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Combination of IFNα and poly-I:C reprograms bladder cancer microenvironment for enhanced CTL attraction

Combination of IFNα and poly-I:C reprograms bladder cancer microenvironment for enhanced CTL... Background: BCG is a prototypal cancer immunotherapeutic factor currently approved of bladder cancer. In attempt to further enhance the effectiveness of immunotherapy of bladder cancer and, potentially, other malignancies, we evaluated the impact of BCG on local production of chemokines attracting the desirable effector CD8 T cells (CTLs) and undesirable myeloid-derived suppressor cell (MDSCs) and regulatory T(reg) cells, and the ability of bladder cancer tissues to attract CTLs. Methods: Freshly resected bladder cancer tissues were either analyzed immediately or cultured ex vivo in the absence or presence of the tested factors. The expression of chemokine genes, secretion of chemokines and their local sources in freshly harvested and ex vivo-treated tumor explants were analyzed by quantitative PCR (Taqman), ELISAs and immunofluorescence/confocal microscopy. Migration of CTLs was evaluated ex vivo, using 24-transwell plates. Spearman correlation was used for correlative analysis, while paired Students T test or Wilcoxon was used for statistical analysis of the data. Results: Bladder cancer tissues spontaneously expressed high levels of the granulocyte/MDSC-attractant CXCL8 and T -attractant CCL22, but only marginal levels of the CTL-attracting chemokines: CCL5, CXCL9 and CXCL10. Baseline reg CXCL10 showed strong correlation with local expression of CTL markers. Unexpectedly, BCG selectively induced only the undesirable chemokines, CCL22 and CXCL8, but had only marginal impact on CXCL10 production. In sharp contrast, the combination of IFNα and a TLR3 ligand, poly-I:C (but not the combinations of BCG with IFNα or BCG with poly-I:C), induced high levels of intra-tumoral production of CXCL10 and promoted CTL attraction. The combination of BCG with IFNα + poly-I:C regimen did not show additional advantage. Conclusions: The current data indicate that suboptimal ability of BCG to reprogram cancer-associated chemokine environment may be a factor limiting its therapeutic activity. Our observations that the combination of BCG with (or replacement by) IFNα and poly-I:C allows to reprogram bladder cancer tissues for enhanced CTL entry may provide for new methods of improving the effectiveness of immunotherapy of bladder cancer, helping to extend BCG applications to its more advanced forms, and, potentially, other diseases. Keywords: Tumor microenvironment, Immunomodulation, Chemokines, BCG, TLR3, Poly-I :C, IFNα, Bladder cancer, Effector T cells, Regulatory T cells * Correspondence: kalinskip@upmc.edu Departments of Sugery, University of Pittsburgh, Pittsburgh, PA 15213, USA Department of Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, PA 15213, USA Full list of author information is available at the end of the article © 2015 Muthuswamy et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 2 of 10 Background analysis for the chemokines previously implicated in Bladder cancer, originating from the transitional cells of CTL or MDSC/T attraction to tumor lesions [26-30]. reg the bladder urothelium, accounts for an estimated We observed that bladder tumors uniformly expressed 72,570 new cases and 15,210 deaths in the US. Though only low levels of CTL attracting chemokines: CCL5, bladder cancer is highly treatable if found early, it CXCL9, and CXCL10 (respective ligands for CTL- becomes increasingly difficult to treat at later stages. expressed CCR5 and CXCR3 [19]). In a striking contrast Intravesicular BCG administration has been the stan- to the above CTL-attractants, the chemokines implicated dard therapy for treatment of bladder cancer, but its in attracting MDSCs and T , CCL2 (MCP-1), CCL22 regs effectiveness is limited only to superficial bladder (MDC) and CXCL8 (IL-8) were highly expressed chemo- cancers [1-4]. kines (Figure 1A), with CXCL8 uniformly expressed at the Tumor infiltration with effector CD8 T cells (CTL) highest levels. The prevalence of high CCL22 and CXCL8 has been associated with good prognosis in various can- in bladder tumors at baseline is consistent with previous cers [5-18], therefore immunotherapies able of enhan- studies [31-33] which showed the abundance of these cing intra-tumoral CTL levels may be also effective for undesirable chemokines, particularly in patients with poor invasive bladder cancers. Previous studies by us [19] and prognosis. High intra-tumoral expression of CXCL8 others [10,20-25] have shown that intra-tumoral expres- was confirmed by confocal analysis of tumor samples sion of chemokines regulate local levels of CTL infiltra- (Figure 1C). Taqman analysis for different markers of im- tion, suggesting that T cell-targeting immunotherapies mune infiltrate (Figure 1B), using primers for CD4, CD8, can benefit from modulating tumor-associated chemo- GZMB and T-bet (effector cell markers), CD33 (myeloid kine microenvironments in order to enhance local CTL marker), GITR (Treg marker) and NCF2 (myeloid or infiltration. neutrophil marker) revealed that NCF2 expression was do- Since myeloid-derived suppressor cell (MDSCs) and minant, corresponding to the high IL-8 levels (see below). regulatory T cells are both known to protect tumors (reg) from CTL-mediated elimination and to promote tumor Intra-tumoral expression of CCL5, CXCL9, and CXCL10 growth, ideal immunotherapies should be able to se- shows a strong correlation with CTL markers CD8 and lectively enhance tumor production of CTL-attracting Granzyme B chemokines, without enhancing local levels of CXCL8, In order to test if the spontaneous expressed nominal CXCL12 and CCL22, the chemokines mediating local CTL-attracting chemokines indeed predict local CTL attraction of MDSC and T to tumors [26-30]. Prom- reg infiltration in bladder cancer tissues, we attempted to pted by the above considerations, we evaluated the correlate intra-tumoral mRNA expression of CCR5 ability of BCG and alternative adjuvants to reprogram ligands and CXCR3 ligand (CCL5, CXCL9 and CXCL10) local chemokine milieu in bladder cancer to enhance with Intra-tumoral mRNA expression of CTL markers. the overall magnitude of local production of CTL- As shown in Figure 2, Spearman correlative analysis of attracting chemokines in relation to MDSC/T -attrac- reg the chemokines and CTL markers revealed strong cor- tants. Unexpectedly, we observed that BCG, used alone, relation between CD8 and Granzyme B with CCL5, not only failed to enhance local expression of CTL- CXCL9 and CXCL10. As expected, neither of these attracting chemokines, CCL5 and CXCL10, but selec- markers was correlated with the local expression of tively enhanced MDSC- and T -attracting chemokines, reg aT -attractant, CCL22 (data not shown). The levels reg CCL22 and CXCL8. These undesirable side-effects of IL-8 expression were strongly correlated with could be reversed by the combination of IFNα and poly- NCF2 expression (neutrophil marker; Additional file 1: I:C (TLR3 ligand), raising the possibility of enhancing Figure S1). the effectiveness of the BCG-based and other forms of immunotherapy of bladder cancer and potentially other malignancies. BCG treatment of bladder tumors up regulates CXCL8, CCL22 expression, but not CXCL10 Results Using CXCL10 as a representative of CTL-attracting Bladder cancer tissues spontaneously produce MDSC- and chemokines, involved in attraction of CXCR3 CTL im- T -attracting CXCL8 and CCL22, but not effector plicated with good prognosis for cancer patients reg T cell-attracting chemokines [7,11,12,15], we tested whether BCG treatment can en- In order to evaluate the spontaneous chemokine ex- hance intra-tumoral production CXCL10. Unexpect- pression and determine the baseline chemokine pro- edly, we observed that, while BCG strongly up regulated duction pattern in bladder tumors, we isolated RNA the secretion of CXCL8 (P < 0.05) and CCL22 (P < 0.05) from resected tumors of bladder cancer patients by ex vivo-treated bladder cancer explants (n = 11 pa- (N = 20) and performed real-time PCR (Taqman) tients), it did not enhance CXCL10 secretion (Figure 3). Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 3 of 10 N= 2 0 T CKs T /M D S C C K s eff re g N= 2 0 Anti-tum or Pro -tu m o r nd 2 Ab nd 2 Ab CXCL8, CXCL10 & Nuclei Tumor-2 Tumor-1 Figure 1 Bladder cancer tissues spontaneously express high levels of MDSC/T - attracting chemokines CCL22 and CXCL8, but only reg marginal levels of CTL- attractants, CCL5, CXCL9 and CXCL10. A. Spontaneous chemokine mRNA expression by bladder tumors (N = 20 different patients) by real-time PCR analysis (Taqman). B. Spontaneous mRNA expression analysis for markers of immune filtrate in bladder tumors (N = 20 different patients) by real-time PCR analysis (Taqman). The levels of chemokine and immune filtrate markers were normalized to HPRT1 mRNA (housekeeping gene). C. Confocal analysis for CXCL8 (Red), CXCL10 (Green) protein expression in 2 representative bladder tumors. Nuclei were stained with Sytox orange (Blue) and specificity controls (secondary antibody only) are shown in insets. Combination of IFNα + poly-I:C reverses the BCG-driven CTL chemokines in colorectal cancer tissues [19], we enhancement of undesirable chemokines tested whether their addition to BCG can enhance its Since we have previously demonstrated that the combi- effectiveness. In a preliminary set of experiments we nation of IFNα with poly-I:C effectively up regulated used an in vitro model system involving TS4 bladder CC L 5 C X C L9 C XC L 10 CX CL 12 CCL 2 C CL 22 CX CL 8 CD8 GZ M B C D 4 T -B E T C D 33 G IT R N CF 2 mR N A mR NA Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 4 of 10 N=20 10 rh o = 0.87, P < 0.0 00 1 rho = 0.89, P < 0.0 00 1 0.1 0.1 0.01 0.01 0.001 0.001 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 CCL5 CCL5 10 10 rho = 0.84, P < 0 .0 00 1 rh o = 0 .8 9 , P < 0 .0 0 0 1 1 1 0.1 0.1 0.01 0.01 0.001 0.001 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 CX CL9 CX CL9 10 rh o = 0 .7 6 , P < 0 .0 0 0 1 rh o= 0.68 , P <0 .0 0 1 0.1 0.1 0.01 0.01 0.001 0.001 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 CX CL10 CX CL10 Figure 2 CCR5- and CXCR3-binding chemokines (CCL5, CXCL9 and CXCL10) show high correlation with CTL markers in bladder cancer tissues. Spearman (Rho) analysis of the correlations between the spontaneous expression (mRNA) of CTL markers (CD8 and Granzyme B) and CTL attracting chemokines (CCL5, CXCL9 and CXCL10). Values on the scale are log10 transformations of relative mRNA levels for each of the markers were evaluated using real time PCR (Taqman). cancer cells, blood isolated monocytes and fibroblasts In accordance with these observations, our experi- (Additional file 1: Figure S3) to directly compare mul- ments performed in the tumor tissue explant model tiple combinatorial adjuvants in a single experiment, (n = 6 patients), demonstrated that in contrast to BCG, without the limitation imposed by the amount of blad- the combination of IFNα and poly-I:C strongly elevated der cancer tissues available from resections and their tumor secretion of CXCL10. The combination of BCG variability between different patients. In accordance with with IFNα + poly-I:C resulted in only marginal or no fur- the data from our bladder cancer explant cultures, BCG ther enhancement of CXCL10 secretion, but was asso- alone was completely ineffective in promoting CXCL10 ciated with the undesirable elevation of CCL22 (Figure 4). secretion in such cell cultures. In contrast, we observed strong synergy between IFNα and poly-I:C in promoting IFNα + poly-I:C-treated tumors show enhanced attraction CXCL10 secretion, both in the absence and in the pres- of effector CD8 cells ence of BCG (Additional file 1: Figure S3). Importantly, To test whether the modified chemokine production neither the combination of BCG with poly-I:C nor the patterns in BCG- and IFNα + poly-I:C-treated bladder combination of BCG with IFNα was effective, which cancer tissues result in their differential ability to attract may explain the limited effectiveness of that later com- CTLs, supernatants of the differentially-treated bladder bination in the recently-completed clinical trial [4,34,35]. cancer tissues were tested for their ability to attract C D 8 CD8 CD8 GZMB GZMB G Z M B Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 5 of 10 Figure 3 BCG exposure of bladder tissues does not induce N= 1 1 CXCL10, but further enhances tumor production of MDSC/T - reg attracting CCL22 and CXCL8. Bladder tumor biopsies were cultured in the absence or presence of BCG (2 × 10 CFU) for 24 hours. CXCL10, CCL22 and CXCL8 proteins in tumor supernatants were measured by ELISA and expressed as ng/ml. The results were evaluated using two- tailed, paired Student’s t test. Statistically ** significant differences between groups are highlighted by * (P < 0.05). NS-Not significant. N=6 (-) B C G 40 N= 1 1 * * (-) B C G N= 1 1 ns Figure 4 Combination of IFNα with poly-I:C is a powerful inducer of CXCL10 in bladder cancer lesions in the absence or presence of BCG. Bladder tumors biopsies were cultured for 0 24 hours in the absence or presence of 10,000 units IFNα +20 μg/ml specific poly-I:C, with or without BCG (2 × 10 CFU). The levels of (-) B C G CXCL10 and CCL22 in tumor supernatants were measured by specific ELISAs. The results were evaluated using two- tailed, paired Wilcoxon Test. Statistically significant differences between groups are highlighted by * (P < 0.05). (-) IF N + poly(I:C) BC G BC G+IF N +p o ly(I:C ) CX C L10 (ng/m l) C C L22 (ng /m l) CX CL8 (ng /m l) CX C L 10 (n g/m l) CC L 22(ng /m l) Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 6 of 10 ex vivo-induced effector CD8 T cells (pre-activated by chemokines (CCL2, CCL22 and CXCL8, respective li- SEB-loaded LPS + IFNγ matured DCs, capable of high gands for CCR2, CCR4 and CXCR1/2), but only low levels IL-12 production [36,37]). As expected, bladder cancer of CTL- attracting chemokines (CCL5, CXCL9-10; re- tissues (n = 3 patients) treated with IFNα + poly-I:C spective ligands for CCR5 and CXCR3), suggesting that significantly attracted more of the effector CD8 T cells the chemokine imbalance can contribute to the pathogen- than untreated or BCG alone treated tumors (Figure 5). esis of bladder cancer and may limit the effectiveness of The combination of BCG with IFNα + poly-I:C didn’t its immunotherapies. Unexpectedly, despite the docu- further increase the CTL attraction. These data indicate mented beneficial role of BCG in bladder cancer, our data that BCG by itself is insufficient to reprogram the indicate that its ability to modify the microenvironment of bladder cancer-associated chemokine environment for bladder cancer ex vivo is largely limited to the enhance- enhanced CTL attraction, but that such goal can be ment of local production of T -and MDSC-attracting reg achieved by the combination of BCG with (or its re- chemokines, CCL22 and CXCL8, without inducing placement by) IFNα plus poly-I:C. CXCL10 or facilitating CTL attraction. Our current data is consistent with the previous observations that bladder Discussion cancer tissues do not produce the desirable chemokine BCG is a prototypal cancer immunotherapeutic factor CXCL10 within the first week of BCG treatment, although which has been widely demonstrated to be effective in can produce this factor after 3 weekly doses of BCG [38]. the treatment of superficial bladder cancer [1-4]. In They are also consistent with the previous observations attempt to further enhance the effectiveness of the that bladder cancer lesions typically produce high levels of immunotherapy of bladder cancer, including its more CCL22 and CXCL8 at baseline [31-33,39,40], which may advanced stages, and potentially other malignancies, we be further amplified by treatment of patients with BCG evaluated the impact of BCG on local production of the and particularly by treatment with by BCG combined with chemokines attracting the desirable effector CD8 T cells chemotherapy [32], constituting the undesirable side- and undesirable MDSCs and T cells. effect of treatment [33]. Since intratumoral expression of reg We observed that bladder cancer tissues spontaneously CXCL8 has been shown to be a negative prognostic expressed high levels of T - and MDSC- recruiting marker [31], these observations highlight a potential for reg N=3 (-) BC G IF N + p o ly - I : C ** BC G + IF N + po ly -I:C B la dder tum ors tre ate d w ith Figure 5 Bladder cancer lesions exposed to IFNα + poly-I:C or BCG + IFNα + poly-I:C show strongly-enhanced CTL-attracting activity. + + Day 6 effector Granzyme B /CD8 T cells (induced by SEB-loaded LPS + IFNγ-matured DC [36,37]) were harvested and allowed to migrate for 2 hours towards the supernatants from the differentially-treated bladder cancer tissues in 24 trans-well system. Migrated cells in the bottom + + chamber were harvested and stained for CD8, Granzyme B. The counts of Granzyme B CD8 T cells were analyzed by FACS. The results were evaluated using two- tailed, paired Student’s t test. Statistically significant differences between groups are highlighted by * (P < 0.05) or ** (P < 0.01). (-) B CG IFN +p o ly - I : C BC G+ I F N + p oly- I : C + + Migrated C D 8 GZMB Tcells Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 7 of 10 targeting the bladder cancer-associated chemokine micro- BCG in effective reprogramming of cancer tissues for en- environments to improve the outcomes of BCG-treatment hanced CTL entry, may help to improve the effectiveness and chemotherapy of this cancer type. of immunotherapy of bladder cancer and enhance the ap- This selective activity of BCG may result from its plications of BCG to its more advanced forms, as well as dominant pattern of TLR2-mediated signaling [41], other malignancies or other disease states (such a myco- which has been shown to induce significantly less type-1 bacterial infections), where the current BCG-based thera- interferons compared to other TLR ligands [42,43]. In peutic regimens are not effective. accordance with this possibility, our observations sug- gests that the combination of IFNα with poly-I:C Methods (a TLR3 ligand) or BCG + IFNα + poly-I:C (although nei- Ex vivo culture of bladder tumor explant tissues ther BCG + IFNα nor BCG + poly-I:C) are highly effect- Tumor from patients was obtained by informed consent ive in enhancing intra-tumoral production of CXCL10 in under the IRB approved protocol UPCI 86–022. Patient bladder cancer tissues and promoting CTL infiltration. clinical characteristics and sets of patients used for spe- Interestingly, both these effective combinations showed cific types of experiments are given in Table 1 below. at least partial selectivity in inducing CXCL10 (rather Using a 2 mm biopsy punch knife, uniform 2 mm than CCL22) in cancer tissues. Our current experiments cubes of resected tumor tissue were made. Tumor ex- aim to determine if the additional inclusion of COX in- plants were assorted as 3 × two mm cubes/wells in 48 hibitors (which proved to be effective in suppressing well plate respectively and cultured in IMDM plus 10% local production of CCL22 in metastatic colon cancer le- FBS, either untreated or treated with 2 × 10 CFU of sions [19]) with the currently-evaluated BCG + IFNα + BCG (TICE® BCG, Schering Plough, NJ) and/or 10,000 poly-I:C combination will further potentiate its effective- units of IFNα (Merck, NJ), 20 μg/ml of poly-I:C (Sigma ness by suppressing COX-2 dependent production of Aldrich, St. Louis, MO). Tumor tissue biopsies were CCL22, CXCL8 and boosting induction of CXCL10 and harvested at 0 and 24 hours for mRNA and confocal other CTL-attracting factors. microscopy analysis and culture supernatants were har- vested at 24 hours for ELISA. Conclusion Our current data suggest that the suboptimal ability of Bladder cancer cell line-macrophage-fibroblast co-culture BCG to reprogram cancer-associated chemokine environ- system ments may represent a factor limiting its clinical activity, In order to compare multiple combinatorial adjuvants in especially in more advanced stages of bladder cancer, and a single experiment, we established a model system in- may represent an area of additional therapeutic interven- volving 1 × 10 each of TS4 (bladder cell line; ATCC, tion. Our identification of the combination of IFNα and Manassas, VA), blood isolated monocytes (isolated by poly-I:C, as factors which may supplement (or replace) CD14 microbeads; Miltenyi Biotech.) and fibroblasts Table 1 Bladder cancer patients Patient number N (%) Total qPCR analysis ELISA analysis Chemotaxis Patients 23(100) 20(100) 11(100) 3(100) Male 15(65) 14(70) 6(55) 1(33) Female 8(35) 6(30) 5(45) 2(67) Tumor stage: Stage 0 4(17) 4(20) 2(18) Stage 0is 1(4) 1(5) Stage 1 6(26) 5(25) 3(27) 1(33) Stage 2 2(9) 2(10) 1(9) Stage 3 8(35) 6(30) 4(36) 2(67) Stage 4 2(9) 2(10) 1(9) Prior treatment: None 15(65) 13(65) 7(64) 2(67) BCG 5(22) 5(25) 2(18) Other treatment 3(13) 2(10) 2(18) 1(33) Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 8 of 10 (Life technologies, Grand Island, NY) in 48 well plates. ELISA primary and secondary antibody pairs for the ana- The cells were treated with the indicated permutation lysis of CCL5, CXCL8 and CXCL10, protein secretion were combinations of celecoxib (10 μM, Biovision, Milpitas, purchased from Peprotech, Rocky Hill, NJ. ELISA plates CA), IFNα (10000 units/ml), poly-I:C (20 μg/ml) and (Corning Inc, Corning, NY) were coated overnight at RT BCG (2 × 10 CFU) for 24 hours and supernatants were with 100 μL of primary antibody at 1 μg/mL, followed by harvested for ELISA analysis of various chemokines. In washing and blocking with PBS + 4% BSA for 1 hour. preliminary experiments, we used macrophages gene- 50 μL of samples were added to the wells and incubated rated from healthy donors’ monocytes in 6 day-long for 1 hr and subsequently washed and incubated with GM-CSF-supplemented cultures, to directly compare 50 μL of biotinylated secondary antibodies at 0.5 μg/mL the chemokine-modulating impact of increasing doses of for 1 hour. The plates were washed and incubated BCG in the same experiment. As shown in Additional for 30 minutes with streptavidin-HRP conjugate (Pierce file 1: Figure S2, different doses of BCG (0.4, 2 and Biotechnology Inc, Rockford, IL), diluted 1:10000 in wash 10 × 10 CFU), all induced high levels of CXCL8, but not buffer (50 mM Tris, 0.2% Tween). The plates were washed CXCL10. and detected with 50 μL of TMB substrate (Pierce). Reactions were stopped with 2% H SO and 450 nm ab- 2 4 Taqman analysis of mRNA expression in tumor sorbance was measured, using Wallac 1420 Victor 2 mi- Tumor biopsies were placed into lysing Matrix E tubes croplate Reader (Perkin Elmer, Waltham, MA). (MP Biologicals, Solon, OH), containing RLT buffer (RNAeasy kit, Qiagen, Valencia, CA), agitated in FP120 Generation of effector T cells homogenizer (MP Biologicals). Debris-free supernatants Naïve CD8 T cells were purified from peripheral blood from the lysis matrix tubes were transferred into new of normal donor using EasySep naïve CD8 enrichment tubes and total RNA was extracted using the RNAeasy kit (#19158, Stemcell Tech, Vancouver, Canada). Isolated kit (Qiagen,MA). 1 μg of RNA extracted by above naïve CD8 T cells were stimulated with SEB (1 ng/ml) method was used for cDNA synthesis using Quanta bio- pulsed, LPS + IFNγ-matured DC for 6 days [36]. On 6th sciences synthesis kit and 25–50 ng of the resulting day, effector CD8 T cells were harvested, cells counted, cDNA was used to do mRNA expression analysis by density-adjusted to 10 cells per ml and used in chemo- Taqman, using primers and equipment (Step One Plus taxis assays. system) from (Life Technologies, Long Island, NY). Chemotaxis Confocal microscopy analysis of tumor sections In a 24 trans-well plates with membrane with 5 μM pore 5 + Tumor biopsies were embedded in OCT medium con- size (#3421, Corning, CA), 2 × 10 (200ul) effector CD8 taining cryomolds and immediately frozen in 2-methyl- T cells were loaded onto top chambers and allowed to butane. 5 μm frozen sections of the tissues were made migrate for 2 hours [19,36,44] towards 400 μl of super- using the cryostat and layered on superfrost® plus slides natants generated from 24 hour bladder cancer explant (Thermo Scientific, Rockford, IL). The slides were fixed cultures. Migrated cells were harvested from the bottom in 4% para-formaldehyde for 15 minutes, washed and chambers and stained for CD8 and Granzyme B (GZMB). blocked for 60 minutes at room temperature. The slides Number of migrated GZMB positive effector CD8 Tcells were then stained for 3 hours at room temperature (RT) were quantified by fixed 100 μl run on Accuri C6 (BD with antibodies for CXCL10 (ab9807) and CXCL8 Biosciences, San Jose, CA). (ab18672), both from Abcam, Cambridge, MA. The slides were washed 5 times with 1 × PBS and incubated Statistical analysis with secondary antibodies anti-rabbit-Alexa 488, anti- Spearman rank correlations (rho) between the mRNA of mouse-Alexa 647 (both from Cell Signal, Danvers, MA) chemokine genes and cytotoxic T cell markers were and with nuclear dye Sytox orange (Invitrogen, Carlsbad, calculated. Comparisons of continuous variables between CA) for 30 minutes at RT. The slides were washed 5 groups were performed by 2 tailed, paired t test or times with 1 × PBS. Cover slips were mounted on the paired Wilcoxon test using graphPad Prism 6 software. sections using prolong gold anti-fade solution (Life The values of P <0.05 were considered as significant. Technologies). Confocal analyses of stained slides were performed using a LEICA TCS SL DMRE Microsystems. Additional file ELISA analysis of chemokines in tumor ex vivo culture or Additional file 1: Figure S1. Intratumoral CXCL8 expression shows in cell co-cultures strong correlation with the neutrophil marker, NCF2. Spearman (rho) analysis of the correlation between intra-tumoral mRNA expression of 24 hour culture supernatants from tumor ex vivo culture CXCL8 (IL-8) with Intra-tumoral expression of NCF2 (Neutrophil marker). were analyzed by ELISA for chemokine proteins expressed. Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 9 of 10 10. Mlecnik B, Tosolini M, Charoentong P, Kirilovsky A, Bindea G, Berger A, et al. Figure S2. BCG dose titration analysis and its impact on chemokine Biomolecular network reconstruction identifies T-cell homing factors associated secretion by macrophages. Macrophages, generated by culturing with survival in colorectal cancer. Gastroenterology. 2010;138:1429–40. monocytes in presence of GMCSF for 6 days, were exposed to increasing 11. Dudley ME, Gross CA, Langhan MM, Garcia MR, Sherry RM, Yang JC, et al. doses of BCG (0.4, 2, 10 × 106 CFU) for 24 hrs. Secretion of CXCL8 and CD8+ enriched “young” tumor infiltrating lymphocytes can mediate CXCL10 was measured by specific ELISAs. Figure S3. BCG combination regression of metastatic melanoma. Clin Cancer Res. 2010;16:6122–31. with IFNα + poly-I:C reduces CXCL10 levels in co-cultures of bladder 12. Pages F, Kirilovsky A, Mlecnik B, Asslaber M, Tosolini M, Bindea G, et al. cancer cells, macrophages and fibroblasts. TS4(Bladder cell line) was co In situ cytotoxic and memory T cells predict outcome in patients with cultured with macrophages and Fibroblasts in ratio 1:1:1 and treated with early-stage colorectal cancer. J Clin Oncol. 2009;27:5944–51. different permutations of celecoxib, IFNα, poly-I:C, and BCG. CXCL10 13. Sharma P, Shen Y, Wen S, Yamada S, Jungbluth AA, Gnjatic S, et al. CD8 secretion was measured in 24 hour supernatants by ELISA. CCL22 and tumor-infiltrating lymphocytes are predictive of survival in muscle-invasive CXCL8 were below detection levels. urothelial carcinoma. Proc Natl Acad Sci U S A. 2007;104:3967–72. 14. Piersma SJ, Jordanova ES, van Poelgeest MI, Kwappenberg KM, van der Competing interests Hulst JM, Drijfhout JW, et al. High number of intraepithelial CD8+ tumor- The authors declare that they have no competing interests. infiltrating lymphocytes is associated with the absence of lymph node metastases in patients with large early-stage cervical cancer. Cancer Res. 2007;67:354–61. Authors’ contributions 15. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, RM was involved in concept, design and performing of experiments, and wrote et al. Type, density, and location of immune cells within human colorectal the manuscript. LW was involved in performing experiments. JP was involved in tumors predict clinical outcome. Science. 2006;313:1960–4. performing experiments; JRG participated in the study design, provided patient 16. Sato E, Olson SH, Ahn J, Bundy B, Nishikawa H, Qian F, et al. Intraepithelial material and analyzed patient-related data, and was involved in preparation of CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell the manuscript. PK conceived the study, was involved in concept and design of ratio are associated with favorable prognosis in ovarian cancer. Proc Natl experiments, critically involved in writing and editing of the manuscript. All Acad Sci U S A. 2005;102:18538–43. authors read and approved the final manuscript. 17. Pages F, Berger A, Camus M, Sanchez-Cabo F, Costes A, Molidor R, et al. Effector memory T cells, early metastasis, and survival in colorectal cancer. Acknowledgements N Engl J Med. 2005;353:2654–66. This study was supported by the NIH grants P01 CA132714 and P30 CA047904. 18. Prall F, Duhrkop T, Weirich V, Ostwald C, Lenz P, Nizze H, et al. Prognostic Liwen Wang was partially supported by the Tsinghua University – University of role of CD8+ tumor-infiltrating lymphocytes in stage III colorectal cancer Pittsburgh Joint Education Program and China Scholarship Council. with and without microsatellite instability. Hum Pathol. 2004;35:808–16. 19. Muthuswamy R, Berk E, Junecko BF, Zeh HJ, Zureikat AH, Normolle D, et al. 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Combination of IFNα and poly-I:C reprograms bladder cancer microenvironment for enhanced CTL attraction

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Copyright © 2015 by Muthuswamy et al.; licensee BioMed Central.
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Medicine & Public Health; Oncology; Immunology
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2051-1426
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25806105
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Abstract

Background: BCG is a prototypal cancer immunotherapeutic factor currently approved of bladder cancer. In attempt to further enhance the effectiveness of immunotherapy of bladder cancer and, potentially, other malignancies, we evaluated the impact of BCG on local production of chemokines attracting the desirable effector CD8 T cells (CTLs) and undesirable myeloid-derived suppressor cell (MDSCs) and regulatory T(reg) cells, and the ability of bladder cancer tissues to attract CTLs. Methods: Freshly resected bladder cancer tissues were either analyzed immediately or cultured ex vivo in the absence or presence of the tested factors. The expression of chemokine genes, secretion of chemokines and their local sources in freshly harvested and ex vivo-treated tumor explants were analyzed by quantitative PCR (Taqman), ELISAs and immunofluorescence/confocal microscopy. Migration of CTLs was evaluated ex vivo, using 24-transwell plates. Spearman correlation was used for correlative analysis, while paired Students T test or Wilcoxon was used for statistical analysis of the data. Results: Bladder cancer tissues spontaneously expressed high levels of the granulocyte/MDSC-attractant CXCL8 and T -attractant CCL22, but only marginal levels of the CTL-attracting chemokines: CCL5, CXCL9 and CXCL10. Baseline reg CXCL10 showed strong correlation with local expression of CTL markers. Unexpectedly, BCG selectively induced only the undesirable chemokines, CCL22 and CXCL8, but had only marginal impact on CXCL10 production. In sharp contrast, the combination of IFNα and a TLR3 ligand, poly-I:C (but not the combinations of BCG with IFNα or BCG with poly-I:C), induced high levels of intra-tumoral production of CXCL10 and promoted CTL attraction. The combination of BCG with IFNα + poly-I:C regimen did not show additional advantage. Conclusions: The current data indicate that suboptimal ability of BCG to reprogram cancer-associated chemokine environment may be a factor limiting its therapeutic activity. Our observations that the combination of BCG with (or replacement by) IFNα and poly-I:C allows to reprogram bladder cancer tissues for enhanced CTL entry may provide for new methods of improving the effectiveness of immunotherapy of bladder cancer, helping to extend BCG applications to its more advanced forms, and, potentially, other diseases. Keywords: Tumor microenvironment, Immunomodulation, Chemokines, BCG, TLR3, Poly-I :C, IFNα, Bladder cancer, Effector T cells, Regulatory T cells * Correspondence: kalinskip@upmc.edu Departments of Sugery, University of Pittsburgh, Pittsburgh, PA 15213, USA Department of Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, PA 15213, USA Full list of author information is available at the end of the article © 2015 Muthuswamy et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 2 of 10 Background analysis for the chemokines previously implicated in Bladder cancer, originating from the transitional cells of CTL or MDSC/T attraction to tumor lesions [26-30]. reg the bladder urothelium, accounts for an estimated We observed that bladder tumors uniformly expressed 72,570 new cases and 15,210 deaths in the US. Though only low levels of CTL attracting chemokines: CCL5, bladder cancer is highly treatable if found early, it CXCL9, and CXCL10 (respective ligands for CTL- becomes increasingly difficult to treat at later stages. expressed CCR5 and CXCR3 [19]). In a striking contrast Intravesicular BCG administration has been the stan- to the above CTL-attractants, the chemokines implicated dard therapy for treatment of bladder cancer, but its in attracting MDSCs and T , CCL2 (MCP-1), CCL22 regs effectiveness is limited only to superficial bladder (MDC) and CXCL8 (IL-8) were highly expressed chemo- cancers [1-4]. kines (Figure 1A), with CXCL8 uniformly expressed at the Tumor infiltration with effector CD8 T cells (CTL) highest levels. The prevalence of high CCL22 and CXCL8 has been associated with good prognosis in various can- in bladder tumors at baseline is consistent with previous cers [5-18], therefore immunotherapies able of enhan- studies [31-33] which showed the abundance of these cing intra-tumoral CTL levels may be also effective for undesirable chemokines, particularly in patients with poor invasive bladder cancers. Previous studies by us [19] and prognosis. High intra-tumoral expression of CXCL8 others [10,20-25] have shown that intra-tumoral expres- was confirmed by confocal analysis of tumor samples sion of chemokines regulate local levels of CTL infiltra- (Figure 1C). Taqman analysis for different markers of im- tion, suggesting that T cell-targeting immunotherapies mune infiltrate (Figure 1B), using primers for CD4, CD8, can benefit from modulating tumor-associated chemo- GZMB and T-bet (effector cell markers), CD33 (myeloid kine microenvironments in order to enhance local CTL marker), GITR (Treg marker) and NCF2 (myeloid or infiltration. neutrophil marker) revealed that NCF2 expression was do- Since myeloid-derived suppressor cell (MDSCs) and minant, corresponding to the high IL-8 levels (see below). regulatory T cells are both known to protect tumors (reg) from CTL-mediated elimination and to promote tumor Intra-tumoral expression of CCL5, CXCL9, and CXCL10 growth, ideal immunotherapies should be able to se- shows a strong correlation with CTL markers CD8 and lectively enhance tumor production of CTL-attracting Granzyme B chemokines, without enhancing local levels of CXCL8, In order to test if the spontaneous expressed nominal CXCL12 and CCL22, the chemokines mediating local CTL-attracting chemokines indeed predict local CTL attraction of MDSC and T to tumors [26-30]. Prom- reg infiltration in bladder cancer tissues, we attempted to pted by the above considerations, we evaluated the correlate intra-tumoral mRNA expression of CCR5 ability of BCG and alternative adjuvants to reprogram ligands and CXCR3 ligand (CCL5, CXCL9 and CXCL10) local chemokine milieu in bladder cancer to enhance with Intra-tumoral mRNA expression of CTL markers. the overall magnitude of local production of CTL- As shown in Figure 2, Spearman correlative analysis of attracting chemokines in relation to MDSC/T -attrac- reg the chemokines and CTL markers revealed strong cor- tants. Unexpectedly, we observed that BCG, used alone, relation between CD8 and Granzyme B with CCL5, not only failed to enhance local expression of CTL- CXCL9 and CXCL10. As expected, neither of these attracting chemokines, CCL5 and CXCL10, but selec- markers was correlated with the local expression of tively enhanced MDSC- and T -attracting chemokines, reg aT -attractant, CCL22 (data not shown). The levels reg CCL22 and CXCL8. These undesirable side-effects of IL-8 expression were strongly correlated with could be reversed by the combination of IFNα and poly- NCF2 expression (neutrophil marker; Additional file 1: I:C (TLR3 ligand), raising the possibility of enhancing Figure S1). the effectiveness of the BCG-based and other forms of immunotherapy of bladder cancer and potentially other malignancies. BCG treatment of bladder tumors up regulates CXCL8, CCL22 expression, but not CXCL10 Results Using CXCL10 as a representative of CTL-attracting Bladder cancer tissues spontaneously produce MDSC- and chemokines, involved in attraction of CXCR3 CTL im- T -attracting CXCL8 and CCL22, but not effector plicated with good prognosis for cancer patients reg T cell-attracting chemokines [7,11,12,15], we tested whether BCG treatment can en- In order to evaluate the spontaneous chemokine ex- hance intra-tumoral production CXCL10. Unexpect- pression and determine the baseline chemokine pro- edly, we observed that, while BCG strongly up regulated duction pattern in bladder tumors, we isolated RNA the secretion of CXCL8 (P < 0.05) and CCL22 (P < 0.05) from resected tumors of bladder cancer patients by ex vivo-treated bladder cancer explants (n = 11 pa- (N = 20) and performed real-time PCR (Taqman) tients), it did not enhance CXCL10 secretion (Figure 3). Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 3 of 10 N= 2 0 T CKs T /M D S C C K s eff re g N= 2 0 Anti-tum or Pro -tu m o r nd 2 Ab nd 2 Ab CXCL8, CXCL10 & Nuclei Tumor-2 Tumor-1 Figure 1 Bladder cancer tissues spontaneously express high levels of MDSC/T - attracting chemokines CCL22 and CXCL8, but only reg marginal levels of CTL- attractants, CCL5, CXCL9 and CXCL10. A. Spontaneous chemokine mRNA expression by bladder tumors (N = 20 different patients) by real-time PCR analysis (Taqman). B. Spontaneous mRNA expression analysis for markers of immune filtrate in bladder tumors (N = 20 different patients) by real-time PCR analysis (Taqman). The levels of chemokine and immune filtrate markers were normalized to HPRT1 mRNA (housekeeping gene). C. Confocal analysis for CXCL8 (Red), CXCL10 (Green) protein expression in 2 representative bladder tumors. Nuclei were stained with Sytox orange (Blue) and specificity controls (secondary antibody only) are shown in insets. Combination of IFNα + poly-I:C reverses the BCG-driven CTL chemokines in colorectal cancer tissues [19], we enhancement of undesirable chemokines tested whether their addition to BCG can enhance its Since we have previously demonstrated that the combi- effectiveness. In a preliminary set of experiments we nation of IFNα with poly-I:C effectively up regulated used an in vitro model system involving TS4 bladder CC L 5 C X C L9 C XC L 10 CX CL 12 CCL 2 C CL 22 CX CL 8 CD8 GZ M B C D 4 T -B E T C D 33 G IT R N CF 2 mR N A mR NA Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 4 of 10 N=20 10 rh o = 0.87, P < 0.0 00 1 rho = 0.89, P < 0.0 00 1 0.1 0.1 0.01 0.01 0.001 0.001 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 CCL5 CCL5 10 10 rho = 0.84, P < 0 .0 00 1 rh o = 0 .8 9 , P < 0 .0 0 0 1 1 1 0.1 0.1 0.01 0.01 0.001 0.001 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 CX CL9 CX CL9 10 rh o = 0 .7 6 , P < 0 .0 0 0 1 rh o= 0.68 , P <0 .0 0 1 0.1 0.1 0.01 0.01 0.001 0.001 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 CX CL10 CX CL10 Figure 2 CCR5- and CXCR3-binding chemokines (CCL5, CXCL9 and CXCL10) show high correlation with CTL markers in bladder cancer tissues. Spearman (Rho) analysis of the correlations between the spontaneous expression (mRNA) of CTL markers (CD8 and Granzyme B) and CTL attracting chemokines (CCL5, CXCL9 and CXCL10). Values on the scale are log10 transformations of relative mRNA levels for each of the markers were evaluated using real time PCR (Taqman). cancer cells, blood isolated monocytes and fibroblasts In accordance with these observations, our experi- (Additional file 1: Figure S3) to directly compare mul- ments performed in the tumor tissue explant model tiple combinatorial adjuvants in a single experiment, (n = 6 patients), demonstrated that in contrast to BCG, without the limitation imposed by the amount of blad- the combination of IFNα and poly-I:C strongly elevated der cancer tissues available from resections and their tumor secretion of CXCL10. The combination of BCG variability between different patients. In accordance with with IFNα + poly-I:C resulted in only marginal or no fur- the data from our bladder cancer explant cultures, BCG ther enhancement of CXCL10 secretion, but was asso- alone was completely ineffective in promoting CXCL10 ciated with the undesirable elevation of CCL22 (Figure 4). secretion in such cell cultures. In contrast, we observed strong synergy between IFNα and poly-I:C in promoting IFNα + poly-I:C-treated tumors show enhanced attraction CXCL10 secretion, both in the absence and in the pres- of effector CD8 cells ence of BCG (Additional file 1: Figure S3). Importantly, To test whether the modified chemokine production neither the combination of BCG with poly-I:C nor the patterns in BCG- and IFNα + poly-I:C-treated bladder combination of BCG with IFNα was effective, which cancer tissues result in their differential ability to attract may explain the limited effectiveness of that later com- CTLs, supernatants of the differentially-treated bladder bination in the recently-completed clinical trial [4,34,35]. cancer tissues were tested for their ability to attract C D 8 CD8 CD8 GZMB GZMB G Z M B Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 5 of 10 Figure 3 BCG exposure of bladder tissues does not induce N= 1 1 CXCL10, but further enhances tumor production of MDSC/T - reg attracting CCL22 and CXCL8. Bladder tumor biopsies were cultured in the absence or presence of BCG (2 × 10 CFU) for 24 hours. CXCL10, CCL22 and CXCL8 proteins in tumor supernatants were measured by ELISA and expressed as ng/ml. The results were evaluated using two- tailed, paired Student’s t test. Statistically ** significant differences between groups are highlighted by * (P < 0.05). NS-Not significant. N=6 (-) B C G 40 N= 1 1 * * (-) B C G N= 1 1 ns Figure 4 Combination of IFNα with poly-I:C is a powerful inducer of CXCL10 in bladder cancer lesions in the absence or presence of BCG. Bladder tumors biopsies were cultured for 0 24 hours in the absence or presence of 10,000 units IFNα +20 μg/ml specific poly-I:C, with or without BCG (2 × 10 CFU). The levels of (-) B C G CXCL10 and CCL22 in tumor supernatants were measured by specific ELISAs. The results were evaluated using two- tailed, paired Wilcoxon Test. Statistically significant differences between groups are highlighted by * (P < 0.05). (-) IF N + poly(I:C) BC G BC G+IF N +p o ly(I:C ) CX C L10 (ng/m l) C C L22 (ng /m l) CX CL8 (ng /m l) CX C L 10 (n g/m l) CC L 22(ng /m l) Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 6 of 10 ex vivo-induced effector CD8 T cells (pre-activated by chemokines (CCL2, CCL22 and CXCL8, respective li- SEB-loaded LPS + IFNγ matured DCs, capable of high gands for CCR2, CCR4 and CXCR1/2), but only low levels IL-12 production [36,37]). As expected, bladder cancer of CTL- attracting chemokines (CCL5, CXCL9-10; re- tissues (n = 3 patients) treated with IFNα + poly-I:C spective ligands for CCR5 and CXCR3), suggesting that significantly attracted more of the effector CD8 T cells the chemokine imbalance can contribute to the pathogen- than untreated or BCG alone treated tumors (Figure 5). esis of bladder cancer and may limit the effectiveness of The combination of BCG with IFNα + poly-I:C didn’t its immunotherapies. Unexpectedly, despite the docu- further increase the CTL attraction. These data indicate mented beneficial role of BCG in bladder cancer, our data that BCG by itself is insufficient to reprogram the indicate that its ability to modify the microenvironment of bladder cancer-associated chemokine environment for bladder cancer ex vivo is largely limited to the enhance- enhanced CTL attraction, but that such goal can be ment of local production of T -and MDSC-attracting reg achieved by the combination of BCG with (or its re- chemokines, CCL22 and CXCL8, without inducing placement by) IFNα plus poly-I:C. CXCL10 or facilitating CTL attraction. Our current data is consistent with the previous observations that bladder Discussion cancer tissues do not produce the desirable chemokine BCG is a prototypal cancer immunotherapeutic factor CXCL10 within the first week of BCG treatment, although which has been widely demonstrated to be effective in can produce this factor after 3 weekly doses of BCG [38]. the treatment of superficial bladder cancer [1-4]. In They are also consistent with the previous observations attempt to further enhance the effectiveness of the that bladder cancer lesions typically produce high levels of immunotherapy of bladder cancer, including its more CCL22 and CXCL8 at baseline [31-33,39,40], which may advanced stages, and potentially other malignancies, we be further amplified by treatment of patients with BCG evaluated the impact of BCG on local production of the and particularly by treatment with by BCG combined with chemokines attracting the desirable effector CD8 T cells chemotherapy [32], constituting the undesirable side- and undesirable MDSCs and T cells. effect of treatment [33]. Since intratumoral expression of reg We observed that bladder cancer tissues spontaneously CXCL8 has been shown to be a negative prognostic expressed high levels of T - and MDSC- recruiting marker [31], these observations highlight a potential for reg N=3 (-) BC G IF N + p o ly - I : C ** BC G + IF N + po ly -I:C B la dder tum ors tre ate d w ith Figure 5 Bladder cancer lesions exposed to IFNα + poly-I:C or BCG + IFNα + poly-I:C show strongly-enhanced CTL-attracting activity. + + Day 6 effector Granzyme B /CD8 T cells (induced by SEB-loaded LPS + IFNγ-matured DC [36,37]) were harvested and allowed to migrate for 2 hours towards the supernatants from the differentially-treated bladder cancer tissues in 24 trans-well system. Migrated cells in the bottom + + chamber were harvested and stained for CD8, Granzyme B. The counts of Granzyme B CD8 T cells were analyzed by FACS. The results were evaluated using two- tailed, paired Student’s t test. Statistically significant differences between groups are highlighted by * (P < 0.05) or ** (P < 0.01). (-) B CG IFN +p o ly - I : C BC G+ I F N + p oly- I : C + + Migrated C D 8 GZMB Tcells Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 7 of 10 targeting the bladder cancer-associated chemokine micro- BCG in effective reprogramming of cancer tissues for en- environments to improve the outcomes of BCG-treatment hanced CTL entry, may help to improve the effectiveness and chemotherapy of this cancer type. of immunotherapy of bladder cancer and enhance the ap- This selective activity of BCG may result from its plications of BCG to its more advanced forms, as well as dominant pattern of TLR2-mediated signaling [41], other malignancies or other disease states (such a myco- which has been shown to induce significantly less type-1 bacterial infections), where the current BCG-based thera- interferons compared to other TLR ligands [42,43]. In peutic regimens are not effective. accordance with this possibility, our observations sug- gests that the combination of IFNα with poly-I:C Methods (a TLR3 ligand) or BCG + IFNα + poly-I:C (although nei- Ex vivo culture of bladder tumor explant tissues ther BCG + IFNα nor BCG + poly-I:C) are highly effect- Tumor from patients was obtained by informed consent ive in enhancing intra-tumoral production of CXCL10 in under the IRB approved protocol UPCI 86–022. Patient bladder cancer tissues and promoting CTL infiltration. clinical characteristics and sets of patients used for spe- Interestingly, both these effective combinations showed cific types of experiments are given in Table 1 below. at least partial selectivity in inducing CXCL10 (rather Using a 2 mm biopsy punch knife, uniform 2 mm than CCL22) in cancer tissues. Our current experiments cubes of resected tumor tissue were made. Tumor ex- aim to determine if the additional inclusion of COX in- plants were assorted as 3 × two mm cubes/wells in 48 hibitors (which proved to be effective in suppressing well plate respectively and cultured in IMDM plus 10% local production of CCL22 in metastatic colon cancer le- FBS, either untreated or treated with 2 × 10 CFU of sions [19]) with the currently-evaluated BCG + IFNα + BCG (TICE® BCG, Schering Plough, NJ) and/or 10,000 poly-I:C combination will further potentiate its effective- units of IFNα (Merck, NJ), 20 μg/ml of poly-I:C (Sigma ness by suppressing COX-2 dependent production of Aldrich, St. Louis, MO). Tumor tissue biopsies were CCL22, CXCL8 and boosting induction of CXCL10 and harvested at 0 and 24 hours for mRNA and confocal other CTL-attracting factors. microscopy analysis and culture supernatants were har- vested at 24 hours for ELISA. Conclusion Our current data suggest that the suboptimal ability of Bladder cancer cell line-macrophage-fibroblast co-culture BCG to reprogram cancer-associated chemokine environ- system ments may represent a factor limiting its clinical activity, In order to compare multiple combinatorial adjuvants in especially in more advanced stages of bladder cancer, and a single experiment, we established a model system in- may represent an area of additional therapeutic interven- volving 1 × 10 each of TS4 (bladder cell line; ATCC, tion. Our identification of the combination of IFNα and Manassas, VA), blood isolated monocytes (isolated by poly-I:C, as factors which may supplement (or replace) CD14 microbeads; Miltenyi Biotech.) and fibroblasts Table 1 Bladder cancer patients Patient number N (%) Total qPCR analysis ELISA analysis Chemotaxis Patients 23(100) 20(100) 11(100) 3(100) Male 15(65) 14(70) 6(55) 1(33) Female 8(35) 6(30) 5(45) 2(67) Tumor stage: Stage 0 4(17) 4(20) 2(18) Stage 0is 1(4) 1(5) Stage 1 6(26) 5(25) 3(27) 1(33) Stage 2 2(9) 2(10) 1(9) Stage 3 8(35) 6(30) 4(36) 2(67) Stage 4 2(9) 2(10) 1(9) Prior treatment: None 15(65) 13(65) 7(64) 2(67) BCG 5(22) 5(25) 2(18) Other treatment 3(13) 2(10) 2(18) 1(33) Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 8 of 10 (Life technologies, Grand Island, NY) in 48 well plates. ELISA primary and secondary antibody pairs for the ana- The cells were treated with the indicated permutation lysis of CCL5, CXCL8 and CXCL10, protein secretion were combinations of celecoxib (10 μM, Biovision, Milpitas, purchased from Peprotech, Rocky Hill, NJ. ELISA plates CA), IFNα (10000 units/ml), poly-I:C (20 μg/ml) and (Corning Inc, Corning, NY) were coated overnight at RT BCG (2 × 10 CFU) for 24 hours and supernatants were with 100 μL of primary antibody at 1 μg/mL, followed by harvested for ELISA analysis of various chemokines. In washing and blocking with PBS + 4% BSA for 1 hour. preliminary experiments, we used macrophages gene- 50 μL of samples were added to the wells and incubated rated from healthy donors’ monocytes in 6 day-long for 1 hr and subsequently washed and incubated with GM-CSF-supplemented cultures, to directly compare 50 μL of biotinylated secondary antibodies at 0.5 μg/mL the chemokine-modulating impact of increasing doses of for 1 hour. The plates were washed and incubated BCG in the same experiment. As shown in Additional for 30 minutes with streptavidin-HRP conjugate (Pierce file 1: Figure S2, different doses of BCG (0.4, 2 and Biotechnology Inc, Rockford, IL), diluted 1:10000 in wash 10 × 10 CFU), all induced high levels of CXCL8, but not buffer (50 mM Tris, 0.2% Tween). The plates were washed CXCL10. and detected with 50 μL of TMB substrate (Pierce). Reactions were stopped with 2% H SO and 450 nm ab- 2 4 Taqman analysis of mRNA expression in tumor sorbance was measured, using Wallac 1420 Victor 2 mi- Tumor biopsies were placed into lysing Matrix E tubes croplate Reader (Perkin Elmer, Waltham, MA). (MP Biologicals, Solon, OH), containing RLT buffer (RNAeasy kit, Qiagen, Valencia, CA), agitated in FP120 Generation of effector T cells homogenizer (MP Biologicals). Debris-free supernatants Naïve CD8 T cells were purified from peripheral blood from the lysis matrix tubes were transferred into new of normal donor using EasySep naïve CD8 enrichment tubes and total RNA was extracted using the RNAeasy kit (#19158, Stemcell Tech, Vancouver, Canada). Isolated kit (Qiagen,MA). 1 μg of RNA extracted by above naïve CD8 T cells were stimulated with SEB (1 ng/ml) method was used for cDNA synthesis using Quanta bio- pulsed, LPS + IFNγ-matured DC for 6 days [36]. On 6th sciences synthesis kit and 25–50 ng of the resulting day, effector CD8 T cells were harvested, cells counted, cDNA was used to do mRNA expression analysis by density-adjusted to 10 cells per ml and used in chemo- Taqman, using primers and equipment (Step One Plus taxis assays. system) from (Life Technologies, Long Island, NY). Chemotaxis Confocal microscopy analysis of tumor sections In a 24 trans-well plates with membrane with 5 μM pore 5 + Tumor biopsies were embedded in OCT medium con- size (#3421, Corning, CA), 2 × 10 (200ul) effector CD8 taining cryomolds and immediately frozen in 2-methyl- T cells were loaded onto top chambers and allowed to butane. 5 μm frozen sections of the tissues were made migrate for 2 hours [19,36,44] towards 400 μl of super- using the cryostat and layered on superfrost® plus slides natants generated from 24 hour bladder cancer explant (Thermo Scientific, Rockford, IL). The slides were fixed cultures. Migrated cells were harvested from the bottom in 4% para-formaldehyde for 15 minutes, washed and chambers and stained for CD8 and Granzyme B (GZMB). blocked for 60 minutes at room temperature. The slides Number of migrated GZMB positive effector CD8 Tcells were then stained for 3 hours at room temperature (RT) were quantified by fixed 100 μl run on Accuri C6 (BD with antibodies for CXCL10 (ab9807) and CXCL8 Biosciences, San Jose, CA). (ab18672), both from Abcam, Cambridge, MA. The slides were washed 5 times with 1 × PBS and incubated Statistical analysis with secondary antibodies anti-rabbit-Alexa 488, anti- Spearman rank correlations (rho) between the mRNA of mouse-Alexa 647 (both from Cell Signal, Danvers, MA) chemokine genes and cytotoxic T cell markers were and with nuclear dye Sytox orange (Invitrogen, Carlsbad, calculated. Comparisons of continuous variables between CA) for 30 minutes at RT. The slides were washed 5 groups were performed by 2 tailed, paired t test or times with 1 × PBS. Cover slips were mounted on the paired Wilcoxon test using graphPad Prism 6 software. sections using prolong gold anti-fade solution (Life The values of P <0.05 were considered as significant. Technologies). Confocal analyses of stained slides were performed using a LEICA TCS SL DMRE Microsystems. Additional file ELISA analysis of chemokines in tumor ex vivo culture or Additional file 1: Figure S1. Intratumoral CXCL8 expression shows in cell co-cultures strong correlation with the neutrophil marker, NCF2. Spearman (rho) analysis of the correlation between intra-tumoral mRNA expression of 24 hour culture supernatants from tumor ex vivo culture CXCL8 (IL-8) with Intra-tumoral expression of NCF2 (Neutrophil marker). were analyzed by ELISA for chemokine proteins expressed. Muthuswamy et al. Journal for ImmunoTherapy of Cancer (2015) 3:6 Page 9 of 10 10. Mlecnik B, Tosolini M, Charoentong P, Kirilovsky A, Bindea G, Berger A, et al. Figure S2. BCG dose titration analysis and its impact on chemokine Biomolecular network reconstruction identifies T-cell homing factors associated secretion by macrophages. Macrophages, generated by culturing with survival in colorectal cancer. Gastroenterology. 2010;138:1429–40. monocytes in presence of GMCSF for 6 days, were exposed to increasing 11. Dudley ME, Gross CA, Langhan MM, Garcia MR, Sherry RM, Yang JC, et al. doses of BCG (0.4, 2, 10 × 106 CFU) for 24 hrs. Secretion of CXCL8 and CD8+ enriched “young” tumor infiltrating lymphocytes can mediate CXCL10 was measured by specific ELISAs. Figure S3. BCG combination regression of metastatic melanoma. Clin Cancer Res. 2010;16:6122–31. with IFNα + poly-I:C reduces CXCL10 levels in co-cultures of bladder 12. Pages F, Kirilovsky A, Mlecnik B, Asslaber M, Tosolini M, Bindea G, et al. cancer cells, macrophages and fibroblasts. TS4(Bladder cell line) was co In situ cytotoxic and memory T cells predict outcome in patients with cultured with macrophages and Fibroblasts in ratio 1:1:1 and treated with early-stage colorectal cancer. J Clin Oncol. 2009;27:5944–51. different permutations of celecoxib, IFNα, poly-I:C, and BCG. CXCL10 13. Sharma P, Shen Y, Wen S, Yamada S, Jungbluth AA, Gnjatic S, et al. CD8 secretion was measured in 24 hour supernatants by ELISA. CCL22 and tumor-infiltrating lymphocytes are predictive of survival in muscle-invasive CXCL8 were below detection levels. urothelial carcinoma. Proc Natl Acad Sci U S A. 2007;104:3967–72. 14. Piersma SJ, Jordanova ES, van Poelgeest MI, Kwappenberg KM, van der Competing interests Hulst JM, Drijfhout JW, et al. High number of intraepithelial CD8+ tumor- The authors declare that they have no competing interests. infiltrating lymphocytes is associated with the absence of lymph node metastases in patients with large early-stage cervical cancer. Cancer Res. 2007;67:354–61. Authors’ contributions 15. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, RM was involved in concept, design and performing of experiments, and wrote et al. Type, density, and location of immune cells within human colorectal the manuscript. LW was involved in performing experiments. JP was involved in tumors predict clinical outcome. Science. 2006;313:1960–4. performing experiments; JRG participated in the study design, provided patient 16. Sato E, Olson SH, Ahn J, Bundy B, Nishikawa H, Qian F, et al. Intraepithelial material and analyzed patient-related data, and was involved in preparation of CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell the manuscript. PK conceived the study, was involved in concept and design of ratio are associated with favorable prognosis in ovarian cancer. Proc Natl experiments, critically involved in writing and editing of the manuscript. 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Journal for ImmunoTherapy of Cancer – Springer Journals

Published: Mar 24, 2015

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