BALB/c mice were born, grown and maintained in the Laboratory of Animal Experimentation of the Autonomous University of Nuevo Leon. Male mice aged 4 to 8 weeks were used to isolate BM-MSCs. A total of 72 female BALB/c mice (12 weeks old) weighing 19–22 g were used to induce solid lymphomas and evaluate the in vivo anti-lymphoma efficacy. From mice birth and throughout the experiments until the last mouse was euthanized, all animals were kept in individually ventilated cages at 25°C with sterilized air with EPA filters (UREAtac; LAB&Bio), with 50% humidity, ≤500 ppm of CO₂, ≤15 ppm of NH₃ and a light/dark cycle of 12/12 h. Access to food (Labdiet 5001; LabSupply) and purified sterile water was provided ad libitum.

The thymic lymphoma cell line L5178Y was used to produce lymphomas; the clone that was used (3.7.2C) was derived from the L5178YS cell line. In vitro, these cells grow in suspension; in vivo, when inoculated intramuscularly, they produce solid lymphomas, and when injected intraperitoneally, they grow abundantly and produce ascites. The L5178Y cell line was purchased from the American Type Culture Collection (cat. no. CRL-9518™; ATCC^®) and cultured as instructed by the manufacturer. The culture medium was Dulbecco's modified Eagles medium (DMEM) high glucose medium (4.5 g/l), 0.1% pluronic, 100 µg gentamycin/ml, 2.5 µg amphotericin B/ml, and 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.).

Lentiviral vectors were produced and provided by Cyagen Biosciences, Inc. as a gene delivery tool, named as follows: pLV[Exp]-EGFP/Neo-EF1A>{sMurTRAIL}, pLV[Exp]-EGFP/Neo-EF1A>mIfnb1[NM_010510.1], pLV[Exp]-EGFP/Neo-EF1A>{MurIL12-70p}. The lentiviral vectors were of 3rd generation and also included an integration cassette for G-418 (geneticin; Gibco; Thermo Fisher Scientific, Inc.) resistance for cell selection and a green fluorescent protein (GFP) gene to evaluate transgene integration.

BM-MSCs were obtained and characterized as previously described (22). Briefly, six mice were placed inside a CO₂ chamber to sacrifice them (70% vol/min); immediately after, their femurs and tibias were dissected under aseptic conditions. Their epiphysis was removed, and using a hypodermic needle (27 gauge), their BM was obtained by perfusing the shafts inner barrel with 500-1,000 µl DMEM/Nutrient Mixture F-12 (DMEM F-12) medium supplemented (s) with 10% FBS, 100 µg gentamicin/ml, and 2.5 µg amphotericin B/ml (Gibco; Thermo Fisher Scientific, Inc.) and poured into T-25 cell culture flasks (Corning, Inc.). The first cell passage was accomplished as follows: The aforementioned preparations were incubated for 24 h at 37°C in a 5% CO₂ humid atmosphere. The expended medium and the non-adherent cells were discarded. The culture flasks with the adherent cells were replenished with 4 ml fresh medium and incubated as before until the cell monolayer reached 80% confluence (7–10 days). The second passage was performed by discarding the spent medium and incubating the cell monolayer with a solution of 0.25% trypsin and 0.1% EDTA (Gibco; Thermo Fisher Scientific, Inc.) in sterile phosphate-buffered saline (PBS), washing trice with PBS and inoculating 5×10⁵ cells in a new culture flask with fresh 5 ml of supplemented DMEM F-12 and incubating it as before. The procedure described for the second passage was repeated in each of the following passages.

The characterization of MSCs and the assays performed with them were accomplished with cell cultures from passage 3 or 4. MSCs were characterized by immunocytochemistry and their multipotency was evaluated (Fig. 1). Cells were fixed with methanol-acetone (1:1) at 4°C per 10 min. Regarding immunocytochemistry, CD90 and CD105 surface markers were investigated using specific monoclonal antibodies included in the mouse and rabbit specific HRP/DAB (ABC) detection IHC kit (cat. no. ab64264; Abcam). Antibodies were diluted in PBS to 1:200 (CD90) and 1:25 (CD105). MSC multipotency was examined by inducing the cells to differentiate to osteoblasts or chondroblasts, using the appropriate reagents in the Mouse Mesenchymal Stem Cell Functional Identification kit (cat. no. SC010; R&D Systems, Inc.) and following the manufacturer's instructions. The osteoblasts were identified by staining with the Von Kossa technique and chondroblasts with Alcian blue. Cells were fixed with 4% paraformaldehyde for 20 min at ambient temperature. In Von Kossa staining, slides were submerged in saturated lithium carbonate solution for 20 min, and then washed with water and submerged in a 5% silver nitrate solution. Slides were exposed to bright light for 60 min and then were submerged in 5% sodium thiosulfate solution for 5 min. Finally, the slides were counterstained with neutral red. In Alcian blue staining, the colorant was added at ambient temperature for 10 min, and then the slides were washed with water and counterstained with neutral red at ambient temperature for 2 min.

All transgene constructs contained murine sequences of the genes of interest, a promotor cassette for Elongation Factor 1-Alpha (EF1-α) to allow a constitutive, high protein expression, and the Kozak consensus sequence (GCC GCC ACC) (23). The constructs used in the present study were designed with Vector Builder software (Cyagen Biosciences, Inc.). The design of the sTRAIL transgene is presented in Fig. 2A. This design contained the sTRAIL nucleotide sequence codifying the amino acid fragment 118 to 291; an isoleucine zipper sequence to facilitate in situ TRAIL's trimerization (24,25), a furin specific cleavage site (CGCACCAAACGC), a signal sequence (SS) derived from Gaussia sp. (a copepod) luciferase (TGG GAG TCAAAGTTCTGTTTGCCCTGATCTGCATCGCTG-TGGCCGAGGCC) to conduct protein secretion (26) and Kozak's sequence. As revealed in Fig. 2B, besides EF1-α and Kozak's sequence, the construct used to perform the transduction of BM-MSCs with the flTRAIL transgene contained a nucleotide sequence coding its 1–291 amino acids. The entire flTRAIL was retracted from GeneBank (accession number U37522, URL: http://www.ncbi.nlm.nih.gov/nuccore/U37522). Regarding IFNβ, the murine IFNβ gene sequence was retracted from GeneBank (accession number NM_010510, URL: http://www.ncbi.nlm.nih.gov/nuccore/NM_010510). As demonstrated in Fig. 2C, the scheme of the IFNβ transgene design was as follows: The complete IFNβ nucleotide sequence was included and added with Kozak's sequence.

BM-MSCs (5×10⁵) were seeded into T-25 culture flasks (Corning, Inc.) containing 5 ml DMEM F-12 supplemented with 10% FBS and incubated overnight under standard conditions, 50% confluency. Then, protamine sulfate (Sigma-Aldrich), 5 µg/ml were added to the BM-MSC cultures. Each lentivirus preparation carrying sTRAIL, flTRAIL, or INFβ transgenes was added to separate BM-MSC cultures. The multiplicity of infection (MOI) employed was two viral particles/one BM-MSC (27). The BM-MSCs infected with the lentivirus vectors were incubated overnight, to allow viral infection. Then, spent media was discarded and replenished with fresh DMEM-F12 and incubated 48 h before fluorescence analysis. Lentivirus integration was validated with fluorescence emitted by GFP, which was visualized with a fluorescence microscope (Eclipse 10i; Nikon Corporation) and a representative field photographed with a Sight DS-2MV (Nikon Corporation) digital camera. The selection of transduced BM-MSCs was accomplished by adding 200 µg of geneticin/ml to the culture medium. Transduction efficiency was calculated by applying the following equation: % Transduction efficiency=BM-MSC-GFP/total BM-MSC ×100. Where BM-MSC-GFP are transduced cells that exhibit a green fluorescent signal under the epifluorescence microscope. Total BM-MSC is the total number of cells detected by counting nuclei by fluorescence with DAPI (blue signal). Mounting medium Vectashield with DAPI (7 µl) per slide was applied (Vecta Mount; Vector Laboratories, Inc.).

Gene expression was validated by western blot analysis. BM-MSCs (5×10⁵) transduced with sTRAIL, flTRAIL or INFβ were placed, separately, into T-75 flasks (Corning, Inc.), containing 10 ml of DMEM-F12 and incubated at 37°C with 5% CO₂ for 48 h and allowing 70% confluency. The spent medium was discarded, and then the cell culture flasks were replenished with 10 ml fresh supplemented media and incubated at 37°C with 5% CO₂ for 48 h. For soluble proteins (sTRAIL and IFNβ), the spent medium was centrifuged at 4,000 × g for 5 min, concentrated 40 times with an Amicon filtration unit of 10 kDa MW cut-off (Merck KGaA). FlTRAIL is a transmembrane protein that was extracted and purified. FlTRAIL-BM-MSCs were cultured as aforementioned with the sTRAIL-BM-MSCs or IFNβ-BM-MSCs until flTRAIL-BM-MSCs culture reached 80% confluency. Then, flTRAIL-BM-MSCs were harvested and washed twice with PBS. Subsequently, 1×10⁶ cells were resuspended in 50 µl of lysis buffer [Triton X-100 (50 ml), Tris-HCl 1 M pH 7.5 (5 ml), KCl 2 M (12.5 ml), MgCl₂ 1 M (1 ml), DTT (1 µl, Invitrogen; Thermo Fisher Scientific, Inc.) and a protease inhibitor cocktail (5 µl; Sigma-Aldrich; Merck KGaA)]. The mixture was adjusted at 500 µl with ultrapure sterile water, chilled in ice for 20 min, centrifuged at 16,000 × g at 4°C for 5 min, and the supernatant was collected.

The aforementioned protein concentrates were quantified with Bradford reagent (Bio-Rad Laboratories, Inc.). After, 40 µg protein concentrate (5 µl) was submitted to 2% sodium dodecyl sulfate (SDS), 4% stacking and 12% resolving polyacrylamide gel electrophoresis (PAGE) at 100 V for 120–150 min and the protein bands were electro-transferred to a 0.45-µm mesh polyvinylidene fluoride membrane (Bio-Rad Laboratories, Inc.) at 100 V for 60 min. Protein was Electro-transfer to a PVDF membrane. The membrane was blocked for 1 h with 5% skimmed milk in Tris-buffered saline with Tween-20 (TBST) at 4°C. The protein bands of sTRAIL, flTRAIL, or IFNβ were identified with rabbit monoclonal antibodies anti-murine TRAIL (1:200; cat. no. ab10516; Abcam) or 1:500 rabbit polyclonal anti-murine IFNβ (cat. no. ab85803; Abcam), as primary antibodies, incubated overnight at 4°C. The secondary antibody was an HRP-conjugated anti-rabbit polyclonal antibody (1:10,000; cat. no. W4011; Promega Corporation), incubated for 2.5 h at 4°C. Dilutions of all antibodies were performed with Tris-buffered saline (TBS buffer) in 5% skimmed milk.

The marked protein bands were revealed with the luminol kit Clarity Western ECL blotting substrate (Bio-Rad Laboratories, Inc.) following the manufacturer's protocol and analyzed with the Molecular Imager Chemidoc XRS+ Imaging System (Bio-Rad Laboratories, Inc.) equipped with the Image Lab software.

Commercial recombinant proteins were used as positive controls in all western blot assays; soluble TRAIL recombinant protein (cat. no. 315-19; PeproTech, Inc.) which consists in the extracellular fraction of murine TRAIL, weighing 20 kDa; ~200 ng of recombinant protein was loaded into the gel well. IFNβ standard (cat. no. IF011; Merck KGaA) consisted of recombinant murine IFNβ weighing 22 kDa; ~2 kU was loaded into the gel well.

L5178Y cells (5×10⁵) were inoculated in the right gastrocnemius with a 27-gauge syringe. The cells were suspended in 100 µl of saline solution for a total of 72 female BALB/c mice. The mice exhibited mild discomfort immediately after inoculation; however, no sign of discomfort was observed overnight. The implanted leg of each mouse was measured with a Vernier calibrator to calculate the gastrocnemius volume (mm³), using the following equation: Vol=π/6 (LxWxh); where Vol is the volume of the leg, expressed in mm³, π is a radius constant (3.1416), L is the leg's length, measured from the ankle to the knee. W is the width of the thickest portion of the leg, and h is the height, measuring from the rear leg section (gastrocnemius) to the front (tibialis anterior). These measurements were performed every third day throughout the experiment. When legs carrying lymphomas showed an increment in the leg volume of 1–2 mm³, mice were implanted with the transduced BM-MSCs.

Mice with lymphomas were divided into nine groups of eight mice each. Transduced BM-MSCs were implanted to one of these groups (G) as follows: G1, BM-MSCs-sTRAIL; G2, BM-MSCs-IFNβ plus-flTRAIL; G3, BM-MSCs-IFNβ; G4, flTRAIL; G5, sTRAIL plus IFNβ, G6, not transduced or naïve BM-MSC plus-IFNβ plus-flTRAIL; G7, naïve BM-MSC; G8, mice not implanted with BM-MSCs but injected with isotonic saline; G9, mice not implanted with BM-MSCs. In addition, the experiment was repeated with groups G5, sTRAIL plus IFNβ, and G6, not transduced BM-MSC to confirm results. However, mice were reduced to five animals per group with the same characteristics. In all implanted mice, 1×10⁶ viable BM-MSCs, suspended in 100 µl isotonic sterile saline, were distributed into the entire volume of each tumor. The number of BM-MSCs transduced separately with two transgenes or two transgenes plus not transduced MSCs were inoculated with an appropriate proportion of BM-MSCs to sum a total of 1×10⁶ cells. For example, G5 mice were implanted with 5×10⁵ BM-MSCs-sTRAIL and 5×10⁵-INFβ and mice from G6 were implanted with 3.33×10⁵ not transduced BM-MSC, plus 3.33×10⁵ BM-MSCs IFNβ plus 3.33×10⁵ flTRAIL. All inoculations were performed in aseptic conditions with a new, sealed sterile 27-gauge needle for each mouse. Before and throughout the experiments, mice behavior, their ability to eat and drink, and pain indicators were monitored. When a mouse showed intense suffering as previously described (28), these signs included bent down low ears, orbital tightening, bristly hair, sluggishness, or a lack of response to external stimuli. When all signs were detected, the mouse was euthanized. These animals were considered deceased due to their induced lymphoma. Euthanasia was practiced by cervical dislocation. Tumor volume and mouse weight measuring were performed thrice a week, as at the beginning of the experiment.

Tumor biopsies of ~5 mm³ were dissected from recently deceased animals. The samples from each lymphoma were preserved with Tissue-Tek optimum cutting temperature (O.C.T.) (Sakura Finetek Europe B.V.), freezing the blocks with liquid nitrogen and stored at −80°C.

Frozen samples were cut with a cryostat (model 2230; Shenyang Roundfin Trade Co., Ltd.), stained with hematoxylin and eosin, and observed with a clear field using a light microscope (model DM IL LED; Leica Microsystems GmbH) at 100–400 amplification diameters. Images were captured with a camera (DFC295; Leica Microsystems GmbH) attached to the microscope.

The number of deceased mice from all groups was monitored daily. When the last not-implanted mouse with BM-MSCs carrying lymphomas succumbed, the percentage of surviving mice from each group and the number of days elapsed since the lymphoma cells were inoculated were registered.

The survival rate was analyzed with a Kaplan-Meier estimator with a Mantel-Cox test to assess statistical significance with SPSS software (SPSS Statistics for Windows; version 22.0; IBM Corp.). Statistical analysis was conducted using GraphPad Prism version 7.00 for Windows (GraphPad Software, Inc.). Differences between the analyzed groups were determined with the t-test and one-way ANOVA (followed by Tukey's post hoc test). P<0.05 was considered to indicate a statistically significant difference.

The present study was approved (approval no. BI15-005) by the Scientific Research and Bioethics Committee and the Institutional Animal Care Committees of the School of Medicine of the Autonomous University of Nuevo Leon (Monterrey, Mexico). The mice were handled according to the Mexican Standard NOM-062-ZOO-1999. The mice were kept in the animal facility of the Department of Immunology in individually ventilated cages (Lab & Bio) that maintain filtered air flow and a barrier that protects them from contact with external agents. The system records pressure, oxygen, CO₂, humidity, temperature, and ammonia concentration. Furthermore, this system maintains all these parameters in uniform conditions. The cages were cleaned every 2–3 days by the technical personnel of the vivarium. The model was euthanized if the tumor exceeded 10% of the normal mouse body weight according to the international manual Institutional Animal Care and Use Committee (2002) and would be euthanized by inhalation of CO₂ with subsequent cervical dislocation and monitoring of vital signs. They were also euthanized if they showed signs of severe pain according to the Rodent Management Laboratory Manual accepted by the American Association for the Accreditation of Animal Care Laboratories (29): decreased intake of water and food, weight loss (>20%), bristly coat, dry skin, lacerations, posture, abnormal gait, head tilt, lethargy, bloating, diarrhea, seizures, discharge from body orifices and dyspnea. The experimental groups were kept under close care from the day of inoculation until the last mouse in the control group succumbed. The mice succumbed due to tumor growth. However, the mice in the experimental groups that survived until the last mouse in the untreated group were sacrificed on the same day that the last untreated mouse lived.

BM-MSC isolated from mice presented fibroblast morphology in culture and were characterized by expression of CD90 and CD105 superficial markers that presented >95% positive cell staining (Fig. 1A-C). In addition, BM-MSC differentiated to chondroblasts and osteoblasts, showing positivity to Alcian blue and Von Kossa stains, respectively (Fig. 1D and E). A representative field of transduced BM-MSCs is shown in Fig. 2D. All cells fluoresced intensely in green due to expression of the GFP gene and geneticin selection, denoting an efficient integration of each gene construct. The percentage of transduction efficiency of BM-MSC-sTRAIL and -flTRAIL was 90% and for BM-MSC-IFNβ was 95%. For protein expression, western blot results are shown in Fig. 2E and F. The protein bands, whose images are demonstrated in Fig. 2E, correspond to sTRAIL and an sTRAIL standard, respectively, with both weighing ~20 kDa (control). The image of flTRAIL, revealing a ladder of five bands; the smallest one weighs ~20 kDa, which matches the 2 soluble TRAIL standard control; however, 4 heavier bands of ~30, ~39, ~45, and ~60 kDa were also revealed. In Fig. 2F, two IFNβ bands are demonstrated, one weighing ~22 kDa and a slightly heavier ~24 kDa band. Alongside as control, the recombinant IFNβ standard shows a single band weighing ~22 kDa.

On day 7 post-inoculation (PI), a slight inflammation was observed in all inoculated subjects. PI leg volumes on day 9, comparing the non-inoculated and inoculated groups are revealed in Fig. 3; considering one standard deviation as a reference, it displays the totality of the study subjects with tumor engraftment.

First, deceased mice from the untreated group were registered by day 26 PI; therefore, the last tumor measure with full subjects' groups was on day 23 PI. Fig. 4A highlights the most relevant results: the untreated group shows the largest tumor (average 2.5 cm³) and first deceased mouse at day 26 PI; on the other hand, the group with combined treatment of IFNβ and sTRAIL showed the smallest tumor (1.2 cm³) by day 23, and the first mouse succumbed by day 29 PI. Notably, separated treatments of both sTRAIL and IFNβ also showed considerably smaller tumors (compared with the untreated group) but slightly larger than the combined one (1.3 and 1.5 cm³, respectively).

During the study, mice weights were documented. Any mouse that exhibited a loss of ≥20% of the baseline weight was withdrawn from the study and euthanized. The weight of all groups during the study is revealed in Fig. 4B; mice showed stable weight by day 19 PI; however, important weight variability was registered thereafter. All mice were carefully observed to detect a disability to eat or drink; however, despite massive tumors, mice were eating and drinking; thus, weight variability was associated with tumor growth. All subjects gained weight according to the developed tumor mass.

In addition, tumor tissues were analyzed with a light microscope. A section of the tumor of untreated mice was compared with tissue of the inoculated leg of surviving mice. Images of histological sections are revealed in Fig. 5. It was demonstrated how the tumor tissue almost completely displaced muscle tissue (Fig. 5B and D), exhibiting basophilic predominance of lymphocyte (tumoral cell) nuclei, which in contrast to a surviving mouse, most of the tissue was acidophilic with skeletal muscle tissue (Fig. 5A and C). It was also observed that a few nuclei dispersed over the muscle tissue, indicating an active immune reaction. The statistical analysis of the infiltration rate and morphology observed in treated tissues is revealed in Fig. 5E and F. Chi-square showed differences between the control and the treatment group (P<0.001); however, in morphology, no statistical differences were identified.

The last untreated mouse died at day 41 PI; thus, a cut-off point was established. All groups comparing each treated group against the untreated group were analyzed. In Fig. 6A, a Kaplan-Meier estimator with the most relevant results is demonstrated: 100% of untreated mice succumbed due to the tumor by day 41 PI. On the other hand, the combined treatment of IFNβ and sTRAIL showed a 62.5% survival rate. Additionally, the separated groups of IFNβ and sTRAIL showed 25 and 50% survival, respectively, in addition to initial tumor mass, which showed a similar result, suggesting an additive effect. The log-rank test showed significant differences between the groups (P<0.05). All P-values are included in Table I.

To confirm these results, the combination treatment of BM-MSC expressing sTRAIL and IFNβ was repeated. The results are presented in Fig. 6B. The control treatment of naïve BM-MSCs was added. At first, both treatments reduced tumor growth in the right gastrocnemius; however, near day 20, mice of the groups treated with naïve BM-MSCs presented an increase in tumor volume. On day 36 PI, the last mouse of the naïve BM-MSC group was euthanized, and 40% of survivors remained in the group treated with sTRAIL plus IFNβ. The log-rank test showed significant differences between the groups (P<0.001). Mice were kept monitored until day 50; nevertheless, mice did not present visually solid tumors in the right gastrocnemius.

All treatments showed anti-tumorigenic activity to some extent. Independent treatments showed poorer antitumor activity than combined ones. IFNβ exhibited the lowest therapeutic activity with a 25% survival rate, sTRAIL showed 37.5%. Combined treatment showed 37.5% survival rate, indicating a non-additive effect since it remained nearly the same as when treatment was performed with sTRAIL alone. Notably, saline solution had a 25% survival rate. A combined treatment of MSC naïve, IFNβ and flTRAIL showed a 50% survival rate, indicating a slight improvement over flTRAIL and IFNβ alone, suggesting MSC antitumoral effect.