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. 2018 Jan 26:8:6.
doi: 10.3389/fonc.2018.00006. eCollection 2018.

Improvement of Antitumor Therapies Based on Vaccines and Immune-Checkpoint Inhibitors by Counteracting Tumor-Immunostimulation

Paula Chiarella  1 , Mónica Vermeulen  2 , Daniela R Montagna  1 , Pablo Vallecorsa  3 , Ariel Ramiro Strazza  1 , Roberto P Meiss  3 , Oscar D Bustuoabad  4 , Raúl A Ruggiero  1 , Richmond T Prehn  5
Affiliations

Improvement of Antitumor Therapies Based on Vaccines and Immune-Checkpoint Inhibitors by Counteracting Tumor-Immunostimulation

Paula Chiarella et al. Front Oncol. .

Abstract

Immune-checkpoint inhibitors and antitumor vaccines may produce both tumor-inhibitory and tumor-stimulatory effects on growing tumors depending on the stage of tumor growth at which treatment is initiated. These paradoxical results are not necessarily incompatible with current tumor immunology but they might better be explained assuming the involvement of the phenomenon of tumor immunostimulation. This phenomenon was originally postulated on the basis that the immune response (IR) evoked in Winn tests by strong chemical murine tumors was not linear but biphasic, with strong IR producing inhibition and weak IR inducing stimulation of tumor growth. Herein, we extended those former observations to weak spontaneous murine tumors growing in pre-immunized, immune-competent and immune-depressed mice. Furthermore, we demonstrated that the interaction of specifical T cells and target tumor cells at low stimulatory ratios enhanced the production of chemokines aimed to recruit macrophages at the tumor site, which, upon activation of toll-like receptor 4 and p38 signaling pathways, would recruit and activate more macrophages and other inflammatory cells which would produce growth-stimulating signals leading to an accelerated tumor growth. On this basis, the paradoxical effects achieved by immunological therapies on growing tumors could be explained depending upon where the therapy-induced IR stands on the biphasic IR curve at each stage of tumor growth. At stages where tumor growth was enhanced (medium and large-sized tumors), counteraction of the tumor-immunostimulatory effect with anti-inflammatory strategies or, more efficiently, with selective inhibitors of p38 signaling pathways enabled the otherwise tumor-promoting immunological strategies to produce significant inhibition of tumor growth.

Keywords: antitumor vaccines; immune-checkpoints inhibitors; immunosurveillance; murine tumors; tumor-immunostimulation.

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Figures

Figure 1
Figure 1
(A,B) Idealized linear (A) and biphasic (B) antitumor immune reaction curve relating the immune reactants/tumor cells ratio inoculated into test mice (x-axis) with tumor growth (y-axis). Letters a, b, c, d, e, f, and g indicate different ratios between immune reactants and target tumor cells. Tumor growth was expressed as a percentage of control tumor growth which was that observed with tumor cells alone and is represented by the horizontal dashed line. (C–E) Real biphasic antitumor immune response curve evaluated in Winn tests relating the immune spleen cells/tumor cells ratio inoculated into euthymic or nude test mice (x-axis) with tumor growth (y-axis). Data from both euthymic and nude test mice were very similar and, in consequence, were pooled. The tumor cells were obtained from the strongly immunogenic MC-C tumor (C), and from the tumors of undetectable immunity CEI (D) and LB (E). Spleen cells were obtained from normal mice or mice immunized against tumor cells (MC-C, CEI, or LB) by pretreatment with X-lethally irradiated homologous tumor cells that had been either untreated [full line: (▪)] or treated, before being irradiated, with an inhibitor of programmed death-ligand 1 (PD-L1) expression (JQ1, 200 nM in culture for 48 h) [dashed line: (▴)]. For MC-C, both curves were virtually identical, and for simplicity only the normal line was shown. Tumor growth, initiated in all cases with 1 × 105 tumor cells, was expressed as a percentage of control tumor growth which was that observed with tumor cells alone or mixed with normal spleen cells and is represented by the horizontal line in 100% (for simplicity, SE of control—that was lower than 10% of the mean value, in the three tumor models—was not represented in the Figure). Differences were observed throughout tumor growth. By simplicity, values at day 35 of tumor growth for MC-C and CEI and at day 18 for LB were registered in the Figure taking into account that MC-C and CEI tumors grow significantly lower than LB tumor. It is worth noting that tumor mass at these days predicted the survival time: the larger the tumor mass, the shorter the survival time. The curves represent the mean ± SE of five (for MC-C) and four (for CEI and LB) independent experiments. Each point of each experiment represents the mean of 4–6 mice. Statistics: αp < 0.05; βp < 0.02; γp < 0.01; δp < 0.001, as compared with control. (F–H) Expression of PDL-1. Representative experiment showing the content of surface PDL-1 in MC-C (F), CEI (G), and LB (H) tumor cells using flow cytometry.
Figure 2
Figure 2
Evaluation of tumor growth in mice displaying different degrees of immune competence. Different strains of mice were used: euthymic, thymectomized at birth (Tx), nude, and Nod Scid Gamma (NSG) that were challenged with different doses of tumor cells to assesses the tumor dose 50 (TD50). The tumors used were: MC-C, LB, CEI, and C7HI. MC-C: mean ± SE of six experiments for euthymic, Tx, and nude mice and mean ± SE of two experiments for NSG mice; LB: mean ± SE of six experiments for euthymic, Tx, and nude mice and mean ± SE of two experiments for NSG mice; CEI: mean ± SE of three experiments for euthymic and nude mice; C7HI: mean ± SE of four experiments for euthymic and nude mice. In each experiment, 12–16 mice were utilized. Statistics: αp < 0.05, γp < 0.01, δp < 0.001.
Figure 3
Figure 3
Role of inflammatory components in the immune-mediated tumor-stimulatory effect. (A) In vitro proliferation of 1 × 105 MC-C tumor cells alone (MCC alone), or admixed with anti-MC-C immune spleen cells (ISC) [obtained from mice immunized against MC-C by pretreatment with X-lethally irradiated MC-C tumor cells] or with normal spleen cells (NSC) at 1:1 or 50:1 ISC/MCC and NSC/MCC ratios or with inflammatory macrophages at 1:1 ratio, as evaluated by using a 18 h 3[H]-thymidine uptake assay. Macrophages were collected from the peritoneum of mice that had received 1 ml of 3% of the pro-inflammatory thyoglycollate by the i.p. route, 3 days earlier. The overall 3H-thymidine uptake by attached cells was attributed to MC-C tumor cells since 3[H]-uptake by macrophages alone was negligible. Number of determinations per group, n = 6. Statistics: βp < 0.02 as compared with MCC alone, ISC/MCC (1:1), NSC/MCC (1:1), and NSC/MCC (50:1); δp < 0.001 as compared with the other groups. (B) Expression of RANTES and MIP-α. Increase in the 24h-conditioned medium of the mixture ISC/MC-C cultured at 1:1 ratio as compared with that of NSC/MCC (1:1) and MC-C alone. The conditioned medium was obtained after culturing 24 h 5 × 106 MC-C tumor cells alone or mixed with 5 × 106 ISC or NSC. The number of determinations was six for RANTES and three for MIP-α. Statistics: αp < 0.05; βp < 0.02; γp < 0.01 as compared with ISC/MC-C (1:1). (C) Growth of MC-C tumor, 8 days after the s.c. implantation of 1 × 105 MC-C tumor cells admixed with 1 × 105 anti-MC-C ISCs (stimulatory mixture) (a,c) or with 1 × 105 NSCs (control) (b,d). (D) Immunohistochemistry of MC-C tumor. Increase of CD11b+/F4/80+ (a,c) and CD3+ (b,d), 8 days after the s.c. implantation of stimulatory mixture. Up to day 8, lymphocytes were mainly CD3+ T cells but afterward, CD20+ B220+ B cells were also observed (not shown).
Figure 4
Figure 4
Low or undetectable immune-mediated tumor-stimulatory effect in mice displaying low inflammatory responses. Evaluation of the immune response curve using Winn test in macrophage-depleted mice, indomethacin-treated mice, toll-like receptor 4 (TLR4)-KO mice, p38 locally deficient mice or B-cell-depleted mice (▪). For comparative purposes, we have, in each figure, added the control group carried out using recipient euthymic or nude mice (▫) [data were very similar and for simplicity, were pooled]. The dose of indomethacin (0.5 mg/kg) was diluted in 0.015 M NaCl and was inoculated in the i.p. route 1 day before tumor inoculation. The selective p38 inhibitor SB202190 was inoculated four consecutive days (30 μg/kg/day) at the site of tumor implantation, starting at the day of tumor inoculation. Normal spleen cells (NSCs) were obtained from normal mice and immune spleen cells were obtained from mice immunized against MC-C by pretreatment with X-lethally irradiated MC-C tumor cells. Tumor growth, initiated in all cases with 1 × 105 tumor cells, was expressed as a percentage of control tumor growth which was that observed in euthymic and nude test mice that received tumor cells alone or mixed with NSCs and is represented by the horizontal line in 100% (for simplicity, SE of control—that was lower than 10% of mean value—was not represented in the Figure). Differences were observed throughout tumor growth. By simplicity, values at day 35 of tumor growth were registered in the Figure. Each point of each experiment represents the mean of 4–6 mice. The curves represent the mean ± SE of three independent experiments. Statistics: αp < 0.05 as compared with control and 1/1 ratio; δp < 0.001 as compared with control.
Figure 5
Figure 5
(A) Expression of phosphorylated (p)-38 (p38) by Western blotting. Macrophages (3 × 106 cells) collected surrounding the s.c. tumor place (one experiment) or from the peritoneum (two experiments), 3 days after s.c. or i.p. implantation of 1 × 105 MC-C tumor cells alone (T), or mixed with normal spleen cells (NSCs) at 1/1 ratio [N:T (1:1)], with immune spleen cells (ISCs) at 1/1 ratio [I:T (1:1)] or with ISCs at 1/1 ratio plus the p38 inhibitor (days 0–3, 30 μg/kg/day) at the place of tumor inoculum [I:T (1:1) + α-p38]. Intraperitoneal macrophages from normal mice were used as negative controls of p38 expression (Ct−). Pervanadate (OVAN) was used as positive phosphorylation controls (Ct+). Macrophages collected from mice that received 1 ml of 3% thyoglycollate (tg) by the i.p. route, 3 days earlier, served as control of non-specific inflammation. At the left, a representative experiment is shown. At the right, histograms show levels of p38 in the different groups, normalized with beta-actin densitometric units, representing the mean ± SE of three independent experiments. NSCs were obtained from normal mice and ISCs were obtained from mice immunized against MC-C tumor by pretreatment with X-lethally irradiated MC-C tumor cells. Statistics: βp < 0.02; γp < 0.01; δp < 0.001 as compared with the I:T (1:1) group. (B) Expression of TNF-α, IL-1β, and IL-6 pro-inflammatory cytokines in the 24-h conditioned medium of macrophages collected from the peritoneum of mice that had received, 3 days before, an i.p. inoculation of 1 × 105 MC-C tumor cells alone or mixed with stimulatory mixture at 1/1 ratio [I:T (1:1)]. Statistics: control versus I:T (1:1); βp < 0.02; γp < 0.01; mean ± SE of three experiments. (C) Effect of macrophages activated by the stimulatory mixture on the growth of MC-C tumor. Euthymic test mice received by the s.c. route 1 × 105 MC-C tumor cells mixed with peritoneal macrophages (1 × 105) of mice that had received i.p. 3 days earlier the stimulatory mixture [at 1/1 ratio, (●), n = 6]. As a control of non-specific inflammation, MC-C tumor cells were mixed with peritoneal macrophages of mice that received 3% thyoglycollate (tg) [(▫); n = 4, i.p. 3 days earlier]. The control groups (○) were, mice that received MC-C tumor cells alone (1 × 105, n = 4), or mixed with macrophages collected from mice that had received the normal mixture (at 1/1 ratio, n = 4), or none (n = 4). The data of the control groups were almost identical and were pooled. Tumor growth was registered in all groups. Statistics: Stimulatory mixture group (●) versus Control groups (○): αp < 0.05; γp < 0.01; and δp < 0.001; Stimulatory mixture group (●) versus tg group (▫): αp < 0.05 and γp < 0.01 and tg group (▫) versus control groups (○): βp < 0.02.
Figure 6
Figure 6
Therapeutic antitumor immunological schedules. (A) Vaccines and Immune-depressors. Tumor growth was initiated at day 0 with a s.c. inoculum of 1 × 105 MC-C or LB tumor cells in the right flank. Afterward, at selected times after tumor inoculation, different groups of mice received in the left flank an antitumor vaccine [4 × 106 X-lethally irradiated tumor cells or 3 × 105 dendritic cells loaded with tumor lysate (both vaccines rendered similar results and in consequence their data were pooled)] or the immune-depressor Cyclosporine A [inoculated i.p. every day (15 mg/kg/day) starting at day 3 or 18 of MC-C tumor growth or at day 1 or 10 of LB tumor growth]. The group of mice that did not receive any treatment served as control. The Figure shows representative experiments (one for MC-C and one for LB) out of four experiments for MC-C and three experiments for LB that rendered similar results. Six mice per group were utilized in the representative experiments and data were expressed as mean (mm3) ± SE of tumor volume. Statistics: Differences versus control group: αp < 0.05; βp < 0.02; γp < 0.01; δp < 0.001. (B) Immune-checkpoint inhibitors. Tumor growth was initiated at day 0 with a s.c. inoculum of 1 × 105 MC-C or LB tumor cells in the right flank. Afterward, different groups of mice received blocking anti-cytotoxic T lymphocyte-antigen 4 (CTLA-4) (inoculated i.p. three times a week), blocking anti-programmed death-ligand 1 (PD-L1) (inoculated i.p. for 13 consecutive days) or both blocking anti-CTLA-4 plus blocking anti-PD-L, starting at selected times (indicated in the Figure) after tumor inoculation. The group of tumor-bearing mice that did not receive any treatment served as control. The Figure shows representative experiments (one for MC-C and one for LB) out of two experiments for MC-C and three experiments for LB that rendered similar results. Four to six mice per group were utilized in the representative experiments and data were expressed as mean (mm3) ± SE of tumor volume. Statistics: Differences versus control group: αp < 0.05; γp < 0.01; δp < 0.001. (C) Counteraction of the tumor-immunostimulatory effects observed on well-established tumors (well-established meaning solid tumors that are ≥400 mm3). MC-C tumor: tumor growth was initiated at day 0 with a s.c. inoculum of 1 × 105 MC-C tumor cells in the right flank. At day 18 of tumor growth, different groups of mice received an antitumor vaccine alone (X-irradiated tumor cells or dendritic cells loaded with tumor lysate), an antitumor vaccine plus indomethacin, anti-CTLA-4 alone, anti-CTLA-4 plus indomethacin, or anti-CTLA-4 plus SB202190. Anti-CTLA-4 was inoculated i.p. three times a week starting at day 18 of tumor growth. Indomethacin (0.5 mg/kg) was inoculated i.p. once at day 17. SB202190 (30 μg/kg/day) was inoculated i.p. for four consecutive days starting at day 18 of tumor growth. The group of tumor-bearing mice that did not receive any treatment served as control. LB tumor: tumor growth was initiated at day 0 with a s.c. inoculum of 1 × 105 LB tumor cells in the right flank. At day 1 of tumor growth, two groups of mice received an antitumor vaccine alone (X-irradiated tumor cells or dendritic cells loaded with tumor lysate), or the antitumor vaccine plus indomethacin (inoculated i.p. at day 0). At day 10 of tumor growth other groups of mice received anti-PD-L1 alone (inoculated i.p. for 13 consecutive days starting at day 10 of tumor growth), or in combination with indomethacin (inoculated i.p. at day 9 of tumor growth) or with SB202190 (inoculated i.p. for four consecutive days starting at day 10 of tumor growth). The group of tumor-bearing mice that did not receive any treatment served as control. The Figure shows representative experiments (one for MC-C and one for LB) out of three experiments for MC-C and three experiments for LB that rendered similar results. Four to six mice per group were utilized in the representative experiments and data were expressed as mean (mm3) ± SE of tumor volume. Statistics: differences versus control group: αp < 0.05; βp < 0.02; γp < 0.01; δp < 0.001. Difference between anti-CTLA-4 plus indomethacin group versus anti-CTLA-4 plus SB202190 group: *p < 0.02. Difference between anti-PD-L1 plus indomethacin group versus anti-PD-L1 plus SB202190 group: **p < 0.05. y-axis: tumor volume (mm3). (A,B) x-axis: days of tumor growth at the onset of treatment. For MC-C tumor, days 3, 7, 12, 18, and 28 of tumor growth corresponded to tumor volumes of <10, 50, 250, 600, and 1,500 mm3, respectively. For LB tumor, days 1, 6, and 10 of tumor growth corresponded to tumor volumes of <10, 300, and 700 mm3, respectively. (C) x-axis: treatments. Differences were observed throughout tumor growth. By simplicity, values at day 35 of MC-C tumor growth and at day 18 of LB tumor growth were registered in the figures taking into account that MC-C tumor grows significantly slower than LB.
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References

    1. Prehn RT, Main JM. Immunity to methylcholanthrene-induced sarcomas. J Natl Cancer Inst (1957) 18:769.tyjl–78.tyjl. - PubMed
    1. Janeway CA, Travers P, Walport M, Shlomchik MJ. Manipulation of the immune response. In: Janeway CA, editor. Immunobiology. New York: Garland; (2001). p. 566–77.
    1. Chiarella P, Vulcano M, Bruzzo J, Vermeulen M, Vanzulli S, Maglioco A, et al. Anti-inflammatory pretreatment enables an efficient dendritic cell-based immunotherapy against established tumors. Cancer Immunol Immunother (2008) 57:701–18.10.1007/s00262-007-0410-4 - DOI - PMC - PubMed
    1. Wiedermann U, Davis AB, Zielinski CC. Vaccination for the prevention and treatment of breast cancer with special focus on Her-2/neu peptide vaccines. Breast Cancer Res Treat (2013) 138:1–12.10.1007/s10549-013-2410-8 - DOI - PubMed
    1. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer (2012) 12:252–64.10.1038/nrc3239 - DOI - PMC - PubMed

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