Heather H. Cornnell

Acidity generated by the tumor microenvironment drives local invasion

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.

Systemic Buffers Inhibit Carcinogenesis in TRAMP Mice

PURPOSE Hypoxia and acidosis develop in in situ tumors as cellular expansion increases the diffusion distance of substrates and metabolites from blood vessels deep to the basement membrane. Prior studies of breast and cervical cancer revealed that cellular adaptation to microenvironmental hypoxia and acidosis is associated with the transition from in situ to invasive cancer. We hypothesized that decreased acidosis in intraductal tumors would alter environmental selection pressures for acid adapted phenotypes and delay or prevent evolution to invasive cancer. MATERIALS AND METHODS A total of 37 C57BL/6 TRAMP mice were randomized to a control group or to 1 of 4 treatment groups. In the latter groups 200 mM sodium bicarbonate were added to drinking water starting between ages 4 and 10 weeks. RESULTS In all 18 controls prostate cancer developed that was visible on 3-dimensional ultrasound at a mean age of 13 weeks. They died within 52 weeks (median 37). When sodium bicarbonate therapy commenced before age 6 weeks in 10 mice, all reached senescence (age 76 weeks) without radiographic evidence of prostate cancer. Histological sections of the prostates in this cohort showed hyperplasia but no cancer in 70% of mice and minimal well differentiated cancer in the remainder. When therapy commenced after age 6 weeks in 9 mice, prostate cancer development was no different from that in controls. CONCLUSIONS Immunohistochemical staining for carbonic anhydrase 9 in regions of ductal hyperplasia showed increased expression in controls vs the early treatment group. Regional pH perturbation in in situ tumors may be a simple, inexpensive and effective cancer prevention strategy.

Free Base Lysine Increases Survival and Reduces Metastasis in Prostate Cancer Model

BACKGROUND Malignant tumor cells typically metabolize glucose anaerobically to lactic acid even under normal oxygen tension, a phenomenon called aerobic glycolysis or the Warburg effect. This results in increased acid production and the acidification of the extracellular microenvironment in solid tumors. H+ ions tend to flow along concentration gradients into peritumoral normal tissue causing extracellular matrix degradation and increased tumor cell motility thus promoting invasion and metastasis. We have shown that reducing this acidity with sodium bicarbonate buffer decreases the metastatic fitness of circulating tumor cells in prostate cancer and other cancer models. Mathematical models of the tumor-host dynamics predicted that buffers with a pka around 7 will be more effective in reducing intra- and peri-tumoral acidosis and, thus, and possibly more effective in inhibiting tumor metastasis than sodium bicarbonate which has a pKa around 6. Here we test this prediction the efficacy of free base lysine; a non-bicarbonate/non-volatile buffer with a higher pKa (~10), on prostate tumor metastases model. METHODS Oxygen consumption and acid production rate of PC3M prostate cancer cells and normal prostate cells were determined using the Seahorse Extracellular Flux (XF-96) analyzer. In vivo effect of 200 mM lysine started four days prior to inoculation on inhibition of metastasis was examined in PC3M-LUC-C6 prostate cancer model using SCID mice. Metastases were followed by bioluminescence imaging. RESULTS PC3M prostate cancer cells are highly acidic in comparison to a normal prostate cell line indicating that reduction of intra- and perit-tumoral acidosis should inhibit metastases formation. In vivo administration of 200 mM free base lysine increased survival and reduced metastasis. CONCLUSION PC3M prostate cancer cells are highly glycolytic and produce large amounts of acid when compared to normal prostate cells. Administration of non-volatile buffer decreased growth of metastases and improved survival indicating acidity plays a significant role in growth and invasion in-vivo.

Development and in vivo quantitative magnetic resonance imaging of polymer micelles targeted to the melanocortin 1 receptor.

Recent emphasis has focused on the development of rationally designed polymer-based micelle carriers for drug delivery. The current work tests the hypothesis that target specificity can be enhanced by micelles with cancer-specific ligands. In particular, we describe the synthesis and characterization of a new gadolinium texaphyrin (Gd-Tx) complex encapsulated in an IVECT micellar system, stabilized through Fe(III) cross-linking and targeted with multiple copies of a specific ligand for the melanocortin 1 receptor (MC1R), which has been evaluated as a cell-surface marker for melanoma. On the basis of comparative MRI experiments, we have been able to demonstrate that these Gd-Tx micelles are able to target MC1R-expressing xenograft tumors in vitro and in vivo more effectively than various control systems, including untargeted or un-cross-linked Gd-Tx micelles. Taken in concert, the findings reported herein support the conclusion that appropriately designed micelles are able to deliver contrast agent payloads to tumors expressing the MC1R.

Imaging biomarkers to monitor response to the hypoxia-activated prodrug TH-302 in the MiaPaCa2 flank xenograft model

TH-302, a hypoxia-activated anticancer prodrug, was evaluated for antitumor activity and changes in dynamic contrast-enhanced (DCE) and diffusion-weighted (DW) magnetic resonance imaging (MRI) in a mouse model of pancreatic cancer. TH-302 monotherapy resulted in a significant delay in tumor growth compared to vehicle-treated controls. TH-302 treatment was also associated with a significant decrease in the volume transfer constant (K(trans)) compared to vehicle-treated controls 1 day following the first dose measured using DCE-MRI. This early decrease in K(trans) following the first dose as measured is consistent with selective killing of the hypoxic fraction of cells which are associated with enhanced expression of hypoxia inducible transcription factor-1 alpha that regulates expression of permeability and perfusion factors including vascular endothelial growth factor-A. No changes were observed in DW-MRI following treatment with TH-302, which may indicate that this technique is not sensitive enough to detect changes in small hypoxic fractions of the tumor targeted by TH-302. These results suggest that changes in tumor permeability and/or perfusion may be an early imaging biomarker for response to TH-302 therapy.

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.

MR Imaging Biomarkers to Monitor Early Response to Hypoxia-Activated Prodrug TH-302 in Pancreatic Cancer Xenografts

TH-302 is a hypoxia-activated prodrug known to activate selectively under the hypoxic conditions commonly found in solid tumors. It is currently being evaluated in clinical trials, including two trials in Pancreatic Ductal Adenocarcinomas (PDAC). The current study was undertaken to evaluate imaging biomarkers for prediction and response monitoring of TH-302 efficacy in xenograft models of PDAC. Dynamic contrast-enhanced (DCE) and diffusion weighted (DW) magnetic resonance imaging (MRI) were used to monitor acute effects on tumor vasculature and cellularity, respectively. Three human PDAC xenografts with known differential responses to TH-302 were imaged prior to, and at 24 h and 48 hours following a single dose of TH-302 or vehicle to determine if imaging changes presaged changes in tumor volumes. DW-MRI was performed at five b-values to generate apparent diffusion coefficient of water (ADC) maps. For DCE-MRI, a standard clinically available contrast reagent, Gd-DTPA, was used to determine blood flow into the tumor region of interest. TH-302 induced a dramatic decrease in the DCE transfer constant (Ktrans) within 48 hours after treatment in the sensitive tumors, Hs766t and Mia PaCa-2, whereas TH-302 had no effect on the perfusion behavior of resistant SU.86.86 tumors. Tumor cellularity, estimated from ADC, was significantly increased 24 and 48 hours after treatment in Hs766t, but was not observed in the Mia PaCa-2 and SU.86.86 groups. Notably, growth inhibition of Hs766t was observed immediately (day 3) following initiation of treatment, but was not observed in MiaPaCa-2 tumors until 8 days after initiation of treatment. Based on these preclinical findings, DCE-MRI measures of vascular perfusion dynamics and ADC measures of cell density are suggested as potential TH-302 response biomarkers in clinical trials.

Evaluation of the ''Steal'' Phenomenon on the Efficacy of Hypoxia Activated Prodrug TH-302 in Pancreatic Cancer

Pancreatic ductal adenocarcinomas are desmoplastic and hypoxic, both of which are associated with poor prognosis. Hypoxia-activated prodrugs (HAPs) are specifically activated in hypoxic environments to release cytotoxic or cytostatic effectors. TH-302 is a HAP that is currently being evaluated in a Phase III clinical trial in pancreatic cancer. Using animal models, we show that tumor hypoxia can be exacerbated using a vasodilator, hydralazine, improving TH-302 efficacy. Hydralazine reduces tumor blood flow through the “steal” phenomenon, in which atonal immature tumor vasculature fails to dilate in coordination with normal vasculature. We show that MIA PaCa-2 tumors exhibit a “steal” effect in response to hydralazine, resulting in decreased tumor blood flow and subsequent tumor pH reduction. The effect is not observed in SU.86.86 tumors with mature tumor vasculature, as measured by CD31 and smooth muscle actin (SMA) immunohistochemistry staining. Combination therapy of hydralazine and TH-302 resulted in a reduction in MIA PaCa-2 tumor volume growth after 18 days of treatment. These studies support a combination mechanism of action for TH-302 with a vasodilator that transiently increases tumor hypoxia.

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