Inhibitory Effects of Doxycycline on Tumor Progression In vitro and on Metastases in Early-Stage Osteosarcoma Xenografts Mice.

doi:10.21203/rs.3.rs-1019005/v1

PDF

Research

Inhibitory Effects of Doxycycline on Tumor Progression In vitro and on Metastases in Early-Stage Osteosarcoma Xenografts Mice.

https://doi.org/10.21203/rs.3.rs-1019005/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted 08 Nov, 2021

You are reading this older preprint version

Read the latest preprint version →

Background

Osteosarcoma (OS) is the commonest primary osseous malignant tumor with a high propensity to metastasize in lungs. Pulmonary widespread micrometastatic lesions are present in up to 80% of patients at initial diagnosis and they are associated with significantly worse prognosis. Doxycycline (Dox) is a synthetic tetracycline that has been shown to have anti-cancer properties in vitro and in vivo, and inhibit angiogenesis, effects that may prove beneficial for several types of cancer. The aim of the present work was to study how Dox affects OS cells’ growth in vitro and in vivo and OS-driven pulmonary metastasis in vivo.

Methods

In vitro, the effect of Dox was measured in MG-63 and 143B human OS cells’ viability, apoptosis, and migration. In vivo, highly metastatic143B cells were orthotopically implanted into the tibia of SCID mice and tumor growth as well as pulmonary metastases between Dox treated and untreated, non-amputated and early amputated xenografts were examined.

Results

Dox decreased the viability, inhibited the migration, and induced the apoptosis of OS cells in vitro. In vivo, Dox significantly enhanced tumor necrosis at primary OS sites, similarly to its in vitro effect. It also decreased the expression of Ki67, metalloproteinases 2 and 9 (MMP2 and MMP9), vascular endothelial growth factor A (VEGFA) and Ezrin in primary tumors. It also decreased the circulating VEGFA and MMP9 protein levels, in line with the decreased metastatic burden in Dox-treated mice in both non-amputated and early amputated xenografts.

Conclusions

Our results suggest that adjuvant administration of Dox may decrease OS growth and development of pulmonary metastases. Administration of Dox in combination with surgical resection and standard chemotherapeutic protocols in the early-stages of OS treatment is also supported. Moreover, Dox administration prior to the development of clinically detectable pulmonary macrometastases, is associated with enhanced clinically benefits from its anti-metastatic effect.

Oncology

doxycycline

osteosarcoma

VEGF

MMP

xenograft

metastasis

Osteosarcoma (OS) is the most common primary malignant bone tumor with an estimated incidence of 1.7 - 4.4 per million people being diagnosed every year [1]. While OS frequently affects people between the age of 5 and young adulthood, it can also occur in people over 65 [2]. The distal femoral metaphysis and the proximal tibia are the most usual sites for primary OS to develop [3]. Usually, OS presents as a high-grade intramedullary bone lesion with malignant mesenchymal cells, which invade surrounding tissues and produce immature bone, known as osteoid [3]. Approximately 80% of newly diagnosed OS cases have subclinical pulmonary micro metastases without radiologic evidence [4], leading to a significant decrease in the overall 5-year survival compared to the metastases-free OS patients (overall 5-year survival rate 30% compared to 70% for those free of distant metastases) [5]. Currently, the treatment protocol for OS includes neo-adjuvant chemotherapy (cisplatin, doxorubicin, ifosfamide, methotrexate) followed by wide surgical resection of the primary tumor and adjuvant chemotherapy [6]. Although the suggested OS treatment protocol presents the practice of choice for the treatment of OS, it does not seem to affect the metastatic rate of this very aggressive disease [7].

Dox is a synthetic third-generation tetracycline with a broad antibiotic spectrum. Its anti-cancer effects in several types of cancer have been previously examined with promising results [8, 9, 10, 11]. There is also limited in vitro evidence obtained from OS cell lines suggesting that Dox may be a potential OS anticancer agent [12, 13, 14]. One of the mechanisms through which Dox may exert its anticancer effects has been suggested to be its ability to inhibit matrix metalloproteinases (MMPs) due to calcium chelation [15]. MMPs are calcium-dependent, zinc-containing endopeptidases secreted by tumor cells with fundamental involvement in the degradation of the extracellular matrix (ECM), cancer cell invasion, metastasis and angiogenesis [16, 17]. MMPs may be also important for OS progression and targeting MMPs has been suggested as a potential therapeutic approach [18]. Experimental trials have suggested that MMPs interfere with the secretion of vascular endothelial growth factor A (VEGFA) from cancer cells [19]. MMPs and VEGFA help OS micro metastatic disease to stimulate local angiogenesis and remodel the microenvironment to support tumor neovascularization. In end-stage disease, micro metastases expand to become clinically detectable and give rise to lethal macro metastases [20].

The aim of the present study was to determine the in vitro and in vivo antitumor effect of Dox in OS and its potential for the prevention or limitation of pulmonary metastases in early-stage OS xenografts.

2.1 Cell cultures

143B (ATCC® CRL-8303) and MG-63 (ATCC® CRL-1427) human OS cell lines were purchased from the American Type Culture Collection [ATCC; (www.lgcstandards-atcc.org)]. OS cells were grown in Eagle’s minimal essential medium (ΕMEM) supplemented with 10% fetal bovine serum (FBS) and 100 u/ml penicillin - 100 mg/ml streptomycin. Cells were kept at 37°C in a humidified atmosphere of 5% CO₂.

2.2 Cell viability Assay (Trypan blue exclusion)

OS cells were plated at a cell density of 3.5 × 10⁴ cells/well in 6-well plates in EMEM supplemented with 10% FBS. Twenty-four and 48 h after seeding cells, trypan blue exclusion assay was used to determine the number of living MG-63 and 143B cells exposed to 0, 5, 10 and 20 µg/ml of Dox [21].

2.3 Cell migration assay (Wound healing assay)

OS cells cultured in EMEM supplemented with 10% FBS were seeded into 24-well tissue culture plate wells at a density of 2×10⁶ cells/well so that after 24 h of growth, they should reach 90-95% confluency as a monolayer. The monolayers were scratched vertically with a sterilized 200 µl pipette tip across the center of the well. After scratching, the wells were washed twice with 1x phosphate-buffered saline (PBS) to remove the detached cells and replenished with fresh medium containing Dox (20 µg/ml) or PBS (control). Cells were photographed at 0, 8, 16, and 24 h after scratching, using an Olympus Bx40 microscope. TScratch software version 7.8 (Computational Science and Engineering Laboratory, Swiss Federal Institute of Technology, Zurich, Switzerland) was then used to perform image analysis and measure the gap areas [21, 22].

2.4 Apoptosis Assay (Fluorescence-activated cell sorting/FACS)

The effect of Dox in OS cell lines apoptosis was carried out using flow cytometry. Briefly, 143B and MG-63 cells were fixed overnight at 4⁰ C in 70% ethanol. The fixed cells were stained with RNase-containing propidium iodide (PI) and Annexin V FITC solution (TACS Annexin V FITC, Apoptosis Detection Kit, Gaithersburg, MD, USA). Cells were then separated as early apoptotic (Annexin V FITC-stained), late apoptotic (Annexin V FITC and PI-stained), necrotic (PI-stained) or non-apoptotic/live cells (no staining). DNA content was analyzed using a FACS caliber flow cytometer (Becton Dickinson, San Jose, CA) and MoD Fit software (Verity Software House, Topsham, ME) [23].

2.5 In vivo orthotopic implantation of 143B human OS cells in mice xenografts

143B is a highly tumorigenic OS cell line with high metastatic rate [24, 25, 26]. The injection of 143B cells in mice tibias generates either spontaneous (tumor cells interact with their native microenvironment, invade local vessels, and move to distant sites) or experimental (direct seeding of tumor cells in lungs during the injection procedure) pulmonary metastases [27, 28, 29].

Thirty-two 6 to 8-week-old female severe combined immunodeficient (SCID) mice were obtained from the National Center for Scientific Research (Demokritos, Greece). Mice were acclimatized for 10 days without any interventions after transportation to the Laboratory for Experimental Surgery and Surgical Research “N.S. Christeas”, Medical School, University of Athens, Greece, where the in vivo study was conducted. The animals were housed individually in clean metabolic cages placed in a well-ventilated house with optimum conditions (temperature 23 ± 1°C; photoperiod 12 h natural light and 12 h dark; humidity 45-50%) with access to food and water ad libitum during the entire study period. On day 0, the animals were anesthetized using a ketamine (100 mg/kg, Imalgene 1000; Merial, France) - xylazine (10 mg/kg Rompun; Bayer Animal Health GmbH, Germany) mixture injected intraperitoneally (IP)[30]. 143B OS cells (1 x 10⁶ in 100 µl of PBS) were injected into the left proximal tibia of SCID mice using a 25-G needle [31]. All procedures were approved by the Regional Veterinary Service (no:366107/July 9, 2019) the Ethics Committee of the NKUA/Medical School (no:163/September 18, 2019) and were in accordance with National Legislation and European Directive 63/2010.

2.6 Study design

Mice were randomly divided into four groups. Mice in group A, Dox⁻/Amp⁻ (n=7), comprised the untreated control group. Mice in group B, Dox⁺/Amp⁻ (n=8), received Dox hyclate 50 mg/kg (D9891; Sigma Aldrich Chemical Co., St Louis, MO, USA) diluted in water and injected IP daily for 28 consecutive days [32]. Mice in group C, Dox⁻/Amp⁺ (n=7), underwent transfemoral amputation using a standardized method (Day 5) [33]. Mice in group D, Dox⁺/Amp⁺ (n=6), underwent transfemoral amputation (Day 5) and received Dox hyclate (50 mg/kg) IP daily for 28 consecutive days.

Four weeks after tumor inoculation (Day 28), all animals were anesthetized, blood was drawn from the orbital sinus and mice were euthanized via cervical dislocation [34, 35]. During the inoculation process 4/32 mice died after the acute onset of tachypnea, possibly due to pulmonary embolism. The left tibias (primary tumor sites) were evaluated with x-rays (Chirana type RK 75-10 EKv 2mm AL, Film Agfa 100NIF 25x30, Cassette Agfa CR MD 4.0 General) three weeks after engraftment of 143B cells to assess tumor formation at primary sites.

Resected xenograft tibias and tumor dimensions were measured using a digital caliper according to the formula: volume = (L + W) (L) (W) (0.2618). The value assigned as width (W) was the average between the anterior - posterior and medial - lateral planes of the proximal tibia. The value assigned as length (L) was the distance between the most proximal and the most distal tumor margin [28]. The lung volumes were calculated with a digital weight scale, and lung tissues were investigated for macroscopically detectable metastases using a stereoscope.

2.7 Surgical technique

Mice were anesthetized using a ketamine - xylazine mixture, as described above, injected IP [30]. A small animal heating pad was used during surgery to maintain normothermia. While under anesthesia, the left tibia of the mouse was shaved, cleaned with povidone iodine, and then rinsed with alcohol. The knees were flexed in 90⁰ and tibias were removed after midshaft femur cut to achieve tumor-free margins. The skin was closed with size 4-0 monofilament nylon sutures [33]. During this procedure, a mouse from group C died due to excessive bleeding (group C, new n=6). Buprenorphine (0.1 mg/kg, IP every 8 h; Bupaq® 0.3, Neocell Ltd.) was used for pain control over the first 24 h after amputation (Day 5) [36].

2.8 Quantitative measurement of blood biomarkers MMP9 and VEGFA

On day 28, blood samples were aspirated from mice under anesthesia with retro-orbital technique using fine-walled glass Pasteur pipettes (diameter:150mm). The blood samples were collected in heparin coated microhematocrit tubes and stored on crushed ice for no longer than 30 min before being centrifuged at 8000 rpm at 4°C for 10 min. Mouse serum was examined for MMP9 and VEGFA protein levels by ELISA (#ab100610 and #ab100662 respectively, Abcam, Cambridge, UK), according to manufacturer’s instructions. The cell line we used to generate the in vivo OS model was human and we used human anti-VEGFA and anti-MMP9 ELISA kits to avoid cross-reaction with mouse VEGFA and MMP9 [37].

2.9 Histological Examination And Immunohistochemistry (Ihc)

Primary tumors and lungs were collected from mice, fixed in 4% paraformaldehyde in Tris-buffered saline (TBS) at 4°C for 18 h. Paraffin embedded tissue specimens were sectioned at a thickness of 3.0 µm, followed by deparaffinization in xylene and dehydration in a graded series of ethanol solutions

Histological examination

Each section was photographed in its entirety and subsequently, digitally processed utilizing the IpWin6 program. Tumorous areas were manually demarcated, and the program proceeded to assess the percentage of tumor areas to overall tissue surface. The same procedure was conducted to assess the percentage of necrotic tumor areas to the overall tumor surface in primary sites.

IHC: Antigen retrieval was performed by heating the samples for 20 min at 95°C in citrate buffer (pH 6.0), and endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 min at room temperature. Afterwards, the sections were incubated with anti-MMP9 antibody (rabbit polyclonal, #ab38898, Abcam), anti-MMP2 antibody (rabbit polyclonal, #ab97779, Abcam), anti-vimentin antibody (rabbit monoclonal [EPR3776], #ab92547, Abcam), anti-E-cadherin antibody (mouse monoclonal [4A2], #ab231303, Abcam), anti-Ki67 antibody (rabbit polyclonal, #ab15580, Abcam), anti-Ezrin antibody (mouse monoclonal, #ab4069, Abcam), anti-VEGFA antibody (rabbit polyclonal, cat. #ab46154; Abcam), all at a dilution of 1:300 at 4°C overnight. After washing with PBS, the sections were incubated with biotinylated secondary antibody (goat anti-mouse IgG or goat anti-rabbit IgG, cat. no. 20775; Millipore, Burlington, USA) for 10 min at room temperature (RT). They were then incubated with Streptavidin HRP (#20774, Millipore, Burlington, USA) for 10 min at RT, and the reaction was visualized using 3,3'-diaminobenzidine (#D12384, Sigma-Aldrich, Gillingham, UK). Eventually, the specimens were counterstained with Mayer’s hematoxylin at RT for 1 min. Images were photographed using a Nikon Eclipse 80i microscope equipped with a digital camera image system (Cellsens). The immunostaining of antibodies was scored on the scale of semi-quantitative assessment by evaluating the intensity and percentage of positively stained cells. The intensity of antibodies staining was scored as follows: 0, none; 1, weak; 2, moderate; and 3, strong. Samples were blindly inspected by two independent experienced pathologists [38].

2.10 Statistical analysis

All statistical analysis was performed using SPSS version 26 (IBM SPSS Statistics for Windows; IBM Corp., Armonk, NY). All data were tested for normality using a Kolmogorov–Smirnov test and displayed parametric or nonparametric distributions (p < 0.05). The measurements of blood biomarkers, number of lung metastases, lung weight and quantitative IHC in lungs were analyzed using one-way analysis of variance (ANOVA) (Kolmogorov–Smirnov significance p >0.05) followed by a post-hoc Bonferroni test. The measurements for the tumor volume, tumor weight, tumor necrosis and quantitative IHC in primary tumors were analyzed using an independent samples t test, as the data showed normality. P<0.05 was considered as a statistically significant difference.

3.1 In vitro studies

3.1.1 Doxycycline inhibits viability and induces early and late apoptosis of MG-63 and 143B human OS cells

Exogenous administration of Dox decreased the number of both MG-63 and 143B viable cells in a concentration-dependent manner. The decrease was evident at 24 h with the higher used Dox concentrations and was significant even with the lower Dox concentrations at 48 h (Fig. 1A).

The apoptotic effect of Dox on the same cells was examined by Annexin V-FITC and PI staining following a 24 h exposure of cells to either 20 µg/ml Dox or solute (PBS). Dox significantly enhanced late apoptosis in both types of OS cells, but also induced necrosis of these cells (Fig. 1B), in line with its inhibitory effect on the number of viable cells shown in Fig. 1A.

3.1.2 Doxycycline inhibits migration of MG-63 and 143B human OS cells

The in vitro effect of Dox in the migration capacity of OS cells was examined by a wound healing assay. Both Dox treated OS cell lines (MG-63 and 143B) presented a significant reduction in their mobility even at 8 h after the onset of treatment (p < 0.0001 in both cases) (Fig. 2).

3.2 In vivo studies

3.2.1 Endpoint metrics in 143B OS xenografts

The volume (cm³) and weight (g) of primary tumors in left tibias were measured in the Dox⁻/Amp⁻ and Dox⁺/Amp⁻ groups. No differences in primary tumor volume and weight were found between Dox treated and non-treated xenografts (Fig. 3A). The percentage of primary tumor necrosis in the Dox⁺/Amp⁻ group was significantly increased (P < 0.0001) compared to the Dox⁻/Amp⁻ group (Fig. 3B). Histopathologic evaluation of primary tumors with hematoxylin and eosin (H&E) staining revealed malignant tumor and stroma cells with destruction of normal bone (Fig. 3C).

3.2.2 Dox decreases the expression of the prognostic factors Ki67, VEGFA, MMP2, MMP9 and Ezrin in primary tumors

Based on IHC staining, the primary tumors of both Dox⁻/Amp⁻ and Dox⁺/Amp⁻ xenografts were vimentin positive and E-cadherin negative (Fig. 4A), verifying the mesenchymal nature of OS [39, 40, 41].

The Ki67 nuclear protein is a well-established prognostic and predictive indicator in OS biopsies [42, 43]. We found a statistically significant decrease (P <0.041) in the expression of Ki67 in primary tumors of the Dox⁺/Amp⁻ group (20.62 ± 8.21%) compared to the Dox⁻/Amp⁻ group (45.00 ± 21.90%) (Fig. 4A).

Using quantitative IHC analysis, we found that the expression of MMP2 in Dox⁺/Amp⁻ group (7.50 ± 8.01%) was significantly decreased (P < 0.002) compared to the Dox⁻/Amp⁻ group (22.85 ± 6.98%). Similarly, the expression of MMP9 in primary tumors of the Dox⁺/Amp⁻ group (4.25 ± 4.68%) was significantly (P < 0.0001) decreased compared to the Dox⁻/Amp⁻ group (25.71 ± 5.34%). The expression of VEGFA in Dox⁺/Amp⁻ group (2.12 ± 7.03%) was also significantly decreased (P < 0.0001) compared with the Dox⁻/Amp⁻ (72.85 ± 13.49%). Finally, based on the notion that Ezrin expression may be a marker and a target in OS [44], we looked for its expression in primary tumors and found that it was significantly decreased (P < 0.003) in the Dox⁺/Amp⁻ group (8.12 ± 8.83%) compared to the Dox⁻/Amp⁻ group (26.42 ± 10.29%) (Fig. 4).

3.2.3 Doxycycline prevents the formation of pulmonary macro metastatic disease

In all groups, abnormal lesions were recognized on the lung surfaces of numerous mice and they were recorded as metastatic sites. We used H&E staining (Fig. 5A) to evaluate the percent metastatic surface / total lung surface ratio for each xenograft from our groups. As shown in Fig. 5B, the number of metastases was significantly lower in the Dox⁺/Amp⁻ compared to the Dox⁻/Amp⁻ group. Similarly, the number of metastases was significantly lower in the Dox⁻/Amp⁺ compared to the Dox⁻/Amp⁻ group. The decrease observed in the Dox⁺/Amp⁺ group compared to the Dox⁻/Amp⁺ group did not reach statistical significance. Early amputation also resulted in a significantly lower lung weight independently of Dox treatment (Fig. 3C).

3.2.4 The expression of markers in pulmonary macrometastatic lesions

The expression of Ki67, MMP2, MMP9, VEGFA and Ezrin in metastatic lesions was high in all xenografts and no evidence of statistically significant difference between the Dox-treated and untreated groups was found (Fig. 6 and Table 1). The metastatic lesions were vimentin positive and E-cadherin negative, similarly to the primary tumors shown in Fig. 4.

Table 1

The intensity of antibodies staining in pictures like those presented in Fig. 6 was scored as described in Methods and results are expressed as mean ± SD of the percentage of tumor cells which express the marker to the overall tumor surface. Statistical analysis was performed by one-way Anova.
	Dox+/Amp+ (n=6)	Dox-/Amp+ (n=6)	Dox+/Amp- (n=8)	Dox-/Amp- (n=6)	P value
ΜMP2	6.66 ± 12.11	2.50 ± 6.12	2.12 ± 4.01	5.83 ± 9.17	0.670
MMP-9	8.33 ± 13.29	8.33 ± 13.29	12.50 ± 14.63	18.33 ± 17.22	0.607
VEGFA	26.66 ± 41.31	30.00 ± 46.47	50.00 ± 41.74	55.00 ± 42.77	0.573
Ki-67	28.33 ± 44.00	29.16 ± 45.21	50.00 ± 41.74	58.33 ± 45.68	0.546
Ezrin	13.33 ± 32.65	13.33 ± 24.22	16.87 ± 27.11	16.66 ± 18.61	0.990

3.2.5 Blood levels of VEGFA and MMP9 correlate with the number of experimental metastases

The hypothesis that blood levels of VEGFA could have a predictive and prognostic value in OS patients has been previously assessed, showing that VEGF blood levels are significantly higher in OS patients compared with healthy counterparts and correlate with response to chemotherapy or metastasis [45]. The predictive role of MMP9 blood levels has been discussed for several types of tumors [46], but not yet in OS patients. In the present work, we evaluated the correlation between VEGFA and MMP9 blood levels and response to Dox. Both VEGFA and MMP9 blood levels were high in the non-treated Dox⁻/Amp⁻ control group and they were significantly decreased in all other groups (P < 0.0001, one way ANOVA). The decrease observed in the Dox⁺/Amp⁺ group compared to the Dox⁻/Amp⁺ group did not reach statistical significance (Fig. 7), similarly to what has been observed in the formation of pulmonary metastases shown in Fig. 5B.

Concurring to the literature [12, 13, 14], our in vitro evidence suggests a dose-dependent effect of Dox in decreasing the number and the migration of OS cell lines. In addition, Dox effectively induced both apoptosis and necrosis of OS cell lines, in line with the decreased number of cells and the enhanced necrosis observed in vivo. The in vitro anti-tumor effect of Dox on MG-63 and 143B cells was studied at a dose between 5 to 20 µg/ml. To our knowledge, this in vitro treatment range has been proposed as the equivalent human dosage for Dox [11–13].

Since lung is the most common site of metastasis in OS patients and pulmonary metastasis is associated with a significant worse prognosis in OS patients, we also aimed to assess the use of Dox as a supportive therapy in OS patients for the prevention of pulmonary metastasis. In our in vivo human 143B OS xenograft model, doxycycline hyclate at 50 mg/kg/d was intraperitoneally administered for 28 days in dox-treated mice. A study by Luccheti J et al, revealed that the intraperitoneal injection of Dox in C57BL76 mice at a dose of 10 - 100 mg/kg had led in a peak plasma concentration of 2-10 µg/mL which is superimposable to the established oral treatment of Dox at 100-200 mg per day in humans [47]. In addition, we designed our study in accordance with a previously conducted study which reported a great tolerance of Dox when administered at a dose of 50 mg/kg/d in breast BALB/c xenografts [32]. The only study which evaluated the suppressing effect of Dox in tumor progression of human OS- or rhabdomyosarcoma- implanted athymic mice had used Dox at ∼30 mg/kg/d in drinking water with promising results [48]. However, in our opinion the administration of Dox ‘ad libitum’ via drinking water is not well controlled and this consists a limitation of the experimental study by Dickens D et al. Other limitations of that study are that the effect of Dox in the prevention of pulmonary metastatic disease was not examined in lung tissue biopsies, and the design of that study had not mimicked the standard treatment strategy of human OS, which includes neoadjuvant and adjuvant administration of chemotherapeutic agents accompanied with surgical excision of primary site [6]. Our study is the first experimental trial that has mimicked the clinical therapeutic strategy as in human OS. We administered Dox preoperatively, as well as postoperatively, after wide resection of the primary tumor in one of our study groups (Dox⁺/Amp⁺) and the results were compared with control groups.

In our study, we observed that treatment with 50 mg/kg Dox resulted in a smaller number of metastases compared with the numerous metastatic lesions in the Dox⁻/Amp⁻ control group. In addition, the mice treated with neo-adjuvant Dox, early amputation and adjuvant administration of Dox were almost free of metastases. Dox has been suggested to have anti-cancer properties through inhibition of MMPs, but the timing of an MMP inhibitor application in cancer is critical to achieve the desired therapeutic effect [49]. However, previously conducted phase I, II and III clinical trials that have evaluated the effect of MMP-inhibitors (e.g., the broad-spectrum MMP inhibitor Marimastat and the chemically modified tetracycline Col-3) as chemotherapeutic agents in advanced end-stage metastatic cancers (e.g., breast, pancreatic, gastric) [50, 51, 52, 53] were not encouraging. This may have resulted from the fact that these MMP inhibitors had not been used at early-stage cancers before the establishment of macro metastatic disease. A novel proposal of our in vivo study is the administration of Dox in early-stage OS that seems to prevent the progression of micro metastatic to macro metastatic lethal disease.

The present study demonstrated that circulating levels of human VEGFA and MMP9, secreted by the human OS cells, were decreased by Dox in line with the fewer macro metastases, resulting in better prognosis. The circulating levels of human VEGFA and MMP9 in the Dox⁻/Amp⁺ and Dox⁺/Amp⁺ groups were much lower compared to the groups that did not undergo early amputation. In these groups, both VEGFA and MMP9 were most likely secreted by the OS cells that formed pulmonary micro metastases and could be used as a biomarker even in cases when these metastases cannot be detected.

Similarly, to the in vitro model, the Dox-treated mice developed increased tumor necrosis in primary tumors, supporting the prognostic value of tumor necrosis in primary site for the development of systemic disease in OS [54]. Furthermore, Dox treated mice depicted fewer metastases compared to non-treated mice. On the contrary, the tumor size and the tumor volume were not affected by Dox administration and were not correlated with a higher or a lower metastatic surface / total lung surface ratio. This finding is not in accordance with bibliography which tends to recognize the significance of primary tumor size as a prognostic factor for subsequent lung metastases in OS [55]. The reason for this discrepancy was that some of our non-amputated mice developed limping and skin ulcers on primary sites and had to be euthanized early from the 28th day respectful to their welfare. Consequently, a significant difference in tumor size between Dox-/Amp- and Dox+/Amp- groups could not be entirely assessed.

The effect of Dox on EMT reversal in our in vivo model is verified by the immunohistochemical evaluation of E-cadherin (epithelial marker) and Vimentin (mesenchymal marker) [56]. In our study, primary tumors as well as pulmonary metastatic lesions were Vimentin positive and E-Cadherin negative. No significant difference in the intensity of the signal was recognized among Dox-treated and non -treated xenografts. Subsequently, we concluded that Dox prevents metastatic spreading to the lungs in OS, but this effect is not related to an EMT process, which has been also described in other tumors, such as lung cancer [57].

In Dox-treated mice, the expression of VEGFA, MMP2, MMP9, Ki67 and Ezrin in primary tumors were all downregulated and were strictly associated with a better prognosis. The association between VEGFA, MMP2, MMP9, Ki67 and prognosis is well known for many types of cancer [58, 59]. Ezrin has been shown to positively regulate the expression of MMPs and VEGFA in tumor cells and promote the metastatic potential of tumor cells [60, 61]. In pulmonary metastases, the expression of these markers was decreased in the amputated compared to the non-amputated groups but were not affected by Dox treatment. This may be due to the well described differences between metastatic cell clones and primary tumor cells that include their chemosensitivity to anticancer agents [62, 63].

Despite our effort to provide a close approximation of human OS using a humanized mouse model and provide therapeutic outcomes of Dox with clinical relevance, our study has two main limitations. The first limitation is that orthotopic implantation of allogeneic OS cells in SCID mice do not fully recapitulate the human immune and stroma-tumor interactions occurring in de novo tumors [64]. Secondly, the administration of Dox in Dox⁺/Amp⁻ and Dox⁺/Amp⁺ mice had started the same day with 143B cells intramedullary engraftment. Certainly, in most human cases the diagnosis does not coincide with the onset of treatment. However, tumor progression could not have been accurately monitored in mice to start treatment immediately prior the development of poorly controlled numerous metastases and therefore, we have chosen to start the administration of Dox from a baseline time for all treated mice. Nevertheless, the achievement of experimental metastases from the start points (seeding of OS cells in lungs during their engraftment in tibias) remains a strong point of our study design, as it replicates the clinical scenario for 80% of humans who suffer from pulmonary OS micro metastases already from the day in which diagnosis is made and chemotherapy is given.

Our data encourage the design of further clinical studies with adjuvant administration of Dox in non-metastatic OS disease (IA-IIB), supporting the use of Dox in combination with surgical resection and standard chemotherapeutic protocols in the early stages of OS treatment, prior to the development of clinically detectable pulmonary macro metastases.

Osteosarcoma, Dox:Doxycycline

VEGF

Vascular Endothelial Growth Factor

MMP

Metalloproteinase

SCID

Severe Combined Immunodeficient

Amp

Amputation

ECM

extracellular matrix

IHC

Immunohistochemistry

H&E

Hematoxylin and Eosin

Intraperitonially

NKUA

National and Kapodistrian University of Athens

Ethics approval: Our in vivo experimental trial in osteosarcoma xenograft mice followed the standard criteria of the ‘NC3Rs primates’ (replacement, reduction, refinement) guidelines. All procedures were approved by the Regional Veterinary Service (no:366107/July 9,2019), the Ethics Committee of the NKUA/Medical School (no:163/September 18,2019) and were in accordance with national legislation and European Directive 63/2010.

Consent for publication: Not applicable

Availability of data and materials: This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Competing interests: The authors declare that they have no competing interests.

Funding: This study was supported financially for necessary equipment purchase by Implantcast Hellas and DMPS LTD (Developed and Medical Products and Services). The sponsors were not involved in the study design, collection, analysis, and interpretation of data as well as in the writing of the report and the decision to submit the article for publication.

Author’s contributions: ACH, AFF, EP and AA contributed to the design, management and writing of this study. ACH and AA designed and performed the in vitro assays and ACH, CP and IC the in vivo trials. Histopathology and immunohistochemistry were examined by ALP, ST, PS. EP, AMP, AFM, ODS,AK and PJP provided the appropriate study database and guided the authorship of this study. All authors read and approved the submitted manuscript.

Acknowledgements

We are grateful to Dr. Athanasios Antonopoulos (Senior Consultant-Chief of the 3rd Department of Orthopedic surgery at KAT Hospital where I work) for his support during this study and Miss Magdalini Christopoulou (Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, 26504, Patra, Greece) for her technical efforts.

Mirabello L, Rebecca JT, Savage SA. Osteosarcoma incidence and survival improvement. Cancer. 2009;115(7):1531–43.
Lindsey BA, Markel JE, Kleinerman ES. Osteosarcoma Overview. Rheumatol Ther. 2017;4(1):25–43.
Durfee RA, Mohammed M, Luu HH. Review of Osteosarcoma and Current Management. Rheumatol Ther. 2016;3(2):221–43.
Bruland ØS, Høifødt H, Hall KS, Smeland S, Fodstad Ø. Bone marrow micrometastases studied by an immunomagnetic isolation procedure in extremity localized non-metastatic osteosarcoma patients. In: Jaffe N., Bruland O. BS, editor. Pediatric and Adolescent Osteosarcoma Cancer Treatment and Research [Internet]. Springer, Boston, MA; 2009. p. 509–15. Available from: https://pubmed.ncbi.nlm.nih.gov/20213412/
Huang X, Zhao J, Bai J, Shen H, Zhang B, Deng L, et al. Risk and clinicopathological features of osteosarcoma metastasis to the lung: A population-based study. J Bone Oncol. 2019;16(November 2018).
Nataraj V, Rastogi S, Khan SA, Sharma MC, Agarwala S, Vishnubhatla S, et al. Prognosticating metastatic osteosarcoma treated with uniform chemotherapy protocol without high dose methotrexate and delayed metastasectomy: a single center experience of 102 patients. Clin Transl Oncol. 2016;18(9):937–44.
Zhang Y, Yang J, Zhao N, Wang C, Kamar S, Zhou Y, et al. Progress in the chemotherapeutic treatment of osteosarcoma. Oncol Lett. 2018;16(5):6228–37.
Galván-Salazar HR, Soriano-Hernández AD, Montes-Galindo DA, Espíritu GC, Guzman-Esquivel J, Rodríguez-Sánchez IP, et al. Preclinical trial on the use of doxycycline for the treatment of adenocarcinoma of the duodenum. Mol Clin Oncol. 2016;5(5):657–9.
Lin CC, Lo MC, Moody RR, Stevers NO, Tinsley SL, Sun D. Doxycycline targets aldehyde dehydrogenasepositive breast cancer stem cells. Oncol Rep. 2018 Jun;39(6):3041–3047.
Matsumoto T, Uchiumi T, Monji K, Yagi M, Setoyama D, Amamoto R, Matsushima Y, Shiota M, Eto M, Kang D. Doxycycline induces apoptosis via ER stress selectively to cells with a cancer stem cell-like properties: importance of stem cell plasticity. Oncogenesis. 2017 Nov 29;6(11):397.
Scatena C, Roncella M, Di Paolo A, Aretini P, Menicagli M, Fanelli G, Marini C, Mazzanti CM, Ghilli M, Sotgia F, Lisanti MP, Naccarato AG. Doxycycline, an Inhibitor of Mitochondrial Biogenesis, Effectively Reduces Cancer Stem Cells (CSCs) in Early Breast Cancer Patients: A Clinical Pilot Study. Front Oncol. 2018 Oct 12;8:452.
Cakir Y HK. Direct action by doxycycline against canine osteosarcoma cell proliferation and collagenase (MMP-1) activity in vitro. In Vivo (Brooklyn) [Internet]. 1999;13(4):327–31. Available from: https://pubmed.ncbi.nlm.nih.gov/10586373/.
Fife RS, Rougraff BT, Proctor C, Sledge GW Jr. Inhibition of proliferation and induction of apoptosis by doxycycline in cultured human osteosarcoma cells. J Lab Clin Med. 1997 Nov;130(5):530–4.
Roomi MW, Kalinovsky T, Rath M, Niedzwiecki A. In vitro modulation of MMP-2 and MMP-9 in pediatric human sarcoma cell lines by cytokines, inducers and inhibitors. Int J Oncol. 2014;44(1):27–34.
Lipowsky HH, Sah R, Lescanic A. Relative roles of doxycycline and cation chelation in endothelial glycan shedding and adhesion of leukocytes. Am J Physiol - Hear Circ Physiol. 2011;300(2):415–23.
Siddhartha R, Garg M. Molecular and clinical insights of matrix metalloproteinases into cancer spread and potential therapeutic interventions. Toxicol Appl Pharmacol. 2021 Sep 1;426:115593.
Quintero-Fabián S, Arreola R, Becerril-Villanueva E, Torres-Romero JC, Arana-Argáez V, Lara-Riegos J, et al. Role of Matrix Metalloproteinases in Angiogenesis and Cancer. Front Oncol. 2019;9(December):1–21.
Tang H, Tang Z, Jiang Y, Wei W, Lu J. Pathological and therapeutic aspects of matrix metalloproteinases: Implications in osteosarcoma. Asia Pac J Clin Oncol. 2019;15(4):218–24.
Ebrahem Q, Chaurasia SS, Vasanji A, Qi JH, Klenotic PA, Cutler A, et al. Cross-talk between vascular endothelial growth factor and matrix metalloproteinases in the induction of neovascularization in vivo. Am J Pathol [Internet]. 2010;176(1):496–503. Available from: http://dx.doi.org/10.2353/ajpath.2010.080642
Hurst RE, Bastian A, Bailey-Downs L, Ihnat MA. Targeting dormant micrometastases: Rationale, evidence to date and clinical implications. Ther Adv Med Oncol. 2016;8(2):126–37.
Armakolas A, Kaparelou M, Dimakakos A, Papageorgiou E, Armakolas N, Antonopoulos A, et al. Oncogenic role of the Ec peptide of the IGF-1Ec isoform in prostate cancer. Mol Med. 2015;21:167–79.
Liang CC, Park AY, Guan JL. In vitro scratch assay: A convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc. 2007;2(2):329–33.
Rieger AM, Nelson KL, Konowalchuk JD, Barreda DR. Modified annexin V/propidium iodide apoptosis assay for accurate assessment of cell death. J Vis Exp. 2011;(50):3–6.
Hong ES, Burkett SS, Morrow J, Lizardo MM, Osborne T, Li SQ, et al. Characterization of the metastatic phenotype of a panel of established osteosarcoma cells. Oncotarget. 2015;6(30):29469–81.
Lauvrak SU, Munthe E, Kresse SH, Stratford EW, Namløs HM, Meza-Zepeda LA, Myklebost O. Functional characterisation of osteosarcoma cell lines and identification of mRNAs and miRNAs associated with aggressive cancer phenotypes. Br J Cancer. 2013 Oct 15;109(8):2228–36. doi: 10.1038/bjc.2013.549.
Jacques C, Renema N, Ory B, Walkley CR, Grigoriadis AE, Heymann D. Murine Models of Bone Sarcomas. Methods Mol Biol. 2019;1914:331–342.
Price JE. Spontaneous and experimental metastasis models: nude mice. Methods Mol Biol. 2014;1070:223–33.
Yuan J, Ossendorf C, Szatkowski JP, Bronk JT, Maran A, Yaszemski M, Bolander ME, Sarkar G, Fuchs B. Osteoblastic and osteolytic human osteosarcomas can be studied with a new xenograft mouse model producing spontaneous metastases. Cancer Invest. 2009 May;27(4):435–42.
Luu HH, Kang Q, Park JK, Si W, Luo Q, Jiang W, Yin H, Montag AG, Simon MA, Peabody TD, Haydon RC, Rinker-Schaeffer CW, He TC. An orthotopic model of human osteosarcoma growth and spontaneous pulmonary metastasis. Clin Exp Metastasis. 2005;22(4):319–29.
Scott MC, Tomiyasu H, Garbe JR, Cornax I, Amaya C, O’Sullivan MG, et al. Heterotypic mouse models of canine osteosarcoma recapitulate tumor heterogeneity and biological behavior. DMM Dis Model Mech. 2016;9(12):1435–44.
Uluçkan Ö, Segaliny A, Botter S, Santiago JM, Mutsaers AJ. Preclinical mouse models of osteosarcoma. Bonekey Rep. 2015 May 6;4:670.
Tang X, Wang X, Zhao YY, Curtis JM, Brindley DN. Doxycycline attenuates breast cancer related inflammation by decreasing plasma lysophosphatidate concentrations and inhibiting NF-ΚB activation. Mol Cancer. 2017;16(1):1–13.
Fourman MS, Mahjoub A, Mandell JB, Yu S, Tebbets JC, Crasto JA, et al. Quantitative primary tumor indocyanine green measurements predict osteosarcoma metastatic lung burden in a mouse model. Clin Orthop Relat Res. 2018;476(3):479–87.
Frohlich JR, Alarcón CN, Toarmino CR, Sunseri AK, Hockman TM. Comparison of Serial Blood Collection by Facial Vein and Retrobulbar Methods in C57BL/6 Mice. J Am Assoc Lab Anim Sci. 2018 Jul 1;57(4):382-391.
Parasuraman S, Raveendran R, Kesavan R. Blood sample collection in small laboratory animals. J Pharmacol Pharmacother. 2010;1(2):87–93.
Jirkof P, Tourvieille A, Cinelli P, Arras M. Buprenorphine for pain relief in mice: repeated injections vs sustained-release depot formulation. Lab Anim. 2015 Jul;49(3):177–87.
Yu Y, Lee P, Ke Y, Zhang Y, Yu Q, Lee J, Li M, Song J, Chen J, Dai J, Do Couto FJ, An Z, Zhu W, Yu GL. A humanized anti-VEGF rabbit monoclonal antibody inhibits angiogenesis and blocks tumor growth in xenograft models. PLoS One. 2010 Feb 5;5(2):e9072.
Kim SW, Roh J, Park CS. Immunohistochemistry for Pathologists: Protocols, Pitfalls, and Tips. J Pathol Transl Med. 2016 Nov;50(6):411–418. doi: 10.4132/jptm.2016.08.08.
Raimondi L, De Luca A, Costa V, Amodio N, Carina V, Bellavia D, Tassone P, Pagani S, Fini M, Alessandro R, Giavaresi G. Circulating biomarkers in osteosarcoma: new translational tools for diagnosis and treatment. Oncotarget. 2017 Aug 3;8(59):100831-100851.
Zhu QS, Rosenblatt K, Huang KL, Lahat G, Brobey R, Bolshakov S, Nguyen T, Ding Z, Belousov R, Bill K, Luo X, Lazar A, Dicker A, Mills GB, Hung MC, Lev D. Vimentin is a novel AKT1 target mediating motility and invasion. Oncogene. 2011 Jan 27;30(4):457–70.
Jolly MK, Ware KE, Xu S, Gilja S, Shetler S, Yang Y, Wang X, Austin RG, Runyambo D, Hish AJ, Bartholf DeWitt S, George JT, Kreulen RT, Boss MK, Lazarides AL, Kerr DL, Gerber DG, Sivaraj D, Armstrong AJ, Dewhirst MW, Eward WC, Levine H, Somarelli JA. E-Cadherin Represses Anchorage-Independent Growth in Sarcomas through Both Signaling and Mechanical Mechanisms. Mol Cancer Res. 2019 Jun;17(6):1391–1402.
Cates JM, Dupont WD. Cytologic anaplasia is a prognostic factor in osteosarcoma biopsies, but mitotic rate or extent of spontaneous tumor necrosis are not: a critique of the College of American Pathologists Bone Biopsy template. Mod Pathol. 2017 Jan;30(1):52–59.
Jong R, Davis AM, Mendes MG, Wunder JS, Bell RS, Kandel R. Proliferative activity (ki-67 expression) and outcome in high grade osteosarcoma: a study of 27 cases. Sarcoma. 2000;4(1-2):47–55.
Ren L, Khanna C. Role of ezrin in osteosarcoma metastasis. Adv Exp Med Biol. 2014;804:181–201.
Rastogi S, Kumar R, Sankineani SR, Marimuthu K, Rijal L, Prakash S, Jalan D, Khan SA, Sharma MC. Role of vascular endothelial growth factor as a tumour marker in osteosarcoma: a prospective study. Int Orthop. 2012 Nov;36(11):2315–21.
Huang H. Matrix Metalloproteinase-9 (MMP-9) as a Cancer Biomarker and MMP-9 Biosensors: Recent Advances. Sensors (Basel). 2018 Sep 27;18(10):3249.
Lucchetti J, Fracasso C, Balducci C, Passoni A, Forloni G, Salmona M, Gobbi M. Plasma and Brain Concentrations of Doxycycline after Single and Repeated Doses in Wild-Type and APP23 Mice. J Pharmacol Exp Ther. 2019 Jan;368(1):32–40.
Dickens DS, Cripe TP. Effect of combined cyclooxygenase-2 and matrix metalloproteinase inhibition on human sarcoma xenografts. J Pediatr Hematol Oncol. 2003 Sep;25(9):709–14.
Fields GB. The Rebirth of Matrix Metalloproteinase Inhibitors: Moving Beyond the Dogma. Cells. 2019 Aug 27;8(9):984.
Sparano JA, Bernardo P, Stephenson P, Gradishar WJ, Ingle JN, Zucker S, Davidson NE. Randomized phase III trial of marimastat versus placebo in patients with metastatic breast cancer who have responding or stable disease after first-line chemotherapy: Eastern Cooperative Oncology Group trial E2196. J Clin Oncol. 2004 Dec 1;22(23):4683-90.
Evans JD, Stark A, Johnson CD, Daniel F, Carmichael J, Buckels J, Imrie CW, Brown P, Neoptolemos JP. A phase II trial of marimastat in advanced pancreatic cancer. Br J Cancer. 2001 Dec 14;85(12):1865-70.
Bramhall SR, Hallissey MT, Whiting J, Scholefield J, Tierney G, Stuart RC, Hawkins RE, McCulloch P, Maughan T, Brown PD, Baillet M, Fielding JW. Marimastat as maintenance therapy for patients with advanced gastric cancer: a randomised trial. Br J Cancer. 2002 Jun 17;86(12):1864–70.
Rudek MA, Figg WD, Dyer V, Dahut W, Turner ML, Steinberg SM, Liewehr DJ, Kohler DR, Pluda JM, Reed E. Phase I clinical trial of oral COL-3, a matrix metalloproteinase inhibitor, in patients with refractory metastatic cancer. J Clin Oncol. 2001 Jan 15;19(2):584–92.
Hanafy E, Al Jabri A, Gadelkarim G, Dasaq A, Nazim F, Al Pakrah M. Tumor histopathological response to neoadjuvant chemotherapy in childhood solid malignancies: is it still impressive? J Investig Med. 2018 Feb;66(2):289–297.
Basile P, Greengard E, Weigel B, Spector L. Prognostic Factors for Development of Subsequent Metastases in Localized Osteosarcoma: A Systematic Review and Identification of Literature Gaps. Sarcoma. 2020 Mar 18;2020:7431549.
Sannino G, Marchetto A, Kirchner T, Grünewald TGP. Epithelial-to-Mesenchymal and Mesenchymal-to-Epithelial Transition in Mesenchymal Tumors: A Paradox in Sarcomas? Cancer Res. 2017 Sep 1;77(17):4556-4561.
Qin Y, Zhang Q, Lee S, Zhong WL, Liu YR, Liu HJ, Zhao D, Chen S, Xiao T, Meng J, Jing XS, Wang J, Sun B, Dai TT, Yang C, Sun T, Zhou HG. Doxycycline reverses epithelial-to-mesenchymal transition and suppresses the proliferation and metastasis of lung cancer cells. Oncotarget. 2015 Dec 1;6(38):40667-79.
Deryugina EI, Quigley JP. Tumor angiogenesis: MMP-mediated induction of intravasation- and metastasis-sustaining neovasculature. Matrix Biol. 2015 May-Jul;44-46:94–112.
Li LT, Jiang G, Chen Q, Zheng JN. Ki67 is a promising molecular target in the diagnosis of cancer (review). Mol Med Rep. 2015 Mar;11(3):1566–72.
Khan K, Long B, Deshpande GM, Fox PL. Bidirectional Tumor-Promoting Activities of Macrophage Ezrin. Int J Mol Sci. 2020 Oct 19;21(20):7716.
Zhang Y, Zhang L, Zhang G, Li S, Duan J, Cheng J, Ding G, Zhou C, Zhang J, Luo P, Cai D, Kuang L, Zhou Y, Tong L, Yu X, Zhang L, Xu L, Yu L, Shi X, Ke A. Osteosarcoma metastasis: prospective role of ezrin. Tumour Biol. 2014 Jun;35(6):5055–9.
PosthumaDeBoer J, Witlox MA, Kaspers GJ, van Royen BJ. Molecular alterations as target for therapy in metastatic osteosarcoma: a review of literature. Clin Exp Metastasis. 2011 Jun;28(5):493–503.
Stefanovic S, Wirtz R, Deutsch TM, Hartkopf A, Sinn P, Varga Z, Sobottka B, Sotiris L, Taran FA, Domschke C, Hennigs A, Brucker SY, Sohn C, Schuetz F, Schneeweiss A, Wallwiener M. Tumor biomarker conversion between primary and metastatic breast cancer: mRNA assessment and its concordance with immunohistochemistry. Oncotarget. 2017 May 19;8(31):51416-51428.
Cekanova M, Rathore K. Animal models and therapeutic molecular targets of cancer: utility and limitations. Drug Des Devel Ther. 2014 Oct 14;8:1911–21.

PDF

Version 1

posted 08 Nov, 2021

You are reading this older preprint version

Read the latest preprint version →

Inhibitory Effects of Doxycycline on Tumor Progression In vitro and on Metastases in Early-Stage Osteosarcoma Xenografts Mice.

Status:

Version 1

Abstract

Figures

1. Background

2. Materials And Methods

2.9 Histological Examination And Immunohistochemistry (Ihc)

3. Results

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Status:

Version 1