Abstract
Ultraviolet radiation (UVR) damages the dermis and fibroblasts; and
increases melanoma incidence. Fibroblasts and their matrix contribute
to cancer, so we studied how UVR modifies dermal fibroblast function,
the extracellular matrix (ECM) and melanoma invasion. We confirmed
UVR-damaged fibroblasts persistently upregulate collagen-cleaving
matrix metalloprotein-1 (MMP1) expression, reducing local collagen
(COL1A1), and COL1A1 degradation by MMP1 decreased melanoma invasion.
Conversely, inhibiting ECM degradation and MMP1 expression restored
melanoma invasion. Primary cutaneous melanomas of aged humans show more
cancer cells invade as single cells at the invasive front of melanomas
expressing and depositing more collagen, and collagen and single
melanoma cell invasion are robust predictors of poor melanoma-specific
survival. Thus, primary melanomas arising over collagen-degraded skin
are less invasive, and reduced invasion improves survival. However,
melanoma-associated fibroblasts can restore invasion by increasing
collagen synthesis. Finally, high COL1A1 gene expression is a biomarker
of poor outcome across a range of primary cancers.
Subject terms: Melanoma, Cancer microenvironment
__________________________________________________________________
Ultraviolet radiation (UVR) increases melanoma incidence. Here, the
authors report that UVR-damaged dermal fibroblasts upregulate MMP1 to
degrade collagen which inhibits melanoma invasion and that aged primary
melanomas in skin with degraded collagen have a better prognosis, while
new collagen synthesis restores invasion and leads to death.
Introduction
UVR is the major environmental risk factor for the development of
melanoma^[56]1–[57]5 and sun exposure is the main cause of rising
disease incidence^[58]3. While the association between UVR and melanoma
incidence is well established^[59]5, there are controversial clinical
studies associating sun exposure, or sun damage to the dermis, with
improved melanoma survival^[60]6–[61]8. However, other studies have
found no association between sun damage and outcome, and clinical
studies show melanomas arising on the scalp and neck, areas likely
chronically sun damaged, are linked to poor outcome^[62]9–[63]11.
UVR damage accumulates with increasing decades of life, and aged
patients have worse melanoma survival^[64]12–[65]15. Therefore, it is
possible that chronic UVR damage may lead to shorter melanoma-specific
survival (MSS). However, in common with some non-hormonal cancers, the
incidence and mortality of melanoma sharply rise after age 60, and then
significantly decrease after age 85 (refs. ^[66]16,[67]17), suggesting
the relationship between cumulative UVR exposure, cutaneous damage, age
and melanoma death is not linear.
Previous studies have shown collagen quantity in the extracellular
matrix (ECM) modifies melanoma cell behaviour^[68]18. Surprisingly,
both increased^[69]19 and decreased^[70]20 deposition of collagen have
been linked to malignant behaviour, suggesting the effect of collagen
on cancer behaviour extends beyond protein level and scaffold function.
In this study, we explore how collagen levels in the dermis, which vary
according to sun damage and age, affect melanoma survival.
Results
Somatic mutation burden in dermal fibroblasts correlates with extracellular
matrix degradation and collagenase expression
The pivotal task of dermal fibroblasts is to regulate ECM remodelling,
including the turnover of collagen^[71]21. In chronically UVR-damaged
skin there is an increase of mutations^[72]22 and degraded collagen
that is not compensated by new collagen synthesis, contributing to
overall ECM degradation. We analysed gene expression in human adult
fibroblasts to compare matched UVR-damaged and UVR-protected dermis
from healthy donors (median age 42, range 19–66, ref. ^[73]23). We used
COSMIC total signature 7 mutations^[74]24, which indicate UVR-induced
damage, as a surrogate marker of accumulated UVR exposure. Strikingly,
the most significantly differentially expressed pathway was the ECM
pathway (Fig. [75]1a, b, Supplementary Data [76]1 and Supplementary
Table [77]1), demonstrating progressive downregulation of collagen
genes (Supplementary Table [78]2) and upregulation of matrix
metalloproteinases (MMP), including matrix metalloproteinase-1 (MMP1),
in UVR-damaged adult donor fibroblasts, with increasing signature 7
mutations. Furthermore, MMP1 was highly expressed in donor fibroblasts
from UVR-exposed calves of adults in the Genotype-Tissue Expression
cohort^[79]25 (median TPM = 51.85, n = 504, median all tissues
TPM = 0.078).
Fig. 1. UVR-driven mutations in adult donor dermal fibroblasts correlate with
ECM degradation and collagenase expression.
[80]Fig. 1
[81]Open in a new tab
a Volcano plot gene expression and b extracellular matrix (ECM) heatmap
by COSMIC signature 7 mutations in skin fibroblasts^[82]23. Colour
scale: red upregulated, blue downregulated genes, sample clustered by
signature 7 mutations: colour scale 0 (white) to 112 (green), red box:
genes upregulated with signature 7 mutations. c Atomic force microscopy
(scale: 5 µm, height colour scale −115 (dark brown), 115 (white)), and
d matrix roughness (Rq) of in vitro ECM of HFF and UV-HFF, two-sided
Mann–Whitney U *p = 0.0286, data represents two measurements from two
biologically independent cell lines per condition. e Quantification of
fibre alignment distribution in human foreskin fibroblasts HFF and
UV-HFF-derived ECM by fibronectin immunofluorescence. f Fraction of
fibres within 10° of mode orientation, two-sided Mann–Whitney U
**p = 0.0022, data represents two biologically independent cell lines
three fields of view per condition. g Immunofluorescence of fibronectin
fibres in decellularised HFF and UV-HFF-derived ECM, colour coded for
orientation of fibre, cyan represents mode, red ±90°, scale bar: 25 µm.
h Masson’s trichrome collagen stain of sun-protected (n = 9, noCSD) and
sun-damaged (n = 7, CSD) dermis, two-sided Mann–Whitney U
****p < 0.0001. i Correlation between SNV load and solar elastosis in
fibroblasts, blue: CSD, pink: noCSD, two-sided Spearman correlation
R = 0.40, p = 0.2, n = 13 biologically independent samples. Error bars:
standard error of the mean (bar).
MMP1 cleaves collagen 1 (COL1A1) after acute UVR
exposure^[83]26,[84]27, so we compared the secretion of MMP1, COL1A1
expression and ECM collagen deposition of isogenic UVR-naive
fibroblasts from a human foreskin fibroblast cell line (HFF) and
UVR-damaged fibroblasts (UV-HFF) 2 weeks after UVR exposure
(Supplementary Fig. [85]1a). We found UV-HFF fibroblasts increased the
secretion of MMP1 (Supplementary Fig. [86]1b) with no compensatory
increase in COL1A1 transcription (Supplementary Fig. [87]1c) or
deposition in the ECM of UV-HFF, compared to UVR-naive fibroblasts
(COL1A1 mean label-free quantification (LFQ) intensity HFF = 33.20,
UV-HFF = 33.47, q value = 0.16, COL1A2 HFF = 32.57, UV-HFF = 32.87, q
value = 0.08, Supplementary Fig. [88]1d). In addition, atomic force
microscopy (AFM) topographic imaging suggested UV-HFF
fibroblast-generated ECM presented more fragmented, sparser and
disorganised matrix fibrils than UVR-naive, HFF fibroblasts. The higher
roughness (Rq) value indicates less symmetry across the ECM surface
plane, in keeping with degradation of UV-HFF fibroblast-generated
ECM^[89]28–[90]30 (Fig. [91]1c, d). Furthermore, immunofluorescent
staining of fibronectin fibres in HFF and UV-HFF-derived ECM, confirmed
that UV-HFF matrices were significantly more disorganised with fewer
aligned fibres than matrices generated by HFF fibroblasts
(Fig. [92]1e–g).
Since UVR damage alters fibroblast function, compromising ECM renewal,
we compared the density of collagen fibres in chronically sun-damaged
and sun-protected healthy skin of aged patients (n = 16, age > 59);
confirming reduction of collagen in UVR-damaged dermis^[93]31
(Fig. [94]1h). In addition, we confirmed fibroblasts from
tumour-adjacent sun-damaged patient dermis (solar
elastosis^[95]31,[96]32) have higher total somatic mutation
burden^[97]22 (n = 13, Fig. [98]1i), indicating that cumulative UVR
leads to dermal ECM degradation and decreased collagen.
Low collagen concentration and reduced collagen integrity decrease melanoma
invasion
To study if UVR damage to fibroblasts driving collagen degradation
affects melanoma progression, we compared melanoma invasion in
spheroids embedded in matrices of increasing collagen concentrations.
Melanoma cell lines present varying degrees of invasion, so we used
three cell lines established from tumours bearing different UVR
mutation signatures, reflecting UVR and non-UVR tumour origins^[99]33
(Supplementary Fig. [100]2a). We found that regardless of the UVR
history of the melanoma cell line, the invasion of the three melanoma
lines was optimal in 1.5 mg/ml collagen, and higher (2.5 mg/ml,
p < 0.0001) and lower collagen concentrations significantly reduced
invasion into the ECM (0.25 mg/ml, p < 0.0001, 0.5 mg/ml, p = 0.03;
Fig. [101]2a, b and Supplementary Fig. [102]2b). In addition, we
quantified the number of melanoma cells detaching from the spheroid and
invading as single cells, and found single cell invasion optimal within
a range of collagen concentrations, decreasing with higher and lower
collagen densities (Fig. [103]2c, d). We generated organotypic dermal
constructs with HFF or UV-HFF foreskin human fibroblasts, seeded with
melanoma cell lines (Fig. [104]2e and Supplementary Fig. [105]2c), and
confirmed UV-HFF constructs presented fewer melanoma cells detaching
from the tumour edge, singly advancing in the dermis (Sk-mel-28
p = 0.04; Fig. [106]2f). UV-HFF constructs replicated the cardinal
features of UVR damage^[107]34–[108]36, with significantly reduced
collagen levels compared to HFF constructs (p < 0.0001, Supplementary
Fig. [109]2d, e). In addition, UV-HFF constructs presented reduced
fibronectin (Supplementary Fig. [110]2f), and no difference in elastin
expression compared to HFF constructs (Supplementary Fig. [111]2g).
These data indicate that melanoma invasion is optimal within a range of
collagen concentrations. Critically, lower collagen concentrations
limit melanoma invasion.
Fig. 2. Low collagen quantity and integrity decrease melanoma cell invasion.
[112]Fig. 2
[113]Open in a new tab
a Mean and individual b melanoma spheroid invasion (Kruskal–Wallis with
Dunn’s multiple comparison tests *p = 0.0317, ****p < 0.0001, n = 8
replicate spheroids for three cell lines across two-independent
experiments), and c single cell invasion (Skmel28) (Kruskal–Wallis with
Dunn’s multiple comparison tests ****p < 0.0001, n = 9 independent
measurements over two experiments) in different collagen
concentrations. d Representative images of spheroid and melanoma single
cell invasion, top: Hoechst (inverted), bottom: invading cells (blue).
e A375 melanoma invasion H&E (scale bar: 10 µm) and f single cell
invasion in organotypic dermal collagen HFF and UV-HFF constructs
(two-sided Mann–Whitney U *p = 0.0485, ns not significant, data
represents 12 fields of view across two-independent experiments). g
Mean and individual h melanoma invasion (Kruskal–Wallis with Dunn’s
multiple comparison tests ***p < 0.001, ****p < 0.0001, n = 8 replicate
spheroids for three cell lines across two-independent experiments), and
i melanoma single cell invasion (Kruskal–Wallis with Dunn’s multiple
comparison tests *p = 0.0426, n = 7 independent measurements over two
experiments) by collagenase I concentration. j Representative images of
spheroid and single cell invasion, top: Hoechst (inverted), bottom:
invading cells (blue). k Melanoma spheroid invasion by adult patient
fibroblast secretome, MMP1 levels (two-sided Pearson correlation, red:
Sk-mel-28 R = −0.39 p = 0.1, blue: A375 R = −0.21 p = 0.4, data
represent eight independent cell lines measured in duplicate) and l by
COL1A1 relative expression (RE) in fibroblasts (two-sided Pearson
correlation, red: Sk-mel-28 R = 0.57 p = 0.02, blue: A375 R = 0.32
p = 0.2, data represent eight independent cell lines measured in
duplicate). Error bars: standard error of the mean (bar).
Since UVR compromises collagen integrity indirectly by damaging
fibroblasts (Fig. [114]1), we exposed melanoma spheroids, embedded in
equal concentrations of collagen matrices, to increasing concentrations
of the enzyme collagenase I to mimic the effects of UVR exposure
(Supplementary Fig. [115]2h). We found that melanoma invasion and
single cell invasion significantly decreased in matrices exposed to
higher doses of collagenase I (5 µg/ml, p = 0.0005, 10 µg/ml,
p < 0.0001; Fig. [116]2g–j and Supplementary Fig. [117]2i). These data
show that collagen quantity and degraded collagen limit melanoma
invasion.
To explore if adult fibroblasts regulate collagen degradation and
melanoma invasion, we harvested adult dermal fibroblasts from different
anatomic sites from tumour-adjacent normal skin of eight patients and
established cells lines (Supplementary Table [118]3, age median 69,
range 34–77). We confirmed the patient dermal fibroblasts express and
secrete varying levels of MMP1 (Supplementary Fig. [119]2j, k), and
embedded melanoma spheroids in matrices of collagen mixed with the
donor fibroblast secretome. We found melanoma spheroids were less
invasive in matrices containing patient fibroblast secretomes with
higher amounts of MMP1 (Fig. [120]2k). Importantly, COL1A1 expression
correlated strongly with melanoma invasion, (Sk-mel-28 p = 0.02,
R = 0.32, Fig. [121]2l). Finally, we confirmed that human fibroblasts,
and not melanoma cells, are the main source of MMP1; and the melanoma
cell lines do not express COL1A1 or COL1A2 (ref. ^[122]37,
Supplementary Fig. [123]2l). Taken together, these data demonstrate
human adult fibroblasts modulate collagen biology and melanoma
invasion.
Inhibition of MMP1 restores melanoma invasion
We studied if adult human fibroblasts from distinct anatomic sites
affect the ECM and melanoma invasion differentially, and established
fibroblast lines from chronic sun-damaged (CSD) and sun-protected, or
non-sun damaged (noCSD) tumour-adjacent skin^[124]32 of two patients
(age CSD = 77, age noCSD = 46). Donor fibroblasts were exposed to low
doses of UVB (8 × 100 J/m^2) to generate isogenic pairs of noCSD and
noCSD-UV fibroblasts, CSD and CSD-UV fibroblasts and allowed 14 days
recovery (Supplementary Fig. [125]2m). We confirmed noCSD-UV
fibroblasts robustly increased MMP1 expression (fold change = 2.03,
p = 0.002) and secretion (median MMP1: noCSD = 6858 pg/ml,
noCSD-UV = 13,292 pg/ml, fold change = 1.98, p = 0.03) compared to
noCSD donor fibroblasts (Fig. [126]3a, b); and CSD-UV fibroblasts
weakly upregulated MMP1 expression (fold change = 1.46, p = 0.39), and
secretion (median MMP1: CSD = 9066 pg/ml, CSD-UV = 13051 pg/ml, fold
change = 1.49, p = 0.03). Importantly, noCSD-UV and CSD-UV fibroblasts
did not increase COL1A1 expression (Supplementary Fig. [127]2n). We
then compared the effect of the fibroblast secretomes on melanoma
spheroid invasion in the presence or absence of the MMP inhibitor
Batimastat, which directly blocks the activity of MMPs. Consistent with
a higher MMP1 expression, the noCSD-UV secretome significantly
decreased melanoma invasion compared to the noCSD secretome
(p = 0.001), and importantly, Batimastat restored melanoma cell
invasion (p = 0.01; Fig. [128]3c). Intriguingly, UVR damage or
Batimastat treatment of the CSD fibroblast model (CSD-UV) only slightly
modulated melanoma invasion (Fig. [129]3d), possibly indicating the
effect of UVR damage to fibroblasts and MMP1 expression is capped, and
higher doses of MMP inhibition are required in highly expressing MMP1,
CSD-UV fibroblasts.
Fig. 3. Inhibition of MMP1 restores melanoma invasion.
[130]Fig. 3
[131]Open in a new tab
a Fold change in MMP1 relative expression (RE) in chronically
UVR-treated adult patient fibroblasts compared to untreated isogenic
cell lines, noCSD no chronic sun damage, pink, CSD chronic sun damage,
blue (two-sided Mann–Whitney U **p = 0.0022, ns not significant, data
represent two samples per condition quantified in triplicate). b Fold
change in secreted MMP1 in chronically UV-treated adult fibroblasts
compared to untreated isogenic cell lines, noCSD no chronic sun damage,
pink, CSD chronic sun damage, blue, (two-sided Mann–Whitney U
*p = 0.0286, data represent two samples per condition quantified in
triplicate). c Melanoma spheroid invasion in noCSD and isogenic
noCSD-UV fibroblast secretomes in the presence of Batimastat or DMSO
vehicle, (two-sided Mann–Whitney U **p = 0.0011, *p = 0.0104, data
represents eight replicate spheroids measured across two-independent
experiments per condition). d Melanoma spheroid invasion in CSD and
isogenic CSD-UV fibroblast secretomes in the presence of 8 nM
Batimastat or DMSO vehicle, (two-sided Mann–Whitney U, ns not
significant). Data represents eight replicate spheroids measured across
two-independent experiments per condition, box plots represent 25th to
75th percentiles with median, whiskers represent minimum and maximum
values. e Representative images of collagen degradation in shCtrl-HFF,
shCtrl-UV-HFF, shMMP1-HFF and shMMP1-UV-HFF fibroblasts. Green: intact
DQ collagen; red: phalloidin; blue: Hoechst. Size bars: 20 µm. f Fold
change in collagen degradation of shCtrl-HFF and shMMP1-HFF (pink), and
their isogenic chronic UVR cell lines shCtrl-UV-HFF and shMMP1-UV-HFF
(blue), (two-sided Mann–Whitney U ***p = 0.0007, ns not significant,
n = 149 scores across two biologically independent cell lines per
condition). g Immunofluorescence of fibronectin fibres in
decellularised shCtrl-HFF, shCtrl-UV-HFF, shMMP1-HFF and
shMMP1-UV-HFF-derived ECM, colour coded for orientation of fibre, cyan
represents mode, red ±90°, scale bar: 25 µm. h Quantification of fibre
alignment distribution in shCtrl-HFF, shCtrl-UV-HFF, shMMP1-HFF and
shMMP1-UV-HFF-derived ECM, data represents z-stacks of three fields of
view per sample. i Quantification of invading melanoma cells into
organotypic dermal collagen constructs made with shCtrl-HFF and
shMMP1-HFF (pink), and isogenic chronic UVR cell lines shCtrl-UV-HFF
and shMMP1-UV-HFF (blue), (two-sided Mann–Whitney U *p = 0.0272, ns not
significant, data represents >8 fields of view for two-independent
experiments in biologically independent cell lines), scoring was
performed on duplicate constructs counting in at least five fields of
view per cell line. j Representative images of A375 invasion into
organotypic constructs stained with H&E (scale bar: 10 µm). Error bars
represent standard error of the mean (bar).
Since MMP1 specifically cleaves COL1A1 (ref. ^[132]26), we generated
isogenic shCtrl-HFF, shCtrl-UV-HFF, shMMP1-HFF and shMMP1-UV-HFF lines
from HFF to compare collagen degradation in the absence of acute UVR
exposure (Supplementary Fig. [133]3b, c). In keeping with a higher
expression of MMP1, shCtrl-UV-HFF fibroblasts degraded more collagen,
while shMMP1-UV-HFF did not increase collagen degradation compared to
shMMP1-HFF fibroblasts (Fig. [134]3e, f). The specific role of MMP1 was
validated with an additional knockdown with an siRNA targeting MMP1
(Supplementary Fig. [135]3d–f). In addition, we found shRNA targeting
MMP2 did not modify collagen degradation (Supplementary Fig. [136]3b,
g, h); and knockdown of MMP1 restored the alignment of fibres in UV-HFF
matrices (Fig. [137]3g, h and Supplementary Fig. [138]3i). Furthermore,
organotypic invasion assays with matrices generated with shCtrl-HFF,
shCtrl-UV-HFF, shMMP1-HFF or shMMP1-UV-HFF fibroblasts, showed melanoma
invasion was decreased in the shCtrl-UV-HFF constructs (p = 0.03), and
not in shMMP1-HFF or shMMP1-UV-HFF fibroblast matrices (Fig. [139]3i,
j). Knockout of MMP1 restored collagen and fibronectin levels in UV-HFF
constructs to similar levels as HFF (Supplementary Fig. [140]3j, k, l
and see also Supplementary Fig. [141]2c, d, f). Altogether, these data
demonstrate fibroblast-secreted MMP1 degrades collagen, limiting
melanoma invasion.
Collagen degradation decreases primary melanoma invasion and improves
survival
If the amount and integrity of collagen restricts single cell invasion,
patients with primary cutaneous melanoma invading in a less collagenous
dermis should live longer than patients with more dermal collagen.
Compared to young fibroblasts and dermis, aged and UVR-protected
fibroblasts in an aged ECM drive melanoma invasion and
metastasis^[142]38,[143]39, so we restricted our study to three
international cohorts of older primary cutaneous melanoma patients
(Supplementary Table [144]4). We determined the proportion of melanoma
cells invading in the ECM at the invasive front (IF), the amount of
collagen and the degree of ECM degradation (solar elastosis) in
tumour-adjacent dermis (Fig. [145]4a and Supplementary Fig. [146]4a).
We found patient samples with more solar elastosis (less collagen in
tumour-adjacent skin), had fewer invading cells at the IF (Fisher exact
test, p = 2.25 × 10^−5 Fig. [147]4b, Supplementary Fig. [148]4b and
Supplementary Data [149]2). Critically, MSS was significantly improved
in patients with less invasion in multivariate analyses (Fig. [150]4c,
Supplementary Fig. [151]4c, d and Supplementary Table [152]5).
Intriguingly, solar elastosis was not as powerfully associated with
better outcome (p = 0.9, Fig. [153]4d, Supplementary Fig. [154]4e, f
and Supplementary Table [155]5). To explain this difference, we
hypothesised that collagen at the IF, rather than collagen degradation
in the tumour-adjacent dermis, would be a better biomarker of survival.
Further analysis confirmed that collagen at the IF strongly correlated
to single cell invasion (Spearman R 0.5, p < 0.0001, Fisher Exact p =
0.002, Fig. [156]4e), MSS and progression-free survival (PFS;
Fig. [157]4f, Supplementary Fig. [158]4g, and Supplementary
Table [159]5). Furthermore, consistent with invasion data in
Fig. [160]2j, TCGA primary cutaneous melanomas expressing low COL1A1
showed improved survival (Fig. [161]4g and Supplementary Table [162]5).
These data suggest that primary melanomas invading in collagen-poor
matrices require new collagen synthesis in order to invade
successfully. To confirm this, we studied collagen at the IF
specifically, in short-term and long-term survivors. We confirmed
melanomas arising at CSD sites with less collagen in the
tumour-adjacent dermis and shorter survival (MSS <5 years), increased
collagen deposition at the IF and tumour cell invasion (Fig. [163]4h, i
and Supplementary [164]5).
Fig. 4. Low collagen correlates with low invasion and improved outcome in
aged melanoma patients.
[165]Fig. 4
[166]Open in a new tab
a H&E top panel: primary cutaneous melanoma and inset (box) with single
cell invasion (arrows) in a collagen-rich dermis with no chronic sun
damage (noCSD), collagen: red (scale bars left: 4000 µm, right:
400 µm). Lower panel: melanoma in a collagen-poor dermis with chronic
sun damage (CSD) and inset (box), no single cell invasion; collagen:
red (scale bars left: 3000 µm, right: 300 µm). b Histogram displaying
melanoma invasion at the invasive front (IF) in noCSD and CSD melanomas
(B and C cohorts, n = 170, two-sided Fisher exact test,
p = 2.25 × 10^−5). c Kaplan–Meier of melanoma-specific survival (MSS)
in prominent (high, red) and minimal (low, blue) melanoma invasion at
the IF (two-sided log-rank test, B and C cohorts, n = 167). d
Kaplan–Meier of MSS in melanoma invading in CSD (blue) and noCSD (pink)
dermis (two-sided log-rank test, B and C cohorts, n = 331). e Histogram
displaying collagen quantity at the IF in highly invasive (red) and
minimally invasive (blue) melanoma (C cohort, n = 89, two-sided Fisher
exact test, p = 0.002). f Kaplan–Meier of MSS by collagen quantity at
the IF (two-sided log-rank test, C cohort, n = 62). g Kaplan–Meier of
MSS by COL1A1 expression in aged (>54) primary cutaneous melanoma
(two-sided log-rank test, TCGA cohort, n = 80). h Fold increase in
collagen deposition at the IF of noCSD and CSD melanomas by MSS
(two-sided Mann–Whitney U ***p = 0.0009 C cohort n = 90). Error bars
represent standard error of the mean (bar). i H&E top panel: left CSD
melanoma, middle: from box inset: tumour-adjacent dermis; right: from
dashed box inset: IF (dashed line, scale bars: 2000, 300, 300 µm).
Bottom: left CSD melanoma, middle: from box inset: tumour-adjacent
dermis; right: from dashed line box inset: IF between dashed lines,
arrows: melanoma invasion, (scale bars: 2000, 70, 200 µm, n = 90). j
Kaplan–Meier of MSS by melanoma-associated fibroblast (MAF) signature
score in aged (>54) primary cutaneous melanoma cohort (two-sided
log-rank test, TCGA cohort, n = 80). k Hazard ratio (centre) and 95% CI
(bars) for OS, and PFS (l) in two-sided univariate Cox regression of
COL1A1 expression by cancer type in PANCAN TCGA, p values unadjusted.
(ACC n = 79, adrenocortical carcinoma, BLCA n = 407, bladder urothelial
carcinoma, CESC n = 304, cervical and endocervical cancers, COAD
n = 448, colon adenocarcinoma, KICH n = 65, kidney chromophobe, KIRC
n = 533, kidney renal clear cell carcinoma, KIRP n = 289, kidney renal
papillary cell carcinoma, LGG n = 514, brain lower grade glioma, LUAD
n = 506, lung adenocarcinoma, MESO n = 86, mesothelioma, PAAD n = 178,
pancreatic adenocarcinoma PRAD n = 497, prostate adenocarcinoma, STAD
n = 409, stomach adenocarcinoma). Risk tables for all Kaplan–Meier
analyses in Supplementary Data [167]2.
To further explore the association between collagen and survival, we
investigated if melanoma-associated fibroblasts (MAFs) in primary
melanomas increase collagen to sustain invasion. For this, we extracted
the gene expression signature from single cell RNAseq^[168]37 and
confirmed COL1A is expressed by MAFs (Supplementary Fig. [169]4h). We
then tested if increased expression of MAFs in primary melanomas
correlates with outcome and were able to demonstrate that a higher
expression of MAF genes is associated with poor survival
(Fig. [170]4j). Furthermore, we show that the expression of collagen
genes specifically within the MAF expression signature is what impacts
survival, as the MAF signature is not significantly prognostic in the
absence of collagen genes (Supplementary Fig. [171]4i and Supplementary
Table [172]5).
Solid cancers synthesise high amounts of ECM proteins and COL1A1; and
ECM remodelling promotes primary tumour progression and
metastasis^[173]40,[174]41. Therefore, we designed a tumour-agnostic
approach to test the potential of COL1A1 expression as a biomarker for
primary pan-cancer survival. This revealed young and aged patients with
primary cancers expressing high levels of COL1A1 are at greater risk of
death and have shorter PFS (Fig. [175]4k, l and Supplementary
Fig. [176]4j–l).
Discussion
Multiple in vivo studies confirm UVR cooperates with oncogenic
mutations to increase the incidence and penetrance of
disease^[177]5,[178]42. However, whether UVR exposure affects the odds
of survival has not been comprehensively investigated, and there are
contradictory studies finding sun exposure inferred by anatomic
site^[179]8, history of sunburn^[180]9,[181]18 or the presence of
UVR-induced dermal degradation^[182]6,[183]10 can affect outcome. The
majority of melanoma deaths affect the elderly^[184]13, and age is
strongly associated with accumulated sun exposure^[185]7. We
investigated if pre-existing UVR damage affects melanoma survival, and
found low collagen quantity and integrity limit melanoma invasion. UVR
damage to fibroblasts degrades collagen and the ECM, delaying melanoma
progression. We confirmed our in vitro results, showing that in aged
primary cutaneous melanomas, single tumour cells invading the dermis
and collagen at the IF robustly predict poor survival. Paradoxically,
this study finds UVR damage to the dermis destroys collagen, limiting
invasion and improving outcome, unless tumours increase the production
of collagen at the IF, providing the structural support for melanoma
invasion. Together with recent work showing UVR-protected aged
fibroblasts^[186]38 and ECM^[187]39 drive melanoma metastasis, this
study strongly implicates the physical composition and structure of the
aged tumour microenvironment, as key to primary melanoma progression.
We therefore infer from these joint studies that excessive old age
mortality particularly affects patients with tumours arising at
anatomic sites with preserved dermal collagen, or sun-protected skin.
In contrast, UVR damage modifies the dermis and decreases collagen
content as we age. As melanomas arising in collagen-poor, sun-damaged
skin require collagen to invade, we show that collagen deposition at
the invasive edge of the tumour is an independent, robust biomarker of
survival. Conveniently, the deposition of collagen at the IF can be
scored simply from haematoxylin and eosin stains, making this an ideal
biomarker.
Melanomas with more UVR damage accumulate more mutations and
neoantigens, possibly eliciting stronger immune
responses^[188]43,[189]44. However, we show a proportion of UVR
melanomas have a better prognosis due to collagen degradation,
independently of the mutation burden, tumour and stromal cell
immunogenicity, which should be considered when evaluating responses to
adjuvant immunotherapy. One possibility is to prioritise adjuvant care
according to single cell invasion, collagen at the IF and risk of
death. Supporting this rationale, recent evidence shows collagen
density modifies the immune milieu of breast cancers, limiting T-cell
responses^[190]45.
The accumulation of a collagenous ECM, increasing stiffness, leads to
poor prognosis and lack of response to therapies in other
cancers^[191]40. Alterations of the ECM dynamics are a hallmark of
cancer, able to deregulate cancer and stromal cells^[192]46,[193]47,
promote cell transformation and the pro-metastatic niche, which becomes
rich in vasculature and tumour-promoting inflammation^[194]48. Clinical
trials with drugs inhibiting MMPs and limiting ECM remodelling have
yielded negative and sometimes deleterious results. One possible
explanation for this failure, based on our study in melanoma, is that
inhibition of collagen degradation may support tumour invasion.
Collagen can drive cancer cell
de-differentiation^[195]19,[196]49–[197]51 and is often found in areas
of active epithelial cancer invasion, facilitating
migration^[198]50,[199]52.
Ageing is associated with less collagen deposition, more degradation
and higher overall cancer incidence and mortality in multiple tissues,
including skin. One possibility is the collagen decrease in aged
tissue, which could lead to less aggressive cancer, is offset by a
decrease in ECM structural fitness, collagen and matrix organisation,
and pro-tumourigenic signalling with age^[200]41. This work suggests
new collagen synthesis by the TME is a critical regulator of aged
primary melanoma progression, a feature that could drive poor outcome
in other aged cancers. Critically, our data shows collagen expression
is associated in multiple solid epithelial and non-epithelial primary
tumours with shorter PFS in all ages, possibly due to direct modulation
of invasion.
Methods
Cell lines and patient fibroblasts
HFF were purchased from ATCC (ATCC SCRC-1041). Three melanoma cell
lines, Sk-mel-28 (ATCC HTB-72), Sk-mel-3 (ATCC HTB-69) and A375 (ATCC
CRL-1619) were purchased from ATCC. All cell lines were cultured in
DMEM (Gibco, 41966-029) supplemented with 10% FCS (Sigma Aldrich,
F7524), 1× Glutamax (Gibco, 35050061), 100 U/ml penicillin and
streptomycin (Gibco, 15140122) and 1 mM sodium pyruvate (Gibco,
11360070). Cells lines were cultured at 37 °C in 5% CO[2] with medium
replaced as required. Cell lines were tested every fortnight for
mycoplasma using LookOut Mycoplasma PCR Detection Kit (Sigma Aldrich,
MP0035). Cell line identity was confirmed using STR profiling.
Patient fibroblasts
A prospective cohort of patient fibroblast cultures was established
from redundant skin acquired during surgical resection of the wide
local excision of healthy skin from melanoma patients treated at the
tertiary referral cancer Christie hospital. Ethical approval to
establish cell lines was granted by the local Biobank committee
(17_AMVI_01), which required signed informed consent from all
participants. The hypodermis of whole skin samples was removed scraping
with a scalpel, and the residual specimen was incubated overnight in
Dispase (Gibco, 17105-041) at 4 °C to separate the epidermis and
dermis. The dermis was digested in collagenase I (Gibco, 17018029) in
DMEM (without FCS) at 37 °C for 6 h, and then filtered through 70 µm
filter to remove the residual debris. Dermal cells were spun at 300 × g
and resuspended in DMEM 20% FCS, cultured in DMEM 20% FCS until they
became confluent and stained for vimentin (Abcam, ab92547). The level
of solar elastosis of the redundant skin collected was scored using
previously well-established methods^[201]32.
Whole-exome sequencing of patient fibroblasts
Whole-exome sequencing of fibroblasts was performed by Novogene
(Novogene (UK) Company Ltd.). Exome capture was performed with the
SureSelect Human All Exon v6 kit (Agilent) and sequenced on the
Illumina HiSeq platform. Sequencing reads were trimmed using
Trimmomatic^[202]53, aligned to the hg38 reference genome using
BWA^[203]54, and duplicate reads were marked using Picard Tools
([204]http://broadinstitute.github.io/picard). Somatic mutations were
called using the Varscan 2 pipeline^[205]55. Identified somatic
variants were annotated using Variant Effect Predictor^[206]56 and
variants present in dbSNP (but not in the COSMIC database) were
excluded.
Lentiviral shRNA transfection
Knockdown of MMP1 expression in HFF cells was performed using shRNA
Lentiviral Particles (Santa Cruz Biotechnology) and siGENOME siRNA
(Horizon Discovery). For MMP1 shRNA knockdown MMP1 shRNA (h) lentiviral
particles (sc-41552-V) were used alongside a scramble control, control
shRNA lentiviral particles A (sc-108080) and copGFP control lentiviral
particles (sc-108084) were used to measure transduction efficiency. A
total of 5 × 10^4 cells were cultured in cell culture media with
5 ug/ml polybrene (Santa Cruz Biotechnology, sc-134220). Lentiviral
particles were added to cells and incubated overnight. Media containing
lentiviral particles and polybrene was removed and incubated in DMEM
overnight before preforming selection of transfected cells using
increasing concentration of puromycin over 72 h. Once cells were stably
growing in puromycin, cells were cultured as normal in DMEM. For MMP1
siRNA siGENOME human MMP1 siRNA (D-005951-02-0002) and non-targeting
siRNA #4 (D-001210-04-05) were used with DharmaFECT 1 transfection
reagent (Horizon Discovery, T-2001-02), according to manufactures
protocols, with a final siRNA concentration of 25 nM. Knockdown of MMP2
was performed with MMP2 shRNA (h) lentiviral particles (sc-29398-V) as
above. Knockdown of all gene expression was validated by qPCR and
western blot.
UV treatment and CSD model
Cell lines were treated with UVB using a Bio-Sun UV irradiation system
(Vilber Loumat). For chronic treatment the dose 100 J/m^2 was used as
it represents a physiologically relevant dose of UVB that would
penetrate the dermis between 1 and 5 minimal erythema dose^[207]57. To
create isogenic in vitro chronic UV-damaged UV-HFF and shMMP1-UV-HFF,
noCSD-UV, CSD-UV cell lines, 1 × 10^6 HFF, shMMP1-HFF or patient dermal
fibroblasts were cultured in 100 mm dishes in phenol-free DMEM 1% FCS.
All fibroblasts were treated every 24 h with 100 J/m^2 UVB for eight
consecutive days. Following the UV treatments, the medium was changed
to DMEM 10% FCS and cultured for 1 week. Isogenic untreated control
cell lines (HFF, shMMP1-HFF, noCSD and CSD) were cultured in identical
conditions without the UVB treatments. Each HFF condition was created
in biological duplicates.
Secretome collection
To collect secretomes 1 × 10^6 cells were plated in a 100 mm dish and
cultured for 72 h in DMEM without FBS to limit cell proliferation.
Secretomes were collected in duplicate, aliquoted and stored at −80 °C
until used.
Batimastat treatment
MMP inhibitor Batimastat (Sigma Aldrich, SML0041) was resuspended in
DMSO at 15 mg/ml and a stock was diluted to 1 mM. Batimastat was added
to fibroblast secretomes to a final concentration of 8 nM in secretome
volume and control secretomes had equal volume of DMSO added as a
vehicle control.
RNA-sequencing data analysis
RNA-seq data from ENA project [208]PRJEB13731; also at
[209]https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-4652/ were
downloaded. Data are single-end RNA-seq from short-term cultivated
fibroblasts sequenced on an Illumina HiSeq 2000 sequencer. Two samples
had been obtained from each individual from different locations
(B = buttock, not UV exposed; S = shoulder, UV exposed). Sequencing
reads were trimmed^[210]53 and aligned to the human reference genome
(GRCh37) using STAR^[211]58. Production of analysis-ready reads was
conducted according to the Broad Institute Best Practices pipeline
([212]https://gatkforums.broadinstitute.org/gatk/discussion/3892/the-ga
tk-best-practices-for-variant-calling-on-rnaseq-in-full-detail) using
GATK v3.2 ([213]http://www.broadinstitute.org/gatk). Somatic single
nucleotide variants (SNV) in each patient matched sample in regions
annotated as protein-coding only (based on Ensembl
Homo_sapiens.GRCh37.87) were identified with the mutation calling
algorithm MuTect v1, using the other sample as the comparator^[214]59.
Identified somatic variants were annotated using Variant Effect
Predictor and common variants were excluded.
COSMIC mutational signatures v2
([215]https://cancer.sanger.ac.uk/cosmic/signatures_v2) were identified
using the MutationalPatterns^[216]60 package (version 1.8.0) in R
(version 3.5.1, RStudio v1.2.5001, RStudio Inc). Differential
expression analysis was performed using the DESeq2 package (version
1.22.2, ref. ^[217]61) in R (version 3.5.1). Reads counts of genes were
filtered for genes expressed in fibroblasts by removing any gene with
<100 counts across all samples. For pathway enrichment analysis genes
that were significantly differentially expressed in by signature 7
mutation count (false discovery rates (FDR) p value < 0.1) were
compared against the Reactome Database^[218]62 using the Molecular
Signatures Database v7.0 (Msigdb, Broad Institute,
[219]https://www.gsea-msigdb.org/gsea/msigdb/index.jsp).
Fibroblast extracellular matrix production
Following ECM construction^[220]63 cell culture dishes were coated with
0.2% sterile gelatin (Sigma Aldrich, G1393), fixed with 1%
glutaraldehyde and quenched with 1 M glycine in PBS (pH 7). Fibroblasts
were cultured on gelatin plates in normal 10% FCS DMEM containing
50 µg/ml ascorbic acid for 8 days. Cells were lysed with extraction
buffer (20 mM NH[4]OH and 0.5% Triton X-100 in PBS), and washed
thoroughly with PBS containing calcium and magnesium. DNA was digested
with 10 µg/ml DNase I (Roche, 04716728001) and washed. For mass
spectrometry the ECM was collected with a lysis buffer (100 mM TrisHCl
pH 7.5, 4% SDS and 100 mM DTT) and collected with scraper, sonicated
and boiled at 95 °C, followed by centrifugation (16,000 × g, 15 min)
and collection of the supernatant, stored at −80 °C until use.
Immunofluorescence and ECM fibre analysis
For immunofluorescence and ECM fibre analysis, fibroblasts derived
matrices constructed as above on glass coverslips, after DNase
digestion matrices, were fixed in 4% paraformaldehyde (PFA), blocked 5%
BSA TBS-T (5% BSA in 1× Tris-buffered saline and 0.1% Tween 20) for
1 h, and stained for Fibronectin^[221]64. Primary antibody to
fibronectin (1:200, F3648, Sigma Aldrich, 5% BSA TBS-T) and secondary
goat anti-rabbit Alexa Fluor 488 (1:2000, Thermo Fisher) were incubated
for 1 h at room temperature. The immunofluorescence imaging was
performed using a Carl Zeiss LSM880 inverted confocal microscope with
63× NA 1.4 oil objectives lens controlled by ZEN black software. The
images were acquired with 488 nm illumination laser line from an Argon
laser (Lasos) and the emission spectrum range from 500 to 550 nm
collected with a PMT detector (Zeiss). Z-series optical sections were
collected with a step size of 0.5 micron driven by Piezo stage (Zeiss).
Fibre orientation analysis was performed using ImageJ OrientationJ
plugin^[222]64,[223]65. Maximal projection of three individual z-stacks
for each condition were analysed.
Mass spectrometry sample preparation and analysis
ECM protein lysates were separated on a 4–12% gradient NuPAGE Novex
Bis-Tris gel (Life Technologies). Each sample was cut into three slices
and in-gel digested with trypsin (Promega)^[224]66,[225]67. Digested
peptides were desalted by C18 StageTip^[226]68, acetonitrile was
removed by speed vacuum, and peptides were resuspended in 1%
trifluoroacetic acid and 0.2% formic acid. Peptides were injected into
an EASY-nLC (Thermo Fisher Scientific) coupled online to an Orbitrap
Fusion Lumos mass spectrometer (Thermo Fisher Scientific), separated
using a 20 cm fused silica emitter (New Objective) packed in house with
reversed-phase Reprosil Pur Basic 1.9 μm (Dr Maisch GmbH) and eluted
with a flow of 300 nl/min from 5 to 30% of buffer (80% ACN and 0.1%
formic acid), in a 90 min linear gradient. MS raw data were acquired
using the XCalibur software (Thermo Fisher Scientific). MS raw files
were processed using MaxQuant software^[227]69 (version 1.6.3.3) and
searched against the human UniProt database (release 2016_07, 70,630
sequences), using the Andromeda search engine^[228]70 with the
following settings: the parent mass and fragment ions were searched
with an initial mass deviation of 4.5 and 20 p.p.m., respectively.
Carbamidomethyl (C) was added as a fixed modification and acetyl
(N-term) and oxidation (M) as variable modifications. The minimum
peptide length was set to seven amino acids and a maximum of two missed
cleavages, and specificity for trypsin cleavage were required. The FDR
at the protein and peptide level were set to 1%. The LFQ setting was
enabled for protein quantification^[229]71. Razor and unique peptides
were used for quantification. Perseus software^[230]72 (version
1.6.2.2) was used for statistical analysis. Data were filtered to
remove potential contaminants, reverse peptides that match a decoy
database and peptides only identified in their modified form. LFQ
intensities were transformed by log[2]. A two-sample t test was used to
determine significantly regulated proteins, with the permutation-based
FDR ≤ 0.05 and S0 = 0.1 being considered significant.
Atomic force microscopy
For imaging purposes, the ECMs prepared as above were fixed with 2% PFA
and stored with PBS containing 1% penicillin/streptomycin at 4 °C. A
day prior to imaging, the dishes were washed with distilled water five
times to wash off any salt and then air dried overnight. Samples were
imaged by intermittent contact mode in air using a Bruker ScanAsyst
9.1. The probe was auto-tuned using Nanoscope software (version 1.4).
Images were taken at 10 × 10 μm and 2 × 2 μm area at least two sites.
Data were processed using Nanoscope analysis software 1.4 prior to
image export. The roughness (Rq) values were determined using the
software. Roughness is the root mean square average of the image and is
calculated based on the height difference per pixel along the sample
length. Rq is used to study the surface topography of various
nanostructures^[231]28,[232]29. Rq provides a quantitative measure of
fibril organisation in dermis and could possibly suggest the integrity
of matrix^[233]30.
Collagen degradation assay
To quantify the degradation of collagen in different fibroblast cell
lines a collagen degradation assay based on ref. ^[234]73 was used. DQ
collagen, type I from bovine skin, fluorescein conjugate (Invitrogen,
[235]D12060) was used to coat the wells of a 96-well plate for 1 h at
37 °C and quenched with 20 mM glycine for 5 min. Next, fibroblasts were
plated at a concentration of 2.5 × 10^3/well and incubated in normal
culture conditions for 18 h. Cells were then fixed with 4% PFA for
20 min at room temperature, permeabilised with Triton 0.1% X-100 for
5 min at room temperature and stained with Alex Fluor 546 Phalloidin
(Invitrogen, A22283) 1:2000 for 1 h and DAPI (Invitrogen, D3571) 1:2000
for 15 min. Wells were imaged with the Opera Phenix High Content
Screening System (Perkin Elmer, Inc.), and collagen degradation was
quantified by either measuring the area, where DQ collagen had been
degraded and normalising to cell number (DAPI) or by scoring of images
by two-independent assessors (one of them blinded), scoring the
intensity of collagen degradation as low (1: peri-cytoplasmic ring of
collagen degradation <1/4 of the cytoplasmic diameter); medium (2:
peri-cytoplasmic ring of collagen degradation ~1/3 of the cytoplasmic
diameter) or high (3: peri-cytoplasmic ring of collagen degradation
~1/2 of the cytoplasmic diameter; Supplementary Fig. [236]3a). To
establish the ratio of degradation between shCtrl-HFF and
shCtrl-UV-HFF, and shMMP1-HFF and shMMP1-UV-HFF cells, we generated a
score (H) of collagen degradation for each condition:
[MATH: H=Σlowintensityimages×1+mediumintensityimages×2+highintensityimages×3÷totalimages :MATH]
We scored, 150 images for shCtrl-HFF, 147 images for shCtrl-UV-HFF, 150
images for shMMP1-HFF and 146 images for shMMP1-UV-HFF fibroblasts. The
ratios were established as: ratio shCtrl-UV-HFF/shCtrl-HFF = H-score
shCtrl-UV-HFF/H-score shCtrl-HFF, and ratio
shMMP1-UV-HFF/shMMP1-HFF = H-score shMMP1-UV-HFF/H-score shMMP1-HFF.
MMP1 ELISA
MMP1 in the secretome of cell lines was quantified with a MMP1 Human
ELISA Kit (Thermo Fisher Scientific, EHMMP1), according to
manufacturer’s protocol. Secretomes collected from cells were diluted
1:10 and standards and samples were measured in triplicate. Samples
were incubated on plate overnight at 4 °C. Absorbance measured on a
Spectra Max M5 plate reader (Molecular Devices).
Quantitative PCR
RNA was collected in duplicate from 1 × 10^6 cells lysed in TRIzol
Reagent (Invitrogen, 15596018) after secretomes were collected. The
aqueous phase of phenol–chloroform separation was collected and RNA
extracted using RNeasy Mini Kit (Qiagen, 74104). Concentration was
determined with the Qubit RNA HS Assay (Invitrogen, [237]Q32852) and
500 ng RNA was reverse transcribed to cDNA using TaqMan Reverse
Transcription Reagents (Thermo Fisher, N8080234), and diluted 1:20 in
nuclease free water. Genes were quantified by qPCR using TaqMan Gene
expression assays and Fast Mastermix on a QuantStudio 3 system. GAPDH
(Hs02758991_g1) and ACTB (Hs01060665_g1) were used as housekeeping
genes. MMP1 (Hs00899658_m1), COL1A1 (Hs00164004_m1) and COL1A2
(Hs01028956_m1) were quantified and normalised to the geometric mean of
both housekeeping genes and relative expression calculated using
2^−Δct.
Western blots
Intracellular protein was extracted from cells using the NucBuster
Protein Extraction Kit (Merck, 77183) and quantified using Pierce BCA
Protein Assay Kit (Thermo Fisher Scientific, 23225). A total of 40 µg
of cytoplasmic protein was diluted in laemmli buffer (Bio-Rad, 1610747)
with beta-mercaptoethanol, denatured at 95 °C for 5 min and loaded onto
Mini-PROTEAN TGX Gels (Bio-Rad, 4568084). Samples were transferred to
nitrocellulose membranes using the TransBlot Tubro system (Bio-Rad,
170–4270) and protein visualised with Ponceau Stain (G-Biosciences).
Membranes were blocked in 5% BSA TBS-T (5% BSA in 1× Tris-buffered
saline and 0.1% Tween 20) for 1 h and incubated with primary antibodies
overnight in 5% BSA TBS-T (MMP1 1:1000 ab137332, MMP2 1:1000 D4M2N Cell
Signalling, B-actin 1:10,000 ab8226, Abcam). Membranes were washed with
TBS-T and incubated in secondary antibodies (Dnk pAb to Rb IgG IRDye
680RD, ab216779, Goat pAb to Ms IgG IRDye 800CW, ab216772, Abcam) for
1 h at room temperature. Membranes were visualised using the Odyssey
CLx system (Licor).
Melanoma spheroid invasion assay
Melanoma cell lines were cultured in U-bottom 96-well plates (Brand,
781900) at 1 × 10^3 cells per well, spun at 200 × g, and spheroids
allowed to form over 72 h. Culture media was removed from the wells and
100 µl collagen (PureCol, Advanced BioMatrix, 5005-100 ML) added to the
wells. Plates were briefly spun for 15 s at 200 × g and incubated at
37 °C for 1 h to set collagen. For concentration gradient collagen was
diluted to 0.25, 0.5, 1, 1.5, 2 or 2.5 mg/ml in phenol-free DMEM
without FCS. For degraded collagen invasion collagen was diluted to
1.5 mg/ml in phenol-free DMEM containing collagenase I (Gibco) to final
concentration in the collagen of 0.5, 1, 5 or 10 µg/ml. For invasion
using fibroblast secretomes collagen was diluted to 1.5 mg/ml using
secretome collected from various fibroblast cell lines instead of DMEM.
Spheroids were allowed to invade over 72 h and light microscopy
photographs were taken. Images were analysed to quantify the invasive
area around the spheroid by creating a layered mask for the spheroid
core and invasive area, and quantifying the invasive area as a
percentage of the total size of the spheroid in ImageJ software
(1.53c). For single cell invasion analysis, spheroids in the above
conditions were imaged using an Opera Phenix (Perkin Elmer) with a 5×
(0.16 NA) lens. Spheroids were stained with Hoechst 33342 for 1 h prior
to imaging. Stacks of images were acquired through the full depth of
the spheroids, and images were analysed using Columbus software (Perkin
Elmer). A custom analysis pipeline was used to detect individually
invading cells. In brief, a maximal intensity projection of the Hoechst
stain was processed (Flatfield correction: Basic, Guassian blur: 2px)
was used to determine the perimeter of the spheroid, and nuclei were
detected in the remaining portion of the image. Experiments with
varying levels of collagen matrices or collagenase were repeated, and
confirmed by an independent laboratory, blinded for matrix composition.
Organotypic 3D invasion models
Melanoma invasion through fibroblast-modified collagen was assayed
using a protocol adapted from Timpson^[238]74. Briefly, equal numbers
of HFF, UV-HFF, shMMP1-HFF and shMMP1-UV-HFF fibroblasts were mixed
with collagen I, rat tail (Corning, 354236) and cultured in 35 mm
culture dishes. Collagen discs were allowed to contract until they fit
in a 24-well plate. Cell suspensions of Sk-mel-28 and A375 at 4 × 10^4
cells/ml were plated on top of each collagen disc in duplicate for each
fibroblast condition. Cells and collagen were cultured as normal for
approximately 5 days. Collagen discs were then transferred to Falcon
3.0 µm high density PET membrane (Corning, 353092) in Falcon six-well
Deep Well TC-treated Polystyrene Plates (Corning, 355467) to create an
air/liquid interface to drive melanoma invasion into collagen. After 10
days constructs were fixed in 4% PFA, and embedded in paraffin and
stained with H&E and stained for Fibronectin with FN1 antibody (F3648,
Sigma Aldrich). For each construct, the number of cells invading into
the collagen was counted in at least five different fields of view
under light microscopy, by two-independent scorers. Leica SCN400 was
used for whole slide imaging alongside ImageScope software v12.3 (Leica
Microsystems).
Second-harmonic generation imaging
Second-harmonic generation imaging of collagen in 3D organotypic models
was performed using a Leica SP8 upright confocal microscope with 25× NA
0.95 water objectives controlled by Leica LAS X software. The images
were acquired with 880 nm illumination laser line from MaiTai
Ti:Sapphire laser (Spectra Physics) and HyD-RLD detector installed
440/20 nm filter cube (Leica), also used 483/32 nm filter (Leica)
collecting autofluorescence signals at the same time. Z-series optical
sections were collected with a step size of 1 micron driven by SuperZ
galvo stage (Leica). Collagen was quantified in ImageJ by measuring the
mean signal intensity of z-stack sum projections in three equal sized
areas across three fields of views for each collagen disc.
Clinical samples
Three international patient cohorts of primary cutaneous melanoma were
used in this paper. The A cohort (n = 31), the B cohort (n = 222) and
the C cohort (n = 113). (A: Salford Royal NHS Foundation Trust, UK, B:
Instituto Valenciano de Oncología, Valencia, Spain, C: Aix-Marseille
University Hospital, France). Comprehensive clinical outcome was
available for the B and C cohorts; and was collected prospectively at
both institutions. All clinical and pathological information assessed
complied with all relevant ethical regulations for work with human
participants in the UK, Spain and France: Salford cohort A: Local
ethics and UK NHS REC regulation approval, IRAS 16/LO/2098
(16/SW/0323); no patient signed consent required; Spanish cohort B:
Internal Review Board of the Instituto Valenciano Oncología in
Valencia, and verbal informed consent was obtained from all patients
who were alive at the time of the study; French cohort C: Internal
Review Board of the Comite de Protection des Personnes Sud Méditerranée
and Aix-Marseile University Hospital approval, and signed informed
consent was obtained. The analyses were performed by at least two
observers (cohort A: observer A.V. and L.M.; cohort B: V.T., E.N. and
A.V.; cohort C: A.V. and L.M.). Discrepancy in cohorts A and B were
jointly reviewed and consensus agreed. There was high kappa
interobserver agreement in cohort C (>0.65), and all scores were done
blinded for clinical outcome.
Comprehensive clinical outcome was available for the B and C cohorts;
and was collected prospectively at both institutions. The correlation
between solar elastosis and invasion of melanoma cells at the IF was
done in the A cohort in patients with invasive primary melanoma, where
a distinct vertical growth was determined in patients aged ≥55 at the
time of diagnosis. The correlation and histological assessments of the
B and C cohorts were done in primary cutaneous melanomas of patients
aged ≥55 at the time of diagnosis with Breslow ≥1 mm. The clinical
characteristics of the cohorts are described in Supplementary
Table [239]4.
Histological and clinical sample analysis
The histological assessment of primary cutaneous melanomas of the A, B
and C (20%) cohorts, from three international centres, was performed by
at least two observers (cohort A: observer 1 and 2; cohort B: 1, 3 and
4; cohort C: 1 and 2). Discrepancy in cohorts A and B were jointly
reviewed and consensus agreed. There was high interobserver agreement
in cohort C (>0.65), and all scores were done blinded for clinical
outcome. The survival analyses were performed by members of the team
who did not score histological variables. We included all samples with
sufficient material to assess the tumour body, IF and tumour-adjacent
skin. Recurrent tumours were excluded. Non-primary melanomas were
excluded. Solar elastosis was scored as described by Landi^[240]32 et
al., and cutoffs for low, moderate and high solar elastosis; or CSD
noCSD established from the original Landi categories. Landi et al.
established a scoring system for the degree of solar elastosis from
absent to severe using an 11-point score, from 0 to 3+. To generate
binary categories, cases are classified as bearing no chronic sun
damage (noCSD), for scores between 0 and 2−, or CSD for scores 2 to 3+.
Cutoffs for absent (range 0, 0+), low (range 1− to 1+), moderate (range
2− to 2+) and high (3− to 3+) were established from the same
range^[241]75. We assessed the inter-reliability of the binary CSD
classification between two scorers using the kappa statistic, which
showed 0.75 concordance for the B cohort (weighted kappa = 0.75, 95%
CI = 0.69–0.79).
The proportion of melanoma cell invasion at the IF in the dermis was
scored in categories. We assessed the front of the melanoma component
in the dermis that is in direct contact with the dermal matrix, and
scored 0/1: no invasion/minimal invasion: <5% of cells in contact with
the dermis are actively invading the matrix, detaching from the IF of
the melanoma; 2: low invasion: 5–25% of melanoma cells at the IF
detaching from the tumour body; 3: moderate invasion: 25–50% of the IF
is actively detaching from the main VGP and entering deeper structures;
4: high invasion: the majority of cells at the IF are independently
interacting with the matrix, detached from the body of the tumour.
Binary categories were then generated with low invasion (scores 0–2)
and high invasion (3–4), a decision taken before performing survival
analyses. We assessed the inter-reliability of the invasion score
binary classification between two scorers using the kappa statistic,
which showed 0.7 concordance for the C cohort (weighted kappa = 0.7,
95% CI = 0.64–0.77).
The amount of collagen at the IF of the tumour and in tumour-adjacent
skin was scored from H&E slides (C cohort) according to abundance of
distinctly formed collagen bundles. Two-independent pathologists
examined collagen on H&E routine-stained sections of normal skin
surrounding the melanomas and the collagen adjacent/enveloping the IF
of the tumour in the dermis at 100–200× magnification. The following
scoring system was used: collagen absent or low (1): when fully formed
collagen bundles were rare, and the visible collagen was distributed in
haphazard smaller fragments or unidentifiable in an amorphous deposit
of elastotic material. Low collagen (2): when well-defined, undulating
fibres of normal dermal length collagen, are scarce, and a pattern of
elastotic (fragmented or aggregate) material predominates. Medium
collagen (3): well-defined, undulating and organised fibres coexist
with aggregate elastotic material. High collagen (4): well-defined
fibres in organised disposition predominating, with minimal or absent
elastotic material interspersed between the tight bundles
(Supplementary Fig. [242]4m). We assessed the inter-reliability of the
collagen score classification between two scorers using the kappa
statistic, which showed 0.78 concordance for the C cohort (weighted
kappa = 0.78, 95% CI = 0.7–0.81). A subset of samples in cohort A
(n = 16), for which tissue was available, were stained with trichrome
of Masson to quantify collagen. We were unable to score the B cohort IF
due to Covid-19 international restrictions.
Statistical analysis
Data collection was performed with Microsoft Excel 2010 (64-bit,
Microsoft). For in vitro studies, statistical analysis was performed in
GraphPad Prism (version 7.01, GraphPad Software, Inc.). For comparisons
between two groups, Mann–Whitney tests were used and for comparisons
between multiple groups, Kruskal–Wallis with Dunn’s multiple comparison
tests. A p value < 0.05 was considered significant, after correcting
for multiple testing where necessary. For human studies, statistical
analysis was performed in R (version 3.5.1, RStudio v1.2.5001, RStudio
Inc). Association between categorical data was performed with Fisher
exact tests. Survival analysis was performed using survival (version
3.1–12) and survminer (version 0.4.6) packages. For all clinical
cohorts, MSS, overall survival (OS) and PFS were calculated from time
of diagnosis. Univariate grouped survival analysis performed with
Kaplan–Meier and log-rank tests, and multivariate analyses with Cox
regression models, with evaluation of the proportional hazard
assumption. Gene expression (log[2](x + 1) normalised RSEM) and
clinical data from the TCGA SKCM and PANCAN data sets was accessed from
the UCSC Xena data portal ([243]https://xenabrowser.net/datapages/).
Samples were grouped into COL1A1 high or low based on the expression
relative to the median expression of all samples. The MAF score for
each sample was determined by calculating the geometric mean of all
genes in a published MAF signature^[244]37. High and low MAF samples
were classified based on the median signature score.
Reporting summary
Further information on research design is available in the [245]Nature
Research Reporting Summary linked to this article.
Supplementary information
[246]Supplementary Information^ (1.3MB, pdf)
[247]Peer Review File^ (4.3MB, pdf)
[248]41467_2021_22953_MOESM3_ESM.pdf^ (68KB, pdf)
Description of Additional Supplementary Files
[249]Supplementary Data 1^ (1.8MB, xlsx)
[250]Supplementary Data 2^ (12.6KB, xlsx)
[251]Reporting Summary^ (85.4KB, pdf)
Acknowledgements