Combination immunotherapy using G-CSF and oncolytic virotherapy reduces tumor growth in osteosarcoma

Abstract

Background: Osteosarcoma is the most common malignant solid tumor that affects bones, however, survival rates of patients with relapsed osteosarcoma have not improved in the last 30 years. Oncolytic virotherapy, which uses viruses designed to selectively replicate in cancer cells, has emerged as a promising treatment for solid tumors. Our group uses mesenchymal stem cells (MSCs) to transport oncolytic adenoviruses (OAds) to the tumor site, a therapeutic strategy called Celyvir. This treatment has been already applied in human patients, canine patients and different mouse models. In parallel, previous results have probed that administration of granulocyte-colony stimulating factor (G-CSF) increased immune infiltration in tumors. We then hypothesized that the mobilization of immune cells by G-CSF may increase the antitumor efficacy of Celyvir treatment by increasing the immune infiltration into the tumors.

Methods: In this study, we use a murine version of Celyvir consisting in murine MSCs carrying the murine OAd dlE102-here called OAd-MSCs-in an immunocompetent model of osteosarcoma. We tested the antitumoral efficacy of the combination of OAd-MSCs plus G-CSF.

Results: Our results show that treatment with OAd-MSCs or the union of OAd-MSCs with G-CSF (Combination) significantly reduced tumor growth of osteosarcoma in vivo. Moreover, treated tumors presented higher tumor infiltration of immune cells-especially tumor-infiltrating lymphocytes-and reduced T cell exhaustion, which seems to be enhanced in tumors treated with the Combination. The comparison of our results to those obtained from a cohort of pediatric osteosarcoma patients showed that the virotherapy induces immunological changes similar to those observed in patients with good prognosis.

Conclusions: The results open the possibility of using cellular virotherapy for the treatment of bone cancers. Indeed, its combination with G-CSF may be considered for the improvement of the therapy.

Trial registration: ClinicalTrials.gov NCT01844661.

Keywords: immunotherapy; lymphocytes; oncolytic virotherapy; programmed cell death 1 receptor; t-lymphocytes; tumor-infiltrating.

Conflict of interest statement

Competing interests: None declared.

© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

In vitro oncolytic effect of oncolytic adenovirus dlE102 in murine osteosarcoma cell lines. (A) For flow cytometry viability assay, cells were infected at different MOI (1–50) and cytotoxicity was measured 7 days later using LIVE/DEAD viability kit, showing red fluorescence for dead cells and green fluorescence for live cells. (B) Percentages refers to dead cells and statistical differences are compared with control group. (C) For luciferase viability assay, K5 cells and p53−/−Rb−/− MSCs were transduced with a lentiviral vector containing a firefly luciferase cassette and infected at MOI 1 and 10. Luminescence of alive cells was quantified 4 days later and expressed as infected/control ratio. (D) Bright field pictures show the cytopathic effect of K5 cells and p53−/−Rb−/− MSCs 4 days after infection with dlE102 at MOI 10. The experiment was repeated twice (N=2). Unpaired t-test. *p<0.05, **p<0.01, ***p<0.001. MOI, multiplicity of infection; MSCs, mesenchymal stem cells.

Figure 2

In vivo antitumor efficacy of OAd-MSCs and Combination in osteosarcoma. (A) Graph represents the experimental design and antitumor effect of OAd-MSCs and Combination in BALB/c mice bearing subcutaneous K5 tumors. Lines represent the mean+SEM of tumor volume in groups treated with PBS (black, n=11), MSCs (gray, n=4), G-CSF (dark gray, n=9), OAd-MSCs (blue, n=9) or Combination (red, n=7). (B) Representative pictures and tumor volumes at end point. Mann-Whitney U test. (C) Follow-up of tumor volume in mice treated with MSCs, G-CSF, OAd-MSCs or Combination (individual values) and PBS group (mean+SEM, black line). (D, E) Tumor weight and tumor growth at end point. Mann-Whitney U test. (F) Kaplan-Meier survival curve of treated mice. Log-rank (Mantel-Cox) test. *p

Figure 3

Tumor-infiltrating immune cells in osteosarcoma. (A) Density of tumor-infiltrating leukocytes, NK cells, and adaptive immune cells at end point expressed as percentage per gram of tumor. CD4+/CD8+ ratio is also represented. (B, C) Percentage of tumor-infiltrating lymphocytes, CD4+ and CD8+ T cells expressing PD-1 or CD137. (D) Density of tumor-infiltrating innate immune cells. (E) Ratio of pro-inflammatory/anti-inflammatory status of macrophages (M1/M2) and neutrophils (N1/N2). Mann-Whitney U test (n=3–9). *p<0.05. G-CSF, granulocyte-colony stimulating factor; LMR, lymphocyte-monocyte ratio; MSCs, mesenchymal stem cells; NLR, neutrophil-lymphocyte ratio; NK, natural killer; OAd-MSCs, MSCs carrying the murine oncolytic adenovirus dlE102; PBS, phosphate-buffered saline; PD-1, programmed cell death protein 1

Figure 4

Leukocyte infiltration and tumor microenvironment in osteosarcoma after treatment with OAd-MSCs and Combination. (A–C) Representative images of immunohistochemistry of CD45+ cells in osteosarcoma tumors treated with PBS (A), OAd-MSCs (B) or Combination (C). Upper panel shows images of the periphery, while lower panel shows images of the core of the tumors. (D) Infiltration of CD45+ cells in tumors quantified in 40× high-power fields. (E) Protein expression of CD45 and TBP in tumors treated with PBS, OAd-MSCs or Combination. (F) Heat-map showing the pro-inflammatory cytokine array performed in tumors treated with PBS, OAd-MSCs or Combination. White represents the lowest expression while dark blue represents the highest expression. (G) Quantification by integrated density of pro-inflammatory cytokines differentially expressed in tumors treated with PBS, OAd-MSCs or Combination. Unpaired t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. OAd-MSCs, mesenchymal stem cells carrying the murine oncolytic adenovirus dlE102; PBS, phosphate-buffered saline; TBP, TATA-box binding protein.

Figure 5

Histopathological features in osteosarcoma after treatment with OAd-MSCs or Combination. (A) Representative X-ray imaging of osteosarcoma tumors treated with PBS, G-CSF, OAd-MSCs or Combination. Mineralized area is marked in red, while cellular area is marked in yellow. (B) Percentage of cellular tumor measured in X-ray images. (C) Representative images of Masson’s-Trichrome staining. (D) Percentage of cellular tumor measured from Masson’s stained slide images. Unpaired t-test. *p

Figure 6

Comparison of preclinical results in the murine osteosarcoma model with clinical osteosarcoma patients. (A) Graph represents the experimental flow followed for the genomic analysis of osteosarcoma patients. (B) Hazard ratio of protective (1, red) regarding immune cell populations present in osteosarcoma tumors. (C) Kaplan-Meier survival curve of osteosarcoma patients regarding high (purple) or low (yellow) levels of different immune biomarkers. Log-rank (Mantel-Cox) test. (D) Violin plots represent differences in NLR, CD4+/CD8+ ratio, T cells and CD8+ T cells in patients according to their current clinical status (dead in orange; live in green). (E) Violin plots represent differences in NLR, CD4+/CD8+ ratio, T cells and CD8+ T cells in mice treated with PBS, OAd-MSCs or Combination. Mann-Whitney U test. *p<0.05. NLR, neutrophil-lymphocyte ratio; OAd-MSCs, mesenchymal stem cells carrying the murine oncolytic adenovirus dlE102; PBS, phosphate-buffered saline.