TRECAN 249 No.

of Pages 12

Review

Nuclear Mechanopathology and Cancer

Diagnosis
Caroline Uhler1 and G.V. Shivashankar2,3,*

Abnormalities in nuclear and chromatin organization are hallmarks of many Highlights

diseases including cancer. In this review, we highlight our understanding of Nuclear morphology is regulated by
both the cytoskeletal architecture
how the cellular microenvironment regulates nuclear morphology and, with it,
and chromatin condensation.
the spatial organization of chromosomes and genes, resulting in cell type-
specific genomic programs. We also discuss the molecular basis for maintain- The nuclear envelope and its interac-
tions with the nuclear lamina are critical
ing nuclear and genomic integrity and how alterations in nuclear mechano- determinants of nuclear morphologies.
transduction pathways result in various diseases. Finally, we highlight the
importance of digital pathology based on nuclear morphometric features com- Defects in nuclear mechanotransduc-
tion, the SUN-KASH domain proteins,
bined with single-cell genomics for early cancer diagnostics. and specific mutations on lamin-asso-
ciated genes lead to altered nuclear
Microenvironmental Control of Nuclear Morphology morphologies.
Cells within the tissue microenvironment are in contact with either the basal membrane,
Nuclear morphology and architecture
extracellular matrix (see Glossary), and/or neighboring cells. For example, epithelial cells as well as chromosomal translocations
and endothelial cells form both the cell matrix as well as cell–cell junctions, whereas connective are a hallmark of cancer.
tissue cells, such as fibroblasts, adhere to the extracellular matrix. Cell morphology, mechanics,
Machine-learning techniques, such as
and behavior are largely dictated by the interactions of the cell with the local microenvironment
deep learning, applied to images of the
[1–3]. An elaborate cytoskeletal network links the cell membrane with the nuclear membrane cell nucleus provide highly accurate
and also shapes the nuclear morphology [4–6]. methods for classifying normal versus
cancer cells based on nuclear
morphology.
Signals experienced by cells within the microenvironment remodel the cytoskeletal network,
thus enabling tight microenvironmental control of nuclear function [7,8]. For example, physical
signals, such as shear, stretch, or compression, activate specific receptors (e.g., integrins, ion
channels, and receptor tyrosine kinases) [9]. The activation of such receptors results in either
calcium uptake or the triggering of secondary messengers to remodel the cytoskeletal network
[10]. Among other things, calcium uptake regulates the polymerization state of actin, whereas
integrin receptors modulate actomyosin contractility and trigger various downstream signaling
pathways [11]. Interestingly, the regulation of microtubule organization by the microenviron-
ment feeds back into actomyosin organization, thus providing subtle control of nuclear
morphology by the cytoskeletal structures [12]. Furthermore, intermediate filaments, such
as vimentins, which act as shock absorbers to the nucleus, are also modulated by microenvi-
ronmental signals [13]. In addition to physical signals, soluble signals, such as growth factors or
1
Department of Electrical Engineering
cytokines, trigger their respective receptors (e.g., TNF-a receptors, TGF-b receptors, NOTCH
& Computer Science, Institute for
receptors, and junctional protein complexes), which can activate elaborate intracellular signal Data, Systems & Society, MIT,
transduction pathways that remodel cytoskeletal structures and nuclear function [14–16]. Cambridge, MA, USA
2
Mechanobiology Institute, National
University of Singapore, Singapore
Recent progress revealed several nuclear membrane proteins that serve to bridge cytoskeletal 3
FIRC Institute of Molecular Oncology
networks, for example nesprin and its various isoforms, linked to all three types of cytoskeleton, (IFOM), Milan, Italy
namely actin, microtubules and intermediate filaments [17,18]. Collectively, the cytoskeletal
networks provide an important link between the extracellular microenvironment and the *Correspondence: dbsgvs@nus.edu.sg
nucleus for both maintaining nuclear morphology and signaling to the nucleus [19]. Defects (G.V. Shivashankar).

Trends in Cancer, Month Year, Vol. xx, No. yy https://doi.org/10.1016/j.trecan.2018.02.009 1

© 2018 Published by Elsevier Inc.
 TRECAN 249 No. of Pages 12

in the coupling between the cytoskeletal network and the nucleus have been associated with Glossary
alterations in nuclear morphology and nuclear mechanotransduction pathways leading to Actomyosin machinery: contractile
several diseases, including cancer [20–22]. protein complexes comprising actin
and myosin.
Deep learning: machine-learning
Impact of Nuclear Morphology on Chromatin Dynamics techniques that perform nonlinear
The morphology of the nucleus is determined by both the cytoskeletal links and the degree of transformations on the data (e.g.,
chromatin condensation within the nucleus [23]. DNA, an approximately 2 m-long flexible nuclear images) for learning a
representation of the data that
polymer chain, is folded into a chromatin fiber by histone and nonhistone proteins and the
admits accurate classification.
associated chemical modifications on the histone proteins [24]. The chromatin fiber, depending Extracellular matrix: matrix of
on such modifications (i.e., either histone acetylation or methylation on specific unstructured protein filaments, such as collagen,
histone tail amino acid residues) dictates the higher-order compaction, thereby implying both to which cells attach.
Integrins: transmembrane receptor
genetic and nongenetic functions of the genome [25]. Recent studies revealed that this higher-
proteins that are sequestered on the
order structure comprises megabase-pair topologically associated domains leading to a plasma membrane for
highly organized chromatin structure [26–28]. Since the DNA is an entropic coil, such con- mechanosensing.
densation results in a balance between outward entropic forces and inward chromatin con- Lamin-associated domains
(LADs): functional chromatin
densation forces [29,30]. However, this description is too simplistic and also needs to
domains that have been found to be
incorporate coupling between chromatin organization with the nucleus, scaffold proteins, linked to the nuclear envelope.
which could represent an additional layer of the nuclear matrix, and the cytoskeletal forces Lamins: nuclear structural proteins
acting on the nucleus [31,32]. that regulate nuclear stiffness and
chromatin condensation.
Nuclear bodies: micromolecular
Within the cellular context, such force balance results in the nucleus being held under a assemblies of proteins and enzymes
prestressed condition. This is counter-balanced by the cytoskeletal network, where actin that are phase separated into
stress fibers have been shown to apply contractile forces on the nucleus, whereas the aggregates within the cell nucleus.
Nuclear mechanotransduction:
microtubules have been shown to apply compressive forces, thereby regulating the shape, the transmission of both mechanical
size, and plasticity of the nucleus. In addition, more recent findings also suggest that apical and biochemical signals to the cell
actin stress fibers also apply compressive forces on the nucleus [33,34]. Hence, modulations of nucleus.
SUN-KASH domain proteins:
the extracellular microenvironment not only remodel the cytoskeletal structures, but also
protein links, including nesprins, that
impinge on nuclear morphology and chromatin organization [35,36]. bridge the outer and inner nuclear
membrane with the cytoskeleton and
In addition, global chromatin-remodeling enzymes that either add or remove acetyl or methyl the chromatin.
Topologically associated
groups to/from the histone proteins shuttle between the cytoplasm and the nucleus, thereby
domains: folding of chromatin into
regulating the condensation state of the chromatin [37]. In addition, there are several chroma- functional domains of approximately
tin-remodeling enzymes that could reside within the nucleus and modulate chromatin conden- 1 Mbp in size.
sation independent of mechanical forces [38]. More acetylated states of chromatin are highly
decondensed and dynamic, whereas more methylated states of chromatin are highly con-
densed and static [39]. The balance between acetylation and methylation states of chromatin
dictates not only chromatin plasticity and accessibility to the DNA sequence, but also the
entropic forces exerted on the nuclear membrane [40]. Alterations in this fine balance have
important implications in exposing functionally important chromatin regions. such as coding or
telomere regions. and can lead to their misregulation and genomic instabilities [41,42].

Furthermore, the nuclear envelope comprises intermediate filaments (i.e., lamin A/C and B),
which have been identified to be the nuclear structural proteins [43]. While lamin B aids in
chromatin condensation, lamin A/C is involved in determining the stiffness of the cell nucleus (i.
e., higher concentrations of lamin A/C result in a stiffer cell nucleus) [44]. Thus, lamin A/C forms
a mechanical intermediate between the chromatin forces and the cytoskeletal forces, thereby
determining the overall nuclear structural properties [45]. Lamin dimers form head-to-tail
polymers of 48 nm. These head-to-tail polymers laterally interact to form protofilaments, which
are anchored both to the nuclear membrane and to chromatin fibers [46]. A recent study
revealed separate roles for chromatin and lamins in determining the nuclear mechanical

2 Trends in Cancer, Month Year, Vol. xx, No. yy

 TRECAN 249 No. of Pages 12

properties [47]. Recent evidence has also shown that lamin A/C levels and their assembly are
highly regulated by the interactions of cells with their local microenvironment [48]. Cells that
attach to rigid substrates have higher levels of lamin A/C, while cells on softer substrates have
lower lamin A/C levels, resulting in cell type-specific mechanical homeostasis of the cell nucleus
[49].

Mechanical Control of Chromosome Organization and Genomic Programs

Several studies have revealed that every chromosome has its own territory and that these
territories are organized within the nucleus in a tissue-specific manner [50–53]. In addition,
small or gene-rich chromosomes are usually located more towards the interior, whereas larger
or gene-poor chromosomes are located more towards the nuclear periphery [54]. At the
nuclear periphery, specific lamin-associated domains (LADs) have been identified to anchor
these chromosomes with the nuclear envelop, thereby providing a mechanism for differentially
arranging chromosomes [55]. However, the exact association of LADs to the nuclear periphery
is currently under intense investigation. While stem cells exhibit highly dynamic chromatin
structures resulting from a more plastic cell nucleus, differentiated cells show lineage-specific
chromosome arrangements and a stiffer cell nucleus [52,56,57].

Interestingly, the 3D organization of chromosomes has also been shown to be correlated with
gene expression [50–54,58]. In particular, the relative organization of chromosomes is
optimized for the respective transcription network topology (i.e., chromosomes that are
more transcriptionally active have been found to be spatially clustered [59–61]). Even more,
chromosomes that harbor [28_TD$IF]coregulated genes have also been found to be clustered. For
example, the immunoglobin genes that are found on different chromosomes are spatially
clustered for their [283_TD$IF]coregulation [62]. In addition, genes of specific transcription pathways are
spatially clustered and [284_TD$IF]colocalized with the transcriptional machinery and the associated
post-translational modifications [63]. These findings suggest that chromosome intermingling
regions are mechanical hotspots for transcription control [64]. Indeed, these intermingling
regions are enriched with activated RNA Pol-II and several other activation marks, including
histone acetylation and transcription factors [65,66]. We speculate that these chromosome
intermingling regions, which undergo active transcription and, thus, experience increased
mechanical stresses due to DNA supercoiling [67], are also important sites where cells
acquire mutations.

Cellular microenvironmental signals modulate not only nuclear morphology and chromatin
plasticity, but also the spatial arrangement of chromosomes [64,68–72]. For example, cells that
are highly polarized exhibit chromosomes that are preferentially oriented with the mechanical
axis. The most-aligned chromosomes have also been found to be more intermingling and
transcriptionally active. Depending on the mechanical context, not only the chromosomes are
reoriented, but there also exists a differential cytoplasmic-to-nuclear transduction of various
transcription factors, their cofactors, and chromatin-remodeling enzymes. For example, in
fibroblast cells on rigid substrates with higher actomyosin contractility the cofactor MKL is
localized in the nucleus, whereas the transcription factor p65 and the chromatin-remodeling
enzyme HDAC3 are localized in the cytosol. However, when these cells are relaxed or on soft
substrates, MKL is shuttled out of the nucleus, whereas p65 and HDAC3 are shuttled into the
nucleus. These factors then integrate with the new spatial chromosome neighborhoods to
induce differential gene expression programs [73]. Taken together, these findings highlight an
important layer of gene regulation through both nuclear mechanotransduction and the spatial
organization of chromosomes [69]. Figure 1 depicts the differences in morphology and the
resulting downstream gene expression programs in normal and abnormal cells.

Trends in Cancer, Month Year, Vol. xx, No. yy 3

 TRECAN 249 No. of Pages 12

Signals

Microtubule

Stretch-acƟvated
channel

Regulatory
molecules Receptor

AcƟn

ECM

Normal Abormal
condiƟon condiƟon
Chromosome Lamina
territory

TranscripƟon
factory

TranscripƟon
factors

ChromaƟn
regulators

Nuclear pore Nucleolus

complex

Normal gene expression Abnormal gene expression

(See figure legend on the bottom of the next page.)

4 Trends in Cancer, Month Year, Vol. xx, No. yy

 TRECAN 249 No. of Pages 12

The dynamic mechanical exposure of chromatin has also been shown to be critical in the
maintenance of genomic stability [74]. Since chromatin plasticity is tightly controlled by physical
and biochemical signals, access to fragile DNA sequences is also regulated by microenviron-
mental inputs. For example, compressive forces on cells result in the redistribution of ATR to the
nuclear envelope and also to increased DNA strand breaks, as observed by gH2AX staining
[75]. Similarly, recent experiments have revealed that cells passing through narrow constric-
tions undergo nuclear squeezing and, more importantly, accumulate DNA strand breaks [76].
Several studies also revealed that either rupture of the nuclear membrane or the phase
separation of chromatin-binding proteins induce DNA strand breaks in cells migrating through
constrictions [77,78]. Since cells in tissue microenvironments undergo migration in a variety of
functional contexts, the regulation of DNA strand breaks through mechanical forces highlights
the essential role of chromatin plasticity in inducing genomic instabilities. Such induction of DNA
strand breaks by mechanical control could be precursors in both oncogenic transitions and the
switching from benign to malignant tumor cells.

Molecular Basis of Nuclear and Chromatin Morphometrics

Collectively, the above findings suggest that nuclear and chromatin organization are critically
dependent on the cell microenvironmental interactions and that alterations in these interactions
can lead to differential regulation of gene expression and potentially the onset of genomic
instabilities resulting in various disease pathologies. A closer look at the molecular basis of
nuclear morphology revealed that a plethora of molecules involving focal adhesion proteins,
actin crosslinking proteins, microtubule dependent proteins, as well as several nuclear mem-
brane proteins that link to the cytoskeletal network, have an essential role in regulating nuclear
morphology and force transmission [79–81]. More precisely, the KASH domain proteins
(nesprins) on the outer nuclear membrane physically link with actin, microtubules, and inter-
mediate filaments. In addition, nesprins are in turn connected to the inner nuclear membrane
through SUN domain proteins, which are further linked to nuclear lamina and the chromatin,
thereby mechanically bridging the cytoskeleton and chromatin organization [82,83]. Recent
studies using secondary RNAi screens revealed that both the nuclear morphology and
chromatin condensation patterns are tightly linked to ablation of any of these linker proteins.
For example, ablating actin-related molecules resulted in a more rounded and softer cell
nucleus and increased chromatin plasticity, whereas ablating microtubule-related proteins
resulted in a more flattened and stiffer cell nucleus and more condensed chromatin. Interest-
ingly, ablating proteins, such as vinculin or talin, that are part of the focal adhesion complexes,
also resulted in a more rounded and softer cell nucleus and increased chromatin plasticity,
thereby exemplifying the importance of the mechanical links between the cell membrane and
the nuclear membrane to control chromatin structural states [84,85]. Figure 2 depicts the
molecular mechanisms that regulate nuclear morphometrics.

Analysis of post-translational modifications of histones in the background of genetic ablations

of such cytoskeletal network proteins revealed the tight control between cytoskeletal remod-
eling and functional states of chromatin assembly. Furthermore, transcriptome analysis in the
background of specific genetic ablations of the linker molecules revealed that the genetic
programs are dependent on the nuclear morphology and the associated chromatin

Figure 1. Differences in Nuclear Morphology and Downstream Gene Expression in Normal and Abnormal Cells. Normal cells comprise an intact cell
nucleus with a nonrandom spatial organization of chromosomes and genes. Such cell type-specific architecture can be altered in various diseases through impaired cell
microenvironmental interactions leading to globulated and indented nuclear morphologies with defective nucleoli and other nuclear bodies. Abbreviations: ECM,
extracellular matrix; ER, endoplasmic reticulum.

Trends in Cancer, Month Year, Vol. xx, No. yy 5

 TRECAN 249 No. of Pages 12

Lamina
ChromaƟn Nucleus
Emerin
SUN protein

KASH
protein
Nuclear
pore
Nesprin 1 Kinesin
complex
Nesprin 1
Nesprin 3
and 2
PlecƟn
KASH
Cytoplasm
Intermediate
filament
F-acƟn

Microtubule

RNAi ablaƟon of
RNAi ablaƟon of
RNAi ablaƟon of nuclear structural
Normal microtubule-related
acƟn-related genes proteins e.g., SUN-KASH
genes
and lamin

Figure 2. Molecular Mechanisms that Regulate Nuclear Morphometrics. Normal cell nuclear morphologies are balanced by contractile forces through actin-
related links to the nucleus, compressive forces through microtubule nuclear links, and entropic forces through chromatin. In addition, intermediate filaments, such as
lamins, act as a mechanical bridge balancing the cytoskeletal and chromatin forces. Defects in actin organization lead to more relaxed and rounded cell nuclei, whereas
defects in microtubule–nuclear coupling result in increased contractile tensions and a more elongated cell nucleus. Finally, defects in intermediate filaments lead to more
globulated and indented cell nuclei.

condensation states [84,85]. In line with these studies, several adaptor proteins have also been
identified that bridge the outer nuclear membrane to the cytoskeletal networks as well as the
inner nuclear membrane to the intermediate filaments, such as lamin A/C and heterochromati-
nized regions of chromosomes [17,86]. In addition, these adaptor proteins, including the SUN-
KASH domain proteins, help the microenvironmental control of genome programs [87].
Thus, these results highlight an important role of the mechanical control of chromatin in
maintaining genomic programs, and even subtle alterations in chromatin architecture have
been correlated with several diseases.

6 Trends in Cancer, Month Year, Vol. xx, No. yy

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Case Studies of Altered Nuclear Morphometrics in Diseases

Several diseases have been associated with defects in nuclear morphology. We here briefly
discuss two specific examples.

Hutchinson–Gilford Progeria Syndrome

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disorder that manifests as
premature aging, where patients have symptoms of aging already at a very young age,
including wrinkled skin, loss of eyesight[285_TD$IF], and cardiovascular diseases [88]. Several studies
revealed that HGPS is associated with defects in the nuclear structural protein lamin A, which
in patients with HGPS affixes to the nuclear rim. This form of lamin is called progerin and
results in abnormal nuclear morphology [89]. In patients with HGPS, the nucleus appears
highly globulated and irregularly shaped. Studies showed that progerin is most commonly
related to a point mutation in the gene encoding lamin A that results in abnormal splicing of the
prelamin A transcript. This in turn leads to the deletion of 150 nucleotides and a mutant
protein that lacks the cleavage site for prelamin A processing, which is critical for maintaining
nuclear integrity [90]. In addition, early drug discovery programs revealed that therapeutic
interventions, for example inhibitors to farnesyl transferase, reverted the nuclear morphology
in HGPS cells to their normal states, suggesting that the defects in lamin A-mediated nuclear
morphology are critical to disease progression [91]. More recent experiments showed that
defects in lamin A result in both altered cytoskeletal structures and chromatin organization.
The alterations in cytoskeletal structures in HGPS cells lead to compromised cell microenvi-
ronmental interactions, whereas the alterations in chromatin organization result in misregu-
lated transcriptional programs. Since lamin A is also important for DNA repair, patients with
HGPS show increased DNA damage, chromosome aberrations, as well as sensitivity to DNA-
damaging agents. A growing number of diseases, termed ‘laminopathies’, have now been
associated with defects in lamin, including Emery–Dreifuss muscular dystrophy, familial partial
lipodystrophy, and Pelger–Huet anomaly. Interestingly, more recent literature revealed that
neurological disorders, such as Alzheimer’s, are also associated with defects in nuclear
lamins [92].

Cancer
Early pathological inspections revealed that tumor progression is associated with alterations in
3D chromosome organization and abnormal nuclear morphologies [93]. More recent studies
also showed that several post-translational modifications of histones and the recruitment of
transcription factors to specific spatial neighborhoods are critical in the onset of tumorigenesis
[94,95]. In a recent study, proto-oncogenic genes were found to cluster in space, which was
determined to be crucial for their expression [96]. Alterations at the chromatin level in cancer
cells are also reflected at the scale of nuclear bodies, where one finds enrichment and
enlargement of specific nuclear organelles, such as the nucleolus and promyelocytic leukemia
(PML) bodies [22]. Furthermore, these changes are also reflected at the scale of the nuclear
lamina (i.e., altered nuclear lamin structures), specific chromosomal translocations, increased
heterochromatinization, as well as misshaped nuclei. Collectively, these findings suggest that
tumor cells are defective in cell–matrix and cell–cell junctions. The defective cell microenviron-
mental interactions regulate the cytoskeletal to nuclear links, thus resulting in impaired nuclear
mechanotransduction and altered expression programs [97]. Interestingly, cancer cells often
originate within the epithelial niche and undergo an epithelial-to-mesenchymal transition, a
switch that appears to be critical for tumor progression in several (but not all) types of cancer
[78]. In this context, recent literature has highlighted the mechanical microenvironment of the
tissue as a critical regulator of oncogenic transitions [98,99].

Trends in Cancer, Month Year, Vol. xx, No. yy 7

 TRECAN 249 No. of Pages 12

Digital Nuclear Mechanopathology for Cancer Diagnosis

Alterations in nuclear morphology have been used as a hallmark of cancer for decades; in fact,
such alterations are one of the major clinical diagnostic approaches used by pathologists to
determine the malignant potential of cancer cells [100–102]. These pathological inspections
relied heavily on observable nuclear and chromatin condensation defects. However, since the
tissue microenvironment is highly heterogeneous, such morphometric assays are currently
subjective and depend on the interpretation of the pathologist. To circumvent this subjectivity,
recent advances in imaging combined with machine learning have provided new avenues
[103,104]. These include parametric machine-learning techniques that use quantitative nuclear
morphometric information, such as nuclear size, shape, nucleus:cytoplasm ratio, and chro-
matin texture, for classifying histopathology images [105,106]. More recently, various non-
parametric methods, including deep learning, have been applied for diagnosing various
cancers, including breast, skin, and thyroid cancer, with high accuracies [107,108]. These
approaches learn the features directly by optimizing classification performance from images,
which can include staining for DNA, transcription factors, DNA repair proteins, nuclear bodies,
nuclear lamina, or specific genes and chromosomes. While this line of research has allowed
accurate late-stage prognosis, early diagnosis remains a major challenge. This is primarily due
to the difficulty that fine-needle biopsies or blood smears represent a heterogeneous population
of normal cells with possibly a small number of abnormal cells. In addition, machine-learning
methods require training samples and it is difficult to obtain samples of very early cancer stages.
Finally, while there are accurate deep-learning approaches for late-stage cancer detection, they
generally do not provide interpretable features or functional annotations that could be used by
pathologists for early disease diagnosis. To overcome these limitations, recent deep-learning
methods have been developed that perform the analysis on small regions within the cell
nucleus, thereby providing novel nuclear mechanomorphometric features at the level of
chromatin condensation that also yield high classification accuracies [109]. Figure 3 depicts
the application of machine-learning algorithms for the analysis of nuclear morphometrics and
chromatin condensation patterns for disease diagnostics.

Current approaches for cancer diagnosis also include genomic analyses to annotate mutations
and alterations in the epigenome [110,111]. While earlier genomic methods relied on population
studies, their efficiency to detect abnormalities in single cells within tissue biopsies was often
low. Therefore, current approaches also include a combination of morphometric assays and
immunohistochemistry, combined with single-cell genomic analyses. In particular, several
genomic studies in recent years have provided detailed single-cell sequencing maps to analyze
cell-to-cell variability in gene expression programs [112]. These methods use innovative
barcoding techniques to obtain massively parallel single-cell gene expression data in a variety
of functional contexts [113]. In addition, several proteomic technologies are currently under
development to assess altered functional states of proteins in the heterogeneous tissue
microenvironment [114].

To accurately determine a small number of abnormal cells in a heterogeneous population as

early as possible, all these different approaches will need to be combined at the level of single
cells within the tissue microenvironment. In particular, low-cost and fully automated machine-
learning methods based on single-cell imaging will have to be combined with single-cell
genomic methods to obtain accurate early cancer diagnostics. Such a combination of methods
is not only attractive for clinical applications to cancer diagnostics, but would also open new
avenues for drug discovery programs as well as the development of therapeutic interventions at
a single-cell resolution.

8 Trends in Cancer, Month Year, Vol. xx, No. yy

 TRECAN 249 No. of Pages 12

Parametric methods
Feature extracƟon

Nuclear
shape
ChromaƟn
condensaƟon
ChromaƟn
Images SegmentaƟon texture

ClassificaƟon
Nonparametric methods and
biomarkers

Neural network:
ConvoluƟonal layers Max pooling layers

Figure 3. Application of Machine-Learning Algorithms for the Analysis of Nuclear Morphometrics and Chromatin Condensation Patterns for Disease
Diagnostics. A series of single-cell nuclear images are fed into a pipeline that first segments the nuclei. These segmented images are then input either into parametric
or nonparametric learning methods. In parametric approaches, classification is performed by learning the weights of given features, such as nuclear size and shape and
chromatin texture. In nonparametric methods, such as deep learning, classification is performed by directly optimizing classification performance between normal and
abnormal training samples. An analysis of the learned weights can be used for inferring biomarkers that are relevant in disease diagnostics.

Concluding Remarks
Taken together, cells within the tissue microenvironment are subjected to a variety of
mechanical and chemical signals that regulate cellular homeostasis. The cell nucleus oper-
ates as an integrator of these signals via an elaborate meshwork of cytoskeletal to nuclear
links. In normal cells, these links maintain the nuclear mechanical homeostasis to regulate
genomic programs. Alterations in physical and chemical signals to the cell nucleus have been
shown to have a critical role in the onset of various diseases. Importantly, cancer cells exhibit
structural, mechanical, and conformational aberrations in their nuclear architecture, including
altered nuclear envelope organization, chromatin arrangements, and telomere stability.
Cancer tissues have been conjectured to arise from single cells within the tissue microenvi-
ronment, in which impaired nuclear mechanotransduction has resulted in altered genomic
programs. While large tissue-scale abnormalities are easier to detect, early diagnostics of
small groups of defective cells, for example in tissue biopsies, has been a major challenge.
While challenging, early disease diagnostics has been identified as a major goal since it carries
the promise of greatly increasing survival rates as well as reducing healthcare costs. In this
context, digital methods combined with genomic and proteomic methods to analyze nuclear
mechanopathological alterations may prove to be effective in early cancer diagnostics (see
Outstanding Questions).

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Acknowledgments Outstanding Questions

C.U. was partially supported by NSF (1651995), ONR (N00014-17-1-2147), and a Sloan Fellowship. G.V.S. was funded by
the Mechanobiology Institute, Singapore, MOE-Tier3 grant Singapore, and IFOM, Milan Italy. We thank members of the
Uhler and Shivashankar laboratories for useful discussions. We also thank Melanie Lee for the schematic drawings.
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