Radiotherapy in bone sarcoma: the quest for better treatment option.

Bone sarcomas are rare tumors representing 0.2% of all cancers. While osteosarcoma and Ewing sarcoma mainly affect children and young adults, chondrosarcoma and chordoma have a preferential incidence in people over the age of 40. Despite this range in populations affected, all bone sarcoma patients require complex transdisciplinary management and share some similarities. The cornerstone of all bone sarcoma treatment is monobloc resection of the tumor with adequate margins in healthy surrounding tissues. Adjuvant chemo- and/or radiotherapy are often included depending on the location of the tumor, quality of resection or presence of metastases. High dose radiotherapy is largely applied to allow better local control in case of incomplete primary tumor resection or for unresectable tumors. With the development of advanced techniques such as proton, carbon ion therapy, radiotherapy is gaining popularity for the treatment of bone sarcomas, enabling the delivery of higher doses of radiation, while sparing surrounding healthy tissues. Nevertheless, bone sarcomas are radioresistant tumors, and some mechanisms involved in this radioresistance have been reported. Hypoxia for instance, can potentially be targeted to improve tumor response to radiotherapy and decrease radiation-induced cellular toxicity. In this review, the benefits and drawbacks of radiotherapy in bone sarcoma will be addressed. Finally, new strategies combining a radiosensitizing agent and radiotherapy and their applicability in bone sarcoma will be presented.

The in vivo radiosensitizing effect of gold nanoparticles based MRI contrast agents.

Owing to the high atomic number (Z) of gold element, the gold nanoparticles appear as very promising radiosensitizing agents. This character can be exploited for improving the selectivity of radiotherapy. However, such an improvement is possible only if irradiation is performed when the gold content is high in the tumor and low in the surrounding healthy tissue. As a result, the beneficial action of irradiation (the eradication of the tumor) should occur while the deleterious side effects of radiotherapy should be limited by sparing the healthy tissue. The location of the radiosensitizers is therefore required to initiate the radiotherapy. Designing gold nanoparticles for monitoring their distribution by magnetic resonance imaging (MRI) is an asset due to the high resolution of MRI which permits the accurate location of particles and therefore the determination of the optimal time for the irradiation. We recently demonstrated that ultrasmall gold nanoparticles coated by gadolinium chelates (Au@DTDTPA-Gd) can be followed up by MRI after intravenous injection. Herein, Au@DTDTPA and Au@DTDTPA-Gd were prepared in order to evaluate their potential for radiosensitization. Comet assays and in vivo experiments suggest that these particles appear well suited for improving the selectivity of the radiotherapy. The dose which is used for inducing similar levels of DNA alteration is divided by two when cells are incubated with the gold nanoparticles prior to the irradiation. Moreover, the increase in the lifespan of tumor bearing rats is more important when the irradiation is performed after the injection of the gold nanoparticles. In the case of treatment of rats with a brain tumor (9L gliosarcoma, a radio-resistant tumor in a radiosensitive organ), the delay between the intravenous injection and the irradiation was determined by MRI.

The In Vivo Radiosensitizing Effect of Gold Nanoparticles Based MRI Contrast Agents.

Owing to the high atomic number (Z) of gold element, the gold nanoparticles appear as very promising radiosensitizing agents. This character can be exploited for improving the selectivity of radiotherapy. However, such an improvement is possible only if irradiation is performed when the gold content is high in the tumor and low in the surrounding healthy tissue. As a result, the beneficial action of irradiation (the eradication of the tumor) should occur while the deleterious side effects of radiotherapy should be limited by sparing the healthy tissue. The location of the radiosensitizers is therefore required to initiate the radiotherapy. Designing gold nanoparticles for monitoring their distribution by magnetic resonance imaging (MRI) is an asset due to the high resolution of MRI which permits the accurate location of particles and therefore the determination of the optimal time for the irradiation. We recently demonstrated that ultrasmall gold nanoparticles coated by gadolinium chelates (Au@DTDTPA-Gd) can be followed up by MRI after intravenous injection. Herein, Au@DTDTPA and Au@DTDTPA-Gd were prepared in order to evaluate their potential for radiosensitization. Comet assays and in vivo experiments suggest that these particles appear well suited for improving the selectivity of the radiotherapy. The dose which is used for inducing similar levels of DNA alteration is divided by two when cells are incubated with the gold nanoparticles prior to the irradiation. Moreover, the increase in the lifespan of tumor bearing rats is more important when the irradiation is performed after the injection of the gold nanoparticles. In the case of treatment of rats with a brain tumor (9L gliosarcoma, a radio-resistant tumor in a radiosensitive organ), the delay between the intravenous injection and the irradiation was determined by MRI.

Cancer characterization using light backscattering spectroscopy and quantitative ultrasound: an ex vivo study on sarcoma subtypes.

Histological analysis is the gold standard method for cancer diagnosis. However, it is prone to subjectivity and sampling bias. In response to these limitations, we introduce a quantitative bimodal approach that aims to provide non-invasive guidance towards suspicious regions. Light backscattering spectroscopy and quantitative ultrasound techniques were combined to characterize two different bone tumor types from animal models: chondrosarcomas and osteosarcomas. Two different cell lines were used to induce osteosarcoma growth. Histological analyses were conducted to serve as references. Three ultrasound parameters and intensities of the light reflectance profiles showed significant differences between chondrosarcomas and osteosarcomas at the 5% level. Likewise, variations in the same biomarkers were reported for the two types of osteosarcoma, despite their similar morphology observed in the histological examinations. These observations show the sensitivity of our techniques in probing fine tissue properties. Secondly, the ultrasound spectral-based technique identified the mean size of chondrosarcoma cells and nuclei with relative errors of about 22% and 9% respectively. The optical equivalent technique correctly extracted scatterer size distributions that encompass nuclei and cells for chondrosarcomas and osteosarcomas ([Formula: see text] and [Formula: see text] respectively). The optical scattering contributions of nuclei were estimated at 52% for the chondrosarcomas and 69% for the osteosarcomas, probably indicating the abundant and the absent extracellular matrix respectively. Thus, the ultrasound and the optical methods brought complementary parameters. They successfully estimated morphological parameters at the cellular and the nuclear scales, making our bimodal technique promising for tumor characterization.

A TLR4 Agonist Induces Osteosarcoma Regression by Inducing an Antitumor Immune Response and Reprogramming M2 Macrophages to M1 Macrophages.

Osteosarcoma (OsA) has limited treatment options and stagnant 5-year survival rates. Its immune microenvironment is characterized by a predominance of tumor-associated macrophages (TAMs), whose role in OsA progression remain unclear. Nevertheless, immunotherapies aiming to modulate macrophages activation and polarization could be of interest for OsA treatment. In this study, the antitumor effect of a liposome-encapsulated chemically detoxified lipopolysaccharide (Lipo-MP-LPS) was evaluated as a therapeutic approach for OsA. Lipo-MP-LPS is a toll-like receptor 4 (TLR4) agonist sufficiently safe and soluble to be IV administered at effective doses. Lipo-MP-LPS exhibited a significant antitumor response, with tumor regression in 50% of treated animals and delayed tumor progression in the remaining 50%. The agent inhibited tumor growth by 75%, surpassing the efficacy of other immunotherapies tested in OsA. Lipo-MP-LPS modulated OsA's immune microenvironment by favoring the transition of M2 macrophages to M1 phenotype, creating a proinflammatory milieu and facilitating T-cell recruitment and antitumor immune response. Overall, the study demonstrates the potent antitumor effect of Lipo-MP-LPS as monotherapy in an OsA immunocompetent model. Reprogramming macrophages and altering the immune microenvironment likely contribute to the observed tumor control. These findings support the concept of immunomodulatory approaches for the treatment of highly resistant tumors like OsA.

The TLR3 L412F polymorphism prevents TLR3-mediated tumor cell death induction in pediatric sarcomas.

Toll-like receptor 3 (TLR3) is a pattern recognition receptor mainly known for its role in innate immune response to infection. Indeed, binding of double-stranded RNA (dsRNA) to TLR3 triggers a pro-inflammatory cascade leading to cytokine release and immune cell activation. Its anti-tumoral potential has emerged progressively, associated with a direct impact on tumor cell death induction and with an indirect action on immune system reactivation. Accordingly, TLR3 agonists are currently being tested in clinical trials for several adult cancers. Meanwhile, TLR3 variants have been linked to auto-immune disorders, and as risk factors of viral infection and cancers. However, aside from neuroblastoma, TLR3 role in childhood cancers has not been evaluated. Here, by integrating public transcriptomic data of pediatric tumors, we unveil that high TLR3 expression is largely associated with a better prognosis in childhood sarcomas. Using osteosarcomas and rhabdomyosarcomas as models, we show that TLR3 efficiently drives tumor cell death in vitro and induces tumor regression in vivo. Interestingly, this anti-tumoral effect was lost in cells expressing the homozygous TLR3 L412F polymorphism, which is enriched in a rhabdomyosarcomas cohort. Thus, our results demonstrate the therapeutic potential associated with the targeting of TLR3 in pediatric sarcomas, but also the need to stratify patients eligible for this clinical approach with respect to the TLR3 variants expressed.

An OMA1 redox site controls mitochondrial homeostasis, sarcoma growth, and immunogenicity.

Aggressive tumors often display mitochondrial dysfunction. Upon oxidative stress, mitochondria undergo fission through OMA1-mediated cleavage of the fusion effector OPA1. In yeast, a redox-sensing switch participates in OMA1 activation. 3D modeling of OMA1 comforted the notion that cysteine 403 might participate in a similar sensor in mammalian cells. Using prime editing, we developed a mouse sarcoma cell line in which OMA1 cysteine 403 was mutated in alanine. Mutant cells showed impaired mitochondrial responses to stress including ATP production, reduced fission, resistance to apoptosis, and enhanced mitochondrial DNA release. This mutation prevented tumor development in immunocompetent, but not nude or cDC1 dendritic cell-deficient, mice. These cells prime CD8+ lymphocytes that accumulate in mutant tumors, whereas their depletion delays tumor control. Thus, OMA1 inactivation increased the development of anti-tumor immunity. Patients with complex genomic soft tissue sarcoma showed variations in the level of OMA1 and OPA1 transcripts. High expression of OPA1 in primary tumors was associated with shorter metastasis-free survival after surgery, and low expression of OPA1, with anti-tumor immune signatures. Targeting OMA1 activity may enhance sarcoma immunogenicity.

The coenzyme A precursor pantethine enhances antitumor immunity in sarcoma.

The tumor microenvironment is a dynamic network of stromal, cancer, and immune cells that interact and compete for resources. We have previously identified the Vanin1 pathway as a tumor suppressor of sarcoma development via vitamin B5 and coenzyme A regeneration. Using an aggressive sarcoma cell line that lacks Vnn1 expression, we showed that the administration of pantethine, a vitamin B5 precursor, attenuates tumor growth in immunocompetent but not nude mice. Pantethine boosts antitumor immunity, including the polarization of myeloid and dendritic cells towards enhanced IFNγ-driven antigen presentation pathways and improved the development of hypermetabolic effector CD8+ T cells endowed with potential antitumor activity. At later stages of treatment, the effect of pantethine was limited by the development of immune cell exhaustion. Nevertheless, its activity was comparable with that of anti-PD1 treatment in sensitive tumors. In humans, VNN1 expression correlates with improved survival and immune cell infiltration in soft-tissue sarcomas, but not in osteosarcomas. Pantethine could be a potential therapeutic immunoadjuvant for the development of antitumor immunity.

Fusion-negative rhabdomyosarcoma 3D organoids to predict effective drug combinations: A proof-of-concept on cell death inducers.

Rhabdomyosarcoma (RMS) is the main form of pediatric soft-tissue sarcoma. Its cure rate has not notably improved in the last 20 years following relapse, and the lack of reliable preclinical models has hampered the design of new therapies. This is particularly true for highly heterogeneous fusion-negative RMS (FNRMS). Although methods have been proposed to establish FNRMS organoids, their efficiency remains limited to date, both in terms of derivation rate and ability to accurately mimic the original tumor. Here, we present the development of a next-generation 3D organoid model derived from relapsed adult and pediatric FNRMS. This model preserves the molecular features of the patients' tumors and is expandable for several months in 3D, reinforcing its interest to drug combination screening with longitudinal efficacy monitoring. As a proof-of-concept, we demonstrate its preclinical relevance by reevaluating the therapeutic opportunities of targeting apoptosis in FNRMS from a streamlined approach based on transcriptomic data exploitation.

Micro-RNA profiles in osteosarcoma as a predictive tool for ifosfamide response.

Micro-RNAs (miRNA) are currently used as cancer biomarkers for hematological cancers and solid tumors. Osteosarcoma is the first primary malignant bone tumor, characterized by a complex genetic and resistance to conventional treatments. For this latter property, the median survival has not been improved since 1990 despite preoperative administration of chemotherapeutic agents. The prediction of tumor response before chemotherapy treatment would constitute a major progress for this pathology. We assessed in this study if miRNA profiling could surpass the current limitations for osteosarcoma diagnosis. We measured the miRNA expression in different osteosarcoma samples: (i) 27 osteosarcoma paraffin-embedded tumors from patients, (ii) human osteosarcoma cell lines, and (iii) tumors from a syngeneic rat osteosarcoma model, recapitulating human osteosarcoma. miRNA profiles were determined using microfluidic cards performing high-throughput TaqMan(®) -based PCR assays, called TaqMan(®) Low Density Arrays. Osteosarcoma of rat and human origins showed a miRNA signature, which could discriminate good from bad responders. In particular, we identified five discriminating miRNAs (miR-92a, miR-99b, miR-132, miR-193a-5p and miR-422a) in patient tumors, which could be easily transferable to diagnosis. These discriminating miRNAs, as well as those identified in rat, targeted the TGFß, the Wnt and the MAP kinase pathways. These results indicate that our platform constitutes a potent diagnostic tool to predict tumor sensitivity to a drug in attempt to better adapt treatment to tumor biological specificities and also to identify new potential therapeutic strategies.

Caspase-8 deficiency induces a switch from TLR3 induced apoptosis to lysosomal cell death in neuroblastoma.

In cancer cells only, TLR3 acquires death receptor properties by efficiently triggering the extrinsic pathway of apoptosis with Caspase-8 as apical protease. Here, we demonstrate that in the absence of Caspase-8, activation of TLR3 can trigger a form of programmed cell death, which is distinct from classical apoptosis. When TLR3 was activated in the Caspase-8 negative neuroblastoma cell line SH-SY5Y, cell death was accompanied by lysosomal permeabilization. Despite caspases being activated, lysosomal permeabilization as well as cell death were not affected by blocking caspase-activity, positioning lysosomal membrane permeabilization (LMP) upstream of caspase activation. Taken together, our data suggest that LMP with its deadly consequences represents a "default" death mechanism in cancer cells, when Caspase-8 is absent and apoptosis cannot be induced.

Aldehyde Dehydrogenase, a Therapeutic Target in Chordoma: Analysis in 3D Cellular Models.

Chordomas are rare, slow-growing tumors of the axial skeleton. These tumors are locally aggressive and refractory to conventional therapies. Radical surgery and radiation remain the first-line treatments. Despite these aggressive treatments, chordomas often recur and second-line treatment options are limited. The mechanisms underlying chordoma radioresistance remain unknown, although several radioresistant cancer cells have been shown to respond favorably to aldehyde dehydrogenase (ALDH) inhibition. The study of chordoma has been delayed by small patient cohorts and few available models due to the scarcity of these tumors. We thus created cellular 3D models of chordoma by using low-adherence culture systems. Then, we evaluated their radiosensitivity using colony-forming and spheroid size assays. Finally, we determined whether pharmacologically inhibiting ALDH increased their radiosensitivity. We found that 3D cellular models of chordoma (derived from primary, relapse, and metastatic tumors) reproduce the histological and gene expression features of the disease. The metastatic, relapse, and primary spheroids displayed high, medium, and low radioresistance, respectively. Moreover, inhibiting ALDH decreased the radioresistance in all three models.

Corrigendum to: ``The immune landscape of chondrosarcoma reveals an immunosuppressive environment in the dedifferentiated subtypes and exposes CSFR1+ macrophages as a promising therapeutic target.'' J Bone Oncol. 2020 Feb; 20: 100271.

[This corrects the article DOI: 10.1016/j.jbo.2019.100271.].

18F-FDG PET SUVmax correlates with osteosarcoma histologic response to neoadjuvant chemotherapy: preclinical evaluation in an orthotopic rat model.

UNLABELLED: Assessment of osteosarcoma response to neoadjuvant chemotherapy is performed by histopathologic analysis after surgical resection of the primary tumor. The purpose of this study was to evaluate whether (18)F-FDG PET could be a noninvasive surrogate to histopathologic analysis and allow for earlier response evaluation to neoadjuvant chemotherapy in osteosarcoma. METHODS: Metabolic response to neoadjuvant chemotherapy was assessed in immunocompetent rats with a preestablished orthotopic osteosarcoma using (18)F-FDG PET before and after receiving 2 doses of ifosfamide. Comparison was then made by assessing histologic responses on euthanized animals. RESULTS: Maximum standardized uptake value (SUVmax) measured by (18)F-FDG PET after 2 doses of chemotherapy was correlated to histologic classification (P < 0.01). An SUVmax less than 15 corresponded to good responders, whereas an SUVmax greater than 15 but less than 20 and an SUVmax greater than 20 corresponded to partial responders or nonresponders, respectively. A 40% decrease in SUVmax between the first and second (18)F-FDG PET scans distinguished between partial and good response to chemotherapy. CONCLUSION: Determination of SUVmax using semiquantitative (18)F-FDG PET predicts response to neoadjuvant chemotherapy earlier than does histologic analysis.

Infiltrating Myeloid Cells Drive Osteosarcoma Progression via GRM4 Regulation of IL23.

The glutamate metabotropic receptor 4 (GRM4) locus is linked to susceptibility to human osteosarcoma, through unknown mechanisms. We show that Grm4-/- gene-targeted mice demonstrate accelerated radiation-induced tumor development to an extent comparable with Rb1+/- mice. GRM4 is expressed in myeloid cells, selectively regulating expression of IL23 and the related cytokine IL12. Osteosarcoma-conditioned media induce myeloid cell Il23 expression in a GRM4-dependent fashion, while suppressing the related cytokine Il12. Both human and mouse osteosarcomas express an increased IL23:IL12 ratio, whereas higher IL23 expression is associated with worse survival in humans. Consistent with an oncogenic role, Il23 -/- mice are strikingly resistant to osteosarcoma development. Agonists of GRM4 or a neutralizing antibody to IL23 suppressed osteosarcoma growth in mice. These findings identify a novel, druggable myeloid suppressor pathway linking GRM4 to the proinflammatory IL23/IL12 axis. SIGNIFICANCE: Few novel systemic therapies targeting osteosarcoma have emerged in the last four decades. Using insights gained from a genome-wide association study and mouse modeling, we show that GRM4 plays a role in driving osteosarcoma via a non-cell-autonomous mechanism regulating IL23, opening new avenues for therapeutic intervention.See related commentary by Jones, p. 1484.This article is highlighted in the In This Issue feature, p. 1469.

Bcl-xl as the most promising Bcl-2 family member in targeted treatment of chondrosarcoma.

Chondrosarcomas are malignant cartilage tumors showing relative resistance to conventional chemo- and radiotherapy. Previous studies showed that chondrosarcoma cells could be sensitized to chemotherapy by inhibiting the Bcl-2 family members Bcl-2, Bcl-xl and Bcl-w using ABT-737. In this study we explored the specific role of Bcl-2 family members to identify the most important player in chondrosarcoma cell survival and chemo resistance. Immunohistochemistry was performed on tissue microarrays containing 137 conventional chondrosarcomas of different grades. Selective inhibition of Bcl-2 (S55746) or Bcl-xl (WEHI-539 or A-1155463) and the combination with doxorubicin or cisplatin was investigated in a panel of 8 chondrosarcoma cell lines using presto blue viability assays and caspase 3/7 glo apoptosis assays. In addition Bcl-2 and Bcl-xl inhibition was investigated in an orthotopic Swarm Rat Chondrosarcoma (SRC) model. Bcl-2 and Bcl-xl were most abundantly expressed in the primary tumors, and expression increased with increasing histological grade. A subset of chondrosarcoma cell lines was sensitive to selective inhibition of Bcl-xl, and synergy was observed with doxorubicin or cisplatin in 3 out of 8 chondrosarcoma cell lines resulting in apoptosis. Conversely, selective inhibition of Bcl-2 was not effective in chondrosarcoma cell lines and could not sensitize to chemotherapy. In vivo, selective inhibition of Bcl-xl, but not Bcl-2 resulted in a decrease in tumor growth rate, even though no sensitization to doxorubicin was observed. These results suggest that among the Bcl-2 family members, Bcl-xl is most important for chondrosarcoma survival. Further research is needed to validate whether single or combination treatment with chemotherapy will be beneficial for chondrosarcoma patients.

Molecular mechanisms regulating the angiogenic phenotype in tumors: clinical impact in the future.

Tumor progression depends on the angiogenic switch. In this review, we recapitulate the molecular mechanisms involved in this angiogenic switch. The rat osteosarcoma model employed would permit further studies in the sequential events leading to initial recruitment of blood vessels and could lead to development of an angiogenesis-based panel of circulating blood cells (endothelial cells, endothelial progenitor cells and accessory cells) that can be quantified and used to detect microscopic tumors or to follow the efficacy of antiangiogenic therapy. Such a result would lead to the possibility of early therapy in cancer progression.