Dominique Abrahams

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.

Noninvasive Detection of Breast Cancer Lymph Node Metastasis Using Carbonic Anhydrases IX and XII Targeted Imaging Probes

Purpose: To develop targeted molecular imaging probes for the noninvasive detection of breast cancer lymph node metastasis. Experimental Design: Six cell surface or secreted markers were identified by expression profiling and from the literature as being highly expressed in breast cancer lymph node metastases. Two of these markers were cell surface carbonic anhydrase isozymes (CAIX and/or CAXII) and were validated for protein expression by immunohistochemistry of patient tissue samples on a breast cancer tissue microarray containing 47 normal breast tissue samples, 42 ductal carcinoma in situ, 43 invasive ductal carcinomas without metastasis, 46 invasive ductal carcinomas with metastasis, and 49 lymph node macrometastases of breast carcinoma. Targeted probes were developed by conjugation of CAIX- and CAXII-specific monoclonal antibodies to a near-infrared fluorescent dye. Results: Together, these two markers were expressed in 100% of the lymph node metastases surveyed. Selectivity of the imaging probes were confirmed by intravenous injection into nude mice-bearing mammary fat pad tumors of marker-expressing cells and nonexpressing cells or by preinjection of unlabeled antibody. Imaging of lymph node metastases showed that peritumorally injected probes detected nodes harboring metastatic tumor cells. As few as 1,000 cells were detected, as determined by implanting, under ultrasound guidance, a range in number of CAIX- and CAXII-expressing cells into the axillary lymph nodes. Conclusion: These imaging probes have potential for noninvasive staging of breast cancer in the clinic and elimination of unneeded surgery, which is costly and associated with morbidities. Clin Cancer Res; 18(1); 207–19. ©2011 AACR.

Development of an Orthotopic Human Pancreatic Cancer Xenograft Model Using Ultrasound Guided Injection of Cells

Mice have been employed as models of cancer for over a century, providing significant advances in our understanding of this multifaceted family of diseases. In particular, orthotopic tumor xenograft mouse models are emerging as the preference for cancer research due to increased clinical relevance over subcutaneous mouse models. In the current study, we developed orthotopic pancreatic cancer xenograft models in mice by a minimally invasive method, ultrasound guided injection (USGI) comparable to highly invasive surgical orthotopic injection (SOI) methods. This optimized method prevented injection complications such as recoil of cells through the injection canal or leakage of cells out of the pancreas into the peritoneal cavity. Tumor growth was monitored in vivo and quantified by ultrasound imaging weekly, tumors were also detected by in vivo fluorescence imaging using a tumor targeted molecular probe. The mean tumor volumes for the USGI and SOI models after 2 weeks of tumor growth were 205 mm3 and 178 mm3 respectively. By USGI of human pancreatic cancer cell lines, human orthotopic pancreatic cancer xenografts were established. Based on ultrasound imaging, the orthotopic human pancreatic cancer xenograft take rate was 100% for both human pancreatic cancer cell lines used, MiaPaCa-2 and Su86.86, with mean tumor volumes of 28 mm3and 30 mm3. We demonstrated that this USGI method is feasible, reproducible, facile, minimally invasive and improved compared to the highly-invasive SOI method for establishing orthotopic pancreatic tumor xenograft models suitable for molecular imaging.

A Mammaglobin-A Targeting Agent for Noninvasive Detection of Breast Cancer Metastasis in Lymph Nodes

Pathologic axillary lymph node (ALN) status is an important prognostic factor for staging breast cancer. Currently, status is determined by histopathology following surgical excision of sentinel lymph node(s), which is an invasive, time consuming, and costly procedure with potential morbidity to the patient. Here, we describe an imaging platform for noninvasive assessment of ALN status, eliminating the need for surgical examination of patients to rule out nodal involvement. A targeted imaging probe (MamAb-680) was developed by conjugation of a mammaglobin-A-specific monoclonal antibody to a near-infrared fluorescent dye. Using DNA and tissue microarray, mammaglobin-A was validated as a cell-surface target that is expressed in ALN-positive patient samples but is not expressed in normal lymph nodes. In vivo selectivity was determined by i.v. injection of MamAb-680 into mice with mammaglobin-A-positive and -negative mammary fat pad (MFP) tumors; and by peritumoral MFP injection of the targeted imaging probe in mice with spontaneous ALN metastases. Fluorescence imaging showed that probe was only retained in positive tumors and metastases. As few as 1,000 cells that endogenously express mammaglobin-A were detected in ALN, indicating high sensitivity of this method. Translation of this approach offers considerable potential as a noninvasive clinical strategy to stage breast cancer.

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.

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

Real-time ultrasound-guided needle injection of the mouse jugular vein.

This unit describes a novel method for direct venous injection into mice that offers potentially significant advantages over commonly used mouse vein injection techniques. This is achieved via percutaneous needle placement into the mouse jugular vein under real-time B-mode ultrasound (US) imaging. Real-time US imaging of the injection process allows for immediate determination of the overall success of injection. Unique, and potentially significant, advantages of this technique over others include: (1) direct visual confirmation of needle tip placement in the lumen of the vein, (2) immediate visual detection of extravascular extravasation of injectate, when compared to blinded techniques, such as tail vein injections, and (3) reduced morbidity and mortality compared to surgical vascular access techniques (i.e., jugular vein cannulation). This technique may lead to more accurate determination of the success of the injection procedure for each mouse, thus improving the quality of acquired data in dependent mouse experiments.