all the counterparts

The interaction between the leukemia (SD-1 and UPF26K) and feeder cells (NHDF and hMSCs) under varying oxygen conditions reveals distinct patterns. The experimental results indicated the positive impact of co-cultivation with the feeders on the behavior of the commercial SD-1 cell line, with the NHDF inducing a more pronounced impact than the hMSCs. The co-cultivation of SD-1 cells with NHDF led to increased SD-1 proliferation while lowering their mitochondrial activities under NX and HX (Fig. 5A). However, a decrease in the 2-DG uptake in the SD-1 cells was observed under NX, while the uptake was enhanced under HX (Fig. 5C), thus suggesting a shift towards a glycolytic metabolism and adaptation to HX stress. The impact of the HX microenvironment, in which leukemic cells prioritize glycolysis, has been reported by Al Tameemi et al. [2] and Huang et al. [90]. The adaptation of SD-1 cells to low oxygen conditions was further indicated by the enhanced HIF1α gene expression (Fig. 8), even for individual cultivation under HX. Typically, HIF1α is not expressed under NX conditions; however, we observed its expression under NX for both leukemic cell types when co-cultivated with NHDF cells. This may be due to the ability of NHDF to create local HX microenvironment conditions (tight interaction between cells) and to secrete factors that stabilize HIF1α, thereby enhancing its expression even under NX conditions [18]. In addition, strong cell-to-cell interaction was apparent between SD-1 and the NHDF feeders regardless of the oxygen level (Fig. 6A) supported by an increase in the expression of the gene responsible for cell interactions (PGE) under NX and, to a greater extent, HX. This indicated enhanced adaptation to HX due to the soluble factors released from the feeders and the direct interaction with them. It is known that NHDFs support cancer cell growth and differentiation via both paracrine signals and extracellular matrix remodeling [69, 73].

Similarly, co-cultivation with hMSCs feeders promoted SD-1 cell proliferation and a metabolic shift to glycolysis, though less than with the NHDFs. Visible cell-to-cell interactions between SD-1 and hMSCs were observed solely under NX (Fig. 6. B). This was not supported by elevated PGE expression levels; however, it was possibly responsible for the slight increase in the SD-1 cell number. Hence, hMSCs, known for their immunomodulatory and regenerative properties [5, 21], only subtly impact the leukemic cell metabolism as compared to NHDFs, as shown in Fig. 5B. Ahani-Nahayati et al. [1] reported that hMSCs do not significantly enhance the proliferation of ALL cells after short-term co-cultivation (up to 1 day) as do fibroblasts, whereas others observed the improved proliferation of ALL cells in co-cultivation with hMSCs after 7 days [48, 72]. Rather than enhancing proliferation, hMSCs are able to create a chemo-resistant niche [32, 80], promote metabolic shifts towards glycolysis [26, 54] and facilitate immune evasion [43, 78]. These interactions revealed the role of hMSCs in terms of enhancing leukemic cell survival and significantly modulating leukemic cell behavior and treatment responses beyond merely supporting proliferation [3, 48]. It is possible that the influence of hMSCs on other cells is not via direct contact, as with NHDFs as suggested by the presented results, but rather through paracrine mechanisms.

Overall, after 2 days of co-cultivation, the NHDF feeders exerted a more pronounced impact on the SD-1 cells by enhancing their proliferation, glycolytic activity and strong direct intercellular communication than on the hMSCs. In contrast, the hMSCs exerted a modest impact, primarily by slightly increasing the cell number and promoting glycolysis under HX only. This is likely due to hMSCs cells requiring more time to establish their impact on SD-1 cells since their immunomodulatory interactions develop gradually over an extended period. As widely reported in the literature [44, 66], hMSCs possess a dual nature– they both suppress and support tumor growth. This duality highlights the complex and multifaceted role of hMSCs in tumor biology, thus rendering their overall impact difficult to predict. Moreover, HX drives SD-1 cells to adapt by enhancing the 2-DG uptake and shift to glycolysis, thereby allowing them to survive and to maintain energy production under low oxygen conditions.

The UPF26K cells evinced a different reaction to co-cultivation. Their cell number was strongly reduced after co-cultivation with both feeder types (Fig. 5A); however, they exhibited increased metabolic (mitochondrial) activity and 2-DG uptake after co-cultivation with NHDF under both oxygen conditions after 2 d (Fig. 5B, C). This suggests enhanced glycolysis and metabolic adaptability, as described by Boraldi et al. and Wierenga et al. [6, 82]. In contrast, their co-cultivation with hMSCs did not elicit a metabolic response, which may indicate a shift towards the quiescent state. The weak binding of UPF26K cells to the feeders (Fig. 6C, D) and the unchanged PGE expression suggest no direct interaction of these cells with the feeders (as demonstrated for the SD-1 cells); thus, they can only react to paracrine stimuli. The observed increase in the 2-DG uptake and the high GLUT1 and HIF1α expressions by the UPF26K cells under both NX and HX conditions may indicate a response to stress or a need for increased energy to ensure survival and growth, which is not associated with the oxygen level but with the presence of feeders. These observations are in line with findings that feeder cells enhance glycolytic activity and HX adaptation in leukemic cells through various growth factors and cytokines [26, 91]. Furthermore, the heterogeneous response of patients with leukemia to the same therapy (e.g. multidrug resistance) described for AML and ALL patients [30, 92], indicates the diverse behavior of leukemic cells and varied responses to therapy for the same diagnosis.

Despite both feeders inhibiting UPF26K growth, the NHDF appear to stimulate the overall metabolic activity of UPF26K cells, whereas hMSCs tend to induce the quiescent state. It also seems that HX plays a role in modulating these interactions by mitigating the negative effects of co-cultivation on the proliferation of UPF26K cells. These findings highlight the complexity of cell-to-cell interactions and the crucial role of the microenvironment.

As also reported in previous studies, co-cultivation affects both leukemic cells and feeders, thus indicating that both cell types contribute to changes in the niche environment (stromal, tumor) [16, 70].

Our results indicate the differing impacts of the selected leukemic cell types on the feeders. The number of NHDF cells was markedly reduced after co-cultivation with the SD-1 cells as was their 2-DG uptake; however, their metabolic (mitochondrial) activity increased under both oxygen conditions (Fig. 5D, E, F). This suggests that interaction with the SD-1 cells induces a metabolic shift in the NHDF cells, which enhances their mitochondrial activity as was also evident from the upregulated SDHA expression and, further, by their denser mitochondrial network with elongated mitochondria under NX conditions (Fig. 7A). These morphological changes correspond to a shift in the metabolic activity, as shown by Liu & Hajnóczky and McCarron et al. [46, 50], This observation is also in line with Katsuno et al. and Nara et al. [39, 57], who reported the reduced CFU of fibroblasts in patients with ALL/AML, which indicated decreased fibroblast proliferation when in the presence of leukemic cells. Moreover, HX conditions enhance the impact of SD-1 cells on NHDF feeders. SD-1 cells significantly lower NHDF proliferation under HX when compared to NX and, most likely, induce metabolic stress by interfering with NHDF receptor signaling, as evidenced by strong cell-to-cell interactions (Fig. 6A) and an increase in the PGE gene expression in SD-1 cells (Fig. 8). In addition, the significant downregulation of the HIF1α expression was observed, which suggests that SD-1 cells may block HX sensing in NHDF cells, thus diminishing their response to HX. This complex adaptive response highlights the aggressive metabolic alterations imposed by SD-1 cells and the limited effectiveness of NHDF cellular adaptations under co-cultivation and HX conditions, which may be compounded by competition for glucose, where the leukemic cell line is successful, which increases its uptake and thus boosts its own proliferation (Fig. 5).

The co-cultivation of NHDF cells with UPF26K impacted them differently to the SD-1 cells. UPF26K cells caused an increase in NHDF proliferation and 2-DG uptake (Fig. 5D, F) while simultaneously causing a decrease in their mitochondrial metabolic activity (Fig. 5E). This may have been due to the release of specific extracellular vesicles from the leukemic cells that enhance glycolytic activity and reduce mitochondrial respiration in fibroblasts, thereby supporting leukemia progression as recently reported [13, 61]. Moreover, the positive impact of the UPF26K cells on the proliferation of NHDF feeders was further enhanced under HX conditions, despite their metabolic (mitochondrial) suppression also being apparent via the fragmented mitochondrial network (Fig. 7C). However, the UPF26K cells caused an increase in the uptake of 2-DG in the NHDF cells (Fig. 5F), which may help fibroblasts survive under low oxygen conditions by modulating the protein expression or other cellular parameters [6, 79]. Interestingly, UPF26K cells do not increase their own proliferation or metabolism, rather the opposite, which may be due to their reliance on maintaining a controlled, stable metabolic state to ensure their long-term survival and dominance in the environment. Such manipulation allows UPF26K cells to create a favorable niche without overexerting their own metabolic resources.

Overall, the SD-1 cells exerted an aggressive and stress-inducing impact on the NHDF feeders; they decreased their cell numbers and 2-DG uptake while increasing their mitochondrial activity, thus leading to significant stress responses. Conversely, the UPF26K cells induced NHDF proliferation and 2-DG uptake but reduced their mitochondrial metabolic activity. These findings highlight the distinct strategies of leukemic cells in terms of altering the metabolic environment and the crucial role of HX in these interactions.

The hMSCs exhibited a weaker and differing response to co-cultivation with the leukemic cells than the NHDF cells. No change in hMSCs proliferation occurred during co-cultivation with the SD-1 cells under NX; however, a reduction in their mitochondrial activity and 2-DG uptake and lower GLUT expression suggests some degree of interference with their metabolism. Moreover, the increased mitochondrial fragmentation indicates increased cellular stress and a compromised mitochondrial function. Interestingly, the hMSCs evinced higher cell numbers when co-cultivated with SD-1 cells under HX than for individual cultivation (Fig. 5D). This increase, despite the decreased mitochondrial activity and 2-DG uptake and fragmented mitochondrial network, indicated a stress response focused on survival, suggesting that HX triggers compensatory mechanisms in hMSCs for them to adapt to low oxygen levels, surprisingly without an elevated HIF1α expression (Fig. 8) as observed in individual cultivations under and indicating adaptation to, HX [36]. It has been shown that leukemia cells are able to modify hMSCs by upregulating pro-inflammatory cytokines and metabolic pathways, thereby affecting their proliferation and metabolic activity [15, 28]. The increased proliferation of hMSCs under HX, despite the metabolic suppression, is in line with these findings and suggests that hMSCs may activate alternative pathways for survival and proliferation in response to the stress induced by leukemic cells.

Co-cultivation with the UPF26K cells stimulated hMSCs proliferation more than did the SD-1 cells, but only under HX, where the mitochondrial activity was markedly reduced and the mitochondria highly fragmented (Fig. 7D), but with an increased 2-DG uptake, thus indicating a shift to glycolysis so as to meet energy demands and support cell growth. The glycolytic switch of hMSCs may reduce glycolytic activity in leukemic cells, thus affecting their proliferation and survival [38]. This metabolic support is vital for certain ALL samples, which illustrates a selective dependency on the stromal metabolism [7]. These findings highlight the dynamic relationship between mitochondrial morphology, metabolic activity and the co-cultivation conditions for feeder cells. Leukemic cells are able to induce mitochondrial fragmentation and dysfunction in feeders and facilitate mitochondrial transfer, which suggests alterations in the cellular metabolism and bioenergetics, as described previously [31, 53].

In summary, the HX environment acts to change the dynamics of hMSCs due most likely to compensatory mechanisms that adapt to low oxygen levels [22]. Increased hMSCs proliferation under HX, despite the overall metabolic suppression by SD-1 cells, suggests an adaptive response that strengthens other metabolic pathways for survival purposes. In contrast, UPF26K cells under HX boost hMSCs proliferation and 2-DG uptake, which indicates a shift to glycolysis and a reduction in mitochondrial respiration. While co-cultivation alone does not significantly impact hMSCs, HX enhances their proliferation with a strong dependency on the type of co-cultivated cells. This points to distinct metabolic responses that are based on the leukemia cell type, thus suggesting variations in terms of nutrient utilization or signaling. The metabolic reprogramming of hMSCs to support leukemic cell survival has significant implications for targeted therapies [55].

Conclusion

The co-cultivation of commercial SD-1 cells with feeders significantly enhanced SD-1 cell proliferation and glycolytic activity. Although intensive contact between the leukemic SD-1 cells and the feeders, particularly NHDF, promoted leukemic cell viability, it exerted an inhibitory impact on the growth and metabolism of the feeders. In contrast, the co-cultivation of patient-derived UPF26K cells with the feeders inhibited UPF26K cell proliferation; however, the NHDF feeders induced an increase in glycolysis and the mitochondrial activity in the cells, whereas the hMSCs triggered the quiescent state. The negative effects of co-cultivation were, however, mitigated by HX. The impact of the leukemic cells on the feeders differed significantly. While the SD-1 cells reduced NHDF proliferation and glycolysis but increased their mitochondrial metabolism, the UPF26K cells stimulated NHDF proliferation and glycolysis but reduced their mitochondrial metabolism. HX further amplified the influence of the leukemic cells on the NHDF. The less pronounced effect of the leukemic cells on the hMSCs was detected. The hMSCs exhibited stable proliferation despite metabolic disruptions and adapted to HX by increasing their proliferation and shifting towards glycolysis, thus demonstrating the resilience and metabolic flexibility of hMSCs. To conclude, it appears that cells that switch to glycolysis during co-cultivation profit most from the conditions (co-cultivation and, particularly, the oxygen level).

The leukemic cells differed significantly in terms of their metabolic profiles, which influenced their responses to co-cultivation with the feeders. HX conditions generally supported leukemic cell survival and adaptation by promoting a glycolytic shift and metabolic flexibility; however, the impact varied across the conditions and cell types.

The distinct metabolic responses of the leukemic cells and the feeders underscore the importance of targeting metabolic and microenvironmental interactions in leukemia therapy. Understanding the role of HX in modulating cell interactions and metabolism has the potential to inform strategies that enable the manipulation of oxygen levels in therapeutic settings, thus improving patient outcomes.

The observed heterogeneity in the leukemic cell responses to co-cultivation highlights the need for personalized treatment approaches based on specific metabolic and microenvironmental profiles. Optimizing these models has the potential to improve both in vitro work with primary leukemic cells and the efficacy of the use of PDX mouse models, thus providing valuable data and enhancing the predictive accuracy of treatment responses.

Materials and methods

Cell preservation conditions

Human dermal fibroblasts

(NHDF-Ad) (Lonza, Switzerland) were grown in phenol red-free DMEM medium (Gibco™, USA) with 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (iFBS, Gibco™, USA) and antibiotics (penicillin 10,000 U/mL, streptomycin 10 µg/mL, Sigma-Aldrich, USA) under NX at 37 °C in 5% CO₂.

Human mesenchymal stem cells

(hMSCs) from healthy bone marrow donors (passages 1 to 5) obtained following ethical committee approval (General University Hospital, Prague) (signature of informed consent and following the Declaration of Helsinki). The phenotypic characterization of these cells has been previously described by Pytlík et al. [66]. hMSCs were cultivated in αMEM medium (Life Technologies, USA) with 10% iFBS and antibiotics under NX at 37 °C in 5% CO₂.

Adult B acute lymphoblastic leukemia

(B-ALL) SD-1 cell line (DSMZ, Germany), derived from adult female peripheral blood with Philadelphia chromosome positive ALL cells and immortalized with Epstein-Barr virus, were cultivated in phenol red-free IMDM medium (Gibco™, USA) with 10% iFBS and antibiotics under NX at 37 °C in 5% CO₂.

Adult B acute lymphoblastic leukemia

(B-ALL) UPF26K (a rare cell line created from patient cells transferred to long-term cultivation) obtained from the Institute of Pathological Physiology following protocols for generating patient-derived cell lines models as described by Dolniková et al. [19]. Cells were initially cultivated in OCI medium with 20% Human Plasma and Heparin, then transferred to phenol red-free IMDM medium (Gibco™, USA) with 10% iFBS and antibiotics maintained under NX at 37 °C in 5% CO₂.

Hypoxic conditions (HX)

The experiments were conducted in an HX chamber Coy O2 control glove box (Accela, Czech Republic) with the precise control of the oxygen environment (settings: 1% O2 and 5% CO2) throughout the experiments. The experiments were performed simultaneously under NX, 20% O2 with 5% CO2 and HX, 1% O2, with 5% CO2 conditions for direct comparison purposes, following the hypoxia protocol described by Daumova et al. [17].

Co-cultivation conditions

Adherent feeder cells were pre-seeded in 24-well tissue culture polystyrene (TCPS) plates (TPP, Switzerland), NHDF at 1 × 10⁴ cells/cm² and hMSCs at 8 × 10³ cells/cm² in a media mix (αMEM + IMDM 1:1) with 10% iFBS and antibiotics for 24 h under NX and HX. Suspension leukemic cells (SD-1 or UPF26K) were then added (to NHDF at 5 × 10⁴ cells/cm² and to hMSCs at 4 × 10⁴ cells/cm²). The ratio of seeded cells was always maintained at 1:5 (feeders: leukemic cells). The cells were separated and processed after 48 h of co-cultivation. The well contents were collected and washed thoroughly and the supernatants from each washing cycle were collected in a single tube. The bond strength and cell distribution were assessed, and images were captured via light microscopy after washing. The feeders were then trypsinized and both cell types were diluted in PBS and analyzed via flow cytometry. The cells designated for RNA isolation were snap-frozen and stored at − 80 °C for subsequent analysis.

Proliferation assay

The feeders were seeded in 96-well TCPS plates at 1 × 10⁴ cells/cm² and cultivated for 24–72 h. Cell proliferation was measured via the fluorometric quantification of DNA using the CyQUANT Proliferation Assay Kit (ThermoFisher Scientific, USA), following the manufacturer’s instructions. The fluorescence intensity (~ 485 nm Ex/~530 nm Em) was recorded using a Spark^® multimode microplate reader (Tecan, Switzerland) for the statistical analysis. The leukemic cell proliferation was measured via flow cytometry.

Metabolic activity measurement (MTS)

The feeders were seeded in 96-well (individual cultivation) or 24-well TCPS plates (co-cultivation) at 1 × 10⁴ cells/cm² and the leukemic cells at 5 × 10⁴ cells/ cm² for 24–72 h. The metabolic activity was assessed using MTS assay (Cell Titer 96 AQueous One Solution Cell Proliferation Assay, Promega, USA) according to the standard instructions. The absorbance (490 nm with a 655 nm reference) of the colored formazan produced by the viable cells’ dehydrogenases of the viable cells was measured using a microplate reader (Synergy™ 2, BioTek, USA). The results were normalized to the untreated control cells in their respective media. Concerning normalization, the metabolic activity of the feeders was compared with their proliferation rate (provided by the CyQUANT proliferation assays); the leukemic cell metabolic activity was normalized to the cell counts (flow cytometry). Both cell types were normalized to the flow cytometry cell count results in the co-cultivation experiments.

Population doubling time (DT)

The population doubling time was determined for the feeders at 1 × 10⁴ cells/cm² and the leukemic cells at 2.5 × 10⁴ cells/cm² seeded in 6-well or 12-well plates in their respective media. The cells were cultivated for 4, 24 and 48 h, harvested and counted using a Bürker chamber. The DT was calculated using the http://www.doubling-time.com/compute.php online tool.

Colony-forming assay (CFU-F)

The feeders were seeded at a low density of 1.5 cells/cm² on Petri dishes (TPP, Switzerland) in their respective media. The new colonies derived from the individual cells were counted after 15 days of incubation then fixed and stained with 1% crystal violet in methanol. Colonies with a diameter of at least 2 mm were counted as CFU according to the formula: (counted CFU-F / cells originally seeded) × 100, as adapted from the protocol described by [77].

Light microscopy

Light microscopy images of the cells were obtained using an Eclipse Ti- S microscope (Nikon, Japan) and a DS-Qi1Mc digital camera (Nikon). Images were acquired with a 4 or 10 × lens and adjusted using ImageJ software (USA, http://imagej.nih.gov/ij/).

Fluorescence staining of the cells

The cells were seeded at a density of 1 × 10⁴ cells/cm² on Cell Imaging Coverglass (Eppendorf, Germany) and incubated for72 h. They were then washed with fresh medium and stained with 10 nM MitoTracker RED CMX/Ros (ThermoFisher, USA) at 37 °C for 30 min. After washing with PBS, they were fixed with cold methanol at room temperature (RT) for 3 min and permeabilized by 0.1% Triton X-100 in PBS (Sigma-Aldrich, USA) at RT for 20 min. The cell nuclei were then stained with DAPI (4′, 6-diamidino-2-phenylindole, Sigma-Aldrich, USA) in PBS at 37 °C for 15 min. The cells were then washed 2x with PBS, left to air dry and mounted by Shandon™ Immu-Mount™ (ThermoFisher, USA), followed by fluorescent microscopy.

Glucose uptake assay

The feeders were seeded in 96-well (individual cultivation) or 24-well TCPS plates (co-cultivation) at 1 × 10⁴ cells/cm² and the leukemic cells at 5 × 10⁴ cells/cm² for 24–72 h. Following incubation, the cells were washed in PBS then resuspended in 100 µl Seahorse XF Base Medium containing 10 µM fluorescent derivative 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-DG) (ThermoFisher Scientific, USA) and incubated for 30 min at 37 °C. The cells were subsequently washed with cold PBS (300 g/4°C/5 min) and stored in ice. The 2-DG fluorescence was analyzed immediately using the FITC channel on a FACS Canto II flow cytometer. The median fluorescence intensity (MFI) of the unstained cell samples was used as a control for comparison with the 2-DG-stained samples.

RNA extraction and RT–PCR

The total RNA from each type of co-cultivated cells (72 h) was isolated using the RNeasy Plus Micro Kit (QIAGEN GmbH, Germany) according to the manufacturer’s recommendations. The RNA quality and quantity were assessed using a NanoDrop spectrophotometer (ND-1000, ThermoFisher Scientific, USA). 100–200 ng of isolated RNA was used for cDNA synthesis with SuperScript III Reverse Transcriptase (ThermoFisher Scientific, USA) following the manufacturer’s instructions.

Gene expression analysis (qRT-PCR)

The quantitative PCR was performed using a QuantStudio™ 7 Pro Real-Time PCR System (Applied Biosystems™, USA) and Biotool™ 2xSYBR Green qPCR Master Mix (BioTools, Jupiter, United States). Diluted cDNA was used as a template and the template-free controls were included in each qPCR reaction. Each sample was tested in triplicate via at least two independent experiments. The threshold cycle (CT) values were averaged, and the relative gene expression was calculated using the 2ex (min cp-cp) method; the gene expression was then normalized to the geometrical mean of the housekeeping genes (HPRT, Ubiquitin C) and presented as the mean of the gene. Design & Analysis Software (ThermoFisher Scientific, USA) was used to identify and remove the outlier replicate of the CT value. The primers used are listed in Table 2. The gene expression in the co-cultivated samples was compared to the control samples from the individual-cell type cultivation.

Table 2 List of primers and their sources used for gene expression analysis

Flow cytometry

Flow cytometry was employed for both the cell counting and staining with 2-DG. The analysis was performed using a FACS Canto II flow cytometer (BD Biosciences, USA) equipped with 405 nm and 488 nm lasers with data acquisition via BD FACSDiva software version 6.1.3. The debris and dead cells were excluded applying FSC-A/SSC-A gating and the cell doublets were identified using FSC-A/FSC-H gating. The samples in 96-well TCPS plates were diluted in PBS for analysis. The flow cytometry data was analyzed using the latest version of FlowJo software (BD Biosciences).

Metabolic analysis applying extracellular flux analysis (Seahorse analysis)

The Real-time ATP rate assay was performed using a Seahorse XFp Analyzer (Agilent, USA). The NHDF (1 × 10⁴ cells/well) and hMSCs (8 × 10³ cells/well) were seeded directly onto Seahorse XFp Cell Culture Miniplates (Agilent, USA) and cultivated for 2 days under HX and NX conditions in their respective media. The cells were pre-seeded in 6-well plate under HX conditions for long-term cultivation (7d HX) purposes. The day before the assay, the cells were harvested with 0.1% trypsin and transferred to Seahorse XFp Cell Culture Miniplates under the respective oxygen conditions. Immediately before the assay, the adherent cell medium was replaced with Seahorse XF DMEM Base Medium (Agilent, USA) containing 1 mM pyruvate (Agilent, USA), 2 mM glutamine (Gibco™, USA) and 10 mM of glucose (Agilent, USA). The cells were cultivated at 37 °C in a non-CO₂ incubator for the time specified in the manufacturer’s instructions. The SD-1 suspension cells and UPF26K cells (5 × 10⁴ cells/well) were seeded directly onto XFp Cell Culture Miniplates pre-coated with Poly-L-lysine (Sigma-Aldrich, USA) after varying cultivation periods (NX, 2d HX, 7d HX) in Seahorse XF RPMI Base Medium (Agilent, USA) with the same supplements. The cells were stained with CyQUANT™ NF Cell Proliferation Assay after the Seahorse analysis for data normalization purposes. The data was statistically evaluated using Wave 2.6.3.5 and Seahorse XF Cell Test Report software (Agilent Technologies, USA).

Statistical analysis

Biological data was obtained from at least two independent experiments with two to four biological replicates. The statistical analysis was conducted using Statistica software (StatSoft CR, s.r.o.) and GraphPad Prism (GraphPad Software, USA).

In our figures, data are presented using boxplots, bar graphs or stacked bar graphs, each accompanied by individual data points where applicable. For boxplots (box and whiskers plots), the box indicates the interquartile range (IQR), the horizontal line marks the median, and the whiskers represent minimum and maximum values; individual data points are also shown. The numbers above the boxplots indicate the mean for each group. For bar graphs (scatter dot plots), the dots show individual values, the horizontal line depicts the mean, and the error bars represent the standard deviation (SD). The numbers above the bars indicate the mean for each group. For stacked bar graphs, the bar height corresponds to the mean value, with error bars indicating the SD. Numbers within each segment denote the mean percentage contribution of that component. Specific details for each figure can be found in the corresponding figure legend. To assess the normality of our datasets, we generated Quantile-Quantile (QQ) plots for each group. Homoscedasticity was evaluated using the Bartlett and Brown–Forsythe tests. Since the data violated either normality or equal variance assumptions, we employed the non-parametric, two-tailed Mann–Whitney U test for all two-sample comparisons (without correction). This approach ensured that our statistical analysis remained robust despite the non-normal distribution of our data. Each statistical comparison reported in the manuscript was conducted using this method. Statistical significance was accepted at p ≤ 0.05. The effect sizes between the two groups were measured using Glass’s Delta, with small (d = 0.20), medium (d = 0.50) and large (d = 0.80) effect sizes according to Cohen’s standards (1992).