Abstract
Patient-derived tumor organoids (PDOs) are a highly promising
preclinical model that recapitulates the histology, gene expression,
and drug response of the donor patient tumor. Currently, PDO culture
relies on basement-membrane extract (BME), which suffers from
batch-to-batch variability, the presence of xenogeneic compounds and
residual growth factors, and poor control of mechanical properties.
Additionally, for the development of new organoid lines from
patient-derived xenografts, contamination of murine host cells poses a
problem. We propose a nanofibrillar hydrogel (EKGel) for the initiation
and growth of breast cancer PDOs. PDOs grown in EKGel have
histopathologic features, gene expression, and drug response that are
similar to those of their parental tumors and PDOs in BME. In addition,
EKGel offers reduced batch-to-batch variability, a range of mechanical
properties, and suppressed contamination from murine cells. These
results show that EKGel is an improved alternative to BME matrices for
the initiation, growth, and maintenance of breast cancer PDOs.
Subject terms: Breast cancer, Biomaterials - cells, Gels and hydrogels,
Breast cancer
__________________________________________________________________
Patient-derived tumour organoids are important preclinical models but
suffer from variability from the use of basement-membrane extract and
cell contamination. Here, the authors report on the development of
mimetic nanofibrilar hydrogel which supports tumour organoid growth
with reduced batch variability and cell contamination.
Introduction
Breast cancer is the most common cancer diagnosed in women worldwide
and is responsible for substantial morbidity and mortality^[52]1,[53]2.
Development of effective breast cancer treatments is hindered by the
lack of efficient preclinical models that recapitulate the complexity
and heterogeneity of breast tumors in vivo. Breast cancer has many
histologic and molecular subtypes, and individual cancers have distinct
genotypes, morphologies, and treatment sensitivities, which are shaped
by prior treatments^[54]3,[55]4. Furthermore, the breast tumor
extracellular matrix (ECM) is highly heterogeneous^[56]5 and its
dynamic structure and physical properties influence tumor progression
and treatment response^[57]6. In spite of this diversity, over the past
several decades, breast cancer research has relied primarily on
two-dimensional culture of only a few dozen clonal cell lines that fail
to fully capture breast cancer heterogeneity, limiting their use in
predicting clinical outcomes^[58]7.
Currently, patient-derived xenografts (PDX), in which human tumor
fragments are transplanted directly into immunocompromised mice, serve
as the gold-standard in fundamental and translational breast cancer
research, as they largely retain the morphology, genomic profile, and
intratumoral heterogeneity of the parental tumor^[59]8. Furthermore,
drug response in PDX models appears to correlate well with the clinical
response of donor patients^[60]9,[61]10. Yet, PDX models present
ethical challenges, and are cost, time, and labor-intensive.
Furthermore, new PDX models can take months to years to develop, and
for hormone-receptor positive breast cancers, tumor engraftment is
highly inefficient.
Patient-derived tumor organoids (PDOs), sub-millimeter
three-dimensional multicellular structures grown from cancer patient
tissue in a three-dimensional matrix, have emerged as a promising model
that bridges the gap between immortalized cell lines and PDX
models^[62]11. In contrast to cell lines, the PDO models capture intra-
and interpatient tumor heterogeneity and are significantly less
resource-intensive than PDX models^[63]12,[64]13. Furthermore, PDOs
have the capacity to maintain the histological features and gene
expression, and, most importantly, drug response of the donor patient
tumor^[65]14–[66]19, thus making them reliable models for preclinical
evaluation of anticancer agents and potentially, personalized cancer
therapies. Over the past 6 years, methods for PDO model generation have
been reported for diverse solid tumors, including colorectal^[67]19,
lung^[68]20, pancreatic^[69]14, ovarian^[70]18, prostate^[71]21,
breast^[72]16, stomach^[73]22, and other solid tumors^[74]11, thereby
rapidly making PDOs indispensable in vitro models.
Currently, pre-clinical PDO applications are hindered by their heavy
reliance on mouse tumor basement membrane extract (BME) (commercially
available as Matrigel, Cultrex BME or Geltrex), a “gold standard”
hydrogel for 3D cell culture^[75]14–[76]19. BME is a gelatinous mixture
of laminin, type IV collagen, entactin, proteoglycans, and growth
factors, which is secreted by Engelbreth-Holm-Swarm mouse sarcoma
cells^[77]23–[78]25. The presence of xenogeneic compounds and residual
growth factors, as well as batch-to-batch variability in BME
composition and properties leads to compromised reliability of BME as a
matrix for PDO growth^[79]16,[80]18,[81]21,[82]22. Furthermore, BME is
not conducive to modifications of its mechanical properties, which
are important for the development of an understanding of the role of
mechanical and structural cues provided by the tumor microenvironment
in cancer progression^[83]6,[84]26–[85]29 and tumor response to
drugs^[86]30–[87]32. Since BME is a physical hydrogel, it has poor
tolerance of flow-induced stresses, thus complicating BME’s use in
microfluidic organoid-on-a-chip platforms. Importantly, for the
development of new organoid lines from patient-derived xenografts
(PDXOs), contamination of murine host cells, which overtakes the human
organoids in culture, poses a problem. Thus, a strong need exists for
alternative biomimetic chemically crosslinked hydrogels for breast PDO
initiation and maintenance, in order to extend the potential
applications of this model system.
Several biologically-derived hydrogels formed by proteins (e.g.,
collagen^[88]33 or fibrin^[89]28,[90]29) and polysaccharides (e.g.,
hyaluronic acid^[91]31,[92]32 or alginate^[93]34), as well as synthetic
matrices^[94]35–[95]39 have been developed as matrices for spheroid
growth from cancer cell lines and organoid culture. These spheroids,
however, often do not reflect the biology and the clinical spectrum of
primary cancer cells and cannot be used for prediction of
patient-specific responses to therapy^[96]7,[97]40. Since these
hydrogels generally do not emulate the composition, structure, and
properties of the extracellular matrix (ECM) in vivo^[98]36,[99]37,
many patient-derived cancer cells that are aggressive in vivo, do not
grow in synthetic matrices in vitro as they lack the appropriate
microenvironment^[100]25,[101]36. In particular, the vast majority of
synthetic hydrogels fail to recapitulate the filamentous architecture
of the breast tumor ECM, which has a significant impact on cell
mechanotransduction, growth factor signaling, long-distance
cell-to-cell communication, and migration^[102]38,[103]41–[104]43. For
the small fraction of hydrogel matrices that have been successfully
used for PDO propagation from patient-derived breast and colorectal
tumor cells^[105]39,[106]44,[107]45, the ability to initiate new PDOs
lines and maintain them over multiple passages, while preserving their
phenotype remains largely unexplored. Recognizing that the ECM in the
breast tumor environment has a filamentous
structure^[108]6,[109]38,[110]46,[111]47 and a Young’s modulus in the
range from 1.2 to 3.7 kPa^[112]48, we aim to design a chemically
crosslinked biomimetic hydrogel recapitulating these properties.
Here we report a nanofibrillar hydrogel with controllable stiffness,
which was prepared by the reaction between chemically modified
cellulose nanocrystals and gelatin. This hydrogel (henceforth referred
to as EKGel) provides the ability to grow and passage organoids
initiated directly from patient tissue (PDOs) and PDX-derived tumor
organoids (PDXOs) for multiple breast cancer subtypes. Comprehensive
testing of PDOs grown in EKGel shows that they exhibit proliferation,
histopathologic features, gene expression, and drug responses that are
similar to those of the original tumors and to PDOs formed in standard
BME. In contrast with BME, the EKGel exhibits strongly reduced
batch-to-batch variability in mechanical properties and stability under
close-to-physiological flow conditions, making it amenable to
microfluidic “organoid-on-a-chip” platforms^[113]49,[114]50. For
generation of new PDO lines from primary patient material, EKGel
matches BME’s initiation rate. However, for development of new organoid
lines from PDXs (PDXOs), EKGel exhibits a distinct advantage. Whereas
organoid culture in BME has been limited by contamination of murine
host cells which can initiate and rapidly overtake the human organoids
in culture^[115]20, here we show that EKGel allows for the initiation
of PDXOs by suppressing contamination from murine cells. In summary,
our results show that EKGel can replace BME matrices in the culture of
breast cancer PDOs, enabling novel applications of organoid models and
unlocking large collections of existing and well-characterized PDX for
the development of breast PDXOs.
Results
The biomimetic EKGel was synthesized from gelatin and rod-like
aldehyde-modified cellulose nanocrystals (a-CNCs) with an average
length and diameter of 176 ± 50 nm and 20 ± 4 nm, respectively.
Figure [116]1a illustrates the hydrogel structure with Schiff base
crosslinks between aldehyde groups on the a-CNC surface and amine
groups of lysine residues in gelatin. Gelatin provided the
arginine-glycine-aspartate integrin receptor-binding motif present in
native ECM proteins, thus facilitating cell-matrix
interactions^[117]51, while assembly of rod-like a-CNCs resulted in a
nanofibrillar structure of the EKGel. EKGel is composed of a network of
fibers (Fig. [118]1b), similar to the architecture of collagen in the
in vivo tumor ECM^[119]38,[120]52–[121]54. The diameter of fibers in
EKGel was from 20 to 105 nm with average fiber diameter of EKGel of
43 ± 17 nm (Supplementary Fig. [122]1), which was comparable with the
dimensions of collagen fibrils in the breast tumor
microenvironments^[123]38,[124]52–[125]54. The significantly larger
pores in EKGel, in comparison with those in BME (Fig. [126]1c), enabled
enhanced convection-driven transport of nutrients, waste products, and
drugs through the EKGel matrix to tumor organoids^[127]38,[128]55. The
Darcy permeability of EKGel (a measure of convective transport) was
1.9 × 10^−11 cm^2, which was more than two orders of magnitude larger
than the reported values for BME, varying from 10^−13 to
10^−14 cm^2 ^[129]56–[130]58.
Fig. 1. Properties of EKGel and BME.
[131]Fig. 1
[132]Open in a new tab
a Schematic of EKGel. b, c Scanning electron microscopy images of EKGel
(b) and BME (c). Scale bars in (b, c) are 1 µm. In b C[a-CNC] = 1 wt%.
d Variation in the storage modulus, G’, of EKGel, plotted as a function
of C[a-CNC]. Data shown as mean ± st. dev for N = 3 samples (error bars
smaller than symbols). The shaded red area shows the standard deviation
(G’ = 43 ± 24 Pa) for three distinct batches of BME. e Relative
temporal volume loss of EKGel and BME under continuous perfusion of
cell culture medium at a flow rate of 96 μm/s. In e data shown as
mean ± st. dev of N = 50 microgels in a single experiment.
The variation in mechanical properties of EKGel was achieved by
changing a-CNC concentration, C[a-CNC], in the hydrogel, while
maintaining gelatin a concentration of 2 wt%. The storage modulus of
EKGel was measured at 37 °C within the range of linear viscoelastic
behavior (frequency of 1 Hz, 1 % strain, Supplementary Fig. [133]2).
The shear storage modulus, Gʹ, of EKGel at 37 °C changed from 8 to 1246
with C[a-CNC] increasing from 0.5 to 3.75 wt% (Fig. [134]1d), which
corresponded to a Young’s modulus from 24 to 3738 Pa, respectively.
This stiffness range covers the stiffness of ECM in breast tumor
biopsies, which have Young’s modulus from 1.2 to 3.7 kPa)^[135]59.
Importantly, for all compositions tested, the standard deviation for
three distinct separately synthesized EKGel batches did not exceed 11%
of the mean, while for BME (Gʹ = 43 Pa) for three separately purchased
batches with different lot numbers the percent standard deviation was
56% (the shaded region in Fig. [136]1d). Notably, EKGel was not
cytotoxic over the entire range of C[a-CNC] in Fig. 1D (Supplementary
Fig. [137]3). For the remainder of this work, including initiation and
culture of breast PDOs we used EKGel with Gʹ = 44 Pa to match the
storage modulus of BME.
Covalent crosslinking of EKGel resulted in enhanced hydrogel stability,
in contrast with BME. Figure [138]1e shows the decrease in EKGel and
BME volumes under the continuous flow of cell culture medium at
96 μm/s. The geometry of the microfluidic device used to measure
hydrogel stability is shown in Supplementary Fig. [139]4 and
experimental details are provided in the [140]Supplementary
Information. Over five days, the relative reduction in BME and EKGel
volume was 60 and 14%, respectively, which indicated higher EKGel
stability under shear-induced stress, thus providing more consistent
stiffness and porosity of the matrix over the relevant time period of
PDO growth. EKGel experiences slow degradation as the imine crosslinks
hydrolyze over time. In contrast, BME, which has no covalent
crosslinking, is rapidly washed away. In the absence of flow, the
reduction in volume for both matrices was minimal (Supplementary
Fig. [141]4). This result indicates that EKGel is amenable for use in
microfluidic organoid-on-a-chip platforms that incorporate
physiological flow^[142]50,[143]60,[144]61.
To explore the versatility of EKGel for initiation and expansion of
PDOs from primary breast cancer cells, we grew organoids from breast
cancer cells with different receptor statuses and tissue sources, which
were derived either from primary breast cancer tissue (PDOs) or from
PDXs (PDXOs). As shown in Table [145]1, Lines 1 and 3 were developed
from breast tumors obtained from patients that underwent surgical
resection under informed consent, whereas Line 2 was PDX-derived.
Breast cancer cells were isolated using a combination of mechanical
disruption and enzymatic digestion (described in “Methods”).
Table 1.
Characteristics of patient-derived breast cancer organoid lines.
Name Abbreviation Diagnosis Tissue Source Receptor Status Initiation
matrix
Line 1 PDO-1 Invasive ductal carcinoma Primary breast cancer tissue
ER+(5–10%)/PR−/HER2− BME
Line 2 PDXO-2 Metastatic breast cancer Patient-derived xenograft
ER−/PR−/HER2− BME
Line 3 PDO-3 Invasive ductal carcinoma Primary breast cancer tissue
ER+(21–30%)/
PR+(21–30%)
/HER2−
BME and EKGel
[146]Open in a new tab
To initiate growth of PDOs, the isolated breast cancer cells were
encapsulated in either EKGel, or BME by suspending cells in the
hydrogel precursor suspension, and subsequently, allowing for gelation
for 2 h. The cell-laden hydrogel was overlaid with breast cancer
organoid media (Supplementary Table [147]1), and the cells were
cultured for 2–3 weeks. To explore the ability of EKGel for PDO
maintenance, PDO-1 and PDXO-2, which had been initiated and passaged 4
times in BME, were transferred into EKGel for subsequent passaging.
PDO-3 was independently initiated in both BME and EKGel in parallel.
Figure [148]2a–c shows brightfield images of the PDOs grown in EKGel
and BME from the Lines listed in Table [149]1. The PDOs in EKGel and
BME grew into organoids consisting of spherical clusters of cells.
Qualitatively, PDOs grown in BME and EKGel from each of three Lines had
similar appearance. Figure [150]2d–f show the corresponding PDOs
stained with antibodies directed against Ki67 and human EpCAM. The PDOs
formed in BME and EKGel expressed human EpCAM on the cell surface,
confirming that the cells are indeed human epithelial cells^[151]62.
Similarly, the cell nuclei were positive for Ki67 staining, indicating
that actively dividing cells are present after >2 weeks of
culture^[152]63. Furthermore, no qualitative difference was observed in
the number of focal adhesions or cytoskeleton organization between BME
and EKGel (Supplementary Fig. [153]5).
Fig. 2. Growth of breast PDOs in EKGel and BME.
[154]Fig. 2
[155]Open in a new tab
a–c Brightfield images of PDOs grown in EKGel and BME from PDO-1
(ER+/PR−/HER2−) (a), PDXO-2 (ER−/PR−/HER2−) (b), and PDO-3
(ER+/PR+/HER2−) (c), as in Table [156]1. Scale bars are 100 µm. d–f
Organoids in (a–c) stained for Ki67 (green), human EpCAM (red), and
nuclei (blue). Scale bars are 50 µm. g–i Diameters of organoids formed
in EKGel (blue) and BME (red) after four passages from PDO-1
(ER+/PR−/HER2−) (g), PDXO-2 (ER−/PR−/HER2−) (h), and PDO-3
(ER+/PR+/HER2−) (i). In (g–i) data shown as mean ± st. dev, with
whiskers representing minimum and maximum values, of N = 100 spheroids
measured over four repeated experiments. No significant difference
between BME and EKGel observed (Student’s t-test, Bonferroni-Dunn
method, two tailed, p > 0.01).
To characterize PDO growth in EKGel and BME quantitatively, we
monitored temporal change in organoid diameter and cell proliferation.
Figure [157]2g–i show the diameters of the organoids formed from the
three lines in BME and EKGel after each of four passages. No
statistically significant difference was observed between the diameters
of PDOs grown in EKGel and BME (Student’s t-test, Bonferroni-Dunn
method, p > 0.01). Furthermore, the organoid diameters for each line
were consistent over four passages, indicating that EKGel is suitable
for long-term passage and maintenance of breast PDOs. Cell
proliferation in BME and EKGel was monitored by counting the number of
cells at each passage for four consecutive passages. The cell
population doubling time for each of the three PDO lines ranged from 77
to 160 h, with no statistically significant difference for PDOs formed
in BME and EKGel (Student’s t-test, Bonferroni-Dunn method, p > 0.01)
(Supplementary Fig. [158]6).
To verify that organoid growth in EKGel does not influence the tumor
initiating capability of the breast cancer cells, we initiated
xenografts in immunocompromised mice from PDOs maintained in each
matrix (Supplementary Fig. [159]7a). Tumors derived from organoids
grown in EKGel and BME grew at similar rates, and both grew to a final
volume of 1.3 cm^3 by 144 days post-injection (Supplementary
Figure [160]7b, N = 1).
Under optimal conditions, PDOs should recapitulate histologic features
of their parental tumors and maintain protein expression of clinically
relevant biomarkers. To further explore the suitability of EKGel for
PDO formation, histology and biomarker immunostaining of organoids,
grown in EKGel and BME, were analyzed and compared to their parental
tumors by an experienced clinical breast pathologist (Analysis of PDO-3
in Fig. [161]3 and PDO-1 and PDXO-2 in Supplementary Fig. [162]8). In
histologic sections for all samples studied, PDOs grown in EKGel and
BME appeared equally well-formed and showed highly similar architecture
and cytomorphology. Characteristic histologic features of the parental
tumor were observed in both types of PDOs, including relative abundance
of eosinophilic to amphophilic cytoplasm, varying degree of cytoplasmic
vacuolization with focal formation of “clear-cells”, round to oval
nuclei with moderate to high nuclear pleomorphism, dispersed chromatin
pattern with focal vacuolation and variable formation of one to
multiple prominent nucleoli (Fig. [163]3). As has been previously
reported in organoid systems, for PDO-3 both EKGel and BME cultures
exhibited reduced ER expression compared to the parental tumor, and was
present in only a minority of organoids^[164]16,[165]64,[166]65.
Similarly, PR expression (which was weak in the clinical specimen) was
reduced in the organoids (Fig. [167]3). We did, however, observe
heterogeneous ER expression in many of the PDO-1 organoids as seen in
the patient tissue (Supplementary Fig. [168]8a).
Fig. 3. Tumor and organoid histology.
Fig. 3
[169]Open in a new tab
Hematoxylin and eosin staining, and immunohistochemistry were performed
on PDO-3 that were independently initiated and then passaged 4 times in
BME and EKGel. Staining was performed in parallel with the primary
tumor from which the organoids were derived. Scale bar is 100 µm.
To evaluate the similarity in gene expression between PDOs grown in
different matrices, we performed RNA sequencing of cells from each
condition (see “Methods”). RNA was extracted from cells isolated from
organoid cultures or from frozen tumor tissue, libraries prepared after
ribosomal RNA depletion and subjected to paired end sequencing to
obtain approximately 80 million reads per sample on an Illumina Novaseq
6000. We then performed systematic analyses to identify any differences
imposed by the growth matrix and contrast these to the originating
tumors. PDOs originating from the same tumor grown in different
matrices clustered together with high similarity (average Spearman
Correlation = 0.89) showing minimal differences in expression patterns
between the growth matrices (Fig. [170]4a). In addition, we observed
high similarities between PDOs and their originating tumors (average
Spearman Correlation = 0.83) supporting the concept that PDOs are good
models to recapitulate patients’ tumors (Fig. [171]4a). Furthermore, we
performed a differential expression analysis to evaluate the
differences between PDOs grown in EKGel and BME matrices at the gene
level. We found only six genes to be significantly different between
the two growth matrices (Fig. [172]4b). Further inspection of these
genes did not reveal any connections to important breast cancer
biological processes or therapeutics. A query of a large and
comprehensive pan-cancer pharmacogenomic database (PharmacoDB)
identified no significant correlations between the expression of any of
these six genes and drug response. Given the low number of
differentially expressed genes between BME and EKGel and even lower
number after multiple hypothesis correction, we applied pathway
enrichment analysis based on a Hypergeometric test in order identify
any biological processes that differ between BME and EKGel and we found
no pathway exhibiting a significant difference (FDR < 0.05) out of 1604
biological pathways curated by REACTOME database^[173]66,[174]67.
Altogether results demonstrated consistency in gene expression between
PDOs grown in EKGel and BME and identified no major alterations in the
expression of relevant genes or pathways that would be expected to
impact the use of models grown in EKGel for basic research or
pharmacologic testing.
Fig. 4. Gene expression of breast cancer organoids in EKGel and BME.
[175]Fig. 4
[176]Open in a new tab
a Pairwise correlation of global gene expression in PDOs grown in EKGel
and BME, as well as associated in vivo xenograft or tumor tissues. For
each Line, gene expression in BME and EKGel organoids grown in vitro or
in vivo are highly correlated (average Spearman Correlation = 0.89). b
Differential gene expression analysis to compare global gene expression
for PDOs grown in vitro in BME vs. EKGel. Only six genes, as shown with
red color, showed differential expression at false discovery rate (FDR)
threshold of 0.05, none of which are recognized as important
determinants of drug response or breast cancer biology.
Next, we performed in vitro drug assays to explore the chemosensitivity
of the organoids Lines listed in Table [177]1 grown in EKGel and BME to
commonly used breast cancer drugs. Figure [178]5a–c show drug
sensitivity measured as area above the curve (AAC) - a metric preferred
for its reproducibility across pharmacogenomic
studies^[179]68,[180]69—for paclitaxel, eribulin, carboplatin, and
doxorubicin in PDO-1, PDXO-2 and PDO-3, respectively. The dose–response
curves used to determine AAC are included in Supplementary Fig. [181]9.
No significant difference in the AAC values was observed between
matrices (Student’s t-test, Bonferroni-Dunn method, p > 0.1).
Fig. 5. Response of breast tumor organoids to drugs.
[182]Fig. 5
[183]Open in a new tab
a–c Area above the curve (AAC) of three different drugs in EKGel and
BME, measured by the cell titer-Glo assay. a AAC of eribulin,
paclitaxel, and doxorubicin for PDO-1. b AAC of eribulin, paclitaxel,
and carboplatin for PDXO-2. c AAC of eribulin, paclitaxel, and
doxorubicin for PDO-3. In (a–c) data shown as mean ± st. dev of N = 3
biological replicates and ns indicates no significant difference
(Student’s t-test, Bonferroni-Dunn method, two tailed, p > 0.01).
To evaluate whether PDO growth in EKGel impacts their chemosensitivity
in vivo, the organoids grown in EKGel and BME from PDXO-2
(ER-/PR-/HER2-) cells were enzymatically digested by TrypLE Express
(Gibco) and the cells were implanted into the mammary fat pad of
non-obese diabetic (NOD)/SCID mice to generate xenografts, which were
subsequently treated with paclitaxel. Supplementary Fig. [184]10A shows
a schematic illustrating this experiment. Once the tumors were
established and reached a volume of ~150 mm^3, paclitaxel treatment was
initiated and delivered intravenously at an established and
clinically-relevant dose (20 mg/kg), on a weekly schedule. The animals
were sacrificed when the tumors reached the humane endpoint
(1500 mm^3). Supplementary Fig. [185]10B, C show the growth of tumors
with and without drug administration in EKGel (EKGel-tumors) and BME
(BME-tumors), respectively. The untreated controls grew at similar
rates in both EKGel and BME. Both the EKGel-tumors and BME-tumors
showed response to paclitaxel, with substantial tumor growth
inhibition, but not regression observed in the drug-treated tumors.
Furthermore, the growth rate of the paclitaxel-treated EKGel-tumors and
BME-tumors were similar, indicating that the in vivo drug response
appears unimpacted by organoid growth in EKGel vs. BME.
After verifying that organoids can be successfully initiated and grown
in the EKGel, while maintaining the phenotype of their tumor of origin,
we explored the potential of EKGel for initiation of new breast cancer
PDOs and PDXOs. We processed multiple tumors from PDXs (N = 17) or
directly from patients (N = 5), plated dissociated single tumor cells
in parallel in EKGel and BME, and monitored organoid initiation. As
shown in Fig. [186]6a, the overall initiation rate for organoids in
both matrices was similar, that is, 88% for PDXO and 80% for PDO.
Notably, one PDXO line (BPDXO.107) was successfully initiated in EKGel,
but not in BME and another line, BPDXO.113, was initiated in BME, but
not in EKGel, indicating that the initiation properties are not
identical.
Fig. 6. Organoid initiation in EKGel and BME.
[187]Fig. 6
[188]Open in a new tab
a Initiation rate of PDXO (N = 17) and PDO (N = 5) in EKGel and BME. b,
c Brightfield microscopy images of PDXOs grown in EKGel (b) and BME (c)
for 14 days. Scale bars are 100 μm. Red arrows indicate contaminating
mouse cell clusters. d Flow cytometry characterization of the content
of human (EpCAM+) and mouse cells (H2K+) of the PDXO shown in (b, c)
initiated in EKGel (right) and BME (left). FITC-H2K was used to stain
mouse cells and APC-EpCAM was used to stain human cells. e Comparison
of the fraction of cells that are H2K+ or EpCAM+ for PDXO initiated in
BME and EKGel in (b, c).
Although not extensively described in the literature, the establishment
of PDXO models is confounded by the presence of murine host cells,
which have the potential to grow and rapidly overtake the human
organoids in culture. We and others have found such contaminations to
be a major hurdle in the ability to establish long-term organoid
cultures from PDX tumors (from breast and other cancers) and have
therefore incorporated a mouse cell depletion step in an attempt to
alleviate this problem^[189]20,[190]70,[191]71. Even with this
purification step, we found a striking difference between organoid
initiation from PDX tumors in BME and EKGel. Out of 17 of the PDX tumor
organoids initiated in BME, 8 (or 47%) had significant mouse cell
contamination, as determined by organoid imaging and/or by flow
cytometry (Fig. [192]6). Mouse cell contamination appears as larger
dark clusters that are indicated by red arrows in Fig. [193]6c.
Supplementary Fig. [194]11 shows immunofluorescence staining for Human
EpCAM and mouse H2K-d that confirms that these clusters are indeed
murine cells. In contrast, all of the same 17 PDX tumor organoids grown
in EKGel were free of such mouse cell contamination. A representative
example is shown in Fig. [195]6b–e. Dissociated, mouse cell depleted
PDX tumor cells were plated in parallel in EKGel (Fig. [196]6b) and BME
(Fig. [197]6c) and grown for 14 days. Flow cytometry was then performed
on the cells to determine the mouse and human cell content.
Figure [198]6d and e shows the fraction of EpCAM+ human cells and H2K+
mouse cells in from the same sample initiated in either EKGel and BME.
The sample initiated in BME was 51.4% H2K+ mouse cells, while the
sample plated in EKGel only contained 0.08% H2K+ mouse cells.
Immunofluorescence staining for human EpCAM and mouse H2K-d confirmed
that when PDX samples are plated in BME, both human organoids and large
clusters of mouse cells are observed (Supplementary Fig. [199]11). In
contrast, when the same PDX sample was plated in EKGel, only human
EpCAM positive organoids, and no clusters of mouse H2K-d positive mouse
cells were observed. Notably, single mouse cells were present when the
PDX sample was plated in EKGel, but they did not proliferate into large
mouse cell cultures that overtook the human organoids, as was observed
in BME. We speculate that the increased proliferation of normal mouse
cells in BME is related to the presence of residual murine growth
factors, including TGF-β, IGF, PGFD, EGF, NGF, and VEGF in BME
matrices^[200]23,[201]72,[202]73.
To further investigate the origins of EKGel’s ability to exclude
contamination of mouse cells, we cultured different types of mouse
cells in EKGel and BME. The PDXs used here are initially grafted into
the mammary fat pad and then grafted subcutaneously for subsequent
passages. To assess whether EKGel inhibits proliferation of normal
(non-cancerous) mouse cells from skin or mammary tissue, we isolated
cells from both a mammary gland and skin samples from mice and cultured
them in both EKGel and BME (Supplementary Figs. [203]12 and [204]13).
Proliferation of both mouse mammary gland and skin cells were
significantly higher in BME than in EKGel. We speculate that the
increased proliferation of normal mouse cells in BME is related to the
presence of residual murine growth factors, including TGF-β, IGF, PGFD,
EGF, NGF, and VEGF in BME matrices^[205]23,[206]72,[207]73. We propose
that the difference in only the proliferation of the normal mouse
cells, and not the human breast cancer cells, is that cancer cells have
mechanisms to escape dependence on growth factors like IGF^[208]72, and
thus do not require their presence for proliferation. To support this
conclusion, we showed that there was no difference in the proliferation
of mouse mammary tumor cells in BME and EKGel (Supplementary
Fig. [209]14).
We next investigated if the mouse cell depletion step could be
eliminated altogether when plating cells directly in EKGel. Five
independent PDX tumors were processed for organoids and depleted and
undepleted cells were plated in parallel in both BME and EKGel. Of the
5 PDX tumors, none of those that were undepleted of mouse cells had
mouse cell contamination EKGel, while 4 out of 5 had contaminating
murine cells when plated in BME (Supplementary Fig. [210]15). This
observation reveals a significant advantage of EKGel over BME, as there
are hundreds of breast cancer PDXs lines that have been established,
characterized and made available through various consortia
worldwide^[211]74. This important property of EKGel creates the
opportunity to leverage the major investments made in these PDX
resources to generate well-annotated organoid models.
Discussion
We have developed a new biomimetic hydrogel for effective initiation
and expansion of breast cancer PDOs and benchmarked its properties
against BME, a “gold standard” hydrogel for 3D cell culture. EKGel had
highly reproducible mechanical properties, with storage modulus
fine-tuned over three orders of magnitude, thus overcoming the
batch-to-batch variability and narrow range of stiffnesses of BME.
While in most of our work we matched EKGel stiffness to that of BME,
the mechanical properties of EKGel can be varied to replicate the
properties of normal breast tissue and breast tumors^[212]48,[213]59.
Furthermore, EKGel has a fibrous structure that mimics the architecture
of the tumor extracellular matrix^[214]47. This is a distinct advantage
of EKGel, as fibrillar architecture of microenvironments have a
significant impact on cell phenotype. Such architecture impacts
mechanotransduction, growth factor signaling, long-distance
cell-to-cell communication, and cancer cell invasion^[215]41–[216]43.
While several filamentous hydrogels have been developed, none have been
validated for culture of breast PDOs to the extent we have shown
here^[217]38. While the simplicity of EKGel preparation is its great
advantage, further chemical functionalization of CNCs with e.g.,
proteoglycans can be readily achieved. Importantly, in comparison with
BME (and other physically crosslinked gels), the EKGel was mechanically
stable under close-to-physiological flow conditions, which enables its
utilization in microfluidic organoid-on-a-chip models that are rapidly
finding a broad range of applications in fundamental cancer research
and drug screening^[218]49,[219]61,[220]75–[221]77. This trend is
driven by a recognition of the influential role that interstitial fluid
flow plays in determining cell phenotype, tumor progression, and drug
response^[222]78–[223]81. The compatibility of EKGel with these
platforms is a significant benefit, relative to BME.
We showed that EKGel supports both initiation and passage of breast
cancer PDOs with different histologic subtypes and from different
source materials (that is, both patient samples and PDXs), thereby
demonstrating the versatility of EKGel as a matrix. Importantly, the
growth, tumorigenicity, and drug response of the breast PDOs were
consistent between EKGel and BME. Furthermore, no major alterations of
the histopathological properties or gene expression patterns were
identified between the source tissues and the PDOs grown in EKGel and
BME. We did, however, observe a loss of ER and PR expression in the
PDO-3 organoids grown in both matrices as compared to the tumor tissue
from which they were derived. Loss or reduction in hormone receptor
expression in breast cancer organoids relative to parental tissue has
been described by others^[224]16,[225]64,[226]65. This phenomenon may
be due to a combination of factors, including but not limited to clonal
selection and media composition. The commonly used media formulation is
serum-free and therefore, lacks estrogen, potentially explaining the
loss/reduction in ER and PR, whose expression are hormonally regulated.
Together, these results support the use of breast cancer organoids
cultured in EKGel as an in vitro model for translational research, with
potential for applications in personalized cancer therapy. In the
future, we hope to validate the relevance of the in vitro drug response
of PDOs to the in vivo and clinical settings. Importantly, such
verification would be possible owing to EKGel’s ability to initiate
PDXOs, as drug response can be compared to data from the source PDX.
While the growth, tumorigenicity, and drug response of the breast PDOs
were consistent between EKGel and BME, these results are limited by
their n-of-1 design, and larger-scale studies will be required to fully
characterize the relationship between in vitro and in vivo behaviors.
Here we demonstrated that PDO properties are maintained in both EKGel
and BME for up to four passages. In future work, it will be valuable to
investigate the stability of PDO properties over long-term passage. We
note that two models including PDO-3 have been initiated and passaged
independently in BME and EKGel for over 10 passages.
Low initiation rates remain a problem with cancer PDOs and developing
new matrices that can increase initiation rates would be a substantial
advance^[227]13. Here, we show that initiation rate of new breast tumor
organoid lines is the same for both EKGel and BME. The combined
initiation rate for both PDOs and PDXOs in this work is 86% (N = 22),
which is slightly higher than the previously reported initiation rate
for breast cancer PDOs (exclusively from clinical samples) of
~80%^[228]16. Notably, the ability to tune the physical properties of
EKGel could provide a route to improve breast cancer organoid
initiation rates in the future. Future work should explore initiation
rates in hydrogels with different stiffnesses. Furthermore, the ability
to vary and control the stiffness of EKGel over a broad range opens the
door for studies on the effect of the mechanical properties of the
matrix on PDO properties.
Importantly, the EKGel used here exhibited a distinct advantage for the
initiation of PDXOs by suppressing the contamination and overgrowth of
murine cells that is commonly observed in BME. This advantage unlocks
the hundreds of well-characterized PDXs around the world as tissue
sources for organoid development, and in vitro applications that are
not feasible to carry out in vivo. In addition, the absence of
xenogenic components in the culture system may facilitate the
application of PDO to study interactions between cancer and immune
cells, a critical need for the ongoing development of immuno-oncology
therapies.
The ability of EKGel to suppress the overgrowth of normal mouse cells
deserves special attention, as it suggests that EKGel may also suppress
overgrowth and contamination by normal human cells. This effect may
offer a crucial advance: when initiating PDOs, normal human cells
present in the source tumor tissue source often overtake cancer cells
in PDO cultures, thus posing a challenge in the application of PDOs in
personalized medicine^[229]13,[230]82. If EKGel can suppress the
overgrowth by normal human cells, it would provide a route to
overcoming this limitation of PDOs. This interesting and potentially,
very useful application of EKGel—under appropriate conditions—will form
the basis of a separate study.
Organoids have been established from a broad range of tissue types,
including both healthy tissue, primary tumors, and metastatic lesions.
Based on our results, EKGel has the potential to serve as a scaffold
for these organoid types, enabling in vitro models that capture intra-
and interpatient heterogeneity that could be used to develop
personalized cancer therapies. Future large-scale evaluation of this
novel biomaterial will permit more detailed characterization of the
degree to which EKGel permits faithful organoid modeling across diverse
breast and other cancer types and permits accurate prediction of
clinical response to diverse drug classes.
Methods
Materials
Type A gelatin (300 g bloom), ethylene glycol (≥99% purity), 25%
glutaraldehyde aqueous solution (≥98% purity), and sodium periodate
(≥99 % purity) were purchased from Sigma-Aldrich, Canada. An aqueous
12.2 wt % CNC suspension was purchased from the University of Maine
Process Development. BME was purchased from Trevigen, Inc. All
chemicals were used as received without further purification unless
otherwise specified. All other reagents (drugs, assay reagents, etc.)
are listed in Supplementary Table [231]3, and antibodies are listed in
Supplementary Table [232]4. Breast tumor material was obtained
following informed consent and used under Research Ethics
Board-approved protocols at the Princess Margaret Cancer Centre (UHN
#15-9481).
Preparation of breast organoid medium
A detailed list of the components is provided in Supplementary
Table [233]1. Organoid media was stored at 4 °C. Breast organoid medium
composition was prepared as described elsewhere^[234]16.
Surface modification of CNC with aldehyde groups
Aldehyde-functionalized CNCs (a-CNCs) were prepared by surface
oxidation of CNCs with sodium periodate, as reported elsewhere (54,
68). Sodium periodate (NaIO[4], 1.2 g) was added to 200 mL of a 1 wt %
CNC suspension. The flask was covered with aluminum foil, and the
mixture was stirred for 2 h at room temperature. The oxidation reaction
was quenched by adding 600 µL of ethylene glycol. The suspension of
a-CNCs was dialyzed for 2 weeks against Milli-Q grade deionized water
(DI, 18.2 MΩ cm resistivity) with the water being changed twice a day
(cellulose membrane, 12 kDa cutoff). The a-CNC suspension was then
concentrated to >3 wt% using rotary evaporation. To adjust the ionic
strength of a-CNC solution, 10× HBSS buffer was added to the a-CNC
suspension in a 1:10 vol. ratio to reach a final concentration of 1×
HBSS.
Preparation of EKGel
EKGel was prepared by thorough vortex mixing of a stock a-CNC
suspension (3 wt% in HBSS) and gelatin solution (10 wt% gelatin in
Advanced DMEM/F12 cell culture medium) with organoid medium to reach a
final composition of 1 wt% a-CNC and 2 wt% gelatin. The stock
suspensions were sterilized under ultraviolet light for at least 10 min
and kept in a 37 °C water bath for at least 20 min prior to gelation,
to ensure that gelatin did not undergo physical gelation at low
temperatures.
Scanning electron microscopy
The structures of EKGel and BME were studied using scanning electron
microscopy (SEM). Supercritical point drying was utilized to prepare
hydrogel samples. EKGel samples were allowed to gel overnight at 37 °C,
fixed by submerging them in 2 wt % glutaraldehyde in HBSS for 24 h and
washed with deionized water three times. Subsequently, the water was
exchanged with ethanol by consecutively submerging the EKGel for 30 min
in 30, 40, 50, 60, 70, 80, and 90 % (v/v) ethanol/water mixtures and
then finally, in pure ethanol. Afterward, the hydrogels were placed in
an Autosamdri-810 Tousimis critical point dryer. The ethanol in the
sample was exchanged with liquid CO[2], which was subsequently brought
to a supercritical state and removed by slow venting. The dried EKGel
was fractured and gold-coated using an SC7640 High Resolution Sputter
Coater (Quorum Technologies). The samples were imaged on a Quanta FEI
scanning electron microscope (10 kV).
Rheology
The rheological properties of the EKGel and BME were characterized
using a rheometer (AR-1000 TA Instruments) with a cone and plate
geometry, with a cone angle and diameter of 0.9675° and 40 mm,
respectively. An integrated Peltier plate was used to control the
temperature, and a solvent trap was utilized to minimize solvent
evaporation. The hydrogels were allowed to equilibrate at 37 °C for 3 h
before experiments. Unless specified, a strain was 1% and frequency of
1Hz were used (within the linear viscoelastic regime) and the
temperature during the measurements was 37 °C.
Determining EKGel permeability
The Darcy permeability coefficient, K[s], of EKGel was determined using
a previously reported method^[235]57,[236]58. Briefly, an EKGel sample
with dimensions 3 mm × 3mm × 13.7 mm (width × height × length) was
prepared in a chamber fabricated in poly(dimethylsiloxane) (PDMS). The
chamber was connected to inlet and outlet reservoirs by
perfluoroalkoxyalkane tubing (IDEX Health & Science). The inlet
reservoir was placed above the outlet reservoir to apply a difference
in pressure, ΔP, to the hydrogel. The HBSS solution was perfused
through the hydrogel under ΔP. The volumetric flow rate, Q[p], of this
solution was determined by measuring the change in the mass of the
outlet reservoir over time. The Darcy permeability coefficient, K[s],
was determined by Eq. [237]1, where L is the hydrogel length (13.7 mm);
ΔP is the pressure difference across the hydrogel, calculated from the
difference in heights of the inlet and outlet reservoirs; η is the
viscosity of HBSS solution (taken as 1.002 cP), and A is the
cross-sectional area of the hydrogel (9 mm^2).
[MATH: KS=ηLQpAΔP :MATH]
1
Patient tumor dissociation
Patient tumor tissue was collected with informed patient consent and
used according to Research Ethics Board at the Princess Margaret Cancer
Centre, University Health Network approved protocols (06-196 and
15-9481). Upon receipt, a portion of tumor was fixed in 10% buffer
formalin for downstream histology, fragments were snap frozen and
stored at −80 °C for genomic analyzes and the remaining tumor was
minced and digested in 5–10 mL of Advanced DMEM/F12 containing 1X
GlutaMAX, 10 mm HEPES and 1× antibiotic-antimycotic (ADF+++) with
500 µg/mL Liberase TH. Samples were incubated at 37 °C with gentle
rocking on a nutator for 1 h. Samples were resuspended with a 5mL
pipette to further dissociate undigested tissue, volume was brought up
to 13 mL with ADF+++ and centrifugation was performed at 400 × g for
15 min, 4 °C. The cell pellet was resuspended in 2–5 mL TrypLE Express,
triturated with a P1000 pipette and incubated at 37 °C for 15 min.
Sample volume was then brought up to 13 mL with ADF+++ and passed over
a 100 µm cell strainer. Centrifugation was performed at 300xg for
5 min, 4 °C. The cell pellet was treated with 1–2 mL Red Cell Lysing
Solution for 5 min on ice; ADF+++ was added to bring volume to 10 mL
and cells were pelleted at 300 × g for 5 min, 4 °C. Cells were counted
and cell viability was determined by Trypan Blue staining. Next, 80,000
viable cells/well were plated in 50 µL/BME per well of a 24-well plate
and were overlaid with 500 µL breast organoid media/well after allowing
BME to gel for 10 min at 37 °C. 480,000 viable cells/well were plated
in 300 µL 1 wt% EKGel/well of a 24-well plate and were overlaid with
750 µL breast organoid media/well after allowing EKGel with cells to
solidify at 37 °C for at least 2 h.
PDX tumor dissociation
PDX tumors were dissociated using the same as for patient tissue,
except that 250 µg/mL Liberase TH was used and tissue was only digested
for 45 min. An additional pellet washing step was included after the
Liberase TH digestion and centrifugation and mouse cells were depleted
from samples prior to plating in BME or EKGel, except where indicated
in text. The gating strategy for flow cytometry is provided in
Supplementary Fig. [238]15. For PDX growth, NSG mice (NOD.Cg NSG),
Females at 4–6 wks of age, were housed in cages containing up to 5
animals on vented racks with 12/12 h light/dark cycle at 21–22 °C and
35–40% relative humidity. Ethics oversight was provided by the Research
Ethics Board at the Princess Margaret Cancer Centre, University Health
Network (UHN, #15-9481 and 06-196).
Mouse cell depletion from PDX tumors
Digested PDX tumor cells were counted and resuspended in Depletion
Buffer (PBS containing 0.5% BSA and 0.1× antibiotic-antimycotic). A
mouse cell depletion was performed using Mouse Cell Depletion Cocktail,
MACS LS columns and magnet as described by the manufacturer (Miltenyi).
Cells were counted prior to and after depletion to determine depletion
efficiency; this was also routinely monitored by flow cytometry. A
fraction of cells were stained with 1:100 dilution
FITC-anti-mouse-H-2K/H-2D (Clone 34-1-2S) and 1:100 dilution
APC-anti-human-CD326 (EpCAM) prior to and after depletion to visualize
mouse and human cell content. Flow cytometry was performed on a Canto
II HTS instrument (BD Biosciences) and analysis was done using FlowJo
software. The gating strategy for flow cytometry is shown in
Supplementary Fig. [239]10.
Organoid culture and passaging
Medium changes were performed every 3–4 days. Organoids were passaged
every 2–3 weeks. For passage, medium was replaced with 1 mL TrypLE
Express (Gibco)/well and the gels (BME or EKGel) were broken apart by
manual shearing with a P1000. Organoids were incubated in TrypLE at
37 °C and triturated every 10 min for no more than an hour until
dissociated to single cells. Cells were then centrifuged at 300 × g for
5 min, and the supernatant was removed. Cells were then suspended in
either BME (50,000 viable cells/well were plated in 50 µL/BME per well)
or in EKGel (300,000 viable cells/well were plated in 300 µL/EKGel per
well) and plated in a 24-well-plate. In both matrices, the cell density
was 1000 cells/µL. Once the matrix had solidified (10–15 min for BME
and min 2 h for EKGel), the encapsulated cells were overlaid with
media. Organoids were passaged every 2–3 weeks.
Organoid and tissue histology
Organoids were removed from 24-well plates using a spatula and embedded
in cryomolds using HistoGel (ThermoScientific). Once the
organoid/HistoGel mix hardened, it was removed from the cryomold and
placed in a histology cassette in 10% buffered formalin. Tissue pieces
were also fixed in 10% formalin. Paraffin embedding and
immunohistochemistry was performed by DDP-AMPL at the University Health
Network. Antibody details and dilutions are included in Supplementary
Table [240]4.
Measurement of organoid diameter
Organoid diameter was measured from brightfield images in the ImageJ
software (NIH). For each organoid, the diameter was determined as the
geometric mean of two orthogonal diameters.
Immunofluorescence staining
Eight-well chamber slides were coated with either BME or EKGel and left
to solidify for at least 10 min or 2 h, respectively. Organoids were
recovered from 24-well plates using Corning Cell Recovery Solution and
plated in coated chamber slides in breast cancer organoid media (with
2% BME for those plated on wells coated in BME). Organoids were washed
with PBS twice and then fixed in 5 wt% formalin for 30 min. Next
organoids were washed three times with 400 µL 0.1 M glycine in PBS
(10 min each wash). 400 µL of 0.5% Triton-X-100 in PBS was then added
to permeabilize the cells for 5 min. The organoids were then washed
three times (10 min each wash) with 400 µL immunofluorescence (IF)
washing solution (IF wash) consisting of 0.05 wt. % NaN[3], 0.1 wt. %
Bovine Serum Albumin, 0.2 vol. % Triton-X-100 and 0.05 vol. % Tween-20
in PBS. Next, organoids were incubated with 400 mL of block solution
(10 wt % goat serum in IF wash) for 90 min at room temperature. The
block solution was replaced with 400 µL of the primary antibodies
[1:800 anti-EpCAM(VU1D9); (Cell Signaling #2929) and 1:1000 anti-Ki67
(Abcam; ab15580)] in the block solution, and incubated overnight at
room temperature. Organoids were then washed three times with IF wash
(20 min each wash) and incubated with the secondary antibody solution
consisted of 1:500 goat anti-rabbit Alexa Fluor 488 (Invitrogen^TM) and
1:500 goat anti-mouse Alexa Fluor 568 (Invitrogen^TM) in blocking
solution for 60 min. The organoids were washed three times with 400 µL
IF wash (20 min each wash). To stain for nuclei, 0.5 ng/mL of DAPI in
PBS was added for 10 min at room temperature. The organoids were
visualized using confocal microscopy (Zeiss LSM700 Confocal Microscope,
Zeiss Zen software version 3.2).
Drug assays
384-well clear-bottomed plates (Greiner) were pre-coated with 8 µL
BME/well or 10 µL EKGel/well and left to solidify for at least 10 min
and 2 h, respectively. Organoids were dissociated to single cells and
3000 cells were plated/well and left to grow for 4 days prior to the
addition of equal volume of 2× drug. Cells were incubated with drugs
for 5 days prior assay development using Cell Titer Glo 3D cell
(Promega). AAC values were determined using PharmacoGx R
package^[241]83.
Establishing PDX from organoids
Organoids were dissociated to single cells using TrypLE Express and 1
million cells were injected into the mammary fat pad of NSG mice
(NOD.Cg NSG), mice in a 100 µL volume (1:1 ratio of cells in organoid
media: BME). Once tumors reached 150 mm^3, they were treated with
either vehicle control or taxol (20 mg/kg weekly). Tumor growth was
monitored and recorded over time; once vehicle controls reached
1000–1500 mm^3, all tumors were harvested and preserved for downstream
characterization. NSG mice (NOD.Cg NSG), Females at 4–6wks of age, were
used for PDX experiments. Mice were housed in cages containing up to 5
animals on vented racks with 12/12 hour light/dark cycle at 21–22 °C
and 35–40% relative humidity. Ethics oversight was provided by the
Research Ethics Board at the Princess Margaret Cancer Centre,
University Health Network (UHN, #15-9481 and 06-196).
RNA-Seq
Organoids were recovered from BME using 1mL Corning Cell Recovery
media/well followed by a 1 h incubation on ice and centrifugation.
Organoids were recovered from EKGel by resuspending in 1mL TrypLE
Express/well, immediately adding ADF+++ and pelleted by centrifugation.
RNA was isolated from recovered organoids and from snap frozen tissue
fragments using NucleoSpin TriPrep. Library preparation was done using
Illumina TruSeq Stranded Total RNA Sample Preparation kit with RiboZero
Gold and samples were subjected to paired end sequencing (~80 million
reads/sample) using the Illumina Novaseq 6000 at the Princess Margaret
Genomics Centre. Gene expression profiles were generated using the
Kallisto pipeline with GRCh38 as human reference^[242]84. Spearman
correlation was used to measure the similarities between the different
samples and DESeq2 R package was used to perform the gene expression
differential analysis^[243]85. Pathway enrichment analysis was
performed using Piano R package utilizing hypergeometric test^[244]86.
Data and code to reproduce these analyses is available at
[245]https://github.com/bhklab/PDO_BME_EKGel.
Statistical analysis
All data in the Results section are presented as mean ± st. dev.,
unless otherwise specified. Student’s t-test (Bonferroni-Dunn method,
two-tailed) was used to determine statistical significance when
comparing PDO diameters, doubling times, and drug AAC values for PDOs
grown in EKGel and BME. Student’s t-test was performed in GraphPad
Prism. The condition for statistical significance was p < 0.01. All
micrographs are representative, and all microscopy experiments were
repeated at least twice. For measurement of PDO diameter, N = 100 PDOs
were measured per passage. For doubling time measurements, N = 4
biological replicates. For AAC measurements, N = 3 biological
replicates.
Reporting summary
Further information on research design is available in the [246]Nature
Research Reporting Summary linked to this article.
Supplementary information
[247]Supporting Information^ (6.5MB, docx)
[248]Reporting Summary^ (258.6KB, pdf)
Acknowledgements