Tumor-induced Bone Destruction Research Articles

Cryoprotective isoliquiritigenin-zein phosphatidylcholine nanoparticles inhibits breast cancer-bone metastasis by targeting JAK-STAT signaling pathways

Breast cancer (BC) is the most common cancer in women and is known for its tendency to spread to the bones, causing significant health issues and mortality. In this study, we aimed to investigate whether cryoprotective isoliquiritigenin-zein phosphatidylcholine nanoparticles (ISL@ZLH NPs) could inhibit BC-induced bone destruction and tumor metastasis in both in vitro and animal models. To evaluate the potential of ISL@ZLH NPs, we conducted various experiments. First, we assessed cell viability, colony formation, transwell migration, and wound healing assays to determine the impact of ISL@ZLH NPs on BC cell behavior. Western blotting, TRAP staining and ALP activity were performed to examine the effects of ISL@ZLH NPs on osteoclast formation induced by MDA-MB-231 cell-conditioned medium and RANKL treated RAW 264.7 cells. Furthermore, we assessed the therapeutic impact of ISL@ZLH NPs on tumor-induced bone destruction using a mouse model of BC bone metastasis. Treatment with ISL@ZLH NPs effectively suppressed BC cell proliferation, colony formation, and motility, reducing their ability to metastasize. ISL@ZLH NPs significantly inhibited osteoclast formation and the expression of factors associated with bone destruction in BC cells. Additionally, ISL@ZLH NPs suppressed JAK-STAT signaling in RAW264.7 cells. In the BCBM mouse model, ISL@ZLH NPs led to a significant reduction in osteolytic bone lesions compared to the control group. Histological analysis and TRAP staining confirmed that ISL@ZLH NPs preserved the integrity of bone structure, preventing invasive metastasis by confining tumor growth to the bone marrow cavity. Furthermore, ISL@ZLH NPs effectively suppressed tumor-induced osteoclastogenesis, a key process in BC-related bone destruction. Our findings demonstrate that ISL@ZLH NPs have the potential to inhibit BC-induced bone destruction and tumor metastasis by targeting JAK-STAT signaling pathways and suppressing tumor-induced osteoclastogenesis. These results underscore the therapeutic promise of ISL@ZLH NPs in managing BC metastasis to the bones.

Multifunctional nanomaterials via cell cuproptosis and oxidative stress for treating osteosarcoma and OS-induced bone destruction

Reactive Oxygen Species (ROS) refers to a highly reactive class of oxidizing species that have the potential to induce cellular apoptosis and necrosis. Cuproptosis, a type of cell death, is primarily associated with the effects of copper ions. However, the specific relationship between ROS, cuproptosis, and osteosarcoma (OS) remains relatively unexplored. Additionally, there is limited research on the use of cuproptosis in conjunction with oxidative stress for treating OS and inhibiting tumor-induced bone destruction. To address these gaps, a novel treatment approach has been developed for OS and neoplastic bone destruction. This approach involves the utilization of glutathione (GSH) and pH-responsive organic-inorganic mesoporous silica nanoparticles@Cu2S@oxidized Dextran (short for MCD). The MCD material demonstrates excellent cytocompatibility, osteogenesis, tumor suppression, and the ability to inhibit osteoclast formation. The specific mechanism of action involves the mitochondria of the MCD material inhibiting key proteins in the tricarboxylic acid (TCA) cycle. Simultaneously, the generation of ROS promotes this inhibition and leads to alterations in cellular energy metabolism. Moreover, the MCD biomaterial exhibits promising mild-temperature photothermal therapy in the second near-infrared (NIR-II) range, effectively mitigating tumor growth and OS-induced bone destruction in vivo.

Re-Evaluating the Role of PTHrP in Breast Cancer.

Parathyroid-hormone-related protein (PTHrP) is a protein with a long history of association with bone metastatic cancers. The paracrine signaling of PTHrP through the parathyroid hormone receptor (PTHR1) facilitates tumor-induced bone destruction, and PTHrP is known as the primary driver of humoral hypercalcemia of malignancy. In addition to paracrine signaling, PTHrP is capable of intracrine signaling independent of PTHR1 binding, which is essential for cytokine-like functions in normal physiological conditions in a variety of tissue types. Pre-clinical and clinical studies evaluating the role of PTHrP in breast cancer have yielded contradictory conclusions, in some cases indicating the protein is tumor suppressive, and in other studies, pro-growth. This review discusses the possible molecular basis for the disharmonious prognostic indications of these studies and highlights the implications of the paracrine, intracrine, and nuclear functions of the protein. This review also examines the current understanding of the functional domains of PTHrP and re-evaluates their role in the unique context of the breast cancer environment. This review will expand on the current understanding of PTHrP by attempting to reconcile the functional domains of the protein with its intracrine signaling in cancer.

Osteosarcoma-enriched transcripts paradoxically generate osteosarcoma-suppressing extracellular proteins.

Osteosarcoma (OS) is the common primary bone cancer that affects mostly children and young adults. To augment the standard-of-care chemotherapy, we examined the possibility of protein-based therapy using mesenchymal stem cells (MSCs)-derived proteomes and OS-elevated proteins. While a conditioned medium (CM), collected from MSCs, did not present tumor-suppressing ability, the activation of PKA converted MSCs into induced tumor-suppressing cells (iTSCs). In a mouse model, the direct and hydrogel-assisted administration of CM inhibited tumor-induced bone destruction, and its effect was additive with cisplatin. CM was enriched with proteins such as calreticulin, which acted as an extracellular tumor suppressor by interacting with CD47. Notably, the level of CALR transcripts was elevated in OS tissues, together with other tumor-suppressing proteins, including histone H4, and PCOLCE. PCOLCE acted as an extracellular tumor-suppressing protein by interacting with amyloid precursor protein, a prognostic OS marker with poor survival. The results supported the possibility of employing a paradoxical strategy of utilizing OS transcriptomes for the treatment of OS.

Mesoporous nanoplatform integrating photothermal effect and enhanced drug delivery to treat breast cancer bone metastasis.

Bone metastatic breast cancer has severely threatened the survival and life quality of patients. Due to the suboptimal efficacy of anti-metastatic chemotherapeutic drugs and the complicated bone marrow microenvironments, effective treatment of metastatic breast cancer remains challenging for traditional clinical approaches. In this work, we developed a mesoporous nanoplatform (m-CuS-PEG) with the co-loading of CuS nanodots and a chemotherapeutic drug cisplatin for the combined photothermal-chemotherapy of bone-metastasized breast cancer. The CuS nanodots were decorated onto mesoporous silica (m-SiO2) surface with dendritic mesoporous channels, into which the cisplatin was accommodated. The carboxyl-terminated poly (ethylene glycol) (PEG) was further functionalized onto the surface to obtain the functional nanoplatform m-CuS-PEG. The drug release of the loaded cisplatin exhibited pH- and thermal-dual responsive manner. The attached CuS nanodots rendered the mesoporous nanoplatform with high photothermal conversion ability. Upon irradiation with a near-infrared laser in the second near-infrared (NIR-II) window, m-CuS-PEG dispersions exhibited rapid temperature elevation and high photostability. The results revealed that m-CuS-PEG had excellent biocompatibility. The cisplatin-loaded m-CuS-PEG not only showed superior cancer cell-killing effects, but also significantly inhibit the growth of metastatic tumors. The tumor-induced bone destruction was also dramatically attenuated by the mesoporous nanoplatform-mediated combined therapy. Overall, the developed functional nanoplatform integrates photothermal therapy and efficient chemotherapeutic drug delivery to offer an alternative approach for combating breast cancer bone metastasis.

Suppression of osteosarcoma progression by engineered lymphocyte-derived proteomes

Cancer cells tend to develop resistance to chemotherapy and enhance aggressiveness. A counterintuitive approach is to tame aggressiveness by an agent that acts opposite to chemotherapeutic agents. Based on this strategy, induced tumor-suppressing cells (iTSCs) have been generated from tumor cells and mesenchymal stem cells. Here, we examined the possibility of generating iTSCs from lymphocytes by activating PKA signaling for suppressing the progression of osteosarcoma (OS). While lymphocyte-derived CM did not present anti-tumor capabilities, the activation of PKA converted them into iTSCs. Inhibiting PKA conversely generated tumor-promotive secretomes. In a mouse model, PKA-activated CM suppressed tumor-induced bone destruction. Proteomics analysis revealed that moesin (MSN) and calreticulin (Calr), which are highly expressed intracellular proteins in many cancers, were enriched in PKA-activated CM, and they acted as extracellular tumor suppressors through CD44, CD47, and CD91. The study presented a unique option for cancer treatment by generating iTSCs that secret tumor-suppressive proteins such as MSN and Calr. We envision that identifying these tumor suppressors and predicting their binding partners such as CD44, which is an FDA-approved oncogenic target to be inhibited, may contribute to developing targeted protein therapy.

Moderate tibial loading and treadmill running, but not overloading, protect adult murine bone from destruction by metastasized breast cancer

Osteolytic bone lesions, which develop in many metastatic breast cancer patients, impair bone integrity and lead to adverse skeletal related events that are difficult to treat and sometimes fatal. Moderate mechanical loading has been shown to suppress osteolysis in young mice with breast cancer. In this study, we aimed to investigate the dose-dependent effects of mechanical loading on protecting the integrity of adult skeletons with breast cancer. Localized tibial loading and aerobic treadmill running with three levels of varying intensity were tested in a syngeneic mammary tumor bone metastasis model. Adult C57BL/6J female mice (14-week-old, N=88 mice) received intra-tibial injections of Py8119 triple-negative murine breast cancer cells or PBS and underwent 4 to 5weeks of exercise or acted as sedentary/non-loaded controls. The bone structure was monitored longitudinally with weekly in vivo micro-computed tomography imaging, while the cellular responses in bone and marrow were examined using immunohistochemistry. Moderate treadmill running (16m/min, 50min/day, 5days/week, and 5weeks) and tibial loading (4.5N, 630 με, 4Hz, 300cycles/day, 5days/week, and 4weeks) suppressed tumor-induced bone destruction, as evaluated by full-thickness perforation of tibial cortex and the volume of osteolytic lesions in the cortex. In contrast, tibial loading at higher magnitude (8N, 1100 με) induced woven bone and accelerated bone destruction, compared with the non-loaded controls. The three exercise regimens differentially affected osteocyte apoptosis, osteocyte hypoxia, osteoclast activity, bone marrow vasculature, and tumor proliferation. In conclusion, the relationship between exercise intensity and the risk of breast cancer-induced osteolysis was found to follow a J-shaped curve in a preclinical model, suggesting the need to optimize exercise parameters in order to harness the skeletal benefits of exercise in metastatic breast cancers.

From Good to Bad: The Opposing Effects of PTHrP on Tumor Growth, Dormancy, and Metastasis Throughout Cancer Progression.

Parathyroid hormone related protein (PTHrP) is a multifaceted protein with several biologically active domains that regulate its many roles in normal physiology and human disease. PTHrP causes humoral hypercalcemia of malignancy (HHM) through its endocrine actions and tumor-induced bone destruction through its paracrine actions. PTHrP has more recently been investigated as a regulator of tumor dormancy owing to its roles in regulating tumor cell proliferation, apoptosis, and survival through autocrine/paracrine and intracrine signaling. Tumor expression of PTHrP in late stages of cancer progression has been shown to promote distant metastasis formation, especially in bone by promoting tumor-induced osteolysis and exit from dormancy. In contrast, PTHrP may protect against further tumor progression and improve patient survival in early disease stages. This review highlights current knowledge from preclinical and clinical studies examining the role of PTHrP in promoting tumor progression as well as skeletal and soft tissue metastasis, especially with regards to the protein as a regulator of tumor dormancy. The discussion will also provide perspectives on PTHrP as a prognostic factor and therapeutic target to inhibit tumor progression, prevent tumor recurrence, and improve patient survival.

Hypoxia in bone metastasis and osteolysis.

Hypoxia is a common feature in tumors, driving pathways that promote epithelial-to-mesenchymal transition, invasion, and metastasis. Clinically, high levels of hypoxia-inducible factor (HIF) expression and stabilization at the primary site in many cancer types is associated with poor patient outcomes. Experimental evidence suggests that HIF signaling in the primary tumor promotes their dissemination to the bone, as well as the release of factors such as LOX that act distantly on the bone to stimulate osteolysis and form a pre-metastatic niche. Additionally, the bone itself is a generally hypoxic organ, fueling the activation of HIF signaling in bone resident cells, promoting tumor cell homing to the bone as well as osteoclastogenesis. The hypoxic microenvironment of the bone also stimulates the vicious cycle of tumor-induced bone destruction, further fueling tumor cell growth and osteolysis. Furthermore, hypoxia appears to regulate key tumor dormancy factors. Thus, hypoxia acts both on the tumor cells as well as the metastatic site to promote tumor cell metastasis.

Tuning Ligand Density To Optimize Pharmacokinetics of Targeted Nanoparticles for Dual Protection against Tumor-Induced Bone Destruction.

Breast cancer patients are at high risk for bone metastasis. Metastatic bone disease is a major clinical problem that leads to a reduction in mobility, increased risk of pathologic fracture, severe bone pain, and other skeletal-related events. The transcription factor Gli2 drives expression of parathyroid hormone-related protein (PTHrP), which activates osteoclast-mediated bone destruction, and previous studies showed that Gli2 genetic repression in bone-metastatic tumor cells significantly reduces tumor-induced bone destruction. Small molecule inhibitors of Gli2 have been identified; however, the lipophilicity and poor pharmacokinetic profile of these compounds have precluded their success in vivo. In this study, we designed a bone-targeted nanoparticle (BTNP) comprising an amphiphilic diblock copolymer of poly[(propylene sulfide)-block-(alendronate acrylamide-co-N,N-dimethylacrylamide)] [PPS-b-P(Aln-co-DMA)] to encapsulate and preferentially deliver a small molecule Gli2 inhibitor, GANT58, to bone-associated tumors. The mol % of the bisphosphonate Aln in the hydrophilic polymer block was varied in order to optimize BTNP targeting to tumor-associated bone by a combination of nonspecific tumor accumulation (presumably through the enhanced permeation and retention effect) and active bone binding. Although 100% functionalization with Aln created BTNPs with strong bone binding, these BTNPs had highly negative zeta-potential, resulting in shorter circulation time, greater liver uptake, and less distribution to metastatic tumors in bone. However, 10 mol % of Aln in the hydrophilic block generated a formulation with a favorable balance of systemic pharmacokinetics and bone binding, providing the highest bone/liver biodistribution ratio among formulations tested. In an intracardiac tumor cell injection model of breast cancer bone metastasis, treatment with the lead candidate GANT58-BTNP formulation decreased tumor-associated bone lesion area 3-fold and increased bone volume fraction in the tibiae of the mice 2.5-fold. Alnconferred bone targeting to the GANT58-BTNPs, which increased GANT58 concentration in the tumor-associated bone relative to untargeted NPs, and also provided benefit through the direct antiresorptive therapeutic function of Aln. The dual benefit of the Aln in the BTNPs was supported by the observations thatdrug-free Aln-containing BTNPs improved bone volume fraction in bone-tumor-bearing mice, while GANT58-BTNPs created better therapeutic outcomes than both unloaded BTNPs and GANT58-loaded untargeted NPs. These findings suggest GANT58-BTNPs have potential to potently inhibit tumor-driven osteoclast activation and resultant bone destruction in patients with bone-associated tumor metastases.

HTLV-1 viral oncogene HBZ drives bone destruction in adult T cell leukemia.

Osteolytic bone lesions and hypercalcemia are common, serious complications in adult T cell leukemia/lymphoma (ATL), an aggressive T cell malignancy associated with human T cell leukemia virus type 1 (HTLV-1) infection. The HTLV-1 viral oncogene HBZ has been implicated in ATL tumorigenesis and bone loss. In this study, we evaluated the role of HBZ on ATL-associated bone destruction using HTLV-1 infection and disease progression mouse models. Humanized mice infected with HTLV-1 developed lymphoproliferative disease and continuous, progressive osteolytic bone lesions. HTLV-1 lacking HBZ displayed only modest delays to lymphoproliferative disease but significantly decreased disease-associated bone loss compared with HTLV-1-infected mice. Gene expression array of acute ATL patient samples demonstrated increased expression of RANKL, a critical regulator of osteoclasts. We found that HBZ regulated RANKL in a c-Fos-dependent manner. Treatment of HTLV-1-infected humanized mice with denosumab, a monoclonal antibody against human RANKL, alleviated bone loss. Using patient-derived xenografts from primary human ATL cells to induce lymphoproliferative disease, we also observed profound tumor-induced bone destruction and increased c-Fos and RANKL gene expression. Together, these data show the critical role of HBZ in driving ATL-associated bone loss through RANKL and identify denosumab as a potential treatment to prevent bone complications in ATL patients.

Biphasic Injectable Bone Cement with Fe3O4/GO Nanocomposites for the Minimally Invasive Treatment of Tumor-Induced Bone Destruction

Minimally invasive surgery will be gradually applied to the surgical treatment of bone tumors. One of the difficulties in the minimally invasive treatment of bone tumors is the lack of injectable materials that can be used to treat tumor-induced bone defects. Therefore, it is imperative to develop an injectable bone filler that can not only be injected into the defect site by minimally invasive methods to provide strong support and repair bone tissue but also inactivate residual tumor cells around the defect. To achieve this aim, in our study, for the first time, we doped Fe3O4/graphene oxide (GO) nanocomposites into α-tricalcium phosphate (α-TCP)/calcium sulfate (CS) biphasic bone cement to prepare an injectable magnetic bone cement (α-TCP/CS/Fe3O4/GO, αCFG), which can be applied in bone tumor minimally invasive surgery and fit ideally even if the area is irregular. The magnetothermal performance of the αCFG bone cement could be well adjusted by altering the content of Fe3O4/GO nanocomposites and the magnetic field parameters, but a 10 wt % Fe3O4/GO content formed the most stable bone cement with excellent magnetothermal performance. The αCFG bone cement not only promotes bone regeneration but also exhibits enhanced tumor treatment effects. Such multifunctional bone cement could provide a promising clinical strategy for the minimally invasive treatment of tumor-induced bone destruction.

1,25(OH)2D3 deficiency increases TM40D tumor growth in bone and accelerates tumor-induced bone destruction in a breast cancer bone metastasis model

Breast cancer is one of the most common malignancies and bone is the commonest site of distant metastases. Evidences indicate that adequate supply of vitamin D will decrease the morbidity and mortality of breast cancer. However, the main role of vitamin D deficiency in breast cancer bone metastases remains unclear. In this study, the relationship between vitamin D and breast cancer bone metastases were evaluated. Results showed that 1,25(OH)2D3 can not only inhibit the proliferation, migration and invasion of breast cancer cell TM40D in vitro, but also attenuate the breast cancer cell TM40D-induced bone destruction in vivo, whose underlying mechanism was at least partially through decreasing the number of the osteoclasts. To our knowledge, this is the first to use 1-alpha-hydroxylase [1α(OH)ase] knockout mice which characterized vitamin D deficiency to establish the breast cancer bone metastases model. Based on this model, we also found that vitamin D deficiency will accelerate the osteolytic lesions, and 1,25(OH)2D3 supplement will restrain osteolytic lesions. Therefore, these findings suggest that vitamin D has the potential capacity to be a therapeutic agent for the breast cancer bone metastases.

Combined treatment with a transforming growth factor beta inhibitor (1D11) and bortezomib improves bone architecture in a mouse model of myeloma-induced bone disease

Multiple myeloma (MM) patients frequently develop tumor-induced bone destruction, yet no therapy completely eliminates the tumor or fully reverses bone loss. Transforming growth factor-β (TGF-β) activity often contributes to tumor-induced bone disease, and pre-clinical studies have indicated that TGF-β inhibition improves bone volume and reduces tumor growth in bone metastatic breast cancer. We hypothesized that inhibition of TGF-β signaling also reduces tumor growth, increases bone volume, and improves vertebral body strength in MM-bearing mice. We treated myeloma tumor-bearing (immunocompetent KaLwRij and immunocompromised Rag2−/−) mice with a TGF-β inhibitory (1D11) or control (13C4) antibody, with or without the anti-myeloma drug bortezomib, for 4weeks after inoculation of murine 5TGM1 MM cells. TGF-β inhibition increased trabecular bone volume, improved trabecular architecture, increased tissue mineral density of the trabeculae as assessed by ex vivo micro-computed tomography, and was associated with significantly greater vertebral body strength in biomechanical compression tests. Serum monoclonal paraprotein titers and spleen weights showed that 1D11 monotherapy did not reduce overall MM tumor burden. Combination therapy with 1D11 and bortezomib increased vertebral body strength, reduced tumor burden, and reduced cortical lesions in the femoral metaphysis, although it did not significantly improve cortical bone strength in three-point bending tests of the mid-shaft femur. Overall, our data provides rationale for evaluating inhibition of TGF-β signaling in combination with existing anti-myeloma agents as a potential therapeutic strategy to improve outcomes in patients with myeloma bone disease.

417. Intratumoral Delivery of Oncolytic Adenovirus Expressing Decorin Inhibits Growth and Metastases of 4T1 Breast Tumors in Syngeneic Immune Competent BALB/c Mice Model

During the advanced stage of breast cancer, majority of the patients develop distal metastases that eventually lead to mortality. Unfortunately, conventional strategies generally invoke limited therapeutic responses in patients with tumor metastases. Therefore, the development of novel and highly effective therapies for the treatment of distal metastases of breast cancer is an unmet need in medicine. Decorin (DCN) is a small leucine-rich proteoglycan and is often down-regulated in tumor stroma of breast cancer patients. Our previous work demonstrated that systemic delivery of Ad.dcn, an oncolytic adenovirus expressing DCN, significantly inhibited skeletal metastases and the tumor induced bone destruction in MDA-MB-231 bone metastasis model. However, the therapeutic responses of Ad.dcn in immune-competent models have never been examined. In this study, we showed that non-replicated adenovirus Ad(E1-). dcn mediated high level expression of human DCN in 4T1 cells, and inhibited the expression of tyrosine kinase receptor MET, β-catenin and vascular endothelial growth factor A. To examine the anti-tumor responses of Ad.dcn, we established subcutaneous 4T1 tumors in BALB/c mice. Ad.dcn was delivered intratumorallly (2.5×1010VPs) on day 7 post-cell inoculation, and a repeat dose was given on day 10. Our data showed that Ad.dcn inhibited the growth of local tumors as measured by the caliper as well as by the Bioluminescence Imaging (BLI). At the terminal time point (day 25) we also observed a significant reduction in the tumor weight. Analysis of BLI in real time also demonstrated that Ad.dcn significantly reduced the tumor metastases to lung. On day 25 Ad.dcn group had fewer metastases (2/8 mice), compared to buffer treated group (7/8 mice). Similar reductions in lung metastases were observed by H&E staining. Moreover, Ad.dcn treatment reduced the expression of Th2 cytokines (IL-2, IL-4 and IL-10) in the tumors, which will potentially activate the antitumor immune responses. Furthermore, Ad.dcn also reduced the expression of N-cadherin and vimentin in the tumors, indicating Ad.dcn could also potentially reduce tumor metastases by inhibiting epithelial-mesenchymal transition (EMT) of the tumor cells. Taken together, our results suggested that Ad.dcn not only inhibited the growth of local tumor, but also prevented the distal metastases of tumor. Combining these results in the 4T1 tumor model, and previously examined MB-MDA-231 bone metastases model, we propose the following model of Ad.dcn-mediated inhibition of tumor growth and metastases. Ad.dcn is taken up by tumor cells after systemic or intratumoral delivery, and then replicates in tumor cells and kills them. Tumor cells produce and release high level of decorin protein into tumor microenvironment. Decorin then targets multiple tumor and stromal components to activate anti-tumor immune responses, decrease angiogenesis, and inhibit EMT. Therefore, Ad.dcn is a potential therapeutic strategy for the treatment of breast cancer and its distal metastases.Y.Y. and W.X. made equal contribution. L.W. and P.S. are the corresponding Authors.

Photothermal ablation of bone metastasis of breast cancer using PEGylated multi-walled carbon nanotubes.

This study investigates therapeutic efficacy of photothermal therapy (PTT) in an orthotropic xenograft model of bone metastasis of breast cancer. The near-infrared (NIR) irradiation on Multi-Walled Carbon Nanotubes (MWNTs) resulted in a rapid heat generation which increased with the MWNTs concentration up to 100 μg/ml. MWNTs alone exhibited no toxicity, but inclusion of MWNTs dramatically decreased cell viability when combined with laser irradiation. Thermographic observation revealed that treatment with 10 μg MWNTs followed by NIR laser irradiation resulted in a rapid increase in temperature up to 73.4±11.98 °C in an intraosseous model of bone metastasis of breast cancer. In addition, MWNTs plus NIR laser irradiation caused a remarkably greater suppression of tumor growth compared with treatment with either MWNTs injection or NIR irradiation alone, significantly reducing the amount of tumor-induced bone destruction. All these demonstrate the efficacy of PTT with MWNTs for bone metastasis of breast cancer.

Does the presence of tumor-induced cortical bone destruction at CT have any prognostic value in newly diagnosed diffuse large B-cell lymphoma?

PurposeTo determine the prognostic value of tumor-induced cortical bone destruction at computed tomography (CT) in newly diagnosed diffuse large B-cell lymphoma (DLBCL).Materials and methodsThis retrospective study included 105 patients with newly diagnosed DLBCL who had undergone CT and bone marrow biopsy (BMB) before R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin, Oncovin, and prednisolone) chemo-immunotherapy. Cox regression analyses were used to determine the associations of cortical bone status at CT (absence vs. presence of tumor-induced cortical bone destruction), BMB findings (negative vs. positive for lymphomatous involvement), and dichotomized National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI) strata (low risk vs. high risk) with progression-free survival (PFS) and overall survival (OS).ResultsUnivariate Cox regression analysis indicated that cortical bone status at CT was no significant predictor of either PFS or OS (p = 0.358 and p = 0.560, respectively), whereas BMB findings (p = 0.002 and p = 0.013, respectively) and dichotomized NCCN-IPI risk strata (p = 0.002 and p = 0.003, respectively) were significant predictors of both PFS and OS. In the multivariate Cox proportional hazards model, only the dichotomized NCCN-IPI score was an independent predictive factor of PFS and OS (p = 0.004 and p = 0.003, respectively).ConclusionsThe presence of tumor-induced cortical bone destruction at CT was not found to have any prognostic implications in newly diagnosed DLBCL.

NGF blockade at early times during bone cancer development attenuates bone destruction and increases limb use.

Studies in animals and humans show that blockade of nerve growth factor (NGF) attenuates both malignant and nonmalignant skeletal pain. While reduction of pain is important, a largely unanswered question is what other benefits NGF blockade might confer in patients with bone cancer. Using a mouse graft model of bone sarcoma, we demonstrate that early treatment with an NGF antibody reduced tumor-induced bone destruction, delayed time to bone fracture, and increased the use of the tumor-bearing limb. Consistent with animal studies in osteoarthritis and head and neck cancer, early blockade of NGF reduced weight loss in mice with bone sarcoma. In terms of the extent and time course of pain relief, NGF blockade also reduced pain 40% to 70%, depending on the metric assessed. Importantly, this analgesic effect was maintained even in animals with late-stage disease. Our results suggest that NGF blockade immediately upon detection of tumor metastasis to bone may help preserve the integrity and use, delay the time to tumor-induced bone fracture, and maintain body weight.

Wnt signaling induces gene expression of factors associated with bone destruction in lung and breast cancer.

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone destruction in bone metastatic tumors. Transforming growth factor-beta (TGF-β) stimulates PTHrP production in part through the transcription factor Gli2, which is regulated independent of the Hedgehog signaling pathway in osteolytic cancer cells. However, inhibition of TGF-β in vivo does not fully inhibit tumor growth in bone or tumor-induced bone destruction, suggesting other pathways are involved. While Wnt signaling regulates Gli2 in development, the role of Wnt signaling in bone metastasis is unknown. Therefore, we investigated whether Wnt signaling regulates Gli2 expression in tumor cells that induce bone destruction. We report here that Wnt activation by β-catenin/T cell factor 4 (TCF4) over-expression or lithium chloride (LiCl) treatment increased Gli2 and PTHrP expression in osteolytic cancer cells. This was mediated through the TCF and Smad binding sites within the Gli2 promoter as determined by promoter mutation studies, suggesting cross-talk between TGF-β and Wnt signaling. Culture of tumor cells on substrates with bone-like rigidity increased Gli2 and PTHrP production, enhanced autocrine Wnt activity and led to an increase in the TCF/Wnt signaling reporter (TOPFlash), enriched β-catenin nuclear accumulation, and elevated Wnt-related genes by PCR-array. Stromal cells serve as an additional paracrine source of Wnt ligands and enhanced Gli2 and PTHrP mRNA levels in MDA-MB-231 and RWGT2 cells in vitro and promoted tumor-induced bone destruction in vivo in a β-catenin/Wnt3a-dependent mechanism. These data indicate that a combination of matrix rigidity and stromal-secreted factors stimulate Gli2 and PTHrP through Wnt signaling in osteolytic breast cancer cells, and there is significant cross-talk between the Wnt and TGF-β signaling pathways. This suggests that the Wnt signaling pathway may be a potential therapeutic target for inhibiting tumor cell response to the bone microenvironment and at the very least should be considered in clinical regimens targeting TGF-β signaling.

Abstract 4858: A novel strategy for the selective and tissue specific inhibition of MMPs in active breast cancer to bone metastases

Abstract Bone metastasis is a common event during breast cancer progression: The delicate balance between bone matrix synthesizing osteoblasts and bone resorbing osteoclasts activities is typically subverted by the presence of tumor cells at the bone site. The resulting incurable lesions cause extensive osteolysis and severely impact the patient's quality of life. Understanding how active breast-to-bone metastases manipulate the bone microenvironment is critical for the development of new therapies to treat the disease. We have recently shown in preclinical animal models of breast to bone metastases that MMP-2 proteolytic activity is critical for the growth of the tumor and the associated osteolysis. We therefore hypothesized that the selective inhibition of MMP-2 at the tumor site would be a potent means through which to prevent the progression of the malignancy. Given that previous broad-spectrum MMP inhibitor (MMPI) trials were unsuccessful mainly because of consistent side effects, we took a novel chemical approach to generate a selective MMPI that would preferentially target the skeleton. To this end, we utilized a bisphosphonate backbone (tiludronate) to synthesize an MMPI (BMMPI) with high specificity for MMP-2 (IC50 0.14± 0.04μM). To test the efficacy of the BMMPI in vivo, we inoculated 6-week old immunocompromized female mice with a luciferase expressing osteolytic breast cancer cell line, PyMT-Luc (105 cells). Following tumor injection, mice (n=6/group) received saline, tiludronate (25mg/kg, to control for bisphosphonate effects) or BMMPI (25mg/kg) sub-cutaneously, twice weekly. Tumor growth was measured by quantitating luminescence over time. Our results show that application of the BMMPI significantly reduced the breast cancer growth over a two-week period (p<0.05). Furthermore, using Faxitron analysis to determine the extent of tumor induced bone destruction, we observed significantly less osteolysis in the tibia images of BMMPI treated mice compared to the control group. Our initial studies also suggest that BMMPIs perform better than clinically used bisphosphonates such as tiludronate. We are currently following up our in vivo/ex vivo analyses with histomorphometrical and histology approaches. In conclusion, our data thus far demonstrate that an MMP-2 specific BMMPI may be an effective means to prevent the progression of breast to bone metastases while at the same time eliminating some of the noted off-target effects of broad-spectrum MMP inhibitors. Since bisphosphonates are well tolerated in the clinical setting, we predict that BMMPIs could be rapidly translated to the patient. Citation Format: Marilena Tauro, Antonio Laghezza, Paolo Tortorella, Conor C. Lynch. A novel strategy for the selective and tissue specific inhibition of MMPs in active breast cancer to bone metastases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4858. doi:10.1158/1538-7445.AM2014-4858