Hodiset al.,Science 376 , eabi8175 (2022) 29 April 2022 7 of 14
Fig. 4. Genome-edited
melanocytic tumors
share expression
programs with
patient melanomas,
with matching
genetic associations.
(A) Experimental
approach to profile
gene expression from
tumor cells from
xenografts with scRNA-
seq. (B) Intra- and
intergenotype variation
of cancer cell states
in vivo. UMAP embed-
ding of engineered
melanocyte scRNA-seq
profiles (dots) colored
and labeled (boxes)
by genotype and
replicate (legend).
#, CBTP rep. 3 is a
mixture of four tumors
from two mice; all
other replicates are
from a single tumor.
(C) Expression programs.
UMAP embedding as
in (B), colored by per-
cell relative usages
(color bar) of each of
the seven expression
programs identified by
cNMF. (D) Programs
reflect key cellular
processes that vary in
usage across genotypes.
Top: Distribution of
relative program usage
(yaxis) in single cells of
each genotype (xaxis,
color legend). Middle:
Aggregate (pseudobulk)
expression level [Z score
of expression level
(log 2 (TP10K)), color
bar] and percent of
expressing single cells
(white circles) of
the 15 top program-
associated genes (rows)
per genotype (columns).
Bottom: Ranked lists
of gene sets (MSigDB hallmark) ( 83 ) enriched in each program (Mann-WhitneyUtest, FDR< 10−^3 ;*FDR<10−^10 ;**FDR<10−^20 ). (E) Correspondence of in vivo
and in vitro programs. Significance of overlap [-log 10 (Pvalue), Fisher’s exact test, colorbar] of top 50 associated genes between in vivo (rows) and in vitro
(columns, as in Fig. 2) programs. Only overlaps withPvalue < 10−^3 are shown to account for multiple hypothesis testing. (F) Correspondence of in vivo
programs and programs in patient melanomas ( 16 ). Significance of overlap [-log 10 (Pvalue), Fisher’s exact test, colorbar] of top 50 associated genes between
in vivo (rows) and patient (column) programs. Only overlaps withPvalue < 10−^3 are shown to account for multiple hypothesis testing. Associations of
expression programs with either p53 or Wnt pathway gene mutations are noted. (G) Similar usage of melanoma ( 16 ) and in vivo model programs across in vivo
melanoma model single-cell profiles. UMAP embedding [as in (B)], colored by per-cell relative usage of patient melanoma expression programs (right) or
sums of relative usages of in vivo melanocyte expression programs (left).
ABDEFGCUMAP 1UMAP 2CBTA rep. 1
CBTA rep. 2
CBTA rep. 3
CBTP rep. 1
CBTP rep. 2
CBTP rep. 3 #Program 1Program 2
Ox-PhosProgram 1
RibosomalProgram 6
Interferon / TNF/ HypoxiaProgram 7
Protein secretionProgram 2 Program 3Program 3
Interferon / TGFProgram 5 Program 6 Program 71.0
0.8
0.6
0.4
0.2
0.0Program 4Program 4
EMTProgram 5
-catenin / MITFCBTP (6 mo.) rep. 1
CBTP (6 mo.) rep. 2
CBTP3 rep. 2
CBTP3 rep. 1
CBTP3 rep. 3
CBTPA rep. 1
CBTPA rep. 2
CBTPA rep. 30 25 50 75 100Expressing cells (%)Program
usageRibosomal genes –2 –1 0 1 2
(by manual review)- Protein secretion
Program 80.60.80.4
0.2
0.0UsageUsageGene set enrichmentGene set enrichmentcNMF1.0
0.8
0.6
0.4
0.2
0.0Program
usageUMAP 1 UMAP 1 UMAP 1 UMAP 1UMAP 2UMAP 2Myc /mTORC1 / Ox-P
hosMelanocyte
Interferon /p53EMT Myc /mTORC1
S PhaseG2 / MitosisOx-Phos
Ribosomal
Protein secretionInterferon / TGF
EMT
Cell cycle3691215Overlap of top 50associated genes
(–logFisher’s exact test)(p-value);4
68Overlap of top 50
associated genes
(–logFisher’s exact test)(p-value);OxPhos
Common
MITF-low0.60.81.00.4
0.2
0.00.60.81.00.4
0.2
0.00.60.81.00.4
0.2
0.00.60.80.4
0.2
0.00.60.80.4
0.2
0.00.60.80.4
0.2
0.0Program 8
Cell cycle
0.6
0.4
0.2
0.0Associated
Wntgenetic pathwayWnt
Wntp53p53
p53p53p53Ox-Phos
+ -catenin / MITF
+ Protein secretionEMT
+ Interferon / TGF
+ Interf. / TNF / HypoxiaUMAP 1UMAP 2UMAP 2In vivo melanoma
model programsIn vitro melanoma model programsOxPhosMITF-lowUMAP 1Patient
melanoma programs
(Alkallas et al. 2020)Patient melanoma programs (Alkallas et al. 2020)CBTA rep. 1CBTA rep. 2
CBTA rep. 3CBTP rep. 1
CBTP rep. 2CBTP rep. 3 #
CBTP (6 mo.) rep. 1CBTP (6 mo.) rep. 2
CBTP3 rep. 2CBTP3 rep. 1CBTP3 rep. 3
CBTPA rep. 1CBTPA rep. 2
CBTPA rep. 3Expression level
(logz-norm pseudo-bulk
2 (TP10K))Mutant melanocytes
cultured in vitro
Injection of cells of a single genotype
into dermis of immunodeficient mouse36–151 daysTumor growth isolationTumor Dissociationof cells scRNA-Seq melanocytesAnalysis:- Myc targets v2
- Xenobiotic metabolism
- mTORC1 signaling*
- Fatty acid metabolism*
- Myc targets v1*
- Cholesterol homeostasis
- DNA repair
- ROS pathway
- Adipogenesis
- Ox-Phos**
- TGF signaling
- Interferon response*
- Interferon response*
- UV response down
- Apical junction
- Epithelial-mesenchymal
transition (EMT)** - DNA repair*
- Myc targets v2*
- Ox-Phos**
- Myc targets v1**
- G2M checkpoint**
- E2F targets**
- ROS pathway
- mTORC1 signaling
- Spermatogenesis
- Fatty acid metabolism
- Mitotic spindle
All: adj. p < 1 × 10–3; *adj. p < 1 × 10–10; **adj. p < 1 × 10–20- IL6 JAK STAT3 signaling
- Inflammatory response
- Hypoxia*
- TNF signaling via NFB**
- Interferon response**
- Allograft rejection
- Interferon response**
LOXL1TGFBI
DCBLD2RCAN1
MEG3CNN1
SLC9A3R2KCNMA1
PTGESRTN1
TAGLNLMO4
PMEPA1TAGLN2
PDLIM7TUBA1BUBE2S
UBE2CCENPF
HMGN2CDKN3
HMGB2CKS1B
HMGB1PTTG1
TOP2APBK
CCNB1BIRC5
CKS2RPS27LVAMP8
NDUFB1AEBP1
MT-ND3S100A4
AHNAK2CCR10
ALDH1A1RPS29
SNHG25SMIM4
ATP5MECD81
GPNMBISG15IER3
LDHAIFI6
TIMP3ISG20
BTG1OASL
IFITM1MX1
IFI44LLY6E
IFIT1IFI27
OAS1SELENONGAL3ST1
VAT1LGPC1
COL22A1FREM2
ANKHAQP1
SLC20A1SDC3
LAMA4DAG1
PCDH9THBS2
LST1LINC00520CTNNB1
MT-ND1NBL1
GUK1MIF
RPS7BRI3
MT-CO1RPL29
MITFSCD
PRKD3RPS2
MYO5ACAPGUBB
GSTP1CTSL
RAB38PMEL
IGFBP7FDPS
GSTO1SKP1
PAEPQPCT
NQO1TPI1
MGST3EEF1A1FABP7
BCANAPOE
RPL23AGNG7
RPS4XRBP7
RPS24CIRBP
SPP1TPT1
SERPINF1FXYD1
NACA-catenin / MITF
Interferon / TNF / HypoxiaOx-PhosRibosomal
Protein secretionInterferon / TGF
EMT
Cell cycle-catenin / MITFInterferon / TNF / HypoxiaIn vivo programsIn vivo programsRESEARCH | RESEARCH ARTICLE