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Figure S6 - Figure.S6 shows combination of rapamycin and BCG increase percentage of effector memory γδ T cells and production of cytokine such as IFNγ within γδ T cells.

Reagent information for flow cytometry and ELISPOT assay.

Legends for all 7 supplementary figures

Bacillus Calmette-Guérin (BCG) is the most effective intravesical agent at reducing recurrence for patients with high-grade, non–muscle-invasive bladder cancer. Nevertheless, response to BCG is variable and strategies to boost BCG efficacy have not materialized. Prior work demonstrated a requirement for either conventional αβ or nonconventional γδ T cells in mediating BCG treatment efficacy, yet the importance of T-cell antigen specificity for BCG's treatment effect is unclear. Here, we provide direct evidence to show that BCG increases the number of tumor antigen–specific αβ T cells in patients with bladder cancer and protects mice from subsequent same-tumor challenge, supporting BCG induction of tumor-specific memory and protection. Adoptive T-cell transfers of antigen-specific αβ T cells into immunodeficient mice challenged with syngeneic MB49 bladder tumors showed that both tumor and BCG antigen–specific αβ T cells contributed to BCG efficacy. BCG-specific antitumor immunity, however, also required nonconventional γδ T cells. Prior work shows that the mTOR inhibitor rapamycin induces the proliferation and effector function of γδ T cells. Here, rapamycin increased BCG efficacy against both mouse and human bladder cancer in vivo in a γδ T cell–dependent manner. Thus, γδ T cells augment antitumor adaptive immune effects of BCG and support rapamycin as a promising approach to boost BCG efficacy in the treatment of non–muscle-invasive bladder cancer.

Flow cytometry gating strategy is outlined. After digestion of tumor infiltrated bladders, samples yielded 1-2 million cells per bladder. A) Live T lymphocyte subpopulations were then gated from spleen tissue, and B) bladder T cells of interest were then selected.

Intravesical bacillus Calmette–Guérin (BCG) immunotherapy is the standard of care in treating non–muscle-invasive bladder cancer, yet its mechanism of action remains elusive. Both innate and adaptive immune responses have been implicated in BCG activity. Although prior research has indirectly demonstrated the importance of T cells and shown a rise in CD4+ T cells in bladder tissue after BCG, T-cell subpopulations have not been fully characterized. We investigated the relationship between effector and regulatory T cells in an immune competent, clinically relevant rodent model of bladder cancer. Our data demonstrate that cancer progression in the N-methyl-N-nitrosourea (MNU) rat model of bladder cancer was characterized by a decline in the CD8/FoxP3 ratio, consistent with decreased adaptive immunity. In contrast, treatment with intravesical BCG led to a large, transient rise in the CD4+ T-cell population in the urothelium and was both more effective and immunogenic compared with intravesical chemotherapy. Whole-transcriptome expression profiling of posttreatment intravesical CD4+ and CD8+ T cells revealed minimal differences in gene expression after BCG treatment. Together, our results suggest that although BCG induces T-cell recruitment to the bladder, the T-cell phenotype does not markedly change, implying that combining T-cell–activating agents with BCG might improve clinical activity. Cancer Immunol Res; 5(7); 594–603. ©2017 AACR.

Supplemental Figure 1. Gene set enrichment analysis. Supplemental Figure 2. TCF21 expression analysis. Supplemental Figure 3. TCF21 expression in cell lines. Supplemental Figure 4. Loss-of-function and gain-of-function studies of TCF21 Supplemental Figure 5. CD24 and CD44 expression in TCF21 OE UM-UC3 and UM-UC13 cells. Supplemental Figure 6. GSEA analysis of common pathways altered between the three cell lines. Supplemental Figure 7. Heatmap of hierarchical clustering of luminal markers showing differential expression after TCF21 upregulation in UC13 LN (A) and UC13 MET (B) cells.

Supplementary Figure Legends

(A) Rats 8 weeks post MNU were treated intravesically with MMC, MMC+BCG, and Cisplatin for 6 weeks (n=5 for each group). Bladders were harvested at week 16 and then digested into single cell suspensions and T cell populations analyzed with flow cytometry. (B) Flow Cytometry was then performed on digested bladders, and T cell subpopulations are reported as percentages, cell numbers, and (C) ratios. A "*" denotes statistical significance. All experiments were performed 2 times.

5 BCG treated rats, 5 untreated rats, and 20 controls rat were sacrificed and bladders were processed in single cell suspensions. CD4 and CD8 T cells were isolated for each sample and RNA was extracted. A gene expression analysis was subsequently performed on sorted CD4 and CD8 cells from each group using an Affymetrix Rat Gene 1.0 ST Array. MVA plot of sorted (A) CD8 and (B) CD4 T cells from MNU rats treated with BCG vs Untreated tumors. Genes selected from the Gene Ontology term "Immune Response" that have

Related Article from The Molecular, the Bad, and the Ugly: Preventing Bladder Cancer via mTOR Inhibition

Supplementary Table 1: Relative Gene Expression of outlier "immune response" genes after BCG vs no treatment in sorted CD8+ cells from bladder cancer. Supplementary Table 2: Relative Gene Expression of outlier "immune response" genes after BCG vs no treatment in sorted CD4+ cells from bladder cancer.

Supplementary figures. Figures S1-S5

COXEN Model development

Purpose:To characterize immune cell expression among patients with non–muscle invasive bladder cancer (NMIBC) treated with Bacillus Calmette-Guerin (BCG).Experimental Design:Patients with NMIBC treated with intravesical BCG (2008–2015) were identified, and a tissue microarray was constructed using paired pre- and post-BCG bladder samples. Among patients undergoing BCG, cystoscopic evaluation began 3 months after initiating BCG treatment to determine therapeutic response. IHC was performed for CD8, CD4, FoxP3, PD-L1 (SP-142 and 22C3), and PD-1. A full slide review of PD-L1+ staining tumors was performed to characterize PD-L1 and CD8 colocalization. RNA-seq was performed on cored tumors from available specimens. We compared immune cell populations between BCG responders and nonresponders, and between pretreatment and postreatment tumor samples. Baseline PD-L1 staining in the BCG naïve population was then validated in a separate cohort.Results:The final cohort contained 63 pretreatment NMIBC cases, including 31 BCG responders and 32 BCG nonresponders. No differences in CD4, CD8, or FoxP3 expression were identified between responders and nonresponders. Baseline PD-L1 expression (22C3 and SP-142) was observed in 25% to 28% of nonresponders and 0% to 4% of responders (P < 0.01). PD-L1+ cells in BCG nonresponders colocalized with CD8+ T cells. In addition, BCG therapy did not increase PD-L1 gene expression (RNA-seq) or protein levels (IHC). The number of pretreatment CD4+ T cells was very low among PD-L1+ nonresponders (12%) and high among PD-L1− nonresponders (50%, P < 0.01). In a separate cohort of 57 patients with NMIBC undergoing BCG, baseline PD-L1 (22C3) staining was similar (26%).Conclusions:One mechanism of BCG failure may be adaptive immune resistance. Baseline tumor PD-L1 expression predicts an unfavorable response to BCG and if validated, could be used to guide therapeutic decisions.

Supplemental Table 1. Calculation of individual drug scores for COXEN

Supplementary Figures S1-S3 from Effects of mTOR Inhibitor Everolimus (RAD001) on Bladder Cancer Cells

Supplemental Table 2. Comparing GC and DDMVAC for Pathologic Response at Cystectomy - intention to treat analysis

Supplementary Data from Nuclear Factor-κB p65/relA Silencing Induces Apoptosis and Increases Gemcitabine Effectiveness in a Subset of Pancreatic Cancer Cells

Purpose:Clinical trials with immune checkpoint inhibition in sarcomas have demonstrated minimal response. Here, we interrogated the tumor microenvironment (TME) of two contrasting soft-tissue sarcomas (STS), rhabdomyosarcomas and undifferentiated pleomorphic sarcomas (UPS), with differing genetic underpinnings and responses to immune checkpoint inhibition to understand the mechanisms that lead to response.Experimental Design:Utilizing fresh and formalin-fixed, paraffin-embedded tissue from patients diagnosed with UPS and rhabdomyosarcomas, we dissected the TME by using IHC, flow cytometry, and comparative transcriptomic studies.Results:Our results demonstrated both STS subtypes to be dominated by tumor-associated macrophages and infiltrated with immune cells that localized near the tumor vasculature. Both subtypes had similar T-cell densities, however, their in situ distribution diverged. UPS specimens demonstrated diffuse intratumoral infiltration of T cells, while rhabdomyosarcomas samples revealed intratumoral T cells that clustered with B cells near perivascular beds, forming tertiary lymphoid structures (TLS). T cells in UPS specimens were comprised of abundant CD8+ T cells exhibiting high PD-1 expression, which might represent the tumor reactive repertoire. In rhabdomyosarcomas, T cells were limited to TLS, but expressed immune checkpoints and immunomodulatory molecules which, if appropriately targeted, could help unleash T cells into the rest of the tumor tissue.Conclusions:Our work in STS revealed an immunosuppressive TME dominated by myeloid cells, which may be overcome with activation of T cells that traffic into the tumor. In rhabdomyosarcomas, targeting T cells found within TLS may be key to achieve antitumor response.

Purpose: Prior clinical trials evaluating cisplatin for non–muscle-invasive bladder cancer (NMIBC) were stopped due to local and systemic toxicity. Currently, there is still a need for improved intravesical therapies, and nanoparticle-based CDDP may be efficacious without the toxicity of free cisplatin observed in the past.Experimental Design: Cisplatin nanoparticles (CDDP NPs) were developed using biocompatible poly(l-aspartic acid sodium salt; PAA), both with and without low and high grafting density of methoxy-polyethylene glycol (PEG). In vitro cytotoxicity studies confirmed activity of CDDP NPs and CDDP solution against a papillary bladder cancer cell line. Local toxicity was assessed by three weekly intravesical administrations of CDDP formulations. CDDP NPs and CDDP solution were evaluated for bladder absorption in murine models 1 and 4 hours after intravesical administration. In vivo efficacy was evaluated in an immunocompetent carcinogen model of NMIBC.Results: CDDP NPs showed decreased local toxicity, as assessed by bladder weight, compared with CDDP solution. Furthermore, >2 μg/mL of platinum was observed in mouse serum after intravesical administration of CDDP solution, whereas serum platinum was below the limit of quantification after intravesical administration of CDDP NPs. CDDP NPs provided significantly increased (P < 0.05) drug levels in murine bladders compared with CDDP solution for at least 4 hours after intravesical administration. In vivo, CDDP NPs reduced cancer cell proliferation compared with untreated controls, and was the only treatment group without evidence of invasive carcinoma.Conclusions: Cisplatin-loaded PAA NPs have the potential to improve intravesical treatment of NMIBC while reducing local and systemic side effects. Clin Cancer Res; 23(21); 6592–601. ©2017 AACR.

Purpose:Dose-dense methotrexate-vinblastine-adriamycin-cisplatin (ddMVAC) and gemcitabine-cisplatin (GC) are accepted neoadjuvant regimens for muscle-invasive bladder cancer. The aim of this study was to validate the score from a coexpression extrapolation (COXEN) algorithm–generated gene expression model (GEM) as a biomarker in patients undergoing radical cystectomy.Patients and Methods:Eligibility included cT2-T4a N0 M0, urothelial bladder cancer, ≥ 5 mm of viable tumor, cisplatin eligible, with plan for cystectomy; 237 patients were randomized between ddMVAC, given every 14 days for four cycles, and GC, given every 21 days for four cycles. The primary objective assessed prespecified dichotomous treatment-specific COXEN score as predictive of pT0 rate or ≤ pT1 (downstaging) at surgery.Results:Among 167 evaluable patients, the OR for pT0 with the GC GEM score in GC-treated patients was 2.63 [P = 0.10; 95% confidence interval (CI), 0.82–8.36]; for the ddMVAC COXEN GEM score with ddMVAC treatment, the OR was 1.12 (P = 0.82, 95% CI, 0.42–2.95). The GC GEM score was applied to pooled arms (GC and ddMVAC) for downstaging with an OR of 2.33 (P = 0.02; 95% CI, 1.11–4.89). In an intention-to-treat analysis of eligible patients (n = 227), pT0 rates for ddMVAC and GC were 28% and 30% (P = 0.75); downstaging was 47% and 40% (P = 0.27), respectively.Conclusions:Treatment-specific COXEN scores were not significantly predictive for response to individual chemotherapy treatment. The COXEN GEM GC score was significantly associated with downstaging in the pooled arms. Additional biomarker development is planned.

Supplemental Figures description. S1-S5

Supplementary Table 1: Nanoparticle physicochemical characteristics. Table S2: IC50 values of various formulations against various bladder cancer cell lines.

Supplementary Figure 1: Transmission electron micrographs of PAA-CDDP NPs, PEGlow-PAA-CDDP NPs and PEGhigh-PAA-CDDP NPs (Scale bars = 500 nm). Supplementary Figure 2. Apoptosis was compared in RT4 cells treated with CDDP and PAA-CDDP NPs. DNA (blue, stained with hoechst33342), apoptotic cells (green, stained with Apoxin), and dead cells (red, stained with propidium iodide). Both formulations induced apoptosis in RT4 cells. Supplementary Figure 3: Images of gross tissue size supported the increased bladder weights in the CDDP treated group after 3 weekly administrations compared to PAA-CDDP NP, PEGlow-PAA-CDDP NPs and untreated control. Supplementary Figure 4. (A) Representative IHC sections stained for cleaved caspase 3 (brown) of rats treated with intravesical CDDP solution or intravesical PAA-CDDP NPs. (B) No significant differences in caspase 3+ cells were identified between treatment groups.

Table components of supplementary figures

Supplementary Figure Legend

Purpose: Nuclear factor κB (NFκB) activity may increase survival and protect cancer cells from chemotherapy. Therefore, NFκB activity may be prognostic, and inhibition of NFκB may be useful for pancreatic cancer therapy. To test these hypotheses, we examined NFκB activity and the effects of inhibiting NFκB in several pancreatic cancer cell lines with differing sensitivities to gemcitabine.Experimental Design: The gemcitabine sensitivity of pancreatic cancer cell lines BxPC-3, L3.6pl, CFPAC-1, MPanc-96, PANC-1, and MIA PaCa-2 were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and fluorescence-activated cell sorting assays. NFκB levels were determined by electrophoretic mobility shift assay and reporter assays. The effects of gemcitabine on NFκB activity were determined in vitro and in vivo. NFκB was inhibited by silencing of the p65/relA subunit using small interfering RNA in vitro and by neutral liposomal delivery of small interfering RNA in vivo, and the effects were evaluated on gemcitabine sensitivity.Results: The cell lines L3.6pl, BxPC-3, and CFPAC-1 were sensitive, whereas MPanc-96, PANC-1, and MIA PaCa-2 were resistant to gemcitabine. No significant correlation was observed between basal NFκB activity and gemcitabine sensitivity. Gemcitabine treatment did not activate NFκB either in vitro or in vivo. Silencing of p65/relA induced apoptosis and increased gemcitabine killing of all gemcitabine-sensitive pancreatic cancer cells. No significant effects, however, were observed on gemcitabine-resistant pancreatic cancer cell lines either in vitro or in vivo.Conclusions: NFκB activity did not correlate with sensitivity to gemcitabine. Silencing of p65/relA was effective alone and in combination with gemcitabine in gemcitabine-sensitive but not gemcitabine-resistant pancreatic cancer cells. Thus, NFκB may be a useful therapeutic target for a subset of pancreatic cancers.

Purpose: We investigated the effect of the mTOR inhibitor RAD001 (everolimus) on human bladder cancer (BC) cells in vitro and in vivo.Experimental Design: The effect of RAD001 on the growth of UM-UC-3, UM-UC-6, UM-UC-9, and UM-UC-14 BC cells were assessed by crystal violet and [3H]thymidine incorporation assays. Flow cytometric cell-cycle analyses were done to measure the apoptotic cell fraction. Protein synthesis was measured using tritium-labeled leucine incorporation assays. The effects of RAD001 on the mTOR pathway were analyzed by Western blotting. To test the effects of RAD001 in vivo, UM-UC-3, UM-UC-6, and UM-UC-9 cells were subcutaneously implanted into nude mice. Tumor-bearing mice were treated orally with RAD001 or placebo. Tumors were harvested for immunohistochemical analysis.Results:In vitro, RAD001 transiently inhibited BC cell growth in a dose-dependent manner. This effect was augmented by re-treatment of cells after 3 days. UM-UC-14 cells were the most sensitive to RAD001, whereas UM-UC-9 cells were the least sensitive. After re-treatment with RAD001, only sensitive cell lines showed G1-phase arrest, with no evidence of apoptosis. RAD001 significantly inhibited the growth of tumors that were subcutaneously implanted in mice. Inhibition of protein synthesis through the S6K and 4EBP1 pathways seems to be the main mechanism for the RAD001-induced growth inhibition. However, inhibition of angiogenesis was the predominant mechanism of the effect of RAD001 on UM-UC-9 cells.Conclusions: The mTOR inhibitor RAD001 inhibits growth of BC cells in vitro. RAD001 is effective in treating BC tumors in an in vivo nude mouse model despite the heterogeneity of in vitro responses. Clin Cancer Res; 17(9); 2863–73. ©2011 AACR.

Supplemental Table 3 Number of Patients with a Given Type and Grade of Adverse Event Adverse Events Unlikely or Not Related to Treatment Excluded

Supplementary Figure Legends 1-3 from Control of HIF-1α Expression by eIF2α Phosphorylation–Mediated Translational Repression

Supplementary Figure 1 from Control of HIF-1α Expression by eIF2α Phosphorylation–Mediated Translational Repression

PDF file, 1717K, qRT-PCR of autotaxin and nfat1 mRNA after silencing nfat1 in A375SM melanoma cells

PDF file, 1628K, Invasion, migration, and soft agar assay of melanoma cells after silencing galectin-3

PDF file, 61K, Oligos used for experiments

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Supplementary Figure 2 from Epithelial to Mesenchymal Transition Contributes to Drug Resistance in Pancreatic Cancer