Additional file 1: Figure S1. of Quantitative assessment of the spatial heterogeneity of tumor-infiltrating lymphocytes in breast cancer

Batch to batch testing. Average AQUAÂŽ scores for serial sections of tonsil whole tissue run along with batch samples to ensure consistency in staining and analysis between batches. Figure S2. Distribution of CD20 in different FOVs from a single core on a single slide. CD20 cells often form tertiary lymphoid structures that give rise to highly heterogeneous FOVs on even a single slide. This is an illustration of a CD20 stain showing a tertiary lymphoid structure on the left and a nearby negative FOV on the right. Figure S3. Distribution of TIL subsets and intratumor heterogeneity in breast cancer. Representative immunofluorescence images showing the heterogeneity of CD3 (red), CD8 (green) and CD20 (magenta) in breast cancer tissue. Fluorescence signal was captured and unmixed using automated quantitative epifluorescence microscopy. Areas with high (left panels), intermediate (center panels), and low (right panels) signal for each marker obtained from the same core are shown although each row is from a separate patient for optimal illustration. Figure S4. Comparison of QIF versus cell count for CD8 and CD20. Graphs comparing cell counts with INform on the Y axis compared to QIF by AQUAÂŽ on the X-axis show a good, but not perfect correlation as the methods measure different tissue parameters. QIF AQUAÂŽ score is more similar to a protein concentration per unit area (FOV) while the cell count shows the absolute cell count in the same FOV (in this case a TMA spot from a lung cancer cohort). (PPTX 994 kb

Kaplan-Meier survival curve comparing progression-free survival estimation between low- and high-expressing E-cadherin, beta-catenin and EGFR groups.

<p>a) Patients with high tumor E-cadherin expression exhibit a higher probability of PFS (59.7% vs. 40.6%, p = 0.04). b) Patients with low expression of beta-catenin trended towards worse PFS (p = 0.057). c) There was no association between EGFR expression and PFS (p = 0.49).</p

Kaplan-Meier survival curve comparing overall survival estimation between low- and high-expressing E-cadherin groups.

<p>a) Patients with high tumor E-cadherin expression exhibit a higher probability of OS (69.6% vs. 44.3%, p = 0.05). b) There was no association between beta-catenin and OS (p = 0.16). c) Patients with high tumor EGFR expression had inferior 5-year overall survival compared with those with low tumor EGFR expression (27.7% vs. 54%, p = 0.029).</p

Multivariable 5-year overall survival (OS) analysis, by Cox regression.

<p>Multivariable 5-year overall survival (OS) analysis, by Cox regression.</p

Demographic, clinical and pathologic characteristics.

<p>Demographic, clinical and pathologic characteristics.</p

Additional file 2: Figure S1. of Effect of neoadjuvant chemotherapy on tumor-infiltrating lymphocytes and PD-L1 expression in breast cancer and its clinical significance

Showing HES of TILs at baseline and post-treatment. A Baseline HES of a case with moderate TIL infiltration at baseline and increased TIL counts following treatment. B Matched post-neoadjuvant HES of the baseline biopsy shown in (A). C Baseline HES of a case that displayed decrease TIL counts following treatment. D. Matched post-neoadjuvant HES of the baseline biopsy shown in (C). 20× Magnification, bar = 200 μm. (TIF 948 kb

Additional file 5: Figure S3. of Effect of neoadjuvant chemotherapy on tumor-infiltrating lymphocytes and PD-L1 expression in breast cancer and its clinical significance

Showing SP142 antibody validation and reproducibility. A Regressions in QIF scores (average) between staining performed in different days in serial sections of a lung cancer TMA (245). B Representative immunostaining for PD-L1 SP142 antibody in control tissues (placenta in upper panel and lung in lower panel) using QIF (left panel; SP142 in Cy5, red and cytokeratin mask in Cy3, green) and conventional IHC staining with DAB (right panel). (TIF 583 kb

Additional file 4: Figure S2. of Effect of neoadjuvant chemotherapy on tumor-infiltrating lymphocytes and PD-L1 expression in breast cancer and its clinical significance

Showing PD-L1 expression in post-neoadjuvant breast cancer specimens. A Distribution of maximal scores of PD-L1 (SP142 antibody) in the tumor (red) and stromal (blue) compartments. The cutoff was set at 500 AQUA units (QIF). B Linear regression of stromal versus tumor PD-L1 AQUA (QIF) scores. (TIF 148 kb

Combinations of either of NVP-BEZ235 and Erlotinib in H2170 (left) and HCC2935 (right) lung cancer cell lines.

<p>H2170 and HCC2935 cells were treated with the PI3K inhibitor NVP-BEZ235 alone or combined with Erlotinib at their respective single drug nanomolar IC₅₀ or IC₂₅ concentrations. Viability was measured at 72 hours with the Cell Titer Glo assay. The bars show viability as a percentage of viable cells relative to untreated cells. Three separate experiments were performed and each condition was measured in three replicate wells.</p

Effects of NVP-BKM120, LY294002 and NVP-BEZ235 on pAKT, pP70S6K, and pS6 expression in HCC2935 and H2170 lung cancer cell lines.

<p><b>Panel A</b>: HCC2935 and H2170 cells were treated with the indicated concentration of NVP-BKM120, LY294002 or NVP-BEZ235 for 1, 6 or 24 hours and harvested at these time points. Cells were lysed and proteins were extracted, electrophoresed and probed for expression of phosporylated AKT (Ser⁴⁷³), phosphorylated p70S6K (Thr³⁸⁹) and phosphorylated S6 Ribosomal Protein (Ser^235/236). Protein gel loading was assessed by expression of β-Actin. <b>Panel B</b>: Effects of NVP-BEZ235 on apoptosis in HCC2935 and H2170 lung cancer cell lines. Treatment of H2170 and HCC2935 cells with ascending concentrations of NVP-BEZ235 resulted in dose-dependent PARP cleavage and caspase-2 induction.</p