Osteosarcoma, a common bone cancer in dogs, affects more than 10,000 dogs in the U.S. each year. While chemotherapy is generally effective at killing some of the cancer cells, the numerous side effects can be painful and often a subset of cancer cells exist that are resistant to chemotherapy.
To offer an alternative, Jeffrey Bryan, a professor at the University of Missouri’s College of Veterinary Medicine, and the veterinary oncology team collaborated with ELIAS Animal Health to create a vaccine from a dog’s own tumor to target and kill cancer cells in dogs suffering from osteosarcoma.
Now, the success of this treatment in dogs has led the Food and Drug Administration (FDA) to grant a rare fast-track designation for ELIAS Animal Health’s parent organization, TVAX Biomedical, to use the ELIAS immunotherapy approach to treat glioblastoma multiforme, a tumorous brain cancer in humans.
“This precision medicine approach uses the patient’s own tumor to make a vaccine, which stimulates the immune system against the abnormal proteins specific to the patient’s tumor, causing the body to generate white blood cells, called lymphocytes,” said Bryan, who also serves as the associate director of comparative oncology for the Ellis Fischel Cancer Center and a faculty research lead for the NextGen Precision Health Institute.
“We then harvest and expand these lymphocytes outside the body, which activates them so they are highly aggressive toward their target. By infusing them back into the patient’s body they can seek out and destroy the harmful cancer cells.”
A just-completed clinical trial at MU’s College of Veterinary Medicine found that dogs receiving this therapy had more than 400 days of cancer survival compared to about 270 days for dogs receiving chemotherapy in a separate study by the National Cancer Institute.
By positively impacting health outcomes in dogs with bone cancer, the FDA granted fast-track designation for this approach to be used in human trials to treat brain cancer in people.
“Both osteosarcoma in dogs and glioblastoma multiforme in people are very aggressive diseases that tend to take the patient’s life quickly, and they both express mutant proteins that can be targets for the immune system,” said Bryan, who serves on the scientific advisory board for ELIAS Animal Health.
“The beauty of this immunotherapy approach is it can be theoretically generalized for any kind of cancer. The advancement to these human trials shows that we can apply this technology to help treat different diseases that are very deadly and have few effective therapies currently.”
ELIAS Animal Health is continuing the development of this immunotherapy for osteosarcoma in pursuit of approval from the U.S. Department of Agriculture so that the treatment can be utilized on dogs across North America.
Also, if TVAX Biomedical’s human trials are successfully able to treat glioblastoma multiforme, the immunotherapy approach could be expanded to treat other cancers in humans.
“My hope is that one day this approach can be used to treat bone cancer in children,” Bryan said. “My overall goal is to be part of discoveries that not only benefit dogs but humans as well.”
Bryan’s research is an example of precision medicine, a key component of the NextGen Precision Health Initiative. By partnering with government and industry leaders, the initiative will help accelerate medical breakthroughs for both patients in Missouri and beyond.
Osteosarcomas (OSs) are the most common primary malignant bone sarcomas, with a bimodal age distribution. The highest incidence is in children and adolescents (median of age of 18), with a second smaller peak of incidence in elderly individuals over 60 years.
Worldwide, the incidence of OS is around one to three cases annually per million individuals . These tumors develop mainly in the long bones (femur, tibia, humerus), close to the growth plate in the bone metaphysis, and less frequently in the skull, jaw, and pelvis.
OSs are characterized by the presence of transformed osteoblastic cells producing osteoid matrix. Nevertheless, the precise identity of the cell at the origin of the tumor remains unknown. Evidence supports the idea of an origin of OS in mesenchymal stem/stromal cells (MSCs) and/or in more committed osteoblastic precursors [2,3].
Since the introduction of chemotherapies to treat OS the late 70s, patients diagnosed with OS receive a neo-adjuvant treatment followed by a post-surgery adjuvant therapy with a cocktail of chemotherapies, i.e., high-dose methotrexate (12 g/m2), etoposide, and ifosfamide for children and young adults (<25 years) in the French OS2006/sarcome-09 study , or other protocols combining doxorubicin, cisplatin, and ifosfamide with or without high-dose methotrexate [5,6,7].
With these therapeutic regimens, the 5 year survival has reached 78% for children and young adults with localized disease, but still remains at only 20% in patients with metastasis at diagnosis or in relapse [1,4].
Moreover, in the last 40 years, survival has not notably improved for patients without metastases and has not improved at all for metastatic patients . Therefore, improving therapy for OS remains a constant and major goal for many worldwide research and clinical groups.
A major characteristic of OSs tumors is their heterogeneity, both at the intra-tumoral level and also between individuals. Therefore, the common genomic initiating biological processes driving osteosarcomagenesis are still not identified.
The complexity of the somatic genome of OS is a major cause of intra-tumoral heterogeneity, characterized by chromosomal aneuploidy, alteration of genes by mutation and/or variation of copy number, genomic instability featured by massive rearrangement through chromotripsis, and the presence of patterns of localized hypermutated regions, named kataegis .
A small set of genes has been found to be recurrently mutated in OS (TP53, RB, MDM2, ATRX, and DLG2) . Recently, a subset of OSs was described with genomic alterations in genes of the DNA repair pathways, reminiscent of BRCA1/2-deficient tumors .
Several inherited syndromes such as Li–Fraumeni, Rothmund–Thomson, Werner, Bloom, and retinoblastoma familial cancers have also been associated with a predisposition to developing OS .
Nevertheless, in the vast majority of cases (95%), OSs appear as sporadic events. Overall, poorly defined oncogenic events associated with high cellular heterogeneity of tumor cells make the development of molecular targeted therapies devoted exclusively to tumor cells difficult.
Bone sarcomas, and in particular OSs, grow in the bone microenvironment, a very specialized, complex, and highly dynamic environment composed of bone cells (osteoclasts, osteoblasts, osteocytes), stromal cells (MSCs, fibroblasts), vascular cells (endothelial cells and pericytes), immune cells (macrophages, lymphocytes), and a mineralized extracellular matrix (ECM).
In physiological conditions, a coordinated and fine-tuned orchestrated activity of bone, vascular, and stromal cells ensures bone homeostasis through intense paracrine and cellular communications.
According to Paget’s theory , tumor cells find in this microenvironment a fertile soil to seed and manage to highjack bone physiological pathways to their advantage in order to survive and grow.
Cross-talk between OS and the bone microenvironment involves numerous environmental signals, induced by multiple cytokines, chemokines, and soluble growth factors , but also conveyed by extracellular vesicles (EVs), considered today to be effective vectors of communication between cells .
In OS, the difficulty of designing and validating new therapies rests on two levels of complexity: first, a high heterogeneity in tumor cells with no evident targetable event, and second, an active and reacting microenvironment composed of active cells, interconnected and intensively communicating through paracrine secretion of soluble factors and EVs.
In this review, we describe the different actors of the OS microenvironment in the context of their complex interaction with tumors cells. We also discuss the past, current, and future therapeutic strategies, regarding the complex ecosystem of OS, with a focus on the emergence of multi-kinase inhibitors (MKI) that target tumor cells and the cells of their microenvironment, and on the role of EVs as essential conveyors of information in bone sarcoma biology.
OS-Induced Bone Remodeling
Osteoclasts and Osteolysis
OS development is associated with para-tumor osteolysis, causing frequent painful bone fragility at the time of the detection of OS in patients. OS aggressiveness has been associated with osteolysis markers in a few clinical cases .
Notably, the binding of the soluble molecule Receptor Activator of Nuclear Factor kappa B Ligand (RANKL), alias TNFSF11, to its receptor (RANK), mainly regulates osteolysis through paracrine regulation.
RANKL is produced by osteoblasts and osteocytes in the bone environment , while RANK is expressed on the cell surface of osteoclast precursors . In OSs, osteoclast activity leads to a vicious cycle between OS cell proliferation and bone degradation, leading to the release of pro-tumor factors such as insulin-like growth factor 1 (IGF1) or transforming growth factor-β (TGF-β) from the bone matrix [13,18].
However, clinical trials using monoclonal antibody therapy to block the IGF receptor tyrosine kinase in patients with OS showed limited and unpredictable response rates, leading to the cessation of this therapy .
The link of osteolysis in the vicious cycle observed in OS has been demonstrated in preclinical studies, using either chemical inhibitors (mainly zoledronic acid, ZOL) [20,21] or RANKL receptor competitors (including osteoprotegerin (OPG) , RANK-Fc ), or RANKL silencing .
Thus, osteolysis inhibition became an attractive therapeutic target in combination with chemotherapeutics to treat OS. However, initiated on the basis of promising preclinical studies, OS2006, a Phase III clinical trial combining ZOL with chemotherapy and surgery gave very disappointing results, with no improvement but slightly worse therapeutic results .
Despite the fact that ZOL has also been described in vitro to have a direct effect on OS cells, its efficacy against OS primary growth and pulmonary metastasis remains controversial .
Direct implication of osteoclast activity in OS development and progression in patients is still difficult to decipher. Indeed, a loss of osteoclasts was associated with increased metastasis in a preclinical model of OS , while co-injection of pre-osteoclasts with human OS cells had no effect on OS local growth and lung metastases in nude mice .
Denosumab, an antibody directed against RANKL, efficiently inhibits osteoclast activity and is currently used to treat bone loss in bone metastasis, multiple myeloma, or giant cell tumors.
However, no clinical results have been reported to date for denosumab in OS patients, except in combination with the MKI sorafenib for one patient [29,30]. Even following a more specific targeting of RANKL, denosumab does not have differentiated action towards different cell types.
Indeed, the RANKL/RANK pathway is involved not only in osteoclasts, but also in many other cells of the tumor environment, including osteoblasts, stromal cells, immune cells (T and B lymphocytes, dendritic cells), and endothelial cells.
Local coupling between bone resorption and formation is essential to preserve bone density and should occur in basic multicellular units, including osteoclasts and osteoblasts, which are covered by bone lining cells forming a canopy, as originally described by Lassen et al. .
Under the canopy, RANKL secreted by osteoblasts induces osteoclast differentiation, as described in a well-demonstrated paradigm. Interestingly, a new paradigm model of intercellular communication of osteoclasts towards osteoblasts may be relevant (Figure 1), as it was recently reported that mature osteoclasts were able to produce EVs bearing RANK, allowing interaction with RANKL on osteoblasts .
RANK-bearing EVs were initially identified in mouse primary osteoclasts and precursors derived from bone marrow . Recently, Ikebuchi et al. effectively demonstrated that RANK-bearing EVs issued from mouse mature osteoclasts were able to interact with RANKL-expressing osteoblasts, and therefore to induce osteoblastic differentiation coupled with bone formation involving RUNX2 signaling .
RANKL-reverse signaling in osteoblasts was demonstrated using RANK-masking on EVs and by creating a mutant mouse model RanklP29A, where RANKL intracellular signaling domain was suppressed.
Consequently, RANK–RANKL interaction appears to be bi-directional, dual, and complementary in the coupling of bone resorption and formation: RANK transduction on osteoclasts and precursors activates osteolysis, while RANKL transduction on osteoblasts and precursors activates osteogenesis.
In the context of OS, bone remodeling is linked to a vicious cycle between osteoclasts and tumor cells , which is established through the release of growth factors from the degraded bone matrix. Nevertheless, this vicious cycle may be additionally enhanced by EVs secreted by osteoclasts and OS cells .
Indeed, EVs secreted by OS cells were able to enhance osteolysis, while RANK-EVs secreted by osteoclasts may activate RANKL expressed on OS cells , suggesting a possible RANK–RANKL reverse signaling in OS, as previously described in normal bone physiology .
In one retrospective clinical study involving 40 patients, RANKL expression was observed in 75% of OS biopsy samples and its high expression level was correlated to a poor patient outcome . Branstetter et al.  detected RANKL in 68% of human OSs, but only 37% OS samples showed more than 10% of tumor cells expressing RANKL.
The same year, it was reported that the proliferation of RANKL-expressing OS cell lines was increased through transduction signaling involving AKT and ERK activation when cells were exposed to OPG .
One could hypothesize that RANKL expressed on the surface of OS cells could have been activated by OPG, as this protein is the decoy and soluble form of RANK that binds RANKL (Figure 2).
Nevertheless, this pro-proliferative effect of OPG was believed to be independent of RANKL because soluble RANK did not induce similar effects. Thus, it was proposed that OPG’s pro-proliferative effect was mediated by an unknown receptor.
In regard to the innovative identification of the RANKL reverse signaling as described above (Figure 1 and Figure 2) , RANKL activation in OS cells should be revisited, as RANK-EVs released by osteoclasts may have an unexpected role in OS through a possible RANK–RANKL reverse signaling in OS cells.
Osteoblasts and Bone Formation
Primary bone tumors have potent local influences on bone and the clinical consequences of these influences can be devastating. OS is characterized by the formation of osteoid matrix surrounding anaplastic tumor cells [39,40], and it can stimulate the formation of various bone structures, such as Codman’s triangles or bone spines, designed as the sunburst periosteal reaction.
The sunburst pattern of bone is due to new layers of collagen fibers stretching out perpendicularly to the bone. This process is mainly due to a deregulation of bone remodeling and in part to the activity of non-tumor osteoblasts, as observed in mouse OS models.
Osteoblastic progenitors are MSCs mainly present in the bone marrow, and more specifically multipotent skeletal stem cells (MSSCs), which are a subset of MSCs that were recently identified .
Under the control of different specific transcription factors, MSCs are able to differentiate into osteoblasts, chondroblasts, myoblasts, and adipocytes, while MSCCs differentiate into osteoblasts and chondroblasts, but not into myoblasts and adipocytes.
However, there is not yet evidence indicating that either MSCs or MSSCs are the most important cells in the pathogenesis of OS. Current knowledge on osteoblastogenesis is based on MSC rather than MSSC differentiation. Briefly, RUNX2 and Osterix or SOX9 transcription factor expression leads to MSC differentiation, respectively towards the osteoblastic and chondroblastic lineages .
The differentiation of MSCs into mature osteoblasts involves a complex series of proliferation and differentiation steps (Figure 3). Briefly, RUNX2 (also known as CBFA1) is a transcriptional factor that binds a consensus site, called OSE2, present along the proximal promoters of many genes including those of the α1 chain of type I collagen (COL1A1), bone sialoprotein (BSP), osteocalcin (OCN), and osteopontin (OPN) [43,44].
RUNX2 is crucial for the early steps of MSC differentiation into pre-osteoblasts and to maintain osteoblastic function, while Osterix (also known as SP7) is involved in osteoblastic differentiation mainly downstream of RUNX2 by allowing the differentiation of pre-osteoblasts into functional mature osteoblasts .
Upstream of those transcriptional factors, a signal transduction cascade has to be activated by cytokines or growth factors such as TGF-βs, fibroblast growth factors (FGFs), or wingless-type MMTV integration site family members (WNTs). Most of these cytokines or growth factors are implicated in OS development.
The TGF-β family comprises at least 30 members in humans . The role of TGF-βs during bone remodeling is complex. Regarding the mesenchymal osteoblastic lineage, TGF-β1 favors bone formation by stimulating the proliferation and migration of MSCs during the early stages of osteoblastogenesis [47,48].
In contrast, during the late stages of osteoblastogenesis, TGF-β1 inhibits the differentiation of MSCs into osteoblasts and the mineralization of mature osteoblasts in culture . Interestingly, TGF-β1 is mainly implicated in OS development during either primary tumor growth or metastatic progression .
Blocking TGF-β activity in OS cells by SMAD7 overexpression has decreased primary tumor growth by affecting the relationships between tumor cells and non-tumor cells .
FGFs are also key regulators of skeletal development . For example, FGF2 is important for the proliferation and maturation of pre-osteoblasts, while FGF18 is essential for mature formation of osteoblasts.
Therefore, FGF receptors are receptor tyrosine kinases that may represent a therapeutic target in OS patients . Indeed, Weekes et al. reported an important decrease of lung metastases upon using the inhibitor AZD4547 to block FGF receptor signaling following OS induction in mice .
WNTs are a family of 19 secreted glycoproteins. The binding of a WNT ligand (i.e., WNT1, WNT3a) to a frizzled (FZD) receptor, and its co-receptor LRP5/6 activates the canonical WNT pathway .
Activation of the WNT signaling cascade leads to the promotion of bone formation and suppression of bone resorption, leading to a balance in bone remodeling . Interestingly, a monoclonal antibody against the WNT signaling inhibitor dickkopf-1 inhibited OS metastasis in a preclinical model of OS .
Evidence is thus emerging for a role of osteoblasts in tumor growth in bone. Osteoblasts directly regulate bone matrix synthesis by their own secretome and indirectly regulate bone resorption through the release of RANKL, which binds RANK on osteoclast precursors as previously presented (Figure 1).
Additionally, RANK is expressed on MSCs and is downregulated during osteoblastogenesis. Intriguingly, Branstetter et al. did not detect RANK expression on tumor cells into OS samples .
Nevertheless, one might address the importance of RANK signaling in OS cells, which derived from cells committed in differentiation pathway between MSCs or pre-osteoblasts towards mature osteoblasts .
In this context, Navet et al. investigated the role of RANK overexpression in OS cell lines and during OS development in immune-deficient mice . Activation of the RANKL–RANK pathway in these OS cell lines did not change cell proliferation or migration, nor tumor growth in vivo.
Such results suggest that RANK activation in OS cells is not involved in tumor growth. However, RANK-overexpressing OS cells induced a significant increase of lung metastases that was prevented with an antibody directed against RANKL.
In another study , whole body deletion of RANKL proteins prevented OS development and lung metastases in genetically predisposed mice while, in contrast, Rank deletion in osteoblasts did not change OS burden, nor lung metastasis. RANKL–RANK pathway activation does not seem to be directly implicated in OS development, but can be indirectly involved in OS progression.
Implication of a potential RANKL reverse signaling in OS cells has not been tested in these studies, but it would be interesting now to take into account the implication of RANKL transduction on osteoblasts  (Figure 1).
Antibodies against RANKL and the whole-body deletion of RANKL could disrupt the coupling between bone resorption and formation and modify the progression of OS by inhibiting the transduction of RANKL on osteoblasts and on OS cells expressing RANKL (Figure 2).
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