Cancer-associated mesenchymal stroma fosters the stemness of osteosarcoma cells in response to intratumoral acidosis via NF-κB activation

The role of mesenchymal stem cells (MSC) in osteosarcoma (OS), the most common primary tumor of bone, has not been extensively elucidated. We have recently shown that OS is characterized by interstitial acidosis, a microenvironmental condition that is similar to a wound setting, in which mesenchymal reactive cells are activated to release mitogenic and chemotactic factors. We therefore intended to test the hypothesis that, in OS, acid-activated MSC influence tumor cell behavior. Conditioned media or co-culture with normal MSC previously incubated with short-term acidosis (pH 6.8 for 10 hr, H+-MSC) enhanced OS clonogenicity and invasion. This effect was mediated by NF-κB pathway activation. In fact, deep-sequencing analysis, confirmed by Real-Time PCR and ELISA, demonstrated that H+-MSC differentially induced a tissue remodeling phenotype with increased expression of RelA, RelB and NF-κB1, and downstream, of CSF2/GM-CSF, CSF3/G-CSF and BMP2 colony-promoting factors, and of chemokines (CCL5, CXCL5 and CXCL1), and cytokines (IL6 and IL8), with an increased expression of CXCR4. An increased expression of IL6 and IL8 were found only in normal stromal cells, but not in OS cells, and this was confirmed in tumor-associated stromal cells isolated from OS tissue. Finally, H+-MSC conditioned medium differentially promoted OS stemness (sarcosphere number, stem-associated gene expression), and chemoresistance also via IL6 secretion. Our data support the hypothesis that the acidic OS microenvironment is a key factor for MSC activation, in turn promoting the secretion of paracrine factors that influence tumor behavior, a mechanism that holds the potential for future therapeutic interventions aimed to target OS

Reduction of metastasis using a non-volatile buffer

The tumor microenvironment is acidic as a consequence of upregulated glycolysis and poor perfusion and this acidity, in turn, promotes invasion and metastasis. We have recently demonstrated that chronic consumption of sodium bicarbonate increased tumor pH and reduced spontaneous and experimental metastases. This occurred without affecting systemic pH, which was compensated. Additionally, these prior data did not rule out the possibility that bicarbonate was working though effects on carbonic anhydrase, and not as a buffer per se. Here, we present evidence that chronic ingestion of a non-volatile buffer, 2-imidazole-1-yl-3-ethoxycarbonylpropionic acid (IEPA) with a pKa of 6.9 also reduced metastasis in an experimental PC3M prostate cancer mouse model. Animals (n = 30) were injected with luciferase expressing PC3M prostate cancer cells either subcutaneously (s.c., n = 10) or intravenously (i.v., n = 20). Four days prior to inoculations, half of the animals for each experiment were provided drinking water containing 200 mM IEPA buffer. Animals were imaged weekly to follow metastasis, and these data showed that animals treated with IEPA had significantly fewer experimental lung metastasis compared to control groups (P < 0.04). Consistent with prior work, the pH of treated tumors was elevated compared to controls. IEPA is observable by in vivo magnetic resonance spectroscopy and this was used to measure the presence of IEPA in the bladder, confirming that it was orally available. The results of this study indicate that metastasis can be reduced by non-volatile buffers as well as bicarbonate and thus the effect appears to be due to pH buffering per se

Evolutionary Mechanisms for Host Resistance to Tumor Growth and Subsequent Cancer Cell Counter-Adaptations

Cancer is well-recognized as an evolutionary system, as first proposed by Cairns and Nowell more than 60 years ago. In an evolutionary context, cancers growing in vivo typically consist of heterogeneous subpopulations of cells that interact with each other and with host cells through selection forces operating at many temporal and spatial scales. Moreover, the tumor environment comprises more than just cancer cells; it includes a rich cancer stroma and cancer-driving molecules such as cytokines and metabolites. The tumor’s environment comprises intratumoral heterogeneity that often leads to therapy resistance attributed to the essential roles of many genetic and nongenetic mechanisms. My dissertation investigated possible outcomes from complex eco-evolutionary interactions between cancer cells and their host organism. By exploring phenotypic, genetic, and epigenetic mechanisms and responses, I discovered that both immune and non-immune resistance strategies are evolutionarily possible. Thus, my findings from three related studies provide novel insights into the evolutionary “arms race” of tumor progression in immune-competent and immune-deficient mice. In the first study involving interactions between the tumor and the host immune system, I identified the consequence of disturbing the equilibrium phase of the dynamic process that consists of immunosurveillance and tumor progression (i.e., cancer immunoediting). This phase is a characteristic of tumor dormancy that is achieved when a complex equilibrium occurs between the tumor cell and the immune system, and the tumor remains in stasis. My studies have shown that perturbation of this equilibrium by a stress stimulus, such as administration of volatile and intravenous anesthetics, enhances tumor growth in immune-competent mice but not in immune-deficient mice. This suggests that the immune system can be a key component in the oncological stress response for those pharmacologic agents for hosts that are not immune compromised. In the second study, I identified different strategies that allow tumors to be resistant to one type of cancer by applying selective breeding over 10 generations to laboratory immune-competent and immune-deficient mice inoculated with subcutaneous tumors. My studies showed that both mice strains evolved greater cancer resistance and suppression mechanisms after 10 generations of selection, but the tumors of these mice responded differently. In the absence of an intact adaptive immune system, the immune-deficient mice evolved with changes in mesenchymal cells that limited resources and cancer cell growth. In contrast, the immune-competent mice evolved with improved immune-mediated killing of cancer cells through changes in immune cell frequency, phenotype, and function. Cancer cells deployed observable counter- responses to the hosts’ cancer suppression mechanisms. These counter-responses included increased proliferation in immune-competent mice and both less cell proliferation and higher necrosis in immune-deficient mice. My studies suggest that host species can rapidly evolve both immunologic and non-immunologic tumor defenses depending on the lineage. However, cancer cells maintain sufficient plasticity to deploy effective phenotypic and population-based counterstrategies quickly. For example, variation in tumor gene expression was largely explained by the differences between the hosts and the fact that the hosts responded differently to selection for resistance to the tumor. In the third study, I examined how transcriptomic responses evolved in the hosts in response to selection for resistance as well as the transcriptomic response of the original cancer cell line. In immune-competent mice compared to immune-deficient mice, I found increased expression in genes enriched for developmental processes, cell migration and movement, and cell membrane composition. The gene with the highest fold expression increase was Semaphorin 3D (Sema3D). This gene is implicated in the development and formation of blood vessels during angiogenesis for the regulation of the epithelial to mesenchymal transition. In addition, genes related to integrin binding, cell adhesion molecular binding, and extracellular matrix (ECM) binding were differentially expressed in immune-deficient mice. Expression levels of extracellular matrix markers, such as collagen type VI (Col18a1), alpha, prolyl 3-hydroxylase 2 (p3h2), and collagen, type XII alpha (Col12a1), were decreased. My future plans include associating the genome-wide differentially expressed genes with methylation changes, as well as how examining how patterns of gene expression and methylation may change across the tumor and the host in response to selection for cancer resistance. The data presented here demonstrates the importance of molecular-level mechanisms that can be effectively targeted for therapeutic benefits. Furthermore, the mice strains developed in these studies can be used to discover more mechanisms of tumor growth resistance and metastasis that may lead to significant advancements in clinical treatments for patients with cancer

Evolutionary Mechanisms for Host Resistance to Tumor Growth and Subsequent Cancer Cell Counter-Adaptations

Cancer is well-recognized as an evolutionary system, as first proposed by Cairns and Nowell more than 60 years ago. In an evolutionary context, cancers growing in vivo typically consist of heterogeneous subpopulations of cells that interact with each other and with host cells through selection forces operating at many temporal and spatial scales. Moreover, the tumor environment comprises more than just cancer cells; it includes a rich cancer stroma and cancer-driving molecules such as cytokines and metabolites. The tumor’s environment comprises intratumoral heterogeneity that often leads to therapy resistance attributed to the essential roles of many genetic and nongenetic mechanisms. My dissertation investigated possible outcomes from complex eco-evolutionary interactions between cancer cells and their host organism. By exploring phenotypic, genetic, and epigenetic mechanisms and responses, I discovered that both immune and non-immune resistance strategies are evolutionarily possible. Thus, my findings from three related studies provide novel insights into the evolutionary “arms race” of tumor progression in immune-competent and immune-deficient mice. In the first study involving interactions between the tumor and the host immune system, I identified the consequence of disturbing the equilibrium phase of the dynamic process that consists of immunosurveillance and tumor progression (i.e., cancer immunoediting). This phase is a characteristic of tumor dormancy that is achieved when a complex equilibrium occurs between the tumor cell and the immune system, and the tumor remains in stasis. My studies have shown that perturbation of this equilibrium by a stress stimulus, such as administration of volatile and intravenous anesthetics, enhances tumor growth in immune-competent mice but not in immune-deficient mice. This suggests that the immune system can be a key component in the oncological stress response for those pharmacologic agents for hosts that are not immune compromised. In the second study, I identified different strategies that allow tumors to be resistant to one type of cancer by applying selective breeding over 10 generations to laboratory immune-competent and immune-deficient mice inoculated with subcutaneous tumors. My studies showed that both mice strains evolved greater cancer resistance and suppression mechanisms after 10 generations of selection, but the tumors of these mice responded differently. In the absence of an intact adaptive immune system, the immune-deficient mice evolved with changes in mesenchymal cells that limited resources and cancer cell growth. In contrast, the immune-competent mice evolved with improved immune-mediated killing of cancer cells through changes in immune cell frequency, phenotype, and function. Cancer cells deployed observable counter- responses to the hosts’ cancer suppression mechanisms. These counter-responses included increased proliferation in immune-competent mice and both less cell proliferation and higher necrosis in immune-deficient mice. My studies suggest that host species can rapidly evolve both immunologic and non-immunologic tumor defenses depending on the lineage. However, cancer cells maintain sufficient plasticity to deploy effective phenotypic and population-based counterstrategies quickly. For example, variation in tumor gene expression was largely explained by the differences between the hosts and the fact that the hosts responded differently to selection for resistance to the tumor. In the third study, I examined how transcriptomic responses evolved in the hosts in response to selection for resistance as well as the transcriptomic response of the original cancer cell line. In immune-competent mice compared to immune-deficient mice, I found increased expression in genes enriched for developmental processes, cell migration and movement, and cell membrane composition. The gene with the highest fold expression increase was Semaphorin 3D (Sema3D). This gene is implicated in the development and formation of blood vessels during angiogenesis for the regulation of the epithelial to mesenchymal transition. In addition, genes related to integrin binding, cell adhesion molecular binding, and extracellular matrix (ECM) binding were differentially expressed in immune-deficient mice. Expression levels of extracellular matrix markers, such as collagen type VI (Col18a1), alpha, prolyl 3-hydroxylase 2 (p3h2), and collagen, type XII alpha (Col12a1), were decreased. My future plans include associating the genome-wide differentially expressed genes with methylation changes, as well as how examining how patterns of gene expression and methylation may change across the tumor and the host in response to selection for cancer resistance. The data presented here demonstrates the importance of molecular-level mechanisms that can be effectively targeted for therapeutic benefits. Furthermore, the mice strains developed in these studies can be used to discover more mechanisms of tumor growth resistance and metastasis that may lead to significant advancements in clinical treatments for patients with cancer

The Genomic Processes of Biological Invasions: From Invasive Species to Cancer Metastases and Back Again

The concept of invasion is useful across a broad range of contexts, spanning from the fine scale landscape of cancer tumors up to the broader landscape of ecosystems. Invasion biology provides extraordinary opportunities for studying the mechanistic basis of contemporary evolution at the molecular level. Although the field of invasion genetics was established in ecology and evolution more than 50 years ago, there is still a limited understanding of how genomic level processes translate into invasive phenotypes across different taxa in response to complex environmental conditions. This is largely because the study of most invasive species is limited by information about complex genome level processes. We lack good reference genomes for most species. Rigorous studies to examine genomic processes are generally too costly. On the contrary, cancer studies are fortified with extensive resources for studying genome level dynamics and the interactions among genetic and non-genetic mechanisms. Extensive analysis of primary tumors and metastatic samples have revealed the importance of several genomic mechanisms including higher mutation rates, specific types of mutations, aneuploidy or whole genome doubling and non-genetic effects. Metastatic sites can be directly compared to primary tumor cell counterparts. At the same time, clonal dynamics shape the genomics and evolution of metastatic cancers. Clonal diversity varies by cancer type, and the tumors’ donor and recipient tissues. Still, the cancer research community has been unable to identify any common events that provide a universal predictor of “metastatic potential” which parallels findings in evolutionary ecology. Instead, invasion in cancer studies depends strongly on context, including order of events and clonal composition. The detailed studies of the behavior of a variety of human cancers promises to inform our understanding of genome level dynamics in the diversity of invasive species and provide novel insights for management

The Genomic Processes of Biological Invasions: From Invasive Species to Cancer Metastases and Back Again

The concept of invasion is useful across a broad range of contexts, spanning from the fine scale landscape of cancer tumors up to the broader landscape of ecosystems. Invasion biology provides extraordinary opportunities for studying the mechanistic basis of contemporary evolution at the molecular level. Although the field of invasion genetics was established in ecology and evolution more than 50 years ago, there is still a limited understanding of how genomic level processes translate into invasive phenotypes across different taxa in response to complex environmental conditions. This is largely because the study of most invasive species is limited by information about complex genome level processes. We lack good reference genomes for most species. Rigorous studies to examine genomic processes are generally too costly. On the contrary, cancer studies are fortified with extensive resources for studying genome level dynamics and the interactions among genetic and non-genetic mechanisms. Extensive analysis of primary tumors and metastatic samples have revealed the importance of several genomic mechanisms including higher mutation rates, specific types of mutations, aneuploidy or whole genome doubling and non-genetic effects. Metastatic sites can be directly compared to primary tumor cell counterparts. At the same time, clonal dynamics shape the genomics and evolution of metastatic cancers. Clonal diversity varies by cancer type, and the tumors’ donor and recipient tissues. Still, the cancer research community has been unable to identify any common events that provide a universal predictor of “metastatic potential” which parallels findings in evolutionary ecology. Instead, invasion in cancer studies depends strongly on context, including order of events and clonal composition. The detailed studies of the behavior of a variety of human cancers promises to inform our understanding of genome level dynamics in the diversity of invasive species and provide novel insights for management

Intermittent hypoxia selects for genotypes and phenotypes that increase survival, invasion, and therapy resistance.

Hypoxia in tumors correlates with greater risk of metastases, increased invasiveness, and resistance to systemic and radiation therapy. The evolutionary dynamics that links specific adaptations to hypoxia with these observed tumor properties have not been well investigated. While some tumor populations may experience fixed hypoxia, cyclical and stochastic transitions from normoxia to hypoxia are commonly observed in vivo. Although some phenotypic adaptations to this cyclic hypoxia are likely reversible, we hypothesize that some adaptations may become fixed through mutations promoted by hypoxia-induced genomic instability. Here we seek to identify genetic alterations and corresponding stable phenotypes that emerge following cyclic hypoxia. Although these changes may originate as adaptations to this specific environmental stress, their fixation in the tumor genome may result in their observation in tumors from regions of normoxia, a condition known as pseudohypoxia. We exposed several epithelial cell lines to 50 cycles of hypoxia-normoxia, followed by culture in normoxia over a period of several months. Molecular analyses demonstrated permanent changes in expression of several oncogenes and tumor-suppressors, including p53, E-cadherin, and Hif-1α. These changes were associated with increased resistance to multiple cytotoxins, increased survival in hypoxia and increased anchorage-independent growth. These results suggest cycles of hypoxia encountered in early cancers can select for specific and stable genotypic and phenotypic properties that persist even in normoxic conditions, which may promote tumor progression and resistance to therapy

Mechanisms of buffer therapy resistance

Many studies have shown that the acidity of solid tumors contributes to local invasion and metastasis. Oral pH buffers can specifically neutralize the acidic pH of tumors and reduce the incidence of local invasion and metastatic formation in multiple murine models. However, this effect is not universal as we have previously observed that metastasis is not inhibited by buffers in some tumor models, regardless of buffer used. B16-F10 (murine melanoma), LL/2 (murine lung) and HCT116 (human colon) tumors are resistant to treatment with lysine buffer therapy, whereas metastasis is potently inhibited by lysine buffers in MDA-MB-231 (human breast) and PC3M (human prostate) tumors. In the current work, we confirmed that sensitive cells utilized a pH-dependent mechanism for successful metastasis supported by a highly glycolytic phenotype that acidifies the local tumor microenvironment resulting in morphological changes. In contrast, buffer-resistant cell lines exhibited a pH-independent metastatic mechanism involving constitutive secretion of matrix degrading proteases without elevated glycolysis. These results have identified two distinct mechanisms of experimental metastasis, one of which is pH-dependent (buffer therapy sensitive cells) and one which is pH-independent (buffer therapy resistant cells). Further characterization of these models has potential for therapeutic benefit

Immunomodulatory and pro-oncologic effects of ketamine and isoflurane anesthetics in a murine model.

IntroductionVolatile and intravenous anesthetics may worsen oncologic outcomes in basic science animal models. These effects may be related to suppressed innate and adaptive immunity, decreased immunosurveillance, and disrupted cellular signaling. We hypothesized that anesthetics would promote lung tumor growth via altered immune function in a murine model and tested this using an immunological control group of immunodeficient mice.MethodsLewis lung carcinoma cells were injected via tail vein into C57BL/6 immunocompetent and NSG immunodeficient mice during exposure to isoflurane and ketamine versus controls without anesthesia. Mice were imaged on days 0, 3, 10, and 14 post-tumor cell injection. On day 14, mice were euthanized and organs fixed for metastasis quantification and immunohistochemistry staining. We compared growth of tumors measured from bioluminescent imaging and tumor metastasis in ex vivo bioluminescent imaging of lung and liver.ResultsMetastases were significantly greater for immunocompromised NSG mice than immunocompetent C57BL/6 mice over the 14-day experiment (partial η2 = 0.67, 95% CI = 0.54, 0.76). Among immunocompetent mice, metastases were greatest for mice receiving ketamine, intermediate for those receiving isoflurane, and least for control mice (partial η2 = 0.88, 95% CI = 0.82, 0.91). In immunocompetent mice, significantly decreased T lymphocyte (partial η2 = 0.83, 95% CI = 0.29, 0.93) and monocyte (partial η2 = 0.90, 95% CI = 0.52, 0.96) infiltration was observed in anesthetic-treated mice versus controls.ConclusionsThe immune system appears central to the pro-metastatic effects of isoflurane and ketamine in a murine model, with decreased T lymphocytes and monocytes likely playing a role