Dose-dense chemotherapy improves mechanisms of antitumor immune response

Abstract

Dose-dense (DD) regimens of combination chemotherapy may produce superior clinical outcomes, but the basis for these effects are not completely clear. In this study, we assessed whether a DD combinatorial regimen of low-dose cisplatin and paclitaxel produces superior immune-mediated efficacy when compared with a maximum tolerated dose (MTD) regimen in treating platinum-resistant ovarian cancer as modeled in mice. Immune responses generated by the DD regimen were identified with regard to the immune cell subset responsible for the antitumor effects observed. The DD regimen was less toxic to the immune system, reduced immunosuppression by the tumor microenvironment, and triggered recruitment of macrophages and tumor-specific CD8(+) T-cell responses to tumors [as determined by interleukin (IL)-2 and IFN-γ secretion]. In this model, we found that the DD regimen exerted greater therapeutic effects than the MTD regimen, justifying its further clinical investigation. Fourteen patients with platinum-resistant relapse of ovarian cancer received DD chemotherapy consisting of weekly carboplatin (AUC2) and paclitaxel (60-80 mg/m(2)) as the third- or fourth-line treatment. Serum was collected over the course of treatment, and serial IFN-γ and IL-2 levels were used to determine CD8(+) T-cell activation. Of the four patients with disease control, three had serum levels of IL-2 and IFN-γ associated with cytotoxic CD8(+) T-cell activity. The therapeutic effect of the DD chemotherapy relied on the preservation of the immune system and the treatment-mediated promotion of tumor-specific immunity, especially the antitumor CD8(+) T-cell response. Because the DD regimen controlled drug-resistant disease through a novel immune mechanism, it may offer a fine strategy for salvage treatment.

Figures

Figure 1. Dose dense (DD) chemotherapy exhibited better therapeutic efficacy against cisplatin- resistant tumor, R HM-1

In order to mimic the treatment of ovarian cancer in a clinical setting, we designed two treatment protocols for a mouse ovarian cancer model to which DD or maximum-tolerated dose (MTD) chemotherapies were administered. (A) DD chemotherapy exhibited better anti-tumor effect in mice bearing R HM-1 cell tumor. R HM-1 cells (1· 106) were injected subcutaneously (s.c.) into the female (C57BL/6, C3H/He) F1 mice (5 in each group, day 0). On day 4, mice started chemotherapy with paclitaxel and cisplatin delivered intraperitoneally (i.p.) in either DD (paclitaxel 5 mg/kg plus cisplatin 3 mg/kg in a 3-day interval for 7 courses) or MTD (paclitaxel 12 mg/kg plus cisplatin 7 mg/kg in a10- day interval for 3 courses) regimen. Control group mice were treated with PBS in 3-day interval. Significant therapeutic efficacy was noted in mice treated by DD chemotherapy (#p=0.017, control versus DD), which was better than that of MTD (p=0.039, MTD versus DD). (B) The specific anti- tumor effect of DD chemotherapy on R HM-cells was abolished in nude mice, suggesting host immunity might be involved in the tumor elimination. Cisplatin-resistant HM-1 cells (1· 106) were injected s.c. into female athymic nude mice (5 in each group, day 0). On day 4, mice began intra-peritoneal administration of paclitaxel and cisplatin chemotherapy given in either DD (paclitaxel 5 mg/kg plus cisplatin 3 mg/kg in a 3-day interval for 7 courses) or MTD (paclitaxel 12 mg/kg plus cisplatin 7 mg/kg in a10-day interval for 3 courses) format. Control group mice were treated by PBS in a 3-day interval. There is no difference in the tumor size between the different groups of immune-deficient mice.

Figure 2. MTD chemotherapy caused significant myelosuppression while DD chemotherapy preserved the major immune cells and decreased myeloid-derived suppressor cells (MDSC)

(A) Female (C57BL/6 C3H/He) F1 mice (without tumor) were treated with different formats of chemotherapy. MTD chemotherapy-treated mice were severely depleted of CD8+, CD4+, NK, CD11b+, CD11c+ and F4/80 cells. Conversely, DD chemotherapy preserved the numbers of CD8+, CD4+ and CD11b+ cells. The suppression of NK, CD11C+ and F4/80 cells by DD chemotherapy is much less than that by MTD chemotherapy (***p<0.0001, **p<0.001 and **p<0.001, respectively). (B) Female (C57BL/6 C3H/He) F1 mice (with or without R HM-1 tumor) were treated with different formats of chemotherapy. In tumor-free mice, the different treatments did not significantly change the numbers of MDSCs. In R HM-1 tumor-bearing mice, DD chemotherapy drastically reduced the number of MDSCs in comparison to the control and MTD groups (***p<0.0001, control versus DD; **P<0.001, DD versus MTD). (C) DD and MTD regimen similarly reduced the number of Treg cells in tumor- bearing mice (percentage of CD4+CD25+ cells, p=0.048, control versus DD).

Figure 3. DD chemotherapy resulted in the significant induction of peritoneal macrophage and antitumor effect could be abrogated by macrophage depletion

(A) DD chemotherapy induced a large amount of F4/80+ cells in the peritoneum of tumor-bearing mice. Cells were obtained through peritoneal lavage with 10 ml of PBS in R HM-1 tumor-bearing mice treated with different formats of chemotherapy. Mononuclear cells were separated by Ficoll- Paque gradient. Representative flow cytometry data demonstrated a larger population of F4/80+ cells was elicited by DD chemotherapy (12690 in 20000 cells analyzed), whereas in mice receiving MTD chemotherapy, the population of F4/80+ cells became scant (1571 in 20000 cells) (**p<0.001, control versus DD, control versus MTD). (B) Tumor growth curve showed diminishing anti-tumor effect of DD chemotherapy when macrophage was depleted by injection of clodronate liposome (macrophage inhibitor) into mice. R HM-1 cells (1· 106) were injected s.c. into female (C57BL/6 C3H/He) F1 mice (5 in each group, day 0). On day 4, mice began DD chemotherapy (paclitaxel 5 mg plus cisplatin 3 mg every 3 days for 7 doses). Control group mice were treated by PBS. Clodronate liposome and control liposome began on day 1 (1.5 mg/mice) and was intraperitoneally administered at a 5-day interval. Administration of clodronate liposome reduced the antitumor effect by DD chemotherapy in comparison to the vehicle (liposome) control (*p=0.01, clodronate liposome versus liposome only).

Figure 4. DD therapeutic effect on drug-resistant tumor is immune-dependent and mediated by CD8+ effector cells

(A) The use of neutralizing antibodies for depletion of lymphocyte sub-populations revealed that CD8+ T cells are essential for tumor eradication. R HM-1 cells (1· 106) were injected s.c. into female (C57BL/6, C3H/He) F1 mice (5 in each group, day 0). Neutralizing antibodies were given i.p. (100 μg/mice/day initiated on D1, given every other day for 2 weeks, then 200 μg/mice/day every week). On day 4, Mice began DD format chemotherapy. Control group mice were treated by PBS. Tumor growth curve demonstrated that anti-tumor effect was abolished when CD8+ cells were depleted by anti-CD8 antibody (**p<0.001, anti-CD8 versus rat IgG), while the anti-CD4 and anti-NK antibodies did not affect tumor growth. (B) In the absence of CD8+ T lymphocytes, MTD demonstrates moderately more therapeutic effect than DD. R HM-1 cells (1· 106) were injected s.c. into female (C57BL/6, C3H/He) F1 mice (5 in each group, day 0). Neutralizing antibodies were given i.p. (100 μg/mice/day initiated on D1, given every other day for 2 weeks, then 200 μg/mice/day every week). Lymphocyte depletion assay failed to identify a specific population of effectors cells that is associated with anti-tumor effects of MTD chemotherapy for R HM-1 tumor. (C) In tumor-bearing mice, DD chemotherapy elicited anti-tumor CD8+IFN-γ+ T lymphocytic reaction. Mice bearing R HM-1 tumor were treated with different formats of chemotherapies. After treatment, spleens were obtained and the single-cell splenocyte suspension was prepared by tissue dissociation and enzyme digestion. Cells were then stained with anti-mouse CD8 and anti-mouse IFN-γ antibodies before getting analyzed by flow cytometry. Representative flow data showed that CD8+IFN-γ+ cells were found in greater numbers in the mice receiving DD chemotherapy (**p<0.001, DD versus control and MTD). (D) Representative flow data demonstrated more tumor-infiltrating CD8+IFN-γ+ T lymphocytes were also induced in mice treated by DD (**p<0.001, DD versus control and MTD).

Figure 5. DD chemotherapy elicited the greatest number of IFN-γ-secreting CD14+F4/80+ macrophage and subsequent CD8+IFN-γ+ tumor-infiltrating lymphocytes (TILs) inside the peritoneal cavity of tumor-bearing mice

(A) In this i.p. tumor model, DD chemotherapy elicited and recruited largest number of CD14+F4/80+ macrophages inside the peritoneal cavity of R ID8 tumor-bearing mice (**p<0.001, control versus DD, *p<0.01, DD versus MTD). (B) The proportion of macrophage did not change in tumor-naïve mice regardless of chemotherapy. (C) Flow cytometry analysis indicated more activated macrophages, as determined by IFN-γ secretion, after DD chemotherapy. Following treatment, cells from the peritoneal lavage of R ID8 tumor-bearing mice were cultured in medium with protein transporter inhibitor BD GolgiPlug. Cells were stained with anti-mouse F4/80 PE, as well as anti-mouse IFN-γ FITC antibodies before analysis by flow cytometry. Representative data showed the number of F4/80+ IFN-γ+ cells increased in mice receiving DD chemotherapy but not in the mice receiving MTD and PBS (control) (8.16 % in DD versus 2.11% in MTD and 2.31% in Control, both ***p<0.0001). (D) In ID8 tumor-bearing mice, DD chemotherapy elicited CD8+IFN-γ+ tumor-infiltrating lymphocytes (TILs). Mice bearing R ID8 tumor were treated with different formats of chemotherapies. After treatment, peritoneal cells were obtained by lavage and prepared for single-cell suspension. Cells were then stained with anti-mouse CD8 and anti-mouse IFN-γ antibodies before getting analyzed by flow cytometry. Representative flow data showed that peritoneal CD8+IFN-γ+ cells were found in greater numbers within the tumors of mice receiving DD chemotherapy (**p<0.001, control versus DD and DD versus MTD). (E) CD8+ T cells from peritoneal lavage in mice treated with different formats of chemotherapies were isolated and examined for the expression of IFN-γ and interleukin-2 (IL-2) by Q- PCR. A significant enhancement in the expressions of IFN-γ and IL-2 was noted in the peritoneal CD8+ cells in DD group mice (IFN-γ, *p<0.01, control versus DD, DD versus MTD; IL-2, *p<0.01, control versus DD, #p=0.034, DD versus MTD).

Figure 6. Patients who responded to DD chemotherapy had higher serum levels of IFN-γ and IL-2

Fourteen patients with platinum-resistant recurrence of ovarian cancer received DD chemotherapy with weekly carboplatin (AUC 2) plus paclitaxel (80 mg/m2) as their third or fourth line of treatment. Their serum levels of IFN-γ and IL-2 were measured before treatment (PreDD), one month after (DD1), and two months after (DD2) the start of DD chemotherapy. (A) Of the four patients whose disease was controlled, three had higher serum levels of IFN-γ and IL-2 (red), whereas patients with no response had low or undetectable levels of IFN-γ or IL-2 (black) (DD2 for IFN-γ, **p<0.001, responsive versus unresponsive; DD1 and DD2 for IL-2, **p<0.001 responsive versus unresponsive).