Tumor cell behaviour modulation by mesenchymal stromal cells

In order to determine, whether AT-MSC exhibit tumor supportive or inhibitory effect on melanoma cells, we first admixed AT-MSC to melanoma cell doses with 100% tumor penetrance. AT-MSC coimplanted with M4Beu melanoma cells significantly decreased time to 100%-tumor onset in comparison to M4Beu alone. Average tumor burden was higher in M4Beu/AT-MSC (ratio 5:1) group in comparison to the control group (Fig. 1A and 1B). Similarly, A375/AT-MSC (5:1 ratio) injected group of animals also exhibited shortened time to tumor onset from 10 days in A375 alone-group to 3 days concomitantly exhibiting tendency to higher average tumor volume in AT-MSC coinjected groups (Fig. 1C and 1D).

Next, we aimed to determine whether AT-MSC could affect tumor incidence for limited amounts of tumor cells, which leave almost all animals long term tumor free (1 × 10⁵ A375). AT-MSC coinjection significantly increased tumor incidence and tumor growth in groups coinjected with 10:1 or 1:1 AT-MSC to A375 cell ratio (Fig. 2A). Moreover, soluble AT-MSC produced factors were sufficient to increase tumor incidence, if AT-MSC conditioned cell-free medium was used for the A375 cell resuspension, which indicated a role of paracrine factors in tumor-promoting action. Tumor volume as a measure of tumor burden within the treatment groups was proportional to the amount of coinjected AT-MSC and significantly higher in comparison to tumor burden in control group due to earlier latency abrogation (Fig. 2B-C). Thus we conclude that AT-MSC could abrogate tumor latency for as low as 100,000 melanoma A375 cells, which would not produce tumors if injected alone in immunocompromised host.

Coculture experiments in vitro were designed to characterize the interaction between tumor cells A375 and AT-MSC to unravel mechanism responsible for the protumorigenic effect. In order to determine the effect on tumor cell proliferation, A375 cells stably expressing EGFP (EGFP-A375) were mixed with increasing amounts of AT-MSC or conditioned medium produced from corresponding amount of AT-MSC cells. Output fluorescence was proportional to the number of EGFP-A375 cells and the amount of admixed AT-MSC cells did not interfere with output fluorescence. Soluble factors supported EGFP-A375 proliferation even in serum-limiting culture conditions although to much lesser extent in comparison to directly cocultured cells (Fig. 3A). AT-MSC also protected tumor cells from serum-deprivation induced apoptosis (A375/AT-MSC ratio 10:1, Fig. 3B). Direct cocultures of melanoma cells with AT-MSC (ratio 10:1) did not exhibit change in effector caspase activation induced by cytotoxic drugs in standard serum concentrations (not shown). However, doxorubicin and cisplatin treatment under serum deprivation conditions resulted in AT-MSC-mediated significant decrease in effector caspase-3/7 activation consequently leading to decrease in proportion of apoptotic and dead cells (Fig. 3C and 3D). Our data suggest that AT-MSC may assist tumor cells to sustain cellular stress such as nutrient deprivation and/or cytotoxicity. Indeed, in the presence of AT-MSC there was a significant increase of A375 colony-forming ability even in the absence of cell-cell contact in vitro (Fig. 4A). No such effect was observed when AT-MSC were added to the cultures three days later, so AT-MSC seemed to initiate colony growth at early stage. Indirect cocultures of melanoma and AT-MSC cells enabled us to analyze the expression of growth factors and receptors that was previously implicated to play a role in AT-MSC/tumor cell interactions. Quantitative analysis unravelled increased CCL5 production from AT-MSC in response to melanoma cells (Fig. 4B). Sustained expression of several potential prosurvival and proangiogenic factors and their cognate receptors in AT-MSC and A375 was demonstrated even upon 3 day coculture (Fig. 4C).

Multiplex cytokine analysis was performed in order to quantitatively evaluate a paracrine signalling in tumor/AT-MSC cocultures. A wide plethora of cytokines and chemokines was detected to be secreted from both cell types. Combined coculture of these cells exhibited additive or slightly synergistic effects for most of them, nevertheless significantly increased secretion of G-CSF was detected, apparently as a response of melanoma cells, and increased VEGF production proportional to the AT-MSC number in cocultures (Fig. 5A, B). Taken together mutual crosstalk between melanoma and AT-MSC within the tumor microenvironment results in formation of proinflammatory and proangiogenic cellular milieu resulting in melanoma growth promotion in vivo. Next, in order to confirm relevance of the VEGF increased secretion in vivo we injected group of animals with mixtures of A375/AT-MSC (2:1 ratio) and treated them with neutralizing antibody against human VEGF (antiVEGF, Avastin). This treatment decreased tumor incidence in comparison to antiVEGF untreated A375/AT-MSC group to some extent and also resulted in lower average tumor burden confirming the role of VEGF in the AT-MSC mediated tumor growth support (Fig. 5C).

In order to evaluate whether AT-MSC-mediated tumor supporting effect is dependent on direct co-implantation and paracrine stimulation only, we decided to use different delivery route for AT-MSC - systemic intravenous administration frequently used in a clinical setting. AT-MSC intravenous administration concomitant with the implantation of low A375 melanoma dose s.c. (2 × 10⁵) lead to tumor growth in 7 out of 8 animals in contrast to no tumors growing without AT-MSC treatment (0/4, p = 0.00552, data not shown). Even half of the melanoma cell dose was sufficient to mediate tumor growth in 67% of AT-MSC i.v. treated animals in contrast to 12.5% A375 alone s.c. inoculations (Fig. 6A). Consistently with previously published findings [17], we were not able to detect substantial proportion of EGFP expressing AT-MSC in subcutaneous A375 xenografts post-systemic administration at experiment endpoint by flow-cytometric analysis of single-cell suspension (data not shown). This might be caused by transient and/or early AT-MSC homing at the tumor site, detection limit of the system due to the outnumbering by rapidly proliferating tumor cells and/or limited AT-MSC proliferation within the tumor xenotransplant. However, we searched for the potential key mediator(s) that could have affected early homing/incorporation of AT-MSC into xenotrasplant implantation site. Role of SDF-1α/CXCR4 axis in this process was recognized [9‐11]. We have confirmed sustained SDF-1α production from AT-MSC (1,756.5 pg ± 108.2 pg per 50,000 AT-MSC). Moreover, 28.5% of A375 cells isolated from A375 xenograft expressed CXCR4 on cell surface (Fig. 6B). We hypothesized that even though the substantial amount of AT-MSC could not be detected in tumors, they actually might have homed very early into the site of tumor growth and SDF-1α/CXCR4 axis could have been responsible for the homing. SDF-1α signaling can be blocked by a CXCR4 antagonist AMD3100 - small molecule inhibitor, which enables to unravel role of this axis in the protumorigenic effects observed in vivo. In an attempt to abrogate tumor supportive effect of systemic AT-MSC administration on A375 xenograft, animals injected with A375s.c./AT-MSC i.v. were treated with 1.25 mg/kg AMD3100 every other day. Average tumor volume was decreased in AMD3100 treated group in comparison to control, but tumor incidences remained unaffected (Fig. 6C). These data indicated that SDF-1α/CXCR4 axis contributed to AT-MSC mediated tumor growth support; however additional mechanism(s) might be responsible for tumor dormancy abrogation upon systemic injection of AT-MSC.

In order to determine whether AT-MSC have tumor supportive effect on different tumor cell type, we have performed coculture experiments in vitro with glioblastoma cells 8MGBA. 8MGBA cells stably expressing EGFP were mixed with increasing amounts of AT-MSC or conditioned medium produced from corresponding amount of AT-MSC cells. Output fluorescence was proportional to the number of EGFP-8MGBA cells and was not influenced by the amount of admixed AT-MSC cells. Soluble factors did not support EGFP-8MGBA proliferation and directly cocultured EGFP-8MGBA/AT-MSC cocultures exhibited proliferation inhibition at highest 8MGBA/AT-MSC proportions (Fig. 7A). Neither local coinjection nor systemic AT-MSC administration promoted glioblastoma xenograft growth in vivo. Implantation of high glioblastoma cell dose s.c. (10⁷ 8MGBA) concomitantly with systemic AT-MSC administration resulted in 37.5% tumor incidence by day 55 (3 out of 8), which represented significant tumor growth suppression (p = 0.0304). AT-MSC showed tendency to decrease tumor incidence upon admixing to low 8MGBA glioblastoma cells dose (Fig. 7B), even though expression analysis has shown similar expression pattern for the 8MGBA except for constitutive CXCR4 expression in comparison to melanoma cells A375 (Fig. 7C). Quantitative analysis has unravelled lower cMet receptor expression and significantly higher SDF-1α mRNA level in 8MGBA (Fig. 7C). Multiplex cytokine analysis was performed in order to quantitatively evaluate a paracrine signalling in tumor/AT-MSC cocultures. Combined coculture of 8MGBA/AT-MSC (2:1) exhibited increased secretion of IL-6, IFN-γ, G-CSF and additive effects for most of them. Overall outcome demonstrated several fold higher cytokine levels in 8MGBA/AT-MSC cocultures which might have been responsible for the observed inhibitory effect due to the synergistic action of these soluble factors (Fig. 7D).

Taken together, we have demonstrated both protumorigenic and antitumorigenic effect AT-MSC on malignant cell behaviour dependent on the mutual interplay between malignant and stromal cells to each other.

Human melanoma cell lines A375 (ECACC No. 88113005), M4Beu, human fibroblasts (kindly provided by Dr. J. Bizik, CRI SAS, Bratislava), and glioblastoma multiforme 8MGBA (kindly provided by Dr. Perzelova, Med. School, Comenius University, Bratislava) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% fetal calf serum (FCS) and Antibiotic-Antimycotic mix (GIBCO BRL, Gaithesburg, MD). EGFP-A375 and EGFP-8MGBA cells lines stably expressing enhanced green fluorescent protein were prepared as described elsewhere [49] and cultured as above. Cells were kept in humidified atmosphere and 5% CO₂ at 37°C. AT-MSC were derived by plastic adherence technique as previously described in [24]. Briefly, AT-MSC cells were expanded from adherent cells obtained from stromal-vesicular fraction upon collagenase type VIII digestion of lipoaspirate obtained from healthy persons, who provided an informed consent. Cells were expanded in low glucose (1,000 mg/ml) DMEM supplemented with 10% Mesenchymal stem cell stimulatory supplement (human, MSCSS) (StemCell Technologies, Grenoble, France) and Antibiotic-antimycotic (GIBCO BRL, Gaithesburg, MD). AT-MSC were characterized by surface marker expression as CD44+, CD73+, CD90+, CD105+, CD14-, CD34-, CD45- population and AT-MSC were capable of differentiation into adipocytes and osteoblasts. Conclusions were drawn from similar results of experiments performed with two different isolates if not specified otherwise.

All chemicals were purchased from Sigma (St. Louis, MO) if not stated otherwise.

For proliferation evaluation, triplicates of 4,000 EGFP-A375 or EGFP-8MGBA tumor cells/well were seeded in black-walled 96-well plates (Greiner Bio-One Intl. AG) with or without increasing numbers of AT-MSC for overnight. Same amounts of AT-MSC in triplicates were seeded in parallel 96-well plates in 1% and 2.5% FBS containing DMEM for preparation of AT-MSC conditioned media. This media after overnight incubation was transferred into corresponding wells to evaluate tumor cell proliferation in AT-MSC conditioned medium. Medium was replaced every day and relative proliferation was evaluated by PolarStar OPTIMA reader (BMG Labtechnologies, Offenburg, Germany) on day 3. Values were expressed as means of relative fluorescence ± SD, where EGFP tumor cell fluorescence in appropriate medium DMEM without AT-MSC was set to 100% by default. It was previously determined, that there was linear correlation between fluorescence intensity and number of EGFP expressing cells under these experimental culture conditions.

For apoptosis evaluation, quadruplicates of 15,000 tumor cells/well were seeded either alone or into wells containing 1,500 AT-MSC/well in 96-well plates for overnight. Cells were washed and treated with 0%, 0.1% and 0.5% serum containing media for 3 days to determine the extent of serum-deprivation induced apoptosis. Cells were treated with 1 μg/ml doxorubicin, 5 μg/ml cisplatin or 50 μg/ml 5-fluorouracil in serum-free or 5% serum containing media for 16 hrs to evaluate extent of cytotoxicity. Caspase-3/7 activation was determined by Caspase-Glo^® 3/7 Assay (Promega, Madison, WI) on LUMIstar GALAXY reader (BMG Labtechnologies, Offenburg, Germany). Values were expressed as fold increase in relative luminescence units (RLU) in comparison to tumor cells maintained in media alone.

For flow cytometric determination of apoptotic and necrotic cell proportions in direct cocultures, adherent AT-MSC were labelled with 5 μM carboxy-fluorescein diacetate, succinimidyl ester (CFDA-SE, Molecular Probes, Eugene, OR) in a serum-free DMEM for 15 min at 37°C. Medium was replaced for standard culture medium for overnight incubation. 100,000 tumor cells/well were seeded with or without 10,000 CFDA-SE-AT-MSC/well in duplicates in 24-well plates and serum-deprived for overnight. Cells were treated with 200 ng/ml doxorubicin, 5 μg/ml cisplatin or 50 μg/ml 5-fluorouracil in 5% serum containing media for 20 hrs. Apoptotic cells were stained with Phycoerythrin-labelled Annexin V (Invitrogen, Carlsbad, CA); dead cells were detected with 7-AAD viability dye. Stained cells were analyzed using an EPICS ALTRA flow cytometer (Beckman Coulter, Fullerton, CA) equipped with Expo 32 program.

Colony-formation ability was evaluated in indirect tumor cell coculture with AT-MSC or fibroblasts. Tumor cells (280/cm² in 6-well plates) were plated into wells and combined with AT-MSC or fibroblasts physically separated in upper compartment and seeded on 0.4 μm cell culture inserts (5 × 10⁴ cell/insert) (Nalge Nunc International, Rochester, NY). Cells were maintained for 9 days in standard culture media. Average total number of A375 colonies per well was counted after Giemsa-Romanowski staining. Conclusions were drawn from three independent experiments.

Tumor cells were cultured with or without AT-MSC seeded on 0.4 μm inserts (10⁵ cells/insert) (Nalge Nunc International, Rochester, NY) for 3 days. Total RNA was isolated from 0.5 × 10⁶ 8MGBA, A375, AT-MSC, A375 co-cultured cells and AT-MSC co-cultured cells collected from inserts by RNeasy mini kit (Qiagen, Hilden, Germany) and treated with RNase-free DNase (Qiagen, Hilden, Germany). RNA was reverse transcribed with RevertAid™ H minus First Strand cDNA Synthesis Kit (Fermentas, Hanover, MD). 200 ng of cDNA was subject to standard PCR performed in 1× PCR Master Mix (Fermentas, Hanover, MD) with 35 cycles and gel resolved on 2% agarose or 4% MetaPhor^® Agarose (Lonza, Rockland, ME, USA) for qualitative analysis.

Quantitative PCR was performed in 1× ABsolute™ QPCR SYBR^® Green Mix (ABgene, Surrey, UK), 0.16 μM primers and 500 ng of template cDNA on RotorGene 2000 (Corbett Research, Sydney, Australia) and analyzed by RotorGene Software version 4.6. Primer sequences were used as previously published [15, 27]. Relative gene expression change was calculated according to the formula Fold increase = (reaction efficiency*2)^ΔΔCt, where ΔΔCt = [{Ct_{GOI(control cells)}-Ct_{GAPDH(control cells)}}-{Ct_{GOI(treated cells)}-Ct_{GAPDH(treated cells)}}]. GAPDH expression was taken as endogenous reference gene (GOI = gene of interest). Analysis was performed twice in triplicates and data expressed as means ± SE.

CXCR4 surface expression was analyzed in A375 cultures, or A375 cells isolated from tumor xenografts. Tumors were treated with 0.15% collagenase VIII, and 0.05 mg/ml DNAse I for 45 min at 37°C. Cells were sieved through 30 μm pre-separation filters (Miltenyi Biotec, Bergisch Gladbach, Germany) to obtain single-cell suspension and immunomagnetically separated by EasySep human FITC selection kit (StemCell Technologies, Vancouver, BC, Canada) by positive selection with FITC conjugated human specific anti-CD44 antibody (Millipore, Billerica, USA). CD44 positive cells were stained with PE-conjugated anti CXCR4 antibody (Invitrogen, Carlsbad, CA), anti-IgG2a isotype control (Invitrogen, Carlsbad, CA) and by flow cytometer. Representative result out of three independent experiments is shown.

50,000 A375 or 8MGBA, 25,000 AT-MSC cells alone plated in wells, 10,000 AT-MSC cells alone, 50,000 A375 (or 8MGBA) cells mixed with AT-MSC (ratio 5:1 or 2:1) were cultured in complete culture medium for three days. Cell-free supernatants were collected and subjected to human Bio-Plex™ 27-plex Cytokine Assay (Bio-Rad Laboratories Inc, Hercules, CA). Measurements were performed on Luminex 100 System (Luminex Corporation, Austin, TX) in duplicates with two different AT-MSC isolates. Results were expressed as means and relative cytokine expression was calculated by comparison to cytokine production from A375 (8MGBA) cells respectively.

SDF-1α level was determined in cell free supernatants prepared as above by human SDF1-α Quantikine Immunoassay (R&D Systems Inc.) on PolarStar OPTIMA reader (BMG Labtechnologies, Offenburg, Germany) as recommended by manufacturer.

Six weeks old athymic nude mice (Balb/c-nu/nu) were used in accordance with institutional guidelines under the approved protocols. It was determined in preliminary studies that 10⁶ M4Beu cells, 1.5 × 10⁶ A375 cells or 10⁷ 8MGBA cells injected s.c. exhibit 100% tumor incidence. Following cell suspensions were injected in high dose coinjection studies: 1.5 × 10⁶ A375 cells, 1.5 × 10⁶ A375 + 1.5 × 10⁵ AT-MSC (10%AT-MSC), 1.5 × 10⁶ A375 + 3 × 10⁵ AT-MSC (20% AT-MSC), 1 × 10⁶ M4Beu cells, 1 × 10⁶ M4Beu + 1 × 10⁵ AT-MSC (10% AT-MSC) (in 100 μl of PBS s.c. into the flank). In an independent study animals received low tumor cell dose (tumor incidence 1/10) of 1 × 10⁴, 1 × 10⁵ or 2 × 10⁵ A375 cells s.c as indicated. Groups of animals were directly coinjected with 1 × 10⁶, 1 × 10⁵, 1 × 10⁴ AT-MSC in admixture s.c., or s.c administered 1 × 10⁵ tumor cells were resuspended in AT-MSC conditioned media prior to injection. Independent group of animals was systemically administered with 1 × 10⁶ AT-MSC i.v. into the lateral tail vein concomitantly with s.c. administration of A375 cells alone. Animals were subsequently treated with 1.25 mg/kg AMD3100 every other day s.c. or 1 mg/kg Avastin (Bevacizumab, Roche, kindly provided by National Cancer Institute, Bratislava) i.p. twice a week where indicated.

For glioblastoma xenograft study animals received low (1.5 × 10⁶) 8MGBA cell dose cells s.c. Experimental groups of animals were directly coinjected with 1 × 10⁶ or 1 × 10⁵ AT-MSC in admixture s.c. Independent group of animals was systemically administered with 1 × 10⁶ AT-MSC i.v. into the lateral tail vein concomitantly with s.c. administration of 8MGBA high cell dose (10⁷).

Animals were regularly inspected for tumor incidence and designated tumor-free when no palpable rigid structures exceeding 1 mm in diameter could have been detected. Growing tumors were measured by calliper and volume was calculated according to formula volume = length × width²/2. Animals were sacrificed at the point, when the tumors exceeded 1 cm in diameter. Results were evaluated as mean volume ± SE.

Student's t test was used for comparison between the groups, differences in tumor incidences were evaluated by log-rank test, P value < 0.05 was considered significant.