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Supplementary Figure Legends
Supplementary Figure S1. Plasmid map of the pCI-neo-mCitrine expression vector.
The human cytomegalovirus (hCMV) promoter provides constitutive expression of mCitrine
fluorescent protein.
A neomycin-resistance cassette is included to select for transgene
expression.
Supplementary Figure S2. Gal-1 expression is positively correlated with histological grade
in human glioma.
(A) Patient survival data from the REpository for Molecular BRAin Neoplasia DaTa
(REMBRANDT) database stratified by gal-1 expression among 343 patients diagnosed with
glioma. Gliomas with upregulated gal-1 expression (red curve) carry worse prognosis compared
to those with intermediate (yellow curve; p<0.00000001 vs. upregulated) and downregulated
(green curve; p<0.00000001 vs. upregulated) gal-1 expression. (B) Table comparing LGALS1
expression from the 343 patients represented in panel A stratified by histological grade. As
LGALS1 expression increases the proportion of patients carrying grade IV gliomas increases.
Supplementary Figure S3.
Validation of GL26-Cit cells independently transduced with
gal-1-specific or control shRNA.
(A) Western blot for gal-1 in GL26-Cit cell-lines demonstrating the lack of gal-1 knockdown in
each control cell-line (i.e. EV1, EV2, NT1, NT2; independently transduced replicates) compared
to efficient knockdown in three independent transduction experiments where single-cell GL26Cit-gal1i clones were isolated (i.e. C01; -84.85% knockdown vs. NT, C02; -76.52% knockdown
vs. NT, and C03; -79.55% knockdown vs. NT). (B) GL26-Cit-gal1i clones (C01, C02, C03)
NK Cells Eradicate Gal-1 Deficient Glioma
implanted into the striatum of RAG1-/- mice. Like the mixed GL26-Cit-gal1i population, each
clone also fails to diffusely invade the brain after 48hrs in-vivo and is characterized by a column
of tumor cells that fail to leave the initial site of tumor implantation.
Supplementary Figure S4. GL26-Cit-gal1i cells do not undergo spontaneous cell-death invitro.
GL26-Cit-gal1i and GL26-Cit-NT cells were initially seeded at 1x105 cells/T25 tissue culture
flask (3 independent flasks per group), which were sequentially harvested and analyzed by
Typan blue dye exclusion every 24hrs for 3-days.
Supplementary Figure S5. Morphometric analysis of 48hr post-implantation intracranial
GL26-Cit gliomas (shown in Fig. 1G).
Glioma morphologies were characterized as being either compact (gal-1-), or diffuse (gal1+), as
illustrated by the cartoons to the left and right of the radar chart, respectively. Total tumor area
was subdivided into color-coded cell-clusters based on size, and graphed on the radar chart
shown at center. Color-coded lines on the chart represent cell-clusters of various sizes as
indicated by the key below.
Radial black arrows correspond to a fluorescence confocal
micrograph of the indicated glioma. The point at which each color-coded line intersects the
black arrow specifies the percentage of total tumor area made up of cell-clusters of that size.
Cell-clusters of each glioma sum to 100%. The four GL26-Cit-gal1i tumors (left-hand side of
chart) display a skewed distribution towards large cell-clusters  104 pixels in size (blue colorcoded line) (***p<0.001; 66.10 ± 3.73% in gal-1- tumors vs. 47.10 ± 3.188% in gal-1+ tumors;
two-way ANOVA followed by Tukey’s post-test). Conversely,
the five GL26-Cit tumors (right
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NK Cells Eradicate Gal-1 Deficient Glioma
hand side of chart) exhibit a significantly higher proportion of total tumor area made up of
smaller cell clusters  104 -  103 pixels in size (red color-coded line) (***p<0.001; 17.86 ±
4.07% in gal-1+ tumors vs. 3.42 ± 2.18% in gal-1- tumors; two-way ANOVA followed by
Tukey’s post-test).
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Supplementary Figure S6.
Validating immunodepletion of NK cells and basophils in
RAG1-/- mice.
(A) Flow cytometric analysis with antibodies against NK1.1 and CD3ε, makers of NK cells and
T-cells, respectively, on whole splenocytes harvested from RAG1-/- mice treated with two doses
of normal rabbit serum (left dot plot) or anti-asialo GM1 (right dot plot) each separated by 3-days
(n=6; 3 mice/treatment group). Mice were euthanized 5-days after the initiation of treatment.
The average percentage of NK1.1+/CD3ε- cells (i.e. NK cells) present within each treatment
group as shown. (B) Treatment with anti-asialo GM1 led to a ~86% reduction in splenic-derived
NK cells compared to mice treated with normal rabbit serum after two doses of anti-asialo GM1
(*p=0.0356; 8.42 ± 2.26% normal rabbit serum vs. 1.20 ± 0.49% anti-asialo GM1; unpaired, twotailed, Student’s t-test), thus validating anti-asialo GM1 as an in-vivo NK depletion agent in our
model. (C) Basophil depletion with anti-CD200R3 monoclonal antibodies in RAG1-/- mice
bearing GL26-Cit-gal1i cells after 10dpi. Top epifluorescence show the brains of 3 mice treated
with rat IgG2b isotype control antibodies. Bottom epifluorescence show the brains of 3 mice
treated with anti-CD200R3 (n=6; 3mice/treatment group). Both agents failed to permit gal-1
deficient glioma growth in RAG1-/- mice.
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NK Cells Eradicate Gal-1 Deficient Glioma
Supplementary Figure S7.
Anti-CD3ε immunolabeled brain tissue sections from mouse
A1 (top image) and mouse N2 (bottom image) shown in Fig. 5I.
Numerous tumor-infiltrating CD3ε+ cells can be seen in the brains of mice treated with ASGM1,
while NRS treated mice lack these cells.
Supplementary Figure S8. Splenic-derived RAG1-/- NK cell purity assessment.
Representative histogram of post-sort purity assessment of NK1.1+/CD3ε- FACS sorted RAG1-/splenocytes used for in-vitro NK-mediated cytotoxcity assays.
Isolated NK cells were
consistently >95% pure and >99% viable by Trypan dye exclusion.
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