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Mouse Models of Advanced Metastatic Disease: Development, Challenges and Utility with a Key Role for In Vivo Molecular Imaging July 21, 2016 Bryan Q. Spring Assistant Professor Northeastern University Boston, Massachusetts Interdisciplinary team PHYSICS CHEMISTRY Tayyaba Hasan, PhD Xiang Zheng R. Bryan Sears Ruth Goldschmidt S. Sibel Erdem Akilan Palanisami PhD PhD PhD PhD PhD Wellman Center for Photomedicine Harvard Medical School and Massachusetts General Hospital COLLABORATORS BIOLOGY Adnan Abu-Yousif PhD Imran Rizvi PhD Zhiming Mai Sriram Anbil Lawrence Mensah PhD HHMI clinical fellow PhD Brian Pogue Steve Pereira PhD FRCP PhD Dartmouth University College College London Funding from the National Institutes of Health Hasan: NIH R01-AR40352, RC1-CA146337, R01-CA160998, & P01-CA084203 Spring: NIH F32-CA144210 (NRSA Postdoctoral Fellowship) & K22-CA181611 (NCI Transition Career Development Award) First, brief background on photomedicine and biomedical optics Photodynamic therapy (PDT) Celli et al. Chem. Rev. 110, 2795–2838 (2010). PS = photosensitizer (nontoxic PDT agent) PDT agents can be used for fluorescence-guided surgery Glioblastoma (brain cancer) Stummer et al. Lancet Oncol 7, 392–401 (2006) Eljamel, Goodman & Moseley. Lasers Med Sci 23, 361–7 (2007) Dartmouth group: David Roberts & Keith Paulson Ovarian cancer van Dam et al. Nat Med 17, 1315–19 (2011) No doubt more tumor is removed…but recurrence is still a problem. Mopping up danger zones using PDT Fluorescenceguided surgery 80% local recurrence PDT to mop up danger zone (within 2 cm of original tumor volume)1 (treatment depths up to 1 cm possible in brain with conventional PDT)2 Fiber optic light delivery in the clinic 1. Milano et al. Int J Radiation Oncology Biol Phys 78, 1147–1155 (2010). 2. For example: 630 nm light has an attenuation coefficient (1/e, 37%) of 1 to 4 mm in most tissues, about 2 to 3 mm in brain with tumor. Necrosis occurs at depths up to about 3x the penetration depth — 6 to 12 mm (source: Neuro-onclology: The Essentials, edited by Mark Bernstin, Mitchel S. Berger, Chapter 25 Photodynamic therapy). http://www.moderncancerhospital.com http://www.medlight.com/ Metastatic ovarian cancer 80-95% of recurrence in peritoneal cavity1 Primary tumor Micrometastasis Problems leading to recurrence: (1) residual, occult microscopic disease missed by surgery (2) radio- and chemoresistant cancer stem-like cells2,3 1. Gadducci et al. Int J Gynecol Cancer 17, 21–31 (2007). 2. Kulkarni-Datar et al. Cancer Letters 339, 237–46 (2013). 3. Foster, Buckanovich & Rueda. Cancer Letters 338, 147–57 (2013). Mopping up the danger zones of recurrence PDT in the peritoneal cavity wide-field irradiation of disseminated metastases Bowel toxicity was the major hurdle in first human studies1,2 1. 2. Hahn et al. Clin Cancer Res 12, 2517–25 (2006). Cengel et al. Cancer Treat Res 134, 493–514 (2007). Model we used… Mouse model of metastatic ovarian cancer (peritoneal carcinomatosis) Cell line xenograft model: intraperitoneal (i.p.) injection of ~10∙106 human OVCAR5 cancer cells into athymic Swiss female Nu/Nu mice (other cell lines tested formed ascites but not metastases) Barbara Goff Kelly Molpus Marcela Del Carmen Linda Duska Developed and applied by past gynecologic oncology clinical fellows Molpus et al. Int J Cancer 68, 588–95 (1996) Wellman Center and Vincent Center, Massachusetts General Hospital Goff et al. Br J Cancer 70, 474–80 (1994) Dusk et al. J Natl Cancer Inst 91, 1557–63 (1999) Del Carmen et al. J Natl Cancer Inst 97, 1516–24 (2005) Micrometastatic disease forms in ~10 days Challenges Poor reproducibility Pearson correlation r = 0.99, n = 5 mice, P = 0.0006 no tumor control, n = 4 mice no treatment, n = 30 mice Developed qRT-PCR assay to count human cancer cells per gram tissue in entire peritoneal cavity Large variance in metastatic burden 2 weeks post cancer cell inoculation (variable human-versus-graft response?) Other challenges & open questions (1) Why the need to implant tens of millions of cells? Seeding efficiency appears to be less than 10% (or a very slow doubling time)—innate immune response and/or most cells not able to initiate a tumor? (2) How can we track tumor growth and treatment response in multifocal, microscopic tumors deep inside the body? (3) For spontaneous metastasis models starting with an orthotropic tumor implant—how can we achieve long enough survival to study metastasis? (Surgical removal of primary tumor helps; Francia et al. 2011, Nat Rev Cancer 11, 135–41). (4) How to build in patient derived tissues to better recapitulate heterogeneity of human 1. cancer and the intricate tumor microenvironment? Molecular imaging to improve reproducibility • Direct visualization of tumor growth and treatment response over time • Developed micrometastasis imaging and applied it to monitor efficacy of a new therapy Imaging probe/combination therapeutic: molecular targeting + fluorescence activation Savellano et al. Photochem Photobiol 77, 431–439 (2003). Synergistic tumor reduction when used as separate, unconjugated agents: del Carmen et al. J Natl Cancer Inst 97, 1516–1524 (2005). Cet-BPD synthesis & anti-EGFR activity in vitro: Savellano et al. Photochem Photobiol 77, 431–439 (2003). Savellano et al. Clin Cancer Res 11, 1658–1668 (2005). Abu-Yousif et al. Cancer Letters 321, 120–127 (2012). Tumor-targeted, activatable photoimmunotherapy (taPIT) Cancer cell activation via lysosomal proteolysis Cet-BPD Abu-Yousif et al. Cancer Letters 321, 120–127 (2012). free BPD (cell lysates) 1h Cet-BPD (cell lysates) 40h 1h 4h 17h 40h proteolysis: Cet-BPD fragments Savellano et al. Clin Cancer Res 11, 1658–1668 (2005). Selective visualization/damage of microscopic tumors Improved tumor-to-bowel ration from 1–2 in the clinical trial (Hahn et al, 2006 Clin Cancer Res 12, 2517–25) to 19 (Spring et al, 2014 PNAS 111, E933–42) Microendoscopic imaging of micrometastases in vivo $50,000! monolayer 3D culture camera always-on BPD $2 light emitting diode 100 µm 100 µm mouse peritoneal wall Zhong & Celli et al. Br J Cancer 101, 2015–2022 (2009). 100 µm Validation of in vivo imaging Exclusion of treated mice Spearman correlation r = 0.70 (P = 0.004) tumor recognition sensitivity = 100% and specificity = 100%, n = 15 mice Spring et al. PNAS 111, E933–42 (2014) Spearman correlation r = 0.59 (P = 0.0001) tumor recognition sensitivity = 86% and specificity = 73%, n = 37 mice Selectivity at the microscale freshly excised tissues ex vivo no-tumor control 1 mm 100 μm leukocyte uptake gives false positive Cet-BPD activation OvCa mouse 1 mm 100 μm Always-on BPD: 86% sensitivity & 59% specificity1 Cet-BPD for micrometastases > 30 μm: 93% sensitivity & 93% specificity2 Cet-BPD + leukocyte (CD45) imaging: 99.9% sensitivity & 98% specificity single cancer cells3 ROC area under the curve (AUC) = 0.961, n = 12 mice (296 fields). 1. 2. 3. Zhong et al. Br J Cancer 101, 2015–2022 (2009). Spring et al. PNAS 111, E933–42 (2014). Spring et al. J Biomed Opt 19, 066006 (2014). Putting it all together: taPIT with longitudinal monitoring of micrometastases Molecular imaging of microscopic tumors no tumor control Cet-BPD(1:7) no treatment control taPIT Longitudinal monitoring of micrometastases 100 µm Cet-BPD (no light, 2 cycles), n = 8 mice taPIT (50 J・cm−1, 2 cycles), n = 8 mice ***P <0.001 (Mann-Whitney U test) Spring et al. PNAS. 111, E933–42 (2014) A short aside on outcomes of the photomedicine study taPIT phototoxicology Maximum tolerated dose (100% survival of ovarian cancer mice) [PS] × light dose Total PDT dose “always-on” PDT1 0.25 mg・kg−1 PS (BPD) × 8 J・cm−1・quadrant−1 =2 “always-on” PIT2–4 1 mg・kg−1 PS × 6 J・cm−1・quadrant−1 =6 taPIT 2 mg・kg−1 PS (BPD) × 50 J・cm−1・quadrant−1 = 100 PS = photosensitizing agent for photodynamic therapy (PDT; e.g., BPD) 1. 2. 3. 4. Molpus et al. Cancer Res 56, 1075–82 (1996). Goff et al. Br J Cancer 70, 474–80 (1994). Molpus et al. Gynecologic Oncology 76, 397–404 (2000). Rizvi et al. Isr J Chem 52, 776–87 (2012). taPIT enables a ~17–50× increase in total PDT dose High-dose taPIT efficacy taPIT is effective and potentiates chemotherapy benchmarks for comparison tumor reduction chemo (1 cycle)a,1 3% “always-on” PIT (1 cycle) + chemo (2 cycles)1 66% taPIT (2 cycles) 90% taPIT + chemo (1 cycle) 97% achemo: cisplatin (3 mg·kg-1) + paclitaxel (10 mg·kg-1); poor response may be due to intrinsic chemoresistance2,3 1. 2. 3. Rizvi et al. Isr. J. Chem. 52, 776–87 (2012). Roberts et al. Br J Cancer 92, 1149–58 (2005). Meirelles et al. PNAS 109, 2358–63 (2012). Now that selectivity is worked out, we will focus on evaluating and optimizing efficacy in future studies. Summary • • A simple cell line xenograft mouse model is challenging to implement in practice: • needs lots of cells (inefficient seeding of metastases) • wide variance in tumor burden • micrometastatic disease too small for standard techniques (tumor weight, bioluminescence, PET, MRI, etc.) Development of molecular imaging/cellular-resolution microendoscopy enables monitoring of tumor progression and treatment response (at least in select sites)— overcoming intrinsic biological variability in metastatic models Next steps • Challenges remain—how to monitor and target multiple cell types and heterogenous tumors (e.g., patient-derived xenograft models)? The Transition Career Development Award (K22) • Goal is to guide taPIT of multiple chemoresistant phenotypes Potential points of collaboration • Can we find ways to enrich certain phenotypes that form metastases (“metastasisinitiating cells”) in culture? • Develop a chemoresistant, humanized patient-derived tumor xenograft mouse model that generates metastatic ovarian cancer—building on success of Bankert et al. 2011, PLoS ONE 6, e24420. Tumor/molecular-targeted activation reduced bowel fluorescence/toxicity “always-on” BPD tumor-to-bowel ratio = 2.8 “tumor-targeted, activatable” Cet-BPD tumor-to-bowel ratio = 18.6 Clinical Benchmark “always-on” PDT clinical trial1 tumor-to-bowel ratio = 1.1–2.1 1. Hahn et al. Clin Cancer Res 12, 2517–25 (2006). Cet-BPD(1:7) (n = 10 mice) always-on BPD (n = 10 mice) *P <0.05 **P <0.01 ***P <0.001 ****P <0.0001 (two-tailed unpaired t-test)