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PDF file - 65K, Supplemental Figure 1. Body and prostate gland weights of C57BL/6J and TRAMP mice: A. Body weight change of C57BL/6J. B. Prostate weight of C57BL/6J. C. Body weight change of TRAMP. D. Prostate weight of TRAMP. Supplemental Figure 2. Mean distribution-adjusted lesion grade of different lobes (anterior, dorsal, lateral and ventral) of the prostate in control TRAMP mice and mice treated with low, medium and high doses of Polyphenon E.

Prostate cancer treatment is often accompanied by untoward side effects. Therefore, chemoprevention to reduce the risk and inhibit the progression of prostate cancer may be an effective approach to reducing disease burden. We investigated the safety and efficacy of Polyphenon E, a green tea extract, in reducing the progression of prostate cancer in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. A total of 119 male TRAMP and 119 C57BL/6J mice were treated orally with one of 3 doses of Polyphenon E (200, 500, and 1,000 mg/kg/day) in drinking water ad libitum replicating human achievable doses. Baseline assessments were performed before treatments. Safety and efficacy assessments during treatments were performed when mice were 12, 22, and 32 weeks old. The number and size of tumors in treated TRAMP mice were significantly decreased compared with untreated animals. In untreated 32 weeks old TRAMP mice, prostate carcinoma metastasis to distant sites was observed in 100% of mice (8/8), compared with 13% of mice (2/16) treated with high-dose Polyphenon E during the same period. Furthermore, Polyphenon E treatment significantly inhibited metastasis in TRAMP mice in a dose-dependent manner (P = 0.0003). Long-term (32 weeks) treatment with Polyphenon E was safe and well tolerated with no evidence of toxicity in C57BL/6J mice. Polyphenon E is an effective chemopreventive agent in preventing the progression of prostate cancer to metastasis in TRAMP mice. Polyphenon E showed no toxicity in these mouse models. Our findings provide additional evidence for the safety and chemopreventive effect of Polyphenon E in preventing metastatic progression of prostate cancer. Cancer Prev Res; 7(4); 435–44. ©2014 AACR.

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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.

Simultaneous video of four simulations of tumor growth with accompanying phenotypic "flow" diagram, Top left: untreated; top right: low dose, early; bottom left: high dose, late; bottom right: low dose, late. The appearance of the white box in each of the treated windows indicates when the buffer treatment was applied (all vessels secrete buffer). In the phenotype flow plot, the cyan dot shows the median phenotype of all tumor cells, black points indicate distribution of phenotypes present in the tumor at each time point. The simulations show that the buffer therapy reverses the evolution towards the glycolytic, acid-resistant phenotype, with early or high dose having the stronger effect. A late, low dose does have an effect on the progression, but the invasive phenotype persists despite the therapy and will eventually dominate.

PDF file - 241K, Figure S7: Segmentation and analysis of vasculature In vivo. In vivo vessels and tumors were independently segmented and quantified within four ordinal quadrants emanating from the tumor centroid as described in Methods.

PDF file - 81K, Figure S10: Inhibition of tumor growth as a result of NaHCO3 treatment. Tumor area was measured at days 4 and 13 by fluorescent pixel number using Image-Pro Plus v6.2. The mean growth illustrated a significant increase in size of the control tumor over 2 weeks (p

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. Cancer Res; 76(6); 1381–90. ©2015 AACR.

PDF file - 43K, Figure S5: Relation between tumor growth and pH for all 5 mice. Data for all 5 mice were parsed as in figure 2D wherein the growth (in pixels) and pH were calculated every 22.5o of arc. These data show that growth increased with decreasing pH for all animals, and that there was a threshold of pH above which no growth occurred. Lines represent the trend below threshold for each mouse.

PDF file - 77K, Figure S4: Quantification of tumor growth. Images are shown for mouse tumor #23 on day 4 (green) and day 14 (red). Images were co-registered using the ring clip of the DWC as a fiducial. The centroid of the tumor on day 4 was calculated and radial lines were drawn every 22.5o of arc, beginning at the ring. Growth was calculated as the number of pixels that the day 14 tumor extended beyond the edge of the day 4 tumor using Definiens Developer XD�.

PDF file - 45K, Figure S2: Comparison of normal vs tumor cells by size. Using these phase contrast images, ROI were chosen and area of cells was determined by pixel number using Image-Pro Plus v6.2. Once area was measured, data analysis revealed statistical significance (p

PDF file - 50K, Figure S6: Ex vivo comparison of vessel density in stroma vs tumor tissue. Using the Image Scope v7.1 regions of viable tumor were manually selected. 200�m micrometers were placed around the circumference of the tumor region perpendicular to the tumor edge. A second manually selected region was drawn around the perimeter of the micrometers. This effectively segments the entire tissue section into three regions of interest; the tumor, the edge within 200�m of the tumor and the distant stromal region. The Aperio (Vista, CA, USA) microvessel algorithm v1.0 was used with the following parameters (smoothing 2; dark threshold160; light threshold 210; regioning 6; completion 7) to identify the vascular density which was defined as the vessel area divided by total area (�m2).

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PDF file - 45K, Figure S8: In vivo relation between vessel density and tumor growth. The data from Figure S5 were used to compare tumor growth (change in number of pixels from day 4 to day 13) to vessel density (on day 4) in each of the 4 quadrants.

PDF file - 46K, Figure S3: Growth of MDAmb231 tumor within the dorsal window chamber. Mean growth of MDAmb23/GFP tumors within the dorsal window chamber. Cells were embedded in a 0.8 mg/mL collagen type 1 matrix followed by inoculation into the window chamber. Tumor area was measured at days 2, 3, and 16 by counting the number of pixels. Tumor area measured by fluorescence pixel number obtained using Image-Pro Plus 6.2.

Supplemental Figure S1: Inhibition of PI3 kinase signaling leads to loss of T cell function. Activated T cells were cultured at pH 6.6 or 7.4 for 24h in presence or absence of the PI3K Supplemental Figure S2: OT-1 T cell metabolism. OT-1 T cells were cultured with OVASIINFEKL peptide for 48 hours at either pH 6.6 or 7.4. Cells were subsequently metabolically profiled using a Seahorse XF-96 analyzer. Supplemental Figure S3: Knocking out the acid sensor, TDAG8, does not rescue IFN-gamma production by T cells. T cells were isolated from TDAG8 k/o or wild-type (WT) mice and stimulated in the presence of 10 ug/ml plate-bound anti-CD3 antibodies for 48 hours at pH 6.6 or 7.4. Supernatants were collected and IFN-gamma was measured by ELISA. Supplemental Figure S4: Representative B16 tumors in mice treated with anti-CTLA4 antibodies with or without buffer therapy. Supplemental Figure S5: Buffer therapy has little effect on systemic T cell activity in B16- bearing mice treated with anti-PD-1 antibody. inhibitor BEZ-235.

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Supplementary Figures 1-5, Tables 1-3 from Bicarbonate Increases Tumor pH and Inhibits Spontaneous Metastases

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.

Tumor acidosis plays a major role in tumor aggressiveness, invasion, and resistance. Therefore, it is considered an important target for novel anticancer strategies. In this study, we tested the efficacy of a transdermal treatment applied directly to the tumor area to reduce interstitial acidosis. The product involved is a patented transdermal cream formulation (DYV800) produced by Dyve Biosciences that contains 33% sodium bicarbonate and serves as a buffer medicament. C57BL/6 mice bearing B16-F10 melanoma subcutaneous tumors were treated with DYV800, and pH was measured at different time points after cream application. The MRI pH imaging technique used in this study is the Chemical Exchange Saturation Transfer (CEST) method, which involves the injection of an iodinated contrast agent, iopamidol (Isovue, Bracco Imaging), to measure extracellular pH (pHe) in vivo. In the first experiment to examine the time-course effect of DYV800 on tumor acidosis, mice (N=30, 15 untreated and 15 treated) were treated only once with 100 µL of cream. pH acquisitions were done with untreated mice for baseline pH measurements (N=15) and at different time points after cream application in another set of mice (1, 2, 4 and 24 hours; N=15). As revealed by the pH measurements, the highest increase in pH was reported after 2 hours of the cream application (6.71 vs. 6.84, untreated vs. post 2hrs, p-value 0.025 ANOVA with Bonferroni comparison test), and although pH was higher for all the other time points compared to baseline values, no statistical significance was found (post 1hr 6.75; post 4hrs 6.78; post 24hrs 6.75). In a second experiment, mice (N=10) were untreated (N=3) or received prolonged treatment that lasted 5 days, with one group of mice treated once-a-day (N=3) and the second group of mice treated twice-a-day (N=4) with 100 µL of cream for each application. MRI pH imaging was performed the next day after the treatment regimen was completed. Baseline measurements confirmed that B16 tumors have an acidic pH of 6.7 that increased to 6.82 in the once-a-day treated group (p-value 0.037 unpaired t-test vs. baseline) and to 6.77 in the twice-a-day treated group (not significant compared to baseline). To complete the pH evaluations, untreated mice reported a slight reduction in pHe (6.65) after 6 days of the baseline acquisitions. These experiments demonstrate for the first time that the extracellular pH of tumors can be increased upon application of a buffer formulation transdermally delivered to mice. Future experiments are expected to provide insights into the effect of DYV800 cream application on tumor growth and immune cell migration and activation in the tumor microenvironment. Citation Format: Pietro Irrera, Bruna Victorasso Jardim-Perassi, Dominique Abrahams, Thomas Sherlock, Samantha Byrne, Andrew Ojeda, Justin Y. Lau, Nathan Fitzsimmons, Audrene Rice, Chuck Harbert, Renee Arakawa, Arig Ibrahim-Hashim, Ryan Beal, Shari Pilon-Thomas. Transdermal alkalinization treatment induces pH changes in a murine melanoma model as measured with MRI-CEST pH imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2391.

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.

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.

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., T-cell activation primarily occurs in the lymph nodes, highly organized and specialized secondary lymphoid organs. Here the authors show that the acidic extracellular pH in lymph node paracortical zones limits cytokine production by effector T-cells, but does not alter their activation by antigen-presenting cells.

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.

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.

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.

Tumors rapidly ferment glucose to lactic acid even in the presence of oxygen, and coupling high glycolysis with poor perfusion leads to extracellular acidification. Here we demonstrate that acidity, independent from lactate, augments the pro-tumor phenotype of macrophages. We used zwitterionic buffers to show that 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 neutralizing intratumoral acidity reduced the pro-tumor phenotype of macrophages, while also decreasing tumor 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 modeling shows that acid-resistant macrophages can limit tumor progression. In summary, this study suggests that tumor acidity contributes to prostate carcinogenesis by altering the state of macrophage activation.

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.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.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.This study suggests that tumour acidity contributes to prostate carcinogenesis by altering the state of macrophage activation.

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.

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 hours, 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.

“Pseudohypoxia” is a process wherein cells express hypoxia (i.e., low oxygen)-associated proteins, regardless of the oxygen status. Notably, this phenotype is often observed at the tumor–stromal interface in locally invading tumors. This may be an important feature of tumor physiology, and is being corroborated by mathematical modeling and clinical histology. Until now, little attention has been given to the physiology of the tumor–stromal interface. We propose that pseudohypoxia at the invasive edge provides an evolutionary fitness advantage for tumors that becomes selected during clonal expansion within the tumor. Pseudohypoxic cells are more aggressive, acid producing, and glycolytic, due to upregulation of proteins, such as glucose transporter 1 (GLUT-1) and carbonic anhydrase nine (CA-IX). Investigation of this phenotype is untapped, and may lead to a deeper understanding of basic tumor physiology and potentially illuminate targets that could disrupt this vitally important tumor survival system.

Cancer is a complex, adaptive, and dynamic system, made up of cancer cells interacting with an ever-changing microenvironment. Interactions among components of the cancer system over time and space are often multiscalar and typically nonlinear. Each transition, such as expression of a new gene product, is governed by a cell’s content and history as well as local microenvironmental properties and selection forces. Hence, cancer is an open system, as it freely interacts with the local host tissue. This permits normal tissue to affect tumor but also allows tumor populations to alter the local environment in ways that promote tumor growth—an adaptive strategy termed “niche construction.” Because these dynamic interactions between the host and the tumor are both complex and nonlinear, even subtle perturbations of the environment can have profound effects on the tumor. Here we focus on the tumor–host interactions that give rise to metabolic heterogeneity, which includes the following: (1) tumor oxygenation, governed by variations in delivery due to temporal and spatial heterogeneity in blood flow, which in part is governed by tumor-derived angiogenic factors; and (2) tumor extracellular pH (pHe), governed by both blood flow and upregulation of fermentative glucose pathways in regions of hypoxia and normoxia, known as aerobic glycolysis (i.e., the Warburg effect; see subsequent text), which aids the tumor by blunting the immune response and promoting invasive growth. Importantly, these dynamics can be exploited or manipulated to treat tumors. For example, tumor cells in regions of low oxygen can be targeted with specific hypoxia-activated prodrugs. Similarly, blood flow can be altered with antiangiogenesis drugs and tumor cells in acidic regions can be targeted by specific metabolic inhibitors and buffer therapies. Furthermore, these metabolic factors can significantly affect the coevolution of cancers with the innate and adaptive immune systems. Here we focus on the complex and dynamic interactions between the microenvironmental factors and tumor cells, and their impact on carcinogenesis and metastasis.

Prostate cancer treatment is often accompanied by untoward side effects. Therefore, chemoprevention to reduce the risk and inhibit the progression of prostate cancer may be an effective approach to reducing disease burden. We investigated the safety and efficacy of Polyphenon E, a green tea extract, in reducing the progression of prostate cancer in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. A total of 119 male TRAMP and 119 C57BL/6J mice were treated orally with one of 3 doses of Polyphenon E (200, 500, and 1,000 mg/kg/day) in drinking water ad libitum replicating human achievable doses. Baseline assessments were performed before treatments. Safety and efficacy assessments during treatments were performed when mice were 12, 22, and 32 weeks old. The number and size of tumors in treated TRAMP mice were significantly decreased compared with untreated animals. In untreated 32 weeks old TRAMP mice, prostate carcinoma metastasis to distant sites was observed in 100% of mice (8/8), compared with 13% of mice (2/16) treated with high-dose Polyphenon E during the same period. Furthermore, Polyphenon E treatment significantly inhibited metastasis in TRAMP mice in a dose-dependent manner (P = 0.0003). Long-term (32 weeks) treatment with Polyphenon E was safe and well tolerated with no evidence of toxicity in C57BL/6J mice. Polyphenon E is an effective chemopreventive agent in preventing the progression of prostate cancer to metastasis in TRAMP mice. Polyphenon E showed no toxicity in these mouse models. Our findings provide additional evidence for the safety and chemopreventive effect of Polyphenon E in preventing metastatic progression of prostate cancer. Cancer Prev Res; 7(4); 435–44. ©2014 AACR.

As first proposed by Cairns and Nowell more than 60 years ago, cancer is a Darwinian process so that cancer progression and resistance to therapy are governed by natural selection and eco-evolutionary dynamics. This creates an “evolutionary arms race” between the cancer cells and the tumor suppressor mechanisms of the host. To mechanistically investigate these dynamics we have conducted an experiment in which we selected for resistance to growth of implanted Lewis lung (mouse) cancer tumor (LL/2-Luc-M38) cells in immuno-deficient (SCID) mice and immuno-competent (C57BL/6). At each round a fixed numbers of LL/2-Luc-M38 cells were implanted in 10 male SCID and 10 male C57BL/6 mice. Tumor growth was measured and the two animals that exhibited the slowest tumor growth were bred and their male progeny were used as host in the subsequent round. After 12 generations, the tumors grew at approximately 1/10th the rate compared their initial generation. We observed that the hosts evolved cancer suppression through two general strategies. The evolved SCID mice suppressed tumor growth via mechanical restriction with increased in collagen deposition in and around the tumor. In contrast BL/6 mice enhance their immunologic response. We investigated the molecular properties of tumor cells growing in the different cohorts suing microarray. We identified genes significantly different in their expression of cancer cells harvested at different days following injection in Wild Type and Evolved SCID and BL/6 mice using Principle component analysis (PCA) method. The genes were functionally classified based on Gene Ontology and the majority was found to be related to integrin binding, cell adhesion molecular binding, and extra cellular matrix (ECM) binding in SCID mice with collagen, type XII, alpha (Col12a1) being the significantly decreased (results validated with RT-PCR (**p=0.009)). However, in BL/6 mice Molecular studies, contrary to expectation, did not show significant changes in expression of immune-related genes. Rather we found a decrease in expression of genes associated with the angiogenesis and ATP production as well as increased expression of genes regulating EMT. Thus, our results highly suggest that the counter adaptive strategies deployed by the cancer cells in response to the host suppressive mechanisms is host dependent and can be a possible target for therapy. Citation Format: Arig A. Ibrahim Hashim, Dominique Abrahams, Kim Luddy, Robert Gillies, Christina L. Richards, Joel Brown, Robert Gatenby. Investigating the tumor-host evolutionary arms race as a possible strategy for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3773.

The pH of solid tumors is acidic due to increased fermentative metabolism and poor perfusion. It has been hypothesized that acid pH promotes local invasive growth and metastasis. The hypothesis that acid mediates invasion proposes that H+ diffuses from the proximal tumor microenvironment into adjacent normal tissues where it causes tissue remodeling that permits local invasion. In the current work, tumor invasion and peritumoral pH were monitored over time using intravital microscopy. In every case, the peritumoral pH was acidic and heterogeneous and the regions of highest tumor invasion corresponded to areas of lowest pH. Tumor invasion did not occur into regions with normal or near-normal extracellular pH. Immunohistochemical analyses revealed that cells in the invasive edges expressed the glucose transporter-1 and the sodium–hydrogen exchanger-1, both of which were associated with peritumoral acidosis. In support of the functional importance of our findings, oral administration of sodium bicarbonate was sufficient to increase peritumoral pH and inhibit tumor growth and local invasion in a preclinical model, supporting the acid-mediated invasion hypothesis. Cancer Res; 73(5); 1524–35. ©2012 AACR.

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.