Analysis of intracellular Transferrin trafficking . Fibroblasts fed with fluorescently labeled Transferrin were co - stained with LysoTracker ( G ) or an antibody against LAMP2 ( H ) . Representative images ( left ) of fibroblasts transduced with control virus , with magnified areas demonstrating co - localized puncta ( arrowheads ) . Scale bars 10 μm or 2 μm ( inserts ) . Quantifications ( right ) of the co - localization between Transferrin and LysoTracker or LAMP2 , respectively , expressed as Pearson correlation coefficients . Colored horizontal bars indicate the median values and whiskers 5 and 95 percentiles ( n = 10 optical sections from 3 independent experiments ) .

O O O B-GENEPROD O O B-CELL_TYPE O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O B-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of intracellular Transferrin trafficking. Fibroblasts fed with fluorescently labeled Transferrin were co-stained with LysoTracker (G) or an antibody against LAMP2 (H). Representative images (left) of fibroblasts transduced with control virus, with magnified areas demonstrating co-localized puncta (arrowheads). Scale bars 10 μm or 2 μm (inserts). Quantifications (right) of the co-localization between Transferrin and LysoTracker or LAMP2, respectively, expressed as Pearson correlation coefficients. Colored horizontal bars indicate the median values and whiskers 5 and 95 percentiles (n=10 optical sections from 3 independent experiments).


Confocal micrographs of fibroblasts transduced with control or VPS16 - expressing lentivirus and labeled with LysoTracker . Scale bar , 20 μm . Quantification of LysoTracker - stained puncta number and intensities ( ca 80 cells analyzed for each of n = 3 biological replicates ) .

B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O B-GENEPROD O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

Confocal micrographs of fibroblasts transduced with control or VPS16-expressing lentivirus and labeled with LysoTracker. Scale bar, 20 μm. Quantification of LysoTracker-stained puncta number and intensities (ca 80 cells analyzed for each of n=3 biological replicates).


Representative immunoblots ( bottom ) and summary quantifications ( top ) of the levels of LAMP1 ( C ) and LAMP2 ( D ) in fibroblasts , normalized to levels of actin ( ACTB ) and expressed as % of controls ( n = 3 biological replicates ) . Analysis of lysosomal Cathepsin D ( CtsD ) processing by immunoblotting . Immunoblot to detect CtsD isoforms ( left ) and quantification ( right ) of processing , calculated as the percentage of CtsD divided by the sum of all isoforms ( n = 3 biological replicates ) .

O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

Representative immunoblots (bottom) and summary quantifications (top) of the levels of LAMP1 (C) and LAMP2 (D) in fibroblasts, normalized to levels of actin (ACTB) and expressed as % of controls (n=3 biological replicates). Analysis of lysosomal Cathepsin D (CtsD) processing by immunoblotting. Immunoblot to detect CtsD isoforms (left) and quantification (right) of processing, calculated as the percentage of CtsD divided by the sum of all isoforms (n=3 biological replicates).


Analysis of autophagosome numbers in fibroblasts transfected to express mRFP - GFP - LC3 . ( F ) Representative confocal micrographs ( optical sections ) of fibroblasts imaged live under basal or serum - starved conditions . Scale bar , 20 μm .

O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O

Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. (F) Representative confocal micrographs (optical sections) of fibroblasts imaged live under basal or serum-starved conditions. Scale bar, 20 μm.


Analysis of autophagosome numbers in fibroblasts transfected to express mRFP - GFP - LC3 . Quantification of autophagosomes , defined as green + red puncta ( G ) and autophago - lysosomes , calculated as the number of red ( but not green ) puncta divided by the total number of puncta . Colored horizontal bars denote median values and whiskers 5 and 95 percentiles ( n = 50 - 113 cells from a total of 3 independent experiments ; for exact values see Appendix Table S1 ) .

O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. Quantification of autophagosomes, defined as green + red puncta (G) and autophago-lysosomes, calculated as the number of red (but not green) puncta divided by the total number of puncta. Colored horizontal bars denote median values and whiskers 5 and 95 percentiles (n=50-113 cells from a total of 3 independent experiments; for exact values see Appendix Table S1).


Myelination , as reported using the Tg ( mbp : gfp - caax ) transgenic , is significantly reduced in vps16 crispants . The yellow box indicates the region assessed in D . Scale bar 100 μm . Bar graphs represent data as mean ± SEM . Statistical comparisons by Mann - Whitney U tests ( n = 6 ) . * * p < 0 . 01 .

B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Myelination, as reported using the Tg(mbp:gfp-caax) transgenic, is significantly reduced in vps16 crispants. The yellow box indicates the region assessed in D. Scale bar 100 μm. Bar graphs represent data as mean ±SEM. Statistical comparisons by Mann-Whitney U tests (n=6). ** p < 0.01.


LysoTracker signal colocalizes with the astroglial reporter slc1a2b : citrine and does not appear to overlap with the pan - neuronal reporter elavl3 : GCaMP5G .

B-SMALL_MOLECULE O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-SMALL_MOLECULE O

LysoTracker signal colocalizes with the astroglial reporter slc1a2b:citrine and does not appear to overlap with the pan-neuronal reporter elavl3:GCaMP5G.


b . In vitro activity . Left y - axis : 13C2 - oxalate in mM , right y - axis : 13C - formate in mM , x - axis : time in minutes . SYNB8802 ( blue , circles , solid line : oxalate , dotted line : formate ) The control EcN ( pink , triangle , solid line : oxalate , dotted line : formate ) . Three biological replicates were run and plotted separately .

O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O

b. In vitro activity. Left y-axis: 13C2-oxalate in mM, right y-axis: 13C-formate in mM, x-axis: time in minutes. SYNB8802 (blue, circles, solid line: oxalate, dotted line: formate) The control EcN (pink, triangle, solid line: oxalate, dotted line: formate). Three biological replicates were run and plotted separately.


c . In vitro simulation ( IVS ) . Left y - axis : Rate of oxalate degradation in μmol / h / 109 cells . X - axis = time in hours . Left X - axis 0 - 4 hours , Right X - axis 6 - 48 h . Three biological replicates were run and plotted separately . Data points in the orange box represent incubation in simulated gastric fluid ( SGF ) . Data points in the light blue box represent incubation in simulated intestinal fluid ( SIF ) . Data points in the pink box represent incubation in simulated colonic fluid ( SCF ) .

O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O

c. In vitro simulation (IVS). Left y-axis: Rate of oxalate degradation in μmol/h/109 cells. X-axis = time in hours. Left X-axis 0 - 4 hours, Right X-axis 6 - 48 h. Three biological replicates were run and plotted separately. Data points in the orange box represent incubation in simulated gastric fluid (SGF). Data points in the light blue box represent incubation in simulated intestinal fluid (SIF). Data points in the pink box represent incubation in simulated colonic fluid (SCF).


b . Acute mouse study of UOx lowering with SYNB8802 . The y - axis shows the urinary 13 C2 - oxalate normalized to creatine . The x - axis shows the two groups , EcN ( control , pink ) vs SYNB8802AbxR ( light blue ; n = 15 mice per group ) . Individual dots represent each metabolic cage ( n = 3 mice per metabolic cage ) . Error bars are calculated as SEM . ( unpaired t - test with Welch ' s correction , * * * * p < 0 . 0001 )

O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O B-TISSUE B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

b. Acute mouse study of UOx lowering with SYNB8802. The y-axis shows the urinary 13 C2-oxalate normalized to creatine. The x-axis shows the two groups, EcN (control, pink) vs SYNB8802AbxR (light blue; n = 15 mice per group). Individual dots represent each metabolic cage (n = 3 mice per metabolic cage). Error bars are calculated as SEM. (unpaired t-test with Welch's correction, **** p < 0.0001)


d . Nonhuman primate model development study . The y - axis shows urinary oxalate normalized to creatine . X - axis shows baseline urinary oxalate levels after overnight fast , and urinary oxalate measured 6 hours post administration of either a spinach preparation ( triangle , pink ) or water ( circle , blue ) ( n = 6 for each group ) . Individual dots represent individual animals . Error bars are calculated as SEM . ( Two - way ANOVA followed by Sidak ' s multiple comparison analysis , * * * * p < 0 . 0001 ) .

O O O B-ORGANISM O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

d. Nonhuman primate model development study. The y-axis shows urinary oxalate normalized to creatine. X-axis shows baseline urinary oxalate levels after overnight fast, and urinary oxalate measured 6 hours post administration of either a spinach preparation (triangle, pink) or water (circle, blue) (n=6 for each group). Individual dots represent individual animals. Error bars are calculated as SEM. (Two-way ANOVA followed by Sidak's multiple comparison analysis, **** p < 0.0001).


f . Urinary recovery of oxalate in nonhuman primates . The y - axis shows change in urinary oxalate from vehicle control . The x - axis shows vehicle ( control , grey ) and increasing doses of SYNB8802AbxR ( n = 24 for vehicle , n = 6 for treatment groups ) . Individual dots represent normalized urinary oxalate for each individual animal . Error bars are calculated as SEM . ( One - way ANOVA followed by Tukey ' s multiple comparison analysis , * p < 0 . 05 )

O O B-TISSUE O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f. Urinary recovery of oxalate in nonhuman primates. The y-axis shows change in urinary oxalate from vehicle control. The x-axis shows vehicle (control, grey) and increasing doses of SYNB8802AbxR (n = 24 for vehicle, n = 6 for treatment groups). Individual dots represent normalized urinary oxalate for each individual animal. Error bars are calculated as SEM. (One-way ANOVA followed by Tukey's multiple comparison analysis, * p < 0.05)


g . Urinary recovery of 13C2 oxalate in nonhuman primates . The y - axis shows change in urinary 13C2 - oxalate from vehicle control . The x - axis shows vehicle ( control , grey ) and increasing doses of SYNB8802AbxR ( n = 24 for vehicle , n = 6 for treatmentgroups ) . Individual dots represent normalized urinary 13C2 - oxalate for each individual animal . Error bars are calculated as SEM . ( One - way ANOVA followed by Tukey ' s multiple comparison analysis , * * p < 0 . 01 )

O O B-TISSUE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O B-TISSUE B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

g. Urinary recovery of 13C2 oxalate in nonhuman primates. The y-axis shows change in urinary 13C2-oxalate from vehicle control. The x-axis shows vehicle (control, grey) and increasing doses of SYNB8802AbxR (n = 24 for vehicle, n = 6 for treatmentgroups). Individual dots represent normalized urinary 13C2-oxalate for each individual animal. Error bars are calculated as SEM. (One-way ANOVA followed by Tukey's multiple comparison analysis, ** p < 0.01)


( B ) Time constants of decay , τdecay , of the STC isolated pharmacologically in WT and FHM2 KI mice . Top , superimposed representative traces of the inward current evoked in an astrocyte ( held at - 90 mV ) by a single pulse stimulation ( indicated by the arrow ) in a WT slice , before ( trace a ) and after ( trace b ) application of a saturating concentration of the GluT inhibitor TFB - TBOA ( TBOA ) . The STC was obtained by subtracting the residual current remaining in the presence of TBOA from the total inward current ( trace a - b ) ; the decay of the STC was best fitted by a single exponential function with τdecay = 6 . 53 ms ( in red ) . The bar plot shows the average values of τdecay of the STC isolated pharmacologically in cortical slices ( n = 13 ) from P22 - 23 WT ( N = 7 ) and KI mice ( n = 9 ; N = 3 ) . STC τdecay is 17 % higher in KI compared to WT astrocytes ( unpaired t - test : P = 0 . 003 ) . Hereafter , n indicates the number of slices and N indicates the number of mice .

O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O

(B) Time constants of decay, τdecay, of the STC isolated pharmacologically in WT and FHM2 KI mice. Top, superimposed representative traces of the inward current evoked in an astrocyte (held at -90 mV) by a single pulse stimulation (indicated by the arrow) in a WT slice, before (trace a) and after (trace b) application of a saturating concentration of the GluT inhibitor TFB-TBOA (TBOA). The STC was obtained by subtracting the residual current remaining in the presence of TBOA from the total inward current (trace a-b); the decay of the STC was best fitted by a single exponential function with τdecay = 6.53 ms (in red). The bar plot shows the average values of τdecay of the STC isolated pharmacologically in cortical slices (n=13) from P22-23 WT (N=7) and KI mice (n=9; N=3). STC τdecay is 17% higher in KI compared to WT astrocytes (unpaired t-test: P = 0.003). Hereafter, n indicates the number of slices and N indicates the number of mice.


( C ) τdecay of the STC isolated using an exponential waveform approximating the average TBOA - insensitive current in WT and KI mice . Top trace , average normalized TBOA - insensitive current obtained by pooling the normalized TBOA - insensitive currents recorded in 16 WT and KI cells . This current was best fitted by an exponentially rising function ( 1 - exp ( - t / τrise ) ) with τrise = 2 . 35 ms ( in green ) . The STC was obtained by subtracting from the total current elicited in the astrocyte ( a : same representative trace as in ( B ) ) the exponential function A ( 1 - exp ( - t / τrise ) ) with τrise = 2 . 35 ms and A equal to the maximal current measured in the astrocyte at about 60 ms after stimulation ( trace c ) ; the decay of the STC ( trace a - c ) was best fitted by a single exponential function with τdecay = 6 . 49 ms ( in red ) . The bar plot shows the average values of τdecay of the STC isolated as shown in the top panel in cortical slices from P22 - 23 WT ( n = 28 ; N = 11 ) and KI mice ( n = 27 ; N = 9 ) . STC τdecay is 21 % higher in KI compared to WT astrocytes ( unpaired t - test : P < 0 . 0001 ) .

O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O

(C) τdecay of the STC isolated using an exponential waveform approximating the average TBOA-insensitive current in WT and KI mice. Top trace, average normalized TBOA-insensitive current obtained by pooling the normalized TBOA-insensitive currents recorded in 16 WT and KI cells. This current was best fitted by an exponentially rising function (1-exp (-t/τrise)) with τrise= 2.35 ms (in green). The STC was obtained by subtracting from the total current elicited in the astrocyte (a: same representative trace as in (B)) the exponential function A(1-exp (-t/τrise)) with τrise = 2.35 ms and A equal to the maximal current measured in the astrocyte at about 60 ms after stimulation (trace c); the decay of the STC (trace a-c) was best fitted by a single exponential function with τdecay = 6.49 ms (in red). The bar plot shows the average values of τdecay of the STC isolated as shown in the top panel in cortical slices from P22-23 WT (n = 28; N=11) and KI mice (n = 27; N=9). STC τdecay is 21% higher in KI compared to WT astrocytes (unpaired t-test: P < 0.0001).


( A ) Isolation of the STC elicited by the last pulse of a high - frequency train of action potentials . Left : superimposed representative traces of the inward current evoked in an astrocyte ( held at - 90 mV ) by extracellular stimulation with a train of 10 pulses ( trace a : black ) and a train of 9 pulses ( trace b : brown ) at 50 Hz in a WT cortical slice . The inward current elicited by the 10th pulse was obtained by subtracting the current elicited by 9 pulses from that elicited by 10 pulses ( trace a - b ) . Right : The STC elicited by the 10th pulse ( trace a - b - c ) was obtained by subtracting the exponential function that simulates the TBOA - insensitive current elicited by a single pulse ( trace c , obtained as in Figure 1C ) to the 10 - 9 pulses difference current ( trace a - b ) . The decay of the STC elicited by the 10th pulse was best fitted by a single exponential function with τdecay = 8 . 04 ms ( in red ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Isolation of the STC elicited by the last pulse of a high-frequency train of action potentials. Left: superimposed representative traces of the inward current evoked in an astrocyte (held at -90 mV) by extracellular stimulation with a train of 10 pulses (trace a: black) and a train of 9 pulses (trace b: brown) at 50 Hz in a WT cortical slice. The inward current elicited by the 10th pulse was obtained by subtracting the current elicited by 9 pulses from that elicited by 10 pulses (trace a-b). Right: The STC elicited by the 10th pulse (trace a-b-c) was obtained by subtracting the exponential function that simulates the TBOA-insensitive current elicited by a single pulse (trace c, obtained as in Figure 1C) to the 10-9 pulses difference current (trace a-b). The decay of the STC elicited by the 10th pulse was best fitted by a single exponential function with τdecay = 8.04 ms (in red).


( B ) τdecay of the STC elicited by the 10th pulse of 50 Hz ( τ10 ( 50 Hz ) , left panel ) and 100 Hz ( τ10 ( 100 Hz ) , right panel ) trains in layer 1 astrocytes in acute cortical slices from P22 - 23 WT ( n = 23 ; N = 10 for 50 Hz ; n = 18 ; N = 8 for 100 Hz ) and KI ( n = 21 ; N = 9 for 50 Hz and n = 14 ; N = 7 for 100 Hz ) mice . The STC elicited by the 10th pulse was obtained as described in ( A ) . STC τ10 ( 50 Hz ) and τ10 ( 100 Hz ) are 30 % and 37 % higher in KI compared to WT mice , respectively ( unpaired t - test : P < 0 . 0001 in both cases ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

(B) τdecay of the STC elicited by the 10th pulse of 50 Hz (τ10 (50 Hz), left panel) and 100 Hz (τ10 (100 Hz), right panel) trains in layer 1 astrocytes in acute cortical slices from P22-23 WT (n = 23; N=10 for 50 Hz; n = 18; N=8 for 100 Hz) and KI (n = 21; N=9 for 50 Hz and n = 14; N=7 for 100 Hz) mice. The STC elicited by the 10th pulse was obtained as described in (A). STC τ10 (50 Hz) and τ10 (100 Hz) are 30% and 37% higher in KI compared to WT mice, respectively (unpaired t-test: P < 0.0001 in both cases).


( A ) Simultaneous visualization of GLT - 1a + ( green ) and VGLUT1 + puncta ( red ) in first somatic sensory cortex ( SI ) of a WT and a KI mouse ( P35 ) . Arrows point to some GLT - 1a + puncta overlaying with VGLUT1 + puncta ( i . e . , GLT - 1a / VGLUT1 related puncta ) ; framed regions ( enlarged below ) are examples of GLT - 1a / VGLUT1 related puncta ( arrowheads ) . All microscopic fields are from layers II / III . Scale bars : 3 . 5 µm for left and right panels and 1 µm for enlarged framed areas . ( B ) Percentage and size of GLT - 1a + puncta overlaying with VGLUT1 in P35 WT and KI mice . Left , percentage of GLT - 1a + puncta overlaying with VGLUT1 is comparable in WT and KI mice ( data were obtained from 14 and 18 fields of 20 µm x 20 µm from 2 WT and 2 KI mice ( 4 sections / animal ) , respectively ) ( unpaired t - test : P = 0 . 52 ) . Right , size of GLT - 1a / VGLUT1 related puncta is 19 % reduced in KI mice ( 169 GLT - 1a + puncta analyzed from 2 mice ) compared to WT ( 174 GLT - 1a + puncta analyzed from 2 mice ) ( Mann - Withney U test : P = 0 . 016 ) .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O

(A) Simultaneous visualization of GLT-1a+ (green) and VGLUT1+ puncta (red) in first somatic sensory cortex (SI) of a WT and a KI mouse (P35). Arrows point to some GLT-1a+ puncta overlaying with VGLUT1+ puncta (i.e., GLT-1a/VGLUT1 related puncta); framed regions (enlarged below) are examples of GLT-1a/VGLUT1 related puncta (arrowheads). All microscopic fields are from layers II/III. Scale bars: 3.5 µm for left and right panels and 1 µm for enlarged framed areas.(B) Percentage and size of GLT-1a+ puncta overlaying with VGLUT1 in P35 WT and KI mice. Left, percentage of GLT-1a+ puncta overlaying with VGLUT1 is comparable in WT and KI mice (data were obtained from 14 and 18 fields of 20 µm x 20 µm from 2 WT and 2 KI mice (4 sections/animal), respectively) (unpaired t-test: P = 0.52). Right, size of GLT-1a/VGLUT1 related puncta is 19 % reduced in KI mice (169 GLT-1a+ puncta analyzed from 2 mice) compared to WT (174 GLT-1a+ puncta analyzed from 2 mice) (Mann-Withney U test: P = 0.016).


( A ) Distribution of GLT - 1agold particles in astrocytic processes contacting asymmetric synapses ( perisynaptic astrocytic processes : PAP ) in SI of 5 WT and 5 KI ( 2 male , 3 female ) mice ( P34 - 35 ) . In cortical PAP of KI mice the density ( particles / µm2 ) of total and membrane - associated gold particles ( arrowheads ) are reduced compared to WT mice , whereas the density of cytoplasmicgold particles ( arrows ) is comparable to that in WT ( cf Table I for numerical values and statistics ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O B-SUBCELLULAR O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-TISSUE B-SUBCELLULAR O O B-ORGANISM O O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O

(A) Distribution of GLT-1agold particles in astrocytic processes contacting asymmetric synapses (perisynaptic astrocytic processes: PAP) in SI of 5 WT and 5 KI (2 male, 3 female) mice (P34-35). In cortical PAP of KI mice the density (particles/µm2) of total and membrane-associated gold particles (arrowheads) are reduced compared to WT mice, whereas the density of cytoplasmicgold particles (arrows) is comparable to that in WT (cf Table I for numerical values and statistics).


( B ) Distribution of GLT - 1agold particles in excitatory axon terminals forming asymmetric synaptic contact ( AxT ) in SI of P34 - 35 WT and KI mice . In cortical AxT of KI and WT mice , GLT - 1a density is comparable ( arrowheads : membrane - associated gold particles ; arrows : cytoplasmicgold particles ) ( cf Table I ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-ORGANISM O O B-TISSUE O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O

(B) Distribution of GLT-1agold particles in excitatory axon terminals forming asymmetric synaptic contact (AxT) in SI of P34-35 WT and KI mice. In cortical AxT of KI and WT mice, GLT-1a density is comparable (arrowheads: membrane-associated gold particles; arrows: cytoplasmicgold particles) (cf Table I).


Left , superimposed representative traces of the normalized inward current evoked in a WT ( black trace ) and a KI corticalastrocyte ( blue trace ) by extracellular stimulation with a train of 10 pulses at 50 Hz in cortical slices . The inset shows in an expanded time scale the portion of the traces indicated by the dotted line . The slowly decaying current ( τdecay = 2 . 00 s and 3 . 04 s for the WT and KI trace , respectively ) is largely a [ K + ] e - dependent K + current ( IK ) whose decay kinetics provide a measure of the rate of K + clearance by astrocytes ( see text ) . Right , time constant of decay of IK elicited by trains of 10 pulses at 50 Hz in corticalastrocytes from P22 - 23 WT ( n = 21 ; N = 12 ) and KI mice ( n = 20 ; N = 8 ) . IK τdecay is 22 % higher in KI compared to WT astrocytes ( unpaired t - test : P = 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O

Left, superimposed representative traces of the normalized inward current evoked in a WT (black trace) and a KI corticalastrocyte (blue trace) by extracellular stimulation with a train of 10 pulses at 50 Hz in cortical slices. The inset shows in an expanded time scale the portion of the traces indicated by the dotted line. The slowly decaying current (τdecay = 2.00 s and 3.04 s for the WT and KI trace, respectively) is largely a [K+]e-dependent K+current (IK) whose decay kinetics provide a measure of the rate of K+ clearance by astrocytes (see text). Right, time constant of decay of IK elicited by trains of 10 pulses at 50 Hz in corticalastrocytes from P22-23 WT (n = 21; N=12) and KI mice (n = 20; N=8). IK τdecay is 22% higher in KI compared to WT astrocytes (unpaired t-test: P = 0.001).


( A ) Images of a cortical slice before ( t = 0 ) and after ( t = 15 . 6 s ) pressure ejection of a high KCl pulse that elicits a CSD ( top panels ) , showing that the propagating CSD is associated with a change in light transmittance . Scale bar : 500 µm . The traces below show representative changes in intrinsic optical signal ( IOS ) relative to background measured in a WT and a KI cortical slice during CSD propagation at increasing times and distances from the KCl puff , as indicated in color code in the right image on the top . The velocity of CSD propagation , obtained from the rate of horizontal spread of the change in IOS , in these two representative WT and KI slices is 3 . 11 and 4 . 14 mm / min , respectively . ( B ) Stimulation threshold for CSD induction ( CSD threshold ) and rate of CSD propagation ( CSD velocity ) in WT ( n = 24 ; N = 3 ) and KI ( n = 27 ; N = 8 ) cortical slices from P22 - 23 mice . CSD threshold is expressed as duration of the first KCl pulse eliciting a CSD . CSD threshold is 28 % lower ( Mann Whitney U test , P < 0 . 0001 ) and CSD velocity 21 % higher ( unpaired t - test : P < 0 . 0001 ) in KI compared to WT mice .

O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O

(A) Images of a cortical slice before (t = 0) and after (t = 15.6 s) pressure ejection of a high KCl pulse that elicits a CSD (top panels), showing that the propagating CSD is associated with a change in light transmittance. Scale bar: 500 µm. The traces below show representative changes in intrinsic optical signal (IOS) relative to background measured in a WT and a KI cortical slice during CSD propagation at increasing times and distances from the KCl puff, as indicated in color code in the right image on the top. The velocity of CSD propagation, obtained from the rate of horizontal spread of the change in IOS, in these two representative WT and KI slices is 3.11 and 4.14 mm/min, respectively.(B) Stimulation threshold for CSD induction (CSD threshold) and rate of CSD propagation (CSD velocity) in WT (n = 24; N=3) and KI (n = 27; N=8) cortical slices from P22-23 mice. CSD threshold is expressed as duration of the first KCl pulse eliciting a CSD. CSD threshold is 28% lower (Mann Whitney U test, P < 0.0001) and CSD velocity 21% higher (unpaired t-test: P< 0.0001) in KI compared to WT mice.


( A ) Western blottings of GLT - 1a in cortical crude synaptic membranes of P39 WT mice following Cef treatment for 8 days . GLT - 1a levels are significantly increased in mice treated with Cef ( Cef WT , N = 4 ) compared to control saline - injected mice ( Ctr WT , N = 4 ) ( Mann - Withney U test : P = 0 . 028 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O B-SUBCELLULAR I-SUBCELLULAR O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O

(A) Western blottings of GLT-1a in cortical crude synaptic membranes of P39 WT mice following Cef treatment for 8 days. GLT-1a levels are significantly increased in mice treated with Cef (Cef WT, N = 4) compared to control saline-injected mice (Ctr WT, N = 4) (Mann-Withney U test: P = 0.028).


( B ) Visualization of GLT - 1a + puncta ( green ) and VGLUT1 + puncta ( red ) in sections from SI of P45 - 46 WT mice that were treated with saline ( Ctr WT ) or Cef ( Cef WT ) . Arrows point to some GLT - 1a / VGLUT1 related puncta . Framed regions ( enlarged below ) are examples of GLT - 1a / VGLUT1 related puncta ( arrowheads ) . Right , Cef treatment increased significantly the size of GLT - 1a + puncta overlaying VGLUT1 ( 142 and 186 GLT - 1a + puncta analyzed from 4 Ctr WT and 4 Cef WT mice , respectively ; 3 sections / animal ) ( Mann - Withney U test : P < 0 . 0001 ) . All microscopic fields are from layers II / III . Scale bars : 3 . 5 µm for left and right panels and 1 µm for enlarged framed areas .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Visualization of GLT-1a+ puncta (green) and VGLUT1+ puncta (red) in sections from SI of P45-46 WT mice that were treated with saline (Ctr WT) or Cef (Cef WT). Arrows point to some GLT-1a/VGLUT1 related puncta. Framed regions (enlarged below) are examples of GLT-1a/VGLUT1 related puncta (arrowheads). Right, Cef treatment increased significantly the size of GLT-1a+ puncta overlaying VGLUT1 (142 and 186 GLT-1a+ puncta analyzed from 4 Ctr WT and 4 Cef WT mice, respectively; 3 sections/animal) (Mann-Withney U test: P < 0.0001). All microscopic fields are from layers II/III. Scale bars: 3.5 µm for left and right panels and 1 µm for enlarged framed areas.


( C ) CSD threshold and CSD velocity in cortical slices from P30 - 33 KI mice that were injected with saline ( Ctr KI , n = 38 ; N = 7 ) or Cef ( Cef KI , n = 31 ; N = 6 ) . CSD threshold is 12 % higher ( Mann - Withney U test : P = 0 . 02 ) in Cef - treated compared to saline - treated KI mice . CSD velocity is not altered by Cef treatment ( P = 0 . 90 ) .

O O O O O O O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

(C) CSD threshold and CSD velocity in cortical slices from P30-33 KI mice that were injected with saline (Ctr KI, n = 38; N=7) or Cef (Cef KI, n = 31; N=6). CSD threshold is 12% higher (Mann-Withney U test: P = 0.02) in Cef-treated compared to saline-treated KI mice. CSD velocity is not altered by Cef treatment (P = 0.90).


( A ) Distribution of GLT - 1agold particles in PAP of saline - injected ( Ctr KI , N = 4 ) and Cef - treated KI mice ( Cef KI , N = 4 ) ( P45 - 46 ) . Cef treatment does not modify the density of total , cytoplasmic ( arrows ) and membrane - associated ( arrowheads ) gold particles in PAP of KI mice ( cf Table II ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O B-ORGANISM O O O O O O

(A) Distribution of GLT-1agold particles in PAP of saline-injected (Ctr KI, N=4) and Cef-treated KI mice (Cef KI, N=4) (P45-46). Cef treatment does not modify the density of total, cytoplasmic (arrows) and membrane-associated (arrowheads) gold particles in PAP of KI mice (cf Table II).


( B ) Distribution of GLT - 1agold particles in AxT in SI of Ctr KI and Cef KI KI mice . The density of the membrane - associated gold particles ( arrowheads ) is increased in Cef - treated KI mice ( cf Table II ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O O

(B) Distribution of GLT-1agold particles in AxT in SI of Ctr KI and Cef KI KI mice. The density of the membrane-associated gold particles (arrowheads) is increased in Cef-treated KI mice (cf Table II).


( C ) Western blottings of GLT - 1a in cortical crude synaptic membranes of P39 KI mice following Cef treatment . GLT - 1a levels are similar in Cef - treated ( Cef KI , N = 4 ) and and saline - injected ( Ctr KI , N = 4 ) ( Mann - Withney U test : P = 0 . 83 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O B-SUBCELLULAR I-SUBCELLULAR O O O B-ORGANISM O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Western blottings of GLT-1a in cortical crude synaptic membranes of P39 KI mice following Cef treatment. GLT-1a levels are similar in Cef-treated (Cef KI, N=4) and and saline-injected (Ctr KI, N=4) (Mann-Withney U test: P = 0.83).


( D ) Visualization of GLT - 1a + puncta ( green ) and VGLUT1 + puncta ( red ) in KI mice that received saline ( Ctr KI ) and in Cef - treated KI mice ( Cef KI ) ( P45 - 46 ) . Arrows point to some GLT - 1a / VGLUT1 related puncta ; framed regions are examples of GLT - 1a / VGLUT1 related puncta ( arrowheads ) . Right , Cef treatment does not increase the size of GLT - 1a + puncta overlaying with VGLUT1 ( 160 and 175 GLT - 1a + puncta analyzed from the same 4 Ctr KI and 4 Cef KI used for post - embedding electron microscopy analysis ; 3 sections / animal ) ( Mann - Withney U test : P = 0 . 31 ) . Scale bar : 3 . 5 µm for left and right panels and 1 µm for enlarged framed areas . All microscopic fields are from layers II / III .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Visualization of GLT-1a+ puncta (green) and VGLUT1+ puncta (red) in KI mice that received saline (Ctr KI) and in Cef-treated KI mice (Cef KI) (P45-46). Arrows point to some GLT-1a/VGLUT1 related puncta; framed regions are examples of GLT-1a/VGLUT1 related puncta (arrowheads). Right, Cef treatment does not increase the size of GLT-1a+ puncta overlaying with VGLUT1 (160 and 175 GLT-1a+ puncta analyzed from the same 4 Ctr KI and 4 Cef KI used for post-embedding electron microscopy analysis; 3 sections/animal) (Mann-Withney U test: P = 0.31). Scale bar: 3.5 µm for left and right panels and 1 µm for enlarged framed areas. All microscopic fields are from layers II/III.


( A ) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22 - 23 WT mice before ( Ctr WT ) and after application of 2 . 5 µM DL - TBOA ( TBOA 2 . 5 ) ( n = 4 ; N = 2 ) ( right panel ) . The traces on the left are the corresponding average normalized STCs , isolated as in Figure 1C . The STC τdecay in TBOA 2 . 5 is 32 ± 2 % higher than in Ctr WT ( paired t - test : P = 0 . 002 ) . The left top panel shows the time course of τdecay of the transient component ( due to the STC ) of the current recorded in an astrocyte during a representative experiment in which TBOA 2 . 5 was applied at the time indicated by the horizontal bar . The steady - state effect was reached within 10 - 15 minutes from the beginning of the drug perfusion .

O O O O O O O O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22-23 WT mice before (Ctr WT) and after application of 2.5 µM DL-TBOA (TBOA 2.5) (n = 4; N=2) (right panel). The traces on the left are the corresponding average normalized STCs, isolated as in Figure 1C. The STC τdecay in TBOA 2.5 is 32 ± 2 % higher than in Ctr WT (paired t-test: P = 0.002). The left top panel shows the time course of τdecay of the transient component (due to the STC) of the current recorded in an astrocyte during a representative experiment in which TBOA 2.5 was applied at the time indicated by the horizontal bar. The steady-state effect was reached within 10-15 minutes from the beginning of the drug perfusion.


( B ) CSD threshold and velocity measured in cortical slices from P22 - 23 WT mice after perfusion for 20 minutes with extracellular solution without ( Ctr WT : n = 23 ; N = 15 ) and with 2 . 5 µM DL - TBOA ( TBOA 2 . 5 : n = 25 ; N = 8 ) . CSD threshold in TBOA 2 . 5 is 36 % lower than in Ctr WT ( Mann - Withney U test test : P < 0 . 0001 ) and CSD velocity is 20 % higher ( unpaired t - test : P < 0 . 0001 ) .

O O O O O O O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) CSD threshold and velocity measured in cortical slices from P22-23 WT mice after perfusion for 20 minutes with extracellular solution without (Ctr WT: n = 23; N = 15) and with 2.5 µM DL-TBOA (TBOA 2.5: n = 25; N = 8). CSD threshold in TBOA 2.5 is 36 % lower than in Ctr WT (Mann-Withney U test test: P < 0.0001) and CSD velocity is 20% higher (unpaired t-test: P < 0.0001).


( C ) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22 - 23 WT mice before ( Ctr WT ) and after application of 1 . 5 µM DL - TBOA ( TBOA 1 . 5 ) ( n = 4 ; N = 4 ) . The STC τdecay in TBOA 1 . 5 is 22 ± 3 % higher than in Ctr WT ( paired t - test : P = 0 . 004 ) .

O O O O O O O O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22-23 WT mice before (Ctr WT) and after application of 1.5 µM DL-TBOA (TBOA 1.5) (n = 4; N=4). The STC τdecay in TBOA 1.5 is 22 ± 3 % higher than in Ctr WT (paired t-test: P = 0.004).


( D ) CSD threshold and velocity measured in cortical slices from P22 - 23 WT mice after perfusion for 20 minutes with extracellular solution without ( Ctr WT : n = 23 ; N = 15 ) and with 1 . 5 µM DL - TBOA ( TBOA 1 . 5 : n = 18 ; N = 6 ) . CSD threshold in TBOA 1 . 5 is 23 % lower than in Ctr WT ( Mann - Withney U test test : P < 0 . 0001 ) and CSD velocity is 13 % higher ( unpaired t - test : P = 0 . 003 ) .

(D) CSD threshold and velocity measured in cortical slices from P22-23 WT mice after perfusion for 20 minutes with extracellular solution without (Ctr WT: n = 23; N = 15) and with 1.5 µM DL-TBOA (TBOA 1.5: n = 18; N = 6). CSD threshold in TBOA 1.5 is 23 % lower than in Ctr WT (Mann-Withney U test test: P < 0.0001) and CSD velocity is 13 % higher (unpaired t-test: P = 0.003).


( A Total PD - L1 , PARP from colon ( Colo - 205 ) , lung ( A549 ) , pancreatic ( PANK1 ) lysates treated with indicated DR5 agonist antibodies named Lexa ( lexatumumab ) , KMTR2 GAPDH is loading

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-TISSUE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-TISSUE O B-CELL_LINE O O B-TISSUE O B-GENEPROD O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE B-GENEPROD O O

(A Total PD-L1, PARP from colon (Colo-205), lung (A549), pancreatic (PANK1) lysates treated with indicated DR5 agonist antibodies named Lexa (lexatumumab), KMTR2 GAPDH is loading control


Total PD - L1 , PARP from triple negative breast cancer cell ( MDA - MB - 436 ) lysates treated with indicated DR5 agonist antibodies named Lexa ( lexatumumab ) , KMTR2 , BaCa , GAPDH is loading

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Total PD-L1, PARP from triple negative breast cancer cell (MDA-MB-436) lysates treated with indicated DR5 agonist antibodies named Lexa (lexatumumab), KMTR2, BaCa, GAPDH is loading control


C ) Total PD - L1 , PARP , cleaved caspase - 3 from ovarian cell ( Cavo - 3 ) lysates treated with indicated DR5 agonist antibodies named tigatuzumab . GAPDH is loading

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C) Total PD-L1, PARP, cleaved caspase-3 from ovarian cell (Cavo-3) lysates treated with indicated DR5 agonist antibodies named tigatuzumab. GAPDH is loading control


( D ) Total PD - L1 , CD47 , Calreticulin , PARP from MDA - MB - 436 cell lysates treated with indicated DR5 agonist antibodies ± caspase inhibitor Z - VAD . GAPDH is loading control .

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(D) Total PD-L1, CD47, Calreticulin, PARP from MDA-MB-436 cell lysates treated with indicated DR5 agonist antibodies ± caspase inhibitor Z-VAD. GAPDH is loading control.


( E ) Total PD - L1 western blotting signal from the lysates of OVCAR - 3 ( n = 3 ) and MDA - MB - 436 ( n = 3 ) cells after 6 hrs treatment of 100nM lexa ( DR5 agonist antibody ) was normalized to GAPDH .

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(E) Total PD-L1 western blotting signal from the lysates of OVCAR-3 (n=3) and MDA-MB-436 (n=3) cells after 6 hrs treatment of 100nM lexa (DR5 agonist antibody) was normalized to GAPDH.


( F ) Surface biotinylation of PD - L1 from indicated tumor cells after indicated DR5 agonist treatments . Cleaved caspase - 3 and PARP indicates activation of DR5 signaling . In lane 3 and 4 , KMTR2 was pre - neutralized either with recombinant DR5 ( rDR5 ) or recombinant FOLR1 ( rFOLR1 ) .

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(F) Surface biotinylation of PD-L1 from indicated tumor cells after indicated DR5 agonist treatments. Cleaved caspase-3 and PARP indicates activation of DR5 signaling. In lane 3 and 4, KMTR2 was pre-neutralized either with recombinant DR5 (rDR5) or recombinant FOLR1 (rFOLR1).


( G ) Representative flow cytometry plots of PD - L1 from two different tumor cell lines treated with indicated DR5 antibodies . Secondary alone and IgG1 control are included .

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(G) Representative flow cytometry plots of PD-L1 from two different tumor cell lines treated with indicated DR5 antibodies. Secondary alone and IgG1 control are included.


( H ) Relative surface PD - L1 % cells , after DR5 agonist treatments Relative signal is normalized to surface PD - L1 in IgG1 treated cells in corresponding tumor cells and horizontal line in samples indicated mean ( n = 3 - 4 ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

(H) Relative surface PD-L1 % cells, after DR5 agonist treatments Relative signal is normalized to surface PD-L1 in IgG1 treated cells in corresponding tumor cells and horizontal line in samples indicated mean (n=3-4).


( J ) Relative surface PD - L1 % cells in indicated tumors after DR5 agonist treatments Relative signal is normalized to surface PD - L1 in IgG1 treated tumors and horizontal line in samples indicated mean ( n = 2 - 6 )

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O

(J) Relative surface PD-L1 % cells in indicated tumors after DR5 agonist treatments Relative signal is normalized to surface PD-L1 in IgG1 treated tumors and horizontal line in samples indicated mean (n=2-6)


( K ) ER ( - ) , PR ( - ) and HER2 ( - ) UCD52 patient derived tumor tissue was xenografted in breast fat pad of NOD . Cg - Prkdcscid Il2rgtm1Wjl / SzJ mice , following by treatment with IgG1 or KMTR2 ( 50μg , 4 doses ) . Harvested tumors were analyzed for PD - L1 , CD47 and PARP in lysates .

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(K) ER (-), PR (-) and HER2 (-) UCD52 patient derived tumor tissue was xenografted in breast fat pad of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, following by treatment with IgG1 or KMTR2 (50μg, 4 doses). Harvested tumors were analyzed for PD-L1, CD47 and PARP in lysates.


( L ) KMTR2 treated ( 50μg , 4 doses ) UCD52 TNBC PDX tumors were stained for PD - L1 using immunohistochemistry ( IHC ) .

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(L) KMTR2 treated (50μg, 4 doses) UCD52 TNBC PDX tumors were stained for PD-L1 using immunohistochemistry (IHC).


( M ) Total PD - L1 and CD47 blotting analysis from DR5 resistant MDA - MB - 436 cell lines after indicated DR5 agonist treatment ( 50nM , 6hr ) .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O O O O O

(M) Total PD-L1 and CD47 blotting analysis from DR5 resistant MDA-MB-436 cell lines after indicated DR5 agonist treatment (50nM, 6hr).


( C ) Luciferase activity of reporter lines from tumor cell - Jurkat cell co - culture assay using MDA - MB - 436 and OVCAR3 cells after treatment ( 50nM ) with indicated DR5 agonists ( tiga : tigatuzumab , AMG : AMG655 , KMTR1 , Lexa : Lexatumumab ) . The background luciferase signal from untreated cells ( due to basal surface PD - L1 ) was subtracted . Various controls treatments ( IgG1 , anti - PD - L1 , anti - EGFR ) are also shown ( n = 3 ) . ( D ) Same as ( C ) except tumor cells were pre - treated with indicated inhibitors for AKT , ERK , mTOR , MEK , STAT3 , PARP - i and NF - Kβ along with DR5 agonist KMTR2 antibody prior to co - culture .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_LINE I-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O

(C) Luciferase activity of reporter lines from tumor cell-Jurkat cell co-culture assay using MDA-MB-436 and OVCAR3 cells after treatment (50nM) with indicated DR5 agonists (tiga: tigatuzumab, AMG: AMG655, KMTR1, Lexa: Lexatumumab). The background luciferase signal from untreated cells (due to basal surface PD-L1) was subtracted. Various controls treatments (IgG1, anti-PD-L1, anti-EGFR) are also shown (n=3). (D) Same as (C) except tumor cells were pre-treated with indicated inhibitors for AKT, ERK, mTOR, MEK, STAT3, PARP-i and NF-Kβ along with DR5 agonist KMTR2 antibody prior to co-culture.


MDA - MB - 436 cells were treated with TNFα and indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 , CSN5 GAPDH is loading control in

B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O

MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1, CSN5 GAPDH is loading control in E


MDA - MB - 436 cells were treated with TNFα and indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 phosphorylated p65 , total p65 , cleaved caspase - 3 and PARP . Tubulin is loading control in F .

B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O

MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1 phosphorylated p65, total p65, cleaved caspase-3 and PARP. Tubulin is loading control in F.


MDA - MB - 436 cells were treated with TNFα and indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 , CSN5 , phosphorylated p65 and PARP . GAPDH is loading control in G .

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MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1, CSN5, phosphorylated p65 and PARP. GAPDH is loading control in G.


( H ) Flow cytometry analysis of MDA - MB - 436 cells treated with TNFα ± MG132 ( top ) and indicated DR5 agonist ± MG132 ( bottom ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O

(H) Flow cytometry analysis of MDA-MB-436 cells treated with TNFα ± MG132 (top) and indicated DR5 agonist ± MG132 (bottom).


( I ) Total PD - L1 from OVCAR3 cell lysates was analyzed after treatment of indicated DR5 agonist ± MG132 .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O

(I) Total PD-L1 from OVCAR3 cell lysates was analyzed after treatment of indicated DR5 agonist ± MG132.


( J ) Flow cytometry surface PD - L1 analysis of OVCAR - 3 cells treated with indicated DR5 agonist ± MG132 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-SMALL_MOLECULE O

(J) Flow cytometry surface PD-L1 analysis of OVCAR-3 cells treated with indicated DR5 agonist ± MG132.


( K ) MDA - MB - 436 cells were treated with MG132 for indicated times and total PD - L1 from lysates was analyzed . GAPDH is loading control .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O

(K) MDA-MB-436 cells were treated with MG132 for indicated times and total PD-L1 from lysates was analyzed. GAPDH is loading control.


( L ) Immunoblotting of DR5 confirming generation of knock out ( DR5 - KO ) cell lines .

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(L) Immunoblotting of DR5 confirming generation of knock out (DR5-KO) cell lines.


( M ) Cell viability analysis of DR5 - KO and DR5 - WT cells ( n = 3 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O

(M) Cell viability analysis of DR5-KO and DR5-WT cells (n=3).


( N ) DR5 - KO and WT cells were treated with TNFα and indicated DR5 agonist followed by flow cytometry using PD - L1 specific antibodies .

O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O

(N) DR5-KO and WT cells were treated with TNFα and indicated DR5 agonist followed by flow cytometry using PD-L1 specific antibodies.


( O ) TNBC WT and DR5 - KO ( MDA - MB - 436 ) cells were treated with either DR5 agonist or TNFα as indicated . Lysates were analyzed for PD - L1 , S5a and DR5 . GAPDH is loading control .

O O O B-DISEASE O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O

(O) TNBC WT and DR5-KO (MDA-MB-436) cells were treated with either DR5 agonist or TNFα as indicated. Lysates were analyzed for PD-L1, S5a and DR5. GAPDH is loading control.


( P ) OVCAR - 3 and MDA - MB - 436 ( WT and DR5 - KO ) cells were treated with KMTR2 followed by ubiquitin and S5a immunoblotting from total lysates . GAPDH is loading control .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O

(P) OVCAR-3 and MDA-MB-436 (WT and DR5-KO) cells were treated with KMTR2 followed by ubiquitin and S5a immunoblotting from total lysates. GAPDH is loading control.


ApoEVs isolated after IgG1 and KMTR2 treatment ( OVCAR - 3 ) were blotted against CD63 and PD - L1 in dot blots .

B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O

ApoEVs isolated after IgG1 and KMTR2 treatment (OVCAR-3) were blotted against CD63 and PD-L1 in dot blots.


ApoEVs isolated after IgG1 and tigatuzumab treatment ( MDA - MB - 436 ) were run on western and blotted against CD63 and PD - L1 .

B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O

ApoEVs isolated after IgG1 and tigatuzumab treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1.


ApoEVs isolated after IgG1 and lexatumumab ( D ) treatment ( MDA - MB - 436 ) were run on western and blotted against CD63 and PD - L1 .

B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O

ApoEVs isolated after IgG1 and lexatumumab (D) treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1.


( F ) ApoEVs isolated from DR5 sensitive tumor cells grown in Met - HPG + media were added on to DR5 - KO cells ( growing in regular media ) . After 48 hrs flow cytometry analysis was carried out with HPG catalyzing dye . HPG incorporation from flow cytometry data confirms ApoEVs transfer from cells growing in Met - HPG + ( DR5 - WT ) to DR5 - KO cells .

O O O B-SUBCELLULAR O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O O O O

(F) ApoEVs isolated from DR5 sensitive tumor cells grown in Met-HPG+ media were added on to DR5-KO cells (growing in regular media). After 48 hrs flow cytometry analysis was carried out with HPG catalyzing dye. HPG incorporation from flow cytometry data confirms ApoEVs transfer from cells growing in Met-HPG+ (DR5-WT) to DR5-KO cells.


( G ) ApoEVs isolated from DR5 - WT OVCAR - 3 cells ( KMTR2 treated ) were added on to DR5 - KO ( MDA - MB - 231 ) cells for indicated times ( 24 - 96 hrs ) to analyze PD - L1 transfer kinetics from

O O O B-SUBCELLULAR O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O

(G) ApoEVs isolated from DR5-WT OVCAR-3 cells (KMTR2 treated) were added on to DR5-KO (MDA-MB-231) cells for indicated times (24-96 hrs) to analyze PD-L1 transfer kinetics from ApoEVs


after addition of MDA - MB - 436 derived ApoEVs ( treated with combination of KMTR2 + lexatumumab DR5 agonists or IgG1 ) on to DR5 - KO MDA - MB - 231 cells , total lysates were immunoblotted for PD - L1 after 24 hrs . KMTR2 + lexatumumab ( n = 3 ) , IgG1 ( n = 2 ) .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-GENEPROD O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O

after addition of MDA-MB-436 derived ApoEVs (treated with combination of KMTR2+lexatumumab DR5 agonists or IgG1) on to DR5-KO MDA-MB-231 cells, total lysates were immunoblotted for PD-L1 after 24 hrs. KMTR2+lexatumumab (n=3), IgG1 (n=2).


( I ) Left : PD - L1 surface histogram shows direct antibody treatment on DR5 - KO cells . Right : PD - L1 surface histogram from DR5 - KO cells after treatment ( 24 hrs ) with ApoEVs isolated from DR5 sensitive cells after either Lexa or KMTR2 treatment .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O

(I) Left: PD-L1 surface histogram shows direct antibody treatment on DR5-KO cells. Right: PD-L1 surface histogram from DR5-KO cells after treatment (24 hrs) with ApoEVs isolated from DR5 sensitive cells after either Lexa or KMTR2 treatment.


ApoEVs treated tumor cells were analyzed in PD - 1 reporter co - culture assays for luciferase

B-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY

ApoEVs treated tumor cells were analyzed in PD-1 reporter co-culture assays for luciferase signal


( K ) MDA - MB - 436 cells were treated with KMTR2 for 6 hrs and along with ZDEVD for indicated times . - ZDEVD shows final time of KMTR2 exposure to cells in absence inhibitor . Lysates were immunoblotted for cleaved caspase - 3 , 8 , PARP , PD - L1 , S5a , ROCK1 and GAPDH ( n = 3 ) . u . c : uncleaved , c : cleaved , n . s : non - specific

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

(K) MDA-MB-436 cells were treated with KMTR2 for 6 hrs and along with ZDEVD for indicated times. - ZDEVD shows final time of KMTR2 exposure to cells in absence inhibitor. Lysates were immunoblotted for cleaved caspase-3, 8, PARP, PD-L1, S5a, ROCK1 and GAPDH (n=3). u.c: uncleaved, c: cleaved, n.s: non-specific band


( L ) Relative caspase - 8 and caspase - 3 activity assays caspase - 8 maintained steady activity , while caspase - 3 activity required at least 40 ( + ) mins of DR5 agonist treatment without ZDEVD ( n = 3 ) .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O

(L) Relative caspase-8 and caspase-3 activity assays caspase-8 maintained steady activity, while caspase-3 activity required at least 40 (+) mins of DR5 agonist treatment without ZDEVD (n=3).


( M ) After indicated treatments with KMTR2 ( DR5 agonist ) and ZDEVD ( caspase - 3 preferred inhibitor ) , cells were allowed to grow 24 hrs , followed by cell viability analysis .

O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O

(M) After indicated treatments with KMTR2 (DR5 agonist) and ZDEVD (caspase-3 preferred inhibitor) , cells were allowed to grow 24 hrs, followed by cell viability analysis.


( A KMTR2 , KMTR2 + Z - VAD treated MDA - MB - 436 lysates for indicated early time points were analyzed for caspase - 8 , caspase - 3 , ROCK1 PARP as indicated .

O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O

(A KMTR2, KMTR2+Z-VAD treated MDA-MB-436 lysates for indicated early time points were analyzed for caspase-8, caspase-3, ROCK1 PARP as indicated.


B ) lexatumumab treated OVCAR3 lysates for indicated early time points were analyzed for caspase - 3 , ROCK1 , pMLC , PARP , PD - L1 and CMTM6 etc . as indicated .

O O B-SMALL_MOLECULE O B-CELL_LINE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O

B) lexatumumab treated OVCAR3 lysates for indicated early time points were analyzed for caspase-3, ROCK1, pMLC, PARP, PD-L1 and CMTM6 etc. as indicated.


( C ) OVCAR3 cells were treated with pMLC activating ionophore A23187 ( positive control ) and lexatumumab ± ROCK1 inhibitors or ± rDR5 or ± rFOLR1 . Lysates were later analyzed for ROCK1 , pMLC , caspase - 3 and PARP with GAPDH as loading control . Blue arrows indicate cleaved and activated ROCK1 and caspase - 3 .

O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O

(C) OVCAR3 cells were treated with pMLC activating ionophore A23187 (positive control) and lexatumumab ± ROCK1 inhibitors or ± rDR5 or ± rFOLR1. Lysates were later analyzed for ROCK1, pMLC, caspase-3 and PARP with GAPDH as loading control. Blue arrows indicate cleaved and activated ROCK1 and caspase-3.


( D ) MDA - MB - 436 and OVCAR3 cells were treated with indicated ROCK1 inhibitors 2 hours prior to DR5 agonist KMTR2 treatment ( 20nM ) . After 6 hours flow cytometry was used to analyze surface PD - L1 . ( GSK269 : GSK269962A , GSK429 : GSK429286A )

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O O B-GENEPROD O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

(D) MDA-MB-436 and OVCAR3 cells were treated with indicated ROCK1 inhibitors 2 hours prior to DR5 agonist KMTR2 treatment (20nM). After 6 hours flow cytometry was used to analyze surface PD-L1. (GSK269: GSK269962A, GSK429: GSK429286A)


( E ) MDA - MB - 436 cell survival assay after treatment with DR5 agonist antibody KMTR2 ± ROCK1 inhibitors , error bars indicate SD ( n = 2 ) . ( GSK269 : GSK269962A , GSK429 : GSK429286A )

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

(E) MDA-MB-436 cell survival assay after treatment with DR5 agonist antibody KMTR2 ± ROCK1 inhibitors, error bars indicate SD (n=2). (GSK269: GSK269962A, GSK429: GSK429286A)


Cultured MDA - MB - 436 cells were treated with KMTR2 ( IgG1 - Fc ) or IgG1 control ± ROCK1i ( GSK269962 ) for 6 hrs . After 6 hrs , 500nM anti - PD - L1 avelumab ( IgG4 - Fc ) was added to the media for additional 1 hr . Cellular lysates were pulled down with anti - hu - IgG4 specific beads . ( G ) Immunoprecipitated lysates and leftover supernatant after various treatments were run at the same time followed by blotting with ROCK1 , PD - L1 and CMTM6 . Total lysates as a control were also generated in exactly similar conditions and run next to supernatant and IP samples . Blue arrows indicate cleaved and activated ROCK1 .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O

Cultured MDA-MB-436 cells were treated with KMTR2 (IgG1-Fc) or IgG1 control ± ROCK1i (GSK269962) for 6 hrs. After 6 hrs, 500nM anti-PD-L1 avelumab (IgG4-Fc) was added to the media for additional 1 hr. Cellular lysates were pulled down with anti-hu-IgG4 specific beads. (G) Immunoprecipitated lysates and leftover supernatant after various treatments were run at the same time followed by blotting with ROCK1, PD-L1 and CMTM6. Total lysates as a control were also generated in exactly similar conditions and run next to supernatant and IP samples. Blue arrows indicate cleaved and activated ROCK1.


( H ) Immunoprecipitation assays with anti - PD - L1 ( avelumab ) using OVCAR - 3 cell lines . Blue arrow indicates cleaved and activated ROCK1 .

O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-GENEPROD O

(H) Immunoprecipitation assays with anti-PD-L1 (avelumab) using OVCAR-3 cell lines. Blue arrow indicates cleaved and activated ROCK1.


( J ) Relative luciferase activity ata is from 4 different DR5 agonist antibodies used in combination of two different ROCK1i inhibitors ( n = 3 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O

(J) Relative luciferase activity ata is from 4 different DR5 agonist antibodies used in combination of two different ROCK1i inhibitors (n=3).


( K ) Tumor regression efficacy of MD5 - 1 ( 50μg ) , GSK269962A ( < 2mg / kg ) and MD5 - 1 + GSK269962A ( 50μg + 2mg / kg ) 4T1 tumors ( n = 4 - 7 ) . 6 - 8 weeks old BALB / c mice bearing 4T1 tumors ( ~ 100mm3 ) were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . GSK269962A ( ROCK1i ) was injected directly into the tumors . Tumor volumes were quantified at indicated days by caliper measurements .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-ORGANISM O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

(K) Tumor regression efficacy of MD5-1 (50μg), GSK269962A (<2mg/kg) and MD5-1+ GSK269962A (50μg + 2mg/kg) 4T1 tumors (n=4-7). 6-8 weeks old BALB/c mice bearing 4T1 tumors (~100mm3) were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). GSK269962A (ROCK1i) was injected directly into the tumors. Tumor volumes were quantified at indicated days by caliper measurements.


( B - D ) FACs plots confirming expression of human DR5 ( huDR5 ) and Chi - G4S - DR5 in mouse 4T1 cells . Chi - DR5 was not expressed on cell surface ( C ) .

O O O O O B-EXP_ASSAY O O O O B-ORGANISM B-GENEPROD O O O O O O O O B-GENEPROD O B-ORGANISM B-CELL_LINE O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O

(B-D) FACs plots confirming expression of human DR5 (huDR5) and Chi-G4S-DR5 in mouse 4T1 cells. Chi-DR5 was not expressed on cell surface (C).


( E ) Cell viability analysis of huDR5 and Chi - G4S - DR5 stable 4T1 cells with indicated human DR5 agonists lexatumumab and KMTR2 ( n = 3 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-CELL_LINE O O O B-ORGANISM B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O

(E) Cell viability analysis of huDR5 and Chi-G4S-DR5 stable 4T1 cells with indicated human DR5 agonists lexatumumab and KMTR2 (n=3).


( F ) Comparison of tumor growth of grafted huDR5 and Chi - G4S - DR5 stable 4T1 cells in Balb / c mice ( n = 3 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O B-ORGANISM O O O O O O

(F) Comparison of tumor growth of grafted huDR5 and Chi-G4S-DR5 stable 4T1 cells in Balb/c mice (n=3).


( G ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 150 - 200mm3 ) were treated with 4 doses ( 100μg ) of IgG1 , lexatumumab , lexatumumab + avelumab followed by tumor recovery and surface PD - L1 analysis using flow

O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY

(G) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~150-200mm3) were treated with 4 doses (100μg) of IgG1, lexatumumab, lexatumumab +avelumab followed by tumor recovery and surface PD-L1 analysis using flow cytometry


( H ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . Isolated tumors were imaged together ( n = 4 - 5 ) . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections ( 2mg / kg ) .

O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg).


( H ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections ( 2mg / kg ) . average tumor weight is shown ( n = 4 ) .

O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg). average tumor weight is shown (n=4).


( J - K ) Confirmation of selective CD8 + T cell population depletion in mice spleen and grafted tumors after injecting animals with either anti - PD - L1 avelumab alone or avelumab + anti - CD8a antibody . CD4 + T - cells remained unchanged .

O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM B-TISSUE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O

(J-K) Confirmation of selective CD8+ T cell population depletion in mice spleen and grafted tumors after injecting animals with either anti-PD-L1 avelumab alone or avelumab+anti-CD8a antibody. CD4+ T-cells remained unchanged.


( L ) Average of tumor weights ( harvested at same time ) from mice treated ( i . p . ) with IgG1 , lexatumumab , ROCK1i , lexatumumab + ROCK1i and anti - CD8 + lexatumumab + ROCK1i ( n = 4 - 6 , 50μg Lexatumumab , 2mg / kg ROCK1i , 6 doses ) . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections . Various treatments were started when tumors were ~ 100mm3 size .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

(L) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, ROCK1i, lexatumumab + ROCK1i and anti-CD8+ lexatumumab + ROCK1i (n=4-6, 50μg Lexatumumab, 2mg/kg ROCK1i, 6 doses). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections. Various treatments were started when tumors were ~100mm3 size.


( M ) Average of tumor weights ( harvested at same time ) from mice treated ( i . p . ) with IgG1 , lexatumumab , avelumab , lexatumumab + avelumab and anti - CD8 + lexatumumab + avelumab ( n = 4 - 6 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O

(M) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, avelumab, lexatumumab +avelumab and anti-CD8+ lexatumumab +avelumab (n=4-6).


( N ) 6 - 8 weeks old C57BL / 6 bearing Chi - G4S - DR5 expressing MC38 tumors were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . Various treatments were started when tumors were ~ 100mm3 size . Tumor volumes were quantified at indicated days by caliper measurements .

O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

(N) 6-8 weeks old C57BL/6 bearing Chi-G4S-DR5 expressing MC38 tumors were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). Various treatments were started when tumors were ~100mm3 size. Tumor volumes were quantified at indicated days by caliper measurements.


Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . Isolated tumors were imaged together ( n = 4 - 5 ) . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections ( 2mg / kg ) . ( O ) KMTR2 ( 8 doses ) antibody used as the DR5 agonist for the experiment . ( P ) Average of tumor weights of data shown in ( O ) .

O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg). (O) KMTR2 (8 doses) antibody used as the DR5 agonist for the experiment. (P) Average of tumor weights of data shown in (O).


( Q ) Kaplan - Meier plot depicting the survival of syngeneic Chi - G4S - DR5 4T1 tumor bearing animals injected i . p . with 100μg of indicated antibodies such as IgG1 , KMTR2 and avelumab . Animals were injected with GSK269962A 2mg / kg ( in PBS ) directly into tumors wherever ROCK1i is indicated .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O B-GENEPROD B-CELL_LINE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O

(Q) Kaplan-Meier plot depicting the survival of syngeneic Chi-G4S-DR5 4T1 tumor bearing animals injected i.p. with 100μg of indicated antibodies such as IgG1, KMTR2 and avelumab. Animals were injected with GSK269962A 2mg/kg (in PBS) directly into tumors wherever ROCK1i is indicated.


( A ) Chi - G4S - DR5 stable 4T1 tumors harboring mice were treated lexatumumab , lexatumumab + ROCK1i , and avelumab + lexatumumab and other controls as indicated . Antibodies were treated i . p at 100μg dose ( 6 total ) , ROCK1i ( in PBS ) was injected directly into tumors at 2mg / kg dose ( 6 total ) . Various treatments were started when tumors were ~ 400mm3 size . Harvested tumors were grouped together and sized matched ( 3 independent sets : n = 2 - 6 tumors in each set ) followed by TIL isolation CD8 / CD45 and CD4 / CD45 expressing cells were measured by flow cytometry .

O O O O O O O B-GENEPROD O B-CELL_LINE O O B-ORGANISM O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(A) Chi-G4S-DR5 stable 4T1 tumors harboring mice were treated lexatumumab, lexatumumab +ROCK1i, and avelumab + lexatumumab and other controls as indicated. Antibodies were treated i.p at 100μg dose (6 total), ROCK1i (in PBS) was injected directly into tumors at 2mg/kg dose (6 total). Various treatments were started when tumors were ~400mm3 size. Harvested tumors were grouped together and sized matched (3 independent sets: n=2-6 tumors in each set) followed by TIL isolation CD8/CD45 and CD4/CD45 expressing cells were measured by flow cytometry.


( B ) Plots showing % of total double positive TILs ( CD8 + CD45 + + CD4 + CD45 + ) in right upper quadrant after combining 3 independent experiments . ( C ) Ratio of CD8 + CD45 + / CD4 + CD45 + isolated TILs from tumors in each indicated treatment ( n = 3 ) .

O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O

(B) Plots showing % of total double positive TILs (CD8+CD45+ + CD4+CD45+) in right upper quadrant after combining 3 independent experiments. (C) Ratio of CD8+CD45+/CD4+CD45+ isolated TILs from tumors in each indicated treatment (n=3).


Chi - G4S - DR5 stable MC38 tumors harboring mice were treated ( 6 total doses ) lexatumumab , avelumab , ROCK1i , lexatumumab + ROCKi , and avelumab + lexatumumab and IgG1 control as indicated . Harvested tumors homogenized followed by quantitation . Protein lysates were run on SDS - Page followed by immunoblotting using indicated CD8 , CD4 , Foxp3 , caspase - 3 and granzyme - b antibody . GAPDH is loading control . Red arrows indicated cleaved caspase - 3 p19 and p17 fragments .

O O O O B-GENEPROD O B-CELL_LINE O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

Chi-G4S-DR5 stable MC38 tumors harboring mice were treated (6 total doses) lexatumumab, avelumab, ROCK1i, lexatumumab +ROCKi, and avelumab + lexatumumab and IgG1 control as indicated. Harvested tumors homogenized followed by quantitation. Protein lysates were run on SDS-Page followed by immunoblotting using indicated CD8, CD4, Foxp3, caspase-3 and granzyme-b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase-3 p19 and p17 fragments.


( H ) Cell killing assay of 4T1 cells treated with murine DR5 agonist MD5 - 1 and bispecific avelu - MD5 antibody .

(H) Cell killing assay of 4T1 cells treated with murine DR5 agonist MD5-1 and bispecific avelu-MD5 antibody.


( I ) Balb / c mice harboring luciferase stable 4T1 tumors were i . p . injected with indicated antibodies . 50μg dose and mice were imaged after 5 doses .

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(I) Balb/c mice harboring luciferase stable 4T1 tumors were i.p. injected with indicated antibodies. 50μg dose and mice were imaged after 5 doses.


( J ) 6 - 8 weeks old C57BL / 6 mice bearing MC38 tumors were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . Indicated treatments were started when tumors were ~ 100mm3 size . Tumor volumes were quantified at indicated days by caliper measurements .

O O O O O O O O O O O B-ORGANISM O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

(J) 6-8 weeks old C57BL/6 mice bearing MC38 tumors were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). Indicated treatments were started when tumors were ~100mm3 size. Tumor volumes were quantified at indicated days by caliper measurements.


( K ) ~ 150 - 200mm3 size MC38 tumor bearing C57LB / 6 mice were treated with indicated MD5 - 1 , avelumab and bispecific antibodies along with control IgG1 , 6 total doses . Harvested tumors were homogenized followed by quantitation . Protein lysates were run on SDS - Page followed by immunoblotting using indicated CD8 , CD4 , caspase - 3 and granzyme - b antibody . GAPDH is loading control . Red arrows indicated cleaved caspase - 3 p19 and p17 fragments .

O O O O O O O O B-CELL_LINE O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

(K) ~150-200mm3 size MC38 tumor bearing C57LB/6 mice were treated with indicated MD5-1, avelumab and bispecific antibodies along with control IgG1, 6 total doses. Harvested tumors were homogenized followed by quantitation. Protein lysates were run on SDS-Page followed by immunoblotting using indicated CD8, CD4, caspase-3 and granzyme-b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase-3 p19 and p17 fragments.


( L ) 6 - 8 weeks old C57BL / 6 mice were injected with MC38 cells . When tumors reached ~ 150 - 200mm3 , animals were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day . On day 18 , tumors were harvested , sized matched and pooled by treatment group , exposed to collagenase / DNase and were single cell suspensions enriched for CD8 + cells . Enriched CD8 + T - cells from various treatments were restimulated with anti - CD3 ( OKT3 ) antibody for 4 additional hours . CD8 gated cells were next analyzed for IFN - γ intracellular expression using flow cytometry .

O O O O O O O O O O O B-ORGANISM O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

(L) 6-8 weeks old C57BL/6 mice were injected with MC38 cells. When tumors reached ~150-200mm3, animals were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day. On day 18, tumors were harvested, sized matched and pooled by treatment group, exposed to collagenase/DNase and were single cell suspensions enriched for CD8+ cells. Enriched CD8+ T-cells from various treatments were restimulated with anti-CD3 (OKT3) antibody for 4 additional hours. CD8 gated cells were next analyzed for IFN-γ intracellular expression using flow cytometry.


( M ) Percentage of IFN - γ + CD8 + double positive cells from 3 independent experiments .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O

(M) Percentage of IFN-γ+CD8+ double positive cells from 3 independent experiments.


Median CSF protein abundance distribution as calculated from MS - intensities of quantified peptides of each protein . The top ten most abundant proteins and hemoglobin proteins are highlighted .

O B-TISSUE O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O

Median CSF protein abundance distribution as calculated from MS-intensities of quantified peptides of each protein. The top ten most abundant proteins and hemoglobin proteins are highlighted.


A - C Protein AD / non - AD fold changes plotted vs statistical significance for Sweden ( A ) , Magdeburg / Kiel ( B ) , and Berlin ( C ) cohort . Proteins associated with the GO annotation neuron projection labelled in orange . Proteins above the dashed green line are statistically significant ( p < 0 . 05 ) and those above the black curves have a q - value below 0 . 05

O O O O B-DISEASE O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C Protein AD/non-AD fold changes plotted vs statistical significance for Sweden (A), Magdeburg/Kiel (B), and Berlin (C) cohort. Proteins associated with the GO annotation neuron projection labelled in orange. Proteins above the dashed green line are statistically significant (p < 0.05) and those above the black curves have a q-value below 0.05


A - C Protein AD / non - AD fold changes plotted vs statistical significance for Sweden ( A ) , Magdeburg / Kiel ( B ) , and Berlin ( C ) cohort . Proteins associated with the GO annotation neuron projection labelled in orange . Proteins above the dashed green line are statistically significant ( p < 0 . 05 ) and those above the black curves have a q - value below 0 .

O O O O B-DISEASE O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


Correlation of protein AD / non - AD fold changes in pairwise combinations of two cohorts each . Combinations are Sweden vs Magdeburg / Kiel Proteins included differ significantly ( p < 0 . 05 ) and consistently in abundance by AD status in both cohorts each .

O O O B-DISEASE O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O

Correlation of protein AD/non-AD fold changes in pairwise combinations of two cohorts each. Combinations are Sweden vs Magdeburg/Kiel Proteins included differ significantly (p < 0.05) and consistently in abundance by AD status in both cohorts each.


Correlation of protein AD / non - AD fold changes in pairwise combinations of two cohorts each . Combinations are Sweden vs Berlin ( D ) , and Magdeburg / Kiel vs Berlin ( E ) . Proteins included differ significantly ( p < 0 . 05 ) and consistently in abundance by AD status in both cohorts each .

O O O B-DISEASE O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O

Correlation of protein AD/non-AD fold changes in pairwise combinations of two cohorts each. Combinations are Sweden vs Berlin (D), and Magdeburg/Kiel vs Berlin (E). Proteins included differ significantly (p < 0.05) and consistently in abundance by AD status in both cohorts each.


Proteins that differ significantly ( p - value < 0 . 05 in E ; q - value < 0 . 05 in F ) in abundance by AD status across all three cohorts . Z - scored abundances of proteins in the AD and non - AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non - AD groups . Pyruvat kinase PKM ( PKM ) was quantified in two isoforms and UniProt IDs are given in parenthesis . Black frames highlight proteins with consistent AD / non - AD fold changes across cohorts .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O B-DISEASE O O O B-DISEASE O O O O O O O O O O O O B-DISEASE O O O B-DISEASE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O B-DISEASE O O O O O

Proteins that differ significantly (p-value < 0.05 in E; q-value < 0.05 in F) in abundance by AD status across all three cohorts. Z-scored abundances of proteins in the AD and non-AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non-AD groups. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis. Black frames highlight proteins with consistent AD/non-AD fold changes across cohorts.


A - C ) Correlation of proteins with ELISA - measured t - tau concentration across samples within the Sweden ( A ) , Magdeburg ( B ) and Berlin ( C ) cohorts . Proteins with a q - value below 0 . 05 are labeled in yellow . Proteins of the 40 protein signature are colored in red for those with higher abundance in AD CSF and in blue for those with higher abundance in non - AD CSF .

O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE B-TISSUE O O O O O O O O O O O B-DISEASE B-TISSUE O

A-C) Correlation of proteins with ELISA-measured t-tau concentration across samples within the Sweden (A), Magdeburg (B) and Berlin (C) cohorts. Proteins with a q-value below 0.05 are labeled in yellow. Proteins of the 40 protein signature are colored in red for those with higher abundance in AD CSF and in blue for those with higher abundance in non-AD CSF.


D ) Three cohort summary of proteins significantly correlating with ELISA - measured t - tau . Protein names given for the 29 proteins out of the 40 protein signature with significant ( p < 0 . 05 ) correlation in each of the three cohorts . Pyruvat kinase PKM ( PKM ) was quantified in two isoforms and UniProt IDs are given in parenthesis .

O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

D) Three cohort summary of proteins significantly correlating with ELISA-measured t-tau. Protein names given for the 29 proteins out of the 40 protein signature with significant (p < 0.05) correlation in each of the three cohorts. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis.


Protein abundance distribution of CSF showing the abundances of AD - modulated CSF proteins . Proteins of our 40 protein signature are highlighted in red ( elevated abundance in AD ) and blue ( elevated abundance in non - AD ) . Proteins linked to glucose metabolism are highlighted in purple and labelled .

O O O O B-TISSUE O O O O B-DISEASE O O B-TISSUE O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O B-DISEASE O O O O O B-SMALL_MOLECULE O O O O O O O O

Protein abundance distribution of CSF showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled.


Protein abundance distribution of brain showing the abundances of AD - modulated CSF proteins . Proteins of our 40 protein signature are highlighted in red ( elevated abundance in AD ) and blue ( elevated abundance in non - AD ) . Proteins linked to glucose metabolism are highlighted in purple and labelled .

Protein abundance distribution of brain showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled.