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

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

Transcriptome of interstitial cells of Cajal reveals unique and selective gene signatures

<div><p>Transcriptome-scale data can reveal essential clues into understanding the underlying molecular mechanisms behind specific cellular functions and biological processes. Transcriptomics is a continually growing field of research utilized in biomarker discovery. The transcriptomic profile of interstitial cells of Cajal (ICC), which serve as slow-wave electrical pacemakers for gastrointestinal (GI) smooth muscle, has yet to be uncovered. Using copGFP-labeled ICC mice and flow cytometry, we isolated ICC populations from the murine small intestine and colon and obtained their transcriptomes. In analyzing the transcriptome, we identified a unique set of ICC-restricted markers including transcription factors, epigenetic enzymes/regulators, growth factors, receptors, protein kinases/phosphatases, and ion channels/transporters. This analysis provides new and unique insights into the cellular and biological functions of ICC in GI physiology. Additionally, we constructed an interactive ICC genome browser (<a href="http://med.unr.edu/physio/transcriptome" target="_blank">http://med.unr.edu/physio/transcriptome</a>) based on the UCSC genome database. To our knowledge, this is the first online resource that provides a comprehensive library of all known genetic transcripts expressed in primary ICC. Our genome browser offers a new perspective into the alternative expression of genes in ICC and provides a valuable reference for future functional studies.</p></div

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

Identification of <i>Thbs4</i> as ICC-specific.

<p>(A) A genomic map view of three <i>Thbs4</i> variants (V1-3) expressed in JICC and CICC. Three alternative start exons (E1, E8 and E15) are circled (red). (B) Expression levels of total <i>Thbs4</i> mRNAs in JICC and CICC. (C) Expression levels of <i>Thbs4</i> transcriptional vaiants in JICC and CICC. (D) PCR validation of <i>Thbs4</i> exons with different transcriptional initiation sites in isolated ICC and in the muscularis of the murine jejunum and colon. NTC is non-template control. Primer sets were designed from variant exons in the regions of interest (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176031#pone.0176031.s020" target="_blank">S11 Table</a> for primer sequences). (E) Expression levels of V1, V1+V2, and V1+V2+V3 mRNAs in isolated ICC and muscularis of the murine jejunum and colon were measured by qPCR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176031#pone.0176031.s020" target="_blank">S11 Table</a> for primer sequences). Expression levels of <i>Thsb4</i> variants were normalized using the endogenous control, <i>Gapdh</i>. (F) Western blot analysis using an N-terminal THBS4 antibody, showing THBS4 protein expressed in the muscularis of the murine jejunum and colon (1, 1 month and 2, 2 months). (G) Detection of THBS4 protein in colonic and jejunal ANO1⁺ ICC-MY, ICC-SMP, and ICC-DMP. Cryosection images (top 2 panels) showing THBS4 was detected in ANO1⁺ ICC-MY (arrows in colon and jejunum), ICC-SMP (arrow heads in colon) and ICC-DMP (arrow heads in jejunum). Whole mount images (bottom 2 panels) also showing the protein was found in ANO1⁺ ICC-MY and ICC-DMP. All scale bars are 50 μm.</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

Identification of ICC-specific <i>Hcn4</i>.

<p>(A) A genomic map view of <i>Hcn4</i> variants expressed in JICC and CICC. Three alternative initial exons (V1-3) are circled in red and the differential last exon (E8) is boxed in blue. (B) Expression levels of total <i>Hcn</i> isoform genes in JICC and CICC. (C) Expression levels of <i>Hcn4</i> transcriptional vaiants in JICC and CICC. (D) A topological map of HCN4 variants. Each circle denotes a single amino acid. Colors on amino acid sequence show distinct regions and domains. Green represents start codons found in alternatively initiated variants (V1-3). Six transmembrane domains (S1-6) and a pore region are shown. Red represents voltage sensor residues in S4. Two cAMP binding sites are in purple. (E) Western blot analysis showing HCN4 protein expressed in multiple tissues including stomach, jejunum, and colon muscularis. (F) Cryosection images of HCN4 protein in jejunum and colon. HCN4 was detected in the KIT⁺ serosal layer, myenteric plexus, and deep muscular plexus in jejunum. It was also detected in the serosal layer and submuscular plexus in colon with much lower levels. (G) Whole mount images of HCN4 protein in jejunum (top panels) and colon (bottom panels) showing the protein found in KIT⁺ ICC-SS, ICC-MY, and ICC-DMP in jejunum, and KIT⁺ ICC-SS AND ICC-SMP in colon. All scale bars are 50 μm.</p

Identification of ICC-specific genes through expression profiles and histone modifications.

<p>(A) Specificity of ICC-enriched genes. Cell specificity was determined by comparative analysis of gene expression profiles among ICC, SMC, and PDGFRα⁺ cells: ICC^{expression level (FPKM)}/[SMC^{expression level (FPKM)} + PDGFRα⁺ cells^{expression level (FPKM)}]. (B and C) Comparison of JICC- and CICC-enriched gene expression. (D) Comparison of histone modifications on ICC-enriched genes. The signal value is the average of the mininum and maxium values of the ChIP-seq signals in the small intestine for each histone modification.</p