Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a Schematic diagram showing the establishment of humanized mice (humice) with human immune system reconstituted in NIKO mice. The presence of human CD45 + cells, NK cells, CD4 + and CD8 + T cells in the mice’s peripheral system was validated by flow cytometry. b Primary LM2 tumor size in humice (control, n = 14; Keytruda, n = 14; ENT, n = 12; PX478, n = 14; ENT + Keytruda, n = 16; PX478 + Keytruda, n = 16) and NIKO mice (control, n = 10; ENT + Keytruda, n = 10; PX478 + Keytruda, n = 10), at Day 21 of treatments. c Lung metastasis of humice (control, n = 6; Keytruda, n = 6; ENT, n = 6; PX478, n = 6; ENT + Keytruda, n = 7; PX478 + Keytruda, n = 7) and NIKO mice (control, n = 5; ENT + Keytruda, n = 5; PX478 + Keytruda, n = 5) bearing LM2 tumors at Day 35 assessed by bioluminescence (BLI) measurement. d Representative bioluminescence (BLI) images showing the lung metastasis of humice and NIKO mice. e Flow cytometric analysis of LM2 tumors harvested from humanized mice. IFNγ, TNFα, and granzyme B expression was examined in tumor-infiltrating human CD8 + T cells and NK cells. N = 5 for each group. f Flow cytometry analysis of LM2 tumors harvested from humanized mice. Expressions of human PD-L1 and PD-L2 were examined in total living cells dissociated from LM2 tumors. N = 5 for each group. Quantification data of flow cytometry ( e , f ) are presented as a box and whiskers, with median values and whiskers of minimum and maximum values. Data for b and c were presented as mean ± SD . P values were determined by one-way ( e , f ) or two-way ( b , c ) ANOVA with Turkey’s test. Source data are provided as a source data file.

Article Snippet: The following antibodies were used for staining, anti-activated pimonidazole FITC antibody (Hypoxyprobe, CAT# HP2-200kit, dilution 1:200), anti-mouse HIF1α APC antibody (R&D Systems, CAT# IC1935A, dilution 1:50), anti-mouse CD3 BV421 antibody (BD Biosciences, CAT# 564008, dilution 1:100), anti-mouse CD45 Percp-Vio700 antibody (Miltenyi Biotec, CAT# 130-110-663, dilution 1:100) anti-mouse CD8 APC-Vio770 antibody (Miltenyi Biotec, CAT# 130-120-737, dilution 1:100), anti-mouse Nkp46 APC antibody (Miltenyi Biotec, CAT# 130-112-202, dilution 1:100), anti-mouse CD4 BV650 antibody (Biolegend, CAT# 563747, dilution 1:100), anti-mouse TIM-3 BV711 antibody (Biolegend, CAT# 119727, dilution 1:100), anti-mouse PD-1 PE-Vio770 (Miltenyi Biotec, CAT# 130-120-391, dilution 1:100), anti-mouse IFNγ PE (Miltenyi Biotec, CAT# 130-117-352, dilution 1:100), anti-mouse TNFα BV711 (BD Biosciences, CAT# 563944, dilution 1:100), anti-mouse/human granzyme B FITC (Miltenyi Biotec, Cat#130-118-430, dilution 1:100), anti-mouse PD-L1 BV786 antibody (BD Biosciences, CAT# 741014, dilution 1:100), anti-mouse PD-L2 FITC antibody (Miltenyi Biotec, Cat# 130-102-222, dilution 1:100), anti-human CD45 FITC antibody (BD Biosciences, CAT# 304006, dilution 1:100), anti-human CD3 PE antibody (Biolegend, CAT# 300308, dilution 1:100) anti-human CD8 APC-Cy7 antibody (BD Biosciences, CAT# 557834, dilution 1:100), anti-human CD56 BV711 antibody (Biolegend, CAT# 318336, dilution 1:100), anti-human CD4 APC antibody (Biolegend, CAT# 300514, dilution 1:100), anti-human IFNγ BV785 (Biolegend, CAT# 502542, dilution 1:100), anti-human TNFα BV650 (Biolegend, CAT# 502398, dilution 1:100), anti-human Granzyme B BV421 (BD Biosciences, Cat# 563389, dilution 1:100), anti-human PD-L1 PE-Cy7 antibody (Biolegend, CAT# 374506, dilution 1:100), anti-human PD-L2 PE antibody (Miltenyi Biotec, CAT# 130-098-530, dilution 1:100).

Techniques: Flow Cytometry, Control, Expressing

Journal: Molecular imaging and biology

Article Title: An analysis of isoclonal antibody formats suggests a role for measuring PD-L1 with low molecular weight PET radiotracers

doi: 10.1007/s11307-020-01527-3

Figure Lengend Snippet: A summary of the kinetic constants for the C4 minibody, scFv and their respective DFO-conjugates. The binding was assayed against the ectodomain of recombinant human PD-L1 using biolayer interferometry. The data are representative of two independent experiments. In the case of the K D and the K dis for the C4 minibody, the constants approached the limit of detection and the instrument was not capable of reporting error calculations.

Article Snippet: Kinetic constants for the minibody and scFv antibody against human and mouse PD-L1 (Sino Biological Inc.) were determined via biolayer interferometry with an Octet RED384 instrument (ForteBio) using a previously described approach[ 11 ].

Techniques: Binding Assay, Recombinant

Journal: Molecular imaging and biology

Article Title: An analysis of isoclonal antibody formats suggests a role for measuring PD-L1 with low molecular weight PET radiotracers

doi: 10.1007/s11307-020-01527-3

Figure Lengend Snippet: A. DAR and anti-PDL1 immunohistochemistry (co-stained with hematoxylin) of a liver section from a 1 year old female Alb Cre; MYCTg; KRASG12D genetically engineered mouse model of heptacellular carcinoma. Multiple tracer avid foci are detected against the background of normal liver. At right are shown the merged images with the PDL1 immunohistochemistry magnified at 40X in selected fields of view to show the concordance between radiotracer binding and PD-L1 expression in tumor. Additional fields of view and tumor slices are shown in Supplemental Figure 1. B. Biodistribution data showing the uptake of 89Zr-scFv in an orthotopic hepatocellular tumor established from a mouse cell line derived from the Alb Cre; MYCTg; KRASG12D GEM model.

Article Snippet: Kinetic constants for the minibody and scFv antibody against human and mouse PD-L1 (Sino Biological Inc.) were determined via biolayer interferometry with an Octet RED384 instrument (ForteBio) using a previously described approach[ 11 ].

Techniques: Immunohistochemistry, Staining, Binding Assay, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor resistant HER2-positive breast cancer

doi: 10.1101/671198

Figure Lengend Snippet: ( A ) Waterfall plots showing percent change of tumor volume with 14-day’s treatment in MMTV-Neu mice (n=8,6,6 and 9 for Ctrl, Ab, Pal and Ab+Pal). Ctrl, vehicle treated; Ab, anti-Her2/Neu antibody; Pal, CDK4/6 inhibitor Palbociclib. ( B ) Tumor volume curves showing tumors rebounded during sustained Ab+Pal combination treatment (n=12 for Ctrl and n=10 for Ab+Pal). ( C ) Schematic for sample processing, enrichment of tumor cells and Drop-seq based single-cell RNA sequencing. ( D ) t-distributed stochastic neighbor embedding (t-SNE) plots colored by treatment groups and clustering of 4711 tumor cells derived from Ctrl, Ab or Pal alone, responsive/residual tumors (APP) and resistant tumors (APR) with Ab+Pal treatment. Each point represents a single cell. ( E ) Abundance of each cell cluster in tumors with indicated treatment as presented and classified in panel D. ( F ) Volcano plots comparing ssGSEA enrichment score of 1053 canonical pathways/gene sets of the C2 collection of Molecular Signatures Database between APR and Ctrl single cell RNA-Seq data. Each point represents one pathway/gene set. X-axis, mean difference of single cell ssGSEA enrichment score; Y-axis, −log10 (P-value by t-test). ( G ) ssGSEA enrichment score violin plots for single cells in each group for indicated signatures. ( H ) Volumes of Ab+Pal resistant tumors after treatment combining immune checkpoint blockades (n=7 for Ab+Pal and n=6 for Ab+Pal+ICB). ICB, anti-CTLA4 and anti-PD-1 antibody cocktail. P-value by two-tailed Student’s t-test.

Article Snippet: The following pre-conjugated antibodies purchased from Fluidigm were used in this study: CD45-089Y (3089005B, 30-F11); Ly-6G-141Pr (3141008B, 1A8,); CD11c-142Nd (3142003B, N418); CD45R-144Nd (3144011B, RA3-6B2); CD4-145Nd (3145002B, RM4-5); CD11b-148Nd (3148003B, M1/70); CD44-150Nd (3150018B, IM7); CD25-151Eu (3151007B, 3C7); CD3e-152Sm (3152004B, 145-2C11); PD-L1-153Eu (3153016B, 10F.9G2); CTLA-4-154Sm (3154008B, UC10-4B9); PD-1-159Tb (3159024B, 29F.1A12); Ly-6C-162Dy (3162014B, HK1.4); CX3CR1-164Dy (3164023B, SA011F11); NK1.1-165Ho (3165018B, PK136); c-Kit-166Er (3166004B, 2B8); CD8a-168Er (3168003B, 53-6.7); CD86-172Yb (3172016B, GL1); I-A/I-E-209Bi (3209006B, M5/114.15.2).

Techniques: RNA Sequencing Assay, Derivative Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor resistant HER2-positive breast cancer

doi: 10.1101/671198

Figure Lengend Snippet: (A) Enrichment analysis of cabozantinib target genes across single TILs grouped and plotted by different phenotypes (left) or by different immune cell types (right) as annotated in . P -value by Student’s t-test (two-tailed). (B) Expression distribution of Kit and Met on t-SNE plot (left) and quantification of Kit and/or Met expressing cells among different immune celltypes as annotated (right). P -value by three-sample Chi-square test. (C) Abundance of Kit and/or Met expressing IMCs among tumors with different phenotypes. P -value by three-sample Chi-square test. (D) Growth of Ab+Pal resistant tumors with Ab+Cabo treatment (n=7 for Ab+Pal, n=6 for Cabo and n=7 for Ab+Cabo). (E) Representative images and quantification of CD3 immunohistochemistry staining for tumors with Ab+Pal or Ab+Cabo treatment. Scale bar, 20 μm. P -value by Student’s t-test. (F) T-cell depletion during Ab+Cabo treatment against Ab+Pal resistant tumors. P -value by Student’s t-test. (G) Relative volumes of Ab+Pal resistant tumors after treatment with Cabo and ICB. Ab+Pal resistant tumors were transplanted to recipient MMTV-Neu mice and first treated with Ab+Pal to acquire the resistance phenotype (left), then treated with Ab+Pal (n=6), Ab+Pal+ICB (n=6), Ab+ICB (n=8), Ab+Cabo (n=5), or Ab+Cabo+ICB (n=7) for 2 weeks (right). (H) Growth of Ab+Pal resistant tumors after sequential treatment with Ab+Pal+ICB and Ab+Cabo+ICB (n=9). Ab+Pal resistant tumors were first treated with Ab+Pal+ICB for 1 week then switched to Ab+Cabo+ICB treatment for 3 weeks. (I) Survival time to doubled tumor volume from experiment in (D). P -value by log-rank (Mantel-Cox) test. Cabo, protein kinase inhibitor cabozantinib. ICB, immune checkpoint blockades cocktail with anti-CTLA4 and anti-PD-1 mAb.

Article Snippet: The following pre-conjugated antibodies purchased from Fluidigm were used in this study: CD45-089Y (3089005B, 30-F11); Ly-6G-141Pr (3141008B, 1A8,); CD11c-142Nd (3142003B, N418); CD45R-144Nd (3144011B, RA3-6B2); CD4-145Nd (3145002B, RM4-5); CD11b-148Nd (3148003B, M1/70); CD44-150Nd (3150018B, IM7); CD25-151Eu (3151007B, 3C7); CD3e-152Sm (3152004B, 145-2C11); PD-L1-153Eu (3153016B, 10F.9G2); CTLA-4-154Sm (3154008B, UC10-4B9); PD-1-159Tb (3159024B, 29F.1A12); Ly-6C-162Dy (3162014B, HK1.4); CX3CR1-164Dy (3164023B, SA011F11); NK1.1-165Ho (3165018B, PK136); c-Kit-166Er (3166004B, 2B8); CD8a-168Er (3168003B, 53-6.7); CD86-172Yb (3172016B, GL1); I-A/I-E-209Bi (3209006B, M5/114.15.2).

Techniques: Two Tailed Test, Expressing, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor resistant HER2-positive breast cancer

doi: 10.1101/671198

Figure Lengend Snippet: ( A ) Representative images of H&E staining for tumors with indicated treatment and quantification of hypocellularity. Scale bar, 100 μm. ( B ) Representative images and quantification of Ki67 immunohistochemistry staining tumors with indicated treatment. Scale bar, 20 μm. ( C ) Relative volumes of Ab+Pal resistant tumors after sequential treatment with Ab+Pal+ICB and Ab+Cabo+ICB (as in ). Ab+Pal resistant tumors were first treated with Ab+Pal+ICB for 1 week then switched to Ab+Cabo+ICB treatment for 3 weeks. Cabo, protein kinase inhibitor cabozantinib; ICB, immune checkpoint blockades, cocktail of anti-CTLA4 and anti-PD-1 antibodies. P -value by one-way ANOVA with Tukey’s test.

Article Snippet: The following pre-conjugated antibodies purchased from Fluidigm were used in this study: CD45-089Y (3089005B, 30-F11); Ly-6G-141Pr (3141008B, 1A8,); CD11c-142Nd (3142003B, N418); CD45R-144Nd (3144011B, RA3-6B2); CD4-145Nd (3145002B, RM4-5); CD11b-148Nd (3148003B, M1/70); CD44-150Nd (3150018B, IM7); CD25-151Eu (3151007B, 3C7); CD3e-152Sm (3152004B, 145-2C11); PD-L1-153Eu (3153016B, 10F.9G2); CTLA-4-154Sm (3154008B, UC10-4B9); PD-1-159Tb (3159024B, 29F.1A12); Ly-6C-162Dy (3162014B, HK1.4); CX3CR1-164Dy (3164023B, SA011F11); NK1.1-165Ho (3165018B, PK136); c-Kit-166Er (3166004B, 2B8); CD8a-168Er (3168003B, 53-6.7); CD86-172Yb (3172016B, GL1); I-A/I-E-209Bi (3209006B, M5/114.15.2).

Techniques: Staining, Immunohistochemistry