Agenda

1. Quantification of cellular nanoarchitecture during early carcinogenesis using partial wave spectroscopic microscopy *Dhwanil Damania1, Yolanda Stypula1, Christine Will2, Hariharan Subramanian1, Jonathan Licht2, John Marko3, Vadim Backman1 Northwestern PS-OC Project 3 1Biomedical Engineering Department, Northwestern University, Evanston, IL 60208, USA 2Division of Hematology/Oncology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA 3Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA June 19, 2010

2. Agenda • Partial Wave Spectroscopic (PWS) Microscopy • Motivation • Brief Introduction • Results on biological cells • Biological mechanisms for disorder strength • PS-OC Cell lines • Microscopic appearance • PWS results

3. 1 µm Motivation • Cells have highly heterogeneous and complex organization spanning length scales from nanometers to microns. • Spatially continuous fluctuation of intracellular density (and, therefore, refractive index). • If a cell is histologically “normal”, this does not necessarily imply that the cell’s nanoarchitecture is also normal!

4. 100 nm Complex level of Cell Organization: From Microscale to Nanoscale • Cellular level: ~ 10 μm • Organellar level: ~1 μm • Macromolecular level: < 0.1 μm What is Cell nano-architecture ? • Fundamental building blocks of a cell • Most of the cell volume is densily occupied by macromolecular structures • Cytoplasm: ribosomes, cytoskeletal componenets, intracellular membranes, etc. • Nucleus: nucleosomes, 30 nm chromatin fibers, nuclear matrix, higher-order chromatin structure, cytoskeletal proteins (such as β-actin) etc. Membranes Actin filaments Ribosomes

5. Existing methodologies?? • Conventional microscopy and histopathology do not enable analysis of cell structure at nanoscale. • Diffraction limited resolution (~ 300 nm) • In most light scattering measurements in tissue, • Scattering from individual cell is obscured. • Scattering from nanometer scale structures are weighted down. • 3D scattering: • 1D scattering: • Solution: Partial wave spectroscopic microscopy* * H. Subramanian, PNAS, 105(51), 20124-20129 (2008)

6. Partial wave spectroscopic (PWS) Microscopy: Instrumentation Spectrometer based system • White light illumination: λ=400-700 nm • Scanning in x-direction • Low NA of illumination • Medium NA of collection • Illumination spot size ~120 m >> cell size Faster Imaging system • White light illumination: λ=400-700 nm • Spectral Scanning - LCTF • Low NA of illumination • Medium NA of collection • Illumination spot size ~120 m >> cell size

7. virtual aperture Heterogeneous slab – 1D Uniform slab Introduction - Partial wave spectroscopy • 1D propagating waves: • Non self averaging. • Sensitive to arbitrarily small refractive index fluctuations. Surface waves 1 D propagating wave 1D Spectrum • Uniform medium: • Internal refractive index fluctuation. • Δn = 0. • Frequency α thickness • Heterogeneous medium: • Internal refractive index fluctuation. • Δn = 0.05, lc = 5nm • Amplitude α alterations in internal refractive index.

8. 1 µm 1 µm Disorder Strength (Ld) Ld ~ <(Dn)2>lc • Mesoscopic Light Transport theory • PWS measures Disorder Strength (Ld) for a particular part of a cell • Disorder strength quantifies the spatial fluctuations of the local concentration of cellular material at the nanoscale (DNA, RNA, proteins, lipids, etc.) • Ld = variance of the concentration of cellular material • X • average size of intracellular (nanoscale) “building blocks”

9. Validation of PWS • The sensitivity of PWS to nanoarchitecture was tested in • Simulations: rigorous computations using FDTD • Experiments: Nano-structured models • Cell lines: genetic variants of HT-29 cell line • Animal studies: AOM-treated rat and MIN-mouse models of intestinal carcinogenesis • Human studies: colon, pancreas and lung cancer studies • Lesson 1: PWS is indeed sensitive to essentially arbitrarily small length scales of refractive index fluctuations. • Lesson 2: Higher disorder strength of a cell is associated with neoplastic behavior. • Lesson 3: The values of disorder strength in cells correspond to Lc~10-100 nm.

10. PWS Microscopy of Colonic Cell Lines Microscopically-identical HT-29 cell constructs Disorder strength Ld (mm) H. Subramanian et al, PNAS, 105(51) (2008)

11. Field Carcinogenesis • Genetic/environmental milieu that results in a neoplastic lesion in a particular tissue site that should be detectable farther away from this location • Conventional wisdom: a cell from a tumor is abnormal, a cell from surrounding tissue is normal. • This is only an approximation! • Field effect reported in lung, head and neck, colon, breast, ovarian, esophageal, stomach, liver and other cancers. • Opportunity: detecting carcinogenesis by analysis of normal appearing cells from an accessible part of an organ.

12. Human Colon Cancer Resection Study

13. Human Colon Cancer Resection Study

14. Lung cancer: Brushings from buccal mucosa (135 patients). PWS as a screening technique 4 3 2 Normalized Disorder Strength 1 0 Control Adenoma Advanced Adenoma HNPCC • Pancreatic cancer: • Brushings from periampullary duodenal mucosa (35 patients). • Colon cancer: Brushings from rectum mucosa(65 patients). * * p-value < 0.0001 * * * H. Subramanian et al, Cancer Research (2009)

15. 1. What is the origin of these nanoscale architectural changes that we are sensitive to?2. Are the disorder strength changes localized or distributed throughout a cell? Need to probe a macromolecular compound which is ubiquitous throughout a cell

16. Cytoskeleton • Back-bone of any cell ; maintains the shape & structure of a cell • Important for inter- and intra-cellular transport of molecules • Important for gene transcription, signal transduction, chromosome structure Cytoskeleton Cytochalasin D No Treatment Colchicine Intermediate Filaments Actin Filaments Micro-tubules Affected Compartments Nucleus and Cytoplasm Only Cytoplasm No Effect and No change Affected Compartments

17. Summary of the results of different treatments μ1and μ2 are mean disorder strength while σ1 and σ2 are its standard deviation for HT29 and CSK constructs • Cytochalasin disrupts actinfilaments in both cytoplasm and the nucleus  Ld normalizes in both compartments • Colchicine disrupts microtubules in the cytoplasm only  Ld normalizes in the cytoplasm but not in the nucleus Red  Statistically significant ; Black  Statistically non-significant D. Damania et al, Biophysical Journal, (2010)

18. Histone Deacetylase (HDAC) • HDAC protein expression  marker of nuclear structural changes • HDAC  up-regulated in tumors (including colorectal) • HDACi affects expression of other genes (ex: VEGF, p21) • Class I HDAC (localized to the nucleus) HDAC2 expression in human colon resection samples CSK shRNA HT29 NIH3T3 HDAC2 (60kDa)

19. S/TEM PWS PWS PWS Molecular Determinants of Nanoscale Disorder and Crowding Project 3 goals: Looking at Chromosomal changes • Molecular correlation of high-order chromatin alterations: • TEM: heterochromatin compaction • Consistent with macromolecular crowding • Partially controlled by nuclear actin • Partially controlled by histone deacetylases (HDACs) over-expression

20. PS-OC Cell lines

21. Phase Contrast Microscopy (60x phase) MCF-10A, passage 3 MDA-MB231, passage 3

22. X 10 -4 2.6 * 2.2 Disorder Strength (μm) 1.8 1.4 1.0 MCF10A MDA-MB-231 Nuclear Disorder Strength Differences *P-value ~ 0.04 n = 40 n = 42

23. PWS Quasi Bright Field PWS Disorder Strength Map MCF10A MDA-MB-231 There appears an increase in nuclear disorder strength (more dark red patches) in MDA-MB-231 cells compared to MCF10A cells

24. Conclusions • PWS results are promising in differentiating between the nuclear morphological changes between the two NCI cell lines : MCF10A (‘non-tumorigenic’) and MDA-MB-231 (‘tumorigenic’) • Although both the cells look microscopically differentiable, PWS can quantify these difference in terms of intracellular mass density and can provide information about spatial mass-density fluctuations • There appears to be higher variability in MDA-MB-231(~ 86%) cells in terms of shape, cell morphology compared to MCF10A cells (~ 56%) • Future Work: • Performing experiments similar to the HDAC ones on these cell lines and then studying the nuclear changes • Electron microscopy analysis of the cell lines and looking at the nuclear architectural modifications

25. Summary • PWS provides unprecedented insights into the cellular nano-architecture – disorder strength. • Disorder strength is proportional to the • <(Δn)2> - variance of refractive index fluctuation within the cell • lc - correlation length of refractive index fluctuation. • PWS can detect field effect associated with carcinogenesis in colon, pancreas and lung. • Potential for cancer screening/risk-stratification. • Changes in disorder strength are contributed to in part by cytoskeletal organization in nucleus and cytoplasm • Higher order chromatin structures play an important role in determining nuclear disorder

26. Acknowledgement • Northwestern University • Prof. VadimBackman • Prof. John Marko • Prof. Jonathan Licht • Hariharan Subramanian, Ph.D. • PrabhakarPradhan, Ph.D. • Yolanda Stypula • Christine Will Northshore University Health System • Hemant K. Roy, M.D. • Ashish Kumar Tiwari • DhananjayKunte Funding • National Cancer institute • National Science Foundation • National Institutes of Health • Coulter Foundation

27. Thank You

30. NCI MDA-MB231 cells (60x phase) MDA-MB231, passage 3 MDA-MB231, passage 3 Courtesy: Prof. John Marko

31. Micro-RNA analysis • To further understand changes in the cytoskeleton which PWS is sensitive to and the biological relevance in carcinogenesis EB1 (end binding protein 1) as candidate protein: • Reported to be over-expressed in Esophageal and gastric cancer cells • Role in microtubule polymerization, chromosomal stability • Our results suggest increase expression in both HT29-CSK cell-lines and in AOM (Azoxy-methane) treated rat models compared to Saline treated rats EB1 (35kDa) AOM Saline

32. Macromolecular Crowding/Re-arrangement • The vast majority of the intracellular environment is occupied by macromolecules, which then alters the properties of molecules and biological processes. • Crowding alters chromatin structure  gene expression • Side-effect of genetic/epigenetic events or a necessary step in carcinogenesis? • How can we measure crowding?  Qualitatively and Quantitatively Image from Richter et al, J Cell Sci, 2007

33. Origins of increased disorder in the cell nucleus – Alterations in nuclear chromatin organization • Heterochromatin (HT): dense regions • Enriched for nonessential regions of DNA - genes present in HT are generally inactive • Localized to nuclear periphery • Euchromatin: less dense regions • Rich in actively transcribed genes • Loosely packed in loops • Three different parameters were used to quantify chromatin organization • % Heterochromatin within a nucleus. • Run length - size of heterochromatin clumps. • Dissociation length - detachment of heterochromatin from nuclear membrane. Nucleus of a normal patient Nucleus of an adenoma patient

34. Effect size = 50% P-value < 0.0001 Effect size = 60% P-value < 0.0001 Effect size = 90% P-value < 0.0001 Run length Dissociation length (from nuclear membrane) % Heterochromatin Effect size = 46% P-value = 0.1 Effect size = 100% P-value < 0.0001 Run length Dissociation length (from nuclear membrane) % Heterochromatin Effect size = 0.4 P-value = 0.4 TEM Analysis of Nuclei From Rectal Cells: From Cell Lines: The 3 different parameters were able to differentiate between control and adenoma nuclei

35. p=0.006 Disorder Strength 6 HT29 HT29 +320mM 5 CSK CSK +320mM 4 p=0.005 Average Ld (um) 3 2 1 0 Crowding - PWS • Induced crowding as before and measured nuclear Ld • Conclusions: • Introducing crowding agents into HT29 and CSK cells significantly increased Ld • Trends similar to HDAC2 expression levels X 10 -4