Arig Ibrahim Hashim

Bicarbonate Increases Tumor pH and Inhibits Spontaneous Metastases

The external pH of solid tumors is acidic as a consequence of increased metabolism of glucose and poor perfusion. Acid pH has been shown to stimulate tumor cell invasion and metastasis in vitro and in cells before tail vein injection in vivo. The present study investigates whether inhibition of this tumor acidity will reduce the incidence of in vivo metastases. Here, we show that oral NaHCO(3) selectively increased the pH of tumors and reduced the formation of spontaneous metastases in mouse models of metastatic breast cancer. This treatment regimen was shown to significantly increase the extracellular pH, but not the intracellular pH, of tumors by (31)P magnetic resonance spectroscopy and the export of acid from growing tumors by fluorescence microscopy of tumors grown in window chambers. NaHCO(3) therapy also reduced the rate of lymph node involvement, yet did not affect the levels of circulating tumor cells, suggesting that reduced organ metastases were not due to increased intravasation. In contrast, NaHCO(3) therapy significantly reduced the formation of hepatic metastases following intrasplenic injection, suggesting that it did inhibit extravasation and colonization. In tail vein injections of alternative cancer models, bicarbonate had mixed results, inhibiting the formation of metastases from PC3M prostate cancer cells, but not those of B16 melanoma. Although the mechanism of this therapy is not known with certainty, low pH was shown to increase the release of active cathepsin B, an important matrix remodeling protease.

Imaging pH and metastasis

Metastasis is a multistep process that culminates in the spread of cells from a primary tumor to a distant site or organs. For tumor cells to be able to metastasize, they have to locally invade through basement membrane into the lymphatic and the blood vasculatures. Eventually they extravasate from the blood and colonize in the secondary organ. This process involves multiple interactions between the tumor cells and their microenvironments. The microenvironment surrounding tumors has a significant impact on tumor development and progression. A key factor in the microenvironment is an acidic pH. The extracellular pH of solid tumors is more acidic in comparison to normal tissue as a consequence of high glycolysis and poor perfusion. It plays an important role in almost all steps of metastasis. The past decades have seen development of technologies to non‐invasively measure intra‐ and/or extracellular pH. Most successful measurements are MR‐based, and sensitivity and accuracy have dramatically improved. Quantitatively imaging the distribution of acidity helps us understand the role of the tumor microenvironment in cancer progression. The present review discusses different MR methods in measuring tumor pH along with emphasizing the importance of extracelluar tumor low pH on different steps of metastasis; more specifically focusing on epithelial‐to‐mesenchymal transition (EMT), and anti cancer immunity. Copyright

Targeting the Metabolic Microenvironment of Tumors

The observation of aerobic glycolysis by tumor cells in 1924 by Otto Warburg, and subsequent innovation of imaging glucose uptake by tumors in patients with PET-CT, has incited a renewed interest in the altered metabolism of tumors. As tumors grow in situ, a fraction of it is further away from their blood supply, leading to decreased oxygen concentrations (hypoxia), which induces the hypoxia response pathways of HIF1α, mTOR, and UPR. In normal tissues, these responses mitigate hypoxic stress and induce neoangiogenesis. In tumors, these pathways are dysregulated and lead to decreased perfusion and exacerbation of hypoxia as a result of immature and chaotic blood vessels. Hypoxia selects for a glycolytic phenotype and resultant acidification of the tumor microenvironment, facilitated by upregulation of proton transporters. Acidification selects for enhanced metastatic potential and reduced drug efficacy through ion trapping. In this review, we provide a comprehensive summary of preclinical and clinical drugs under development for targeting aerobic glycolysis, acidosis, hypoxia and hypoxia response pathways. Hypoxia and acidosis can be manipulated, providing further therapeutic benefit for cancers that feature these common phenotypes.

Safety and Chemopreventive Effect of Polyphenon E in Preventing Early and Metastatic Progression of Prostate Cancer in TRAMP Mice

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.

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.

Abstract 5932: Targeting tumor acidity with the LDHA inhibitor (FX11) and CAIX inhibitor (DH348) overcomes resistance to PD-1 blockade and inhibits metastasis in a pancreatic cancer model

Prior work by us and others has demonstrated that the extracellular pH (pHe) of solid tumors is acidic, due to a combination of increased fermentative metabolism, resulting in lactic acid production and poor perfusion. This acidity promotes tumor progression and metastasis formation. Recently we have shown in melanoma and pancreatic cancer models that acidity inhibits antitumor immunity by preventing T-cell activation. Reversal of acidity with buffer therapy (200mM NaHCO3) synergized with checkpoint blockade (anti-CTLA4 and anti-PD1) and adoptive T-cell therapy has resulted in cures. While this is promising, concerns are high regarding the ingestion of such large amounts of sodium bicarbonate, which makes clinical translation a challenge. Hence, we hypothesize that alternative pharmacological interventions can neutralize the pHe of tumors and remove this immunosuppressive effect. To study this we first investigated a series of agents for their ability to inhibit metastasis in the PC3 prostate cancer model, which is exquisitely sensitive to inhibition with buffer therapy. In this study, male SCID mice were grouped in to 6 groups (n=5) and treated with tap water, 200 mM bicarbonate ad lib, 30 mg/kg daily (q.d.) intraperitoneal (i.p.) Acetazolamide (CA inhibitor), 1.2 mg/kg q.d.i.p. Furosemide (diuretic), 10 mg/kg q.d.i.p. DH348 (selective CAIX inhibitor) or 2.1 mg/kg q.d.i.p. FX-11 (LDHA inhibitor). Mice were intravenously injected with 5*10^6 PC3M-luc cells and ventral bioluminescence images were acquired at time 0 and weekly thereafter. Our results showed that FX-11, acetazolamide, DH348 and bicarbonate were able to effectively (p Citation Format: Arig A. Ibrahim Hashim, Dominique Abrahams, Liping Xu, Barbra Centeno, Enakshi Sunassee, Rasha Abddelgader, Ludwig Dubois, Philippe Lambin, Robert A. Gatenby, Robert J. Gillies. Targeting tumor acidity with the LDHA inhibitor (FX11) and CAIX inhibitor (DH348) overcomes resistance to PD-1 blockade and inhibits metastasis in a pancreatic cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5932. doi:10.1158/1538-7445.AM2017-5932

Abstract 34: Microenvironmental independence or co-option: How tumour-stromal interactions influence prostate cancer lethal outcomes

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The role of the tumour-microenvironment interactions in tumor progression has been the extensively studied in the last few years. Recently, the key role that stromal interactions in both cancer initiation and progression has become an important focus. Both of these interactions, cell-cell and cell-microenvironment, can fundamentally affect the outcome of tumour progression. In prostate cancer this is particularly true, with the tumour microenvironment being an independent predictor of patient prognosis. There are two main lethal outcomes for prostate cancer: (i) early in its genesis the tumour acquires a highly malignant lethal phenotype, growing rapidly and displacing the original stromal population. These less common aggressive tumours are independent of much of the local microenvironment. (ii) the tumour co-opts the local stroma taking on a classic stromagenic phenotype where the local microenvironment facilitates cancer growth. Using an integrated approach we have investigated the influence of tumour stroma interactions in driving these outcomes. With the help of a mathematical model based on evolutionary game theory, we considered three distinct cellular populations: stromal cells, tumour cells that are self-reliant in terms of microenvironmental factors and tumour cells that depend on the environment for resources (such as growth factors, nutrients and space) but can also co-opt stroma. The results from this mathematical model highlight the importance of carcinoma associated fibroblasts in stromagenic outcomes and that the resulting cooperation between certain stromal cells and the tumour could be disrupted as a result of changes in the microenvironment leading to a stromal independent outcome. Subsequently we have employed a biological model using TRAMP mice (TRansgenic Adenocarcinoma of the Mouse Prostate). TRAMP mice develop prostate cancer spontaneously after a few weeks and recapitulate many features of prostate cancer in humans. This mouse model allowed us to investigate the differences between the tumour populations driving the two distinct outcomes as well as the importance of microenvironmental changes in diverting this progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 34. doi:10.1158/1538-7445.AM2011-34

Abstract B51: Tumor cell evolutionary strategies to overcome immune response

Introduction : The human immune system is complex, dynamic, and highly effective with system tools ranging from simplistic barrier formation to innate and adaptive cellular responses. In turn, the organisms subject to the immune response can evolve a number of different adaptive strategies. Here we examine these evolutionary dynamics in host immune response to cancer focusing on available strategies that permit cancer cells to evade the immune response. Materials and Methods : Two rapidly proliferating human cell lines, SW620 colon cancer and MDA-MB-231 triple negative breast cancers were subjected to repeated exposure to either immune conditioned media created by lipopolysaccharide stimulated immune cells or intermittent direct culturing with human peripheral blood leucocytes. Resulting phenotypes were evaluated for alterations in growth dynamics, immune resistance, and gene expression. Results : Conditioned media had only slight effects on tumor cell death. However, after 4 months selected cells have an increased resistance to T cell mediated killing. Co-culturing with immune cells at high effector to target ratios resulted in strong selection with greater than 50% tumor cell death. After 15 rounds we evolved cancer cells that were resistant to this killing. Interestingly, In-vitro selection of SW620, a more rapidly proliferating cell line, resulted in an anti-apoptotic strategy when confronted with immune cells while MDA-MB-231 cells increased fecundity. Conclusion : Utilizing different arms of the immune system resulted in different styles and strengths of selection force. Additionally, we demonstrate that the two different cell lines employed distinctly different strategies to overcome host immune response. The MDA-MB-231 population adapts to immune attack by accelerating proliferation so that it exceeds the death rate imposed by the immune system. Interestingly, this has been observed clinically as some tumors show explosive growth during immunotherapy. In contrast, the SW620 cells upregulate anti-apoptotic cellular machinery which appears to be phenotypically costly so that proliferation of resistant cells is significantly diminished. As clinical applications of immunotherapy continue to grow it is imperative that we do not ignore the evolutionary consequences of immune selection on the tumor phenotype. While investigations of immune evasive strategies in tumor cells has led to a growing list of specific mechanisms, here we look to not only expand this list but to exploit it. Detailed understanding of the specific adaptive strategy for each tumor population may reveal phenotypic vulnerabilities to second line treatments. Citation Format: Kimberly A. Luddy, Jan Poleszczuk, Arig Ibrahim Hashim, Mehdi Damaghi, Robert Gillies, Joel Brown, Robert Gatenby. Tumor cell evolutionary strategies to overcome immune response. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B51.

Abstract 3941: Manipulating the tumor microenvironment for therapeutic benefit: identifying mechanisms of buffer resistance.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The acidic microenvironment is a common physiological phenomenon of most solid tumors that can be attributed to increased fermentative metabolism combined with poor perfusion. Acidic niches provide a highly selective environment promoting carcinogenesis and malignancy. We previously demonstrated that neutralizing acidosis with buffers reduced spontaneous or experimental metastasis in-vivo. This effect is due to buffering, because by reducing the buffering capacity of lysine five-fold by titrating the solution from pH 10 to pH 8.4 (below the second of three pKas), we observed a correlating decrease in efficacy in-vivo. In these studies, some tumor types, notably LL/2 and B16-F12 cells are resistant to buffer therapy. To examine the mechanisms that lead to resistance, we first determined their metabolic profiles using a Seahorse XF96® to measure glucose-dependent acidification rates (ECAR) and oxygen consumption rates (OCR). Contrary to our hypothesis, these data showed that the resistant lines, LL/2 and B16-F12 had significantly higher basal OCR and lower glucose-stimulated ECAR, compared to PC3M cells (buffer sensitive). PC3M cells also had low spare glycolytic capacity, suggesting they are at or near maximal glycolysis in their basal state. Glucose consumption assays confirm increased rates of glucose consumption by PC3M cells, confirming a glycolytic phenotype. Hence, resistance to buffer therapy is not simply a consequence of higher metabolic proton production rates. Acidosis has been shown to stimulate lysosomal redistribution and protease secretion in-vitro. Thus, an alternative hypothesis is that protease activation may be a mechanism by which low pH stimulates metastasis in-vivo. To observe protease activity in vivo, MMPsense® 680 and Prosense® 750EX activatable probes, which measure MMP and cysteine cathepsin activity respectively, were injected intravenously into nu/nu mice bearing bilateral LL/2 and PC3M tumors. Mice were provided either tap water or 200 mM lysine, ad lib for the duration of the experiment. Mice were imaged 24 hours following probes injection using a Perkin-Elmer FMT 2500. LL/2 tumors (buffer resistant) had significantly higher basal cysteine cathepsin activity than PC3M tumors in control mice, and these decreased upon lysine treatment. MMP activity between LL/2 and PC3M tumors was not different in control mice, however, MMP activity increased significantly in LL/2 tumors with buffer therapy. In vitro 3D-DQ-collagen IV degradation assays correlate with protease activity seen in vivo. These data suggest that buffer-sensitive cells rely on acid production from glycolysis for metastatic potential, whereas resistant lines metastasize through a pH-independent proteolytic mechanism. Citation Format: Kate M. Bailey, Jonathan W. Wojtkowiak, Arig A. Ibrahim Hashim, Robert J. Gillies. Manipulating the tumor microenvironment for therapeutic benefit: identifying mechanisms of buffer resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3941. doi:10.1158/1538-7445.AM2013-3941

Abstract 3520: The use of vasodilators to enhance efficacy of hypoxia or acid activated prodrugs in pancreatic cancers

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The vasculature of tumors is torturous and immature and in many cases lacks tone (the ability to self-regulate pressure). The administration of vasodilating agents induces a drop in systemic blood pressure which is not matched in the tumor, resulting in decreased intratumoral perfusion due to the “steal phenomenon”. This research uses this phenomenon to induce decreased tumor perfusion which results in increased tumor acidity and hypoxia. We hypothesize that this change in the tumor microenvironment can be used in combination with hypoxia- and acid-activated prodrugs (HAPs and AAPs) to enhance their efficacy, by increasing both the depth and distribution of hypoxia or acidosis within the tumor. Specifically, we have investigated the administration of hydralazine in several pancreatic xenograft tumors and used a needle microelectrode to measure decreases in pH as a result of decreased perfusion. We have also employed a computational math model to investigate the timing of the delivery of both hydralazine and HAPs to optimize the cell death. Preliminary computational results suggest that multiple rounds of vasodilators may be given with a single round of HAPs, depending on several factors including duration of vasodilation effect and half-life of the HAP. The pH investigation showed a substantial pH drop in one tumor type (MiaPaca2), but no change in two others (HS766t and SU8686), suggesting that the ‘steal’ phenomenon is not universal. Preliminary data suggest that combination therapy in the ‘steal’-sensitive models will increase efficacy. We are continuing research with the combination therapy, employing the timing strategies suggested by the computational models. We are also investigating different imaging modalities to monitor the effects of the steal phenomenon, including Ultrasound (for perfusion) and MRI (for bulk tumor changes and monitoring pH). In conclusion, the preliminary data supports the hypothesis that vasodilatation agents may be used to induce increases in acidosis and hypoxia within the microenvironment of some (but not all) tumors. These effects can be taken advantage of through combination with HAPs or AAPs to enhance antitumor effects. Further, computational math models can be employed to help determine the best timing for the combination therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3520. doi:10.1158/1538-7445.AM2011-3520