Mechanical Disruption of Tumors by Iron Particles and Magnetic Field Application Results in Increased Anti-Tumor Immune Responses

<div><p>The primary tumor represents a potential source of antigens for priming immune responses for disseminated disease. Current means of debulking tumors involves the use of cytoreductive conditioning that impairs immune cells or removal by surgery. We hypothesized that activation of the immune system could occur through the localized release of tumor antigens and induction of tumor death due to physical disruption of tumor architecture and destruction of the primary tumor <em>in situ</em>. This was accomplished by intratumor injection of magneto-rheological fluid (MRF) consisting of iron microparticles, in Balb/c mice bearing orthotopic 4T1 breast cancer, followed by local application of a magnetic field resulting in immediate coalescence of the particles, tumor cell death, slower growth of primary tumors as well as decreased tumor progression in distant sites and metastatic spread. This treatment was associated with increased activation of DCs in the draining lymph nodes and recruitment of both DCs and CD8(+)T cells to the tumor. The particles remained within the tumor and no toxicities were observed. The immune induction observed was significantly greater compared to cryoablation. Further anti-tumor effects were observed when MRF/magnet therapy was combined with systemic low dose immunotherapy. Thus, mechanical disruption of the primary tumor with MRF/magnetic field application represents a novel means to induce systemic immune activation in cancer.</p> </div

Augmented immune activation and inhibition of metastasis with MRF/magnetic treatments in comparison to cryoablation.

<p>(A) Experimental design of MRF and magnet treatments and cryoablation. BALB/c mice received 4T1 orthotopically on one side of the mammary fat pad. When tumors reached 6–7 mm in size, some mice received one of the following: PBS i.t, MRF i.t, MRF i.t and magnet treatment (5 min/day for 5 days) or cryoablation once. (B) Lung cells were collected and plated for tumor CFUs (after 4 days of magnet application). (C–D) Percentage of activated DCs (MHC II⁺ CD83⁺ of CD45⁺ CD11c⁺ CD19⁻) in the DLNs (B) and NDLNs (C). (E) Percentage of CD8(+) T cells (gated on CD3+ CD45+) in primary tumor in each group relative to 4 or 7 days of magnet treatments. Values represent the mean ± SEM of one of two experiments with similar results (n = 3–5 mice/group). One-way or Two-way ANOVA. * P<0.05, ** P<0.01, *** P<0.001, n.s: not significant.</p

DC expansion and activation after MRF and magnetic field treatment.

<p>4T1 tumors were injected into the mammary fat pad of female Balb/c mice as illustrated in Fig. 1A. Briefly, primary tumors were injected with 100 µL of 60% MRF i.t in the treatment groups, some mice received no further treatments and some mice received direct magnet application over the tumor for a total of 5 days. Control group received 100 µL PBS i.t. Five days after magnet treatments, DLN, NDLN, tumor and spleen were excised and analyzed by flow cytometry for (<b>A–B</b>) total number of nucleated cells in (<b>A</b>) DLN and NDLN or (<b>B</b>) tumor and spleen. All four organs were analyzed for CD83, MHC II expression gating on CD45⁺ CD11c⁺ CD19⁻ as follow: (<b>C–D</b>) represents both the total percentage and numbers of activated DCs in the tumor, (<b>E–F</b>) in the DLN, (<b>G–H</b>) in the NDLN and (<b>I–J</b>) in the spleen. Data (mean ± SEM) representative of one of four experiments (n = 3–4 mice/group). One-way ANOVA. * P<0.05, *** P<0.001. n.s: not significant.</p

Combination of MRF/magnetic field application with immunotherapy results in heightened systemic anti-tumor responses.

<p>4TI breast cancer cells were implanted into the mammary fat pad of BALB/c mice. Treatment was initiated when tumors reached an average volume of 6–7 mm. Mice received one of the following 5 treatments: 1) PBS i.t, 2) MRF i.t alone, 3) MRF and magnet, 4) MRF+ magnet + anti-CD40 (25 ug) and recombinant human IL2 (2.5 ×10^∧5 IU) i.p or 5) anti-CD40 and IL2 alone. Anti-CD40 was given for 5 consecutive days starting the same day as the magnetic field treatments and IL2 was administered on days 2, 5, 9 and 11 post MRF injections. (A–G) Immune effects of combination of low dose anti-CD40/IL2 with MRF/magnet after 5 days of magnet treatment and anti-CD40/IL2. (A, D) Total nucleated cells in the DLN and NDLNs, respectively. (B) Total percentage of DCs (CD11c⁺ CD19⁻ CD45⁺) in DLNs and (E) NDLNs. (C) Total CD3+ CD8(+) T cells in DLNs or (F) NDLNs or (G) in the tumor. Data representative of one of three experiments with similar results (n = 3–4 mice/group). (H) Experimental model for systemic anti-tumor effects: 4TI breast cancer cells were implanted s.c. on the right and left side of the mammary fat pad of BALB/c mice. Only the right tumors were injected with MRF or PBS. Some groups were further treated for 5 consecutive days with magnetic field. Other groups received anti-CD40 (25 ug) on the same days of magnet treatment and rh-IL2 (2.5×10⁵ IU) (days 2, 5, 9 and 11 post MRF injections). (I-J) Tumor volumes after 28 days post tumor inoculation or equivalent to 12 days after start of magnet treatments are represented for (J) primary tumors that received MRF or (J) the contralateral untreated tumors. Data representative of one of five experiments with similar results (n = 5–7 mice/group). One-way ANOVA. * P<0.05, ** P<0.01, *** P<0.001. n.s: not significant. IT: anti-CD40/IL2.</p

MRF and magnetic field application results in antigen-specific T cell accumulation.

<p>(<b>A</b>) Experimental model for antigen-specific responses following MRF and magnet treatments: BALB/c mice received Renca-HA on the right flank s.c. When tumors reached the desired size, mice first received 2×10⁶ CD8(+)-HA-Tet⁺ cells i.v. One day following Tg-CD8(+) T cell transfer, mice were administered MRF or PBS intratumor. 24 h later, some groups are left untreated or receive magnetic treatments for 5 min a day for 5 consecutive days. After 5 days of daily magnetic field treatments, tumors or tumor-DLNs were collected and assayed by flow cytometry. (<b>A</b>) Percentage and (<b>B</b>) total number of transgenic HA-Tg-CD8(+)-CD25+ T cells in Renca-HA tumor. (<b>C</b>) DLNs from the same mice in (<b>A–B</b>) were analyzed for homing of HA-Tg-CD8(+)CD25+ T cells. Data representative of one single experiment (n = 3 mice/group in PBS or MRF group and n = 5 mice in the MRF/magnet group). One-way ANOVA. ** P<0.01, *** P<0.001, n.s: not significant.</p

Inhibition of local and systemic tumor growth as a result of increased primary tumor necrosis after MRF implantation and magnetic field treatment.

<p>4T1 tumors were established into the mammary fat pad of female Balb/c mice as illustrated in Fig. 1A. When tumors reached 6–7 mm, 100 µL of 60% MRF was injected i.t (day 17) for treatment groups or 100 µl PBS in control group. One group was treated 24 hours after MRF injection with direct magnetic field application for 5 consecutive days (days 18–22). (A) Tumor volume during and after MRF/Magnet treatments. Data is representative of one of six experiments (n = 9 mice/group). (B) Total number of tumor cells by flow cytometry after 5 days of magnet treatment showing smaller tumor load after magnet treatment and (C) tumor CFUs of bone marrow. MRF/magnet treatments result in inhibition of growth of metastatic disease. (D) Representative flow staining for tubulin and 7AAD in the tumor. (E) Percentage of tubulin⁺7AAD⁻ (necrotic) cells in the tumor gated on CD45⁻ cells. (B–E) Data representative of one of two experiments with similar results (n = 3–4 mice/group). One-way or Two-way ANOVA. * P<0.05, ** P<0.01, *** P<0.001.</p

Bystander Activation and Anti-Tumor Effects of CD8+ T Cells Following Interleukin-2 Based Immunotherapy Is Independent of CD4+ T Cell Help

<div><p>We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent “bystander-activated” (CD8⁺CD44^high) T cells displaying a CD25⁻NKG2D⁺ phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4+ T cell help for antigen-specific CD8+ T cell expansion, little is known regarding the role of CD4+ T cells in antigen-nonspecific bystander-memory CD8+ T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8+ T cells upregulated PD-1 in the absence of CD4+ T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8+ T cells. Interestingly, compared to CD8+ T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8+ response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8+ T cell expansion, CD4+ T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8+ T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion.</p></div

Anti-tumor effects of IT in CD4 knockout mice.

<p>3LL tumor bearing WT or CD4 knockout (B6.129S2-CD4^tm1Mak/J) mice were treated with IT or PBS/rIgG (control) and survival and tumor growth were measured. For <i>in vivo</i> tumor studies one million 3LL cells were administered by s.c. injection into the flank of C57BL/6 mice seven days prior to initiation of therapy. (<b>a</b>) Survival. (<b>b</b>) Mean tumor volume with SEM. (<b>c–f</b>) Growth plots of individual tumors in each group. N = 12 mice per group. (*<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.001).</p

Memory CD8+ T cell function after IT in CD4+ T cell deficient models.

<p>Control or CD4 deficient (depleted or knockout) C57BL/6 mice were treated with IT or PBS/rIgG (control) and assessed for function of memory CD8+ T cells 11 days after the initiation of IT. NKG2D and granzyme B expression were quantified by flow cytometric analysis. Interferon gamma production was quantified by flow cytometric analysis after <i>in vitro</i> restimulation of splenocytes with PMA/Ionomycin (0.16/1.6 ug/ml) for one hour followed by incubation with golgi stop (0.7 ug/ml) for three hours. CD8+ T cell killing function was assayed by scintillation counting using an <i>in vitro</i> redirected lysis assay with ⁵¹Cr labeled P815 target cells incubated for 30 minutes with 10 ug/mL anti-CD3e. (<b>a,f</b>) NKG2D expression on memory CD8+ T cells in CD4 depletion (<b>a</b>) and knockout (<b>f</b>) models. Representative dot plots for NKG2D+ CD25− gating are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102709#pone-0102709-g001" target="_blank">Figure 1</a>. (<b>b</b>) Representative dot plots for IFNγ+ gating on CD8+ CD44^high cells in the spleens of CD4+ depletion model mice. (<b>c,g</b>) Interferon gamma production by memory CD8+ T cells in CD4 depletion (<b>c</b>) and knockout (<b>g</b>) models. (<b>d</b>) Representative dot plots for Granzyme B+ gating on CD8+ CD44^high cells in the spleens of CD4+ depletion model mice. (<b>e,h</b>) Granzyme B expression by memory CD8+ T cells in CD4 depletion (<b>e</b>) and knockout (<b>h</b>) models. Killing function of splenocytes from CD4 depleted (<b>i</b>) or CD knockout (<b>j</b>) mice expressed as percentage of maximal lysis. Results are representative of two (CD4 knockout) or three (CD4 depletion) independent experiments with a minimum of three mice per group. (*<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.001).</p

PD-1 expression on central and effector memory CD8+ T cells after IT in CD4+ T cell depleted mice.

<p>Control or CD4+ depleted C57BL/6 mice were treated with IT or PBS/rIgG (control). CD62L and PD-1 expression on memory T-cells was quantified by flow cytometric analysis 11 days after the initiation of IT. (<b>a,b</b>) Examples of the gating strategy for PD-1 expression on the CM and EM components of the memory CD8+ T cell compartment. The majority of cells in the memory compartment are CM in untreated mice (<b>a</b>) and EM in IT treated mice (<b>b</b>). (<b>c</b>) PD-1 expression on CM and EM cells. (<b>d</b>) The composition of the memory CD8+ T cell compartment in control or CD4 depleted mice treated with IT or PBS/rIgG. PD-1+ CM cells (<b>e</b>) and PD-1+ EM cells (<b>f</b>) as a percentage of the total memory CD8+ T cell compartment. Results are representative of two to three independent experiments with 3 mice per group (*<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.001, ****<i>P</i><.0001).</p