Osteosarcoma cells exhibit functional interactions with stromal cells, fostering a lung microenvironment conducive to the establishment of metastatic tumor cells.

BACKGROUND: Osteosarcoma (OS) stands out as the most common bone tumor, with approximately 20% of the patients receiving a diagnosis of metastatic OS at their initial assessment. A significant challenge lies in the frequent existence of undetected metastases during the initial diagnosis. Mesenchymal stem cells (MSCs) possess unique abilities that facilitate tumor growth, and their interaction with OS cells is crucial for metastatic spread. METHODS AND RESULTS: We demonstrated that, in vitro, MSCs exhibited a heightened migration response toward the secretome of non-metastatic OS cells. When challenged to a secretome derived from lungs preloaded with OS cells, MSCs exhibited greater migration toward lungs colonized with metastatic OS cells. Moreover, in vivo, MSCs displayed preferential migratory and homing behavior toward lungs colonized by metastatic OS cells. Metastatic OS cells, in turn, demonstrated an increased migratory response to the MSCs' secretome. This behavior was associated with heightened cathepsin D (CTSD) expression and the release of active metalloproteinase 2 (MMP2) by metastatic OS cells. CONCLUSIONS: Our assessment focused on two complementary tumor capabilities crucial to metastatic spread, emphasizing the significance of inherent cell features. The findings underscore the pivotal role of signaling integration within the niche, with a complex interplay of migratory responses among established OS cells in the lungs, prometastatic OS cells in the primary tumor, and circulating MSCs. Pulmonary metastases continue to be a significant factor contributing to OS mortality. Understanding these mechanisms and identifying differentially expressed genes is essential for pinpointing markers and targets to manage metastatic spread and improve outcomes for patients with OS.

Up-regulation of pro-angiogenic molecules and events does not relate with an angiogenic switch in metastatic osteosarcoma cells but to cell survival features.

Osteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children. Metastases represent a major clinical challenge and an estimated 80% would present undetectable micrometastases at diagnosis. The identification of metastatic traits and molecules would impact in micrometastasis management. We demonstrated that OS LM7 metastatic cells secretome was able to induce microvascular endothelium cell rearrangements, an angiogenic-related trait. A proteomic analysis indicated a gain in angiogenic-related pathways in these cells, as compared to their parental-non-metastatic OS SAOS2 cells counterpart. Further, factors with proangiogenic functions like VEGF and PDGF were upregulated in LM7 cells. However, no differential angiogenic response was induced by LM7 cells in vivo. Regulation of the Fas-FasL axis is key for OS cells to colonize the lungs in this model. Analysis of the proteomic data with emphasis in apoptosis pathways and related processes revealed that the percentage of genes associated with those, presented similar levels in SAOS2 and LM7 cells. Further, the balance of expression levels of proteins with pro- and antiapoptotic functions in both cell types was subtle. Interestingly and of relevance to the model, Fas associated Factor 1 (FAF1), which participates in Fas signaling, was present in LM7 cells and was not detected in SAOS2 cells. The subtle differences in apoptosis-related events and molecules, together with the reported cell-survival functions of the identified angiogenic factors and the increased survival features that we observed in LM7 cells, suggest that the gain in angiogenesis-related pathways in metastatic OS cells would relate to a prosurvival switch rather to an angiogenic switch as an advantage feature to colonize the lungs. OS metastatic cells also displayed higher adhesion towards microvascular endothelium cells suggesting an advantage for tissue colonization. A gain in angiogenesis pathways and molecules does not result in major angiogenic potential. Together, our results suggest that metastatic OS cells would elicit signaling associated to a prosurvival phenotype, allowing homing into the hostile site for metastasis. During the gain of metastatic traits process, cell populations displaying higher adhesive ability to microvascular endothelium, negative regulation of the Fas-FasL axis in the lung parenchyma and a prosurvival switch, would be selected. This opens a new scenario where antiangiogenic treatments would affect cell survival rather than angiogenesis, and provides a molecular panel of expression that may help in distinguishing OS cells with different metastatic potential.

Thyroid status regulates the tumor microenvironment delineating breast cancer fate.

The patient's hormonal context plays a crucial role in the outcome of cancer. However, the association between thyroid disease and breast cancer risk remains unclear. We evaluated the effect of thyroid status on breast cancer growth and dissemination in an immunocompetent mouse model. For this, hyperthyroid and hypothyroid Balb/c mice were orthotopically inoculated with triple-negative breast cancer 4T1 cells. Tumors from hyperthyroid mice showed an increased growth rate and an immunosuppressive tumor microenvironment, characterized by increased IL-10 levels and decreased percentage of activated cytotoxic T cells. On the other hand, delayed tumor growth in hypothyroid animals was associated with increased tumor infiltration of activated CD8+ cells and a high IFNγ/IL-10 ratio. Paradoxically, hypothyroid mice developed a higher number of lung metastasis than hyperthyroid animals. This was related to an increased secretion of tumor CCL2 and an immunosuppressive systemic environment, with increased proportion of regulatory T cells and IL-10 levels in spleens. A lower number of lung metastasis in hyperthyroid mice was related to the reduced presence of mesenchymal stem cells in tumors and metastatic sites. These animals also exhibited decreased percentages of regulatory T lymphocytes and myeloid-derived suppressor cells in spleens but increased activated CD8+ cells and the IFNγ/IL-10 ratio. Therefore, thyroid hormones modulate the cellular and cytokine content of the breast tumor microenvironment. A better understanding of the mechanisms involved in these effects could be a starting point for the discovery of new therapeutic targets for breast cancer.

Hyaluronan Metabolism is Associated with DNA Repair Genes in Breast and Colorectal Cancer. Screening of Potential Progression Markers Using qPCR.

In this work, we compared mRNA levels of Hyaluronan (HA) metabolism members and BRCA genes, known to be involved in the tumoral process, between tumor and non-tumor adjacent tissue and its correlation with previously proposed biomarkers (ER, PR, HER2 and KI67) in order to assess their value as a progression biomarkers. We show alteration in HA metabolism in colorectal but not breast cancer. However, we found a decrease in Hyaluronidase 1 HYAL1 levels in the breast but not colorectal cancer. We also show lower HA levels in tumor compared with normal tissue that could indicate a possible influence of tumor on its surrounding "normal" tissue. In both breast and colorectal cancer, CD44 and BRCA2 showed a strong positive correlation. Besides, our results show first indicators that qPCR of the analyzed genes could be used as an easy and low cost procedure for the evaluation of molecular markers we propose here.

Investigación traslacional en microbioma (InTraMic): un área de interés común y creciente en el IMTIB, Hospital Italiano e Instituto Universitario / Translational research in microbiome (InTraMic): an area of common and growing interest at IMTIB, Hospital Italiano and Instituto Universitario

Se estima que aproximadamente 100 trillones de microorganismos (incluidos bacterias, virus y hongos) residen en el intestino humano adulto y que el total del material genético del microbioma es 100 veces superior al del genoma humano. Esta comunidad, conocida como microbioma se adquiere al momento del nacimiento a través de la flora comensal de la piel, vagina y heces de la madre y se mantiene relativamente estable a partir de los dos años desempeñando un papel crítico tanto en el estado de salud como en la enfermedad. El desarrollo de nuevas tecnologías, como los secuenciadores de próxima generación (NGS), permiten actualmente realizar un estudio mucho más preciso de ella que en décadas pasadas cuando se limitaba a su cultivo. Si bien esto ha llevado a un crecimiento exponencial en las publicaciones, los datos sobre las poblaciones Latinoamérica son casi inexistentes. La investigación traslacional en microbioma (InTraMic) es una de las líneas que se desarrollan en el Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB). Esta se inició en 2018 con la línea de cáncer colorrectal (CCR) en una colaboración con el Colorectal Cancer Research Group del Leeds Institute of Medical Research en el proyecto Large bowel microbiome disease network: Creation of a proof of principle exemplar in colorectal cancer across three continents. A fines de 2019 se cumplió el objetivo de comprobar la factibilidad de la recolección, envío y análisis de muestras de MBF en 5 continentes, incluyendo muestras provenientes de la Argentina, Chile, India y Vietnam. Luego de haber participado de capacitaciones en Inglaterra, se ha cumplido con el objetivo de la etapa piloto, logrando efectivizar la recolección, envío y análisis metagenómico a partir de la secuenciación de la región V4 del ARNr 16S. En 2019, la línea de enfermedad de hígado graso no alcohólico se sumó a la InTraMic iniciando una caracterización piloto en el marco de una colaboración con el laboratorio Novartis. Los resultados de ese estudio, así como el de cáncer colorrectal, están siendo enviados a publicación. En 2020, con la incorporación de la línea de trasplante alogénico de células progenitoras hematopoyéticas, fue presentado un proyecto para un subsidio del CONICET que ha superado la primera etapa de evaluación. En el presente artículo se brinda una actualización sobre la caracterización taxonómica de microbioma y se describen las líneas de investigación en curso. (AU)

It is estimated that approximately 100 trillion microorganisms (including bacteria, viruses, and fungi) reside in the adult human intestine, and that the total genetic material of the microbiome is 100 times greater than that of the human genome. This community, known as the microbiome, is acquired at birth through the commensal flora of the mother's skin, vagina, and feces and remains relatively stable after two years, playing a critical role in both the state of health and in disease. The development of new technologies, such as next-generation sequencers (NGS), currently allow for a much more precise study of it than in past decades when it was limited to cultivation. Although this has led to exponential growth in publications, data on Latin American populations is almost non-existent. Translational research in microbiome (InTraMic) is one of the lines developed at the Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB). This started in 2018 with the Colorectal Cancer Line (CRC) in a collaboration with the Colorectal Cancer Research Group of the Leeds Institute of Medical Research in the project "Large bowel microbiome disease network: Creation of a proof of principle exemplar in colorectal cancer across three continents". At the end of 2019, the objective of verifying the feasibility of collecting, sending and analyzing MBF samples on 5 continents, including samples from Argentina, Chile, India and Vietnam, was met. After having participated in training in England, the objective of the pilot stage has been met, achieving the collection, delivery and metagenomic analysis from the sequencing of the V4 region of the 16S rRNA. In 2019, the non-alcoholic fatty liver disease line joined InTraMic, initiating a pilot characterization in the framework of a collaboration with the Novartis laboratory. The results of that study, as well as that of colorectal cancer, are being published. In 2020, with the incorporation of the allogeneic hematopoietic stem cell transplantation line, a project was presented for a grant from the CONICET that has passed the first stage of evaluation. This article provides an update on the taxonomic characterization of the microbiome and describes the lines of ongoing research. (AU)

Acquisition of stem associated-features on metastatic osteosarcoma cells and their functional effects on mesenchymal stem cells.

BACKGROUND: Osteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children and young adults. Metastases are a major clinical challenge in OS. In this context, 20% of OS patients are diagnosed with metastatic OS, but near 80% of all OS patients could present non-detectable micrometastases at the moment of diagnosis. METHODS: Osteogenic differentiation; doxorubicin exclusion assay; fluorescence microscopy; RT-qPCR; proteomic analysis. RESULTS: Our results suggest that metastatic OS cells possess a diminished osteoblastic differentiation potential with a gain of metastatic traits like the capacity to modify intracellular localization of chemodrugs and higher levels of expression of stemness-related genes. On the opposite hand, non-metastatic OS cells possess bone-associated traits like higher osteoblastic differentiation and also an osteoblastic-inducer secretome. OS cells also differ in the nature of their interaction with mesenchymal stem cells (MSCs), with opposites impacts on MSCs phenotype and behavior. CONCLUSIONS: All this suggests that a major trait acquired by metastatic cells is a switch into a stem-like state that could favor its survival in the pulmonary niche, opening new possibilities for personalized chemotherapeutic schemes. GENERAL SIGNIFICANCE: Our work provides new insights regarding differences among metastatic and non-metastatic OS cells, with particular emphasis on differentiation potential, multidrug resistance and interaction with MSCs.

Metastatic Niches and the Modulatory Contribution of Mesenchymal Stem Cells and Its Exosomes.

Mesenchymal stem cells (MSCs) represent an interesting population due to their capacity to release a variety of cytokines, chemokines, and growth factors, and due to their motile nature and homing ability. MSCs can be isolated from different sources, like adipose tissue or bone marrow, and have the capacity to differentiate, both in vivo and in vitro, into adipocytes, chondrocytes, and osteoblasts, making them even more interesting in the regenerative medicine field. Tumor associated stroma has been recognized as a key element in tumor progression, necessary for the biological success of the tumor, and MSCs represent a functionally fundamental part of this associated stroma. Exosomes represent one of the dominant signaling pathways within the tumor microenvironment. Their biology raises high interest, with implications in different biological processes involved in cancer progression, such as the formation of the pre-metastatic niche. This is critical during the metastatic cascade, given that it is the formation of a permissive context that would allow metastatic tumor cells survival within the new environment. In this context, we explored the role of exosomes, particularly MSCs-derived exosomes as direct or indirect modulators. All this points out a possible new tool useful for designing better treatment and detection strategies for metastatic progression, including the management of chemoresistance.

Co-treatment of tumor cells with hyaluronan plus doxorubicin affects endothelial cell behavior independently of VEGF expression.

Hyaluronan, the main glycosaminoglycan of extracellular matrices, is concentrated in tissues with high cell proliferation and migration rates. In cancer, hyaluronan expression is altered and it becomes fragmented into low-molecular-weight forms, affecting mechanisms associated with cell proliferation, invasion, angiogenesis and multidrug resistance. Here, we analyzed the effect of low-molecular-weight hyaluronan on the response of T lymphoma, osteosarcoma, and mammary adenocarcinoma cell lines to the antineoplastic drug doxorubicin, and whether co-treatment with hyaluronan and doxorubicin modified the behavior of endothelial cells. Our aim was to associate the hyaluronan-doxorubicin response with angiogenic alterations in these tumors. After hyaluronan and doxorubicin co-treatment, hyaluronan altered drug accumulation and modulated the expression of ATP-binding cassette transporters in T-cell lymphoma cells. In contrast, no changes in drug accumulation were observed in cells from solid tumors, indicating that hyaluronan might not affect drug efflux. However, when we evaluated the effect on angiogenic mechanisms, the supernatant from tumor cells treated with doxorubicin exhibited a pro-angiogenic effect on endothelial cells. Hyaluronan-doxorubicin co-treatment increased migration and vessel formation in endothelial cells. This effect was independent of vascular endothelial growth factor but related to fibroblast growth factor-2 expression. Besides, we observed a pro-angiogenic effect on endothelial cells during hyaluronan and doxorubicin co-treatment in the in vivo murine model of T-cell lymphoma. Our results demonstrate for the first time that hyaluronan is a potential modulator of doxorubicin response by mechanisms that involve not only drug efflux but also angiogenic processes, providing an adverse tumor stroma during chemotherapy.

IL-8, GRO and MCP-1 produced by hepatocellular carcinoma microenvironment determine the migratory capacity of human bone marrow-derived mesenchymal stromal cells without affecting tumor aggressiveness.

New therapies are needed for advanced hepatocellular carcinoma (HCC) and the use of mesenchymal stromal cells (MSCs) carrying therapeutic genes is a promising strategy. HCC produce cytokines recruiting MSCs to the tumor milieu and modifying its biological properties. Our aim was to study changes generated on human MSCs exposed to conditioned media (CM) derived from human HCC fresh samples and xenografts. All CM shared similar cytokines expression pattern including CXCL1-2-3/GRO, CCL2/MCP-1 and CXCL8/IL-8 being the latter with the highest concentration. Neutralizing and knockdown experiments of CCL2/MCP-1, CXCL8/IL-8, CXCR1 and CXCR2 reduced in vitro MSC migration of ≥20%. Simultaneous CXCR1 and CXCR2 neutralization resulted in 50% of MSC migration inhibition. MSC stimulated with CM (sMSC) from HuH7 or HC-PT-5 showed a 2-fold increase of migration towards the CM compared with unstimulated MSC (usMSC). Gene expression profile of sMSC showed ~500 genes differentially expressed compared with usMSC, being 46 genes related with cell migration and invasion. sMSC increased fibroblasts and endothelial cells chemotaxis. Finally, sMSC with HuH7 CM and then inoculated in HCC tumor bearing-mice did not modify tumor growth. In this work we characterized factors produced by HCC responsible for the changes in MSC chemotactic capacity with would have an impact on therapeutic use of MSCs for human HCC.

Integrin αvß3 acting as membrane receptor for thyroid hormones mediates angiogenesis in malignant T cells.

The interaction of lymphoid tumor cells with components of the extracellular matrix via integrin αvß3 allows tumor survival and growth. This integrin was demonstrated to be the membrane receptor for thyroid hormones (THs) in several tissues. We found that THs, acting as soluble integrin αvß3 ligands, activated growth-related signaling pathways in T-cell lymphomas (TCLs). Specifically, TH-activated αvß3 integrin signaling promoted TCL proliferation and angiogenesis, in part, via the upregulation of vascular endothelial growth factor (VEGF). Consequently, genetic or pharmacologic inhibition of integrin αvß3 decreased VEGF production and induced TCL cell death in vitro and in human xenograft models. In sum, we show that integrin αvß3 transduces prosurvival signals into TCL nuclei, suggesting a novel mechanism for the endocrine modulation of TCL pathophysiology. Targeting this mechanism could constitute an effective and potentially low-toxicity chemotherapy-free treatment of TCL patients.

Human umbilical cord perivascular cells exhibited enhanced migration capacity towards hepatocellular carcinoma in comparison with bone marrow mesenchymal stromal cells: a role for autocrine motility factor receptor.

Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. Unfortunately, the incidence and mortality associated with HCC are increasing. Therefore, new therapeutic strategies are urgently needed and the use of mesenchymal stromal cells (MSCs) as carrier of therapeutic genes is emerging as a promising option. Different sources of MSCs are being studied for cell therapy and bone marrow-derived cells are the most extensively explored; however, birth associated-tissues represent a very promising source. The aim of this work was to compare the in vitro and in vivo migration capacity between bone marrow MSCs (BM-MSCs) and human umbilical cord perivascular cells (HUCPVCs) towards HCC. We observed that HUCPVCs presented higher in vitro and in vivo migration towards factors released by HCC. The expression of autocrine motility factor (AMF) receptor, genes related with the availability of the receptor on the cell surface (caveolin-1 and -2) and metalloproteinase 3, induced by the receptor activation and important for cell migration, was increased in HUCPVCs. The chemotactic response towards recombinant AMF was increased in HUCPVCs compared to BM-MSCs, and its inhibition in the conditioned medium from HCC induced higher decrease in HUCPVC migration than in BM-MSC. Our results indicate that HUCPVCs could be a useful cellular source to deliver therapeutic genes to HCC.

Increased migration of human mesenchymal stromal cells by autocrine motility factor (AMF) resulted in enhanced recruitment towards hepatocellular carcinoma.

BACKGROUND AND AIMS: Several reports described the migration of human mesenchymal stromal cells (MSCs) towards tumor-released factors. Autocrine motility factor (AMF) is produced by several tumors including hepatocellular carcinoma (HCC). The aim of this study was to analyze AMF involvement on MSC migration towards human HCC. METHODS: Production of AMF by HCC tumors was evaluated by western analysis. The effects of AMF on MSCs from different sources (bone marrow, adipose tissue and perivascular cells from umbilical cord) were analyzed using in vitro migration assay; metalloproteinase 2 (MMP2) activity and expression of critical genes were studied by zymography and qRT-PCR, respectively. To assess AMF involvement on the in vivo MSC migration, noninvasive fluorescence imaging was performed. To test the effect of AMF-primed MSCs on tumor development, in vitro proliferation and spheroids growth and in vivo tumor volume were evaluated. RESULTS: AMF produced by HCC was found to induce migration of different MSCs in vitro and to enhance their MMP2 activity. Stimulation of MSCs with recombinant AMF (rAMF) also induced the in vitro adhesion to endothelial cells in coincidence with changes in the expression levels of MMP3, AMF receptor, caveolin-1, and -2 and GDI-2. Importantly, stimulation of MSCs with rAMF increased the in vivo migration of MSCs towards experimental HCC tumors. AMF-priming of MSCs did not induce a pro-tumorigenic effect on HCC cells neither in vivo nor in vitro. CONCLUSION: AMF plays a role in MSC recruitment towards HCC. However, its ability to increase MSC migration to HCC for therapeutic purposes merits further evaluation.

A specific subpopulation of mesenchymal stromal cell carriers overrides melanoma resistance to an oncolytic adenovirus.

The homing properties of mesenchymal stromal cells (MSCs) toward tumors turn them into attractive tools for combining cell and gene therapy. The aim of this study was to select in a feasible way a human bone marrow-derived MSC subpopulation that might exhibit a selective ability to target the tumor mass. Using differential in vitro adhesive capacities during cells isolation, we selected a specific MSC subpopulation (termed MO-MSCs) that exhibited enhanced multipotent capacity and increased cell surface expression of specific integrins (integrins α2, α3, and α5), which correlated with an enhanced MO-MSCs adhesiveness toward their specific ligands. Moreover, MO-MSCs exhibited a higher migration toward conditioned media from different cancer cell lines and fresh human breast cancer samples in the presence or not of a human microendothelium monolayer. Further in vivo studies demonstrated increased tumor homing of MO-MSCs toward established 578T and MD-MBA-231 breast cancer and A375N melanoma tumor xenografts. Tumor penetration by MO-MSCs was highly dependent on metallopeptidases production as it was inhibited by the specific inhibitor 1,10 phenantroline. Finally, systemically administered MO-MSCs preloaded with an oncolytic adenovirus significantly inhibited tumor growth in mice harboring established A375N melanomas, overcoming the natural resistance of the tumor to in situ administration of the oncolytic adenovirus. In summary, this work characterizes a novel MSC subpopulation with increased tumor homing capacity that can be used to transport therapeutic compounds.

Hepatocellular carcinoma cells and their fibrotic microenvironment modulate bone marrow-derived mesenchymal stromal cell migration in vitro and in vivo.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third cause of cancer-related death. Fibrogenesis is an active process characterized by the production of several proinflammatory cytokines, chemokines and growth factors. It involves the activation of hepatic stellate cells (HSCs) which accumulate at the site of injury and are the main source of the extracellular matrix deposits. There are no curative treatments for advanced HCC, thus, new therapies are urgently needed. Mesenchymal stromal cells (MSCs) have the ability to migrate to sites of injury or to remodeling tissues after in vivo administration; however, in several cancer models they demonstrated limited efficacy to eradicate experimental tumors partially due to poor engraftment. Thus, the aim of this work was to analyze the capacity of human MSCs (hMSCs) to migrate and anchor to HCC tumors. We observed that HCC and HSCs, but not nontumoral stroma, produce factors that induce hMSC migration in vitro. Conditioned media (CM) generated from established HCC cell lines were found to induce higher levels of hMSC migration than CM derived from fresh patient tumor samples. In addition, after exposure to CM from HCC cells or HSCs, hMSCs demonstrated adhesion and invasion capability to endothelial cells, type IV collagen and fibrinogen. Consistently, these cells were found to increase metalloproteinase-2 activity. In vivo studies with subcutaneous and orthotopic HCC models indicated that intravenously infused hMSCs migrated to lungs, spleen and liver. Seven days post-hMSC infusion cells were located also in the tumor in both models, but the signal intensity was significantly higher in orthotopic than in subcutaneous model. Interestingly, when orthotopic HCC tumors where established in noncirrhotic or cirrhotic livers, the amount of hMSCs localized in the liver was higher in comparison with healthy animals. A very low signal was found in lungs and spleens, indicating that liver tumors are able to recruit them at high efficiency. Taken together our results indicate that HCC and HSC cells produce factors that efficiently induce hMSC migration toward tumor microenvironment in vitro and in vivo and make MSCs candidates for cell-based therapeutic strategies to hepatocellular carcinoma associated with fibrosis.

Overexpression of SPARC obliterates the in vivo tumorigenicity of human hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related death worldwide. Current treatments are extremely disappointing. SPARC (Secreted protein, acidic and rich in cysteine) is a matricellular glycoprotein with differential expression in several tumors, including HCC, which significance remains unclear. We infected HCC cells (HepG2, Hep3B and Huh7) with an adenovirus expressing SPARC (AdsSPARC) to examine the role of SPARC expression on HCC cells and its effect on tumor aggressiveness. The in vitro HCC cells substrate-dependent proliferation and cell cycle profile were unaffected; however, SPARC overexpression reduced HCC proliferation when cells were grown in spheroids. A mild induction of cellular apoptosis was observed upon SPARC overexpression. SPARC overexpression resulted in spheroid growth inhibition in vitro while no effects were found when recombinant SPARC was exogenously applied. Moreover, the clonogenic and migratory capabilities were largely decreased in SPARC-overexpressing HCC cells, altogether suggesting a less aggressive HCC cell phenotype. Consistently, AdsSPARC-transduced cells showed increased E-cadherin expression and a concomitant decrease in N-cadherin expression. Furthermore, SPARC overexpression was found to reduce HCC cell viability in response to 5-FU-based chemotherapy in vitro, partially through induction of apoptosis. In vivo experiments revealed that SPARC overexpression in HCC cells inhibited their tumorigenic capacity and increased animal survival through a mechanism that partially involves host macrophages. Our data suggest that SPARC overexpression in HCC cells results in a reduced tumorigenicity partially through the induction of mesenchymal-to-epithelial transition (MET). These evidences point to SPARC as a novel target for HCC treatment.

Expression of granulocyte-colony-stimulating factor and its receptor in human Ewing sarcoma cells and patient tumor specimens: potential consequences of granulocyte-colony-stimulating factor administration.

BACKGROUND: Ewing sarcoma (ES) is a highly vascular malignancy. It has been demonstrated that both angiogenesis and vasculogenesis contribute to the growth of ES tumors. Granulocyte-colony-stimulating factor (G-CSF), a cytokine known to stimulate bone marrow (BM) stem cell production and angiogenesis, is routinely administered to ES patients after chemotherapy. Whether ES cells and patient tumor samples express G-CSF and its receptor (G-CSFR) and whether treatment with this factor enhances tumor growth was examined. METHODS: Human ES cell lines were analyzed for expression of G-CSF and G-CSFR in vitro and in vivo. Sixty-eight paraffin-embedded and 15 frozen tumor specimens from patients with ES were also evaluated for the presence of G-CSF and G-CSFR. The in vivo effect of G-CSF on angiogenesis and BM cell migration was determined. Using a TC/7-1 human ES mouse model, the effect of G-CSF administration on ES tumors was investigated. RESULTS: G-CSF and G-CSFR protein and RNA expression was identified in all ES cell lines and patient samples analyzed. In addition, G-CSF was found to stimulate angiogenesis and BM cell migration in vivo. Tumor growth was found to be significantly increased in mice treated with G-CSF. The average tumor volume for the group treated with G-CSF was 1218 mm(3) compared with 577 mm(3) for the control group (P = .006). CONCLUSIONS: The findings that ES cells and patient tumors expressed both G-CSF and its receptor in vitro and in vivo and that the administration of G-CSF promoted tumor growth in vivo suggest that the potential consequences of G-CSF administration should be investigated further.

Suppression of Ewing's sarcoma tumor growth, tumor vessel formation, and vasculogenesis following anti vascular endothelial growth factor receptor-2 therapy.

PURPOSE: We previously showed that bone marrow cells participate in new tumor vessel formation in Ewing's sarcoma, and that vascular endothelial growth factor 165 (VEGF(165)) is critical to this process. The purpose of this study was to determine whether blocking VEGF receptor 2 (VEGFR-2) with DC101 antibody suppresses tumor growth, reduces tumor vessel formation, and inhibits the migration of bone marrow cells into the tumor. EXPERIMENTAL DESIGN: An H-2 MHC-mismatched bone marrow transplant Ewing's sarcoma mouse model was used. Bone marrow cells from CB6F1 (MHC H-2(b/d)) mice were injected into irradiated BALB/cAnN mice (MHC H-2(d)). TC71 Ewing's sarcoma cells were s.c. injected 4 weeks after the bone marrow transplantation. Mice were then treated i.p. with DC101 antibody or immunoglobulin G (control) twice a week for 3 weeks starting 3 days after tumor cell injection. RESULTS: DC101 antibody therapy significantly reduced tumor growth and tumor mean vessel density (P < 0.05) and increased tumor cell apoptosis. Decreased bone marrow cell migration into the tumor was also shown after DC101 therapy as assessed by the colocalization of H-2K(b) and CD31 using immunohistochemistry. DC101 inhibited the migration of both human and mouse vessel endothelial cells in vitro. CONCLUSION: These results indicated that blocking VEGFR-2 with DC101 antibodies may be a useful therapeutic approach for treating patients with Ewing's sarcoma.