Tumor-Microenvironment Characterization of the MB49 Non-Muscle-Invasive Bladder-Cancer Orthotopic Model towards New Therapeutic Strategies

Bacillus Calmette-Guérin (BCG) instillations for the treatment of non-muscle-invasive bladder cancer patients can result in significant side effects and treatment failure. Immune checkpoint blockade and/or decreasing tumor-infiltrating myeloid suppressor cells may be alternative or complementary treatments. Here, we have characterized immune cell infiltration and chemoattractant molecules in mouse orthotopic MB49 bladder tumors. Our data show a 100-fold increase in CD45+ immune cells from day 5 to day 9 tumors including T cells and mainly myeloid cells. Both monocytic myeloid-derived suppressor-cells (M-MDSC) and polymorphonuclear (PMN)-MDSC were strongly increased in day 9 tumors, with PMN-MDSC representing ca. 70% of the myeloid cells in day 12 tumors, while tumor associated macrophages (TAM) were only modestly increased. The kinetic of PD-L1 tumor expression correlated with published data from patients with PD-L1 expressing bladder tumors and with efficacy of anti-PD-1 treatment, further validating the orthotopic MB49 bladder-tumor model as suitable for designing novel therapeutic strategies. Comparison of chemoattractants expression during MB49 bladder tumors grow highlighted CCL8 and CCL12 (CCR2-ligands), CCL9 and CCL6 (CCR-1-ligands), CXCL2 and CXCL5 (CXCR2-ligands), CXCL12 (CXCR4-ligand) and antagonist of C5/C5a as potential targets to decrease myeloid suppressive cells. Data obtained with a single CCR2 inhibitor however showed that the complex chemokine crosstalk would require targeting multiple chemokines for anti-tumor efficacy.


Introduction
Bladder cancer (BCa) is the 10th most common malignancy, with more than 500,000 new cases diagnosed each year [1]. About 70% of BCa are diagnosed as nonmuscle-invasive (NMIBC) [2] and according to specific tumor-stage and grade characteristics; intravesical immunotherapy with Bacillus Calmette-Guerin (BCG) is used to prevent recurrence and/or progression [3]. However, treatment failure may occur in 30-40% of cases [4,5], emphasizing the necessity to evaluate alternative or complementary therapies.
Among the numerous regulatory mechanisms generated during bladder tumor development [6], the presence of immune checkpoints, as well as myeloid regulatory cell subsets, have emerged as promising targets in BCa. Thus, blockade of PD-1/PD-L1 axis recently led to breakthrough clinical successes in muscle invasive BCa and several trials are ongoing in NMIBC [7]. In addition, the local balance between T lymphocytes and myeloid derived suppressor-cells (MDSC) may be predictive of BCG failure in the patients [8]. Decreasing Int. J. Mol. Sci. 2023, 24, 123 2 of 11 the numbers of infiltrating MDSC and tumor associated macrophages (TAM) in bladder tumors, alone or in conjunction with BCG therapy, may also diminish recurrence and progression rates in NMIBC patients.
Here, we will focus on the targeting of TAM and/or MDSC chemoattractant axes, as it has become an attractive way to decrease those immunosuppressive cells. The chemoattractant CCL2 molecule has been involved in several cancers for inducing MDSC/TAM tumor infiltration and targeting this chemokine with anti-CCL2 antibodies and/or inhibitors of its major ligand, CCR2, have improved tumor regression in pre-clinical models of several cancers [9][10][11]. Although CCL2 was associated with poor survival in muscle-invasive or metastatic BCa [12], the CCL2-CCR2 axis was not previously targeted in NMIBC patients or in animal models of NMIBC. Other chemokine targets includes CCL20-CCR6 [13], CCL5-CCR5 [14] and/or CXCL12-CXCR4 [15] signaling which have been associated with TAM accumulation in breast cancer or glioma [10]. Chemokine receptors CXCR1/CXCR2 and their ligands (CXCL1-3, 5, 8) have been associated with tumor associated neutrophil (TAN) and/or polymorphonuclear-MDSC (PMN-MDSC) infiltration in various tumors [16], including BCa [17], and targeted in patients with breast cancer and prostate cancer [16].
Here, to design chemoattractant targeting, we have selected an orthotopic murine model, where syngeneic bladder tumor cells (MB49, a tumor cell line derived from a chemically induced urothelial carcinoma in a male C57BL/6 mouse [18]) are intravesically instilled, so that tumor deposition and seeding onto the mouse urothelium more closely reproduces NMIBC of patients. We characterized the immune microenvironment of growing MB49 bladder tumors including the kinetic of TAM and MDSC tumor infiltration, but also of the expression of the immune checkpoint PD-1/PD-L1. The kinetic of PD-L1 tumor expression correlated to the efficacy of an anti-PD-1 treatment in agreement with published results obtained in patients with PD-L1 expressing bladder tumors [19], thus suggesting that data obtained in the MB49 bladder tumor model may be informative for future therapeutic strategies. Our chemokine analysis highlighted the chemoattractants that may be targeted to decrease the immune myeloid suppressive cells in NMIBC, but also revealed a complex chemokine crosstalk, which deserves further attention to design novel anti-tumor-treatments.

Characterization of the Immune Cell Infiltration in MB49 Bladder Tumors and Model Evaluation
Immune cell infiltration from naïve bladder to growing MB49-bladder tumors (day 5, 9 and 12, Figure 1A) was examined after antibody staining of recovered cells by flowcytometry (Supplementary Figure S1). A significant 100-fold increase of CD45 + immune cells/mg of tumor was observed from day 5 to day 9 tumors (Figure 1B), including CD3 + T-cells ( Figure 1C), and myeloid-cells (CD11b high , Figure 1D). Myeloid-cell subtypes [20] ( Figure 1E) included CD11b high Ly6G high (a phenotype of PMN-MDSC), CD11b high Ly6C high (a phenotype of M-MDSC), and CD11b high Ly6C low Ly6G low F480 + (a phenotype of TAM), with half of these TAM being CD206 + (a phenotype of immunosuppressive M2 TAM). In day 5 MB49 tumors a first significant influx of PMN-MDSC was observed (p < 0.001), while a significantly greater increase (ca. 100-fold, p < 0.001) of both M-MDSC and PMN-MDSC occurred in day 9 tumors. In contrast, TAM (ca. half of them being M2 TAM in naïve and in day 5 tumors) were only modestly increased at day 9 (ca. 5-fold, p < 0.05) and only represented <5% of the myeloid cell subtypes, which were dominated by PMN-MDSC (>70% at day 12, Figure 1F). Noteworthy, a positive correlation between PMN-MDSC infiltration and tumor size was also found in patients with BCa [21]. PMN-MDSC as the predominant type of MDSC was also previously reported in mouse models of thymoma, mammary carcinoma, melanoma, colon cancer and hepatocellular carcinoma, often associated with CCL2 and/or CCL9 chemoattractants [22,23]. predominant type of MDSC was also previously reported in mouse models of thymoma, mammary carcinoma, melanoma, colon cancer and hepatocellular carcinoma, often associated with CCL2 and/or CCL9 chemoattractants [22,23]. Expression of the PD-1 and PD-L1 immune checkpoint receptor and ligand were examined in day 5 and day 9 MB49 tumors. PD-1 was already expressed in >50% of T cells and in a minority of myeloid cells (Figure 2A and Supplementary Figure S2A), while PD-L1 expression was only expressed in myeloid cell at day 5, but significantly increased at day 9 in both T cells (ca. 50%) and myeloid cells (ca. 50%, Figure 2B and Supplementary Figure S2B). To characterize the kinetic of PD-L1 expression on tumor cells, MB49 cells expressing the green fluorescent protein (gfp) were used for intravesical tumor implantation ( Figure 2C,D and supplementary Figure S2C). Data showed no detectable expression of PD-L1 in day 5 gfp+ tumor cells, followed by a progressive and significant increase of both percentage of cells and intensity of expression reaching a relative fold increase (RFI) of ca. 20-fold in day 9 tumors (p < 0.0001, Figure 2D). Accordingly, a single anti-PD-1 i.p. injection (200 μg/mouse) performed in MB49-tumor bearing mice at day 9 or at day 12 was sufficient to provide a significant anti-tumor efficacy with >70% survival at long term, while a single treatment at day 5 did not significantly improve mouse survival ( Figure  2E), suggesting tumor expression of PD-L1 was necessary for anti-tumor efficacy. A similar and significant mouse survival of 70-80% was also obtained in our model after four  Figure S2C). Data showed no detectable expression of PD-L1 in day 5 gfp+ tumor cells, followed by a progressive and significant increase of both percentage of cells and intensity of expression reaching a relative fold increase (RFI) of ca. 20-fold in day 9 tumors (p < 0.0001, Figure 2D). Accordingly, a single anti-PD-1 i.p. injection (200 µg/mouse) performed in MB49-tumor bearing mice at day 9 or at day 12 was sufficient to provide a significant anti-tumor efficacy with >70% survival at long term, while a single treatment at day 5 did not significantly improve mouse survival ( Figure 2E), suggesting tumor expression of PD-L1 was necessary for anti-tumor efficacy. A similar and significant mouse survival of 70-80% was also obtained in our model after four consecutive intravesical instillations of BCG (1 week-apart, starting 1 day after tumor implantations [24,25], while a single BCG instillation at day 5 was ineffective [24] thus highlighting the efficacy of the single dose anti-PD-1 treatment. Of note, a significant, although lower, survival rate was previously reported with an anti-PD-L1 treatment performed from day 9 to day 15, in a similar orthotopic MB49 tumor model [26]. More importantly, our data are in agreement with efficacy results obtained in patients with PD-L1 positive, but not PD-L1 negative, bladder tumors [19], suggesting that the data obtained in the MB49 bladder tumor model may be informative for designing future therapeutic strategies.
consecutive intravesical instillations of BCG (1 week-apart, starting 1 day after tumor implantations [24,25], while a single BCG instillation at day 5 was ineffective [24] thus highlighting the efficacy of the single dose anti-PD-1 treatment. Of note, a significant, although lower, survival rate was previously reported with an anti-PD-L1 treatment performed from day 9 to day 15, in a similar orthotopic MB49 tumor model [26]. More importantly, our data are in agreement with efficacy results obtained in patients with PD-L1 positive, but not PD-L1 negative, bladder tumors [19], suggesting that the data obtained in the MB49 bladder tumor model may be informative for designing future therapeutic strategies.
CCL9 was previously shown to recruit MDSCs in intestinal tumors [28] and hepatocellular carcinoma [29]. CCL6 has recently been shown to be secreted by eosinophil to promote metastatic tumor growth [30]. Pro-tumorigenic eosinophil have been associated with BCa [31] and whether the human CCL6 homologue, CCL23 [30], may be targeted to decrease pro-tumorigenic eosinophils deserve further investigation. The increase of the CXCR2 ligands CXCL2 and CXCL5, as well as of the CXCR4 ligand CXCL12 in day 15 MB49 bladder tumors is in agreement with their higher expression in BCa patients correlating with a poor prognosis associated with MDSC recruitment for CXCL2 [17] or to higher tumor cell migration/invasiveness for CXCL5 and CXCL12 [32][33][34]. The greater increase of complement C5/C5a in day 15 MB49 tumor, was also in agreement with higher expression of C5aR in BCa with poor prognosis [35]. The ability of C5a/C5aR blockade to decrease MDSC and tumor progression in a variety of cancers [36] thus deserve further investigation in bladder cancer. Interestingly, CCL2 chemokine levels did not vary during tumor growth in our NMIBC model, in contrast to increased levels of CCL8 and CCL12 (a murine chemokine) which are alternate ligands of CCR2 [23,27], suggesting that a CCR2 inhibitor (CCR2i) may decrease monocyte infiltration. We therefore tested in our MB49 bladder tumor model an available CCR2i that was previously shown to decrease TAM and inflammatory monocytes in a murine model of pancreatic cancer [37]. However, the data showed no significant alteration in immune cell infiltration ( Figure 3A) neither for T or myeloid cells ( Figure 3B), TAM or MDSC ( Figure 3C), nor of tumor growth ( Figure 3D) and mice survival ( Figure 3E).
MB49 bladder tumors is in agreement with their higher expression in BCa patien lating with a poor prognosis associated with MDSC recruitment for CXCL2 [1 higher tumor cell migration/invasiveness for CXCL5 and CXCL12 [32][33][34]. The gr crease of complement C5/C5a in day 15 MB49 tumor, was also in agreement wit expression of C5aR in BCa with poor prognosis [35]. The ability of C5a/C5aR blo decrease MDSC and tumor progression in a variety of cancers [36] thus deserve investigation in bladder cancer. Interestingly, CCL2 chemokine levels did not vary tumor growth in our NMIBC model, in contrast to increased levels of CCL8 and C murine chemokine) which are alternate ligands of CCR2 [23,27], suggesting that inhibitor (CCR2i) may decrease monocyte infiltration. We therefore tested in ou bladder tumor model an available CCR2i that was previously shown to decrea and inflammatory monocytes in a murine model of pancreatic cancer [37]. Howe data showed no significant alteration in immune cell infiltration ( Figure 3A) neith or myeloid cells ( Figure 3B), TAM or MDSC ( Figure 3C), nor of tumor growth (Fig  and mice survival (Figure 3E).  Chemokine analysis of MB49 tumors upon CCR2i treatment, as compared to controltreatment ( Table 2), showed that some myeloid cell chemoattractants were significantly increased by CCR2i (C5/C5a, CCL11, CCL12, CCL8 and CX3CL1), while other were decreased (CXCL1, CXCL5, and CCL9/10), possibly explaining the status quo situation of the tumor immune cell infiltration. a Chemokine level at day 9 in vehicle-treated MB49 tumor bearing mice (control) are expressed as mean pixel density, when significantly higher than background signal + 3SD. b relative change in chemokine levels (when detectable) after a CCR2i treatment as compared to vehicle-treated (control) mice. Increased/decreased chemokine levels between treatment and control ≥1.3 and ≤0.7 (i.e., ≤ and ≥ to the 99% confidence interval of the mean fold-increases) are indicated in bold. c ND = not detectable.
The lack of effects observed in the MB49 bladder tumor model using a CCR2i differed from reports in pancreatic, liver, breast, prostate, renal, endometrial and ovarian cancer models [10,37,38], but may be in line with Fridlender et al., that reported that a CCL2 blockade was unable to reduce the influx of macrophages in s.c. human papillomavirus oncogene expressing TC-1 tumors, possibly due to other chemoattractants replacing CCL2/CCR2i axis [9]. This complex chemokine cross-talk thus suggest the necessity of targeting multiple chemokines for efficiently decreasing myeloid suppressor cells tumor infiltration. Of additional interest may also be the targeting of chemokines in combination with anti-PD-1/PDL1 treatment, which may however be stalled by the severe adverse events observed in recent clinical trials in pancreatic cancer (targeting CXCL12/CXCR4 + PD-1) and in colorectal, pancreatic, lung and hepatic cancer (targeting CCL2/CCR2 + PD-1) [39]. However, toxicity may be decreased by using the intravesical route of administration in NMIBC patients, as recently reported for selected patients receiving an anti-PD1 treatment [40], suggesting that other combination therapies may be similarly administered in NMIBC patients.

Conclusions
Our data point to the orthotopic MB49 BCa model as a suitable NMIBC model to design novel anti-tumor strategies. Chemokine analysis highlighted CCR2, CXCR2, CXCR4 and C5aR ligands/chemoattractants as possible targets to decrease the immune myeloid suppressive cells in NMIBC, but also revealed a complex chemokine crosstalk, which deserve particular attention to design novel anti-tumor-treatments.
The MB49 orthotopic bladder tumor model. Seven to ten-week-old female C57BL/6 wild-type mice (Envigo, Gannat, france) were used and all experiments were performed in accordance with Swiss law and with approval of the Cantonal Veterinary Office of Canton de Vaud, Switzerland. Bladder tumors were established in deeply anesthetized mice that were urethrally catheterized using Introcan 24Gx3/4 catheters (Braun, Melsungen, Germany) as previously described [25]. A 15 min pre-treatment with 100 µL 22% ethanol was performed before instillation of 500,000 MB49-luc or MB49-gfp cells in 50 µL. MB49luc tumor growth was monitored by bioluminescence 15 min after intraperitoneal (i.p.) injection of D-luciferin (Promega, Dübendorf, Switzerland, L8220, 150 µg/g of body weight) in the Xenogen imaging system (Xenogen/IVIS Caliper Life Science, kindly provided by cellular imaging facility, CIF/UNIL, Lausanne, Switzerland). 100% of the mice will develop bladder tumors and monitoring of MB49-luc tumors establishment and growth can be efficiently assessed during the first 3 weeks. Uncontrolled loss of luminescence of the growing tumors can then often appear [41], requiring additional monitoring by palpation, hematuria and overall health status of the mice, that were euthanized when they reached humane endpoints.
Treatments. A single intraperitoneal injection of 200 µg of an anti-PD1 mAb (10 mg/kg, clone RPM1.14) or of its isotype control (an IgG2a, clone 2A3), both kindly provided by Roche, Penzberg, Germany, was performed at the indicated time points. CCR2 inhibitor (CCR2i, PF-413609, Med Chem Express, obtained from Roche, Penzberg, Germany, 10 mg/mL in 5% DMSO, 0.33% Tween80, in 0.9% NaCl.. Mice received 2 mg (100 mg/kg) subcutaneously in the flank, twice a day, starting 24 h after MB49 tumor cell instillation and for a duration of 10 days in tumor protection assays or until sacrifice for chemokine analysis. Control animals received similarly the vehicle alone.
Immunostaining and flow cytometry analysis. Mice were sacrificed by CO 2 inhalation to collect the bladders. Single-cell suspensions were obtained by mincing in DLdithiothreitol (D9779, Sigma-Aldrich, Schaffhausen, Switzerland) and by subsequent digestion with 1 mg/mL collagenase/dispase (Roche, 11097113001) and 0.1 mg/mL DNAse I (D5025, Sigma-Aldrich, Schaffhausen, Switzerland) with 20% Fetal Calf Serum Chemokine array. Bladders were homogenized in 500-1000 µL PBS with protease inhibitors (10 µg/mL Aprotinin from bovine lung, 10 µg/mL Leupeptin hemisulfate salt, and 10 µg/mL Pepstatin A; all from Sigma-Aldrich). TritonX-100 (final concentration 1%, Sigma-Aldrich) was added after homogenization, and after two freeze-thaw cycle's samples were centrifuged at 10,000× g for 5 min to remove debris. Protein concentration was assessed using BCA protein assay (Thermo Fisher Scientific). Chemokines were detected using the Proteome profiler array: mouse chemokine array kit (R&D Systems), according to the manufacturer instructions. Briefly, 150 µg of protein (pooled from three bladders, 50 µg each) of each condition were used for the assay. Detection of chemokine levels was performed using ImageJ software (NIH, Bethesda, MD, USA) and expressed as mean pixel density. Significant detection was determined as > mean + 3SD of pixel density of negative spots. Increased chemokine levels between the different conditions and controls were considered significant when ≥ to the 99% confidence interval of the mean fold-increases examined.
Statistics. Statistical analyses were performed using Prism 9.00 for Windows (Graph-Pad software, San diego, CA USA). Multiple comparisons were performed using one-way ANOVA and Tukey's or Sidak post-test or log-rank test as indicated in the figure legends.