Download Mouse Models of Advanced Metastatic Disease: Development

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)