Ines Lohse

Targeting of metastasis-promoting tumor-associated fibroblasts and modulation of pancreatic tumor-associated stroma with a carboxymethylcellulose-docetaxel nanoparticle.

Pancreatic ductal adenocarcinomas are characterized by the desmoplastic reaction, a dense fibrous stroma that has been shown to be supportive of tumor cell growth, invasion, and metastasis, and has been associated with resistance to chemotherapy and reduced patient survival. Here, we investigated targeted depletion of stroma for pancreatic cancer therapy via taxane nanoparticles. Cellax-DTX polymer is a conjugate of docetaxel (DTX), polyethylene glycol (PEG), and acetylated carboxymethylcellulose, a construct which condenses into well-defined 120nm particles in an aqueous solution, and is suitable for intravenous injection. We examined Cellax-DTX treatment effects in highly stromal primary patient-derived pancreatic cancer xenografts and in a metastatic PAN02 mouse model of pancreatic cancer, focusing on specific cellular interactions in the stroma, pancreatic tumor growth and metastasis. Greater than 90% of Cellax-DTX particles accumulate in smooth muscle actin (SMA) positive cancer-associated fibroblasts which results in long-term depletion of this stromal cell population, an effect not observed with Nab-paclitaxel (Nab-PTX). The reduction in stromal density leads to a >10-fold increase in tumor perfusion, reduced tumor weight and a reduction in metastasis. Consentingly, Cellax-DTX treatment increased survival when compared to treatment with gemcitabine or Nab-PTX in a metastatic PAN02 mouse model. Cellax-DTX nanoparticles interact with the tumor-associated stroma, selectively interacting with and depleting SMA positive cells and macrophage, effects of which are associated with significant changes in tumor progression and metastasis.

Assessment of Hypoxia in the Stroma of Patient-Derived Pancreatic Tumor Xenografts

The unusually dense stroma of pancreatic cancers is thought to play an important role in their biological aggression. The presence of hypoxia is also considered an adverse prognostic factor. Although it is usually assumed that this is the result of effects of hypoxia on the epithelial component, it is possible that hypoxia exerts indirect effects via the tumor stroma. We therefore measured hypoxia in the stroma of a series of primary pancreatic cancer xenografts. Nine patient-derived pancreatic xenografts representing a range of oxygenation levels were labeled by immunohistochemistry for EF5 and analyzed using semi-automated pattern recognition software. Hypoxia in the tumor and stroma was correlated with tumor growth and metastatic potential. The extent of hypoxia varied from 1%–39% between the different models. EF5 labeling in the stroma ranged from 0–20% between models, and was correlated with the level of hypoxia in the tumor cell area, but not microvessel density. Tumor hypoxia correlated with spontaneous metastasis formation with the exception of one hypoxic model that showed disproportionately low levels of hypoxia in the stroma and was non-metastatic. Our results demonstrate that hypoxia exists in the stroma of primary pancreatic cancer xenografts and suggest that stromal hypoxia impacts the metastatic potential.

Targeting hypoxic microenvironment of pancreatic xenografts with the hypoxia-activated prodrug TH-302

Previous reports have suggested that the hypoxic microenvironment provides a niche that supports tumor stem cells, and that this might explain clinical observations linking hypoxia to metastasis. To test this, we examined the effects of a hypoxia-activated prodrug, TH-302, on the tumor-initiating cell (TIC) frequency of patient-derived pancreatic xenografts (PDX). The frequencies of TIC, measured by limiting dilution assay, varied widely in 11 PDX models, and were correlated with rapid growth but not with the levels of hypoxia. Treatment with either TH-302 or ionizing radiation (IR), to target hypoxic and well-oxygenated regions, respectively, reduced TIC frequency, and the combination of TH-302 and IR was much more effective in all models tested. The combination was also more effective than TH-302 or IR alone controlling tumor growth, particularly treating the more rapidly-growing/hypoxic models. These findings support the clinical utility of hypoxia targeting in combination with radiotherapy to treat pancreatic cancers, but do not provide strong evidence for a hypoxic stem cell niche.

Activity of the novel polo-like kinase 4 inhibitor CFI-400945 in pancreatic cancer patient-derived xenografts.

Background Polo-like kinase 4 PLK4 plays a key role in centriole replication. Hence PLK4 inhibition disrupts mitosis, and offers a novel approach to treating chromosomally unstable cancers, including pancreatic cancer. CFI-400945 is a first in class small molecule PLK4 inhibitor, currently undergoing early phase clinical trials. Results Treatment with CFI-400945 significantly reduced tumor growth and increased survival in four out of the six models tested. Consistent with PLK4 inhibition, we observed reduced expression of the proliferation marker Ki-67 associated with an increase in nuclear diameter during treatment with CFI-400945. Additionally, treatment with CFI-400945 resulted in a significant reduction of tumor-initiating cells. Discussion These results support the further investigation of PLK4 as a drug target in pancreatic cancer. Methods Sensitivity to CFI-400945 was tested in a series of six patient-derived pancreatic cancer xenografts, selected to represent the range of growth characteristics, genetic features, and hypoxia found in pancreatic cancer patients.

Abstract B282: The PLK-4 inhibitor CFI-400945 reduces tumor growth in patient-derived pancreatic xenografts.

The polo-like kinase 4 (PLK-4) has recently emerged as a target that strongly correlates with aggressive tumor growth, high levels of hypoxia and metastasis in patient-derived pancreatic xenografts. In contrast to other members of the PLK family, the role of PLK-4 in cancer remains mostly elusive. Overexpression of PLK-4 in tumors has been suggested to contribute to chromosomal instability due to its crucial role in centriole duplication and therefore presents an interesting molecular target for the development of new therapeutic options for pancreatic cancer patients. The newly developed PLK-4 inhibitor CFI-400945 has been shown to specifically target PLK-4, resulting in incorrect centriole duplication and is currently developed for clinical trials. We used 6 orthotopically implanted patient-derived pancreatic xenograft models with varying growth rates and levels of tumor hypoxia in order to investigate the efficiency of the PLK-4 inhibitor CFI-400945 as a mono-therapy or in combination with Gemcitabine. Animals were treated with CFI-400945 using a daily dose of 7.5mg/kg for 21 days for experiments evaluating activity as a mono-therapy or with 13.5mg/kg according to a 2-day-on-5-days-off schedule in combination bi-weekly 100mg/kg Gemcitabine for 21 days. Tumor weight after completion of the treatment and overall survival were evaluated. Immunofluorescence (IF) staining of xenograft sections was used to evaluate PLK-4 inhibition, tumor hypoxia and proliferation. Treatment with CFI-400945 as a mono-therapy for 21 days significantly reduced tumor weight in 4 of the 6 tested patient-derived pancreatic xenograft models and an increase in overall survival in a number of responsive models. The strongest response to CFI-400945 treatment was observed in the highly hypoxic fast growing xenograft models OCIP51 where medium survival was 103days in the treatment group when compared to 54 days in vehicle treated animals. Experiments investigating the benefit of the combination of CFI-400945 with Gemcitabine using a clinically relevant treatment schedule are currently in progress in 2 xenograft models and will be completed shortly. We furthermore examined the correlation between tumor hypoxia, proliferation and response to CFI-400945 treatment in order to establish biological correlates for treatment response. Preliminary results show that although tumor weight was reduced in most of the tested models, the strongest response to CFI-400945 as a mono-therapy was observed in highly hypoxic models when compared to medium or low hypoxic models. These findings suggest that the PLK-4 inhibitor CFI-400945 may provide an effective treatment strategy for pancreatic cancer patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B282. Citation Format: Ines Lohse, Pinjiang Cao, Trevor Do, Emin Ibrahimov, Ming-Sound Tsao, David W. Hedley. The PLK-4 inhibitor CFI-400945 reduces tumor growth in patient-derived pancreatic xenografts. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B282.

Naturally occurring compounds as pancreatic cancer therapeutics

Naturally occurring small molecule compounds have long been in the spotlight of pancreatic cancer research as potential therapeutics to prevent cancer progression and sensitize chemoresistant tumors. The hope is that terminal pancreatic cancer patients receiving aggressive chemotherapy can benefit from an increase in treatment efficacy without adding further toxicity by way of utilizing natural compounds. While preclinical studies on a number of natural compounds, such as resveratrol, curcumin, rapalogs and cannabinoids, show promising preclinical results, little has translated into clinical practice, though a number of other compounds hold clinical potential. Nevertheless, recent advances in compound formulation may increase the clinical utility of these compounds.

Abstract A102: Combination treatment with hypoxia-activated prodrug TH-302 and radiation reduce pancreatic tumor initiating cells and tumor growth in patient-derived xenografts

Rationale: Using orthotopically-grown primary pancreatic cancer xenografts we recently identified an association between hypoxia and spontaneous metastasis formation, and we hypothesized that metastatic potential is conferred by the presence of tumor-initiating cells (TIC). We therefore investigated the relationship between hypoxia, tumor growth and TIC, and examined the effect of the hypoxia-activated prodrug TH-302 and ionizing radiation (IR) on pancreatic TIC and tumor growth. Methods: Eleven patient-derived pancreatic cancer xenograft models were used to examine tumor-initiation potential in relation to the tumor phenotype. To examine the effect of TH-302 and IR on TIC, tumor-bearing mice were treated with 50 mg/kg or 150 mg/kg TH-302, 10Gy of IR, or with the combination of TH-302 and IR. To examine the effect of TH-302 and IR on tumor growth, mice were treated with either TH-302 or IR alone, or with the combination treatment according to a clinically relevant schedule. Animals were treated with 50mg/kg TH-302 injected ip on days 1, 5 and 9 and/or 2Gy irradiation per day on days 2, 3 and 4. Tumor hypoxia was measured by EF5 administration and immunostaining. Results: TIC frequency varied across the patient-derived xenograft models and rapid tumor growth was strongly correlated with high TIC frequency but not hypoxia. Treatment with TH-302 in combination with IR led to a significant reduction in TIC frequency compared with either treatment alone in all models tested. Treatment-induced reduction in TIC frequency was independent of the magnitude of tumor hypoxia. Treatment-induced reduction of tumor growth was only observed in fast-growing hypoxic models. Additionally, TH-302 treatment also induced DNA damage in tumor tissue adjacent to the hypoxic zone which is consistent with the previously demonstrated bystander effect of TH-302. Conclusions: These results suggest that TIC can reside in both the oxic and the hypoxic microenvironment and that the combination of TH-302 and IR may present a novel therapeutic strategy to reduce the frequency of TIC and tumor growth rate of pancreatic tumors. Citation Format: Ines Lohse, Joanna Rasowski, Pinjian Cao, Melania Pintilie, Trevor Do, Emin Ibrahimov, Ming-Sound Tsao, Richard P. Hill, David W. Hedley. Combination treatment with hypoxia-activated prodrug TH-302 and radiation reduce pancreatic tumor initiating cells and tumor growth in patient-derived xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A102.

Abstract A106: BRCA mutation sensitizes pancreatic tumors to treatment with cisplatin

Rationale: Recent clinical observations in pancreatic cancer suggest that cancers arising in germline BRCA1/2 mutation carriers are highly sensitive to Cisplatin chemotherapy. Both BRCA1 and BRCA2 are required for DNA double-strand break repair by homologous recombination (HR). Platinum chemotherapy generates interstrand cross-links that can only be adequately repaired by HR-based DNA repair, and consequently BRCA1/2-deficient cells are highly sensitive to platinum chemotherapy both in vitro and in vivo. With a strong biological rationale and favourable clinical observations, we aim to investigate whether BRCA mutation indeed sensitize to Cisplatin treatment and if the addition of the PARP inhibitor ABT-888 further reduces tumor growth in patient-derived pancreatic xenografts Methods: Primary xenografts were derived from three patients with known BRCA2 germline mutations (2 from surgical resections, 1 from ascites fluid). All 3 patients experienced a major response when treated with cisplatin. These models were used to assess the in vivo sensitivity to the drugs Cisplatin, Gemcitabine and the PARP inhibitor ABT-888, all of which are being used in the clinic. Treatment sensitivities were compared to 2 primary xenografts that were BRCA w/t. For single treatments, animals were treated with 4mg/kg Cisplatin weekly, 100mg/kg Gemcitabine biweekly or 12.5mg/kg ABT-888 daily for 30 days. For the combination treatment, animals were treated with a single dose of 4mg/kg Cisplatin followed by 30 days of 12.5mg/kg ABT-888 daily. Results: Preliminary results show no difference in tumor growth between BRCA2 wt and mt models was observed in response to treatment with ABT-888 alone. Treatment with cisplatin or gemcitabine on the other hand significantly reduced tumor growth and increased survival in all 3 BRCA2 mutated xenograft models, while being mostly ineffective in BRCA w/t tumors. The combination of Cisplatin and ABT-888 significantly reduced tumor growth when compared to ABT-888 alone. Conclusions: The results suggest that BRCA1/2 mt pancreatic xenograft models are highly sensitive to cisplatin. The availability of these models facilitates “near-clinical” testing of alternative strategies for optimizing the individualized treatment of patients carrying these rare mutations, and for the exploration of protocols incorporating novel agents. Citation Format: Ines Lohse, Pinjian Cao, Emin Ibrahimov, Ming-Sound Tsao, David W. Hedley. BRCA mutation sensitizes pancreatic tumors to treatment with cisplatin. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A106.

Estimating tumour volume in a primary orthotopic mouse model of human pancreatic cancer using rapid acquisition magnetic resonance imaging

Pancreatic cancer has a mortality rate that closely approximates its incidence. This statistic highlights a dismal prognosis that has not improved despite decades of clinical trials. There is optimism that more sophisticated in vivo studies will better predict for clinical efficacy. This includes utilizing more relevant, orthotopic, primary xenograft models and incorporating imaging for tumour burden assessment. There has been hesitation in the routine incorporation of imaging in preclinical studies related primarily to the length and cost of imaging protocols. We hypothesized that rapid acquisition MR scans that balance efficiency with tumour visualization, could be effectively used to provide accurate and timely tumour size estimates. Mice bearing orthotopic primary pancreatic xenografts were study subjects. Animals were imaged on a 7 Tesla preclinical MR scanner, using the most rapid acquisition that provided adequate assessment of tumour size. Two different tumour volume estimates were made -one calculated from 2 dimensions obtained on imaging, and the other a software-based (MIPAV) volumetric analysis. Both were compared with tumour mass (range: 237 to 2575 mg). Although both calculated tumour volume (V=½ (LxW 2 )) and software-based volumetric analysis underestimated tumour size, they correlated well with tumour mass (correlation co-efficient 0.88 (p<0.0001) and 0.94 (p<0.0001) respectively). Our data demonstrate that rapid acquisition MR protocols can provide accurate tumour volume estimates to allow for longitudinal assessment of growth kinetics. This data has implications for the routine adoption of small animal imaging for preclinical studies using orthotopic pancreatic xenograft models.