L-DOS47 Elevates Pancreatic Cancer Tumor pH and Enhances Response to Immunotherapy.

Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds to CEACAM6, a cell-surface protein that is highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO2, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Using chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) to measure the tumor extracellular pH (pHe), we confirmed that L-DOS47 raises the tumor pHe from 4 h to 96 h post injection in acidic tumors (average increase of 0.13 units). Additional studies showed that combining L-DOS47 with anti-PD1 significantly increases the efficacy of the anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

Experimental and phylogenetic evidence for correlated gene expression evolution in endometrial and skin fibroblasts.

Gene expression change is a dominant mode of evolution. Mutations, however, can affect gene expression in multiple cell types. Therefore, gene expression evolution in one cell type can lead to similar gene expression changes in another cell type. Here, we test this hypothesis by investigating dermal skin fibroblasts (SFs) and uterine endometrial stromal fibroblasts (ESFs). The comparative dataset consists of transcriptomes from cultured SF and ESF of nine mammalian species. We find that evolutionary changes in gene expression in SF and ESF are highly correlated. The experimental dataset derives from a SCID mouse strain selected for slow cancer growth leading to substantial gene expression changes in SFs. We compared the gene expression profiles of SF with that of ESF and found a significant correlation between them. We discuss the implications of these findings for the evolutionary correlation between placental invasiveness and vulnerability to metastatic cancer.

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

INTRODUCTION: Volatile 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. METHODS: Lewis 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. RESULTS: Metastases 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. CONCLUSIONS: The 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.

L-DOS47 enhances response to immunotherapy in pancreatic cancer tumor.

Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds CEACAM6, a cell surface protein highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO2, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Our results demonstrate that combining L DOS47 with anti-PD1 significantly increases the efficacy of anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

Intraperitoneal Delivery of Iopamidol to Assess Extracellular pH of Orthotopic Pancreatic Tumor Model by CEST-MRI.

The extracellular pH (pHe) of solid tumors is often acidic, as a consequence of the Warburg effect, and an altered metabolic state is often associated with malignancy. It has been shown that acidosis can promote tumor progression; thus, many therapeutic strategies have been adopted against tumor metabolism; one of these involves alkalinization therapies to raise tumor pH to inhibit tumor progression, improve immune surveillance, and overcome resistance to chemotherapies. Chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) is a noninvasive technique that can measure pH in vivo using pH-sensitive contrast agents. Iopamidol, an iodinated contrast agent, clinically used for computed tomography (CT), contains amide group protons with pH-dependent exchange rates that can reveal the pHe of the tumor microenvironment. In this study, we optimized intraperitoneal (IP) delivery of iopamidol to facilitate longitudinal assessments of orthotopic pancreatic tumor pHe by CEST-MRI. Following IV-infusion and IP-bolus injections, we compared the two protocols for assessing tumor pH. Time-resolved CT imaging was used to evaluate the uptake of iopamidol in the tumor, revealing that IP-bolus delivered a high amount of contrast agent 40 min postinjection, which was similar to the amounts reached with the IV-infusion protocol. As expected, both IP and IV injection protocols produced comparable measurements of tumor pHe, showing no statistically significant difference between groups (p=0.16). In addition, we showed the ability to conduct longitudinal monitoring of tumor pHe using CEST-MRI with the IP injection protocol, revealing a statistically significant increase in tumor pHe following bicarbonate administration (p < 0.001). In conclusion, this study shows the capability to measure pHe using an IP delivery of iopamidol into orthotopic pancreatic tumors, which is important to conduct longitudinal studies.

Back to basic: Trials and tribulations of alkalizing agents in cancer.

"Dysregulated" metabolism is a characteristic of the cancer cell phenotype. This includes persistent use of glycolytic metabolism in normoxic environments (Warburg effect) leading to increased acid production and accumulation of protons in the interstitial space. Although often thought to be disordered, altered cancer metabolism is the outcome of intense Darwinian selection and, thus, must have evolved to maximize cancer cell fitness. In an evolutionary context, cancer-induced acidification of the microenvironment represents a niche construction strategy to promote proliferation. Ecological advantages conferred on the cancer population included remodeling of the extracellular matrix to promote local invasion, suppression of potential competitive proliferation of fibroblasts, and suppression of host immune response. Preclinical data demonstrates that increasing the serum buffering capacity (through, for example, oral sodium bicarbonate and TRIS) can neutralize the acidic tumor microenvironment with inhibition local invasion and proliferation which can be synergistic with the effects of chemotherapy and immunotherapy agents. Here, we describe the proton dynamics in cancer and their influence on tumor progression and metastasis. Additionally, we will discuss targeting the tumor acidosis with alkalizing agents including our bicarbonate clinical trial results. Clinical Trial Registration: clinicaltrials.gov, identifier NCT01350583, NCT01198821 and NCT01846429.

A simulation of parental and glycolytic tumor phenotype competition predicts observed responses to pH changes and increased glycolysis after anti-VEGF therapy.

Clinical cancers are typically spatially and temporally heterogeneous, containing multiple microenvironmental habitats and diverse phenotypes and/or genotypes, which can interact through resource competition and direct or indirect interference. A common intratumoral evolutionary pathway, probably initiated as adaptation to hypoxia, leads to the "Warburg phenotype" which maintains high glycolytic rates and acid production, even in normoxic conditions. Since individual cancer cells are the unit of Darwinian selection, intraspecific competition dominates intratumoral evolution. Thus, elements of the Warburg phenotype become key "strategies" in competition with cancer cell populations that retain the metabolism of the parental normal cells. Here we model the complex interactions of cell populations with Warburg and parental phenotypes as they compete for access to vasculature, while subject to direct interference by Warburg-related acidosis. In this competitive environment, vasculature delivers nutrients, removes acid and necrotic detritus, and responds to signaling molecules (VEGF and TNF-α). The model is built in a nested fashion and growth parameters are derived from monolayer, spheroid, and xenograft experiments on prostate cancer. The resulting model of in vivo tumor growth reaches a steady state, displaying linear growth and coexistence of both glycolytic and parental phenotypes consistent with experimental observations. The model predicts that increasing tumor pH sufficiently early can arrest the development of the glycolytic phenotype, while decreasing tumor pH accelerates this evolution and increases VEGF production. The model's predicted dual effects of VEGF blockers in decreasing tumor growth while increasing the glycolytic fraction of tumor cells has potential implications for optimizing angiogenic inhibitors.

Frequency-dependent interactions determine outcome of competition between two breast cancer cell lines.

Tumors are highly dynamic ecosystems in which diverse cancer cell subpopulations compete for space and resources. These complex, often non-linear interactions govern continuous spatial and temporal changes in the size and phenotypic properties of these subpopulations. Because intra-tumoral blood flow is often chaotic, competition for resources may be a critical selection factor in progression and prognosis. Here, we quantify resource competition using 3D spheroid cultures with MDA-MB-231 and MCF-7 breast cancer cells. We hypothesized that MCF-7 cells, which primarily rely on efficient aerobic glucose metabolism, would dominate the population under normal pH and low glucose conditions; and MDA-MB-231 cells, which exhibit high levels of glycolytic metabolism, would dominate under low pH and high glucose conditions. In spheroids with single populations, MCF-7 cells exhibited equal or superior intrinsic growth rates (density-independent measure of success) and carrying capacities (density-dependent measure of success) when compared to MDA-MB-231 cells under all pH and nutrient conditions. Despite these advantages, when grown together, MCF-7 cells do not always outcompete MDA-MB-231 cells. MDA-MB-231 cells outcompete MCF-7 cells in low glucose conditions and coexistence is achieved in low pH conditions. Under all conditions, MDA-MB-231 has a stronger competitive effect (frequency-dependent interaction) on MCF-7 cells than vice-versa. This, and the inability of growth rate or carrying capacity when grown individually to predict the outcome of competition, suggests a reliance on frequency-dependent interactions and the need for competition assays. We frame these results in a game-theoretic (frequency-dependent) model of cancer cell interactions and conclude that competition assays can demonstrate critical density-independent, density-dependent and frequency-dependent interactions that likely contribute to in vivo outcomes.

Artificial selection for host resistance to tumour growth and subsequent cancer cell adaptations: an evolutionary arms race.

BACKGROUND: Cancer progression is governed by evolutionary dynamics in both the tumour population and its host. Since cancers die with the host, each new population of cancer cells must reinvent strategies to overcome the host's heritable defences. In contrast, host species evolve defence strategies over generations if tumour development limits procreation. METHODS: We investigate this "evolutionary arms race" through intentional breeding of immunodeficient SCID and immunocompetent Black/6 mice to evolve increased tumour suppression. Over 10 generations, we injected Lewis lung mouse carcinoma cells [LL/2-Luc-M38] and selectively bred the two individuals with the slowest tumour growth at day 11. Their male progeny were hosts in the subsequent round. RESULTS: The evolved SCID mice suppressed tumour growth through biomechanical restriction from increased mesenchymal proliferation, and the evolved Black/6 mice suppressed tumour growth by increasing immune-mediated killing of cancer cells. However, transcriptomic changes of multicellular tissue organisation and function genes allowed LL/2-Luc-M38 cells to adapt through increased matrix remodelling in SCID mice, and reduced angiogenesis, increased energy utilisation and accelerated proliferation in Black/6 mice. CONCLUSION: Host species can rapidly evolve both immunologic and non-immunologic tumour defences. However, cancer cell plasticity allows effective phenotypic and population-based counter strategies.

T-cells produce acidic niches in lymph nodes to suppress their own effector functions.

The acidic pH of tumors profoundly inhibits effector functions of activated CD8 + T-cells. We hypothesize that this is a physiological process in immune regulation, and that it occurs within lymph nodes (LNs), which are likely acidic because of low convective flow and high glucose metabolism. Here we show by in vivo fluorescence and MR imaging, that LN paracortical zones are profoundly acidic. These acidic niches are absent in athymic Nu/Nu and lymphodepleted mice, implicating T-cells in the acidifying process. T-cell glycolysis is inhibited at the low pH observed in LNs. We show that this is due to acid inhibition of monocarboxylate transporters (MCTs), resulting in a negative feedback on glycolytic rate. Importantly, we demonstrate that this acid pH does not hinder initial activation of naïve T-cells by dendritic cells. Thus, we describe an acidic niche within the immune system, and demonstrate its physiological role in regulating T-cell activation.

Correction: Acidity promotes tumour progression by altering macrophage phenotype in prostate cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Acidity promotes tumour progression by altering macrophage phenotype in prostate cancer.

BACKGROUND: Tumours rapidly ferment glucose to lactic acid even in the presence of oxygen, and coupling high glycolysis with poor perfusion leads to extracellular acidification. We hypothesise that acidity, independent from lactate, can augment the pro-tumour phenotype of macrophages. METHODS: We analysed publicly available data of human prostate cancer for linear correlation between macrophage markers and glycolysis genes. We used zwitterionic buffers to adjust the pH in series of in vitro experiments. We then utilised subcutaneous and transgenic tumour models developed in C57BL/6 mice as well as computer simulations to correlate tumour progression with macrophage infiltration and to delineate role of acidity. RESULTS: Activating macrophages at pH 6.8 in vitro enhanced an IL-4-driven phenotype as measured by gene expression, cytokine profiling, and functional assays. These results were recapitulated in vivo wherein neutralising intratumoural acidity reduced the pro-tumour phenotype of macrophages, while also decreasing tumour incidence and invasion in the TRAMP model of prostate cancer. These results were recapitulated using an in silico mathematical model that simulate macrophage responses to environmental signals. By turning off acid-induced cellular responses, our in silico mathematical modelling shows that acid-resistant macrophages can limit tumour progression. CONCLUSIONS: This study suggests that tumour acidity contributes to prostate carcinogenesis by altering the state of macrophage activation.

Acidosis and cancer: from mechanism to neutralization.

The extracellular pH of solid tumors is unequivocally acidic due to a combination of high rates of lactic acid production (a consequence of fermentative glycolytic metabolism) and poor perfusion. This has been documented by us and others in a wide variety of solid tumor models, primarily using magnetic resonance spectroscopic imaging (MRSI). This acidity contributes to tumor progression by inducing genome instability, promoting local invasion and metastases, inhibiting anti-tumor immunity, and conferring resistance to chemo- and radio-therapies. Systemic buffer therapies can neutralize tumor acidity and has been shown to inhibit local invasion and metastasis and improve immune surveillance in a variety of cancer model systems. This review will revisit the causes and consequences of acidosis by summarizing strategies used by cancer cells to adapt to acidosis, and how this acidity associated with carcinogenesis, metastasis, and immune function. Finally, this review will discuss how neutralization of acidity can be used to inhibit carcinogenesis and metastasis and improve anti-cancer immunotherapy.

Acid Suspends the Circadian Clock in Hypoxia through Inhibition of mTOR.

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.

Hypoxia and acidosis: immune suppressors and therapeutic targets.

Due to imbalances between vascularity and cellular growth patterns, the tumour microenvironment harbours multiple metabolic stressors including hypoxia and acidosis, which have significant influences on remodelling both tumour and peritumoral tissues. These stressors are also immunosuppressive and can contribute to escape from immune surveillance. Understanding these effects and characterizing the pathways involved can identify new targets for therapy and may redefine our understanding of traditional anti-tumour therapies. In this review, the effects of hypoxia and acidosis on tumour immunity will be summarized, and how modulating these parameters and their sequelae can be a useful tool for future therapeutic interventions is discussed.

Tris-base buffer: a promising new inhibitor for cancer progression and metastasis.

Neutralizing tumor external acidity with oral buffers has proven effective for the prevention and inhibition of metastasis in several cancer mouse models. Solid tumors are highly acidic as a result of high glycolysis combined with an inadequate blood supply. Our prior work has shown that sodium bicarbonate, imidazole, and free-base (but not protonated) lysine are effective in reducing tumor progression and metastasis. However, a concern in translating these results to clinic has been the presence of counter ions and their potential undesirable side effects (e.g., hypernatremia). In this work, we investigate tris(hydroxymethyl)aminomethane, (THAM or Tris), a primary amine with no counter ion, for its effects on metastasis and progression in prostate and pancreatic cancer in vivo models using MRI and bioluminescence imaging. At an ad lib concentration of 200 mmol/L, Tris effectively inhibited metastasis in both models and furthermore led to a decrease in the expression of the major glucose transporter, GLUT-1. Our results also showed that Tris-base buffer (pH 8.4) had no overt toxicity to C3H mice even at higher doses (400 mmol/L). In conclusion, we have developed a novel therapeutic approach to manipulate tumor extracellular pH (pHe) that could be readily adapted to a clinical trial.

Defining Cancer Subpopulations by Adaptive Strategies Rather Than Molecular Properties Provides Novel Insights into Intratumoral Evolution.

Ongoing intratumoral evolution is apparent in molecular variations among cancer cells from different regions of the same tumor, but genetic data alone provide little insight into environmental selection forces and cellular phenotypic adaptations that govern the underlying Darwinian dynamics. In three spontaneous murine cancers (prostate cancers in TRAMP and PTEN mice, pancreatic cancer in KPC mice), we identified two subpopulations with distinct niche construction adaptive strategies that remained stable in culture: (i) invasive cells that produce an acidic environment via upregulated aerobic glycolysis; and (ii) noninvasive cells that were angiogenic and metabolically near-normal. Darwinian interactions of these subpopulations were investigated in TRAMP prostate cancers. Computer simulations demonstrated invasive, acid-producing (C2) cells maintain a fitness advantage over noninvasive, angiogenic (C3) cells by promoting invasion and reducing efficacy of immune response. Immunohistochemical analysis of untreated tumors confirmed that C2 cells were invariably more abundant than C3 cells. However, the C2 adaptive strategy phenotype incurred a significant cost due to inefficient energy production (i.e., aerobic glycolysis) and depletion of resources for adaptations to an acidic environment. Mathematical model simulations predicted that small perturbations of the microenvironmental extracellular pH (pHe) could invert the cost/benefit ratio of the C2 strategy and select for C3 cells. In vivo, 200 mmol/L NaHCO3 added to the drinking water of 4-week-old TRAMP mice increased the intraprostatic pHe by 0.2 units and promoted proliferation of noninvasive C3 cells, which remained confined within the ducts so that primary cancer did not develop. A 0.2 pHe increase in established tumors increased the fraction of C3 cells and signficantly diminished growth of primary and metastatic tumors. In an experimental tumor construct, MCF7 and MDA-MB-231 breast cancer cells were coinjected into the mammary fat pad of SCID mice. C2-like MDA-MB-231 cells dominated in untreated animals, but C3-like MCF7 cells were selected and tumor growth slowed when intratumoral pHe was increased. Overall, our data support the use of mathematical modeling of intratumoral Darwinian interactions of environmental selection forces and cancer cell adaptive strategies. These models allow the tumor to be steered into a less invasive pathway through the application of small but selective biological force. Cancer Res; 77(9); 2242-54. ©2017 AACR.

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.

Neutralization of Tumor Acidity Improves Antitumor Responses to Immunotherapy.

Cancer immunotherapies, such as immune checkpoint blockade or adoptive T-cell transfer, can lead to durable responses in the clinic, but response rates remain low due to undefined suppression mechanisms. Solid tumors are characterized by a highly acidic microenvironment that might blunt the effectiveness of antitumor immunity. In this study, we directly investigated the effects of tumor acidity on the efficacy of immunotherapy. An acidic pH environment blocked T-cell activation and limited glycolysis in vitro. IFNγ release blocked by acidic pH did not occur at the level of steady-state mRNA, implying that the effect of acidity was posttranslational. Acidification did not affect cytoplasmic pH, suggesting that signals transduced by external acidity were likely mediated by specific acid-sensing receptors, four of which are expressed by T cells. Notably, neutralizing tumor acidity with bicarbonate monotherapy impaired the growth of some cancer types in mice where it was associated with increased T-cell infiltration. Furthermore, combining bicarbonate therapy with anti-CTLA-4, anti-PD1, or adoptive T-cell transfer improved antitumor responses in multiple models, including cures in some subjects. Overall, our findings show how raising intratumoral pH through oral buffers therapy can improve responses to immunotherapy, with the potential for immediate clinical translation.